Discovery of Novel Pyridone-Conjugated Monosulfactams as Potent and Broad-Spectrum Antibiotics for Multidrug-Resistant Gram-Negative Infections

Article abstract of DOI:10.1021/acs.jmedchem.6b01261

Conjugating a siderophore to an antibiotic is a promising strategy to overcome the permeability-mediated resistance of Gram-negative pathogens. On the basis of the structure of BAL30072, novel pyridone-conjugated monosulfactams incorporating diverse substituents into the methylene linker between the 1,3-dihydroxypyridin-4(1H)-one and the aminothiazole oxime were designed and synthesized. Structure-activity relationship studies revealed that a variety of substituents were tolerated, with isopropyl (compound 12c) and methylthiomethyl (compound 16a) showing the best efficacy against multidrug-resistant (MDR) Gram-negative pathogens. In addition, compound 12c exhibits a good free fraction rate in an in vitro human plasma protein binding test, along with a low clearance and favorable plasma exposure in vivo. In a murine systemic infection model with MDR Klebsiella pneumoniae, compound 12c shows an ED₅₀ of 10.20 mg/kg. Taken together, the results indicate that compound 12c is a promising drug candidate for the treatment of serious infections caused by MDR Gram-negative pathogens.

Full text of DOI:10.1021/acs.jmedchem.6b01261

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Article
Discovery of Novel Pyridone-Conjugated Monosulfactams as Potent and
Broad-Spectrum Antibiotics for Multidrug-Resistant Gram-Negative Infections
Liang Tan, Yunliang Tao, Ting Wang, Feng Zou, Shuhua Zhang, Qunhuan Kou,
Ao Niu, Qian Chen, Wenjing Chu, Xiaoyan Chen, Haidong Wang, and Yushe Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01261 • Publication Date (Web): 13 Mar 2017
Downloaded from http://pubs.acs.org on March 14, 2017
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Discovery of Novel PyridoneꢀConjugated
Monosulfactams as Potent and BroadꢀSpectrum
Antibiotics for MultidrugꢀResistant GramꢀNegative
Infections
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†,‡
§
#
#
#
†,‡
Liang Tan, Yunliang Tao, Ting Wang, Feng Zou, Shuhua Zhang, Qunhuan Kou,
Ao
||
†,‡
†,‡
†,‡
,§
,†,‡
Niu, Qian Chen, Wenjing Chu,
Xiaoyan Chen, Haidong Wang, * and Yushe Yang*
†
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203, China.
‡
University of Chinese Academy of Sciences, Beijing 100049, China.
§
College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing, Zhejiang
Province 314001, China.
#
Department of Microbiology, Sichuan Primed BioꢀTech Group Co., Ltd, Chengdu, Sichuan
Province 610041, China.
||
Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing,
Jiangsu Province 210009, China.
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ABSTRACT: Conjugating a siderophore to an antibiotic is a promising strategy to overcome the
permeabilityꢀmediated resistance of Gramꢀnegative pathogens. Based on the structure of
BAL30072, novel pyridoneꢀconjugated monosulfactams incorporating diverse substituents into
the methylene linker between the 1,3ꢀdihydroxypyridinꢀ4(1H)ꢀone and the aminothiazole oxime
were designed and synthesized. Structure–activity relationship studies revealed that a variety of
substituents were tolerated, with isopropyl (compound 12c) and methylthiomethyl (compound
0
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16a) showing the best efficacy against multidrugꢀresistant (MDR) Gramꢀnegative pathogens. In
addition, compound 12c exhibits a good free fraction rate in an in vitro human plasma protein
binding test, along with a low clearance and favorable plasma exposure in vivo. In a murine
systemic infection model with MDR Klebsiella pneumoniae, compound 12c shows an ED of
50
10.20 mg/kg. Taken together, the results indicate that compound 12c is a promising drug candidate
for the treatment of serious infections caused by MDR Gramꢀnegative pathogens.
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ꢀ
INTRODUCTION
Infections caused by multidrugꢀresistant (MDR) bacteria, particularly by Gramꢀnegative
pathogens, represent a serious and growing threat to human health. The “ESKAPE” pathogens,
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such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas
1, 2
aeruginosa, are emerging as threatening pathogens.
In particular, the carbapenemꢀresistant
Enterobacteriaceae (CRE, mostly contributed by K. pneumoniae) represent the most worrisome
antibiotic resistance crisis, which typically exhibit extremely drugꢀresistant (XDR) phenotypes,
2
and CRE infections are associated with high mortality rates. Despite the dramatic increase
prevalence of highly resistant Gramꢀnegative pathogens, there are few treatment options for
related infections, besides polymyxins, an old antibiotic class with serious nephrotoxicity
2
, 3
issues.
Furthermore, the number of new Gramꢀnegative antibiotic candidates under
development is limited and these candidates are not capable of providing coverage against all of
4, 5
the four serious pathogens described above.
broadꢀspectrum Gramꢀnegative antibiotics.
Therefore, there is an urgent need for new and
The reduction of outer membrane permeability represents a major mechanism of resistance of
6ꢀ8
Gramꢀnegative pathogens, and one strategy to circumvent the penetration issue is to exploit the
9
iron uptake pathway of Gramꢀnegative pathogens by conjugating a siderophore to an antibiotic.
Siderophores are small ironꢀchelating molecules secreted by bacteria to acquire iron from the
1
0, 11
environment.
In response to icon deficiency, siderophores are excreted and sequester iron
(III), the resulting complexes are recognized by specific outer membrane receptors and
translocated into the microbial cell. A plenty of studies have been reported in the past in
1
2ꢀ18
understanding the iron binding and recognition processes mentioned above.
