Antimalarial efficacy of hydroxyethylapoquinine (SN-119) and its derivatives

Article abstract of DOI:10.1128/AAC.01704-13

Quinine and other cinchona-derived alkaloids, although recently supplanted by the artemisinins (ARTs), continue to be important for treatment of severe malaria. Quinine and quinidine have narrow therapeutic indices, and a safer quinine analog is desirable, particularly with the continued threat of antimalarial drug resistance. Hydroxyethylapoquinine (HEAQ), used at 8 g a day for dosing in humans in the 1930s and halving mortality from bacterial pneumonias, was shown to cure bird malaria in the 1940s and was also reported as treatment for human malaria cases. Here we describe synthesis of HEAQ and its novel stereoisomer hydroxyethylapoquinidine (HEAQD) along with two intermediates, hydroxyethylquinine (HEQ) and hydroxyethylquinidine (HEQD), and demonstrate comparable but elevated antimalarial 50% inhibitory concentrations (IC50) of 100 to 200 nM against Plasmodium falciparum quinine-sensitive strain 3D7 (IC50, 56 nM). Only HEAQD demonstrated activity against quinine-tolerant P. falciparum strains Dd2 and INDO with IC50s of 300 to 700 nM. HEQD had activity only against Dd2 with an IC50 of 313 nM. In the lethal mouse malaria model Plasmodium berghei ANKA, only HEQD had activity at 20 mg/kg of body weight comparable to that of the parent quinine or quinidine drugs measured by parasite inhibition and 30-day survival. In addition, HEQ, HEQD, and HEAQ (IC50>90 μM) have little to no human ether-a-go-go-related gene (hERG) channel inhibition expressed in CHO cells compared to HEAQD, quinine, and quinidine (hERG IC50s of 27, 42, and 4 μM, respectively). HEQD more closely resembled quinine in vitro and in vivo for Plasmodium inhibition and demonstrated little hERG channel inhibition, suggesting that further optimization and preclinical studies are warranted for this molecule. Copyright

Full text of DOI:10.1128/AAC.01704-13

Antimalarial Efficacy of
Hydroxyethylapoquinine (SN-119) and Its
Derivatives
Natalie G. Sanders, David J. Meyers and David J. Sullivan
Antimicrob. Agents Chemother. 2014, 58(2):820. DOI:
1
0.1128/AAC.01704-13.
Published Ahead of Print 18 November 2013.
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Antimalarial Efficacy of Hydroxyethylapoquinine (SN-119) and Its
Derivatives
a
b
a
Natalie G. Sanders, David J. Meyers, David J. Sullivan
a
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA ; Johns
b
Hopkins University School of Medicine, Department of Pharmacology and Molecular Sciences, Baltimore, Maryland, USA
Quinine and other cinchona-derived alkaloids, although recently supplanted by the artemisinins (ARTs), continue to be impor-
tant for treatment of severe malaria. Quinine and quinidine have narrow therapeutic indices, and a safer quinine analog is desir-
able, particularly with the continued threat of antimalarial drug resistance. Hydroxyethylapoquinine (HEAQ), used at 8 g a day
for dosing in humans in the 1930s and halving mortality from bacterial pneumonias, was shown to cure bird malaria in the
1940s and was also reported as treatment for human malaria cases. Here we describe synthesis of HEAQ and its novel stereoiso-
mer hydroxyethylapoquinidine (HEAQD) along with two intermediates, hydroxyethylquinine (HEQ) and hydroxyethylquini-
dine (HEQD), and demonstrate comparable but elevated antimalarial 50% inhibitory concentrations (IC ) of 100 to 200 nM
50
against Plasmodium falciparum quinine-sensitive strain 3D7 (IC , 56 nM). Only HEAQD demonstrated activity against
50
quinine-tolerant P. falciparum strains Dd2 and INDO with IC s of 300 to 700 nM. HEQD had activity only against Dd2 with an
50
IC50 of 313 nM. In the lethal mouse malaria model Plasmodium berghei ANKA, only HEQD had activity at 20 mg/kg of body
weight comparable to that of the parent quinine or quinidine drugs measured by parasite inhibition and 30-day survival. In ad-
dition, HEQ, HEQD, and HEAQ (IC50 > 90 ␮M) have little to no human ether-à-go-go-related gene (hERG) channel inhibition
expressed in CHO cells compared to HEAQD, quinine, and quinidine (hERG IC s of 27, 42, and 4 ␮M, respectively). HEQD
50
more closely resembled quinine in vitro and in vivo for Plasmodium inhibition and demonstrated little hERG channel inhibi-
tion, suggesting that further optimization and preclinical studies are warranted for this molecule.
alaria, caused by the Plasmodium parasite, is a devastating depolarization and, in the case of quinidine, repolarization, lead-
M
disease that has plagued mankind for centuries and contin- ing to prolonged QRS and QT intervals, respectively (11). Blind-
ues to wreak havoc across continents, with almost half of the ness is another severe adverse event associated with quinine over-
global population being at risk for the disease every year (1). Ma- dose, while other side effects include tinnitus, hearing loss,
laria is the leading cause of death of children under 5 years of age headache, and loss of taste sensation (8). In light of these quinine-
in sub-Saharan Africa and was responsible for over 1 million associated toxicities, a novel compound, similar to quinine but
deaths in Africa alone in 2010 (1, 2). Due to the large reservoir of less toxic, would be ideal as either a partner drug with artemisinin-
asymptomatic cases and the spread of antimalarial drug resis- related compounds or a replacement for other antimalarial drugs
tance, new strategies of intervention and effective treatment are that have become ineffective due to resistance.
rapidly becoming more urgent to achieve disease elimination. In
Hydroxyethylapoquinine (HEAQ), also known as hydroxy-
particular, new, economically feasible drugs that can rapidly kill ethylapocupreine, was discovered in the 1930s and cited as a com-
the parasite are especially crucial in light of the fact that definitive pound “as good as, or better than quinine” with noted antipneu-
drug resistance or delayed parasite clearance has been reported for mococcal activity but was not pursued due to the advent of
all classes of antimalarials available, including artemisinin-based penicillin and chloroquine (CQ) as successful antibacterial and
combined therapy (ACT) (3–6).
antimalarial drugs. Specifically, HEAQ was used at doses of 8
Quinine was discovered 4 centuries ago as the first antimalarial grams per day as an effective antibacterial compound from 1936 to
drug and was recently supplanted by the artemisinins as the gold 1939 in the treatment of over 500 pneumonia cases in Pittsburgh,
standard in the treatment of severe malaria. Presently, quinine or PA, resulting in a 50% reduction in mortality, with no visual dis-
quinidine is still widely used for treating severe malaria because of turbances or severe adverse effects, including atrial fibrillation,
the lack of availability of intravenous artesunate (7–9). Periodic noted for the drug (12–14). Additional studies confirmed the
reports of drug resistance to quinine do exist, mainly in Southeast lower toxicity of HEAQ than quinine and other alkaloids such as
Asia and South America, but the development of resistance is slow ethylapocupreine, showing no visual toxicity in dogs (15). Hegner
and not sustained, classified as “low grade,” with no “high-grade” et al. reported the efficacy of HEAQ in the treatment of three
resistance being noted for severe malaria cases (7). In many cases,
quinine treatment failure is due to noncompliance to a treatment
regimen rather than an actual increase in the 50% inhibitory con-
centration (IC ) (8, 10).
