U.S. patent application number 15/335926 was filed with the patent office on 2017-04-06 for novel lactams.
The applicant listed for this patent is UNILEVER PLC. Invention is credited to George ISKANDER, Naresh Kumar.
Application Number | 20170096391 15/335926 |
Document ID | / |
Family ID | 38308775 |
Filed Date | 2017-04-06 |
United States Patent
Application |
20170096391 |
Kind Code |
A1 |
Kumar; Naresh ; et
al. |
April 6, 2017 |
NOVEL LACTAMS
Abstract
The present invention relates to novel lactams of formulae (I)
and (II). The present invention further relates to the use of these
compounds in the treatment of microbial infections and microbial
contamination of surfaces, particularly infections and surface
contaminations characterised by biofilm formation. Further provided
are compounds of formula (I) and (II) substituted with acrylate or
methacrylate groups and their attachment to surfaces or polymers to
inhibit microbial contamination.
Inventors: |
Kumar; Naresh; (Maroubra,
AU) ; ISKANDER; George; (Oakhurst, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNILEVER PLC |
London |
|
GB |
|
|
Family ID: |
38308775 |
Appl. No.: |
15/335926 |
Filed: |
October 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12087862 |
Jun 15, 2012 |
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PCT/AU2007/000060 |
Jan 24, 2007 |
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15335926 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/00 20180101;
C07D 207/36 20130101; C07D 207/44 20130101; C07D 207/273 20130101;
A61P 31/04 20180101; A01N 43/36 20130101; A01N 43/36 20130101; C08F
26/06 20130101; C07D 207/38 20130101; C07D 207/27 20130101; A01N
25/10 20130101; A61P 33/00 20180101 |
International
Class: |
C07D 207/44 20060101
C07D207/44; C07D 207/38 20060101 C07D207/38; A01N 43/36 20060101
A01N043/36 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 2006 |
AU |
2006900343 |
Claims
1-20. (canceled)
21. A compound of formula I: ##STR00045## wherein: R.sub.1 is
hydrogen; R.sub.2 is selected from the group consisting of
heterocyclyl, heteroaryl, aryl and arylalkyl; R.sub.3 is selected
from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl,
alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl,
aryl, arylalkyl, and --C(O)CR.sub.6.dbd.CH.sub.2; R.sub.4 and
R.sub.5 are each hydrogen; R.sub.6 is selected from the group
consisting of hydrogen or methyl.
22. The compound of claim 21, wherein R.sub.3 is selected from
hydrogen, methyl and phenyl.
23. The compound of claim 21, wherein R.sub.2 is phenyl.
24. The compound of claim 21, wherein R.sub.2 is mono-substituted
phenyl.
25. A compound of formula II: ##STR00046## wherein R.sub.1 is
hydrogen; R.sub.2 is mono-substituted phenyl; R.sub.3 is selected
from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl,
alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl,
aryl, arylalkyl, and --C(O)CR.sub.6.dbd.CH.sub.2; R.sub.4 and
R.sub.5 are each hydrogen; R.sub.6 is selected from the group
consisting of hydrogen or methyl. R.sub.7 is hydrogen.
26. The compound of claim 25, wherein R.sub.3 is selected from
hydrogen, methyl and phenyl.
27. A method of treating a microbial infection in a subject, the
method comprising administering a compound according to claim 21 to
the subject.
28. A method according to claim 27 wherein the infection is a
bacterial infection.
29. A method according to claim 27 wherein the infection is
characterised by biofilm formation.
30. A method of inhibiting microbial contamination of a surface,
the method comprising administering a compound according to claim
21 to the surface.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel lactams, methods for
their synthesis and uses of these compounds.
BACKGROUND OF THE INVENTION
[0002] In International Patent Application No. WO 2004/016588 (the
disclosure of which is hereby incorporated by reference), the
present applicant disclosed a method of synthesising lactams of
formulae A and B by reacting an appropriate 5-halomethylene
substituted furanone with an amine under mild conditions to give
lactams of general formula A, optionally followed by dehydration to
give lactams of general formula B.
##STR00001##
[0003] Each of the lactams exemplified in the specification of WO
2004/016588 included one or more bromine substituents, usually at
position R.sub.3 or R.sub.4. Positions R.sub.1 and R.sub.2 were
substituted with halo, alkyl (substituted or unsubstituted) or
hydrogen. The lactams were shown to have antibacterial properties
and to act as quorum sensing inhibitors.
SUMMARY OF THE INVENTION
[0004] The present invention is directed to members of a novel
class of lactams comprising a heterocyclyl, heteroaryl, aryl or
arylalkyl substituent at the 3 or 4 position of the lactam ring and
which are not brominated on the exocyclic methylene group. The lack
of bromination on the exocyclic methylene group improves the
stability of these compounds relative to the compounds exemplified
in WO 2004/016588. In addition, the presence of a heteroaryl, aryl
or arylaklyl substituent at the 3 or 4 position provides additional
in vivo stability when compared to the compounds exemplified in WO
2004/016588 that bear an alkyl group. The compounds have been shown
to have antibacterial properties. Further, at least some of the
compounds of the present invention have surprisingly improved
antibacterial activity and/or surprisingly reduced cytotoxicity
when compared to the certain lactams exemplified in the
specification of WO 2004/016588. The present inventors have also
found that analogues of these compounds can be substituted with
acrylate and methacrylate groups so that these compounds can be
readily polymerised, copolymerised or attached to surfaces which
bear functional groups by, for example, the Michael addition
reaction.
[0005] Accordingly, in a first aspect, the present invention
provides a compound of formula I:
##STR00002##
[0006] wherein:
[0007] R.sub.1 and R.sub.2 are each independently selected from the
group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkoxy,
oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl, and
arylalkyl;
[0008] R.sub.3 is selected from the group consisting of hydrogen,
hydroxy, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl,
heterocyclyl, heteroaryl, cycloalkyl, aryl, arylalkyl, and
--C(O)CR.sub.6.dbd.CH.sub.2;
[0009] R.sub.4 and R.sub.5 are each selected from the group
consisting of hydrogen, aryl, heterocyclyl, heteroaryl, and
arylalkyl provided that at least one of R.sub.4 and R.sub.5 is
hydrogen;
[0010] R.sub.6 is selected from the group consisting of hydrogen or
methyl;
[0011] with the proviso that at least one of R.sub.1 and R.sub.2 is
selected from the group consisting of heterocyclyl, heteroaryl,
aryl and arylalkyl.
[0012] In a second aspect, the present invention provides a
compound of formula (II):
##STR00003##
[0013] wherein
[0014] R.sub.1 and R.sub.2 are each independently selected from the
group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkoxy,
oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl, and
arylalkyl;
[0015] R.sub.3 is selected from the group consisting of hydrogen,
hydroxy, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl,
heterocyclyl, heteroaryl, aryl, arylalkyl, and
--C(O)CR.sub.6.dbd.CH.sub.2;
[0016] R.sub.4 is selected from the group consisting of hydrogen,
aryl, heterocyclyl, heteroaryl and arylalkyl;
[0017] R.sub.6 is selected from the group consisting of hydrogen or
methyl;
[0018] R.sub.7 is selected from the group consisting of H and
--C(O)CR.sub.6.dbd.CH.sub.2;
[0019] with the proviso that at least one of R.sub.1 and R.sub.2 is
selected from the group consisting of heterocyclyl, heteroaryl,
aryl and arylalkyl.
[0020] In a third aspect, the present invention provides a method
of treating or preventing a microbial infection of a subject, the
method comprising administering a compound of Formula I or a
compound of Formula II to the subject.
[0021] In a fourth aspect, the present invention provides a method
of inhibiting or preventing microbial contamination of a surface,
the method comprising administering the compound of Formula I or a
compound of Formula II to the surface.
[0022] In a fifth aspect, the present invention provides a
pharmaceutical formulation comprising a compound of Formula I or a
compound of Formula II and a carrier.
[0023] In a sixth aspect, the present invention provides a compound
of Formula I wherein R.sub.3 is --C(O)CR.sub.6.dbd.CH.sub.2.
[0024] In a seventh aspect, the present invention provides a
compound of Formula II wherein at least one of R.sub.3 and R.sub.7
is selected from --C(O)CR.sub.6.dbd.CH.sub.2.
[0025] Preferably, each of R.sub.3 and R.sub.7 is selected from
--C(O)CR.sub.6.dbd.CH.sub.2.
[0026] In an eighth aspect, the present invention provides a
compound according to the sixth or seventh aspect when used to form
an oligomer or polymer
[0027] In a ninth aspect, the present invention provides a polymer
or oligomer formed by oligomerising or polymerising a compound
according to the sixth or seventh aspect directly or with one or
more other monomers.
[0028] In a tenth aspect, the present invention provides a compound
of the sixth or seventh aspect when the terminal vinyl group of the
compound is reacted with a functional group.
[0029] In an eleventh aspect, there is provided a compound
according to the sixth or seventh aspect when attached to a
surface.
