Synthesis and Evaluation of Chirally Defined Side Chain Variants of 7-Chloro-4-Aminoquinoline To Overcome Drug Resistance in Malaria Chemotherapy

ABSTRACT

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

INTRODUCTION

Malaria is a major infectious disease affecting mainly tropical and subtropical areas. Of the five species of Plasmodium which are responsible for human malaria, Plasmodium falciparum causes the most severe form of the disease (1). There were an estimated 207 million malaria cases in 2012 and an estimated 627,000 deaths; 90% of malaria deaths occur in sub-Saharan Africa, and 77% occur in children under 5 years of age (2). For several decades, chloroquine (CQ), a 4-aminoquinoline, has been the frontline drug in malaria chemotherapy because of its therapeutic efficacy, ease of use, and low cost (3, 4). However, due to the emergence of P. falciparum and P. vivax strains resistant to commonly used drugs, such as CQ, amodiaquine, and artemisinin (Fig. 1), there is an urgent need to develop new chemical entities with the goal of overcoming parasite resistance.

FIG 1.

Antimalarial drugs.

Aminoquinolines, such as chloroquine, were the most important antimalarials for more than 4 decades because of their efficacy against all species of human malaria parasites and their safety profile. The emergence of resistance to this class of compounds during the 1980s created a genuine health crisis in the developing world. Studies toward elucidation of the mechanism of resistance and general trends emerging from structure-activity relationship (SAR) studies revealed that chloroquine resistance does not involve any change to the target of this class of drugs but involves compound-specific efflux mechanisms (5). Based on this premise, a number of research groups have developed short-chain analogs of 4-aminoquinoline which are active against CQ-resistant (CQ-R) strains of P. falciparum. However, these derivatives undergo a biotransformation (dealkylation) that significantly affects the lipid solubility of the drug and decreases the biological efficacy (6). In view of this background information, it was surmised that a focused library of small molecules based on a 4-aminoquinoline scaffold with suitable functionalities would result in molecules with improved antiplasmodial activity against CQ-resistant parasites. Therefore, modifications employing side chain refinement and conformational rigidity were considered. The seminal finding of our study is that a new chemical entity with significant activity against CQ-resistant parasites has been identified, and the results are discussed here.

Stocks et al. synthesized and evaluated a series of short-chain CQ derivatives, by replacement of diethyl amino function with more metabolically stable side chain (tert-butyl) as well as heterocyclic ring (piperidyl, pyrrolidino, and morpholino) modifications, that led to a substantial increase in antimalarial activity against CQ-resistant parasite strains (7). Madrid et al. replaced diethyl amino functionality by using one propyl group as a constant and replacing the other ethyl group with a bulky or aromatic ring, and the results indicated that some of these analogs are active against multidrug-resistant (MDR) strains (8). Some annals suggest that 4-aminoquinoline analogs with an altered side chain, such as N′-(7-chloro-quinolin-4-yl)-N,N-diethyl-propane-1,3-diamine, show potential as leads for the development of new drugs (9). Ryckebusch et al. evaluated a new series [1,4-bis(3-aminopropyl) piperazine derivatives] against CQ-resistant strains of P. falciparum. The compounds displayed moderate to good activity when quinoline and/or aryl moieties were attached to the above-mentioned linker. In this series, compounds containing a piperazine moiety were found to be active against CQ-resistant strains of P. falciparum (10–12).

Based on these annotations, Solomon et al. from our laboratory previously explored different modifications at the pendant nitrogen of the CQ lateral side chain that led to compounds with improved activity, particularly against CQ-resistant strains (13–18). By taking into account the above facts, Sinha et al. from our laboratory more recently developed a generic methodology for the synthesis of chiral chloroquine and its analogs. The key feature of this methodology is that it enables the use of amino acids to generate 4-aminoquinolines with chirally defined substituted side chains and also to address the role of hydrophobic substitution at the chiral center. It was inferred from the antiplasmodial activity data that analogs containing N-methylpiperazine at the terminal part of the side chain show excellent in vitro activity against resistant strains with reference to CQ. Sinha et al. also examined chain length variation by homologation of selected α-amino acids to obtain the corresponding β³- and γ-amino acids (19). Most of the derivatives displayed excellent in vitro antiplasmodial activity, and a few compounds in the in vivo studies showed 100% parasitemia suppression on day 4 (20). The objective of the present study was to synthesize a new series of 4-aminoquinoline derivatives with reduced side chain amide bonds, with a view to increase in vivo stability, and also to synthesize compounds with different substitutions at the chiral center. These compounds have been evaluated for antiplasmodial activity against chloroquine-sensitive (CQ-S) and chloroquine-resistant (CQ-R) strains, and the results are reported below.

RESULTS AND DISCUSSION

Chemistry.

The target compounds envisaged in the present study, with the general structure shown in Fig. 2, were obtained by strategies using α-amino acids and β-amino acids as shown in the schemes depicted in Fig. 3 and 4.

FIG 2.

General structure of synthesized compounds.

FIG 3.

Synthesis of compounds 7a to 7r (scheme 1). Reagents and conditions are as follows. (i) (Boc)₂O, NaOH-dioxane, 0°C, 2 to 3 h, and/or benzylchloroformate, NaOH, 0°C, 2 to 3 h. (ii) NMM, IBCF, NaBH₄, dry THF, −15°C, 1 h. (iii) Triethylamine, methane sulfonyl chloride, THF, 45 min. (iv) N-Methylpiperazine, acetonitrile, N₂, 48 h. (v) 20% HCl-dioxane and/or H₂/Pd, methanol, 1 h. (vi) 4,7-Dichloroquinoline and/or 4-chloro-7-(trifluoromethyl)quinoline amines (compounds 6a to 6j), phenol, 140 to 155°C, 4 to 6 h.

FIG 4.

Synthesis of compounds 15a to 15c (scheme 2). Reagents and conditions are as follows. (i) Benzylchloroformate, NaOH, 0°C, 2 to 3 h. (ii) NMM, IBCF, CH₂N₂, dry THF, −15°C, 1 h. (iii) Silver benzoate, methanol, 70°C, 1 h. (iv) Methanol, NaBH₄, THF, 55 to 60°C, 45 min. (v) Triethylamine, methane sulfonyl chloride, THF, 45 min. (vi) N-Methylpiperazine, acetonitrile, N₂, 2 days. (vii) H₂/Pd/C, methanol, 1 h. (vii) 4,7-Dichloroquinoline and/or 4-chloro-7-(trifluoromethyl)quinoline amines (compounds 14a and 14b), phenol, 140 to 155°C, 4 to 6 h.

Synthesis of compounds 7a to -r.

Synthesis of compounds 7a to -r involved the following steps, starting from the preparation of Boc- and/or Cbz-protected α-amino acids. Compounds 1a to -j were converted to the corresponding Boc and/or Cbz derivatives 2a to -j in quantitative yields (20, 21). The Boc/Cbz-protected amino acids were reduced to the corresponding alcohols by use of a mixed-anhydride protocol (22). Boc/Cbz amino alcohols were subjected to mesylation to afford compounds 4a to -j in good yields (22). The mesylated products were treated with N-methylpiperazine under a nitrogen atmosphere to give compounds 5a to -j (23). Boc deprotection was done by using 20% HCl-dioxane at room temperature in quantitative yields, and the Cbz group was removed by using 10% Pd/C catalyst to afford free amines 6a to -j (20, 22). Compounds 6a to -j thus obtained were fused to 4,7-dichloroquinoline or 4-chloro-7-(trifluoromethyl)quinoline in the presence of phenol to obtain compounds 7a to -r (24) (Fig. 3).

Synthesis of compounds 15a to -c.

Synthesis of compounds 15a to -c involved the following steps, starting from the preparation of N-Cbz-protected amino acids (scheme 2). Amino acids 1a and -i were converted to the corresponding N-Cbz derivatives 8a and -b in quantitative yields (21, 25). N-Cbz-protected amino acids were converted into the corresponding β-amino esters by the Arndt-Eistert reaction. The reaction involved two steps. In the first step, the Cbz-amino acids were converted into the corresponding diazoketones 9a and -b by a mixed-anhydride reaction and treatment of the mixed-anhydride intermediate with diazomethane in tetrahydrofuran (THF) at −15°C. Diazoketones were purified by silica gel column chromatography and characterized by ¹H nuclear magnetic resonance (NMR) analysis. Purified diazoketone derivatives were converted into the corresponding β-amino esters via Wolf rearrangement in the presence of silver benzoate to give compounds 10a and -b (26). The β-amino esters were converted to β-amino alcohols 11a and -b (27), and subsequently the alcohols were transformed to mesylates 12a and -b, followed by replacement of the mesyl group with N-methylpiperazine to obtain compounds 13a and -b (23). The N-Cbz protecting group was deprotected by using Pd/C to give amines 14a and -b (22). The amines so obtained were fused with 4,7-dichloroquinoline or 4-chloro-7-(trifluoromethyl)quinoline in the presence of phenol, resulting in compounds 15a to -c (24). The details of the synthetic steps and the reaction conditions are depicted in Fig. 4 (scheme 2).

Preparation of phosphate salt of compound 7g (compound 16).

A cold solution of 400 mg of compound 7g (>99% pure) in methanol (MeOH) was added to a methanolic solution of phosphoric acid made from 85% phosphoric acid (3.0 eq). After complete conversion of free base as seen by thin-layer chromatography (TLC), isopropyl alcohol was added to the reaction mixture and the phosphate salt separated as oil. The alcohol layer was decanted; acetone was added to the oil and triturated/stirred until it solidified. The mixture was then filtered, and the phosphate salt thus obtained was washed with acetone and quickly placed in a vacuum desiccator. Further, it was inferred by differential scanning calorimetry (DSC) (Fig. 5) that no free base (7g) was present in the phosphate salt (compound 16). The yield was quantitative, with a melting point (mp) of 195 to 198°C.

FIG 5.

DSC curve for compound 7g and its phosphate salt (compound 16).

In vitro antiplasmodial activity.

The synthesized compounds 7a to -r and 15a to -c were screened against the 3D7 (CQ-S) and K1 (CQ-R) strains of P. falciparum in vitro for antiplasmodial activity, and the results are presented in Table 1. Most of the compounds displayed excellent antiplasmodial activity, in the nanomolar range. As expected, reduction of the amide bond led to a substantial increase in antiplasmodial activity against both strains. Our data (Table 1) suggest that compounds derived from glycine, leucine, and phenylalanine, i.e., 7a, 7b, 7g, 7j, 7o, and 7p, displayed similar antiplasmodial activities against both strains when the chloro substituent was replaced with a trifluoromethyl group, whereas some compounds exhibited moderate to substantial differences in antiplasmodial activity.

TABLE 1.

Biological and biophysical data on the synthesized compounds^e

^a Minimum concentration of compound inducing 50% parasitic cells.

^b Selectivity index (SI) = IC₅₀ for cytotoxicity to Vero cells/IC₅₀ for antimalarial activity.

^c Log K value for 1:1 (compound:hematin) complex formation in 40% aqueous DMSO, 20 mM HEPES buffer, pH 7.5, at 25°C. Data are expressed as means ± SD for at least three different experiments performed in duplicate.

^d The IC₅₀ represents the millimolar equivalents of test compounds, relative to hemin, required to inhibit β-hematin formation by 50%. Data are expressed as means ± SD for at least three different experiments.

^eND, not done.

Among the 21 compounds tested (7a to -r and 15a to -c), 14 compounds, namely, 7a to -c, 7e, 7g to -k, 7o to -q, 15a, and 15c, exhibited potent antiplasmodial activity, in the range of 3.27 to 25.1 nM, against the CQ-S strain, with a 50% inhibitory concentration (IC₅₀) range of 9.79 to 167.4 nM against the CQ-R strain. Seven compounds, 7d, 7f, 7l to -n, 7r, and 15b, displayed antiplasmodial activity against the CQ-S strain in the IC₅₀ range of 81.22 to 723 nM. One compound, 7r, showed comparable activity against the CQ-R strain, with an IC₅₀ of 259 nM. Two compounds, 7d and 15b, exhibited IC₅₀s of 548 and 585 nM, and four compounds, 7f and 7l to -n, demonstrated no detectable antiplasmodial activity against the K1 strain at the highest concentration tested (IC₅₀ > 1,000 nM). On the other hand, compounds with an iso-butyl group (7g to -j) or a benzyl group (7o, 7p, and 15c) at the chiral center were found to be the most active in this series. Moreover, the enantiomeric pair of 7g and 7h and the racemic compound 7i did not show any difference in the activities against both the 3D7 and K1 strains. There was a >1.7-fold increase in the activity against the CQ-S strain for compound 7a (3.27 nM) compared to CQ (5.46 nM). In fact, compounds 7g to -j, 7p, 15a, and 15c exhibited almost 20- to 28-fold increases in the activity against the CQ-resistant strain, with IC₅₀s of 13.57, 11.16, 11.79, 12.13, 11.42, 9.79, and 11.52 nM, compared to that of chloroquine. In this study, we also explored the effect of chain length variation by homologation of selected α-amino acids to obtain the corresponding β-amino acids. Increases in chain length showed positive effects on the antiplasmodial activities against both the 3D7 and K1 strains of P. falciparum. A compound derived from glycine (15a) (IC₅₀, 9.79 nM) showed 10-fold higher activity than that of CQ in the case of the resistant strain, whereas in the case of phenylalanine (15c) (IC₅₀, 11.52 nM), the activity was increased 4-fold with respect to that of CQ.

In vitro cytotoxicity.

The cytotoxicity of all the synthesized molecules against the Vero cell line was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Table 1). Our target compounds showed selectivity indices ranging from 83.20 to 37,281. Compounds 7a, 7g, and 15c exhibited excellent selectivity indices, i.e., 37,281, 22,727, and 9,852, respectively, whereas compounds 7e, 7k, and 7p displayed selectivity indices of 8,538, 7,026, and 8,024, respectively, compared to chloroquine. Thus, these compounds demonstrated the most promising safety profiles.

Biophysical studies.

It is well established that the mode of action of 4-aminoquinoline-based antimalarial compounds, such as chloroquine, is by interaction with heme, leading to inhibition of hemozoin formation. The association constant (log K) for the drug-ferriprotoporphyrin (FP) ring provides valuable information about the antimalarial activity of the synthesized molecules. Another biophysical study which involves the in vitro inhibition of hemozoin formation provides the possible mode of action of the synthesized compounds. This involves inhibition of in vitro polymerization of hematin to β-hematin. The term β-hematin is used for chemically or in vitro-synthesized hemozoin pigments, while the term hemozoin is used for the biosynthetic malaria pigment. The inhibition of β-hematin formation takes place due to blockage of the growing face of the crystal by a capping effect by the majority of CQ-like compounds.