Received 7 August 2013 Returned for modification 11 September 2013
Accepted 12 November 2013
5
0
Published ahead of print 18 November 2013
Although quinine and quinidine have proven to be effective
antimalarial drugs, both compounds have narrow therapeutic in-
dices; that is, the effective dose is very close to doses associated
with toxicity. In particular, higher doses of quinine and quinidine
may result in cardiotoxicity associated with delayed ventricular
Address correspondence to David J. Sullivan, dsulliva@jhsph.edu.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01704-13
820 aac.asm.org
Antimicrobial Agents and Chemotherapy p. 820–827
February 2014 Volume 58 Number 2

Antimalarial Efficacy of SN-119 Derivatives
strains of bird malaria, demonstrating that HEAQ was as effective ambient temperature, an off-white crystalline solid formed in the still
as but less potent than quinine against Plasmodium lophurae, P. pot and was filtered via vacuum filtration to obtain 6.85 g (72% yield).
The product was consistent with previously reported characterization
relictum, and P. cathemerium (16–18). Most intriguing is a report
by W. W. G. Machlachlan in 1963, in which he states, “We were
aware of the fact that in malaria hydroxyethylapocupreine
1
data. H NMR (500 MHz, CD OD) ␦ 8.60 (d, J ϭ 4.56 Hz, 1H), 7.91 (d,
3
J ϭ 9.12 Hz, 1H), 7.63 (d, J ϭ 4.56 Hz, 1H), 7.34 (dd, J ϭ 2.52, 9.12 Hz,
1
1
H), 7.30 (d, J ϭ 2.52 Hz, 1H), 5.76 (ddd, J ϭ 7.47, 10.14, 17.29 Hz,
H), 5.54 (d, J ϭ 3.14 Hz, 1H), 4.99 (td, J ϭ 1.49, 17.13 Hz, 1H), 4.92
[HEAQ] was as effective as quinine as observed in some clinical
cases in veterans from Korea and also in Venezuela, in addition to
experimental studies recorded by Hegner et al.” (19). Because no
formal human trials have been conducted for the use of HEAQ
(
2
td, J ϭ 1.34, 10.38 Hz, 1H), 3.68 to 3.80 (m, 1H), 3.08 to 3.20 (m, 2H),
.68 to 2.82 (m, 2H), 2.40 (br. s., 1H), 1.85 to 1.97 (m, 2H), 1.79 to 1.85
(m, 1H), 1.56 to 1.68 (m, 1H), 1.46 (tdd, J ϭ 3.30, 10.06, 13.20 Hz, 1H).
against Plasmodium falciparum, we sought to examine activity in 13
C NMR (126 MHz, CD OD) ␦ 158.1, 149.6, 147.6, 144.1, 142.4, 131.6,
3
vitro with human P. falciparum and in vivo with the mouse malaria
128.5, 123.5, 120.0, 115.3, 105.3, 72.0, 61.1, 57.5, 44.5, 40.8, 29.2, 28.0,
model as well as the important chemical property of quinolines to 21.7.
inhibit human ether-à-go-go-related gene (hERG) channels.
Preparation of demethyl quinidine. The same procedure for the con-
Here we resynthesized and tested HEAQ, in addition to three version of quinine to demethyl quinine was used for the conversion of
quinidine (5.00 g; 15.4 mmol) to demethyl quinidine, but the product did
novel compounds, hydroxyethylquinine (HEQ) and the diastereom-
not crystallize. Oil was purified via flash chromatography (gradient of
ers hydroxyethylquinidine (HEQD) and hydroxyethylapoquinidine
99% CHCl plus 1% Et N to 10% methanol [MeOH]–89% CHCl plus
3 3 3
(HEAQD), againstP. falciparuminvitroaswellasinamurinemalaria
1
% Et N) to obtain 3.47 g of a yellow glass. This material contained ap-
3
model to determine the efficacy of these drugs compared to those of
the parent compounds quinine and quinidine. Further characteriza-
tion in regard to heme crystal inhibition and chemical properties of
these derivatives was also conducted along with cytotoxicity and
hERG channel studies using a human fibroblast cell line and Chinese
hamster ovary (CHO) cells, respectively.
proximately 10% quinidine and was carried on to the next step without
further purification.
Preparation of hydroxyethylapoquinine (HEAQ) and hydroxyethyl-
apoquinidine (HEAQD) was carried out as described previously by Carl-
son and Cretcher (21), as follows.
Preparation of HEQ. Ethylene carbonate (5.25 g, 59.6 mmol, 20.0
equivalents), potassium carbonate (824 mg, 5.96 mmol, 2.00 equivalents),
demethyl quinine 2 (926 mg, 2.98 mmol, 1.00 equivalent), and 5 ml an-
hydrous tert-butanol were added to a single-necked 50-ml round-bottom
flask equipped with a reflux condenser. The reaction mixture was placed
into a preheated oil bath (95°C) for 1 h. The reaction mixture was then
poured while still hot onto ice and approximately 10 to 20 ml of 5 M
aqueous NaOH. The reaction mixture was extracted with dichlorometh-
ane, and volatiles were removed to obtain a brown oil. This material was
purified via flash chromatography (gradient of 99% CHCl plus 1% Et N
MATERIALS AND METHODS
General synthesis information. All reagents and solvents were used as
supplied by commercial sources, without further purification. All dry sol-
vents were purchased from Aldrich as Sure Seal bottles. Reactions involv-
ing air- and/or moisture-sensitive reagents were carried out under an
argon atmosphere using glassware that was dried under a vacuum with a
heat gun. The evacuated flask was then filled with argon. The reactions
were monitored by thin-layer chromatography using Analtech chroma-
tography plates (silica gel hard-layer inorganic binder plus calcium sulfate
with fluorescent indicator UV254 [GHLF], 250 ␮m). Visualization was
performed by UV light (254 and 365 nm) and/or by staining with potas-
sium permanganate. Flash chromatography was performed by using a
Grace Reveleris flash purification system and Grace silica cartridges (av-
3
3
to 10% MeOH–89% CHCl plus 1% Et N) to obtain 854 mg of a brown
3
3
glass. The brown glass was dissolved in ca. 5 ml of hot 95% ethyl alcohol
EtOH), allowed to cool to ambient temperature, and then placed into a
(
Ϫ20°C freezer. Pink-tan crystals formed (726 mg) and were collected by
1
vacuum filtration. H NMR (500 MHz, CD OD) ␦ 8.66 (d, J ϭ 4.56 Hz,
3
1
13
1H), 7.96 (d, J ϭ 9.12 Hz, 1H), 7.68 (d, J ϭ 4.56 Hz, 1H), 7.42 to 7.51 (m,
erage particle size, 40 ␮m). H (500-MHz) and C (125-MHz) nuclear
2H), 5.76 (ddd, J ϭ 7.62, 10.14, 17.29 Hz, 1H), 5.58 (d, J ϭ 3.14 Hz, 1H),
magnetic resonance (NMR) spectra of compounds were obtained by us-
1
4.97 (td, J ϭ 1.49, 17.13 Hz, 1H), 4.90 (td, J ϭ 1.34, 10.37 Hz, 1H), 4.20 to
4.29 (m, 2H), 3.93 to 4.01 (m, 2H), 3.69 (dddd, J ϭ 2.36, 5.03, 10.65, 13.24
Hz, 1H), 3.06 to 3.17 (m, 2H), 2.64 to 2.78 (m, 2H), 2.36 (br. s., 1H), 1.83
to 1.96 (m, 2H), 1.76 to 1.83 (m, 1H), 1.54 to 1.65 (m, 1H), 1.46 (tdd, J ϭ
ing a Varian Mercury spectrometer. H NMR spectra recorded in deuter-
13
ated methanol (CD OD) were referenced to 3.310 ppm. C NMR spectra
3
recorded in CD OD were referenced to 39.15 ppm. Accurate mass deter-
3
minations were recorded by the Mass Spectrometry Facility located at the
University of California, Riverside.
