Preparation, gram-negative antibacterial activity, and hydrolytic stability of novel siderophore-conjugated monocarbam diols

Article abstract of DOI:10.1021/ml200012f

A novel series of monocarbam compounds exhibiting promising antibacterial activity against multidrug resistant Gram-negative microorganisms is reported, along with the synthesis of one such molecule MC-1 (1). Also reported are structure-activity relationships associated with the in vitro and in vivo efficacy of 1 and related analogues in addition to the hydrolytic stability of such compounds and possible implications thereof.

Full text of DOI:10.1021/ml200012f

LETTER
pubs.acs.org/acsmedchemlett
Preparation, Gram-Negative Antibacterial Activity, and Hydrolytic
Stability of Novel Siderophore-Conjugated Monocarbam Diols
Mark E. Flanagan,*,† Steven J. Brickner,^ Manjinder Lall,† Jeffrey Casavant,† Laura Deschenes,‡
Steven M. Finegan,§ David M. George,§ Karl Granskog,† Joel R. Hardink,|| Michael D. Huband,§
Thuy Hoang,† Lucinda Lamb,§ Andrea Marra,# Mark Mitton-Fry,† John P. Mueller,§ Lisa M. Mullins,§
Mark C. Noe,† John P. O'Donnell,§ David Pattavina,‡ Joseph B. Penzien,§ Brandon P. Schuff,† Jianmin Sun,†
David A. Whipple,r Jennifer Young,† and Thomas D. Gootzþ
†Worldwide Medicinal Chemistry, ‡Pharmaceutical Sciences, §Antibacterials Research Unit, and Pharmacokinetics, Dynamics and
Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States
^SJ Brickner Consulting, LLC, Ledyard, Connecticut 06339, United States
#Rib-X Pharmaceuticals, New Haven, Connecticut 06511, United States
rDel Shankel Structural Biology Center, University of Kansas, Lawrence, Kansas 66047, United States
þThomas Gootz Consulting, LLC, Estes Park, Colorado 80517, United States
S Supporting Information
b
ABSTRACT: A novel series of monocarbam compounds exhibiting promising antibacterial
activity against multidrug resistant Gram-negative microorganisms is reported, along with
the synthesis of one such molecule MC-1 (1). Also reported are structure-activity
relationships associated with the in vitro and in vivo efficacy of 1 and related analogues in
addition to the hydrolytic stability of such compounds and possible implications thereof.
KEYWORDS: Monocarbam, monobactam, β-lactam, MC-1, siderophore-conjugate,
antibiotic
ultidrug resistance among Gram-negative nonfermenters
such as Pseudomonas aeruginosa is a significant public health
U-78608 (2) is an example of the latter and was used as a
comparator agent for these studies (Figure 1).16 Monocarbams
are monocyclic β-lactams that are unique from other members of
this class, such as aztreonam (3), in that they contain a carbon-
centered activating group attached to the β-lactam nitrogen.
Siderophore-conjugated monocarbams like 2 and pirazmonam
(4)17 were not progressed beyond early development despite
impressive in vitro activity. This was likely due to their projected
high cost of goods, lack of Gram-positive activity, and insufficient
market potential, which at the time was dominated by effective
and less structurally complex, broad spectrum β-lactams. Today,
however, because of the changing landscape of the Gram-
negative arena driven largely by the antibiotic resistance de-
scribed above, this siderophore conjugate approach has received
renewed attention.18 Herein, we report on a novel series of
siderophore-conjugated monocarbam analogues and describe
the synthesis of one such molecule MC-1 (1), along with
structure-activity relationships (SAR) associating physical
property attributes with in vivo biological outcomes.
M
problem.1 Particularly concerning is the growing resistance that
these pathogens now exhibit to multiple classes of frontline
antibiotics.2 While broad-spectrum antibiotics such as carbape-
nems and fluoroquinolones still maintain activity against most
strains, there is a continual erosion of the effectiveness of
members of these classes against multidrug-resistant (MDR)
P. aeruginosa.3,4 Combining this fact with results of multiple
surveillance studies suggesting that up to 50% of all hospital
infections are MDR Gram-negative in nature makes the need for
new agents capable of addressing this medical threat readily
apparent.5,6 Few new introductions of antibiotics with signifi-
cantly improved activity against such MDR strains have occurred
over the past decade. Modifications of existing classes of anti-
biotics have proven unsuccessful in providing potent activity
against MDR strains that possess multiple resistance mechan-
isms. Focus in this field has shifted to efforts at finding anti-
microbials that enter MDR pathogens by novel mechanisms, thus
avoiding many common efflux and low permeability mutations.
