Selective antibacterial agents

57 Pages·2013·2.31 MB·English

US 20030105143A1
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2003/0105143 A1
Ammendola et al. (43) Pub. Date: Jun. 5, 2003
(54) SELECTIVE ANTIBACTERIAL AGENTS A1 and A2 each independently represent an optionally
substituted Cl-Czo-alkyl group Which may contain
(76) Inventors: Aldo Ammendola, Munchen (DE);
one or more group(s) Z, or a mono cyclic or poly
Bernd Kramer, Aachen (DE); Wael
cyclic optionally substituted aromatic or non-aro
Saeb, Pinnegg Martinsried (DE)
matic ring system Which may contain one or more
Correspondence Address: group(s) X, and in case of a polycyclic ring system,
Carol A. Schneider said system contains at least one aromatic ring;
IRELL & MANELLA LLP
Suite 900 Z is selected from the group consisting of S, O, N, NR4,
1800 Avenue of the Stars CO, CO2, CS, SO or SO2
Los Angeles, CA 90067 (US)
X is selected from the group consisting of S, O, N, NR4,
(21) Appl. No.: 10/094,301 SO or $02;
(22) Filed: Mar. 8, 2002 said substituted ring system carries a substituent R3 on
one or more of the carbon atoms of said ring system;
Related US. Application Data
said substituted Cl-Czo-alkyl group carries a substitu
(63) Continuation of application No. PCT/EP01/012875,
ent R3 on one or more of the carbon atoms of said
?led on Nov. 7, 2001. alkyl group;
Publication Classi?cation R3 is independently H, OR4, SR4, hydroxyalkyl,
hydroxyalkylamino, cycloalkyl, halogen, haloalkyl,
(51) Int. c1.7 ....................... .. A61K 31/44; A61K 31/40; haloalkyloxy, N02, CN, SO2NR4R5, CO2NR4R5,
A61K 31/137; A61K 31/13
COR4, CO2R4, SO2R4, SO3R4, NR4R5, alkyl, aryl or
(52) US. Cl. ....................... .. 514/357; 514/649; 514/408;
heteroaryl;
514/674
R4 is H, alkyl, cycloalkyl, aryl or heteroaryl;
(57) ABSTRACT
R5 is H, O-alkyl, O-aryl, alkyl, heteroaryl or aryl;
The present invention relates to the use of compounds of the
general Formula (I)
Y1 and Y2 are independent from each other C=O,
c=s, so2 or C=NR5;
pis0,mis0,nis0;
orpis0,mis1,nis1;
for the regulation of the quorum sensing system of micro orpis1,mis0,nis1;
organisms Wherein in Formula (I),
orpis0,mis0,nis1;
R is H, alkyl, cycloalkyl, aryl or heteroaryl;
R1 is H, alkyl, cycloalkyl, aryl or heteroaryl; orpis1,mis0,nis0;
R2 is H, alkyl, cycloalkyl, aryl or heteroaryl; orpis1,mis1,nis1.
Patent Application Publication Jun. 5, 2003 Sheet 1 0f 6 US 2003/0105143 A1
Figure 1: General N-acyl-L-homoserine lactone structure (R = acyl side chain)
Patent Application Publication Jun. 5, 2003 Sheet 2 of 6 US 2003/0105143 A1
3,0
control
2,5 - #27
#42
#43
2,0 - #36
OD600nm
1,5 -
1,0 -
0,5 -
0,0 -
10
time (hours)
Figure 2: In?uence of representative compounds on the growth of E. coli MT102
(pSB403).
Patent Application Publication Jun. 5, 2003 Sheet 3 of 6 US 2003/0105143 A1
120
100 -
RPAe[rlcoattt%eiiavvseie] t y on O |
a: o
|
8
20
Figure 3: Inhibitory effect of several compounds on the protease production of
P. aeruginosa PAOl -J P2.
Patent Application Publication Jun. 5, 2003 Sheet 4 of 6 US 2003/0105143 A1
3,5
30 _ _.—— control
—-——v——— #42
2,5 ' —— —l — _ #36
—-—El—-—— #6
2,0 -
E ———0—— #7
5 ——<>— #32
3 1’5 #65
O
0
1,0 -
0,5 -
0,0 -
l I I I I I I
time (hours)
Figure 4: In?uence of the tested compounds on the growth of P. aeruginosa PAO-JPZ.