(i) Association constant for hematin and 4-aminoquinoline derivatives (log K).

Heme interaction has remained the unequivocal target for antimalarial activity of the 4-aminoquinoline class of compounds. Accordingly, the association constant (log K) of this interaction was calculated by titrating hematin with different concentrations of compounds in a 40% dimethyl sulfoxide (DMSO) solution, and the log K values obtained were in the range of 4.23 to 6.37. There was a linear correlation between the induced hypochromic effects and the concentrations of the compounds. Among all the compounds reported in the present study, compounds 7c, 7e, 7g, 7h, 7k, 7o, 7q, 15a, and 15c have shown very strong binding to hematin. This result is concurrent with the generally accepted mechanism of action of this class of compounds.

(ii) Assay of inhibition of β-hematin formation.

The results presented in Table 1 indicate that the derivatives inhibited β-hematin formation in a concentration-dependent manner. The IC₅₀s calculated for all the compounds were in the range of 0.14 to 0.27 mM. Most of the synthesized compounds were good inhibitors of β-hematin formation, and some of them showed moderate antimalarial activity against CQ-S and CQ-R strains of P. falciparum. The most potent inhibitors in the hemozoin inhibition assay were compounds 7a, 7c, 7e, 7g to -k, 7o to -q, 15a, and 15c. These results are consistent with the observed antiplasmodial activity.

In vitro efficacy of compound 7g.

From the data presented in Table 1, it may be inferred that the enantiomeric pair of 7g and 7h and the racemic compound 7i displayed excellent antiplasmodial activity against the CQ-R strain. Additionally, we also evaluated the in vitro parasite killing efficacy against CQ-S and CQ-R strains. In the case of sensitive parasites, after 12 h and 24 h, a marginal difference was observed compared to the efficacy of CQ, whereas in the case of resistant parasites, the lead molecule 7g rapidly killed the parasite, which is a major prerequisite for antimalarial drug-like candidates. It displayed an excellent IC₉₀ against the K1 strain. The results are shown in Table 2 and Fig. 6.

TABLE 2.

In vitro efficacy of compound 7g

Compound and parameter	Value (nM) for strain
	3D7	K1
7g
IC50	6.7–11.1	11.8–20.4
IC90	23.4–52.9	24.3–47.8
CC50	>200 μM
CQ
IC50	3.9–7.0	220–279
IC90	11.8–22.3	477–675
CC50	125 μM

FIG 6.

In vitro efficacy of compound 7g compared to CQ.

In vivo antimalarial activity.

On the basis of in vitro potency and structural diversity, compounds 7a, 7b, 7g to -j, 7o, 7p, 15a, and 15c were tested for in vivo activity against chloroquine-resistant P. yoelii (strain N-67) in albino mice of the Swiss background. Initially, the in vivo activities of selected molecules were determined through the oral route, using a dose of 100 mg/kg of body weight administered once daily for four or seven consecutive days postinfection and monitoring parasitemia reduction and survival of mice until day 28 postinfection. Parasitemia reductions for multidose regimens are reported in Table 3. All compounds were administered as hydrochloride salts, except for compound 7g, which was administered as a hydrochloride salt and a phosphate salt (compound 16).

TABLE 3.

In vivo antimalarial activity against CQ-resistant strain N-67 in albino mice of the Swiss background

Compound	Dose (mg/kg [× indicated time]) (p.o.)c	% suppression on day 4	Survivala	Cureb
7a	100 × 7 days	100	5/6	0/6
	50 × 7 days	100	4/6	0/6
7b	100 × 7 days	100	5/5	0/5
	50 × 4 days	99.9	5/5	0/5
7g	100 × 7 days	100	5/5	5/5
	50 × 7 days	100	5/5	5/5
	25 × 7 days	100	5/5	5/5
	25 × 4 days	100	5/5	5/5
	10 × 4 days	100	5/5	4/5
7h	100 × 7 days	100	5/5	5/5
	50 × 7 days	100	5/5	5/5
	25 × 7 days	100	5/5	5/5
	10 × 4 days	100	5/5	3/5
7i	50 × 7 days	100	5/5	5/5
	25 × 7 days	100	5/5	5/5
	10 × 4 days	100	5/5	5/5
7j	100 × 4 days	100	5/5	5/5
	25 × 4 days	100	5/5	5/5
	12.5 × 4 days	100	4/5	2/5
7o	100 × 7 days	100	5/6	4/6
	50 × 7 days	100	0/5	0/5
7p	100 × 4 days	100	5/5	5/5
	50 × 4 days	100	3/5	2/5
	25 × 4 days	100	0/5	0/5
15a	100 × 7 days	100	5/5	5/5
	50 × 7 days	99.9	3/5	0/5
15c	100 × 7 days	100	5/5	5/5
16	25 × 4 days	100	5/5	5/5
	10 × 4 days	100	8/8	8/8
	12.5 × 4 days	100	5/5	5/5
	6.25 × 4 days	100	5/5	2/5
	5 × 4 days	100	6/8	0/8
CQ	20 × 4 days	99.0	5/5	0/5
Arteether	5 × 4 days (i.m.)	100	5/5	5/5

^aNumber of mice that survived until day 28 postinfection/total number of mice in the group.

^bNumber of mice without parasitemia (cured) through day 28 postinfection/total number of mice in the group.

^cp.o., oral; i.m., intramuscular.

Compound 7a showed 100% parasitemia suppression on day 4 at doses of 100 and 50 mg/kg given for 4 days, with survival rates of 83 and 66%, respectively, up to day 28, but none of the animals were cured. While compound 7b displayed 100% parasitemia suppression on day 4 at a dose of 100 mg/kg given for 4 days, at 50 mg/kg the same compound displayed 99.9% parasitemia suppression and all the mice survived up to day 28, but none of them were cured. The enantiomeric pair compounds 7g and 7h and the racemic compound 7i showed 100% parasitemia suppression at doses of 100, 50, and 25 mg/kg, and all mice survived to day 28 as well as being cured. At a dose of 10 mg/kg, the racemic compound (7i) displayed 100% survival and curative effect, whereas the enantiomeric pair (7g and 7h) exhibited a 100% survival rate and 80% and 60% curative effects, respectively. The trifluoro-substituted compound (7j) showed 100% parasitemia suppression on day 4 at doses of 100 and 25 mg/kg, and all mice survived and were cured at the same dose levels. However, the same compound at 12.5 mg/kg exhibited 100% parasitemia suppression on day 4, with four of five mice surviving and two of five mice being cured. These results are encouraging compared to those with the standard drug CQ, which showed a 90% curative effect at a dose of 100 mg/kg but no curative effect at a dose of 25 mg/kg. Compound 7o was also administered in multiple dose regimens of 100 and 50 mg/kg, and at these two doses, this compound showed 100% parasitemia suppression on day 4. At 100 mg/kg, five of six mice survived and four of six mice were cured, while at 50 mg/kg none of the mice survived to day 28. Compound 7p exhibited 100% inhibition of parasitemia on day 4 at multiple doses (100, 50, and 25 mg/kg) given for four consecutive days, but at 100 mg/kg, all mice survived and were cured, while at 50 mg/kg, three of five mice survived and two mice were cured, and at 25 mg/kg, none of the mice survived. Compound 15a inhibited 100% of parasitemia on day 4, all mice survived to day 28, and all mice were cured, while at the 50-mg/kg dose, there was 99.9% suppression of parasitemia and a 60% survival rate, and none of the mice were cured. Compound 15c at a dose of 100 mg/kg showed 100% parasitemia suppression on day 4, all mice survived up to day 28, and all mice were cured.

Compound 16, which is a phosphate salt of compound 7g and was administered orally at doses of 25, 12.5, 10, 6.25, and 5 mg/kg, showed a curative effect at doses of 25.0, 12.5, and 10.0 mg/kg. At lower doses, there was 100% suppression of parasitemia but no curative effect. The effective curative dose of compound 16 is 10.0 mg/kg. It may be inferred from the above-mentioned data that compound 16 is nearly 2 times more active than its corresponding free base compound 7g, possibly because of improved gastrointestinal (GI) absorption. This is consistent with the pharmacokinetic data discussed below.

Dose-response studies were done for the enantiomeric pair of 7g and 7h and the racemic compound 7i against a chloroquine-resistant strain of P. yoelii (N-67), and the results are shown in Table 4. There was no difference observed in the in vivo activities of the (S)- and (R)-isomers and the racemic compound. Therefore, compound 7g was chosen for preclinical studies, considering the easy availability of l-amino acids and the statutory requirement of a chirally defined center as opposed to a racemate in the drug discovery chain.

TABLE 4.

Dose-response studies of an enantiomeric pair (7g and 7h) and a racemic compound (7i) against P. yoelii strain N-67, a chloroquine-resistant strain^a

Compound	Dose (mg/kg [× 4 days]) (oral route)	No. of cured mice/no. of treated mice
7g [(S)-isomer]	100	5/5
	50	5/5
	25	5/5
	10	4/5
7h [(R)-isomer]	100	5/5
	50	5/5
	25	5/5
	10	3/5
7i (racemic compound)	100	5/5
	50	5/5
	25	5/5
	10	5/5

^aThere was 100% suppression on day 4 for all compound and dose regimens tested.

In order to evaluate the broad spectrum of activity of the identified molecule 7g, its in vivo activity against a multidrug-resistant (MDR) strain was carried out. Based on their in vivo potency against the P. yoelii strain (N-67), compounds 7g to -i were further selected for determination of their in vivo antimalarial activity against a P. yoelii multidrug-resistant strain in albino mice of the Swiss background (Table 5). Initially, compound 7g, which is an (S)-enantiomer, was screened at multiple doses via the oral route in the form of a hydrochloride salt at doses of 100, 50, and 25 mg/kg given for 7 days. This compound suppressed 100% of parasitemia on day 4 at all doses, all mice survived and were cured at 100 and 50 mg/kg, and at the dose of 25 mg/kg, three of five mice were cured. Similar results were obtained with the (R)-enantiomer 7h and the racemic compound 7i. In addition to this, the broad spectrum of activity of 7g was evaluated against P. vinckei, which gives a model very close to the human disease. The results are shown in Tables 5 and 6.

TABLE 5.

In vivo antimalarial activity against a P. yoelii MDR strain in albino mice of the Swiss background

Compound	Dose (mg/kg [× indicated time])	% suppression on day 4	Survivala	Curedb
7g	100 × 7 days	100	5/5	5/5
	50 × 7 days	100	5/5	5/5
	25 × 7 days	100	3/5	3/5
	25 × 4 days	99.9	0/5	0/5
7h	50 × 7 days	100	5/5	5/5
7i	50 × 7 days	100	3/5	3/5
CQ	20 × 7 days	99.3	3/5	0/5

^aNumber of mice that survived until day 28 postinfection/total number of mice in the group.

^bNumber of mice without parasitemia (cured) through day 28 postinfection/total number of mice in the group.

TABLE 6.

In vivo antimalarial activity of compound 7g [(S)-isomer] against different rodent malaria models

Parasite	Drug sensitivity	Total curative dose (mg/kg [× 4 days])
P. yoelii	CQ resistant	25
	MDR	50
P. vinckei	CQ resistant	25

Screening against Plasmodium cynomolgi-rhesus monkey model.

Dose-response studies with compound 7g against a simian model showed that a regimen of 10 mg/kg given 3 times was curative against P. cynomolgi in monkeys. Four treated animals with initial parasitemia levels of 8,000 to 15,000/mm³ showed parasite clearance within 48 h, and no recrudescence was recorded during the 70-day posttreatment observation period. Treatment of two monkeys with a regimen of 5 mg/kg given 3 times showed parasite clearance in 72 h. While one of the monkeys showed recrudescence on day 13, the other was cured. A chloroquine regimen of 10 mg/kg given 3 times is also curative in this model.

Chromatographic conditions for checking the chiral purity of 7g and 7h.

The lead molecule 7g identified in the present study has a chiral center; it was therefore important to establish its chiral purity before proceeding further with preclinical investigations. Toward this objective, a high-pressure liquid chromatography (HPLC) method was successfully developed. HPLC separation of compound 7g and its enantiomer, 7h, was achieved on a Lux 5μ cellulose-1 (250 mm × 4.60 mm × 5 μm) chiral column (Phenomenex) at 25 ± 3°C, utilizing a mobile phase consisting of a mixture of hexane, isopropanol, and methanol (95:4.5:0.5) with the addition of triethylamine (TEA) (0.8%), at a flow rate of 2.0 ml/min and with a detection wavelength of 254 nm. Injections were given in triplicate for each isomer and the racemate. The HPLC profiles of the enantiomers vis-à-vis the racemate clearly suggest that compounds 7g and 7h are chirally pure and that there is no cross contamination of the enantiomers (Fig. 7).

FIG 7.

HPLC spectra of the enantiomers 7g and 7h and the racemic compound 7i.

Pharmacokinetic studies. (i) In vitro stability studies.

The in vitro pharmacokinetic data are shown in Table 7. To evaluate the stability of compound 7g under different conditions encountered after oral administration, in vitro simulated gastric fluid (SGF), simulated intestinal fluid (SIF), metabolic, and plasma stability studies were performed. The results of these studies indicate that the candidate molecule is stable in the GI tract before being absorbed. The compound was found to be more than 97% stable under both acidic (SGF) and basic (SIF) conditions for up to 2 h. In vitro metabolic stability assays were performed in rat liver microsomes to assess the contribution of the liver to the total clearance of the compound from the body. Testosterone was used as a positive control to assess the activity of the microsomes. The half-life of testosterone was in agreement with literature reports. The half-life of 7g was found to be 154.54 min. The compound was found to have a low clearance. The plasma stability of 7g was found to be 96.13% after 2 h (Table 7).

TABLE 7.

In vitro pharmacokinetic parameters of compound 7g

Parameter	Value (n = 3)
Simulated gastric fluid stability (% remaining, 2 h)	96.03 ± 2.29
Simulated intestinal fluid stability (% remaining, 2 h)	97.72 ± 1.64
Plasma stability (% remaining, 2 h)	96.14 ± 3.15
Metabolic stability (half-life [t1/2]) (min)	154.54 ± 15.67

(ii) In vivo pharmacokinetics.

