In vitro antibacterial activity of CE-156811, a novel analog derived from hygromycin A

Article abstract of DOI:10.1128/AAC.01326-07

We evaluated a novel truncated hygromycin A analog in which the furanose ring was replaced with a 2-fluoro-2-cyclopropylethyl substituent for its activity against multidrug resistant gram-positive bacteria and compared its activity to the activities of linezolid, quinupristin-dalfopristin, and vancomycin. CE-156811 demonstrated robust in vitro activity against gram-positive bacteria that was comparable to that of linezolid. Copyright

Full text of DOI:10.1128/AAC.01326-07

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2008, p. 2663–2666
0066-4804/08/$08.00ϩ0 doi:10.1128/AAC.01326-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 7
In Vitro Antibacterial Activity of CE-156811, a Novel Analog Derived
from Hygromycin Aᰔ
Gregory G. Stone,1* Dennis Girard,1 Steve Finegan,1 Joan Duignan,1 John Schafer,1
Meghan Maloney,1 Richard P. Zaniewski,1 Steven J. Brickner,1 Sarah K. Wade,1
Phuong T. Le,3 and Michael D. Huband2
Pfizer Global Research & Development, Groton/New London Laboratories, Pfizer Inc., Groton, Connecticut 063401;
Pfizer Global Research & Development, Ann Arbor Laboratories, Pfizer Inc., Ann Arbor, Michigan 481052; and
Pfizer Global Research & Development, La Jolla Laboratories, Pfizer Inc., La Jolla, California 921213
Received 15 October 2007/Returned for modification 6 November 2007/Accepted 9 April 2008
We evaluated a novel truncated hygromycin A analog in which the furanose ring was replaced with a
2-fluoro-2-cyclopropylethyl substituent for its activity against multidrug resistant gram-positive bacteria
and compared its activity to the activities of linezolid, quinupristin-dalfopristin, and vancomycin. CE-
156811 demonstrated robust in vitro activity against gram-positive bacteria that was comparable to that
of linezolid.
The continuing emergence of resistance to existing antibac-
terial agents in gram-positive organisms has created the need
for the discovery of novel antibacterial agents that possess
activity against these resistant bacteria (1, 5, 9). The isolation
of hygromycin A from Streptomyces hygroscopicus was first re-
ported in 1953 (10), and it was demonstrated to possess weak
activity against gram-positive organisms (6). The mechanism of
action of hygromycin A has been demonstrated to be protein
synthesis inhibition through interference with peptide bond
formation (2).
The current study was designed to evaluate the in vitro
antibacterial activity of CE-156811 against various gram-posi-
tive and gram-negative isolates in comparison to the activities
of other marketed agents.
(This study was presented in part at the 46th Interscience
Conference on Antimicrobial Agents and Chemotherapy, San
Francisco, CA, 27 to 30 September 2006 [4, 11].)
CE-156811 is a novel truncated hygromycin A analog with
potent activity against multidrug-resistant Streptococcus pneu-
moniae, vancomycin-resistant enterococci (VRE), methicillin-
resistant Staphylococcus aureus (MRSA), community-acquired
MRSA, and vancomycin-intermediate S. aureus. The chemical
structures of CE-156811 and hygromycin A are displayed in
Fig. 1. All bacterial strains used in this study were from the
Pfizer collection of clinical bacterial cultures and were ob-
tained between 2000 and 2004 from several sources, including
various surveillance studies and clinical trials. Transcription
and translational inhibition studies of protein synthesis were
performed as described previously (7). Susceptibility testing
was performed by the broth microdilution method, as de-
scribed by the CLSI (formerly the NCCLS) (8). In vitro killing
kinetics studies were performed with 25-ml volumes of Muel-
FIG. 1. Structures of hygromycin A and CE-156811.
ler-Hinton broth (supplemented with 3% lysed horse blood for
streptococci) with agitation. CE-156811 was tested at 1ϫ, 2ϫ,
4ϫ, and 8ϫ the MIC; and samples were plated for colony
count determinations at 0, 2, 4, 8, and 24 h. A Ն3-log reduction
from the original inoculum at 24 h was considered bactericidal.
CE-156811 was obtained by alkylation of the phenol {3-(2,5-
difluoro-4-hydroxy-phenyl)-2-methyl-N-[(3aS,4R,5R,6S,7R,7aR)-
4,6,7-trihydroxy-hexahydro-benzo[1,3]dioxol-5-yl]-acrylamide}
with (R)-methanesulfonic acid 2-cyclopropyl-2-fluoro-ethyl
ester and potassium carbonate in anhydrous N,N-dimethylform-
amide at 75°C for 5 days. The phenol can be derived from the
corresponding O-allyl ether (3) by a Pd(Ph3P)4 (where Ph is
phenyl)-catalyzed deallylation with dimedone. CE-156811 was
found to undergo slow hydrolysis at 25°C in water (to the
extent of 4% decomposition over 24 h) to generate the corre-
sponding cyclopropyl alcohol (validated by independent syn-
thesis) resulting from the hydrolysis of the cyclopropyl alkyl
* Corresponding author. Present address: Infection Biosciences,
AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Waltham, MA
02451. Phone: (781) 839-4425. Fax: (781) 839-4800. E-mail: gregory
.stone@astrazeneca.com.
ᰔ Published ahead of print on 21 April 2008.
2663

