Development of (6 R)-2-Nitro-6-[4-(trifluoromethoxy)phenoxy]-6,7-dihydro-5 H -imidazo[2,1- b] [1,3]oxazine (DNDI-8219): A New Lead for Visceral Leishmaniasis

Article abstract of DOI:10.1021/acs.jmedchem.7b01581

Discovery of the potent antileishmanial effects of antitubercular 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]oxazoles and 7-substituted 2-nitro-5,6-dihydroimidazo[2,1-b][1,3]oxazines stimulated the examination of further scaffolds (e.g., 2-nitro-5,6,7,8-tetrahydroimidazo[2,1-b][1,3]oxazepines), but the results for these seemed less attractive. Following the screening of a 900-compound pretomanid analogue library, several hits with more suitable potency, solubility, and microsomal stability were identified, and the superior efficacy of newly synthesized 6R enantiomers with phenylpyridine-based side chains was established through head-to-head assessments in a Leishmania donovani mouse model. Two such leads (R-84 and R-89) displayed promising activity in the more stringent Leishmania infantum hamster model but were unexpectedly found to be potent inhibitors of hERG. An extensive structure-activity relationship investigation pinpointed two compounds (R-6 and pyridine R-136) with better solubility and pharmacokinetic properties that also provided excellent oral efficacy in the same hamster model (>97% parasite clearance at 25 mg/kg, twice daily) and exhibited minimal hERG inhibition. Additional profiling earmarked R-6 as the favored backup development candidate.

Full text of DOI:10.1021/acs.jmedchem.7b01581

Subscriber access provided by Universitaetsbibliothek | Johann Christian Senckenberg
Article
Development of (6R)-2-nitro-6-[4-(trifluoromethoxy)phenoxy]-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (DNDI-8219): a new lead for visceral leishmaniasis
Andrew M. Thompson, Patrick D. O'Connor, Andrew J. Marshall, Adrian Blaser, Vanessa
Yardley, Louis Maes, Suman Gupta, Delphine Launay, Stephanie Braillard, Eric Chatelain,
Baojie Wan, Scott G. Franzblau, Zhenkun Ma, Christopher B. Cooper, and William A. Denny
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01581 • Publication Date (Web): 20 Feb 2018
Downloaded from http://pubs.acs.org on February 20, 2018
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Development of (6R)ꢀ2ꢀnitroꢀ6ꢀ[4ꢀ
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(
trifluoromethoxy)phenoxy]ꢀ6,7ꢀdihydroꢀ5Hꢀ
imidazo[2,1ꢀb][1,3]oxazine (DNDIꢀ8219): a new
lead for visceral leishmaniasis
1,*
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Andrew M. Thompson, Patrick D. O’Connor, Andrew J. Marshall, Adrian Blaser,
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Vanessa Yardley, Louis Maes, Suman Gupta, Delphine Launay, Stephanie Braillard, Eric
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Chatelain, Baojie Wan, Scott G. Franzblau, Zhenkun Ma, Christopher B. Cooper, and
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William A. Denny
1
Auckland Cancer Society Research Centre, School of Medical Sciences, The University of
2
Auckland, Private Bag 92019, Auckland 1142, New Zealand; Faculty of Infectious &
Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London
3
WC1E 7HT, United Kingdom; Laboratory for Microbiology, Parasitology and Hygiene,
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp,
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Universiteitsplein 1, Bꢀ2610 Antwerp, Belgium; Division of Parasitology, CSIRꢀCentral
5
Drug Research Institute, Lucknow 226031, India; Drugs for Neglected Diseases initiative,
6
1
5 Chemin Louis Dunant, 1202 Geneva, Switzerland; Institute for Tuberculosis Research,
College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago,
7
Illinois 60612, United States; Global Alliance for TB Drug Development, 40 Wall Street,
New York 10005, United States
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Abstract
Discovery of the potent antileishmanial effects of antitubercular 6ꢀnitroꢀ2,3ꢀ
dihydroimidazo[2,1ꢀb][1,3]oxazoles and 7ꢀsubstituted 2ꢀnitroꢀ5,6ꢀdihydroimidazo[2,1ꢀ
b][1,3]oxazines stimulated the examination of further scaffolds (e.g., 2ꢀnitroꢀ5,6,7,8ꢀ
tetrahydroimidazo[2,1ꢀb][1,3]oxazepines) but the results for these seemed less attractive.
Following the screening of a 900 compound pretomanid analogue library, several hits with
more suitable potency, solubility and microsomal stability were identified, and the superior
efficacy of newly synthesized 6R enantiomers with phenylpyridineꢀbased side chains was
established through headꢀtoꢀhead assessments in a Leishmania donovani mouse model. Two
such leads (Rꢀ84 and Rꢀ89) displayed promising activity in the more stringent Leishmania
infantum hamster model but were unexpectedly found to be potent inhibitors of hERG. An
extensive SAR investigation pinpointed two compounds (Rꢀ6 and pyridine Rꢀ136) having
better solubility and pharmacokinetics that also provided excellent oral efficacy in the same
hamster model (>97% parasite clearance at 25 mg/kg, twice daily) and exhibited minimal
hERG inhibition. Additional profiling earmarked Rꢀ6 as the favoured backup development
candidate.
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INTRODUCTION
Visceral leishmaniasis (VL) is a particularly lethal sandflyꢀborne parasitic disease that is
prevalent in more than 60 countries, where it mostly affects underprivileged people in remote
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ꢀ3
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rural areas who have limited access to diagnosis and treatment. Major outbreaks of VL in
East Africa have been attributed to waves of forced migration during periods of conflict and
such epidemics are exacerbated by weak healthcare systems, malnutrition, and HIV/AIDS
4,5
coinfection. Moreover, in this region, the firstꢀline drug combination of paromomycin and
sodium stibogluconate was found to be unsuitable for VL patients over 50 or those with HIV,
6
,7
and no other therapies have shown adequate efficacy. Failure of the most recently evaluated
8
new agent, fexinidazole, in a phase II clinical trial for VL in Sudan has now left the clinical
9
pipeline empty, underlining the compelling need to develop more satisfactory medications.
The target product profile (TPP) of an optimised new chemical entity for the treatment of
VL requires: (i) effectiveness against all causative species, in all endemic areas, in both
immunocompetent and immunosuppressed individuals, with a clinical efficacy of >95%; (ii)
activity against resistant strains; (iii) no adverse safety events requiring monitoring and no
contraindications; (iv) no drugꢀdrug interactions (suitable for combination therapy); (v) oral
administration once per day for a maximum of 10 days (or intramuscular dosing three times
over 10 days); (vi) stability in relevant climates (3 years), and (vii) affordable cost (<$80,
10
ideally <$10 per course). However, new drug discovery for VL faces formidable
challenges, such as inadequate investment, a lack of validated targets, poor translation of in
vitro activity into in vivo models, and meagre hit rates (<0.1%) for phenotypic screening of
1
1ꢀ13
compound libraries.
The latter may be due in part to the concealed location of parasites in
14
acidic parasitophorous vacuoles within macrophages. Furthermore, the unique glycolipidꢀ
15
rich cell surface of the amastigotes presents an additional barrier to chemotherapy. Another
issue is that many cellularly active hits may never meet TPP and progression criteria, even
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after valiant optimisation attempts.
Nevertheless, drug development efforts spearheaded
by the Drugs for Neglected Diseases initiative (DNDi) have now shown encouraging progress
12,17,18
in several novel classes, including oxaboroles and aminopyrazoles.
Novartis has also
disclosed a triazolopyrimidine preclinical lead with utility in vivo against both leishmanial
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and trypanosomal infections.
The 2ꢀnitroimidazooxazines are best known for their potent effects against Mycobacterium
2
0
tuberculosis (M. tb), the causative agent of tuberculosis (TB). The first drug candidate from
this class, pretomanid (PAꢀ824, Sꢀ1; Figure 1), has shown excellent safety and bactericidal
21
22
efficacy in phase II clinical trials for TB, leading to its ongoing combination assessment,
while our collaborative work with the TB Alliance on second generation analogues of Sꢀ1
23
culminated in the advancement of TBAꢀ354 (Sꢀ2) into phase I studies. We have recently
described the investigation of a novel 7ꢀsubstituted 2ꢀnitroimidazooxazine class, which in
addition to possessing considerable potential against TB has also demonstrated exciting
activity against both VL and Chagas disease, resulting in the selection of preclinical VL lead
24
3
.
This followed an inꢀdepth analysis of the structurally related 6ꢀnitroimidazooxazole
25
class, where phenotypic screening of some of our initial examples by DNDi had enabled the
26
27
discovery of previous development nominee 4 (DNDIꢀVLꢀ2098). The latter was found to
be activated by a novel leishmanial nitroreductase (NTR2). In comparison to 4, candidate 3
exhibited an improved safety profile and had similarly notable efficacy in two animal models
24
of VL. Furthermore, while the new TB drug delamanid (5) has also been suggested as a
28
possible VL therapy, it is noteworthy that 3 was substantially more effective than this agent
24,25
in the highly stringent chronic infection hamster model.
As part of our VL lead optimization program with DNDi, it was considered important to
develop a few efficacious backup compounds having good physicochemical/pharmacological
profiles and better safety, in order to mitigate development risks. Given the encouraging
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results with nitroimidazooxazoles and 7ꢀsubstituted 2ꢀnitroimidazooxazines, we first
evaluated various other pretomanidꢀrelated scaffolds for VL, including those with a reversed
29,30
linker at Cꢀ6
and novel nitroimidazooxazepines. We then assessed our larger collection of
pretomanid analogues via the mediumꢀthroughput screening of about 900 compounds at the
Institut Pasteur Korea (IPK). Finally, a more systematic synthetic approach was employed to
redevelop the 6ꢀsubstituted 2ꢀnitroimidazooxazine class for VL, taking into consideration
both enantiomer forms. We now report the findings from these wideꢀranging SAR studies,
including the detailed in vitro/in vivo profiling of selected new leads, which resulted in our
identification of the title compound as a very promising VL backup candidate.