1
3
3
1
6
.22, 9.96, 13.15 Hz, 1H). C NMR (126 MHz, CD OD) ␦ 159.1, 150.7,
3
48.3, 144.9, 142.8, 131.6, 128.2, 123.8, 120.2, 115.1, 103.4, 72.3, 71.4, 61.7,
1.2, 57.7, 44.3, 41.0, 29.3, 28.3, 21.8. high-resolution mass spectrometry
HRMS) (m/z): [MH ] calculated for C H N O 355.2016, found
Preparation of demethyl quinine. Preparation of demethyl quinine
(
cupreine) was a modified procedure adapted from procedures reported
ϩ
(
by Xu et al. (36) and Furuya et al. (37). A 500-ml three-necked flask
equipped with an argon inlet, a reflux condenser, and a large magnetic stir
bar (38 by 16 mm) was charged with N,N-dimethylformamide (DMF)
21 27 2 3
355.2012.
Preparation of HEQD. Ethylene carbonate (19.7 g, 224 mmol, 20.0
equivalents), potassium carbonate (3.09 g, 22.4 mmol, 2.00 equivalents),
crude demethyl quinidine (3.47 g, 11.2 mmol, 1.00 equivalent), and 18.6
ml anhydrous tert-butanol were added to a single-necked 100 ml RBF
(
100 ml) and 95% sodium hydride (5.92 g, 247 mmol, 8 equivalents).
Ethanethiol (Stench!) (18.3 ml, 247 mmol, 8 equivalents) was added drop-
wise with cooling with an ice water bath. Note that after the addition of
approximately 10 ml of ethanethiol, stirring became difficult. The reac- equipped with a reflux condenser. The reaction mixture was placed into a
tion mixture was allowed to warm to room temperature, and quinine preheated oil bath (95°C) for 3 h. The reaction mixture was then poured
(
10.0 g, 30.8 mmol, 1 equivalent) was added in one portion. Once the while still hot onto ice and 10 to 20 ml of 5 M aqueous NaOH. The
reaction mixture could be efficiently stirred, the remainder of the ethane- reaction mixture was extracted with dichloromethane to obtain a brown
thiol was added dropwise at room temperature. Once the addition of oil, which was purified via flash chromatography (gradient of 99% CHCl
ethanethiol was complete, the reaction mixture was stirred at 100°C for 24 plus 1% Et N to 10% MeOH–89% CHCl plus 1% Et N) to obtain a
3
3
3
3
h. The reaction mixture was cooled to ambient temperature and quenched reddish-orange glass. This material was crystallized from hot ethyl acetate
1
with saturated aqueous NH Cl and water, and the aqueous layer was ex- to obtain 1.79 g of an off-white crystalline solid. H NMR (500 MHz,
4
tracted with ethyl acetate (3 by 200 ml). The organic phase was dried with CD OD) ␦ 8.66 (d, J ϭ 4.56 Hz, 1H), 7.95 (d, J ϭ 9.12 Hz, 1H), 7.69 (d, J ϭ
3
anhydrous MgSO , and approximately 300 ml of volatiles was removed by 4.56 Hz, 1H), 7.41 to 7.48 (m, 2H), 6.14 to 6.21 (m, 1H), 5.63 (d, J ϭ 3.14
4
simple distillation in a well-ventilated hood (Stench!). During cooling to Hz, 1H), 5.05 to 5.14 (m, 2H), 4.20 to 4.27 (m, 2H), 3.96 (t, J ϭ 4.72 Hz,
February 2014 Volume 58 Number 2
aac.asm.org 821

Sanders et al.
2H), 3.56 (ddd, J ϭ 2.12, 7.78, 13.52 Hz, 1H), 3.05 (dt, J ϭ 2.59, 9.08 Hz, a function of the nanomoles of monomeric heme not crystallized divided
1H), 2.88 to 2.95 (m, 2H), 2.77 to 2.86 (m, 1H), 2.20 to 2.35 (m, 2H), 1.73 by the total nanomoles of heme present in the assay mixture.
(
br. s., 1H), 1.53 to 1.64 (m, 2H), 1.08 (ddd, J ϭ 4.09, 9.43, 13.52 Hz, 1H).
Fluorescence determination. To verify that the quinoline derivatives
1
3
C NMR (126 MHz, CD OD) ␦ 159.0, 150.9, 148.3, 144.9, 142.0, 131.5, retained fluorescent chemical properties similar to those of quinine and
3
1
2
3
28.2, 123.7, 120.1, 115.2, 103.3, 72.6, 71.4, 61.7, 60.9, 51.0, 50.5, 41.6, quinidine, 1 M drug stocks of quinine, quinidine, HEAQ, and HEAQD
ϩ
9.9, 27.4, 21.3. HRMS (m/z): [MH ] calculated for C H N O
were made in 1 M sulfuric acid. The solutions were diluted 1:100 in dis-
tilled water, and 5 dilutions of these stocks were made by using 0.05 M
21
27
2
3
55.2016, found 355.2022.
The general procedure for the preparation of HEAQ or HEAQD was sulfuric acid. The fluorescence of these compounds was determined at 350
carried out as described by Portlock et al. (analytical-scale double-bond and 450 nm.
isomerization) (38).
Seventy-two-hour SYBR green I parasite inhibition assay. A 72-h
HEAQ. To a solution of HEQ (250.0 mg, 0.705 mmol, 1.00 equivalent) SYBR green assay (20, 24) was used to determine the sensitivity of three
dissolved in 12 ml of 50% aqueous ethanol and concentrated (37%, 12 N) strains of P. falciparum (3D7, INDO, and Dd2, obtained from the Malaria
HCl (0.59 ml, 7.05 mmol, 10.0 equivalents) was added 12.5 mg of 5 wt% Research Reference Reagent Resource) to quinine, quinidine, ART, CQ,
rhodium catalyst on activated carbon. The mixture was heated to reflux and the derivatives HEQ, HEAQ, HEQD, and HEAQD. Drug stocks were
for 24 h. After allowing the reaction mixture to cool to ambient temper- prepared at 10 mM concentrations in DMSO or water, filter sterilized, and
ature, the reaction mixture was vacuum filtered through Celite, and the stored at Ϫ20°C. Dilutions were made in RPMI 1640 medium to the
volatiles of the filtrate were removed in vacuo. The residue was taken up in appropriate starting concentration, followed by serial 2-fold dilutions to
water, and the pH was made basic with concentrated NH OH. The result- generate 5 to 10 concentrations for each drug. Drugs (10 ␮l) were ali-
4
ing white precipitate was vacuum filtered and dried under high vacuum to quoted in triplicate into 96-well plates (catalog number 3595; Costar) at
1
obtain 192 mg (76%) of a white amorphous solid. H NMR (500 MHz, 10 times the final concentration. P. falciparum cultures were synchronized
CD OD) ␦ 8.66 (d, J ϭ 4.56 Hz, 1H), 7.95 (d, J ϭ 8.80 Hz, 1H), 7.68 (d, J ϭ and diluted to 2% ring stage and adjusted to 1% hematocrit with unin-
3
4.56 Hz, 1H), 7.36 to 7.58 (m, 2H), 5.60 to 5.72 (m, 1H), 5.08 to 5.28 (m, fected red blood cells (RBCs). The cultures (90 ␮l) were then added to the
1H), 4.15 to 4.39 (m, 2H), 3.89 to 4.06 (m, 2H), 3.69 to 3.87 (m, 1H), 3.38 assay plates, and the plates were incubated in a gassed chamber at 37°C for
to 3.59 (m, 2H), 3.02 to 3.15 (m, 1H), 2.68 to 2.87 (m, 1H), 2.35 (br. s., 72 h until the no-drug control parasitemia reached between 10 and 15%
H), 2.07 to 2.19 (m, 1H), 1.83 to 1.98 (m, 1H), 1.56 to 1.71 (m, 1H), 1.53 (trophozoite or schizont stage). Assay plates were frozen at Ϫ80°C for at
1
13
(
d, J ϭ 6.76 Hz, 1H), 1.47 (d, J ϭ 6.76 Hz, 2H), 1.16 to 1.38 (m, 1H).
