1,3-Dioxane-Linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity and Reduced hERG Inhibition

Article abstract of DOI:10.1021/acsinfecdis.8b00375

The development of new therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) is needed to counteract the significant threat that MRSA presents to human health. Novel inhibitors of DNA gyrase and topoisomerase IV (TopoIV) constitute one highly promising approach, but continued optimization is required to realize the full potential of this class of antibiotics. Herein, we report further studies on a series of dioxane-linked derivatives, demonstrating improved antistaphylococcal activity and reduced hERG inhibition. A subseries of analogues also possesses enhanced inhibition of the secondary target, TopoIV.

Full text of DOI:10.1021/acsinfecdis.8b00375

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Article
1,3-Dioxane-linked Bacterial Topoisomerase Inhibitors with
Enhanced Antibacterial Activity and Reduced hERG Inhibition
Linsen Li, Antony Akura Okumu, Sheri Nolan, Anthony English, Sandip Vibhute, Yanran Lu,
Katherine Hervert-Thomas, Justin Seffernick, Lovette Azap, Serena L. Cole, D. Shinabarger,
Laura Koeth, Steffen Lindert, Jack Yalowich, Daniel J. Wozniak, and Mark Joseph Mitton-Fry
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00375 • Publication Date (Web): 01 May 2019
Downloaded from http://pubs.acs.org on May 3, 2019
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1
,3-Dioxane-linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity
and Reduced hERG Inhibition
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Linsen Li, Antony A. Okumu, Sheri Nolan, Anthony English, Sandip Vibhute, Yanran Lu,
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Katherine Hervert-Thomas, Justin T. Seffernick, Lovette Azap, Serena L. Cole, D.
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Shinabarger, Laura M. Koeth, Steffen Lindert, Jack C. Yalowich, Daniel J. Wozniak, and
Mark J. Mitton-Fry1,*
1Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University, 500 W. 12th
2
Ave., Columbus, OH 43210. Microbial Infection and Immunity, The Ohio State University, 460
th
3
W. 12 Ave., Columbus, OH 43210.. Division of Pharmacology, The Ohio State University, 500
th
4
W. 12 Ave., Columbus, OH 43210. Department of Chemistry, Ohio Wesleyan University, 61 S.
5
Sandusky St., Delaware, OH 43015 USA. Department of Chemistry and Biochemistry, The Ohio
th
6
State University, 100 W. 18 Ave., Columbus, OH 43210. Micromyx, 4717 Campus Drive,
7
Kalamazoo, MI 49008 USA. Laboratory Specialists, Inc., 26214 Center Ridge Rd., Westlake, OH
8
th
4
4145 USA. Department of Microbiology, The Ohio State University, 484 W. 12 Ave.,
Columbus, OH 43210.
Corresponding author
*
E-mail: mitton-fry.1@osu.edu. Telephone: +1-614-292-1903
Keywords: methicillin-resistant Staphylococcus aureus, novel bacterial topoisomerase
inhibitors, gyrase
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The development of new therapies to treat methicillin-resistant Staphylococcus aureus (MRSA)
is needed to counteract the significant threat that MRSA presents to human health. Novel
inhibitors of DNA gyrase and topoisomerase IV (TopoIV) constitute one highly promising
approach, but continued optimization is required to realize the full potential of this class of
antibiotics. Herein, we report further studies on a series of dioxane-linked derivatives,
demonstrating improved anti-staphylococcal activity and reduced hERG inhibition. A subseries
of analogs also possesses enhanced inhibition of the secondary target, TopoIV.
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Infections caused by multidrug-resistant bacteria pose a significant challenge to the
1
practice of modern medicine. MRSA infection occurs in a wide variety of clinical settings,
including bacteremia, endocarditis, pneumonia, and skin infection as well as in individuals living
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,3
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with cystic fibrosis. The number of deaths attributed to MRSA infection is particularly high as
4
compared to other multidrug-resistant pathogens. While recent years have seen a number of
approvals for new antibiotics with potent anti-MRSA activity, these compounds derive from
existing classes of antibiotics,5-9 and resistance has already been observed in some cases.10
Consequently, new antibiotic classes are urgently needed. Novel bacterial topoisomerase
inhibitors (NBTIs),11,12 which inhibit the same targets as the fluoroquinolones but avoid cross-
1
3
resistance, are one particularly promising option. The NBTI class possesses potent anti-
14
staphylococcal activity. A recent elegant study of the pharmacology of gepotidacin, an NBTI
that has advanced to Phase 2 clinical trials,15,16 demonstrated that it potently inhibits DNA gyrase
in S. aureus through the induction of stable single-stranded breaks in DNA. Nevertheless, the
translational development of many of these agents has been hampered by cardiovascular safety
concerns11,17-20 (primarily hERG inhibition) and by resistance observed via single amino acid
substitutions in DNA gyrase.11,21-24 Consequently, additional research efforts directed at
optimization of this promising novel antibiotic class are highly warranted.
We recently disclosed a series of 5-amino-1,3-dioxane linked NBTIs with potent anti-
25
13,14,26
staphylococcal activity. In line with previously reported crystallographic studies,
we
propose that compounds such as 1 form a ternary complex with DNA and DNA gyrase (Figure
1), with the quinoline moiety binding to DNA and the dihydrobenzodioxine moiety filling a
small hydrophobic pocket in DNA gyrase. The dioxane serves to link these two motifs and acts
as a handle for the modulation of physicochemical properties such as lipophilicity and basicity.25
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The majority of NBTIs employ a fused bicyclic enzyme-binding moiety, as seen in 1.
Exceptions include certain anti-tubercular NBTIs28-31as well as anti-staphylococcal NBTIs with
2
1
carboxypiperidine linkers, for example NXL-101 and the previously reported arylcyclobutane
derivatives.22,32 In this new series of dioxane-linked NBTIs, fused bicyclic enzyme-binding
moieties such as that of 1 were also advantageous. Intriguingly, however, monocyclic 4-
chlorophenyl derivatives (such as in 2) also proved surprisingly potent against S. aureus.
Additional advantages to dioxane-linked NBTIs were observed when comparing hERG
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inhibition versus a series of matched pair controls with either piperidine or cyclohexyl linkers,
2
5
consistent with the design hypothesis. The current work focuses on efforts to establish a
broader understanding of structure-activity relationships of the enzyme binding motif, to enhance
the whole cell antibacterial activity further, to improve the inhibition of the secondary enzyme
target (TopoIV), and to reduce hERG inhibition further.
