Synthesis and evaluation of 7-substituted 4-aminoquinoline analogs for antimalarial activity

Abstract

We previously reported that substituted 4-aminoquinolines with a phenylether substituent at the 7-position of the quinoline ring and the capability of intramolecular hydrogen bonding between the protonated amine on the side chain and a hydrogen bond acceptor on the amine’s alkyl substituents exhibited potent antimalarial activity against the multi-drug resistant strain P. falciparum W2. We employed a parallel synthetic method to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogs, in the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 and cytotoxicity mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset showed consistently good potency against the drug-resistant K1strain good selectivity with regard to mammalian cytotoxicity. Overall, our data indicate that the biaryl-containing series contains promising candidates for further study.

Introduction

Malaria, a devastating infectious disease caused by five species of Plasmodium, affects 200–500 million people and causes over 800,000 deaths annually.¹ The most lethal form of malaria is caused by Plasmodium falciparum.² Since the development of quinine, drugs containing the quinoline nucleus have been a mainstay of antimalarial therapy.^3,4 However, the spread of chloroquine (CQ) and mefloquine resistant strains of P. falciparum has curbed the usefulness of this class of drugs.^5,6 In the past two decades, a number of new potent quinolines that overcome chloroquine resistance have been reported.^7–9 Most contain the 7-chloroquinoline nucleus of chloroquine and vary in the length and nature of their basic amine side chain.^10–12

We have reported extensive studies on the role of side chains for the antimalarial activity and efficacy.^{13, 14} Additional work in our laboratory systematically examined modification of the quinoline ring and the basic side chain of 4-aminoquinolines (1) ^{13, 14,15, 16} demonstrating that intramolecular hydrogen bonding (2) between the protonated amine and the hydroxyl group on the tertiary basic amine is crucial for the potency against CQ-resistant strains. In a similar systematic survey we found that most modifications to the quinoline nucleus resulted in lowered potency and had little effect on relative potency in multi-drug resistant (MDR) strains, such as W2.^{15, 16} This finding was in line with the consensus model from the literature that requires an small, electron withdrawing group at this position (chlorine or trifluoromethyl). From these studies, one finding that did stand out was the high potency of the 7-PhO substitution in the context of a shorter amino containing side chain, which was generically more potent against MDR strains. In addition other studies showed that exchanging the quinoline ring system with an acridine ring system in the context of the same side chain gave improved antimalarial potency against both CQ-sensitive and CQ-resistant strains, suggesting that increased bulk and/or hydrophobicity might be advantageous.¹⁷ Given these previous SAR results, we designed a new array of 4-aminoquinoline analogs (3) to explore this hypothesis by using parallel medicinal chemistry. Here we report the design, synthesis, and biological evaluation of this array.

Our strategy for the design of this compound array is shown in Figure 1. We fixed the side chain portion using a propyl spacer (three carbons) between two amines because such shorter side chain variants have consistently given better antimalarial potency than longer chains (> four carbons).¹⁶ A fixed set of substitutions on the terminal alkyl amine was chosen to allow for combinatorial interactions with the quinoline nucleus within the highly potent “chemical space.” This diversity set was based on previously obtained SAR for chloroquine analogs. The 7-position of the quinoline ring system was varied through a range of substituents that were accessible by the Ullmann coupling,^{18, 19} Suzuki coupling,^{20, 21} and Negishi coupling reactions,²² which respectively generate diaryl ether, biaryl, and alkylaryl substitutions. These three sub-series were chosen to systematically vary the size, hydrogen bonding capabilities, electrostatics, and hydrophobicity at the 7-position. Overall this study was expected to firmly establish which types of substitutions are tolerated at the 7-position and how this position interacts with the side chain in defining potency in CQ-resistant Plasmodium species.

Figure 1. Rational design for 7-substituted 4-aminoquinolines.

The three carbon spacer between two amines and the terminal secondary amine was selected since this pair consistently gave high potency in previous work. To explore substitution on the quinoline ring at the 7-position metal assisted-coupling reactions including the Ullmann, Suzuki, and Negishi reactions were applied to generate diarylether, biaryl, and alkylaryl subsets, respectively.

Chemistry

The general approach used to produce this compound array followed our previously established route (Scheme 1). The main challenge in this work was the development of a robust, general route to 7-bromo-4-chloroquinoline 7a, which served as the key intermediate for the diversification. Since chloro and bromo substituted carbons in general have strongly differing reactivity, this intermediate allowed for the selective synthesis of all targeted compounds. The synthetic route to the key intermediate 7a followed a previously reported method.¹⁶ Condensation of 3-bromoaniline with Meldrum’s acid 4 and trimethyl orthoformate gave enamine 5. Cyclization with microwave acceleration gave regioisomers 6a and 6b, as a 2:1 mixture that was inseparable by both flash column chromatography and crystallization. Chlorination of the mixture with POCl₃ allowed the successful separation of 7-bromo-4-chloroquinoline 7a, the key intermediate, from its isomer 5-bromo-4-chloroquinoline 7b, by column chromatography. Condensing 7b with 20 equivalents of 1,3-propyldiamine afforded compound 8, which was used in the next coupling reaction without purification.

Scheme 1. Synthesis of the compound array 3{x,y,z}.

Reagents and conditions: (a) 3-bromoaniline, triethylorthoformate, reflux, 1h; (b) 225 °C, MW, Ph₂O, 5 min; (c) POCl₃, toluene, 100 °C, 1 h; (d) 1,3-aminopropane, K₂CO₃, TEA, NMP, 140 °C, 1 h; (e) for Ullmann reagent: phenols, CuI, Cs₂CO₃, Ligand, 1,4-dioxane, 4 Å MS, reflux, 24 h; for Suzuki reagents: boronic acids, PdCl₂(dppf), Cs₂CO₃, toluene, 4 Å MS, 100 °C, 24 h; for Negishi reagents: zinc halides, Pd(PPh₃)₄, THF, reflux, 16 h.

Intermediate 8 was coupled with three sets of building blocks (diversity sets shown in Figure 2): phenols (x=1, y=1–8), boronic acids (x=2, y=1–8), and alkyl/benzyl zinc halides (x=3, y=1–12) in the presence of the corresponding metal catalyst. Each diversity set was designed to sample widely in sterics, electrostatics, and hydrophobicity. This parallel diversification step resulted in the production of the array of 7-substituted-4-aminoquinolines 9{x,y}. The final array was produced by an orthogonal diversification at the terminal amine. Each member of array 9{x,y} was treated in parallel with the four aldehydes defined from our previous studies (z=1–4) to give 4,7-disubstituted quinoline analogs 3{x,y,z}.

