Discovery of amodiachins, a novel class of 4-aminoquinoline antimalarials active against multidrug-resistant Plasmodium falciparum

Abstract

A structure activity relationship study was conducted to optimize metabolic stability and in vivo activity of amodiaquine analog antimalarials. Antiplasmodial activity of synthesized amodiachins (ADCs) were evaluated against drug-sensitive and drug-resistant strains of Plasmodium falciparum. Select compounds were tested in Plasmodium yoelli murine models. Structural modifications, including introduction of a piperidine ring and varied N-alkyl substitutions enhanced antiparasitic activity, metabolic stability, and in vivo efficacy. Compound 43 (ADC-028) emerged as a standout candidate, exhibiting nanomolar activity against drug-sensitive and multidrug-resistant P. falciparum, minimal cytotoxicity, and favorable murine microsomal stability (t_1/2 = 48.2 min). In vivo, 43 achieved a non-recrudescence dose at 16 mg/kg/d and a single dose cure at 50 mg/kg. Pharmacokinetic analysis of 43 showed excellent metabolic stability (T_1/2 = 84 h) and oral bioavailability (F = 76%). This study identifies 43 as a potential candidate for further evaluation as a novel compound to overcome drug-resistant malaria.

Graphical Abstract

INTRODUCTION

Malaria is a devastating disease caused by the parasitic infection of Plasmodium spp. and is spread from host-to-host through infected female Anopheles mosquitoes.^1–5 Compared to pre-pandemic estimates in 2019, malaria cases have risen from 229 million to a staggering 263 million cases worldwide in 2023 while deaths have increased from 409,000 individuals in 2019 to an estimated 567,000 people in 2023.^6,7 The emergence and spread of resistant strains of the deadliest species, P. falciparum (Pf), threaten global prevention, treatment, and eradication efforts.⁸ Therefore, the discovery and development of novel therapies that overcome existing resistance mechanisms of P. falciparum are desperately needed.

The discovery of the natural product quinine and its use to treat malaria inspired research programs that ultimately led to the discovery of successful 4-aminoquinoline antimalarials: chloroquine (1), amodiaquine (2), and piperaquine in particular (Figure 1).⁹

Figure 1:

Chemical structures of the antimalarials quinine and select 4-aminoquinolines: chloroquine (1), amodiaquine (2), and isoquine (3). Representative structure (4) of initial rationale for the amodiachin SAR study described henceforth.

Unfortunately, chloroquine was deployed as a monotherapy in worldwide efforts to prevent and cure malaria, and now chloroquine resistance in Plasmodium falciparum has spread to virtually all disease endemic areas around the globe.¹⁰ Further development of the 4-aminoquinoline scaffold gave rise to amodiaquine (2), which was shown to overcome chloroquine resistant strains of Pf.^11,12 Despite decades of use, amodiaquine’s clinical use has been restricted due to its connection with hepatotoxicity and agranulocytosis.^13,14 The observed toxicity appears to be associated with the P-450-catalyzed oxidation of the para-hydroxyl moiety of amodiaquine.^11,15 Work to eliminate this metabolic liability resulted in an amodiaquine regioisomer, isoquine (3).¹¹ When the para-hydroxyl and meta-Mannich base were interchanged, isoquine exhibited an improved toxicological profile, and increased activity in vitro and in vivo compared to amodiaquine; however, during preclinical evaluation isoquine underwent a high-rate of first pass metabolism – posing a significant challenge to further development and compromising isoquine activity in chloroquine resistant (CQR) Plasmodium parasites.¹⁶ While the advancement of isoquine for clinical trials was halted, this work highlights exploitable chemical space that overcomes drug resistance to previous 4-aminoquinoline analogs. In our group’s previous work of 4-aminoquinolines, structure activity relationship (SAR) studies focusing on the 3-methyl position of the quinoline in the sontochin-like series named “pharmachins” indicated improved activity against CQR strains when replaced by a trifluoromethoxy phenyl.¹⁷ This result suggested that increasing rigidity at the 3-position by adding aromatic rings and/or heterocycles improves potency against CQR and multidrug-resistant (MDR) strains. We decided to adopt the same strategy to rigidify the 4-position side chain of amodiaquine to overcome drug resistance and to address the toxicity issue by eliminating conversion to a toxic quinoneimine metabolite. Focusing on the aryl substitution pattern of amodiaquine we sought to replace the labile hydroxy group and diethylaminomethyl feature with basic substituted heterocycles (4, Figure 1) as a countermeasure to chloroquine resistance and to increase metabolic stability. Herein, we introduce a new series termed “amodiachins” (ADCs) by describing the associated structure activity profile against MDR P. falciparum parasites, and for select molecules we provide the results from testing for metabolic stability, cytotoxicity, pharmacokinetics, and efficacy in a murine model of malaria infection.

CHEMISTRY

The initial compound design was focused on creating an amodiaquine-like structure with a para-substituted heterocycle, while incorporating a trifluoromethoxy phenyl at the 3-position of the quinoline ring, similar to the pharmachins. Compounds 7-21 were synthesized using the procedures outlined in Scheme 1 and Scheme 2. Starting materials 5 and 6a were commercially available, however 6b was synthesized in-house using previously described methods.¹⁸ Respective 4-aminoquinolines were prepared using either, (i) ethanol and catalytic 12 M hydrochloric acid at reflux, or (ii) 2-ethoxyethanol and phenol at 150 °C. Compounds 12-16 were prepared from the respective para-substituted bromobenzene (9a and 9b) and either alkylated with iodoethane or benzyl bromide (10a-10c). 11a-11c were prepared through amination of the para-substituted bromobenzene intermediates using a Buchwald-Hartwig cross-coupling reaction to afford the para-substituted anilines.¹⁹ Final compounds 12-16 were prepared through reaction conditions (ii). Using the previously described methods, compound 17 was afforded from commercially available 9c.

Scheme 1: Synthesis of initially designed amodiachins.^a.

^aReagents and conditions: (i) aniline, 4-chloroquinoline, EtOH, fuming HCl, reflux 16 h. (ii): aniline, 4-chloroquinoline, phenol, EGEE, 150 °C, 12 h. (iii): substituted bromobenzene, 2M K₂CO₃, bromobenzene, DMF, 50 °C, 12 h. (iv): substituted bromobenzene, iodoethane, DIPEA, DMF, rt, 48 h. (v): substituted bromobenzene, Pd(t-Bu₃P)₂, Pd(dba)₂, LiHMDS, toluene, 70 °C, 18 h, HCl.

Scheme 2: Synthesis of compounds to explore the contribution of substituents around benzenoid ring of the quinoline core.^b.

^bReagents and conditions: (i) aniline, 4-chloroquinoline, EtOH, fuming HCl, reflux 16 h. (vi) aniline, 4-chloroquinoline, phenol, DMF, 150 °C, 2 h.

Scheme 2 depicts the chemical synthesis of compounds 20-26 using methods (i) and (vi) to examine the contribution of the quinoline ring substituents to antiplasmodial activity. Commercially available 18a and 18b were used in the preparation of compounds 20-26 using a previously described method (i) or an alternative method (vi) where the appropriate 4-chloro containing quinoline was mixed with the appropriate aniline in the presence of phenol in DMF and heated to 150 °C for 45 min in a microwave reactor.

Target compounds 31-45 were prepared by the synthesis outlined in Scheme 3 to assess the contribution of the heterocycle to antiplasmodial activity and later the contribution of the tertiary amine’s alkyl substituent (R⁶, R⁷) to in vivo antimalarial efficacy. Compounds 31 and 32 were synthesized from commercially available alkyl piperazines (28a and 28b) and 27 with trimethylamine in DMSO at 120 °C for 16 h (vii). The resulting intermediates were reduced to the appropriate aniline and then used in the previously described procedure (i) with 6a to afford the final product. Commercially available 30c-30e and 6a were used in previously described procedures (vi) to produce compounds 33-35. Compounds 36 and 37 were synthesized from 30f and 30g in the presence of 6a in THF at 120 °C for 20 minutes (ix). To afford compounds 40-45, 36 and 37 were deprotected in the presence of trifluoroacetic acid in dichloromethane (x) and then reacted with the appropriate alkyl halide and either (xi) DIPEA in DMF at room temperature for 16 h or (xii) potassium carbonate in DMF at 85 °C for 16 h.

Scheme 3: Synthesis of amodiachins that explored the contribution of the heterocycle and its alkyl substituents to antiplasmodial activity.^c.

^cReagents and conditions: (vii): 27, substituted piperazine, TEA, DMSO, 120 °C, 16 h. (viii) substituted nitrobenzene, Pd/C, MeOH,12 h. (i) aniline, 4-chloroquinoline, EtOH, fuming HCl, reflux 16 h. (vi) aniline, 4-chloroquinoline, phenol, DMF, 150 °C, 2 h. (ix) aniline, 4-chloroquinoline, THF, 120 °C, 20 m. (x): 4-analinoquinoline, TFA, DCM, 2 h. (xi): 4-analinoquinoline, iodoethane, DIPEA, DMF, rt, 16 h. (xii): 4-analinoquinoline, alkyl halide (1.2 equiv.), K₂CO₃, DMF, 85 °C, 16 h.

RESULTS AND DISCUSSION

Compounds were assessed for antiplasmodial activity against drug-sensitive (D6) and multidrug-resistant (Dd2) strains of Plasmodium falciparum in a fluorescence-based antiplasmodial assay, and are reported in Tables 1, 2. 3 and 5.^20,21 The P. falciparum strain Dd2 is reported to be resistant to mefloquine, cycloguanil, pyrimethamine, and chloroquine.^22,23

Table 1.

Markush structure (above) of select analogs and associated antiplasmodial activity against PfD6 and PfDd2 and cytotoxicity in HepG2 mammalian cells.^d

Compound	R1	R2	IC50 v D6 ± SD (nM)	IC50 v Dd2 ± SD (nM)	CC50 v HepG2 (μM)	cLogP
1	-	-	9.6 ± 2	106 ± 10	3824	5.06
7	--H		≥ 250	≥ 250	> 200	4.60
8			≥ 250	≥ 250	> 200	7.61
12	--H		≥ 250	≥ 250	> 200	6.59
13	--H		69 ± 28	≥ 250	> 200	5.25
14			≥ 250	≥ 250	> 200	8.26
15	--H		101 ± 15	≥ 250	38	6.45
16			≥ 250	≥ 250	> 200	9.46
17	--H		20 ± 3	26 ± 9	> 200	6.88
20	--H		10 ± 3	26 ± 12	> 200	5.69
21	--H		14 ± 5	23 ± 9	185	5.16
38	--H		51 ± 66	30 ± 33	54	4.58

^dclogP values calculated using ChemDraw software (version 22). Antiplasmodial activity (IC₅₀) values and standard deviations (SD) were derived from the average of at least three independent experiments, each performed in quadruplicate. Cytotoxicity (CC₅₀) assay was performed using human hepatoma derived HepG2 cells across an initial concentration of 200 μM to 10 nM in triplicate. Details of assays performed can be found in the Experimental Section.

Table 2.