The in vivo pharmacokinetic studies were performed using plasma as the matrix. This decision was made after preliminary studies in plasma as well as blood as a bioanalytical matrix. The biological levels were comparable, and we could study the complete pharmacokinetic profile in plasma. Whole-blood partitioning (erythrocyte uptake studies) and plasma and blood stability studies using blood originating from healthy as well as infected mice as the matrix were also performed to check the suitability of plasma as a matrix (data not given). The other reason for choosing plasma was to obtain much cleaner and reproducible results for the pharmacokinetic studies, as we could eliminate interfering matrix. The analytical method used for the analysis was sensitive enough to detect systemic levels for up to 2 days of exposure, which suggests the usefulness of plasma as a matrix. The mean plasma concentration-time profile of 7g is shown in Fig. 8. Upon oral administration, 7g was rapidly absorbed and showed a double-peak phenomenon in the plasma concentration-time profile (Fig. 8). The peak plasma concentrations of 7g were 20.36 ± 13.92 and 22.3 ± 9.18 ng/ml at 0.44 ± 0.38 and 3.67 ± 0.58 h, respectively. This behavior may be due to solubility constraints of the compounds or absorption from multiple sites and enterohepatic recirculation. The oral bioavailability (%F) of 7g was found to be 25.30%. The discrepancy in the bioavailability data may be because of the poor solubility of 7g. Therefore, it was considered appropriate to prepare the phosphate salt of 7g (compound 16), and as expected, the pharmacokinetic parameters were significantly improved compared to those of the free base (7g). The mean plasma concentration-time profile of compound 16 is shown in Fig. 8. Upon oral administration, the salt form was rapidly absorbed and showed the double-peak phenomenon in the plasma concentration-time profile (Fig. 8), similarly to its free base. In the case of the salt form (compound 16), the peak plasma concentrations were 104.9 ± 5.46 and 47.5 ± 1.83 ng/ml at 0.25 and 4 h, respectively, indicating improved absorption from the intestine. The oral bioavailability (%F) of 7g was found to be 64.47% when the salt form (compound 16) was administered. This indicates that the salt form has better pharmacokinetic properties than the free base.

FIG 8.

Intravenous (IVPK; 10 mg/kg) and oral (SDOPK; 15 mg/kg) pharmacokinetic profiles of compounds 7g and 16 in male Sprague-Dawley rats (n = 4).

Pharmacological safety, toxicity, detailed pharmacokinetic (including multiple-dose pharmacokinetic, tissue distribution, and excretion studies), and toxicokinetic studies are in progress. In vitro hERG assay results indicate that compound 16 (phosphate salt of compound 7g) does not bind to the hERG ion channel at concentrations of up to 10 μM. However, we observed binding at the highest tested concentration, i.e., 33 μM. E-4031, a known hERG ligand, was used as a positive control in the assay. These studies are part of our next report, which addresses mainly regulatory considerations and investigational new drug (IND)-enabling endeavors.

Molecular docking studies.

The ability of compound 7g to form a complex with Fe(III)FPIX was investigated by molecular modeling studies. A database was prepared by using the SYBYL-X 1.3 (Tripos Inc., St. Louis, MO) modeling package. Compound 7g was drawn via the sketch module in SYBYL. Structures were minimized further by adding Gasteiger-Huckel charges along with distance-dependent dielectric and the Powell conjugate gradient algorithms, with a convergence criterion of 0.001 kcal/mol. All structures were put into a database and finally aligned with each other by way of the “fit atom” method. The obtained docking values are reported in Table 8. As shown in Fig. 9, chloroquine binds with the carboxylic acid of the porphyrin ring via single hydrogen bonding with a C score of 4, whereas compound 7g interacts with another free carboxylic group of heme with a C score of 5. The docking results complement the biophysical data, confirming that the mechanism of antiplasmodial activity is through heme binding.

TABLE 8.

Docking scores for compound 7g and for CQ

Compound	Total score	Crash score	Polarity	D score	PMF score	G score	Chem score	C score	Global score
7g	1.31	−0.87	1.36	161.25	−35.35	−87.17	−19.64	5	5
CQ	1.78	−0.79	1.47	130.29	−19.22	−70.26	−20.59	4	3

FIG 9.

Molecular docking studies with heme (compounds CQ and 7g).

In silico properties of compound 7g.

In order to evaluate the drug-likeness of compound 7g, we used Schrodinger/QikProp/software, and it is apparent from the data shown in Table 9 that the selected compound does not violate the limitations. The molecular weight, solvent-accessible surface area, rotatable bonds, and H-bond donors and acceptors are within the accepted ranges. More importantly, it does not violate the Lipinski rule of 5 (no more than 5 hydrogen bond donors [total number of nitrogen-hydrogen and oxygen-hydrogen bonds], no more than 10 hydrogen bond acceptors [all nitrogen or oxygen atoms], a molecular mass of <500 Da, and an octanol-water partition coefficient [log P] that is no greater than 5). Compound 7g complies with all parameters (Table 9). The in silico evaluation confirmed that compound 7g has “drug-like” properties.

TABLE 9.

Schrodinger/QikProp/predictions for CQ, AQ-13, and compound 7g

Principal descriptor	Value or description			Range for 95% of drugs
	Chloroquine	AQ-13	Compound 7g	Minimum	Maximum
Mol wt	319.876	291.823	360.929	130	725
Total solvent-accessible surface area (SASA)	649.001	603.348	667.701	300	1,000
No. of rotatable bonds	8	7	6	0	15
No. of H-bond donors	1	1	1	0	6
No. of H-bond acceptors	4	4	6	2	20
QP log P for octanol-water	4.508	3.674	3.578	−2	6.5
QP log K HSA for serum protein binding	0.604	0.341	0.469	−1.5	1.5
No. of Lipinski rule of 5 violations	0	0	0	Maximum is 4
% human oral absorption in GI tract (±20%)	100	100	93	<25% is poor
Qualitative model for human oral absorption	High	High	High	>80% is high

Conclusions.

In conclusion, we synthesized a novel series of chirally pure 4-aminoquinoline derivatives by using amino acids as building blocks for the side chain modifications. These analogs displayed excellent in vitro as well as in vivo antimalarial activities against P. falciparum, with oral efficacy against chloroquine-resistant parasites in a P. yoelii mouse model. Based on detailed antiparasitic tests, the speed of parasitic reduction upon administration of the compound (in vitro), the propensity of the parasites to recrudesce following administration (measured over 28 days), and finally, efficacy validation of the compound in a P. falciparum-simian model, compound 16 has been identified as a preclinical candidate molecule. Furthermore, in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) assays carried out on compound 16 have shown that compound 16 has excellent physicochemical properties, an acceptable pharmacokinetic profile, and moderate metabolic clearance in rat liver microsomes. Overall, compound 16 has “drug-like” properties. These results are in consonance with the in silico ADME predictions and Medicines for Malaria Venture (MMV) criteria for selection of antimalarial compounds. Currently, the compound is under toxicological and regulatory pharmacological evaluations.

MATERIALS AND METHODS

Chemistry.

Melting points (mp) were taken in open capillaries on a Complab melting point apparatus and are uncorrected. The ¹H NMR (200 and 300 MHz) and ¹³C NMR (50 and 75 MHz) spectra were recorded in CDCl₃ on DPX-200 and 300 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 (electrospray ionization-mass spectrometry [ESI-MS]) and high-resolution mass spectra (ESI-HRMS) were recorded on Jeol (Japan)/SX-102 and Agilent 6520 Q-TOF spectrometers, respectively. Analytical thin-layer chromatography (TLC) was carried out on Merck's precoated silica gel 60 F₂₅₄ plates, and spots were visualized by irradiation with UV light (254 nm). Iodine was used as a developing agent and/or by spraying with Dragendorff's reagent. Column chromatographic purification was performed over neutral alumina and silica gel (silica gel 60 to 120, 100 to 200, and 230 to 400 mesh), using a gradient solvent system (with n-hexane/ethyl acetate [EtOAc], dichloromethane [DCM]/hexane, or chloroform/methanol as the eluent unless otherwise specified). All chemicals and reagents were obtained from Aldrich (United States), Lancaster (United Kingdom), and Spectrochem Pvt. Ltd. (India) and were used without further purification. Analytes were eluted using an isocratic mobile phase of methanol and 0.05% trifluoroacetic acid (TFA) in water (60:40) and detected at 254 nm. The purities of compounds submitted for biological evaluation were >98% as determined by HPLC. Reported yields are not optimized.

General procedure for synthesis of compounds 2a to 2j.

Amino acids, namely, glycine, phenylalanine, tryptophan, and methionine (1.0 eq), were dissolved in a dioxane-water mixture (1:1), and the reaction mixture was stirred at room temperature. Addition of 2 N NaOH to this reaction mixture dissolved the reactant, thereby affording a miscible solution. The reaction mixture was then cooled to 0°C and stirred for 15 min. Finally, (Boc)₂O (1.1 eq) was added, and the reaction mixture was allowed to stir at 0°C for 10 min. The ice bath was then removed, and the temperature of the reaction mixture was allowed to rise to room temperature. On completion of the reaction, the reaction mixture was concentrated under reduced pressure. The aqueous layer was acidified with citric acid (pH 2 to 3) and extracted with EtOAc, and the organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The yields were quantitative.

Amino acids, namely alanine, valine, leucine, and isoleucine (1.0 eq), in a 2 N aqueous NaOH solution (17 ml) were cooled in an ice bath to 0°C. Under vigorous stirring, benzyl chloroformate (1.1 eq) and a 2 N aqueous NaOH solution were simultaneously added within 2 min. The mixture was stirred for 20 min at room temperature and extracted with diethyl ether. The aqueous layer was separated and acidified with concentrated hydrochloric acid to a pH of 2 to 3. The resulting emulsion was extracted with EtOAc. The organic phases were combined, washed with brine, and dried with Na₂SO₄. The yields were quantitative.

(i) 2-(tert-Butoxycarbonylamino) acetic acid (2a).

Compound 2a was obtained as a white solid in quantitative yield, with the following data: mp, 86 to 88°C; ¹H NMR (CDCl₃, 300 MHz), δ 1.46 [s, 9H, C (CH₃)₃], 3.96 (s, 2H, CH₂ COOH); ESI-MS, (m/z) 176 (M + H)⁺.

(ii) (S)-2-(Benzyloxycarbonylamino) propanoic acid (2b).

Compound 2b was obtained as a white solid in quantitative yield, with the following data: mp, 46 to 48°C; ¹H NMR (300 MHz, CDCl₃), δ 1.49 (d, J = 6.7 Hz, 3H, CH CH₃), 4.44 (br s, 1H, CHCH₃), 5.15 (s, 2H, CH₂C₆H₅), 7.37 (m, 5H, C₆H₅); ESI-MS, (m/z) 224 (M + H)⁺.

(iii) (S)-2-(Benzyloxycarbonylamino)-3-methylbutanoic acid (2c).

Compound 2c was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.92 to 1.01 [m, 6H, CH(CH₃)₂], 2.31 to 2.22 [m, 1H, CH(CH₃)₂], 4.43 [br s, 1H, CHCH(CH₃)₂], 5.12 (s, 2H, CH₂C₆H₅), 7.35 (s, 5H, C₆H₅); ESI-MS, (m/z) 252 (M + H)⁺.

(iv) (S)-2-(Benzyloxycarbonylamino)-4-methylpentanoic acid (2d).

Compound 2d was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.98 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.55 to 1.61 [m, 1H, CH(CH₃)₂], 1.70 to 1.75 [m, 2H, CH₂CH(CH₃)₂], 4.43 (br s, 1H, NCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 266 (M + H)⁺.

(v) (R)-2-(Benzyloxycarbonylamino)-4-methylpentanoic acid (2e).

Compound 2e was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.98 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.55 to 1.61 [m, 1H, CH(CH₃)₂], 1.70 to 1.75 [m, 2H, CH₂CH(CH₃)₂], 4.43 (br s, 1H, NCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 266 (M + H)⁺.

(vi) 2-(Benzyloxycarbonylamino)-4-methylpentanoic acid (2f).

Compound 2f was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.98 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.55 to 1.61 [m, 1H, CH(CH₃)₂], 1.70 to 1.75 [m, 2H, CH₂CH(CH₃)₂], 4.43 (br s, 1H, NCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 266 (M + H)⁺.

(vii) (2S,3S)-2-(Benzyloxycarbonylamino)-3-methylpentanoic acid (2g).

Compound 2g was obtained as a gummy substance in quantitative yield, with the following data: ¹H-NMR (300 MHz, CDCl₃), δ 0.93 to 1.08 (m, 6H, CHCH₃CH₂CH₃), 1.15 to 1.45 (m, 2H, CH₂CH₃), 1.45 to 1.54 (m, 1H, CHCH₃), 4.42 (br s, NHCH), 5.28 (d, J = 8.3 Hz, NHCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 266 (M + H)⁺.

(viii) (S)-2-(tert-Butoxycarbonylamino)-4-(methylthio)butanoic acid (2h).

Compound 2h was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (CDCl₃, 300 MHz), δ 1.46 [s, 9H, C (CH₃)₃], 1.97 to 2.05 (m, 2H, CHCH₂), 2.13 (s, 3H, SCH₃), 2.51 to 2.60 (m, 2H, CH₂ SCH₃); ESI-MS, (m/z) 250.3 (M + H)⁺.

(ix) (S)-2-(tert-Butoxycarbonylamino)-3-phenylpropanoic acid (2i).

Compound 2i was obtained as a white solid in quantitative yield, with the following data: mp, 85 to 87°C; ¹H NMR (CDCl₃, 300 MHz), δ 1.41 [s, 9H, C(CH₃)₃], 2.73 to 3.04 (m, 2H, CH₂C₆H₅), 4.07 (br s, 1H, NCH), 7.17 to 7.27 (m, 5H, C₆H₅); ESI-MS, (m/z) 266.5 (M + H)⁺.

(x) (S)-2-(tert-Butoxycarbonylamino)-3-(1H-indol-3-yl)propanoic acid (2j).

Compound 2j was obtained as a white solid in quantitative yield, with the following data: mp, 134 to 136°C; ¹H NMR (CDCl₃, 300 MHz), δ 1.42 [s, 9H, C(CH₃)₃], 3.21 to 3.31 (m, 2H, CH₂-Ind), 4.64 (br s, 1H, NCH), 5.06 (br s, 1H, NHBoc), 7.02 (s, 1H, Ind-2H), 7.08 to 7.21 (m, 2H, Ind-5, 6-H), 7.33 (d, J = 7.5 Hz, 2H, Ind-4H), 7.59 (d, J = 7.8 Hz, 2H, Ind-7H), 8.11 (br s, 1H, Ind-NH).

General procedure for synthesis of compounds 3a to 3j.