Figure 2.

Diversity Sets used in Generating the Compound Array.

The crude products were purified by flash chromatography using a SP-1 (Biotage) purification system with dichloromethane and methanol mixture (1 -> 50% gradient condition). Each purified product was then dissolved in a saturated methanolic solution of hydrochloric acid and then evaporated to dryness to afford the HCl salts. Purity and identity were verified for all compounds by UPLC/MS and ¹H-NMR. The yield, purity, and NMR data of final compounds are available in the Supporting Information. All purified products were dissolved in DMSO to a standard concentration of 10 mM for biological testing.

Biological and Pharmacological Testing

The compounds in array 3{x,y,z} were tested against CQ-sensitive (3D7) and CQ- resistant (K1) strains of P. falciparum in concentration-respons e experiments in a three-fold dilution schema spanning 15 μM to 0.7 nM, using a previously established method.²³ All experiments were carried out in triplicate and each triplicate experiment being replicated on two separate days, for a total of 9 replicates. Data are reported as mean EC₅₀ values with 95% confidence limits.

The compounds in array 3{x,y,z} were also tested for growth inhibition against four human cell lines: Raji, a Burkett’s lymphoma derived line; BJ, an immortalized foreskin fibroblast derived line; HEK 293, an embryonic kidney fibroblast derived line; and HepG2, a hepatocellular carcinoma derived line. The cytotoxicity was determined by detection of total cellular ATP levels (Cell Titer Glo). The same concentration-response plates were used for both antimalarial and growth proliferation experiments. All experiments were carried out in triplicate with one replicate for a total of 3 replicates. Data are reported as mean EC₅₀ values with 95% confidence limits. Finally, the kinetic solubility and passive permeability of the molecules in array 3(x,y,z) were investigated to provide guidance in interpreting cell based assay results and to inform considerations about the development potential of the compounds. Solubility was determined using the pIon method by dilution from DMSO stocks into PBS buffer containing citric acid at neutral pH (7.4).²⁴ Permeability was assessed using the pIon implementation of the parallel artificial membrane permeation assay (PAMPA) in PBS buffer containing citric acid at neutral pH (7.4), thus reflecting the conditions required for activity in cell-based assays.²⁵ Both assays emulate the conditions used to determine cytotoxicity and antimalarial activity. The results of this testing schema are discussed below, broken out by subset of the analogs at the 7-position of the quinoline nucleus.

Diaryl Ether Series

Thirty two diaryl ethers derived from the Ullmann reaction were synthesized and evaluated. The data is summarized in the heat map in Table 1, which was produced by using the EC₅₀ values determined by fitting curves to the concentration-response experiments described above. The tabulated experimental values and errors can be found in the supplemental materials. Of 32 diaryl ethers, 21 compounds showed reasonable potency according to the MMV lead criteria (EC₅₀ < 50 nM) against the CQ-sensitive 3D7 strain. However, only two compounds (3{1,4,1} and 3{1,7,4}) exhibited highly potent activity (20 nM and 31 nM EC₅₀, respectively) against the CQ-resistant strain K1. This is in stark contrast to the results with the analogous chloro compounds, which are all equivalent compounds are roughly equipotent against the two strains.¹³ One set of compounds stands out: 3{1,1,4}, 3{1,3,4}, and 3{1,5,4}, all carrying the 3-F-6-MeO-Bn alkyl amine substituent, showed enhanced potency in strain K1. Generally the furfuryl (z=1) and 3-F-6-MeO-Bn (z=4) substituted alkylamines in this series exhibited better potency against strain K1 than the 2-HO-3-MeO-Bn (z=2) and piperonyl (z=3) substituents. All of 2-HO-3-MeO-Bn (z=2) substituted alkylamines had poor antimalarial activity against strain K1, whereas they were highly potent against strain 3D7. Electronic effects were not strongly evident for the diaryl ethers, but electron-donating substituents showed slightly better activity against strain 3D7. The compounds were tested against a panel of four mammalian cell lines (HepG2, HEK293, Raji, and BJ). In general HepG2 cells were the most sensitive, while other lines were less sensitive against compounds (EC₅₀ >5 uM). Table 1 shows only HepG2 data, while all cytotoxicity data is summarized in the Supporting Information. Most of the compounds active against strain 3D7 (< 50 nM EC₅₀) showed moderate cytotoxicity (EC₅₀ 3–15 μM) in HepG2, resulting in a reasonable average selectivity for the parasite (> 100-fold). Actives against strain K1 (< 50 nM EC₅₀) also showed at least 100-fold selectivity. All compounds in this series had reasonable solubility (14.5–46 μM) and good permeability (317–1309 × 10⁻⁶ cm/s) in pH 7.4 buffer (Table 1 in Supporting Information).

Table 1.

Biological and pharmacological evaluation of diaryl ethers: antimalarial activity and cytotoxicity.

