Synthesis of a Bicyclic Azetidine with in Vivo Antimalarial Activity Enabled by Stereospecific, Directed C(sp3)-H Arylation

Article abstract of DOI:10.1021/jacs.7b06994

The development of new antimalarial therapeutics is necessary to address the increasing resistance to current drugs. Bicyclic azetidines targeting Plasmodium falciparum phenylalanyl-tRNA synthetase comprise one promising new class of antimalarials, especially due to their activities against three stages of the parasite's life cycle, but a lengthy synthetic route to these compounds may affect the feasibility of delivering new therapeutic agents within the cost constraints of antimalarial drugs. Here, we report an efficient synthesis of antimalarial compound BRD3914 (EC₅₀ = 15 nM) that hinges on a Pd-catalyzed, directed C(sp³)-H arylation of azetidines at the C3 position. This newly developed protocol exhibits a broad substrate scope and provides access to valuable, stereochemically defined building blocks. BRD3914 was evaluated in P. falciparum-infected mice, providing a cure after four oral doses.

Full text of DOI:10.1021/jacs.7b06994

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Article
Synthesis of a bicyclic azetidine with in vivo antimalarial
3
activity enabled by stereospecific, directed C(sp)-H arylation
Micah Maetani, Jochen Zoller, Bruno Melillo, Oscar Verho,
Nobutaka Kato, Jun Pu, Eamon Comer, and Stuart L. Schreiber
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06994 • Publication Date (Web): 21 Jul 2017
Downloaded from http://pubs.acs.org on July 22, 2017
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Synthesis of a bicyclic azetidine with in vivo antimalarial activity
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enabled by stereospecific, directed C(sp )-H arylation
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Micah Maetani , Jochen Zoller , Bruno Melillo , Oscar Verho , Nobutaka Kato , Jun Pu ,
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†‡
§
Eamon Comer , and Stuart L. Schreiber *
†
Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
Massachusetts 02138, United States
‡
Broad Institute, Cambridge, Massachusetts 02142, United States
§
Howard Hughes Medical Institute, Cambridge, Massachussetts 02138, United States
║
These authors contributed equally to this work. *email: stuart_schreiber@harvard.edu
Abstract:
The development of new antimalarial therapeutics is necessary to address the increasing
resistance to current drugs. Bicyclic azetidines targeting Plasmodium falciparum phenylalanylꢀ
tRNA synthetase comprise one promising new class of antimalarials, especially due to their
activities against three stages of the parasite’s life cycle, but a lengthy synthetic route to these
compounds may affect the feasibility of delivering new therapeutic agents within the cost
constraints of antimalarial drugs. Here, we report an efficient synthesis of antimalarial compound
3
BRD3914 (EC50 = 15 nM) that hinges on a Pdꢀcatalyzed, directed C(sp )ꢀH arylation of
azetidines at the C3 position. This newly developed protocol exhibits a broad substrate scope and
provides access to valuable, stereochemically defined building blocks. BRD3914 was evaluated
in P. falciparumꢀinfected mice, providing a cure after four oral doses.
Introduction
Malaria is caused by infection with Plasmodium species and transmitted by female
1
Anopheles mosquitoes. While substantial progress has been made over the past two decades in
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reducing both mortality and burden associated with the disease, the emergence of parasite
resistance to firstꢀline antimalarial treatments highlights the need for safe and effective new
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therapies. The lifecycle of the malaria parasite includes three distinct stages – the asexual
blood stage, the liver stage, and the gametocyte blood stage – and nextꢀgeneration antimalarial
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therapies should ideally target all three.
We recently identified antimalarial compounds originating from diversityꢀoriented
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synthesis (DOS), highlighted by phenylalanylꢀtRNA synthetase inhibitor BRD7929 (2S,3R,4R)
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(Figure 1). BRD7929 exhibits activity in all stages of the parasite life cycle and is curative in
multiple in vivo mouse models. However, a lengthy synthetic route to bicyclic azetidine
BRD7929 (21 steps) hampers the ability to evaluate analogs systematically in vivo. Analysis of
stereochemistryꢀbased structureꢀactivity relationships (SAR) revealed that two of eight possible
stereoisomers of parent compound BRD3444 – (2S,3R,4R) and (2R,3R,4R) – were active against
a multidrugꢀresistant strain of malaria (P. falciparum, strain Dd2). This observation suggested
that substituents at one of the stereocenters (C2) were not necessary for activity. Indeed,
BRD3914, an analog lacking substitution at C2, was found to retain substantial antimalarial
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activity in vitro (EC = 13 nM, Dd2 strain).