25
Figure 1. Previously reported dioxane-linked NBTIs (left) and proposed binding mode of
compound 1 to S. aureus DNA gyrase (right, DNA rendered semi-transparent for clarity).
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RESULTS AND DISCUSSION
Encouraged by our previous results, a diverse set of compounds with both monocyclic
and fused bicyclic enzyme-binding moieties was synthesized using the previously described
1
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route. A 6-methoxyquinoline DNA-binding motif was employed in the initial survey due to
ease of synthesis. Minimum inhibitory concentrations (MICs) were determined according to
3
4
Clinical and Laboratory Standards Institute (CLSI) guidelines using S. aureus ATCC 29213 as
a reference strain. Compounds with MICs ≤ 64 g/mL were also assayed using a ciprofloxacin-
25
resistant USA 300 strain of MRSA, as we described previously. Given the objective to enhance
dual-targeting activity so as to reduce the rate of resistance emergence, biochemical assays were
also employed on a subset of compounds (minimally those with S. aureus ATCC 29213 MICs ≤
2
g/mL) to measure inhibition of both DNA gyrase and TopoIV.
Based on the 4-chlorophenyl derivative 2, a variety of analogs bearing a 4-substituted
phenyl ring were prepared and assayed (Table 1). Analogs possessing methyl (3), trifluoromethyl
(4), methoxy (5), and trifluoromethoxy (6) groups possessed S. aureus MICs ranging from 1-4
2
5
g/mL, a dramatic improvement from the unsubstituted analog (7, > 64 g/mL ). The smaller
fluoro (8) and more polar cyano (9) analogs lacked activity, whereas the large and lipophilic tert-
butyl moiety (10) was tolerated. Additionally, none of these compounds tested for TopoIV
activity (2-5, 9, and 10) potently inhibited this enzyme (IC50 values > 100 M). The more polar
methylpyridine analog 11 was moderately less potent than the analogous phenyl compound 3.
Cyclohexyl analog 12 had modest activity that was somewhat improved by incorporation of a 4-
methyl substituent as in 13 (MICs of 8 and 2 g/mL, respectively).
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Bax and coworkers elucidated the binding mode of the NBTI class of antibiotics by
determination of the X-ray crystal structure of GSK299423 in ternary complex with DNA and
DNA gyrase. 13 Their structure suggested the presence of an unusual hydrogen bond between the
methylene of the oxathiolopyridine ring and the backbone carbonyl of A68 in DNA gyrase. We
hypothesized that a similar non-traditional hydrogen bond might also be achieved by a
difluoromethoxy (14, Figure 2) or difluoromethyl (15) substituent. Unfortunately, while both
compounds showed MICs ≤ 8 g/mL, neither proved to be an improvement over the non-
fluorinated (5 and 3) or fully fluorinated (6 and 4) parent molecules.
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Figure 2. Non-traditional hydrogen-bonding observed
1
3
for GSK299423 and proposed for novel analog 14.
2
5
In line with our prior experience, 3-substituted phenyl analogs (16, 17, 18, 19)
displayed only very modest MICs (16-64 g/mL), and the 2-substituted derivatives (20, 21, 22)
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were wholly devoid of activity (Table 1). A variety of dihalogenated compounds were also
synthesized, but only the 4-chloro-3-fluoro derivative (23, 2 g/mL) and the isomeric 3-chloro-
4
-fluoro analog (24, 8 g/mL) showed single-digit MICs (Table 1).
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2
3
4
5
6
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8
9
0
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4
5
6
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0
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2
3
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Table 1. Antibacterial and biochemical activity of NBTIs with 6-methoxyquinoline moiety.
Cmpd.
(R , R , R
S. aureus
S. aureus
(MRSA) USA
300
S. aureus DNA S. aureus
1
2
3
)
(MSSA) ATCC
gyrase
TopoIV
c
2
9213
IC (M)
IC50
5
0
a,b
a,b
d
MIC (g/mL)
MIC (g/mL)
(M)
2
5
1 (see above)
225
0.25-1
0.5
4
0.10
1.2
14
(Cl, H, H)
1
>100
>100
~100
>100
18e
3
(Me, H, H)
4 (CF , H, H)
(OMe, H, H)
(OCF , H, H)
1
1
0.22
0.3
3
1
2
5
6
4
8
1.5
3
2
4
2.1
2
5
7 (H, H, H)
>64
>64
NT
NT
NT
NT
NT
8
(F, H, H)
NT
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9
1
(CN, H, H)
>64
2
NT
4
13
>100
>100
NT
0 (t-Bu, H, H)
0.63
NT
NT
0.77
11 (see above)
4-8
8
16
8
0
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0
1
2
3
4
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7
8
9
0
1
1
2 (R
3 (R
1
1
= H)
NT
= Me, mix
2
2
67f
of cis/trans)
1
4 (OCF
2
H, H,
4-8
8
NT
NT
H)
15 (CHF
2
, H, H)
2
4
0.36
NT
>100
NT
1
1
6 (H, CN, H)
64
4-16
16
32
>64
>64
>64
2
128
32
32
>32
NT
NT
NT
1
7 (H, OMe, H)
NT
NT
18 (H, Me, H)
NT
NT
1
2
9 (H, F, H)
NT
NT
0 (H, H, OMe)
NT
NT
21 (H, H, Me)
NT
NT
2
2
2 (H, H, F)
3 (Cl, F, H)
NT
NT
0.95
2.1
>100
>100
NT
24 (F, Cl, H)
8
8
2
2
2
2
2
5 (F, F, H)
64
2
64
4
NT
6 (see above)
7 (see above)
8 (see above)
9 (see above)
0.17
0.52
0.69
0.81
74
1
2
>100
6.3e
1
2
1
2
13e
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>100f
3
3
0 (Fig. 3)
1 (Fig. 3)
>8
>8
7.7
0.5
1
1.1
>200e
4.0
ciprofloxacin
vancomycin
0.125-0.5
NT
16-32
0.5-2
13.3
NT
1
1
1
1
1
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1
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NT
a
34
Assays were conducted according to CLSI broth microdilution guidelines (n=3, minimum).
b
c
Ranges of observed values provided where appropriate. Determined at Inspiralis (Norwich,
d
UK) (n=2, minimum). Method A (see Experimental Section), determined at Inspiralis (Norwich,
e
f
UK) (n=2, minimum). Method B (see Experimental Section, n=2). Method B (n=1).
