Diversity-oriented synthesis-facilitated medicinal chemistry: Toward the development of novel antimalarial agents

Article abstract of DOI:10.1021/jm500994n

Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure-activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead.

Full text of DOI:10.1021/jm500994n

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Article
Diversity-Oriented Synthesis-Facilitated Medicinal Chemistry:
Towards the Development of Novel Antimalarial Agents
Eamon Comer, Jennifer A Beaudoin, Nobutaka Kato, Mark E. Fitzgerald, Richard Wayne Heidebrecht,
Maurice duPont Lee, Daniela Masi, Carol Mulrooney, Marion Mercier, Giovanni Muncipinto, Ann Rowley,
Keila Crespo-Llado, Adelfa E Serrano, Amanda k Lukens, Roger C. Wiegand, Dyann F Wirth, Michelle
A Palmer, Michael A Foley, Benito Munoz, Christina A Scherer, Jeremy R Duvall, and Stuart L Schreiber
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Sep 2014
Downloaded from http://pubs.acs.org on October 6, 2014
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Diversity-Oriented Synthesis-Facilitated Medicinal
Chemistry: Towards the Development of Novel
Antimalarial Agents
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Eamon Comer,*† Jennifer A. Beaudoin, † Nobutaka Kato, † Mark E. Fitzgerald, † Richard W.
Heidebrecht, † Maurice duPont Lee IV, † Daniela Masi, † Marion Mercier, † Carol Mulrooney, †
Giovanni Muncipinto, † Ann Rowley, † Keila Crespo-Llado, ‡ Adelfa E. Serrano,‡ Amanda K.
Lukens , ¥ †† Roger C. Wiegand,¥ Dyann F. Wirth, ¥†† Michelle A. Palmer,† Michael A. Foley,†
Benito Munoz,† Christina A. Scherer,† Jeremy R. Duvall,† Stuart L. Schreiber.†§
†Center for the Science of Therapeutics, Howard Hughes Medical Institute, Broad Institute, 7
Cambridge Center Cambridge, MA 02142, United States.
§Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
Massachusetts 02138, United States.
‡University of Puerto Rico-School of Medicine Department of Microbiology and Medical
Zoology P.O. Box 365067 San Juan, PR 00936-5067.
¥
Infectious Disease Initiative, Broad Institute, 7 Cambridge Center, Cambridge, MA 02142,
United States.
†† Harvard School of Public Health, Huntington Avenue, Boston, Massachusetts 02115, United
States.
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ABSTRACT:
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Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis
(DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent
that derived from the synthetic pathway. Structure–activity relationships that focused on both
appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-
stage growth with improved solubility and microsomal stability and reduced hERG binding. The
build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening
library, facilitated medicinal chemistry optimization of the antimalarial lead.
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INTRODUCTION:
Malaria is a major public health threat responsible for high mortality and morbidity burdens in
endemic countries.1 The disease in humans is caused by five species of mosquito-borne
Plasmodium parasites, with P. falciparum being the most virulent and deadly.2 Resistance has
become a major challenge and recently artemisinin and its derivatives such as artesunate, the
mainstays of malaria treatment,3 have shown reduced clinical efficacy to populations at the
Cambodia-Thai border,4,5 indicating that it may only be a matter of time before these too become
ineffective. Given the lack of an effective vaccine,6 the discovery of safe and effective
antimalarial therapeutics is urgent. While the search for antimalarials is centuries old,7 no new
class of antimalarial has been introduced into clinical practice since 1996.8 Given that resistance
to the traditional chemotypes is an increasing problem, there is a pressing need for the discovery
of new classes of compounds, with unique core structures, more likely to have novel mechanisms
of action.9 We have developed a screening collection of 100,000 diverse small molecules that
combine the complexity of natural products and the efficiency of high-throughput synthesis10,11,12
and subsequently reported the performance of a small subset of this collection (~8,000
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compounds) in the phenotypic screening of P. falciparum blood-stage parasites.13 Given the need
for new antimalarial chemotypes we sought to develop upon our previous reported inhibitor of
the viability of P. falciparum. Our approach, outlined in Figure 1 uses a ‘build/couple/pair’
(B/C/P) strategy of DOS,14 originally developed during library synthesis10 to manipulate almost
every position of the reported 14-membered macrocycle allowing us to investigate the biological
relevance of most core atoms. This report describes the DOS-facilitated medicinal chemistry and
subsequent in vivo studies of antimalarial macrocycles.
