Synthesis and anti-staphylococcal activity of novel bacterial topoisomerase inhibitors with a 5-amino-1,3-dioxane linker moiety

Article abstract of DOI:10.1016/j.bmcl.2018.06.003

Novel bacterial type II topoisomerase inhibitors (NBTIs) constitute a promising new class of antibacterial agents. We report a series of NBTIs with potent anti-staphylococcal activity and diminished hERG inhibition. Dioxane-linked compound 9 demonstrated MICs ≤1 μg/mL against both methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA), accompanied by reduced hERG inhibition as compared to cyclohexane- or piperidine-linked analogs.

Full text of DOI:10.1016/j.bmcl.2018.06.003

Accepted Manuscript
Synthesis and Anti-staphylococcal Activity of Novel Bacterial Topoisomerase
Inhibitors with a 5-Amino-1,3-dioxane Linker Moiety
Linsen Li, Antony Okumu, Sheri Dellos-Nolan, Zoe Li, Soumendrakrishna
Karmahapatra, Anthony English, Jack C. Yalowich, Daniel J. Wozniak, Mark
J. Mitton-Fry
PII:
S0960-894X(18)30480-3
https://doi.org/10.1016/j.bmcl.2018.06.003
BMCL 25884
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 March 2018
14 May 2018
1 June 2018
Please cite this article as: Li, L., Okumu, A., Dellos-Nolan, S., Li, Z., Karmahapatra, S., English, A., Yalowich,
J.C., Wozniak, D.J., Mitton-Fry, M.J., Synthesis and Anti-staphylococcal Activity of Novel Bacterial
Topoisomerase Inhibitors with a 5-Amino-1,3-dioxane Linker Moiety, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.003
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Synthesis and Anti-staphylococcal Activity of Novel Bacterial Topoisomerase Inhibitors with a 5-
Amino-1,3-dioxane Linker Moiety
Linsen Li,1 Antony Okumu,1 Sheri Dellos-Nolan,2 Zoe Li,3 Soumendrakrishna Karmahapatra,3 Anthony
English,3 Jack C. Yalowich,3 Daniel J. Wozniak,2,4 and Mark J. Mitton-Fry*,1
*Corresponding author. Tel. (614) 292-1903, email address mitton-fry.1@osu.edu
2
3
1Division of Medicinal Chemistry and Pharmacognosy. Microbial Infection and Immunity. Division of
Pharmacology. 4Department of Microbiology. The Ohio State University, Columbus, OH 43210 USA.
Abstract
Novel bacterial type II topoisomerase inhibitors (NBTIs) constitute a promising new class of antibacterial
agents. We report a series of NBTIs with potent anti-staphylococcal activity and diminished hERG
inhibition. Dioxane-linked compound 9 demonstrated MICs ≤ 1 g/mL against both methicillin-
susceptible (MSSA) and –resistant Staphylococcus aureus (MRSA), accompanied by reduced hERG
inhibition as compared to cyclohexane- or piperidine-linked analogs.