This
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siderophoreꢀmediated iron uptake mechanism is very efficient, microbially selective and
essential for bacteria, making it an important target to circumvent the permeabilityꢀmediated
9
, 19
resistance of Gramꢀnegative pathogens through a “Trojan Horse” approach.
βꢀlactam
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antibiotics target the penicillinꢀbinding proteins (PBPs), which are located in the periplasm, thus
only the outer membrane has to be crossed by βꢀlactam antibiotics for these compounds to be
effective antibacterial agents. Therefore, siderophoreꢀconjugated βꢀlactam antibiotics comply with
9
the “Trojan Horse” strategy perfectly. Natural siderophores most often contain three bidentate
1
1
ligands for stoichiometric binding of iron (III) . In order to reduce this inherent complexity, a
large number of simplified conjugates, which incorporate a single bidentate ligand such as a
2
0ꢀ22
catechol or a hydroxypyridone, have been studied since 1980s.
One precedent example is
2
3
compound 1 (Sꢀ649266, Figure 1), a catecholꢀconjugated cephalosporin, which has progressed
24, 25
into phase II/III clinical trials.
Among all βꢀlactam antibiotics, the monocyclic βꢀlactam subclass is supposedly a preferred
drug moiety to conjugate to a siderophore because of their potent antimicrobial activity against
Gramꢀnegative bacteria, as well as their resistance to βꢀlactamases, including the
2
6
metalloꢀβꢀlactamases. Structures of representative pyridoneꢀconjugated monocyclic βꢀlactams
2
7
28
29
are shown in Figure 1. Compounds such as 2 (Uꢀ78608), 3 (MCꢀ1) and 4 are potent
3
0
monocarbams against P. aeruginosa and resistant to βꢀlactamases. However, the therapeutic
potential of these compounds might be limited by their poor hydrolytic stability and high plasma
28
protein binding (PPB). Monobactam 5 (MBꢀ1) demonstrates excellent in vitro activity against P.
aeruginosa along with an improved PPB profile; however, it was reported to be ineffective in
31, 32
vivo.
None of the these compounds have progressed into clinical trials. Compound 6
33
(
BAL30072, Figure 2), a monosulfactam discovered by Basilea Pharmaceutica, has ever been
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investigated in phase I clinical studies. It exhibits potent activity against MDR P. aeruginosa and
3
5
A. baumannii, including carbapenemꢀresistant strains. In a rat soft tissue infection model,
36
compound 6 was active against most of tested A. baumannii strains. However, it was reported to
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be ineffective against some Enterobacteriaceae, especially for K. pneumoniae,
limit its application in the treatment of associated infections.
which would
Figure 1. Compound 1 and pyridoneꢀconjugated monocyclic βꢀlactams 2–5.
Figure 2. Design of new compounds based on compound 6.
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Discovered from products of saprophytic bacteria in 1979, monobactam is a new class of
3
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βꢀlactams characterized by 2ꢀoxoazetidineꢀ1ꢀsulfonic acid moiety.
Initially, extensive
structural modifications around the Cꢀ3 amide side chain and Cꢀ4 positions of the azetidinone
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ring led to the discovery of aztreonam. Subsequently, other acidic and electronegative groups
such as phosphonate and Oꢀsulfate groups were investigated for their activating potential at the
4
1ꢀ43
Nꢀ1 position.
As shown in Figure 2, compound 7 (tigemonam) is a monosulfactam activated
4
4
by an Oꢀsulfate group, and it exhibits high level of oral absorption in laboratory animals.
Compound 6 was derived from 7 through conjugating 1,3ꢀdihydroxypyridinꢀ4(1H)ꢀone to the
33
aminothiazole oxime side chain. To date, there has been limited structure–activity relationships
3
3, 45
(
SAR) generated on 6.
The clinical potential of 6, along with its drawbacks, which have been
discussed above, encourages further optimization of its structure, which we anticipate would
provide opportunities to discover better drug candidates. In this article, we report our recent
efforts on this topic, which include the following two parts: (1) siderophore mimics other than
1
,3ꢀdihydroxypyridinꢀ4(1H)ꢀone were explored to optimize that part of the molecule; and (2) the
linker connecting the pyridone moiety and the βꢀlactam core, which has been reported to influence
significantly the antibacterial activity and the physicochemical properties of
2
8ꢀ31, 46
pyridoneꢀconjugates,
was extensively studied to determine the SAR. As a result, a number
of novel compounds with excellent and broadꢀspectrum antibacterial activity have been
discovered, including a preclinical candidate 12c, which qualifies for further evaluation.
ꢀ RESULTS AND DISCUSSION
Structure–Activity Relationship of monosulfactams. To better correlate the in vitro activity
of the new compounds with their future clinical application, the tested bacterial strains were all
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clinical isolates. These strains include extended spectrum βꢀlactamases (ESBLs) producing E.
coli, Klebsiella pneumoniae carbapenemase (KPCꢀ2) producing MDR K. pneumoniae,
oxacillinase (OXAꢀ23) producing MDR A. baumannii, and Imipenemase (IMPꢀ4) producing
MDR P. aeruginosa, with five strains in each family, respectively. All new compounds were
phenotypically screened against these strains and their activities are presented as minimum
inhibitory concentrations (MICs). The results are shown in Table 1–3 as ranges of MIC values and
the details can be found in Supporting Information.
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Bacteria express a wide variety of siderophores with different structural types, thus
9
hydroxamic acid as a common structural type of siderophore was introduced initially to the
aminothiazole oxime of 6 to replace the 1,3ꢀdihydroxypyridinꢀ4(1H)ꢀone, affording the
hydroxamate analogue 8. As shown in Table 1, compound 8 showed moderate activity against E.
coli but a significant loss of activity against A. baumannii and P. aeruginosa when compared with
that of 6. Further modifications exemplified by the replacement of the 1,3ꢀdihydroxypyridinꢀ
4(1H)ꢀone with other plausible ironꢀchelating groups provided analogues 9, 10 and 11; however,
all three compounds also displayed marked loss of activity against A. baumannii and P.
