Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.jmedchem.5b01833

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.

Full text of DOI:10.1021/acs.jmedchem.5b01833

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Article
Novel cephalosporins selectively active on
non-replicating Mycobacterium tuberculosis
Ben Gold, Robert Smith, Quyen Nguyen, Julia Roberts, Yan Ling, Landys Lopez Quezeda,
Selin Somersan-Karakaya, Thulasi Warrier, David Little, Maneesh Pingle, David Zhang, Elaine
Ballinger, Matthew Zimmerman, Veronique Dartois, Paul Ronald Hanson, Lester A. Mitscher,
Patrick Porubsky, Steven Rogers, Frank J. Schoenen, Carl F. Nathan, and Jeffrey Aubé
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01833 • Publication Date (Web): 04 May 2016
Downloaded from http://pubs.acs.org on May 5, 2016
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Novel cephalosporins selectively active on non-replicating Mycobacterium
‡
tuberculosis
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¡
∆,±
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†
Ben Gold , Robert Smith , Quyen Nguyen , Julia Roberts Yan Ling , Landys Lopez
,
†
∞
†
†
†
Quezada , Selin Somersan , Thulasi Warrier , David Little , Maneesh Pingle , David
†
†
‡
‡
∫,∆
Zhang , Elaine Ballinger , Matthew Zimmerman , Véronique Dartois , Paul Hanson ,
√
∆
±
¡
Lester A. Mitscher , Patrick Porubsky , Steven Rogers , Frank J. Schoenen , Carl
†
,*
¡,∆,√,±,*
Nathan , Jeffrey Aubé
Running title: Cephalosporins active on nonꢀreplicating M. tuberculosis
†
∞
Departments of Microbiology & Immunology and Medicine, Weill Cornell Medical
‡
College, New York, NY, 10065; Public Health Research Institute, New Jersey Medical
¡
School, Rutgers, the State University of New Jersey, Newark, NJ 07013, University of
∆
Kansas Specialized Chemistry Center,
Chemical Methodologies and Library
∫
√
Development Center, Department of Chemistry and
Department of Medicinal
±
Chemistry, University of Kansas, Lawrence, Kansas, 66047; Division of Chemical
Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of
North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
‡
Dedicated to the memory of Lester A. Mitscher
*
Coꢀsenior authors
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Abstract
We report two series of novel cephalosporins that are bactericidal to Mycobacterium
tuberculosis alone of the pathogens tested, that only kill M. tuberculosis when its
replication is halted by conditions resembling those believed to pertain in the host, and
whose bactericidal activity is not dependent on or enhanced by clavulanate, a βꢀlactamase
inhibitor. The two classes of cephalosporins bear an ester or, alternatively, an oxadiazole
isostere, at Cꢀ2 of the cephalosporin ring system, a position that is almost exclusively a
carboxylic acid in clinically used agents in the class. Representatives of the series kill M.
tuberculosis within macrophages without toxicity to the macrophages or other
mammalian cells.
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Keywords: cephalosporins, βꢀlactams, Mycobacterium tuberculosis, nonꢀreplication,
structureꢀactivity relationship, ester, oxadiazole
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Introduction
Antibiotics that rapidly kill Mycobacterium tuberculosis in axenic culture require
months to years to produce the same result in tuberculosis (TB) patients. Accordingly,
TB treatment lags far behind that of other bacterial diseases in terms of treatment
duration, number of antibiotics required, toxicity to the host, and cure rates. While new
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drugs such as oxazolidinones (linezolid Pfizer), diarylquinolines (bedaquiline, Janssen),
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and nitroimidazoles (e.g. delamanid) offer hope of shortening TB therapy and reducing
mortality in patients whose TB is resistant to the standard regimen, there is an urgent
need to discover additional antiꢀTB drugs.
Even when M. tuberculosis is genetically sensitive to existing drugs, a small
fraction of a replicating population survives exposure to each such drug in vitro. Such
4
bacteria are termed “persisters”. They display class I phenotypic tolerance, in that when
the drug is removed and the persisters are allowed to replicate, application of the drug at
the same concentration again kills the vast majority. Mechanisms of class I persistence
5
range from temporary nonꢀreplication of a small subpopulation to heterogeneous
expression of proteins that activate prodrugs and misincorporation of amino acids into
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proteins. Genetically susceptible M. tuberculosis can also display class II phenotypic
tolerance when external stresses, such as those imposed by the host immune system,
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, 10ꢀ12
prevent most of the population from replicating.
Ideally, TB should be treated with
a combination of drugs such that bacteria displaying class I phenotypic tolerance to any
one of them are killed by at least one of the others, and at least one of the drugs can kill
nonꢀreplicating M. tuberculosis that display class II phenotypic tolerance.
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Some of the conditions in the host that can drive M. tuberculosis into replication
arrest and class II phenotypic tolerance include residence in interferon γ (IFNγ)ꢀactivated
macrophages that traffic the bacilli to acidified phagosomes and expose them to
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nitrosative and oxidative stress
or release of M. tuberculosis into the hypoxic milieu
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of necrotic granulomas. The search for compounds active against nonꢀreplicating M.
17ꢀ30
tuberculosis has been pursued in a variety of in vitro nonꢀreplicating models.
We
recently developed a high throughput screening platform to identify small molecules that
kill class II persistent M. tuberculosis that are rendered nonꢀreplicating by a combination
13ꢀ15, 21, 31ꢀ35
of four hostꢀrelevant conditions:
low pH (5.0), a flux of nitric oxide (generated
from 0.5 mM nitrite at that pH), hypoxia (1% O ), and low concentrations of a fatty acid
2
(0.05% butyrate) as the carbon source, instead of the conventional carbon sources,
2
1, 36
dextrose and glycerol.
