Overcoming chloroquine resistance in malaria: Design, synthesis, and structure-activity relationships of novel hybrid compounds

Article abstract of DOI:10.1128/AAC.02476-15

Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge. Reversal of acquired resistance can be achieved using agents that resensitize resistant parasites to a previously efficacious therapy. Building on our initial work describing novel chemoreversal agents (CRAs) that resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA scaffold to chloroquine produces hybrid compounds with restored potency toward a range of resistant malaria parasites. A preferred compound, compound 35, showed broad activity and good potency against seven strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane permeability, and in vitro toxicity in a hepatocyte line and a cardiomyocyte line indicates that compound 35 has a good therapeutic window and favorable drug-like properties. This study provides initial support for CQ-CRA hybrid compounds as a potential treatment for resistant malaria.

Full text of DOI:10.1128/AAC.02476-15

AAC Accepted Manuscript Posted Online 7 March 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.02476-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Overcoming Chloroquine Resistance in Malaria: Design, Synthesis and Structure-Activity
Relationships of Novel Hybrid Compounds
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Aicha Boudhar,a,b Xiao Wei Ng,a Chiew Yee Loh,a Wan Ni Chia,a Zhi Ming Tan,a Francois Nosten,c,d
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Brian W. Dymock,b# Kevin S. W. Tana#
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Laboratory of Molecular and Cellular Parasitology, Department of Microbiology and Immunology,
National University of Singapore, Singaporea; Department of Pharmacy, National University of
Singapore, Singaporeb; Shoklo Malaria Research Unit, Mae Sot, Tak, Thailandc; Center for Clinical
Vaccinology and Tropical Medicine, Headington, Oxford, United Kingdomd
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Running Head: Synthesis and SAR of New Hybrid Antimalarial Compounds
14 #Address correspondence to Kevin S. W. Tan, mictank@nus.edu.sg, and Brian W. Dymock,
15 phadbw@nus.edu.sg.
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16 ABSTRACT
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Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge.
18 Reversal of acquired resistance can be achieved using agents which resensitize resistant parasites to a
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previously efficacious therapy. Building on our initial work describing novel chemoreversal agents
(CRAs) which resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single
agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA
scaffold to chloroquine produces hybrid compounds with restored potency towards a range of resistant
malaria parasites. A preferred compound, 35, showed broad activity and good potency against seven
strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane
25 permeability and in vitro toxicity, in a hepatocyte and a cardiomyocyte cell line, indicates that 35 has a
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good therapeutic window and favourable drug-like properties. This study provides initial support for
CQ-CRA hybrid compounds as a potential treatment for resistant malaria.
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INTRODUCTION
Malaria is a global infectious disease, caused by a parasitic protozoan of the genus Plasmodium (P.
31 falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi) which exhibits a complex life cycle involving
32 an insect vector (mosquito) and a vertebrate host (human)(1). Following WHO estimates there were
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34
214 million cases and 438,000 deaths in 2015 (2). Early diagnosis and treatment of malaria reduces
disease burden, prevents death and contributes to reducing malaria transmission. Historically,
35 chloroquine (CQ, 1, Fig. 1) was a widely used drug due to its good efficacy, low toxicity and
36 affordability. However P. falciparum, the predominant species infecting humans, has developed
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mechanisms to effectively neutralize the effects of 1 and as a result became resistant to the effects of 1.
38 The current best available treatment, particularly for P. falciparum malaria, is an artemisinin-based
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combination therapy (ACT). Unfortunately, ACT has already shown prolonged parasite clearance
times in Thailand and Cambodia (3-5). The constant threat of developing resistance against
antimalarial drugs has led to the search for innovative therapies, such as new compounds with novel
mechanisms of action, or new combination therapies to prevent drug resistance (6, 7).
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In our previous work we have described the reversal of chloroquine resistance with novel
chemoreversal agents (CRAs) (8). Therein, we demonstrated the use of a fluorescent probe in screens
for new chemosensitizing compounds (8, 9), the design of analogues of hit compounds as well as their
46 activity and improved potential as CRAs (8).
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Herein we report our results developing single agent hybrid compounds based on these CRAs. The
concept of hybrid compounds, combining more than one biological activity in a single compound, is a
relatively unexplored concept in antimalarial resistance related drug discovery (10). Hybrid
50 compounds have several advantages, including a lower risk of drug-drug interactions, simpler
51 pharmacokinetics and dosing regimens compared to combinations of single-mechanism drugs. Our aim
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was to achieve the combined bioactive effects of each of the pharmacophores via linking, fusing or
merging. One example of a combination strategy to create single molecule drugs is that of artemisinin
and quinine (2) (11-13), another being the combination of antimalarial agents with other scaffolds such
as 3 (Fig. 2) (14, 15). There are also a few reported examples of CRAs combined to 1, aiming to have
antimalarial activity of CQ and at the same time blocking the chloroquine resistance transporter
(PfCRT) with the CRA (16-18).
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With CRAs 4-6 (Fig. 3) at hand, we investigated their utility in hybrid compounds with 1 as the
antimalarial component, seeking novel structures retaining good physicochemical properties with
improved potency against resistant strains. Thus a series of hybrid compounds were designed, linking
the scaffold from 1 via its terminal secondary or tertiary amine with CRAs 4-6 (Fig. 1 and Fig. 3). The
4-aminoquinoline scaffold has been shown to be essential for the binding of CQ to free heme (19), and
both the terminal tertiary amino group and the basicity of the quinolyl nitrogen are key elements (20,
21). Ideally the length of the aliphatic side chain should be 2-4 carbons, however the methyl group has
65 little influence on the activity and was thus not retained.
66 MATERIALS AND METHODS
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All reagents purchased from commercial sources were of the highest purity grade available and were
used without further purification. Commercially available AR grade solvents or anhydrous solvents
packed in resealable bottles were used as received. All reaction temperatures stated in the procedures
are external bath temperatures. Non-aqueous reactions were performed under a positive pressure of
nitrogen in oven-dried glassware. Yields refer to chromatographically and spectroscopically
homogeneous materials, unless otherwise stated. Reaction progress was monitored by analytical thin
layer chromatography (TLC) with 0.25 mm Merck pre-coated silica gel plates (60F-254) using UV
light (254 nm) as visualizing agent, and ceric ammonium molybdate or potassium permanganate
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solutions as developing stains. Flash chromatography was performed on silica gel 60 (0.040 –
0.063 mm) purchased from SiliCycle or Merck. The structures of synthesized compounds were
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1
verified by H NMR, 13C NMR, and mass spectrometry. H (400 MHz) and 13C (101 MHz) NMR
78 spectra were measured in CDCl3 on a Bruker Avance III 400 (Ultrashield Plus) spectrometer.
79
Chemical shifts are reported in parts per million (ppm) using the residual CDCl3 peak at 7.26 (1H) or
1
80 77.16 (13C) as internal standard. H NMR coupling constants (J) are reported in Hertz (Hz), and
81
multiplicities are presented as follows: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad).
82 Mass spectra were obtained on a Bruker amaZonX for the nominal mass or on a Bruker micrOTOFQII
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spectrometer for the high resolution mass analysis. Purity of the compounds was assessed by high
pressure liquid chromatography detecting at 254 nm using an Agilent 1200 series HPLC system with a
85 Zorbax SB-C18 5 micron 4.6 x 250 mm column using a gradient elution starting from a 5:95 solution
86 of acetonitrile/water, with 1% trifluoroacetic acid (TFA) to 100% acetonitrile and 1% TFA with
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flowrate 0.5 mL per minute over 15 mins. HPLC purity is greater than 95% unless stated. All
compounds synthesized were stored in a −20 °C freezer.
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Chemistry
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Synthesis of precursors 13 to 17 is described in the Supporting Information.
N1-(7-chloroquinolin-4-yl)-N4-(quinuclidin-3-yl)butane-1,4-diamine (24)
92
To a stirred solution of quinuclidin-3-one hydrochloride (23) (154 mg, 0.95 mmol, 1.0 equiv.) in dry
93 methanol (1.40 mL), under inert conditions, was added a 1 M solution of ZnCl2 in Et2O (0.19 mL,
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95
0.19 mmol, 0.2 equiv.). After stirring at room temperature for 30 min was added N1-(7-chloroquinolin-
4-yl)butane-1,4-diamine (19c) (476 mg, 1.91 mmol, 2.0 equiv.). After stirring another hour at room
96 temperature, solid sodium cyanoborohydride (120 mg, 1.91 mmol, 2.0 equiv.) was added in portions.
97
98
The reaction was then stirred for 4 h at room temperature and quenched by addition of water (about
5 mL). The quenched reaction was partitioned between 5 M NaOH(aq.) and DCM. The aqueous layer
5

