Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914)

Article abstract of DOI:10.1021/acs.jmedchem.5b00863

A novel class of bacterial type-II topoisomerase inhibitor displaying a spiropyrimidinetrione architecture fused to a benzisoxazole scaffold shows potent activity against Gram-positive and fastidious Gram-negative bacteria. Here, we describe a series of N

Full text of DOI:10.1021/acs.jmedchem.5b00863

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Article
Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones
-
Benzisoxazole Fusion with N-Linked Oxazolidinone
Substituents Leading to a Clinical Candidate (ETX0914)
Gregory S. Basarab, Peter Doig, Vincent Galullo, Gunther Kern, Amy Kimzey,
Amy Kutschke, Joseph Newman, Marshall Morningstar, John Mueller,
Linda Otterson, Karthick Vishwanathan, Fei Zhou, and Madhu Gowravaram
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00863 • Publication Date (Web): 09 Jul 2015
Downloaded from http://pubs.acs.org on July 11, 2015
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Discovery of Novel DNA Gyrase Inhibiting
Spiropyrimidinetriones - Benzisoxazole Fusion
with N-Linked Oxazolidinone Substituents
Leading to a Clinical Candidate (ETX0914)
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Gregory S. Basarab,* Peter Doig, Vincent Galullo, Gunther Kern, Amy Kimzey, Amy Kutschke,
Joseph P. Newman, Marshall Morningstar, John Mueller, Linda Otterson, Karthick
Vishwanathan, Fei Zhou and Madhusudhan Gowravaram
AstraZeneca R&D Boston, Infection Innovative Medicines, 35 Gatehouse Drive, Waltham,
Massachusetts 02451, USA
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ABSTRACT
A novel class of bacterial type-II topoisomerase inhibitor displaying a spiropyrimidinetrione
architecture fused to a benzisoxazole scaffold shows potent activity against Gram-positive and
fastidious Gram-negative bacteria. Here, we describe a series of N-linked oxazolidinone
substituents on the benzisoxazole that improve upon the antibacterial activity of initially
described compounds of the class, show favorable PK properties and demonstrate efficacy in an
in vivo Staphylococcus aureus infection model. Inhibition of the topoisomerases DNA gyrase
and topoisomerase IV from both Gram-positive and a Gram-negative organisms was
demonstrated. Compounds showed a clean in vitro toxicity profile, including no genotoxicity and
no bone marrow toxicity at the highest evaluated concentrations or other issues that have been
problematic for some fluoroquinolones. Compound 1u was identified for advancement into
human clinical trials for treatment of uncomplicated gonorrhoea based on a variety of beneficial
attributes including the potent activity and the favorable safety profile.
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INTRODUCTION
There is a critical need to discover and develop antibacterial agents with a novel mode-of-action
that address highly problematic resistance issues across the most widely used classes of
antibacterials including, but not limited to β-lactam and glycopeptide antibiotics that inhibit cell-
wall biosynthesis, aminoglycoside and macrolide antibiotics that disrupt ribosome function, and
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-4
fluoroquinolone topoisomerase inhibiting antibacterials that impede DNA replication.
Multidrug resistant Staphylococcus aureus, for example, represents one of a series of resistant
pathogens encountered routinely in hospital settings where treatment options have become quite
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limited. Recently, an alarming spread of drug resistant Neisseria gonorrhoeae
10, 11
has caught
12
the attention of the World Health Organization, the Centers for Disease Control and
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Prevention and the European Centre for Disease Control and Reponse, which have implored
the development of novel treatment options. To address such problems of resistance, we
previously described a novel class of antibacterial agents with a benzisoxazole scaffold fused to a
six-membered ring that displays a spirocyclic pyrimidinetrione pharmacophore (e.g. Compound
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1u, ETX0914, Figure 1 and Table 1).
Compound 1u showed activity against Gram-positive
bacteria including Streptococcus pneumoniae, Streptococcus pyogenes and methicillin-resistant
fluoroquinolone-resistant (MRQR) S. aureus as well as against fastidious Gram-negative bacteria
including Haemophilus influenzae and N. gonorrhoeae. Like fluoroquinolones (e.g.
ciprofloxacin 2, Figure 1), 1u and other spiropyrimidinetriones inhibit type II topoisomerases,
DNA gyrase and topoisomerase IV (Topo IV), but through a different mode of inhibition as
suggested by their potent activity against clinical bacterial strains resistant to the
fluoroquinolones. Compound 1u was also fully active against laboratory strains of S. aureus and
S. pneumoniae resistant to novobiocin (3, Figure 1), which binds to the ATP site of DNA gyrase
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and Topo IV.
It was also more active against S. aureus including MRSA (methicillin
resistant S. aureus) than linezolid (4, Figure 1), a current standard of care for skin infections
caused by the organism. Developing novel drugs that operate by a novel DNA gyrase and Topo
IV inhibitory mechanism would ultimately expand the arsenal of drugs available to practicing
physicians to treat bacterial infections. Preliminary structure-activity relationships around the
benzisoxazole scaffold showed that the oxazolidinone substituent averted bone marrow toxicity
and genotoxicity seen with other substituents, which led to the selection of 1u for development
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through Phase 1 clinical trials (Clinicaltrials.gov Identifier: NCT01929629). The introduction
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of the oxazolidinone sp centers enabled the incorporation of asymmetric centers that were not
possible with a wide variety of aromatic substituents linked through a carbon or nitrogen atom to
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the benzisoxazole. Clinical success has been correlated with increased fraction sp (Fsp ) more
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generally in drug candidates attributed, in part, to the potential to increase solubility and decrease
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off-target activity. Herein, we describe a more in depth analysis of benzisoxazole
spiropyrimidinetriones with N-linked oxazolidinone substituents that generated considerable
import due to favorable antibacterial, physical property and pharmacokinetic (PK) attributes.
Presented are the associated structure-activity relationships, pharmacology and toxicology
properties that led to the selection of 1u for human clinical trials.
RESULTS AND DISCUSSION
Synthesis. The general synthesis of fluorobenzisoxazoles with oxazolidinone and
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imidazolidinone substituents (Scheme 1) utilized either compound 5a or 5b (see Supporting
Information), which can be made in multigram quantities in five steps from commercial starting
materials. Chlorobenzisoxazole 5c (Supporting Information) was made similarly to 5a and was
carried on in the reaction sequence of Scheme 1. Using the chiral trans (2R,4R)-
dimethylmorpholine in 5a, 5b and 5c enabled the enantiospecific synthesis of final products.
Treatment of the compounds with a variety of chiral oxazolidinones (X = O) and
imidazolidinones (X = NH, NCH ) and sodium hydride afforded 6a-q, the products of S Ar
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N
displacement of the 3-position chloride. These were generally converted to spiropyrimidinetrione
final products 1a-q by heating with barbituric acid in acetic acid-water. For two compounds, 1d
and 1i with pendant hydroxyl groups, the final conversion was better carried out in an ethanol-
aqueous HCl co-solvent to avoid by-product acylation seen with the acetic acid conditions.
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Overall, the sequence of Scheme 1 enabled the efficient late stage introduction and survey of
oxazolidinone/imidazolidinone substituents for SAR purposes. Compounds 1a-q (as well as all
final products herein) were purified predominately by chiral Supercritical Fluid Chromatography
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(
SFC) to remove 7a-q, a minor diastereomer produced in each of the reactions. Though
diastereomers and not enantiomers are being separated, purifications were best achieved via such
chiral chromatography. The final reaction proceeded by an in situ ketal hydrolysis to the
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aldehyde, a Knövenagel condensation and a tertiary amino effect reaction (T-reaction).
The
T-reaction in its current form was pioneered by Reinhoudt in the 1980s and involves an
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irreversible internal redox or [1,5]-hydride shift reaction to form a zwitterion intermediate.
The morpholine methyl substituent next to the iminium species can epimerize accounting for the
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configuration seen in compounds 1a-p.
The cyclization of the zwitterion iminium species
under the reaction conditions can occur above and below the plane of either the cis- or trans-
dimethylmorpholine rings in a reversible fashion to allow for four possible diastereomers. Only
diastereomers 1 and 7 were seen in the crude reaction mixture, and their configurations represent
the thermodynamically lowest energy of the four possible with 1 being favored over 7 in a 9:1
ratio.