Here we report that this screening protocol has led to the
discovery of the first cephalosporins, to our knowledge, that are selectively active against
bacteria in a nonꢀreplicating state. We describe an initial analysis of their structure–
activity relationship.
Results
Identification of cephalosporins active on non-replicating M. tuberculosis. A
highꢀthroughput screening campaign against replicating and nonꢀreplicating M.
tuberculosis was carried out using a library of compounds from an inꢀhouse screening
collection assembled at the University of Kansas. This screen led to the identification of
3
7
three cephalosporin esters (1–3, Figure 1a) whose activity was unique to nonꢀreplicating
M. tuberculosis. For comparison, cephalexin (4), a broadꢀspectrum antibiotic in clinical
use, was also tested in the multiꢀstress nonꢀreplicating model, but found inactive. The
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three hit molecules were reꢀsynthesized to >96% purity, and displayed MIC ’s against
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nonꢀreplicating M. tuberculosis of 1.8ꢀ2.7 ꢀg/mL and 0.5ꢀ1.1 ꢀg/mL during exposures
lasting 3 or 6 days, respectively (Table 1). Initial results were obtained with a strain of M.
tuberculosis whose dual auxotrophy for pantothenate and lysine increases its safety for
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laboratory personnel
and key results for select molecules were verified using virulent,
wildꢀtype M. tuberculosis H37Rv. Given the propensity of βꢀlactams for inoculum
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effects we also determined the MIC ’s against nonꢀreplicating M. tuberculosis using a
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tenꢀfold lower inoculum of A580 of 0.01. The results at 3 days (0.7ꢀ3.1 ꢀg/mL) were
similar to those found for the higher inoculum cultures exposed for 6 days. Activity
against replicating M. tuberculosis and against human HepG2 hepatoma cells was not
seen up to the highest concentration tested (100 ꢀg/mL) and the best selectivity index
was ≥250. Thus, the activity of these compounds against M. tuberculosis was directly
dependent on time of exposure and the state of nonꢀreplication, inversely dependent on
concentration of the bacteria, and selective for M. tuberculosis over human cells.
Stability in cell-free PBS and non-replicating medium. Since some molecules
21, 35, 36
are chemically unstable in the multiꢀstress model of nonꢀreplication,
compounds 1
and 2 were tested and found stable for up to 7 days in cellꢀfree PBS and nonꢀreplicating
medium containing or omitting NaNO (Figure 2a, 2b). However, 3 was unstable in cellꢀ
2
free nonꢀreplicating medium containing NaNO (Figure 2c; summarized in Table 1). For
2
comparison, cephalexin was partially unstable in cellꢀfree PBS and stable in cellꢀfree
nonꢀreplicating medium either containing or lacking NaNO (Figure 2d).
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Structure–Activity Relationship (SAR) Studies. These promising results
prompted us to undertake an initial structure–activity relationship survey. For each new
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analog, we determined the activity against Mtb under both nonꢀreplicating (NR) and
replicating (R) conditions. In addition, each compound was assayed for cytotoxicity
against HepG2 cells. For the present discussion, analogs are presented in Tables 2–5
according to the chemical class investigated.
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To test if the ester moiety in compounds 1–3 was essential for activity, we
synthesized five free acid analogues of 1 (9) or related molecules and tested three
commercially available cephalosporins bearing Cꢀ2 carboxylic acids (cephalexin,
cefdinir, cephalothin). The activity of all the cephalosporin carboxylates tested against
nonꢀreplicating M. tuberculosis ranged from ~50 ꢀg/mL to > 100 ꢀg/mL (Table 2),
signifying the importance of an ester moiety at Cꢀ2. To determine if additional functional
groups could be tolerated at this position, analogues containing various amides, alcohols,
and ethers at Cꢀ2 were prepared and tested (Table 2, compounds 5–18). All were found to
be inactive. However, replacement of the ester moiety by the isosteric 1,2,4ꢀoxadiazole in
analogue 5 resulted in a compound that was close in activity to 1 (Figure 1c and Table 2).
Moreover, compound 5 was stable in cellꢀfree nonꢀreplicating medium containing NaNO2
(Figure 3).
To determine if simply adding ester groups or an oxadiazole to Cꢀ2 of a classical
cephalosporin would confer activity against nonꢀreplicating M. tuberculosis, we made
cephalexin analogues 4a-c bearing such modifications. Testing determined that analogues
4
a and 4c were poorly active against nonꢀreplicating M. tuberculosis and completely
inactive against replicating M. tuberculosis (Table 3). nꢀPropyl ester 4b was slightly more
active, with an NRꢀMIC of 15.5 ꢀg/mL.
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Given these results, we chose to concentrate on exploring the effect of Cꢀ2 and Cꢀ
amino substitution in two series of analogues, the esters (Table 4) and the oxadiazoles
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Table 5). All compounds were synthesized as summarized in Scheme 1. Esters were
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generated from commercially available 7ꢀaminodeacetoxycephalosporanic acid (7ꢀ
ADCA) by amidation, followed by esterification. The oxadiazole series was prepared by
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installing the desired oxadiazole onto the Cꢀ2 of carboxylic acid A as above.
Compounds were typically purified by massꢀdirected HPLC fractionation and rendered in
purities of ≥95% for biological evaluation.
