Long-Lasting and Fast-Acting in Vivo Efficacious Antiplasmodial Azepanylcarbazole Amino Alcohol

Article abstract of DOI:10.1021/acsmedchemlett.7b00391

With ～429,000 deaths in 2016, malaria remains a major infectious disease where the need to treat the fever symptoms, but also to provide relevant post-treatment prophylaxis, is of major importance. An azepanylcarbazole amino alcohol is disclosed with a long- and fast-acting in vivo antiplasmodial efficacy and meets numerous attributes of a desired post-treatment chemoprophylactic antimalarial agent. The synthesis, the parasitological characterization, and the animal pharmacokinetics and pharmacodynamics of this compound are presented along with a proposed target.

Full text of DOI:10.1021/acsmedchemlett.7b00391

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Letter
Long-lasting and fast-acting in vivo efficacious
antiplasmodial azepanylcarbazole amino alcohol
Nada Abla, Sridevi Bashyam, Susan A. Charman, Béatrice Greco, Philip Hewitt, Maria Belén
Jiménez-Díaz, Kasiram Katneni, Holger Kubas, Didier Picard, Yuvaraj Sambandan, Laura X
Sanz, Dennis A. Smith, Tai Wang, Paul A. Willis, Sergio Wittlin, and Thomas Spangenberg
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00391 • Publication Date (Web): 28 Nov 2017
Downloaded from http://pubs.acs.org on December 4, 2017
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Longꢀlasting and fastꢀacting in vivo efficacious antiplasmodial azeꢀ
panylcarbazole amino alcohol.
Nada Abla,1,3 Sridevi Bashyam,4 Susan A. Charman,8 Béatrice Greco,1 Philip Hewitt,2 Maria Belén Jiꢀ
ménezꢀDíaz,6† Kasiram Katneni,8 Holger Kubas,2 Didier Picard,9 Yuvaraj Sambandan,4 Laura Sanz,6
Dennis Smith,7 Tai Wang,9†† Paul Willis,3 Sergio Wittlin,5 Thomas Spangenberg1*
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1Merck Global Health Institute, Ares Trading S.A., a subsidiary of Merck KGaA (Darmstadt, Germany), Coinsins, Switzerꢀ
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land, Merck, KGaA, Darmstadt, Germany, 3Medicines for Malaria Venture, Geneva, Switzerland, 4Syngene International,
Bangalore, India, 5Swiss TPH, Basel, Switzerland, 6GSK,Tres Cantos, Spain, 7MMV Expert Scientific Advisory Committee;
8Centre for Drug Candidate Optimisation, Monash University, Parkville, VIC, Australia ; 9Département de Biologie Celluꢀ
laire, Université de Genève, Sciences III, 30 quai ErnestꢀAnsermet, CHꢀ1211, Genève 4, Switzerland.
KEYWORDS antiplasmodial • carbazole • amino alcohol • pharmacokinetics • pharmacodynamics • Hsp90
ABSTRACT: With ~429,000 deaths in 2016, malaria remains a major infectious disease where the need to treat the fever sympꢀ
toms, but also to provide relevant postꢀtreatment prophylaxis, is of major importance. An azepanylcarbazole amino alcohol is disꢀ
closed with a longꢀ and fastꢀacting in vivo antiplasmodial efficacy and which meets numerous attributes of a desired postꢀtreatment
chemoprophylactic antimalarial agent. The synthesis, the parasitological characterization, the animal pharmacokinetics and pharꢀ
macodynamics of this compound are presented along with a proposed target.