C
least 1 h or overnight, followed by thawing at 37°C for 1 to 2 h. Immedi-
NMR (126 MHz, CD OD) ␦ 159.1, 150.8, 148.3, 144.9, 141.5, 140.4, ately after the freeze-thaw, 100 ␮l of 2ϫ SYBR green I in lysis buffer (20
3
131.5, 128.1, 123.7, 120.1, 115.9, 115.7, 103.4, 103.4, 72.3, 71.4, 71.4, 62.0, mM Tris [pH 7.5], 5 mM EDTA, 0.008% [wt/vol] saponin, 0.08% [vol/
6
1.7, 61.7, 59.9, 57.6, 45.0, 45.0, 34.7, 28.6, 28.4, 27.5, 27.3, 27.2, 12.9, 12.6. vol] Triton X-100) was added to each well for a total volume of 200 ␮l/well
ϩ
HRMS (m/z): [MH ] calculated for C H N O 355.2016, found and mixed by pipetting up and down. The plates were allowed to incubate,
21
27 2 3
355.2017.
protected from light, for at least 1 h. Fluorescence was measured at 485
HEAQD. The same procedure was used for HEQD to obtain 264 mg and 535 nm by using an HTS 7000 Plus BioAssay reader, adjusting the gain
1
(
88%) of a white amorphous solid. H NMR (500 MHz, CD OD) ␦ 8.65 between 80 and 90 for optimal reads. Data were exported to Microsoft
3
(
d, J ϭ 4.56 Hz, 1H), 7.95 (d, J ϭ 9.59 Hz, 1H), 7.59 to 7.73 (m, 1H), 7.39 Excel, where background fluorescence from the positive controls (1 mM
to 7.53 (m, 2H), 5.52 to 5.72 (m, 1H), 5.09 to 5.32 (m, 1H), 4.17 to 4.40 chloroquine) was subtracted from each sample, and percent inhibitions
m, 3H), 3.36 (d, J ϭ 17.13 Hz, 1H), 3.14 to 3.28 (m, 1H), 2.88 to 3.04 (m, were calculated by dividing the sample fluorescence by the that of no-drug
(
1
H), 2.70 to 2.88 (m, 1H), 2.35 (br. s., 1H), 1.98 to 2.13 (m, 1H), 1.58 to controls and multiplying by 100. IC s were calculated as the concentra-
50
13
1.71 (m, 3H), 1.55 (d, J ϭ 6.76 Hz, 3H), 1.32 to 1.50 (m, 1H). C NMR tion of drug required to inhibit 50% of the parasite growth in the no-drug
(
126 MHz, CD OD) ␦ 159.0, 159.0, 150.7, 150.7, 148.3, 144.9, 144.9, control. At least three replicates were completed for each strain of P.
3
1
1
3
42.5, 141.4, 131.5, 128.2, 128.2, 123.8, 123.7, 120.2, 120.1, 114.3, 114.0, falciparum and each drug unless otherwise noted.
03.4, 103.3, 72.6, 72.5, 71.4, 71.4, 61.7, 60.7, 60.7, 53.3, 52.2, 51.8, 51.0,
Murine malaria model. We obtained 70 C57BL/6 mice, 5 weeks old
ϩ
4.9, 28.2, 28.0, 27.4, 27.4, 27.0, 12.9, 12.6. HRMS (m/z): [MH ] calcu- and weighing between 15 to 23 g, from the Jackson Laboratory for our
lated for C H N O 355.2016, found 355.2029.
experiment (n ϭ 5 mice per group; 14 groups [4 mice were used in the
2
1
27 2 3
Heme crystallization inhibition assay. The heme crystallization as- artesunate-alone group]). Mice were kept in the Johns Hopkins
say was designed to mimic hemozoin crystal formation in the parasite Bloomberg School of Public Health mouse facility according to ACUC
digestive vacuole, using acidic pH and lipids to initiate crystallization of animal protocol number MO09H401. Mice were infected intraperitone-
7
monomeric heme (22, 23). Drug dilutions were made from a 10 mM stock ally with Plasmodium berghei ANKA (1 ϫ 10 infected erythrocytes), and
with 100 mM sodium acetate (pH 4.8) and aliquoted in a 96-well plate drugs were administered orally twice a day by using sterile plastic feeding
(
catalog number 3595; Costar) in triplicate, with five serial, 2-fold dilu- tubes (catalog number FTP 2038; Instech Solomon Scientific) for 5 days
tions per drug. A heme stock (10 mM) was made in dimethyl sulfoxide beginning at 24 h postinfection (25, 26). All quinoline salts were dissolved
DMSO) and was diluted to 50 ␮M with 100 mM sodium acetate (pH 4.8). in distilled water (8 mg/ml or 2 mg/ml), and artesunate was dissolved in
(
A 10 mM 1-monooleoyl-rac-glycerol (MOG) stock was made in ethanol 100% ethanol (100 mg/ml) and diluted 1:100 in distilled water. The effects
and sonicated before addition to a 50 ␮M heme stock to make 25 ␮M of compounds on parasite levels and mouse survival were determined by
MOG–50 ␮M heme in 100 mM sodium acetate (pH 4.8). The 25 ␮M measuring weekly parasitemia levels by Giemsa-stained blood smears and
MOG–50 ␮M heme solution was sonicated and added to the assay plate at checking mouse survival daily for up to 30 days.
1
00 ␮l/well. The plates were incubated at 37°C for 2 h to allow crystalli-
zation, followed by addition of 100 ␮l 100 mM sodium bicarbonate (pH nels were stably expressed in Chinese hamster ovary (CHO) cells which
.1) to solubilize any remaining monomeric heme. After incubation for 30 were held at Ϫ70 mV, and hERG currents were evoked by two voltage
hERG channel inhibition Ionworks patch clamp assay. hERG chan-
9
min at room temperature, the amount of solubilized monomeric heme pulses (27). During the first pulse, cells were depolarized to ϩ40 mV for 2
was determined by measuring the absorbance at 405 nm and calculating s and hyperpolarized to Ϫ30 mV for 2 s. This was repeated after a 3-s hold
the nanomoles of heme based on a previously determined standard curve. at Ϫ70 mV. The difference of tail currents at the second pulse between
Finally, 20 ␮l of 1 M sodium hydroxide was added to the plates to dissolve pre-compound and post-compound addition was used to measure com-
any crystals that formed, and the absorbance was read at 405 nm to deter- pound activity, with dofetilide and buffer as positive and negative con-
mine the total amount of heme present in each well. Data were exported to trols, respectively. Compounds were dissolved in DMSO and diluted 1:3
Microsoft Excel, and inhibition of heme crystallization was determined as in an 8-point gradient with the maximum concentration at 100 ␮M. In
822 aac.asm.org
Antimicrobial Agents and Chemotherapy

Antimalarial Efficacy of SN-119 Derivatives
FIG 1 Scheme for synthesis of derivatives.
order to be classified as a hERG inhibitor, the compounds caused Ͼ3 HEAQD were effective only at IC s above 50 ␮M (Fig. 2). In addi-
50
standard deviations of reduction in hERG currents.
tion, the derivatives HEQ, HEAQ, HEQD, and HEAQD were found
to fluoresce at 465 nm when excited at 360 nm, similarly to quinine
and quinidine. Fluorescence at 350/450 nm does not interfere with
SYBR green I fluorescence at 485/535 nm.