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Scheme 1. Synthetic route for preparation of ester and oxadiazole analogues
(a)
HO
O
N
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Ester series 1
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DCC, 2,4-dinitrophenol
. 110 °C
N
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Oxadiazole series 2
Numerous analogues in both series of compounds were active against nonꢀ
replicating M. tuberculosis. The activity profiles of these molecules were responsive to
these basic chemical changes, which suggested that the compounds were exerting their
activity through action at a discrete cellular target. For example, a preference for longish
and unbranched esters at Cꢀ2 was generally observed, with ethyl, propyl, and butyl esters
being preferred. Activity was sharply diminished for analogues containing propargyl
groups at this position (e.g., compounds 4, 6, and 22), although benzyl esters were
tolerated (compounds 14, 24 and 29). Recalling that cephalexin analogues 4a and 4b
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containing a side chain bearing a primary amine were poorly active, we concentrated our
initial SAR on neutral Cꢀ7 amide moieties (although the single Cꢀ7 propyl amine
examined, compound 3, did have significant activity). Most of the compounds examined
bore a substituted 3ꢀphenylpropamide side chain or its ethereal analogue (e.g.,
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2
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compounds 13). We also observed that moving the double bond from the ꢁ to the ꢁ
had only a modest effect (cf. compounds 34 and 35).
Among the Cꢀ2 oxadiazoles (Table 5), similar trends were observed with a few
addenda. Here, a wider range of carbamates, including a single carbothioate, were
prepared and found active (compounds 14ꢀ17). In this series, we noted that while
compounds bearing para electronꢀwithdrawing substituents retained excellent potency, in
many cases measurable levels of cellular toxicity were also observed. Particularly notable
examples included some pꢀCl and 3,4ꢀdichloro analogues (compounds 5, 12, 21 and 29).
Physicochemical properties and metabolic stability. Compounds 1 and 5 were
chosen as representative molecules of the alkyl ester and oxadiazole classes of
cephalosporins that are active against nonꢀreplicating M. tuberculosis, while cephalexin,
cefdinir, and cephalothin were chosen as representatives of cephalosporins lacking such
activity. The active cephalosporins shared higher values for clogP and pKa, whereas
other properties such as Hꢀbond donors, Hꢀbond acceptors, molecular weight, heavy atom
count, and rotatable bonds were similar (Table 6).
We next determined the hydrolytic stability of these compounds under strongly
acidic conditions, such as would be encountered in the stomach. Both 1 and 5 were more
stable at pH 2 (100% remaining after 4 hours) than cephalexin (ca. 74% remaining)
(
Table 7), whereas all three compounds were stable at pH 7 and degraded in base (pH
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2). Compound 5 and cephalexin were soluble at 84 ꢀM and 76 ꢀM at pH 7.4,
respectively, while 1 was less soluble at 23 ꢀM (Table 7).
Parallel artificial membrane permeability assays (PAMPA) predicted that both 1
and 5 would be membrane permeable (Table 8). However, unlike cephalexin, both 1 and
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were rapidly metabolized by mouse liver microsomes (Table 8). Compounds 1 and 5
were less susceptible to metabolism by human liver microsomes, with halfꢀlives of ~ 80
minutes and CLint values suggestive of slow metabolism (Table 8).
Next, we assessed the stability of compounds 1 and 5 in mouse plasma to
determine the feasibility of testing these compounds for activity in a mouse model of
tuberculosis. Both compounds 1 and 5 were completely transformed in mouse plasma in
<
5 minutes (Figure 4a). In human plasma, compounds 1 and 5 had halfꢀlives of
approximately 2ꢀ3 hours (Figure 4b). Cephalexin (4) was stable in plasma from both
species (Figures 4a and 4b).
Selective bactericidal action of cephalosporins on non-replicating M.
tuberculosis. Narrow spectrum bactericidal activity is preferred for TB drugs for two
reasons. First, TB treatment is protracted, and longꢀterm exposure to broadꢀspectrum
antibacterial agents can precipitate severe and sometimes fatal intestinal dysfunction,
such as that caused by overgrowth of Clostridium difficile. Second, efficacy of a given
drug against other bacterial infections can prompt its use in the community, including in
people who have undiagnosed TB. Monotherapy of TB often selects for emergence of
genetically resistant strains. The spread of such strains in the community would render
the new drug progressively less useful for the treatment of TB. Hence it was important to
test the antimicrobial spectrum of the new cephalosporins against other bacteria.
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Compounds 1 and 5 had MIC ’s > 100 ꢀg/mL against replicating Pseudomonas
9
0
aeruginosa, Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis and
Mycobacterium bovis BCG, as well as against the fungus Candida albicans (Figure S1).
To determine the extent of bacterial kill, we exposed nonꢀreplicating wildꢀtype M.
tuberculosis at an OD580 of 0.01 (low inoculum) to 1 and 5 for 7 days (Figure 5). At ~
10
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0
.7ꢀ0.8 ꢀg/mL, both compounds reduced colonyꢀforming units (CFUs) by 2 log10.
Compounds 1 and 5, at 3 ꢀg/mL and 10 ꢀg/mL, respectively, reduced CFU to the extent
that there were no recoverable colonies when 10 ꢀL of undiluted sample was plated (≥
3.4 log kill). Thus, no class I phenotypic tolerance was observed. Bactericidal activity
1
0
of 1 was not enhanced by the addition of a βꢀlactamase inhibitor, clavulanate, and
addition of clavulanate did not lead to activity of 3, 2, or 1 against replicating M.
tuberculosis (data not shown). In contrast, clavulanate enhanced the replicating MIC of
9
0
meropenem fourꢀfold.