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Figure 1. Overview of B/C/P strategy showing points of manipulation and appendage sites.
RESULTS
We have previously reported the 14-membered macrolactam 2 with potent antimalarial activity
against both wild type 3D7 and multidrug-resistant Dd2 strains (Table 1) along with initial
ADME data such as plasma protein binding (PPB, 99% bound).13 Further analysis of this
compound revealed a potential hERG liability (95% displacement of control ([3H]astemizole) at
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10 uM of 2, see SI), poor mouse microsomal stability (<5% remaining after 1 hour) and low
solubility in the PBS medium (<5 uM). Given that these ADME properties can lead to
developmental issues such as low bioavailability and high-dose administrations in the case of
insolubility, our goal was to increase microsomal stability, address the potential cardiotoxicity
(hERG) liability and obtain a compound with PBS solubility at least >20 uM while maintaining
potency. Macrolactam 2 contains three appendage-diversification sites, namely: the exocyclic
dimethyl amine (Table 1, R1), the exocyclic sulfonamide and the urea at the aniline nitrogen
(Table 1, R2). Our strategy sought to explore two of these sites while taking advantage of the
modularity of the B/C/P process to investigate almost every atom in our lead structure. By
systematically accessing each fragment of 2, we aimed to identify the minimum pharmacophore
required for activity. These analogs were accessed through the pathways illustrated in Figure 2.
The modular nature of the pathway enables changes to be introduced into the final analogs by
varying individual ‘building blocks’. Several features of these medicinal chemistry efforts are
worth noting: 1) the introduction of heteroatoms within the aromatic ring; 2) removal of both
methyl groups within the macrocycle; and 3) systematic variation of the ring size from a 14-
membered through an 8-membered ring.
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Figure 2. Build/Couple/Pair pathway for achieving appendage and skeletal SAR.
We initially focused on two of the three appendage sites since we had previously established
that the sulfonamide was essential for antimalarial activity13. We hypothesized that attenuation of
the nitrogen pKa at R1, structural changes to both R1 and R2, and the introduction of functionality
to lower log D would impart more favorable DMPK. The p-fluorophenyl urea 3 had similar
potency to the parent (2) but lacked microsomal stability (<5% remaining after 1 hour) (Table 1).
Solubilizing groups such as the oxetane (4) had a dramatic effect on PBS solubility (>100 uM)
and while 4 was approximately 60 fold less potent than 2 it still showed good activity (GI50 = 32
nM). We reasoned that replacement of the phenyl urea with the 3,5-dimethylisoxazole urea
derivative would improve solubility through the introduction of heteroatoms and the disruption
of planarity.15 This afforded the more soluble derivative 5 (PBS solubility > 100 µM) with good
potency (GI50 = 4.2 nM). Further profiling of 5 also showed significantly lowered hERG binding
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relative to the parent (13% displacement of control) and reduced plasma protein binding (8% free
fraction in mouse plasma).
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Table 1. Key physicochemical properties from SAR studies of the R1 and R2 positions of the 14-
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membered macrocycle.
We then investigated substituents on R1 while keeping R2 constant. The previously
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prepared alcohol analogue 7
gave diminished potency (GI50 = 240 nM) and microsomal
stability (<5% remaining), as did the isopropyl analogue 8 (<5% remaining). The steric bulk of
the amine functionality was increased with the diethylamine and neopentane analogues (9 and
11, respectively), however, this did not significantly improve microsomal stability. Reducing the
basicity of the amine functionality as in the trifluoroethylamine 12 and morpholine derivative 13
resulted in active compounds (GI50 = 9 and 12 nM, respectively) but with similar microsomal
stability to 2 (<5% remaining). Both the N-oxide amine 10 and the mono-methyl amine analog 6
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did show improved solubility (> 100 µM), microsomal stability (35% and 51% remaining,
respectively) along with a lower LogD, but at the expense of potency (GI50 = 160 and 320 nM,
respectively).