P. falciparum activity. Quinoline derivatives were evaluated
for antimalarial efficacy against three strains of P. falciparum in a
standard 72-h SYBR green assay, and results are shown in Table 1.
All derivatives were effective at inhibiting a quinine-sensitive
Forty-eight-hour alamarBlue assay with human foreskin fibro-
blasts. Human foreskin fibroblasts (ATCC CRL-1635) were grown and
harvested at log phase (day 3 after passage). Cells were plated at 10,000
cells per well in 200 ␮l of minimal essential medium (MEM) supple-
mented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin,
and 1% L-glutamine in a 96-well plate (catalog number 3595; Costar) and
allowed to incubate for 1 day at 37°C in 5% CO until cells again reached
2
log phase. After 24 h, 100 ␮l MEM was removed and replaced with fresh
medium. After another 24-h incubation, 150 ␮l medium was removed strain, 3D7, at Ͻ300 nM, with an IC50 approximately three to five
from wells, and 150-␮l drug dilutions made in MEM from 10 mM stocks times higher than that of the parent compound quinine or quin-
were added to the assay plate in triplicate, with four 2-fold serial dilutions idine. Of note, the quinidine derivative HEQD (IC , 111 nM) had
50
of drug. Plates were incubated for 2 days at 37°C in 5% CO . After 48 h, 20
2
the most activity against 3D7, comparable with quinine at an IC50
of 56 nM. Against quinine-tolerant strains Dd2 and INDO, HEAQ
and HEQ had ␮M inactivity, while HEAQD had IC s of 333 nM
and 725 nM for Dd2 and INDO, respectively, with HEQD having
only submicromolar activity for Dd2 at an IC50 of 313 nM. Arte-
misinin functioned as a control, consistently inhibiting all three
strains at low-nanomolar concentrations.
␮l of alamarBlue was added to each well, including a no-drug control as
well as a blank well with medium only. Sample fluorescence was read on
an HTS 700 Plus Bio Assay reader at 550 and 595 nm, and data were
exported to Microsoft Excel. Cytotoxicity of drugs was calculated as the
percentage of the no-drug growth control after 48 h.
50
RESULTS
In vivo murine malaria model. Due to previous data on
Synthesis. We modified the original synthetic approach of Butler
and Cretcher in 1937 and 1938 (39, 40) to prepare HEAQ from qui-
nine and HEAQD from quinidine (Fig. 1). According to Xu et al. (36)
and Furuya et al. (37), quinine underwent demethylation in the pres-
ence of sodium ethanethiolate to form cupreine. Formation of the
hydroxyethyl ether was accomplished by reacting cupreine with eth-
ylenecarbonateandpotassiumcarbonate, yieldingHEQaccordingto
methods described by Carlson and Cretcher (21). Finally, rhodium-
catalyzed positional isomerization of the terminal alkene of HEQ re-
sulted in the final product, HEAQ(E,Z), as a mixture of E and Z
geometric isomers. A similar approach was used to produce HEQD
and HEAQD(E,Z), using quinidine as the parent compound. The
crystalline alkaloids of HEAQ, HEQD, and HEAQD were initially
obtained from solution as free bases and later converted to hydro-
chloride salts for use in animal studies.
Heme crystal inhibition. Like other cinchona alkaloids with
antimalarial activity, quinolines accumulate in the parasite diges-
tive vacuole and are thought to inhibit Plasmodium growth by
forming an intramolecular hydrogen bond with heme, disrupting FIG 2 Heme crystallization inhibition by quinine, quinidine, and their deriv-
formation of the hemozoin crystals in the digestive vacuole, re- atives. Only the compound HEQD appreciably inhibited heme crystallization
similarly to quinine and quinidine after 16 h. Two to five independent exper-
iments were completed for each compound with each concentration in tripli-
sulting in more free heme, which is toxic to the parasite (28, 29)
(
Fig. 2). Only HEQD (IC , 24 ␮M) demonstrated activities com-
50
cate per experiment, reported with the corresponding standard errors of the
means. Mean IC s calculated for all compounds in the order listed in graph
parable to those of quinine and quinidine (both at an IC of 16 ␮M)
50
5
0
in inhibiting heme crystallization in vitro, while HEQ, HEAQ, and were 16.3, 16.0, 208, 59.3, 24.0, and 155 ␮M, respectively.
February 2014 Volume 58 Number 2
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Sanders et al.
TABLE 1 Average IC s of quinine and quinidine derivatives against
days 20 to 25 compared to the early day 9 deaths for nontreated
controls (Fig. 4). In combination with artesunate at 10 mg/kg, 20
mg/kg HEQD enabled 3 of 5 mice to live parasite free for 30 days,
compared to 4 of 5 mice in the group treated with the combination
of 80 mg/kg HEAQD and artesunate. Quinidine at 20 mg/kg plus
artesunate cured 1 of 5 mice by day 30. Only HEQD at 20 mg/kg
was similar to quinine and quinidine in this mouse malaria model.
hERG channel inhibition. The most compelling evidence re-
ported for HEAQ, besides its antimalarial efficacy, is the decreased
toxicity associated with its historic human use compared to that of
other quinine derivatives. An important clinical adverse drug re-
action of the quinoline class is prolongation of the QRS heart
interval associated with inhibition of the hERG channel (11).
hERG inhibition studies were conducted by using Chinese ham-
ster ovary (CHO) cells expressing hERG channels and an Ion-
works automatic patch clamp to determine hERG IC s for all
5
0
three strains of P. falciparum determined by using a 72-h SYBR green
a
assay
Avg IC50 (nM) (SEM)
Compound
3D7
INDO
Dd2
Quinine
HEQ
HEAQ
HEAQ-HCl salt
Quinidine
HEQD
HEAQD
HEAQD-HCl salt
Artemisinin
a
56 (6)
263 (25)
7,100 (276)
3,470 (429)
—
153 (279)
1,875 (427)
725 (170)
—
92.0 (10)
258 (33)
255 (29)
240 (77)
24 (2)
111 (22)
168 (25)
148 (55)
8.3 (1)
2,800 (363)
1,133 (133)
1,250 (189)
42.5 (1)
b
313 (13)
333 (44)
233 (17)
5.5 (1)
5.1 (1)
Three or more biological replicates were completed for all compounds unless
otherwise noted, with each compound in triplicate per experiment, reported with
corresponding standards error of the means. —, no data for these compounds.
50
b
derivatives, HEQ, HEAQ, HEQD, and HEAQD, compared to the
parent compounds (Table 2 and Fig. 5). Strikingly, the com-
pounds HEQ, HEAQ, and HEAQD demonstrated IC s of ap-
Two independent experiments were completed for this compound.