One third of the world’s population is at risk of malaria, a
disease caused by protozoan parasites of the genus Plasmodi-
um upon an infectious mosquito bite. Although the burden of
malaria has reduced by 37% since 2000, the number of victims
remains unacceptable with ~429,000 deaths in 2016 where a
vast majority are children below 5 years of age.1 Cases of
malaria are seasonal due to the seasonal difference in the
abundance of the mosquito vector and subjects can undergo
multiple malaria infections/reꢀinfections in a year, therefore
highlighting the need to treat the acute infection and also to
provide relevant postꢀtreatment prophylaxis. This target comꢀ
pound profile has been framed as being a longꢀacting blood
stage antimalarial.2 Ideally, this compound should be able to
maintain its efficacious plasma concentration for at least four
weeks, which is typically needed in high transmission areas
with high reinfection rates, thus providing significant postꢀ
treatment prophylaxis.2 Identifying molecules which have a
very long halfꢀlife is challenging as they are typically highly
lipophilic and are often bases, both of which increase the
affinity for tissue membranes and thereby increase the volume
of distribution and resulting halfꢀlife. Consequently, such
molecules have a higher risk to be promiscuous for human
receptors and cause adverse events.3,4 Examples of long acting
blood stage antimalarial agents are piperaquine (4ꢀ
aminoquinoline) and mefloquine (amino alcohol) with halfꢀ
lives of 33 and 14 to 28 days, respectively.5,6
numerous attributes of a desired fast and long acting antimaꢀ
larial agent. Also as opposed to its enantiomer, (ꢀ)ꢀ1 showed
reduced binding on monoamine transporters (i.e. norepinephꢀ
rine, dopamine and 5ꢀHT, see Supporting Information). Carꢀ
bazole scaffolds are found in nature, with reported antiplasꢀ
modial activity.8.9.10 Also synthetic analogues have been deꢀ
scribed with such activity along with BAX channel modulaꢀ
tion and more recently as neuroprotective agents.11,12,13,14
Compound (ꢀ)ꢀ1 [(3S,4S)ꢀ4ꢀ(3,6ꢀbis(trifluoromethyl)ꢀ9Hꢀ
carbazolꢀ9ꢀyl)azepanꢀ3ꢀol] is a representative member of the
series and has an 3S,4S absolute stereochemistry configuraꢀ
tion. Figure 1 depicts the chemical route for the rapid access to
optically active (ꢀ)ꢀ1 in 5 linear steps and one chiral resolution
on a multiꢀgram scale.
The 3,6ꢀbis(trifluoromethyl)ꢀ9Hꢀcarbazole, 6, was obtained
efficiently in two steps using palladiumꢀmediated couplings.
First, pꢀ(trifluoromethyl)aniline was assembled with pꢀ
(trifluoromethyl)iodobenzene through a BuchwaldꢀHartwig
crossꢀcoupling reaction to produce the desired adduct, 7, in
96% yield. Then an intramolecular palladium(II)ꢀcatalyzed
coupling via C−H activation of the diarylamine, 7, yielded the
desired carbazole, 6, in 56%.15 In parallel, the use of standard
alkylating procedures enabled the formation of the bisꢀolefin,
5, with a yield of 87%. Next, a 5% loading of Grubbs I cataꢀ
lyst afforded the cyclic olefin, 4, by ring closing metathesis in
57% yield. The latter product was reacted with mꢀCPBA to
form the desired epoxide, 3, with a yield of 44%. Finally, the
carbazole, 6, and the epoxide, 3, were treated with cesium
carbonate in DMF to allow – with a favorable regioselectivity
of 2:1 – the slow formation of the desired transꢀisomer, 2,
resulting in an isolated yield of 39%. At that stage no residue
(<5 ppm) of Ru or Pd could be detected. Subsequently, the
Based on previous work on antiplasmodial acyclic and raꢀ
cemic aminoꢀalcohol carbazoles,7 we disclose the synthesis,
the parasitological characterization, the animal pharmacokinetꢀ
ics and pharmacodynamics of compound (ꢀ)ꢀ1, a member of a
novel azepanyl amino alcohol carbazole series (Figure 1). The
latter has shown improvement of the acyclic series as it met
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Boc protecting group was quantitatively removed by treatment
with HCl in dioxane, followed by a chiral supercritical fluid
chromatography, which allowed the isolation of the desired
enantiomer. An Xꢀray diffraction of the monoꢀcrystal indicatꢀ
ed unambiguously the absolute stereochemistry to be 3S,4S.
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Figure 1. Access to optically active (ꢀ)ꢀ1 and display of the singleꢀcrystal Xꢀray diffraction.