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These results demonstrate our ability to improve PBS solubility and reduce hERG
binding while maintaining acceptable activity through modification of different appendage
groups. Although 5 is a viable candidate for in vivo efficacy studies, we sought to improve its
microsomal stability by focusing on modulation of the core molecule itself. Metabolite
identification studies on 5 and 6 revealed that both compounds were extensively metabolized via
N-demethylation at R1, hydroxylation, and combined events. In the case of 5 six major
metabolites were identified and while the exact point of hydroxylation could not be determined,
at least one hydroxylation appeared to take place within the core molecule (See SI Figure S2).
We first directed our attention to the removal of N,N’-diaryl urea (Figure 3, Table 2 and
SI Schemes S3-S7) to identify the minimum pharmacophore. This was achieved using a series of
alkene containing des-urea acids in the ‘coupling’ phase of the B/C/P pathway. Coupling linear
amine 1 followed by deprotection of the secondary alcohol and subsequent allylation gave the
ring closing metathesis (RCM) precursor. RCM was efficiently achieved for most substrates to
give compounds 14-26.
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Figure 3. General synthesis of the des-urea series of compounds (See SI Schemes S3-S7 for
expended schemes).
Reagents and conditions: a) PyBOP/BOPCl, DIEA, Ar/AlkylCO2H; b) TBAF; c) NaH, allylBr; d) Grubbs-Hoveyda
catalyst II; e) H2, 10% Pd/C; f) Pd(OH)2, H2
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Table 2. SAR of the des-urea series of compounds.
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As shown in Table 2, removal of the aromatic group (14) (see SI Scheme S3 for
expanded synthetic route) led to an inactive substrate (GI50 = 3410 nM). Replacement of the
N,N’-diaryl urea at the 4-position with a fluorine atom (15) or CF3 group (result not shown) also
resulted in a loss in potency (GI50 = 162 and 138 nM, respectively).16 The 5-fluoro isomer 16
showed good activity as did the corresponding 5-trifluoromethyl analog 17 (GI50 = 38 and 48
nM, respectively). We prepared both the 3- and 6-pyridyl analogues (18 and 19) (see SI Scheme
S4 for expanded synthetic route) and while the former provided a less active compound, the 6-
pyridyl macrocycle provided good potency (GI50 = 59 nM). Building on this result, both pyrazine
20 and substituted pyridine 21 were prepared but proved less potent than 19 (GI50 = 214 and 162
nM, respectively). Replacement of the aromatic derivative with five-membered aromatic
fragments was also investigated; however, both thiophene 25 and pyrazole 26 obviated much of
the activity. With both the pyridyl and fluoro-substituted analogs showing similar potency within
the des-urea series, the pyridyl series was pursued due to its perceived physicochemical property
advantage. Removal of the OPMB group in 19 and installation of the dimethylamine group (22)
did not preserve potency (GI50 = 231 nM) as was the case with the N,N’-diaryl urea series;
however, it did afford excellent solubility (>100 uM). A number of derivatives of the 6-pyridyl
macrocycle were prepared. Here, we found that the (2,5-difluorophenyl)methanamine analogue
24 was highly potent (GI50 = 10 nM) but lacked PBS solubility (<5 uM). Further profiling of both
22 and 23 showed no improvement in microsomal stability (< 5% remaining). With reduced
potency and no improvement in microsomal stability, we decided to shift our focus back to lead
5 rather than pursue the des-urea series further.
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Figure 4. Synthesis of C-5 and C-12 desmethyl analogues.
Reagents and conditions: a) BH3-SMe2; b) PyBOP, DIEA, 2-alkyl-5-nitrobenzoic acid; c) PyBOP, DIEA, 2-fluoro-
5-nitrobenzoic acid; d) TBAF; e) NaH, allyl-Br; f) Grubbs-Hoveyda catalyst II; g) H2, 10% Pd/C; h) ArNCO; i)
DDQ; j) DPPA, DBU, THF; k) PPh3, H2O; l) CH2O, MgSO4,H2O; then NaBH(OAc)3; m) DIEA, but-3-en-1-amine;
n) di-tert-butyl dicarbonate, DMAP, THF, reflux; o) TBS-OTf, lutidine then HF-pyridine; p) SnCl2-2H2O.
