Diversity-oriented synthesis yields a novel lead for the treatment of malaria

Article abstract of DOI:10.1021/ml200244k

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure-activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Full text of DOI:10.1021/ml200244k

Letter
pubs.acs.org/acsmedchemlett
Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment
of Malaria
Richard W. Heidebrecht, Jr.,*,†,§ Carol Mulrooney,† Christopher P. Austin,‡ Robert H. Barker, Jr.,○
Jennifer A. Beaudoin,† Ken Chih-Chien Cheng,‡ Eamon Comer,† Sivaraman Dandapani,† Justin Dick,§
Jeremy R. Duvall,† Eric H. Ekland,∥ David A. Fidock,∥,⊥ Mark E. Fitzgerald,† Michael Foley,†
Rajarshi Guha,‡ Paul Hinkson,○ Martin Kramer,○ Amanda K. Lukens,†,§ Daniela Masi,†
Lisa A. Marcaurelle,† Xin-Zhuan Su,# Craig J. Thomas,‡ Michel Weïwer,† Roger C. Wiegand,†,§
Dyann Wirth,†,§ Menghang Xia,‡ Jing Yuan,# Jinghua Zhao,‡ Michelle Palmer,† Benito Munoz,†
and Stuart Schreiber†,∇
†The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142, United States
‡Chemical Genomics Center, National Institutes of Health, Bethesda, Maryland 20892, United States
§Harvard School of Public Health, Huntington Avenue, Boston, Massachusetts 02115, United States
∥Department of Microbiology and Immunology, Colombia University, New York, New York 10032, United States
⊥Department of Medicine, Division of Infectious Diseases, Colombia University, New York, New York 10032, United States
#National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, United States
∇Howard Hughes Medical Institute, Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138,
United States
○Genzyme Corporation, 153 Second Avenue, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: Here, we describe the discovery of a novel antimalarial agent using
phenotypic screening of Plasmodium falciparum asexual blood-stage parasites.
Screening a novel compound collection created using diversity-oriented synthesis
(DOS) led to the initial hit. Structure−activity relationships guided the synthesis of
compounds having improved potency and water solubility, yielding a subnanomolar
inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an
excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable
in human plasma. This compound is available via the molecular libraries probe
production centers network (MLPCN) and is designated ML238.
KEYWORDS: diversity-oriented synthesis, malaria, macrocycle, high-throughput screening, phenotypic screen, infectious disease,
molecular libraries probe production centers, stereochemical structure−activity relationships
alaria afflicts approximately 225 million people and leads
densities, and insufficient health care funding. These factors have
combined to present a challenging clinical environment.8
Combination therapies have been used to mitigate the rate of
resistance in several diseases.9 As such, new small molecules with
novel mechanism(s) of action have the greatest potential to
enable a successful path to the treatment of drug-resistant
malarial infections.10,11
M
to ∼780 000 deaths per year.1,2 The apicomplexan
Plasmodium parasite responsible for this condition is contracted
via Anopheles mosquito bites and lives a part of its life cycle
within this vector.3 Endemic areas are limited to the tropics,
where continuous mosquito breeding is possible. Several species
of Plasmodium can infect mammalian hosts, and Plasmodium
falciparum is the most prevalent cause of fatal outcomes in
malaria patients.4
Chemotherapy over a period of decades has likely contributed
to the high degree of genetic diversity within these parasites.5
The emergence of resistance to antimalarial drugs has led to poor
treatment outcomes.6 The current best practice is a combina-
tion therapy with the artemisinin family of antimalarial agents.7
In addition to drug resistance, malaria is prevalent in under-
developed nations with poor infrastructures, high population
Diversity-oriented synthesis (DOS)12−14 yields small mole-
cules having diverse stereochemistries and skeletons. Short,
modular synthetic pathways, for example, using the build/
couple/pair (B/C/P) strategy, facilitate downstream efforts
using medicinal chemistry. These novel compounds effectively
Received: October 14, 2011
Accepted: December 14, 2011
Published: December 22, 2011
© 2011 American Chemical Society
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Table 1. SARs Derived from the Broad Compound
Collection
Figure 1. Graphical depiction of the HTS triage.
We initiated the search for novel antimalarial agents via HTS
of an 8000-membered DOS 'informer' collection in a phenotypic
blood-stage malaria assay.25 This representative set of com-
pounds was computationally selected for maximum diversity
representing all libraries in our collection while maintaining the
full complement of all possible stereoisomers of each compound.
HTS was conducted in triplicate at a 5 μM compound con-
centration using a growth inhibition assay (72 h, DAPI) with
Figure 2. Structure of lead compound 1, potency in two strains of
blood-stage malaria parasites, and the SSAR of the hit.
complement traditional screening collections.15−21 Our recent
efforts22 have focused on incorporating both structure−activity
relationships (SAR) and stereochemical structure−activity
relationships (SSAR) into the library design, thereby enabling
quick prioritization of hit clusters from primary screens.