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Production cost constitutes an important consideration in the development of antimalarial
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therapeutics. We recognized in BRD3914 an opportunity to achieve an efficient synthesis of this
class of antimalarials that proceeds with high regioꢀ and stereoselectivity of key transformations
in fewer steps than the previous synthesis, and that minimizes the number of chromatographic
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purifications, which are known to drive both cost and waste output. To this end, we developed a
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stereospecific C(sp )ꢀH arylation of azetidines, enabling the preparation of all four stereoisomers
of members of this series. The (2R,3S) stereoisomer of this building block led to an expeditious
synthesis of antimalarial BRD3914. Subsequent in vivo evaluation in P. falciparumꢀinfected
humanized mice showed parasite clearance and lack of recrudescence after four oral doses,
resulting in a durable cure.
A key step in our original synthesis of BRD3914 (14 steps) and other bicyclic azetidines
comprises a baseꢀmediated intramolecular cyclization from 1 that affords azetidineꢀ2ꢀcarbonitrile
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a,8
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with little or no diastereoselectivity (ca. 1:1, Figure 2a). To date, intramolecular cyclization
from complex starting materials remains the predominant approach to access substituted
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azetidines. Following azetidine ring formation, nitrile reduction with DIBAL, protection with oꢀ
nitrobenzenesulfonyl chloride, and allylation gives the precursor for the key ringꢀclosing
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metathesis to generate the eightꢀmembered ring (Figure S1). While the previous DOS route
provided a versatile starting point for an initial SAR assessment focused, for example, on the 4ꢀ
methoxyurea region, it limited the ability to assess other regions of the scaffold. Notably, various
aryl and heteroaryl derivatives at the C3 position of the azetidine require the execution of the
entire 14ꢀstep synthesis, and the introduction of functional groups on the eightꢀmembered ring
relies on olefin chemistry and thus presents regioꢀ and stereoselectivity challenges.
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In a revised retrosynthetic analysis (Figure 2a), we reasoned that a bifunctional linker
could be used in an advantageous way to form the eightꢀmembered ring (diamine 5). This
approach would rely on cisꢀsubstituted azetidine 4 as a precursor, which we envisioned arising
from a directed CꢀH arylation using commerciallyꢀavailable
Dꢀazetidineꢀ2ꢀcarboxylic acid 3.
Since the first examples exploiting 8ꢀaminoquinoline as a directing group by Daugulis and coꢀ
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workers, C(sp )ꢀH functionalization methods have grown and been applied to a number of
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challenging scaffolds, including cyclopropanes,
cyclobutanes
, cyclopentanes,
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pyrrolidines, and piperidines. There are few reports of the use of azetidines as a substrate or
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product in CꢀH functionalization, and to our knowledge the C3 arylation of azetidines has not
been described.
Results & Discussion
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Unfortunately, initial attempts to extend C(sp )ꢀH arylation reactions to azetidines using
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known conditions exploiting 8ꢀaminoquinoline as a directing group resulted in trace amounts of
product (Table S1). Following substantial optimization on a model system (Figure 2b, Table
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S2-S9), we found that an unconventional Nꢀtrifluoroacetamide (NꢀTFA) protecting group and
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the additive (BnO) PO H were critical to reaction efficiency. Under the optimized conditions –
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aryl iodide (3 equiv), Pd(OAc) (10 mol%), (BnO) PO H (20 mol%), AgOAc (2 equiv), DCE
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(
1.0 M), 110 °C – model product 7a was formed in 72% yield as determined by H NMR with
internal standard. The reaction exhibited optimal performance under N atmosphere, though it
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afforded product in 52ꢀ56% yield under air or O2.
NꢀTFA is known to undergo hydrolysis readily, releasing the corresponding amine in
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mild alkaline solutions, raising the possibility that the azetidine could be deprotected directly
after the arylation. We were delighted to find that upon completion of the CꢀH arylation, the Nꢀ
TFA group could be cleaved in the crude reaction mixture by the addition of 7.0 N NH in
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MeOH to afford the free azetidine. The oneꢀpot CꢀH arylation/deprotection sequence was
performed on a range of aryl iodides to demonstrate the broad applicability of this method
(Table 1). The reaction tolerated a variety of 3ꢀ and 4ꢀsubstituted aryl iodides (8a-m), including
both electronꢀdonating and electronꢀwithdrawing groups. Halogenated arylꢀiodide derivatives
performed well and provide sites for further diversification. Exceptions include 2ꢀfluoroꢀ
iodobenzene and 2ꢀmethyliodobenzene, which gave 32% isolated product (8n) and no product
(8o), respectively, likely due to steric congestion around a palladacycle intermediate as proposed
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in previous studies.