Patent Application Publication Jun. 5, 2003 Sheet 5 of 6 US 2003/0105143 A1
A B
120
RAe[lamt%ivne] m
Figure 5: Inhibitory effect of several compounds (0,4 mM) on the bio?lm formation of
Burkholderia cepacia H111. A: statistical data of at least ?ve separate
representative experiments; B: microtitre dish image.
Patent Application Publication Jun. 5, 2003 Sheet 6 0f 6 US 2003/0105143 A1
3,5
3 o —O— control _ .9»?
2,5 -
2,0 -
1,5 -
1,0 -
0,5 -
0,0 -
time (hours)
Figure 6: In?uence of the tested compounds (0,4 mM) 0n the growth of Burkholderia
cepacia H111.
US 2003/0105143 A1 Jun. 5, 2003
SELECTIVE ANTIBACTERIAL AGENTS These molecules, also referred to as “pheromones” or “quo
romones”, comprise a homoserine lactone moiety linked to
[0001] The present invention relates to the use of com
an acyl side chain. Bacteria use this signaling system to
pounds such as amide, carbaZide, hydraZide, urea, and
monitor their population cell density in a process referred to
guanidine derivatives as selective inhibitors of bacterial
pathogens. In particular the invention refers to a family of as “quorum sensing”. In each cell of a population an HSL
compounds that block the quorum sensing system of Gram synthase from usually the LuxI family of proteins produce a
negative bacteria, a process for their manufacture, pharma low basal level of diffusible HSLs. The HSL concentration
ceutical compositions containing them and to their use for increases with bacterial population density until a threshold
the treatment and prevention of microbial damages and concentration is reached which results in expression of
diseases, in particular for diseases where there is an advan various HSL-dependent genes through an HSL-receptor
tage in inhibiting quorum sensing regulated phenotypes of protein belonging generally to the LuxR family of transcrip
pathogens. tional regulators. This HSL-receptor protein complex serves
[0002] Many microorganisms, including bacteria, fungi, not only as positive transcription regulator of quorum sens
ing regulated genes but also as positive regulator for the
protoZoa and algae cause severe damages or diseases in
HSL synthesis itself. Therefore, the entire system is ampli
different areas such as industry, agriculture, environment
and medicine. Especially bacteria as human pathogens cause ?ed via a process of autoinduction.
tremendous costs in public health systems worldwide. The
[0004] This system was ?rst discovered in the biolumi
continuing emergence of multiple-drug-resistant bacterial
nescent marine bacteria Vibrio harveyi and V ?scheri where
strains has necessitated ?nding new compounds that can be
it is used to control bioluminescence expression. In recent
used in antibacterial treatment. There are two broad strate
years it has become apparent that many Gram-negative
gies for the control of bacterial infection: either to kill the
bacteria employ one or more quorum sensing systems com
organism or to attenuate its virulence such that it fails to
prising HSL derivatives with different acyl side chains to
adapt to the host environment. The latter approach has,
regulate in a cell-density dependent manner a wide variety
however, lacked speci?c targets for rational drug design.
of physiological processes such as swarming motility, bio
The discovery that Gram-negative bacteria employ a signal
?lm formation, pathogenicity, conjugation, bioluminescence
transduction pathway comprising a small molecule to glo
or production of pigments and antibiotics (Table 1, for
bally regulate the production of virulence determinants
reviews and further references see, e.g.: Fuqua et al., Ann.
offers such a novel target.
Rev. Microbiol. 50:727-51, 1996; Fuqua & Greenberg, Curr.
[0003] A wide variety of Gram-negative bacteria produce Opinion Microbiol. 11183-89, 1998; Eberl, Syst. Appl.
N-acyl-L-homoserine lactone (AHL or HSL, FIG. 1) deriva Microbiol. 22:493-506, 1999; De Kievit & Iglewski, Infect.
tives as signal molecules in intercellular communication. Immun. 68:4839-49, 2000).