Compound (ꢀ)ꢀ1 was first assessed in the standard in vitro P.
falciparum infected erythrocytes (72
incubation, 3Hꢀ
EC50 generated a similar profile with a PRR of 4.5 Log units,
h
suggesting that the minimum parasiticidal concentration correꢀ
sponds to 3 times EC50 or higher. Compared to chloroquine, a
24 h lag time was observed suggesting that the mode of action
is likely to be different. In synchronized P. falciparum culꢀ
tures, compound (ꢀ)ꢀ1 showed a slightly (~2 fold) more proꢀ
nounced activity on schizonts than on ring stages (see Supꢀ
porting Information).19 Finally, it has not been possible to
generate resistant parasites with compound (ꢀ)ꢀ1 using 2 times
the EC90 at various in vitro parasite levels (106ꢀ109 P. falcipa-
rum Dd2 parasites, in triplicates).20 In addition, attempts using
a drug ramping protocol to generate resistant mutants have
failed [data not shown].
hypoxanthine incorporation readout). On a highly sensitive
strain, such as P. falciparum NF54, compound (ꢀ)ꢀ1 displayed
a parasite growth inhibition with an EC50 of 2.4 nM and
freeEC50 of 0.17 nM when correcting for albumax® media bindꢀ
ing (92.9% bound). Similar data were obtained for the enantiꢀ
omer (+)ꢀ1. As crossꢀresistance to existing antimalarials is
highly undesirable, compound (ꢀ)ꢀ1 was then studied in a
panel of strains showing resistance to antimalarial agents. EC50
values ranged from 0.4 to 3.4 nM, with relative variations to
the NF54 strains ranging from 0.2 to 1.4 fold, where EC90
values were found to be ~2 fold the EC50s, suggesting that no
existing mechanism of resistance is to be expected (Table
1).16,17
P. falciparum strain
EC50 in nM (72 EC50/EC50 (relaꢀ
h,
3Hꢀ tive to NF54)
hypoxanthine
incorporation)
2.4 (2.3, 2.5)
0.7 (0.6, 0.8)
2.1 (1.5, 2.7)
0.4 (0.3, 0.5)
3.0 (3.0, 3.0)
3.4 (4.2, 2.5)
1.3 (1.6, 0.95)
3.2 (2.3, 4.1)
3.0 (2.9, 3.1)
1.0
0.3
0.9
0.2
1.2
1.4
0.5
1.3
1.3
NF54
K1
HB3
7G8
TM90C2B
D6
V1/S
Dd2
FCB
Figure 2. Parasite Reduction Ratio against P. falciparum 3D7A
strain for compound (±)ꢀ1, artemisinin, chloroquine, atovaquone,
pyrimethamine and at 10 times the EC50 and/or 3 times the EC50.
Table 1. Parasite growth inhibition expressed as EC50 in nM (72 h
incubation, Hꢀhypoxanthine incorporation readout, mean (indiꢀ
vidual data, n=2) on a panel of P. falciparum strains for (ꢀ)ꢀ1.
3
Next a full characterization of compound (ꢀ)ꢀ1 was perꢀ
formed and Table 2 summarizes the physicochemical, the in
vitro drug metabolism and pharmacokinetic (DMPK) properꢀ
ties in human and preclinical species, as well as the in vivo
pharmacokinetic (PK) profiles in mouse, rat and dog. The
compound has clear drugꢀlike properties, but sits on the lipoꢀ
philic edge. The measured pKa of the azepane was 8.05 allowꢀ
ing acceptable kinetic solubility in acidic pH regions. With
respect to in vitro metabolism, compound (ꢀ)ꢀ1 had excellent
microsomal stability across different species and hepatocyte
With this information in hand, compound (±)ꢀ1 was then asꢀ
sessed in a Parasite Reduction Ratio (PRR) assay to evaluate
the rate of killing.18 In brief, this method measures the in vitro
direct effect of antimalarial compounds on the parasite viabilꢀ
ity, based on a limiting serial dilution of treated parasites and
reꢀgrowth monitoring. Figure 2 shows the in vitro results, and
it can be concluded that (±)ꢀ1 affects parasite viability and
achieves a rapid parasite clearance after 48 h, similar to chloꢀ
roquine with a reduction ratio of 4.8 Log units at 10 times the
EC50. Also, a reduction of the concentration to 3 times the
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data showed a similar trend of low turnover. As a result, no
across the doses tested. The effect of treatment on the parasites
was measured by flow cytometry. Under the experimental
conditions used, compound (ꢀ)ꢀ1 was efficacious against P.
falciparum. It was evident that 10 and 30 mg·kgꢀ1 resulted in
similar levels of efficacy with an initial 24 h lag phase prior to
reduction of parasites and reaching the lower limit of quantifiꢀ
cation at day 4 post treatment. Therefore, the maximum killing
rate was obtained with a dose ≥ 10 mg·kgꢀ1.