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CHEMISTRY
Scheme 1 outlines the synthetic methods used to prepare eight novel racemic analogues of
Sꢀ1 featuring changes to the original nitroimidazooxazine core (14, 22, 23, 32, 33, 39, 40, and
47). A common strategy (based on the original, wellꢀvalidated route to Sꢀ1 and simple
3
1,32
derivatives)
proved effective for the first five of these (Scheme 1AꢀC), involving the
33
34
initial reaction of functionalized epoxides (9, 17, and 27 ) with 2,4ꢀdinitroimidazole (8),
followed by THP protection of the derived alcohols (10, 18, and 28). In the shorter chain
cases (11 and 19), subsequent cleavage of the TBS ether (TBAF) enabled in situ annulation,
whereas in the latter instance (29), oxazepine ring formation required additional treatment
with a strong base (NaH). Removal of the THP group with methanesulfonic acid and standard
alkylation chemistry on alcohols 13, 21, and 31 then gave the aforeꢀmentioned targets.
24
For the isomeric oxazepines (Scheme 1D), THP protection of alcohol 34 and desilylation
(TBAF) similarly furnished the nonꢀcyclised alcohol 36, which was ring closed (NaH, DMF)
and THPꢀdeprotected (HCl) to produce alcohol 38. However, both final step alkylations of 38
unexpectedly cogenerated significant quantities (20ꢀ36%) of the isomeric 7ꢀsubstituted
oxazine derivative (41 or 42), together with the desired oxazepine ether (39 or 40). Although
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unconfirmed, it is postulated that this rearrangement may involve an intramolecular S Ar
reaction, with attack by the oxazepine alkoxide anion at the imidazole ring junction carbon 9a
and subsequent alkylation of the released 2ꢀnitroimidazooxazine 7ꢀalkoxide (but it should be
emphasized that no similar rearrangements were detected during the derivatisation of 13, 21,
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31, or the alcohol precursor to Sꢀ1, and that Sꢀ1 itself has shown excellent safety and a
35
lengthy 16ꢀ18 h halfꢀlife in clinical trials for TB, suggesting that 2-nitroimidazooxazineꢀ
based VL leads would be unlikely to demonstrate an excessive reactivity toward biological
nucleophiles. This is further supported by an observed tolerance of the latter ring system
toward several basic nucleophiles in the chemistry reported below). The remaining scaffold,
nitrotriazolooxazine 47, was accessed from 5ꢀbromoꢀ3ꢀnitroꢀ1,2,4ꢀtriazole (43) and iodide
36
4
4, via desilylation/in situ annulation of the major adduct 46, as above (Scheme 1E).
The assembly of new biaryl and heterobiaryl side chain variants of Sꢀ1 and its enantiomer
3
7,38
Rꢀ1
was relatively straightforward (Schemes 2 and 3). Biphenyl analogues (Rꢀ58, Rꢀ60,
and Rꢀ62) were created by Suzuki coupling reactions on halobenzyl ether derivatives of the
32
39
key 6ꢀR alcohol 56, while combination of 56 with bromide 63 afforded the terminal
32
pyridine Rꢀ64 (Scheme 2A). Alternative alkylation of 56 or its 6ꢀS equivalent 65 with
39
various isomeric bromomethyl bromopyridines (66, 71, 75, 82, and 97) then set up the
Suzukiꢀbased manufacture of novel orthoꢀlinked phenylpyridines (Sꢀ69, Rꢀ69, Sꢀ74, and Rꢀ
74; Scheme 2B,C) and both metaꢀ and paraꢀlinked congeners (Schemes 2D and 3A,C). This
latter work was further expanded to include examples containing a pyridazine, pyrazine, or
3
9
pyrimidine ring (Rꢀ109, Rꢀ112, and Rꢀ115), using similar chemistry (Scheme 3D), and to a
40
novel racemic nitrotriazole counterpart of Rꢀ84 (88; Scheme 3B), starting from alcohol 86.
Lastly, a buffered mꢀCPBA oxidation of Rꢀ84 supplied pyridine N-oxide Rꢀ85 (Scheme 3A).
38
32
Several reference benzyl ethers (Rꢀ1, Rꢀ7, Rꢀ118, and Rꢀ119) were sourced through
32
direct alkylations of 6ꢀR alcohol 56 (Scheme 4A). Three extended ether targets (Rꢀ122, Rꢀ
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123, and Rꢀ124) were also formed by Sonogashira reactions on the propargyl ether Rꢀ121,
41
derived from the coupling of 56 with bromide 120. Next, the orthogonally diprotected triol
4
2
1
25 was employed in complementary syntheses of the novel R and S enantiomers of
42
racemic ether 6 (Scheme 4B,C). Following a Mitsunobu reaction of 125 with 4ꢀ
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(trifluoromethoxy)phenol, selective removal of the PMB group (DDQ), iodination of the
resulting alcohol 127, and reaction with 2ꢀbromoꢀ4ꢀnitroimidazole (129) gave silyl ether 130.
Treatment of the latter with TBAF and sodium hydrideꢀinduced ring closure then delivered S-
6. Conversely, successive cleavage of the TIPS group from 126, iodination, and then reaction
with 129 provided PMB ether 134. Oxidative debenzylation of 134 with DDQ in the absence
of water unexpectedly led to partial acetalization of the alcohol product with 4ꢀ
methoxybenzaldehyde, but this mixture was cleanly converted back to 135 via acid
hydrolysis (TsOH/MeOH). Baseꢀassisted annulation of alcohol 135 (NaH) then furnished the
second enantiomer, R-6. In subsequent work, 2ꢀpyridinyl ether analogues of both R-6 (Rꢀ136,
32
Rꢀ137, and Rꢀ138) and triazole 47 (139) were respectively accessed from alcohols 56 and
4
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6
via sodium hydrideꢀcatalyzed S Ar displacement reactions on halopyridines (Scheme
N
D), while Cu(I)ꢀinduced coupling of 56 with aryl isocyanates generated the Oꢀcarbamates Rꢀ
40 and Rꢀ141 (Scheme 4E). Finally, piperazine carbamate Rꢀ143 was synthesized by
chloroformylation
trifluoromethoxy)phenyl]piperazine (142) (Scheme 4F).
Further linker diversity was accessed through the transformation of 6ꢀS alcohol 65 into
of
alcohol
56
and
in
situ
reaction
with
1ꢀ[4ꢀ
43
(
3
2
the novel 6ꢀR amine hydrochloride, 146 (Scheme 5A). Following tosylation of 65 and azide
displacement, reduction of 6ꢀR azide 145 with propaneꢀ1,3ꢀdithiol gave the required amine,
which was converted to its hydrochloride salt for improved stability. From this intermediate,
reductive alkylation with benzaldehydes (using NaBH CN), acylation with benzoyl chlorides,
3
or treatment with phenyl isocyanates in the presence of catalytic dibutyltin diacetate yielded
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the expected benzylamine, benzamide, or phenyl urea derivatives (Scheme 5A,B). Then, to
conclude this study, a variety of shorter Oꢀlinked heterobiaryl side chains were constructed
using Suzuki couplings on haloheteroaryl ether precursors (156, 159, 167, 170, and 173;
Scheme 5CꢀE). The latter were obtained directly from alcohol 56, via S Ar reactions on
N
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fluoropyridines or chloropyrimidines, or, in the case of 167, from the diprotected triol 125
and 6ꢀbromopyridinꢀ3ꢀol, using the same methodology as described above for Rꢀ6.
RESULTS AND DISCUSSION
To establish the SARs against kinetoplastid diseases, 76 new (and several known)
pretomanid analogues derived from successive projects with TB Alliance and DNDi were
retrospectively tested in replicate assays conducted at the University of Antwerp (LMPH;
Tables 1ꢀ4). These assays measured activity versus the intracellular amastigote forms of both
Leishmania infantum (L. inf) and Trypanosoma cruzi (T. cruzi) and against the bloodstream
form of Trypanosoma brucei (T. brucei); cytotoxicity toward human lung fibroblasts (MRCꢀ5
44
cells, the host for T. cruzi) was also assessed. Much of our VL lead optimization work with
DNDi was earlier guided by the findings from single IC determinations against Leishmania
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donovani (L. don) in a mouse macrophageꢀbased luciferase assay carried out at the Central
Drug Research Institute (CDRI, India), and by followꢀup evaluations of in vitro microsomal
stability and efficacy in the mouse VL model (Figure 2A). The best leads were then advanced
to further appraisal in the more stringent hamster VL model. Overall, while excellent in vivo
efficacy was a key goal for secondꢀgeneration VL drug candidates, we also aspired to: a)
minimise compound lipophilicity (estimated using ACD LogP/LogD software, version 14.04;
Advanced Chemistry Development Inc., Toronto, Canada) to lessen toxicity risks, b) increase
aqueous solubility (as judged by kinetic data on dry powder forms of active leads) for optimal
24
oral bioavailability, and c) reduce hERG inhibition potential (cf. 4) to improve safety.