3
1,
33, 46
aeruginosa (Table 1). Together with previous results,
we considered
1,3ꢀdihydroxypyridinꢀ4(1H)ꢀone as the optimal siderophore mimic for monocyclic βꢀlactams.
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Table 1. Antibacterial Activity of Monosulfactams 8–11
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a
MIC (ꢁg/mL)
Compd
Eco
4−32
Kpn
32−>64
4−>64
16−64
8−64
Aba
16−64
4−64
Pae
≥64
8
9
0.25−4
1−8
64
1
1
0
1
16−64
8−64
16−64
64
1−>64
0.25−1
0.5−32
0.125−8
0.5−2
6
0.25−>64
>64
0.5−>64
≥64
1−4
aztronam
meropenem
4−64
32−64
>64
32−>64
32−>64
64
ceftizoxime sodium
32−>64
a
Eco: E. coli, 5 strains; Kpn: MDR K. pneumoniae, 5 strains; Aba: MDR A. baumannii, 5 strains;
Pae: MDR P. aeruginosa, 5 strains.
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We next turned our attention to the modification of the methylene linker between the
aminothiazole oxime and the 1,3ꢀdihydroxypyridinꢀ4(1H)ꢀone. The gemꢀdimethyl group is a
common structural feature for βꢀlactam drugs such as aztronam and ceftazidime, and it has been
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reported to play an important role in drugꢀPBP3 interactions. However, all of our attempts to
introduce a gemꢀdimethyl group to the linker failed to afford any compound. Thus, compounds
containing a monosubstitution on the linker were designed and synthesized. As shown in Table 2,
all the alkyl substituent analogues 12a–e displayed improved potency against Enterobacteriaceae
strains and equivalent activity against A. baumannii and P. aeruginosa strains compared with that
of 6. Especially, the isopropyl analogue 12c showed the most excellent antibacterial activity, with
a >100ꢀfold boost in activity against K. pneumoniae compared with that of 6. Aromatic
substituents such as the phenyl analogue 13a and heterocyclic aryl analogues (13b and 13c) also
showed excellent antibacterial activity, albeit moderate activity against K. pneumoniae was
4
8
observed. Since free drug exposure is a key factor for the efficacy of βꢀlactams, the PPB profile
was chosen as an important parameter to enable compound selection for advanced studies. As
shown in Table 2, all tested alkyl analogues (12a–d) showed decreased free fractions relative to 6
in the human PPB test, likely because of the increase in lipophilicity of these compounds.
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Table 2. Antibacterial Activity and Physiochemical Properties of Monosulfactams 12–16
0
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a
c
MIC (ꢁg/mL)
Human
Stability
Compd
b
Eco
Kpn
Aba
Pae
PPB (fu)
(degradation)
1
2a
2b
2c
12d
2e
13a
3b
13c
4a
4b
4c
5a
5b
5c
16a
6b
0.06−0.25
0.25−2
0.03−64
0.5−64
0.25−2
0.25−8
0.125−4
0.25−1
0.25−4
0.5−2
0.5−2
0.5−2
1−2
0.41 ± 0.03
0.35 ± 0.02
0.35 ± 0.06
0.39 ± 0.03
24%
27%
29%
24%
NT
1
0.03−>64
1
<0.03−0.25
0.06−4
<0.03−0.5 0.125−1
0.06−64 0.5−2
d
1
<0.03−0.125 <0.03−>64 0.25−2
NT
<0.03−0.06
<0.03−0.25
<0.03−8
0.06−16
0.5−2
0.5−2
0.5−2
0.5
NT
NT
NT
NT
1
NT
<0.03−0.125 <0.03−8
NT
e
1
0.125−4
0.25−1
1−4
ND
ND
1
0.25−1
0.25−2
0.5−1
0.25−1
0.5−2
0.125−1
1−8
1−4
0.61 ± 0.03
0.69 ± 0.05
0.59 ± 0.10
0.67 ± 0.03
NT
66%
36%
50%
57%
NT
1
0.06−4
0.25−2
4
1
0.06−4
0.25−>64
0.06−2
0.25−16
1−4
1
<0.03−0.25
<0.03−0.25
<0.03−0.25
1
0.25−>64
<0.03−1
0.5−8
0.125−8
0.25−4
1−32
1−4
0.125−1
0.25−2
0.25−2
0.5−>64
0.32 ± 0.01
0.21 ± 0.01
0.64 ± 0.05
0.47 ± 0.02
28%
34%
34%
12%
1
<0.03−0.125 0.06−1
16c
6
<0.03−2
0.25−1
0.06−2
0.25−>64
a
Eco: E. coli, 5 strains; Kpn: MDR K. pneumoniae, 5 strains; Aba: MDR A. baumannii, 5 strains;
b
Pae: MDR P. aeruginosa, 5 strains; n = 4, data represent as mean ± standard deviation; Fu,
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c
fraction unbound; stability: the degradation percentage after 4 h incubation in human plasma at
d
e
3
7 °C; NT: not tested; ND: not determined.
To further explore the SAR of this series of compounds and increase the free drug fraction in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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26
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plasma, several polar functionalities were introduced. As shown in Table 2, all the carboxylic acid
derivatives 14a–c displayed wellꢀbalanced antibacterial activity against all the tested
microorganisms. Hydroxymethyl analogue 15a exhibited good antibacterial activity across all
strains except K. pneumoniae, and methylation of the hydroxymethyl group of 15a to
methoxymethyl (15b) resulted in a boost in potency against K. pneumoniae. However, further
increasing the size of the substituent by replacing the methoxymethyl with isopropoxymethyl
resulted in substantially reduced activity against K. pneumoniae (15c vs 15b). It is of interest that
introduction of a sulfur atom led to improved antibacterial activity, especially against K.
pneumonia, as shown with 16b in comparison to 15c. Oxidization of the methylthiomethyl to
sulfonyl afforded analogue 16c, which exhibited desirable activity against E. coli and K.
pneumoniae, albeit moderate activity against P. aeruginosa was observed.