In an alternative synthetic sequence utilized for the syntheses of 1r-u (Scheme 2), the
benzisoxazole and oxazolidinone were assembled before incorporation of the chiral morpholine
unit, the latter introduced in the penultimate step. This proved advantageous for the scale-up of
individual compounds for three reasons. First, the chiral morpholine accounted for much of the
cost associated with the synthesis, which would be minimized by its late stage introduction.
Second, the late stage introduction allowed for more facile diversification away from the
dimethylmorpholine with alternate amines for the analog program (work not included herein).
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Finally, overall yields of the reaction sequence were higher. The key intermediate 10 was
prepared in four steps starting with protection of aldehyde 8 to set up a fluorine-directed ortho-
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lithiation
followed by DMF quench to form aldehyde 9. The aldehyde was converted to
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oximoyl chloride 10 to be followed by chloride displacement with chiral aminoalcohols and
cyclization to benzisoxazoles 11r-u on treatment with strong base. The oxazolidinone was made
by reaction with carbonyl diimidazole (CDI), and the acetal was hydrolyzed to the aldehyde with
acid affording 12 r-u. S Ar dimethylmorpholine displacement of the fluoride adjacent to the
N
aldehyde afforded 13 r-u, which was followed by Knövenagel condensation and T-reaction with
barbituric acid. As before, a 9:1 ratio of diastereomers was obtained with the major materials 1r-
u being separated from the minor diastereomers. In two cases, 1t and 1u, the minor
diastereomers were isolated and fully characterized analytically and biologically. As described
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earlier for other spiropyrimidinetriones,
the configurations of 1 and 7 were determined by
NMR including detailed NOESY correlations around the morpholine ring (see Supporting
Information for the NOESY spectra of 1u and 7u). The morpholine ring of both 1u and 7u exist
primarily in a chair conformation in solution, with both methyl groups equatorial for the former
and one methyl group axial, the other equatorial for the latter. A crystal structure of 1u as a 1:1
methanol solvate confirmed the configuration, mirrored the solution conformation and showed
the attachment of the oxazolidinone ring to be nearly co-planar with the benzisoxazole scaffold
with a 1˚ torsion (CCDC 1025296, Figure 2). Finally, the enantiomers ent-1t and ent-1u of 1t
and 1u, respectively, were synthesized in an independent synthetic sequence using meso cis-
dimethylmorpholine and chiral oxazolidinones. The sequence therefore required a the separation
of (2R,4S,4aS)- and (2S,4R,4aR)-morpholine diastereomers in the final products (see Supporting
Information).
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Structure-activity relationships Compounds were evaluated for inhibition of DNA gyrase
activity using the Escherichia coli isozyme amenable (versus other isozymes) to a robust
fluorescence polarization (FP) anisotropy-based DNA supercoiling assay (Table 1) that is
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considerably higher through-put than typically used gel-based assays. Antibacterial activity,
determined as the MIC (Table 1), did not correlate well with inhibitory potency for E. coli
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Figure 3A) as ascertained by the R factor from regression analysis being only 0.16. Deviations
(
from linearity were likely due to variable membrane permeability and the capability of the
compounds to also inhibit Topo IV (see below). However, as the logD range narrows further to
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reflect compounds that might have similar permeability, the R continued to increase: for
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example, a narrow logD range of 1.5-1.9 led to an R of 0.91 (Figure 3B). It was within this
narrow range that the aggregate of multivariate compound properties proved best toward
identifying a candidate drug. Among such properties are solubility, protein binding and in vivo
clearance in addition to antibacterial activity. Four compounds (1h, 1o, 1t, and 1u) showed MIC
values <10 µM versus E. coli, though the values were not sufficiently low to anticipate efficacy
against the pathogen at reasonable doses in an in vivo situation. Nonetheless, the robust spread of
MIC data seen for E. coli offered a measure of differentiation among the compounds and a tool
for understanding parameters that account for activity. Compounds with higher antibacterial
activity against E. coli generally showed higher activity against the other bacteria in Table 1
demonstrating the utility of the FP assay for understanding the SAR. In contrast to E. coli, the
other bacteria did show sufficient susceptibility to anticipate demonstration of in vivo efficacy.
These include the fastidious Gram-negative pathogens H. influenzae and N. gonorrhoeae and
four Gram-positive pathogens, namely S. pneumoniae, Streptococcus pyogenes and two strains
of S. aureus, a drug susceptible strain (MSSA) and a methicillin and fluoroquinolone resistant
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strain (MRQR). Two comparators, ciprofloxacin and linezolid are included in Table 1;
ciprofloxacin provides a frame of reference as a fluoroquinolone DNA gyrase inhibitor, and
linezolid serves as a standard of care for Gram-positive skin infections. Resistance to
fluoroquinolones in the MRQR strain is due to two topoisomerase mutations, S85P in GyrA and
S80Y in ParC. As a result, the MIC for ciprofloxacin against the MSSA strain was 0.78 µM and
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>50 µM versus the MRQR strain. In contrast, the MIC values for the spiropyrimidinetriones of
Table 1 versus the MSSA and MRQR strains were similar with variations only 1- to 4-fold. This
supports the differential mode-of-inhibition for spiropyrimidinetriones relative to
fluoroquinolones. A balanced set of MIC values for the compounds of Table 1 were also
obtained across the Staphylococcus and Streptococcus species. Compound 1t, for example,
showed MIC values of 0.2-0.4 µM across S. pneumoniae, S. pyogenes and MSSA in contrast to
initial benzisoxazole spiropyrimidinetriones that showed notably higher antibacterial activity
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against Staphylococci relative to Streptococci. Overall, the compounds with more favorable
physical and PK properties critical for advancing a drug candidate also showed sufficient
antibacterial potency against S. aureus and S. pyogenes to address skin infections, against S.
aureus, S. pneumoniae and H. influenzae to address respiratory tract infections and against N.
gonorrhoeae to address uncomplicated gonorrhea.
As comparators, the opposite enantiomers (ent-1t and ent-1u) of two compounds, 1t and
1u, were made, and the corresponding minor diastereomers 7t and 7u were separated for
biological and physical property profiling (Table 2). As shown in previous publications
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describing spiropyrimidinetriones,
stereochemistry around the morpholine ring had a
profound impact on inhibitory potency and thereby antibacterial activity, and compounds with
the (R,S,S)-morpholine configuration as diagrammed for 1 displayed much higher activity than
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the enantiomeric antipodes (ent-1t and ent-1u) and minor diastereomers (7t and 7u). Importantly,
there was also a recognition element for human plasma protein binding (PPB) as the more active
diastereomers 1t and 1u showed a 6- to 9-fold higher free fraction (or fraction unbound, f ) than
u
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those of the respective enantiomers or the respective minor diastereomers. A higher f would be
u
expected to afford higher activity in an in vivo situation as unbound drug is thought to be
necessary for interaction with the target and the pathogen. As an in vitro indication of this, MIC
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values were determined for MSSA in the presence of 50% human serum (Table 1). For the
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most part, the shift to a higher MIC was around 2-fold. However, more highly protein bound
compounds such as 1m with f = 0.01 displayed a notably larger 16-fold MIC increase. On the
u
other hand, PPB can increase compound exposure in vivo due to its role in protecting a
compound from clearance mechanisms; compounds with both a high f and a low clearance
u
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would be thus best suited as drug candidates.
Realizing maximal drug exposure and efficacy
in a disease model thus relates to the free AUC with the preference being for compounds with a
higher f and AUC (lower clearance) and a lower MIC.
u
Structure insights of DNA gyrase or Topo IV could not be used for optimization of
spiropyrimidinetrione analogs as their binding environment has not been determined to date by
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X-ray diffraction or NMR as has been done for fluoroquinolones,
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topoisomerase inhibitors termed NBTIs (novel bacterial topoisomerase inhibitors),
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and other ATP competitive inhibitors. Hence, the latitude for
aminocoumarins
substitution on the oxazolidinone and imidazolidinone ring as seen in Table 1 was determined
through SAR correlations and was shown to be quite broad. Both saturated carbon positions of
the oxazolidinone (4- and 5-positions, see 1a of Table 1 for numbering) tolerated substitutions in
either possible configuration. However in every matched pair, substituents below the plane of the
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oxazolidinone ring as drawn in Table 1 were preferred over those above. This includes the
matched pairs 1t and 1u that showed higher inhibitory potency and antibacterial activity than 1b
and 1g, respectively. Substituents at each of the oxazolidinone 4- and 5-positions showed similar
activity; hence, 1b-f and 1t with 5-position substituents showed similar activity to 1g-k and 1u
that have respectively identical 4-position substituents. Substitution at both the 4- and 5-positions
with methyl groups followed the trend that overall highest potency was seen with the orientation
of both methyl groups below the plane of the oxazolidinone ring as seen by comparing 1o with
1n. More polar substituents, as incorporated in 1d, 1l, 1p, 1q and 1r, diminished antibacterial
activity presumably due to decreased target potency and/or bacterial membrane permeability.