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Scaffold Modification: Initial Hits. As part of our earlier TB studies, we had examined
some fundamental changes to the nitroimidazooxazine “warhead”, including replacement of
the nitroimidazole ring by nitropyrazole or nitrotriazole (e.g., 48ꢀ50; Table 1) and exchange
40
of the 8ꢀoxygen for sulfur or nitrogen. We had also explored reversal of the Cꢀ6 linker (e.g.,
1
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24ꢀ26)
and transposition of the side chain to the 7ꢀposition (e.g., 41 and 42). In a further
extension to this work (seeking improved metabolic stability and new active scaffolds), the
novel methylated derivatives 14, 22, and 23 were investigated, together with
nitroimidazooxazepines 32, 33, 39 and 40. Unfortunately, except for the 7,7ꢀdimethyl
derivative 22 (which showed comparable antitubercular potency to 1 and excellent stability
toward human liver microsomes, HLM: 92% after 1 h; Table 5), these compounds proved
unattractive for TB. Nevertheless, in preliminary antiparasitic screening at the Swiss Tropical
Institute, 23, 25 and 26 demonstrated encouraging utility against L. don in a mouse
29
macrophage assay (IC50s 1.2ꢀ1.5 µM), and triazole 49 exhibited striking activity versus
Chagas disease (T. cruzi IC 0.084 µM). For greater clarity, we will focus the discussion first
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on the intended main application (VL) and discuss the other parasite data in a closing section.
26
Followꢀup testing of a larger set of compounds at CDRI identified 1, 6, 24, and 40 as
superior for VL (L. don IC50s 0.26ꢀ0.46 µM; Table 1), although such hits were still an order
2
4,25
of magnitude less potent than 4 and the 7ꢀsubstituted oxazine 41 (IC50s 0.03 µM).
These results, together with evidence for the reduced solubility and more rapid metabolism
3
6,42
of analogues with 4ꢀbenzyloxybenzyl side chains,
prompted the further appraisal of 24 in
an L. don infection VL mouse model. Disappointingly, 24 displayed weak activity (31%
inhibition at 25 mg/kg, dosing po daily for 5 d; Table 5), despite its reasonable mouse PK
profile (50% oral bioavailability, moderate exposure, and 2 h halfꢀlife; Table 6). This
outcome implied the need to significantly boost in vitro potencies in this class. However, two
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highly effective phenylpyridine analogues of 24, 116 and 117 (L. don IC s 0.02ꢀ0.05 µM;
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Table 2) also failed to deliver useful activity in this in vivo assay under the same dosing
regimen (23ꢀ49% inhibition). Analysis of their mouse PK data identified low oral
bioavailability (11ꢀ15%) as a contributing factor here because greater oral exposure led to
better efficacy (116; Table 6 and Supporting Information, Figure S1). A related concern for
both compounds was poor aqueous solubility (0.13ꢀ0.27 µg/mL at pH 7; Table 5), while
retrospective testing against L. inf later revealed suboptimal potency (IC50s ~6 µM). Taken
together, these findings reinforced the importance of improving both potency and in vivo PK
properties, in order to achieve suitable efficacy in VL models. To this end, we returned to our
sizeable pretomanid analogue library, where we had already amassed key solubility and
DMPK information from extensive earlier studies with the TB Alliance.
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Library Screening and Hit to Lead Assessments for VL. To assist the identification of
more active leads from the 6ꢀsubstituted nitroimidazooxazine class, an 898 member library
was screened against L. don amastigotes in a 7ꢀpoint 3ꢀfold dilution macrophage assay at the
45
Institut Pasteur Korea (midꢀ2010). In total, 248 compounds (28%) showed >50% inhibition
at 10 µg/mL, although only 89 (36%) of these displayed >50% inhibition at 3.3 µg/mL, and
known actives from the nitroimidazooxazole and 7ꢀsubstituted oxazine classes were dispersed
across both groupings. By eliminating examples from previously inspected classes (including
the “reversed Cꢀ6 linker” series above), a starting set of 169 hits was obtained. This set was
further refined by excluding compounds with a higher propensity for metabolic and/or
solubility issues, based on established trends, to give 42 hits, which were retested at CDRI.
The most relevant results have been summarised in Figure 3, with almost all of the remaining
hits manifesting weaker potencies (L. don IC50s 0.5ꢀ11 µM). We also screened Sꢀ1 and Sꢀ2
but both had only modest activities (L. don IC50s 3.9 and 2.6 µM, respectively). Nevertheless,
in view of the 10ꢀfold higher potency of racemic 1 (IC 0.39 µM; Table 1), this result for Sꢀ1
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was highly significant as it implied that 6R enantiomers (which have little activity against M.
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tb
) may be the more active chiral form for VL. Therefore, we synthesized a trial set of 10
compounds (Rꢀ1, Rꢀ2, Rꢀ7, R-58, R-60, R-62, Rꢀ77, Rꢀ81, Rꢀ84, and Rꢀ94) for assessment. In
9
/10 cases, these 6R enantiomers exhibited 1.1ꢀ to 12ꢀfold superior potencies (L. don IC50
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.06ꢀ1.4 µM; see the Supporting Information, Tables S2 and S3), hence, the 6R counterparts
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of selected hits in Figure 3 were also targeted (in line with the approach in Figure 2A).
In an effort to further prioritise the library screening hits for in vivo evaluation, 5
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47
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compounds of high lipophilicity (CLogP >4: Sꢀ53, Sꢀ54, Sꢀ55, Sꢀ58, and Sꢀ64 ) were
omitted from further study and the remaining 12 were assessed for aqueous solubility and
48
microsomal stability (Table 5 and cited references for Figure 3). Both the amide Sꢀ151 and
urea Sꢀ155 provided encouraging solubility data (132 and 22 µg/mL) but the urea
unexpectedly showed poor stability toward mouse liver microsomes (MLM: 43% parent after
49
3
0 min). Conversely, both the lipophilic arylthiazole Sꢀ51 (CLogP ~4.0) and the
4
2
arylpyrimidine Sꢀ171 were considered of borderline interest due to their modest solubility
values (0.9ꢀ1.6 µg/mL). While some phenylpyridine hits (e.g., Sꢀ77, Sꢀ89, Sꢀ91, Sꢀ92, and Sꢀ
3
9
9
9) were not substantially more soluble than this at pH 7 (1.4ꢀ4.0 µg/mL), these compounds
have demonstrated greatly superior results at pH 1 (211ꢀ1780 µg/mL). It has been recognized
that the low pH of gastric fluid (typically ~1ꢀ2) can enhance the dissolution and oral
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absorption of such weak bases. Furthermore, close analogue Sꢀ2 was advanced to clinical
studies for TB partly on the basis of its superior in vivo PK properties in comparison to those
2
3
of delamanid (5), which are absorption limited. Concordant with this, the most potent
phenylpyridine hits in Figure 3 (Sꢀ77, Sꢀ81, Sꢀ89, Sꢀ91, and Sꢀ92) also displayed broadly
acceptable HLM and MLM stabilities (>70% remaining after 30 min), suggesting their
suitability for in vivo studies.
The best 6 screening hits above (phenylpyridines Sꢀ77, Sꢀ81, Sꢀ89, Sꢀ91, and Sꢀ92, and
amide Sꢀ151) were then evaluated in the mouse VL model alongside a similar set of 6R
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enantiomers (Rꢀ77, Rꢀ84, Rꢀ89, Rꢀ91, Rꢀ92, Rꢀ94, and Rꢀ151), dosing at 50 mg/kg orally, once
daily for 5 days. Results for the metaꢀlinked phenylpyridines Rꢀ77, Sꢀ77, and Sꢀ81 were not
particularly impressive (52%, 44% and 35% inhibition; Table 5), although the level of
activity did track with their respective L. don IC s (0.06, 0.12, and 0.24 µM). In contrast,
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pairwise comparison of paraꢀlinked phenylpyridines Sꢀ89, Sꢀ91, and Sꢀ92 with their 6R
counterparts unequivocally confirmed the latter as superior (Rꢀ89: 99.5% vs Sꢀ89: 45%; Rꢀ91:
99.8% vs Sꢀ91: 69%; Rꢀ92: 94% vs Sꢀ92: 37%), notwithstanding their slightly higher rates of
metabolism (e.g., Rꢀ91: 81% parent after 30 min in MLM vs Sꢀ91: 100%). In this series, the
4ꢀfluoro analogue Rꢀ94 (L. don IC50 0.18 µM) showed reduced utility (83%), whereas the
apparently less potent 4ꢀtrifluoromethoxy congener Rꢀ84 (IC50 0.32 µM in the same CDRI
assay) provided excellent efficacy (99.4%), despite having lower microsomal stability (36%
vs 66% after 1 h in MLM). Overall, the most effective (6S) screening hit was the soluble
amide Sꢀ151 (72%) but its 6R form (Rꢀ151) was unexpectedly poor (5%). However, for this
6ꢀNꢀlinked benzamide class, it was later discovered that the 6S enantiomers had the stronger
in vitro potencies (e.g., L. inf IC s 5.6 vs 12 µM for Sꢀ151 and Rꢀ151), suggesting the need
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for a more systematic investigation of the SAR (Figure 2B). For better clarity, we will
describe this analysis next, before summarising the results from additional in vivo
assessments on all of the most active new VL leads.