One strategy for circumventing the resistance exhibited by
these pathogens involves exploiting the essential iron uptake
pathway of these Gram-negative microorganisms by conjugating
a siderophore to a β-lactam antibiotic.7-11 This approach was
investigated by a number of groups in the 1980s, with siderophore
conjugates pursued primarily withinthe cephalosporin and mono-
bactam β-lactam subclasses.12-15 The Upjohn monocarbam
On the basis of the encouraging in vitro data reported for 2, we
initiated a program with this compound as our lead. Early in vitro
evaluation of 2 in our hands showed it to be a potent Gram-
negative agent, exhibiting a minimum inhibitory concentration
Received: January 14, 2011
Accepted: February 19, 2011
Published: March 02, 2011
r
2011 American Chemical Society
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LETTER
Table 1. P. aeruginosa MICs for Initial Triazalone Substituted
Monocarbam Analogues
Figure 1. MC-1 (1) and related monobactam antibiotics.
(MIC)90 of 2 μg/mL against 91 clinical isolates of P. aeruginosa as
compared to >64 μg/mL for 3 and meropenem (Table 3). This
robust anti-Pseudomonal activity translated into excellent efficacy
in a murine in vivo systemic infection model [intraperitoneally
infected (IP infection model), Table 3]. However, somewhat
surprisingly, 2 did not perform well in a murine model of
respiratory tract infection (RTI PD50 > 150 mg/kg). This was
concerning since the lung is considered to be an essential organ
in which to demonstrate efficacy for any nosocomial, Gram-
negative agent. Reasons for the poor RTI performance exhibited
by 2 were not initially apparent; however, subsequent investiga-
tion provided possible explanations.
The hydrolytic instability of these monocarbam antibiotics
(discussed in more detail later) presented complications in
determining certain end points in assays requiring extended
aqueous incubations, such as plasma protein binding (PPB).
However, efforts to measure PPB for 2 suggested moderately
high binding despite the polar nature of this compound (fu-0.26
in mouse). As a result, we postulated that modulation of the
physical properties around 2 might affect PPB, thereby increasing
free drug concentrations available for activity in the lung. Because
most infections take place within the extravascular space, it is the
free concentrations within this compartment that are most
relevant for efficacy. Total drug distribution into the lung was
generally equivalent for all monocarbam analogues and similar to
that observed for aztreonam (lung/plasma ratios of 0.2-0.3).19
While total drug concentrations in tissue homogenates relative to
the plasma can provide some indication of penetration, these
data are often misleading, since uniform distribution of drug
concentrations is assumed. Concentrations observed in tissue
homogenates reflect an average of intracellular and extracellular
concentrations, as well as drug that is bound to protein. This
value could grossly underestimate the actual free drug concen-
trations in the extravascular space. In the absence of active
a For additional MICs, see ref 28.
transport processes, equilibrium between free drug in the
tissue and the plasma occurs passively at steady state. This has
been demonstrated in microdialysis experiments using several
β-lactam antibiotics.20 Cefpodoxime, for instance, is 25% bound
to human plasma and demonstrates greater than 2-fold higher
peak unbound concentrations in muscle tissue, relative to
cefixime (65% PPB). Thus, a strategy to reduce PPB to increase
free concentration in the tissue was pursued in an attempt to
increase pulmonary efficacy.
Integral to this strategy, was the need to find a position on
these molecules to manipulate physicochemical property space,
while maintaining adequate in vitro potency. We considered a
number of synthetically tractable positions on 2, before settling
on the triazalone (methyl) side chain. On the basis of earlier
reports, this was an unexplored part of the molecule that at the
same time was distal to portions that had previously been
optimized, such as the siderophore and aminothiazole-contain-
ing substructure (also present in 3). Efforts to incorporate new
functionality at this position of the triazalone were facilitated by
enablement of the oxadiazalone intermediate 10 (Scheme 1A).
This compound was prepared in good overall yield from
commercially available kojic acid (5), in six chemical steps.