99 was extracted with DCM (3x), and the combined organic layers were dried with Na2SO4, filtered and
100
concentrated. The crude residue was purified by flash chromatography (eluent DCM/MeOH = 95/5 to
8/2), yielding 9.9 mg (3 %) of N1-(7-chloroquinolin-4-yl)-N4-(quinuclidin-3-yl)butane-1,4-diamine
(24) as a yellow oil. 263 mg (55 %) of the starting material N1-(7-chloroquinolin-4-yl)butane-1,4-
diamine (19c) were also isolated in a second fraction.
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103
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105
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Rf (DCM/MeOH = 95/5) = 0.20; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.50 (1 H, d, J = 5.3 Hz,
H-3), 7.94 (1 H, d, J = 2.0 Hz, H-8), 7.74 (1 H, d, J = 9.0 Hz, H-5), 7.33 (1 H, dd, J = 9.0 Hz,
J = 2.0 Hz, H-7), 6.40 (1 H, d, J = 5.3 Hz, H-2), 5.42 (1 H, bs, NH), 3.34 (2 H, m, H-10), 3.20 (2 H, m,
H-13), 3.30 (4 H, m, H-16), 2.62 (3 H, m, H-14 and H-15), 1.82 (12 H, m, H-12, H-11, H-17, H-18 and
108 NH); 13C NMR (400 MHz, CDCl3) δ (ppm) = 151.7 (C-3), 150.1 (C-1), 148.8 (C-4), 135.2 (C-6),
109
110
128.5 (C-5), 125.5 (C-8), 121.5 (C-7), 117.3 (C-9), 99.1 (C-2), 60.5 (C-15), 53.5 (C-14), 52.8 (C-10),
52.2 (C-13), 47.0 (C-16), 43.3 (C-16), 28.0 (C-11), 26.7 (C-12), 25.1 (C-18), 23.9 (C-17), 18.9 (C-17);
111 MS (IT-TOF) m/z = 359.2 [M+H]+.
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4-((2-((7-chloroquinolin-4-yl)amino)ethyl)(ethyl) amino)-2,2-diphenylbutanenitrile (26)
To a solution of N1-(7-chloroquinolin-4-yl)-N2-ethylethane-1,2-diamine (21a) (426 mg, 1.71 mmol,
1.0 equiv.) and 4-bromo-2,2-diphenylbutanenitrile 11 (512 mg, 1.71 mmol, 1.0 equiv.) in dry MeCN
(5.70 mL), under inert conditions, was added DIPEA (0.91 mL, 5.13 mmol, 3.0 equiv.). After stirring
at reflux for 5 d, the solvent was evaporated under reduced pressure. The crude residue was purified by
flash chromatography (eluent DCM/MeOH = 95/5 to 9/1), yielding 321 mg (40 %) of 4-((2-((7-
118 chloroquinolin-4-yl)amino)ethyl)(ethyl)amino)-2,2-diphenylbutanenitrile (26) as a yellow oil.
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Rf (DCM/MeOH = 9/1) = 0.53; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.54 (1 H, d, J = 5.3 Hz,
H-3), 7.98 (1 H, d, J = 2.0 Hz, H-8), 7.72 (1 H, d, J = 9.0 Hz, H-5), 7.39 (1 H, dd, J = 9.0 Hz,
J = 2.0 Hz, H-7), 7.27 (10 H, m, H-Ph), 6.31 (1 H, d, J = 5.3 Hz, H-2), 5.89 (1 H, bs, NH), 3.14 (2 H,
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q, J = 5.6 Hz, H-12), 2.79 (2 H, t, J = 5.7 Hz, H-10), 2.64 (4 H, m, H-14 and H-11), 2.55 (2 H, m,
H-15), 1.06 (3 H, t, J = 7.0 Hz, H-13); 13C NMR (400 MHz, CDCl3) δ (ppm) = 151.2 (C-3), 150.0
(C-1), 148.8 (C-4), 139.8 (C-18), 135.1 (C-6), 129.1 (C-19), 128.9 (C-5), 128.2 (C-21), 126.7 (C-20),
125.5 (C-8), 122.4 (C-9), 121.4 (C-7), 117.6 (C-17), 99.61 (C-2), 51.7 (C-11), 50.0 (C-16), 49.4
126 (C-14), 47.5 (C-12), 40.1 (C-10), 37.3 (C-15), 12.0 (C-13); MS (ESI) m/z = 469.1 [M+H]+; HRMS
127
(ESI) m/z = 469.2168 [M+H]+, calc.: 469.2154, Diff.: 3.1 ppm.
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N1-(3-(2-chloro-10H-phenothiazin-10-yl)propyl)-N3-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N3-
ethylpropane-1,3-diamine (42)
2-Chlorophenothiazine (39) (1.01 g, 4.30 mmol, 1.0 equiv.) was dissolved under nitrogen in 14.3 mL
of dry THF with the aid of heat. This solution was added to a suspension of NaH (60 w% in oil,
172 mg, 4.30 mmol, 1.0 equiv.) under nitrogen in 7.41 mL of dry DMSO and 1.48 mL of dry THF.
The mixture was stirred at 0 °C for 30 min and then added to a solution of 1-chloro-3-iodopropane (33)
(0.48 mL, 4.52 mmol, 1.05 equiv.) under nitrogen in 1.48 mL of DMSO. The reaction mixture was
stirred at room temperature for 5 h. The reaction mixture was then poured into ice water and was
136 extracted with DCM (3x). The combined extracts were washed with water, dried over Na2SO4, filtered
137 and concentrated under reduced pressure to a pink oil. The crude was purified by flash
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chromatography (eluent Hexane/DCM = 97/3 to 8/2), yielding 615 mg (58 %) of 2-chloro-10-(3-
chloropropyl)-10H-phenothiazine (40) as a colorless oil. 119 mg of the starting material 39 were also
isolated in a second fraction (brsm yield 66 %).
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Rf (Hexane/DCM = 9/1) = 0.33; H-NMR (400 MHz, CDCl3) δ (ppm) = 7.17 (2 H, m, H-2 and
H-3), 7.05 (1 H, d, J = 9.0 Hz, H-5), 6.92 (4 H, m, H-4, H-8, H-9 and H-11), 4.06 (2 H, t, J = 6.9 Hz,
H-13), 3.67 (2 H, t, J = 6.0 Hz, H-15), 2.24 (2 H, m, H-14). The analytical data matched the published
144 data (Harrold et al., J. Med. Chem. 1987, 30, 1631-1635).
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Compound 41 was obtained using the same conditions as for 14, employing N1-(7-chloroquinolin-4-
yl)-N2-ethylethane-1,2-diamine (21a) (209 mg, 0.84 mmol, 1.0 equiv.), (9H-fluoren-9-yl)methyl (3-
147 oxopropyl)carbamate (13) (297 mg, 1.01 mmol, 1.2 equiv.), dry DCM (21 mL) and NaBH(OAc)3
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150
(356 mg, 1.68 mmol, 2.0 equiv.). The reaction mixture was stirred for 5 h and the crude compound was
purified by flash chromatography (eluent DCM/MeOH = 95/5 to 9/1), yielding 363 mg (82 %) of
desired Fmoc-protected amine as a white foam.
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152
153
154
155
Rf (DCM/MeOH = 9/1) = 0.35; H NMR (400 MHz, CDCl3) δ (ppm) = 8.49 (1 H, d, J = 5.3 Hz,
H-3), 7.95 (1 H, s, H-8), 7.72 (3 H, m, H-5 and H-Fluorene), 7.54 (2 H, m, H-Fluorene), 7.32 (5 H, m,
H-Fluorene and H-Ar), 6.34 (1 H, d, J = 5.3 Hz, H-2), 5.98 (1 H, bs, NH), 5.20 (1 H, bs, NH), 4.39
(2 H, d, J = 6.7 Hz, H-18), 4.16 (1 H, t, J = 6.7 Hz, H-19), 3.32 (4 H, m, H-10 and H-16), 2.79 (2 H,
m, H-12), 2.56 (4 H, m, H-11 and H-14), 1.69 (2 H, m, H-15), 1.04 (3 H, t, J = 6.9 Hz, H-13); MS
156 (ESI) m/z = 529.2 [M+H]+; HRMS (ESI) m/z = 529.2364 [M+H]+, calc.: 529.2365, Diff.: 0.2 ppm.
157
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To a stirred solution of the Fmoc-protected amine (880 mg, 1.66 mmol, 1.0 equiv.) in dry DCM
(33 mL), under nitrogen and at room temperature, was then added piperidine (0.82 mL, 8.30 mmol,
5.0 equiv.). The reaction mixture was stirred for 36 h and the solvent was removed. The crude residue
was purified by flash chromatography (eluent DCM/MeOH = 9/1 to 8/2 + 0.1 % TEA), yielding
437 mg (86 %) of the desired free amine 41 as a colourless oil.
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Rf (DCM/MeOH = 9/1) = 0.35; H NMR (400 MHz, CDCl3) δ (ppm) = 8.50 (1 H, d, J = 5.3 Hz,
H-3), 7.93 (1 H, d, J = 2.1 Hz, H-8), 7.70 (3 H, d, J = 8.8 Hz, H-5), 7.33 (1 H, dd, J = 8.8 Hz,
J = 2.2 Hz, H-7), 6.35 (1 H, d, J = 5.3 Hz, H-2), 6.14 (1 H, bs, NH), 3.22 (2 H, m, H-10), 2.78 (4 H, m,
165 H-12 and H-16), 2.59 (4 H, m, H-11 and H-14), 2.26 (2 H, bs, NH2), 1.63 (2 H, m, H-15), 1.06 (3 H, t,
166
167
J = 7.2 Hz, H-13); 13C NMR (400 MHz, CDCl3) δ (ppm) = 152.2 (C-3), 150.1 (C-1), 149.2 (C-4),
134.9 (C-6), 128.8 (C-5), 125.4 (C-8), 121.4 (C-7), 117.6 (C-9), 99.4 (C-2), 51.5 (C-11), 51.1 (C-14),
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168
169 HRMS (ESI) m/z = 307.1688 [M+H]+, calc.: 307.1684, Diff.: 1.4 ppm.
47.2 (C-12), 40.7 (C-10), 40.1 (C-16), 31.0 (C-15), 12.0 (C-13); MS (ESI) m/z = 307.2 [M+H]+;
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A mixture of free amine 41 (64 mg, 0.21 mmol, 1.0 equiv.), 2-chloro-10-(3-chloropropyl)-10H-
phenothiazine (40) (65 mg, 0.21mmol, 1.0 equiv.) and K2CO3 (29 mg, 0.21 mmol, 1.0 equiv.) in
4.2 mL MeOH, under nitrogen, was heated and stirred at reflux for 4 d. After cooling, the solvent was
removed. The crude residue was purified by flash chromatography (eluent DCM/MeOH = 9/1 to 8/2 +
0.1 % TEA), yielding 53 mg of the desired compound with major impurities. This was further purified
by HPLC, yielding 14 mg (12 %) of N1-(3-(2-chloro-10H-phenothiazin-10-yl)propyl)-N3-(2-((7-
176 chloroquinolin-4-yl)amino)ethyl)-N3-ethylpropane-1,3-diamine (42) as a yellowish oil.
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Rf (DCM/MeOH = 8/2) = 0.14; H NMR (400 MHz, CDCl3) δ (ppm) = 8.53 (1 H, d, J = 5.1 Hz,
H-3), 7.95 (1 H, d, J = 2.1 Hz, H-8), 7.68 (3 H, d, J = 8.8 Hz, H-5), 7.33 (1 H, dd, J = 8.8 Hz,
J = 2.1 Hz, H-7), 7.11 (2 H, m, H-21 and H-22), 7.00 (1 H, d, J = 7.9 Hz, H-24), 6.87 (4 H, m, H-23,
H-27, H-28 and H-30), 6.36 (1 H, d, J = 5.1 Hz, H-2), 6.11 (1 H, bs, NH), 3.82 (2 H, t, J = 6.8 Hz,
181 H-19), 3.24 (2 H, m, H-10), 2.77 (2 H, t, J = 5.2 Hz, H-14), 2.55 (8 H, m, H-11, H-12, H-16 and H-17),
182 1.85 (2 H, m, H-18), 1.62 (2 H, m, H-15), 1.06 (3 H, t, J = 6.8 Hz, H-13); 13C NMR (400 MHz,
183
184
CDCl3) δ (ppm) = 152.3 (C-3), 150.0 (C-1), 149.3 (C-4), 146.7 (C-31), 144.7 (C-20), 134.9 (C-6),
144.7 (C-29), 128.9 (C-5), 128.1 (C-22), 127.7 (C-24), 127.6 (C-27), 125.4 (C-8), 125.2 (C-25), 123.9
185 (C-26), 123.1 (C-23), 122.5 (C-28), 121.5 (C-7), 117.6 (C-9), 116.0 (C-21), 115.9 (C-30), 99.5 (C-2),
186
187
188
51.5 (C-11), 51.3 (C-14), 48.6 (C-12), 47.5 (C-19), 47.3 (C-10), 45.5 (C-16), 40.1 (C-17), 27.7 (C-15),
27.1 (C-18), 12.1 (C-13); MS (ESI) m/z = 580.1 [M+H]+; HRMS (ESI) m/z = 580.2059 [M+H]+,
calc.: 580.2063, Diff.: 0.8 ppm.
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Method A for the synthesis of hybrid compounds
190 tert-butyl 3-((2-((7-chloroquinolin-4-yl)amino)ethyl)amino)piperidine-1-carboxylate (27a)
191
192
193
194
195
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Under inert conditions, tert-butyl piperidin-4-ylcarbamate (199 mg, 0.99 mmol, 1.0 equiv.) and
2-((7-chloroquinolin-4-yl)amino)ethyl methanesulfonate (22a) (298 mg, 0.99 mmol, 1.0 equiv.) were
suspended in 4.97 mL THF in a microwave tube, then TEA (0.28 mL, 1.98 mmol, 2.0 equiv.) was
added. The tube was sealed and the reaction mixture was heated in a microwave reactor at 120 °C for
4 h. The reaction mixture was poured into saturated K2CO3 (aq.) and extracted with DCM (3x). The
united organic phase was concentrated under reduced pressure and the crude residue was purified by
flash chromatography (eluent DCM/MeOH = 99/1 to 9/1), yielding 136 mg (34 %) of tert-butyl 3-((2-
((7-chloroquinolin-4-yl)amino)ethyl)amino)piperidine-1-carboxylate (27a) as a yellow oil.
1
199
200
Rf (DCM/MeOH = 9/1) = 0.48; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.49 (1 H, d, J = 5.2 Hz,
H-3), 7.93 (1 H, d, J = 1.8 Hz, H-8), 7.72 (1 H, d, J = 8.8 Hz, H-5), 7.34 (1 H, dd, J = 8.8 Hz,
201 J = 1.8 Hz, H-7), 6.36 (1 H, d, J = 5.2 Hz, H-2), 6.01 (1 H, bs, NH), 3.90 (1 H, m, H-13b), 3.66 (1 H,
202
203
204
205
206
207
m, H-13b), 3.32 (2 H, m, H-10), 3.07 (3 H, m, H-11 and NH), 2.89 (1 H, m, NH), 2.64 (1 H, m, H-12),
2.15 (2 H, m, H-14), 1.92 (1 H, m, H-15a), 1.69 (1 H, m, H-15b), 1.46 (11 H, bs, H-19 and H-16); 13
C
NMR (400 MHz, CDCl3) δ (ppm) = 155.1 (C-17), 151.8 (C-3), 150.2 (C-1), 148.8 (C-4), 135.1 (C-6),
128.5 (C-5), 125.5 (C-8), 121.6 (C-7), 117.4 (C-9), 99.3 (C-2), 79.8 (C-17), 53.4 (C-12), 48.9 (C-13),
45.0 (C-10), 44.9 (C-14), 44.2 (C-11), 31.6 (C-15), 28.4 (C-19), 23.5 (C-16); MS (ESI) m/z = 405.1
[M+H]+; HRMS (ESI) m/z = 405.2067 [M+H]+, calc.: 405.2052, Diff.: 3.7 ppm.
208 tert-butyl 3-((3-((7-chloroquinolin-4-yl)amino)propyl)amino)piperidine-1-carboxylate (27b)
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Compound 27b was obtained by Method A, using the same conditions as for 27a: employing tert-
butyl piperidin-4-ylcarbamate (7 in Part I) (199 mg, 0.99 mmol, 1.05 equiv.), 3-((7-chloroquinolin-4-
yl)amino)propyl methanesulfonate (22b) (298 mg, 0.94 mmol, 1.0 equiv.) and TEA (0.28 mL,
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212
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1.98 mmol, 2.1 equiv.) in 4.95 mL THF. The reaction mixture was heated in a microwave reactor at
120 °C for 5 h and the crude residue was purified by flash chromatography (eluent
214 DCM/MeOH = 95/5 to 8/2), yielding a 136 mg fraction of the desired compound 27b together with
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216
217
some unreacted starting material 7 (yield 27b: 28 %, based on 86 w%, calculated with the H-NMR).
This was further purified by HPLC, yielding 40 mg of tert-butyl 3-((3-((7-chloroquinolin-4-
yl)amino)propyl)amino)piperidine-1-carboxylate (27b) as a yellowish oil.
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219
220
Rf (DCM/MeOH = 9/1) = 0.48; H-NMR (400 MHz, MeOH-D4) δ (ppm) = 8.43 (2 H, m, H-3 and
H-8), 7.90 (1 H, m, H-5), 7.69 (1 H, m, H-7), 6.93 (1 H, m, H-2), 4.09 (1 H, m, H-14a), 3.73 (3 H, m,
H-14b and H-15), 3.58 (1 H, m, NH), 3.42-2.95 (5 H, m, H-10, H-12 and NH), 2.22 (4 H, m, H-13,
221 H-11 and H-16a), 1.69 (3 H, m, H-16b and H-17), 1.46 (9 H, bs, H-20); 13C NMR (400 MHz, MeOH-
222 D4) δ (ppm) = 163.0 (C-18), 157.8 (C-1), 144.0 (C-3), 141.1 (C-4), 140.1 (C-6), 128.8 (C-5), 126.1
223 (C-8), 120.3 (C-7), 117.1 (C-9), 99.8 (C-2), 81.9 (C-19), 53.1 (C-13), 44.9 (C-14), 44.7 (C-10), 44.1
224
225
(C-15), 43.9 (C-12), 41.7 (C-17), 28.5 (C-20), 26.0 (C-11), 21.5 (C-16); MS (ESI) m/z = 419.2
[M+H]+; HRMS (ESI) m/z = 419.2218 [M+H]+, calc.: 419.2208, Diff.: 2.4 ppm.
226
227
228
229
230
231
232
233
234
tert-butyl 3-((3-((2-((7-chloroquinolin-4-yl)amino)ethyl)amino)propyl) (2-iodobenzyl)amino)-
piperidine-1-carboxylate (28)
Compound 28 was obtained by Method A, using the same conditions as for 27a: employing crude
tert-butyl 3-((3-aminopropyl)(2-iodobenzyl)amino)piperidine-1-carboxylate (15) (79 mg, 0.11 mmol,
1.0 equiv.), 3-((7-chloroquinolin-4-yl)amino)ethyl methanesulfonate (22a) (33 mg, 0.11 mmol,
1.0 equiv.) and TEA (30 μL, 0.22 mmol, 2.0 equiv.) in 2.20 mL THF. The reaction mixture was heated
in a microwave reactor at 120 °C for 5 h and the crude residue was purified by HPLC, yielding 15 mg
(21 % over 2 steps) of tert-butyl 3-((3-((2-((7-chloroquinolin-4-yl)amino)ethyl)amino)propyl) (2-
iodobenzyl)amino)-piperidine-1-carboxylate (28) as a yellowish oil.
11