Importantly, addition of the substituents onto the oxazolidinone generally increased solubility
(compare with 1a, Table 1) presumably due to re-organization of crystal packing arrangements.
The X-ray structure of 1u showed the oxazolidinone methyl group to be nearly perpendicular
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(81˚) to the plane encompassing the benzisoxazole and oxazolidinone (Figure 2) without π-π
stacking within the crystal lattice. Fluorine and chlorine substituents on the benzisoxazole ring
were about equipotent; the increased lipophilicity of chlorine decreased f and solubility. Finally,
u
replacement of the oxazolidinone ring oxygen atom with nitrogen (1p and 1q) decreased but did
not eliminate activity.
IV pharmacokinetic (PK) parameters were determined in the rat for select compounds
with higher antibacterial activity, solubility and f (clearances shown in Table 1). Low clearance
u
(Cl) was generally seen with methyl substituents on the oxazolidinone ring. Methoxymethyl
substituents generally afforded higher Cl as seen with 1e, 1j and 1k. Replacement of the fluorine
atom with chlorine usually lowered Cl, as in the instances of 1c, 1f and 1h relative to 1t, 1e and
1u, respectively, correlating with the lower f . Distilling f , antibacterial activity and rat Cl
u u
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(
normalizing AUC to a 1 mg dose) into a single number fAUC/MIC (f *AUC/dose*MIC),
u
showed favorable values for three compounds, 1o, 1t and 1u relative to other compounds of
Table 1. These three compounds were evaluated in rat and dog for both IV and oral PK (Table
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3
). The Cl, associated fAUC/MIC, and bioavailability for the three compounds in both species
were sufficiently high that, taking cross-species hepatocyte stability into account, the expected
human exposure would be favorable.
In vitro genotoxicity attributes. A link has been made between the inhibition of mammalian
topoisomerase IIα (TopoIIα) and genotoxicity as manifested in a mouse micronucleus
40
chromosome aberration assay (MMA) and a mouse lymphoma assay (MLA). Indeed, the use of
the fluoroquinolone gemifloxacin, a 20 µM inhibitor of TopoIIα (Table 4), has been limited due
to clastogenicity in the MLA, an in vitro human lymphocyte chromosome aberration assay and
41
an in vivo rat micronucleus chromosome aberration assay. Compounds 1t and 1u were profiled
4
2
43
for inhibition of human TopoIIα, more broadly for human topoisomerase IIβ (TopoIIβ) and
for mammalian in vitro genotoxicity in the MMA and MLA (Table 4). Gemifloxacin was
included as a genotoxic agent and ciprofloxacin as a fluoroquinolone with a clinically acceptable
genotoxicity profile. Neither 1t nor 1u showed evidence of genotoxicity at the highest
concentrations tested in both assays and a more favorable profile than both ciprofloxacin and
gemifloxacin. The data in Table 4 reflects experiments done in the absence of metabolizing S9
liver fractions. Compounds 1t and 1u were also shown to be negative for genotoxicity in the
presence of S9 liver fractions (data not shown). Moreover, 1t and 1u were less active against the
human TopoIIα and TopoIIβ with multiples ranging from 210- to 2350-fold relative to inhibition
of E. coli DNA gyrase. Overall, the data of Table 4 indicate a correlation between potent
TopoIIα inhibition and mammalian in vitro genotoxicity as measured by the MMA and MLA.
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Both 1t and 1u were tested at sub-lethal concentrations for Ames mutagenicity against a standard
five strain Salmonella typhimurium LT2 panel (TA1535, TA1537, TA100, TA98, TA102) with
and without added S9. Similar to fluoroquinolones, the compounds were positive in only the
DNA repair proficient TA102 strain as would be expected with the topoisomerase mode-of-
action. Consistent with these in vitro results, compound 1u was negative in an in vivo rat
micronucleus assay at the maximum tolerated oral dose of 500 mg/kg/day for two days.
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Mammalian cellular toxicity. Given the low, but measurable inhibitory activity of 1t and 1u
against the human topoisomerases, toxicity to mammalian cell lines was also investigated. Cross
screening against mammalian cell cultures, oftentimes immortalized, offers an avenue to mitigate
toxicity. Neither 1t nor 1u registered high anti-proliferative activity against the human A459 cell
line, a human THLE liver cell line or a THP-1 monocyte white blood cell line. Assessing the
THLE cell line was particularly important as hepatic toxicity is a major factor leading to the
4
4-46
termination of new drug development.
Furthermore, neither 1t nor 1u lysed sheep red blood
cells at the highest concentrations tested. The safety indicated from these assays is not unlike
other on the market antibacterials including linezolid, levofloxacin and gemifloxacin as seen in
Table 5. However, it should be noted that longer term dosing of linezolid and gemifloxacin in the
clinic has been limited due to bone marrow suppression characterized by thrombocytopenia,
4
1, 47,
which would not be foreseen monitoring cytotoxicity against mature mammalian cell lines.
4
8
Hence, the toxicity against immature bone-marrow myeloid and erythroid cell lines was
evaluated (Table 5). The bone marrow suppression seen in the clinic with linezolid and
gemifloxacin would be predictable based on the IC50 measurements against myeloids and
erythroids, while 1u notably showed no activity against either cell line at the highest 100 µM
concentration tested. This is similar to the profile seen for levofloxacin, which does not suffer
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from issues of thrombocytopenia in clinical use. In our opinion, the inhibition of bone marrow
cell growth should be assessed more routinely as a more sensitive gauge of potential toxicity
issues associated with longer term use of antibacterial agents targeting enzymes or receptors with
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human homologues similar to what is routinely done for oncology cytotoxic drugs.
In vivo efficacy. Compounds 1t and 1u were evaluated for efficacy in a S. aureus neutropenic
thigh infection model in mice (Figure 4) using the MSSA strain of Tables 1 and 2 (S. aureus
5
1
ARC516). A higher Cl of 57 mL/min/kg was measured for 1u in the mouse, despite it having a
lower mouse hepatic Clint (6.9 µl/min/mg). By comparison, the mouse hepatic Clint (15
µl/min/mg) for 1t was higher than that for 1u while the in vivo mouse Cl (20 mL/min/kg) was
lower. The mechanism of clearance for the two compounds in the mouse has not been fully
5
2
elucidated. However, co-administration of the P450 inhibitor 1-aminobenzotriazole (ABT) with
1
u in mice lowered Cl to 15 mL/min/kg suggesting a susceptibility to oxidative metabolism.
Hence, ABT was co-administered for an efficacy study with 1u and was not used for 1t. The
untreated control typically showed about a 3.0 log (1000-fold) growth in CFU relative to the
5
level of 5x10 CFU inoculum achieved at the time zero. Gratifyingly, both compounds
demonstrated efficacy in the models. Compound 1u showed a static response at about 10
mg/kg/day, and 1t between 10 and 25 mg/kg/day. Maximal responses of nearly a 2-log reduction
in CFU were seen at 25 and 50 mg/kg/day for 1t and 1u, respectively, and higher doses did not
improve efficacy. Overall, 1t and 1u could not be differentiated from one another based on the
pharmacology, and extensive side-by-side rat and dog tolerability studies (data not shown) along
with the aforementioned somewhat better cytotoxicity profile (Table 5) led to 1u being selected
for pre-clinical development.
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In vitro mode-of-inhibition. The selection of 1u for pre-clinical development instigated studies
to validate the mode-of-action in line with what was observed for previous
1
6, 53
spiropyrimidinetriones.