Representative cephalosporins (compounds 5, 12e, 12i, 18d, 19d, 21b, 22c and
3) from the preliminary SAR campaign (Tables 2 – 5) were tested for activity against
2
nonꢀreplicating wildꢀtype M. tuberculosis. Analogues derived from compounds 1 and 5
were bactericidal in this assay (Supplementary Figure 2).
Reactive nitrogen species enhance bactericidal activity of 1 and 5 against
non-replicating M. tuberculosis. The activity of 1 against nonꢀreplicating M.
tuberculosis increased in relation to the concentration of NaNO (Figure 6a), while that of
2
rifampicin did not at ≤ 0.5 mM NaNO (Figure 6b). At 1 mM NaNO , double the
2
2
concentration used in the nonꢀreplicating screening, we observed nitriteꢀdependent killing
with rifampicin as well. We then tested both 1 and 5 for nitriteꢀdependence by coupling
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the outgrowth to a CFUꢀsurrogate assay (charcoal agar resazurin assay; CARA) that
determines the approximate concentration of compound leading to ≥ 2ꢀ3 log10 CFU
reduction as reflected by the ability of survivors to convert resazurin to a fluorescent
product. Both 1 and 5 decreased fluorescence in a doseꢀdependent manner that was
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strongly enhanced by the addition of NaNO (Figures 6c and 6d). As observed for many
2
of the βꢀlactams in this study, the activities of both 1 and 5 were more potent at a 10ꢀfold
lower inoculum of 0.01 and 7ꢀday exposure (Figure 5d). Both compounds displayed
nitriteꢀindependent activity at the lower inoculum (Figures 5c and 5d). Thus, nitrite
contributed to a 32ꢀ to 64ꢀfold enhancement of 1’s activity, but activity was not strictly
dependent on an exogenous source of nitrite (Figure 6d).
Non-replicating-active cephalosporins kill M. tuberculosis in macrophages.
Wildꢀtype M. tuberculosis is typically growthꢀarrested, or replicates slowly, in activated
macrophages, due in part to phagosomal acidification and macrophage production of
13, 14
reactive nitrogen species (RNS).
The multiꢀstress nonꢀreplicating assay conditions
2
1, 35
were designed in part to mimic this phagosomal microenvironment.
We hypothesized
that cephalosporins active in the nonꢀreplicating model might be bactericidal against
intracellular M. tuberculosis. To test this, we stimulated mouse bone marrow derived
macrophages with IFNγ, or left them unstimulated, infected them with wildꢀtype M.
tuberculosis and treated them with 1, 5, or with diluent alone. We observed
approximately 1ꢀ2 log10 CFU reduction of intracellular M. tuberculosis in activated
macrophages treated with 1 or 5, with no apparent toxicity to the macrophages.
Compound 5’s bactericidal activity against intracellular M. tuberculosis was strictly
IFNγꢀdependent (Figures 7a and 7b).
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Discussion
To our knowledge, this is the first report of βꢀlactams that only kill a given
bacterium when it is nonꢀreplicating, and the first report of βꢀlactams with activity
against any one bacterial species that lack broadꢀspectrum antiꢀbacterial activity. Early
studies by Tuomanen et al. demonstrated that while many βꢀlactams lack activity against
10
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nonꢀgrowing cells, a minority killed starved, nonꢀreplicating Escherichia coli and
43, 44
Streptococcus pneumonia.
Similar findings were recently observed in M.
tuberculosis, as the combination of meropenem and the βꢀlactamase inhibitor,
45
clavulanate, killed both replicating and hypoxic, nonꢀreplicating M. tuberculosis.
Meropenemꢀclavulanate lacked activity against nonꢀreplicating M. tuberculosis in the
conditions studied here. In addition to hypoxia, our conditions included a low pH, a flux
of reactive nitrogen species, and a fatty acid carbon source. Faropenem was also reported
46
to kill both replicating and nonꢀgrowing M. tuberculosis. Like meropenem, faropenem
was inactive in our multiꢀstress model of nonꢀreplication. The novel cephalosporins
described here did not acquire activity against replicating M. tuberculosis when we
included clavulanate in the assays.
Structurally, the two main classes of compounds explored herein differ from
clinicallyꢀused cephalosporins by the lack of a carboxylic acid moiety at Cꢀ2 (a notable
exception being the prodrug cefuroxime axetil), and indeed we showed that carboxylic
acid 9 is inactive against nonꢀreplicating Mtb. Early in this project, we considered
whether the screening hit propyl ester was functioning as a prodrug, but the successful
replacement of this moiety with the corresponding oxadiazole renders this possibility
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unlikely. A hydrophobic moiety at this position is tolerated, as seen with the nꢀpropyl
ester and oxadiazole, and possibly preferred, given the fact that the Cꢀ2 hydroxymethyl
analogue 10 is inactive. Polarity at this position also plays a role, given the inactivity of
the amide analogous to the active esters. In the preliminary SAR pursued to date, we have
also ascertained that biological activity is affected by the amide moiety attached to the
central cephalosporin nucleus, with chains ending in electronꢀpoor aromatic rings being
preferred and a moderate dependence of activity on the length of the chain leading to this
point. The role of the βꢀlactam itself is currently ambiguous; while we know that
hydrolytic cleavage of this ring results in an inactive compound, it is not clear whether
this is because the βꢀlactam is essential per se, i.e., in analogy to the generally accepted
0
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mechanism of most βꢀlactams, which involve covalent binding of this group to the target
protein, or because of a structural alignment resulting from the cephalosporin ring
system.