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metabolites were found in human hepatocytes even after inꢀ
creasing the incubation time (96% of parent remaining after 5
h incubation). The permeability of the compound in Cacoꢀ2
cells was determined in the presence of human plasma in both
donor and acceptor sides in order to decrease the nonꢀspecific
binding observed with this lipophilic compound. Following
correction for the unbound concentration under these condiꢀ
tions, the permeability was excellent, with apparent permeabilꢀ
ity (Papp) values of 120x10ꢀ6 cm/s. The fraction unbound in
plasma was in the range of 1.3ꢀ3.5% across all species. After
iv administration in the mouse (0.2 mg·kgꢀ1), compound (ꢀ)ꢀ1
demonstrated a low clearance (~3% liver blood flow, Qh),
with a high volume of distribution (~10 L/kg) and a very long
halfꢀlife (~90 h). The maximum concentration, Cmax, was
achieved 6 h after po administration (0.5 mg·kgꢀ1) and plasma
concentrations declined thereafter with a halfꢀlife similar to
that after iv administration (~86 h). The bioavailability was
~97%, in keeping with the low clearance. Compound (ꢀ)ꢀ1
overall shows a trend for a low clearance, high volume of
distribution and a very long t1/2 in mouse (iv and po). The
corresponding enantiomer (data not shown) has a similar PK
profile. The PK profiles in rat and dog also led to very long
halfꢀlives after oral administration (160 and 69 h in rat and
dog, respectively). Additionally, compound (ꢀ)ꢀ1 was shown to
cross the bloodꢀbrainꢀbarrier without accumulating. Brain
binding was high based on in vitro data (>99.9%), resulting in
low calculated free brain concentrations and expected high
safety margins vs. brain targets.
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Figure 3. PK and PD plot for (ꢀ)ꢀ1 after a single oral dose treatꢀ
ment at 1 (green), 3 (blue), 10 (yellow) and 30 (red) mg·kgꢀ1.
Solid lines indicate blood concentration in µM and dashed lines
indicate % parasitemia on a Log10 scale. Lowest limit of quantiꢀ
fication of parasitemia by Flow Cytometry is 0.01. Vehicleꢀ
treated group showed an appropriate growth (1.18% parasitemia
at day 7, n=3); Chloroquine was used as positive control with an
ED50 of 2.8 mg·kgꢀ1.
MW (g/mol)
416.36
LogP / LogD pH 4.6 / 4.1
7.4*
pKa*
8.05
Solubility in µg/mL
1.5 (PBS., pH 7.4); 10 (FaSSIF
pH 6.5); 83.5 (FeSSIF pH 5)
Microsomal CLint in Human <10, Mouse <10, Rat <10,
µL/min/mg prot
Monkey <10, Dog <10
Cacoꢀ2 Permeability 120 (efflux ratio: 0.8)
(Papp x 10ꢀ6 cm/s)
The estimated ED90, the dose that reduces parasitemia at day
7 after infection by 90 % with respect to vehicleꢀtreated mice,
was 3.6 mg·kgꢀ1. The AUCED90, which is the average daily
exposure in whole blood necessary to reduce P. falciparum
parasitemia in peripheral blood at day 7 after infection by 90
% with respect to vehicleꢀtreated mice, was 2.2 ꢁg·h·mLꢀ
1·dayꢀ1. At the dose giving the maximum efficacy (10 mg·kgꢀ
1), the maximum concentration, Cmax, was ~1 µM and this
concentration was sustained over several days (> 5 days).
Plasma Protein
binding: Fub in %
Blood/Plasma ratio
Human=3.5, Dog=1.3, Rat=2.5, Mouse=2.6
Human=1.5; Mouse=1.6
14ꢀday in vivo PK**
po t1/2 (h)
Mouse
86
Rat
160
Dog
69
CL (L/h/Kg)
0.156
(3% Qh)
10
0.0963
(3% Qh)
12.5
0.179
(10% Qh)
10
Vss (L/Kg)
F%
Finally, structurally similar acyclic amino alcohol carbaꢀ
zoles have recently been described as potential speciesꢀ
selective inhibitors of the molecular chaperone Hsp90 of P.