SAR of 6-Substituted 2-Nitroimidazooxazines for VL. Following the discovery that
many 6R enantiomers had superior in vitro and in vivo activities against VL, a more extensive
lead optimisation study was initiated to develop additional backup candidates to 4 possessing
an advantageous solubility, PKꢀPD, and safety profile. In light of the high potency of orthoꢀ
linked biphenyl hit Sꢀ58 (L. don IC50 0.18 µM), we first sought to establish the optimal
linking position for biaryl side chains. Comparison of Rꢀ58, Rꢀ60, and Rꢀ62 in both L. don
and L. inf assays (Table 2) unexpectedly indicated that an orthoꢀlinkage was most preferred
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and that paraꢀlinkage was least preferred. Therefore, the novel orthoꢀlinked phenylpyridines
Sꢀ69, Rꢀ69, Sꢀ74, and Rꢀ74 were studied. Here, Rꢀ69 and Rꢀ74 were equal best, although 1.8ꢀ
fold less effective than Rꢀ58 (L. inf IC50s 2.0 vs 1.1 µM). Interestingly, these two
phenylpyridine isomers showed major differences in both solubility and microsomal stability,
with the more soluble Rꢀ74 (78 vs 0.51 µg/mL) being metabolised extremely rapidly in all
three microsome species (0.1ꢀ8% remaining after 1 h; Table 5), whereas Rꢀ69 was more
stable than the paraꢀlinked analogue Rꢀ84 above (44% vs 36% after 1 h in MLM).
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In the metaꢀlinked phenylpyridine series, two new compounds (Rꢀ79 and Rꢀ80) having
terminal ring substituents favoured in paraꢀlinked isomers above (2ꢀF, 4ꢀOCF and 4ꢀ
3
OCF H) were marginally more potent than Rꢀ77 (4ꢀOCF ) and Rꢀ81 (4ꢀF) (L. inf IC s 0.45
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3
50
and 0.51 vs 0.63 and 0.73 µM; Table 2). Here, replacement of the proximal phenyl ring in Rꢀ
60 by 2ꢀpyridine (Rꢀ77) resulted in a 3.7ꢀfold improvement in activity (L. inf IC50s 2.3 and
0.63 µM). This enhancement by 2ꢀpyridine was even more pronounced in the paraꢀlinked
phenylpyridine series (L. inf IC s 0.71 and >64 µM for Rꢀ84 and Rꢀ62), clarifying that with
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this heterocycle, orthoꢀlinkage was less useful than metaꢀ and paraꢀlinkage. However, in the
paraꢀlinked set, we considered that the 2’ꢀnitrogen would have less steric protection against
oxidation, perhaps accounting for the modest microsomal stabilities of some analogues.
Indeed, we could form the Nꢀoxide derivative of Rꢀ84 (Rꢀ85), which was found to be 2.3ꢀfold
less potent (L. inf IC 1.6 µM). Because microsomal stability varied significantly with the
50
substituents in the terminal ring, we evaluated three new congeners (Rꢀ90: 3ꢀF, 4ꢀOCF , Rꢀ
3
3
93: 4ꢀCF , and Rꢀ96: 2,4ꢀdiF). The most promising of these was Rꢀ96 (L. inf IC50 1.2 µM, ~2ꢀ
fold weaker activity than early leads Rꢀ84, Rꢀ89, Rꢀ91, and Rꢀ92), which exhibited a better
stability profile in MLM and HLM (57ꢀ59% vs 16ꢀ40% for Rꢀ84, Rꢀ89, and Rꢀ91; Table 5).
As suggested by the screening data (Figure 3), replacement of the proximal phenyl ring in
Rꢀ62 by 3ꢀpyridine (Rꢀ2) was less favourable (L. inf IC 4.1 µM vs 0.71 µM for Rꢀ84; Table
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2). Nevertheless, the 6R enantiomers of two hits (Rꢀ99: 2ꢀF, 4ꢀOCF and Rꢀ101: 2ꢀCl, 4ꢀ
3
OCF ) and the novel 2,4ꢀdifluoro analogue Rꢀ106 all displayed good potencies (L. inf IC50s
1.1, 0.61, and 0.85 µM) and at least comparable microsomal stabilities to their 2ꢀpyridine
counterparts, although Rꢀ101 was cytotoxic (MRCꢀ5 IC50 17 µM). By extension, we
examined three less lipophilic diaza proximal rings (Rꢀ109, Rꢀ112, and Rꢀ115) that had
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proven to be very effective in our TB studies but these turned out to be of lower interest (L.
inf IC50s 1.4ꢀ2.8 µM). In summary, several new phenylpyridines provided profiles that were
attractive for in vivo evaluation but we had yet to investigate other linker groups. Therefore,
we next turned our attention to simpler monoaryl side chains to explore these changes. For
this part of the study we restricted our focus to linkers that had shown particular promise
either in the initial screening (e.g., 6ꢀO, 6ꢀNHCO, 6ꢀNHCONH) or in our earlier TB work.
Commencing with the enantiomer of pretomanid (Rꢀ1: L. inf IC50 4.7 µM; Table 3),
variation of the trifluoromethoxy position identified that ortho substitution was best (Rꢀ119:
L. inf IC 1.4 µM), followed by meta substitution (Rꢀ118), mimicking findings for biphenyl
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linkage. Switching to a propargyl ether (Rꢀ122) produced much greater activity (L. inf IC
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0.33 µM), which was largely retained in trifluoromethylpyridine replacements for the aryl
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ring (Rꢀ123 and Rꢀ124: L. inf IC50s 0.44 and 0.53 µM). Conversely, removal of the benzylic
methylene (Rꢀ6) also enabled high potency (L. inf IC50 0.53 µM, L. don IC50 0.19 µM), while
critically allowing the retention of good aqueous solubility (12 µg/mL) and high microsomal
stability (79ꢀ81% parent after 1 h in MLM and HLM; Table 5). Therefore, we similarly
investigated trifluoromethylpyridine analogues of Rꢀ6 (Rꢀ136, Rꢀ137, and Rꢀ138) and while
there was a ca. 2ꢀfold loss in activity, Rꢀ136 demonstrated a 9ꢀfold improvement in aqueous
solubility (110 µg/mL), together with a slower rate of metabolism (90ꢀ92% parent after 1 h in
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MLM and HLM). However, changing to an Oꢀcarbamate linker (Rꢀ140, Rꢀ141, and Rꢀ143)
proved less satisfactory, with moderate to low potencies observed (L. inf IC s 2.1ꢀ17 µM).
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Another option to improve solubility was to replace the ether linkage at Cꢀ6 with nitrogenꢀ
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based linkers. The 6ꢀamino analogue of Rꢀ1 (Rꢀ147) had a 5ꢀfold better solubility value at
pH 7 (84 µg/mL) and was >2000 times more soluble at pH 1. Nevertheless, this compound
was less stable toward microsomes (e.g., 61% vs 86% in MLM) and showed 2.6ꢀfold lower
activity (L. inf IC 12 vs 4.7µM), which was not sufficiently improved by varying the ring
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substituent position (Rꢀ148 and Rꢀ149). Alternatively, with a carboxamide or urea linker, the
enantiomer preference was reversed, with the original 6S forms (Sꢀ150, Sꢀ151, Sꢀ152, Sꢀ154,
and Sꢀ155) being clearly superior but not particularly potent (L. inf IC50s ~6ꢀ10 µM). Hence,
the only compounds with useful antileishmanial activity were ether linked at Cꢀ6, although it
was apparent that the original OCH linkage was not optimal and that removal of the benzylic
2
methylene may have metabolic stability and potency advantages. To investigate this further, a
small set of Oꢀlinked phenylpyridine and phenylpyrimidine derivatives was evaluated (Table
4
). In the phenylpyridine series (Rꢀ157 to Rꢀ169), activity against L. inf was similar or better
than for Rꢀ6, with the 3ꢀpyridine isomers preferred (Rꢀ168 and Rꢀ169: IC s 0.13ꢀ0.20 µM).
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These latter compounds exhibited microsomal stabilities comparable to those of the parent
linker series above but their solubility values were inferior (0.36ꢀ1.2 µg/mL at pH 7). Finally,
contrary to the screening data for Sꢀ171, a proximal pyrimidine ring was tolerated only when
it was paraꢀlinked (Rꢀ174 and Rꢀ175; L. inf IC50s 0.65ꢀ0.83 µM). Generally, potency against
24,25
L. inf was more discriminating and tended to better correlate with in vivo outcomes.
Throughout the course of these studies, 10 more candidates (Rꢀ1, Rꢀ6, Rꢀ69, Rꢀ96, Rꢀ99, Rꢀ
06, Rꢀ136, Rꢀ147, Rꢀ168, and Rꢀ169) were screened for activity in the mouse VL model at
0 mg/kg (dosing po daily for 5 d; Table 5). The orthoꢀlinked phenylpyridine Rꢀ69 was
1
5
disappointing (20% inhibition) but the new paraꢀlinked analogues Rꢀ96, Rꢀ99 and Rꢀ106
displayed high efficacies (91ꢀ97%). More heartening still, shorter chain phenylpyridines Rꢀ
1
68 and Rꢀ169 gave essentially complete clearance of the parasite infection (99.9%), as did
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their monoaryl counterparts, Rꢀ6 and Rꢀ136. However, both Rꢀ1 and its aminoꢀlinked
equivalent Rꢀ147 were unsatisfactory (50% and 12%, respectively), consistent with their
38
weaker in vitro potencies. During the concluding stages of this project, Patterson et al.
reported that Rꢀ1 was a potential oral treatment for VL on the basis of its in vivo activity in a
comparable mouse model at the much higher dosage of 100 mg/kg twice daily, but it is clear
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from these results and other studies that Rꢀ1 may not be the optimal development candidate.