2
5
Our previous report employed several prototypical fused bicyclic enzyme binding
motifs, which universally delivered potent MICs, so additional analogs in this subseries were
35
also synthesized and assayed (Table 1). Ring contraction of 1 as in benzodioxole 26 resulted in
somewhat poorer MICs. Difluorination as in 27 slightly improved activity. Unfortunately,
neither of these analogs potently inhibited TopoIV. The fully unsaturated lipophilic 2-naphthyl
analog 28 was similar in anti-staphylococcal potency and inhibited TopoIV with an IC50 of 6.5
23

M. Compound 29, bearing the oxathiinopyridazine moiety, was similar to compound 1 in
terms of both anti-staphylococcal activity and TopoIV inhibition.
Inspection of several ternary crystal structures of DNA, DNA gyrase, and NBTIs reported
by GSK13,36,37 (PDB codes 2XCS, 4BUL, and 5IWI) revealed two alternate NBTI binding poses
as a result of the symmetric nature of the hydrophobic pocket formed from the two GyrA
domains. Superposition of these ligands (silver and black in Figure 3) suggested that the entrance
to the hydrophobic pocket may be sufficiently wide to accommodate moieties such as the 1-
naphthyl group of 30. Unfortunately, the compound was a poor inhibitor of gyrase (IC50 of 7.7

M) and failed to demonstrate antibacterial activity at concentrations up to 8 g/mL (Table 1).
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The SAR of the monocyclic derivatives (vide supra) suggested, however, that gyrase inhibition
and antibacterial activity may require a substituent that extends further into the hydrophobic
pocket. Consequently, acenaphthylene derivative 31 was synthesized and evaluated. 31
demonstrated gyrase inhibition (IC50 of 1.1 M) and antibacterial activity (S. aureus ATCC
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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
9213 MIC = 0.5 g/mL) similar to 28, suggesting that the lack of activity in 30 originated not
from its width but from the absence of binding deeper in the enzyme pocket. Unfortunately, 31
showed no inhibition of TopoIV (Table 1). That observation, coupled with its extreme
lipophilicity (cLogP = 5.7) led to deprioritization of additional efforts on this analog.
Figure 3. Design of “wider” NBTIs informed by published crystal structures.
Having established a general understanding of structure-activity relationships for the
enzyme-binding moiety of these dioxane-linked NBTIs, we sought to enhance the antibacterial
activity through incorporation of a 7-fluoro-2-methoxy-1,5-naphthyridine DNA-binding
moiety.20,23,26,32,38,39 Compounds were synthesized via a modification of our previously published
2
5
route (Scheme 1). Briefly, naphthyridinol 32 was brominated with PBr to afford the
3
bromonaphthyridine derivative 33 in 88% yield. Hydroboration of alkene 34 with 9-BBN
followed by Suzuki coupling with 33 afforded intermediate 35 in 54% yield. Deprotection of the
phthalimide was accomplished using ethanolamine in refluxing ethyl acetate as previously
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reported25,40 to afford the primary amine 36 as a key intermediate. Final analogs (37-42) were
synthesized by reductive amination under our previously described conditions.25
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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5
6
0
1
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4
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8
9
0
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0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1
As expected, compounds 37-40 bearing previously demonstrated enzyme-binding
2
5
moieties displayed very potent MICs (≤ 0.25 g/mL) against both the ATCC S. aureus and
MRSA strains, as did the oxathiinopyridazine23,39 analog 41 (Table 2). Naphthyridine analogs
with suboptimal enzyme-binding moieties such as the 3,4-difluorophenyl derivative 42 displayed
markedly improved MICs as compared to the 6-methoxyquinoline analog 25 (2 versus 64
g/mL, respectively), although the activity was still modest in comparison to 37-41. Although
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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
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
we did not prepare a comprehensive set of matched pair analogs, we observed that the SAR in
the naphthyridine series largely mirrored that of the quinoline series, with consistently enhanced
whole cell activity.
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
Table 2. Antibacterial and biochemical activity of NBTIs with 7-fluoro-2-
methoxynaphthyridine moiety.
Cmpd.
S. aureus
MSSA) ATCC
9213
S. aureus
(MRSA) USA
300
S. aureus DNA S. aureus
gyrase TopoIV
(
c
d
2
IC (M)
IC (M)
50
50
a,b
a,b
MIC (g/mL)
≤0.25, 0.25
0.25
MIC (g/mL)
0.125
0.125
0.25
0.98, 1.0e
3
7
0.03
0.04
0.16
0.42
0.07
4.43
0.10
13.3
NT
51, 2.4e
38
5.3, 0.71e
3
4
9
0
≤0.25, 0.125
0.125
0.125-0.25
0.25
18, 4.3e
0.76f
>100
14
41
0.25
4
2
2
4
125
0.25-1
0.5
ciprofloxacin 0.125-0.5
vancomycin NT
16-32
0.5-2
4.0, 8.6f
NT
a
34
Assays were conducted according to CLSI broth microdilution guidelines (n=3, minimum).
b
c
Ranges of observed values provided where appropriate. Determined at Inspiralis (Norwich,
d
UK) (n=2, minimum). Method A (see Experimental Section), determined at Inspiralis (Norwich,
e
f
UK) (n=2, minimum). Method B (see Experimental Section , n=2, minimum). Method B (n=1).
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Inhibition of DNA gyrase and TopoIV was also generally improved for this series of
naphthyridine derivatives (Table 2). Particularly exciting was compound 37, with a TopoIV IC50
= 0.98 M, representing a significant step forward in the effort to discover potent dual-target
1
1
1
1
1
1
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1
1
1
2
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2
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0
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8
9
0
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0
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3
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9
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3
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9
0
1
2
3
4
5
6
7
8
9
0
inhibitors. Compound 38 showed less potent TopoIV inhibition (IC50 = 51 M) using the
standard conditions (Method A, see Experimental section for details), but additional experiments
suggested that solubility issues may have confounded that assessment. Consequently, further
studies were conducted in-house for compound 38 using a slightly modified method (Method B)
with careful monitoring for solubility, revealing an IC50 = 2.4 M. Compound 37 was used to
validate the in-house assay; the IC50 values determined by both methods were highly consistent.
Compounds 37-41showed potent TopoIV inhibition similar to 37 using Method B.
In order to better understand the ability of compound 37 and related analogs to inhibit
TopoIV, we constructed a computational homology model and examined a potential binding
pose for 37 (see Experimental Section for additional details). As shown in Figure 4, the overall
binding mode is similar to that proposed for compound 1 binding to DNA gyrase (Fig. 1). Efforts
toward further refinement of this model, along with its use in the prospective design of superior
TopoIV inhibitors, is ongoing.