Markush structure (above) of a series of quinoline analogs including associated antiplasmodial activity against PfD6 and PfDd2 and cytotoxicity in HepG2 mammalian cell.^d

Compound	R3	IC50 v D6 ± SD (nM)	IC50 v Dd2 ± SD (nM)	CC50 v HepG2 (μM)	cLogP
20		10 ± 3	27 ± 12	> 200	5.69
22		44 ± 27	90 ± 38	106	4.88
23		48 ± 30	53 ± 19	> 200	5.69
24		18 ± 10	26 ± 9	> 200	6.31
25		9 ± 4	14 ± 8	48	5.84
26		15 ± 4	29 ± 10	> 200	5.93

^dclogP values calculated using ChemDraw software (version 22). Antiplasmodial activity (IC₅₀) values and standard deviations (SD) were derived from the average of at least three independent experiments, each performed in quadruplicate. Cytotoxicity (CC₅₀) assay was performed using human hepatoma derived HepG2 cells across an initial concentration of 200 μM to 10 nM in triplicate. Details of assays performed can be found in the Experimental Section.

Table 3.

Markush structure (above) of nitrogen containing heterocycle analogs with varying substituents and the associated antiplasmodial activity against PfD6 and PfDd2 and cytotoxicity in mammalian cells.^d

Compound	R4	IC50 v D6 ± SD (nM)	IC50 v Dd2 ± SD (nM)	CC50 v HepG2 (μM)	cLogP
36		69 ± 34	217 ± 57	> 200	6.57
32		54 ± 27	95 ± 41	> 200	7.77
33		27 ± 22	68 ± 54	> 200	5.53
31		11 ± 13	11 ± 8	> 200	6.39
34		197 ± 75	200 ± 71	> 200	5.98
39		9 ± 2	71 ± 15	58	5.34
40		5 ± 2	11 ± 3	111	6.32
35		4 ± 2	10 ± 6	> 200	6.63

^dclogP values calculated using ChemDraw software (version 22). Antiplasmodial activity (IC₅₀) values and standard deviations (SD) were derived from the average of at least three independent experiments, each performed in quadruplicate. Cytotoxicity (CC₅₀) assay was performed using human hepatoma derived HepG2 cells across an initial concentration of 200 μM to 10 nM in triplicate. Details of assays performed can be found in the Experimental Section.

Table 5:

Markush structure (above) of N-terminal alkyl analogs with their associated antiplasmodial activity against PfD6 and PfDd2 and cytotoxicity in mammalian cells (below).^d

Compound	R4	X	IC50 v D6 ± SD (nM)	IC50 v Dd2 ± SD (nM)	CC50 v HepG2 (μM)	cLogP
40		C	5 ± 2	11 ± 3	111	6.32
35		C	4 ± 2	10 ± 6	> 200	6.63
41		C	8 ± 3	15 ± 6	> 200	6.85
42		C	4 ± 4	11 ± 13	> 200	7.37
43		C	7 ± 7	10 ± 11	> 200	7.24
44		C	2.6 ± 1.7	9 ± 9	> 200	7.15
45		N	8 ± 3	12 ± 6	> 200	6.61

^dclogP values calculated using ChemDraw software (version 22). Antiplasmodial activity (IC₅₀) values and standard deviations (SD) were derived from the average of at least three independent experiments, each performed in quadruplicate. Cytotoxicity (CC₅₀) assay was performed using human hepatoma derived HepG2 cells across an initial concentration of 200 μM to 10 nM in triplicate. Details of assays performed can be found in the Experimental Section.

Compounds 7, 8, and 12-16 had limited activity (PfD6 and PfDd2 IC₅₀ ≥ 250 nM, Table 1), with compounds 13 and 15 exhibiting minimal antiplasmodial activity against PfD6, IC₅₀ = 69 ± 28 nM and 101 ± 15 nM, respectively. However, this level of activity was not maintained against PfDd2 strain (Table 1). Moving away from the morpholine heterocycle, a piperazine ring was next implemented as in 17. Promising activity against drug- sensitive PfD6 IC₅₀ = 20 ± 3 nM and drug-resistant PfDd2 IC₅₀ = 26 ± 9 nM, was observed with this compound. Concerned that this molecule might be unstable to microsomal metabolism it was decided to replace the terminal aromatic ring with an ethyl (20) and methyl (21) group. The structure activity profile was further investigated by varying the substituents at the 6- and 7-positions of the quinoline ring (Table 2). Interestingly, loss of the 7-position chlorine in 22 reduces potency in combination with decreased susceptibility against PfDd2. However, introducing a chlorine atom at the 6-position (23) returns activity across both strains, but reduction of potency is still observed. It was not until the 7-position chlorine was reintroduced, as in 24, that we observed the potency return to the low nanomolar (≤ 20 nM) range. Clearly, the 7-position chlorine was essential for potent activity and therefore substitutions were varied at the critical 7-position to include bromine (25), as well as trifluoromethyl (26). The importance of the 7-position chlorine to the antimalarial activity of some 4-aminoquinolines is well established across drug discovery campaigns and is suggested as playing a vital role in the 4-aminoquinoline mechanism of action.²⁵ Herein we can see that although activity against PfD6 and PfDd2 is maintained with both derivatives, 25 was more toxic to mammalian cells (CC₅₀ = 48) than its progenitor 20. Furthermore, the trifluoromethyl derivative, 26, exhibited a slight decrease in antiplasmodial activity against the drug-sensitive PfD6 strain.

Having established that the 7-position chlorine atom was an essential element for antiplasmodial activity in this 4-aminoquinoline SAR campaign, we returned our focus to the 4-position sidechain. Indeed, from the results presented in Table 1 the attachment of a piperazine ring to the aromatic phenyl group greatly improves antiparasitic activity. We therefore decided to explore the effect of structural variation of the N-terminal group on antiplasmodial activity, results reported in Table 3. The N-Boc protected analog (36) exhibited diminished antiplasmodial activity, which may relate to the weakened basicity of the carbamate nitrogen. This is consistent with the poor activity of 38 where the same nitrogen atom carries only a hydrogen atom. Use of a large aromatic trifluoromethylphenyl group (38) also diminished the activity relative to 20. In hopes of enhancing basicity and metabolic stability at the outermost ring nitrogen we introduced cyclopropyl (33) and tert-butyl (31) groups. While the results for 33 showed relatively modest antiplasmodial activity, we found that the potency of 31 was in low nanomolar range (≤ 20 nM) against both PfD6 and PfDd2. Following these results, we explored the contribution of the individual piperazine nitrogen atoms to antiplasmodial activity. This led to the design and synthesis of two novel piperidine derivatives – one with a nitrogen atom attached directly to the aromatic ring (34) and another with a nitrogen atom at the outermost position (39) as a secondary amine. The activity of 34 was severely reduced in both strains of Pf, but when the piperidine nitrogen was presented as a secondary amine (39) activity in PfD6 was maintained compared to 20, but susceptibility of PfDd2 was decreased to 71 ± 15 nM. This same effect was observed in the similar compound 38 (Table 1). Next, we decided to explore the impact of converting the outer secondary amine of 39 to a tertiary amine by addition of an ethyl group. That strategy proved effective for when adorned with an ethyl substituent, compound 40 provided impressively low nM IC₅₀’s with an increase in activity against multidrug-resistant PfDd2. It is noteworthy that 40 exhibits a modest level of cytotoxicity against mammalian cells in culture (CC₅₀ = 111 μM). We therefore decided to explore the impact of increasing length and branching of the terminal alkyl group as the addition of the ethyl substituent in 40 appeared to decrease the cytotoxicity compared to 39 (CC₅₀ = 58 μM). Excitingly the addition of an N-isopropyl in 35 further decreased cell cytotoxicity (CC₅₀ > 200 μM) while exhibiting equipotency against PfD6 and PfDd2. In summary, these results demonstrate that amodiachin derivatives containing either a 4-aminophenyl piperazine bridge or a 4-aminophenyl piperidine bridge exhibit excellent potency against both sensitive and multidrug-resistant strains of Pf. Furthermore, the presence, length, and degree of branching of the N-alkyl substituent can significantly influence potency against drug-sensitive and drug-resistant strains of Pf, and degree of cytotoxicity.

At this point, amodiachins were selected for use in further studies based on antiplasmodial activity (PfD6 IC₅₀ < 20 nM), susceptibility in the drug-resistant strain (PfD6 IC₅₀ / PfDd2 IC₅₀ < 2.5), and cytotoxicity (CC₅₀ > 100 μM). Compounds were next evaluated for metabolic stability in pooled liver murine microsomes (Table 4), from which intrinsic metabolic stability (t_1/2) was calculated.

Table 4:

Microsomal stability, parasite burden suppression, and in vivo efficacy of select amodiachins.^e

Compound	t1/2 (min), CLint (mL/min/kg)	% Py Suppression (2.5 mg/kg/d)	ED50 v Py (mg/kg/d)	ED90 v Py (mg/kg/d)	NRD (mg/kg/d)
17	13.92, 391.9	0%	-	-	-
20	1.86, 2930	60%	1.5	8.4	ND (10)
21	1.44, 3784	-	-	-	-
31	∞, 0.00	30%	4.0	7.6	ND (20)
40	11.0, 496.7	13%	1.8	13	ND (10)
35	44.1, 123.6	48%	1.1	4.1	ND (10)

^et_1/2 and CL_int determined from HPLC UV traces from pooled murine liver microsomes incubated with drug. Full procedure for microsomal stability and in vivo assays can be found in the Experimental Section. (−) = not performed. ND() = not detected at corresponding dosage. All animal studies used group sizes of n = 4 per dose regimen.

The difference of either methyl or ethyl in the N-alkyl substituent (21 and 20) from the outermost nitrogen of piperazine did not enhance metabolic stability (t_1/2 = 1.44 and 1.86 min respectively). However, when capped with a benzyl moiety as in 17, stability was noticeably increased (t_1/2 = 13.9 min). Interestingly, when adorned with a heavily branched tert-butyl group found in 31, no detectable amount of compound was metabolized over the course of the experiment. Evaluating piperidine containing analogs led to a similar observation, increasing the N-alkyl branching by a single carbon in 40 increases stability (t_1/2 = 11.0 to 44.1 min) as seen in 35. All compounds except 21 were down-selected for in vivo assessment of antiplasmodial activity in a modified Peter’s 4-day murine malaria model against P. yoelii.²⁶ Each analog was tested at a fixed dose of 2.5 mg/kg/d and parasitemia on the fifth day was determined from direct microscopic analysis of Giemsa-stained blood smears. Percent parasite suppression (Table 4) was calculated from average parasitemia of experimental groups compared to average parasitemia of the vehicle-only control group. All compounds except 17 exhibited modest suppression at this dose and were selected as candidates for further evaluation to establish the effective dose to reduce parasitemia by 50% (ED₅₀) and 90% (ED₉₀) as well as attempts to determine a non-recrudescence dose (NRD). Compounds 20 and 40 exhibited a relatively large gap between ED₅₀ and ED₉₀ suggesting rapid metabolism, as previously indicated by the results of the microsomal stability assay, or possibly due to poor absorption. The most effective antimalarial from this screen was 35, with an ED₅₀ and ED₉₀ of 1.1 and 4.1 mg/kg/d, respectively. Since 35 exhibited a high degree of antiplasmodial activity in addition to superior in vivo efficacy and metabolic stability, we thought to investigate the interplay of the piperidyl N-alkyl substituent and the resulting pharmacodynamic and pharmacokinetic profile.