Increased yields were obtained with 4ꢀiodobiphenyl (8p) and 2ꢀ
iodonaphthalene (8q). Arylation with 2ꢀiodothiophene (8s) proceeded in 80% yield while other
heterocyclic iodides were found to react less efficiently (8t-v). Encouragingly, the homologous
pyrrolidine (9) and piperidine (11) analogs yielded products in 95% and 72% isolated yields,
respectively (Figure 3a), demonstrating the utility of these reaction conditions in the installation
of C3 aryl groups on other cyclic amines.
Next, we explored reaction conditions to remove the directing group (Figure 3b) to
maximize stereochemical diversity. This would enable access to broad, wellꢀdefined chemical
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space in only a few steps from commercial material. We chose aryl bromide 8f as the substrate
for these diversification studies given the possibility for further elaboration of the aromatic ring.
Global Boc installation on (+)ꢀ8f (i.e. the azetidine and amide nitrogens) followed by a
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LiOH/H O ꢀmediated cleavage
of the 8ꢀaminoquinoline auxiliary provided cisꢀ(+)ꢀ13 in
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69% yield with retention of stereochemistry at the αꢀposition. Alternatively, monoꢀBoc
protection followed by treatment with NaOH/EtOH at 110 °C afforded transꢀ(−)ꢀ14 in 81%
yield, thus achieving both removal of the auxiliary and epimerization at the αꢀcarbon. These
conditions were then applied to (−)ꢀ8f and provided (−)ꢀ13 and (+)ꢀ14 in 72% and 77% yield,
respectively. All four stereoisomers can be accessed from (+)ꢀ and (−)ꢀ8f without the need for
chromatographic purification. Thus, any one of the four stereoisomers could be prepared in three
steps with the right choice of substrate (
removal conditions.
Lꢀ or Dꢀazetidineꢀ2ꢀcarboxylic acid) and auxiliary
Having established conditions for the CꢀH arylation and removal of the directing group,
we turned our attention to the synthesis of BRD3914. TFAꢀprotection and installation of 8ꢀ
aminoquinoline on
D
ꢀazetidineꢀ2ꢀcarboxylic acid (+)ꢀ3 (10.0 g) afforded 26.3 g of (+)-6a in 82%
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yield and could be achieved in a single reaction vessel (Figure 4). The use of only 0.95
equivalents of DIPEA appeared critical to avoid epimerization at the αꢀposition during the
HATUꢀmediated amide coupling. The key oneꢀpot arylation and deprotection sequence was
performed on a 10ꢀg scale with 1ꢀbromoꢀ4ꢀiodobenzene and furnished (−)ꢀ8f in 52% yield with
both relative and absolute stereochemistry confirmed by Xꢀray crystallography. Of note, we
found that the reaction performed equally well on 0.3, 3.0, and 30.1 mmol scale (100 mg to 10.0
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g, Table S10), although incomplete TFAꢀdeprotection under standard NH in MeOH conditions
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was observed on ≥3.0 mmol scale. In these cases, modified conditions using K CO in 9:1
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MeOH/H O completely removed the protecting group. Directing group cleavage from (−)ꢀ8f
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with retention of cisꢀstereochemistry afforded gram quantities of the synthetically useful
carboxylic acid building block (−)ꢀ13.