A question of pressing interest is the molecular target that renders these
cephalosporins profoundly active against M. tuberculosis in a nonꢀreplicating state.
Experiments to address this are underway by a variety of approaches but have not yet
yielded an answer, although we note that our observation of structure–activity
relationships is consistent with action through one or more specific targets. The canonical
role of βꢀlactams in killing replicating bacteria has been widely accepted as the arrest of
peptidoglycan biosynthesis. Disruption of the balance between new peptidoglycan
47, 48
synthesis and peptidoglycan cleavage by hydrolases leads to cellular lysis
due to a
4
9
futile cycle in the synthetic pathway. The sensitivity of hypoxic M. tuberculosis to
meropenem and clavulanate, albeit significantly less than the sensitivity of replicating M.
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tuberculosis, suggests that M. tuberculosis may require peptidoglycan biosynthesis to
4
5
survive the hypoxic state. In addition to classical D,Dꢀtranspeptidases that catalyze the
formation of 4’ ꢀ 3’ peptidoglycan crossꢀlinks, M. tuberculosis may use at least five
L,Dꢀtranspeptidases (LdtMT1 – LdtMT5; LDTs) for peptidoglycan 3’ ꢀ 3’ crossꢀlinking.
The 3’ ꢀ 3’ crossꢀlinks account for ~80% of peptidoglycan extracted from M.
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tuberculosis in stationary phase, a form of nonꢀreplication. One of the nonclassical L,Dꢀ
transpeptidases, LdtMT2, plays a role in M. tuberculosis virulence in a mouse model of
5
1
52
infection. Meropenem and other carbapenems bind recombinant Ldt
. However,
MT1,2,4,5
there are additional covalent targets of βꢀlactams, such as signal peptidases and
5
3ꢀ55
proteases.
Thus, we anticipate that betaꢀlactams that specifically target nonꢀ
replicating populations may have either single or multiple canonical or nonꢀcanonical
targets.
Two of the cephalosporins with activity against nonꢀreplicating M. tuberculosis
were stable in PBS and in nonꢀreplicating medium, whether or not the medium contained
NaNO . An equipotent compound, 3, was unstable. Thus, 3 may have entered the bacilli
2
or otherwise exerted its bactericidal effect before its structure was transformed in the
extracellular medium.
5
6
In animal and human tuberculosis, M. tuberculosis often resides in macrophages
and the ability to kill intracellular bacilli is an important feature of antiꢀmycobacterial
compounds. Two of the nonꢀreplicating active cephalosporins, compounds 1 and 5,
killed intracellular M. tuberculosis but with differential dependence on immune
activation. Immune activation of M. tuberculosisꢀinfected macrophages leads to profound
changes of the phagosomal microenvironment that are anticipated to lead to growth
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arrest. These changes include phagosomal acidification to approximately pH 4.5 and
1
4,15
induction of iNOS, which produces nitric oxide.
M. tuberculosis exhibits variable
behavior in mouse bone marrow derived macrophages, ranging from subꢀexponential
replication in nonꢀactivated macrophages to slower replication, no net change in CFU, or
a modest decline in CFU in activated macrophages. An inhibitor of dihydrolipoamide
acyltransferase (DlaT) selectively kills M. tuberculosis and M. bovis BCG in vitro when
they are nonꢀreplicating, and this compound is effective against BCG in activated
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macrophages. Likewise, compound 5 killed intracellular M. tuberculosis when the
macrophages were immune stimulated, consistent with our hypothesis that compound 5
exerts activity in an acidic, nitrosative phagosome. However, compound 1 killed
intracellular M. tuberculosis both in the absence or presence of IFNγ activation and thus
may possess some activity against replicating M. tuberculosis at the concentrations
tested.
Summary
The potential ability of βꢀlactams to treat TB has been suggested for many years
but only recently has gained substantial notice with the report of promising results using
4
5, 46
meropenem in human trials.
It would be of considerable interest to test the role of
betaꢀlactams that target nonꢀreplicating M. tuberculosis in TB therapy in combination
with agents active against replicating M. tuberculosis. Cephalosporins with activity
against nonꢀreplicating M. tuberculosis identified in this study, 5 and 1, were nonꢀtoxic,
stable in cellꢀfree medium, stable at pH 2 and 7, soluble at pH 7.4, predicted to be
membraneꢀpermeable, active in macrophages, and inactive against the other bacterial and
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yeast species tested. Compounds 1 and 5 were relatively stable when incubated with
human liver microsomes. While compounds 1 and 5 were highly labile in mouse plasma,
they were more stable in human plasma, with halfꢀlives of 2ꢀ3 h. Some analogues of
compound 5 were active in the ng/mL range. We are continuing to study the SAR of 5
while seeking its targets.
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Experimental Section
General Procedure for Synthesis of Esters. Propyl (6R,7R)-3-methyl-8-oxo-7-
3
7
(2-phenoxyacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (1).