Figure 4. Structure and binding mode of compound 37 with TopoIV, as predicted using
computational homology modeling (DNA rendered semi-transparent for clarity).
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As a result of their distinctive binding mode, NBTIs are expected to maintain activity in
1
3
the presence of mutations that confer resistance to fluoroquinolones. The S84L amino acid
substitution in gyrase is one prototypical example.21,23,39 We tested the inhibitory activity of three
representative naphthyridine analogs (37, 38, and 41) and ciprofloxacin against S. aureus gyrase
with the S84L substitution (Table 3, for ease of comparison, the IC50 values against the wild type
gyrase from Table 2 are duplicated here). Compounds 37 and 41 showed equipotent inhibition of
the S84L gyrase, whereas 38 showed slightly diminished activity (4-fold loss). Ciprofloxacin, in
contrast, showed ~15-fold loss of activity. Moreover, gyrA sequencing of the USA 300 MRSA
isolate used in the current work demonstrated that this strain contains the S84L substitution (see
Experimental Section). The consistent MICs for our NBTIs between the S. aureus ATCC 29213
and USA 300 strains are further evidence of the lack of cross-resistance.
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
Table 3. Inhibition of wild-type and fluoroquinolone-resistant (S84L) DNA gyrase.
Cmpd.
S. aureus wild-type
S. aureus S84L
a
a
DNA gyrase IC50 (M)
DNA gyrase IC50 (M)
37
0.03
0.04
0.07
13.3
0.018
0.16
3
4
8
1
0.065
209.9
ciprofloxacin
aDetermined at Inspiralis (Norwich, UK) (n=2, minimum).
Representative potent fluoronaphthyridine-series analogs 37-40 were also assayed in
spontaneous frequency of resistance (FoR) studies using S. aureus ATCC 29213 as the test
organism and ciprofloxacin as a control. These compounds displayed especially potent MICs by
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the agar dilution method (Table 4); FoR values were determined at 4X and 8X multiples of the
-7
agar MIC. As shown, FoR values in the 10 range were observed across the series, with only
modest improvements at the 8X concentrations. The observed frequencies are similar to those
3
9
-8
41
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
reported for other NBTIs against this strain: ACT-387042 (3.0x10 at 4X) and AM-8722
-7
(
6.7x10 at 4X). We conclude that further optimization of TopoIV inhibitory potency is needed
to afford substantially reduced FoR values.
Table 4. Spontaneous Frequency of Resistance.a
Cmpd.
S. aureus ATCC
Spontaneous
Spontaneous
b
2
9213 MIC (g/mL) mutation frequency
mutation frequency
(8X MIC)
(4X MIC)
(
n=2)
1.53x10-7
2.31x10-7
6.75x10-7
4.5x10-8
37
0.015
0.015
0.015
0.03
1.62x10-7
1.67x10-7
3
3
8
9
2.26x10-7
1.08x10-7
40
1.15x10-8
<7.69x10-10
ciprofloxacin
0.25
a
b
See Experimental for full details. Assays were conducted according to CLSI agar dilution
guidelines.34,42
The potent anti-staphylococcal activity observed for these dioxane-linked NBTIs
prompted an assessment of the potential for broader spectrum activity as well as a more thorough
test of the activity against fluoroquinolone-resistant S. aureus. Table 5 shows MICs against a
variety of both Gram-positive and Gram-negative pathogens for 37, the aza-analog 40, and the
oxathiinopyridazine 41. All three compounds showed potent whole cell activity for Gram-
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2
2
2
2
2
2
2
2
2
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3
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positive pathogens, including fluoroquinolone-resistant MRSA, vancomycin-resistant
Enterococcus faecium (VRE) and penicillin-resistant Streptococcus pneumoniae. Compound 41
demonstrated a potent MIC90 value of 0.12 g/mL when tested against 11 strains of
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
fluoroquinolone-resistant S. aureus (as compared to ciprofloxacin, with an MIC90 of 32 g/mL).
Additionally, anti-Gram-negative activity was also seen for Pseudomonas aeruginosa and
especially for compound 41 against Acinetobacter baumannii. These results raise the exciting
prospect that further structural optimization might yield new leads for treating these critically
important pathogens, as has been reported for other series of NBTIs.43,44
Table 5. Broad spectrum antibacterial activity of select NBTIs.
Strain
Cmpd. 37 Cmpd. 40 Cmpd. 41 Ciprofloxacin
MIC
MIC
MIC
MIC
a
a
a
a
(
g/mL) (g/mL) (g/mL) (g/mL)
S. aureus ATCC 29213 (n=3)
MRSA DRL-3161
≤0.06b
≤0.06
≤0.06
≤0.06b
≤0.06
≤0.06
0.5
≤0.06b
≤0.06
≤0.06
0.25
0.25
>64
0.25
2
Streptococcus pyogenes LSI-1235
Streptococcus pneumoniae LSI-4234c 0.25
Streptococcus pneumoniae LSI-4246d ≤0.06
≤0.06
0.5
≤0.06
0.25g
0.25
0.5
Enterococcus faecium DRL-4193e
Enterococcus faecium LSI-3340h
Enterococcus faecalis ATCC 29212
Fluoroquinolone-resistant S. aureus
0.12f
0.12
0.5
0.25
16
0.25
1
0.5
0.5
NT
NT
0.12
32
(11 strains)
(MIC90
)
(MIC90)
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Escherichia coli ATCC 25922 (n=2)
Pseudomonas aeruginosa LSI-2946
Acinetobacter baumannii LSI-2957
Klebsiella pneumoniae LSI-2999
Enterobacter cloacae LSI-2943
16
4
64
8
32
≤0.06
0.5
8
2
1
≤0.06
>64
>64
0.25
0.25
≤0.06
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
>64
32
>64
>64
aDetermined according to CLSI broth microdilution guidelines.34 bVery small pinpoint growth up
c
d
to 4 µg/mL, no viable growth from pinpoint wells. Penicillin-susceptible. Penicillin-resistant.
e
f
g
Vancomycin-susceptible. Trailing observed; no viable growth within trailing wells. Pinpoint
h
growth through 64 µg/mL. Vancomycin-resistant.
As an initial assessment of compound safety, we measured hERG inhibition (vide infra)
and assayed for potential mammalian cytotoxicity with human leukemia K562 cells as previously
2
5
reported (Table 6). By comparing MIC values from Tables 1 and 2 with K562 growth
inhibition IC50 values (Table 6), we calculated a preliminary selectivity index (after converting to
the same concentration units). Naphthyridine analogs 38-40 displayed a substantial selectivity
index (50- to 100-fold), whereas 37 and 41 were more modest (7- and 15-fold, respectively).