Next, deprotection of (−)ꢀ13 using 4.0 M HCl in dioxane followed by reductive
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amination with bifunctional linker 15 proceeded smoothly in a single flask, delivering (+)ꢀ16 in
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1% yield. Fmoc was removed using SiliaBond Piperazine, and sequential intramolecular
amide bond formation led to lactam (−)ꢀ17 (structure confirmed by Xꢀray crystallography) in
9% isolated yield in a single flask. The lactam reduction proved remarkably difficult as
conventional reagents such as LiAlH4 or BH ·Me S resulted in either competing
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protodebromination or formation of intractable adducts. Ultimately, we found that modified
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conditions reported by Reeves and colleagues
using Ru (CO)
and 1,1,3,3ꢀ
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tetramethyldisiloxane (TMDS) in toluene at 90 °C reduced the lactam; the resulting diamine
being trapped in situ with 4ꢀmethoxyphenyl isocyanate to afford the corresponding urea in 57%
yield. Finally, a Pdꢀcatalyzed Heck alkynylation with phenylacetylene delivered BRD3914 in
92% isolated yield. Overall, the synthesis of BRD3914 required seven singleꢀflask
transformations (five chromatographic separations) and proceeded in 10% overall yield from
commercially available (+)ꢀ3, representing a major improvement over our previous work (14
6a
steps).
The short synthesis permitted further evaluation of the biological activity of BRD3914.
First, the compound was confirmed to be potent in vitro (EC = 15 nM) against P. falciparum
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parasites (Dd2 strain), in line with previously measured activity. Pleasingly, BRD3914
exhibited low in vitro cytotoxicity to human cell lines (A549 CC = 38 µM, HEK293 CC > 50
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µM, HepG2 CC >50 µM). We then sought to evaluate the in vivo efficacy of this compound for
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obese diabetic/severe combined immunodeficiency (NOD/SCID) Il2rγ mice engrafted with
human erythrocytes (huRBC NSG). This mouse model has been shown to correlate well with
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human malaria challenge models.
huRBC NSG mice were inoculated with parasites (ꢀ48 h)
before administration with either 25 mg/kg or 50 mg/kg BRD3914 (single dose at 0 h, or
multiple dosing at 0, 24, 48, and 72 h, formulated in 70% PEG300/30% solution of 5%
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dextrose/H O) and monitored for 30 days (Figures 5, S2). Chloroquine was used as a positive
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control. After a single 50 mg/kg dose of BRD3914, a reduction in parasiteꢀassociated
bioluminescence was observed in mice, but recrudescence occurred around day three.
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Gratifyingly, huRBC NSG mice treated with four q.d. (quaque die) doses (25 mg/kg or 50
mg/kg) of BRD3914 were parasiteꢀfree after 30 days based on bioluminescent imaging. These
results are striking compared to chloroquine treatment (4 × 25 mg/kg), where recrudescence of
infection was observed in one of two mice.
While BRD3914 has a dosing shortcoming in vivo when compared to BRD7929, overall
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the compound succeeds in curing mice under a traditional Peters’ test, retains in vitro activity
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against P. falciparum parasites, and, most importantly, exhibits a more attractive safety profile.
In our in vivo studies, BRD3914 outperformed the antimalarial chloroquine, one of the most
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successful antimalarial agents to date.
The biological studies presented herein were facilitated by the development of a Pdꢀ
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catalyzed C(sp )ꢀH arylation strategy, which allowed the efficient preparation of BRD3914 in
seven steps with five chromatographic separations. This represents a significant improvement
over previous syntheses of this class of inhibitors with respect to step count, chromatographic
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separations, and waste minimization. Notable features of the synthetic approach are the
stereospecificity of the arylation and the controlled cleavage of the directing group, which
affords any one of four C3ꢀarylated azetidine carboxylic acids with judicious choice of substrate
and reaction conditions. The CꢀH arylation was performed on up to a 10ꢀgram scale without a
significant decrease in yield and provides a general strategy to C3ꢀarylated azetidines,
pyrrolidines, and piperidines. Given the substrate scope and the ability to explore stereochemical
space, we envision that this method can be applied to the synthesis of other promising azetidineꢀ
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containing antimalarials and antimicrobials in addition to finding general use for the
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preparation of diverse fragments for chemical libraries or peptidomimetics.
Conclusions
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In summary, we developed a Pdꢀcatalyzed, directed C(sp )ꢀH arylation of azetidines,
which exhibited a broad substrate scope and permitted the preparation of stereochemically
defined, synthetically useful carboxylic acid building blocks. We used this method in the short
synthesis of antimalarial compound BRD3914, which cured P. falciparum infection in a mouse
model after four oral doses. Collectively, our results arise from interplay between chemical
methodology, stereochemicallyꢀoriented diversification, synthesis of a structurally complex
scaffold, and biological studies.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
XXXXXXXXXXXXXX.
Supplementary figures, experimental details, compound characterization, and abbreviation,
(PDF)
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: stuart_schreiber@harvard.edu.