falciparum (PfHsp90), a characteristic which is likely to conꢀ
tribute to their antiplasmodial activity.7,22 Hsp90 is a highly
abundant and conserved cytosolic protein, which is essential to
maintain protein homeostasis in eukaryotes, including protoꢀ
zoan pathogens.23,24,25 Using a similar in silico methodology to
previous work22, compound (ꢀ)ꢀ1, a conformationally restricted
analogue, underwent docking studies and showed that a simiꢀ
lar binding mode is possible to the acyclic amino alcohol
carbazole (see Supporting Information). Therefore one could
speculate about a contribution of PfHsp90 inhibition by (ꢀ)ꢀ1
in the overall antiplasmodial activity.
97
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100
*measured (see Supporting Information); **single iv and po
administration
Table 2. PhysChem properties & PK parameters for (ꢀ)ꢀ1 in
mouse, rat and dog.
Subsequently, compound (ꢀ)ꢀ1 was assessed in a murine
model of Plasmodium falciparum malaria, whereby SCID
mice engrafted with human erythrocytes are used to determine
its oral therapeutic efficacy against Plasmodium falciparum
3D7.21 In brief, efficacy was assessed following administration
of a single oral dose of compound (ꢀ)ꢀ1 (1, 3, 10 and 30
mg·kgꢀ1) at day 3 after infection (Figure 3). The blood levels
of compound (ꢀ)ꢀ1 were measured during the 120 h period
after administration in all mice. The PK appeared to be linear
In conclusion, a novel long and fast acting antiplasmodial
azepanyl amino alcohol carbazole series, represented by comꢀ
pound (ꢀ)ꢀ1, has been disclosed. The compound was rapidly
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Assistance from Christian Scheurer and Sibylle Sax from the
Swiss Tropical Institute and Public Health is gratefully acknowlꢀ
edged.
accessed in 5 linear steps followed by a final chiral resolution.
The parasitological characterization showed an EC50 in the low
nanomolar range against a panel of resistant P. falciparum
mutants along with low propensity to develop mutant paraꢀ
sites. In addition to being active against parasites it showed a
rate of kill similar to chloroquine, with an additional 24 h lag
phase. From a pharmacokinetic and pharmacodynamic perꢀ
spective, compound (ꢀ)ꢀ1 has met numerous attributes of a
desired fast and long acting antimalarial agent with a t1/2 in
mouse (iv and po) that is above 24 h and an ED90 of 3.6 mg·kgꢀ
1. The lowest dose giving the fastest clearance after a single
oral administration was 10 mg·kgꢀ1, which corresponds to a
total Cmax of ~1 µM. Based on previous in silico work, one
could speculate the antiplasmodial activity of compound (ꢀ)ꢀ1
could partly be due to the inhibition of P. falciparum Hsp90.
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ABBREVIATIONS
BAX, Bclꢀ2ꢀassociated X protein; SCID mice, severe combined
immunodeficient mice. hERG, the human EtherꢀàꢀgoꢀgoꢀRelated
Gene.
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associated with increased risk of QT interval prolongation
which may increase the risk of episodes of torsades de
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Supporting Information
Synthesis and analytical data of intermediates and compound (ꢀ)ꢀ
1, details for P. falciparum in vitro and in vivo assays, mouse
pharmacokinetic studies, in vitro physicochemical, DMPK and
pharmacology assays, and in silico docking.
This material is available free of charge via the Internet at
http://pubs.acs.org
Supporting Information_1 (PDF)
AUTHOR INFORMATION
Corresponding Author
*thomas.spangenberg@merckgroup.com
Present Addresses
†current address: The Art of Discovery, SL, Biscay Science and
Technology Park, Spain. ††current address: Chemical Biology
Program, Memorial Sloan Kettering Cancer Center,417 East 68th
Street, New York, NY 10065, USA.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
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[17] Identification and Deconvolution of Antimalarial Compounds
CrossꢀResistance Signals using MultidrugꢀResistant Plasmodium
falciparum Strains. Chugh, M.; Scheurer, C.; Sax, S.; Bilsland, E.;
Van Schalkwyk, D. , A.; Wicht, K. J.; Brand, F.; Sharma,
Funding Sources
This research was generously supported by Merck KGaA and
MMV donors.
ACKNOWLEDGMENT
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