Aminoindoles, a novel scaffold with potent activity against Plasmodium falciparum

Article abstract of DOI:10.1128/AAC.01714-10

This study characterizes aminoindole molecules that are analogs of Genz-644442. Genz-644442 was identified as a hit in a screen of ～70,000 compounds in the Broad Institute's small-molecule library and the ICCB-L compound collection at Harvard Medical Scho

Full text of DOI:10.1128/AAC.01714-10

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2011, p. 2612–2622
0066-4804/11/$12.00 doi:10.1128/AAC.01714-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 6
Aminoindoles, a Novel Scaffold with Potent Activity against
Plasmodium falciparumᰔ†
Robert H. Barker, Jr.,1* Sameer Urgaonkar,2 Ralph Mazitschek,2,3 Cassandra Celatka,1 Renato Skerlj,1
Joseph F. Cortese,2 Erin Tyndall,2 Hanlan Liu,1 Mandy Cromwell,1 Amar Bir Sidhu,6 Jose E. Guerrero-Bravo,4
Keila N. Crespo-Llado,4 Adelfa E. Serrano,4 Jing-wen Lin,5 Chris J. Janse,5 Shahid M. Khan,5
Manoj Duraisingh,6 Bradley I. Coleman,6 Inigo Angulo-Barturen,7 María Bel´en Jim´enez-Díaz,7
Noem´ı Mag´an,7 Vanesa Gomez,7 Santiago Ferrer,7 María Santos Martínez,7 Sergio Wittlin,8,9
Petros Papastogiannidis,8,9 Thomas O’Shea,1 Jeffrey D. Klinger,1 Mark Bree,1 Edward Lee,1
Mikaela Levine,1 Roger C. Wiegand,2 Benito Munoz,2 Dyann F. Wirth,6 Jon Clardy,10
Ian Bathurst,11 and Edmund Sybertz1
Genzyme Corporation, 153 Second Avenue, Waltham, Massachusetts 024511; the Broad Institute, Infectious Disease Initiative Program,
7 Cambridge Center, Cambridge, Massachusetts 021422; Center for Systems Biology, Massachusetts General Hospital, Boston,
Massachusetts 021143; University of Puerto Rico School of Medicine, Department of Microbiology and Medical Zoology,
P.O. Box 365067, San Juan, Puerto Rico 00936-50674; Leiden Malaria Research Group, Parasitology, Center of
Infectious Diseases, Leiden University Medical Center (LUMC, L4-Q), Albinusdreef 2, 2333 ZA Leiden, Netherlands5;
Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue,
Bldg. 1, Rm. 704, Boston, Massachusetts 021156; GlaxoSmithKline, Medicines Development Campus, Diseases of the
Developing World, Severo Ochoa 2, Tres Cantos 28760, Madrid, Spain7; Swiss Tropical and Public Health Institute,
Socinstrasse 57, CH-4002 Basel, Switzerland8; University of Basel, 4056 Basel, Switzerland9; Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue,
C-643, Boston, Massachusetts 0211510; and Medicines for Malaria Venture,
Route de Pre-Bois, 1215 Geneva, Switzerland11
Received 9 December 2010/Returned for modification 4 February 2011/Accepted 17 February 2011
This study characterizes aminoindole molecules that are analogs of Genz-644442. Genz-644442 was identified as
a hit in a screen of ϳ70,000 compounds in the Broad Institute’s small-molecule library and the ICCB-L compound
collection at Harvard Medical School. Genz-644442 is a potent inhibitor of Plasmodium falciparum in vitro (50%
inhibitory concentrations [IC50s], 200 to 285 nM) and inhibits P. berghei in vivo with an efficacy of >99% in an
adapted version of Peters’ 4-day suppressive test (W. Peters, Ann. Trop. Med. Parasitol. 69:155–171, 1975).
Genz-644442 became the focus of medicinal chemistry optimization; 321 analogs were synthesized and were tested
for in vitro potency against P. falciparum and for in vitro absorption, distribution, metabolism, and excretion
(ADME) properties. This yielded compounds with IC50s of approximately 30 nM. The lead compound, Genz-668764,
has been characterized in more detail. It is a single enantiomer with IC50s of 28 to 65 nM against P. falciparum in
vitro. In the 4-day P. berghei model, when it was dosed at 100 mg/kg of body weight/day, no parasites were detected
on day 4 postinfection. However, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in
cures of 3/5 animals. The compound had comparable activity against P. falciparum blood stages in a human-
engrafted NOD-scid mouse model. Genz-668764 had a terminal half-life of 2.8 h and plasma trough levels of 41
ng/ml when it was dosed twice a day orally at 55 mg/kg/day. Seven-day rat safety studies showed a no-observable-
adverse-effect level (NOAEL) at 200 mg/kg/day; the compound was not mutagenic in Ames tests, did not inhibit the
hERG channel, and did not have potent activity against a broad panel of receptors and enzymes. Employing
allometric scaling and using in vitro ADME data, the predicted human minimum efficacious dose of Genz-668764
in a 3-day once-daily dosing regimen was 421 mg/day/70 kg, which would maintain plasma trough levels above the
IC90 against P. falciparum for at least 96 h after the last dose. The predicted human therapeutic index was
approximately 3, on the basis of the exposure in rats at the NOAEL. We were unable to select for parasites with
>2-fold decreased sensitivity to the parent compound, Genz-644442, over 270 days of in vitro culture under drug
pressure. These characteristics make Genz-668764 a good candidate for preclinical development.
Malaria continues to be a major global health burden, en-
demic in 87 countries with 2.5 billion people at risk (11).
Widespread resistance to current antimalarials such as chloro-
quine (29, 40), atovaquone (33), pyrimethamine (39), and sul-
fadoxine (37) and, more recently, reduced efficacy of artemis-
inin derivatives (7, 38) emphasize the urgent need for new
antimalarial drugs. At least in part because the majority of
people affected live in the poorest parts of the world, the
response of the pharmaceutical industry has been sparse or
irregular. New collaborations combining the expertise of aca-
demia, industry, and public-private partnerships have been
suggested to overcome these obstacles, and it is in that spirit
* Corresponding author. Mailing address: Genzyme Corporation,
153 Second Avenue, Waltham, MA 02451. Phone: (781) 434-3425. Fax:
(781) 434-3400. E-mail: Robert.barker@genzyme.com.
† Supplemental material for this article may be found at http://aac
.asm.org/.
ᰔ Published ahead of print on 21 March 2011.
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AMINOINDOLES ACTIVE AGAINST PLASMODIUM
2613
was washed once with complete culture medium by centrifugation (450 ϫ g, 8
min), and then erythrocytes were resuspended in complete culture medium at 1
to 3% parasitemia and 1% hematocrit and were mixed with serial solutions of the
drugs in complete culture medium (final volume, 1 ml/well in 24-well plates).
Plates were incubated for 24 h under standardized in vitro culture conditions
(14), allowing the ring forms/young trophozoites to develop into mature schiz-
onts. After incubation, 250 ␮l of the cell suspension from each well was trans-
ferred to Eppendorf tubes and cells were pelleted by centrifugation (13,000 ϫ g
for 1 min). Supernatant was removed and cells were lysed by addition of 100 ␮l
cell culture lysis reagent (luciferase assay system kit; Promega). Ten microliters
of lysate was transferred to wells of a 96-well plate, 100 ␮l luciferase assay
substrate (luciferase assay system kit; Promega) was added, and the lumines-
cence spectrum of each well was read for 10 s using a multiplate reader (Wallac
1420 multilabel counter; Perkin Elmer). Measurements of luciferase activity
were expressed in relative light units (RLUs) and represent the average of
triplicate samples at each drug dilution. Wells containing phosphate-buffered
saline (PBS) (instead of infected cells) were used as negative controls.