Additional Assessments to Determine the Best VL Lead. Doseꢀresponse experiments on
7 of the 10 best compounds (Rꢀ6, Rꢀ84, Rꢀ89, Rꢀ91, Rꢀ92, Rꢀ96, and Rꢀ99) in the mouse VL
model yielded ED values of 7.5, 12, 14, 28, 20, 28, and 19 mg/kg, respectively (Table 5 and
50
Figure 4). The more recent Oꢀlinked leads, Rꢀ136, Rꢀ168 and Rꢀ169, were also evaluated at
the lower dose of 6.25 mg/kg, and provided parasite burden reductions of 30%, 99.7%, and
72%, respectively. The most efficacious of these (Rꢀ168) produced 84% inhibition at 3.13
2
5,26
mg/kg, representing activity at a similar level to the nitroimidazooxazole 4,
albeit
marginal aqueous solubility (0.85 µM at pH 7) deterred its advanced assessment. Instead, we
elected to focus initially on the monoaryl ethers Rꢀ6 and Rꢀ136, together with
phenylpyridines Rꢀ84 and Rꢀ89, which all produced favourable mouse PK data, including
excellent oral exposure levels and halfꢀlives of 6ꢀ30 h (Table 6 and Supporting Information,
Figure S1).
The selected candidates were further assessed in the chronic infection hamster model,
which is considered the bona fide experimental model for VL because it mimics many
51
features of progressive human disease. Pleasingly, at 50 mg/kg twice daily for 5 d,
phenylpyridine Rꢀ84 achieved 99.9ꢀ100% L. inf clearance in all three target organs, and Rꢀ89
was almost as good (Table 7 and Figure 5), although both compounds gave less inhibition in
bone marrow at 25 mg/kg b.i.d. (81ꢀ88%). The monoaryl ethers Rꢀ6 and Rꢀ136 were even
more effective at both dose levels, enabling a 97ꢀ99% parasite kill at 25 mg/kg b.i.d. (for
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comparison, this efficacy level was similar to that observed for 3 at 12.5 mg/kg b.i.d. ).
Pyridinyl ether Rꢀ136 additionally demonstrated fully curative activity (100% parasite
clearance in all three organs) at 25 mg/kg b.i.d. in an L. don infection hamster model. Three
more phenylpyridines (Rꢀ96, Rꢀ99, and Rꢀ106) were similarly evaluated in the L. inf hamster
model but only Rꢀ99 (the 3ꢀpyridyl isomer of Rꢀ89) showed any promise (86ꢀ98% at 50
mg/kg b.i.d.), paralleling efficacy trends in the mouse model. These results were also broadly
in line with the hamster PK data, where Rꢀ96 and Rꢀ106 displayed inferior oral exposures and
much lower oral bioavailabilities than the other leads (21ꢀ37% vs 64ꢀ100%; Table 6 and
Supporting Information, Figure S2).
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To better discriminate between the four preferred candidates, we next considered key safety
features, starting with measuring their interactions with the hERG channel. While the
monoaryl ethers Rꢀ6 and Rꢀ136 posed minimal risk (hERG IC50s >30 µM), unfortunately,
phenylpyridines Rꢀ84 and Rꢀ89 both caused potent inhibition (IC50s 0.81 and 0.92 µM),
5
2
53,54
indicating a strong likelihood of QT prolongation (lead optimization criteria
mandate an
IC >10 µM). This outcome was not anticipated, as 6S counterparts had generated much less
5
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concern. The remaining two compounds were then checked for any evidence of mutagenicity
in the Ames test. Here, phenyl ether Rꢀ6 was negative but the more soluble pyridinyl ether Rꢀ
1
36 unexpectedly yielded a positive result. Although several other nitroimidazole drugs are
55
Ames positive (e.g., metronidazole and fexinidazole), this outcome effectively ruled Rꢀ136
out of contention because it would face a more difficult path to achieve regulatory
16,53
approval.
Thus Rꢀ6 was identified as the optimal VL lead.
Further appraisal of VL lead R-6. Additional properties of Rꢀ6 were measured and
weighed against those of the initial preclinical candidate 4 (Table 8). The two compounds
were comparable in terms of molecular weight (345 vs 359 Da) and were both highly
48
permeable, but Rꢀ6 had a lower measured Log D value (2.59 vs 3.10 for 4 and 2.52 for Sꢀ1 ),
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superior thermodynamic solubility (23 vs 2.8 µM), and a reduced propensity to bind to
plasma proteins in various species (82ꢀ87% vs 92ꢀ96% for 4). This lead also showed only
weak CYP3A4 activity (IC50 >40 µM) and produced a notably favourable rat PK profile, with
prolonged exposure and 100% oral bioavailability (Table 6 and Supporting Information,
Figure S2). These attributes reinforced our conclusion that Rꢀ6 (DNDIꢀ8219) was indeed a
very promising backup candidate for VL.
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A larger scale synthesis of Rꢀ6 has recently provided a single 170 g batch of high quality
material (HPLC purity >99.9% and 97.2% ee) in reasonable overall yield (8% over 9 linear
steps, starting from commercial Rꢀsolketal). However, the current synthetic route would still
require significant improvement to deliver a scalable, robust and costꢀeffective chemical
process, in line with the stated TPP objectives for an affordable drug. Possible alternatives
5
6
include using the known enantiomer of orthogonally diprotected triol 125 and following the
pathway described for S-6 in Scheme 4B because this enantiomer could be obtained from the
57
cheap, optically pure starting material Dꢀmannitol (via the less expensive Sꢀsolketal).
Mechanistic studies on the closely related nitroimidazooxazine Rꢀ1 demonstrated that it
was not activated by the previously identified type I nitroreductase (NTR1) in Leishmania
which mediates the cidal effects of monocyclic nitroheterocyclic drugs such as fexinidazole
38,58
and nifurtimox.
Instead, the activity of Rꢀ1 was solely triggered by the same novel flavin
mononucleotide dependent NADH oxidoreductase (NTR2) in Leishmania that was employed
2
7
by nitroimidazooxazoles, such as 4 and 5. This new mode of action was elucidated through
a combination of quantitative proteomics and whole genome sequencing of susceptible and
drug resistant L. don promastigotes, the latter being generated via culture in the continuous
27
presence of Rꢀ1 for 80 days (leading to reduced expression of NTR2). The further
observation that Rꢀ1 and fexinidazole sulfone displayed additive effects against drug
susceptible L. don sparked the suggestion of combination therapy between monocyclic and
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bicyclic nitro drugs to reduce the likelihood of any future clinical drug resistance. However,
we consider that it may be more preferable to look for alternative partner drugs for 3 (or Rꢀ6)
with greater diversity in their mechanism of action.
One final aspect to consider with Rꢀ6 was its efficacy against a wider range of VL and
cutaneous leishmaniasis (CL) strains. Overall, Rꢀ6 displayed potent broadꢀspectrum activity
against both reference strains and clinical isolates (Table 9), comparing favourably with the
standard agents sodium stibogluconate, paromomycin and miltefosine. This lead was also
effective against the drug resistant clinical isolates L. don BHU1, L. inf LEM5159, and L. inf
MHOM/FR/96/LEM3323 (IC s 1.3ꢀ5.4 µM), as well as the miltefosine resistant laboratory
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strain L. inf MHOM/FR/96/LEM3323 C14 MIL4 (IC 0.59 µM). These data confirm that Rꢀ
5
0
6
has excellent potential as a therapy for VL and may have an additional application for the
1
treatment of CL (the more common skin lesion form of leishmaniasis).
SAR of 6-Substituted 2-Nitroimidazo- (or 2-Nitrotriazolo-)oxazines for Chagas
Disease. While the primary objective of this reinvestigation of pretomanid analogues was to
develop a backup drug candidate for VL, retrospective screening against the protozoan
parasites T. cruzi and T. brucei presented an opportunity to assess the possible capacity of
these compounds to treat Chagas disease and human African trypanosomiasis (HAT),
respectively. A brief inspection of Tables 1ꢀ4 found that only one compound (116) had
submicromolar activity against HAT (T. brucei IC50 0.63 µM), and this hit could be
disregarded on the basis of its less favourable mouse PK profile, inferior aqueous solubility,
3
6
and poor MDCKꢀMDR1 cell permeability.
Conversely, like 7ꢀsubstituted 2ꢀ
24
nitroimidazooxazines, the majority of compounds displayed interesting potencies against T.
cruzi (IC50s 0.025ꢀ1 µM). The selective antiꢀChagas activity of nitrotriazolooxazines (e.g.,
4
9: T. cruzi IC50 0.25 µM vs >64 µM against L. inf) was particularly striking, being
2
5
reminiscent of that of a nitrotriazolooxazole analogue of 4. However, further scrutiny
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revealed that the enantiomers of 48 were roughly equipotent (Table 3), while the racemic
nitrotriazole counterparts of key VL leads Rꢀ6, Rꢀ84, and Rꢀ136 (47, 88, and 139) were 9ꢀ to
68ꢀfold less active, suggesting limited utility for this class.
Aside from the less suitable reversed linker congeners (24ꢀ26, 116 and 117) and Oꢀlinked
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biaryls (Rꢀ157, Rꢀ160, Rꢀ161, Rꢀ168, and Rꢀ171), the most potent 6ꢀsubstituted 2ꢀ
nitroimidazooxazine antiꢀChagas leads were paraꢀlinked phenylpyridines (e.g., Rꢀ84, Rꢀ89,
Rꢀ90, Rꢀ92, and Rꢀ93; IC50s 0.025ꢀ0.078 µM). Here, the 6R enantiomers were clearly superior
(by 15ꢀ to >400ꢀfold) and the 2ꢀpyridine isomer was preferred, although Nꢀoxidation of the
pyridine ring (Rꢀ85) was strongly deactivating (77ꢀfold). In the monoaryl subset,
arylpropargyl ether Rꢀ122 and phenyl ether Rꢀ6 seemed highly promising (IC s 0.11 and
5
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0.15 µM), with the latter favoured on PK and solubility grounds, whereas similarly active
36
benzylamines Rꢀ148 and Rꢀ149 were expected to show rapid metabolism. Overall, given the
significant hERG liability and higher plasma protein binding of the phenylpyridines (e.g.,
97.9% for Rꢀ84 in mice), Rꢀ6 was also regarded as the best new lead for Chagas disease,
although its mechanism of action for this application remains to be determined.