A solution of compounds 2a to 2j (1.0 eq) in dry THF was cooled to −15°C, and after 10 min, N-methylmorpholine (NMM) (1.2 eq) and isobutylchloroformate (IBCF) (1.2 eq) were added with stirring. After 15 min, a solution of NaBH₄ (2 eq) in water (10 ml) was added to this reaction mixture. The reaction mixture was stirred for 1 h. After completion of the reaction (as monitored by TLC), water (100 ml) was added to quench the reaction. The solvent was evaporated under reduced pressure. The oily residue was taken in EtOAc, and the organic layer was washed with 5% NaHCO₃ and, finally, with brine. The organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The products obtained were purified by silica gel column chromatography, using a mixture of EtOAc and hexane as the eluent to afford compounds 3a to 3j.

(i) tert-Butyl 2-hydroxyethylcarbamate (3a).

Compound 3a was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.46 [s, 9H, C (CH₃)₃], 3.30 (d, J = 4.8 Hz, 2H, CH₂CH₂OH), 3.72 (d, J = 3.5 Hz, 2H, CH₂CH₂OH), 4.96 (s, 1H, CH₂CH₂OH).

(ii) (S)-Benzyl 1-hydroxypropan-2-ylcarbamate (3b).

Compound 3b was obtained as a white solid in quantitative yield, with the following data: mp, 134 to 136°C; ¹H NMR (300MHz, CDCl₃), δ 1.17 [d, J = 6.7 Hz, 3H, CH(CH₃)], 3.53 to 3.68 (m, 2H, CHCH₂OH), 3.80 to 3.85 (m, 1H, CHCH₂OH), 4.88 (br s, 1H, CHCH₂OH), 5.10 (s, 2H, CH₂C₆H₅),7.35 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 209.9 (M + H)⁺.

(iii) (S)-Benzyl 1-hydroxy-3-methylbutan-2-ylcarbamate (3c).

Compound 3c was obtained as a white solid in quantitative yield, with the following data: mp, 114 to 116°C; IR (KBr), 3,360, 2,975, 2,881, 1,688, 1,523, 1,456, 1,226, 1,057, 738 cm⁻¹; ¹H NMR (300 MHz, CDCl₃), δ 0.94 to 0.99 [d, J = 6.7 Hz, 6H, CH(CH₃)₂], 1.84 to 1.89 [m, 1H, CH(CH₃)₂], 3.52 to 3.66 (m, 2H, CHCH₂OH), 3.68 to 3.75 (m, 1H, CHCH₂OH), 4.91 (br s, 1H, CHCH₂OH), 5.13 (s, 2H, CH₂C₆H₅), 7.37 (m, 5H, C₆H₅); ESI-MS, (m/z) 260.2 (M + Na)⁺.

(iv) (S)-Benzyl 1-hydroxy-4-methylpentan-2-ylcarbamate (3d).

Compound 3d was obtained as a gummy substance in quantitative yield, with the following data: IR (undiluted), 3,523, 2,925, 1,713, 1,517, 1,466, 1,251, 1,106 cm⁻¹; ¹H NMR (300 MHz, CDCl₃), δ 0.98 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.61 to 1.55 [m, 2H, CH₂CH(CH₃)₂], 1.75 to 1.70 [m, 1H, CH(CH₃)₂], 4.43 (br s, 1H, NCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 251.9 (M + Na)⁺.

(v) (R)-Benzyl 1-hydroxy-4-methylpentan-2-ylcarbamate (3e).

Compound 3e was obtained as a gummy substance in quantitative yield, with the following data: IR (undiluted), 3,523, 2,925, 1,713, 1,517, 1,466, 1,251, 1,106 cm⁻¹; ¹H NMR (300 MHz, CDCl₃), δ 0.98 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.61 to 1.55 [m, 2H, CH₂CH(CH₃)₂], 1.75 to 1.70 [m, 1H, CH(CH₃)₂], 4.43 (br s, 1H, NCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 260.2 (M + Na)⁺.

(vi) Benzyl 1-hydroxy-4-methylpentan-2-ylcarbamate (3f).

Compound 3f was obtained as a gummy substance in quantitative yield, with the following data: IR (undiluted), 3,523, 2,925, 1,713, 1,517, 1,466, 1,251, 1,106 cm⁻¹; ¹H NMR (300 MHz, CDCl₃), δ 0.98 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.61 to 1.55 [m, 2H, CH₂CH(CH₃)₂], 1.75 to 1.70 [m, 1H, CH(CH₃)₂], 4.43 (br s, 1H, NCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 260.2 (M + Na)⁺.

(vii) Benzyl (2S,3S)-1-hydroxy-3-methylpentan-2-ylcarbamate (3g).

Compound 3g was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (CDCl₃, 300 MHz), δ 0.88 to 0.97 (m, 6H, CHCH₃CH₂CH₃), 1.12 to 1.21 (m, 2H, CH₂CH₃), 1.44 to 1.50 (m, 1H, CHCH₃), 3.60 to 3.77 (m, 3H, CHCH₂OH), 4.94 (br s, CH₂OH), 5.66 (br s, NHCH), 5.14 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 252.0 (M + H)⁺.

(viii) (S)-tert-Butyl 1-hydroxy-4-(methylthio)butan-2-ylcarbamate (3h).

Compound 3h was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (CDCl₃, 300 MHz), δ 1.46 [s, 9H, C(CH₃)₃], 1.78 to 1.85 (m, 2H, CHCH₂CH₂SCH₃), 2.13 (s, 3H, SCH₃), 2.53 to 2.61 (m, 2H, CH₂CH₂SCH₃), 3.61 to 3.78 (m, 3H, NCHCH₂OH); ESI-MS, (m/z) 236.4 (M + H)⁺.

(ix) (S)-tert-Butyl 1-hydroxy-3-phenylpropan-2-ylcarbamate (3i).

Compound 3i was obtained as a white solid in quantitative yield, with the following data: mp, 95 to 97°C; IR, 3,356, 2,980, 2,925, 1,684, 1,526, 1,456, 1,316, 1,269, 1,168, 1,007, 885, 775 cm⁻¹; ¹H NMR (300 MHz, CDCl₃), δ 1.42 [s, 9H, C(CH₃)₃], 2.84 (d, J = 7.0 Hz, 2H, CH₂C₆H₅), 3.49 to 3.70 (m, 2H, CH₂OH), 3.87 (br s, 1H, NCH), 4.69 (t, J = 7.2 Hz, 1H, CH₂OH), 7.20 to 7.30 (m, 5H, C₆H₅).

(x) (S)-tert-Butyl 1-hydroxy-3-(1H-indol-3-yl)propan-2-ylcarbamate (3j).

Compound 3j was obtained as a white solid in quantitative yield, with the following data: mp, 118 to 120°C; ¹H NMR (300 MHz, CDCl₃), δ 1.42 [s, 9H, C(CH₃)₃], 2.99 (d, J = 6.72 Hz, CH₂-Ind), 3.57 to 3.72 (m, 2H, CH₂OH), 3.98 (br s, 1H, NCH), 4.79 (br s, 1H, CH₂OH), 7.05 (s, 1H, Ind-2H), 7.10 to 7.22 (m, 2H, Ind-5,6H), 7.36 (d, J = 7.9 Hz, 2H, Ind-4H), 7.65 (d, J = 7.6 Hz, 2H, Ind-7H), 8.09 (br s, 1H, Ind-NH).

General procedure for synthesis of compounds 4a to 4j.

To a suspension of compounds 3a to 3j (1.0 eq) in anhydrous THF under a nitrogen atmosphere was added triethylamine (3.0 eq). The mixture was cooled to below 0°C. Methanesulfonyl chloride (3.0 eq) was added slowly, with the temperature kept below 5°C, and the reaction mixture was stirred in an ice bath for 45 min. After completion of the reaction as monitored by TLC, the reaction mixture was diluted with a saturated NaHCO₃ solution, and the reaction mixture was extracted with DCM. The combined organic extracts were dried over Na₂SO₄ and concentrated under reduced pressure to afford compounds 4a to 4j. These intermediates were used without further purification.

(i) 2-(tert-Butoxycarbonylamino)ethyl methanesulfonate (4a).

Compound 4a was obtained as a yellow gummy substance in quantitative yield, with the following data: ¹H NMR (CDCI₃, 300 MHz), δ 1.46 [s, 9H, C(CH₃)₃], 3.05 (s, 3H, SO₂CH₃), 3.49 (d, J = 5.4 Hz, 2H, CH₂CH₂OSO₃), 4.30 (t, J = 9.9 Hz, 2H, CH₂CH₂OSO₃); ESI-MS, (m/z) 240 (M + H)⁺.

(ii) (S)-2-(Benzyloxycarbonylamino) propyl methanesulfonate (4b).

Compound 4b was obtained as a white solid in quantitative yield, with the following data: mp, 84 to 86°C; ¹H NMR (300 MHz, CDCl₃), δ 1.28 [d, J = 6.6 Hz, 3H, CH(CH₃)], 2.99 (s, 3H, SO₂CH₃), 4.01 to 4.26 (m, 3H, CHCH₂OSO₂CH₃), 4.89 (br s, 1H, NHCO), 5.12 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 309 (M + Na)⁺.

(iii) (S)-2-(Benzyloxycarbonylamino)-3-methylbutyl methanesulfonate (4c).

Compound 4c was obtained as a white solid in quantitative yield, with the following data: mp, 62 to 64°C; ¹H NMR (300 MHz, CDCl₃), δ 0.89 to 0.97 [2d, J = 6.6 Hz, 6H, CH(CH₃)₂], 1.68 to 1.77 [m, 1H, CH(CH₃)₂], 2.96 (s, 3H, SO₂CH₃), 3.57 to 3.68 (m, 1H, NCH), 4.28 (d, J = 4.1 Hz, 2H, SOCH₂), 4.92 (br s, 1H, NH), 5.12 (s, 2H, CH₂C₆H₅), 7.35 (s, 5H, C₆H₅); ESI-MS, (m/z) 338.9 (M + Na)⁺.

(iv) (S)-2-(Benzyloxycarbonylamino)-4-methylpentyl methanesulfonate (4d).

Compound 4d was obtained as a yellow solid in quantitative yield, with the following data: mp, 55 to 57°C; ¹H NMR (CDCl₃, 300 MHz), δ 0.95 [d, J = 3.9 Hz, 6H, CH(CH₃)₂], 1.51 to 1.59 [m, 2H, CH₂CH(CH₃)₂], 1.60 to 1.70 [m, 1H, CH(CH₃)₂], 2.96 (s, 3H, SO₂CH₃), 3.91 to 3.99 (m, 1H, NCHCH₂OSO₃), 4.01 to 4.47 (m, 2H, CHCH₂OSO₃), 5.11 (s, 2H, CH₂C₆H₅), 5.44 (br s, 1H, NHCO), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 352.1 (M + Na)⁺.

(v) (R)-2-(Benzyloxycarbonylamino)-4-methylpentyl methanesulfonate (4e).

Compound 4e was obtained as a yellow solid in quantitative yield, with the following data: mp, 55 to 57°C; ¹H NMR (CDCl₃, 300 MHz), δ 0.95 [d, J = 3.9 Hz, 6H, CH(CH₃)₂], 1.51 to 1.59 [m, 2H, CH₂CH(CH₃)₂], 1.60 to 1.70 [m, 1H, CH(CH₃)₂], 2.96 (s, 3H, SO₂CH₃), 3.91 to 3.99 (m, 1H, NCHCH₂OSO₃), 4.01 to 4.47 (m, 2H, CHCH₂OSO₃), 5.11 (s, 2H, CH₂C₆H₅), 5.44 (br s, 1H, NHCO), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 352.1 (M + Na)⁺.

(vi) 2-(Benzyloxycarbonylamino)-4-methylpentyl methanesulfonate (4f).

Compound 4f was obtained as a yellow solid in quantitative yield, with the following data: mp, 55 to 57°C; ¹H NMR (CDCl₃, 300 MHz), δ 0.95 [d, J = 3.9 Hz, 6H, CH(CH₃)₂], 1.51 to 1.59 [m, 2H, CH₂CH(CH₃)₂], 1.60 to 1.70 [m, 1H, CH(CH₃)₂], 2.96 (s, 3H, SO₂CH₃), 3.91 to 3.99 (m, 1H, NCHCH₂OSO₃), 4.01 to 4.47 (m, 2H, CHCH₂OSO₃), 5.11 (s, 2H, CH₂C₆H₅), 5.44 (br s, 1H, NHCO), 7.37 (s, 5H, CH₂C₆H₅); ESI-MS, (m/z) 352.1 (M + Na)⁺.

(vii) (2S,3S)-2-(Benzyloxycarbonylamino)-3-methylpentyl methanesulfonate (4g).

Compound 4g was obtained as a yellow solid in quantitative yield, with the following data: mp, 55 to 57°C; ¹H NMR (CDCl₃, 300 MHz), δ 0.87 to 0.95 (m, 6H, CHCH₃CH₂CH₃), 1.07 to 1.19 (m, 2H, CH₂CH₃), 1.31 to 1.50 (m, 1H, CHCH₃), 2.90 (s, 3H, SO₂CH₃), 3.65 to 3.73 (m, 1H, CHCH₂OSO₃), 4.08 to 4.46 (m, 2H, CHCH₂OSO₃), 5.24 (s, 2H, CH₂C₆H₅), 5.39 (br s, 1H, NHCO),7.38 (s, 5H, CH₂C₆H₅).

(viii) (S)-2-(tert-Butoxycarbonylamino)-4-(methylthio)butyl methanesulfonate (4h).

Compound 4h was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (CDCl₃, 300 MHz), δ 1.43 [s, 9H, C(CH₃)₃], 1.81 to 2.01 (m, 2H, CHCH₂CH₂SCH₃), 2.17 (s, 3H, SCH₃), 2.53 to 2.61 (m, 2H, CH₂CH₂SCH₃), 3.67 (s, 3H, SO₂CH₃), 3.82 to 3.95 (m, 1H, NCH), 4.27 (d, J = 4.0 Hz, 2H, SOCH₂); ESI-MS, (m/z) 313.5 (M + H)⁺.

(ix) (S)-2-(tert-Butoxycarbonylamino)-3-phenylpropyl methanesulfonate (4i).

Compound 4i was obtained as a yellow gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.43 [s, 9H, C(CH₃)₃], 2.84 to 2.97 (m, 2H, CH₂C₆H₅), 3.03 (s, 3H, SO₂CH₃), 3.85 (br s, 1H, NCH), 4.12 to 4.27 (m, 2H, SOCH₂), 4.71 (br s, NH), 7.22 to 7.33 (m, 5H, C₆H₅).

(x) (S)-2-(tert-Butoxycarbonylamino)-3-(1H-indol-3-yl)propyl methanesulfonate (4j).