There are limitations to this type of preliminary analysis due to differences between the assays,
such as the presence of a small amount of serum and direct cell counting in the K562 assay.
Nevertheless, the comparison demonstrates the promise of selective toxicity toward bacterial
cells. Furthermore, compounds were also evaluated using K/VP.5 cells, an etoposide-resistant
45,46
subclone with reduced expression of human topoisomerase II.
Similar inhibitory activity
was seen in both K562 and K/VP.5 cells, suggesting a lack of inhibition of the orthologous
human target. This finding was further supported by biochemical assays using the isolated
human topoisomerase II. No compounds displayed >50% inhibition of decatenation at 100 M
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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concentration. Given the lack of significant human topoisomerase II inhibition at this high
concentration, IC50 values against the human target were not determined.
Cardiovascular safety, as manifested by hERG inhibition, has presented a significant
obstacle to the successful development of the NBTI class of antibiotics.11,17-20,43,44 We designed
dioxane-linked NBTIs with the hypothesis that the two electronegative oxygen atoms would
reduce nitrogen basicity,25,47 and our initial studies demonstrated reduced hERG inhibition by
0
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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
5
matched pair analysis with other linkers. Table 6 provides hERG inhibition data for selected
compounds from the current study. Within the quinoline series, the lipophilic 4-substituted
phenyl analogs were potent hERG inhibitors, with IC50 ≤ 5 M across the series. Direct
2
5
comparison of various naphthyridine derivatives (37-40) with the previously reported quinoline
analogs showed only minor differences (< two-fold change in either direction), and the oxathiino
analogs 29 (quinoline) and 41 (naphthyridine) followed the same trend. The improved
antibacterial activity of the naphthyridines vis-à-vis the quinolines suggests an improved
therapeutic index. For example, compound 40 is a modestly less potent hERG inhibitor with
roughly eight-fold improvement in MICs as compared to the previously reported quinoline
2
5
analog (S. aureus ATCC 29213 MIC 0.125 versus 1-2 g/mL and hERG IC50 15 versus 9.5
M). We have targeted a hERG IC50 of ~100 M as being consistent with a favorable profile for
a lead compound.32,44 Thus, while the dioxane moiety presents an advantage as compared with
2
5
other linkers, additional improvements are clearly necessary.
Table 6. In vitro safety assessment of selected NBTIs.
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Cmpd.
hERG
K562 IC50 K/VP.5 IC50 hTopoII (% inhibition
b
IC50
(M)
(M)
at 100 M)
a
(M)
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
3
4
6
4.4
3.4
3.2
4.7
8.6
5.0
12
17
25
12
14
6.3
26
14
20
3.1
25
46
62
7.6
NT
11.5
1.8
8.9
13
3.8
1
2
2
2
0
6
7
9
7.6
25
-2.1
14.4
0.9
14
28
-3.1
45.0
5.1
37
8.0
7.9
9.3
15
3.4
29
3
3
8
9
53
20.2
30.4
3.6
40
69
4
1
13
7.5
NT
cisapridec
0.012-
NT
0
.021
etoposide
NT
0.3
NT
5.1
NT
63.9 (n=6)
12.3 (n=6)
ciprofloxacin NT
a
b
Determined using IonWorks Barracuda (Charles River, Cleveland, OH). Percent inhibition
c
calculated as 100 minus percent activity (Inspiralis, Ltd., Norwich, UK). Cisapride serves as the
positive control for the hERG study.
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Three approaches were taken to mitigate this issue: 1) use of a more polar DNA-binding
moiety, 2) replacement of the amine by an amide, and 3) removal of the secondary amine. In the
4
8
first instance, four N-linked quinoxalinone analogs (43-46, Table 7) were synthesized (see
supporting information). Unfortunately, these compounds displayed inferior antibacterial
activity. Compound 43 demonstrated reduced DNA gyrase inhibition (IC50 = 2.0 M), no
meaningful TopoIV inhibition (IC50 > 100 M), and no inhibition of human topoisomerase II
0
1
2
3
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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
(2.5% inhibition at 100 M). Moreover, compound 43 showed a hERG IC50 = 6.6 M, i.e. no
improvement as compared to earlier compounds. Three N-linked analogs containing the recently
4
9
reported isoxazoloquinolinone DNA-binding moiety (47-49) were also synthesized (see
supporting information). Compounds 47 and 48 had sub-micromolar gyrase IC50 values and
MICs of 1-2 g/mL against S. aureus ATCC 29213 and MRSA. Limited inhibition of human
topoisomerase II was demonstrated at 100 M (16% for 47 and 38% for 48). Compounds 47
and 48 also afforded modestly improved hERG profiles, but solubility limitations precluded
determination of an IC50 in both cases. Additionally, compounds 47 and 48 did not inhibit
TopoIV at concentrations up to 200 M (Table 7).
Limited literature precedent supports the replacement of the NBTI secondary amine by a
secondary amide.35,50-52 Additionally, the replacement of the basic nitrogen would be expected to
confer diminished hERG inhibition. Compounds 50 and 51 (Table 7) were synthesized to test
this hypothesis. Both analogs, especially 51, displayed potent MICs against S. aureus ATCC
2
9213 and MRSA, and the enhanced antibacterial activity of 51 was mirrored by more potent
gyrase inhibition as compared to 50 (IC50 of 0.59 and 3.7 M, respectively). Whereas compound
0 showed essentially no hERG inhibition (IC50 > 100 M), compound 51 was a surprisingly
5
potent inhibitor, with an IC50 = 3.2 M. The precise origin of this significantly different behavior
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is not understood at the present time, although it is worth noting that 51 is substantially more
lipophilic than 50. Neither compound inhibited human topoisomerase II by >3% at 100 M.
Additional research directed at this intriguing series of amide analogs is underway and will be
reported in due course.
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
In the third strategy, we sought to remove the secondary amine entirely, replacing it with
a methylene unit. Compound 52 (Table 7) was synthesized as a roughly 3:1 mixture of cis:trans
isomers (see supporting information). In contrast to the analogous amine derivative (3, Table 1),
5
2 was wholly devoid of antibacterial activity. Consequently, it was neither separated nor
assayed for hERG inhibition. These results are consistent with the accepted NBTI binding mode
with S. aureus DNA gyrase, in which an amine or secondary amide interact with aspartate
3.13,26,27
8
Table 7. NBTIs designed for reduced hERG inhibition with associated assay data.