3{x,y,z}	R1	R2	3D7a EC50 (μM)	K1a EC50 (μM)	K1/3D7	HepG2b EC50 (μM)	HepG2/3D7	HepG2/K1
CQ	--	--	0.03	1.0	33	25	833	33
3{1,4,1}	4-MeO-PhO	Furfuryl	0.004	0.020	5.6	5.3	1479	265
3{1,7,4}	3-Me2N-PhO	3-F-6-MeO-Bn	0.007	0.031	4.7	3.3	514	110
3{1,1,3}	PhO	Piperonyl	0.018	0.078	4.3	2.7	149	34
3{1,2,1}	2-MeO-PhO	Furfuryl	0.001	0.087	66.9	6.5	5014	75
3{1,5,1}	4-F-PhO	Furfuryl	0.026	0.096	3.6	16.0	611	167
3{1,6,4}	4-Cl-PhO	3-F-6-MeO-Bn	0.031	0.109	3.5	4.5	143	41
3{1,5,4}	4-F-PhO	3-F-6-MeO-Bn	0.297	0.135	0.5	6.6	22	49
3{1,1,4}	PhO	3-F-6-MeO-Bn	0.162	0.138	0.9	1.5	9	11
3{1,3,1}	3-MeO-PhO	Furfuryl	0.009	0.153	17.4	15.5	1763	102
3{1,7,1}	3-Me2N-PhO	Furfuryl	0.027	0.155	5.7	16.0	588	103
3{1,8,2}	4-TERTBu-PhO	2-HO-3-MeO-Bn	0.054	0.249	4.6	1.5	28	6
3{1,3,4}	3-MeO-PhO	3-F-6-MeO-Bn	2.360	0.296	0.1	7.1	3	24
3{1,6,3}	4-Cl-PhO	Piperonyl	0.038	0.320	8.4	7.2	189	22
3{1,4,2}	4-MeO-PhO	2-HO-3-MeO-Bn	0.004	0.375	91.5	5.5	1347	15
3{1,2,3}	2-MeO-PhO	Piperonyl	0.014	0.517	37.7	5.9	427	11
3{1,3,3}	3-MeO-PhO	Piperonyl	0.012	0.600	50.0	14.1	1178	24
3{1,4,4}	4-MeO-PhO	3-F-6-MeO-Bn	0.054	0.660	12.2	7.0	129	11
3{1,5,3}	4-F-PhO	Piperonyl	0.015	0.667	44.5	16.0	1067	24
3{1,6,2}	4-Cl-PhO	Furfuryl	0.011	0.785	70.7	5.7	514	7
3{1,6,1}	4-Cl-PhO	Furfuryl	0.040	0.793	19.8	7.8	194	10
3{1,2,4}	2-MeO-PhO	3-F-6-MeO-Bn	0.022	0.960	44.0	6.4	293	7
3{1,4,3}	4-MeO-PhO	Piperonyl	0.046	1.424	30.9	7.9	172	6
3{1,1,2}	PhO	2-HO-3-MeO-Bn	0.015	1.843	122.0	6.8	450	4
3{1,7,2}	3-Me2N-PhO	2-HO-3-MeO-Bn	0.010	2.044	215.1	7.3	765	4
3{1,8,3}	4-TERTBu-PhO	Piperonyl	0.137	2.451	17.9	0.9	7	0.4
3{1,8,4}	4-TERTBu-PhO	3-F-6-MeO-Bn	0.106	2.780	26.2	7.2	68	3
3{1,3,2}	3-MeO-PhO	2-HO-3-MeO-Bn	0.072	3.026	42.0	5.3	74	2
3{1,5,2}	4-F-PhO	2-HO-3-MeO-Bn	0.011	3.132	282.2	11.0	990	4
3{1,7,3}	3-Me2N-PhO	Piperonyl	1.363	3.930	2.9	16.0	12	4
3{1,8,1}	4-TERTBu-PhO	Furfuryl	1.110	4.809	4.3	3.1	3	1
3{1,2,2}	2-MeO-PhO	2-HO-3-MeO-Bn	0.009	5.701	626.4	11.2	1227	2
3{1,1,1}	PhO	Furfuryl	0.314	5.971	19.0	15.2	48	3

Heat map	3D7 EC50 (μM)	K1 EC50 (μM)	K1/3D7	HepG2 EC50	HepG2/3D7	HepG2/K1
	<0.01	<0.01	>100	>15	>1000	>1000
	<0.05	<0.05	>50	>10	>500	>500
	<0.1	<0.1	>10	>5	>100	>100
	<0.5	<0.5	>1	>1	>50	>50
	>0.5	>0.5	<1	<1	<50	<50

Activity summary of the compounds was sorted by EC₅₀ value against K1 strain.

^aValues are the mean of three independent experiments in triplicate.

^bValues are means of one experiment in triplicate.

Biaryl Series

Next we evaluated the biological activity of the biaryl series derived from the Suzuki reaction. All data for this series is summarized in the Supporting Information and selected data are shown in Table 2. Most compounds in this series showed high potency against strain 3D7. Interestingly, many of them were also highly potent (<50 nM EC₅₀) against strain K1. Thus there is a stark contrast between the diarylether and the biaryl series with respect to their activity against strain K1, which expresses the PfCRT transporter, the primary mediator of CQ resistance. However, compounds 3{2,1,2} and 3{2,5,2}, the most potent against 3D7 (both 9 nM EC₅₀), only showed weak potency against K1 (850 nM and 1950 nM EC₅₀ values, respectively) resulting in high selectivity for 3D7 (93 and 213-fold). As was the case with the diaryl ether series, all 2-HO-3-MeO-Bn substituted alkylamines showed very good activity in strain 3D7. A notable trend was observed: increasing hydrophobicity in this series was accompanied by decreasing antimalarial activity. For example, all tert-Bu-Ph (y=6) substituted, naphthyl substituted (y=3), and 3,5-CF₃-Ph substituted (y=2) compounds showed poor activity in both 3D7 and K1. In contrast to the diaryl ether series, compounds with 2-HO-3-MeO-Bn (z=2) and piperonyl (z=3) substituents on the terminal alkylamine exhibited good activity against strain K1 (EC₅₀ < 50 nM). Although this series displayed moderate overall cytotoxicity (0.7–15 μM EC₅₀ in HepG2), all compounds with low nanomolar EC₅₀’s against 3D7 and K1 had a good selectivity for the parasite relative to mammalian cells. All compounds in this series also have good solubility (32–42 μM) and good permeability (237–2101 × 10⁻⁶ cm/s) in pH 7.4 buffer (Table 2 in Supporting Information)

Table 2.

Biological and pharmacological evaluation for biaryls: antimalarial activity and cytotoxicity.