[0030] In a twelfth aspect, there is provided a surface comprising
one or more compounds of the sixth or seventh aspect attached to
the surface.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1. shows the lung bacterial loads between two groups of
mice after challenge with Pseudomonas aeruginosa PAO1 (wt)
5.2.times.10.sup.6 CFU/lung (13.times.10.sup.8CFU/ml) at day 5. The
first group was treated with
5-methylene-4-phenyl-dihydro-pyrrol-2-one (C219) as 12 .mu.g/g body
weight, given as injections twice a day for three days and the
second group were treated with vehicle. (p=0.0008).
DETAILED DESCRIPTION OF THE INVENTION
[0032] In a first aspect, the present invention provides a compound
of formula I:
##STR00004##
[0033] wherein:
[0034] R.sub.1 and R.sub.2 are each independently selected from the
group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkoxy,
oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl, and
arylalkyl;
[0035] R.sub.3 is selected from the group consisting of hydrogen,
hydroxy, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl,
heterocyclyl, heteroaryl, cycloalkyl, aryl, arylalkyl, and
--C(O)CR.sub.6.dbd.CH.sub.2;
[0036] R.sub.4 and R.sub.5 are each selected from the group
consisting of hydrogen, aryl, heterocyclyl, heteroaryl, and
arylalkyl provided that at least one of R.sub.4 and R.sub.5 is
hydrogen;
[0037] R.sub.6 is selected from the group consisting of hydrogen or
methyl;
[0038] with the proviso that at least one of R.sub.1 and R.sub.2 is
selected from the group consisting of heterocyclyl, heteroaryl,
aryl and arylalkyl.
[0039] The present invention also provides for the starting
materials used in the preparation of the compounds of Formula I.
These starting materials have been found to have antibacterial
activity in their own right.
[0040] Accordingly, in a second aspect, the present invention
provides a compound of formula II:
##STR00005##
[0041] wherein
[0042] R.sub.1 and R.sub.2 are each independently selected from the
group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkoxy,
oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl, and
arylalkyl;
[0043] R.sub.3 is selected from the group consisting of hydrogen,
hydroxy, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl,
heterocyclyl, heteroaryl, aryl, arylalkyl, and
--C(O)CR.sub.6.dbd.CH.sub.2;
[0044] R.sub.4 is selected from the group consisting of hydrogen,
aryl, heterocyclyl, heteroaryl and arylalkyl;
[0045] R.sub.6 is selected from the group consisting of hydrogen or
methyl;
[0046] R.sub.7 is selected from consisting of H and
--C(O)CR.sub.6.dbd.CH.sub.2;
[0047] with the proviso that at least one of R.sub.1 and R.sub.2 is
selected from the group consisting of heterocyclyl, heteroaryl,
aryl and arylalkyl.
[0048] Preferably, each of R.sub.4 and R.sub.5 is hydrogen.
[0049] Preferably, R.sub.2 is selected from the group consisting of
heterocyclyl, heteroaryl, aryl and arylalkyl.
[0050] In a preferred form, the compound is selected from the group
consisting of the following compounds set out in the examples:
5-methylene-4-phenyl-dihydro-pyrrol-2-one,
1-methyl-5-methylene-4-phenyl-dihydro-pyrrol-2-one,
5-methylene-1,4-diphenyl-dihydro-pyrrol-2-one,
4-(4'-bromophenyl)-5-methylene-2-dihydro-pyrrol-2-one,
4-benzyl-5-methylene-dihydro-pyrrol-2-one,
4-(4'-methoxyphenyl)-5-methylene-dihydro-pyrrol-2-one,
5-methylene-4-(4'-fluorophenyl)-dihydro-pyrrol-2-one,
5-methylene-4-(4'-trifluoromethylphenyl)-dihydro-pyrrol-2-one,
5-methylene-4-(3'-trifluoromethylphenyl)-dihydro-pyrrol-2-one,
5-methylene-4-(2'-fluorophenyl)-dihydro-pyrrol-2-one and
5-methylene-4-(3'-fluorophenyl)-dihydro-pyrrol-2-one.
[0051] In a third aspect, the present invention provides a method
of treating a microbial infection in a subject, the method
comprising administering a compound of Formula I or Formula II to
the subject.
[0052] The microbial infection may be a bacterial, protozoal or
fungal infection. Preferably, the infection is a bacterial
infection.
[0053] Suitable methods of administration are disclosed in WO
2004/016588, the disclosure of which is hereby incorporated by
reference. Examples of the types of bacterial infections that may
be treated by the methods of the present invention are also
disclosed in WO 2004/016588.
[0054] The compounds of the present invention can act as quorum
sensing inhibitors. The compounds therefore find use in arty
application where the inhibition of quorum sensing is desired. For
example, the compounds of the present invention may have use in
preventing the establishment of biofilms and expression of
virulence by microorganisms through the inhibition of quorum
sensing systems and/or other extracellular systems (eg see, WO
01/47681, the disclosure of which is incorporated herein in its
entirety).
[0055] Quorum sensing pathways (such as those involving homoserine
lactones) are present in a wide range of bacteria. The formation of
biofilms is one instance of quorum sensing.
[0056] The following is a non-exhaustive list of groups of
Grain-Negative bacteria that have members which use homoserine
lactones for cell-cell communication: anaerobic Gram Negative
Straight, Curved and Helical Rods; Bacteroidaceae; The Rickettsias
and Chlamydias; Dissimilatory Sulfate--or Sulfur--Reducing
Bacteria; the Mycoplasmas; The mycobacteria; Budding and/or
Appendaged Bacteria; Sheathed Bacteria; Nocardioforms; and
Actinomycetes, for example. See Bergey's Manual of Systematic
Bacteriology, First Ed., John G. Holt, Editor in Chief (1984),
incorporated herein by reference.
[0057] Further microbial infections that may be treated by the
compounds of the present invention include bacterial infections
caused by Staph. aureus, Staph epidermis, Serratia spp., Vibrio
spp., and Strep. pneumonia; protozoal infections caused by
Acanthamoeba; and fungal infections caused by Fusarium spp.
[0058] Preferably, the method of the third aspect may be used to
treat or prevent a microbial infection in a subject that is
characterised by biofilm formation.
[0059] The present invention is suitable for biofilms originating
from a single type of organism and for mixed biofilms. By "mixed
biofilms" is meant biofilms created by more than one type of
microorganism. It is envisioned that mixed biofilms could be
created by at least two organisms from the group consisting of
bacteria, algae, fungi, and protozoa.
[0060] Non-limiting examples of human infections involving biofilms
include dental caries, periodontitis, otitis media, muscular
skeletal infections, necrotising fascitis, biliary tract infection,
osteomyelitis, bacterial prostatitis, native valve endocarditis,
cystic fibrosis pneumonia, meloidosis, and nosocomial infections
such as ICU pneumonia, urinary catheter cystitis, peritoneal
dialysis (CAPD) peritonitis, and biliary stent blockage. Biofilm
formation can affect sutures, exit sites, arteriovenous sites,
scleral buckles, contact lenses, IUDs, endotracheal tubes, Hickman
catheters, central venous catheters, mechanical heart valves,
vascular grafts, orthopedic devices, penile prostheses. Further
applications are described in Costerton J et al, (1999) Vol. 284,
Science pp 1318-1322 and Costerton J and Steward, (2001) Battling
Biofilms, Scientific American pp 75-81, the disclosures of which
are incorporated herein by reference.
[0061] Other locations in which biofilms may form include dental
plaque which may lead to gum disease and cavities, contact lenses
which may lead to eye infections, ears which may lead to chronic
infection and lungs which may lead to pneumonia.
[0062] The infection may be cystic fibrosis. The infection may be
that resulting from a skin infection, burn infection and/or wound
infection. The method and composition of the invention may be
particularly suitable for the treatment of infection in immuno
compromised individuals.
[0063] The compounds of the present invention have been shown to be
particularly effective in preventing microbial contamination of
surfaces, in particularly by preventing the formation of
biofilms.
[0064] Accordingly, in a fourth aspect, the present invention
provides a method of inhibiting microbial contamination of a
surface, the method comprising administering a compound of Formula
I or Formula II to the surface.
[0065] The microbial contamination may be protozoal, fungal or
bacterial contamination.
[0066] In a preferred form, the microbial contamination is a
bacterial contamination. In a more preferred form, the bacterial
contamination is a biofilm.
[0067] The surface may be any natural or artificial surface. By
"artificial surface", is meant any surface that is not naturally
occurring. In one embodiment, the surface is not an external
surface (eg skin) or an internal surface of a human being or
animal. In another embodiment, the surface is an external surface
or an internal surface of a human being or animal. Examples of
surfaces that might be treated using the compounds of Formula I or
Formula II are disclosed in WO 2004/016588.
[0068] The compound may be administered by any suitable means. For
instance, the compound may be attached to the surface using the
techniques and surfaces described in WO 2004/016588.
[0069] Suitable surfaces include the surfaces of articles for which
it is desirable to prevent bacterial contamination. These include:
medical devices, for example, implantable biomedical devices such
as urinary catheters, percutaneous access catheters, stents,
orthopaedic implants, bone and dental ceramics and polymers as well
as non-implantable devices such as contact lenses, contact lens
storage cases, and the like.
[0070] Other suitable surfaces include the interiors of pipes and
vessels used to distribute oil and gas. The interior surfaces of
oil pipelines can be readily contaminated by biofilms which impede
the flow of oil in the pipeline and hence its efficiency.