(R)-3-Aminopropane-1,2-diol (11) was introduced in a two-step
sequence involving addition of the amine, followed by cycliza-
tion to form triazalone 12 (while Scheme 1A is specific for
construction of triazalone 12, many other triazalones of
interest were prepared from 10 in this or a related manner21).
Triazalone 12 was then activated as previously described16
by per-silylation using methylsilyltrifluoroacetamide (MSTFA)
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LETTER
Table 2. Additional Compounds Prepared To Study the Role
of Physical Properties in RTI Efficacy
in THF (Scheme 1B). Separately, monobactam precursor 1622
was treated with chlorosulfonylisocyanate in dichloromethane,
forming a sulfamoylchloride adduct; this intermediate was then
mixed with the activated triazalone 12, resulting in construc-
tion of the monocarbam moiety and affording 17 as the product.
Stepwise deprotection of 17 and reverse-phase chromato-
graphic purification of 1 was followed by treatment of this
material with 2 equiv of sodium bicarbonate in water. Final
isolation of the disodium salt of 1 was then accomplished by
lyophilization.23
Early SARs for compounds prepared in this manner are shown
in Table 1. The organism used for screening of MICs24,25 was a
virulent (non-MDR) strain of P. aeruginosa (1091-05) also used
for in vivo efficacy studies. The SAR data from this initial survey
suggested that the binding site was quite forgiving regarding
aspects of polarity or moderate size for this triazalone side chain.
Larger groups than methyl were accommodated well (e.g.,
compounds 18 and 19) in addition to compounds with polar
functionality such as a basic amine (20) or an alcohol
(compounds 21 and 22). The only side chains studied that were
not well tolerated were those containing very large groups (data
not shown) or those possessing aromatic functionality (e.g.,
compounds 23 and 24). This SAR is consistent with recent
crystallographic studies of 1 bound to penicillin binding protein 3
(PBP3), indicating a relatively large pocket in the protein where
this side chain resides.26
Encouraged by the flexibility in the SAR associated with this
side chain, we selected a set of compounds for more extensive
evaluation to address the question of triazalone side chain
properties as a function of in vivo performance. Because essen-
tially all compounds tested exhibiting good MICs against
P. aeruginosa 1091-05 (including 2) also worked well in the IP
infection model, we focused instead on RTI performance to
differentiate compounds. The compounds selected are shown
in Table 2. Compounds 1 and 25-28 all contained polar
functionality on this side chain, including at least one hydrogen
a R2 side chain clogP values calculated using ChemDraw.
Table 3. In Vitro and in Vivo Results for Study Compounds MC-1 (1) and 25 through 32 along with Comparator Agents
Aztreonam (3), Meropenem, and U-78608 (2)
MIC (μg/mL)
K. pneumoniae
A. baumannii
P. aeruginosa
MIC90a (μg/mL)
IP PD50 (mg/kg) P. aeruginosa
RTI PD50 (mg/kg)
compd
1000-02
AB-3167
1091-05
P. aeruginosa
1091-05 (95% CI)
P. aeruginosa 1091-05 (95% CI)
aztreonam (3)
>64
<1
0.5
0.5
0.25
0.5
0.5
1
32
0.25
1
8
>64 (n = 138)
>50
>200
meropenem
0.5
>64 (n = 138)
1.30 (0.4-2.1)
1.04 (0.6-1.4)
>150
U-78608 (2)
0.5
2
4 (2.4-5.7)
MC-1 (1)
25
0.5
1
0.125
0.125
0.125
0.125
0.125
0.5
0.5 (n = 138)
<1
20.6 (8.7-32.5)
15.7 (8.45-23.0)
18.6 (8.94-28.3)
32.7 (23.7-42.0)
25.0 (24.8-25.2)
>70.8
1
NT
26
0.5
1
1
NT
27
1
<1
NT
28
1
1
1
29
0.5
0.5
0.5
1
4
NT
30
31 b
2
0.5
NT
2.8 (2.1-3.4)
NT
>100
1.4 (0.9-1.9) c
2
0.25
0.5 (n = 51)
32
4
0.25
0.5 (n = 51)
NT
21.3 (13.0-30.0)
a Ninety-one clinical isolates tested unless otherwise indicated. b PPB for 31, fu-0.51. c Lower inoculum was used [PD50 for 1 was 6.9 mg/kg (5.0-8.8) in
this experiment]. NT, not tested.