Funding Sources
This work was supported by the Bill & Melinda Gates Foundation (grant OPP1032518). S.L.S. is
an investigator at the Howard Hughes Medical Institute. M.M. was supported by a fellowship
from the National Science Foundation (DGE1144152) and from Harvard University’s Graduate
Prize Fellowship. J.Z. was supported by a postdoctoral fellowship from the German Academic
Exchange Service DAAD. B.M. was supported by a postdoctoral fellowship from Harvard
University. O.V. was supported by a postdoctoral fellowship from the WennerꢀGren Foundations.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We thank the Broad Institute analytical team for highꢀresolution mass spectrometry data, Tom
Clinckemaillie (Broad Institute) for assistance with SFC, Drs. Peter Müller and Jonathan Becker
(Massachusetts Institute of Technology) for Xꢀray crystallographic structural analysis, and Drs.
Michael F. Mesleh and Jenna B. Yehl (Broad Institute) for assistance with NMR.
References
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http://www.who.int/malaria/publications/world_malaria_report_2016/en/.
[
2] Wells, T. N. C.; van Huijsduijnen, R. H.; Van Voorhis, W. C. Nat. Rev. Drug Discovery
2015, 14, 424ꢀ442.
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Figure 1. (a) Structures and in vitro antiplasmodial activities of representative bicyclic
azetidines. (b) Stereochemistryꢀbased structureꢀactivity relationships (SAR).
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Figure 2. (a) Comparison of ring formation and CꢀH arylation approaches to BRD3914. (b)
Model system and optimized protocol for the CꢀH arylation of azetidines.
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Table 1. Substrate scope of C-H arylation method.
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Figure 3. (a) CꢀH arylation method to access homologous series of CꢀH arylation products. (b)
Varying conditions for directing group removal to access selectively cis and trans key building
blocks; all compounds are isolated as single stereoisomers with dr >20:1 and ee >99%.
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Figure 4. Reaction conditions: a. Ethyl trifluoroacetate, DIPEA, DMF, 50 °C, 15 h; evaporate
volatile materials, then 8ꢀaminoquinoline, HATU, DIPEA, DMF, 0 °C, 2 h (82%); b. Pd(OAc)2
(
10 mol%), AgOAc (2.0 equiv), (BnO) PO H (20 mol%), 1ꢀbromoꢀ4ꢀiodobenzene (3.0 equiv),
2 2
DCE, 110 °C, 24 h; evaporate volatile materials, then K CO (3.0 equiv), 9:1 MeOH/H O (52%);
2
3
2
c. Boc O (3.0 equiv), MeCN, 50 °C, 15 min; then DMAP (0.1 equiv), 2 h; evaporate volatile
2
materials, then H O (10.0 equiv), LiOH (6.0 equiv), THF/MeOH (2:1), 0 °C, then 50 °C, 20 h
2
2
(72%); d. HCl (4.0 M in dioxane, 15.0 equiv), DCM, 2 h; evaporate volatile materials, then 15
(
2.5 equiv), NaBH(OAc) (3.0 equiv), DCM, 12 h (71%); e. SiliaBond® Piperazine (3.0 equiv.),
3
DMF, 50 °C, 2 h; then DIPEA (5.0 equiv), HATU (1.5 equiv), 20 min (89%); f. Ru (CO) (10
3
12
mol%), 1,1,3,3ꢀtetramethyldisiloxane (15.0 equiv), toluene, 90 °C, 24 h; then 4ꢀ
methoxyphenylisocyanate (1.0 equiv), THF, 10 min (57%); g. XPhos Pd G3 (10 mol%),
phenylacetylene (5.0 equiv), Et N (4.0 equiv), MeCN, 70 °C, 2 h (92%).
3
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HLH/BRD
Figure 5. huRBC NSG mice were inoculated with P. falciparum (3D7
) bloodꢀstage
parasites 48 h before treatment, and BRD3914 was administered as a single dose (25 mg/kg or
0 mg/kg) at 0 h, or multiple dosing at (25 mg/kg or 50 mg/kg) at 0, 24, 48, and 72 h (n = 2 for
5
each group, this study was conducted once). Chloroquine (CQ) was used as a positive control.
Infections were monitored using the in vivo imaging system (IVIS). Bioluminescent intensity
was quantified from each mouse and plotted against time. The dotted horizontal line represents
the mean bioluminescence intensity level obtained from all the animals before the parasite
inoculation.
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