In terms of the PPB profile and plasma stability, compound 14b, 14c, 15a and 15b exhibited
favorable free fractions (0.61, 0.69, 0.59 and 0.67, respectively, Table 2), however, all of them
suffer from plasma instability except for 14c. Compounds containing a sulfur atom showed higher
PPB but better plasma stability compared with their Oꢀcounterparts. For example, the
methylthiomethyl analogue 16a showed marked improvement in plasma stability compared with
that of 15b (degradation, 28% for 16a and 57% for 15b, Table 2). As expected, as the lipophilicity
of the substituent increases, the free fraction decreases (0.32 for 16a and 0.67 for 15b). The
sulfonyl compound 16c showed a greatly enhanced free fraction (0.64) compared with that of 16a.
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6
Since small changes of the aminothiazole of monocyclic βꢀlactams or cephalosporins have been
4
9,
reported to affect significantly their antibacterial activity and their physicochemical properties,
5
0
compounds 12a and 12c were further explored by modifying the thiazole moiety. As shown in
0
1
2
3
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7
8
9
0
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9
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3
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9
0
Table 3, the chlorothiazole analogues showed comparable potency to the thiazole analogues (17 vs
12a, 19 vs 12c), and it is particularly interesting that compound 17 demonstrated a marked increase
in potency against K. pneumoniae when compared with that of 12a. The thiadiazole analogues
exhibited diminished antibacterial activity compared with the thiazole analogues (18 vs 12a, 20 vs
12c). On the other hand, neither chlorothiazole analogues nor thiadiazole analogues showed
improvement in human PPB profiles. However, their stability in plasma was improved, especially
for the thiadiazole analogues, which showed comparable stability to that of 6 (degradation, 13%
for 18, 11% for 20, and 12% for 6, Table 3).
Finally, as a result of the wellꢀbalanced antibacterial activity and favorable plasma stability of
compounds 12c, 16a, 19 and 20, the influence of the stereochemistry of the methylene linker on
antibacterial activity and physicochemical properties was studied. As shown in Table 3, the
diastereomers (12c, 16a, 19 and 20) exhibit the best overall potency and spectrum, and the
preferred configuration depends on the tested panels. For example, as for E. coli, A. baumannii and
P. aeruginosa, the Sꢀisopropyl analogues (21, 23 and 25) and Rꢀmethylthiomethyl analogue 27
showed better MICs. This tendency is especially obvious for A. baumannii that the Sꢀisopropyl
analogues demonstrated significantly superior activity when compared with the Rꢀisopropyl
analogues (21 vs 22, 23 vs 24, 25 vs 26). However, in terms of K. pneumoniae, the Rꢀisopropyl
analogues (22 and 26) and Sꢀmethylthiomethyl analogue 28 displayed greater activity than the
other isomers, and a marked decrease in activity against K. pneumoniae was observed particularly
for the Rꢀmethylthiomethyl analogue 27. The reasons for this phenomenon are not clear, and it
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could be attributed to different Gramꢀnegative bacteria possessing their own unique special and
sophisticated resistance mechanisms. On the other hand, similar human PPB profiles and subtle
differences of plasma stability between diastereomers and their single isomers (12c vs 21, 19 vs 23,
10
11
12
13
14
15
16
17
18
19
20
21
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0 vs 25, 16a vs 27, Table 3) indicate that the stereochemistry of the linker has little impact on
human PPB and plasma stability.
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2
2
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3
4
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4
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5
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5
5
5
6
Table 3. Antibacterial Activity and Physiochemical Properties of Monosulfactams 17–28
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
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2
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4
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7
8
9
0
a
c
MIC (ꢁg/mL)
Human
Stability
Compd
b
Eco
Kpn
Aba
Pae
PPB (fu)
(degradation)
1
2a
0.06−0.25
<0.03−0.25
<0.03−1
<0.03−0.25
0.06−1
<0.03−64
0.06−1
0.5−64
0.25−2
2−64
0.25−8
0.5−4
0.5−64
0.25−1
0.25−8
0.5−32
0.125−1
1−4
0.41 ± 0.03
0.20 ± 0.01
0.37 ± 0.02
0.35 ± 0.06
0.23 ± 0.02
0.28 ± 0.01
0.27 ± 0.01
24%
20%
13%
29%
22%
11%
23%
NT
1
7
1
8
0.25−64
1
2c
<0.03−0.5 0.125−1
1
9
0.06−0.5
0.06−1
0.06−2
0.5−4
2
0
0.06−2
21
0.06−0.25
0.06−2
0.125−1
0.06−0.5
0.25−0.5
0.5−2
1−2
d
2
2
0.25−64
0.5−1
NT
23
0.06−1
0.06−1
0.5−4
1−2
0.23 ± 0.02
NT
33%
NT
2
4
0.125−1
0.5−2
0.25−>64
1−4
25
1−4
0.27 ± 0.01
NT
16%
NT
2
6
0.5−8
0.5−2
0.5−64
0.125−1
0.5−4
1−4
1
6a
<0.03−0.25
<0.03−0.25
<0.03−0.5
0.25−1
<0.03−1
0.06−>64
0.125−0.5
0.25−>64
0.125−1
0.5−2
1−8
0.32 ± 0.01
0.29 ± 0.01
NT
28%
39%
NT
2
7
2
8
0.5−2
6
0.5−>64
1−4
0.47 ± 0.02
12%
a
Eco: E. coli, 5 strains; Kpn: MDR K. pneumoniae, 5 strains; Aba: MDR A. baumannii, 5 strains;
b
Pae: MDR P. aeruginosa, 5 strains; n = 4, data represent as mean ± standard deviation; Fu,
c
fraction unbound; stability: the degradation percentage after 4 h incubation in human plasma at
d
3
7°C; NT: not tested.