DNA gyrase is a large tetrameric complex of two GyrA and two
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GyrB subunits operating as a molecular machine to alter the topological state of DNA necessary
for replication. Topo IV is highly homologous to DNA gyrase containing two analogous ParC
and ParE (to GyrA and GyrB, respectively) subunits. The primary function of DNA gyrase is to
introduce negative supercoils ahead of the DNA replication fork while the primary role of Topo
IV is to decatenate DNA strands. As with other type II topoisomerases, DNA gyrase and Topo
IV cleave both strands of duplex DNA to engineer the change in topological state. Agarose gel
assays from E. coli and S. aureus (Gram-negative and Gram-positive isozymes) were set up to
track the conversion of relaxed to supercoiled plasmid DNA mediated by DNA gyrase and of
catenated to decantanated DNA mediated by Topo IV (Table 6). Compound 1u and the
comparator ciprofloxacin inhibited E. coli and S. aureus DNA gyrase supercoiling and Topo IV
decatenation activity causing double-stranded cleaved DNA to accumulate. This is in contrast to
54
the NBTI class, for which mostly single stranded DNA cleavage was observed, and to
5
5
novobiocin, which does not induce DNA strand cleavage. In the gel based DNA gyrase assay,
only fully supercoiled DNA is quantitatively assessed, and therefore a high amount of
supercoiling needs to be introduced into the test DNA. In the FP assay, a DNA oligomer’s ability
to form triplexes with double-stranded DNA is measured, and the amount of supercoiled DNA
that abrogates triplex formation has not been determined. Hence, though the IC values between
5
0
the two assays will differ, they both serve to rank order relative inhibitor potency. The gel-based
assays showed that, like fluoroquinolones, 1u has the potential for dual-target inhibition in both
Gram-negative and Gram-positive bacteria. Ultimately, analysis of mutations in the targets and
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their capability to confer resistance in cell growth assays is required to dissect the relative
importance of the two enzyme targets to inhibition by 1u. Indeed, spontaneous mutants to 1u in
S. aureus and N. gonorrhoeae shown to have GyrB modifications in the DNA cleavage domain
are not cross-resistant to fluoroquinolone antibacterials. The data supported DNA gyrase over
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6, 57
Topo IV being the primary target of inhibition for 1u for the two organisms.
CONCLUSION
1
5, 58
Compound 1u
was given the designation ETX0914 as it progressed through extensive pre-
clinical investigations including toxicology, formulations development and microbiological
characterization toward entering human clinical trials. The activity and pharmacokinetics of the
compound support utility against skin and skin structure infections caused by Gram-positive
bacteria, S. aureus and S. pyogenes in particular. The spectrum of activity suggests coverage
against respiratory tract infections caused by S. aureus, S. pneumoniae, H. influenzae, Moraxella
catarrhalis and various atypical bacterial pathogens. The high activity against N. gonorrhoeae
including resistant isolates supports treatment of sexually transmitted uncomplicated
5
9
gonorrhea. To identify better analogs, optimization work closely monitored antibacterial
activity, solubility, PPB and PK attributes in a multivariate fashion. In parallel, a variety of in
vitro toxicology parameters were monitored to mitigate safety issues that might occur in clinical
use. Among these were ion channel inhibition including hERG, bone marrow toxicity and
mammalian genotoxicity. Genotoxicity in particular became of paramount concern as early
spiropyrimidinetrione analogs showed positive in vitro activity; hence, the clean genotoxicity
profile for 1u and in fact, for the other oxazolidinone analogs herein that were evaluated, bodes
well for progression of the drug candidate. Ultimately, gonorrhea was chosen as a gateway
indication to evaluate 1u in clinical trials to address a pressing medical need and to enable a
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relatively rapid route to evaluate human tolerability and clinical efficacy, both particularly
important for a novel drug class without precedence in medicine.
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EXPERIMENTAL SECTION
General Considerations All of the solvents and reagents used were obtained commercially and
1
used as such unless noted otherwise. H NMR spectra were recorded in CDCl or DMSO-d
3
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solutions at 300 K using a Bruker Ultrashield 300 MHz instrument or a Bruker Ultrashield 400
13
MHz instrument. C NMR spectra were recorded in DMSO-d solutions at 300 K and 126 MHz
6
using a Bruker DRX-500 500 MHz instrument with a QNP cryoprobe or at 101 MHz using a
Bruker Ultrashield 400 MHz instrument or at 75.5 MHz using a Brucker Ultrashield 300 MHz
1
instrument. F NMR spectra were recorded at 282 MHz in CDCl or DMSO-d solutions at 300
9
3
6
K using a Bruker Ultrashield 300 MHz instrument. Chemical shifts are reported as parts per
1
million relative to TMS (0.00) for H and C NMR and CFCl for F NMR. High-resolution
13
19
3
mass spectra (HRMS) were obtained using a hybrid quadrupole time-of-flight mass spectrometer
+
microTOFq II, Bruker Daltonics) in ESI mode. Silica gel chromatographies were performed on
(
an ISCO Combiflash Companion Instruments using ISCO RediSep Flash Cartridges (particle
size: 35-70 microns) or Silicycle SiliaSep Flash Cartridges (particle size: 40-63 microns).
Preparative reverse phase HPLC was carried out using YMC Pack ODS-AQ (100 × 20 mm ID,
S-5 ꢀ particle size, 12 nm pore size) on Agilent instruments. When not indicated, compound
intermediates and reagents were purchased from chemical supply houses. All final compounds
(Compounds 7a-u) were determined to be greater than 95% pure via analysis by reversed phase
UPLC-MS (retention times, RT, in minutes) with a Waters Acquity UPLC instrument with DAD
and ELSD and a UPLC HSS T3, 2.1 x 30 mm, 1.8 um column and a gradient of 2 to 98%
acetonitrile in water with 0.1% formic acid over 2.0 minutes at 1 mL/min. Injection volume was
1 µL and the column temperature was 30 °C. Detection was based on electrospray ionization
(ESI) in positive and negative polarity using Waters ZQ mass spectrometer (Milford, MA, USA),
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diode-array UV detector from 210 to 400 nm, and evaporative light scattering detector (Sedex
75, Sedere, Alfortville Cedex, France).
3-(5-(Dimethoxymethyl)-6-((2R,6R)-2,6-dimethylmorpholino)-7-fluorobenzo[d]isoxazol-3-
yl)oxazolidin-2-one (6a) A solution of 2-oxazolidinone (728 mg, 8.36 mmol) in 1 mL DMF was
added slowly to a stirred suspension of NaH (60% oil dispersion, 290 mg, 7.25 mmol) in 1 mL
DMF at 0 °C. The mixture was stirred at rt for 10 min, and a solution of 5b (0.25 g, 0.7 mmol,
see Supplementary Materials) in 3 mL DMF was added. This mixture was heated at 80 °C for 5
0
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h, poured into ice cooled aqueous NH Cl, and extracted twice with EtOAc. The organic layers
4
were dried (Na SO ) and concentrated. The residue was chromatographed on silica gel (20-50%
4
2
EtOAc gradient in CHCl ) to afford 500 mg starting material 5b and 800 mg (35%) of the title
3
1
compound. H NMR (300 MHz, DMSO-d ) δ 8.35 (s, 1H), 5.74 (s, 1H), 4.45-4.73 (m, 2H), 3.93-
6
+
.25 (m, 4H), 2.71-2.93 (m, 2H), 3.06-3.35 (m, 8H), 1.23 (br s, 6H); MS (ES) MH : 410.4 for
4
C H FN O .
6
19 25
3
(5S)-3-{6-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]-5-(1,3-dioxolan-2-yl)-7-fluoro-1,2-
benzoxazol-3-yl}-5-methyl-1,3-oxazolidin-2-one (6b) NaH (60% dispersion) (0.157 g, 3.92
6
mmol) was added to a solution of (S)-5-methyloxazolidin-2-one (0.397 g, 3.92 mmol) in 6 mL
0
DMF at rt. After stirring for 30 min, 5a (0.7 g, 1.96 mmol) was added, and the mixture was
heated at 90 °C for 4 h. The mixture was quenched with aqueous NH Cl and solvent was
4
removed. The residue was taken up in Et O, which was washed 3 times with water and once with
2
brine. The combined aqueous layers were extracted with Et O, which was washed twice more
2
with water and once with brine. The combined Et O layers were dried (MgSO ) and concentrated
2
4
to give an oil that was chromatographed on silica gel (0-30% gradient of EtOAc in CH Cl ) to
2
2
1
afford 230 mg starting material 5b and 250 mg (30%) of the title compound. H NMR (400
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MHz, CDCl ) δ 8.52 (d, J=1.0 Hz, 1H), 6.35 (s, 1H), 4.79-5.13 (m, 1H), 4.33 (dd, J=8.0, 9.8 Hz,
3
1H), 4.12-4.26 (m, 4H), 3.97-4.12 (m, 2H), 3.84 (dd, J=7.0, 10.0 Hz, 1H), 3.00 (dd, J=5.5, 11.0
1
9
Hz, 2H), 1.62 (d, J=6.3 Hz, 3H), 1.35 (br. s., 6H); F NMR (282 MHz, CDCl ) δ -144.46; MS
3
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+
ES) MH : 422.1 for C H FN O .