Compounds 41-44 were synthesized to explore the effect of piperidyl N-alkyl substituents on in vitro activity and in vivo efficacy by including structural isomers of 3- and 4-carbon alkyl chains. All compounds exhibited similar in vitro activities against PfD6 and PfDd2 (Table 5) and sustained minimal cytotoxicity (CC₅₀ > 200 μM). When examined for murine microsomal stability (Table 6), 41 demonstrated superior stability (t_1/2 = 70 minutes) compared to compounds 42, 43, and 44 which displayed similar stability to 35. Given the excellent in vitro profile of 41-44, all compounds were selected for Py parasite suppression (Table 6). Surprisingly, 41 produced the lowest degree of parasitemia suppression among the tested analogs with 39% suppression compared to control, whereas 43 reduced parasitemia by 91% compared to control. Although 41-44 met the selection thresholds, in vivo efficacy was assessed for 43 alone, due to the high degree of Py suppression (Table 4). Compound 43 revealed an impressive in vivo efficacy profile, albeit higher ED₅₀ (2.1 mg/kg/d) than compounds 20, 40, and 35 (ED₅₀ = 1.5, 1.8, and 1.1 mg/kg/d, respectively), but an ED₉₀ = 2.5 mg/kg/d was the lowest compared to all previous tested compounds. When given at 16 mg/kg/d, 43 eliminated the parasite burden by Day 5, and recrudescence was prevented for the remainder of the experiment (Day 30). It should be noted that 3 out of 4 mice were cured at the end of the 30-day experiment when dosed at 8 mg/kg/d. Due to its excellent in vivo efficacy, 43 was tested in a single dose cure experiment at 50 mg/kg. At this dose, parasites remained undetectable throughout the 30-day experiment, demonstrating a single dose cure.

Table 6:

Microsomal stability, parasite burden suppression, and in vivo efficacy of select amodiachins.^e

Compound	t1/2 (min), CLint (mL/min/kg)	% Py Suppression (2.5 mg/kg/d)	ED50 v Py (mg/kg/d)	ED90 v Py (mg/kg/d)	NRD (mg/kg/d)	SDC (mg/kg)
35	44.1, 123.6	48%	-	-	-	-
41	70.0, 78.0	39%	-	-	-	-
42	42.1, 129.7	53%	-	-	-
43	48.2, 113.3	91%	2.3	2.5	16	50
44	55.5, 98.3	71%	-	-	-	-
45	6.47, 842.9	30%	-	-	-	-

^et_1/2 and CL_int determined from HPLC UV traces from pooled murine liver microsomes incubated with drug. Full procedure for microsomal stability and in vivo assays can found in the Experimental Section. (−) = not performed. All animal studies used group sizes of n = 4 per dose regimen.

Having demonstrated that the iso-butyl group unlocks superior in vivo efficacy in the amodiachin series, we prepared a piperazine mimetic of 43 for the purpose of direct comparison (45). Although 45 exhibited similar antiplasmodial activity in vitro against cultured parasites (PfD6 and PfDd2 IC₅₀ = 8 ± 3 and 12 ± 6 nM), the compound displayed relatively poor metabolic stability (t_1/2 = 6.47 min), which was observed for the other piperazine containing amodiachins in the series. When evaluated for parasitemia suppression at 2.5 mg/kg, parasite burden was only reduced by 30% compared to control. This result compares poorly to the 91% parasite suppression observed in piperidine analog 43. Taken together, our data suggests that 43 has superior in vivo efficacy compared to all other compounds in this series. To further explore the developmental potential of 43 we investigated its pharmacokinetic profile in mice following a single oral dose of 10.0 mg/kg and an IV injection of 0.5 mg/kg in PEG-400 (Figure 2 and Table 7).

Figure 2:

Mean plasma concentration-time profiles of 43 after PO doses at 5 mg/kg in male CD1 mice (n=3).

Table 7:

Pharmacokinetic properties of 43 in male CD1 mice (n=3) after PO dose of 10 mg/kg and IV dose of 0.5 mg/kg. The last three time points obtained in the IV experiment (t = 72, 96, and 120 h) were below the lower limit of quantification (LLOQ = 1 ng/mL) and were therefore removed from pharmacokinetic analysis.

Administration	Parameter	Calculated value	Administration	Parameter	Calculated Value
0.5 mg/kg - IV	CL	9.78 mL/min/kg	10.0 mg/kg - PO	Tmax	8.0 h
	Vss	62.4 L/kg		Cmax	304 ng/mL
	T1/2	74 h		T1/2	85 h
	AUCINF	851 h*ng/mL		AUCINF	13603 h*ng/mL
	MRTINF	100 h		MRTINF	82.8 h
				F	76 %

It is noteworthy that 43 appeared to be well absorbed with an absolute bioavailability of 76% (F) and a maximum blood-stream concentration reached in 8 hours (T_max) achieving a maximum concentration of 304 ng/mL (C_max). Half-life of 43 given through IV administration compared to PO administration agreed well across the two experiments (T_1/2 = 74 h and 85 h, respectively). These results taken together with the ability of 43 to overcome multidrug-resistance mechanisms present in the Dd2 strain of Pf, its relatively long metabolic stability in murine microsomes, and its ability to completely cure an infection in this challenging P. yoelii murine malaria model – highlight 43 as a potent compound with potential as a novel antimalarial against multidrug-resistant Plasmodium falciparum parasites.

SUMMARY AND CONCLUDING REMARKS

Our interest in revisiting amodiaquine stems from its rapid schizonticidal activity and the relatively unexplored chemical space surrounding its 4-aminophenyl side chain. In this study, we synthesized analogs bearing diverse nitrogen containing heterocycles linked through this side chain and found that introducing a piperazine ring at this position unlocked potent antiplasmodial activity. Further exploration into the quinoline ring confirmed the superiority of a 7-position chlorine atom as it contributed to excellent low nanomolar potency against both drug sensitive and multidrug-resistant strains of P. falciparum with limited cytotoxicity. The superior qualities of the piperazine containing amodiachins led us to expand the structure-activity profiling to replace it with the structurally related piperidine. When assessed for metabolic stability and parasite suppression in vivo, the piperidine containing derivative 35 hinted at the improved potential of this new subseries. Structural isomers of 35 were prepared containing linear and branched 3- and 4-carbon alkyl chains. In vitro and in vivo assessment of these derivatives revealed that compound 43 possessed impressive in vitro antiplasmodial IC₅₀ values and superior efficacy in vivo compared to the other members of this series. In vivo efficacy of 43 was remarkable with a very sharp action curve against murine malaria as evidenced by ED₅₀ and ED₉₀ values of 2.3 and 2.5 mg/kg/day, respectively, following 4 days of oral dosing (Figure S1). A NRD of 16 mg/kg/day for 43 over the same time interval was also a distinguishing positive attribute over other derivatives in this series, along with the single dose cure of 50 mg/kg. Pharmacokinetic analysis of 43 revealed rapid oral absorption and extended pharmacokinetics. It’s interesting to note that the lead-likeness of the series was not improved as a result of the SAR study optimization effort.^27,28 Structural modifications that improved in vitro activity often reduced lead-likeness properties, while efforts to enhance lead-likeness tended to diminish in vitro potency. Ultimately, compounds that were effective in vivo also demonstrated strong in vitro activity, despite lacking conventional lead-like characteristics. Collectively, these findings highlight the potential of amodiachins, such as 43, as viable 4-aminoquinoline replacement candidates.

Historically, 4-aminoquinoline antimalarials have been shown to exhibit antiplasmodial activity through inhibition of hemozoin formation. Like chloroquine and amodiaquine, we believe our most active compounds accumulate in the digestive vacuole of the parasite during the intra-erythrocytic stage and inhibits this vital process. Although the exact role of hemozoin inhibition in 4-aminoquinoline activity is still not fully understood, measuring heme complexation and in vitro hemozoin inhibition provides a means to further elucidate the mechanism of action of this series. Future work will focus on mechanistic studies of the current frontrunner molecule, 43, alongside ongoing efforts to optimize oral formulation for potential human use through evaluation of various salt forms and particle size reduction.

The persistent global burden of malaria and the evolving landscape of antimalarial drug resistance compel efforts to discover new therapeutics that address gaps in the treatment of acute and severe infections. The compounds described here, particularly 43 (ADC-028), represent promising candidates to overcome these challenges and offer the potential to advance global malaria eradication initiatives.

EXPERIMENTAL SECTION

Chemistry

Materials and Instruments.

All solvents, starting materials, and reagents were acquired from commercial sources, including (but not limited to): Fisher Scientific, TCI Chemicals, Combi Blocks, Enamine, Sigma Aldrich. Reaction progress was monitored by TLC, GCMS, or HPLC when permitted. Microware reactions were performed with a Biotage Initiator+. Both reverse phase and normal phase flash chromatography was performed using Biotage Isolera and columns including Sfär Silica, Sfär Silica HC, Sfär Silica-Duo, Sfär Silica-KP Amino, and Sfär C18-Duo. ¹H NMR spectra were taken on a Bruker 400 MHz instrument, and chemical shifts are reported relative to TMS (0.0 ppm) and NMR Solvent (CDCl₃, DMSO-d₆, or CD₃OD). Final compounds were measured to be >95% pure by high performance liquid chromatography (HPLC) using an Agilent Technologies 1260 Infinity II system (unless otherwise noted). High-resolution mass spectrometry (HRMS) using electrospray ionization was performed by the Portland State University BioAnalytical Mass Spectrometry Facility and by Oregon Health & Science University’s Bioanalytical Shared Resource/Pharmacokinetics (BSR/PK) Core Facility for additional structure verification.

General Procedure (i):

To a round bottom flask a mixture of aniline (1.1 equiv.) and 4-chloroquinoline (1 equiv.) was dissolved in ethanol (0.1 M) and catalytic fuming HCl (0.05 equiv.) added. The resulting mixture was refluxed for 16 h, where upon the resulting 4-analinoquinoline precipitates as a HCl salt. The reaction mixture was condensed in vacuo and the resulting 4-analinoquinoline was resuspended in methylene chloride and aqueous 2 M sodium hydroxide. The resulting organic layer was extracted with water, brine, dried with magnesium sulfate, concentrated in vacuo, and purified by flash chromatography.

General Procedure (ii):

To a Carius tube a mixture of aniline (3 equiv.), 4-chloroquinoline (1 equiv.), and phenol (0.05 equiv.) was dissolved in 2-ethoxyethanol (0.1 M). The resulting mixture was heated to 150 °C for 12 hours. The reaction mixture was condensed in vacuo and was resuspended in methylene chloride and aqueous 2 M sodium hydroxide. The resulting organic layer was extracted with water, brine, dried with magnesium sulfate, concentrated in vacuo, and purified by flash chromatography.

General Procedure (iii):

To a round bottom flask equipped with a stir bar para substituted bromobenzene (1 equiv.) was dissolved in DMF followed by the addition of 2M potassium carbonate (2 equiv.) and bromobenzene (1.1 equiv.). The flask was fixed with a septa and stirred at 50 °C for 12 h. Reaction mixture was diluted with water and extracted with ethyl acetate. Organic layers were combined, treated with aqueous 2M sodium hydroxide, and washed with water and a saturated sodium chloride solution. Organic layer was separated, dried with magnesium sulfate, and gravity filtered. Filtrate was condensed in vacuo and purified by flash chromatography.