We investigated the aliphatic portion of the macrocycle with the continued aim of
establishing a minimum pharmacophore (Figures 4, 5 and Table 3). The methyl substituents at
C-5 and C-12 (Figure 4, Table 3) were probed with focus on the the latter as its removal would
simplify the chemistry in the investigation of macrocycle ring size. While HTS included all 16
stereoisomers of our original hit,13 the effect of removing individual stereogenic centers had only
been investigated with respect to the excocyclic C-2 methyl group, which lost approximately 5-
fold potency upon deletion.13 Similar potency was observed between 31 and 40 (GI50 = 6 nM and
0.88 nM respectively) suggesting that the C-12 methyl is not essential in this series. Removal of
the C-5 methyl group (29) resulted in a slight loss in activity (GI50 = 20 nM). The influence of
the aryl ether moiety was then tested by changing the O-13 oxygen for the nitrogen analogue
(33), which showed diminished potency (GI50 = 76 nM) and no improvement in solubility (<5
µM). Interestingly, attempted macrocyclization leading to 33 gave no desired product when the
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N-13 nitrogen heteroatom was protected as N-Boc, but the RCM reaction proceeded efficiently
with the compound lacking this protecting group (see SI Scheme S10). We then systematically
reduced the size of the 14-membered macrocycle 31 to 13, 12, 11 and 8-membered ring analogs
(Figure 5, Table 3). As the synthesis of 9- and 10-membered macrocycles would require the
preparation of peroxides or acetals, these analogues were omitted from the study.
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The 13-membered ring analog 34 was prepared from linear amine 1 using the RCM reaction
(Figure 5, SI Scheme S11). In this case, macrocyclization was not as efficient as the 14-
membered analog and the transformation was best accomplished using the first-generation
Grubbs catalyst. Preparation of the 12- and 11-membered lactams was achieved by either
Michael addition or alkylation of 1 with methyl acrylate (SI scheme S12) or 2-bromoacetate,
respectively (Figure 5). Both analogues contained methyl esters that were reduced to the
corresponding alcohols followed by intermolecular SnAr reactions with tert-butyl 2-fluoro-5-
nitrobenzoate to install the aromatic esters. Simultaneous deprotection of the ester and amine
protecting groups followed by intramolecular coupling yielded the 12- and 11-membered
macrocycles (37 and 38). The synthesis of 8-membered ring analog 39 followed the method
reported by Marcaurelle et. al.10 A clear SAR trend emerged from analogs 34-39 with potency
decreasing with the size of the macrocycle ring (Table 3). While the complete removal of the
aliphatic backbone obviated all activity (39, GI50 >5000 nM), potency was maintained in the 13-
membered analogue (34, GI50 = 17 nM). Similar to previous experiments, analogs 29, 31 and 34
were converted to the dimethyl amine analogs at R1 (30, 32 and 36 respectively) to assess
potency, solubility and microsomal stability. All three analogs showed excellent solubility as
expected, along with somewhat reduced potency relative to their respective PMB protected
analogs. Unfortunately, microsomal stability was still low, with only the 13-membered ring 36
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showing modest gains in microsomal stability (15% remaining). While greater microsomal
stability was observed in the NHMe analogue 35 (65% remaining), this was accompanied by a
significant loss in potency (GI50 = 624 nM).
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Figure 5. Synthesis of the macrocycle ring size analogues.
Reagents and conditions: a) PyBOP, DIEA, 2-fluoro-5-nitrobenzoic acid; b) NaH, prop-2-en-1-ol; c) TBAF; d) NaH
allylBr; e) Grubbs I; f) H2, 10% Pd/C; g) Boc2O, NEt3; h) Cs2CO3, Methyl Acrylate; i) NaHMDS, methyl 2-
bromoacetate; j) DIBAL; k) tert-butyl 2-fluoro-5-nitrobenzoate, TBAF; l) TBS-OTf, lutidine then HF-pyridine; m)
DIEA, BOP-Cl; n) SnCl2 then ArNCO; o) CsF; p) ArNCO; q) DDQ; r) DIAD, PPh3, o-NsNHMe; s) K2CO3, PhSH;
t) CH2O, MgSO4,H2O; then NaBH(OAc)3
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Table 3. SAR of the macrocycle ring size, des methyl and heteroatom exchange analogues.