235
236
237
238
239
1H NMR (400 MHz, MeOH-D4) δ (ppm) = 8.49 (1 H, d, J = 6.9 Hz, H-3), 8.43 (1 H, d, J = 9.0 Hz,
H-8), 7.93 (2 H, m, H-26 and H-5), 7.71 (1 H, m, H-7), 7.61 (1 H, m, H-28), 7.46 (1 H, m, H-27), 7.13
(1 H, m, H-29), 6.99 (1 H, d, J = 6.9 Hz, H-12), 4.31 (3 H, m, H-16 and H-17a), 3.99 (3 H, m, H-23
and H-17b), 3.44 (2 H, m, H-10), 3.14 (4 H, m, H-11 and H-12), 2.24 (1 H, m, H-15), 2.11 (2 H, m,
H-19), 1.83 (2 H, m, H-18), 1.42 (11 H, bs, H-22 and H-13); MS (ESI) m/z = 678.1 [M+H]+; HRMS
240 (ESI) m/z = 678.2093 [M+H]+, calc.: 678.2066, Diff.: 3.9 ppm.
241
242
243
244
N1-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N3-(2,2-diphenylethyl)-N3-(2-iodobenzyl)propane-1,3-
diamine (29)
Compound 29 was obtained by Method A, using the same conditions as for 27a: employing
N1-(2,2-diphenylethyl)-N1-(2-iodobenzyl)propane-1,3-diamine (17) (48 mg, 0.11 mmol, 1.0 equiv.), 3-
245 ((7-chloroquinolin-4-yl)amino)ethyl methanesulfonate (22a) (50 mg, 0.16 mmol, 1.5 equiv.) and TEA
246
247
(30 μL, 0.22 mmol, 2.0 equiv.) in 2.10 mL THF. The reaction mixture was heated in a microwave
reactor at 120 °C for 10 h and the crude residue was purified by HPLC, yielding 24 mg (34 %) of N1-
248 (2-((7-chloroquinolin-4-yl)amino)ethyl)-N3-(2,2-diphenylethyl)-N3-(2-iodobenzyl)-propane-1,3-
249 diamine (29) as a yellowish oil.
250
251
1H-NMR (400 MHz, CDCl3) δ (ppm) = 8.53 (1 H, d, J = 5.4 Hz, H-3), 7.94 (1 H, d, J = 2.1 Hz,
H-8), 7.71 (1 H, dd, J = 7.9 Hz, J = 1.0 Hz,H-26), 7.58 (1 H, d, J = 8.9 Hz, H-5), 7.34 (1 H, dd,
252 J = 8.9 Hz, J = 2.1 Hz, H-7), 7.24 (4 H, m, H-Ar), 7.17 (7 H, m, H-Ar), 6.98 (2 H, m, H-Ar), 6.34
253
254
255
256
257
(1 H, d, J = 5.4 Hz, H-2), 5.79 (1 H, bs, NH), 4.16 (1 H, m, H-16), 3.64 (2 H, s, H-23), 3.15 (4 H, m,
H-15 and H-10), 2.83 (2 H, t, J = 5.7 Hz, H-11), 2.62 (2 H, t, J = 6.5 Hz, H-14), 2.50 (2 H, t,
J = 6.5 Hz, H-12), 1.63 (3 H, m, H-13 and NH); 13C NMR (400 MHz, CDCl3) δ (ppm) = 151.2 (C-3),
150.0 (C-1), 149.3 (C-4), 143.6 (C-17), 141.5 (C-24), 139.3 (C-26), 134.9 (C-6), 130.5 (C-28), 128.9
(C-5), 128.7 (C-27), 128.5 (C-19), 128.4 (C-18), 128.0 (C-8), 126.5 (C-20), 125.4 (C-29), 121.4 (C-7),
12