Mammalian cytotoxicity. Compounds were tested at 10 dilutions against nor-
mal human renal proximal tubule cells (CC-2553; Clonetics) and dermal fibro-
blasts (normal human dermal fibroblasts [NHDFs]; CC-2509; Clonetics) by in-
cubation for 4 days, until cell confluence. Viability was measured using the
alamarBlue assay (Trek Diagnostic Systems). Cytotoxicity IC50s were required to
be Ն20-fold the potency against P. falciparum in the in vitro assay as a criterion
for advancing a compound through distribution/metabolism/pharmacokinetic
(DMPK) assays.
that the Broad Institute, Genzyme Corporation, and Medi-
cines for Malaria Venture have sought to develop novel ther-
apies for malaria.
The aminoindole Genz-644442 was identified as a hit in a
screen of ϳ70,000 compounds in the Broad Institute’s small-
molecule library and the ICCB-L compound collection at Har-
vard Medical School, showing 50% inhibitory concentrations
(IC50s) of 200 to 285 nM against Plasmodium falciparum blood
stages in vitro (2, 36). In vivo, in an adapted version of the 4-day
suppressive test (27, 28), Genz-644442 inhibited P. berghei with
an efficacy of Ͼ99% (36). Subsequent in vitro studies showed
that selectivity, drug-like properties, and metabolic stability
were promising (see below). On the basis of these results and
initial exploratory structure-activity relationship (SAR) analy-
sis, the aminoindole series became the focus of hit-to-lead
medicinal chemistry optimization which resulted in selection of
Genz-668764 as a candidate for preclinical development. We
demonstrate that this compound shows significantly greater
potency against P. falciparum than the parent, is well tolerated
in mice and rats, and can cure P. berghei infections.
Erythrocyte lysis. Compounds were tested at 10 dilutions against fresh human
erythrocytes at 1% hematocrit in Dulbecco’s PBS in V-bottom plates for 24 h at
37°C. Plates were then centrifuged at 2,000 rpm for 5 min, and 50 ␮l of super-
natant was transferred to a fresh flat-bottom plate. Hemoglobin was measured
using a QuantChem hemoglobin assay kit (DIHB-250; BioAssay Systems).
In vitro absorption, distribution, metabolism, and excretion (ADME) proper-
ties. Solubility, passive permeability, metabolic stability, log of the distribution
coefficient (D), percent protein bound, and cytochrome P450 (CYP450) assays
were performed as previously described (5).
hERG channel activity. CHOK1 cells that stably overexpress the hERG ion
channel were tested in a whole-cell voltage clamp procedure at Genzyme by
measuring the ability of a drug to inhibit the peak current flowing through the
hERG channel upon depolarization of the membrane potential. Initially, record-
ings were made in the presence of control saline to establish a baseline (zero
inhibition). This was followed by addition of four increasing concentrations of
test drug. Each drug was tested in at least three different cells (n Ն 3), and the
effect of each drug concentration was compared with the baseline to obtain a
percent inhibition value. The dose-dependent percent inhibition was fitted by a
Blotzmann function to generate a dose-response curve for the compound, and its
IC50 was determined.
In vitro resistance selection. Studies to select for resistance were performed
utilizing successive cycles of drug exposure in which parasites were exposed to
drug for 1 week at 10ϫ the IC50, followed by 2 weeks of culture in the absence
of drug. P. falciparum was grown in four T75 flasks containing 1 ϫ 109 Dd2
parasites (5% hematocrit; 5% parasitemia in 25 ml of medium). Test compound
was added at 10ϫ the IC50 and maintained during cell feeding every other day
for a week; parasitemia was monitored daily by microscopy. During periods of
drug exposure, parasites were generally not detectable after the 4th day. Follow-
ing 1 week of exposure, drug pressure was removed and cultures were fed every
other day until parasites reemerged (1 to 3 weeks). Drug pressure (10ϫ IC50) was
then resumed, repeating the same protocol. Periodically, parasites were removed
and assayed for altered susceptibility to the test compound. For Genz-644442,
this exposure cycle was repeated ϳ10 times over 270 days; for Genz-668764 this
was done for ϳ3 cycles (65 days).
In vivo studies: tolerability/plasma exposure. To guide dose selection for
efficacy studies, tolerability studies were conducted at Genzyme in CD-1 mice.
Protocols were IACUC approved, and animals were housed in AAALAC-ac-
credited facilities. Compounds were dosed orally (p.o.) twice, 8 h apart, to groups
of 3 female 4-week-old CD-1 mice at 50, 100, or 200 mg/kg of body weight/day,
in addition to a vehicle control group. The aminoindole analogs were formulated
in 1.6% lactic acid mixed sodium salt, 1% Tween 80, 9% ethanol, and 20%
hydroxypropyl ␤-cyclodextrin (Cerestar; Wacker Chemie) in water. Animals
were observed for signs of overt toxicity/poor tolerability every 15 min for the
first hour postdosing and then hourly for up to 4 h after the first dose. Blood was
collected 1 h after each dose and 18 h after the final dose to measure plasma
concentrations.
MATERIALS AND METHODS
Chemical synthesis. Details of chemical synthesis are presented in the sup-
plemental material and reference 36.
In vitro P. falciparum viability assay. Potency against P. falciparum in vitro was
assessed using a modified version of the method of Plouffe and coworkers (30).
Parasites were cultured in the presence of drug in RPMI 1640 (Sigma) containing
4.16 mg/ml lipid-rich bovine serum albumin (Albumax II) in a total volume of 50
␮l at a 2.5% hematocrit and an initial parasitemia of 0.3% in black Greiner GNF
clear-bottom plates. Cultures were incubated for 72 h at 37°C under 95% N2, 4%
CO2, and 3% O2. At the end of the incubation, SYBR green was added to a
dilution of 1:10,000 and plates were stored overnight (or until they were ready to
be read) at Ϫ80°C. Just before they were read, plates were centrifuged at 700
rpm and fluorescence was read using 480-nm excitation and 530-nm emission
frequencies. Compound concentrations that inhibit parasite replication reduce
the fluorescence intensity of SYBR green bound to parasite DNA.
In vitro P. knowlesi viability assay. Selected compounds were tested against P.
knowlesi H1 strain parasites cultured in Rhesus blood cells as a surrogate for P.
vivax infections as previously described (19). Briefly, P. knowlesi parasites were
cultured in 2% Rhesus macaque erythrocytes (New England Primate Research
Center) in RPMI 1640 culture medium containing 10% human type O-positive
serum (Interstate Blood Bank). Schizont-stage parasites were purified by flota-
tion in 60% Percoll (GE Life Sciences) and allowed to reinvade to generate a
synchronous population of ring-stage parasites. Drug assays were performed by
plating ring-stage parasites at 0.5% parasitemia in triplicate in RPMI 1640
containing 2.5 ␮g/ml hypoxanthine. Parasites were incubated for 24 h with seri-
ally diluted test compounds. After 24 h, thin smears were made to confirm that
reinvasion had occurred, 0.5 ␮Ci 3H-labeled hypoxanthine (Perkin Elmer) was
added to each well, and parasites were allowed to progress through S phase to
early schizonts. Cells were then harvested via glass filter plates, and 3H incor-
poration was measured by scintillation counting. Values were normalized to a
percentage of the values for controls containing no drug, and IC50s were gener-
ated (GraphPad Prism).