The continuous evolution and dissemination of multidrug-resistant bacteria presents a significant threat
to human health.1 Within this context, methicillin-resistant Staphylococcus aureus (MRSA) is a
particularly important pathogen in both community and hospital settings,2 and the incidence of MRSA in
individuals with cystic fibrosis is also increasing.3 Indeed, according to a 2013 report issued by the
Centers for Disease Control, MRSA ranks as the number one cause of mortality from antibiotic-resistant
infections in the United States.4 Several recent drug approvals hold promise for the treatment of MRSA,
including ceftaroline,5 tedizolid,6 and delafloxacin.7 Nevertheless, these antibiotics are analogs of well-
established drug classes, and fully addressing the threat of multidrug-resistant bacterial infections requires
the development of entirely new therapeutic classes.8 Unfortunately, achieving this goal has proved
particularly challenging, especially in light of reduced pharmaceutical investment.9
Against this backdrop, Novel Bacterial Type II Topoisomerase Inhibitors (NBTIs) have emerged as a
promising new class.10 The NBTIs exert antibacterial activity through inhibition of both DNA gyrase and
topoisomerase IV (TopoIV), the biological targets of the well-established fluoroquinolone antibiotics.
Crystallographic11 and mutational12,13 studies have elucidated a binding mode for NBTIs that is distinct
from the fluoroquinolones, thus circumventing resistance to these important medicines. Finally, several
recent reports have raised the exciting possibility that, in addition to excellent activity against Gram-
positive bacteria, NBTI spectrum of activity might also be extended to Gram-negative pathogens.14,15
X-ray crystallographic studies of the NBTI GSK299423 (1, Figure 1) in ternary complex with S. aureus
DNA gyrase and DNA revealed a three part pharmacophore.11 The bicyclic quinoline moiety binds DNA
and is connected to the enzyme-binding oxathiolopyridine motif through a linker domain. Additional
structural efforts by these authors16,17 and others18,19 have established the generality of this binding mode
for NBTIs. Intriguingly, the linker does not appear to make critical contacts with either the DNA or
enzyme, with the exception of a characteristic amine that contacts an aspartate residue (D83) at the
entrance of a hydrophobic pocket of DNA gyrase. Consequently, the linker has been the focus of
substantial medicinal chemistry efforts and has proved amenable to considerable structural innovation.
Figure 1. Examples of previously reported NBTIs: GSK299423 (1) and gepotidacin (2).
Several NBTIs have been advanced to human clinical trials.10,17,20-22 Gepotidacin (2, Figure 1) has
recently demonstrated efficacy in a Phase 2 trial involving acute bacterial skin and skin structure
infections, with a substantial proportion of isolates being MRSA.23 Potential limitations of gepotidacin
include thrice daily dosing and the clinical observation of S. aureus strains with reduced susceptibility,
likely through a single amino acid substitution in DNA gyrase (D83N). This mutation has been reported
to confer resistance to a number of NBTIs, which commonly inhibit DNA gyrase much more potently
than TopoIV.24-28 Interestingly, although gepotidacin only minimally inhibits hERG (IC50 = 588 g/mL),
significant (>10 ms) prolongation of the cardiac QT interval has been observed in clinical trials.29 At least
one NBTI has previously been eliminated from clinical trials due to QT-prolongation.30 The minimization
of hERG inhibition has thus been a key objective of multiple medicinal chemistry programs. Of particular
interest is the report by Reck and colleagues, who showed attenuation of hERG inhibition by NBTIs
through modulation of physicochemical properties such as amine basicity and lipophilicity.22
Ndubaku and coworkers described the use of a 5-amino-1,3-dioxane moiety as a more polar and less
basic replacement for a piperidine in a series of PAK1 inhibitors, in part to address hERG activity.31
Noting the observed reductions in hERG inhibition,31 we hypothesized that a trans-configured dioxane

ring would serve as a suitable surrogate for previously reported linkers and that its reduced amine basicity
would confer potential safety benefits through diminished hERG inhibition. Prior reports describing
NBTIs with tetrahydropyran15,24,26 and oxabicyclooctane18,19,27 linkers suggested that oxygenation in this
domain would be well tolerated. Facile synthetic access was expected to be highly enabling to the
development of structure-activity relationships (SAR). In this regard, improved dual-target inhibition,
through optimization of TopoIV activity would be expected to confer advantages in terms of a reduced
propensity for resistance emergence.32
In order to establish proof of principle, a series of NBTIs possessing a 6-methoxyquinoline DNA
binding moiety and the 5-amino-1,3-dioxane linker was prepared using a short synthetic route (Scheme
1). Quinoline intermediate 3 was synthesized on multigram scale via bromination33 of commercially
available 4-hydroxy-6-methoxyquinoline with PBr3 and was isolated by simple filtration in high yield
(95%) and purity (>95%). Serinol (4) was efficiently protected as the phthalimide derivative (5) in good
yield (91%) and purity (95%) on > 75 g scale without chromatographic purification. Condensation of diol
5 with acrolein proceeded smoothly to afford alkene 6. Hydroboration of alkene 6 with 9-BBN and
Suzuki-Miyaura reaction of the resulting intermediate with quinoline bromide 3 afforded the coupled
intermediate 7. Deprotection of the phthalimide with ethanolamine in refluxing ethyl acetate34 yielded
primary amine 8 as the key intermediate. Finally, reductive amination installed a series of enzyme-binding
motifs in compounds 9-17.
Scheme 1.
Minimal inhibitory concentrations (MICs) for all analogs were determined using the S. aureus reference
strain ATCC 29213 (Table 1). Ciprofloxacin served as a positive control in each assay. A variety of
bicyclic enzyme-binding moieties were explored in the initial proof of concept efforts, since they have
shown considerable promise in prior reports.35 The following general trends were notable. Benzodioxine 9
afforded potent whole cell activity (≤ 1 g/mL) against the control S. aureus strain that was eroded only
slightly in the aza analogs 10 and 11. Pyridothiazinone 12 and pyridooxazinone 13 also showed potent
whole cell activity. In contrast, the monocyclic phenyl analog 15 and 3-pyridyl derivative 16 lost all
activity, while the 2-pyridyl analog 17 demonstrated only a modest MIC (8 g/mL).