[0071] The material from which the article is formed can be a
metal, a ceramic, a solid synthetic polymer, or a solid natural
polymer, for example a solid biopolymer. Examples of useful
materials for this invention are titanium, hydroxyapatite,
polyethylene (which are useful materials for orthopaedic implants),
polyurethanes, organosiloxane polymers, perfluorinated polymers
(which are useful materials for instance for catheters, soft tissue
augmentation, and blood contacting devices such as heart valves),
acrylic hydrogel polymers, HEMA/GMA polymers and silicon/siloxane
hydrogel polymers (for instance for contact lens and intraocular
lens applications), and the like, and any combination thereof.
Further included are resin composites, componers and resin-modified
glass ionomers used in oral care. The surfaces of these materials
can be chemically inert or contain reactive functional groups.
[0072] Further examples of articles include archival documents,
antiques and art, rare and valuable seeds intended for storage (e.
g. seed banks of conservation groups), etc in which case the
substrate may be paper, material, or other natural or synthetic
material.
[0073] The article may be a shell fish or aquaculture apparatus,
for example, that described in WO 99/05227, the disclosure of which
is incorporated herein by reference.
[0074] The surface of the article may be any hard surface such as
metal, organic and inorganic polymer surface, natural and synthetic
elastomers, board, glass, wood, paper, concrete, rock, marble,
gypsum and ceramic materials which optionally are coated, eg with
paint, enamel etc; or any soft surface such as fibres of any kind
(yarns, textiles, vegetable fibres, rock wool, hair etc.); or
porous surfaces; skin (human or animal); keratinous materials
(nails etc.). The hard surface can be present in process equipment
or components of cooling equipment, for example, a cooling tower, a
water treatment plant, a dairy, a food processing plant, a chemical
or pharmaceutical process plant. The porous surface can be present
in a filter, eg. a membrane filter.
[0075] Particular examples of articles whose surfaces may be
treated in accordance with the invention include, but are not
limited to, toilet bowls, bathtubs, drains, highchairs, counter
tops, vegetables, meat processing rooms, butcher shops, food
preparation areas, air ducts, air-conditioners, carpets, paper or
woven product treatment, nappies (diapers), personal hygiene
products (eg sanitary napkins) and washing machines. The compounds
may be formulated in the form of a toilet drop-in or spray-on
devices for prevention and removal of soil and under rim cleaner
for toilets. The compounds of the present invention also have
applications in cleaning of Industrial surfaces such as floors,
benches, walls and the like and these and other surfaces in medical
establishments such as hospitals (eg surfaces in operating
theatres), veterinary hospitals, and in mortuaries and funeral
parlours.
[0076] Further examples of surfaces which may be treated include
hard, rigid surfaces such as drain pipes, glazed ceramic,
porcelain, glass, metal, wood, chrome, plastic, vinyl and formica
or soft flexible surfaces such as shower curtains, upholstery,
laundry and carpeting. It is also envisioned that both woven and
non woven and porous and non-porous surfaces would be suitable.
[0077] The compound may be administered by any suitable means. For
instance, the compound may be attached to the surface using the
techniques and surfaces described in WO 2004/016588. Examples
include providing the compounds of the present invention as part of
an oligomer or polymer by, for instance, co-polymerising the
compound with other monomers or attaching the compound to the
polymer backbone by techniques well known to those in the art.
[0078] Methods for the covalent immobilization of organic molecules
onto solid surfaces are well known to those skilled in the art.
Interfacial reactions leading to the formation of covalent
interfacial bonds are derived from well known organic-synthetic
reactions. The choice of immobilization reaction depends on both
the nature of the substrate material and the chemical composition
of the compound of the present invention that is desired for a
particular application.
[0079] For example, a compound that contains a hydroxyl group in a
side chain distal to the ring system, can be linked covalently onto
surfaces using epoxide chemistry analogous to the reaction pathway
described for the immobilization of polysaccharides onto epoxidated
surfaces in Li et al., Surface Modification of Polymeric
Biomaterials (B D Ratner and D G Castner, Eds), Plenum Press, NY,
1996 pages 165-173 (the disclosure of which is incorporated herein
in its entirety), through isocyanate groups attached to the surface
to produce stable urethane linkages through thermal processes, or
through carboxylic acid groups or their equivalents, such as acid
chlorides, on the surface to produce ester linkages. A compound
that contains an aldehyde group can be linked onto surface amine
groups using a reductive animation reaction. A compound that
contains a carboxylic acid group can be linked onto surface amine
groups using carbodiimide chemistry.
[0080] Interfacial coupling reactions must of course be selected
not only for their ability to achieve the desired covalent linkage
but also for avoidance of adverse effects on the furanone compound
(s) to be attached. Particularly, the furanone ring system tends to
be labile to alkaline conditions. Such limitations are well known
to those skilled in the art. Among the many possible interfacial
coupling reactions known in the art, there is sufficient scope for
selection of reactions that proceed in a suitable pH range and with
furanones substituted with various functional groups in various
positions.
[0081] Some solid substrate materials possess reactive surface
chemical groups that can undergo chemical reactions with a partner
group on a compound and thereby form a covalent interfacial linkage
directly.
[0082] Alternatively, in situ covalent linkage can be made directly
through the addition of a doubly functionalised linker molecule to
the active surface in the presence of an appropriate compound, or
stepwise by sequential addition of doubly functionalised linker
molecules and then an appropriate compound. It is not always
possible to immobilize furanone compounds directly onto solid
substrate materials; in these cases, surface activation or one or
more interfacial bonding layer (s) is used to effect covalent
immobilization of the compounds.
[0083] Surface activation of solid substrate materials can be
achieved in a number of ways. Examples are corona discharge
treatment or low pressure plasma treatment of polymers. These
methods are well known to introduce a variety of functional groups
onto polymeric surfaces.
[0084] An alternative approach is to provide an interfacial bonding
layer interspersed between the solid substrate material or medical
device and the compound layer. The application of a thin
interfacial bonding layer can be done using methods such as dip
coating, spin coating, or plasma polymerization. The chemistry of
the bonding layer is selected such that appropriate reactive
chemical groups are provided on the surface of this layer, groups
that then are accessible for reaction with compound of the
invention.
[0085] Particularly versatile is the subsequent application of
multiple thin interfacial bonding layers; this method can provide a
very wide range of desired chemical groups on the surface for the
immobilization of a wide range of functionalized furanones and
enables usage of compounds optimized for their biological
efficacy.
[0086] By providing a thin, surface-coated layer of compounds, the
optical quality of antibacterial devices of this invention is not
reduced, which makes the invention applicable to transparent
ophthalmic devices such as contact lenses and intraocular
lenses.
[0087] The present invention provides thin surface coatings that
provide antimicrobial properties and/or antifungal properties to
solid, materials onto which the coatings have been applied. More
particularly, the coatings may be designed to reduce or prevent
colonization of biomedical devices by bacteria that cause adverse
effects on the health of human users of biomedical devices when
such devices are colonized by bacteria.
[0088] Alternatively, the compound may be administered in the form
of a formulation.
[0089] Accordingly, in a fifth aspect, the present invention
provides a formulation comprising a compound of Formula I or
Formula II and a carrier.
[0090] Examples of the types of carrier that may be used with the
compounds of Formula I or Formula II are disclosed in WO
2004/016588.
[0091] The formulations may be in any suitable form. The
formulation may include a carrier or diluent. The carrier may be
liquid or solid. For example, the compositions may be in the form
of a solution or suspension of at least one of the compounds in a
liquid. The liquid may be an aqueous solvent or a non-aqueous
solvent. The liquid may consist of or comprise a one or more
organic solvents. The liquid may be an ionic liquid. Particular
examples of carrier or diluents include, but are not limited to,
water, polyethylene glycol, propylene glycol, cyclodextrin and
derivatives thereof.
[0092] The composition may be formulated for delivery in an aerosol
or powder form.
[0093] The composition may include organic or inorganic polymeric
substances. For example, the compound of the invention may be
admixed with a polymer or bound to, or adsorbed on to, a
polymer.
[0094] When the composition is to be formulated as a disinfectant
or cleaning formulation, the composition may include conventional
additives used in such formulations. Non-limiting examples of the
physical form of the formulations include powders, solutions,
suspensions, dispersions, emulsions and gels.
[0095] A compound of the invention may be incorporated into
epidermal bandages and lotions. Alternatively, the compounds of the
invention may be incorporated into cosmetic formulations, for
example, aftershave lotions, skin creams, deodorants and
anti-dandruff shampoos.
[0096] Compositions of the present invention may be in the form of
an aqueous solution or suspension containing a cleaning-effective
amount of the active compound described above. The cleaning
composition may be in the form of a spray, a dispensable liquid, or
a toilet tank drop-in, under-rim product for prevention, removal
and cleaning of toilets and other wet or intermittently wet
surfaces in domestic or industrial environments.
[0097] The compositions of the present invention may additionally
comprise a surfactant selected from the group consisting of
anionic, non-ionic, amphoteric, biological surfactants and mixtures
thereof. Most preferably, the surfactant is sodium dodecyl
sulfate.