In vitro P. berghei viability assay. Selected compounds were tested against P.
berghei in an in vitro P. berghei viability assay using transgenic parasites containing
a luciferase gene (8). This assay measures luciferase activity (luminescence),
using a luciferase assay system kit (Promega), from transgenic blood-stage par-
asites that express LucIAV luciferase under the control of the schizont-specific
(ama-1) promoter (transgenic parasite RMgm-32; http://www.pberghei.eu/index
.php?rmgm ϭ 32). In these studies, ring-stage parasites (13) were incubated for
24 h at 37°C in 24-well plates in complete culture medium (RPMI 1640 with 20%
fetal calf serum [13]) containing serial dilutions of drugs. Synchronized ring-form
parasites for these assays were prepared as previously described (14). Briefly,
cultured and purified schizonts/merozoites (13) were injected intravenously (i.v.)
into the tail veins of mice. In these animals, merozoites invade within 4 h after
injection, giving rise to synchronized in vivo infections with a parasitemia of 0.5
to 3% containing mainly (Ͼ90%) ring-form parasites. Infected blood collected
by cardiac puncture 4 h after the injection of the purified schizonts/merozoites
PK studies. Pharmacokinetic (PK) evaluation of compounds was first con-
ducted in mice (the host species for the P. berghei efficacy model) and then for

2614
BARKER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
selected compounds in rats, dogs, and monkeys. For studies in mice, the in-life
portion was performed at Mispro Biotech Services, Inc., Montreal, Quebec,
Canada. For dog studies, the in-life portion was performed at Absorption Sys-
tem, San Diego, CA, while rat and monkey studies were performed at Xeno-
metrics, Stillwell, KS. All of these laboratories utilized IACUC-approved proto-
cols and are AAALAC-approved facilities. Bioanalytical work was performed at
Genzyme. In mice, once-per-day (QD) or twice-per-day (b.i.d.) dosing studies
were performed using a group of 3 male CD-1 mice per dosing group. The test
article was administered following a 2-h fast, and mice were given food access
approximately 45 min postadministration. At specified intervals (0.083, 0.25, 0.5,
1, 2, 6, and 24 h) blood (25 to 30 ␮l per time point) was collected into K2EDTA
and processed to plasma. In the rat study, three (group 1) and two (group 2)
Sprague-Dawley male rats (6 to 8 weeks old) were administered the test article
at a dose of 3 mg/kg via oral gavage and a dose of of 1 mg/kg i.v., respectively.
Vehicle was 0.5% lactic acid, 0.89% sodium lactate, 0.95% Tween 80, 4.5% ethyl
alcohol, 9.5% hydroxypropyl ␤-cyclodextrin, and 83% water. The dose volumes
were 3 ml/kg and 2 ml/kg for the p.o. and i.v. groups, respectively. Each animal
was weighed and dosed following an overnight fast. Following dose administra-
tion, whole-blood samples (approximately 250 ␮l) were collected via a jugular
vein catheter from each animal at the following time points: predose and 0.033
(i.v. only), 0.083, 0.167 (p.o. only), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose. Food
was returned to the animals at approximately 1 h postdose. Whole-blood samples
were collected into K2EDTA and processed to plasma. For 0- to 24-h intervals
following dose administration, each of the animals was housed in a metabolism
cage to collect urine.
Dog PK studies were conducted in a crossover design (3-day washout between
two p.o. doses and a 7-day washout for the i.v. dose). Three beagle male dogs (2
to 4 years of age, body weight of 8 to 12 kg at dosing, and at least 2 weeks free
of any treatment prior to this study initiation) were administered the test article
at 2 or 3 ml/kg for the p.o. doses and 1 ml/kg for the i.v. dose. Each animal was
weighed and dosed following an overnight fast. Following dose administration,
whole-blood samples (approximately 0.6 ml) were collected via from the jugular,
cephalic, or saphenous vein from each animal at the following time points:
predose and 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, and 72 h
postdose. Food was returned to the animals at approximately 4 h postdose.
Whole-blood samples were collected into K2EDTA and processed to plasma.
During the 24 h prior to and 0- to 4-h, 4- to 8-h, 8- to 24-h, 24- to 48-h, and 48-
to 72-h intervals following dose administration, each of the animals was housed
in a metabolism cage to collect urine. To evaluate pharmacokinetics in nonhu-
man primates, three cynomolgus male monkeys (weight, 4 to 6 kg) were used in
a crossover study design similar to that use for the dog study. Genz-668764 was
administered at 1 mg/kg (i.v.) and 5 or 20 mg/kg (p.o.) in the same vehicle
formulation used for the rat PK study.
PK studies on selected compounds were also conducted in P. berghei-infected
mice. Malaria infection has been shown to alter quinine protein binding (20),
reduce metabolic clearance of dihydroartemisinin (3), and affect the elimination
half-life of primaquine, so it was important to determine whether metabolism/
availability of the aminoindole compounds is also influenced by infection.
Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was
employed for the analysis of samples from all PK studies described above.
Seven-day safety study in rats. The in-life portion of the 7-day safety study in
rats was performed at Lab Research, Inc., Laval, Quebec, Canada, using
IACUC-approved protocols in AAALAC-accredited facilities. Four groups of 8
rats were dosed orally with vehicle alone (0.5% methylcellulose in water) or with
Genz-668764 (the more active enantiomer from the Genz-666984 racemic mix-
ture) at 100, 200, or 300 mg/kg/day QD for 7 days. Within each dosing group,
animals were divided into safety assessment (5 animals) and toxicokinetic (TK)
groups (3 animals). Clinical observations were made twice daily, and body
weights were measured before treatment; at days 3, 6, 8, 9, and 13; and at final
necropsy. Animals in the 100- and 200-mg/kg/day treatment groups were nec-
ropsied on day 8, as were 5 animals in the control and 300-mg/kg/day groups.
Clinical chemistry and hematology parameters were assessed, while bone mar-
row, kidney, liver, and lymph nodes were observed for gross abnormalities and
then were analyzed for histopathology. The remaining 3 animals in the 300-mg/
kg/day and control groups were observed for an additional 7 days posttreatment
and then were necropsied and analyzed as described above. To assess TK, blood
samples (approximately 0.4 ml) were collected from the jugular vein into tubes
containing K3EDTA at the following time points: 0.5, 1, 2, 8, and 24 h after
dosing on study days 1 and 7. Serial blood samples were collected from 3
animals/group/time point and were processed to plasma for subsequent LC-
MS/MS analysis.
were tested at the University of Puerto Rico (UPR) against parasites of the P.
berghei N strain; however, in select cases, compounds were also tested at the
Swiss Tropical and Public Health Institute (SwissTPH) against P. berghei ANKA
strain parasites to confirm comparability of data across different laboratories and
to examine efficacy against different parasite strains.
At UPR, all studies were conducted in accordance with the Guide for the Care
and Use of Laboratory Animals (24) and the Public Health Service (PHS) guide-
lines under protocols approved by the IACUC of the Medical Sciences Campus,
University of Puerto Rico, and all work was conducted in accordance with the
Guide for the Care and Use of Laboratory Animals (24) and regulations of the PHS
Policy on Humane Care and Use of Laboratory Animals (http://grants.nih.gov
/grants/olaw/references/phspolicylabanimals.pdf). Animals were maintained ac-
cording to NIH guidelines and were allowed to acclimatize for 1 week prior to
the commencement of studies. On day 0, groups of 4- to 6-week-old female Swiss
albino mice (n ϭ 5) were infected by tail vein injection with 0.2 ml heparinized
blood diluted to contain 1 ϫ 107 N-clone parasites. The aminoindole analogs
were formulated in 1.6% lactic acid mixed sodium salt, 1% Tween 80, 9%
ethanol, and 20% hydroxypropyl ␤-cyclodextrin (Wacker Chemie), and the for-
mulations were administered by oral gavage. On day 0, a single dose was given
at 6 h after initial infection, and over the subsequent 3 days the dose was split and
administered b.i.d., with 6 h between doses. Animals in the control group re-
ceived vehicle alone. Dose concentration and frequency of dosing were based
upon preliminary tolerability/exposure studies. On day 4 postinfection (5th day
of assay), blood was collected by tail nick, and thin-smear microscope slides were
prepared and stained using Diff Quick stain. Parasitized erythrocytes were
counted and compared with the total number of erythrocytes per microscopic
field to determine the percent parasitemia. A minimum of 350 erythrocytes was
counted. Animals showing no detectable parasites on day 4 were examined every
2 to 3 days to determine whether cure was sterile. Animals with no detectable
parasites 28 days after cessation of dosing (study day 33) were considered cured;
animals were euthanized at the end of the study.