2664
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Susceptibilities of gram-positive clinical isolates to CE-156811 and comparator agents
MIC (␮g/ml)
Organism (no. of isolates)
Test agent
Range
50%
90%
Staphylococcus aureus, methicillin susceptible (28)
Staphylococcus aureus, methicillin resistant (18)
Staphylococcus aureus, glycopeptide intermediate (8)
Staphylococcus epidermidis, methicillin susceptible (23)
Other coagulase-negative staphylococci (23)
Enterococcus faecalis, vancomycin susceptible (19)
Enterococcus faecalis, VRE (28)
CE-156811
0.25–1
1–4
0.25–2
0.25–2
0.5
2
0.5
0.5
1
Linezolid
2
Quinupristin-dalfopristin
Vancomycin
0.5
1
CE-156811
Linezolid
Quinupristin-dalfopristin
Vancomycin
0.25–1
1
0.06–0.25
0.5–2
0.5
1
0.125
1
0.5
1
0.25
2
CE-156811
Linezolid
Quinupristin-dalfopristin
Vancomycin
0.125–0.5
0.5–1
0.06–0.25
4–8
CE-156811
Linezolid
Quinupristin-dalfopristin
Vancomycin
0.06–0.5
0.25–4
0.06–1
0.25–2
0.12
0.5
0.06
1
0.25
1
0.06
2
CE-156811
Linezolid
Levofloxacin
Vancomycin
0.5–2
1–2
0.06–64
1–4
1
2
0.25
2
2
2
32
2
CE-156811
Linezolid
Quinupristin-dalfopristin
Vancomycin
0.25–1
0.5–2
4–32
1–4
1
2
8
2
2
2
32
4
CE-156811
Linezolid
Quinupristin-dalfopristin
Vancomycin
0.5–2
0.5–16
4–16
1
2
8
2
2
16
8–Ͼ32
Ͼ32
Ͼ32
Enterococcus faecium, vancomycin susceptible (13)
Enterococcus faecium, VRE (49)
CE-156811
0.5–2
2–4
0.5–4
1
1
2
1
1
2
2
2
1
Linezolid
Quinupristin-dalfopristin
Vancomycin
CE-156811
Linezolid
Quinupristin-dalfopristin
Vancomycin
0.5–2
1–4
0.25–4
8–Ͼ32
1
2
1
2
4
2
Ͼ32
Ͼ32
Streptococcus pneumoniae, penicillin susceptible (17)
CE-156811
Linezolid
0.12–0.5
0.5–1
0.25
1
0.5
1
Quinupristin-dalfopristin
Vancomycin
Penicillin
0.06–0.5
0.25
0.015–0.06
0.12
0.25
0.015
0.25
0.25
0.015
Streptococcus pneumoniae, penicillin nonsusceptible (21)
Streptococcus pyogenes (28)
CE-156811
0.06–0.5
0.25–1
0.25
0.5
0.25
0.25
2
0.5
1
Linezolid
Quinupristin-dalfopristin
Vancomycin
Penicillin
0.12–1
1
0.12–0.25
0.12–8
0.25
4
CE-156811
Linezolid
0.5–1
1–4
0.5
2
1
2
Quinupristin-dalfopristin
Vancomycin
Levofloxacin
0.25–1
0.5–2
0.25–2
0.5
0.5
0.5
0.5
0.5
2
Listeria monocytogenes (10)
CE-156811
Linezolid
Levofloxacin
Vancomycin
4–8
2
0.5–1
1
4
2
1
1
8
2
1
1
Continued on facing page