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Pharmacokinetics. Having identified a number of monosulfactams with excellent in vitro
antibacterial activity and physicochemical properties, pharmacokinetic evaluations were
conducted in rats following intravenous (iv) administration at a dose of 1 mg/kg, and the results are
summarized in Table 4. Compound 12c exhibited favorable plasma exposure (AUC0ꢀt = 1730
ng·h/mL) and relatively low clearance (CL = 11.0 mL/min/kg), which were comparable to that of
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11
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14
15
16
17
18
19
20
21
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43
44
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49
50
51
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53
54
55
56
57
58
59
60
6
. Compound 16a displayed poor pharmacokinetic properties with low plasma exposure (AUC =
0ꢀt
661 ng·h/mL) and relatively high clearance (CL = 26.1 mL/min/kg), whereas the sulfonyl
analogue 16c showed 2ꢀfold higher plasma exposure and a marked decrease of clearance when
compared with that of 16a. Interestingly, the chlorothiazole analogue 19 exhibited the longest
elimination half-life (t1/2 = 1.5 h) and largest volume of distribution (Vss = 1.41 L/kg) among all
these monosulfactams, while its plasma exposure was very low (AUC0ꢀt = 634 ng·h/mL).
Although the optically pure form 21 demonstrated equivalent antibacterial activity when
compared with that of 12c, it showed a much higher clearance and lower plasma exposure.
Therefore, the racemic form 12c was chosen for further in vivo studies.
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6
a
Table 4. Pharmacokinetic Properties of Selected Monosulfactams
Compd
AUC (ng·h/mL)
t1/2 (h)
0.27
0.45
0.25
0.33
1.50
0.26
CL (mL/min/kg)
Vss (L/kg)
0.14
0
ꢀt
0
1
2
3
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7
8
9
0
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2
3
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5
6
7
8
9
0
1
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3
4
5
6
7
8
9
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8
9
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2
3
4
5
6
7
8
9
0
6
1714
9.8
11.0
26.1
9.3
1
2c
1730
661
0.30
1
6a
0.37
1
6c
1890
634
0.21
19
26.9
28.5
1.41
2
1
675
0.36
a
Sprague Dawley rat (male), iv, vehicle was water, 1 mg/kg, n = 3. Data are the mean values.
Abbreviations: AUC : area under the concentrationꢀtime curve up to last sampling time; t :
0
ꢀt
1/2
elimination half-life; CL: clearance; Vss: volume of distribution at steady state.
In Vivo Efficacy of Compounds 12c, 16c and 19. Compound 12c, 16c and 19 were evaluated
for their in vivo efficacy using a murine systemic infection model. Compound 6 and meropenem
were chosen as positive controls. As shown in Table 5, compounds 12c and 16c displayed
comparable in vivo efficacy (ED = 3.37, and 5.32 mg/kg, respectively) with that of 6 (ED =
5
0
50
4
.19 mg/kg) against clinical E. coli 210737. Compound 19 demonstrated an efficacy (ED = 6.94
50
mg/kg) inferior to that of 6 in the E. coli infection model, and this might be attributed to its low
plasma exposure and reduced in vitro antibacterial activity. Based on its excellent in vivo efficacy
in the E. coli infection model, compound 12c was selected for further evaluation in the MDR K.
pneumoniae 212232 infection model. As shown in Table 5, compound 12c also displayed a potent
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in vivo efficacy (ED = 10.20 mg/kg), whereas 6 exhibited extremely low activity in this model.
5
0
This excellent in vivo efficacy of 12c makes it an ideal preclinical candidate for further studies.
Table 5. In Vivo Efficacy of Compound 12c, 16c and 19 in the Mouse Systemic Infection Models
a
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
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42
43
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b
c
E. coli 210737
K. pneumoniae 212232
d
compd
d
ED (mg/kg )
50
MIC
ED (mg/kg )
MIC
5
0
(ꢁg/mL)
(95% CI)
(ꢁg/mL)
(
95% CI)
12c
<0.03
0.06
1
3.37 (1.12–6.85)
5.32 (2.99–9.51)
6.94 (3.85–13.7)
4.19 (1.99–11.25)
2.04 (1.05–3.75)
<0.03
0.06
0.06
0.25
>64
10.20 (5.28–18.23)
e
16c
NT
19
NT
f
6
0.25
0.125
>100
f
meropenem
>100
a
Mice were inoculated intraperitoneally, and the drug was given subcutaneously at 0.5 and 4.0 h
b
c
postꢀinfection. E. coli 210737, clinically isolated. K. pneumoniae 212232, MDR clinically
d
isolated. The efficacy criterion, ED , was calculated as the dose at which mice survival rate was
5
0
e
f
50%. Numbers in parentheses are 95% confidence ranges. NT: not tested. Dosing at 100 mg/kg
resulted in 40% survival for 6 and 0% survival for meropenem.