To
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(
6R,7R)ꢀ3ꢀmethylꢀ8ꢀoxoꢀ7ꢀ(2ꢀphenoxyacetamido)ꢀ5ꢀthiaꢀ1ꢀazabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀ
carboxylic acid (197.0 mg, 0.57 mmol) was added sodium bicarbonate (48 mg, 0.57
mmol) and a minimal amount of water (6.0 mL) to dissolve the starting material. The
mixture was stirred at rt for 30 minutes until all solids were dissolved, frozen, and
lyophilized. DMF (10.0 mL) was slowly added followed by 1ꢀIodopropane (0.55 mL, 5.7
mmol). The reaction was stirred at rt for 16 h, then quenched with water and extracted
twice with Et O. The Et O layers were combined and then washed 3 times with water and
2
2
once with brine solution. The organic layer was dried (Na SO ), filtered, and
2
4
concentrated. The residue was purified via MPLC (silica, 30% EtOAc/hexanes → 70%
25
EtOAc/hexanes) to afford the title compound (130.0 mg, 59% yield). [α]D = +71.2 (c =
ꢀ1 1
1.45, CH Cl ); IR (film) ν = 1780, 1721, 1687, 1524, 1494, 1228, 755 cm ; H NMR
2
2
max
(
500 MHz, CDCl ) δ 7.34 (m, 2H), 7.30 (s, 1H), 7.05 (tt, J = 7.7, 1.0 Hz, 1H), 6.94 (m,
3
2
H), 5.88 (dd, J = 9.2, 4.7 Hz, 1H), 5.04 (d, J = 4.8 Hz, 1H), 4.58 (s, 2H), 4.24 (m, 2H),
3.53 (dd, J = 18.3, 1.0 Hz, 1H), 3.22 (d, J = 18.3 Hz, 1H), 2.15 (s, 3H), 1.75 (h, J = 7.4
13
Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H); C NMR (126 MHz, CDCl ) δ 168.80, 164.03, 162.35,
3
1
57.03, 130.88, 129.96, 129.80, 123.01, 122.52, 115.05, 114.90, 67.51, 67.25, 58.44,
+
56.94, 30.27, 22.03, 20.18, 10.61. HRMS (ESIꢀTOF) calcd for C H N O SNH [M +
1
9
22
2
5
4
+
NH ] : 408.1588, found 408.1605.
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General Procedure for Synthesis of Oxadiazoles. N-((6R,7R)-3-Methyl-2-(3-
methyl-1,2,4-oxadiazol-5-yl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-7-yl)-2-
phenoxyacetamide (5). To a solution of 2,4ꢀdinitrophenol (1.03 g, 5.61 mmol) in CH Cl
2
2
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(
10 mL) was sequentially added (6R,7R)ꢀ3ꢀmethylꢀ8ꢀoxoꢀ7ꢀ(2ꢀphenoxyacetamido)ꢀ5ꢀ
thiaꢀ1ꢀazabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylic acid (1.92 g, 5.5 mmol) in a minimal
amount of 1,4ꢀdioxane (8 mL), and DCC (1.15 g, 5.6 mmol) in 8 mL CH Cl . The
2
2
mixture was stirred at rt for 30 min, after that the mixture was filtered through a plug of
cotton to remove the urea. To the filtrate was then added ethylamidoxime (411.0 mg, 5.6
mmol) in CH Cl (7 mL) and the mixture was stirred at rt for 4 h. The mixture was then
2
2
washed twice with sat. aq. NaHCO , filtered, and concentrated. The residue was then
3
placed in a vacuum oven at 110 °C for 16 h and the resulting residue purified via MPLC
(silica, 100% hexanes → 60% EtOAc/hexanes) to afford the title compound as orange
24
solid (703.2 mg, 52% yield). [α]D = +79.6 (c = 0.72, CH Cl ); IR (film) ν = 1775,
max
2
2
–
1 1
1
493, 1331, 1216, 754, 732, 690 cm ; H NMR (400 MHz, CDCl ) δ 7.35 (m, 3H), 7.06
3
(tt, J = 7.4, 1.0 Hz, 1H), 6.96 (m, 2H), 5.95 (dd, J = 9.1, 4.8 Hz, 1H), 5.16 (d, J = 4.7 Hz,
1H), 4.60 (s, 2H), 3.61 (d, J = 18.4 Hz, 1H), 3.35 (d, J = 18.3 Hz, 1H), 2.49 (s, 3H), 2.25
13
(s, 3H); C NMR (126 MHz, CDCl ) δ 168.79, 167.63, 164.56, 156.99, 130.93, 129.98,
3
1
22.56, 117.45, 114.90, 67.25, 58.73, 57.25, 30.02, 20.40, 11.92. HRMS (ESIꢀTOF)
+
+
calcd for C H N O S [M + H] : 387.1122, found 387.1088.
1
8
19
4
4
Strains and growth conditions. Mycobacterial strains and media were prepared
2
1, 35
as described.
Briefly, wildꢀtype M. tuberculosis H37Rv was cultivated at 20% O and
2
5
% CO in Middlebrook 7H9 bacteriologic medium containing 0.2% glycerol, tyloxapol
2
2
(0.02%) and 10% OADC supplement and the M. tuberculosis strain, mc 6220
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(
∆panCD∆lysA)
was grown in similar medium with minor modifications: additional
glycerol (final: 0.5%), OADC supplement, casamino acids (0.05 %), Lꢀlysine (240
µg/mL) and pantothenate (24 µg/mL). Cells were rendered nonꢀreplicating at 1% O , 5%
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CO in a Sauton’sꢀbased medium (per liter: 0.5 g KH P0 , 0.5 g MgS0 , 0.05 g ferric
2
2
4
4
ammonium citrate, BSA (0.5%), NaCl (0.085%), tyloxapol (0.02%), Lꢀlysine (240
µg/mL), pantothenate (24 µg/mL), butyrate (0.05%), and 0.5 mM NaNO2).
High throughput screen. Molecules from the University of Kansas inꢀhouse
2
1, 35
library were screened using a reported protocol
with minor modifications. For the
replicating screen, 500 nL test agent was added to 50 ꢀL replicating M. tuberculosis
2
mc 6220 at an OD = 0.01, giving a final concentration of 20 ꢀg/mL and 1% DMSO.