In vivo studies at the Swiss Tropical and Public Health Institute were per-
formed under a protocol reviewed and approved by the local veterinary author-
ities of Canton Basel-Stadt, Switzerland. NMRI mice were infected with 2 ϫ 107
ANKA strain GFP MRA-865 parasites containing a constitutively expressed
GFP gene (transgenic parasite RMgm-5; http://www.pberghei.eu/index
.php?rmgmϭ5) (9). Dosing was twice/day by oral gavage, using the same formu-
lation as that used at the University of Puerto Rico. Parasitological assessments
were made by resuspending 1 ␮l tail blood in 1 ml PBS buffer and counting
fluorescent cells using a FACScan apparatus (Becton Dickinson), examining a
total of 100,000 red blood cells. Animals that had no detectable parasites on day
4 were followed through day 30 before being deemed cured of infection. In the
SwissTPH study, 100-␮l blood samples were collected at 5 time points to assess
plasma exposure.
In vivo efficacy studies, P. falciparum. Genz-668764 was tested for efficacy
against P. falciparum Pf3D70087/N9 growing in immunodeficient NOD-scid IL-
2R␥null (NSG) mice (generously made available through a collaboration with
Leonard D. Shultz from the Jackson Laboratory) engrafted with human eryth-
rocytes (generously provided by the Spanish Red Cross Bank in Madrid, Spain)
(1, 17). Briefly, groups of 3 animals were infected intravenously on day 0 with 2 ϫ
107 P. falciparum parasites and were treated orally once per day for 4 days with
5, 25, and 100 mg/kg/day formulated as described above beginning on day 3
postinfection. In parallel, Genz-668764’s efficacy was also tested against P. ber-
ghei, with nonengrafted NSG mice infected on day 0. Groups of 3 animals were
infected intravenously on day 0 with 1 ϫ 107 P. berghei ANKA strain parasites on
day 0 and received the same doses beginning 1 h after infection (18). Parasitemia
was assessed by fluorescence-activated cell sorter analysis as previously described
(16). All the experiments performed were approved by the Diseases of the
Developing World (DDW) Ethical Committee on Animal Research, performed
at the DDW Laboratory Animal Science facilities accredited by AAALAC, and
conducted according to European Union legislation and GlaxoSmithKline policy
on the care and use of animals.
Pharmacokinetic studies, infected mice. Single-dose PK evaluation in infected
NSG mice was also performed to determine the time course of Genz-668764
clearance in these animals. Groups of 3 erythrocyte-engrafted NSG mice were
infected with P. falciparum via oral gavage as described above. Blood samples
were collected at 5, 15, and 30 min and 1, 3, 6, 8, 24, and 48 h after administra-
tion, were mixed 1:1 with 0.1% saponin and water for lysis, and were stored at
Ϫ70°C until LC-MS/MS analysis. The same experimental design was followed to
assess the compound’s clearance in nonengrafted NSG mice administered Genz-
668764 1 h after infection with P. berghei as described above.
In vivo efficacy studies, P. berghei. The P. berghei acute-infection model in
rodents is adapted from Peters’ 4-day suppressive test (27, 28). Most compounds

VOL. 55, 2011
AMINOINDOLES ACTIVE AGAINST PLASMODIUM
2615
RESULTS
665848 (see Fig. S2D in the supplemental material) and lower
plasma clearance (i.e., 8 ml/min/kg) than that of Genz-665848
(i.e., 37 ml/min/kg) in mice.
Identification and characterization of Genz-644442. The
aminoindole Genz-644442 was identified as a hit in a screen of
ϳ70,000 compounds tested against P. falciparum strains Dd2
and 3D7 (2, 36). In vitro potency of Genz-644442 ranged from
285 nM (Dd2) to 200 nM (3D7). This activity against P. fal-
ciparum was selective since cytotoxicity IC50s against mamma-
lian cells were 800-fold higher than the IC50s against P. falcip-
arum (Table 1). In vitro ADME assays (Table 2) showed that
Genz-644442 was quite soluble, highly membrane permeant,
and stable in liver microsomes and hepatocytes from rats and
humans. In addition, the IC50 against the major human drug-
metabolizing CYP450 enzymes was Ͼ5 ␮M for all isozymes
tested, indicating a low likelihood of significant drug-drug in-
teractions mediated by CYP450. On the basis of these charac-
teristics (Tables 1 and 2), the aminoindole Genz-644442 was
analyzed further for tolerability and plasma exposure in mice,
for efficacy against P. berghei in mice, and for PK properties. In
initial studies for tolerability and efficacy of Genz-644442, we
used intraperitoneal (i.p.) administration to ensure systemic
exposure. At 50 mg/kg/day there was complete suppression of
parasitemia with no detectable parasites on day 4 (see Fig. S1A
in the supplemental material), while dosing at 25 and 15 mg/
kg/day suppressed parasitemia by 86% and 54%, respectively,
resulting in a 50% effective dose (ED50) of 15 mg/kg/day.
However, all mice dosed with 50 mg/kg/day showed recrudes-
cent parasites by day 9 (see Fig. S1B in the supplemental
material), indicating that this dose did not result in cure of the
infection.
Screening analogs of Genz-644442: identification of lead
compound Genz-668764. Observed differences in metabolic
stability, in vivo systemic clearance, and efficacy of individual
enantiomers shaped the flow of the medicinal chemistry lead
optimization effort (C. Celatka et al., submitted for publica-
tion). Compounds were first screened against P. falciparum in
vitro (Tables 1 and 2) and subsequently in vivo against P.
berghei using 2 doses/compound (usually 100 and 200 mg/kg/
day b.i.d.; Table 3). Compounds showing the best activity and
in vitro metabolic properties (Table 2) were synthesized at a
large scale (50 g), and enantiomers were separated by super-
critical chiral chromatography for PK studies and for addi-
tional efficacy studies. These studies resulted in selection of
Genz-668764 (enantiomer of Genz-666984) for further char-
acterization on the basis of in vivo efficacy and preliminary
safety studies (see below). This compound showed low IC50s
against P. falciparum, P. berghei, and P. knowlesi in vitro (Table
1). P. knowlesi infects both humans and nonhuman primates
(15) and is used for in vitro drug susceptibility testing as a
surrogate for the human parasite P. vivax (6, 19).
Pharmacological properties of Genz-668764. Pharmacoki-
netic parameters of the lead compound Genz-668764 in mice,
rats, and cynomolgus monkeys are shown in Table 4. Clearance
in rodents was high (57.6 and 45.8 ml/min/kg for mouse and
rat, respectively) compared to that in monkeys (13.1 ml/min/
kg), while the half-life ranged from 7.3 h (mouse) to 4.3 h
(monkey). Bioavailability ranged from 20% (monkey) to 33%
(rat). Pharmacokinetic studies of Genz-668764 were per-
formed in both infected and uninfected mice (Fig. 1). The dose
levels and administration were not identical between the 2
studies: infected animals received a single oral dose of 50
mg/kg, while uninfected animals received 2 split doses of 27.5
mg/kg 7 h apart via oral gavage. However, no significant dif-
ferences in area under the curve (AUC) were observed. At
24 h postdosing, plasma concentrations ranged from 70 to 100
ng/ml by 24 h after dosing (Fig. 1), which is only ϳ2 times the
ED90 for P. falciparum in vitro.