(
2
0
25
3
6
(R)-3-(7-Chloro-6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-dioxolan-2-yl)benzo[d]isoxazol-
6
1
3-yl)-5-methyloxazolidin-2-one (6c) A solution of (R)-5-methyloxazolidin-2-one (271 mg,
2.68 mmol) in 4 mL DMF was added slowly to a stirred suspension of NaH (60% oil dispersion,
107 mg, 2.68 mmol) in 3 mL DMF at 0 °C. The mixture was stirred at rt for 10 min, and a
solution of 5c (1.0 g, 2.68 mmol, see Supporting Information) in 5 mL DMF was added. The
resulting mixture was heated at 85 °C for 2 h, poured into ice cooled aqueous NH Cl, and
4
extracted twice with EtOAc. The organic layers were dried (Na SO ) and concentrated. The
2
4
residue was chromatographed on silica gel (25% EtOAc gradient in hexanes) to afford 500 mg
1
starting material 5c and 800 mg (35%) of the title compound. H NMR (300 MHz, DMSO-d ) δ
6
8.58 (s, 1H), 6.22 (s, 1H), 4.87-5.17 (m, 1H), 3.48 - 4.34 (m, 9H), 2.67-3.20 (m, 3H), 1.49 (d,
+
J=6.4 Hz, 3H), 1.32 (br. s., 3H), 1.13 (br. s., 3H); MS (ES) MH : 438 for C H ClN O .
2
0
25
3
6
(S)-5-((tert-Butyldiphenylsilyloxy)methyl)-3-(6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-
dioxolan-2-yl)-7-fluorobenzo[d]isoxazol-3-yl)oxazolidin-2-one A solution of (S)-5-((tert-
butyldiphenylsilyloxy)methyl)oxazolidin-2-one (3.0 g, 8.4 mmol, described in Supplementary
Material) in 10 mL DMF was added slowly to a stirred suspension of NaH (60% dispersion, 0.37
g, 8.4 mmol) in 10 mL DMF at 0°C over 10 min. The mixture was stirred at rt for 30 min, and a
15
solution of 5a (3.0 g, 8.4 mmol) in 10 mL DMF was added. This mixture was heated at 80 °C
for 2 h and poured into ice-cooled aqueous NH Cl before being extracted twice with EtOAc. The
4
organic layers were dried (Na SO ) and concentrated to give a residue that was chromatographed
2
4
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over silica gel (40-50% EtOAc gradient in pet. ether) to afford the title compound. Yield 2.13 g
1
(37%). H NMR (300 MHz, DMSO-d ) δ 8.47 (s, 1H), 7.30-7.70 (m, 10H), 6.19 (s, 1 H), 4.95-
6
5.11 (m, 1H), 4.30 (t, J=9.3 Hz, 1H), 3.83-4.14 (m, 9H), 3.17-3.27 (m, 2H), 2.90 (dd, J=10.83,
0
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+
.4 Hz, 2H), 1.23 (d, J=5.4 Hz, 6H), 0.89 (s, 9H); MS (ES) MH : 676 for C H FN O Si.
5
3
6
43
3
7
(S)-3-(6-((2R,6R)-2,6-Dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-fluorobenzo[d]isoxazol-
-yl)-5-(hydroxymethyl)oxazolidin-2-one (6d) Acetic acid (0.89 mL, 15.5 mmol) and a
3
solution of 1M TBAF (3.1 mL, 3.1 mmol) in THF were added sequentially to a solution of the
preceding compound (2.1 g, 3.11 mmol) in 15 mL THF. The mixture was stirred at rt for 18 h
before being diluted with water and extracted with EtOAc. The organic layer was dried (Na SO )
2
4
and concentrated to afford material that was purified on a silica gel column (50-70% EtOAc
1
gradient in hexanes) to give the title compound (1.33 g, 98% yield). H NMR (300 MHz,
DMSO-d ) δ 8.4 (s, 1H), 6.2 (s, 1H), 4.7-5.05 (m, 1H), 3.9-4.3 (m, 8H), 3.5-3.8 (m, 2H), 2.8-3.3
6
+
(m, 4H), 1.2 (d, J= 5.8 Hz, 6H). MS (ES) MH : 438.1 for C H FN O .
2
0
25
3
7
(S)-3-(6-((2R,6R)-2,6-Dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-fluorobenzo[d]isoxazol-
3-yl)-5-(methoxymethyl)oxazolidin-2-one (6e) Prepared as described for 6c with (S)-5-
(methoxymethyl)oxazolidin-2-one (294 mg, 2,24 mmol), NaH (60% dispersion, 90 mg, 2.24
1
mmol) and 5a (800 mg, 2.24 mmol) to afford 480 mg (47% yield) of the title compound. H
NMR (300 MHz, DMSO-d ) δ 8.44 (s, 1H), 6.18 (s, 1H), 4.91-5.19 (m, 1H), 4.22 (t, J= 8 Hz,
6
1H), 2 3.6-4.3 (m, 9H), 3.34 (s, 3H), 3.16-3.28 (m, 2H), 2.7-2.90 (m, 2H), 1.23 (d, J=6.2 Hz,
+
H); MS (ES) MH : 452 for C H FN O .
6
2
1
27
3
7
(S)-3-(6-((2R,6R)-2,6-Dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-chlorobenzo[d]isoxazol-
3-yl)-5-(methoxymethyl)oxazolidin-2-one (6f) Prepared as described for 6h with 1.05 g (8.04
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5
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7
8
9
mmol) (S)-5-(methoxymethyl)oxazolidin-2-one, NaH (60% dispersion, 322 mg, 8.04 mmol) and
5c (1.0 g, 2.68 mmol) (Supplementary materials) to afford 600 mg (48%) of the title compound.
1
H NMR (300 MHz, CDCl ) δ 8.68 (s, 1H), 6.35 (s, 1 H), 4.9 (m, 1H), 4.1-4.35 (m, 6H), 3.9-4.1
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
m, 2H), 3.70 (dd, J=10.7, 4.0 Hz, 1H), 3.67 (dd, J=10.7, 4.0 Hz, 1H), 3.45 (s and m, 4H), 3.25
+
(m, 1H), 3.05 (m, 1H), 2.7-2.85 (m, 1H), 1.45 (br. s, 3H), 1.15 (s. br, 3H); MS (ES) MH : 468.2
for C H ClN O .
2
1
27
3
7
(4R)-3-{6-[(2R,6R)-2,6-Dimethylmorpholin-4-yl]-5-(1,3-dioxolan-2-yl)-7-fluoro-1,2-
benzoxazol-3-yl}-4-methyl-1,3-oxazolidin-2-one (6g) A solution of (4R)-4-methyl-1,3-
6
oxazolidin-2-one (1.0 g, 9.9 mmol) in 10 mL DMF was added slowly to a stirred suspension of
2
NaH (60% dispersion, 0.24 g, 9.9 mmol) in 10 mL DMF at 0 °C over 10 min. The mixture was
15
stirred at rt for 30 min, and a solution of 5a (1.1 g, 3.1 mmol) in 3 mL DMF was added. This
mixture was heated at 80 °C for 12 h and poured into ice-cooled water before being extracted
twice with EtOAc. The organic layers were dried (Na SO ) and concentrated to give a residue
2
4
that was chromatographed over silica gel (EtOAc gradient in pet. ether) to afford the title
+
compound. Yield: 0.15 g (12%). MS (ES) MH : 422.4 for C H FN O .