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6
ꢀ
CHEMISTRY
The synthesis of monosulfactams 8–11 is shown in Scheme 1. Condensation of alkoxyamines
5
1
31a–d with 2ꢀoxoꢀ2ꢀ(2-(tritylamino)thiazolꢀ4ꢀyl) acetic acid 29 provided oxime acids 32a–d,
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
which were coupled with amine 30 to give intermediates 33a–d. Subsequent deprotection of the
52
protecting groups provided the target compounds 8–11. Alkoxyamine 31a was prepared
5
3
according to a published procedure, and details for the synthesis of alkoxyamines 31b–d can be
found in Supporting Information.
a
Scheme 1. Synthesis of Compounds 8–11
a
Reagents and conditions: (a) MeOH/CH Cl , rt; (b) 30, HATU, NaHCO , DMSO; (c) TFA,
2
2
3
Et SiH, CH Cl .
3
2
2
The syntheses of monosulfactams 12a, 12b, 12d and 12e are depicted in Scheme 2. Selective
protection of the phenolic hydroxyl group of kojic acid (34) with diphenyldiazomethane, followed
by heating with hydroxylamine and then reacting with chlorodiphenylmethane afforded
5
4
intermediate 35, which was oxidized to give aldehyde 36 under Parikh–Doering conditions.
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Reactions of various Grignard reagents (RMgBr) with intermediate 36 provided alcohols 37a–b
and 37d–e in good yields. Mitsunobu reactions between alcohols (37a–b and 37d–e) and
Nꢀhydroxyphthalimide produced phthalimides 38a–b and 38d–e, and subsequent removal of the
phthaloyl groups with hydrazine hydrate obtained the key alkoxyamines 39a–b and 39d–e. Then
the alkoxyamines were converted to target monosulfactams 12a, 12b, 12d and 12e using a similar
method as described above.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Scheme 2. Synthesis of Compounds 12a, 12b, 12d and 12e
a
Reagents and conditions: (a) (i) diphenyldiazomethane, EtOH, 40 °C; (ii) hydroxylamine
hydrochloride, sodium acetate trihydrate, EtOH/H O, 60 °C; (iii) chlorodiphenylmethane, NaI,
2
K CO , DMSO, rt; (b) pyridine–SO , DMSO, NEt , CH Cl ; (c) RMgBr, THF; (d)
2
3
3
3
2
2
Nꢀhydroxyphthalimide, PPh , DEAD, THF; (e) N H , MeOH, rt; (f) 29, MeOH/CH Cl , rt; (g) 30,
3
2
4
2
2
HATU, NaHCO , DMSO; (h) for 12a, 12b, and 12e: TFA, Et SiH, CH Cl , −15 °C; for 12d:
3
3
2
2
HCOOH, 0 °C.
When the R group is changed to an isopropyl (Scheme 2), we failed to get the corresponding
phthalimide analogue through Mitsunobu conditions, and we speculated that an increase in steric
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2
2
2
2
2
2
2
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3
3
3
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3
3
3
3
3
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4
4
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5
5
5
5
5
5
5
6
hindrance could be the reason. Thus, compound 12c was synthesized in a modified way as
described in Scheme 3. Intermediate 42 was prepared according to published procedures with
5
5
kojic acid (34) as starting material, which was oxidized to give 43 in excellent yields.
Nucleophilic addition of 43 with isopropylmagnesium bromide afforded alcohol 44, which was
converted to 45 with mꢀCPBA. Subsequent deprotection with borane trichloride and reprotection
with diphenyldiazomethane, led to the formation of intermediate 47, which lacked the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
characteristic peak of a carbonyl group at about δ 170 ppm in the C NMR spectra (see Supporting
Information). After the Mitsunobu reaction with Nꢀhydroxyphthalimide and the removal of the
phthaloyl group, the key alkoxyamine 49 was obtained. Alkoxyamine 49 was converted to the
target compound 12c using the same method as described above.
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a
Scheme 3. Synthesis of Compound 12c
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
a
Reagents and conditions: (a) BnCl, NaOH, MeOH, 60 °C; (b) NH OH, MeOH, 55 °C; (c) BnCl,
4
K CO , DMSO, 50 °C; (d) pyridine–SO , DMSO, NEt , CH Cl ; (e) iꢀPrMgBr, THF; (f) mꢀCPBA,
2
3
3
3
2
2
CH Cl , rt; (g) BCl , CH Cl , −10 °C; (h) diphenyldiazomethane, MeOH/CH Cl , rt; (i)
2
2
3
2
2
2
2
Nꢀhydroxyphthalimide, PPh , DIAD, THF; (j) N H , MeOH, rt; (k) 29, MeOH/CH Cl , rt; (l) 30,
3
2
4
2
2
HATU, NaHCO , DMSO; (m) TFA, Et SiH, CH Cl , −15 °C.
3
3
2
2
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2
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2
2
2
2
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4
4
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4
4
4
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4
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5
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5
5
5
5
5
5
5
6
The synthesis of monosulfactams 13a–c and 15a–c was achieved using an alternative route. As
illustrated in Scheme 4, intermediate 50 was prepared using similar procedures as described above
for 43. Nucleophilic addition of 50 with phenylmagnesium chloride or aryl lithium afforded
alcohols 52a–c, which were further converted to 53a–c through Mitsunobu conditions.
Oxidization of 53a–c with mꢀCPBA followed by removal of the phthaloyl groups gave the key
alkoxyamines 55a–c. Final target compounds 13a–c were then obtained using the same method as
described above. Alternatively, intermediate 50 was subjected to Corey–Chaykovsky epoxidation
to generate 51, and subsequent nucleophilic ring opening of 51 with alkoxides provided alcohols
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56a–c, which were then converted to the target monosulfactams 15a–c by the same manner as
described for 13a–c.
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a
Scheme 4. Synthesis of Compounds 13a–c and 15a–c
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a
Reagents and conditions: (a) for 52a: phenylmagnesium chloride, THF, –20 °C; for 52b and 52c:
aryl lithium, THF, –78 °C; (b) N-hydroxyphthalimide, PPh , DEAD or DIAD, THF; (c) mꢀCPBA,
3
CH Cl , rt; (d) N H , MeOH, rt; (e) 29, MeOH/CH Cl , rt; (f) 30, HATU, NaHCO , DMSO; (g)
2
2
2
4
2
2
3
+
ꢀ
TFA, Et SiH, CH Cl , 0 °C; (h) Me S (O)I , NaH, DMSO, 0 °C; (i) alkoxides.