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9
1
1
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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
Cmpd.
S. aureus
MSSA)
S. aureus
hERG
S. aureus
DNA
S. aureus
TopoIV
(
(MRSA)
USA 300
MIC
IC50
c
e
ATCC 29213
(M)
gyrase
IC (M)
5
0
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
MIC
IC (M)
5
0
a,b
a,b
(
g/mL)
(g/mL)
4-32
8-32
8-16
4-16
1
32
6.6
2.0
NT
NT
NT
>100f
NT
43
4
4
4
5
32
NT
NT
NT
>10
>30
NT
>100
3.2
32
NT
46
>64
1
NT
4
4
4
5
5
5
7
8
9
0
1
2
0.32
0.83
NT
>200
>200
NT
1
2
4
2
1
1
3.7
>200
>200
NT
0.125
>64
0.25-1
0.125
NT
0.5
16-32
0.5-2
NT
0.59
NT
NT
5.1
125
0.10
13.3
NT
14
f
e
ciprofloxacin 0.125-0.5
vancomycin NT
NT
NT
NT
4.0, 8.6
NT
NT
cisaprideg
0.012-0.021
NT
a
34
Assays were conducted according to CLSI broth microdilution guidelines (n=3, minimum).
b
c
Ranges of observed values provided where appropriate. Determined using IonWorks Barracuda
d
(
Charles River, Cleveland, OH). Determined at Inspiralis (Norwich, UK) (n=2, minimum).
e
f
Method B (see Experimental Section, n=1). Method A (see Experimental Section), determined
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g
at Inspiralis (Norwich, UK) (n=2, minimum). Cisapride serves as the positive control for the
hERG study.
CONCLUSION
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
In summary, we have synthesized and further investigated a diverse series of dioxane-
linked NBTIs using a variety of biochemical, microbiological, and safety assays. Future studies
will be necessary to characterize other important drug-like properties such as metabolic stability.
Secondary amine derivatives bearing a monocyclic enzyme-binding moiety with a lipophilic
para substituent possessed potent anti-staphylococcal activity. Incorporation of a fluorinated
naphthyridine DNA-binding motif robustly enhanced the antibacterial activity and TopoIV
inhibition of compounds 37-41 with minimal impact on the hERG profile. In contrast,
quinoxalinone and isoxazoloquinolinone derivatives did not positively differentiate from the
quinoline series. It was found that hERG inhibition could be substantially reduced without loss
of antibacterial activity in amide analog 50. Dioxane-linked NBTIs thus offer promise for
further optimization into novel therapeutics for treating antibiotic-resistant infections.
EXPERIMENTAL SECTION
General Chemistry Information. Moisture and/or air-sensitive reactions were conducted in
oven-dried glassware under an atmosphere of nitrogen or argon unless otherwise noted.
Dichloromethane (DCM), toluene, tetrahydrofuran (THF), and N,N-dimethylformamide (DMF)
were dried before use by passage through activated alumina under nitrogen. Flash
+
1
chromatography was performed using a Teledyne-ISCO Combiflash-Rf . H NMR spectra were
recorded at either 300 MHz or 400 MHz using residual protiated solvent as the internal
reference: CDCl
3
(7.26 ppm), CD
3
OD (3.31), DMSO-d (2.50 ppm). Assayed compounds had a
6
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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
13
purity of >90% as determined by H NMR analysis. C NMR spectra were recorded at 75 MHz
or 100 MHz using the solvent signal as the internal reference: CDCl
(49.00), DMSO-d (39.52 ppm). High resolution mass spectrometry was performed using
electrospray ionization. The equatorial protons at C and C of the trans-dioxane ring typically
3
(77.16 ppm), CD
3
OD
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
4
6
appear as an apparent doublet of doublets at ca. 4.1-4.2 ppm in the analogs described below.53
Careful inspection in some cases reveals additional partially resolved splitting, arising from W-
coupling of these magnetically non-equivalent protons as well as virtual coupling. The additional
splitting can be effectively replicated using computer simulation of non-first order effects,54
using a W-coupling constant of 1.8 Hz. For the sake of clarity and consistency, this peak is
labeled a doublet of doublets in the characterization data provided below.
Synthesis and Characterization of Compounds 37-42 (Scheme 1).
8
-bromo-7-fluoro-2-methoxy-1,5-naphthyridine (33). To a solution of commercial 3-fluoro-6-
methoxy-1,5-naphthyridin-4-ol (32, 0.8625 g, 4.442 mmol, 1.0 equiv) in DMF (10 mL) was
dropped phosphorus tribromide (466 μL, 5.0 mmol) at 0 °C under N atmosphere. The reaction
mixture was stirred for 1 hour, then water (200 mL) and aqueous sodium hydroxide solution (6N,
2
7
40 μL) were added. The reaction mixture was stirred for 2 hours, and the precipitated solid was
1
collected by filtration to afford the title compound as a tan solid (1.03 g, 90% yield). H NMR
(
300 MHz, CDCl ) δ 8.62 (s, 1H); 8.21 (d, J = 9.0 Hz, 1H); 7.13 (d, J = 9.0 Hz, 1H); 4.17 (s,
3
3
H).
-(2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-yl)isoindoline-1,3-
2
2
5
dione (35). To a solution of 2-(2-vinyl-trans-1,3-dioxan-5-yl)isoindoline-1,3-dione (34) (155.6
mg, 0.6002 mmol, 1.2 equiv relative to 33) in THF (3 mL) was added dropwise 9-
borabicyclo[3.3.1]nonane solution (1.2 mL, 0.5 M in THF, 0.6 mmol, 1.0 equiv relative to 34) at
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room temperature under N
into next step.