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LETTER
Scheme 1. Representative Monocarbam Synthesis: Preparation of MC-1 (1)a,b
a (A) Preparation of the substituted triazalone intermediate 12. Reagents and conditions: (a) Benzylchloride, K2CO3, DMF. (b) CrO3, H2SO4, H2O/
acetone, -5 ꢀC to room temperature. (c) NH4OH, H2O, 80 ꢀC. (d) Benzylchloride, K2CO3, DMF, 80 ꢀC. (e) Hydrazine monohydrate, methanol,
65 ꢀC. (f) Carbonyldiimidazole, THF. (g) Compound 11, THF, 60 ꢀC. (h) Aqueous KOH, 100 ꢀC. b (B) Construction of the monocarbam moiety
followed by stepwise deprotection leading to 1 disodium salt. Reagents and conditions: (i) H2, 10% Pd/C, toluene/ethanol (assumed quant., used
directly in 20% theoretical excess). (j) Toluene/ethanol, 5 h. (k) MSTFA, THF, 12, concentrate at 60 ꢀC. (l) ClSO2NCO, 16, CH2Cl2, 0 ꢀC. (m) Add 12
in fresh THF to 16, 0 ꢀC to room temperature. (n) Pd black, H2, acetic acid, THF. (o) Trifluoroacetic acid, CH2Cl2, 0 ꢀC. (p) NaHCO3 (2 equiv), H2O.
(q) Freeze at -80 ꢀC and lyophilize.
bond donor. However, the side chains of compounds 29 and 30
were more representative of the methyl group of 2 in terms of
their hydrophobicity while still maintaining an overall size
comparable to the other compounds being investigated. All of
the compounds studied exhibited excellent MICs against an
extended spectrum β-lactamase (ESBL)-producing strain of
Klebsiella pneumoniae (1000-02) and a recent clinical isolate of
Acinetobacter baumannii (AB-3167), in addition to P. aeruginosa
(1091-05); furthermore, these compounds also displayed com-
parable MIC90 values against multiple clinical isolates of
P. aeruginosa (Table 3).
The results of this study confirmed that compounds contain-
ing more polar triazalone functionality were superior in terms of
their RTI PD50 values. By contrast, compounds 29 and 30
behaved more like 2 in that they did not provide significant
RTI protection within the same dose range. These data are
consistent with free fraction determinations (PPB), which gen-
erally increased with the inclusion of a polar functionality [e.g.,
fu-0.46 for 1 vs 0.26 for 2 (mouse)]. Somewhat fortuitously, the
increase in free fraction did not result in a proportional increase
in drug clearance. This is likely due to the fact that drug
excretion/clearance for these compounds is not exclusively
mediated by glomerular filtration of unbound drug into the
urine. Taken collectively, these findings were consistent with the
observed RTI PD50 data, suggesting that improved tissue activity
is achieved through higher free drug concentrations at the site of
infection. Lack of differentiation and very low PD50 values in the
IP model were likely a reflection of the high free drug concentra-
tions obtained systemically for all analogues. Combining these
results with studies of resistance frequency27 and additional MIC
testing, 1 emerged as a compound of interest for further profiling.
As alluded to earlier, one complicating factor associated with
studying 1 was its hydrolytic instability. The instability of
monocarbams is largely a manifestation of the electron-with-
drawing nature of the sulfonylurea activating group. In addition
to complicating certain assays requiring longer-term incubations,
the instability of the monocarbam moiety also impeded the
synthetic scale-up of 1. Despite this obstacle, sufficient com-
pound (1) was produced to conduct additional pharmacology28
(Table 4) and toxicology29 studies. The pH dependence asso-
ciated with the hydrolytic ring-opening of the β-lactam moiety of
1 was measured, and the results are as follows: at pH 6.0, 1 was
relatively stable; however, at pH 8.0, 50% degradation occurred
over 100 h at 37 ꢀC (see the Supporting Information, Figure 1).