TABLE 1
Summary of HSL-based guorum sensing systems
Regulatory
Bacterium proteins Major HSL HSL-regulated phenotype
Aeromonas hydrophila AhyR, AhyI C4-HSL Extracellular protease,
bio?lm formation
Aeromonas salmonicida AsaR, AsaI C4-HSL Extracellular protease
Agrobacterium tumefaciens TraR, TraI 3-oxo-C8-HSL Conjugal transfer
Burkholderia cepacia CepR, CepI C8-HSL Protease, lipase, ornibactin
synthesis, bio?lm formation,
swarming motility
Chromobacterium violaceum CviR, CviI C6-HSL Antibiotics, violacein,
exoenzymes, cyanide
Enterobacter agglomerans EagR, EagI 3-oxo-C6-HSL Unknown
Erwinia carotovora CarR, (CarI) 3-oxo-C6-HSL Carbapenem antibiotics,
ExpR, ExpI exoenzyme production
Erwinia chrysanthemi ExpR, ExpI 3-oxo-C6-HSL Pectinase expression
(EchR, EchI)
Escherichia coli SdiA Unknown Cell division, virulence
factor production
Nitrosomonas europaea Unknown 3-oxo-C6-HSL Emergence from lag phase
Obesumbacterium proteus OprR, OprI 3-oxo-C6-HSL Unknown
Pantoea stewartii EsaR, EsaI 3-oxo-C6-HSL Exopolysaccharide
production, virulence factor
production
Pseudomonas aeruginosa LasR, LasI 3-oxo-C12- Extracellular virulence
HSL factors, Xcp, bio?lm
formation, RpoS, RhlR
Pseudomonas aeruginosa RhlR, RhlI C4-HSL Extracellular virulence
factors, cyanide, lectins,
pyocyanin, rhamnolipid, type
4 pili, twitching motility
US 2003/0105143 A1 Jun. 5, 2003
TABLE 1-continued
Summary of HSL-based guorum sensing systems
Regulatory
Bacterium proteins Major HSL HSL-regulated phenotype
Pseudamanas aureafaciens PhzR, PhzI C6-HSL Phenazine antibiotics
Pseudamanas ?uarescens HdtS 3-hydroxy-7- Unknown
cis-C14-HSL
Ralstania salanacearum So1R, So1I C8-HSL Unknown
Rhizabium etli RaiR, Rail 7 HSLs Root nodulation
Rhizabium leguminasarum RhiR 3-hydroxy-7- Nodulation, bacteriocin,
cis-C14-HSL stationary phase survival
Rhizabium leguminasarum RhiR, RhiI C6-HSL, rhizome interactions
CS-HSL
Rhadabacter sphaeraides CerR, CerI 7-cis-C14-HSL Clumping factor
Serratia liquefaciens SwrR, SwrI C4-HSL Swarming motility, protease,
serrawettin W2, lipase
Vibria anguillarum VanR, VanI 3-oxo-C10- Unknown
HSL
Vibria anguillarum VanM, C6-HSL, Unknown
VanN 3-.hydroxy-C6
HSL
Vibria ?scheri LuxR, LuxI 3-oxo-C6-HSL Bioluminescence
Vibria harveyi LuxM, 3-hydroxy-C4- Bioluminescence, PHB
LuxN HSL synthesis
Xenarhabdus nemataphilus Unknown 3-hydroxy-C4- Virulence
HSL
Yersinia enteracalitica YenR, YenI C6-HSL, Unknown
3-oxo-C6-HSL
Yersinia pestis YpeR, YpeJ Unknown Unknown
Yersinia pseudamberculasis YpsR, YpsI 3-oxo-C6-HSL Motility, clumping
Yersinia pseudamberculasis YtbR, YtbI C8-HSL Unknown
Yersinia ruckeri YukR, YukI Unknown Unknown
[0005] With regard to bacteria that utilize HSL-based 1996). Several mechanisms for bio?lm-mediated resistance
quorum sensing as part of their lifestyle, Pseudamanas development have been proposed (Costerton et al., Science
aeruginosa is perhaps the best understood in terms of the 28411318-22, 1999).