258
259
117.5 (C-9), 100.3 (C-25), 99.3 (C-2), 63.7 (C-14), 60.3 (C-15), 52.8 (C-23), 49.8 (C-16), 47.9 (C-10),
47.5 (C-11), 41.9 (C-15), 27.4 (C-13); MS (ESI) m/z = 675.3 [M+H]+; HRMS (ESI) m/z = 675.1760
260 [M+H]+, calc.: 675.1746, Diff.: 2.2 ppm.
261 N1-(7-chloroquinolin-4-yl)-N2-(2,2-diphenylethyl)ethane-1,2-diamine (30)
262
263
264
Compound 30 was obtained by Method A, using the same conditions as for 27a: employing
2,2-diphenylethan-1-amine (9 in Part I) (145 mg, 0.48 mmol, 1.0 equiv.), 3-((7-chloroquinolin-4-
yl)amino)ethyl methanesulfonate (22a) (95 mg, 0.48 mmol, 1.0 equiv.) and TEA (0.13 mL,
265 0.96 mmol, 2.0 equiv.) in 4.80 mL THF. The reaction mixture was heated in a microwave reactor at
266
267
268
120 °C for 7 h and the crude residue was purified by flash chromatography (eluent
DCM/MeOH = 95/5 to 8/2), yielding a 80 mg (42 %) of N1-(7-chloroquinolin-4-yl)-N2-(2,2-
diphenylethyl)ethane-1,2-diamine (30) as a yellowish oil.
1
269
270
271
Rf (DCM/MeOH = 9/1) = 0.44; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.49 (1 H, d, J = 5.1 Hz,
H-3), 7.93 (1 H, d, J = 1.7 Hz, H-8), 7.24 (10 H, m, H-Ar), 6.31 (1 H, d, J = 5.1 Hz, H-2), 5.76 (1 H,
bs, NH), 4.17 (1 H, t, J = 8.1 Hz, H-16), 3.32 (2 H, d, J = 8.1 Hz, H-12), 3.26 (2 H, m, H-10), 3.05
272 (2 H, m, H-11), 1.58 (1 H, bs, NH); 13C NMR (400 MHz, CDCl3) δ (ppm) = 151.8 (C-3), 150.1 (C-1),
273
274
275
148.8 (C-4), 142.8 (C-14), 135.1 (C-6), 128.8 (C-16), 128.4 (C-5), 128.1 (C-15), 126.8 (C-17), 125.4
(C-8), 117.4 (C-9), 99.2 (C-2), 53.8 (C-12), 51.5 (C-13), 47.3 (C-10), 47.8 (C-11); MS (ESI)
m/z = 402.2 [M+H]+; HRMS (ESI) m/z = 402.1746 [M+H]+, calc.: 402.1732, Diff.: 3.6 ppm.
276
277
278
279
280
7-chloro-N-(2-(4-(10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazin-1-yl)ethyl)quinolin-4-amine
(31)
Compound 31 was obtained by Method A, using the same conditions as for 27a: employing
1-(10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazine (56 mg, 0.19 mmol, 1.0 equiv.), 2-((7-
chloroquinolin-4-yl)amino)ethyl methanesulfonate (22a) (57 mg, 0.19 mmol, 1.0 equiv.) and TEA
13

281
282
283
284
(53 μL, 0.38 mmol, 2.0 equiv.) in 1.90 mL THF; the mixture was heated in a microwave reactor at
120 °C for 7 h and the crude residue was purified by flash chromatography (eluent
DCM/MeOH = 95/5 to 9/1). Thus were obtained 21 mg of the hybrid compound 31 as a fluffy yellow
solid as well as 26 mg of a second impure fraction (31/impurity ~ 4/1, overall yield ~44 %).
1
285
286
287
288
289
290
291
292
293
Rf (DCM/MeOH = 9/1) = 0.17; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.53 (1 H, d, J = 5.3 Hz,
H-3), 7.96 (1 H, d, J = 2.1 Hz, H-8), 7.72 (1 H, d, J = 8.5 Hz, H-5), 7.62 (1 H, dd, J = 7.9 Hz,
J = 1.4 Hz, H-23), 7.52 (1 H, dd, J = 7.7 Hz, J = 1.2 Hz, H-7), 7.40 (2 H, m, H-17 and H-18), 7.22
(3 H, m, H-25, H-24 and H-22), 7.10 (2 H, m, H-16 and H-19), 6.37 (1 H, d, J = 5.3 Hz, H-2), 5.98
(1 H, bs, NH), 4.05 (1 H, m, H-14), 3.91 (1 H, m, H-27a), 3.30 (2 H, m, H-10), 3.21 (1 H, m, H-27b),
2.73 (6 H, m, H-13 and H-11), 2.54 (4 H, m, H-12); 13C NMR (400 MHz, CDCl3) δ (ppm) = 152.2
(C-3), 150.0 (C-1), 149.2 (C-4), 142.3 (C-20), 140.3 (C-21), 137.1 (C-26), 135.4 (C-6), 137.1 (C-15),
132.6 (C-23), 131.6 (C-18), 131.4 (C-25), 129.7 (C-24), 128.9 (C-16), 128.6 (C-17), 127.2 (C-5),
126.9 (C-19), 126.5 (C-22), 125.5 (C-8), 121.3 (C-7), 117.5 (C-9), 99.4 (C-2), 65.9 (C-14), 55.7
294 (C-11), 53.4 (C-12), 50.0 (C-13), 39.1 (C-10), 33.6 (C-27); MS (ESI) m/z = 501.1 [M+H]+; HRMS
295
(ESI) m/z = 501.1885 [M+H]+, calc.: 501.1874, Diff.: 2.1 ppm.
296
297
298
299
300
301
302
7-chloro-N-(2-(4-(8-chloro-10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazin-1-yl)ethyl)quinolin-4-
amine (32)
Compound 32 was obtained by Method A, using the same conditions as for 27a: employing
1-(8-chloro-10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazine (36 mg, 0.12 mmol, 1.0 equiv.), 2-((7-
chloroquinolin-4-yl)amino)ethyl methanesulfonate (22a) (36 mg, 0.12 mmol, 1.0 equiv.) and TEA
(33 μL, 0.24 mmol, 2.0 equiv.) in 2.40 mL THF; the mixture was heated in a microwave reactor at
120 °C for 7 h and the crude residue was purified by flash chromatography (eluent
303 DCM/MeOH = 95/5 to 9/1), yielding a 37 mg (58 %). of hybrid compound 32 as a colourless oil.
14