Compound 4j was obtained as a yellow gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.43 [s, 9H, C(CH₃)₃], 2.98 (s, 3H, SO₂CH₃), 2.72 to 2.86 (m, CH₂-Ind), 3.01 to 3.13 (m, 2H, SOCH₂), 4.28 (br s, 1H, NCH), 5.15 (br s, 1H, NHBoc), 7.05 (s, 1H, Ind-2H), 7.10 to 7.22 (m, 2H, Ind-5,6H), 7.36 (d, J = 7.9 Hz, 2H, Ind-4H), 7.65 (d, J = 7.6 Hz, 2H, Ind-7H), 8.17 (br s, 1H, Ind-NH); ESI-MS, (m/z) 369.0 (M + H)⁺.

General procedure for synthesis of compounds 5a to 5j.

To a suspension of compounds 4a to 4j (1.0 eq) in acetonitrile under nitrogen atmosphere was added triethylamine (2.0 eq) and N-methyl piperazine (4.0 eq). The reaction mixture was stirred for 40 h at room temperature. After completion of the reaction (as monitored by TLC), the solvent was evaporated under reduced pressure. The residue was dissolved in DCM, and the organic layer was washed with 10% citric acid. Finally, the aqueous layer was basified with NaHCO₃ and extracted with DCM, and the organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The products obtained were used for the next step without further purification.

(i) tert-Butyl 2-(4-methylpiperazin-1-yl) ethylcarbamate (5a).

Compound 5a was obtained as a gummy substance in 79% yield, with the following data: ¹H NMR (CDCl₃, 300 MHz), δ 1.46 [s, 9H, C(CH₃)₃], 2.29 (s, 3H, NCH₃), 2.45 to 2.48 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.23 [d, J = 4.8 Hz, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃].

(ii) (S)-Benzyl 1-(4-methylpiperazin-1-yl) propan-2-ylcarbamate (5b).

Compound 5b was obtained as a gummy substance in 89% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.19 to 1.21 [d, J = 6.3 Hz, 3H, CH(CH₃)], 2.30 (s, 3H, NCH₃), 2.35 to 2.58 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.75 to 3.78 (m, 1H, CH₃CHCH₂), 5.11 (s, 2H, CH₂C₆H₅), 7.32 to 7.35 (m, 5H, CH₂C₆H₅); ESI-MS, (m/z) 292.3 (M + H)⁺.

(iii) (S)-Benzyl 3-methyl-1-(4-methylpiperazin-1-yl) butan-2-ylcarbamate (5c).

Compound 5c was obtained as a gummy substance in 83% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.87 to 0.94 [2d, J = 6.8 Hz, 6H, CH(CH₃)₂], 2.27 (s, 3H, NCH₃), 2.32 to 2.61 [m, 11H, CH(CH₃)CH₂cycN(CH₂CH₂)₂NCH₃], 3.60 to 3.80 (m, 1H, NHCH), 5.13 (s, 2H, CH₂C₆H₅), 7.37 (s, 5H, C₆H₅); ESI-MS, (m/z) 320.2 (M + H)⁺.

(iv) (S)-Benzyl 4-methyl-1-(4-methylpiperazin-1-yl) pentan-2-ylcarbamate (5d).

Compound 5d was obtained as a white solid in 89% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.91 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.35 to 1.28 [m, 2H, CH₂CH(CH₃)₂], 1.73 to 1.64 [m, 1H, CH(CH₃)₂], 2.28 (s, 3H, NCH₃), 2.59 to 2.30 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.81 to 3.76 (m, 1H, NHCH), 4.70 (br s, 1H, NHCO), 5.11 (s, 2H, CH₂C₆H₅), 7.36 to 7.30 (m, 5H, CH₂C₆H₅); ESI-MS, (m/z) 334.2 (M + H)⁺.

(v) (R)-Benzyl 4-methyl-1-(4-methylpiperazin-1-yl) pentan-2-ylcarbamate (5e).

Compound 5e was obtained as a white solid in 89% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.91 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.35 to 1.28 [m, 2H, CH₂CH(CH₃)₂], 1.73 to 1.64 [m, 1H, CH(CH₃)₂], 2.28 (s, 3H, NCH₃), 2.59 to 2.30 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.81 to 3.76 (m, 1H, NHCH), 4.70 (br s, 1H, NHCO), 5.11 (s, 2H, CH₂C₆H₅), 7.36 to 7.30 (m, 5H, CH₂C₆H₅); ESI-MS, (m/z) 334.2 (M + H)⁺.

(vi) Benzyl 4-methyl-1-(4-methylpiperazin-1-yl) pentan-2-ylcarbamate (5f).

Compound 5f was obtained as a white solid in 89% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 0.91 [d, J = 6.5 Hz, 6H, CH(CH₃)₂], 1.35 to 1.28 [m, 2H, CH₂CH(CH₃)₂], 1.73 to 1.64 [m, 1H, CH(CH₃)₂], 2.28 (s, 3H, NCH₃), 2.59 to 2.30 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.81 to 3.76 (m, 1H, NHCH), 4.70 (br s, 1H, NHCO), 5.11 (s, 2H, CH₂C₆H₅), 7.36 to 7.30 (m, 5H, CH₂C₆H₅); ESI-MS, (m/z) 334.2 (M + H)⁺.

(vii) Benzyl (2S,3S)-3-methyl-1-(4-methylpiperazin-1-yl) pentan-2-ylcarbamate (5g).

Compound 5g was obtained as a gummy substance in 89% yield, with the following data: ¹H NMR (CDCl₃, 300 MHz), δ 0.87 to 1.05 (m, 6H, CHCH₃CH₂CH₃), 1.08 to 1.18 (m, 2H, CH₂CH₃), 1.40 to 1.48 (m, 1H, CHCH₃), 2.27 (s, 3H, NCH₃), 2.33 to 2.61 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.63 to 3.77 (m, 1H, NHCH), 4.90 (br s, 1H, NHCH), 5.13 (s, 2H, CH₂C₆H₅), 7.32 to 7.38 (m, 5H, CH₂C₆H₅); ESI-MS, (m/z) 334.2 (M + H)⁺.

(viii) (S)-tert-Butyl 1-(4-methylpiperazin-1-yl)-4-(methylthio) butan-2-ylcarbamate (5h).

Compound 5h was obtained as an off-white solid in 85% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.97 to 2.05 (m, 2H, CHCH₂), 2.13 (s, 3H, SCH₃), 2.25 (s, 3H, NCH₃), 2.39 to 2.67 [m, 12H, CH₂SCH₃CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.81 to 3.88 (m, 1H, NHCH); ESI-MS, (m/z) 318.0 (M + H)⁺.

(ix) (S)-tert-Butyl 1-(4-methylpiperazin-1-yl)-3-phenylpropan-2-ylcarbamate (5i).

Compound 5i was obtained as an off-white solid in 86% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.42 [s, 9H, C(CH₃)₃], 2.28 (s, 3H, NCH₃), 2.30 to 2.61 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH3], 2.85 (d, J = 5.8 Hz, 2H, CH₂C₆H₅), 3.91 to 4.21 (m, 1H, NCH), 4.59 (br s, 1H, NH), 7.26 to 7.30 (m, 5H, C₆H₅); ESI-MS, (m/z) 334.2 (M + H)⁺.

(x) (S)-tert-Butyl 1-(1H-indol-3-yl)-3-(4-methylpiperazin-1-yl) propan-2-ylcarbamate (5j).

Compound 5j was obtained as an off-white solid in 86% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.43 [s, 9H, C (CH₃)₃], 2.27 (s, 3H, NCH₃), 2.29 to 2.44 [m, 12H, Ind-CH₂CH₂cycN(CH₂CH₂)₂], 4.02 (br s, 1H, NCH), 4.66 (br s, 1H, NHBoc), 7.02 (s, 1H, Ind-2H), 7.08 to 7.20 (m, 2H, Ind-5,6H), 7.37 (d, J = 7.8 Hz, 2H, Ind-4H), 7.66 (d, J = 7.3 Hz, 2H, Ind-7H), 8.09 (br s, 1H, NH-Ind); ESI-MS, (m/z) 373.2 (M + H)⁺.

General procedure for synthesis of compounds 6a to 6j.

To a suspension of compounds 5a, 5h, 5i, and 5j in methanol (1.0 eq in 10.0 ml) was added 20% HCl-dioxane, and the mixture was stirred for 1 h at room temperature. After completion of the reaction, the solvent was evaporated under reduced pressure. The product was purified by trituration with diethyl ether. The hydrochloride salt was basified with triethylamine, and the products obtained were used for the next step without further purification.

To a solution of compounds 5b, 5c, 5d, 5e, 5f, and 5g in MeOH (1.0 eq in 10.0 ml), 10% (wt/wt) Pd/C was added and flushed two times with hydrogen gas, and the reaction mixture was agitated at room temperature for 2 h under hydrogen gas at 30 lb/in². After complete deblocking of the protecting group (as monitored by TLC), the reaction mixture was filtered through Celite and concentrated under reduced pressure to afford the product as a gummy residue. The products obtained were used for the next step without further purification.

(i) 2-(4-Methylpiperazin-1-yl)-ethanamine (6a).

Compound 6a was obtained as a gummy substance in quantitative yield.

(ii) (S)-1-(4-Methylpiperazin-1-yl)propan-2-amine (6b).

Compound 6b was obtained as a gummy substance in quantitative yield.

(iii) (S)-3-Methyl-1-(4-methylpiperazin-1-yl)butan-2-amine (6c).

Compound 6c was obtained as a gummy substance in quantitative yield.

(iv) (S)-4-Methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6d).

Compound 6d was obtained as a gummy substance in quantitative yield.

(v) (R)-4-Methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6e).

Compound 6e was obtained as a gummy substance in quantitative yield.

(vi) 4-Methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6f).

Compound 6f was obtained as a gummy substance in quantitative yield.

(vii) (2S,3S)-3-Methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6g).

Compound 6g was obtained as a gummy substance in quantitative yield.

(viii) (S)-1-(4-Methylpiperazin-1-yl)-4-(methylthio)butan-2-amine (6h).

Compound 6h was obtained as a gummy substance in quantitative yield.

(ix) (S)-1-(4-Methylpiperazin-1-yl)-3-phenylpropan-2-amine (6i).

Compound 6i was obtained as a gummy substance in quantitative yield.

(x) (S)-1-(1H-Indol-3-yl)-3-(4-methylpiperazin-1-yl) propan-2-amine (6j).

Compound 6j was obtained as a gummy substance in quantitative yield.

General procedure for synthesis of compounds 7a to 7r.

The free amines 6a to 6j (2.0 eq) were heated with 4,7-dichloroquinoline (1.0 eq) in the presence of phenol. After completion of the reaction (as monitored by TLC), the reaction mixture was dissolved in chloroform. The organic layer was washed with a 10% aqueous NaOH solution and, finally, with brine. The organic layer was dried over Na₂SO₄, and the solvent was concentrated under reduced pressure. The crude products obtained were purified by column chromatography, using methanol-chloroform-triethylamine as the eluent.

(i) 7-Chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)quinolin-4-amine (7a).

Compound 7a was obtained as a white solid in 78% yield, with the following data: mp, 104 to 106°C; ¹H NMR (300 MHz, CDCl₃), δ 2.34 (s, 3H, NCH₃), 2.52 to 2.60 [m, 8H, CH₂cycN(CH₂CH₂)₂NCH₃], 2.79 to 2.83 [t, J = 6.2 Hz, CH₂cycN(CH₂CH₂)₂NCH₃], 3.34 [s, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 6.07 (br s, NH), 6.39 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.39 to 7.43 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.70 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.98 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 45.9, 52.5, 55.3, 99.2, 117.2, 121.1, 125.4, 128.4, 135.0, 148.6, 150.0, 151.6; HRMS calculated for [C₂₀H₂₇ClN₄ + H]⁺ 305.1528, found 305.1521; ESI-MS, (m/z) 305.1 (M + H)⁺.

(ii) N-(2-(4-Methylpiperazin-1-yl)ethyl)-7-(trifluoromethyl)quinolin-4-amine (7b).

Compound 7b was obtained as a white solid in 78% yield, with the following data: mp, 72 to 74°C; ¹H NMR (300 MHz, CDCl₃), δ 2.34 (s, 3H, NCH₃), 2.52 [br s, 4H, CH₂cycN(CH₂)₂], 2.61 [br s, 4H, CH₂cycN(CH₂)₂NCH₃], 2.83 [t, J = 5.8 Hz, 2H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.35 [d, J = 4.6 Hz, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 6.09 (br s, NH), 6.48 (d, J = 6.8 Hz, 1H, Ar-H quinoline), 7.64 (d, J = 8.5 Hz, 1H, Ar-H quinoline), 7.88 (d, J = 8.5 Hz, 1H, Ar-H quinoline), 8.29 (s, 1H, Ar-H quinoline), 8.63 (d, J = 5.0 Hz, 1H, Ar-H quinoline); ¹H NMR (50 MHz, CDCl₃), δ 38.9, 45.9, 52.5, 55.1, 100.1, 120.6, 121.0, 126.7, 127.4, 130.4, 131.1, 147.5, 149.6, 152.2; HRMS calculated for [C₂₀H₂₁F₃N₄ + H]⁺ 339.1791, found 339.1788; ESI-MS, (m/z) 339.1 (M + H)⁺.

(iii) (S)-7-Chloro-N-(1-(4-methylpiperazin-1-yl)propan-2-yl)quinolin-4-amine (7c).

Compound 7c was obtained as a white solid in 70% yield, with the following data: mp, 96 to 98°C; ¹H NMR (300 MHz, CDCl₃), δ 1.31 [d, J = 6.0 Hz, 3H, CH(CH₃)], 2.26 (s, 3H, NCH₃), 2.42 to 2.67 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.66 to 3.74 (m, 1H, CHCH₃CH₂), 5.99 (br s, 1H, NHCH), 6.44 (d, J = 5.2 Hz, 1H, Ar-H quinoline), 7.37 to 7.40 (dd, J = 1.9, 8.9 Hz, 1H, Ar-H quinoline), 7.72 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 1.8 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 5.2 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 18.8, 45.9, 50.0, 55.1, 62.7, 99.6, 117.9, 121.2, 125.3, 128.6, 134.8, 149.1, 149.8, 151.9; HRMS calculated for [C₁₇H₂₃ClN₄ + H]⁺ 319.1684, found 319.1694; ESI-MS, (m/z) 319.2 (M + H)⁺.

(iv) (S)-N-(1-(4-Methylpiperazin-1-yl)propan-2-yl)-7-(trifluoromethyl)quinolin-4-amine (7d).