Coupling this initial SAR with the ability to synthesize analogues
rapidly using modular synthetic pathways provides an efficient
process to pursue hits identified using high-throughput
screening (HTS). These principles were highlighted recently
by an aldol-based B/C/P strategy, resulting in compounds
having medium-sized and macrocyclic rings derived from a
common linear intermediate.23,24 This report describes an HTS
that yielded novel compounds having antimalarial activity and
subsequent medicinal chemistry that yielded a promising and
novel subnanomolar inhibitor of the major etiologic agent of
severe malaria, P. falciparum.
Figure 3. Preparation of derivatives in the solution phase.
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Table 2. Initial SARs of the Aryl Substituent
Table 3. Initial SARs at the Amine Substitutent
multidrug-resistant Dd2 P. falciparum parasites (Figure 1). A
total of 560 compounds showed greater than 90% growth
inhibition, and titration at a four concentrations further narrowed
the list to 26 candidates (>50% inhibition at 280 nM). Of these,
20 compounds were from the 'ring-closing metathesis' (RCM)
library, and this scaffold was selected for further studies.26
The most active hit (compound 1, Figure 2) was independently
synthesized and iteratively titrated in a 12-point assay to confirm a
potency (GI50) of 120 nM against Dd2 intraerythrocytic parasites
(GI50 values were obtained with Dd2 parasites unless otherwise
specified). This lead has a similar potency in the drug-sensitive
3d7 parasite strain and does not cause hemolysis of erythrocytes
at up to 40 μM concentration; unfortunately, it is largely insoluble
in aqueous solution (<0.5 μM in water). The stereochemical
SAR (SSAR) analysis of all 16 possible isomers of compound 1
demonstrates that interesting biological activity is predominantly
located in two stereoisomeric compounds that are epimeric
outside the macrocyclic ring (C2, Figure 2).
In the first round of analogue testing, we searched the Broad
Institute chemical compound collection for analogues of hit 1
that had the same SRRS configuration (C2C5C6C12) and retained
one of the two (phenyl urea or 4-fluorophenyl sulfonamide) side
chain diversity elements. This exercise resulted in more than
50 available analogues. The results of selected titrations are
reported in Table 1. The first five structures show changes to the
amine side chain. Alternatively substituted and unsubstituted
sulfonamides 2 and 3 showed similar potency to the lead
(GI50 = 160 and 350 nM, respectively), while the corresponding
(GI50 = >5000 nM) led to an inactive substrate, while the cor-
responding phenyl-sulfonamide 9 (GI50 = 2400 nM) also
provided a less biologically interesting substrate. The presence
of both reasonable SAR via the presence of close structural
analogues in the compound collection combined with promising
SSAR prompted additional investigation into this lead.
Independent preparation of interesting compounds and the
synthesis of additional derivatives closely followed the strategy
originally reported by Marcaurelle et al.24 These syntheses were
performed in the solution phase. Protected intermediate 10 was
converted into the requisite derivatives via iterative capping and
deprotection steps (Figure 3). When the aniline group required
masking, a 9-fluorenylmethyloxycarbonyl (Fmoc) group was
used. Details of the synthetic steps used to prepare these
compounds can be found in the Supporting Information.
Our preliminary studies of the SAR of this novel lead
revolved around monitoring potency in the growth inhibition
assay while improving aqueous solubility by the addition of
polar functional groups and attempting to remove hydrophobic
bulk. We first directed our attention to the N,N′-diaryl urea
(Table 2). Formal truncation of the urea group provides the
corresponding aniline 11 (GI50 = 990 nM). Aniline 11, hetero-
cyclic aminobenzoxazole 12 (GI50 = 580 nM), and the
isopropyl urea 6 (GI50 = 400 nM) led to modest decreases in
potency. We followed on the observation that the isopropyl
phenyl amide 4 (GI50 = 2600 nM) and benzyl amine 5 (GI50
=
1100 nM) demonstrated a loss in potency.
Alteration of the urea substituent from phenyl (1, L/D = 120 nM)
to isopropyl (6, GI50 = 400 nM) or 2,4-dimethylisoxazolyl (7,
GI50 = 240 nM) afforded compounds with somewhat diminished
potency. Formal truncation of the urea to the dimethylaniline 8
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a
Table 4. Initial SARs at the Amide Substitutent
Scheme 1. Synthesis of Probe 27 from Initial Lead 1
a
Reagents and conditions: (a) DPPA, DBU, THF, 87%. (b) PPh3,
H2O, THF, 76%. (c) CH2O, MgSO4, H2O, DCM; then NaBH(OAc)3,
87%.
functional groups expected to improve water solubility
(Scheme1). Reduction provided soluble primary amine 25
(GI50 = 370 nM, water solubility = 268 μM). The corresponding
monomethylamine 26 showed no improvements over its parent
(GI50 = 79 nM, water solubility = 401 μM), while dimethylamino
urea 6 showed a modest increase in water solubility (water
solubility =2.2 μM) by independent preparation of the
heterocyclic and substituted 4-amino-3,5-dimethylisoxazolyl
urea 7. This afforded a soluble derivative (water solubility =
45 μM) with some loss of potency (GI50 = 240 nM).