No	R1	R2	3D7a EC50 (μM)	K1a EC50 (μM)	K1/3D7	HepG2b EC50 (μM)	HepG2/3D7	HepG2/K1
CQ	--	--	0.03	1.0	33	25	833	33
3{2,8,1}	4-MeO-Ph	Furfuryl	0.011	0.023	2.2	6.00	565.7	261.9
3{2,7,4}	Piperonyl	3-F-6-MeO-Bn	0.031	0.024	0.8	6.04	194.7	255.8
3{2,4,2}	4-CF3-Ph	2-HO-3-MeO-Bn	0.013	0.024	1.8	2.21	166.2	91.0
3{2,8,3}	4-MeO-Ph	Piperonyl	0.029	0.026	0.9	5.63	192.9	218.3
3{2,7,3}	Piperonyl	Piperonyl	0.026	0.027	1.0	4.79	185.0	178.8
3{2,8,4}	4-MeO-Ph	3-F-6-MeO-Bn	0.096	0.029	0.3	5.96	62.1	205.5
3{2,5,3}	Ph	Piperonyl	0.017	0.029	1.7	4.07	234.0	138.9
3{2,1,3}	4-F-Ph	Piperonyl	0.018	0.035	2.0	8.77	495.4	252.0
3{2,5,4}	Ph	3-F-6-MeO-Bn	0.011	0.045	4.2	2.96	276.4	66.3
3{2,7,1}	Piperonyl	Furfuryl	0.019	0.046	2.4	6.50	340.2	142.2
3{2,7,2}	Piperonyl	2-HO-3-MeO-Bn	0.008	0.050	6.0	1.14	135.4	22.7
3{2,1,1}	4-F-Ph	Furfuryl	0.032	0.069	2.1	7.76	240.4	113.2
3{2,8,2}	4-MeO-Ph	2-HO-3-MeO-Bn	0.013	0.078	6.0	1.60	122.9	20.6
3{2,4,4}	4-CF3-Ph	3-F-6-MeO-Bn	0.054	0.089	1.7	5.19	96.8	58.4
3{2,1,4}	4-F-Ph	3-F-6-MeO-Bn	0.018	0.097	5.5	0.77	44.0	7.9
3{2,4,3}	4-CF3-Ph	Piperonyl	0.025	0.098	3.9	1.89	74.5	19.2
3{2,5,1}	Ph	Furfuryl	0.020	0.128	6.3	5.92	291.6	46.4
3{2,2,2}	3,5-CF3-Ph	2-HO-3-MeO-Bn	0.029	0.209	7.2	0.94	32.4	4.5
3{2,4,1}	4-CF3-Ph	Furfuryl	0.020	0.223	11.0	3.47	171.0	15.5
3{2,6,1}	4-TERT-Bu-Ph	Furfuryl	0.704	0.325	0.5	3.15	4.5	9.7
3{2,6,2}	4-TERT-Bu-Ph	2-HO-3-MeO-Bn	0.251	0.418	1.7	0.69	2.8	1.7
3{2,6,3}	4-TERT-Bu-Ph	Piperonyl	0.223	0.490	2.2	1.37	6.2	2.8
3{2,3,3}	1-Naphtyl	Piperonyl	0.598	0.538	0.9	6.57	11.0	12.2
3{2,2,3}	3,5-CF3-Ph	Piperonyl	0.284	0.604	2.1	1.16	4.1	1.9
3{2,2,4}	3,5-CF3-Ph	3-F-6-MeO-Bn	0.092	0.812	8.8	0.87	9.4	1.1
3{2,1,2}	4-F-Ph	2-HO-3-MeO-Bn	0.009	0.854	92.8	6.84	742.9	8.0
3{2,2,1}	3,5-CF3-Ph	Furfuryl	0.285	0.972	3.4	2.41	8.5	2.5
3{2,3,4}	1-Naphtyl	3-F-6-MeO-Bn	1.154	1.034	0.9	16.00	13.9	15.5
3{2,3,2}	1-Naphtyl	2-HO-3-MeO-Bn	0.033	1.119	33.9	2.52	76.3	2.3
3{2,3,1}	1-Naphtyl	Furfuryl	0.031	1.295	41.2	5.60	178.2	4.3
3{2,6,4}	4-TERT-Bu-Ph	3-F-6-MeO-Bn	0.144	1.594	11.0	0.62	4.3	0.4
3{2,5,2}	Ph	2-HO-3-MeO-Bn	0.009	1.936	212.7	3.21	353.0	1.7

Heat map	3D7 EC50 (μM)	K1 EC50 (μM)	K1/3D7	HepG2 EC50 (μM)	HepG2/3D7	HepG2/K1
	<0.01	<0.01	>100	>15	>1000	>1000
	<0.05	<0.05	>50	>10	>500	>500
	<0.1	<0.1	>10	>5	>100	>100
	<0.5	<0.5	>1	>1	>50	>50
	>0.5	>0.5	<1	<1	<50	<50

Activity summary of the compounds was sorted by EC₅₀ value against K1 strain.

^aValues are the mean of three independent experiments in triplicate.

^bValues are means of one experiment in triplicate.

Alkylaryl Series

The alkylaryl series, derived from Negishi couplings, was evaluated in same test cascade. This set of compounds also showed good antimalarial activity against strain 3D7. However, their activity against strain K1 was very weak (EC₅₀ > 200 nM). Most of the active compounds in this series showed moderate to strong cytotoxicity (0.4–16 μM) in HepG2, resulting in a reasonable selectivity relative to mammalian cells (>100). However, given the poor potency on K1, there was almost no selectivity relative to mammalian cells that strain. Interestingly, the most potent compound 3 with 4 nM EC₅₀ against 3D7 was quite toxic (403 nM EC₅₀) to HepG2 cell, but it still gave good selectivity (>100-fold) between parasite and mammalian cells. Overall this series is the least promising with respect to potential for development. In general the compounds in this series tended to be less soluble and less permeable than the other series studied. They had values ranging from modest to good solubility (4.2–41 μM) and modest to good permeability (23–2110 × 10⁻⁶ cm/s) in pH 7.4 buffer (Table 3 in Supporting Information)

Discussion

In earlier work we identified a previously unappreciated aspect to the structure-activity relationships of the 4-aminoquinoline antimalarials in which the 7-chloro substituent of the quinolone A-ring could be replaced by a hydrophobic substituent with retention of potency. In order to more fully explore this observation, we applied three different coupling chemistries to a common synthetic intermediate to give access to fairly widely ranging substituents at this position (R₁ group). This array of compounds provided a wide range of physico-chemical properties for the quinoline including variant pKa of the quinoline nitrogen; overall electrostatics and sterics, based on the Topliss and Hammett approaches; and overall lipophilicity.