[0098] One or more adjuvant compounds may be added to the cleaning
solution of the present invention. They may be selected from one or
more of biocides, fungicides, antibiotics, and mixtures thereof to
affect planktonics. pH regulators, perfumes, dyes or colorants may
also be added. In addition, the adjuvant could be a cell
permeabilisation agent such as EDTA or FDS.
[0099] In a preferred from, "cleaning-effective amount of active
compound" means the amount of the compound required to remove at
least 10% of bacteria from a biofilm as determined by a reduction
in numbers of bacteria within the biofilm when compared with a
biofilm not exposed to the active compound.
[0100] Preferably, the formulation is a pharmaceutical
formulation.
[0101] Formulations for pharmaceutical uses may incorporate
pharmaceutically acceptable carriers, diluents and excipients known
to those skilled in the art. The formulations make be formulated
for parenteral or non-parenteral administration. The formulations
may be formulated for methods of introduction including, but not
limited to, topical, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, ophthalmic, and
oral routes. They may be formulated for administration by any
convenient route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration may
be localized or systemic. The formulation may be formulated for
intraventricular and intrathecal injection.
[0102] Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0103] In certain preferred embodiments the formulation further
comprises other active agents such as antibiotics and cleaning
agents.
[0104] In other embodiments of the present invention, the
formulation may be formulated as a dentifrice, a mouthwash or a
composition for the treatment of dental caries. The composition may
be formulated for acne treatment or cleaning and disinfecting
contact lenses (eg as a saline solution).
[0105] The present inventors have also devised methods of preparing
analogues of the compounds of Formula I and Formula II which are
functionalised with a vinyl group. The vinyl group allows the
functionalised compounds to be readily incorporated in polymers
and/or attached to surfaces.
[0106] Accordingly, in a sixth aspect, the present invention
provides a compound of Formula I wherein R.sub.3 is
--C(O)R.sub.6.dbd.CH.sub.2.
[0107] Further, in a seventh aspect, the present invention provides
a compound of Formula II wherein at least one of R.sub.3 and
R.sub.7 is selected from --C(O)CR.sub.6.dbd.CH.sub.2.
[0108] In an eighth aspect, the present invention provides a
compound of the sixth or seventh aspect when used to form an
oligomer or polymer.
[0109] Preferably, the oligomer or polymer is formed by
polymerisation of the terminal vinyl group of the compound of the
sixth or seventh aspect.
[0110] In a ninth aspect, the present invention provides a polymer
or oligomer formed by oligomerising or polymerising a compound of
the sixth or seventh aspect directly or with one or more other
monomers.
[0111] The one or more other monomers may be any suitable
polymerisable comonomer eg acrylate ester such as alkyl,
hydroxyalkyl, aminoalkyl, or substituted aryl, acrylates or
methacrylates, crotonates, substituted or unsubstituted
acrylonitriles, vinyl alcohols or acteates, and styrenes.
[0112] The vinyl group of the compounds of the sixth and seventh
aspects allows for polymerisation or reaction with other
unsaturated systems. It also allows for reaction with other
functional groups by, for example, Michael addition. Other
strategies for reacting a vinyl group with a functional group to
create a covalent bond are well known to those skilled in the
art.
[0113] Accordingly, in a tenth aspect, the present invention
provides a compound of the sixth or seventh aspects when the
terminal vinyl group of the compound is reacted with a functional
group.
[0114] The functional group may be, for instance, an amine or a
thiol group. Preferably, the reaction is with an amine. More
preferably, a primary amine.
[0115] Surfaces comprising a compound of the sixth or seventh
aspect attached thereto would be expected to prevent or inhibit
colonisation of the surface by bacteria.
[0116] Accordingly, in an eleventh aspect, there is provided a
compound of the sixth or seventh aspect when attached to a
surface.
[0117] As mentioned above, suitable surfaces to which a compound
according to the present invention may be attached are detailed in
WO 2004/016588. In a preferred form, the surface is the surface of
a contact lens.
[0118] Preferably, the compound of the sixth or seventh aspect is
attached to the surface by reaction of the terminal vinyl group of
the compound with a functional group.
[0119] More preferably, the functional group is a primary
airline.
[0120] In a twelfth aspect, there is provided a surface comprising
one or more compounds of Formula II attached to the surface.
[0121] The term "alkyl" is taken to mean both straight chain and
branched alkyl groups such as methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, sec-butyl, tertiary butyl, and the like.
[0122] Preferably the alkyl group is a lower alkyl of 1 to 10
carbon atoms, more preferably 1 to 6 carbon atoms.
[0123] In certain embodiments, the carbon chain of the alkyl group
is interrupted with one or more heteroatoms. For instance, a
polyethylene glycol group of the form --(CH.sub.2CH.sub.2O).sub.nH
is to be understood to be an alkyl group of such an embodiment.
[0124] The term "cycloalkyl" as used herein refers to cyclic
hydrocarbon groups. Suitable cycloalkyl groups include but are not
limited to cyclopropyl, cyclobutyl and cyclohexyl.
[0125] The term "alkoxy" denotes straight chain or branched
alkyloxy, preferably C.sub.1-10 alkoxy. Examples include methoxy,
ethoxy, n-propoxy, isopropoxy and the different butoxy isomers.
[0126] The term "alkenyl" includes groups formed from straight
chain, branched or mono- or polycyclic alkenes and polyenes.
Substituents include mono- or poly-unsaturated alkyl or cycloalkyl
groups as previously defined, preferably C.sub.2-10 alkenyl.
Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl,
iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl,
1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl,
1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl,
2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl,
1,4-pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl,
1,4-hexadienyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl,
1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, or
1,3,5,7-cyclooctatetraenyl.
[0127] The term "alkynyl" as used herein, refers to straight chain
or branched hydrocarbon groups containing one or more triple bonds.
Suitable alkynyl groups include, but are not limited to ethynyl,
propynyl, butynyl, pentynyl and hexenyl.
[0128] The term "halogen" includes fluorine, chlorine, bromine or
iodine, preferably bromine or fluorine.
[0129] The term "heteroatoms" denotes O, N, S or Si.
[0130] The term "acyl" used either alone or in compound words such
as "acyloxy", "acylthio", "acylamino" or diacylamino" denotes an
alkanoyl, aroyl, heteroyl, carbamoyl, alkoxycarhonyl,
alkanesulfonyl, arysulfonyl, and is preferably a C.sub.1-10
alkanoyl. Examples of acyl include carbamoyl; straight chain or
branched alkanoyl, such as formyl, acetyl, propanoyl, butanoyl,
2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl,
heptanoyl, octanoyl, nonanoyl, decanoyl; alkoxycarbonyl, such as
methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl,
t-pentyloxycarbonyl or heptyloxycarbonyl; cycloalkanecarbonyl such
as cyclopropanecarbonyl cyclobutanecarbonyl, cyclopentanecarbonyl
or cyclohexanecarbonyl; alkanesulfonyl, such as methanesulfonyl or
ethanesulfonyl; alkoxysulfonyl, such as methoxysulfonyl or
ethoxysulfonyl; heterocycloalkanecarbonyl; heterocyclyoalkanoyl,
such as pyrrolidinylacetyl, pyrrolidinylpropanoyl,
pyrrolinylacetyl, pyrrolylacetyl, pyrrolidinylbutanoyl,
pyrrolidinylpentanoyl, pyrrolidinylhexanoyl or thiazolidinylacetyl;
heterocyclylalkenoyl, such as heterocyclylpropenoyl,
heterocyclylbutenoyl, heterocyclylpentenoyl or
heterocyclylhexenoyl; or heterocyclylglyoxyloyl, such as,
thiazolidinylglyoxyloyl or pyrrolidinylglyoxyloyl.
[0131] The term "aryl" refers to aryl groups having 6 through 10
carbon atoms and includes, for example, phenyl, naphthyl, indenyl.
Preferably, the aryl group is phenyl or naphthyl.
[0132] The term "arylalkyl" includes groups such as benzyl and
phenethyl groups which comprise an alkyl chain with an aryl
substituent.
[0133] The term "heterocyclyl" includes monocyclic, polycyclic,
fused or conjugated hydrocarbon residues, preferably C3-6, wherein
one or more carbon atoms (and where appropriate, hydrogen atoms
attached thereto) are replaced by a heteroatom so as to provide a
non-aromatic residue. Suitable heteroatoms include O, N, and S.
When two or more carbon atoms are replaced, this may be by two or
more of the same heteroatom or by different heteroatoms. Suitable
examples of heterocyclic groups include pyrollodinyl, piperidyl,
piperazinyl, morpholino, quinolinyl, isoquinolinyl, thiomorpholino,
dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, and
tetrahydropyrrolyl.
[0134] The term "heteroaryl" includes a 5- or 6-membered
heteroaromatic ring containing one or more heteroatoms selected
from O, N and S. Suitable examples of heteroaryl groups include
tetrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, imiidazolyl,
pyrazolyl, pyridinyl, pyrimidinyl, oxazolyl, and oxadiazolyl.
[0135] Each alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl,
heterocyclyl, heteroaryl, aryl, and arylalkyl group may optionally
be substituted by one or more groups selected from alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, arylalkyl, heterocyclyl,
heteroaryl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxy,
substituted or unsubstituted alkoxy, alkenyloxy, haloalkoxy,
haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl,
nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino,
alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino,
diacylamino, acyloxy, alkylsulfonyloxy, heterocyclyl,
heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulfenyl,
alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups
such as phosphono and phosphinyl.