Inspired by a recent disclosure of NBTIs with anti-mycobacterial activity,36 we also incorporated a 3,4-
dichlorophenyl moiety in compound 18. Gratifyingly, this analog showed whole cell activity on par with
the bicyclic analogs. The monosubstituted 4-chlorophenyl analog (19) was similarly potent, whereas the
3-chloro (20) and 2-chloro (21) derivatives were inferior. Finally, we prepared the substituted pyridyl
analog 22 as a hybrid of compounds 17 and 19; its potency was similar to that of the unsubstituted pyridyl
derivative 17.
MICs for selected compounds were also determined using a USA 300 strain of MRSA (Table 1).37
Vancomycin was used as an additional control. Susceptibility to ciprofloxacin eroded considerably for the
MRSA strain (ca. 64-fold increase in MIC). In contrast, MICs for the novel NBTIs showed no significant
increases, highlighting the promise of NBTIs to tackle pre-existing forms of drug resistance.
Table 1. Minimum inhibitory concentrations (MICs) and target inhibition.
Compound
S. aureus (MSSA) S. aureus (MRSA) S. aureus DNA
S. aureus
ATCC 29213
USA 300
gyrase
TopoIV
MIC (g/mL)a,b
MIC (g/mL)a,b
IC50 (M) (n=2)c IC50 (M) (n=2)d
0.25-1
1-2
0.5
2
0.10
0.36
0.33
0.59
0.78
0.74
NT
14
9
50
10
1
2
25-50
>100
>100
>100
NT
11
0.5
0.5
1
12
0.5
13
1-2
1
14
>64
>64
8
NTe
NT
NT
0.5
4
15
NT
NT
16
NT
NT
17
≤0.25-1
1
0.28
1.2
>100
>100
NT
18
19
32
NT
NT
NT
16-32
1-2
NT
20
>64
8
NT
NT
21
NT
NT
22
ciprofloxacin
vancomycin
0.125-0.5
NT
4.80 (n=4)
14.3 (n=4)
NT
NT
b
aAssays were conducted under CLSI guidelines (minimum n=3). Ranges of observed values are given
where appropriate. cSupercoiling inhibition assay, Inspiralis, Ltd. (Norwich, UK). dDecatenation
inhibition assay, Inspiralis, Ltd. (Norwich, UK). eNT=Not tested.
A subset of analogs was used to assess the degree of inhibition of DNA gyrase or TopoIV, both to
establish the mechanism of action and to evaluate the relative balance of inhibitory activities. Analogs 9-

14, 18, and 19 potently inhibited DNA gyrase, whereas inhibition of TopoIV was substantially inferior.
These results are consistent with previously reported NBTIs, where DNA gyrase inhibition in S. aureus is
superior to TopoIV.10 Efforts to improve TopoIV inhibitory activity, as well as to generate and characterize
mutant strains with resistance to this new series of NBTIs, are ongoing and will be reported in due course.
Central to the design of the dioxane-linked NBTIs was the hypothesis that reduced basicity and
enhanced polarity resulting from the two electronegative oxygen atoms would attenuate hERG
inhibition.22,31 A series of matched pair analogs were prepared to interrogate this hypothesis. Compounds
23-26 differed only from the corresponding dioxane-linked analogs (9, 10, 13, and 18) by the use of a
cyclohexane linker moiety, and 27-30 likewise differed through a piperidine linker (as seen in
gepotidacin). hERG IC50 values were determined using a high-throughput patch clamp assay (Table 2).38
All four matched dioxane-cyclohexane pairs showed the dioxanes to be superior, with 2- to 20-fold
improvements. For example, dioxane-linked compound 9 inhibited hERG 20-fold less potently (IC50 =
5.11 M), than the corresponding cyclohexane-linked analog 23 (IC50 = 0.26 M). pKa calculations using
two different software packages (Table 2) revealed the dioxane-linked compounds to be 1.4-to 2.4 log
units less basic than the corresponding cyclohexane-linked compounds, lending credence to the design
hypothesis that reduced basicity would improve the hERG profile. Additionally, the more polar
pyridodioxine moiety proved consistently superior to the more lipophilic benzodioxine (ca. 2-fold
improvement in each series).
Three of four dioxane-piperidine pairs also demonstrated improvements (5- to 6-fold), although the
dioxane-linked pyridooxazinone 13 was marginally inferior to 29. The analysis of the relationship
between hERG inhibition and basicity is complicated for the piperidine-linked compounds by the
presence of an additional basic center (the piperidine nitrogen). Both software packages showed the most
basic amine in the dioxane-linked analogs to be less basic (by 1 to 3.1 log units) than in the corresponding
piperidine-linked analogs (Table 2). In contrast, the secondary amine of the dioxane-linked analogs was
generally calculated to be similar to or modestly more basic than (up to 1.3 log units, see Supplementary
Table 1, supporting information) that of the piperidine-linked analogs. Consequently, the origins of the
generally improved hERG values are not necessarily as straightforward.
While additional reductions in hERG inhibition are clearly necessary, these data lend strong support to
our design rationale. Disappointingly, incorporation of a pyridooxazinone or pyridothiazinone enzyme-
binding moiety proved detrimental (i.e. increased hERG inhibition), in line with previously reported
studies.24,26
Table 2. Preliminary in vitro safety evaluation data and comparison with structure-matched analogs.
Compound
Linker
hERG
K562 IC50
K/VP.5
IC50
(M)
hTopoII b,c ACD Labs
pKad,e
IC50 (M)a (M)
/
ChemDraw
pKad,f
dioxane
dioxane
dioxane
5.1
9.5
12
7.4
40
32
8.8
48
40
84.18
95.12
93.81
7.2 / 8.1
5.5 / 7.5
5.9 / 7.5
9
10
11