CONCLUSIONS
In response to a compelling clinical need for more satisfactory VL treatments, recent efforts
have been made to reposition leads from other therapeutic areas, seeking to accelerate new
drug development. Promising results with antitubercular nitroimidazooxazoles and 7ꢀ
substituted 2ꢀnitroimidazooxazines encouraged us to evaluate additional scaffolds, e.g.,
nitroimidazooxazepines and methylated or reversed Cꢀ6 linker analogues of pretomanid, but
these lacked sufficient potency and/or suitable PK and efficacy in the L. don mouse model.
However, phenotypic screening of our pretomanid analogue library and followꢀup IC50
testing unveiled more active hits spanning a wide lipophilicity range (CLogP 1.2ꢀ4.5),
including several with better solubility and microsomal stability, e.g., phenylpyridines and
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benzamide Sꢀ151. This work also pointed to the generally improved activities of novel 6R
enantiomers, which was confirmed for phenylpyridines through comparative appraisal in the
mouse VL model. Further studies in this series established that a 4ꢀtrifluoromethoxy phenyl
substituent, paraꢀlinkage, and a proximal 2ꢀpyridine ring were preferred for good in vivo PK
and efficacy, with two such leads (Rꢀ84 and Rꢀ89) giving ≥99% parasite clearance in the L.
inf hamster model at 50 mg/kg, b.i.d. These compounds also showed high potencies against
T. cruzi but unexpectedly high hERG inhibition ultimately terminated their development.
Meanwhile, investigation of the Cꢀ6 linker group revealed that the parent ether moiety in Rꢀ
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(OCH ) was nonꢀoptimal for VL, with shorter and longer chain variants (O or OCH C≡C)
2 2
improving potency against L. inf (whereas Oꢀcarbamate and Nꢀlinked alternatives were poor).
Although Oꢀlinked phenylpyridine Rꢀ168 displayed superb activity in the mouse VL model
(99.7% reduction in parasite burden at 6.25 mg/kg, similar to 4), we elected to focus instead
on two less lipophilic monoaryl leads (Rꢀ6 and pyridine Rꢀ136), having superior solubility
values (12ꢀ110 µg/mL), low hERG risk, and excellent PK profiles in three species (mouse,
rat, and hamster). Both compounds delivered high efficacies in the chronic infection hamster
model (≥97% inhibition at 25 mg/kg, b.i.d.) and showed diminished binding to plasma
proteins, although a positive Ames test for pyridine Rꢀ136 dissuaded its further advancement
and earmarked Rꢀ6 as the favoured VL backup candidate to 3. Finally, like the
phenylpyridines, Rꢀ6 also demonstrated interesting activity against T. cruzi, whereas
nitrotriazolooxazine congeners of such leads were less effective. These results provide new
insights on the exciting potential of bicyclic nitroimidazoles to treat neglected diseases.
EXPERIMENTAL SECTION
Combustion analyses were performed by the Campbell Microanalytical Laboratory,
University of Otago, Dunedin, New Zealand. Melting points were determined using an
Electrothermal IA9100 melting point apparatus and are as read. NMR spectra were measured
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13
on a Bruker Avance 400 spectrometer at 400 MHz for H and 100 MHz for C and were
4
referenced to Me Si or solvent resonances. Chemical shifts and coupling constants were
recorded in units of ppm and hertz, respectively. Highꢀresolution electron impact (HREIMS),
chemical ionisation (HRCIMS), and fast atom bombardment (HRFABMS) mass spectra were
determined on a VGꢀ70SE mass spectrometer at nominal 5000 resolution. Highꢀresolution
electrospray ionisation (HRESIMS) mass spectrometry was conducted on a Bruker
micrOTOFꢀQ II mass spectrometer. Lowꢀresolution atmospheric pressure chemical ionisation
(APCI) mass spectra were obtained for organic solutions using a ThermoFinnigan Surveyor
MSQ mass spectrometer connected to a Gilson autosampler. Optical rotations were measured
on a Schmidt + Haensch Polartronic NH8 polarimeter. Column chromatography was
performed on silica gel (Merck 230ꢀ400 mesh). Chromatographed compounds were typically
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further purified by crystallization from two solvent combinations, e.g., CH
2
Cl
2
and nꢀhexane,
O was
EtOAc and nꢀhexane, Et O and nꢀpentane, or CH Cl and nꢀpentane (occasionally, Et
2
2
2
2
added to the latter combination to induce solidification, while some compounds required
cooling at ꢀ20 °C); more polar compounds were first dissolved in a minimum of 10%
MeOH/CH
chromatography was carried out on aluminiumꢀbacked silica gel plates (Merck 60 F254), with
visualization of components by UV light (254 nm), I , or KMnO staining. Tested compounds
including batches screened in vivo) were all ≥95% pure, as determined by combustion
analysis (results within 0.4% of theoretical values) and/or by HPLC conducted on an Agilent
100 system with diode array detection, using a 150 mm x 3.2 mm Altima 5 µm reversed
phase C18 column or a 150 mm x 4.6 mm Zorbax Eclipse XDB 5 µm C8 column and eluting
with a gradient (40ꢀ100%) of 80% CH CN/water in 45 mM ammonium formate buffer (pH
.5). Finally, the chiral purity of lead Rꢀ6 was assessed by HPLC carried out on a Shimadzu
2 2
Cl and slowly diluted with nꢀhexane to give the solid product. Thinꢀlayer
2
4
(
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2010 system with diode array detection, employing a 150 mm x 4.6 mm CHIRALPAK AYꢀH
5 µm analytical column and isocratic elution with 20% EtOH/nꢀheptane.
Compounds of Table 1. The following section details the syntheses of compounds 14, 39,
and 41 of Table 1, via representative procedures and key intermediates, as described in
Scheme 1. For the syntheses of the other compounds in Table 1, please refer to the
Supporting Information.
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Synthesis of 14 (Scheme 1A):
Procedure A: 1-[(tert-Butyldimethylsilyl)oxy]-3-(2,4-dinitro-1H-imidazol-1-yl)-2-
methylpropan-2-ol (10). A mixture of 2,4ꢀdinitroꢀ1Hꢀimidazole (8) (1.00 g, 6.33 mmol) and
33
tertꢀbutyldimethyl[(2ꢀmethyloxiranꢀ2ꢀyl)methoxy]silane (9) (1.81 g, 8.94 mmol) under N2
was stirred at 75 °C for 18 h. The resulting cooled mixture was diluted with EtOAc (250 mL)
and washed with aqueous NaHCO
3
(150 mL) and brine (125 mL), backꢀextracting with
SO ) and then evaporated to dryness
EtOAc (150 mL). The combined extracts were dried (Na
2
4
under reduced pressure and the remaining oil was chromatographed on silica gel. Elution
1
with 10ꢀ33% EtOAc/petroleum ether gave 10 (0.92 g, 40%) as a yellow oil; H NMR
(CDCl
3
) δ 7.98 (s, 1 H), 4.75 (d, J = 14.0 Hz, 1 H), 4.53 (d, J = 14.0 Hz, 1 H), 3.54 (d, J =
10.2 Hz, 1 H), 3.46 (d, J = 10.1 Hz, 1 H), 2.52 (br s, 1 H), 1.17 (s, 3 H), 0.91 (s, 9 H), 0.09 (2
+
3 25 4 6
s, 2 x 3 H); HRCIMS (NH ) calcd for C13H N O Si m/z [M + H] 361.1543, found 361.1545.
Procedure B: 1-{3-[(tert-Butyldimethylsilyl)oxy]-2-methyl-2-[(tetrahydro-2H-pyran-2-
yl)oxy]propyl}-2,4-dinitro-1H-imidazole (11). A mixture of alcohol 10 (248 mg, 0.688
mmol), 3,4ꢀdihydroꢀ2Hꢀpyran (0.32 mL, 3.51 mmol) and PPTS (95 mg, 0.378 mmol) in
anhydrous CH
diluted with CH
mL) and brine (50 mL), backꢀextracting with CH Cl (50 mL). The combined extracts were
2
Cl
2
(10 mL) under N
2
was stirred at 20 °C for 5 h. The resulting solution was
Cl
2 2
(100 mL) and washed with aqueous NaHCO
3
(2 x 50 mL), water (50
2
2
2 4
dried (Na SO ) and then evaporated to dryness under reduced pressure and the remaining oil
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2
2
2
2
2
2
2
3
3
3
3
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4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
was chromatographed on silica gel. Elution with 10% EtOAc/petroleum ether gave 11 (234
1
mg, 76%) as a yellow oil (a 1:1 mixture of diastereomers); H NMR (CDCl ) δ 8.25, 7.99 (2
3
s, 1 H), 4.88 (d, J = 13.8 Hz, 0.5 H), 4.87 (d, J = 14.0 Hz, 0.5 H), 4.84ꢀ4.72 (m, 1 H), 4.62 (d,
J = 14.0 Hz, 0.5 H), 4.54 (d, J = 13.8 Hz, 0.5 H), 3.73ꢀ3.65 (m, 1 H), 3.60ꢀ3.36 (m, 2 H), 3.29
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(d, J = 10.6 Hz, 0.5 H), 3.26 (d, J = 10.7 Hz, 0.5 H), 1.95ꢀ1.35 (m, 6 H), 1.33, 1.25 (2 s, 3 H),
3
0.94, 0.87 (2 s, 9 H), 0.10 (2 s, 3 H), 0.04, 0.02 (2 s, 3 H); HRCIMS (NH ) calcd for
+
C
18
H
33
N
4
O
7
Si m/z [M + H] 445.2119, found 445.2125.