50
HEAQ suggesting that this compound was much less toxic than proximately 100 ␮M, compared to 42.2 ␮M for quinine, while the
quinine, we next evaluated the in vivo antimalarial parasite inhi- compound HEAQD was seven times less inhibitory than quini-
bition and 30-day survival of HEAQ, HEQD, and HEAQD in a dine, inhibiting 50% of the CHO hERG channels at 27 ␮M, com-
murine malaria model at doses similar to those of the parent pared to 4 ␮M quinidine. Both quinine and quinidine had a less
drugs, at 20 mg/kg of body weight, and 4 times higher, at 80 mg/kg. steep slope, with the IC25 and IC75 being more apart than for the
Seventy C57BL/6 mice were infected intraperitoneally with P. ber- derivatives with a steeper slope.
7
ghei ANKA (1 ϫ 10 infected erythrocytes), and compounds were
Human fibroblast cytotoxicity. In vitro cell toxicity studies
administered orally twice a day for 5 days beginning at 24 h postin- were also completed by observing the effects of different ␮M con-
fection. Parasitemia was reduced by all derivatives except 20 centrations of quinine, quinidine, and the derivatives on human
mg/kg HEAQ at 3 and 6 days (Fig. 3). HEQD at 20 mg/kg was foreskin fibroblasts using a 48-h alamarBlue assay to determine
comparable in parasitemia inhibition to quinine at 20 mg/kg. cell viability. While all compounds showed no toxicity at 100 ␮M,
Only 80 mg/kg HEAQD and 20 mg/kg HEQD, both in combina- quinine had a 50% lethal dose (LD ) of 200 ␮M. Quinidine and
50
tion with artesunate, retained parasite suppression at day 13 sim- HEAQ were slightly less toxic but comparable to quinine, while
ilar to that of quinidine and artesunate in combination. As single HEQD and HEAQD showed no cytotoxicity (100% growth com-
compounds, the derivatives all delayed death to approximately pared to the control) at 200 ␮M.
FIG3 Dose-dependent clearance of P. berghei ANKA by the compounds quinine (QN), HEAQ, quinidine (QND), HEQD, and HEAQD, alone at either 20 mg/kg
or 80 mg/kg and in combination with artesunate (AS) (10 mg/kg), in C57BL/6 mice. Percent parasitemia was calculated as the percentage of infected RBCs out
of 500 RBCs for Ն1% parasitemia and out of 10,000 for Ͻ1% parasitemia. All compounds are graphed with standard errors of the means. Compounds are listed
first, with dosages following (e.g., QN 20 mg/kg). (A) Day 3 parasitemia, after 2 days of dosing. (B) Day 6 parasitemia, 1 day after the final dose was administered.
(C) Day 13 parasitemia, 1 week after the final dose was administered.
824 aac.asm.org
Antimicrobial Agents and Chemotherapy

Antimalarial Efficacy of SN-119 Derivatives
HEAQD, and we also produced intermediates without the isomer-
ized vinyl group, HEQ and HEQD.
Our in vitro and in vivo antimalarial results correspond with
data previously reported by Hegner et al. for the activity of HEAQ
against three strains of bird malaria, with the quinine and quini-
dine derivatives displaying decreased activity per mg/kg but which
at 4-fold-higher doses had action comparable to that of the parent
compounds (18). In the present work, HEQ and HEAQ were less
potent than quinine in vitro against quinine-sensitive strain 3D7,
but HEAQ was equally effective at controlling parasitemia in the
in vivo mouse malaria model at 4-fold-higher doses. Similar re-
sults were noted for HEQD and HEAQD compared to quinidine.
Regarding drug resistance, HEQD and HEAQD demonstrated
promising submicromolar activity against quinine-tolerant iso-
lates, with IC s of Ͻ400 nM for Dd2 and below 700 nM for INDO
50
for HEAQD, which are below or within reported cutoffs for qui-
nine tolerance that range from either above 500 or 800 nM (30–
32). Overall, HEQD showed the greatest efficacy in the in vivo
model when combined with artesunate, with no adverse side ef-
fects being observable in the mice, making it a promising potential
alternative to quinine or quinidine as an antimalarial drug.
The reported hERG channel inhibition data suggest that all
four derivatives have less hERG channel activity than the parent
compound quinine or quinidine, with HEAQ, HEQ, and HEQD
inhibiting 50% of hERG channels at approximately 100 ␮M, twice
the IC50 of quinine (42 ␮M). Interestingly, both the intermediates
HEQ and HEQD had higher IC s for hERG channels than did
50
HEAQ and HEAQD. These results suggest that isomerization of
the vinyl group did not decrease this cardiac potassium channel
inhibition, whereas the hydroxyethyl substituent may slightly de-
crease parasite activity while also importantly decreasing the
hERG potassium channel inhibition. Cretcher and Renfrew pre-
viously commented on the effect of the resulting modifications on
antipneumococcal activity, stating that “greater antipneumococ-
cic action appears to be associated with the ethylidene group,” and
they observed an increased dosage of drug required to obtain the
same toxic effects of quinine (33). While we did not observe in-
creased potency with the ethylidene or isomerized vinyl group, we
did observe a decrease in hERG channel inhibition associated with
FIG 4 Dose-dependent survival of C57BL/6 mice infected with P. berghei
ANKA after treatment with quinine, HEAQ, quinidine, HEQD, and HEAQD,
alone or in combination with AS (10 mg/kg). (A) HEAQ at either 20 mg/kg or
8
0 mg/kg was comparable to quinine in survival, with all mice dying between the hydroxyethyl substitution, suggesting that this modification
days 20 and 25. (B) HEAQD and HEQD were comparable to quinidine, with
mice surviving to days 20 to 25 as with quinine and HEAQ. Despite the finding
that mice treated with 80 mg/kg of HEAQD had a lower parasitemia on day 16,
these mice had a surge in parasitemia and died quickly compared to mice
and not isomerization of the vinyl group can be credited for the
great reduction in overall cardiac disturbances for these com-
pounds seen in historic manuscripts in the 1930s and 1940s. Our
treated with the lower dose of 20 mg/kg HEAQD. (C) Drugs in combination fibroblast cell line cytotoxicity data also agree with data from
with AS demonstrated that 3 of 5 mice given 20 mg/kg of HEQD survived, previous studies on cell lines conducted by Kominos and
comparable to the group given 80 mg/kg of HEAQD, with 4 of 5 mice surviving
without parasites until day 30. Only 1 mouse of 5 treated with quinidine at 20
mg/kg survived until day 30 but did not clear parasites, unlike treatment with
HEAQD and HEQD.
TABLE 2 Inhibition by quinine, quinidine, and derivatives of hERG
a
channels expressed in CHO cells using the Ionworks patch clamp assay
IC50 (␮M)
Hill
Experimental
(SD)
coefficient
(SD)
% at max
concn
hERG
inhibitor
DISCUSSION
b
Compound
Dofetilide
Reported
Antimalarial drug resistance continues to threaten global public
health measures to treat, contain, and eventually eliminate malaria. Quinine
0.1
57
0.05 (0.01)
42.2 (7.41)
Ͼ100
109 (20.9)
3.95 (0.75)
94.3 (8.12)
27.3 (7.57)
2.27 (0.79)
1.3 (0.3)
Ϫ100
Ϫ83.4
Ϫ35.2
Ϫ49.4
Ϫ95.8
Ϫ70.1
Ϫ87.0
Yes
Yes
No
Yes
Yes
Yes
Yes
HEQ
Building on research that began in the 1930s but was not pursued due
HEAQ
to the advent of chloroquine and other effective antimalarials, we
Quinidine
1.8 (0.83)
0.96 (0.18)
4 (0)
4.6
sought to determine whether HEAQ and three derivatives would be
HEQD
effective against P. falciparum in an era of increasing antimalarial HEAQD
3 (0)
drug resistance and also to test hERG channel inhibition. We were
successful in synthesizing HEAQ as well as the novel diastereomer
a
Compounds were tested in quadruplicate. SD, standard deviation.