2
atmosphere. The mixture was stirred for 3 hours and used directly
To a mixture of 33 (128.5 mg, 0.5000 mmol, 1.0 equiv), cesium carbonate (325.8 mg, 1.000
mmol), and [1,1`-bis(diphenylphosphino)ferrocene] dichloropalladium(II) (13.4 mg, 0.0183
mmol, 0.04 equiv) in THF (3 mL) was added dropwise the fresh solution described above at
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
room temperature under N atmosphere. The reaction mixture was stirred overnight. The solvent
2
was then removed in vacuo, and the residue was dissolved in DCM and washed with brine. The
organic layer was concentrated and purified by chromatography on silica gel with hexane/ethyl
1
acetate (3:1) to give the title compound as a white solid (126.4 mg, 58% yield). H NMR (300
MHz, CDCl
3
) δ 8.61 (s, 1H); 8.16 (d, J = 9.0 Hz, 1H); 7.84-7.80 (m, 2H); 7.77-7.68 (m, 2H);
7.06 (d, J = 9.0 Hz, 1H); 4.73 (t, J = 5.0 Hz, 1H); 4.69-4.54 (m, 1H); 4.39 (t, J = 10.6 Hz, 2H);
4
2
.11 (s, 3H); 4.02 (dd, J = 10.7, 4.8 Hz, 2H); 3.32 (t, J = 7.3 Hz, 2H); 2.13 (td, J = 7.7, 5.2 Hz,
H).
2
-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (36). A mixture
of 35 (218.7 mg, 0.5000 mmol, 1.0 equiv), ethanolamine (46 μL, 7.6 mmol, 1.5 equiv) and ethyl
acetate (4 mL) was stirred and heated at 70 °C overnight. The solvent was removed, and the
residue was dissolved in DCM and washed with brine. The organic layer concentrated, and the
crude product was purified by chromatography on silica gel with DCM/methanol (15:1) to give
1
the title compound as an oil (123.1 mg, 80% yield). H NMR (CDCl
3
) δ 8.58 (s, 1H); 8.14 (d, J =
9
4
.0 Hz, 1H); 7.03 (d, J = 9.0 Hz, 1H); 4.41 (t, J = 5.1 Hz, 1H); 4.14-4.06 (m, 5H); 3.26-3.14 (m,
H); 3.08-3.00 (m, 1H); 2.09-1.97 (m, 2H); 1.09 (br s, 2H).
Reductive amination general procedure: To a solution of amine 36 (0.2 mmol) in methanol (2
mL) was added the requisite aldehyde (0.2 mmol) and zinc chloride (2 mg, 0.015 mmol, 0.07
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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
equiv). The mixture was stirred at room temperature for 30 min, followed by addition of sodium
cyanoborohydride (40 mg, 0.6 mmol, 3.0 equiv). The reaction mixture was stirred at room
temperature overnight and then purified by chromatography on silica gel with DCM/methanol
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
(50:1). On occasion, the product obtained from flash chromatography was contaminated by a
1
BH
3
adduct, seen in the H NMR spectrum as four broad peaks from 0.00-0.88 ppm. In these
instances, the column-purified material was dissolved in methanol (2 mL) and stirred overnight
at ambient temperature with Amberlite IRA743 free base (ca. 100 mg). The pure title compound
was then obtained by removal of the resin by filtration and removal of the solvent under reduced
pressure.
N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-
yl)ethyl)-trans-1,3-dioxan-5-amine (37). The title compound was prepared in 52% yield
1
following the general method and obtained as a white solid. H NMR (300 MHz, CDCl
3
) δ 8.59
(s, 1H); 8.15(d, J=9.0 Hz, 1H); 7.04 (d, J=9.0 Hz, 1H); 6.71-6.81 (m, 3H); 4.44 (t, J=5.0 Hz,
1
H); 4.23 (s, 4H); 4.15 (dd, J=11.1, 4.7 Hz, 2H); 4.07 (s, 3H); 3.67 (s, 2H); 3.21-3.27 (m, 4H);
1
3
2.90-2.95 (m, 1H); 2.05 (td, J=7.6, 5.2 Hz, 2H);. C NMR (75 MHz, CDCl
3
) δ 162.3, 158.8,
1
1
55.4, 143.5, 142.7, 141.6, 141.5, 140.1, 138.48, 138.45, 138.1, 137.7, 133.5, 131.8, 131.6,
20.9, 117.2, 116.8, 115.11, 115.07, 101.6, 71.6, 64.4, 64.3, 53.8, 50.8, 49.6, 33.5, 18.3. HRMS
+
(ESI) m/z calc’d for C24
H27FN
3
O
5
[M+H] : 456.1934; found: 456.1910.
N-(3,4-dichlorobenzyl)-2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-
5-amine (38). The title compound was prepared in 45% yield following the general method and
1
obtained as a white solid. H NMR (300 MHz, CDCl
3
) δ 8.60 (s, 1H); 8.16 (d, J=9.0 Hz, 1H);
7
.37-7.42 (m, 2H); 7.13 (dd, J=8.2, 1.8 Hz, 1H); 7.06 (d, J=9.0 Hz, 1H); 4.45 (t, J=5.0 Hz, 1H);
4.16 (dd, J=11.2, 4.7 Hz, 2H); 4.08 (s, 3H); 3.76 (s, 2H) 3.24-3.35 (m, 4H); 2.88-2.95 (m, 1H);
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2
.07 (td, J=7.6, 5.3 Hz, 2H);. C NMR (75 MHz, CDCl
3
) δ 162.3, 158.8, 155.4, 141.6,141.5,
1
40.5, 140.1, 138.51, 138.48, 138.1, 137.7, 132.6, 131.7, 131.5, 131.1, 130.4, 129.8, 127.2, 115.14,
115.10, 101.6, 71.4, 53.8, 50.1, 49.9, 33.4, 18.3. HRMS (ESI) m/z calc’d for C22
H23Cl
2
FN
3
O
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
[
M+H] : 466.1100; found: 466.1088.
N-((3,4-dihydro-2H-pyrano[2,3-c]pyridin-6-yl)methyl)-2-(2-(3-fluoro-6-methoxy-1,5-
naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (39). The title compound was prepared in
1
7
7% yield following the general method and obtained as a thin film. H NMR (300 MHz,CDCl
3
)
δ 8.58 (s, 1H); 8.14 (d, J=9.0 Hz, 1H); 8.05 (s, 1H); 7.04 (d, J=9.0 Hz, 1H); 6.95 (s, 1H); 4.47 (t,
J=5.0 Hz, 1H); 4.22-4.15 (m, 4H); 4.07 (s, 3H); 3.81 (s, 2H); 3.37 (t, J=10.8 Hz, 2H); 3.26 (t,
1
3
J=7.6 Hz, 2H); 3.02-2.95 (m, 1H); 2.76 (t, J=6.5 Hz, 2H); 2.08-2.00 (m, 4H). C NMR (75
MHz, CDCl
3
) δ 162.3, 158.8, 155.37, 151.35, 148.9, 141.6, 141.5, 140.1, 138.48, 138.45, 138.1,
1
1
37.7, 131.7, 131.6, 122.9, 115.14, 115.10, 101.6, 70.9, 66.6, 53.8, 51.4, 50.0, 33.4, 24.3, 21.5,
+
8.3. HRMS (ESI) m/z calc’d for C24
H27FN
4
NaO [M+Na] : 477.1914; found: 477.1895.