3
2
2
3
The synthesis of monosulfactams 14a–c is depicted in Scheme 5. Compound 43 was treated
with sodium cyanide to give the αꢀhydroxynitrile 60, which was converted to ester 61 by
alcoholysis with methanol under hydrochloric conditions. Intermediate 61 was oxidized with
mꢀCPBA, followed by deprotection with borane trichloride and reprotection with
diphenyldiazomethane to give intermediate 64b. Hydrolysis of 64b and subsequent protection
with diphenyldiazomethane furnished intermediate 64a, whereas aminolysis of 64b with
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methylamine provided 64c. Intermediates 64a–c were then converted to the target
monosulfactams 14a–c by the same manner as described for 12c.
a
Scheme 5. Synthesis of Compounds 14a–c
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Reagents and conditions: (a) NaCN, NaHSO ,THF/H O, 0 °C; (b) 4 M HCl, MeOH and then
3
2
NaHCO aqueous; (c) mꢀCPBA, CH Cl , rt; (d) BCl , CH Cl , −10 °C; (e) diphenyldiazomethane,
3
2
2
3
2
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MeOH/CH Cl , rt; (f) Nꢀhydroxyphthalimide, PPh , DIAD, THF; (g) N H , MeOH, rt; (h) 29,
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2
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MeOH/CH Cl , rt; (i) 30, HATU, NaHCO , DMSO; (j) TFA, Et SiH, CH Cl , −15 °C; (k) LiOH,
2
2
3
3
2
2
THF/H O, rt; (l) methylamine, MeOH, rt.
2
The synthesis of monosulfactams 16a–c is outlined in Scheme 6. The starting material 67 was
synthesized using the same method as compound 51. Oxidization of 67 with mꢀCPBA provided
68, which was converted to 69a and 69b subsequently by nucleophilic ring opening reactions.
Deprotection of 69a and 69b with borane trichloride and treatment with diphenyldiazomethane
afforded 71a and 71b, respectively. Compounds 71a and 71b were then converted to 72a and 72b
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through Mitsunobu conditions as described above. Oxidization of 72a with mꢀCPBA afforded 72c.
The final monosulfactams 16a–c were then prepared in a manner similar to that described for 12c.
a
Scheme 6. Synthesis of Compounds 16a–c
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a
Reagents and conditions: (a) mꢀCPBA, CH Cl , rt; (b) for 69a: sodium thiomethoxide, MeOH, rt;
2
2
for 69b: 2ꢀpropanethiol, NaH, dioxane, rt; (c) BCl , CH Cl , −10 °C; (d) diphenyldiazomethane,
3
2
2
MeOH/CH Cl , rt; (e) N-hydroxyphthalimide, PBu , DIAD, THF, −10 °C; (f) N H , MeOH, rt; (g)
2
2
3
2
4
2
9, MeOH/CH Cl , rt; (h) 30, HATU, NaHCO , DMSO; (i) TFA, Et SiH, CH Cl , −15 °C.
2 2 3 3 2 2
The preparation of the chiral building blocks for compounds 21–28 is illustrated in Scheme 7. A
Parikh–Doering treatment of compound 47 generated the chiral precursor 74, which was further
converted to chiral isobutanols (R)–75 and (S)–75 through the Noyori asymmetric reduction of 74
using a pair of chiral catalysts. Greater than 98% ee values were achieved for both enantiomers
after recrystallization in ethyl acetate. The absolute configuration of (S)–75 was confirmed by
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Xꢀray crystallography (see Supporting Information). Mitsunobu reactions of these intermediates
were conducted at −10 °C to provide phthalimides with good ee values, which were subsequently
converted to compounds (R)–76 and (S)–76 after hydrazinolysis with hydrazine hydrate. The same
synthetic procedures were used for the preparations of (R)–79 and (S)–79.
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a
Scheme 7. Asymmetry synthesis of chiral fragments (R)–76, (S)–76, (R)–79 and (S)–79
a
Reagents and conditions: (a) pyridine–SO , DMSO, Et N, CH Cl ; (b) HCOOH, Et N,
3
3
2
2
3
dichloro(p-cymene)ruthenium(II) dimer, (1S, 2S)-(+)-N-(4-toluenesulphonyl)-1, 2-ethane
diamine, MTBE/DMF, rt; (c) HCOOH, Et N, dichloro(p-cymene)ruthenium(II) dimer, (1R,
3
2
R)-(+)-N-(4-toluenesulphonyl)-1, 2-ethane diamine, MTBE/DMF, rt; (d) N-hydroxyphthalimide,
PBu , DIAD, THF, −10 °C; (e) N H , MeOH, rt.
3
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ꢀ
CONCLUSION
In summary, based on the structure of compound 6, our exploration of its pyridone moiety and
optimization of the linker between the pyridone and the βꢀlactam core have led to the discovery of
a series of novel pyridoneꢀconjugated monosulfactams. Various substituents were tolerated on the
methylene linker and a number of new compounds showed potent antibacterial activity against a
variety of clinical MDR Gramꢀnegative pathogens, including K. pneumoniae. The
isopropylꢀsubstituted compound 12c and the methylthiomethylꢀsubstituted compound 16a
exhibited excellent and broadꢀspectrum antibacterial activity, whereas the Nꢀmethyl formamide
analogue 14c and sulfonyl analogue 16c demonstrated more favorable PPB profiles. Among all of
these compounds, 12c showed favorable plasma exposure and relatively low clearance in rats.