It is known that the pKa of the ring and the basic side chain affect the activity against both CQ-sensitive and CQ-resistant strains of Plasmodium.^{12, 26,27} We calculated overall lipophilicity and pKa of quinoline nitrogens (data summarized in Supporting Information).²⁸ Surprisingly, the biaryl series (x=2) and alkylaryl series (x=3) showed identical pKa values (9.47) on the nitrogen of the quinoline ring, whereas the diaryl ether series (x=1) showed a slightly lower pKa value (9.28). Overall this did not appear to correlate with activity trends. Interestingly, no strong stereo-electronic effects were observed for substituents on the R₁ group. Unfortunately, we did not find a good correlation between antimalarial activity and pKa value.

Kaschula et al. reported that a more lipophilic group at the 7-position of 4-aminoquinoline enhanced antimalarial activity by increasing hematin affinity.²⁷ Therefore, correlations with ALogP were explored to elucidate the relationship between activity and lipophilicity. (Supporting Information, Figure 2) Lipophilic compounds in our tests exhibited slightly weaker antimalarial activity in both the 3D7 and K1 strains when all series were combined (Figure 3A). The same trend was observed in the diarylether series alone (Figure 3B) and in the biaryl series (Figure 3C). However, the trend was reversed in the alkylaryl series (Figure 3D). We noted further more that in the diarylether series the antimalarial activity in strain 3D7 was more sensitive to lipophilicity than in strain K1. However, none of these trends was strong enough to have any real interest or to warrant further mechanistic studies with hematin.

Figure 3.

Relationship between lipophilicity and antimalarial activity (3D7 strain (○) and K1 strain (▲)). (A) ALogP vs LogEC₅₀ - complete compound array; (B) ALogP vs LogEC₅₀ – Diarylethers; (C) ALogP vs LogEC₅₀ - Biaryls; (D) ALogP vs LogEC_{50 -} Alkylaryls.

Overall, there were no strong trends in activity that could be easily explained using any conventional models for chloroquine mechanism of action.

Another possible explanation for variations in activity would be variant cellular bioavailability of the compounds. The quinolines are well known to accumulate in the acidic food vacuole of the parasite, so permeability is usually a poor predictor of potency, beyond a requirement that the compounds be able to cross a membrane. As expected we observed no strong correlations between permeability or solubility and potency. The most striking finding was that the alkyl aryl series had fairly extreme cross-resistance with CQ-resistant strains. Presumably, this substitution at the 7-position increases transport by PfCRT.

Conclusions

In conclusion, a parallel synthetic strategy was employed to generate a rationally designed array of 7-substituted 4-aminoquinolines. Utilizing 7-bromo-4-chloroquinoline as a key intermediate enabled the exploration of two-dimensional diversity, using a fixed propyl spacer between the quinoline and the distal basic center and a small set of amine substituents previously shown to be active in CQ- resistant Plasmodium strains when used with the chloroquine nucleus. The diversity explored at the 7- position included diarylethers, biaryls, and alkylaryls and was produced using the Ullmann, Suzuki, and Negishi reactions, respectively. The compounds were screened against CQ-sensitive and CQ-resistant strains of P. falciparum and a panel of mammalian cell line to establish antimalarial potency and selectivity for mammalian cells.

Physico-chemical properties of 4-aminoquinolines such as pKa and lipophilicity can be influenced by the substitution at the 7-position, resulting in changes of the drug-sensitivity against antimalarial. However in the series explored, the calculated pKa of the compounds was not significantly changed by substitution at 7-position, and no correlation was observed between pKa and antimalarial activity. However, lipophilicity did weakly correlate with antimalarial activity. Strikingly, less lipophilic compounds were generally more potent against both the CQ-sensitive (3D7) and CQ-resistant strain (K1) an effect that was especially strong for the biaryl compounds. Taken as a whole, this study highlights the potential for developing novel 4-aminoquinolines including a biaryl at the 7-position of the quinoline. Further exploration in preclinical studies is warranted.

Experimental Section

General

All materials were obtained from commercial suppliers and used without further purification. All solvents were dried using an aluminum oxide column. Thin-layer chromatography was performed on pre-coated silica gel 60 F254 plates. Purification of synthesized compounds was carried out by normal phase column chromatography (SP1 [Biotage], Silica gel 230–400 mesh) followed by evaporation (HT-4X evaporator [Genevac]. Initiator [Biotage] was used for microwave reaction. ¹H NMR spectra were recorded using a Bruker 400-MHz spectrometer, in CDCl₃ or DMSO-d6 solvent. Chemical shifts were reported as parts per million (ppm) downfield from solvent reference. Coupling constants (J values) were measured in Hertz (Hz). NMR peaks were assigned by MestReNova (5.2.2) Identity of all compounds was confirmed by proton and carbon NMR and by mass spectrometry. The purity of all compounds was assessed using LC/MS/UV/ELSD/CLND with the purity being assigned as the average determined by UV/ELSD/CLND. All compounds used for subsequent studies had a minimum purity of 95%.

Chemistry

5-((3-Bromophenylamino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (5)

A solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (4) (21.6 g, 150 mmol) in triethyl orthoformate (100 mL) was refluxed for 1h. After cooling down to room temperature 3-bromoaniline (17.2 g, 100 mmol) was added to the solution. The mixture was refluxed for 2 h, then cooled down to room temperature. The precipitates were collected and washed with ether to give compound 5 (32.8 g, quant. yield) as pale yellow crystals. 1H NMR (CDCl₃) δ ppm 11.51–10.89 (m, 1H), 8.60 (d, J = 14.1 Hz, 1H), 7.47–7.37 (m, 2H), 7.31 (t, J = 8.0 Hz, 1H), 7.21–7.15 (m, 1H), 1.76 (s, 6H).

7-Bromoquinolin-4-ol (6a) and 5-bromoquinolin-4-ol (6b)

Compound 5 (1.0 g, 3.1 mmol) and diphenyl ether (10 mL) were irradiated by microwave at 225 °C for 5 minutes. Ether (10 mL) was added to the mixture, and the resulting brown precipitates were collected and washed with ether to give a mixture of compounds 6a and 6b. Replicating this process 16 times in parallel gave 9.4 g (85% yield) of the product mixture. Caution: The reaction vessel develops high pressure. 1H NMR (DMSO-d6) δ ppm 11.79 (brs, 1H), 7.99 (d, J = 8.6 Hz, 2/3H), 7.92 (d, J = 7.4 Hz, 2/3H), 7.82 (d, J = 7.4 Hz, 1/3H), 7.75 (d, J = 1.9 Hz, 2/3H), 7.52 (dd, J = 1.2, 8.0 Hz, 1/3H), 7.48 (dd, J = 1.4, 8.0 Hz, 1/3H), 7.45 (dd, J = 1.8, 8.6 Hz, 2/3H), 7.44 (dd, J = 8.0, 8.4 Hz, 1/3H), 6.05 (d, J = 7.5 Hz, 2/3H), 6.01 (d, J = 7.5 Hz, 1/3H).