Compound 7d was obtained as a white solid in 66% yield, with the following data: mp, 88 to 90°C; ¹H NMR (300 MHz, CDCl₃), δ 1.33 [d, J = 6 Hz, 3H, CH(CH₃)], 2.26 (s, 3H, NCH₃), 2.42 to 2.75 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.67 to 3.79 (m, 1H, CHCH₃CH₂), 6.08 (br s, 1H, NHCH), 6.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.61 (d, J = 8.6 Hz, 1H, Ar-H quinoline), 7.89 (d, J = 8.5 Hz, 1H, Ar-H quinoline), 8.27 (s, 1H, Ar-H quinoline), 8.61 (d, J = 5.1 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 75 MHz), δ 18.7, 45.1, 45.9, 52.9, 55.1, 62.7, 100.6, 120.1, 121.0, 121.2, 122.2, 125.8, 127.6, 130.5, 131.0, 147.7, 149.6, 152.2; HRMS calculated for [C₁₈H₂₃F₃N₄ + H]⁺ 353.1948; found 353.1946; ESI-MS, (m/z) 353.2 (M + H)⁺.

(v) (S)-7-Chloro-N-(3-methyl-1-(4-methylpiperazin-1-yl)butan-2-yl)quinolin-4-amine (7e).

Compound 7e was obtained as a white solid in 78% yield, with the following data: mp, 95 to 97°C; ¹H NMR (300 MHz, CDCl₃), δ 0.96 to 1.06 [2d, J = 6.8 Hz, 6H, CH(CH₃)₂], 2.24 (s, 3H, NCH₃), 2.31 to 2.70 [m, 11H, CH(CH₃)CH₂cycN(CH₂CH₂)₂NCH₃], 3.53 to 3.59 (m, 1H, NHCH), 5.99 (br s, 1H, NHCH), 6.43 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.38 to 7.42 (dd, J = 2.0, 8.9 Hz, 1H, Ar-H quinoline), 7.76 (d, J = 8.8 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 1.9 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 17.3, 18.3, 29.6, 45.9, 53.1, 54.3, 55.1, 56.7, 99.4, 117.8, 121.1, 125.2, 128.7, 134.85, 150.2, 151.9; HRMS calculated for [C₁₉H₂₇ClN₄ + H]⁺ 347.1997, found 347.2003; ESI-MS, (m/z) 347.2 (M + H)⁺.

(vi) (S)-N-(3-Methyl-1-(4-methylpiperazin-1-yl)butan-2-yl)-7-(trifluoromethyl)quinolin-4-amine (7f).

Compound 7f was obtained as an off-white solid in 78% yield, with the following data: mp, 87 to 89°C; ¹H NMR (300 MHz, CDCl₃), δ 0.97 to 1.07 [2d, J = 6.8 Hz, 6H, CH(CH₃)₂], 2.25 to 2.29 [m, 1H, CH(CH₃)₂], 2.34 (s, 3H, NCH₃), 2.56 to 2.82 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.54 to 3.64 (m, 1H, NHCH), 6.0 (br s, 1H, NHCH), 6.54 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.64 (d, J = 8.3 Hz, 1H, Ar-H quinoline), 8.05 (d, J = 8.7 Hz, 1H, Ar-H quinoline), 8.29 (s, 1H, Ar-H quinoline), 8.60 (d, J = 5.5 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 17.3, 18.4, 28.4, 29.2, 29.6, 45.8, 53.0, 54.4, 55.1, 56.7, 100.0, 120.1, 120.9, 121.1, 122.6, 125.3, 127.6, 130.7, 131.0, 147.8, 150.0, 152.2; HRMS calculated for [C₂₀H₂₇F₃N₄ + H]⁺ 381.2161, found 381.2278; ESI-MS, (m/z) 381.2 (M + H)⁺.

(vii) (S)-7-Chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine (7g).

Compound 7g was obtained as a white solid in 72% yield, with the following data: mp, 103 to 105°C; ¹H NMR (300 MHz, CDCl₃), δ 0.96 [d, J = 6.0 Hz, 3H, CH(CH₃)], 1.06 [d, J = 6.2 Hz, 3H, CH(CH₃)], 1.44 to 1.53 [m, 2H, CH₂CH(CH₃)₂], 1.67 to 1.83 [m, 1H, CH(CH₃)₂], 2.27 (s, 3H, NCH₃), 2.63 to 2.41 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.68 (d, J = 5.7 Hz, 1H, NCH), 5.71 (d, J = 4.1 Hz, 1H, NH), 6.44 (d, J = 5.3 Hz, Ar-H quinoline), 7.37 to 7.41 (dd, J = 2.0, 8.8 Hz, 1H, Ar-H quinoline), 7.51 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.97 (dd, J = 2.0 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 22.5, 23.3, 42.6, 45.9, 53.3, 55.2, 61.2, 99.2, 117.7, 121.1, 125.3, 128.7, 134.8, 149.2, 149.8, 151.9; HRMS calculated for [C₂₀H₂₉ClN₄ + H]⁺ 361.2154, found 361.2151; ESI-MS, (m/z) 361.2 (M + H)⁺.

(viii) (R)-7-Chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine (7h).

Compound 7h was obtained as a white solid in 72% yield, with the following data: mp, 103 to 105°C; ¹H NMR (300 MHz, CDCl₃), δ 0.96 [d, J = 6.15 Hz, 3H, CH(CH₃)], 1.06 [d, J = 6.18 Hz, 3H, CH(CH₃)], 1.44 to 1.51 [m, 2H, CH₂CH(CH₃)₂], 1.66 to 1.75 [m, 1H, CH(CH₃)₂], 2.25 (s, 3H, NCH₃), 2.40 to 2.61 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.66 (d, J = 5.8 Hz, 1H, NCH), 5.70 (d, J = 4.1 Hz, 1H, NH), 6.43 (d, J = 5.3Hz, Ar-H quinoline), 7.36 to 7.39 (dd, J = 1.9, 8.9 Hz, 1H, Ar-H quinoline), 7.69 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.95 (dd, J = 1.8 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 5.2 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 22.4, 23.3, 24.9, 42.6, 45.9, 53.3, 55.2, 61.2, 99.2, 117.7, 121.1, 125.2, 128.7, 134.7, 149.2, 149.7, 151.9; HRMS calculated for [C₂₀H₂₉ClN₄ + H]⁺ 361.2154, found 361.2175; ESI-MS, (m/z) 361.2 (M + H)⁺.

(ix) 7-Chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine (7i).

Compound 7i was obtained as a white solid in 72% yield, with the following data: mp, 103 to 105°C; ¹H NMR (300 MHz, CDCl₃), δ 0.96 [d, J = 6.0 Hz, 3H, CH(CH₃)], 1.06 [d, J = 6.2 Hz, 3H, CH(CH₃)], 1.44 to 1.53 [m, 2H, CH₂CH(CH₃)₂], 1.67 to 1.83 [m, 1H, CH(CH₃)₂], 2.27 (s, 3H, NCH₃), 2.63 to 2.41 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.68 (d, J = 5.7 Hz, 1H, NCH), 5.71 (d, J = 4.1 Hz, 1H, NH), 6.44 (d, J = 5.3 Hz, Ar-H quinoline), 7.37 to 7.41 (dd, J = 2.0, 8.8 Hz, 1H, Ar-H quinoline), 7.51 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.97 (dd, J = 2.0 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 22.5, 23.3, 42.6, 45.9, 53.3, 55.2, 61.2, 99.2, 117.7, 121.1, 125.3, 128.7, 134.8, 149.2, 149.8, 151.9; HRMS calculated for [C₂₀H₂₉ClN₄ + H]⁺ 361.2154, found 361.2151; ESI-MS, (m/z) 361.2 (M + H)⁺.

(x) (S)-N-(4-Methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-7-(trifluoromethyl)quinolin-4-amine (7j).

Compound 7j was obtained as a white solid in 72% yield, with the following data: mp, 96 to 98°C; ¹H NMR (300 MHz, CDCl₃), δ 0.97 [d, J = 6.2 Hz, 3H, CH(CH₃)], 1.07 [d, J = 6.1 Hz, 3H, CH(CH₃)], 1.50 to 1.52 [m, 2H, CH₂CH(CH₃)₂], 1.69 to 1.74 [m, 1H, CH(CH₃)₂], 2.27 (s, 3H, NCH₃), 2.43 to 2.65 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.70 (d, J = 5.3 Hz, 1H, NHCH), 5.81 (br s, 1H, NH), 6.53 (d, J = 5.19 Hz, Ar-H quinoline), 7.36 to 7.39 (dd, J = 1.9, 8.9 Hz, 1H, Ar-H quinoline), 7.89 (d, J = 8.4 Hz, 1H, Ar-H quinoline), 8.28 (s, 1H, Ar-H quinoline), 8.63 (d, J = 5.1 Hz, 1H, Ar-H quinoline); ¹³C NMR (50 MHz, CDCl₃), δ 22.4, 23.3, 24.9, 42.4, 45.9, 53.3, 55.2, 61.2, 100.2, 120.0, 120.9, 127.6, 130.4, 131.1, 147.8, 149.5, 152.2; HRMS calculated for [C₂₁H₂₉F₃N₄ + H]⁺ 395.2417, found 395.2410; ESI-MS, (m/z) 395.2 (M + H)⁺.

(xi) 7-Chloro-N-(2S,3S)-3-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine (7k).

Compound 7k was obtained as a white solid in 66% yield, with the following data: mp, 101 to 103°C; ¹H NMR (300 MHz, CDCl₃), δ 0.92 (d, J = 6.7 Hz, 3H, CHCH₃), 1.02 (t, J = 7.3 Hz, 3H, CH₂CH₃), 1.39 to 1.48 (m, 2H, CH₃CH₂CHCH₃), 1.50 to 1.71 (m, 1H, CH₃CH₂CHCH₃), 2.26 (s, 3H, NCH₃), 2.74 to 2.42 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.68 to 3.63 (m, 1H, NHCH), 5.92 (br s, 1H, NHCH), 6.45 (d, J = 5.6 Hz, 1H, Ar-H quinoline), 7.42 to 7.45 (t, J = 6.9 Hz, 1H, Ar-H quinoline), 7.88 (d, J = 1.7 Hz, 1H, Ar-H quinoline), 8.03 (d, J = 1.7 Hz, 1H, Ar-H quinoline), 8.50 (d, J = 5.4 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 12.3, 14.0, 25.9, 35.8, 45.7, 55.0, 56.0, 99.2, 117.7, 121.5, 125.4, 127.9, 135.2, 148.3, 150.5, 151.1; HRMS calculated for [C₂₀H₂₉ClN₄ + H]⁺ 361.2154, found 361.2172; ESI-MS, (m/z) 361.2 (M + H)⁺.

(xii) N-(2S,3S)-3-Methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-7(trifluoromethyl)quinolin-4-amine (7l).

Compound 7l was obtained as a pale yellow solid in 60% yield, with the following data: mp, 97 to 99°C; ¹H NMR (300 MHz, CDCl₃), δ 0.95 (d, J = 6.7 Hz, 3H, CHCH₃), 1.05 (t, J = 7.2 Hz, 3H, CH₂CH₃), 1.33 to 1.44 (m, 2H, CH₃CH₂CHCH₃), 1.51 to 1.60 (m, 1H, CH₃CH₂CHCH₃), 2.31 (s, 3H, NCH₃), 2.50 to 2.80 [m, 10H, CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.70 (d, J = 4.5 Hz, 1H, NHCH), 5.94 (br s, 1H, NHCH), 6.52 (d, J = 5.2 Hz, 1H, Ar-H quinoline), 7.64 (d, J = 7.9 Hz, 1H, Ar-H quinoline), 7.99 (dd, J = 8.2 Hz, 1H, Ar-H quinoline), 8.29 (s, 1H, Ar-H quinoline), 8.62 (d, J = 5.3 Hz, 1H, Ar-H quinoline); ¹³C NMR (50 MHz, CDCl₃), 12.3, 13.9, 25.9, 35.6, 45.7, 53.0, 55.0, 56.0, 100.3, 120.1, 121.1, 122.6, 125.3, 129.3, 130.7, 131.0, 147.6, 149.9, 152.0; HRMS calculated for [C₂₁H₂₉F₃N₄ + H]⁺ 395.2417, found 395.2410; ESI-MS, (m/z) 395.2 (M + H)⁺.

(xiii) (S)-7-Chloro-N-(1-(4-methylpiperazin-1-yl)-4-(methylthio)butan-2-yl)quinolin-4-amine (7m).

Compound 7m was obtained as an off-white solid in 65% yield, with the following data: mp, 92 to 94°C; ¹H NMR (300 MHz, CDCl₃), δ 1.97 to 2.05 (m, 2H, CHCH₂), 2.12 (s, 3H, SCH₃), 2.25 (s, 3H, NCH₃), 2.37 to 2.66 [m, 12H, CH₂SCH₃CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.80 to 3.87 (m, 1H, NHCH), 5.77 (d, J = 3.78 Hz, 1H, NH), 6.53 (d, J = 4.05 Hz, 1H, Ar-H quinoline), 7.76 (d, J = 6.9 Hz, 1H, Ar-H quinoline), 7.71 (d, J = 6.7 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 1.17 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 4.0 Hz, 1H, Ar-H quinoline); ¹³C NMR (50 MHz, CDCl₃), δ 15.8, 30.2, 32.1, 42.4, 45.4, 45.9, 50.7, 54.5, 55.0, 59.8. 99.0, 117.4, 121.5, 125.5, 128.4, 128.5, 135.1, 149.1, 151.7, 151.8; HRMS calculated for [C₁₉H₂₇ClN₄S + H]⁺ 379.1718, found 379.1716; ESI-MS, (m/z) 379.2 (M + H)⁺.

(xiv) (S)-N-(1-(4-Methylpiperazin-1-yl)-4-(methylthio)butan-2-yl)-7-(trifluoromethyl)quinolin-4-amine (7n).

Compound 7n was obtained as a white solid in 65% yield, with the following data: mp, 89 to 91°C; ¹H NMR (300 MHz, CDCl₃), δ 1.97 to 2.09 (m, 2H, CHCH₂), 2.13 (s, 3H, SCH₃), 2.27 (s, 3H, NCH₃), 2.44 to 2.73 [m, 12H, CH₂SCH₃CHCH₂cycN(CH₂CH₂)₂NCH₃], 3.57 to 3.62 (m, 1H, NHCH), 5.90 (d, J = 3.7 Hz, 1H, NH), 6.63 (d, J = 4.0 Hz, 1H, Ar-H quinoline), 7.60 to 7.63 (dd, J = 1.2, 6.5 Hz, 1H, Ar-H quinoline), 7.91 (d, J = 6.5 Hz, 1H, Ar-H quinoline), 7.97 (s, 1H, Ar-H quinoline), 8.62 (d, J = 4.02 Hz, 1H, Ar-H quinoline); ¹³C NMR (50 MHz, CDCl₃), δ 15.9, 30.7, 32.1, 42.4, 45.4, 45.9, 51.7, 54.5, 55.4, 59.8, 99.0, 117.4, 121.5, 125.5, 128.4, 128.5, 135.1, 149.1, 151.7, 151.8; HRMS calculated for [C₂₀H₂₇F₃N₄S + H]⁺ 413.1981, found 413.1993; ESI-MS, (m/z) 413.2 (M + H)⁺.