4
N-((2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methyl)-2-(2-(3-fluoro-6-methoxy-1,5-
naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (40). The title compound was prepared in
1
4
3% yield following the general method and obtained as a thin film. H NMR (300 MHz, CDCl
3
)
δ 8.58 (s, 1H); 8.14 (d, J=9.0 Hz, 1H); 8.08 (s, 1H); 7.04 (d, J=9.0 Hz, 1H); 6.75 (s, 1H); 4.44 (t,
J=5.0 Hz, 1H); 4.33-4.23 (m, 4H); 4.15 (dd, J=11.2, 4.7 Hz, 2H); 4.06 (s, 3H); 3.75 (s, 2H);
13
3
.35-3.22 (m, 4H); 3.00-2.86 (m, 1H); 2.04 (td, J=7.6, 5.2 Hz, 2H). C NMR (75 MHz, CDCl
3
)
δ 162.3, 158.8, 155.4, 152.9, 150.2, 141.6, 141.5, 140.2, 140.1, 138.9, 138.46, 138.44, 138.1,
137.7, 131.8, 131.6, 115.12, 115.08, 110.6, 101.6, 71.5, 65.0, 64.0, 53.8, 52.0, 49.9, 33.5, 18.3.
+
HRMS (ESI) m/z calc’d for C23
H
26FN
4
O
5
[M+H] : 457.1887; found: 457.1871.
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N-((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl)methyl)-2-(2-(3-fluoro-6-methoxy-1,5-
naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (41). The title compound was prepared in 58
1
% yield following the general method and obtained as light yellow solid. H NMR (300 MHz,
0
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0
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CDCl
3
) δ 8.58 (s, 1H); 8.14 (d, J=9.0 Hz, 1H); 7.27 (s, 1H); 7.04 (d, J=9.0 Hz, 1H); 4.66-4.61
(m, 2H); 4.46 (t, J=4.8 Hz, 1H); 4.20 (dd, J=11.1, 4.7 Hz, 2H); 4.07 (s, 3H); 3.96 (s, 2H); 3.38-
1
3
3
.22 (m, 4H); 3.22-3.17 (m, 2H); 3.03-2.90 (m, 1H); 2.07-2.01 (m, 2H). C NMR (75 MHz,
) δ 162.4, 160.1, 158.9, 156.0, 155.5, 141.7, 141.6, 140.2, 138.61, 138.59, 138.2, 137.8,
31.8, 131.7, 126.0, 125.2, 115.25, 115.22, 101.7, 71.2, 66.3, 53.9, 50.12, 50.09, 33.5, 25.7, 18.4.
CDCl
3
1
+
HRMS (ESI) m/z calc’d for C22
H25FN
5
O S [M+H] : 474.1611; found: 474.1614.
4
N-(3,4-difluorobenzyl)-2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-
5-amine (42). The title compound was prepared in 43% yield following the general method and
1
obtained as a white solid. H NMR (400 MHz, CDCl
3
) δ 8.59 (s, 1H); 8.15 (d, J=9.0 Hz, 1H);
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.18-7.03 (m, 3H); 7.02-6.97 (m, 1H); 4.44 (t, J=5.1 Hz, 1H); 4.18-4.13 (m, 2H); 4.07 (s, 3H);
1
3
3.75 (s, 2H); 3.30-3.22(m, 4H); 2.97-2.89 (m, 1H); 2.11-2.02 (m, 2H). C NMR (100 MHz,
CDCl ) δ 162.3, 158.4, 155.8, 151.7, 151.5, 150.8, 150.7, 149.2, 149.1, 148.4, 148.2, 141.6,
41.5, 140.1, 138.49, 138.47, 138.0, 137.7, 137.32, 137.27, 137.2, 131.7, 131.6, 123.63, 123.60,
3
1
1
23.57, 123.5, 117.2, 117.0, 116.6, 115.2, 115.1, 101.6, 71.5, 53.8, 50.3, 49.8, 33.4, 18.3. HRMS
+
(ESI) m/z calc’d for C22
H
23
F
3
N
3
O
3
[M+H] : 434.1691; found: 434.1682.
The synthesis and characterization of novel compounds 3-6, 8-30, and 43-52 is described in the
supporting information.
Determination of Minimum Inhibitory Concentrations (MICs). MICs against the strains
listed in Tables 1, 2, 5, and 7 were determined by broth microdilution according to the guidelines
established by the Clinical and Laboratory Standards Institute.34
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ACS Infectious Diseases
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Frequency of Spontaneous Resistance Studies. In order to determine the spontaneous mutation
frequency of investigational agents, the MIC was first determined using the agar dilution method
as recommended by the Clinical and Laboratory Standards Institute.34,42 The test agents and
ciprofloxacin were assayed using a drug concentration range of 8 – 0.008 μg/mL. Triplicate
independent inocula of S. aureus ATCC 29213 were evaluated.
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Serial dilutions of the experimental compounds were made in DMSO; ciprofloxacin was made in
water. Agar plates were prepared by mixing 1.25 mL of concentrated drug (40X the agar dilution
MIC) with 48.75 mL of molten (52°C) Mueller-Hinton Agar and pouring into 15-by-150 mm
petri dishes, followed by allowing the plates to solidify and dry at room temperature prior to
inoculation. Duplicate plates were prepared to contain the agents at 4X and 8X the agar dilution
MIC. In addition, a single 4X MIC and 8X MIC plate (standard small petri plates) was prepared
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and inoculated according to CLSI for the agar dilution assay. This was to confirm that the drug
content of the plates was above the MIC, validating that the stock solution of drug(s) was made
properly.
The inoculum for the assay was prepared by streaking S. aureus ATCC 29213 onto
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TSAB plates and growing for approximately 20 hr at 35°C. Using a sterile swab, several well-
isolated colonies were removed and resuspended in sterile saline at a heavy concentration
equivalent to 1.6 on the turbidimeter. This suspension was diluted 1:10 in order to achieve a
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target inoculum of 10 CFU on each spontaneous mutation plate. Each plate was inoculated with
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.2 mL of the cell suspension, resulting in each drug being tested at 4X and 8X the MIC in
duplicate. In addition, a portion of the inoculum was enumerated by making dilutions and
spreading onto TSA, followed by counting colonies after a 24 hr incubation at 35°C.
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