[0136] In order that the nature of the present invention may be
more clearly understood, preferred forms thereof will now be
described with reference to the following non-limiting
examples.
EXAMPLES
[0137] I. Compound Synthesis
[0138] General Procedure:
##STR00006##
Synthesis of 3-phenyl-4-oxo2-pentenoic acid
[0139] Glyoxilic acid (42.3 g; 0.45 mol), phenyl acetone (40.2 g;
0.3 mol) and phosphoric acid 30 ml; 85%) were heated together at
75-80.degree. C. for 5 hrs, after which the mixture was left
standing to cool overnight at room temperature. The dark coloured
reaction mixture was poured into brine (100 ml) and extracted with
CH.sub.2Cl.sub.2:Et.sub.2O (1:1; v/:v) (3.times.50 ml). The
combined extracts was washed with three times with brine, dried
(Na.sub.2SO.sub.4), and evaporated in vacuo leaving a brown syrupy
oil (50 g). The syrup was re-dissolved in dichloromethane (100 ml)
and extracted with saturated sodium bicarbonate (3.times.65 ml).
The organic phase was dried (Na.sub.2SO.sub.4) and evaporated to
yield 5-hydroxy-5-methyl-4-phenyl-2(5H)furanone as a syrupy oil
which solidified on keeping (3.3 g; 6%). Colourless crystals from
CH.sub.2Cl.sub.2/petrol m.p. 105-107.degree. C.
[0140] The combined bicarbonate solution was acidified with 2M
hydrochloric acid and extracted with CH2Cl.sub.2 (3.times.40 ml).
The dichloromethane extracts were washed with brine, dried
(Na.sub.2SO.sub.4) and evaporated to yield
3-phenyl-4-oxo-2-pentenoic acid as a pale yellow oil which
solidifies on standing in the fridge (41 g; 72%). Colourless
crystals from petrol/CH.sub.2Cl.sub.2, m.p. 70.degree. C.
##STR00007##
[0141] The acid (4.57 g; 0.024 mol) was dissolved in thionyl
chloride (20 ml) and heated at reflux for 1.5 hr. Excess thionyl
chloride was removed in vacuo, and the residual oil (5.0 g),
presumably 5-chloro-5-methyl-4-phenyl-2(5H)furanone, was dissolved
in dichloromethane (10 ml) and cooled in an ice-bath. Concentrated
aqueous ammonia (15M, 20 ml) was added dropwise over a period of 1
hr, and the reaction mixture left to stir at room temperature over
night. The mixture was extracted with ethyl acetate (3.times.20 ml)
and the combined organic phase was dried (sodium sulfate) and flash
chromatographed using a plug of silica (EtOAc:CH.sub.2Cl.sub.2;
1:1) to yield of 5-hydroxy-5-methyl-4-phenyl)-dihydro-pyrrol-2-one
(2.83 g; 62.4%) as a pale brown solid.
[0142] A mixture of dihydro-pyrrolone (0.50 g; 2.65 mmol) and
P.sub.2O.sub.5 (0.50 g; 3.52 mmol) in dichloromethane (20 ml) was
stirred at room temperature for 30 min until all of the pyrrolone
has dissolved. The mixture was filtered through a celite/silica
bed; and washed with CH.sub.2Cl.sub.2/EtOAc (1:1). The combined
filtrate and washings were evaporated leaving
5-methylene-4-phenyl-2(5H)-pyrrolone as a pale yellow solid (0.34
g; 75%).
Synthesis of 1-methyl-5-methylene-4-phenyl-dihydro-pyrrol-2-one
##STR00008##
[0144] The acid (0.25 g: 1.32 mmol) was heated at reflux with
SOCl.sub.2 (10 ml) for 3 h. Excess thionyl chloride was removed in
vacuo, and the residue dissolved in dichloromethane (20 ml). The
residue was cooled in an ice-bath and an aqueous solution of
methylamine (10 ml; 24%) was added over a 10 min period. The
mixture was left to stir at room temperature overnight. The organic
phase was separated, dried over anhydrous sodium sulfate, and the
solvent evaporated in vacuo leaving a semi-solid (0.25 g) which was
chromatographed using EtOAc as an eluent to yield
1,5-dimethyl-5-hydroxy-4-phenyl-2(5H)pyrolone (0.21 g; 79%) as
colourless solid. m.p. 148-152.degree. C.
[0145] The alcohol was dissolved in dichloromethane (10 ml) and
stirred with P.sub.2O.sub.5 (1 g) for 1 h. The mixture was filtered
through a celite/silica bed; and washed with CH.sub.2Cl.sub.2/EtOAc
(1:1). The combined filtrate and washings were evaporated and
chromatographed using EtOAc/CH.sub.2Cl.sub.2 (1:19) to yield
1-methyl-5-methylene-4-phenyl-2(5H)pyrolone as colourless solid
(0.24 g; 49%). .sup.1H NMR .delta.(CDCl.sub.3): 7.42-7.44 (m, 5H,
ArH's); 6.21 (s, 1H, C3-H) and 3.18 (s, 3H, N-Me).
Synthesis of 5-methylene-1,4-diphenyl-dihydro-pyrrol-2-one
##STR00009##
[0147] 5-Hydroxy-5-methyl-4-phenyl-2(5H)furanone (0.20 g; 1.05
mmol) was added to a solution of aniline (2 ml) in toluene (7 ml).
The mixture was heated at reflux with stirring for 24 h. After
cooling to room temperature, ethyl acetate (20 ml) was added to the
mixture and the mixture washed with HCl (2M). The solvent was
evaporated in vacuo and chromatographed using
CH.sub.2Cl.sub.2/EtOAc (19:1) to afford
5-hydroxy-5-methyl-1,4-diphenyl-2(5H)pyrolone (0.12 g; 44%).
[0148] The above compound was heated at reflux with a few crystals
of p-TSOH in toluene solution for 0.5 h. The mixture was cooled and
chromatographed to yield
5-methylene-1,4-diphenyl-dihydro-pyrrol-2-one (0.05 g).
Synthesis of
5-methylene-4-(4'-fluorophenyl)-dihydro-pyrrol-2-one
##STR00010##
[0150] The acid (1.5 g; 6.74 mmol) was dissolved in thionyl
chloride (10 ml) and heated at reflux for 3 hr. Excess thionyl
chloride was removed in vacuo, and the residual oil (1.8 g) was
dissolved in dichloromethane (20 ml) and cooled in an ice-bath. Few
crystals of ammonium acetate followed by concentrated aqueous
ammonia (15M, 15 ml) was added dropwise over a period of 1 hr, and
the reaction mixture left to stir at room temperature over night.
The mixture was extracted with ethyl acetate (3.times.20 ml) and
the combined organic phase was dried (sodium sulfate) and
evaporated to yield of
5-hydroxy-5-methyl-4(4'-fluoro-phenyl)-dihydro-pyrrol-2-one (0.70
g) as a pale brown solid. .sup.1H NMR: .delta.
(CDCl.sub.3):7.78-7.83, 2H, m, Ar H's); 7.1-7.14 (2H, t, Ar H's);
6.67 (s, 1H, --NH); 6.10-6.11(d, 1H, C3-H); 3.65 (br, 1H,
--OH).
[0151] The lactam (0.18 g; 0.87 mmol) was dissolved is dry
dichloromethane (10 ml) and cooled in an ice bath, whilst
BF.sub.3.Et.sub.2O (0.15 ml) was added drop wise. The mixture was
stirred in ice for 1 h, then left to stir at room temperature
overnight. The solution was evaporated in vacuo. The residue
re-dissolved in CH.sub.2Cl.sub.2, washed with water and dried over
sodium sulfate. Evaporation of the solvent in vacuo gave
5-methylene-4-(p-fluorophenyl)-2(5H)pyrolone as a pale brown solid
(0.15 g; 91%). .sup.1H NMR: .delta. (CDCl.sub.3): 7.40-7.46 (2H,m,
Ar s); 7.18 (2H, m, Ar H's); 6.25 (s, 1H, C3-H); 5.06 (d, 1H,
C5=CH2) and 5.31 (d, 1H, C5=CH.sub.2).
Synthesis of 4-(4'-bromophenyl)-5-methylene-2(5H)pyrrolone
##STR00011##
[0153] A mixture of
4-(4'-bromophenyl)-5-hydroxy-5-methyl-2(5H)pyrolone (0.17 g; 0.63
mmol) and P.sub.2O.sub.5 (0.50 g) in dichloromethane (20 ml) was
stirred at room temperature for 45 min until all of the pyrrolone
has dissolved. The mixture was diluted with dichloromethane and
filtered through a bed of celite/silica. The Celite bed was washed
with CH.sub.2Cl.sub.2/EtOAc (1:1) and the combined filtrate and
washings were evaporated and chromatographed to yield
4-(4'-bromophenyl)-5-methylene-2(5H)pyrolone, (0.06 g; 35%) as a
pale yellow sonde, m.p. 200.degree. C. (decomp). .sup.1H NMR:
.delta. (CDCl.sub.3): 8.3 (s, 1H, NH); 7.57-7.6 (d, 2H, Ar H's),
7.29-7.32 (d, 2H; Ar H's); 6.23 (s, 1H, C3-H); 5.15 (d, 1H,
C5=CH.sub.2) and 4.95 (d, 1H, C5=CH.sub.2).