Reported values of hERG inhibition from reference 11.
b
February 2014 Volume 58 Number 2
aac.asm.org 825

Sanders et al.
FIG 5 IC s were determined for parent compounds and derivatives against hERG channels expressed in CHO cells, as measured by the Ionworks patch clamp
50
assay. Representative graphs of quinine (A), HEAQ (B), quinidine (C), HEAQD (D), and HEQD (E) are pictured. The positive control, dofetilide, has an IC50 of
0
.053 ␮M. Compounds were tested in quadruplicate in an 8-point gradient with a maximum concentration of 100 ␮M with serial 1:3 dilutions.
Machlachlan, with HEAQ and HEAQD showing less toxicity than strains in some cases, while most resulted in decreased potency
quinine and quinidine and no toxicity at 100 ␮M after 48 h (19). compared to that of parent compounds, including quinine, cin-
Our data suggest that modifications of the methoxy side chain chonine, quinidine, and cinchonidine (35). When the R= group
(R) (Fig. 6) with hydroxylated alkyl groups may be effective at was reduced to the dihydro group (optochin and others), a slight
decreasing cardiotoxic events associated with quinine and quini- decrease in potency was noted. Compound SN-8707 has the di-
dine in addition to the decrease in quinine-associated toxicities such hydro R= group as well as a hydroxyethyl R group, with a reported
as eye damage in dogs, which decreased with hydroxyalkation of R, as quinine ratio of 0.6 in P. lophurae, which is more potent than our
mentioned in other studies. Previous studies have shown that sim- reported derivatives (ϳ0.2 to 0.25). Toxicity studies of SN-8707
ilar alkylations of the side chain without a hydroxyl group such as and the quinidine derivative may provide more insight into the
optochin (RAO-CH -CH ) are more potent but also more toxic role of these modifications in decreasing cardiotoxicity as well as
2
3
than quinine. Interestingly, increased lengths of the alkylated side other quinine-associated toxicities.
chain also increased malaria activity up to 4 to 5 carbons, which
While we focused initially on the compound HEAQ and its ste-
then decreased as the chain length continued (34). In recent stud- reoisomer HEAQD due to previous demonstrations of the use in
ies on P. falciparum, substitutions at the R= group with aromatic humans of HEAQ at high doses with little noted human clinical tox-
groups resulted in increased sensitivity against quinine-resistant icity, our data suggest that fluorescent hydroxyethylquinidine or
HEQD inhibits heme crystallization, does not appreciably inhibit
hERG channels, and is comparable to quinine in vitro against human
P. falciparum and in vivo against mouse P. berghei ANKA, making
it a potential candidate for further derivations to increase potency
against quinine-tolerant strains as well for further in vivo charac-
terizations.
ACKNOWLEDGMENTS
D.J.M. acknowledges NIH Roadmap for Medical Research grant number
UL1 RR 025005 and the Flight Attendants Medical Research Fund. We
thank the Johns Hopkins Malaria Research Institute and The Bloomberg
Family Foundation (D.J.S.).
REFERENCES
1. WHO. 17 December 2012, posting date. World malaria report. WHO,
Geneva, Switzerland. http://www.who.int/malaria/publications/world
_
malaria_report_2012/en/.
FIG 6 Basic structure of levorotatory alkaloids (8S and 9R).
2. Murray CJL, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring
826 aac.asm.org
Antimicrobial Agents and Chemotherapy

Antimalarial Efficacy of SN-119 Derivatives
D, Fullman N, Naghavi M, Lozano R, Lopez AD. 2012. Global malaria
mortality between 1980 and 2010: a systematic analysis. Lancet 379:413–
MS, Wilson EB, Tripathi AK, Gut J, Sharlow ER, Bathurst I, El Mazouni
F, Fowble JW, Forquer I, McGinley PL, Castro S, Angulo-Barturen I,
Ferrer S, Rosenthal PJ, Derisi JL, Sullivan DJ, Lazo JS, Roos DS, Riscoe
MK, Phillips MA, Rathod PK, Van Voorhis WC, Avery VM, Guy RK.
2010. Chemical genetics of Plasmodium falciparum. Nature 465:311–315.
http://dx.doi.org/10.1038/nature09099.
4
31. http://dx.doi.org/10.1016/S0140-6736(12)60034-8.
3
. Frosch AE, Venkatesan M, Laufer MK. 2011. Patterns of chloroquine use
and resistance in sub-Saharan Africa: a systematic review of household
survey and molecular data. Malar. J. 10:116. http://dx.doi.org/10.1186
/
1475-2875-10-116.
24. Johnson JD, Dennull RA, Gerena L, Lopez-Sanchez M, Roncal NE,
Waters NC. 2007. Assessment and continued validation of the malaria
SYBR green I-based fluorescence assay for use in malaria drug screening.
Antimicrob. Agents Chemother. 51:1926–1933. http://dx.doi.org/10
.1128/AAC.01607-06.
4
. O’Brien C, Henrich PP, Passi N, Fidock DA. 2012. Recent clinical and
molecular insights into emerging artemisinin resistance in Plasmodium
falciparum. Curr. Opin. Infect. Dis. 24:570–577. http://dx.doi.org/10
.1097/QCO.0b013e32834cd3ed.
5
. Parija SC, Praharaj I. 2011. Drug resistance in malaria. Indian J. Med. 25. Moon DK, Tripathi A, Sullivan D, Siegler MA, Parkin S, Posner GH.
Microbiol. 29:243–248. http://dx.doi.org/10.4103/0255-0857.83906.
. Sridaran S, McClintock SK, Syphard LM, Herman KM, Barnwell JW.
2011. A single, low, oral dose of a 5-carbon-linked trioxane dimer orthoe-
ster plus mefloquine cures malaria-infected mice. Bioorg. Med. Chem.
Lett. 21:2773–2775. http://dx.doi.org/10.1016/j.bmcl.2010.09.123.
6
2
010. Anti-folate drug resistance in Africa: meta-analysis of reported di-
hydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant 26. Mott BT, Tripathi A, Siegler MA, Moore CD, Sullivan DJ, Posner GH. 2013.
genotype frequencies in African Plasmodium falciparum parasite popula-
tions. Malar. J. 9:247. http://dx.doi.org/10.1186/1475-2875-9-247.
. Achan J, Talisuna AO, Erhart A, Yeka A, Tibenderana JK, Baliraine FN,
Synthesis and antimalarial efficacy of two-carbon-linked, artemisinin-derived tri-
oxane dimers in combination with known antimalarial drugs. J. Med. Chem.
56:2630–2641. http://dx.doi.org/10.1021/jm400058j.
7
Rosenthal PJ, D’Alessandro U. 2011. Quinine, an old anti-malarial drug 27. Zou B, Yu H, Babcock JJ, Chanda P, Bader JS, McManus OB, Li M.
in a modern world: role in the treatment of malaria. Malar. J. 10:144.
http://dx.doi.org/10.1186/1475-2875-10-144.
. Sullivan DJ. 2012. Cinchona alkaloids: quinine and quinidine, p 45–68. In
Staines HM, Krishna S (ed), Treatment and prevention of malaria.
Springer Basel AG, Basel, Switzerland.
2010. Profiling diverse compounds by flux- and electrophysiology-based
primary screens for inhibition of human Ether-a-go-go related gene po-
tassium channels. Assay Drug Dev. Technol. 8:743–754. http://dx.doi.org
/10.1089/adt.2010.0339.