General Procedure (iv):

To a round bottom flask equipped with a stir bar para substituted bromobenzene (1 equiv.) was dissolved in DMF (0.5 M) followed by the addition of iodoethane (1.1 equiv.) and N,N-diisopropylethylamine. The flask was fixed with a septa and stirred at room temperature for 48 h. Reaction mixture was diluted with water and extracted with ethyl acetate. Organic layers were combined and washed with 2M sodium hydroxide, water, and saturated sodium chloride solution. The organic layer was dried with magnesium sulfate and gravity filtered. Filtrate was condensed in vacuo and purified by flash chromatography.

General Procedure (v):

To a dry round bottom flask equipped with a stir bar under inert conditions toluene (0.4 M) was added and degassed with argon for 10 m. bis(tri-tert-butylphosphine)palladium(0) (2.5% molar equiv.) and bis(dibenzylideneacetone)palladium(0) (2.5% molar equiv.) was added to the flask and stirred for 5 m. The substituted aryl halide (1 equiv.) was added, and the reaction vessel was capped with reflux condenser and septa and placed under inert atmosphere. 1M lithium bis(trimethylsilyl)amide in toluene (1.5 equiv.) was added under inert conditions and the reaction was heated to 70 °C for 18 h. Upon consumption of substituted aryl halide, the crude reaction mixture was diluted in ethyl acetate and the newly formed silylamide was deprotected with 1 drop of 1 M hydrochloric acid. The mixture was washed with 2 M sodium hydroxide, water, and brine, and dried with magnesium sulfate. The dried organic layer was filtered and condensed in vacuo and purified by flash chromatography.

General Procedure (vi):

To a microwave reactor flask a mixture of aniline (1 equiv.), 4-chloroquinoline (1.2 equiv.) and phenol (2.0 equiv.) were dissolved in DMF (1.0 M). The flask was sealed and heated to 150 °C for 2 h on high adsorption. The reaction mixture was condensed in vacuo and resuspended in methylene chloride and aqueous 2 M sodium hydroxide. The organic layer was extracted with water, brine, dried with magnesium sulfate, concentrated in vacuo, and purified via flash chromatography to afford desired 4-analinoquinoline.

General Procedure (vii):

To a round bottom flask equipped with magnetic stir bar was charged with 4-fluoronitrobenzene (1 equiv.), substituted piperazine (1.1 equiv.). Solids were dissolved in dimethyl sulfoxide (1.0 M) and trimethylamine (3.5 equiv.) was added the reaction vessel was heated at 120 °C for 16 hours. The resulting mixture was allowed to cool to room temperature and filtered over vacuum filtration using minimal amounts of DMSO to transfer. Resulting solid was recrystallized using ethanol and ethyl acetate, filtered over vacuum filtration, and then air dried.

General Procedure (viii):

A mixture of piperazine substituted nitrobenzene (1.0 equiv.) and wet palladium on carbon (0.1 equiv.) were dissolved in methanol (0.025 M) in a shaker flask. Shaker flask sparged with hydrogen at 45 psi and shaken for 12 hours in Parr Hydrogenator. Pd/C filtered over celite with excess methanol and resulting filtrate condensed in vacuo. Solid resuspended in ethyl acetate and basified with 2 M sodium hydroxide. The organic layer was washed with water and saturated sodium chloride solution and dried with magnesium sulfate. The mixture was condensed in vacuo and purified via flash chromatography to afford piperazine substituted aniline.

General Procedure (ix):

To a microwave reactor flask a mixture of aniline (1 equiv.) and 4-chloroquinoline (1.2 equiv.) were dissolved in THF (1.0 M). The flask was sealed and heated to 120 °C for 20 min on high adsorption. The reaction mixture was condensed in vacuo and resuspended in methylene chloride and aqueous 2 M sodium hydroxide. The resulting organic layer was extracted with water, brine, dried with magnesium sulfate, concentrated in vacuo, and purified via flash chromatography to afford desired 4-analinoquinoline.

General Procedure (x):

Boc-protected 4-anilinoquinoline (1 equiv.) dissolved in methylene chloride (1.0 M) and trifluoroacetic acid added (20 equiv.), reaction stirred at room temperature for 2 hours. Reaction mixture diluted in methylene chloride and aqueous 2 M sodium hydroxide. Resulting precipitate filtered over vacuum filtration and washed with additional methylene chloride and water. Solid air dried to yield desired 4-analinoquinoline, no further purification needed.

General Procedure (xi):

A solution of 4-anilinoquinoline (1.0 equiv.), iodoethane (1.1 equiv.), and N,N-diisopropylethylamine (2.0 equiv.) in dimethylformamide (0.2 M) was stirred at room temperature for 16 h. Upon completion, reaction mixture was poured over ice and resulting solid separated by vacuum filtration. Solid was dissolved in methylene chloride, dried with magnesium sulfate, condensed in vacuo, and purified using flash chromatography to afford alkylated 4-anilinoquinoline.

General Procedure (xii):

A stirred solution of 4-analinoquinoline (1.0 equiv.), alkyl halide (1.2 equiv.), and potassium carbonate (5.0 equiv.) in dimethylformamide (0.2 M) was heated to 85 °C for 16 h. Upon completion, reaction mixture condensed in vacuo. Resulting solid dissolved in methylene chloride and filtered using vacuum filtration. Filtrate extracted with 2 M sodium hydroxide and brine, condensed in vacuo, dry loaded onto celite and purified via flash chromatography. Fractions containing desired product were condensed in vacuo, and resulting solid upon the addition of aqueous 2 M sodium hydroxide was filtered via vacuum filtration and dried in a vacuum oven to afford alkylated 4-anilinoquinoline.

Synthetic Descriptions of Presented amodiachins.

7-chloro-N-(4-morpholinophenyl)quinolin-4-amine (7).

Prepared using general procedure (i) with 7.22 mmol 5, and 21.6 mmol 6a. Yield = 80% (2.452 g). ¹H NMR (METHANOL-d₄, 400 MHz) δ 8.56 (d, 1H, J=9.1 Hz), 8.35 (d, 1H, J=7.1 Hz), 7.95 (d, 1H, J=1.8 Hz), 7.80 (dd, 1H, J=2.1, 9.1 Hz), 7.4–7.4 (m, 2H), 7.2–7.3 (m, 2H), 6.83 (d, 1H, J=7.1 Hz), 3.9–3.9 (m, 4H), 3.3–3.3 (m, 4H). HRMS (ESI) - m/z of [C₁₉H₁₉ClN₃O⁺]: 340.1216, actual: 340.1207.

7-chloro-N-(4-morpholinophenyl)-3-(4-(trifluoromethoxy)phenyl)quinolin-4-amine (8).

Prepared using General Procedure (ii) with 3.00 mmol 5, 1.02 mmol 6b. Yield = 38% (0.195 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.69 (s, 1H), 8.06 (d, 1H, J=2.1 Hz), 7.70 (d, 1H, J=9.1 Hz), 7.45 (d, 2H, J=7.4 Hz), 7.3–7.3 (m, 3H), 7.2–7.3 (m, 1H), 6.79 (s, 4H), 6.00 (s, 1H), 3.8–3.9 (m, 4H, J=3.9, 5.6 Hz), 3.1–3.1 (m, 4H). HRMS (ESI) - m/z of [C₂₆H₂₂ClF₃N₃O₂⁺]: 500.1354, actual: 500.1347.

4-benzyl-3-(4-bromophenyl)morpholine (10a).

Prepared using General Procedure (iii) with 9.89 mmol 9a. Yield = 85% (2.780 g). ¹H-NMR (DMSO-d₆, 400 MHz): δ 7.53–7.50 (m, 2H), 7.35–7.26 (m, 7H), 4.50 (dd, J = 10.2, 2.3 Hz, 1H), 3.94–3.90 (m, 1H), 3.66 (td, J = 11.4, 2.4 Hz, 1H), 3.55–3.47 (m, 2H), 2.85–2.82 (m, 1H), 2.70–2.67 (m, 1H), 2.15 (td, J = 11.5, 3.3 Hz, 1H), 1.94 (dd, J = 11.3, 10.3 Hz, 1H).

3-(4-bromophenyl)-4-ethylmorpholine (10b).

Prepared using General Procedure (iv) with 20.6 mmol 9a. Yield = 60% (4.123 g). ¹H-NMR (CHLOROFORM-d, 400 MHz): δ 7.47 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 3.98–3.94 (m, 1H), 3.80–3.74 (m, 1H), 3.69 (ddd, J = 10.7, 2.8, 1.1 Hz, 1H), 3.38–3.28 (m, 2H), 3.00 (d, J = 11.8 Hz, 1H), 2.60–2.51 (m, 1H), 2.37 (td, J = 11.7, 3.4 Hz, 1H), 2.03 (dq, J = 13.0, 6.6 Hz, 1H), 0.97 (t, J = 7.2 Hz, 3H).

4-benzyl-2-(4-bromophenyl)morpholine (10c).

Prepared using General Procedure (iii) with 3.99 mmol 9b. Yield = 70% (2.809 g). ¹H-NMR (DMSO-d₆, 400 MHz): δ 7.59 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 7.33–7.21 (m, 5H), 3.78 (dt, J = 11.3, 1.5 Hz, 1H), 3.68–3.64 (m, 1H), 3.60 (d, J = 13.4 Hz, 1H), 3.53 (td, J = 11.5, 2.3 Hz, 1H), 3.40 (dd, J = 10.1, 3.3 Hz, 1H), 3.27 (d, J = 11.0 Hz, 1H), 2.93 (d, J = 13.4 Hz, 1H), 2.64 (d, J = 11.8 Hz, 1H), 2.20 (td, J = 11.8, 3.3 Hz, 1H).

1-benzyl-4-(4-bromophenyl)piperazine (10d).

Prepared using General Procedure (iii) with 4.00 mmol 9c. Yield = 35% (0.461 g). ¹H-NMR (DMSO-d₆, 400 MHz): δ 7.35–7.31 (m, 6H), 7.29–7.25 (m, 1H), 6.90–6.86 (m, 2H), 3.52 (s, 2H), 3.14–3.11 (m, 4H), 2.51–2.48 (m, 4H).

4-(4-benzylmorpholin-3-yl)aniline (11a).

Prepared using General Procedure (v) with 7.75 mmol 10a. Yield = 81% (1.638 g) ¹H-NMR (DMSO-d₆, 400 MHz): δ 7.33–7.30 (m, 4H), 7.27–7.23 (m, 1H), 6.95 (d, J = 8.3 Hz, 2H), 6.48 (d, J = 8.5 Hz, 2H), 4.99 (s, 2H), 4.26 (dd, J = 10.2, 2.1 Hz, 1H), 3.87–3.84 (m, 1H), 3.61 (td, J = 11.4, 2.3 Hz, 1H), 3.49 (s, 2H), 2.70–2.65 (m, 2H), 2.12 (td, J = 11.5, 3.3 Hz, 1H), 1.96 (dd, J = 11.2, 10.5 Hz, 1H).

4-(4-ethylmorpholin-3-yl)aniline (11b).