In vitro safety assessment. Genz-668764 was tested in the
ExpressS (Cerep, Redmond, WA) panel of 55 primary molec-
ular targets at 10 ␮M. In only 3 cases (␬ [KOP, kappa opioid
receptor], 93%; ␮ [MOP, mu opioid receptor], 52%; and rat
sodium channel, site 2, 71% inhibition) was inhibition of
Ͼ50% observed, suggesting a low likelihood of off-target ef-
fects. Ames tests were negative, indicating no genotoxicity.
Genz-668764 was also tested for hERG inhibition; the IC50
was 36.6 ␮M for Genz-668764 (Table 1), suggesting a low
potential for arrhythmia for in vivo inhibition of hERG, based
on human efficacious dose predictions.
Preliminary in vivo safety assessment. Three aminoindoles
were evaluated in a preliminary 7-day rat safety study (see
Table S2 in the supplemental material). Because Genz-666984-
treated rats showed fewer clinical adverse findings than vehi-
cle-treated controls, the enantiomer Genz-668764 was tested
in a preliminary safety study in rats. Treatment for 7 days with
300 mg/kg/day Genz-668764 resulted in a 70% reduction in
reticulocytes (Fig. 2A; P Յ 0.01, Dunnett’s test). However,
reticulocytes rebounded during the 8 days following cessation
Enantiomers of Genz-644442. Because of the promising
characteristics of Genz-644442, the aminoindoles became the
focus of medicinal chemistry optimization, generating 321
novel compounds.
The medicinal chemistry approach (C. Celatka et al., sub-
mitted for publication) consisted primarily of exploring various
aspects of the structure to identify essential features while
improving potency and metabolic stability of the new com-
pounds. These molecules were initially synthesized and tested
as racemates around the 3 position of the indole ring of Genz-
644442 (Table 1); therefore, it was essential to determine po-
tency and metabolic stability of individual enantiomers. Genz-
644442 was synthesized as described above, and the 2
enantiomers (Genz-665848 and Genz-665849) were resolved
by supercritical chiral chromatography (see the supplemental
material). Genz-665848 and Genz-665849 were screened for in
vitro potency against P. falciparum and P. berghei, and the IC50s
were in the same range (Table 1). However, when it was tested
against P. berghei in vivo at 25 or 50 mg/kg/day (i.p.) for 4 days,
Genz-665848 was far less effective than Genz-665849 in sup-
pressing parasitemia (see Fig. S2A to C in the supplemental
material). At 50 mg/kg/day, Genz-665849 suppressed para-
sitemia to undetectable levels in all 5 mice, and 2 mice were
cured (see Fig. S2C in the supplemental material). No cure was
obtained with Genz-665848. When Genz-665849 was dosed
orally (see Fig. S3 in the supplemental material), efficacy in
suppressing parasitemia and achieving cures was similar to that
obtained using i.p. dosing (see Fig. S2 in the supplemental
material). The increased efficacy of Genz-665849 appears to be
due to greater plasma exposure compared to that of Genz-

2616
BARKER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. In vitro potency and selectivity
Potency (IC50 ͓nM͔)
Selectivityb
Mammalian cytotoxicity
hERG
concn (␮M)
Genz
no.
Structurea
(IC50 ͓␮M͔)
P. falciparum
P. falciparum
P. knowlesi
P. berghei
ANKA
Dermal
fibroblast
Kidney
epithelial
Eryth.
lysis
CHO
Dd2
3D7
H1
644442
285
200
336
1,560
Ͼ242
Ͼ242
Ͼ155
Ͼ25
665848
665849
644442 enant
644442 enant
1,460
675
665
313
NDc
ND
1,363
1,689
Ͼ242
Ͼ242
Ͼ242
Ͼ242
Ͼ155
Ͼ155
ND
ND
649969
665570
665571
665572
37
455
110
347
30
183
38
ND
318
ND
190
ND
ND
24
24
Ͼ129
Ͼ168
Ͼ147
Ͼ178
ND
ND
ND
ND
Ͼ62.5
Ͼ62.5
Ͼ62.5
Ͼ62.5
Ͼ62.5
Ͼ62.5
ND
108
2,987
667066
37
22
90
ND
Ͼ188
Ͼ188
Ͼ120
19
667664
666984
81
49
21
23
ND
38
ND
ND
Ͼ195
Ͼ195
Ͼ195
Ͼ195
Ͼ125
Ͼ125
ND
21
668762
668764
666984 enant
666984 enant
58
65
18
28
ND
26
342
331
Ͼ195
Ͼ195
Ͼ195
Ͼ195
Ͼ125
Ͼ125
ND
36.6
a The absolute stereochemistry of each enantiomer is currently being determined. 644442 enant, compound is an enantiomer of Genz-644442; structures are shown
for the racemic mixtures.
b Dermal fibroblast, normal human dermal fibroblasts (CC-2509; Clonetics); kidney epithelial, normal human renal proximal tubule cells (CC-2553; Clonetics); Eryth.
lysis, erythrocyte lysis.
c ND, not done.

VOL. 55, 2011
AMINOINDOLES ACTIVE AGAINST PLASMODIUM
TABLE 2. In vitro absorption, distribution, metabolism, and excretion propertiesa
2617
CLpred (ml/min/kg)
Human Rat
Human CYP inhibition IC50 (␮M)
% prot bnd
Perm.
Genz no.
Sol. ␮g/ml
Log D
(10Ϫ6 cm/s)
1A2
2C19
2C9
2D6
3A4
Hum
Mse
␮som
hep
␮som
hep
644442
665848
665849
649969
665570
665571
665572
667066
667664
666984
668762
668764
Ͼ35
Ͼ35
Ͼ35
28
59
48
54
40
63
60
72
111
37
47
43
43
3.0
2.8
2.7
3.6
2.3
2.8
2.4
3.1
2.2
3.1
3.0
3.0
8
12
11
8
7
8
32
34
25
41
40
27
17
34
33
32
39
33
Ͻ12
22
Ͼ5
Ͼ5
Ͼ5
Ͼ5
Ͼ5
Ͼ5
Ͼ5
Ͼ5
Ͼ5
Ͼ5
Ͼ5
89
89
85
97
78
84
67
70
70
89
89
91
93
86
90
97
74
86
69
76
76
92
94
94
6
15
Ͼ5
Ͼ5
Ͼ5
Ͼ5
9
6
10
8
13
6
9
8
Ͻ4
21
ND
30
16
21
ND
18
ND
ND
Ͼ5
Ͼ10
Ͼ5
Ͼ5
Ͼ5
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ5
Ͼ10
Ͼ5
Ͼ5
4.6
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ5
Ͼ10
Ͼ5
Ͼ5
Ͼ5
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ5
Ͼ10
Ͼ5
Ͼ5
Ͼ5
Ͼ10
Ͼ10
Ͼ10
Ͼ10
3.2
20
7
Ͼ10
Ͼ5
Ͼ5
26
20
Ͼ40
15
Ͼ40
Ͼ40
Ͼ40
8
6
9
1.0
8
Ͼ10
4.6
Ͼ10
2.2
11
9
10
a Sol., solubility; Perm., permeation in parallel artificial membrane permeation assay (PAMPA) membranes; CLpred, predicted clearance; ␮som, liver microsomes;
hep, hepatocytes; % prot bnd, percent protein bound in plasma; Hum, human plasma; Mse, mouse plasma; ND, not done.