Procedure C: 6-Methyl-2-nitro-6-[(tetrahydro-2H-pyran-2-yl)oxy]-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (12). A solution of silyl ether 11 (230 mg, 0.517 mmol) in
anhydrous THF (6 mL) under N was treated with TBAF (1.5 mL of a 1 M solution in THF,
.50 mmol) and the mixture was stirred at 20 °C for 1 h. The resulting solution was
2
1
concentrated under reduced pressure, then diluted with EtOAc (50 mL) and washed with
aqueous NaHCO (50 mL) and brine (50 mL), backꢀextracting with EtOAc (2 x 50 mL). The
3
combined extracts were dried (Na SO ) and then evaporated to dryness under reduced
2
4
2 2
pressure and the residue was chromatographed on silica gel. Elution with 5% MeOH/CH Cl
gave 12 (130 mg, 89%) as a white solid (a 1:1 mixture of diastereomers): mp
1
(CH
2
Cl
2
/hexane) 153ꢀ156 °C; H NMR (CDCl
3
) δ 7.40, 7.37 (2 s, 1 H), 5.03ꢀ4.95 (m, 1 H),
4.54 (dd, J = 11.6, 2.3 Hz, 0.5 H), 4.44 (dd, J = 11.8, 2.7 Hz, 0.5 H), 4.34 (dd, J = 12.7, 2.7
Hz, 0.5 H), 4.18 (d, J = 11.6 Hz, 0.5 H), 4.11 (d, J = 11.7 Hz, 0.5 H), 4.09 (dd, J = 12.5, 2.3
Hz, 0.5 H), 3.89 (d, J = 12.6 Hz, 0.5 H), 3.89ꢀ3.81 (m, 1 H), 3.57ꢀ3.44 (m, 1 H), 3.42ꢀ3.33
+
(
m, 0.5 H), 1.92ꢀ1.38 (m, 9 H); APCI MS m/z 284 [M + H] .
Procedure D: 6-Methyl-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ol (13). A
solution of THP ether 12 (120 mg, 0.424 mmol) in MeOH (10 mL) was treated with
methanesulfonic acid (52.6 mg, 0.547 mmol) and the mixture was stirred at 20 °C for 1 h.
3
The resulting solution was neutralised with aqueous NaHCO and then the solvents were
2
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removed under reduced pressure and the residue was chromatographed on silica gel. Elution
with 5% MeOH/CH
2
Cl
2
gave 13 (80 mg, 95%) as a white solid: mp (MeOH/CH
2 2
Cl /hexane)
1
1
4
1
85ꢀ188 °C; H NMR [(CD
3
)
2
SO] δ 8.05 (s, 1 H), 5.50 (br s, 1 H), 4.26 (d, J = 11.1 Hz, 1 H),
.12 (dd, J = 11.1, 2.6 Hz, 1 H), 4.00 (d, J = 12.6 Hz, 1 H), 3.89 (dd, J = 12.6, 2.4 Hz, 1 H),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
.24 (s, 3 H); HREIMS calcd for C
H N O
7 9 3 4
m/z (M ) 199.0593, found 199.0591.
Procedure E: 6-Methyl-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (14). A solution of alcohol 13 (50.7 mg, 0.255 mmol) and 4ꢀ
trifluoromethoxy)benzyl bromide (65 µL, 0.406 mmol) in anhydrous DMF (3 mL) under N
was treated with 60% NaH (20 mg, 0.50 mmol), and the mixture was stirred at 20 °C for 3 h.
The resulting mixture was diluted with EtOAc (150 mL) and washed with aqueous NaHCO
50 mL), water (2 x 50 mL) and brine (50 mL), backꢀextracting with EtOAc (100 mL). The
combined extracts were dried (Na SO ) and then evaporated to dryness under reduced
(
2
3
(
2
4
pressure and the residue was chromatographed on silica gel. Elution with 1ꢀ2%
1
MeOH/CH Cl gave 14 (45 mg, 47%) as a white solid: mp (EtOAc/hexane) 139ꢀ141 °C; H
2
2
NMR [(CD
3
)
2
SO] δ 7.99 (s, 1 H), 7.31 (br d, J = 9.0 Hz, 2 H), 7.28 (br d, J = 9.0 Hz, 2 H),
4.60 (dd, J = 11.9, 2.8 Hz, 1 H), 4.56 (br s, 2 H), 4.37 (d, J = 11.8 Hz, 1 H), 4.24 (dd, J =
1
3
13.2, 2.6 Hz, 1 H), 4.07 (d, J = 13.1 Hz, 1 H), 1.36 (s, 3 H); C NMR [(CD
3
)
2
SO] δ 147.5,
47.0, 142.2, 137.8, 129.0 (2 C), 120.9 (2 C), 120.0 (q, JCꢀF = 255.9 Hz), 117.8, 70.6, 69.2,
2.9, 50.7, 17.0. Anal. Calcd for C15 : C, 48.26; H, 3.78; N, 11.26. Found: C, 48.23;
1
6
14 3 3 5
H F N O
H, 3.98; N, 11.31.
Synthesis of 39 and 41 (Scheme 1D):
-Bromo-4-nitro-1-{3-[(tetrahydro-2H-pyran-2-yl)oxy]-4-
[(triisopropylsilyl)oxy]butyl}-1H-imidazole (35). Reaction of 4ꢀ(2ꢀbromoꢀ4ꢀnitroꢀ1Hꢀ
2
2
4
imidazolꢀ1ꢀyl)ꢀ1ꢀ[(triisopropylsilyl)oxy]butanꢀ2ꢀol (34) with 3,4ꢀdihydroꢀ2Hꢀpyran (4.0
equiv) and PPTS, using procedure B for 1 d (but washing with aqueous NaHCO only and
3
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2
2
2
2
2
2
2
2
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3
3
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3
3
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5
5
6
extracting the product four times with CH
on silica gel, eluting with 25ꢀ50% CH Cl
2 2
Cl ), followed by chromatography of the product
2
2
2 2
/petroleum ether (foreruns) and then with CH Cl ,
1
gave 35 (100%) as a colourless oil (a 1:1 mixture of diastereomers); H NMR (CDCl
3
) δ 8.20,
7.82 (2 s, 1 H), 4.69ꢀ4.58 (m, 1 H), 4.31ꢀ4.10 (m, 2 H), 4.08ꢀ3.99 (m, 1 H), 3.92ꢀ3.43 (m, 4 H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.34ꢀ2.23 (m, 0.5 H), 2.12ꢀ2.01 (m, 1 H), 1.94ꢀ1.41 (m, 6.5 H), 1.18ꢀ0.96 (m, 21 H);
+
3 5
HRFABMS calcd for C21H39BrN O Si m/z [M + H] 522.1822, 520.1842, found 522.1838,
520.1826.
4-(2-Bromo-4-nitro-1H-imidazol-1-yl)-2-[(tetrahydro-2H-pyran-2-yl)oxy]butan-1-ol
(36). Reaction of silyl ether 35 with TBAF (1.1 equiv), using procedure C for 3 h (extracting
the product five times with EtOAc), followed by chromatography of the product on silica gel,
eluting with 33% EtOAc/petroleum ether (foreruns) and then with 33ꢀ50% EtOAc/petroleum
ether, gave 36 (91%) as a white solid (a 3:1 mixture of diastereomers): mp
1
(Et
2
2
O/CH Cl
2
/pentane) 89ꢀ91 °C; H NMR (CDCl
3
) δ 8.16, 7.79 (2 s, 1 H), 4.65ꢀ4.50 (m, 1 H),
4.29ꢀ4.12 (m, 2 H), 4.08ꢀ3.97 (m, 1 H), 3.85ꢀ3.46 (m, 4 H), 2.12ꢀ1.73 (m, 5 H), 1.65ꢀ1.45 (m, 4
H). Anal. Calcd for C12
H18BrN
3
O
5
: C, 39.58; H, 4.98; N, 11.54. Found: C, 39.85; H, 5.12; N,
11.57.
Procedure
F:
2-Nitro-7-[(tetrahydro-2H-pyran-2-yl)oxy]-5,6,7,8-
tetrahydroimidazo[2,1-b][1,3]oxazepine (37). A solution of alcohol 36 (730 mg, 2.00
mmol) in anhydrous DMF (8 mL) under N at 0 °C was treated with 60% NaH (148 mg, 3.70
mmol) and then quickly degassed and resealed under N . The mixture was stirred at 20 °C for
2
2
2 3
3.5 h and then cooled to ꢀ78 °C (CO /acetone), quenched with ice/aqueous NaHCO (50 mL),
and extracted with EtOAc (7 x 50 mL). The extracts were washed with brine (50 mL) and
then evaporated to dryness under reduced pressure (at 30 °C) and the residue was
chromatographed on silica gel. Elution with 0ꢀ33% EtOAc/petroleum ether first gave
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foreruns, and then further elution with 50% EtOAc/petroleum ether gave the crude product,
which was chromatographed again on silica gel. Elution with 0ꢀ2% EtOAc/CH
2
Cl
gave 37 (144 mg, 25%) as a
) δ 7.48 (s, 1 H), 4.83ꢀ4.75 (m,
2
first gave
2 2
foreruns and then further elution with 3ꢀ4% EtOAc/CH Cl
1
white solid: mp (CH
2
Cl
2
/hexane) 158ꢀ160 °C; H NMR (CDCl
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
H), 4.39ꢀ4.04 (m, 4 H), 3.96ꢀ3.82 (m, 2 H), 3.60ꢀ3.50 (m, 1 H), 2.25ꢀ2.02 (m, 2 H), 1.92ꢀ
.71 (m, 2 H), 1.69ꢀ1.50 (m, 4 H). Anal. Calcd for C12 : C, 50.88; H, 6.05; N, 14.83.