(xv) (S)-7-Chloro-N-(1-(4-methylpiperazin-1-yl)-3-phenylpropan-2-yl)quinolin-4-amine (7o).

Compound 7o was obtained as a yellowish white solid in 63% yield, with the following data: mp, 98 to 100°C; ¹H NMR (300 MHz, CDCl₃), δ 2.24 (s, 3H, NCH₃), 2.40 to 2.79 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 2.92 to 3.13 (m, CH₂C₆H₅), 3.83 to 3.90 (m, 1H, NCH), 5.72 (d, J = 4.4Hz, 1H, NHCH), 6.50 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.16 to 7.29 (m, 5H, CH₂C₆H₅), 7.36 to 7.40 (dd, J = 2.0, 8.9 Hz, 1H, Ar-H quinoline), 7.66 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 8.54 (d, J = 5.1 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz),δ 38.1, 45.8, 55.1, 59.8, 99.4, 117.7, 121.7, 125.5, 126.7, 128.5, 129.6, 135.0, 137.0, 138.3, 148.9, 149.6, 151.7; HRMS calculated for [C₂₃H₂₇ClN₄ + H]⁺ 394.1997, found 395.1995; ESI-MS, (m/z) 395.4 (M + H)⁺.

(xvi) (S)-N-(1-(4-Methylpiperazin-1-yl)-3-phenylpropan-2-yl)-7-(trifluoromethyl)quinolin-4-amine (7p).

Compound 7p was obtained as a yellowish white solid in 65% yield, with the following data: mp, 91 to 93°C; ¹H NMR (300 MHz, CDCl₃), δ 2.24 (s, 3H, NCH₃), 2.40 to 2.66 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 2.92 to 3.14 (m, CH₂C₆H₅), 3.92 (s, 1H, NCH), 5.83 (br s, 1H, NHCH), 6.58 (d, J = 5.1 Hz, 1H, Ar-H quinoline), 7.19 to 7.30 (m, 5H, CH₂C₆H₅), 7.60 (d, J = 8.3 Hz, 1H, Ar-H quinoline), 7.84 (d, J = 8.4 Hz, 1H, Ar-H quinoline), 8.28 (s, 1H, Ar-H quinoline), 8.63 (d, J = 5.2 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 38.0, 45.9, 50.3, 52.9, 55.1, 59.8, 100.4, 120.2, 121.03, 126.7, 127.6, 128.5, 129.6, 130.5, 131.2, 136.9, 147.8, 149.2, 152.2; HRMS calculated for [C₂₄H₂₇F₃N₄ + H]⁺ 429.2261, found 429.2272; ESI-MS, (m/z) 429.2 (M + H)⁺.

(xvii) (S)-N-(1-(1H-Indol-3-yl)-3-(4-methylpiperazin-1-yl)propan-2-yl)-7-chloroquinolin-4-amine (7q).

Compound q was obtained as a pale yellow solid in 60% yield, with the following data: mp, 102 to 104°C; ¹H NMR (300 MHz, CDCl₃), δ 2.29 (s, 3H, NCH₃), 2.35 to 2.69 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.16 to 3.30 (m, CH₂-Ind), 4.05 (d, J = 4.7 Hz, 1H, NHCH), 5.75 (br s, 1H, NHCH), 6.56 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.00 (d, J = 2.0 Hz, 1H, Ind-2H), 7.09 to 7.23 (m, 2H, Ind-5,6H), 7.33 to 7.36 (m, 3H, Ind-4H and Ar-H quinoline), 7.61 (d, J = 2.9 Hz, 2H, Ind-7H), 8.00 (d, J = 1.9 Hz, 1H, Ar-H quinoline), 8.21 (d, J = 9.3 Hz, 1H, Ar-H quinoline), 8.54 (d, J = 5.4Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50MHz), δ 29.6, 45.9, 55.1, 60.0, 99.4, 110.7, 111.3, 117.6, 118.8, 119.6, 121.3, 122.1, 123.1, 125.3, 128.0, 128.5, 134.9, 136.2, 149.1, 149.6, 151.8; HRMS calculated for [C₂₅H₂₈ClN₅ + H]⁺ 434.9763, found 434. 9743; ESI-MS, (m/z) 434.3 (M + H)⁺.

(xviii) (S)-N-(1-(1H-Indol-3-yl)-3-(4-methylpiperazin-1-yl)propan-2-yl)-7-(trifluoromethyl)quinolin-4-amine (7r).

Compound 7r was obtained as a pale yellow solid in 60% yield, with the following data: mp, 96 to 98°C; ¹H NMR (300 MHz, CDCl₃), δ 2.26 (s, 3H, NCH₃), 2.41 to 2.65 [m, 10H, CH₂cycN(CH₂CH₂)₂NCH₃], 3.20 to 3.25 (m, 2H, CH₂-Ind), 4.04 (d, J = 4.0 Hz, 1H, NHCH), 5.79 (d, J = 3.7 Hz, NHCH), 6.63 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.09 (d, J = 2.0 Hz, 1H, Ind-2H), 7.10 to 7.23 (m, 2H, Ind-5,6H), 7.54 (d, J = 1.9 Hz, 1H, Ar-H quinoline), 7.60 (d, J = 6.1 Hz, Ind-4H), 7.73 (d, J = 2.9 Hz, 1H, Ind-7H), 8.18 (d, J = 6.4 Hz, 1H, Ar-H quinoline), 8.27 (s, 1H, Ar-H quinoline), 8.61 (d, J = 5.3Hz, 1H, Ar-H quinoline); ¹³C NMR (50 MHz, CDCl₃), δ 29.6, 45.7, 45.8, 49.8, 53.0, 54.9, 55.1, 60.0, 61.1, 110.4, 110.6, 111.3, 118.7, 119.6, 121.0, 121.1, 122.1, 122.6, 123.5, 125.3, 127.4, 127.9, 130.7, 131.0, 136.2, 147.7, 149.4, 152.2; HRMS calculated for [C₂₆H₂₈F₃N₅ + H]⁺ 468.2370, found 434.2368; ESI-MS, (m/z) 468.1 (M + H)⁺.

General procedure for synthesis of compounds 8a and 8b.

Starting from glycine and α-phenylalanine, compounds 8a and 8b were prepared by the procedure described for compound 2b.

(i) 2-(Benzyloxycarbonylamino)acetic acid (8a).

Compound 8a was obtained as a white solid in quantitative yield, with the following data: mp, 119 to 120°C; ¹H-NMR (300 MHz, CDCl₃), δ 3.67 (s, 2H, CH₂), 5.07 (s, 2H, CH₂), 7.35 (s, 5H, OCH₂C₆H₅); ESI-MS, (m/z) 210 (M + H)⁺.

(ii) (S)-2-(Benzyloxycarbonylamino)-3-phenylpropanoic acid (8b).

Compound 8b was obtained as a white solid in quantitative yield, with the following data: mp, 100 to 102°C; ¹H NMR (300 MHz, CDCl₃), δ 2.80 (d, J = 7.0 Hz, 2H, CH₂Ph), 4.50 (m, 1H, NHCH), 4.95 (s, 1H, NHCH), 5.09 (s, 2H, OCH₂Ph), 7.13 to 7.28 (m, 10H, ArH).

General procedure for synthesis of compounds 9a and 9b.

To a stirred solution of N-Cbz-protected amino acids 8a and 8b (1.0 eq) in dry THF at −15°C under a nitrogen atmosphere were successively added isobutylchloroformate (IBCF) (1.1 eq) and N-methylmorpholine (NMM) (1.1 eq). The mixture was stirred for 15 min and then treated dropwise with an ethereal solution of excess CH₂N₂. The yellow solution was allowed to warm to room temperature, and stirring was continued until there was no N-protected amino acid remaining (TLC control). The reaction mixture was concentrated under reduced pressure, and the residue was taken up in EtOAc. The organic phase was washed successively with an aqueous NaHCO₃ solution and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The products were purified by silica gel column chromatography (EtOAc/hexane) to obtain the diazoketones 9a and 9b.

(i) Benzyl 3-diazo-2-oxopropylcarbamate (9a).

Compound 9a was obtained as an off-white solid in 80% yield, with the following data: mp, 68 to 69°C; ¹H NMR (300 MHz, CDCl₃), δ 3.97 (d, J = 4.8 Hz, NHCH₂), 5.12 (s, 2H, CH₂Ph), 5.37 (br s, 1H, CHN₂), 5.54 (br s, 1H, NH), 7.35 (5H, m, C₆H₅).

(ii) (S)-Benzyl 4-diazo-3-oxo-1-phenylbutan-2-ylcarbamate (9b).

Compound 9b was obtained as a yellow solid in 80% yield, with the following data: mp, 80 to 81°C; ¹H NMR (300 MHz, CDCl₃), δ 3.03 (d, 2H, J = 6.8 Hz, CH₂Ph), 4.45 to 4.49 (m, 1H, CHNH), 5.21 (s, 2H, OCH₂Ph), 5.31 (s, 1H, CHN₂), 5.35 (1H, br, NH), 7.18 to 7.42 (m, 10H, ArH).

General procedure for synthesis of compounds 10a and 10b.

A solution of the diazoketones 9a and 9b (1.0 eq) in MeOH at −25°C under N₂, with the exclusion of light, was treated with a solution of silver benzoate (0.11 eq) in Et₃N (2.9 eq). The reaction mixture was allowed to warm to room temperature within 3.0 h in the dark and then concentrated under reduced pressure. The oily residue was dissolved in EtOAc and washed with a brine solution. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

(i) Methyl 3-(benzyloxycarbonylamino) propanoate (10a).

Compound 10a was obtained as a yellow solid in 75% yield, with the following data: mp, 80 to 81°C; ¹H NMR (300 MHz, CDCl₃), δ 2.41 to 2.51 (m, 2H, CH₂COOCH₃), 3.43 to 3.51 (m, 2H, NHCH₂), 3.65 (s, 3H, OCH₃), 5.09 (s, 2H, CH₂Ph), 5.30 (1H, br, NH), 7.35 (m, 5H, ArH).

(ii) (S)-Methyl 3-(benzyloxycarbonylamino)-4-phenylbutanoate (10b).

Compound 10b was obtained as a gummy substance in 75% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 2.49 to 2.53 (m, 2H, CH₂COOCH₃), 2.80 to 2.99 (m, 2H, CH₂Ph), 3.67 (s, 3H, OCH₃), 4.17 to 4.24 (m, 1H, NHCH), 5.07 (s, 2H, OCH₂Ph), 5.29 (d, J = 7.3 Hz, NH), 7.15 to 7.35 (m, 10H, ArH); ESI-MS, (m/z) 328.3 (M + H)⁺.

General procedure for synthesis of compounds 11a and 11b.

MeOH was added dropwise over a period of 20 min to a mixture of esters of N-protected amino acids (10a and 10b) (1.0 eq) and NaBH₄ (2 eq) in THF at 50 to 55°C. The mixture was stirred for 10 to 25 min, and then water was added to the reaction mixture. Organic solvent was concentrated under reduced pressure. The residue was taken in EtOAc and extracted with brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, using hexane-EtOAc as the eluent.

(i) Benzyl 3-hydroxypropylcarbamate (11a).

Compound 11a was obtained as a gummy substance in 75% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.47 to 1.59 (m, 2H, CH₂CH₂CH₂OH), 2.87 to 3.31 (m, CH₂CH₂CH₂OH), 3.47 to 3.61 (m, 3H, CH₂CH₂CH₂OH), 3.94 (br s, 1H, NHCH), 5.09 (s, 2H, O-CH₂Ph), 7.35 (s, 5H, Ar-H).

(ii) (S)-Benzyl 4-hydroxy-1-phenylbutan-2-ylcarbamate (11b).

Compound 11b was obtained as a gummy substance in 80% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.80 to 1.85 (m, 2H, CH₂CH₂OH), 2.82 to 2.87 (m, CH₂C₆H₅), 3.65 (s, 1H, CH₂CH₂OH), 3.94 (br s, 1H, NHCH), 4.72 to 4.75 (m, 2H, CH₂CH₂OH), 5.07 (s, 2H, O-CH₂Ph), 7.15 to 7.35 (m, 10H, Ar-H); ESI-MS, (m/z) 300 (M + Na)⁺.

General procedure for synthesis of compounds 12a and 12b.

Compounds 12a and 12b were prepared by a method similar to that described for compound 4a.

(i) 3-(Benzyloxycarbonylamino)propyl methanesulfonate (12a).

Compound 12a was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.65 to 1.79 (m, 2H, CH₂CH₂CH₂OSO₂CH₃), 2.87 to 3.07 (m, CH₂CH₂CH₂OSO₂CH₃), 3.15 (s, 3H, OSO₂CH₃), 3.91 (br s, 1H, NHCH), 4.07 (t, J = 4.7 Hz, 2H, CH₂CH₂CH₂OSO₂CH₃), 5.07 (s, 2H, O-CH₂Ph), 7.35 (s, 5H, Ar-H).

(ii) (S)-3-(Benzyloxycarbonylamino)-4-phenylbutyl methanesulfonate (12b).

Compound 12b was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.69 to 1.80 (m, 2H, CH₂CH₂OSO₂CH₃), 2.88 to 2.98 (m, CH₂C₆H₅), 3.15 (s, 3H, OSO₂CH₃), 3.96 (br s, 1H, NHCH), 4.26 (t, J = 4.5 Hz, CH₂CH₂OSO₂CH₃), 5.08 (s, 2H, O-CH₂Ph), 7.15 to 7.35 (m, 10H, Ar-H); ESI-MS, (m/z) 400 (M + Na)⁺.

General procedure for synthesis of compounds 13a and 13b.

Compounds 13a and 13b were prepared by a method similar to that described for compound 5a.

(i) Benzyl 3-(4-methylpiperazin-1-yl)propylcarbamate (13a).

Compound 13a was obtained as a gummy substance in quantitative yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.95 [s, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 2.41 (s, 3H, NCH₃), 2.61 to 2.67 [m, 10H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 3.35 to 3.42 (m, 2H, NHCH₂).

(ii) (S)-Benzyl 4-(4-methylpiperazin-1-yl)-1-phenylbutan-2-ylcarbamate (13b).