Synthesis of 4-(4'-methoxyphenyl-5-methylene-2(5H)pyrolone
##STR00012##
[0155] A mixture of
5-hydroxy-5-methyl-4-(4'-methoxyphenyl)-2(5H)pyrolone (0.15 g; 0.68
mmol) and P.sub.2O.sub.5 (1 g; 7 mmol)) in dry dichloromethane (10
ml) was stirred at room temperature for 0.5 h, during which time
the lactam dissolved. The mixture was diluted, with dichloromethane
(20 ml), filtered through a pad of celite/silica. The solvent was
removed in vacuo and the residue chromatographed to yield
4-(4'-methoxyphenyl)-5-methylene-2(5H)pyrolone (0.61 g; 44%) as a
yellow crystalline solid. .sup.1H NMR .delta. (CDCl.sub.3)8.54
(brs; --NH); 7.40 (2H, d, Ar H's); 6.95-6.98 (2H, d, Ar H's); 6.16
(1H, s, C3-H) 5.02 9d, 1H, C5=CH.sub.2); 5.15 (d, C5=CH.sub.2)and
3.85 (3H, s, OMe)
Synthesis of 4-benzyl-5-methylene-dihydro-pyrrol-2-one
##STR00013##
[0157] The acid (3.5 g; 0.017 mol) was dissolved in thionyl
chloride (20 ml) and heated at reflux for 0.5 hr. Excess thionyl
chloride was removed in vacuo, and the residual oil (2.86 g) was
dissolved in dichloromethane (20 ml) and cooled in an ice-bath.
Aqueous ammonia (15M, 7 ml) was added dropwise over a period of 1
hr, and the reaction mixture left to stir at room temperature over
night. The mixture was extracted with ethyl acetate (3.times.20 ml)
and the combined organic phase was dried (sodium sulfate) and
evaporated. The resulting brown oil was chromatographed to yield
5-hydroxy-5-methyl-4-benzyl-dihydro-pyrrol-2-one (0.45 g) as a
colourless solid.
[0158] A mixture of dihydro-pyrrolone (0.45 g) and P.sub.2O.sub.5
(0.50 g) in dichloromethane (20 ml) was stirred at room temperature
for 30 min until all of the pyrrolone has dissolved. The mixture
was filtered through a celite/silica bed; and washed with
CH.sub.2Cl.sub.2/EtOAc (1:1). The combined filtrate and washings
were evaporated leaving 4-benzyl-5-methylene-2(5H)-pyrrolone (0.17
g; 42%) as a pale yellow solid. .sup.1H NMR .delta. (CDCl.sub.3):
8.66 (brs; 1H, NH); 7.18-7.31 (5H; m, Ar H's); 5.80 (s, 1H, C3-H);
4.90-4.95 (d, 2H, C5=CH.sub.2) and 3.78 (s. 2H, --CH.sub.2Ph).
[0159] The compounds of Table 1 were also prepared by the general
synthetic methodology set out above.
TABLE-US-00001 TABLE 1 ##STR00014## ##STR00015## % Yield/Melting
point % Yield/Melting point Substituents degrees Celsius degrees
Celsius R = CH.sub.3; R.sup.1 = H 43; 121-123 49; oil R = Ph;
R.sup.1 = H 98; 249-252 75; >230 R = R.sup.1 = Ph 44; 158-160
47; 210-212 R = Ph; R.sup.1 = CH.sub.3 79; 148-152 49; 112-114 R =
4(F)--Ph; R.sup.1 = H 40; 203-205 66; 95-97 R = 4(F)Ph; R1 = Me 22;
137-140 49; 112-114 R = 3(F)Ph; R.sup.1 = H 19; 160 65; >230 R =
2(F)Ph; R = H 40; 140-143 98; >230 R = PhCH.sub.2; R.sup.1 = H
66; 175-178 42; 110-112 R = 4(Br)Ph; R = H 93; 255 35; >250 R =
4(MeO)Ph; R.sup.1 = H 40; 160-162 70; 175 R = 3(MeO)Ph; R.sup.1 = H
17; 167-168 48; 170 R = 2(MeO)Ph; R.sup.1 = H 11; 151-152 40;
>230 R = 3(CF.sub.3)Ph; R.sup.1 = H 48; >230 36; 190 R =
4(CF.sub.3)Ph; R.sup.1 = H 37; 170-173 41; 170-173
Synthesis of Acrylate and Methacrylate Derivates of Compounds of
Formula I and II
Preparation of methacrylate derivative of
5-hydroxy-5-methyl-4-phenyl-dihydro-pyrrol-2-one
5-Methyl-5-(2'-methylprop-2-enoyloxy)-4-phenyl-dihydropyrrol-2-one
and
5-methyl-1-methylprop-2-enoyl)-(2'-methylprop-2-enoyloxy)-4-phenyl-dihydr-
opyrrol-2-one
##STR00016##
[0161] To a suspension of the pseudo acid lactam (020 g; 1.06 mmol)
in dry CH.sub.2Cl.sub.2 (20 ml), was added triethylamine (1.5 ml;
10.76 mmol) and stirred with cooling in an ice bath. A solution of
methacryloyl chloride (1 ml; 9.56 mmol) in dry CH.sub.2Cl.sub.2 (3
ml) was added drop wise over 15 min. The mixture was left to stir
at this temperature for 2 h. Flash columned with
CH.sub.2Cl.sub.2/petrol (2:1) giving the methacrylates as a viscous
yellow oil (0.25 g; 92%).
5-Methyl-5-(2'-methylprop-2-enoyloxy)-4-phenyl-dihydropyrrol-2-one
[0162] .sup.1H NMR .delta. (CDCl.sub.3): 7.85 (m, 2H, Ar H's);
7.489 (m, 3H, Ar H's); 6.32 (1H, s, C4-H); 5.41 (s, 1H, --NH); 5.36
(s, 1H, .dbd.CH.sub.2); 5.25 (s, 1H, .dbd.CH.sub.2); 2.06 (s, 3H,
Me) and 2.0 (s, 3H, C5-Me).
5-Methyl-1-(2'-methylprop-2-enoyl)-5-(2'-methylprop-2-enoyloxy)-4-phenyl-d-
ihydropyrrol-2-one
[0163] .sup.1H NMR .delta. (CDCl.sub.3): 7.64-7.67 (m, 2H, Ar H's);
7.44-7.47 (m, 3H, Ar H's); 6.48 (1H, s, C4-H); 6.23 (s, 1H,
.dbd.CH.sub.2); 5.66 (d, 1H, .dbd.CH.sub.2); 5.32 (d, 2H,
.dbd.CH.sub.2); 2.1 (s, 3H, Me) and 2.0 (s, 3H, C5-Me), 1.90 (s,
3H, C5-Me).
Preparation of acrylate derivatives of
5-Hydroxy-5-methyl-4-phenyl-dihydro-pyrrol-2-one
5-Methyl-5-(prop-2-enoyloxy)-4-phenyl-dihydropyrrol-2-one and
5-methyl-1-(prop-2-enoyl)-5-(prop-2-enoyloxy)-4-phenyl-dihydropyrrol-2-on-
e
##STR00017##
[0165] To a solution of the pseudo acid lactam (0.23 g; 1.22 mmol)
in dry CH.sub.2Cl.sub.2 (10 ml) and dry THF (1 ml), was added
triethylamine (1.5 m; 1076 mmol) and few crystals of hydroquinone,
while stirring and cooling in an ice bath. A solution of acryloyl
chloride (1 ml; 9.56 mmol) in dry CH.sub.2Cl.sub.2 (3 ml) was added
drop wise over 10 min. The mixture was stirred further for 3 h. The
solvent was removed in vacuo at ca 30.degree. C., and the residual
semi-solid flash columned with CH.sub.2Cl.sub.2 to yield the
acrylates as pale yellow solids (0.21 g; 75%).).
5-Methyl-5-(prop-2-enoyloxy)-4-phenyl-dihydropyrrol-2-one
[0166] .sup.1H NMR .delta. (CDCl.sub.3): 7.88 (m, 2H, Ar H's); 7.48
(m, 3H, Ar H's); 6.62 (d, 1H, .dbd.CH.sub.2); 6.34 (1H, s, C4-H);
5.95 (dd, 1H, .dbd.CH); 5.30 (s, 1H, .dbd.CH.sub.2) 1.92 (s, 3H,
C5-Me).
5-Methyl-1-(prop-2-enoyl)-5-(prop-2-enoyloxy)-4-phenyl-dihydropyrrol-2-one
[0167] .sup.1H NMR .delta. (CDCl.sub.3): 7.65 (m, 2H, Ar H's);
7.43-7.60 (m, 4H, Ar H+CH2); 6.46-6.53 (m, 3H, .dbd.CH.sub.2 and
C3-H); 6.13-6.16 (dd, 1H, --CH--); 5.8-6.0 (m, 2H, .dbd.CH.sub.2)
and 2.1 (s, 3H, C5-Me).