Prepared using General Procedure (v) with 9.30 mmol 10b. Yield = 76% (1.467 g) ¹H-NMR (DMSO-d₆, 400 MHz): δ 6.97 (d, J = 8.3 Hz, 2H), 6.51 (d, J = 8.5 Hz, 2H), 4.97 (s, 2H), 3.82 (dt, J = 11.1, 1.6 Hz, 1H), 3.56 (td, J = 11.4, 2.3 Hz, 1H), 3.50–3.46 (m, 1H), 3.17 (t, J = 10.6 Hz, 1H), 3.02 (dd, J = 10.2, 3.4 Hz, 1H), 2.91–2.87 (m, 1H), 2.49–2.45 (m, 1H), 2.17 (td, J = 11.7, 3.4 Hz, 1H), 1.90 (dq, J = 12.9, 6.6 Hz, 1H), 0.86 (t, J = 7.2 Hz, 3H).

4-(4-benzylmorpholin-2-yl)aniline (11c).

Prepared using General Procedure (v) with 2.72 mmol 10c. Yield = 86% (0.625 g) ¹H-NMR (CHLOROFORM-d, 400 MHz): δ 8.51 (d, J = 5.3 Hz, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.46 (dd, J = 8.9, 2.2 Hz, 1H), 7.23–7.20 (m, 2H), 7.03–6.99 (m, 2H), 6.72 (d, J = 5.3 Hz, 1H), 6.52 (s, 1H), 3.25 (t, J = 5.1 Hz, 4H), 2.60 (t, J = 5.0 Hz, 4H), 2.18 (d, J = 7.4 Hz, 2H), 1.91–1.81 (m, 1H), 0.96 (d, J = 6.6 Hz, 6H).

4-(4-benzylpiperazin-1-yl)aniline (11d).

Prepared using General Procedure (v) with 1.50 mmol 10d. Yield = 69% (0.278 g). ¹H-NMR (METHANOL-d₄, 400 MHz): δ 7.40–7.33 (m, 4H), 7.30 (dd, J = 5.4, 3.5 Hz, 1H), 6.89–6.79 (m, 4H), 3.61 (s, 2H), 3.15 (t, J = 5.0 Hz, 4H), 2.66 (t, J = 5.0 Hz, 4H).

N-(4-(4-benzylmorpholin-3-yl)phenyl)-7-chloroquinolin-4-amine (12).

Prepared using General Procedure (ii) with 0.930 11a and 0.606 mmol 6a. Yield = 24% (0.062 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.56 (d, 1H, J=5.3 Hz), 8.03 (d, 1H, J=2.1 Hz), 7.85 (d, 1H, J=9.0 Hz), 7.5–7.6 (m, 2H), 7.46 (dd, 1H, J=2.2, 8.9 Hz), 7.3–7.3 (m, 6H), 7.2–7.3 (m, 1H), 6.98 (d, 1H, J=5.3 Hz), 6.61 (s, 1H), 3.8–3.9 (m, 2H), 3.81 (d, 1H, J=8.6 Hz), 3.7–3.8 (m, 1H), 3.48 (s, 2H), 2.94 (d, 1H, J=13.4 Hz), 2.8–2.8 (m, 1H, J=11.9 Hz), 2.31 (dt, 1H, J=3.4, 11.8 Hz). HRMS (ESI) - m/z of [C₂₆H₂₅ClN₃O⁺]: 430.1687, actual: 430.1679.

7-chloro-N-(4-(4-ethylmorpholin-3-yl)phenyl)quinolin-4-amine (13).

Prepared using General Procedure (ii) with 0.507 mmol 11b and 1.52 mmol 6a. Yield = 18% (0.062 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.57 (d, 1H, J=5.4 Hz), 8.03 (d, 1H, J=2.1 Hz), 7.85 (d, 1H, J=9.0 Hz), 7.45 (dd, 1H, J=2.2, 8.9 Hz), 7.41 (d, 2H, J=8.3 Hz), 7.27 (s, 1H), 7.24 (s, 1H), 6.98 (d, 1H, J=5.3 Hz), 6.63 (br s, 1H), 3.9–4.0 (m, 1H), 3.76 (dq, 2H, J=2.3, 11.3 Hz), 3.4–3.4 (m, 1H, J=10.9, 10.9 Hz), 3.3–3.4 (m, 1H), 3.00 (br d, 1H, J=11.6 Hz), 2.62 (qd, 1H, J=7.4, 12.7 Hz), 2.37 (dt, 1H, J=3.4, 11.7 Hz), 2.06 (s, 1H), 0.99 (t, 3H, J=7.2 Hz). HRMS (ESI) - m/z of [C₂₁H₂₃ClN₃O⁺]: 368.1529, actual: 368.1522.

7-chloro-N-(4-(4-ethylmorpholin-3-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)quinolin-4-amine (14).

Prepared using General Procedure (ii) with 0.946 11b and 0.605 mmol 6b. Yield = 18% (0.057 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.75 (s, 1H), 8.10 (s, 1H), 7.76 (d, 1H, J=9.0 Hz), 7.4–7.4 (m, 2H), 7.35 (br d, 1H, J=9.0 Hz), 7.3–7.3 (m, 1H), 7.2–7.3 (m, 1H), 7.15 (br d, 2H, J=7.8 Hz), 6.71 (br d, 2H, J=8.1 Hz), 6.08 (s, 1H), 3.93 (br d, 1H, J=11.0 Hz), 3.7–3.8 (m, 1H), 3.6–3.7 (m, 1H), 3.32 (br t, 1H, J=10.8 Hz), 3.2–3.2 (m, 1H), 2.96 (br d, 1H, J=11.9 Hz), 2.5–2.6 (m, 1H), 2.3–2.4 (m, 1H), 1.9–2.0 (m, 1H), 0.95 (br t, 3H, J=7.1 Hz). HRMS (ESI) - m/z of [C₂₈H₂₆ClF₃N₃O₂⁺]: 528.1667, actual: 528.1662.

N-(4-(4-benzylmorpholin-2-yl)phenyl)-7-chloroquinolin-4-amine (15).

Prepared using General Procedure (ii) with 0.904 11c and 0.602 mmol 6a. Yield = 21% (0.054 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.54 (d, 1H, J=5.3 Hz), 8.03 (d, 1H, J=2.1 Hz), 7.85 (d, 1H, J=9.0 Hz), 7.4–7.5 (m, 3H), 7.3–7.4 (m, 5H), 7.26 (s, 2H), 7.2–7.3 (m, 2H), 6.92 (d, 1H, J=5.4 Hz), 6.59 (s, 1H), 4.60 (dd, 1H, J=2.3, 10.2 Hz), 4.0–4.1 (m, 1H), 3.86 (dt, 1H, J=2.4, 11.4 Hz), 3.57 (s, 2H), 2.94 (td, 1H, J=1.9, 11.4 Hz), 2.7–2.8 (m, 1H), 2.31 (dt, 1H, J=3.4, 11.5 Hz), 2.1–2.2 (m, 1H). HRMS (ESI) - m/z of [C₂₆H₂₅ClN₃O⁺]: 430.1687, actual: 430.1682.

N-(4-(4-benzylmorpholin-2-yl)phenyl)-7-chloro-3-(4-(trifluoromethoxy)phenyl)quinolin-4-amine (16).

Prepared using General Procedure (ii) with 0.933 mmol 11c and 0.602 mmol 6b. Yield = 33% (0.117 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.75 (s, 1H), 8.71 (s, 1H), 8.23 (d, 1H, J=9.1 Hz), 8.04 (d, 1H, J=2.1 Hz), 7.61 (dd, 1H, J=2.3, 9.0 Hz), 7.4–7.5 (m, 2H), 7.2–7.4 (m, 7H), 6.92 (d, 2H, J=8.4 Hz), 6.6–6.6 (m, 2H, J=8.6 Hz), 4.27 (dd, 1H, J=1.8, 10.1 Hz), 3.85 (dd, 1H, J=1.9, 11.3 Hz), 3.59 (dt, 1H, J=2.1, 11.4 Hz), 3.4–3.5 (m, 2H), 2.65 (br d, 2H, J=11.6 Hz), 2.09 (dt, 1H, J=3.2, 11.5 Hz), 1.84 (t, 1H, J=10.9 Hz). HRMS (ESI) - m/z of [C₃₃H₂₈ClF₃N₃O₂⁺]: 590.1825, actual: 590.1816.

N-(4-(4-benzylpiperazin-1-yl)phenyl)-7-chloroquinolin-4-amine (17).

Prepared using General Procedure (ii) with 0.904 mmol 11d and 0.642 mmol 6a. Yield = 46% (0.125 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.48 (d, 1H, J=5.3 Hz), 8.01 (d, 1H, J=2.1 Hz), 7.82 (d, 1H, J=9.0 Hz), 7.43 (dd, 1H, J=2.2, 8.9 Hz), 7.3–7.4 (m, 5H), 7.1–7.2 (m, 2H), 6.9–7.0 (m, 2H), 6.69 (d, 1H, J=5.4 Hz), 6.48 (s, 1H), 3.59 (s, 2H), 3.2–3.3 (m, 4H), 2.6–2.7 (m, 4H). HRMS (ESI) – m/z of [C₂₆H₂₆ClN₄⁺]: 429.1840, actual: 429.1840.

7-chloro-N-(4-(4-ethylpiperazin-1-yl)phenyl)quinolin-4-amine (20).

Prepared using General Procedure (i) with 1.50 mmol 18a and 1.68 mmol 6a. Yield = 42% (0.287 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.48 (d, 1H, J=5.3 Hz), 8.00 (d, 1H, J=2.1 Hz), 7.83 (d, 1H, J=9.0 Hz), 7.43 (dd, 1H, J=2.2, 8.9 Hz), 7.2–7.2 (m, 2H), 6.9–7.0 (m, 2H), 6.70 (d, 1H, J=5.4 Hz), 6.50 (s, 1H), 3.2–3.3 (m, 4H), 2.6–2.7 (m, 4H), 2.50 (q, 2H, J=7.2 Hz), 1.15 (t, 3H, J=7.2 Hz). HRMS (ESI) - m/z of [C₂₁H₂₄ClN₄⁺]: 367.1689, actual: 367.1687.

7-chloro-N-(4-(4-methylpiperazin-1-yl)phenyl)quinolin-4-amine (21).

Prepared using General Procedure (i) with 1.50 mmol 18b and 1.65 mmol 6a. Yield = 57% (0.300 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.48 (d, 1H, J=5.3 Hz), 8.00 (d, 1H, J=2.0 Hz), 7.83 (d, 1H, J=9.0 Hz), 7.43 (dd, 1H, J=2.2, 8.9 Hz), 7.2–7.2 (m, 2H), 6.9–7.0 (m, 2H), 6.70 (d, 1H, J=5.4 Hz), 6.53 (s, 1H), 3.2–3.3 (m, 4H), 2.6–2.6 (m, 4H), 2.37 (s, 3H). HRMS (ESI) - m/z of [C₂₀H₂₂ClN₄⁺]: 353.1533, actual: 353.1526.

6-chloro-N-(4-(4-ethylpiperazin-1-yl)phenyl)quinolin-4-amine (22).