2
0
25
3
6
(S)-3-(7-chloro-6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-dioxolan-2-yl)benzo[d]isoxazol-
3-yl)-4-methyloxazolidin-2-one (6h) NaH (60% dispersion, 322 mg, 8.04 mmol) was added to a
62
solution of (S)-4-methyloxazolidin-2-one (1.05 g, 8.04 mmol) in 10 mL DMF. After 10 min
stirring, a solution of 5c (1.0 g, 2.68 mmol, see Supporting Information) in 5 mL DMF was
added, and the mixture was heated at 100 ˚C for 1 h. After quenching with saturated aqueous
NH Cl, The mixture was extracted with EtOAc, which was washed with brine. Drying (Na SO )
4
2
4
and removal of solvent gave a residue that was chromatographed on silica gel (20% acetone in
1
hexanes) to afford 0.6 g (48%) of the title compound as an oil. H NMR (300 MHz, CDCl ) δ
3
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2
2
2
2
2
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3
3
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3
3
3
3
3
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4
4
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5
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5
5
5
5
5
5
5
5
6
8
1
1
.69 (s, 1H), 6.36 (s, 1H), 4.94 (m, 1H), 4.00-4.33 (m, 10H), 3.80-3.91 (m, 1H), 3.71-3.79 (m,
H), 3.63-3.70 (m, 1H), 3.18-3.35 (m, 1H), 3.06 (d, J=10.0 Hz, 1H), 2.79 (d, J=10.7 Hz, 1H),
+
.45 (d, J=5.1 Hz, 3H), 1.19-1.26 (m, 3H); MS (ES) MH : 468.2 for C H ClN O .
21
27
3
7
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
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2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(R)-4-(((tert-butyldimethylsilyl)oxy)methyl)-3-(6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-
dioxolan-2-yl)-7-fluorobenzo[d]isoxazol-3-yl)oxazolidin-2-one (6i) NaH (60% dispersion)
(0.056
g,
1.40
mmol)
was
added
to
a
solution
of
(R)-4-(((tert-
63
butyldimethylsilyl)oxy)methyl)oxazolidin-2-one (0.324 g, 1.40 mmol) in DMF (5 mL) at room
temperature. After stirring for 30 min, 5a (0.5 g, 1.40 mmol) was added, and the mixture was
heated at 80 °C for 6 h. The mixture was quenched with aqueous NH Cl, and solvent was
4
removed. The residue was taken up in Et O, which was washed 3 times with water and once with
2
brine. The combined aqueous layers were extracted with Et O, which was washed twice more
2
with water and once with brine. The combined Et O layers were dried (MgSO ) and concentrated
2
4
to give an oil that was chromatographed on silica gel (0-30% gradient of EtOAc in CH Cl ) to
2
2
isolate two materials. The first eluting material was consistent with starting material 5a and the
1
second eluting material with the title compound as a white solid. Yield 233 mg (30%). H NMR
(300 MHz, DMSO-d ) δ 8.34 (s, 1H), 6.23 (s, 1H), 4.74 (d, J=3.8 Hz, 2H), 4.43-4.54 (m, 1H),
6
3
.96-4.26 (m, 7H), 3.80 (d, J=10.9 Hz, 1H), 3.31 (s, 2H), 3.27 (d, J=10.9 Hz, 2H), 2.95 (dd,
19
J=5.65, 10.7 Hz, 2H), 1.27 (d, J=6.2 Hz, 6H), 0.81 (s, 9H), 0.00 (s, 3H), -0.15 (s, 3H); F NMR
+
(282 MHz, DMSO-d ) δ -145.55; MS (ES) MH : 552.0 for C H FN O Si.
6
26 39
3
7
(
4S)-3-{6-[(2R,6R)-2,6-dimethylmorpholin-4-yl]-5-(1,3-dioxolan-2-yl)-7-fluoro-1,2-
benzoxazol-3-yl}-4-(methoxymethyl)-1,3-oxazolidin-2-one (6j) NaH (60% dispersion, 0.102 g,
.55 mmol) was added to a solution of (S)-4-(methoxymethyl)oxazolidin-2-one in 5 mL DMF at
2
rt. After stirring for 30 min, 5a (0.7 g, 1.96 mmol) was added, and the mixture was heated at 80
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°
C for 6 h. The mixture was quenched with aqueous NH Cl and solvent was removed. The
4
residue was taken up in Et O, which was washed 3 times with water and once with brine. The
2
combined aqueous layers were extracted with Et O, which was washed twice more with water
2
10
11
12
13
14
15
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45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
and once with brine. The combined Et O layers were dried (MgSO ) and concentrated to give an
4
2
oil that was chromatographed on silica gel (0-30% gradient of EtOAc in CH Cl ). Two materials
2
2
were isolated. The first eluting material was consistent with starting material (221 mg) and the
1
second eluting material with the title compound (243 mg, 27%) as a white solid. H NMR (400
MHz, CDCl ) δ 8.45 (d, J=1.0 Hz, 1H), 6.35 (s, 1H), 4.72-4.82 (m, 1H), 4.63-4.69 (m, 1H), 4.57-
3
4.63 (m, 1H), 4.14-4.24 (m, 4H), 4.00-4.10 (m, 2H), 3.91 (dd, J=5.0, 10.3 Hz, 1H), 3.70 (dd,
J=2.6, 10.2 Hz, 1H), 3.37 (s, 3H), 3.25-3.37 (m, 2H), 3.00 (dd, J=5.5, 10.8 Hz, 2H), 1.34 (br. s.,
1
9
+
6
H); F NMR (282 MHz, DMSO-d ) δ -145.51; MS (ES) MH : 452.3 for C H FN O .
6
21 27
3
7
(
S)-3-(7-chloro-6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-dioxolan-2-yl)benzo[d]isoxazol-
-yl)-4-(methoxymethyl)oxazolidin-2-one (6k) Prepared as described for 7c with 703 mg (5.36
mmol) (S)-5-(methoxymethyl)oxazolidin-2-one, 214 mg (60% dispersion, 5.35 mmol) NaH and
3
2.0 g (5.36 mmol) 5c (Supplementary Material) to afford 421 mg (17%) of the title compound.
1
H NMR (300 MHz, DMSO-d ) δ 8.45 (s, 1H), 6.21 (s, 1H), 4.72-4.79 (m, 1H), 4.46 (dd, J=3.5,
6
7.6 Hz, 1H), 3.93-4.15 (m, 8H), 3.87 (dd, J=3.5, 10.5 Hz, 1H), 3.57 (dd, J=2.0, 10.5 Hz, 1H),
3.25 (s, 3H), 3.06 (br. s., 2H), 2.67-2.87 (m, 1H), 1.32 (br. s., 3H), 1.13 (br. s., 3H); MS (ES)
+
MH : 468.2 for C H ClN O .
2
1
27
3
7
(4S)-4-[(Dimethylamino)methyl]-3-{6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-5-(1,3-
dioxolan-2-yl)-7-fluoro-1,2-benzoxazol-3-yl}-1,3-oxazolidin-2-one (6l) Prepared from 5a (335
mg, 0.94 mmol), NaH (60% dispersion, 38 mg, 0.94 mmol) and (135 mg, 0.94 mmol) (4S)-4-
[
(dimethylamino)methyl]-1,3-oxazolidin-2-one (Supporting Information) using the method
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1
1
1
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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
1
described for 6c to afford 102 mg (34%) of the title compound. H NMR (400 MHz, DMSO- d )
6
δ 8.25 (s, 1H), 6.1 (s, 1H), 4.7 (d, 2H), 4.45 (d, 1H), 4.1 (m, 2H), 3.75 (m, 2H), 4.0 (m, 2H), 3.1
+
d, 2H), 2.85 (t, 2H), 2.5 (m, 2H), 2.2 (s, 6H), 1.1 (d, 6H); MS (ES) MH : 465.5 for
(
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
C H FN O .