of dosing and by study day 15 exceeded levels found in control
animals (P Յ 0.01). Treatment with 200 mg/kg/day resulted in
a more modest reduction in reticulocytes on day 8 (33%; P Յ
0.05). Animals in the 300-mg/kg/day group showed a reduced
rate of weight gain compared with controls (P Յ 0.05; Fig. 2B);
however, the rate of weight gain mirrored that of controls
following cessation of dosing. Liver weights were somewhat
elevated in all treatment groups in a dose-dependent fashion
on day 8 (Fig. 2C); however, by day 15, liver weights in the
300-mg/kg/day group returned to those found in control ani-
mals. On day 8, histopathology showed minimal hepatocellular
hypertrophy in all animals treated with 200 or 300 mg/kg/day
Genz-668764 and in one animal treated with 100 mg/kg/day,
but again, by day 15 liver histology of treated animals became
indistinguishable from that of controls. Animals treated with
200 or 300 mg/kg/day Genz-668764 showed significant (P Յ
0.01) thymus weight reductions of 35% and 44%, respectively,
on day 8 (Fig. 2D). In some animals dosed at Ն200 mg/kg/day,
mild lymphoid atrophy was observed on day 8, but by day 15 no
differences in thymus weight or atrophy were observed in
treated versus control animals. On the basis of all these find-
ings, the no-observable-adverse-effect level (NOAEL) for
Genz-668764 in rats is considered to be 200 mg/kg/day.
Results from the toxicokinetic assessment of Genz-668764 in
the 7-day preliminary safety assessment study are shown in
Table 5. Drug appears to accumulate, based on the comparison
of maximum concentration in plasma (Cmax) and AUC within
each dose treatment group for the 200-mg/kg and 300-mg/kg
dose groups on day 1 versus day 7. The Cmaxs were approxi-
mately 1.8- and 2.3-fold higher on day 7 than on study day 1 for
the 200-mg/kg and 300-mg/kg dose groups, respectively. The
areas under the curve from time zero to 24 h (AUC0–24s) were
approximately 1.4- and 2.1-fold higher on day 7 than on study
day 1 for the 200-mg/kg and 300-mg/kg dose groups, respec-
tively.
In vivo efficacy of Genz-668764 against P. berghei and P.
falciparum. The efficacy of compounds was determined in the
4-day suppressive test. Mice infected with the P. berghei N
clone were dosed orally twice/day with the enantiomers Genz-
668762 and Genz-668764, and parasitemia was assessed on day
4 postinfection. The ED50 of Genz-668764 (50 mg/kg/day) was
lower than that of Genz-668762 (72 mg/kg/day) (see Fig. S4A
and B in the supplemental material). Similarly, Genz-668764
had a higher cure rate. All mice treated with 100 mg/kg/day of
Genz-668762 were still parasitemic on day 4, whereas 2 animals
treated with the same dose of Genz-668764 were free of par-
asites on day 4 and did not show recrudescent parasites until
day 14 (see Fig. S4C in the supplemental material).
TABLE 3. In vivo efficacy of aminoindoles against P. berghei
Average day of
recrudescencec
(no. of animals/
total no.)
No. of
animals
curedd/
total no.
Dose
% suppression
on day 5b
Compound
(mg/kg/day)a
644442
665848
665849
50
50
100
80
D13 (5/5)
NAe
D12 (3/5)
(0/5)
D17 (2/5)
(0/5)
D10 (3/5)
D13 (3/5)
D6 (1/5)
NA
D17 (2/5)
D11 (1/5)
D13 (1/5)
NA
D14 (3/5)
D12 (2/5)
0/5
0/5
2/5
5/5
3/5
5/5
0/5
0/5
0/5
0/5
0/5
0/5
3/5
0/5
2/5
3/5
50
100
100
100
100
98
100
100
200
50
100
100
100
200
100
200
100
100f
200g
665570
665572
99
667066
667664
99
95
98
666984
99
99
668762
668764
81
Ͼ99
100
a The dose was administered orally in 2 split doses/day to groups of 5 mice.
b % suppression day 4, percent reduction in parasitemia compared with mean
of untreated controls on day 4, approximately 17 h after the final dose. Values of
100% suppression indicate that no parasites were detected upon microscopic
examination; animals may still contain parasites below the limit of detection.
c Average day of recrudescence is the average study day on which parasites
were detected, preceded by the letter D. Numbers in parentheses are the number
of animals in the dosing group that had recrudescent parasites/total number of
animals.
d No. cured, number of animals with no detectable parasites through day 30 as
a proportion of all the animals in the dosing group.
e NA, not applicable because all animals were parasitemic on day 4.
f Study with ANKA strain.
g Study with N clone of P. berghei.
An additional study was performed with P. berghei ANKA

2618
BARKER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
Genz-668764
TABLE 4. Pharmacokinetic parameters of Genz-644442 and Genz-668764
Genz-644442
Parametera
Mouse
Rat
Dog
Mouse
Rat
Monkey
CL (ml/min/kg)
CLrenal (ml/min/kg)
Vss (liters/kg)
t1/2 (h)
18.1
NAc
5.32
4.21
NA
29.0 Ϯ 18.7b
0.151 Ϯ 0.050
4.73 Ϯ 1.89
6.30 Ϯ 3.65
23 Ϯ 5
6.32 Ϯ 0.32
0.00657 Ϯ 0.00615
2.81 Ϯ 0.15
57.6
NA
13.5
7.30
NA
45.8 Ϯ 0.8
0.121 Ϯ 0.040
6.47 Ϯ 0.14
2.58 Ϯ 0.01
33 Ϯ 8
13.1 Ϯ 3.2
0.0162 Ϯ 0.0126
2.48 Ϯ 0.52
4.26 Ϯ 0.14
21 Ϯ 6
7.34 Ϯ 1.00
F (%)
80 Ϯ 25
a CL, calculated rate of clearance of compound from plasma; CLrenal, clearance through kidney; Vss, steady-state volume of distribution; t1/2, terminal half-life of the
compound; F, oral bioavailability.
b Values are standard errors of the means.
c NA, not available.
strain parasites. In this experiment, cures were observed in 3
out of 5 mice when they were dosed at 100 mg/kg/day b.i.d.
(Fig. 3A), indicating that parasites of the ANKA strain are
somewhat more sensitive to Genz-668764 than parasites of the
N clone, where we did not observe cures at the same dose.
Efficacy of Genz-668764 was also tested in the human-en-
grafted scid mouse model that supports growth of P. falciparum
erythrocytic stages. This study allows direct in vivo comparison
of efficacy against the rodent parasite P. berghei and the human
parasite P. falciparum in the same murine system. We observed
an ED50 of 40 mg/kg/day against P. falciparum, which was
slightly less efficacious than the ED50 of 26 mg/kg/day against
P. berghei ANKA (Fig. 3C; Table 6). This lower efficacy for P.
falciparum was unexpected, since in vitro the blood stages of P.
falciparum show 4- to 10-fold higher susceptibility than P. ber-
ghei blood stages (Table 1).
Human PK parameters and efficacious dose prediction. A
variety of human PK prediction approaches was employed
(12). The predicted human systemic plasma clearance and vol-
ume of distribution ranged from 0.5 to 10 ml/min/kg and 1 to
5 liters/kg, respectively. The highest degree of uncertainty was
in the human clearance prediction. Given the reasonable in
vitro-in vivo correlation between in vitro metabolic stability
in liver microsomes and hepatocytes and in vivo clearance in
preclinical species and differences in metabolism between hu-
man and preclinical species, we utilized the clearance value
predicted from the in vitro human microsomes and hepato-
cytes, which averaged 2 ml/min/kg. The other predicted human
PK parameters used to project the minimum efficacious dose
were 3 liters/kg for volume of distribution at steady state, 17 h
for half-life, 1 hϪ1 for the oral absorption rate constant, and
50% for oral bioavailability. Employing a one-compartment
model with first-order input and output and no lag time, the
predicted human minimum efficacious dose of Genz-668764 in
a 3-day QD dosing regimen was 421 mg/day/70 kg. This main-
tained the plasma trough level above the IC90 against Plasmo-
dium falciparum for 96 h after the last dose.