1
304
305
306
307
308
Rf (DCM/MeOH = 9/1) = 0.55; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.53 (1 H, d, J = 5.4 Hz,
H-3), 7.97 (1 H, d, J = 2.1 Hz, H-8), 7.69 (2 H, m, H-22 and H-5), 7.51 (1 H, d, J = 7.3 Hz, H-23), 7.52
(1 H, dd, J = 8.9 Hz, J = 2.1 Hz, H-7), 7.35 (1 H, d, J = 7.9 Hz, H-18), 7.27 (2 H, m, H-25 and H-24),
7.09 (2 H, m, H-16 and H-19), 6.38 (1 H, d, J = 5.3 Hz, H-2), 5.99 (1 H, bs, NH), 3.92 (2 H, m, H-14
and H-27a), 3.32 (2 H, m, H-10), 3.18 (1 H, m, H-27b), 2.77 (6 H, m, H-13 and H-11), 2.77 (4 H, m,
309 H-12); 13C NMR (400 MHz, CDCl3) δ (ppm) = 152.1 (C-3), 150.0 (C-1), 149.1 (C-4), 142.3 (C-20),
310
311
142.2 (C-21), 136.4 (C-26), 135.1 (C-6), 133.9 (C-15), 133.2 (C-17), 132.9 (C-23), 132.2 (C-18),
131.5 (C-25), 129.7 (C-24), 129.0 (C-16), 128.8 (C-5), 127.0 (C-19), 126.7 (C-22), 125.5 (C-8), 121.3
312 (C-7), 117.5 (C-9), 99.4 (C-2), 65.6 (C-14), 55.6 (C-11), 53.3 (C-12), 48.9 (C-13), 39.2 (C-10), 32.8
313
314
(C-27); MS (ESI) m/z = 535.1 [M+H]+; HRMS (ESI) m/z = 535.1484 [M+H]+, calc.: 535.1484, Diff.:
0.2 ppm.
315
Method B for the synthesis of hybrid compounds
316
317
318
319
320
N1-(3-chloropropyl)-N2-(7-chloroquinolin-4-yl)-N1-ethylethane-1,2-diamine (34)
To a solution of N1-(7-chloroquinolin-4-yl)-N2-ethylethane-1,2-diamine (21a) (580 mg, 2.32 mmol,
1.0 equiv.) and K2CO3 (962 mg, 6.96 mmol, 3.0 equiv.) in dry MeCN (42 mL), under inert conditions,
was added 1-chloro-3-iodopropane (33) (0.50 mL, 4.64 mmol, 2.0 equiv.). After stirring at room
temperature for 4 d, the reaction was quenched with water and extracted with DCM (3x). The
321 combined organic phase was dried over Na2SO4, filtered and the solvent was removed under reduced
322
323
pressure. The crude residue was purified by flash chromatography (eluent DCM/MeOH = 97/3 to 9/1),
yielding 383 mg (51 %) of N1-(3-chloropropyl)-N2-(7-chloroquinolin-4-yl)-N1-ethylethane-1,2-
324 diamine (34) as an off-white solid.
1
325
326
Rf (DCM/MeOH = 9/1) = 0.38; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.53 (1 H, d, J = 5.3 Hz,
H-3), 7.96 (1 H, d, J = 2.1 Hz, H-8), 7.65 (1 H, d, J = 9.0 Hz, H-5), 7.38 (1 H, dd, J = 9.0 Hz,
15

327
328
J = 2.1 Hz, H-7), 6.38 (1 H, d, J = 5.3 Hz, H-2), 5.90 (1 H, bs, NH), 3.61 (2 H, t, J = 6.2 Hz, H-10),
3.29 (2 H, q, J = 5.4 Hz, H-12), 2.84 (2 H, t, J = 5.7 Hz, H-11), 2.70 (2 H, t, J = 7.0 Hz, H-12), 2.61
329 (2 H, q, J = 7.0 Hz, H-16), 1.95 (2 H, m, H-15), 1.08 (3 H, t, J = 7.1 Hz, H-13); 13C NMR (400 MHz,
330 CDCl3) δ (ppm) = 152.2 (C-3), 150.0 (C-1), 149.2 (C-4), 135.0 (C-6), 128.9 (C-5), 125.5 (C-8), 121.1
331 (C-9), 117.5 (C-7), 99.5 (C-2), 51.6 (C-11), 50.0 (C-14), 46.9 (C-12), 43.0 (C-10), 40.0 (C-16), 30.2
332
(C-15), 11.9 (C-13); MS (ESI) m/z = 325.9 [M+H]+.
333
334
335
336
N1-benzyl-N1-((2R,3R)-2-benzylquinuclidin-3-yl)-N3-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N3-
ethylpropane-1,3-diamine (35)
Method B: Under inert conditions, N1-(3-chloropropyl)-N2-(7-chloroquinolin-4-yl)-N1-ethylethane-
1,2-diamine (34) (45 mg, 0.14 mmol, 1.0 equiv.) and 4 (42 mg, 0.14 mmol, 1.0 equiv.) were suspended
337 in 1.40 mL MeCN in a microwave tube, then K2CO3 (39 mg, 0.28 mmol, 2.0 equiv.) and NaI (4 mg,
338
339
340
0.03 mmol, 0.2 equiv.) were added. The tube was sealed and the reaction mixture was heated in a
microwave reactor at 100 °C for 90 min. The solvent was removed under reduced pressure and the
crude residue was purified by flash chromatography (eluent DCM/MeOH = 95/5 to 85/15 + 0.1 %
341 TEA), yielding 35 mg of a fraction with major TEA impurities. This was dissolved in DCM and stirred
342
343
with Amberlyst 21 for 1 H, filtered and the solvent was removed under reduced pressure. Thus were
obtained 19 mg (22 %) of hybrid compound 35 as a colourless oil (with minor impurities).
1
344
345
346
347
348
349
Rf (DCM/MeOH = 9/1) = 0.11; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.52 (1 H, d, J = 5.2 Hz,
H-3), 7.94 (1 H, d, J = 2.1 Hz, H-8), 7.85 (1 H, d, J = 8.8 Hz, H-5), 7.37 (1 H, dd, J = 8.8 Hz,
J = 2.1 Hz, H-7), 7.25 (8 H, m, H-Ph), 7.00 (2 H, m, H-Ph), 6.37 (1 H, d, J = 5.2 Hz, H-2), 5.91 (1 H,
bs, NH), 3.86 (2 H, t, J = 5.4 Hz, H-11), 3.73 (1 H, d, J = 13.0 Hz, H-27a), 3.43 (4 H, m, H-17, H27b
and H-16), 3.22 (2H, m, H-10), 3.01 (3 H, m, H-22a and H-21a), 2.84 (4 H, m, H-18, H-22b and
H-21b), 2.74 (2 H, t, J = 6.0 Hz, H-14), 2.62 (2 H, q, J = 7.1 Hz, H-12), 1.74 (3 H, m, H-15 and H-19),
16