7-Bromo-4-chloroquinoline (7a)

To a suspension of a mixture of compounds 6a and 6b (9.4 g, 42 mmol) in toluene (70 mL) was added phosphorus oxychloride (13 g, 84 mmol). The mixture was stirred for 1 h at 100 °C, then cooled to room temperature. Excess phosphorus oxychloride was quenched by adding a small portion of ice water and then stirred for 1h. Additional water (100 mL) was added and the resulting mixture extracted with dichloromethane. The organic phase was dried over MgSO₄ and the solvent was removed in vacuo. The crude product was purified by flash chromatography (EtOAc in hexane = 5 -> 25%) to give compound 7a (6.2 g, 61% yield) as white crystals and compound 7b (3.0 g, 30% yield) as white crystals. 7a: 1H NMR (CDCl₃) δ ppm 8.79 (d, J = 4.8 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.74 (dd, J = 9.0, 1.9 Hz, 1H), 7.51 (d, J = 4.7 Hz, 1H). 7b: 1H NMR (CDCl₃) δ ppm 8.73 (d, J = 4.6 Hz, 1H), 8.12 (dd, J = 8.4, 1.2 Hz, 1H), 7.96 (dd, J = 7.6, 1.2 Hz, 1H), 7.56 (d, J = 4.4 Hz, 1H), 7.52 (t, J = 8.4, 7.6 Hz, 1H).

N¹-(7-bromoquinolin-4-yl)propane-1,3-diamine (8)

To a mixture of compound 7a (6.18 g, 25.6 mmol) and 1,3-diaminopropane (37.9 g, 512 mmol) in N-methylpyrrolidinone (60 mL) was added triethylamine (777 mg, 7.68 mmol) and K₂CO₃ (1.17 g, 8.45 mmol). The resulting mixture was stirred at 140 °C for 1h and then cooled to room temperature. Water (200 mL) was added to the solution and the product was precipitated by cooling the mixture to 4 °C. The resulting white precipitates were collected, washed with water, and dried to give compound 8 (5.69 g, 80% yield) as white crystals. 1H NMR (DMSO-d6) δ ppm 8.38 (d, J = 5.4 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 2.1 Hz, 1H), 7.54 (dd, J = 9.0, 2.1 Hz, 1H), 7.52 (brs, 1H), 6.48 (d, J = 5.5 Hz, 1H), 3.32 (brs, 2H), 2.68 (t, J = 6.5 Hz, 2H), 1.68–1.77 (m, 2H).

Procedure for parallel synthesis of Compounds 9{x=1}:Ullmann coupling

To a mixture of compound 8 (279 mg, 1 mmol) in 1,4-dioxane (6 mL) was added the phenol reagent (4 mmol), Cs₂CO₃ (1.3 g, 4 mmol), MS 4 Å powder (ca. 280 mg, dried in oven), salicylaldoxime (55 mg, 0.4 mmol) and copper iodide (I) (38 mg, 0.2 mmol). The mixture was heated to reflux and maintained at that temperature for 24 h, and then cooled to room temperature. A solution of dichloromethane (10 mL) and methanol (1 mL) was added to the mixture. After filtration the solvents were removed in vacuo. The crude product was purified by flash chromatography (MeOH/TEA in CH₂Cl₂ = 10%/1% ~ 40%/4% gradient) to give the corresponding compound 9{x=1}.

Procedure for parallel synthesis of Compounds 9{x=2}: Suzuki coupling

To a mixture of compound 8 (279 mg, 1 mmol) in toluene (6 mL) was added the boronic acid reagent (4 mmol), Cs₂CO₃ (1.3 g, 4 mmol), MS 4 Å powder (ca. 280 mg) and PdCl₂(dppf) (41 mg, 0.05 mmol). The mixture was heated to 100 °C for 24 hrs and then cooled to room temperature. Water (50 mL) was added and the resulting mixture extracted with dichloromethane and 10% methanol. The organic phase was dried over MgSO₄, filtered, and dried in vacuo. The crude product was purified by flash chromatography (MeOH/TEA in CH₂Cl₂ = 10%/1% ~ 80%/8% gradient) to give the corresponding compounds 9{x=2}.

Procedure for parallel synthesis of Compounds 9{x=3}: Negishi coupling

To a solution of compound 8 (279 mg, 1 mmol) and Pd(PPh₃)₄ (58 mg, 0.05 mmol) was added the alkyl zinc bromide reagent (8 mL, 0.5 M in THF, 4 mmol). The mixture was refluxed for 16 hrs, then cooled to room temperature (one hour is sufficient for benzyl zinc halide). 1N HCl (10 mL) was added to the mixture and stirred. Additional water (40 mL) was added to the mixture, and the solution then neutralized by adding solid K₂CO₃. The mixture was extracted with 10% methanol in dichloromethane, the organic phase dried over MgSO₄, and concentrated in vacuo. The crude product was purified by flash chromatography (MeOH/TEA in CH₂Cl₂ = 10%/1% ~ 80%/8% gradient) to give the corresponding compounds 9{x=3}. Benzyl zinc bromide solution was prepared as follows: To a suspension of Zn powder (261 mg, 4 mmol) in THF (6 mL) was added benzyl bromide (4 mmol). The solution was heated to 50 °C for 30 min. The solution was used immediately for Negishi coupling.

Procedure for parallel synthesis of Compounds 3{x,y,z}: Reductive amination

To a solution of compound 9{x} (0.1~0.2 mmol) in methanol (1~2 mL) was added 1M aldehyde stock solution in methanol (1 eq.) After stirring for 1 h, sodium borohydride (0.5 eq.) was added to the reaction mixture and the resulting mixture stirred for 30 min. The reaction was quenched by addition of water (2 mL), extracted with CH₂Cl₂ and concentrated in vacuo. The crude product was purified by flash chromotography (1% methanol in CH₂Cl₂ (12 mL) and 30% methanol in CH₂Cl₂ (16 mL)). The solvent was removed in vacuo, and the product was suspended in 1.25 M HCl in methanol solution (1 mL). The solvent and excess of HCl were evaporated in vacuo to give the corresponding HCl salt.