Prepared using General Procedure (vi) with 1.01 mmol 18a and 1.21 mmol 19a. Yield = 62% (0.232 g). ¹H NMR (METHANOL-d₄, 400 MHz) δ 8.32 (d, 1H, J=5.5 Hz), 8.28 (dd, 1H, J=0.8, 8.4 Hz), 7.8–7.9 (m, 1H), 7.71 (ddd, 1H, J=1.3, 6.9, 8.4 Hz), 7.53 (ddd, 1H, J=1.3, 7.0, 8.4 Hz), 7.3–7.3 (m, 2H), 7.1–7.1 (m, 2H), 6.72 (d, 1H, J=5.5 Hz), 3.3–3.3 (m, 3H), 2.7–2.7 (m, 4H), 2.54 (q, 2H, J=7.3 Hz), 1.19 (t, 3H, J=7.3 Hz). HRMS (ESI) - m/z of [C₂₁H₂₅N₄⁺]: 333.2079, actual: 333.2074.

N-(4-(4-ethylpiperazin-1-yl)phenyl)quinolin-4-amine (23).

Prepared using General Procedure (i) with 1.01 mmol 18a and 1.21 mmol 19b. Yield = 11% (0.036 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.48 (d, 1H, J=5.3 Hz), 7.96 (d, 1H, J=9.0 Hz), 7.89 (d, 1H, J=2.3 Hz), 7.60 (dd, 1H, J=2.2, 8.9 Hz), 7.2–7.2 (m, 2H), 7.0–7.0 (m, 2H), 6.73 (d, 1H, J=5.3 Hz), 6.40 (s, 1H), 3.2–3.3 (m, 4H), 2.6–2.7 (m, 4H), 2.50 (q, 2H, J=7.2 Hz), 1.15 (t, 3H, J=7.2 Hz). HRMS (ESI) - m/z of [C₂₁H₂₄ClN₄⁺]: 367.1689, actual: 367.1686. HRMS (ESI) – m/z of [C₂₁H₂₃Cl₂N₄⁺]: 401.1294, actual: 401.1294.

6,7-dichloro-N-(4-(4-ethylpiperazin-1-yl)phenyl)quinolin-4-amine (24).

Prepared using General Procedure (i) with 1.01 mmol 18a and 1.02 mmol 19c. Yield = 6.9% (0.028 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.99 (s, 1H), 8.75 (s, 1H), 8.40 (d, 1H, J=5.4 Hz), 8.07 (s, 1H), 7.2–7.2 (m, 2H, J=8.0 Hz), 7.0–7.1 (m, 2H, J=8.1 Hz), 6.66 (d, 1H, J=5.4 Hz), 3.1–3.2 (m, 4H), 2.4–2.4 (m, 2H), 1.05 (t, 3H, J=7.2 Hz).

7-bromo-N-(4-(4-ethylpiperazin-1-yl)phenyl)quinolin-4-amine (25).

Prepared using General Procedure (vi) with 1.00 mmol 18a and 1.30 mmol 19d. Yield = 22% (0.093 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.93 (s, 1H), 8.3–8.4 (m, 2H), 8.01 (d, 1H, J=2.1 Hz), 7.64 (dd, 1H, J=2.1, 9.0 Hz), 7.20 (d, 2H, J=8.9 Hz), 7.01 (d, 2H, J=9.0 Hz), 6.64 (d, 1H, J=5.4 Hz), 3.1–3.2 (m, 4H), 2.5–2.6 (m, 4H), 2.38 (d, 2H, J=7.3 Hz), 1.04 (t, 3H, J=7.2 Hz). HRMS (ESI) - m/z of [C₂₁H₂₄BrN₄⁺]: 413.1160, actual: 413.1160.

N-(4-(4-ethylpiperazin-1-yl)phenyl)-7-(trifluoromethyl)quinolin-4-amine (26).

Prepared using General Procedure (vi) with 0.63 mmol 18a and 0.64 mmol 19e. Yield = 46% (0.141 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.0–9.1 (m, 1H), 8.6–8.7 (m, 1H), 8.4–8.5 (m, 1H), 8.1–8.2 (m, 1H), 7.76 (dd, 1H, J=1.8, 8.9 Hz), 7.2–7.2 (m, 2H), 7.0–7.1 (m, 2H), 6.7–6.8 (m, 1H), 3.1–3.2 (m, 4H), 2.5–2.5 (m, 4H), 2.3–2.5 (m, 2H), 1.05 (t, 3H, J=7.2 Hz). HRMS (ESI) - m/z of [C₂₂H₂₄F₃N₄⁺]: 401.1953, actual: 401.1948.

1-(tert-butyl)-4-(4-nitrophenyl)piperazine (29a).

Prepared using General Procedure (vii) with 5.5 mmol 27 and 6.0 mmol 28a. Yield = 53% (0.773 g). ¹H-NMR (CHLOROFORM-d, 400 MHz): δ 8.14–8.10 (m, 2H), 6.83–6.79 (m, 2H), 3.42–3.40 (m, 4H), 2.73–2.70 (m, 4H), 1.11 (s, 9H).

1-(4-nitrophenyl)-4-(4-(trifluoromethyl)phenyl)piperazine (29b).

Prepared using General Procedure (vii) with 18.0 mmol 27 and 19.8 mmol 28b. Yield = 20% (1.254 g). ¹H‐NMR (CHLOROFORM-d, 400 MHz): δ 8.21‐8.17 (m, 2H), 7.55 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 6.91‐6.87 (m, 2H), 3.64 (dd, J = 6.5, 4.1 Hz, 4H), 3.51 (dd, J = 6.5, 4.1 Hz, 4H).

4-(4-(tert-butyl)piperazin-1-yl)aniline (30a).

Prepared using General Procedure (viii) with 2.62 mmol 29a. Yield = 56%. ¹H-NMR (CHLOROFORM-d, 400 MHz): δ 6.82 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8 Hz, 2H), 3.41 (s, 1H), 3.06 (t, J = 4.9 Hz, 4H), 2.74 (t, J = 4.9 Hz, 4H), 1.11 (s, 9H).

4-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)aniline (30b).

Prepared using General Procedure (viii) with 3.57 mmol 29b. Yield = = 53% (0.773 g). ¹H‐NMR (CHLOROFORM-d, 400 MHz): δ 7.54‐7.51 (m, 2H), 7.02‐6.98 (m, 2H), 6.90‐6.87 (m, 2H), 6.72‐6.69 (m, 2H), 3.50 (s, 1H), 3.46‐3.43 (m, 4H), 3.22‐3.19 (m, 4H).

N-(4-(4-(tert-butyl)piperazin-1-yl)phenyl)-7-chloroquinolin-4-amine (31).

Prepared using General Procedure (vi) with 0.88 mmol 30a and 1.01 mmol 6a. Yield = 18% (0.072 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.48 (d, 1H, J=5.4 Hz), 8.01 (d, 1H, J=2.1 Hz), 7.83 (d, 1H, J=9.0 Hz), 7.43 (dd, 1H, J=2.2, 8.9 Hz), 7.1–7.2 (m, 2H), 6.9–7.0 (m, 2H), 6.70 (d, 1H, J=5.4 Hz), 6.50 (s, 1H), 3.2–3.3 (m, 4H), 2.7–2.8 (m, 4H), 1.14 (s, 9H). HRMS (ESI) - m/z of [C₂₃H₂₈ClN₄⁺]: 395.2003, actual: 395.2000.

7-chloro-N-(4-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)phenyl)quinolin-4-amine (32).

Prepared using General Procedure (vi) with 0.63 mmol 30b and 0.64 mmol 6a. Yield = 46% (0.141 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.50 (d, 1H, J=5.3 Hz), 8.02 (d, 1H, J=2.1 Hz), 7.84 (d, 1H, J=9.0 Hz), 7.53 (d, 2H, J=8.6 Hz), 7.44 (dd, 1H, J=2.1, 8.9 Hz), 7.2–7.3 (m, 2H), 7.0–7.1 (m, 2H), 7.00 (d, 2H, J=8.6 Hz), 6.72 (d, 1H, J=5.4 Hz), 6.53 (s, 1H), 3.4–3.5 (m, 4H), 3.3–3.4 (m, 4H). HRMS (ESI) - m/z of [C₂₆H₂₃ClF₃N₄⁺]: 483.1564, actual: 483.1562.

7-chloro-N-(4-(4-cyclopropylpiperazin-1-yl)phenyl)quinolin-4-amine (33).

Prepared using General Procedure (i) with 1.50 mmol 30c and 0.78 mmol 6a. Yield = 6% (0.019 g). ¹H‐NMR (DMSO-d₆, 400 MHz): δ 8.91 (s, 1H), 8.41 (d, J = 9.1 Hz, 1H), 8.37 (d, J = 5.4 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.53 (dd, J = 9.0, 2.3 Hz, 1H), 7.22‐7.18 (m, 2H), 7.03‐6.99 (m, 2H), 6.63 (d, J = 5.4 Hz, 1H), 3.11 (t, J = 5.0 Hz, 4H), 2.69 (t, J = 5.0 Hz, 4H), 1.67 (tt, J = 6.7, 3.4 Hz, 1H), 0.48‐0.43 (m, 2H), 0.37‐0.34 (m, 2H). HRMS (ESI) - m/z of [C₂₂H₂₄ClN₄⁺]: 379.1690, actual: 379.1685.

7-chloro-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine (34).

Prepared using General Procedure (i) with 1.00 mmol 30d and 1.14 mmol 6a. Yield = 15% (0.052 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 8.91 (s, 1H), 8.42 (d, 1H, J=9.0 Hz), 8.3–8.4 (m, 1H), 7.85 (d, 1H, J=2.3 Hz), 7.53 (dd, 1H, J=2.3, 9.0 Hz), 7.1–7.2 (m, 2H), 7.0–7.1 (m, 2H), 6.62 (d, 1H, J=5.4 Hz), 3.1–3.2 (m, 4H), 1.64 (br d, 4H, J=5.1 Hz), 1.5–1.6 (m, 2H). HRMS (ESI) - m/z of [C₂₀H₂₁ClN₃⁺]: 338.1424, actual: 338.1419.

7-chloro-N-(4-(1-isopropylpiperidin-4-yl)phenyl)quinolin-4-amine (35).

Prepared using General Procedure (i) with 1.10 mmol 30e and 1.0 mmol 6a. Yield = 29% (0.111 g). ¹H NMR (METHANOL-d₄, 400 MHz) δ 8.35 (d, 1H, J=5.6 Hz), 8.29 (d, 1H, J=9.0 Hz), 7.85 (d, 1H, J=2.0 Hz), 7.49 (dd, 1H, J=2.1, 9.0 Hz), 7.3–7.4 (m, 4H), 6.86 (d, 1H, J=5.6 Hz), 3.1–3.1 (m, 2H), 2.8–2.9 (m, 1H), 2.62 (tt, 1H, J=3.9, 12.1 Hz), 2.4–2.5 (m, 2H), 1.9–2.0 (m, 2H), 1.8–1.9 (m, 2H), 1.16 (d, 6H, J=6.6 Hz). HRMS (ESI) - m/z of [C₂₃H₂₇ClN₃⁺]: 380.1894, actual: 380.1890.

tert-butyl 4-(4-((7-chloroquinolin-4-yl)amino)phenyl)piperazine-1-carboxylate (36).