role quorum sensing plays in pathogenicity. In this human
[0007] In most natural, clinical and industrial settings
opportunistic pathogen, which causes nosocomial infections
bacteria are predominantly found in bio?lms. Drinking
in immunocompromized patients and has an extremely high water pipes, ship hulls, teeth or medical devices represent
potential to develop resistance mechanisms against tradi typical surfaces colonized by bacteria. On the one hand
tional antibiotic treatment, production of many virulence bio?lms decrease the life time of materials through corrosive
factors including several proteases, exotoxin A, rhamno action in the industrial ?eld, a process also referred to as
lipid, pyocyanin, cyanide and chitinase is regulated by two “biofouling”. Furthermore, microbial bio?lms growing for
example on ship hulls increase fuel consumption through
interlinked quorum sensing circuits. Moreover, it has been
enhanced frictional resistance and simultaneously reduce
demonstrated that this signaling system is involved in the
maneuverability. On the other hand two thirds of all bacterial
ability of R aeruginasa to form bio?lms (Davies et al.,
infections in humans are associated with bio?lms (Lewis,
Science 2801295-8, 1998). Recently Huber et al. (Microbi
Antimicrob. Agents Chemother. 451999-1007, 2001).
ology 14712517-28, 2001) demonstrated that bio?lm forma
tion and swarming motility of Burkhala'eria cepacia, like P [0008] Pseudamanas aeruginasa, for example, forms
aeruginosa a human opportunistic pathogen, is also depen infectious bio?lms on surfaces as diverse as cystic ?brosis
lung tissue, contact lenses, and catheter tubes (Stickler et al.,
dent on an HSL-based quorum sensing system.
Appl. Environm. Microbial. 6413486-90, 1998). Burkhald
[0006] Bio?lms are de?ned as an association of microor eria cepacia also forms bio?lms in lungs of cystic ?brosis
ganisms growing attached to a surface and producing a slime patients and is a major industrial contaminant (Govan et al.,
layer of extracellular polymers in which the microbial J. Med. Microbiol. 451395-407, 1996). Since bio?lm forma
tion of both organisms is demonstrated to require an HSL
consortia is embedded in a protective environment (for a
signaling system, inhibition of their quorum sensing systems
review see: Costerton et al.,Ann. Rev. Microbial. 491711-45,
would result in an impaired ability to form bio?lms and
1995). Bio?lms represent a severe problem as bacteria
therefore in an increased susceptability to antibacterial treat
integrated in such a polymer matrix develop resistance to
ment.
conventional antimicrobial agents. P aeruginosa cells, for
example, growing in an alginate slime matrix have been [0009] The discovery that a wide spectrum of organisms
demonstrated to be resistant to antibiotics (e.g., aminogly use quorum sensing to control virulence factor production
cosides, [3-lactam antibiotics, ?uoroquinolones) and disin and other phenotypes such as bio?lm formation makes it an
fectants (Govan & Deretic, Microbiol. Rev. 601539-74, attractive target for antimicrobial therapy. Pathogenic organ
US 2003/0105143 A1 Jun. 5, 2003
isms using this signaling system to control virulence could infectants, antifouling coatings or coatings of medical
potentially be rendered avirulent by blocking this cell-cell devices. In contrast to traditional antibacterial agents (like
communication system. In contrast to traditional antibiotics, amide or 1,2-acylhydraZine derivatives in WO 01/51456; for
the risk of resistance development seems to be very loW, the synthesis of amide or 1,2-acylhydraZine derivatives see
since quorum sensing blocking agents Would not kill the also EP 638545 and EP 982292), the compounds of the
organism but disturb signal transduction pathWays. There present invention do not kill the microorganisms, but render
are several possibilities of interrupting the quorum sensing them avirulent. The advantage of this alternative strategy is
circuit. that the emergence of bacterial resistance against such
antimicrobials is extremely improbable.
[0010] For example, plants expressing an HSL-lactonase
enZyme originally derived from Bacillus sp. have been [0016] In general, the present invention provides com
demonstrated to quench pathogen quorum sensing signaling pounds selectively modulating bacterial cell-cell communi
and to signi?cantly enhance resistance to Erwinia caroto cation. Through inhibition of this communication system the
vora infections (Dong et al., Nature 411:813-7, 2001). An expression of many HSL-dependent virulence genes and
alternative Way to block cell signaling could be to interrupt other phenotypes like sWarming motility and bio?lm forma
the HSL synthesis by using analogs of HSL precursors. tion are signi?cantly reduced or completely abolished ren
dering a bacterial population more susceptible to the host
[0011] HoWever, the most promising possibility to block
immune,-response or to treatment With traditional antibac
quorum sensing is to take advantage of the unique speci?city
terial agents.
the HSLs and HSL-receptor proteins shoW for one another.