8
28. Warhurst DC, Craig JC, Adagu IS, Meyer DJ, Lee SY. 2003. The
relationship of physico-chemical properties and structure to the differen-
tial antiplasmodial activity of the cinchona alkaloids. Malar. J. 2:26. http:
//dx.doi.org/10.1186/1475-2875-2-26.
9
. WHO. 9 March 2010, posting date. Guidelines for the treatment of ma-
laria, 2nd ed. WHO, Geneva, Switzerland. http://www.who.int/malaria
/
publications/atoz/9789241547925/en/index.html.
1
0. Griffith KS, Lewis LS, Mali S, Parise ME. 2007. Treatment of malaria in 29. de Villiers KA, Gildenhuys J, le Roex T. 2012. Iron(III) protoporphyrin
the United States: a systematic review. JAMA 297:2264–2277. http://dx
doi.org/10.1001/jama.297.20.2264.
IX complexes of the antimalarial cinchona alkaloids quinine and quini-
dine. ACS Chem. Biol. 7:666–671. http://dx.doi.org/10.1021/cb200528z.
.
1
1
1. White NJ. 2007. Cardiotoxicity of antimalarial drugs. Lancet Infect. Dis. 30. Pradines B, Hovette P, Fusai T, Atanda HL, Baret E, Cheval P, Mosnier
7
:549–558. http://dx.doi.org/10.1016/S1473-3099(07)70187-1.
J, Callec A, Cren J, Amalvict R, Gardair JP, Rogier C. 2006. Prevalence
of in vitro resistance to eleven standard or new antimalarial drugs among
Plasmodium falciparum isolates from Pointe-Noire, Republic of the
Congo. J. Clin. Microbiol. 44:2404–2408. http://dx.doi.org/10.1128/JCM
.00623-06.
2. Maclachlan WWG, Johnston JM, Bracken MM, Crum GE. 1939. The
treatment of pneumococcic pneumonia by hydroxyethylapocupreine.
Am. J. Med. Sci. 197:31–38. http://dx.doi.org/10.1097/00000441-193901
0
00-00004.
1
3. Maclachlan WW, Bracken MM, Bailey WR, Jr. 1949. Prognosis of 31. Pradines B, Pistone T, Ezzedine K, Briolant S, Bertaux L, Receveur MC,
pneumonia. Am. J. Med. Sci. 217:438–444. http://dx.doi.org/10.1097
00000441-194904000-00012.
4. Maclachlan WWG, Johnston JM, Bracken MM, Pierce LS. 1941. A
comparison of the mortality in pneumococcic pneumonia treated by
hydroxyethylapocupreine and by sulfapyradine. Am. J. Med. Sci. 201:367–
Parzy D, Millet P, Rogier C, Malvy D. 2010. Quinine-resistant malaria in
traveler returning from Senegal, 2007. Emerg. Infect. Dis. 16:546–548.
http://dx.doi.org/10.3201/eid1603.091669.
/
1
32. Pradines B, Mabika Mamfoumbi M, Parzy D, Owono Medang M,
Lebeau C, Mourou Mbina JR, Doury JC, Kombila M. 1999. In vitro
susceptibility of African isolates of Plasmodium falciparum from Gabon to
pyronaridine. Am. J. Trop. Med. Hyg. 60:105–108.
3
74. http://dx.doi.org/10.1097/00000441-194103000-00007.
1
5. Dawson WT, Permar HH, Johnston JM, Maclachlan WWG. 1937. An
experimental study of the variations in the production of visual distur- 33. Renfew AG, Cretcher LH. 1941. Structure and antipneumococcic activity
bance of certain new cinchona derivatives. Am. J. Med. Sci. 193:543–547.
http://dx.doi.org/10.1097/00000441-193704000-00014.
6. Hegner R, Shaw EH, Manwell RD. 1928. Methods and results of exper-
in the cinchona series. Chem. Rev. 30:49–68.
34. Buttle GAH, Henry TA, Solomon W, Trevan JW, Gibbs EM. 1938. The
action of the cinchona and certain other alkaloids in bird malaria.
Biochem. J. 32:47–58.
1
1
1
1
iments on the effects of drugs on bird malaria. Am. J. Hyg. 8:564–582.
7. Hegner R, West E, Dobler M. 1939. A new drug effective against bird 35. Dinio T, Gorka AP, McGinniss A, Roepe PD, Morgan JB. 2012. Inves-
malaria. Am. J. Epidemiol. 33:101–111.
8. Hegner R, West E, Dobler M. 1941. Further studies of hydroxyethylapo-
cupreine against bird malaria. Am. J. Hyg. 34:132–139.
tigating the activity of quinine analogues versus chloroquine resistant
Plasmodium falciparum. Bioorg. Med. Chem. 20:3292–3297. http://dx.doi
.org/10.1016/j.bmc.2012.03.042.
9. Kominos S, Maclachlan WW. 1963. The cytotoxic effect of quinine, 36. Xu F, Corley E, Zacuto M, Conlon DA, Pipik B, Humphrey G, Murry
quinidine and hydroxyethylapocupreine upon mammalian cell cultures.
Am. J. Med. Sci. 245:569–572.
0. Bennett TN, Paguio M, Gligorijevic B, Seudieu C, Kosar AD, Davidson
J, Tschaen D. 2010. Asymmetric synthesis of a potent, aminopiperidine-
fused imidazopyridine dipeptidyl peptidase IV inhibitor. J. Org. Chem.
75:1343–1353.
2
E, Roepe PD. 2004. Novel, rapid, and inexpensive cell-based quantifica- 37. Furuya T, Strom AE, Ritter T. 2009. Silver-mediated fluorination of
tion of antimalarial drug efficacy. Antimicrob. Agents Chemother. 48:
807–1810. http://dx.doi.org/10.1128/AAC.48.5.1807-1810.2004.
1. Carlson WW, Cretcher LH. 1947. Hydroxylalkylation with cyclic alkyl-
functionalized aryl stannanes. J. Am. Chem. Soc. 131:1662–1663.
38. Portlock DE, Naskar D, West L, Seibel WL, Gu T, Krauss HJ, Peng XS,
Dybas PM, Soyke EG, Ashton SB, Burton J. 2003. Positional isomeriza-
tion of quinine and quinidine via rhodium on alumina catalysis: practical
one-step synthesis of D3,10-isoquinine and D3,10-isoquinidine. Tet. Lett.
44:5365–5368.
1
2
ene esters. I. Synthesis of hydroxyethylapocupreine. J. Am. Chem. Soc.
6
9:1952–1956.
2
2. Pisciotta JM, Coppens I, Tripathi AK, Scholl PF, Shuman J, Bajad S,
Shulaev V, Sullivan DJ, Jr. 2007. The role of neutral lipid nanospheres in 39. Butler CL, Hostler M, Cretcher L. 1937. Cinchona alkaloids in pneumo-
Plasmodium falciparum haem crystallization. Biochem. J. 402:197–204.
http://dx.doi.org/10.1042/BJ20060986.
3. Guiguemde WA, Shelat AA, Bouck D, Duffy S, Crowther GJ, Davis PH,
Smithson DC, Connelly M, Clark J, Zhu F, Jimenez-Diaz MB, Martinez
nia. V. Alkyl ethers of apocupreine. J. Am. Chem. Soc. 445:5–6.
40. Butler CL, Renfew AG. 1938. Cinchona alkaloids in pneumonia. VI. A
new method for the hydroxylation of phenolic cinchona alkaloids. J. Am.
Chem. Soc. 60:1472–1475.
2
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