Preparation of methacrylate derivatives of
5-methylene-4-phenyl-dihydro-pyrrol-2-one
Synthesis of
5-Methylene-1-(2'-methylprop-2-enoyl)-4-phenyl-dihydropyrrol-2-one
##STR00018##
[0169] Trifluoroacetic acid (1.5 ml) was added to a solution of
5-methyl-1-(2'-methylprop-2-enoyl)-5-(2'-methylprop-2-enoyloxy)-4-phenyl--
dihydropyrrol-2-one (1.45 g, 4.46 mmol) in dichloromethane. The
mixture was stirred at room temperature for 2 h and washed
successively with saturated sodium bicarbonate solution and water.
The dichloromethane layer was separated, dried over sodium sulfate
and chromatographed on a silica column using dichloromethane as an
eluent to yield
5-methylene-1-(2'-methylprop-2-enoyl)-4-phenyl-dihydropyrrol-2-one
(0.64 g, 60%) as colourless granules.
[0170] .sup.1H NMR .delta. (CDCl.sub.3): 7.48 (m, 5H, Ar H's); 6.23
(s, 1H, .dbd.CH.sub.2); 6.14 (1H, s, C4-H); 5.55 (bs, 1H,
.dbd.CH.sub.2); 5.36 (s, 1H, .dbd.CH.sub.2).
Preparation of acrylate derivatives of
5-methylene-4-phenyl-dihydro-pyrrol-2-one
Synthesis of
5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one
##STR00019##
[0172] Trifluoroacetic acid (1.0 ml) was added to a solution of
5-methyl-1-(prop-2-enoyl)-5-(prop-2-enoyloxy)-4-phenyl-dihydropyrrol-2-on-
e (0.44 g, 1.48 mmol) in dichloromethane (9 ml). The mixture was
stirred at room temperature for 2 h and washed successively with
saturated sodium bicarbonate solution and water. The
dichloromethane layer was separated, dried over sodium sulfate and
chromatographed on a silica column using dichloromethane as an
eluent to yield
5-methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one (0.18 g,
51%) as colourless granules.
[0173] .sup.1H NMR .delta. (CDCl.sub.3): 7.46 (m, 5H, Ar H's); 6.70
(s, 1H, .dbd.CH.sub.2); 6.59 (d, 1H, .dbd.CH.sub.2); 6.15 (1H, s,
C4-H); 5.93 (d, 1H, .dbd.CH.sub.2); 5.41 (s, 1H,
.dbd.CH.sub.2).
[0174] In a similar manner, the acrylate derivatives of other
4-phenyl-5-methylene-dihydropyrrol-2-ones were prepared.
[0175] 2. Antibacterial Assays
[0176] A number of assays were carried out on the compounds of the
present invention. The experimental methodology of these assays is
set out below.
2(a) N-Acylated Homoserine Lactone (AHL) Quorum Sensing Assay
[0177] The present applicant has demonstrated that certain
furanones and furanone analogues can inhibit AHL-mediated quorum
sensing in bacteria. Compounds of the present invention were
compared to compounds of the prior art in order using an AHL-quorum
sensing assay which utilises a reporter strain that expresses Green
Fluorescent Protein (GFP) in the presence of AHL signals. The assay
is performed by measuring GFP output in the presence of the
compound to be measured and comparing the output to a control. By
using multiple samples at varying concentrations of compound and
AHL, an inhibition index of compound activity can be generated. The
inhibition index used in the present example is the relative amount
of compound required to reduce GFP expression to 40% of the
control. The inhibition index is termed AIC40. Lower values of
AIC40 represent better inhibitors of the AHL quorum sensing
system.
[0178] The reporter strain of bacterium used in this assay is E.
coli into which the V. fischeri luxRI system has been engineered. A
gfp gene is fused to the QS controlled luxI promoter as is
described in (Andersen et al., 2001, and Andersen et al, 1998).
[0179] Measurement of AIC40 (ID40 at 3 nM OHHL)
[0180] Determination of the activity of compounds using E. coli
based luxRI construct was performed as follows.
[0181] Inhibition Kinetics
[0182] In a 15 mL plastic tube, mix 3 mL of o.n. culture of the lux
reporter strain with 12 mL fresh medium, incubate at 37.degree. C.
Label six tubes, 10, 20, two 50 and two 100. To each of the tubes,
add OHHL (3-oxohexanoyl homoserine lactone) to a final
concentration of 10, 20, 50, or 100 nM respectively in the AB
medium (add enough medium to distribute across the appropriate
number of wells). To the first row of the microplate (row A), add
200 ul of the OHHL/AB mixture. To the remaining rows (B-H) add 100
ul of the OHHL/AB mixture. To the first row (A) add compounds to be
tested to the 200 ul mixture of OHHL/AB. Make a dilution series in
the first 7 rows by transferring 100 .mu.L from wells in row 1 to
wells in row 2 and so on. Discard the remaining 100 .mu.L from row
7. Add 100 .mu.L diluted lux monitor to each well. Incubate the
plate 2 hours at 37.degree. C. and measure green fluorescence using
the "Victor" plate reader.
[0183] Data Treatment
[0184] Calculate ID.sub.40for each column. To do this, calculate
the relative activity in each well. Each column is calculated
separately, the well that does not contain furanone is set to 100%
activity (the wells in row 8). Make a plot for each concentration
of OHHL vs. the range of compound concentrations used. Calculate
the amount of compound needed to lower the relative activity to
40%, this is termed inhibiting dose 40%, ID.sub.40. For each
compound an inhibition index, AIC.sub.40, is found as follows: plot
ID.sub.40 against its respective AHL concentration; AIC.sub.40 is
slope of the best straight line through the plotted points and
origin.
2(b) LasR Assay
[0185] Compounds of the present invention were also assayed using a
LasR assay. The LasR assay provides a measurement of quorum sensing
inhibition activity. The higher the percentage inhibition. The more
effective the compound. In the LasR assay, an unstable gfp has been
fused to the elastase promoter, so that the amount of Gfp is
regulated by the QS system. The plasmid is put into Pseudomonas
aeruginosa which makes its own AHL signals. The assay is performed
by adding the compounds to be tested to the system at the beginning
of the experiment, at different concentrations. Gfp expression is
measured at different times during growth, ending at the 24 h time
point. The percentage fluorescence was determined at the time point
when the fluorescence reached its maximum in the control, usually
around 11-12 h after inoculation.
Example 2(c)
S. Epidermidis Growth
[0186] The growth inhibition of Staphylococcus epidermidis by the
compounds of the present invention was determined by growing S.
epidermidis in 96 well microtitre plates in Trypticase Soy Broth
plus 0.5% glucose in a Wallac Victor2 microplate reader. Each
compound was added to the wells at different concentrations and a
no-compound control and a set of blank wells were also used. The
OD600 was measured hourly. Growth curves were generated and the
growth of the cells in the presence of the compound is compared to
the controls to determine the concentration of compound that
significantly alters growth rate or final growth yield.
Example 2(d)
Inhibition of Biofilm Formation
[0187] Biofilm formation in petri dishes was measured using the
following protocol as a compromise between flow cells and
microtitre plate based assays. The method increases the number of
replicates (relative to flow cells) that can be tested while still
generating a biofilm that has typical structures, such as
microcolonies, which are important as differentiated structures
within the biofilm. The method consists of placing a surface, such
as a glass slide (for unattached compounds) or a modified plastic
coupon (for covalently attached compounds) on the bottom of a
sterile petri dish and adding growth medium. The bacteria are
inoculated into the dish and allowed to incubate at the appropriate
temperature at 50 rpm. The medium is replaced at 24 h and allowed
to incubate a further 24 h, after which time, the slides are
removed and rinsed to remove loosely attached cells. For monitoring
biofilm formation on glass slides, the cells are either stained
with a suitable fluorescent stain and are visualised using the
confocal microscope and the biofilm (as % surface coverage) is
quantified, or the biofilm is stained with crystal violet, washed
thoroughly to remove excess stain, and subsequently destained and
the absorbance at 540 nm is measured to determine the amount of
biofilm, as a function of crystal violet staining.
[0188] Compound Measurements
[0189] Assay results for a number of compounds according to the
present invention using the assays set out in Examples 2(a)-2(d)
are set out in Tables 2 and 3.