350 1.56 (2 H, m, H-20a), 1.40 (2 H, m, H-20b), 1.07 (3 H, t, J = 7.1 Hz, H-13); 13C NMR (400 MHz,
351
352
353
354
355
356
CDCl3) δ (ppm) = 151.9 (C-3), 149.8 (C-1), 149.1 (C-4), 140.0 (C-28), 139.2 (C-23), 134.9 (C-6),
128.7 (C-24), 128.6 (C-25), 128.3 (C-30), 127.8 (C-29), 126.9 (C-31), 126.2 (C-26), 125.4 (C-7),
125.4 (C-8), 121.6 (C-5), 117.5 (C-9), 98.9 (C-2), 63.9 (C-22), 60.2 (C-17), 54.8 (C-18), 53.4 (C-11),
52.1 (C-14), 51.9 (C-16), 48.9 (C-12), 47.9 (C-10), 41.9 (C-21), 40.6 (C-21), 33.3 (C-22), 28.3 (C-20),
24.6 (C-20), 24.5 (C-19), 19.2 (C-15), 11.7 (C-13); MS (ESI) m/z = 596.4 [M+H]+; HRMS (ESI)
m/z = 596.3527 [M+H] +, calc.: 596.3515, Diff.: 2.1 ppm.
357
358
359
360
361
362
363
364
365
tert-butyl 3-((3-((2-((7-chloroquinolin-4-yl)amino)ethyl)(ethyl)amino)propyl)amino)piperidine-1-
carboxylate (36)
Compound 36 was obtained by Method B, using the same conditions as for 35: employing N1-(3-
chloropropyl)-N2-(7-chloroquinolin-4-yl)-N1-ethylethane-1,2-diamine (34) (102 mg, 0.31 mmol,
1.0 equiv.), tert-butyl piperidin-4-ylcarbamate (63 mg, 0.31 mmol, 1.0 equiv.), K2CO3 (86 mg,
0.63 mmol, 2.0 equiv.) and NaI (9 mg, 0.06 mmol, 0.2 equiv.) in 3.13 mL MeCN. The mixture was
heated in a microwave reactor at 100 °C for 90 min. After flash chromatography (eluent
DCM/MeOH = 95/5 to 8/2 + 0.1 % TEA) and treatment with Amberlyst 21 were thus obtained 81 mg
(44 %) of the hybrid compound 36 as a fluffy yellowish solid with some minor impurities.
1
366
367
368
369
370
Rf (DCM/MeOH = 9/1) = 0.36; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.52 (1 H, d, J = 5.3 Hz,
H-3), 7.94 (1 H, d, J = 2.1 Hz, H-8), 7.70 (1 H, d, J = 8.8 Hz, H-5), 7.36 (1 H, dd, J = 8.8 Hz,
J = 2.1 Hz, H-7), 6.36 (1 H, d, J = 5.3 Hz, H-2), 6.08 (1 H, bs, NH), 3.88 (3 H, m, H-18 and NH), 3.26
(2 H, m, H-10), 2.64 (11 H, m, H-21, H-17, H-11, H-12, H-14 and H-16), 1.65 (6 H, m, H-19, H-20
and H-15), 1.43 (9 H, s, H-24), 1.07 (3 H, t, J = 13.8 Hz, H-13); 13C NMR (400 MHz, CDCl3) δ
371 (ppm) = 154.0 (C-22), 151.6 (C-3), 150.5 (C-1), 148.7 (C-4), 135.3 (C-6), 128.2 (C-7), 125.6 (C-8),
372 122.2 (C-5), 117.6 (C-9), 99.2 (C-2), 80.0 (C-23), 54.0 (C-17), 52.3 (C-11), 51.3 (C-18), 47.8 (C-21),
17

373
47.5 (C-14), 46.1 (C-10), 46.0 (C-16), 40.3 (C-12), 28.6 (C-15), 28.6 (C-24), 26.4 (C-19), 23.5 (C-20),
374 9.3 (C-13); MS (ESI) m/z = 490.2 [M+H]+; HRMS (ESI) m/z = 490.2939 [M+H] +, calc.: 490.2943,
375
Diff.: 0.9 ppm.
376
377
378
379
380
381
382
383
384
N1-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N3-(2,2-diphenylethyl)-N1-ethylpropane-1,3-diamine
(37)
Under inert conditions, N1-(3-chloropropyl)-N2-(7-chloroquinolin-4-yl)-N1-ethylethane-1,2-diamine
(34) (58 mg, 0.18 mmol, 1.0 equiv.) and 2,2-diphenylethylamine (9) (35 mg, 0.18 mmol, 1.0 equiv.)
were suspended in 1.80 mL MeCN in a microwave tube, then K2CO3 (50 mg, 0.36 mmol, 2.0 equiv.)
was added. The tube was sealed and the reaction mixture was heated in a microwave reactor at 100 °C
for 90 min. The solvent was removed under reduced pressure and the crude residue was purified by
flash chromatography (solid deposit, eluent DCM/MeOH = 95/5 to 85/15), yielding 40 mg (46 %) of
N1-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N3-(2,2-diphenylethyl)-N1-ethylpropane-1,3-diamine (37)
385 as a colourless oil. Two additional fractions were also obtained: 15 mg (26 %) of chloride 34 as well as
386 24 mg of a mixture of 34 and 2,2-diphenylethylamine (9).
1
387
388
389
Rf (DCM/MeOH = 9/1) = 0.28; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.53 (1 H, d, J = 5.7 Hz,
H-3), 7.95 (1 H, d, J = 2.1 Hz, H-8), 7.76 (1 H, d, J = 8.8 Hz, H-5), 7.32 (1 H, dd, J = 8.8 Hz,
J = 2.1 Hz, H-7), 7.26 (4 H, m, H-Ph), 7.16 (6 H, m, H-Ph), 6.35 (1 H, d, J = 5.7 Hz, H-2), 6.25 (1 H,
390 bs, NH), 4.10 (1 H, t, J = 7.7 Hz, H-18), 3.18 (4 H, m, H-14 and H-10), 2.74 (4 H, m, H-17 and H-12),
391
392
393
2.48 (4 H, m, H-16 and H-11), 2.03 (1 H, bs, NH), 1.66 (2 H, m, H-15), 0.96 (3 H, t, J = 7.1 Hz, H-13);
+
MS (ESI) m/z = 487.2 [M+H]+; HRMS (ESI) m/z = 487.2644 [M+H] , calc.: 487.2623, Diff.:
4.2 ppm.
18

394
N1-(3-(4-(8-chloro-10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazin-1-yl)propyl)-N2-(7-
395 chloroquinolin-4-yl)-N1-ethylethane-1,2-diamine (38a)
396
397
398
399
400
401
402
Compound 38a was obtained by Method B, using the same conditions as for 35: employing N1-(3-
chloropropyl)-N2-(7-chloroquinolin-4-yl)-N1-ethylethane-1,2-diamine (34) (32 mg, 0.10 mmol,
1.0 equiv.), 1-(10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazine (30 mg, 0.10 mmol, 1.0 equiv.),
K2CO3 (27 mg, 0.20 mmol, 2.0 equiv.) and NaI (3 mg, 0.02 mmol, 0.2 equiv.) in 1.00 mL MeCN. The
mixture was heated in a microwave reactor at 100 °C for 3 h. After flash chromatography (eluent
DCM/MeOH = 95/5 to 8/2 + 0.1 % TEA) were thus obtained 36 mg (62 %) of the hybrid compound
38a as a fluffy yellowish solid with some TEA impurities.
1
403
404
405
406
407
408
Rf (DCM/MeOH = 8/2) = 0.69; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.52 (1 H, d, J = 5.4 Hz,
H-3), 7.94 (1 H, d, J = 2.1 Hz, H-8), 7.67 (1 H, d, J = 8.8 Hz, H-5), 7.58 (1 H, dd, J = 7.6 Hz,
J = 0.9 Hz, H-28), 7.58 (1 H, dd, J = 7.6 Hz, J = 1.1 Hz, H-23), 7.37 (2 H, m, H-7 and H-30), 7.22
(2 H, m, H-29 and H-27), 7.09 (3 H, m, H-21, H-22 and H-24), 6.37 (1 H, d, J = 5.4 Hz, H-2), 6.06
(1 H, bs, NH), 3.94 (1 H, m, H-19), 3.84 (1 H, m, H-32a), 3.23 (2 H, m, H-10), 3.13 (1 H, m, H-32b),
2.81 (6 H, m, H-12, H-17 and H-18), 2.61 (4 H, m, H-17 and H-18), 2.53 (2 H, m, H-11), 2.34 (4 H, m,
409 H-14 and H-16), 1.67 (2 H, m, H-15), 1.06 (1 H, t, J = 7.1 Hz, H-13); 13C NMR (400 MHz, CDCl3) δ
410
411
412
413
414
415
(ppm) = 152.2 (C-3), 150.0 (C-1), 149.2 (C-4), 142.6 (C-25), 140.3 (C-26), 137.0 (C-31), 135.2 (C-6),
134.9 (C-20), 132.6 (C-28), 131.5 (C-23), 131.3 (C-30), 129.6 (C-29), 128.8 (C-21), 128.6 (C-22),
127.1 (C-5), 126.8 (C-24), 126.3 (C-27), 125.4 (C-8), 121.3 (C-7), 117.5 (C-9), 99.4 (C-2), 65.8
(C-19), 56.7 (C-10), 54.0 (C-11), 51.4 (C-14), 51.0 (C-16), 48.5 (C-17), 47.1 (C-12), 46.1 (C-18), 39.1
(C-10), 33.3 (C-32), 24.7 (C-15), 10.0 (C-13); MS (ESI) m/z = 586.2 [M+H]+; HRMS (ESI)
m/z = 586.2777 [M+H]+, calc.: 586.2766, Diff.: 1.9 ppm.
19