We next optimized the amine side chain for potency and
solubility (Table 3). The regioisomeric 2-fluorophenylsulfon-
amide 13 has lower potency (GI50 = 1100 nM) when contrasted
with the parent, and the 4-trifluoromethyl derivative 14 showed
no improvement (GI50 = 880 nM). Hetereocycles such as
3-pyridylsulfonamide 15 were not tolerated (GI50 > 5000 nM),
and benzylic amine 16 also showed a lack of potency in the
primary phenotypic assay (GI50 = 4300 nM). Carbamate
derivatives such as tert-butyl (17), phenyl (18), and methox-
yethoxy (19) showed improvements in solubility but lost
biological activity (all GI50 > 1400 nM).
Exploration of alternate substituents of the amide group began
serendipitously with the submission of protected derivatives to
the growth inhibition assay (Table 4). The para-methoxybenzyl
(PMB) ether 20 showed subnanomolar potency (GI50 = 0.18 nM).
To confirm this result, other less lipophilic groups were
appended to the alcohol. Pivalate (21, GI50 = 7.3 nM), benzoyl
(22, GI50 = 15 nM), and methoxymethyl (23, GI50 = 8.2 nM)
showed similar but somewhat diminished improvements in
potency. Unfortunately, none of these compounds were soluble
in water.
derivative 27 demonstrated both subnanomolar potency (GI50
=
0.54 nM) and solubility in water (water solubility = 120 μM).
With this encouraging result in hand, we revisited the
alternate stereochemical series on the C2 carbon located outside
the macrocycle originally noted in the HTS screen (Table 5).
Independent synthesis and titration of our original HTS hits
showed that the two epimeric alcohols 1 and 28 had similar
potency (GI50 = 120 and 62 nM, respectively), while desmethyl
ethanol derivative 30 lost some activity (GI50 = 630 nM). As
previously noted, the addition of a PMB ether in the S-series
resulted in a 3−4 log improvement in potency (20, GI50 = 0.18
nM, vida supra). When the same formal transformation was
performed on the epimeric (R) diastereomer and desmethyl
examples, no correspondingly large potency improvements
were observed (29, GI50 = 36 nM; and 31, GI50 = 55 nM).
Confident that we were operating on the optimal stereoisomer,
we nominated 27 as the Molecular Libraries Probe Production
Centers Network (MLPCN) probe (ML238) and submitted it
for further testing.
Compound 27 has subnanomolar activity in two P. falciparum
strains, and inhibition of parasite growth is not time dependent
(contrasting 48−96 h time points, Table 6). The compound is
more potent than chloroquine or artesunate and is similar in
potency to atovoquone (GI50 = 0.54 nM, all three known anti-
malarial agents were used as controls). This compound is
Displacement of the sterically hindered hydroxyl group within
1 with azide under Mitsunobu conditions afforded both a potent
derivative (24, GI50 = 3.2 nM) and access to nitrogen-based
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Table 5. SSARs at the Amide Substitutent
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and characterization of new chemical
entities. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 617-714-7309. E-mail: richardh@broadinstitute.org.
Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was funded by the NIH-MLPCN program (1 U54
HG005032-1 awarded to S.L.S. and 5 U54MH08468-1 awarded
to C.P.A.) and the NIGMS-sponsored Center of Excellence in
Chemical Methodology and Library Development (Broad
Institute CMLD; P50 GM069721). Funding for EHE and
DAF was provided in part by the NIH (R01 AI079709).
Funding for specificity testing was provided by Genzyme.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Stephen Johnston and Mike Lewandowski of
the Broad Institute for analytical chemistry support and Norman
Lee of Boston University for high-resolution mass spectroscopy.
ABBREVIATIONS
■
B/C/P, build/couple/pair; DBU; 1,8-diazobicyclo[5.4.0]undec-
7-ene; DCM; dichloromethane; DHODH, dihydroorotate
dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole; DOS,
diversity oriented synthesis; DPPA; diphenylphosphoryl azide;
FMOC, 9-fluorenylmethyloxycarbonyl; HTS, high-throughput
screening; GI50, phenotypic assay GI50 values utilizing Dd2
parasites; MLPCN, Molecular Libraries Probe Production
Centers Network; PMB, para-methoxybenzyl; RCM, ring-
closing metathesis; SAR, structure−activity relationships;
SSAR, stereochemical structure−activity relationship
Table 6. Profile of Malaria Probe 27 (ML238)
assay
result
live/dead (Dd2, GI50, 72 h)
live/dead (3d7, GI50, 96 h)
live/dead (3d7, GI50, 48 h)
solubility (water)
0.54 nM
0.54 nM
3.6 nM
120 μM
>40 μM
>30 μM
99%
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