TABLE-US-00002 TABLE 2 P. S. S. epidermidis aeruginosa epidermidis
E. coli Compd AIC40 LasR (conc) growth biofilm biofilm biofilm
##STR00020## 1.1 80% (50 ug/ml 5 ug/ml 88% (10 ug/ml) NE 95% (5
ug/ml) ##STR00021## 4.61 61% (50 ug/ml) 20 ug/ml 78% (12.5 ug/ml)
40% (10 ug/ml ##STR00022## 52.76 40% (50 ug/ml) ##STR00023## 69.45
61% 50 ug/ml) NE ##STR00024## 5.4 36% (25 ug/ml) 76% (25 ug/ml)
##STR00025## 0.49 45% (50 ug/ml) 1 ug/ml 32% (1 ug/ml) 41% (0.32
ug/ml) 93% (1.5 ug/ml) ##STR00026## 1 54% (25 ug/ml) 1 ug/ml NE
##STR00027## 4.6 40% (25 ug/ml) 0.5 ug/ml NE ##STR00028## 1.3 70%
(25 ug/ml) 0.5 ug/ml NE NE ##STR00029## 1.7 60% (25 ug/ml) 20 ug/ml
NE 60% (0.3 ug/ml)
TABLE-US-00003 TABLE 3 S. epidermidis P. aeruginosa S. epidermidis
E. coil Compd AIC40 LasR (conc) growth biofilm biofilm biofilm
##STR00030## NE 28% (100 ug/ml) 100 ug/ml NE NE ##STR00031## 9.9
40% (100 ug/ml) >100 ug/ml 12% (20 ug/ml) 17% (50 ug/ml)
##STR00032## NE NE >100 ug/ml ##STR00033## NE NE >50 ug/ml
##STR00034## NE 41% (100 ug/ml) >50 ug/ml ##STR00035## NE NE
>100 ug/ml NE, no effect
[0190] All percentages are percent reduction of control at
non-growth inhibitory concentrations
[0191] Numbers in parentheses indicate concentration used.
Example 2(e)
Pulmonary Model of Infection: Mice Challenged with Bacteria/Seaweed
Alginate Through the Trachea
[0192] This model is reminiscent of the situation in CF patients or
patients with a dysfunctional muccociliary escalator.
[0193] Experiment:
[0194] The treatment dosage of
5-methylene-4-phenyl-dihydro-pyrrol-2-one (C219) was 12 .mu.g/g
body weight, given as injections twice a day.
[0195] Mice were challenged with Pseudomonas aeruginosa PAO1 (wt)
5.2.times.10.sup.6 CFU/lung (1.3.times.10.sup.8 CFU/ml). Each group
holds 10 mice, the treatments with C219 and Vehicle lasted for
three days. The mortality was 10% in C219 and 30% in Vehicle
groups. At day 5, the surviving mice were sacrificed. LIMP (the
fraction of the total lung area of the lung that shows
inflammation) and bacterial content in the lungs were
determined.
[0196] Result: [0197] The treatment of C219 significantly reduced
the lung bacterial load compared to Vehicle groups on day 5
(p=0.0008) (FIG. 1). [0198] C219 treatment (two injections per day
each 12 .mu.g/g body weight given for three days) significantly
reduced the severity (LIMP) of P. aeruginosa (PA) lung infection in
mice to placebo (vehicle=control). Therefore, the compounds seem to
exert a positive effect on inflammation.
[0199] 3. Comparative Studies
[0200] The antibacterial activity and cytotoxicity of compounds
according to the present invention were compared to compounds
similar to those exemplified in WO 2004/016588.
3(a) N-Acylated Homoserine Lactone (AHL) Quorum Sensing Assay
[0201] The methodology for this assay is set out in 2(a) above.
[0202] Compounds 219, 257, 294, 295 according to the present
invention and compounds 198 and 205 which are similar to the
compounds exemplified in WO 2004/016588 were assayed and the
results are shown in Table 4, As can be seen compounds 219, 257,
294 and 295 have significantly better AIC40 values than compounds
198 and 205.
TABLE-US-00004 TABLE 4 Comparative results of N-acylated homoserine
lactone (AHL) quorum sensing assay Change to correspond to initial
data AIC40 (ID40 at 3 nM OHHL) ##STR00036## 1.1 (16.2) ##STR00037##
4.61 (17.61) ##STR00038## 52.76 (163.26) ##STR00039## 69.45
(239.79) ##STR00040## 5.4 (61 nM) ##STR00041## 0.49 (21 nM)
##STR00042## 27 ##STR00043## 99
3(b) Cytotoxicity Study
[0203] Compounds were tested for cytotoxic activity using the
following assay:
[0204] Murine L929 cells are established at low density and, during
the testing period, grow to confluency in plastic petri dishes. 24
hours after the dishes are inoculated, the medium on the test
dishes is aspirated and replaced with a medium containing the
compound to be tested.
[0205] The cell monolayer is then cultured for a further 48 hour
period. At the end of the test period, cells are harvested from the
dishes and their numbers assessed and compared with unperturbed
cultures. Differences in cell numbers are expressed as a percentage
inhibition in comparison to non-challenged cultures. An inhibition
of 30% is considered clear indication of cytotoxic potential in the
test compound.
[0206] Cell Line:
[0207] Earle's L Cells--NCTC Clone 929 (Murine) grown in MEM/NA
supplemented with 10% FBS
[0208] The compounds tested were 219, a compound according to the
present invention, and compounds 226 and 223 which are
dibromo-substituted lactams which have the same bromination pattern
as compounds exemplified in WO 2004/016588. The results of the
assay are set out in Table 5.
##STR00044##
TABLE-US-00005 TABLE 5 Treatment Mean Std Dev % Inhibition Null
(NaCl) 4.91E+05 1.66E+04 0.0 4% Ethanol 3.40E+05 1.85E+04 30.7 5%
Ethanol 3.09E+05 1.08E+04 36.9 7.5% Ethanol 7.63E+04 2.36E+04 84.5
219 (40 .mu.g/ml) 4.74E+05 2.09E+04 3.4 219 (20 .mu.g/ml) 4.95E+05
3.04E+04 -1.0 219 (10 .mu.g/ml) 5.41E+05 7.34E+03 -10.3 226 (40
.mu.g/ml) 1.67E+05 5.39E+03 61.6 226 (20 .mu.g/ml) 2.42E+05
1.42E+04 44.4 226 (10 .mu.g/ml) 2.14E+05 1.52E+04 50.9 226 (5
.mu.g/ml) 3.41B+05 2.50E+04 21.8 223 (40 .mu.g/ml) 2.07E+05
6.80E+04 52.4 223 (20 .mu.g/ml) 2.94E+05 2.06E+04 32.5 223 (10
.mu.g/ml) 3.31E+05 2.33E+04 24.1 DMSO 4.97E+05 1.09E+04 -1.3 NB
E+05 is equivalent to .times.10.sup.5
[0209] Compound 219, a compound according to the present invention,
displays surprisingly little or no cytotoxicity when compared with
the structurally related compounds 226 and 223 which are similar to
those exemplified in WO 2004/016588.
[0210] 4. Attachment of Compounds to Surfaces
4(a) Copolymerisation of 219-Methacrylate with HEMA Monomers
[0211] HEMA (hydroxyethylmethacrylate) contact lenses were made
using a HEMA formulation and 219-methacrylate. The copolymer
solution was poured into a two-part contact lens mould and cured
under 365 nm blue/black lamps for 30 minutes followed by an
additional 30 minutes in an oven at 115.degree. C. to complete the
process. Upon cooling, the cured lenses were removed from the
moulds and hydrated in PBS, then rinsed and stored in PBS.
4(b) Surface Attachment of Acrylates and Methacrylates of Compounds
of Formula I and II
[0212] Amino and thiol functionalised surfaces can be reacted with
an acrylate derivative of lactam in a Michael addition fashion.
This strategy is quite versatile and would result in covalent
attachment of the lactam moiety in a single step.
[0213] General Attachment Procedure
[0214] Silicone rubber sheets or catheters are functionalized with
amino groups by techniques known to those skilled in the art. These
techniques include activation of the surface by exposure to plasma
in a plasma chamber and then exposing the surface to heptyamine
vapour and direct reaction of the surface with
aminopropyltriethoxysilane. Other suitable techniques for
functionalisation of silicone surfaces are also known to those
skilled in the art. The sheets or catheters are soaked overnight
with agitation in glass vials containing alcoholic or aqueous
alcoholic solution of a compound of Formula I or II bearing an
acrylate or methacrylate substituent (for example 1 mg/mL in 50%
ethylene glycol, 10% ethanol, 40% Milli Q water) The coated sheets
or catheters are removed from the reaction mixture with tweezers
and washed (agitated for 10 minutes) three times in vials
containing 10 ml of fresh alcohol solution or mixture of 50%
ethylene glycol, 10% ethanol, 40% Milli Q water. The silicone
sheets or catheters are then washed three times with Milli Q water
and once with PBS and stored in PBS.
[0215] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but riot the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0216] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like which has been included in
the present specification is solely for the purpose of providing a
context for the present invention. It is not to be taken as an
admission that arty or all of these matters form part of the prior
art base or were common general knowledge in the field relevant to
the present invention as it existed in Australia or elsewhere
before the priority date of each claim of this application.
[0217] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
REFERENCES
[0218] ANDERSEN, J. B., HEYDORN, A., HENTZER, M., EBERL, L.,
GEISENBERGER, O., CHRISTENSEN, B., BAK, MOLIN, S. & GIVSKOV, M.
(2001) Gfp-based n-acyl homoserine lactone sensor systems for
detection of bacterial communication. Appl. Environ. Microbiol.,
67, 575-585. [0219] ANDERSEN, J. B., STERNBERG, C., POULSEN, L. K.,
BJORN, S. P., GIVSKOV, M. & MOLIN, S. (1998) New unstable
variants of green fluorescent protein for studies of transient gene
expression in bacteria. Appl. Environ. Microbiol., 64,
2240-2246.
* * * * *