17 3 5
H N O
Found: C, 51.08; H, 6.08; N, 14.89.
-Nitro-5,6,7,8-tetrahydroimidazo[2,1-b][1,3]oxazepin-7-ol (38). A solution of THP
ether 37 (137 mg, 0.484 mmol) in MeOH (14 mL) was treated with 3.3 M HCl (0.47 mL,
.55 mmol). The mixture was stirred at 20 °C for 5 h and then cooled to ꢀ20 °C and
neutralised with a solution of NH in MeOH (0.8 mL of 7 M). The resulting mixture was
2
1
3
evaporated to dryness under reduced pressure (at 30 °C) and the residue was
chromatographed on silica gel. Elution with 0ꢀ25% EtOAc/CH Cl first gave foreruns and
2
2
2 2
then further elution with 25% EtOAc/CH Cl gave 38 (91 mg, 94%) as a white solid: mp
1
(
MeOH/CH Cl /hexane) 185ꢀ187 °C; H NMR [(CD ) SO] δ 8.15 (s, 1 H), 5.28 (br s, 1 H),
2
2
3 2
4.22 (ddd, J = 14.1, 9.1, 2.0 Hz, 1 H), 4.19ꢀ4.09 (m, 1 H), 4.02ꢀ3.91 (m, 3 H), 2.09ꢀ1.98 (m, 1
H), 1.88ꢀ1.76 (m, 1 H). Anal. Calcd for C H N O : C, 42.21; H, 4.55; N, 21.10. Found: C,
7
9
3
4
4
2.41; H, 4.72; N, 21.28.
Procedure G:
tetrahydroimidazo[2,1-b][1,3]oxazepine
trifluoromethoxy)benzyl]oxy}methyl)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (41).
A mixture of alcohol 38 (51.0 mg, 0.256 mmol) and 4ꢀ(trifluoromethoxy)benzyl bromide
(0.205 mL, 1.28 mmol) in anhydrous DMF (1.5 mL) under N at 0 °C was treated with 60%
NaH (17.5 mg, 0.438 mmol) and then quickly degassed and resealed under N . The mixture
was stirred at 20 °C for 130 min and then cooled to ꢀ78 °C (CO /acetone), quenched with
2-Nitro-7-{[4-(trifluoromethoxy)benzyl]oxy}-5,6,7,8-
(39) and 2-nitro-7-({[4-
(
2
2
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2
2
2
2
2
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ice/aqueous NaHCO
3
2 2
(10 mL), added to brine (40 mL), and extracted with CH Cl (5 x 50
mL). The combined extracts were evaporated to dryness under reduced pressure (at 30 °C)
and the residue was chromatographed on silica gel. Elution with 0ꢀ50% EtOAc/petroleum
ether first gave foreruns, and then further elution with 50% EtOAc/petroleum ether gave 39
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(37 mg, 39%) as a white solid: mp (CH
2
Cl
2
/pentane) 119ꢀ121 °C; H NMR [(CD
3
)
2
SO] δ
8.18 (s, 1 H), 7.51 (br d, J = 8.8 Hz, 2 H), 7.36 (br d, J = 7.9 Hz, 2 H), 4.66 (s, 2 H), 4.36 (dd,
J = 12.6, 4.6 Hz, 1 H), 4.26ꢀ4.13 (m, 2 H), 4.04 (ddd, J = 14.2, 6.4, 3.0 Hz, 1 H), 3.95ꢀ3.86
13
(m, 1 H), 2.19ꢀ2.02 (m, 2 H); C NMR [(CD
3
)
2
SO] δ 151.1, 147.6 (q, JCꢀF = 1.5 Hz), 140.6,
137.9, 129.3 (2 C), 120.9 (2 C), 120.4, 120.1 (q, JCꢀF = 256.1 Hz), 74.5, 73.3, 68.7, 42.0, 30.1.
Anal. Calcd for C H F N O ⋅0.5H O: C, 47.13; H, 3.96; N, 10.99. Found: C, 46.86; H,
15 14
3
3
5
2
3.63; N, 10.82. HPLC purity: 100%.
Further elution of the above column with 50ꢀ75% EtOAc/petroleum ether gave crude 41,
24
which was chromatographed again on silica gel (as before) to give 41 (34 mg, 36%) as a
24
1
cream solid: mp (EtOAc/hexane) 163ꢀ164 °C (lit. mp 158ꢀ160 °C); H NMR (CDCl ) δ
3
7.42 (s, 1 H), 7.34 (br d, J = 8.7 Hz, 2 H), 7.20 (br d, J = 8.6 Hz, 2 H), 4.61 (s, 2 H), 4.61ꢀ
4.54 (m, 1 H), 4.15 (ddd, J = 12.4, 5.7, 3.7 Hz, 1 H), 4.06 (ddd, J = 12.4, 10.0, 5.8 Hz, 1 H),
3.83 (dd, J = 10.6, 4.4 Hz, 1 H), 3.78 (dd, J = 10.7, 4.9 Hz, 1 H), 2.39ꢀ2.21 (m, 2 H); APCI
+
MS m/z 374 [M + H] ; HPLC purity: 100%.
Compounds of Table 2. The following section details the syntheses of compounds Rꢀ58
and Rꢀ69 of Table 2, via representative procedures and key intermediates, as described in
Scheme 2. For the syntheses of the other compounds in Table 2, please refer to the
Supporting Information.
Synthesis of R-58 (Scheme 2A):
(6R)-6-[(2-Iodobenzyl)oxy]-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(57).
32
Reaction of (6R)ꢀ2ꢀnitroꢀ6,7ꢀdihydroꢀ5Hꢀimidazo[2,1ꢀb][1,3]oxazinꢀ6ꢀol (56) with 2ꢀ
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iodobenzyl chloride (3.0 equiv) and NaH (1.6 equiv), using procedure G for 3.5 h, followed
by chromatography of the product on silica gel, eluting with 0ꢀ0.25% MeOH/CH
Cl
2 2
(
foreruns) and then with 0.25ꢀ0.3% MeOH/CH Cl , gave 57 (91%) as a light yellow solid:
2 2
1
mp (Et
2
O) 118ꢀ119 °C; H NMR (CDCl
3
) δ 7.85 (br d, J = 7.8 Hz, 1 H), 7.40 (s, 1 H), 7.37ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.33 (m, 2 H), 7.08ꢀ7.00 (m, 1 H), 4.72 (d, J = 12.0 Hz, 1 H), 4.72ꢀ4.65 (m, 1 H), 4.64 (d, J =
2.0 Hz, 1 H), 4.40 (br dd, J = 12.1, 0.9 Hz, 1 H), 4.25ꢀ4.19 (m, 3 H). Anal. Calcd for
: C, 38.92; H, 3.02; N, 10.47. Found: C, 39.08; H, 2.98; N, 10.44.
Procedure H: (6R)-2-Nitro-6-{[4'-(trifluoromethoxy)[1,1'-biphenyl]-2-yl]methoxy}-
,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (Rꢀ58). A mixture of iodide 57 (435 mg, 1.08
mmol), [4ꢀ(trifluoromethoxy)phenyl]boronic acid (401 mg, 1.95 mmol), and Pd(dppf)Cl (56
mg, 0.077 mmol) in DMF (6 mL) and aq KHCO (2 mL of 2 M, 4.0 mmol) was degassed,
and then N was added. The resulting mixture was stirred at 75 °C for 4 h, and then cooled,
diluted with brine (100 mL), and extracted with CH Cl (3 x 100 mL). The extracts were
1
13 3 4
C H12IN O
6
2
3
2
2
2
evaporated to dryness under reduced pressure and the residue was chromatographed on silica
gel. Elution with CH
Cl
2 2
2 2
first gave foreruns, and then further elution with 2% MeOH/CH Cl
1
gave Rꢀ58 (218 mg, 46%) as a cream solid: mp (CH Cl /hexane) 133ꢀ135 °C; H NMR
2
2
[(CD
3 2
) SO] δ 8.00 (s, 1 H), 7.50ꢀ7.44 (m, 3 H), 7.44ꢀ7.35 (m, 4 H), 7.33ꢀ7.26 (m, 1 H), 4.57ꢀ
4.49 (m, 2 H), 4.49 (d, J = 10.9 Hz, 1 H), 4.42 (br d, J = 11.8 Hz, 1 H), 4.22ꢀ4.12 (m, 3 H);
13
C NMR [(CD
C), 130.0, 129.9, 128.3, 127.9, 120.6 (2 C), 120.1 (q, JCꢀF = 256.4 Hz), 117.9, 68.1, 67.8,
6.8, 46.5. Anal. Calcd for C20 : C, 55.18; H, 3.70; N, 9.65. Found: C, 55.08; H,
3.66; N, 9.68.
Synthesis of R-69 (Scheme 2B):
3 2
) SO] δ 147.6 (q, JCꢀF = 1.5 Hz), 147.1, 142.1, 140.0, 139.3, 134.5, 130.7 (2
6
16 3 3 5
H F N O
(6R)-6-[(2-Bromopyridin-3-yl)methoxy]-2-nitro-6,7-dihydro-5H-imidazo[2,1-
32
b][1,3]oxazine (68). Reaction of alcohol 56 with 2ꢀbromoꢀ3ꢀ(bromomethyl)pyridine (66)
2
9
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