Prepared using General Procedure (ix) with 5.52 mmol 30f and 5.53 mmol 6a. Yield = 69% (1.672 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.49 (d, 1H, J=5.3 Hz), 8.01 (d, 1H, J=2.1 Hz), 7.84 (d, 1H, J=9.0 Hz), 7.44 (dd, 1H, J=2.1, 9.0 Hz), 7.2–7.2 (m, 2H), 7.0–7.0 (m, 2H), 6.71 (d, 1H, J=5.3 Hz), 6.51 (s, 1H), 3.6–3.7 (m, 4H), 3.1–3.2 (m, 5H), 1.5–1.5 (m, 9H). HRMS (ESI) - m/z of [C₂₄H₂₈ClN₄O₂⁺]: 439.1901, actual: 439.1899.

tert-butyl 4-(4-((7-chloroquinolin-4-yl)amino)phenyl)piperidine-1-carboxylate (37).

Prepared using General Procedure (ix) with 5.80 mmol 30f and 5.81 mmol 6a. Yield = 87% (2.210 g). ¹H‐NMR (DMSO-d₆, 400 MHz): δ 9.04 (s, 1H), 8.45‐8.41 (m, 2H), 7.89 (d, J = 2.2 Hz, 1H), 7.57 (dd, J = 9.0, 2.3 Hz, 1H), 7.33‐7.28 (m, 4H), 6.87 (d, J = 5.4 Hz, 1H), 4.09 (d, J = 11.7 Hz, 2H), 2.83‐2.67 (m, 3H), 1.79 (d, J = 13.2 Hz, 2H), 1.56‐1.45 (m, 2H), 1.43 (s, 9H).

7-chloro-N-(4-(piperazin-1-yl)phenyl)quinolin-4-amine (38).

Prepared using general procedure (x) with 3.42 mmol 36. Yield = 40% (0.464 g) ¹H‐NMR (CHLOROFORM-d, 400 MHz): δ 8.54 (d, J = 5.3 Hz, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.46 (dd, J = 9.0, 2.2 Hz, 1H), 7.30‐7.27 (m, 2H), 7.25‐7.22 (m, 2H), 6.91 (d, J = 5.3 Hz, 1H), 6.55 (dd, J = 2.6, 0.3 Hz, 1H), 3.24‐3.20 (m, 2H), 2.80‐2.74 (m, 2H), 2.66‐2.63 (m, 1H), 1.89‐1.84 (m, 2H), 1.72‐1.62 (m, 2H). HRMS (ESI) - m/z of [C₁₉H₂₀ClN₄⁺]: 339.1376, actual: 339.1372.

7-chloro-N-(4-(piperidin-4-yl)phenyl)quinolin-4-amine (39).

Prepared using general procedure (x) with 3.51 mmol 37. Yield = 69%. ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.54 (d, 1H, J=5.3 Hz), 8.03 (d, 1H, J=2.1 Hz), 7.85 (d, 1H, J=9.0 Hz), 7.45 (dd, 1H, J=2.2, 8.9 Hz), 7.3–7.3 (m, 2H), 7.2–7.3 (m, 2H), 6.91 (d, 1H, J=5.3 Hz), 6.58 (s, 1H), 3.22 (br d, 2H, J=11.8 Hz), 2.77 (dt, 2H, J=2.3, 12.1 Hz), 2.65 (s, 1H), 1.8–1.9 (m, 2H), 1.7–1.7 (m, 2H). HRMS (ESI) - m/z of [C₂₀H₂₁ClN₃⁺]: 338.1424, actual: 338.1419.

7-chloro-N-(4-(1-ethylpiperidin-4-yl)phenyl)quinolin-4-amine (40).

Title compound prepared using General Procedure (xi) with 1.00 mmol 39 and 1.12 mmol iodoethane. Yield =: 30% (0.108 g). ¹H NMR (METHANOL-d₄, 400 MHz) δ 8.37 (d, 1H, J=5.5 Hz), 8.30 (d, 1H, J=9.0 Hz), 7.87 (d, 1H, J=2.1 Hz), 7.51 (dd, 1H, J=2.3, 9.0 Hz), 7.3–7.4 (m, 4H), 6.88 (d, 1H, J=5.5 Hz), 3.15 (br d, 2H, J=11.8 Hz), 2.64 (tt, 1H, J=4.0, 11.9 Hz), 2.54 (q, 2H, J=7.3 Hz), 2.17 (dt, 2H, J=2.6, 11.9 Hz), 1.9–2.0 (m, 2H), 1.8–1.9 (m, 2H), 1.18 (t, 3H, J=7.3 Hz). HRMS (ESI) - m/z of [C₂₂H₂₅ClN₃⁺]: 366.1737, actual: 366.1732.

7-chloro-N-(4-(1-propylpiperidin-4-yl)phenyl)quinolin-4-amine (41).

Title compound was prepared using General Procedure (xii) with 1.00 mmol 39 and 1.2 mmol iodopropane. Yield =: 26% (0.099 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.04 (s, 1H), 8.4–8.5 (m, 2H), 7.88 (d, 1H, J=2.1 Hz), 7.56 (dd, 1H, J=2.2, 9.1 Hz), 7.3–7.3 (m, 4H), 6.86 (d, 1H, J=5.4 Hz), 2.9–3.0 (m, 2H), 2.5–2.5 (m, 1H), 0.00 (t, 2H, J=7.4 Hz), 1.9–2.0 (m, 2H), 1.7–1.8 (m, 2H), 1.6–1.7 (m, 2H), 1.46 (sxt, 2H, J=7.4 Hz), 0.00 (t, 3H, J=7.3 Hz). HRMS (ESI) - m/z of [C₂₄H₂₉ClN₃⁺]: 394.2051, actual: 394.2046.

N-(4-(1-butylpiperidin-4-yl)phenyl)-7-chloroquinolin-4-amine (42).

Title compound was prepared using General Procedure (xii) with 1.00 mmol 39 and 1.2 mmol iodobutane. Yield =: 63% (0.249 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.04 (s, 1H), 8.4–8.5 (m, 2H), 7.88 (d, 1H, J=2.3 Hz), 7.56 (dd, 1H, J=2.2, 9.1 Hz), 7.3–7.3 (m, 4H), 6.86 (d, 1H, J=5.4 Hz), 2.96 (br d, 2H, J=11.4 Hz), 2.4–2.5 (m, 1H), 2.28 (br t, 2H, J=7.1 Hz), 1.9–2.0 (m, 2H, J=2.0, 2.0, 2.0, 9.9, 9.9 Hz), 1.7–1.8 (m, 2H), 1.6–1.7 (m, 2H, J=3.5, 3.5, 3.5, 3.5, 8.6, 8.6, 8.6 Hz), 1.4–1.5 (m, 2H), 1.30 (sxtd, 2H, J=7.2, 14.8 Hz), 0.90 (t, 3H, J=7.3 Hz). HRMS (ESI) - m/z of [C₂₄H₂₉ClN₃⁺]: 394.2051, actual: 394.2046.

7-chloro-N-(4-(1-isobutylpiperidin-4-yl)phenyl)quinolin-4-amine (43).

Title compound was prepared using General Procedure (xii) with 1.00 mmol 39 and 2.03 mmol 1-iodo-2-methylpropane. Yield =: 43% (0.168 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.04 (s, 1H), 8.4–8.5 (m, 2H), 7.88 (d, 1H, J=2.1 Hz), 7.56 (dd, 1H, J=2.3, 9.0 Hz), 7.3–7.3 (m, 4H), 6.86 (d, 1H, J=5.4 Hz), 2.93 (br d, 2H, J=11.4 Hz), 2.4–2.5 (m, 1H), 2.06 (d, 2H, J=7.4 Hz), 1.9–2.0 (m, 2H), 1.7–1.8 (m, 3H), 1.6–1.7 (m, 2H), 0.88 (d, 6H, J=6.5 Hz). HRMS (ESI) - m/z of [C₂₄H₂₉ClN₃⁺]: 394.2051, actual: 394.2045.

N-(4-(1-(sec-butyl)piperidin-4-yl)phenyl)-7-chloroquinolin-4-amine (44).

Title compound was prepared using General Procedure (xii) with 1.01 mmol 39 and 1.2 mmol 2-iodobutane. Yield =: 50% (0.201 g). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.04 (s, 1H), 8.4–8.4 (m, 2H), 7.88 (d, 1H, J=2.3 Hz), 7.56 (dd, 1H, J=2.3, 9.0 Hz), 7.2–7.3 (m, 4H), 6.85 (d, 1H, J=5.4 Hz), 2.7–2.9 (m, 2H), 2.4–2.5 (m, 2H), 2.39 (dt, 1H, J=2.3, 11.6 Hz), 2.1–2.3 (m, 1H), 1.77 (br d, 2H, J=12.9 Hz), 1.6–1.7 (m, 2H), 1.5–1.6 (m, 1H), 1.27 (dsxt, 1H, J=6.4, 7.4 Hz), 0.93 (d, 3H, J=6.5 Hz), 0.87 (t, 3H, J=7.4 Hz). HRMS (ESI) - m/z of [C₂₄H₂₉ClN₃⁺]: 394.2051, actual: 394.2046.

7-chloro-N-(4-(4-isobutylpiperazin-1-yl)phenyl)quinolin-4-amine (45).

Title compound was prepared using General Procedure (xii) with 0.50 mmol 38 and 1.20 mmol 1-iodo-2-methylpropane. Yield =: 19% (0.037 g). ¹H NMR (CHLOROFORM-d, 400 MHz) δ 8.48 (d, 1H, J=5.3 Hz), 8.00 (d, 1H, J=2.1 Hz), 7.83 (d, 1H, J=9.0 Hz), 7.43 (dd, 1H, J=2.1, 8.9 Hz), 7.2–7.2 (m, 2H), 6.9–7.0 (m, 2H), 6.69 (d, 1H, J=5.4 Hz), 6.50 (s, 1H), 3.2–3.3 (m, 4H), 2.6–2.6 (m, 4H), 2.16 (d, 2H, J=7.4 Hz), 1.83 (quind, 1H, J=6.9, 13.6 Hz), 0.94 (d, 6H, J=6.6 Hz). HRMS (ESI) - m/z of [C₂₃H₂₈ClN₄⁺]: 395.2003, actual: 395.1992.

Biology

Plasmodium falciparum Drug Sensitivity:

The following parasite strains were used in this study and obtained through BEI Resources, NIAID, NIH. Plasmodium falciparum, Strain D6 (MRA-285, originally from Sierra Leone, has modest resistance to mefloquine). Plasmodium falciparum, Strain Dd2 (MRA-150, originated from Indochina; derived from W2-mef and is resistant to chloroquine, pyrimethamine, and mefloquine).

Parasite Culture:

P. falciparum parasites were thawed from frozen stock and cultured in suspended human erythrocytes (Lampire Biological Laboratories, Pipersville, PA) not more than 28 days old at 2% hematocrit. The culture medium used: RPMI-1640 supplemented with 25 mM HEPES buffer, 25 mg/L gentamicin sulfate, 45 mg/L hypoxanthine, 10 mM glucose, 2 mM glutamine, and 0.5% Albumax II (complete medium).²⁹ Cultures were maintained in a standard low oxygen atmosphere (5% O₂, 5% CO₂, 90% N₂) in a Forma Series II 3110 environmental chamber and incubated at 37 °C. Cultures were subpassaged every 3–4 days into a fresh culture flask containing complete media and erythrocytes.