Compound 13b was obtained as a gummy substance in 73% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.67 to 1.79 [m, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 2.26 (s, 3H, NCH₃), 2.32 to 2.49 [m, 10H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 2.76 to 3.09 (m, CH₂C₆H₅), 3.94 (br s, 1H, NHCH), 6.0 (br s, 1H, NHCH), 7.15 to 7.35 (m, 10H, Ar-H); ESI-MS, (m/z) 382.3 (M + H)⁺.

General procedure for synthesis of compounds 14a and 14b.

Compounds 14a and 14b were prepared by a method similar to that described for compound 6a.

(i) 3-(4-Methylpiperazin-1-yl) propan-1-amine (14a).

Compound 14a was obtained as a gummy substance and used without further purification.

(ii) (S)-4-(4-Methylpiperazin-1-yl)-1-phenylbutan-2-amine (14b).

Compound 14b was obtained as a gummy substance and used without further purification.

General procedure for synthesis of compounds 15a to 15c.

The final compounds 15a to 15c were prepared by a method similar to that described for compounds 7a to 7r.

(i) 7-Chloro-N-(3-(4-methylpiperazin-1-yl)propyl)quinolin-4-amine (15a).

Compound 15a was obtained as an off-white solid in 75% yield, with the following data: mp, 108 to 110°C; ¹H NMR (300 MHz, CDCl₃), δ 1.97 [s, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 2.41 (s, 3H, NCH₃), 2.62 to 2.68 [m, 10H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 3.37 to 3.43 (m, 2H, NHCH₂), 6.34 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.33 to 7.37 (dd, J = 2.0, 8.8 Hz, 1H, Ar-H quinoline), 7.91 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 1.9 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.2 Hz, 1H, Ar-H quinoline); ¹³C NMR (50 MHz, CDCl₃), δ 23.4, 44.3, 46.2, 53.5, 55.2, 58.6, 98.4, 117.4, 122.4, 124.6, 128.3, 134.7, 148.8, 150.6, 151.8; HRMS calculated for [C₁₇H₂₃ClN₄ + H]⁺ 319.1684, found 319.1683; ESI-MS, (m/z) 319.3 (M + H)⁺.

(ii) N-(3-(4-methylpiperazin-1-yl)propyl)-7-(trifluoromethyl)quinolin-4-amine (15b).

Compound 15b was obtained as an off-white solid in 70% yield, with the following data: mp, 76 to 78°C; ¹H NMR (300 MHz, CDCl₃), δ 1.95 to 2.00 [m, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 2.41 (s, 3H, NCH₃), 2.63 to 2.68 [m, 10H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 3.38 to 3.42 (m, 2H, NHCH₂), 6.41 (d, J = 4.05 Hz, 1H, Ar-H quinoline), 7.53 to 7.56 (dd, J = 1.2, 6.5 Hz, 1H, Ar-H quinoline), 8.07 (d, J = 6.5 Hz, 1H, Ar-H quinoline), 8.25 (s, 1H, Ar-H quinoline), 8.59 (d, J = 4.0 Hz, 1H, Ar-H quinoline); ¹³C NMR (75 MHz, CDCl₃), δ 23.3, 44.4, 46.3, 53.5, 55.2, 58.6, 99.4, 119.3, 119.3, 120.7, 122.2, 125.9, 127.4, 127.5, 130.4, 130.9, 131.3, 147.6, 150.4, 152.4; HRMS calculated for [C₁₈H₂₃F₃N₄ + H]⁺ 353.1948, found 353.1951; ESI-MS, (m/z) 353.2 (M + H)⁺.

(iii) (S)-7-Chloro-N-(4-(4-methylpiperazin-1-yl)-1-phenylbutan-2-yl)quinolin-4-amine (15c).

Compound 15c was obtained as a gummy substance in 45% yield, with the following data: ¹H NMR (300 MHz, CDCl₃), δ 1.67 to 1.89 [m, 2H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 2.28 (s, 3H, NCH₃), 2.36 to 2.47 [m, 10H, CH₂CH₂cycN(CH₂CH₂)₂NCH₃], 2.76 to 3.09 (m, CH₂C₆H₅), 3.91 (br s, 1H, NHCH), 6.40 (d, J = 4.2 Hz, 1H, Ar-H quinoline), 7.11 to 7.27 (m, 5H, CH₂C₆H₅), 7.76 (d, J = 6.9 Hz, 1H, Ar-H quinoline), 7.88 (d, J = 8.7 Hz, 1H, Ar-H quinoline), 7.95 (d, J = 6.5 Hz, 1H, Ar-H quinoline), 8.35 (d, J = 4.0 Hz, 1H, Ar-H quinoline); ¹³C NMR (CDCl₃, 50 MHz), δ 37.9, 40.8, 45.0, 51.7, 55.1, 58.9, 97.6, 117.7, 122.0, 124.6, 125.9, 127.6, 128.6, 128.5, 128.2, 134.6, 138.2, 146.4, 149.0, 149.6; HRMS calculated for [C₁₇H₂₃ClN₄ + H]⁺ 409.2154, found 409.2151; ESI-MS, (m/z) 409.3 (M + H)⁺.

Biological methods. (i) In vitro antimalarial assay.

The compounds were evaluated for antimalarial activity against the 3D7 (CQ sensitive) and K1 (CQ resistant) strains of P. falciparum by using a malaria SYBR green I nucleic acid staining dye-based fluorescence (MSF) assay as mentioned by Singh et al. (28). The stock (5 mg/ml) solution was prepared in DMSO, and test dilutions were prepared in culture medium (RPMI 1640-fetal bovine serum [FBS]). The final concentration of DMSO in Plasmodium cultures was <1%. Chloroquine diphosphate was used as a reference drug.

(a) Test technique.

Fifty microliters of culture medium was dispensed into each well of a 96-well plate, followed by the addition of 50 μl of the highest concentration of test compounds (in duplicate wells) in row B. Subsequent 2-fold serial dilutions were prepared, and finally, 50 μl of a 1.0% parasitized cell suspension containing 0.8% parasitemia was added to each well, except for 4 wells in row A, which received a nonparasitized erythrocyte suspension. The plates were incubated at 37°C in a CO₂ incubator in an atmosphere of 5% CO₂ and air, and 72 h later, 100 μl of lysis buffer containing 2× SYBR green I (in nitrogen) was added to each well and incubated for 1 h at 37°C. The plates were examined at 485 ± 20 nm of excitation and 530 ± 20 nm of emission for the number of relative fluorescence units (RFUs) per well, using a fluorescence plate reader (FLX800; Biotek).

(b) Statistical analysis.

Data were transferred to a graphic program (Excel), and IC₅₀s were obtained by logit regression analysis of dose-response curves by use of a preprogrammed Excel spreadsheet.

(ii) In vitro assay for evaluation of cytotoxic activity.

Cytotoxicity of the compounds was determined using the Vero cell line (C1008; monkey kidney fibroblasts) following the method mentioned by Sinha et al. (19). The cells were incubated with compound dilutions for 72 h, and MTT was used as a reagent for detection of cytotoxicity. The 50% cytotoxic concentration (CC₅₀) was determined using nonlinear regression analysis of dose-response curves in a preprogrammed Excel spreadsheet. The selectivity index (SI) was calculated as follows: SI = CC₅₀/IC₅₀.

(iii) 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 (30). The study received ethical approval from CSIR-Central Drug Research Institute's Institutional Animal Care and Use Committee recognized by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. In addition, selected compounds were also tested on multidrug-resistant P. yoelii and P. vinckei. Mice (22 ± 2 g) were inoculated with 1 × 10⁶ parasitized red blood cells (RBC) on day 0, and treatment was administered to a group of five mice from days 0 to 6, once daily. Aqueous suspensions of compounds were prepared with 0.5% (vol/vol) Tween 80. The efficacy of test compounds was evaluated at 100 mg/kg/day, and the required daily dose was administered in 0.5 ml via the oral route. Parasitemia levels were recorded from thin blood smears at regular intervals of 4 days throughout the 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. In the case of arteether, the drug was dissolved in neutralized ground nut oil and administered via the intramuscular route.

(iv) Determination of hematin-4-aminoquinoline derivative association constant.

Hematin and amino quinoline association constants for the compounds synthesized in the present study were determined by a spectrophotometric titration procedure in aqueous DMSO at pH 7.5 (31). Under the assay conditions, hematin is strictly in a monomeric state, and interpretation of the results is not complicated by the need to consider the hematin disaggregation process. The association constant calculated by this technique is a good reflection of the interaction that would occur in the acidic food vacuole. Use of pH 7.5 improves the stability of hematin solutions and the quality of data.

(v) In vitro inhibition of β-hematin formation.

The ability of the 4-aminoquinoline derivatives to inhibit β-hematin polymerization was induced by 1-oleoyl-rac-glycerol, and measurements were carried out at 405 nm by use of a UV spectrophotometer (32). The triplicate values obtained from the assay were expressed as percentages of inhibition relative to hemozoin formation in a drug-free control. The 50% inhibitory concentration (IC₅₀) values for the compounds were obtained from the sigmoidal dose-response curves, using nonlinear regression curve-fitting analyses with Graph Pad Prism 30 v.3.00 software (29). Each IC₅₀ is the result of at least three separate experiments performed in duplicate.

Molecular docking method. (i) Database preparation.

Compounds were used to perform a docking study against heme. To do a comparison study, we used the chloroquine drug for docking. A database was prepared by using the SYBYL-X 1.3 (Tripos Inc., St. Louis, MO) modeling package (33). All compounds were drawn through the sketch module in SYBYL. Structures were minimized further by adding Gasteiger-Huckel charges along with distance-dependent dielectric and Powell conjugate gradient algorithms, with a convergence criterion of 0.001 kcal/mol. All structures were put into a database and finally aligned with each other by way of the “fit atom” method.

(ii) Protomol-based docking study.

The molecular docking studies of synthesized compounds were performed using the Surflex-Dock module with standard protocols in SYBYL-X 1.3. We extracted the heme molecule from a protein collected from a protein database. Hydrogen atoms were added to the heme structure to obtain a correct configuration. Charges were also added to it by using Gasteiger-Huckel charges, and energy minimizations were performed with a standard protocol, using the Tripos force field with the same Gasteiger-Huckel charges along with distance-dependent dielectric and Powell conjugate gradient algorithms, with a convergence criterion of 0.001 kcal/mol. After that, the automatic Protomol generation method was utilized to create a grid over heme. Molecular docking was performed by placing the molecules, including the reference, into the grid with a reasonable scoring function to score the ligands and Protomol-guided docking. Finally, the Protomol-based method and empirically derived scoring functions (e.g., total score, crash score, polarity, etc.) were used to calculate the binding affinities.

Experimental procedures for pharmacokinetic studies of compounds 7g and 16 (phosphate salt of 7g). (i) In vitro pharmacokinetics of compound 7g.

Simulated gastric fluid (SGF) was prepared by dissolving 234 mg of NaCl and 37.2 mg of KCl in 100 ml triple-distilled water (TDW), and the pH was adjusted to 1.2 with concentrated HCl. Simulated intestinal fluid (SIF) was prepared by dissolving 680 mg of KH₂PO₄ and 90 mg of NaOH in 100 ml triple-distilled water, and the pH was adjusted to 6.8 with orthophosphoric acid. SGF/SIF (2 ml) was taken in a test tube and preincubated in a shaking water bath for 10 to 15 min at 37 ± 2°C. Five microliters of 7g stock solution (100 μg/ml) was used to spike the preincubated SGF/SIF to produce a concentration of 250 ng/ml and immediately subjected to incubation. Fifty microliters of the incubation mixture was sampled at 0, 15, 30, 60, 90, and 120 min, diluted 5 times with acetonitrile, and analyzed by LC-MS/MS. The metabolic stability of 7g was determined in duplicate, using glass tubes. To each tube, 460 μl of phosphate buffer (0.1 M; pH 7.4) and 12.5 μl of microsomal protein (20.0 mg/ml) were added and incubated at 37 ± 0.2°C for 5 min. Next, 2.5 μl of test compound (1 mM) was added. For positive control, testosterone was used as the test compound. The reaction was initiated by addition of 25 μl of NADPH (24 mM), and the reaction mixture was incubated for 0, 5, 10, 15, 30, 45, and 60 min. For negative control, NADPH was replaced by 25 μl of phosphate buffer. The reaction was stopped by addition of 450 μl ice-cold acetonitrile to 50 μl reaction mixture collected at predefined time intervals, followed by centrifugation for 15 min at 10,000 rpm. One hundred microliters of supernatant was directly analyzed by LC-MS/MS. For assessment of plasma stability, a blank rat plasma (2 ml) in a test tube was preincubated in a shaking water bath for 10 min at 37 ± 2°C. Two microliters of 7g stock solution (100 μg/ml) was used to spike the preincubated plasma to produce a concentration of 100 ng/ml and immediately subjected to incubation. Fifty microliters of the plasma was sampled at 0, 5, 10, 15, 30, 60, 90, and 120 min. Plasma proteins were precipitated by addition of 400 μl acetonitrile at predefined time intervals, followed by centrifugation for 15 min at 10,000 rpm. One hundred microliters of supernatant was directly analyzed by LC-MS/MS (34).

(ii) In vivo pharmacokinetic study.

The in vivo pharmacokinetic study was performed with male Sprague-Dawley rats (n = 4). An intravenous formulation of compound 7g was prepared by dissolving an accurately weighed quantity of 7g (20 mg) in 2 ml of DMSO, followed by addition of 400 μl of ethanol and 800 μl of glycerol and vortexing for 2 min. The volume was then made up to 4 ml with TDW, followed by vortexing for 2 min. For the oral formulation, an accurately weighed quantity of 7g (30 mg) was transferred to a mortar and triturated with a pestle, using 200 μl of Tween 20. The volume was then made up to 4 ml with a 0.25% carboxymethyl cellulose (CMC) suspension. The intravenous and oral formulations of the phosphate salt form (compound 16) were prepared by dissolving in distilled water. Blood samples were collected from the retroorbital plexus of rats under light ether anesthesia into microcentrifuge tubes containing heparin as an anticoagulant at 0.083, 0.25, 0.5, 1, 2, 3, 5, 7, 9, 24, 30, and 48 h postdosing for the intravenous study and 0.25, 0.5, 1, 2, 3, 5, 7, 9, 24, 30, and 48 h postdosing for the oral study. Plasma was harvested by centrifuging the blood at 13,000 rpm for 10 min in a Sigma 1-15K centrifuge (Frankfurt, Germany) and was stored frozen at −70 ± 10°C until bioanalysis. Each plasma sample (100 μl) was processed using the protein precipitation method, using 200 μl acetonitrile containing piracetam as an internal standard (IS) as the protein precipitant, and 10 μl of the supernatant was injected for LC-MS/MS (34).