VOL. 52, 2008
NOTES
2665
TABLE 1—Continued
MIC (␮g/ml)
Organism (no. of isolates)
Test agent
Range
50%
90%
Corynebacterium spp. (30)
CE-156811
Linezolid
Levofloxacin
Vancomycin
0.5–1
0.12–1
0.25–64
0.25–1
1
1
1
32
1
1
0.5
0.5
Neisseria spp. (16)
CE-156811
Levofloxacin
0.5–8
0.004–0.125
4
8
0.008
0.008
Moraxella catarrhalis (30)
CE-156811
Levofloxacin
Amoxicillin
0.25–4
0.03–0.06
0.015–16
2
0.06
1
4
0.06
8
Haemophilus influenzae (67)
Enterobacteriaceaea (214)
CE-156811
Levofloxacin
Amoxicillin
1–16
0.004–0.5
0.25–64
4
8
0.015
2
0.25
32
CE-156811
Levofloxacin
Ceftazidime
16–Ͼ64
0.008–Ͼ64
0.06–Ͼ64
Ͼ64
0.06
0.5
Ͼ64
8
Ͼ64
Gram-negative nonfermentersb (82)
CE-156811
Levofloxacin
Ceftazidime
16–Ͼ64
0.06–Ͼ64
1–Ͼ64
Ͼ64
2
16
Ͼ64
16
Ͼ64
Legionella pneumophila (10)
CE-156811
Levofloxacin
0.25–2
0.002–0.015
1
2
0.004
0.015
a Ten Citrobacter diversus, 20 Citrobacter freundii, 17 Enterobacter aerogenes, 21 E. cloacae, 31 E. coli, 13 Klebsiella oxytoca, 30 Klebsiella pneumoniae, 14 extended-
spectrum ␤-lactamase-producing K. pneumoniae, 15 Serratia marcescens, 11 Proteus mirabilis, 6 Proteus vulgaris, 13 Morganella morganii, 1 Providencia rettgeri, 6
Salmonella sp., and 6 Shigella sp. isolates.
b Twenty-three P. aeruginosa, 24 Burkholderia cepacia, 19 Acinetobacter sp., and 17 Stenotrophomonas maltophilia strains.
fluoride. As the hydrolysis presumably involves the intermedi-
acy of the cyclopropyl methyl carbocation, a potential alkylator,
this finding precluded our further development of this com-
pound. The comparator agents were supplied from outside
vendors (vancomycin was purchased from Sigma Chemical
Co.; quinupristin-dalfopristin was provided by Sanofi-Aventis,
France; and linezolid was purchased from Sequoia Research
Products, Pangbourne, United Kingdom).
CE-156811 is structurally diverse from the original parent
molecule of hygromycin A. However, determination of the
inhibition of protein synthesis by a coupled transcriptional-
translational inhibition assay demonstrated that the potency
was very similar to that of hygromycin A. CE-156811 inhibited
transcription and translation at 0.149 ␮M, and hygromycin A
inhibited transcription and translation at 0.190 ␮M. Linezolid
inhibited transcription and translation at 2.6 ␮M, indicating
that CE-156811 may be a more potent inhibitor of ribosome
function than linezolid. The in vitro activities of CE-156811
and the comparator agents are summarized in Table 1 as the
range of MICs, the MIC50s, and the MIC90s. Overall, CE-
156811 had activity similar to or greater than the activities of
the comparator agents (linezolid, vancomycin, and quinupristin-
dalfopristin) against the majority of gram-positive organisms.
CE-156811 had limited activity against gram-negative organ-
TABLE 2. Susceptibilities of linezolid-resistant gram-positive clinical isolates to CE-156811 and comparator agents
MIC (␮g/ml)
Mutant allele
Organism
Strain
(G2576)a
CE-156811
Linezolid
Vancomycin
Q-Db
Chloramphenicol
S. aureus
ATCC 29213
01A1220
01A1221
ATCC 29212
03A1128
03B1073
0/5
5/5
5/5
0/4
4/4
0/6
1/6
2/6
2/6
3/6
5/6
1
16
16
1
2
Ͼ32
Ͼ32
2
0.5
1
0.06
0.06
0.06
4
8
Ͼ32
32
8
S. aureus
S. aureus
1
E. faecalis
E. faecalis
E. faecium
E. faecium
E. faecium
E. faecium
E. faecium
E. faecium
2
1
16
1
Ͼ32
2
Ͼ32
Ͼ32
8
Ͼ32
Ͼ32
Ͼ32
Ͼ32
Ͼ32
0.5
0.5
03B1077
2
8
0.125
0.5
8
03B1074
4
32
32
32
16
32
03B1075
4
32
0.5
03B1078
4
32
0.5
03B1076
8
32
0.25
a The data represent the number of mutant alleles at position G2576 of 23S rRNA/total number of possible mutant alleles.
b Q-D, quinupristin-dalfopristin.