In vitro antiplasmodial activity (IC₅₀) against P. falciparum:

Final compounds were assessed for in vitro antiplasmodial activity against D6 and Dd2 strains of P. falciparum using the previously described fluorescence-based SYBR Green assay.²⁰ In short, compounds were prepared as 10 mM stocks in DMSO. Compounds were evaluated in quadruplicate in flat-bottomed clear 96-well plates and plated at a 2-fold serial dilution with the final column left untreated to span a range of 0.25–250 nM. Asynchronous P. falciparum infected erythrocytes in growth media were added to each well for a total volume of 100 μL, final hematocrit of 2%, and initial parasitemia of 0.2%. Controls included non-infected red blood cells, 10 mM chloroquine and amodiaquine. Plates were incubated in controlled low oxygen atmosphere (5% O₂, 5% CO₂, 90% N₂) at 37 °C. 72 h post inoculation, all wells are simultaneously lysed and dyed using 100 μL of a SYBR Green I dye-detergent solution. Plates were incubated at ambient temperature and atmosphere in the dark for at least 1 h. Fluorescence was read at 497 nm excitation and 520 nm emission bands with a Spectramax iD3 plate reader. Fluorescence values were normalized with respect to the untreated control wells representing normal parasite growth and plotted against the logarithm of drug concentration. An IC₅₀ was determined for each compound by fitting this data to a variable slope nonlinear regression curve using Graphpad Prism software (v. 9).

HepG2 Cytotoxicity Assay:

Final compounds were assessed for mammalian cytotoxicity using an immortalized human liver carcinoma cell line (HepG2) using previously described methods.³⁰ In short, final compounds were prepared in DMSO as 10 mM stock solutions. Human hepatocarcinoma (HepG2) cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C in a humidified 5% CO₂ atmosphere. To 96-well flat-bottomed tissue culture plates, HepG2 cells were added at 2 × 10⁴ density with an additional 160 μL of complete culture media per well and were incubated overnight at 37 °C to allow for adherence. Compound stocks aliquots were applied as 40 μL solutions in complete media to each well in a serial dilution series that ranged from 200–0.2 μL as duplicates. A 10 mM DMSO stock of mefloquine was used as a positive control and prepared as previously described. After drug-treated plates were incubated in 5% CO₂ atmosphere at 37 °C for 24 h, they were aspirated and 200 μL of complete media was added to each well for an additional 24 h incubation in same conditions as previously mentioned. 20 μL of resazurin (Alamar Blue) in PBS buffer was added to each well to a final concentration of 10 μM, and the plates were incubated for 3 h. Fluorescence was measured at 560 nm excitation and 590 nm emission bands using a Spectramax iD3 plate reader. Fluorescence output values were normalized to the untreated control wells and plotted against the logarithm of drug concentration. Cytotoxicity (CC₅₀) was determined for each compound by fitting this data to a variable slope nonlinear regression curve using Graphpad Prism software (v. 9).

In Vivo Parasite Suppression against murine Plasmodium yoelii:

The parasite suppression of select compounds at a fixed dose was measured using a modified 4-day Peters test. Female CF1 mice from Charles River Laboratories were inoculated intravenously with approximately 2.5–5.0 × 10⁴ parasitized erythrocytes (murine malaria Plasmodium yoelii, lethal Kenya strain MR4 MRA-428) from a donor mouse (experiment day zero). On the following 4 days (Days 1–4), solutions of the test compounds in PEG-400 (PEG-400 only for control mice) were administered by oral gavage once daily. Parasitemia of each mouse was determined by microscopic examination of Giemsa-stained blood smears on Day 5. Percent parasite suppression assessed by comparing parasitemia of treated mice relative to untreated controls using Graphpad Prism (v. 9).

In Vivo Efficacy (ED₅₀, ED₉₀, and NRD) against murine Plasmodium yoelii:

The in vivo efficacy of select compounds was measured using a modified 4-day Peters test. Female CF1 mice from Charles River Laboratories were inoculated intravenously with approximately 2.5–5.0 × 10⁴ parasitized erythrocytes (murine malaria Plasmodium yoelii, lethal Kenya strain MR4 MRA-428) from a donor mouse (experiment day zero). On the following 4 days (Day 1–4), solutions of the test compounds in PEG-400 (PEG-400 only for control mice) were administered by oral gavage once daily. Select compounds were assessed at 1.0, 2.5, 5.0, and 10 mg/kg/d, additional experiments outside of the previous range were added if necessary to obtain an interpolated ED₅₀ and ED₉₀ value. Parasitemia of each mouse was determined by microscopic examination of Giemsa-stained blood smears on Day 5. Mice with no observable parasitemia by microscopic analysis on Day 5 were monitored twice weekly for parasitemia until parasites were observed, or until Day 30. In vivo efficacy against infection (ED₅₀ and ED₉₀) assessed by generating dose–response curves of parasitemia of treated mice relative to untreated controls using Graphpad Prism (v. 9). Non-recrudescence dose was identified as minimum dose required to maintain a 0% parasitemia by microscopic analysis until Day 30.

Efficacy of a Single Dose Cure (SDC) of 43 against murine Plasmodium yoelii:

The efficacy of a single dose of 43 was measured using a modified 4-day Peters test. Female CF1 mice from Charles River Laboratories were inoculated intravenously with approximately 2.5–5.0 × 10⁴ parasitized erythrocytes (murine malaria Plasmodium yoelii, lethal Kenya strain MR4 MRA-428) from a donor mouse (Day 0). On the following day (Day 1), a solution of 43 at 50 mg/kg in PEG-400 (PEG-400 only for control mice) was administered by oral gavage. Parasitemia of each mouse was determined by microscopic examination of Giemsa-stained blood smears on Day 5. Mice with no observable parasitemia by microscopic analysis on Day 5 were monitored twice weekly for parasitemia until parasites were observed, or until Day 30. The single dose cure was identified as a dose required to maintain 0% parasitemia by microscopic analysis until Day 30.

Murine Microsomal Stability:

Select compounds were assessed for murine microsomal stability in pooled liver microsomes performed at ChemPartner, Shanghai, China. Compounds were incubated at 37 °C and 1 μM concentration in murine liver microsomes (Corning) for 1 h at a protein concentration of 0.5 mg/mL in potassium phosphate buffer at pH 7.4 containing 1.0 mM EDTA. The metabolic reaction was initiated by NADPH and quenched with ice-cold acetonitrile at 15 min increments up to 1 h. The progress of compound metabolism was followed by LC-MS/MS (ESI positive ion, LC-MS/MS-034(API-6500+) using a C18 stationary phase (ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 μm)) and a MeOH/water mobile phase containing 0.25% FA and 1 mM NH₄OAc. Imipramine or Osalmid were used as internal standards, ketanserin was used as a metabolically unstable control compound. Concentration versus time data for each compound were fitted to an exponential decay function to determine the first-order rate constant for substrate depletion, which was then used to calculate the degradation half-life (t_1/2) and predicted intrinsic clearance value (Cl_int) from an assumed murine hepatic blood flow of 90 mL/min/kg.

Pharmacokinetic Study of 43 in Mice.

The title compound was selected for pharmacokinetic analysis in mice at a PO dose of 10 mg/kg and IV dose of 0.5 mg/kg performed at ChemPartner in Shanghai, China. Three groups of three male CF1 mice (JH Laboratory Animal) were used in each arm of the study and were administer the drug in PEG-400 at 1 mg/mL by oral gavage and in PEG-400 at 0.1 mg/mL by tail vein. At the following time points: 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 120 h post dose administration a single group of mice were manually restrained and approximately 93 μL of blood were taken from the animals via facial vein for semi-serial bleeding into K₂EDTA tubes. Samples were put on ice and centrifuged (2000 G, 5 min at under 4 °C) within 15 minutes of collection. An aliquot of 10 μL sample was added to 20 μL 10mM NH₄OAc and 3 μL internal standard (Diclofenac, 200 ng/mL) in ACN. The mixture was vortexed for 1 m, then added to 800 μL MTBE and continued to vortex for an additional 10 min followed by centrifugation at 14000 rpm for 5 min. A 700 μL aliquot of the supernatant was condensed under nitrogen steam. Samples were reconstituted with 200 μL of 1:1 methanol/water and vortexed for 2 min. 1 μL of solution was injected into LC-MS/MS-21 (Triple Quad 6500) and ran on a Waters ACQUITY UPLC HSS T3 (2.1 × 50 mm, 1.8 μm) column. Pharmacokinetic analysis as a best-fit curve was prepared from drug concentration in plasma as a function of time using non-compartmental analysis as implemented in WinNonlin software (Pharsight – Mountain View, CA). The exposure (AUC_last), half-life (T_1/2), maximum concentration (C_max) and time of maximum concentration (T_max) were determined from the data. Goodness-of-fit was assessed by the r² (linear regression coefficient) of the drug concentration on the terminal phase. Bioavailability (F) was calculated using the following equation, where AUC is area under the curve for oral (po) and tail vein (iv) and D is dose for oral (po) and tail vein (iv):

$$F=100\cdot \frac{AU{C}_{po}\cdot D_{iv}}{AU{C}_{iv}\cdot D_{po}}$$

Ethical Use of Animals Statement.

The procedures involved, together with all matters relating to the care, handling, and housing of the animals used in this study, were approved by the Portland VA Medical Center Institutional Animal Care and Use Committee. Protocol and approval IRBNet #: 1635615–8.

Supplementary Material

Figure S1, and ¹H NMR and HRMS for all final compounds, and HPLC purity for compounds tested in vivo: 17, 20, 31, 35, 40, 41, 42, 43, 44, 45 (.pdf).

Molecular Formula Strings for all final compounds (.csv)

Abbreviations

ACN

acetonitrile

AUC_INF

area under the curve extrapolated to infinity

AUC_last

area under the curve from last time point

Bu

butyl

CD3OD

deuterated methanol

CDCl₃

deuterated chloroform

CL_INF

clearance extrapolated to infinity

CL_int

intrinsic clearance

C_max

maximum serum concentration

cycPr

cyclo-propyl

DIPEA

N,N-diisopropylethylamide

DMSO-d₆

deuterated dimethyl sulfoxide

EtOH

ethanol

FA

formic acid

GCMS

gas chromatography mass spectroscopy

HCl –

hydrochloric acid

i-Bu

iso-butyl

K₂EDTA

dipotassium ethylenediaminetetraacetic acid

LC-MS/MS

liquid chromatography tandem mass spectroscopy

LiHMDS

lithium bis(trimethylsilyl)amide

LLOQ

lower limit of quantification

MeOH

methanol

MRT_INF

mean residence time extrapolated to infinity

ND

not determined

NRD

non-recrudescence dose

Pd/C

palladium on carbon

Pf

Plasmodium falciparum

pfcrt

Plasmodium falciparum chloroquine resistant transport

PfD6

Plasmodium falciparum D6 strain

PfDd2

Plasmodium falciparum Dd2

PhCF₃

phenyl trifluoromethyl

PhOCF₃

phenyl trifluoromethoxide

Py

Plasmodium yoelii

SD

standard deviation

SDC

single dose cure

T_1/2

half-life in mice

TEA

trimethylamine

T_max

time to peak drug concentration

V_ss

apparent volume of distribution at steady state