6
22
30
4
(S)-3-(6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-fluorobenzo[d]isoxazol-
-yl)-4-phenyloxazolidin-2-one (6m) NaH (60% dispersion, 56 mg, 1.40 mmol) was added to a
3
solution of (S)-4-phenyloxazolidin-2-one (0.229 g, 1.40 mmol) in DMF (20 mL) at rt. After
stirring for 30 min, 5a (0.5 g, 1.40 mmol) was added, and the mixture was heated at 80 °C for 24
h. The mixture was quenched with aqueous NH Cl and solvent was removed. The residue was
4
taken up in Et O, which was washed 3 times with water and once with brine. The combined
2
aqueous layers were extracted with Et O, which was washed twice more with water and once
2
with brine. The combined Et O layers were dried (MgSO4) and concentrated to give an oil that
2
was chromatographed on silica gel (0-30% gradient of EtOAc in CH Cl ) to afford two
2
2
components. The first eluting component was consistent with recovered starting material 3a (225
mg). The second eluting component was identified as the title compound and was isolated as a
1
white solid (96 mg, 14%). H NMR (400 MHz, CDCl ) δ 8.41 (d, J=1.0 Hz, 1H), 7.31-7.47 (m,
3
5H), 6.34 (s, 1H), 5.70 (dd, J=6.3, 8.8 Hz, 1H), 4.96 (t, J=8.8 Hz, 1H), 4.47 (dd, J=6.3, 8.8 Hz,
1
H), 3.99-4.27 (m, 7H), 2.97 (dd, J=5.65, 11.2 Hz, 2H), 1.29-1.45 (br. s, 6H); F NMR (282
9
1
+
MHz, CDCl ) δ -145.2; MS (ES) MH : 484.1 for C H FN O .
3
25 27
3
6
(4S,5R)-3-[6-[(2R,6R)-2,6-Dimethylmorpholin-4-yl]-5-(1,3-dioxolan-2-yl)-7-fluoro-1,2-
benzoxazol-3-yl]-4,5-dimethyl-oxazolidin-2-one (6o) and (4R,5S)-3-[6-[(2R,6R)-2,6-
dimethylmorpholin-4-yl]-5-(1,3-dioxolan-2-yl)-7-fluoro-1,2-benzoxazol-3-yl]-4,5-dimethyl-
64
oxazolidin-2-one (6n) A solution of rel-(R,R)-4,5-dimethyloxazolidin-2-one (2.7 g, 18.8
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5
6
7
8
9
mmol) in 50 mL DMF was added slowly to a suspension of NaH (60% dispersion, 752 mg, 18.8
mmol) in 10 mL DMF at rt. After 10 minutes stirring, 5a (6.7 g, 18.8 mmol) was added and the
mixture was heated in the microwave at 100 °C for 1 h. The resulting mixture was cooled and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
33
34
35
36
37
38
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
poured into ice cold aqueous NH Cl, and extracted with EtOAc. The organic layer was washed
4
with water, brine, and dried (Na SO ). After concentration, the residue was purified on a silica
2
4
gel column (elution with 0-5% methanol in CHCl ) to give a solid as a mixture of diastereomers.
3
The diastereomers were separated by chiral HPLC using Chiralpak IC (250 x 4.6mm) column
(hexane:methanol:ethanol (70:15:15) 1.0 mL/min) to afford 2 components. The first eluting
1
1
component was identified as 6o (840 mg, 10% yield). H NMR (300 MHz, DMSO-d ) δ H
6
NMR (300 MHz, DMSO-d ) δ 8.28 (s, 1H), 6.18 (s, 1H), 4.91-5.23 (m, 1H), 4.57-4.77 (m, 1H),
6
4
1
6
.03-4.13 (m, 5H), 3.99 (dd, J=5.0, 8.0 Hz, 2H), 3.24 (m, 1H), 3.20 (m, 1H), 2.91 (d, J=4.90 Hz,
H), 2.88 (d, J=4.7 Hz, 1H), 1.39 (d, J=6.6 Hz, 3H), 1.33 (d, J=6.4 Hz, 3H), 1.23 (d, J=6.0 Hz,
1
9
+
H); F NMR (282 MHz, DMSO-d ) δ -145.46; MS (ES) MH : 436.2 for C H ClN O . The
6
21 27
3
6
65
material matched that made from (R,R)-4,5-dimethyloxazolidin-2-one. The second eluting
1
component was identified as 6n (920 mg, 11% yield). H NMR (300 MHz, DMSO-d ) δ 8.29 (s,
6
1H), 6.18 (s, 1H), 4.90-5.21 (m, 1H), 4.56-4.80 (m, 1H), 3.91-4.15 (m, 7H), 3.28 (m, 1H), 3.24
(m, 1H), 3.20 (m, 1H), 2.91 (d, J=6.4 Hz, 1H), 2.88 (d, J=5.5 Hz, 1H), 1.39 (d, J=6.6 Hz, 3H),
1
9
1.33 (d, J=6.6 Hz, 3H), 1.23 (d, J=5.8 Hz, 6H); F NMR (282 MHz, DMSO-d ) δ -145.47; MS
6
+
ES) MH : 436.2 for C H ClN O .
(
2
1
27
3
6
1-(6-((2R,6R)-2,6-Dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-fluorobenzo[d]isoxazol-3-
yl)imidazolidin-2-one (6p) Prepared from 5a (200 mg, 0.56 mmol), NaH (60% dispersion, 33.6
mg, 0.84 mmol) and imidazolidin-2-one (72.4 mg, 0.84 mmol) using the method described for
1
3b to afford 30 mg (13%) of the title compound. H NMR (300 MHz, CDCl ) δ 8.56 (d, J=1.1
3
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1
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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
Hz, 1H), 6.33 (s, 1H), 5.15 (br. s., 1H), 4.2-4.3 (m, 6H), 4.0-4.1 (m, 2H), 3.7-3.8 (m, 2H), 3.25-
+
.4 (br. s, 2H), 2.9-3.0 (dd, J=11.0, 5.6 Hz, 2H), 1.32 (d, J=6.2 Hz, 6H). MS (ES) MH : 407.1
3
for C H FN O .
4
1
9
24
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1-(6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-fluorobenzo[d]isoxazol-3-
yl)-3-methylimidazolidin-2-one (6q) NaH (60% dispersion, 0.056 g, 1.40 mmol) was added to a
solution of 1-methylimidazolidin-2-one (0.140 g, 1.40 mmol) in DMF (5 mL) at rt. After stirring
for 30 min, 5a (0.5 g, 1.40 mmol) was added, and the mixture was heated at 80 °C for 6 h. The
mixture was quenched with aqueous NH Cl and solvent was removed. The residue was taken up
4
in Et O, which was washed 3 times with water and once with brine. The combined aqueous
2
layers were extracted with Et O, which was washed twice more with water and once with brine.
2
The combined Et O layers were dried (MgSO ) and concentrated to give an oil that was
2
4
chromatographed on silica gel (0-30% gradient of EtOAc in CH Cl ) to afford 216 mg (37%) of
2
2
1
the title compound as a white solid. H NMR (300 MHz, CDCl ) δ 8.65 (s, 1H), 6.35 (s, 1H),
3
1
.11-4.29 (m, 5H), 3.97-4.08 (m, 5H), 3.64 (t, J=7.9 Hz, 2H), 3.00 (m, 5H), 1.34 (br. s., 6H); F
9
4
+
NMR (282 MHz, CDCl ) δ -145.0; MS (ES) MH : 421.9 for C H FN O .
3
20 26
4
5
(R)-2-(5-(1,3-dioxolan-2-yl)-2,3,4-trifluoro-N'-hydroxybenzimidamido)-3-hydroxy-N,N-
dimethylpropanamide Et N (1.5 mL, 10.7 mmol) and 10 (2.5 g, 8.9 mmol, described in
3
Supplementary Materials) were added to a solution of (R)-2-amino-3-hydroxy-N,N-
dimethylpropanamide (1.4 g, 10.7 mmol, described in Supplementary Materials) in 30 mL DMF,
and the mixture was stirred at rt for 16 h. The mixture was poured into water (75 mL) and
extracted with 3 times with EtOAc. The combined organic layers were washed twice with water
and once with brine before being dried (Na SO ). Removal of solvent was followed by
2
4
chromatography on silica gel (3% MeOH in CHCl ) to afford the title compound as a pale yellow
3
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oil. Yield: 3.0 g (90%). H NMR (400 MHz, DMSO-d ) δ 2.72 (s, 3H), 2.79 (s, 3H), 3.40-3.43
6
(
m, 1H), 3.97-4.07 (m, 6H), 4.92 (t, 1H), 5.92 (d, 1H), 6.03 (s, 1H), 7.28 (t, 1H), 10.16 (s, 1H).
1
9
+
F NMR (376.5 MHz, DMSO-d ) δ -134.4 (m), -138.2 (m), -159.48 (m); MS (ES) MH : 378.3
6
10
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for C H F N O .