In vitro selection of resistance to Genz-644442. We at-
tempted to select for parasites (P. falciparum Dd2 strain) that
showed decreased sensitivity to Genz-644442 by cycling 4 in-
dependent cultures between periods of drug exposure (at 10ϫ
the IC50 for 1 week) and outgrowth (for 1 to 3 weeks). At
regular intervals, the IC50s of these parasites were determined.
Over the course of 10 cycles (270 days), we were unable to
select for parasites that showed an IC50 more than 2-fold
higher than that against the wild-type Dd2 strain. Similar stud-
ies are being performed using Genz-668764, and while they
have progressed through only 3 cycles (65 days), thus far there
has been no change in ED50.
DISCUSSION
We have followed two distinct strategies for identifying po-
tential antimalarials. The first consists of screening compound
libraries through whole-cell phenotypic assay to identify inhib-
itors of parasite growth; the second approach first screens
libraries against validated biochemical targets and then co-
optimizes using both biochemical and viability assays to de-
velop SARs. The advantage of using viability screening as the
primary assay is that it is independent of mechanism and
thereby can identify compounds active against new biochemi-
cal pathways previously not associated with known antimalarial
drugs. The disadvantage is that in the absence of a defined
molecular target, target structure is unavailable to guide opti-
mization efforts. The parent aminoindole molecule (Genz-
644442) described here was identified in a live-dead screen of
the small-molecule library at Harvard Medical School
(ICCB-L) and the Broad Institute (36); results from a parallel
program identifying inhibitors of Plasmodium dihydroorotate
dehydrogenase (DHODH) are described elsewhere (5).
FIG. 1. Pharmacokinetics of Genz-668764 in infected and unin-
fected mice. Circles, plasma concentrations of Genz-668764 following
single oral administration (50 mg/kg) to human-engrafted NSG mice
(n ϭ 3) infected with P. falciparum; squares, plasma concentrations of
Genz-668764 following single oral administration (50 mg/kg) to hu-
man-engrafted NSG mice (n ϭ 3) infected with P. berghei; triangles,
plasma concentrations in uninfected CD-1 mice receiving 2 doses (27.5
mg/kg/dose) of Genz-668764 7 h apart; horizontal dashed line, con-
centration required to attain the in vitro IC90 value for P. falciparum.

VOL. 55, 2011
AMINOINDOLES ACTIVE AGAINST PLASMODIUM
2619
FIG. 2. Significant findings for Genz-668764 in preliminary 7-day rat safety study. (A) Total number of reticulocytes in each dosing group on
day 8 (day following last dose) or day 15 (8 days after cessation of dosing). Dose group numbers indicate total daily dose (100, 200, or 300) in
mg/kg/day. Only the vehicle control and the 300-mg/kg/day groups were tested on day 15; ND, not done, and rats were euthanized on study day
8. Error bars indicate standard error of the mean. (B) Average body weight of animals on the study day indicated. Circles, vehicle alone group;
squares, animals receiving 100 mg/kg/day; dashed line, animals receiving 200 mg/kg/day; inverted triangles, animals receiving 300 mg/kg/day.
(C) Average weight of liver on day 8 or day 15. (D) Average weight of the thymus on study day 8 and study day 15.
Genz-644442 was first run through a screening cascade de-
signed to characterize potency, selectivity, and drug-like char-
acteristics, which supported advancing the compound to effi-
cacy studies against P. berghei in vivo, where 4 days’ dosing
either i.p. or orally resulted in significant suppression of par-
asitemia. The two enantiomers Genz-665848 and Genz-665849
did not differ significantly in potency against P. falciparum in
vitro when they were tested individually (Table 1), but unex-
pectedly, Genz-665849 showed higher efficacy against P. ber-
ghei in vivo, most likely due to lower systemic plasma clearance
and higher exposure in vivo.
Three hundred twenty-one aminoindole analogs were syn-
thesized and screened to develop SARs both around in vitro
potency against the parasite and for metabolic stability in
hepatocytes and microsomes. Selected compounds were as-
sessed for in vivo efficacy, for pharmacokinetic parameters, and
in 7-day rat safety studies. This resulted in selection of a lead
compound, Genz-668764. The potency of Genz-668764 against
P. falciparum strain 3D7 is 28 nM, which is 7.1-fold more
potent than Genz-644442 and within the 30 nM considered a
threshold for potency of antimalarial compounds (22). Potency
against the Dd2 strain was approximately 2-fold less, at 65 nM.
TABLE 5. Toxicokinetic parameters of Genz-668764 on study days 1 and 7
a
Cmax (ng/ml)
Cmax/dose (ng/ml)
Tmax (h)
Mean
AUC0-24 (ng ⅐ h/ml)
AUC/dose
Mean target
dose (mg/kg)
Study day
1
Mean
SD
Mean
SD
SD
Mean
SD
Mean
SD
100
200
300
2,450
2,680
2,850
285
172
718
24.5
13.4
9.51
2.85
0.854
2.42
8.0
13.3
18.7
0
9.24
9.24
35,668
50,053
51,425
5,131
6,663
8,247
357
250
171
51.3
33.3
27.5
7
100
200
300
2,180
4,820
6,660
21.2
1,190
381
21.8
24.1
22.2
0.212
5.95
1.31
8.0
8.0
8.0
0
0
0
32,239
68,824
106,973
482
14,748
4,607
322
344
357
4.8
73.7
15.4
a Tmax, time of maximum concentration in plasma.

2620
BARKER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
similar to that for P. falciparum strain 3D7, suggesting that this
aminoindole might be active against blood stages of P. vivax.
Selectivity studies in vitro showed that the potency of Genz-
668764 against P. falciparum parasites was at least 1,900-fold
greater than that against erythrocytes or mammalian primary
cells. Further, the IC50 for hERG inhibition by Genz-668764
was 36.6 ␮M, indicating that this compound is unlikely to
inhibit cardiac hERG channels in vivo.
As was the case for Genz-665848 and Genz-665849 (enan-
tiomers of Genz-644442), the enantiomers Genz-668762 and
Genz-668764 (enantiomers of Genz-666984) differed in their
efficacy against P. berghei in vivo, with Genz-668764 being more
active. The efficacy of Genz-668764 against P. berghei ANKA
strain parasites was slightly greater than that against the N
clone. The strain-specific difference in efficacy was similar to
that observed when the efficacy of Plasmodium DHODH in-
hibitors against P. berghei was studied (5). While the reason for
this is unclear, it may lie in the laboratory history of each
strain: the N clone, derived from the K173 strain, has been
maintained exclusively by syringe passage of blood-stage par-
asites (32), while ANKA strain infections are often maintained
by sporozoite challenge (13). Interestingly, the ED50 of Genz-
668764 against the P. falciparum 3D7 strain in the NSG model
was very similar to that against P. berghei ANKA strain para-
sites in the same model. This was unexpected because blood
stages of P. berghei showed 5- to 12-fold lower sensitivity in
vitro than those of P. falciparum and illustrates the difficulty of
extrapolating from the in vitro potency data.