416 N1-(7-chloroquinolin-4-yl)-N2-(3-(4-(10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazin-1-
417
418
419
yl)propyl)-N2-ethylethane-1,2-diamine (38b)
Compound 38b was obtained by Method B, using the same conditions as for 35: employing
N1-(3-chloropropyl)-N2-(7-chloroquinolin-4-yl)-N1-ethylethane-1,2-diamine (34) (36 mg, 0.11 mmol,
420 1.0 equiv.), 1-(8-chloro-10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazine (34 mg, 0.11 mmol,
421
422
423
1.0 equiv.), K2CO3 (30 mg, 0.22 mmol, 2.0 equiv.) and NaI (3 mg, 0.02 mmol, 0.2 equiv.) in 1.10 mL
MeCN. The mixture was heated in a microwave reactor at 100 °C for 90 min. After flash
chromatography (eluent DCM/MeOH = 95/5 to 8/2 + 0.1 % TEA) and treatment with Amberlyst 21
424 were thus obtained 52 mg (75 %) of the hybrid compound 38b as a yellowish oil.
1
425
426
427
428
429
430
431
432
433
434
435
436
437
Rf (DCM/MeOH = 8/2 + 0.1 % TEA) = 0.57; H-NMR (400 MHz, CDCl3) δ (ppm) = 8.54 (1 H, d,
J = 5.2 Hz, H-3), 7.96 (1 H, d, J = 2.1 Hz, H-8), 7.64 (2 H, m, H-5 and H-28), 7.49 (1 H, m, H-23),
7.37 (1 H, dd, J = 8.8 Hz, J = 2.1 Hz, H-7), 7.33 (1 H, d, J = 8.3 Hz, H-30), 7.22 (2 H, m, H-29 and
H-27), 7.10 (1 H, m, H-21), 7.03 (1 H, m, H-24), 6.38 (1 H, d, J = 5.2 Hz, H-2), 6.03 (1 H, bs, NH),
3.87 (2 H, m, H-19 and H-32a), 3.27 (2 H, m, H-10), 3.12 (1 H, m, H-32b), 2.88 (2 H, t, J = 5.5 Hz,
H-11), 2.89 (8 H, m, H-12, H-14, H-17 and H-18), 2.37 (4 H, m, H-16, H-17 and H-18), 1.68 (2 H, m,
H-15), 1.08 (1 H, t, J = 14.2 Hz, H-13); 13C NMR (400 MHz, CDCl3) δ (ppm) = 152.3 (C-3), 150.0
(C-1), 149.3 (C-4), 142.5 (C-25), 142.3 (C-22), 136.4 (C-26), 154.0 (C-31), 133.8 (C-6), 133.2 (C-20),
132.8 (C-28), 132.2 (C-23), 131.4 (C-30), 129.7 (C-29), 129.0 (C-21), 129.0 (C-5), 127.0 (C-24),
126.6 (C-27), 125.4 (C-8), 121.3 (C-7), 117.6 (C-9), 99.5 (C-2), 65.6 (C-19), 56.7 (C-10), 53.9 (C-11),
51.4 (C-14), 51.0 (C-16), 48.7 (C-17), 47.1 (C-12), 46.1 (C-18), 39.9 (C-10), 32.8 (C-32), 24.8 (C-15),
12.1 (C-13); MS (ESI) m/z = 620.2 [M+H]+; HRMS (ESI) m/z = 620.2377 [M+H]+, calc.: 620.2376,
Diff.: 0.2 ppm.
438
Biology
20

439
Compound preparation
440
441
442
Synthesized compounds for biological testing were diluted in dimethylsulfoxide (DMSO) to create
stock solutions that were stored in aliquots in the dark at −20 ºC. All other drugs were purchased from
Sigma-Aldrich including chloroquine diphosphate, desipramine hydrochloride, loperamide
443 hydrochloride and L703-606 oxalate salt hydrate. Chloroquine and desipramine were dissolved in PBS
444
445
446
and filter-sterilized and stored at −20 °C for up to a month. All individually purchased compounds
were dissolved in DMSO, stored in aliquots at −20 °C and diluted to working concentrations with
PBS. All compounds were protected from light.
447 Inhibitory concentration (IC50) determination for hybrid compounds
448
449
450
451
452
Parasitized cultures, synchronized to rings stage and diluted with fresh erythrocytes and MCM to 1%
parasitemia and 1.25% haematocrit, were incubated with various concentrations of hybrid compounds
for 48 h. Appropriate controls consist of vehicle control (PBS or DMSO), CQ and CQ in combination
with known chemosensitizer. To determine parasitemia, cultures were stained with Hoechst 33342 and
analyzed by flow cytometry. Data obtained from flow analysis were used to plot the sigmoidal dose
453 response curve (i.e. % parasitemia against concentration). The IC50 values, which were the
454
455
concentrations required to inhibit 50% growth could then be determined from the plot.
Hoechst staining
456
457
458
459
Hoechst 33342 stain (Hoechst, Life Technologies) is a DNA-binding fluorescent stain that has an
excitation wavelength of 350 nm (ultraviolet range) and an emission wavelength of 461 nm (blue
fluorescence). After drug treatment, 1 μg/mL of Hoechst stain was added per well for 30 min at 37 °C.
Cells were then washed twice and re-suspended in PBS before flow cytometry analysis.
21

460
Flow cytometric analyses
461
462
463
464
465
466
467
Flow cytometry (DAKO Cytomation Cyan ADP, Fort Collins, CO, USA) was used for Hoechst-
stained cells. Both were excited with a 405 nm violet laser prior to 450/50BP(±25) filter. For the
determination of the proportion of CM-CQ (9) positive parasites, Hoechst-stained duplicate wells were
used to determine parasitemia. To detect both infected erythrocytes and liberated parasites, forward
and side scattering adjustments were made. At least three independent experiments were performed,
unless otherwise stated, to take into account any inter-assay variability. All results are saved in FCS
2.0 format and analyzed using the Flowjo version X (Tree Star) software.
468
469
Determination of the digestive vacuole disruption potency of the hybrid compounds
Parasitized cultures, synchronized to trophozoite stage and diluted with fresh erythrocytes and
470 MCM to 8-10% parasitemia and 2.5% haematocrit, were incubated with either 10 μM or 1 μM of
471
472
473
474
475
476
hybrid compounds for 4 h. Appropriate controls consist of vehicle control (PBS with 0.1% DMSO)
and 3 μM of CQ with 0.1% DMSO. After drug treatment, 1 μg/mL of Hoechst stain and 1 μM of Flu0-
4 AM (Molecular Probes) diluted in MCM were added per well for 30 min at 37 °C. Cells were then
washed twice and re-suspended in PBS to 5% hematocrit before Imagestream X MkII (AMNIS)
analysis. Four independent experiments were performed to take into account any inter-assay
variability.
477
Graphical data plots
478
479
All histograms were generated using Microsoft Excel Starter 2010. All other regression plots were
plotted using Graphpad Prism version 5.0.
22

480
Quantitative analysis
481
482
All assays were done independently at least three times to account for inter-assay variability. After
which, statistical analyses were conducted on the data obtained before making conclusive remarks.
483
Statistical analysis
484
485
486
All data were presented as means ± SEM. Statistical differences were measured using univariate
two-tailed t-test. Conclusive remarks were made based on no difference between independent runs (i.e.
p- value more than 0.05) whereas significant results were indicated as p- value less than 0.05.
487
Ethics statement
488
489
490
491
The blood collection protocol used for in vitro malaria parasite culture was approved by the National
University of Singapore Institutional Review Board (NUS IRB; reference code 11-383, approval
number NUS-1475). Written informed consent was obtained from all of the participants involved in
this study. The clinical isolates used in this study were collected in accordance with the ethical
492 guidelines in the approved protocols (OXTREC reference number 29-09; Center for Clinical
493
494
Vaccinology and Tropical Medicine, University of Oxford, Oxford United Kingdom). The use of field
isolates for work done at the NUS was in accordance with the NUS IRB (reference code 12-369E).
495
496
497
498
RESULTS AND DISCUSSION
Synthesis of hybrid compounds
Simplified analogs of CRA hit compounds 4-6 were used as precursor starting materials for hybrid
499 compound synthesis (compound 7-12, Fig. 4). For compounds 8 and 10, a linker was attached in order
500 to study the influence of the distance of the two attached scaffolds (Fig. 5). After optimization to
23

501
502
identify the most suitable protecting group,1 the Fmoc-protected C3 linker 13 could be installed onto
CRAs 8 and 10 via reductive amination in good yield. Fmoc deprotection with piperidine yielded the
503 free amines 15 and 17.
504
505
Preserving only the key elements of the 4-amino-quinoline scaffold, analogues of 1, 19 and 20, were
obtained by refluxing of the neat amines with 4,7-dichloroquinoline (18, Fig. 6) (16-18). Alcohols 21
506 were transformed to mesylates 22, possessing better leaving groups for subsequent substitution (16-
507 18). New hybrid compounds were obtained by reacting amines 6, 7, 9, 12, 15 and 17 with 22 via a
508
509
510
microwave mediated2 displacement reaction (Fig. 7). This afforded access to hybrid compounds with
features of various CRAs in moderate yields: 27a, 27b, 28, 29, and 30 from analogues of compound 4,
as well as 31 and 32 derived from 6.
511
512
513
514
515
516
In order to explore the structural features necessary to retain chemoreversal activity in the hybrid
compounds, some analogues linking relatively simple precursors of the CRAs were synthesized.
Joining quinuclidinone 23 via reductive amination to amine 19c, the hybrid 24 - with only the
quinuclidine core of 4 attached to the CQ scaffold - was obtained albeit in very poor yield (Fig. 8).
Substitution of bromide 25 with amine 21a gave hybrid 26, possessing the gem-diphenylbutanenitrile
feature of compound 5 (Fig. 9).
517
518
519
520
A further series of hybrid compounds retaining the tertiary ethylamine from 1 was prepared from
intermediate 21a. It has been shown previously that a tertiary amine in this position impacts the
activity of CQ analogues as it influences the diffusion of the compounds in the digestive vacuole (20,
21). Ethyl amine 21a was first connected to a C3 linker, using chloro-iodo-propane (33), leading to
1 Boc and Cbz groups were tested for C2 and C3 linkers, but failed due to difficult deprotection.
2 Reaction times with thermal heating were 7 days or more, this could be significantly reduced to 4- 10
hours by microwave induced heating.
24