Furthermore, 12c displayed a potent in vivo efficacy (ED = 10.20 mg/kg) in a murine systemic
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0
infection model with MDR Klebsiella pneumoniae, whereas the precedent compound 6 showed
very weak efficacy in the same model. The excellent in vitro and in vivo activity of 12c, combined
with its desirable pharmacokinetic properties, make it a promising drug candidate for the treatment
of serious infections caused by MDR Gramꢀnegative pathogens including the CREs. Further
comprehensive evaluations of compound 12c are underway.
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ꢀ
EXPERIMENTAL SECTION
Chemistry. All solvents and chemicals were used as purchased without further purification.
Inert atmosphere operations were conducted under argon in flame-dried glassware. Room
temperature refers to 20–25 °C. All reaction mixtures were monitored using thinꢀlayer
chromatography (TLC) on silica gel Fꢀ254 TLC plates. Column chromatography was carried out
using silica gel (200–300 mesh). Melting points (uncorrected) were determined on an Xꢀ4 melting
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13
point apparatus. H and C NMR spectra were recorded on a Bruker 400 NMR or a Bruker 500
NMR spectrometer using solvent residue as the internal standards. Chemical shifts (δ) are reported
in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). EIꢀMS spectra
were obtained on a Finnigan MAT95 spectrometer and ESIꢀMS spectra were obtained on a Krats
MS 80 mass spectrometer. Purity of all final compounds was determined by analytical HPLC
(PLATISIL ODS 250 × 4.6 mm, particle size 5ꢁm) with methanol/buffer (0.1% CF COOH and
3
0.1% NH OH in water, pH 3.5) as the mobile phase. The ee values were determined using chiral
4
HPLC (CHIRALPAK ADꢀH column 250 × 4.6 mm, particle size 5ꢁm or CHIRALCEL ODꢀH
column 250 × 4.6 mm, particle size 5ꢁm) with ethanol/nꢀhexane as the mobile phase. A purity of
>95% was achieved for all tested compounds. Detailed synthetic procedures and spectral
characterization data for all intermediates is provided in the Supporting Information.
(
S,Z)-3-(2-(2-Aminothiazol-4-yl)-2-((2-(N-hydroxyacetamido)ethoxy)imino)acetamido)-2,
2-dimethyl-4-oxoazetidin-1-yl Hydrogen Sulfate (8). A suspension of 33a (210 mg, 0.25 mmol)
in anhydrous dichloromethane (3 mL) was treated with triethylsilane (0.13 mL, 0.75 mmol) and
cooled to −5 °C. Then trifluoroacetic acid (1.9 mL, 25.0 mmol) was added dropwise. The reacting
mixture was stirred overnight and warmed to 0 °C and stirred for 2 h. After completion of the
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reaction, a mixture solvent of ethyl acetate and hexane (4:1, 15 mL) was added dropwise, the
resulting precipitate was further stirred for 20 min at room temperature and collected by filtration
and then the cake was washed with ethyl acetate, dried in vacuum at room temperature to afford 8
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1
as a white solid (45 mg, 38%), mp: 155 °C decomp. H NMR (400 MHz, DMSOꢀd ) δ 9.96 (s,
6
1H), 9.36 (s, 1H), 6.87 (s, 1H), 4.62 (d, J = 7.9 Hz, 1H), 4.23 (t, J = 6.0 Hz, 2H), 3.78 (t, J = 6.1 Hz,
1
3
2
H), 1.98 (s, 3H), 1.44 (s, 3H), 1.26 (s, 3H). C NMR (126 MHz, DMSOꢀd ) δ 170.83, 169.47,
6
1
62.43, 161.91, 160.45, 148.71, 110.40, 70.75, 66.44, 61.08, 47.35, 23.12, 20.80, 20.06. HRMS
−
(ESI) m/z calcd for C H N O S [M − H] 479.0660, found 479.0668.
1
4
19
6
9 2
(
S,Z)-3-(2-(2-Aminothiazol-4-yl)-2-(((1-hydroxy-2-oxo-1,2-dihydropyridin-4-yl)methoxy)
imino)acetamido)-2,2-dimethyl-4-oxoazetidin-1-yl Hydrogen Sulfate (9). A suspension of 33b
110 mg, 0.12 mmol) in anhydrous dichloromethane (2 mL) was treated with triethylsilane (0.05
(
mL, 0.24 mmol) and cooled to −15 °C. Then trifluoroacetic acid (0.9 mL, 12.1 mmol) was added
dropwise. The reacting mixture was stirred at −15 °C for 5 h. After completion of the reaction, the
reacting mixture was slowly warmed to 0 °C. Then, a mixture solvent ethyl acetate and hexane
(
4:1, 20 mL) was added dropwise, the resulting precipitate was further stirred for 20 min and
collected by filtration and then the cake was washed with ethyl acetate, dried in vacuum at room
1
temperature to afford 9 as a white solid (60 mg, 74%), mp: 178 °C decomp. H NMR (400 MHz,
DMSOꢀd ) δ 9.70 (d, J = 7.8 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 6.84 (s, 1H), 6.49 (s, 1H), 6.15 (dd,
6
1
3
J = 7.2, 2.3 Hz, 1H), 5.03 (s, 2H), 4.65 (d, J = 7.8 Hz, 1H), 1.44 (s, 3H), 1.25 (s, 3H). C NMR (151
MHz, DMSOꢀd ) δ 169.77, 162.14, 161.91, 158.70, 157.98, 149.02, 148.63, 135.86, 116.25,
6
1
11.03, 103.20, 74.18, 68.28, 61.24, 23.87, 20.90. HRMS (ESI) m/z calcd for C H N O S [M −
16 17 6 9 2
−
H] 501.0498, found 501.0492.
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