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After 7 days incubation at 20% O and 5% CO , the OD was determined. For the nonꢀ
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580
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replicating screen, M. tuberculosis mc 6220 was washed 2x in PBS containing tyloxapol
(
0.02%; PBSꢀTyl) and resuspended in nonꢀreplicating medium containing 0.5 mM
NaNO , and 15 µL cells were dispensed into 384ꢀwell tissue culture plates (Greiner,
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reference 781091). Cells were exposed to 150 nL of test compounds in DMSO and plates
were incubated for 7 days at 1% O , 5% CO . After a 3ꢀday exposure to test agents, M.
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tuberculosis in each well was diluted 5ꢀfold by addition of 60 ꢀL fresh replicating
medium using a reagent dispenser (ThermoScientific), which also served to mix cells.
After 7 day outgrowth at 20% O and 5% CO , the OD was determined. Primary
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screening hits and downstream assay data were managed using the CDD Vault from
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Collaborative Drug Discovery (Burlingame, CA. www.collaborativedrug.com) and
JChem for Excel and MarvinView (ChemAxon).
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Activity against replicating and non-replicating M. tuberculosis. For minimal
inhibitory concentration (MIC) assays, compounds were serially diluted twoꢀfold in
DMSO from 10 to 0.04 mM using a Perkin Elmer Janus robot with a P30 row/column
MDT head to make 100X compound source stocks in Greiner compound plates (384ꢀwell
small volume conical well, reference number 784201). Compounds were then distributed
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into 384ꢀwell replicating and nonꢀreplicating assays with M. tuberculosis mc 6220 in
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84ꢀwell microplates as described above. For colony forming unit assays, experiments
were set up using wildꢀtype M. tuberculosis single cell suspensions in 96ꢀwell tissue
culture treated plates (Corning). At select time points, aliquots of cells were serially
diluted in PBSꢀTyl and spread on Middlebrook 7H11 agar plates containing 10% OADC
supplement. Colonies were enumerated ~3 weeks postꢀplating. The minimal bacteriocidal
concentration leading to 99% reduction in colony forming units (MBC ) was
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extrapolated from CFU data.
HepG2 toxicity assays. Toxicity assays using the human hepatoma cell line
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HepG2 were as described. Briefly, HepG2 cells were propagated in Dulbecco’s
Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), pyruvate,
glutamine and nonꢀessential amino acids. HepG2 cells were incubated for 2 days with
DMSO vehicle control or test compounds (≤1% DMSO final) at 3000 cells/well in 384ꢀ
well tissue culture plates (Greiner reference 781091). Cellular viability was determined
after two days by measuring ATP content with a CellTiterꢀGlo kit (Promega).
Microbial spectrum. Select compounds were tested for activity against a panel of
replicating Gram positive and Gram negative bacteria (Mycobacterium smegmatis,
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa) and yeast (Candida
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albicans). Bacteriologic medium and assay conditions were as described. In brief, 200
µL cells at an OD580 of 0.01 in a sterile, clear tissue culture treated Corning 96ꢀwell plate
were exposed to DMSO or drug and growth determined by optical density.
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Stability assay. Compounds were dissolved at 50 ꢀg/mL in cellꢀfree PBS (pH
7.4) or cellꢀfree nonꢀreplicating medium (pH 5.0) containing or not 0.5 mM NaNO2.
Poorly soluble compounds were dissolved at 5 ꢀg/mL and in a 50:50 (vol:vol) solution of
acetonitrile and PBS, or acetonitrile and nonꢀreplicating medium containing or not 0.5
mM NaNO . The nonꢀreplicating medium was as described above except that BSA,
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tyloxapol, lysine and pantothenate were omitted. Solutions containing acetonitrile had
their pH adjusted to 5.0 (the additional acetonitrile increased the pH from 5.0 to 5.8).
Samples were incubated at 37° C and aliquots removed every 12 hours for analysis by
LCꢀMS. Data represent % remaining of the parent compound compared to that at the start
of the experiment.
Cheminformatics. Tanimoto similarity between molecules and cheminformatic
analysis of chemical properties (including ClogP values) were determined in CDD
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Collaborative Drug Discovery (Burlingame, CA. www.collaborativedrug.com) using
ChemAxon software.
Physicochemical, permeability, and metabolism studies. These assays were
performed by BioDuro (Shanghai, China). Mouse and human liver microsomal stability
was assayed at 0, 15, 30, 45 and 60 minutes, in triplicate.
Plasma stability. Cephalosporins 1, 5, and cephalexin, were spiked into lithium
heparin treated human and CDꢀ1 mouse plasma (bioreclammation) at 1 µg/mL. Spiked
samples were incubated at 37 °C, and extractions were performed at 5, 15, 30, 60 and 180
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minutes. The reactions were quenched and proteins precipitated at each time point by
adding 20 µL spiked plasma to 200 µL of extraction solvent containing
methanol:acetonitrile (1:1; vol/vol) and 10 ng/mL of verapamil (Toronto Research
Chemicals, Inc) as an internal standard (IS). In addition 20 µL of 1:1 acetonitrile:water
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(ACN:H O; vol/vol) were added. A reference sample was created by adding 20 µL of
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unspiked plasma to 200 µL of the extraction solvent. After the plasma enzymes were
denatured by the extraction solvent, 20 µL of a 1 µg/mL solution in 1:1 ACN:H O was
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added to the reference sample. Extracted samples were vortexed 5 minutes and then
centrifuged at 3000 RPM for 5 minutes. 100 µL of extract was transferred to 100 µL of
ddH 0 for LCꢀMS analysis. LCꢀMS analysis was performed with an Agilent 1260 liquid
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chromatography system coupled to a 4000 Qtrap mass spectrometer (AB Sciex) in MRM
(
multiple reaction monitoring) mode with positive electrospray ionization (ESI) and an
Agilent column, SBꢀC8, 2.1 x 30mm, 3.5 µm. Mobile phase A was 0.1% formic acid in
00% H O and mobile phase B was 0.1% formic acid in 100% acetonitrile. Injection
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volumes were routinely 2 µL. The ions monitored were: compound 1 (m/z 387.1/195.9),
compound 5 (391.1/199.9), cephalexin (348.1/158.1), and verapamil (455.4/165.2). The
percentage remaining was determined at each time point by dividing the sample
analyte/IS peak area ratio by the reference sample analyte/IS peak area ratio.