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Through systematic chemistry efforts, we were able to develop SAR within lead antimalarial
compound 2. This included removal of the urea and methyl substituents within the ring,
macrocyclic ring contraction and variation at three different appendage sites. After identifying
analogs that showed improved activity/PK profiles along with improved hERG profiles we
investigated the in vivo activity of 5 by intraperitoneal dosing in a P. berghei malaria mouse
model. Our goal was to have in vivo exposure exceeding three times the GI50 of the unbound
compound (not bound to plasma proteins) at its lowest concentration (i.e. exposure > 3 x GI50 x
fu). Intraperitoneal administration of macrocyle 5 at 20 mg/kg showed that sufficient exposure
was observed for approximately 5h (see SI figures S3 and S4). This suggested that it would be
possible to achieve our goal at higher concentrations with intraperitoneal administration. In the
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P. berghei malaria mouse model, animals were infected and administered a total of seven 100
mg/kg intraperitoneal doses every 12 hours over three days. On day four parasitemia was
assessed and 5 produced a two-fold reduction in total parasitemia (p = 0.02). This is a significant
reduction but not to the levels typically seen with standard of care antimalarials such as
chloroquine or artesunate (e.g. artesunate can give parasitemia reductions of 97%). Plasma
samples taken 30 minutes after the final dose on day 3 showed low exposure of the compound
(300 ng/ml of 5 in plasma in this study compared with 768 ng/mL two hours after IP dosing at 20
mg/kg observed during PK study), suggesting that upon dose escalation, compound
pharmacokinetics did not scale. Based on this data, the lack of efficacy in the in vivo assay most
likely speaks to the poor exposure of 5 rather than on the mechanism of action of the compound,
on which we comment below.
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The goal of our study was to use a diverse screening collection to discover antimalarial
chemotypes with new mechanisms of action. In a separate manuscript we discuss the use of
resistance selection to identify the target of 5 as the reductase Qi center of cytochrome b (cytb).17
While inhibition of cytochrome b at Qo has been well studied and is the mechanism of action of
the widely used antimalarial drug atovaquone, inhibition of cytb at Qi has been significantly less
studied.18 An appealing concept is that if used in combination, Qi and Qo site inhibitors would
likely lessen the emergence of resistance. It is conceivable that mutations at both Qi and Qo sites
of the same target would come at a severe cost to cell fitness and would be unlikely to be
tolerated. Given this, Qi site inhibitors acting synergistically with Qo inhibitors have the
potential to be powerful antimalarial agents.
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DISCUSSION AND CONCLUSION
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In this study we prepared analogues with improved potency, solubility, hERG binding
and modestly improved microsomal stability relative to our initial lead using DOS enabled
medicinal chemistry. This allowed for the ready manipulation of our lead antimalarial. While in
vivo efficacy studies gave only moderate reductions in parasitemia, we speculate that this is a
result of insufficient exposure and is not a reflection of the mechanism of action of this
compound. Furthermore, this study has led to the discovery of potent inhibitors of cytochrome b
that do not inhibit this complex through the Qo binding site, but through the comparatively less
studied Qi site and represents a new chemotype and tool to target this promising antimalarial
mechanism of action. These studies encouraged us to expand our studies to the entire Broad
collection of DOS-derived compounds, which has yielded a rich collection of novel antimalarial
compounds acting through varied mechanisms. We are finding that the lessons gained from the
medicinal chemistry studies reported here have been of value as we advance novel antimalarial
agents using this overall drug-discovery strategy.
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ACKNOWLEDGEMENT
We gratefully acknowledge the support of the Broad Compound Management and Analytical
teams. This publication is based on research funded in part by the Bill & Melinda Gates
Foundation (grant OPP1032518 to S.L.S. and OPP1053644 to D.F.W.) and in part by the NIH-
MLPCN program (1U54HG005032-1 awarded to S.L.S.). Additional support came from the
National Institutes of Health (grant AI093716-01A1 to R.C.W. and G12-MD 007600 to A.E.S.).
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ASSOCIATED CONTENT
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SUPPORTING INFORMATION
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Procedures and references for synthesis of compounds 1-39 in addition to NMR data.
Protocols for in vivo studies. This material is available free of charge via the Internet at
http://pubs.acs.org
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Interestingly analogues of 2 placing the phenyl urea in the 5-position showed a significant
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probe targets Plasmodium falciparum cytochrome b ubiquinone reduction site and synergizes
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