dioxane
1.3
31
34
91.14
99.06
93.22
97.84
NTg
NT
5.4 / 7.4
5.3 / 7.4
6.7 / 7.9
7.4 / 8.1
9.5 / 9.6
7.9 / 8.9
7.7 / 8.9
9.0 / 9.3
8.8 / 9.1
8.3 / 9.1
8.4 / 9.1
8.3 / 9.1
NT
12
dioxane
2.5
24
44
13
dioxane
4.8
20
15
18
dioxane
2.0
12
16
19
cyclohexane
cyclohexane
cyclohexane
cyclohexane
piperidine
piperidine
piperidine
piperidine
NA
0.26
0.94
0.41
2.3
4.2
7.5
12
4.5
5.7
11
23
24
NT
25
NT
10
NT
12
NT
26
0.81
1.9
NT
27
28
22
NT
28
3.8
19
16
NT
29
1.0
NT
NT
NT
NT
NT
30
cisaprideh
0.012-
0.021
NT
NT
NA
NA
0.3
NT
5.1
NT
33.6 (n=4) NT
etoposide
ciprofloxacin
NT
85.5 (n=4) NT
aDetermined using IonWorks Barracuda at Charles River (Cleveland, OH). Inspiralis, Ltd. (Norwich,
b
UK). Percent enzyme activity remaining at 100 M. dCalculated for most basic amine. Calculated using
c
e
f
ACD Labs Percepta release 2017.1.3 Classic pKa function. Calculated using ChemDraw Professional
15.0. gNT = not tested (or not calculated). hCisapride serves as the positive control for the hERG assay.
A preliminary assessment of mammalian antiproliferative activity for several analogs was also
conducted in human leukemia K562 cells. For compound 9, the IC50 for human leukemia K562 cell
growth inhibition revealed a modest selectivity margin when compared to the S. aureus MIC (3.5-fold
based on an MIC of 1 g/mL), and compounds 10-13 and 18 showed improved margins. Additionally,
inhibition of the orthologous human target (hTopoII) was assessed both indirectly and directly (Table 2).
For the indirect assessment of hTopoII targeting, a K562 cell subline K/VP.5, with acquired resistance to
the anticancer agent etoposide was utilized. K/VP.5 cells contain 1/5th the level of hTopoII alpha
compared to parental K562 cells and are ~20-fold resistant to etoposide.39,40 Control experiments
performed here demonstrated 17-fold etoposide resistance (Table 2). Nearly identical growth inhibitory
IC50 -values were observed for the novel compounds tested, strongly suggesting that the new NBTIs do
not target hTopoII. In addition, direct in vitro assessment of hTopoII decatenation activity revealed only
minimal (<20%) inhibition of hTopoII decatenation activity at a concentration of 100 M for a range of
analogs, again supporting the conclusion that these compounds do not target the human enzyme.
In summary, this report describes the highly efficient synthesis and preliminary biological evaluation of
a series of 5-amino-1,3-dioxane-linked NBTIs. Potent whole cell anti-staphylococcal activity was
observed for a number of analogs and could be attributed to inhibition of bacterial DNA gyrase.
Consistent with the design hypothesis, dioxane-linked NBTIs demonstrated generally diminished hERG
inhibition. Additional efforts to reduce hERG inhibition further, to enhance antibacterial activity, to
improve TopoIV inhibition, and to optimize drug-like properties are ongoing. Results will be reported in
due course.

Acknowledgment
The authors are grateful for startup funding from The Ohio State University College of Pharmacy and
Discovery Themes Initiative as well as for a grant from the Cystic Fibrosis Foundation.

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A series of dioxane-linked inhibitors of bacterial DNA gyrase was synthesized.
These analogs possess potent whole cell anti-staphylococcal activity.
The dioxane linker results in diminished hERG inhibition by matched-pair analysis.