Preliminary 7-day safety studies of different aminoindole
analogs suggested that at high doses, reduced weight gain and
reticulocytopenia appear to be class effects of the amino-
indoles, while effects on white blood cells (WBCs) and lym-
phocytes are not and instead represent properties of individual
analogs. Since the racemic mixture Genz-666984 had the few-
est potentially adverse effects and had good efficacy (see Table
S3 in the supplemental material), we purified the individual
enantiomers and selected the more efficacious enantiomer,
Genz-668764, for further testing. The 7-day rat safety study
established the NOAEL at 200 mg/kg/day. As had been seen
for the parent compound, the primary findings were reductions
in rate of weight gain and in number of reticulocytes. However,
7 days after cessation of dosing, the rate of weight gain by
animals treated with 300 mg/kg/day then became identical to
FIG. 3. In vivo efficacy against P. berghei ANKA strain and P. fal-
ciparum. Genz-668764 was tested against P. berghei ANKA strain (A
and B) and in the NSG model against P. falciparum and P. berghei
ANKA (C). (A) Dose-response curve showing percent inhibition (rel-
ative to vehicle-treated controls) on study day 4. Solid line, response of
P. berghei ANKA strain (tested at SwissTPH); dotted line, response of
P. berghei N clone (tested at UPR; data are shown for comparison).
The ED50 of Genz-668764 against ANKA was 19 mg/kg/day, whereas
the ED50 against the N clone was 32 mg/kg/day. Error bars are stan-
dard errors of the means. (B) Kaplan-Meier representation of day of
recrudescence. Cont, vehicle-treated controls; dashed line; 100 b.i.d.,
animals dosed at 100 mg/kg/day; dotted line, 100 q.d., animals dosed at
100 mg/kg/day once/day. (C) Dose-response curve showing percent
inhibition of parasitemia 24 h after 4 days of dosing in the human-
engrafted NSG model (P. falciparum; circles, dashed line) and wild-
type nonengrafted NSG mice (P. berghei ANKA strain; squares, solid
line).
TABLE 6. Efficacy of Genz-668764 in different rodent modelsa
ED (mg/kg/day)
SwissTPH
P. berghei
ANKA strain
GFP MRA-865
GSK
UPR
P. berghei
N-clone
GSK P. berghei
ANKA (QD)
P. falciparum
Pf3D70087/N9
(QD)
50%
90%
68
50%
90%
50%
26
90%
48
50%
90%
32
19
30
40
74
a Efficacy in rodent models performed at UPR, SwissTPH, and GlaxoSmith-
Kline (GSK). UPR and SwissTPH studies are nearly identical, but use N-clone
and ANKA strain P. berghei parasites, respectively. In UPR and SwissTPH
studies, animals were dosed b.i.d. GlaxoSmithKline uses NSG mice engrafted
with human erythrocytes for P. falciparum assays and wild-type nonengrafted
NSG mice for P. berghei evaluation. In these studies, animals were dosed QD.
One study done at UPR looked at the effect of administering compound 3
times/day (t.i.d.), every 8 h.
Potency was also assessed against P. knowlesi in vitro. The
latter species infects both humans and nonhuman primates
(15) and is used for in vitro susceptibility testing as a surrogate
for P. vivax (6, 19). For P. knowlesi, the IC50 (26 nM) was very

VOL. 55, 2011
AMINOINDOLES ACTIVE AGAINST PLASMODIUM
2621
that of untreated controls and the number of reticulocytes
rebounded, indicating that these effects were transient. Be-
cause these findings were modest and were fully reversible,
they do not represent an impediment to ongoing development
of Genz-668764.
In vitro testing showed that Genz-668764 inhibited the cyto-
chrome P450 enzyme CYP3A4/5 at 2.2 ␮M (Table 2).
CYP3A4/5 is important in metabolism of antimalarials (34),
including dapsone (21), proguanil (4), quinine (41), and halo-
fantrine (10). To reduce the risk of selecting for resistance, no
antimalarial is likely to be administered as monotherapy.
Therefore, understanding possible drug-drug interactions will
be important in selection of companion drugs, as is the case
with any new antimalarial compound.
antimalarial compounds with good potency against P. falcipa-
rum in vitro. Genz-668764 is efficacious against both P. falcip-
arum and P. berghei in the human-engrafted NSG model, and
cure has been achieved against P. berghei in the adapted Peters
rodent model (27). In vitro activity against P. knowlesi suggests
that Genz-668764 might also be active as a schizontocide
against P. vivax. Preliminary 7-day rat safety studies identified
reversible reductions in weight gain and reticulocytopenia as
the primary findings at exposures above those expected to be
required for dosing in humans. The minimum efficacious hu-
man dose projection for Genz-668764 met the target product
profile: oral daily dosing once/day of less than 1 g per day for
3 days maintaining trough levels above the IC90 for 4 days after
the last dose. The compound exhibited a reasonable predicted
human therapeutic index, based on the plasma exposure in
rats at the NOAEL of 200 mg/kg/day. Finally, while the
mechanism of action is unknown, the repeated failure to
select for drug resistance in vitro over 270 days suggests that
this could be an exciting companion drug for the next gen-
eration of antimalarials.
At present, the mechanism of aminoindole action against
Plasmodium is unknown. On the basis of microscopic exami-
nation of synchronized in vitro P. falciparum cultures, the tro-
phozoite stage appears to be the target (J. F. Cortese, unpub-
lished data). The aminoindoles as a class are 2 to 3 times more
potent in the wild-type 3D7 strain than in the multidrug-resis-
tant Dd2 strain. Preliminary work implicates polymorphisms in
the multidrug-resistant transporter P. falciparum mdr1
(pfmdr1) in this differential activity, and not, for most of the
aminoindoles, including Genz-668764, the chloroquine resis-
tance transporter pfcrt. More detailed studies in progress will
elucidate the precise mechanisms of this differential activity.
The potency of Genz-668764 was shown to be essentially the
same against P. falciparum D10 strain and the transgenic D10-
scDHODH strain (A. B. Sidhu, unpublished data). The D10-
scDHODH strain contains a cytoplasmic copy of Saccharomy-
ces cerevisiae DHODH which can rescue parasites from
compounds that inhibit the mitochondrial electron transport
system (25, 26). The inability of the yeast enzyme scDHODH
to rescue parasites from Genz-668764 suggests that the amino-
indoles do not target the mitochondrial electron transport sys-
tem. Finally, attempts to elucidate the mechanism of action
have been rendered more difficult by our inability to select for
resistance. Other studies have been able to utilize genomic
differences between sensitive and resistant parasites to identify
the putative target (31).
Genz-644442 (the parent compound) was exposed to 10
cycles of culturing parasites in the presence of 10ϫ IC50 for a
week, followed by 2 weeks’ outgrowth in the absence of drug
pressure (270 days total). To date, Genz-668764 has been ex-
posed to 3 cycles (65 days). In both cases, it has not been
possible to select parasites with IC50s Ͼ2 times that of the
unselected parent. While it is hypothetically possible that this
is because the drug is biostatic rather than biocidal, this does
not appear to be the case, based upon microscopic examina-
tion, in which pyknotic forms are observed following drug
exposure. Further, there is significant evidence both in vitro
for pyrimethamine and mefloquine (23) and in vivo for
artemisinin (35) that a subset of parasites may be dormant
or quiescent, giving rise after removal of drug pressure to a
recrudescent population of parasites with unaltered sensi-
tivity to the drug. Whether this is the case for the aminoin-
doles awaits further confirmation, but the outgrowth of re-
crudescent populations in vitro following release from drug
pressure would suggest that it is.
ACKNOWLEDGMENTS
This work was supported by SPARC funds from the Broad Institute,
with funds from Medicines for Malaria Venture, and with support
from the Humanitarian Assistance for Neglected Diseases Initiative at
Genzyme Corporation. Partial support for infrastructure to A.E.S. was
provided by NIH-RCMI-NCRR-G12RR030-51. J.-W. Lin is supported
by a grant from the China Scholarship Council.
We gratefully acknowledge (in memoriam) the superb technical
assistance provided by Miryam Garcia-Rosa.
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