The ability of homoserine lactone-based analogs to inhibit [0017] Thus, in one aspect, the invention refers to a
activation of HSL-receptor proteins has already been dem method for inhibiting an HSL-regulated process in a micro
onstrated in a number of bacteria including Vibrio ?scheri organism by exposing the microorganism to a neW class of
(Schaefer et al., J. Bacteriol. 178:2897-901, 1996), Agro compounds With an inhibitory effect on bacterial signaling.
bacterium tumefaciens (Zhu et al., J. Bacteriol. 1805398
[0018] The present invention therefore refers to com
405, 1998), Chromobacterium violaceum (McLean et al., pounds of the general Formula (I)
Microbiology 143:3703-11, 1997), Aeromonas salmonicida
(Swift et al., J. Bacteriol. 179:5271-81, 1997) and
Pseudomonas aeruginosa (Pesci et al., J. Bacteriol.
179:3127-32, 1997). HoWever, none of these compounds
have been developed as antimicrobial agents, eg in medical
therapy, so far.
[0012] The only described non-HSL-based antimicrobials
Which are supposed to interfere speci?cally With HSL [0019] Wherein
regulated processes are halogenated furanone derivatives
[0020] R is H, alkyl, cycloalkyl, aryl or heteroaryl;
Which are structurally similar to HSLs and have been
isolated from red marine algae Delisea pulchra (WO [0021] R1 is H, alkyl, cycloalkyl, aryl or heteroaryl;
96/29392). Additionally, these substances have been dem
onstrated to inhibit also Gram-positive bacteria (WO [0022] R2 is H, alkyl, cycloalkyl, aryl or heteroaryl;
99/53915). HoWever, the use of most of these compounds is
[0023] A1 and A2 each independently represent an
limited due to their toxicity making them unsuitable for
optionally substituted C1-C20-alkyl group Which
veterinary and medical applications.
may contain one or more group(s) Z, or a monocyclic
[0013] Many target genes involved in bio?lm formation, or polycyclic optionally substituted aromatic or non
methods of screening for compounds to control bio?lm aromatic ring system Which may contain one or more
development and HSL-based compositions to prevent bio group(s) X, and in case of a polycyclic ring system,
?lm formation have been described (WO 99/55368, WO said system contains at least one aromatic ring;
98/57618, WO 99/27786, WO 98/58075), but until noW no
[0024] Z is selected from the group consisting of S,
promising antibacterial drug candidate has been developed
o, N, NR4, C0, C02, cs, so or so2
that is capable of inhibiting bio?lm formation in different
areas, preferentially in the medical ?eld. [0025] X is selected from the group consisting of S,
o, N, NR4, so or $02;
[0014] It is an object of the present invention to provide
compounds blocking speci?cally quorum sensing regulated
[0026] said substituted ring system carries a substitu
processes Without inhibiting bacterial groWth. Furthermore,
ent R3 on one or more of the carbon atoms of said
these compounds should not be structural derivatives of the ring system;
homoserine lactone family of regulatory compounds and
should not exhibit any toxic properties. [0027] said substituted C1-C20-alkyl group carries a
substituent R3 on one or more of the carbon atoms of
[0015] Accordingly, We have been able to ?nd compounds said alkyl group;
that can signi?cantly reduce virulence gene expression and
bio?lm formation of several human pathogens. In contrast to [0028] R3 is independently H, 0R4, SR4, hydroxy
the furanones the compounds of this invention do not shoW alkyl, hydroxyalkylamino, cycloalkyl, halogen,
any toxic effect and are therefore suitable for applications in haloalkyl, haloalkyloxy, N02, CN, SO2NR4R5,
a Wide area. Such applications could be the use of the CO2NR4R5, COR“, CO2R4, SO2R4, SO3R4, NR4R5,
compounds for instance as neW antibiotic therapeutics, dis alkyl, aryl or heteroaryl;