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Charcoal agar resazurin assay (CARA). The CARA was used as described. In
brief, 10 ꢀL aliquots from replicating or nonꢀreplicating MIC90 assay plates were
removed and spotted onto microplates containing 200 ꢀL 7H11ꢀOADCꢀcharcoal agar in
each well. The microplates were then incubated 7ꢀ10 days at 37° C at 20% O and 5%
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CO . The film of bacterial growth (microcolonies) on the microplates was semiꢀ
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quantitated by the addition of 40 ꢀL of a 1:1 (v/v) mixture of Alamar blue™ (AB) and
Tween80 (TW80) and 1 hour of further incubation at 37 °C at 20% O and 5% CO . In
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some cases, if CARA microplate appeared dry, all wells were preꢀwetted with 40 ꢀL PBS
prior to the addition of the AB:TW80 developing solution. Fluorescence was determined
by topꢀread with excitation at 530 nm and emission at 590 nm. The CARAꢀminimal
bactericidal concentration leading to ≥ 99% loss in CFUs (CARAꢀMBC ) was
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estimated as the lowest concentration of drug leading to complete loss of Alamar blue
fluorescence.
Macrophage infections. Primary bone marrow derived macrophage infections
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were performed as described.
In brief, ~ 1 x 10 macrophages isolated from 8ꢀweek
old female C57Bl6 mice were grown in 48 well plates in DMEM supplemented with 4.5
g/l glucose, 0.584 g/l Lꢀglutamine, 1 mM pyruvate, 10% FBS, 10% Lꢀcell conditioned
medium, containing or not 50 ng/mL recombinant mouse IFNγ, and infected with wildꢀ
type M. tuberculosis H37Rv at a multiplicity of infection of 1ꢀ5. Logꢀphase, wildꢀtype
M. tuberculosis was allowed to infect macrophages for 4 hours, after which medium and
extracellular M. tuberculosis were removed by two washes with PBS, and replaced with
fresh medium containing compounds or not at 1% DMSO final. At times indicated,
macrophages were washed and lysed with PBS supplemented with 0.5% TritonꢀX100.
Surviving bacilli were enumerated on 7H11ꢀOADC agar plates. Macrophage supernatants
were assayed for nitrite with the Greiss assay.
ASSSOCIATED CONTENT
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Supporting Information. Figures S1, S2, experimental and analytical details for
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synthetic analogs, and copies of H and C NMR spectra.
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CORRESPONDING AUTHOR INFORMATION
Carl Nathan, Weill Cornell Medical College, 212 746 6505, cnathan@med.cornell.edu,
Jeffrey Aubé, University of North Carolina – Chapel Hill, 919 966 9650, jaube@unc.edu
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ACKNOWLEDGMENTS
We thank S. Brickner (S.J. Brickner Consulting, LLC, Ledyard, CT), A. Mendozaꢀ
Losana, D. Barros, R. Bates and L. Ballell (GlaxoSmithKline, Tres Cantos, Spain), and
K. BurnsꢀHuang and K. Rhee (Weill Cornell Medical College) for insightful discussions
and suggestions. We thank F. Kaya (Public Health Research Institute, New Jersey
Medical School, Rutgers, the State University of New Jersey, Newark, NJ) for assistance
with plasma stability assays, and J. Glasheen (Weill Cornell Medical College) for work
that will be reported elsewhere. This work was supported by the TB Drug Accelerator of
the Bill and Melinda Gates Foundation, the Abby and Howard P. Milstein Program in
Translational Medicine, the National Institute of General Medical Sciences
(P50GM069663), the National Human Genome Research Institute (KU Specialized
Chemistry Center, U54 HG005031) and an NIH TB Research Unit (U19 AI111143). SSK
was supported by NIH grant K08AI108799. The Department of Microbiology and
Immunology is supported by the William Randolph Hearst Foundation.
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ABBREVIATIONS USED
AB, Alamar blue; ACN, acetonitrile; 7ꢀADCA, 7ꢀaminodeacetoxycephalosporanic acid;
CARA, charcoal agar resazurin assay; CDD, Collaborative Drug Discovery; CFUꢀ
surrogate assay; CFU, colonyꢀforming unit; DlaT, dihydrolipoamide acyltransferase;
DMEM, Dulbecco’s modified eagle medium; IFNγ interferon γ; IS, internal standard;
LDT, L,Dꢀtranspeptidase; MDT, modular dispense technology; MRM, multiple reaction
monitoring; Mtb, Mycobacterium tuberculosis; NR, non-replicating; OADC, oleic
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albumin dextrose catalase; PBS-Tyl; PBS containing tyloxapol; R, replicating; RNS,
reactive nitrogen species; TW80, Tween80
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