521
522
523
chloro-propylamine 34 (Fig. 10). Chloride 34 was then attached to various amines via a microwave
mediated Finkelstein reaction, yielding hybrid compounds in moderate to good yields: 35, 36, and 37
with features of 4, as well as 38a and 38b with features of CRA 6.
524
525
526
Additionally, a hybrid compound combining 3 with the scaffold of known chemosensitizer
chlorpromazine was synthesized. Phenothiazine (39) was transformed to chloride 40 using chloro-
iodopropane (33) in moderate yield (22). Chloroquine fragment 21a was attached to a C3 linker with
527 the same method used previously, yielding primary amine 41. Chloride 40 then underwent a
528
529
substitution with amine 41, giving hybrid compound 42 in moderate yield (Fig. 11).
Anti-parasitic testing in CQ/Art sensitive and resistant strains
530
531
532
533
534
535
536
Mechanisms of drug-resistance in malaria depend on the specific drug and are not yet completely
understood. In the case of chloroquine (1) resistance (CQR) it originates in mutations in the PfCRT (P.
falciparum chloroquine resistance transporter) (23, 24). The anti-malarial mechanism of action of 1 is
to hinder heme detoxification of the parasite in the digestive vacuole. In resistant strains the modified
transporters are able to remove 1 from this vacuole. The mutated PfCRT have altered amino acids in
positions 72-76 (CVMNK in the wild type), depending on the geographical origin: CVIET for African
and most South-east Asian resistant strains versus SVMNT for the South American resistant strains
537 (25, 26). The hybrid compounds were initially tested in an IC50 assay, determining the concentration
538
539
540
541
542
required to inhibit the survival of 50% of parasites. Chloroquine sensitive (CQS) parasite strain 3D7,
and resistant (CQR) strain of CVIET haplotype, K1, were used for initial studies (Fig. 12 and Table 1).
Compound 1 was used as a reference compound in all assays, with an IC50 of 28.6 nM for 3D7 and a
higher IC50 of 514 nM for resistant strain K1 (18-fold resistance), both in line with reported data for
these strains (8, 9).
25

543
544 strains. All other hybrid compounds had IC50s below 500 nM for both strains. Gratifyingly,
545 approximately half of the compounds eradicated any effects of resistance, having very similar IC50s
546 against both 3D7 and K1. Compounds with IC50 values of about 200 nM or lower for both strains are
547 27a, 27b, 31, 35, 37, and 38b. Hybrid 35, linking CQ with the most active CRA analogue 4, shows an
548 IC50 of 190 nM for K1, more potent than 1, and retains an excellent anti-malarial effect on 3D7 with an
549 IC50 of 32.4 nM. However, although exhibiting reduced resistance of about 6-fold, hybrid 35 still has a
550 similar pattern to 1 and significant resistance. More in line with our goal to identify compounds with
Hybrids 42 and 29 showed the weakest effects of the tested compounds but similarly active for both
551
552
553
554
555
minimal resistance, 27a and 31 both stand out as they show similarly good effects on both strains,
albeit less potent against 3D7: 27a, combining the chloroquine pharmacophore with model compound
7, possessing the major features of the CRA pharmacophore, shows IC50 values of 98.6 nM and
110 nM for 3D7 and K1, respectively; 31 has IC50s 114 nM for 3D7 and 127 nM for K1, the best
activity for compounds linking the chloroquine pharmacophore with a CRA analogue of series 6
556 compounds based on octoclothepin. Compounds 27b, 37 and 38b were similar but less potent.
557
558
Based on these results, three hybrid compounds representing the most potent hybrids without any
resistance to K1 (27a and 31) and the improved compound with good potency against 3D7 (35) were
559 chosen for further testing on multiple lab strains and field isolates. Strains selected include chloroquine
560
561
562
563
564
sensitive (CQS) or resistant (CQR) strains as well as artemisinin resistant strains (ArtR) and the
intermediate chloroquine resistant strain with SVMNT haplotype, 7G8 (Fig. 13 with data presented in
Table 2). Compound 31 shows a slightly lower IC50 than 1 for three strains, K1, ARS-272 and 7G8.
However, 27a and 35 proved to be efficient on all tested strains. This indicates that aromatic rings,
present in 35 and 31, but not 27a, are not strictly required for broad activity.
565
566
These trends can be seen clearly in Fig. 14 (data presented in Table 3), illustrating the Response
Modification Index (RMI – the ratio of IC50 for new compounds divided by the IC50 for 1) for CQR
26

567
568
569
570
571
572
573
and CQS strains. 31 is 4-5 times more effective against K1 and 7G8 CQR strains (RMI 0.25 and 0.19,
respectively) whereas it is 4-10 fold less effective than 1 against both CQS strains. On the other hand,
27a and 35 show RMIs of 0.2-0.4 for most CQR strains while retaining acceptable activity for CQS
strains (≤100 nM). Hybrid 35 was particularly significant given its consistent broad spectrum activity
for all the resistant strains studied. These strains include not only CQR strains but also artemisinin
resistant strains (ArtR), further evidence that the chemosensitising approach has general applicability
in tackling some of the latest most concerning field isolates. Furthermore, this conclusion is supported
574 by the fact that the CRA component of compound 35, i.e. compound 4, is the most promising CRA
575
576
from our earlier studies of combinations of CRAs with 1. We next carried out preliminary ADME and
toxicity profiling of both of the most promising molecules 27a and 35.
577
Digestive vacuole disruption efficacy of hybrid compounds
578
579
580
581
582
583
The mechanisms of action of chloroquine have been previously elucidated. Apart from heme
polymerization (27), recent studies have shown that chloroquine at micromolar concentrations,
disrupts the digestive vacuole of the parasite, triggering a cascade of programmed cell death (28, 29).
We performed a high content phenotypic assay using the imaging flow cytometer (Imagestream X
MkII) to detect for digestive vacuole disruption after drug treatment. We observed that there was
disruption of the parasite’s digestive vacuole upon treatment with all the hybrid compounds, and the
584 highest amount of disruption occurred after treatment with 35 (Supplementary Fig S1). The digestive
585
586
587
vacuole disrupting activity of all compounds tested were not as potent as chloroquine, suggesting that
they probably work in a similar manner to chloroquine but the potency was reduced possibly due to an
increase in the size of the compound.
588
Cytotoxicity
27

589
The cytotoxicity of 1 and selected hybrids were investigated with a TGF α-transfected mouse
590 hepatocyte (TAMH) cell line, determining the IC50 via an ATP luminescence assay (30). 27a and 1 had
591
592
593
594
595
little effect on cell viability requiring high concentrations, hence very high therapeutic windows. 35
was only weakly toxic with an IC50 of 19.4 µM in TAMH cells representing a therapeutic window of
over 100 fold. However 37, selected for testing due to it being a biaryl pharmacophore analogue of 35
but without the quinuclidine, was more toxic with an IC50 of 2.84 µM and lower therapeutic window of
16 (Table 4).
596
597
598
In addition to TAMH cells, the cardiomyocyte cell line, AC10, was selected to study the potential
cardiotoxic properties of the hybrid compounds (31). Interestingly, in AC10s, 37 had the same toxicity
as in TAMH cells, confirming its lower therapeutic window. 27a was about 10-fold less toxic than 37
599 with a therapeutic window of over 200. Encouragingly, 35 was only weakly toxic with an IC50
600
601
of 71.7 µM equating to a high therapeutic window of 378 in AC10 cells.
Solubility and permeability
602 In a kinetic solubility assay with HPLC quantification, 35 had solubility of 108 µg/mL at pH 7.4
603
604
605
606
following 24 h incubation, similar to the data at 3 h. In a parallel artificial membrane permeability
assay (PAMPA)(32-34) 35 was permeable with an effective permeability (Pe) of 1.10 x 10-6 cms-1 after
a 16 hour equilibration (Table 5). Molecular weight is below 600 and the calculated LogD (pH 7.4)
value of 2.0 is suitable for an orally administered drug.
607
608
Concluding Remarks
In this study we have designed and synthesized new hybrid compounds merging the antimalarial
609 activity of chloroquine (1) with the chemoreversal activity of selected chemoreversal agents (CRAs)
610
611
into a single agent. Linkers of 3 carbons were effective in joining the CRA to a minimum
pharmacophore chloroquine moiety. Hybrids 27a, 31 and 35 were tested in dose response studies
28

612
613
614
against a panel of malaria parasites both sensitive and resistant to 1 and artemisinin. The most
promising hybrid, 35, presents a good solubility, permeability and in vitro toxicity profile. In vivo
safety studies will be conducted before nominating 35 as a clinical candidate. GLP toxicology studies
615 would involve dosing 35 to two species at high, medium and low doses to establish a no-effect level
616
617
dose and to identify the starting dose in humans. This preliminary study provides support for potent
hybrid CQ-CRA compounds as promising potential therapy for the treatment of CQR malaria.
618
619
ACKNOWLEDGEMENTS
620
621
622
623
624
625
626
627
628
Research from KT and BD laboratories has been generously supported by a grant from the National
Medical Research Council (NMRC/1310/2011) and Faculty of Science start-up grant (R-148-000-169-
133), respectively. The authors thank all of the patients and staff of the SMRU for their contribution to
this study. SMRU is sponsored by the Wellcome Trust of Great Britain, as part of the Oxford Tropical
Medicine Research Program of Wellcome Trust–Mahidol University. The authors are also thankful for
the following reagents which were obtained through the MR4 as part of the BEI Resources Repository,
NIAID, NIH: Plasmodium falciparum 3D7, MRA-102, deposited by DJ Carucci; P. falciparum HB3,
MRA-155, deposited by TE Wellems; P. falciparum 7G8, MRA-154, deposited by DE Kyle; P.
falciparum K1, MRA-159, deposited by DE Kyle; P. falciparum Dd2, MRA-156, deposited by TE
629 Wellems. The authors thank the NUS Drug Development Unit (http://ddu.nus.edu.sg/) for technical
630
631
support with solubility, permeability and toxicity assays. We also want to warmly thank Dr. Martin
Lear for helpful discussions.
632
29

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635
636
637
638
639
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