Antimalarial Activity

Two P. falciparum strains were used in this study and were provided by the MR4 Unit of the American Type Culture Collection (ATCC, Manassas, VA). Those two strains were the chloroquine sensitive strain 3D7 and the chloroquine resistant strain K1. Asynchronous parasites were maintained in culture based on the method of Trager²⁹. Parasites were grown in presence of fresh group O-positive erythrocytes (Lifeblood Memphis, TN) in Petri dishes at a hematocrit of 4–6% in RPMI based media(RPMI 1640 supplemented with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM NaHCO₃ (pH 7.3), 100ug/mL hypoxanthine, and 5 Pg/mL gentamycin). Cultures were incubated at 37 °C in a gas mixture of 90% N₂, 5% O₂, 5% CO₂. For IC₅₀ determinations, 20 μL of RPMI 1640 with 5 μg/ml gentamycin were dispensed per well in an assay plate (Corning 384-well microtiter plate, clear bottom, tissue culture treated, cat. 8807BC). 40 nL of compound, previously serial diluted in a separate 384-well white polypropylene plate (Corning, cat 8748BC), were dispensed to the assay plate by hydrodynamic pin transfer (FP1S50H, V&P Scientific Pin Head) and then 20 μl of a synchronized culture suspension (1% rings, 4% hematocrit) were added per well thus making a final hematocrit and parasitemia of 2% and 1%, respectively. Assay plates were incubated for 72 h and the parasitemia was determined by a method previously described ³⁰: Briefly, 10 μl of the following solution in RPMI (10 X Sybr Green I, 0.5% v/v triton, 0.5 mg/ml saponin) were added per well, assay plates were shaken for 30 s, incubated in the dark for 90 min, then read with the Envision spectrophotometer at Ex/Em 485 nm/535 nm. IC₅₀’s were calculated with the robust investigation of screening experiments (RISE) with four parameter logistic equation.

Cytotoxicity in mammalian cells

BJ, HEK293, Hep G2, and Raji cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured according to recommendations. Cell culture media were purchased from ATCC. Cells were routinely tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). Exponentially growing cells were plated in Corning 384 well white custom assay plates, and incubated overnight at 37° C in a humidified, 5% CO₂ incubator. DMSO compound solutions were added the following day to a top final concentration of 25 μM and then diluted 1/3 for a total of ten testing concentrations. Cytotoxicity was determined following a 72-hour incubation using Promega Cell Titer Glo Reagent according to the manufacturer’s recommendation. Luminescence was measured on an Envision plate reader (Perkin Elmer)

Solubility

The solubility assay was carried out on Biomek FX lab automation workstation (Beckman Coulter, Inc.). Ten μL of compound stock was added to 190 μL 1-propanol to make a reference stock plate. Five μL from this reference stock plate was mixed with 70 μL 1-propanol and 75 μL phosphate buffered saline (PBS, pH 7.4) to make the reference plate and the UV spectrum (250 – 500 nm) of the reference plate was measured using a SPECTRAmax PLUS plate reader (Molecular Devices). Six μL of 10 mM test compound stock was added to 600 μL PBS in a 96-well storage plate and mixed. The storage plate was sealed and incubated at room temperature for 18 h. The suspension was then filtered through a 96-well filter plate (pION Inc.). Seventy-five μL of filtrate was mixed with 75 μL 1-propanol to make the sample plate for UV spectroscopic analysis. A single experiment was performed in triplicate for each compound. Solubility was calculated using PSOL Evolution software based on the AUC (area under the curve) of the UV spectrum of the sample plate and the reference plate.

Permeability assay

The Parallel Artificial Membrane Permeability Assay (PAMPA) was carried out on a Biomek FX lab automation workstation (Beckman Coulter, Inc.). Three μL of test compound stock (10 mM in DMSO) was mixed with 600 μL of SSB (system solution buffer, pH 7.4 or 4, pION Inc.) to dilute the test compound. One hundred fifty μL of diluted test compound in SSB was transferred to a UV plate (pION Inc.) and the UV spectrum was measured on a SPECTRAmax PLUS plate reader (Molecular Devices) to establish a reference plate. The membrane on a pre-loaded PAMPA sandwich (pION Inc.) was painted with 4 μL GIT lipid (pION Inc.). The acceptor chamber was then filled with 200 μL ASB (acceptor solution buffer, pION Inc.) and the donor chamber was filled with 180 μL test compound diluted in SSB. The PAMPA sandwich (donor and acceptor chamber) was assembled, placed on the Gut-box (pION Inc.) and stirred for 30 minutes. The Aqueous Boundary Layer was set to 40 μM for stirring and the UV spectrum (250–500 nm) of the donor and the acceptor chambers were read. A single experiment was performed in triplicate for each compound. The permeability coefficient was calculated using PAMPA Evolution 96 Command software (pION Inc.) based on the AUC of the reference, donor, and acceptor plates.

Table 3.

Biological and pharmacological evaluation for alkylaryls: antimalarial activity and cytotoxicity.