2666
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
isms. Of the gram-negative members of the family
Enterobacteriaceae and nonfermentative bacteria, no one par-
ticular species had MICs at the lower end of the range of
MICs. One strain of Acinetobacter species, one strain of Shi-
gella species, one strain of Escherichia coli, and one strain of
Enterobacter cloacae had MICs in the 16- to 32-␮g/ml range,
whereas all the other strains had MICs of Ͼ64 ␮g/ml. We also
investigated the activity of CE-156811 against linezolid-non-
susceptible staphylococci and linezolid-resistant enterococci.
CE-156811 has reduced activity against linezolid-resistant S.
aureus and linezolid-nonsusceptible enterococci (Table 2), in-
dicating the potential for cross-resistance to the oxazolidinone
class of antibacterial agents. The MICs were elevated approx-
imately 16-fold in relation to the MIC for a wild-type strain of
S. aureus. It should be noted that the two staphylococcal iso-
lates were clearly distinct clones, as determined by pulsed-field
gel electrophoresis (data not shown), and had the G2576U
mutation at all five alleles. The linezolid-resistant enterococcal
organisms used in this study were recent clinical isolates that
had an identified number of mutant alleles at G2576 in the 23S
rRNA gene. There were clear increases in the MICs of CE-
156811 with an increasing number of mutant alleles in the
enterococci; however, it is not quite clear if the loss in activity
was due to the G2576U mutations alone or was due to other
factors that may have a role in decreasing the activity of CE-
156811.
The bactericidal activity of CE-156811 against several gram-
positive bacterial strains was evaluated (Fig. 2). CE-156811
treatment resulted in an approximately 1.5-log reduction at
24 h against all the strains tested, indicating that CE-156811 is
bacteriostatic.
Overall, CE-156811 has potent in vitro antibacterial ac-
tivity that is similar to that of linezolid against a wide range
of gram-positive pathogens. It also appears that mutations
of G2576U in the 23S rRNA affect the activity of CE-
156811.
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FIG. 2. Killing kinetics of CE-156811 against several gram-positive
bacterial species: (A) MRSA 01A1095 (MIC, 1 ␮g/ml); (B) E. faecium
03B1022 (VRE; MIC, 1 ␮g/ml); (C) S. pyogenes 02C0203 (MIC, 0.25
␮g/ml); and (D) S. pneumoniae 02J1046 (MIC, 0.125 ␮g/ml).