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ACS Medicinal Chemistry Letters
LETTER
Table 4. Additional MIC50/90 Testing for MC-1 (1) and
Comparator Agents Aztreonam (3) and Meropenem
’ ASSOCIATED CONTENT
S
Supporting Information. In vivo efficacy assays, protein
b
MIC50/MIC90 (μg/mL)
binding and hydrolytic stability protocols, and cumulative % of
susceptibility data for 1, 3, and meropenem, in addition to
experimental procedures and characterization data for com-
pounds 1 and 6-32. This material is available free of charge
via the Internet at http://pubs.acs.org.
organism (no. tested)
MC-1 (1) aztreonam (3) meropenem
Citrobacter freundii (21)
Enterobacter aerogenes (21)
Escherichia coli (79) a
Klebsiella pneumoniae (180) b
Serratia marcescens (25)
P. aeruginosa (138) d
0.5/2
0.25/64
16/64
0.06/0.25
0.06/0.25
0.06/0.06
0.06 c/32 c
0.06/>64
8/>64
0.125/0.25
0.25/2
1/>64
’ AUTHOR INFORMATION
0.5/8
64/>64
0.5/>64
16/>64
>64/>64
32/>64
0.125/0.5
0.125/0.5
Corresponding Author
*E-mail: mark.e.flanagan@pfizer.com.
Stenotrophomonas maltophilia (30) 1/2
>64/>64
1/32
Funding Sources
This research was sponsored by Pfizer, Inc.
Acinetobacter spp. (31) 16/>64
a Includes 34 ESBL, 11 CTX-M (cefotaxime active-Munich, ESBL
subtype), and 34 recent clinical isolates. b Includes 109 ESBL, 22 K.
pneumoniae carbapenemase (KPC), and 49 recent clinical isolates. c N =
98 strains, includes 59 ESBL, 21 KPC, and 18 recent clinical isolates.
d Includes 17 cystic fibrosis isolates, 15 metallo-β-lactamase producers,
and 106 recent clinical isolates.
’ ACKNOWLEDGMENT
We are grateful to Dr. Michael Barbachyn, Prof. Alvin
Crumbliss (Duke University), Dr. Thomas Magee, Dr. Anthony
Marfat, Prof. Marvin Miller (University of Notre Dame), and
Prof. Ute M€ollmann (Hans Kn€oll Institute) for many helpful
conversations. We also acknowledge David Boyles, Dr. Sally Gut-
Ruggeri, and Daniel Widlicka for helpful input on monocarbam
syntheses; Glenn Wilcox and Ruchi Thombre for helpful in-
formation and for performing separations and isolations of
monocarbam products; Jian Lin and Mark Niosi for PPB
measurements; and Hongying Gao for bioanalytical analysis.
As a consequence, one strategy employed to improve the stability
of 1 was to incorporate a methyl group at the C4 position of the
β-lactam ring, such as that, which is present on 3. In the case of 3,
the primary role for the C4-methyl is reported to be improved
stability toward β-lactamases.30 However, other studies also
reveal a role for inductive effects of C4-β-lactam substituents in
activating (or deactivating) the β-lactam ring toward hydroxide
ion.31 To test this hypothesis, compounds 31 and 32 were
prepared by an analogous synthetic sequence as that described
for 1 (Scheme 1). As can be seen from Table 3, these compounds
exhibited comparable in vitro data and in vivo efficacy at least as
good as that determined for 1 and 25.32 Compound 31 was
evaluated for its hydrolytic stability alongside 1 and meropenem.
At pH 6.0 and 37 ꢀC, 31 showed similar stability; however, at pH
8.0, 31 exhibited measurably improved hydrolytic stability as
compared to 1 and meropenem33 over the 100 h incubation
(∼20% degradation as compared to ∼50% for 1 and merope-
nem; see the Supporting Information, Figure 1). Studies are
ongoing to determine if the nature and magnitude of this stability
improvement is sufficient to overcome the obstacles associated
with developing a compound of this complexity.
’ ABBREVIATIONS
MDR, multidrug-resistant; MIC, minimum inhibitory concentra-
tion; SAR, structure-activity relationships; fu, fraction unbound;
IP, intraperitoneal; RTI, respiratory tract infection; PPB, plasma
protein binding; MSTFA, methylsilyltrifluoroacetamide; PBP3,
penicillin binding protein 3; ESBL, extended spectrum β-lacta-
mase; CTX-M, cefotaxime active-Munich ESBL subtype; KPC,
Klebsiella pneumoniae carbapenemase.
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