No	R1	R2	3D7a EC50 (μM)	K1a EC50 (μM)	K1/3D7	HepG2b EC50 (μM)	HepG2/3D7	HepG2/K1
CQ	--	--	0.03	1.0	33	25	833	33
3	1-Et-Pr	2-HO-3-MeO-Bn	0.004	0.273	68.3	0.41	103.3	1.5
3{3,5,2}	3,5-Me-Bn	2-HO-3-MeO-Bn	0.009	1.360	160.0	2.68	315.8	2.0
3{3,5,1}	3,5-Me-Bn	Furfuryl	0.009	0.678	77.0	6.27	712.1	9.2
3{3,3,2}	4-CN-Bn	2-HO-3-MeO-Bn	0.011	5.443	513.5	7.15	674.8	1.3
3{3,4,4}	Bn	3-F-6-MeO-Bn	0.011	1.424	129.5	7.99	726.7	5.6
3{3,6,2}	2-Cl-4-F-Bn	2-HO-3-MeO-Bn	0.013	5.136	404.4	2.35	185.4	0.5
3{3,9,3}	PhEt	Piperonyl	0.013	1.533	117.1	4.98	380.3	3.2
3{3,1,2}	4-F-Bn	2-HO-3-MeO-Bn	0.015	7.150	473.5	4.78	316.6	0.7
3{3,9,2}	PhEt	2-HO-3-MeO-Bn	0.017	5.517	324.5	6.80	400.0	1.2
3{3,4,2}	Bn	2-HO-3-MeO-Bn	0.017	6.971	400.6	5.72	328.6	0.8
3{3,11,2}	cHex	2-HO-3-MeO-Bn	0.019	16.000	864.9	1.08	58.1	0.1
3{3,6,1}	2-Cl-4-F-Bn	Furfuryl	0.020	1.223	62.1	5.73	290.8	4.7
3{3,10,2}	iso-butyl	2-HO-3-MeO-Bn	0.020	8.130	402.5	2.92	144.5	0.4
3{3,7,2}	iso-pentyl	2-HO-3-MeO-Bn	0.022	8.000	365.3	5.39	245.9	0.7
3{3,3,3}	4-CN-Bn	Piperonyl	0.023	3.069	136.4	6.08	270.2	2.0
3{3,2,2}	3-CF3-Bn	2-HO-3-MeO-Bn	0.025	2.189	87.9	1.72	69.0	0.8
3{3,6,3}	2-Cl-4-F-Bn	Piperonyl	0.027	0.953	35.9	16.00	603.8	16.8
3{3,12,3}	1-Et-Pr	Piperonyl	0.027	1.065	39.5	6.35	235.1	6.0
3{3,7,3}	iso-pentyl	Piperonyl	0.027	0.206	7.6	2.80	103.1	13.5
3{3,3,4}	4-CN-Bn	3-F-6-MeO-Bn	0.028	0.770	27.5	6.05	216.2	7.9
3{3,5,4}	3,5-Me-Bn	3-F-6-MeO-Bn	0.034	0.794	23.1	0.94	27.4	1.2
3{3,3,1}	4-CN-Bn	Furfuryl	0.039	0.382	9.7	7.91	201.9	20.7
3{3,2,3}	3-CF3-Bn	Piperonyl	0.039	1.314	33.5	2.20	56.1	1.7
3{3,4,1}	Bn	Furfuryl	0.040	3.550	89.9	16.00	405.1	4.5
3{3,2,4}	3-CF3-Bn	3-F-6-MeO-Bn	0.040	0.323	8.1	0.44	11.0	1.3
3	1-Et-Pr	3-F-6-MeO-Bn	0.040	1.933	48.0	7.86	194.9	4.1
3{3,8,3}	cHexmethyl	Piperonyl	0.041	0.701	17.3	6.29	155.2	9.0
3{3,5,3}	3,5-Me-Bn	Piperonyl	0.041	1.040	25.6	2.25	55.5	2.2
3{3,9,1}	PhEt	Furfuryl	0.046	2.930	63.7	16.00	347.8	5.5
3{3,8,2}	cHexmethyl	2-HO-3-MeO-Bn	0.050	2.610	52.7	0.97	19.6	0.4
3{3,8,1}	cHexmethyl	Furfuryl	0.055	0.621	11.2	1.70	30.7	2.7
3{3,2,1}	3-CF3-Bn	Furfuryl	0.056	0.743	13.2	3.19	56.7	4.3
3{3,1,1}	4-F-Bn	Furfuryl	0.057	1.659	28.9	5.60	97.5	3.4
3{3,10,3}	iso-butyl	Piperonyl	0.066	1.814	27.7	2.38	36.3	1.3
3{3,1,4}	4-F-Bn	3-F-6-MeO-Bn	0.075	0.580	7.8	3.53	47.1	6.1
3{3,12,1}	1-Et-Pr	Furfuryl	0.086	4.300	50.1	6.76	78.7	1.6
3{3,10,4}	iso-butyl	3-F-6-MeO-Bn	0.089	2.119	23.9	2.90	32.7	1.4
3{3,11,4}	cHex	3-F-6-MeO-Bn	0.090	1.596	17.7	2.29	25.4	1.4
3{3,7,4}	iso-pentyl	3-F-6-MeO-Bn	0.116	0.360	3.1	0.89	7.7	2.5
3{3,11,1}	cHex	Furfuryl	0.118	3.900	33.1	4.76	40.4	1.2
3{3,1,3}	4-F-Bn	Piperonyl	0.127	0.256	2.0	5.55	43.7	21.7
3{3,11,3}	cHex	Piperonyl	0.128	4.709	36.7	3.02	23.6	0.6
3{3,9,4}	PhEt	3-F-6-MeO-Bn	0.132	2.143	16.2	4.24	32.1	2.0
3{3,8,4}	cHexmethyl	3-F-6-MeO-Bn	0.138	0.687	5.0	1.83	13.3	2.7
3{3,7,1}	iso-pentyl	Furfuryl	0.176	4.605	26.1	11.24	63.7	2.4
3{3,6,4}	2-Cl-4-F-Bn	3-F-6-MeO-Bn	0.208	1.112	5.4	2.46	11.8	2.2
3{3,10,1}	iso-butyl	Furfuryl	0.581	4.500	7.7	16.00	27.5	3.6
3{3,4,3}	Bn	Piperonyl	0.720	1.237	1.7	6.28	8.7	5.1

Heat map	3D7 EC50 (3M)	K1 EC50 (3M)	K1/3D7	HepG2 EC50 (3M)	HepG2/3D7	HepG2/K1
	<0.01	<0.01	>100	>15	>1000	>1000
	<0.05	<0.05	>50	>10	>500	>500
	<0.1	<0.1	>10	>5	>100	>100
	<0.5	<0.5	>1	>1	>50	>50
	>0.5	>0.5	<1	<1	<50	<50

Activity summary of the compounds was sorted by EC₅₀ value against 3D7 strain.

^aValues are the mean of three independent experiments in triplicate.

^bValues are means of one experiment in triplicate.

Abbreviations

CQ

Chloroquine

MDR

multi-drug resistant

PAMPA

parallel artificial membrane permeation assay

WHO

World Health Organization

MMV

Medicines for Malaria Venture

HTS

High Throughput Screening

SAR

Structure Activity Relationship

Clint’

Intrinsic Clearance