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5
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3
3
5
(R)-2-((5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)amino)-3-hydroxy-N,N-
dimethylpropanamide (11r) A solution of the preceding compound (3.0 g, 7.95 mmol) and
Cs CO (5.7 g, 17.5 mmol) in 10 mL DMF was stirred at rt for 16 h. The mixture was poured
2
3
into water (75 mL) and extracted with 3 times with EtOAc. The combined organic layers were
washed with twice with water and once with brine before being dried (Na SO ) and
2
4
concentrated. The residue was chromatographed on silica gel (1% MeOH in CHCl ) to afford the
3
1
title compound as a pale yellow solid. Yield: 2.0 g (70%). H NMR (400 MHz, DMSO-d ) δ 2.85
6
(s, 3H), 3.14 (s, 3H), 3.63-3.71 (m, 2H), 4.00-4.09 (m, 4H), 4.64-4.69 (m, 1H), 5.03 (t, 1H), 6.09
1
9
(s, 1H), 7.61 (d, 1H), 8.11 (d, 1H); F NMR (376.5 MHz, DMSO-d ) δ -143.2 (d), -161.8 (d);
6
+
MS (ES) MH : 358.3 for C H F N O .
1
5
18
2
3
5
(R)-3-(5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)-N,N-dimethyl-2-
oxooxazolidine-4-carboxamide A solution of 11r (2.0 g, 5.59 mmol) in 10 mL THF was added
to a stirring mixture of NaH (60% dispersion, 223 mg, 5.59 mmol) in 10 mL THF at -10°C, and
stirring was continued for 10 min. A solution of CDI (1.8 g, 11.2 mmol) in 10 mL THF was
added, and after stirring for another 10 min, the reaction mixture was then poured into ice water.
The mixture was extracted twice with EtOAc, and the combined organic layers were washed
with water and brine. Drying (Na SO ) and removal of solvent was followed by chromatography
4
2
1
on silica gel (CHCl ) to afford the title compound as a pale yellow solid. Yield: 2.0 g (93%). H
3
NMR (300 MHz, DMSO-d ) δ 2.86 (s, 3H), 3.07 (s, 3H), 3.98-4.07 (m, 4H), 4.53 (dd, 1H), 4.82
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(
t, 1H), 5.63 (dd, 1H), 6.10 (s, 1H), 8.39 (dd, 1H). F NMR (376.5 MHz, DMSO-d ) δ -140.2 (d,
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19
J=18.8 Hz), -160.9 (d, J=18.8 Hz); F NMR (376.5 MHz, DMSO-d ) δ: -140.2 (d), -160.9 (d);
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+
MS (ES) MH : 384.3 for C H F N O
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(
R)-3-(6,7-difluoro-5-formylbenzo[d]isoxazol-3-yl)-N,N-dimethyl-2-oxooxazolidine-4-
carboxamide (12r) A stirred solution of the preceding compound (1.9 g, 4.96 mmol) in 15 mL
,4-dioxane and 7.5 mL 6N HCl (4.5 mL) was stirred at rt for 8 h. The mixture was poured into
1
ice-cooled water and extracted with twice with EtOAc. The combined organic layers were
washed with water and brine. Drying (Na SO ) and removal of solvent afforded the title product
2
4
1
as a solid. Yield: 1.65 g (98%). H NMR (300 MHz, DMSO-d ) δ 2.86 (s, 3H), 3.08 (s, 3H), 4.57
6
1
dd, 1H), 4.85 (t, 1H), 5.64 (dd, 1H), 8.79 (dd, 1H), 10.19 (s, 1H); F NMR (376.5 MHz,
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(
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9
DMSO-d ) δ -142.9 (d, J=22.6 Hz), -160.2 (d, J=22.6 Hz); F NMR (376.5 MHz, DMSO-d ) δ:
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6
+
140.9 (d), -160.2 (d); MS (ES) MH : 340.3 for C H F N O .
-
14 12 2 3 5
(
R)-3-(6-((2R,6R)-2,6-dimethylmorpholino)-7-fluoro-5-formylbenzo[d]isoxazol-3-yl)-N,N-
dimethyl-2-oxooxazolidine-4-carboxamide (13r) A solution of 12r (1.65 g, 4.80 mmol), DIEA
1.7 mL, 9.6 mmol) and (2R,6R)-2,6-dimethylmorpholine (0.68 g, 5.8 mmol) in 20 mL CH CN
(
3
was heated at 80 ºC for 16 h. After cooling to rt, volatiles were removed and the residue was
chromatographed on silica gel (EtOAc gradient in CHCl ) to give title compound as pale yellow
3
1
solid. Yield: 1.95 g (92%). H NMR (400 MHz, DMSO-d ) δ 10.31 (s, 1H), 8.65 (s, 1H), 5.64
6
(dd, J=3.2, 8.7 Hz, 1H), 4.84 (t, J=8.8 Hz, 1H), 4.55 (dd, J=3.2, 8.8 Hz, 1H), 4.11-4.15 (m, 2H),
19
.38-3.41 (m, 2H), 3.08 (s, 3H), 2.99-3.03 (m, 2H), 2.87 (s, 3H), 1.20 (d, J=6.3 Hz, 6H); F
3
+
NMR (376.5 MHz, DMSO-d ) δ: -146.81; ES MH : 435.4 for C H FN O .
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20 23
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(
2R)-1-[[5-(1,3-Dioxolan-2-yl)-6,7-difluoro-1,2-benzoxazol-3-yl]amino]propan-2-ol (11t) (R)-
1
-aminopropan-2-ol (1.3 ml, 16.6 mmol) was added to a solution of 10 (2.22 g, 7.88 mmol,
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described in Supplementary Materials) 30 mL DMF at rt wherein there was a slow exotherm.
After stirring for 40 min, potassium tert-butoxide (1.77 g, 15.8 mmol) was added all at once.
After stirring for 1 h, the reaction was incomplete by LC-MS analysis and additional potassium
tert-butoxide was added (400 mg, 3.6 mmol). The resulting mixture was stirred at rt for 1 h
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before being quenched with aqueous NH Cl. Solvent was removed, and the solid residue was
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taken up in water while breaking up the solid mass with a spatula, and the mixture was stirred at
rt overnight. The solids were filtered and rinsed through with water before being dried in vacuo
1
to give the title compound. Yield 2.24 g (95%). H NMR (300 MHz, DMSO-d ) δ 8.30-8.55 (m,
6
1H), 6.10 (s, 1H), 4.87-5.21 (m, 1H), 4.27 (t, J=8.9 Hz, 1H), 4.04 (m, 4H), 3.78 (dd, J=7.2, 9.4
1
9
Hz, 1H), 1.49 (d, J=6.2 Hz, 3H); F NMR (282 MHz, DMSO-d ) δ -140.64 (d, J=21.5 Hz), -
6
+
161.34 (d, J=21.5 Hz); ES MH : 301 for C H F N O .
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14
2
2
4
(
R)-3-(5-(1,3-Dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)-5-methyloxazolidin-2-one A
mixture of 11t (500 mg, 1.67 mmol), carbonyl diimidazole (405 mg, 2.50 mmol), and DMAP
102 mg, 0.83 mmol) 10 mL THF was heated at reflux overnight (21 h). The solvent was
(
removed and the residue was taken up in 1N HCl with stirring at rt for 90 min affording solids.
The solids were filtered and rinsed well with water breaking them up with a spatula before
drying in vacuo. The material is consistent with the title compound with about 5% impurity due
1
to hydrolysis of the ketal to corresponding aldehyde. Yield 407 mg (75%). H NMR (300 MHz,
DMSO-d ) δ 8.33-8.55 (m, 1H), 6.10 (s, 1H), 4.86-5.17 (m, 1H), 4.27 (t, J=8.9 Hz, 1H), 4.04 (d,
6
19
J=4.3 Hz, 5H), 3.78 (dd, J=7.2, 9.4 Hz, 1H), 1.49 (d, J=6.2 Hz, 3H); F NMR (282 MHz,
+
DMSO-d ) δ -140.64 (d, J=21.5 Hz), -161.34 (d, J=21.5 Hz); MS (ES) MH : 327 for
6
C H F N O .
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14
2
2
5
(R)-6,7-Difluoro-3-(5-methyl-2-oxooxazolidin-3-yl)benzo[d]isoxazole-5-carbaldehyde (12t)
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