U.S. patent number RE48,335 [Application Number 16/144,967] was granted by the patent office on 2020-12-01 for polymyxin derivatives as antimicrobial compounds.
This patent grant is currently assigned to Monash University. The grantee listed for this patent is Monash University. Invention is credited to Jian Li, Roger Nation, Kade D. Roberts, Philip Thompson, Tony Velkov.
View All Diagrams
United States Patent |
RE48,335 |
Li , et al. |
December 1, 2020 |
Polymyxin derivatives as antimicrobial compounds
Abstract
The present invention relates to antimicrobial compounds and
their uses, and in particular to peptide antibiotics which may be
used in the treatment of bacterial infections such as Gram-negative
bacterial infections, particularly those caused by
multidrug-resistant (MDR) pathogens.
Inventors: |
Li; Jian (Carnegie,
AU), Nation; Roger (Ivanhoe East, AU),
Velkov; Tony (Clarinda, AU), Thompson; Philip
(Northcote, AU), Roberts; Kade D. (Flemington,
AU) |
Applicant: |
Name |
City |
State |
Country |
Type |
Monash University |
Clayton |
N/A |
AU |
|
|
Assignee: |
Monash University (Clayton,
AU)
|
Family
ID: |
54239144 |
Appl.
No.: |
16/144,967 |
Filed: |
September 27, 2018 |
PCT
Filed: |
April 01, 2015 |
PCT No.: |
PCT/AU2015/050149 |
371(c)(1),(2),(4) Date: |
October 17, 2016 |
PCT
Pub. No.: |
WO2015/149131 |
PCT
Pub. Date: |
October 08, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
15301310 |
Apr 1, 2015 |
10047126 |
Aug 14, 2018 |
|
|
Foreign Application Priority Data
|
|
|
|
|
Apr 1, 2014 [AU] |
|
|
2014901182 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
31/04 (20180101); A61P 43/00 (20180101); A61K
38/12 (20130101); C07K 7/62 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
7/62 (20060101); A61K 38/00 (20060101); A61K
38/12 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
2010/35111 |
|
Oct 2010 |
|
TW |
|
2006/045156 |
|
May 2006 |
|
WO |
|
WO 2010/075416 |
|
Jul 2010 |
|
WO |
|
2010/130007 |
|
Nov 2010 |
|
WO |
|
WO 2012/051663 |
|
Apr 2012 |
|
WO |
|
WO 2012/168820 |
|
Dec 2012 |
|
WO |
|
2013/072695 |
|
May 2013 |
|
WO |
|
WO 2015/135976 |
|
Sep 2015 |
|
WO |
|
Other References
Orwa et al, "Isolation and Structural Characterization of Colistin
Components," The Journal of Antibiotics, vol. 54, No. 7, pp.
595-599. (Year: 2001). cited by examiner .
First Office Action in Chinese application No. 201580027296.9 dated
Mar. 4, 2019. cited by applicant .
Magee et al., "Discovery of Dap-3 Polymyxin Analogues for the
Treatment of Multidrug-resistant Gram-negative Nosocomial
Infections", J Med Chem. (2013) 56(12): 5079-5093. cited by
applicant .
Kadar et al., "The Renaissance of Polymyxins," Current Medicinal
Chemistry, 20(30): 3759-3773 (2013). cited by applicant .
Kanazawa et al., "Contribution of Each Amino Acid Residue in
Polymyxin B.sub.3 to Antimicrobial and Lipopolysaccharide Binding
Activity," Chem. Pharm. Bull., 57(3): 240-244 (Mar. 2009). cited by
applicant .
Kimura et al., "Analytical and Preparative Methods for Polymyxin
Antibiotics Using High-Performance Liquid Chromatography with a
Porous Styrene-Divinylbenzene Copolymer Packing," Journal of
Chromatography, 206: 563-572 (1981). cited by applicant .
Niu et al., "Polymyxin P is the active principle in suppressing
phytopathogenic Erwinia spp. by the biocontrol rhizobacterium
Paenibacillus polymyxa M-1," BMC Microbiology, 13(137): 1-13
(2013). cited by applicant .
Terabe et al., "Separation of Polymyxins and Octapeptins by
High-Performance Liquid Chromatography," Journal of Chromatography,
173: 313-320 (1979). cited by applicant .
Van Den Bossche et al., "Identification of impurities in polymyxin
B and colistin bulk sample using liquid chromatography coupled to
mass spectrometry," Talanta, 83: 1521-1529 (2011). cited by
applicant .
Velkov et al., "Teaching `Old` Polymyxins New Tricks:
New-Generation Lipopeptides Targeting Gram-Negative `Superbugs,`"
ACS Chem. Biol., 9: 1172-1177 (2014). cited by applicant.
|
Primary Examiner: Diamond; Alan D
Attorney, Agent or Firm: Knobbe, Martens, Olson & Bear
LLP
Government Interests
.Iadd.STATEMENT OF GOVERNMENT INTEREST.Iaddend.
.Iadd.This invention was made with government support under R01
AI098771 awarded by the National Institute of Allergy and
Infectious Diseases (NIAID), an institute of the National
Institutes of Health (NIH). The government has certain rights in
the invention..Iaddend.
Claims
The claims defining the invention are as follows:
1. A compound of formula (IIb): ##STR00016## wherein: R.sup.1 is
selected from .[.hexanoyl, hepatanoyl, octanoyl, nonanoyl,
decanoyl, dodecanoyl, 7 methyloctanoyl, S-5-methylheptanoyl,
R-5-methylheptanoyl, S,R-5-methylheptanoyl (racemic mixture),.].
4-biphenylcarboxyl, 4-trifluoromethylbenzoyl, 4-ethylbenzoyl,
3,4-dichlorobenzoyl, 4-chlorobenzoyl, 3-chlorobenzoyl,
pentafluorobenzoyl, 4-methylbenzoyl, 4-ethylphenylacetyl,
phenylacetyl, 4-methylphenylacetyl, 4-trifluoromethylphenylacetyl,
pentafluorophenylacetyl, 3,4-dichlorophenylacetyl,
4-chlorophenylacetyl, 3-chlorophenylacetyl, 2-chlorobenzoyl,
2-fluorobenzoyl, 2-methylbenzoyl, 2-chlorophenylacetyl,
2-fluorophenylacetyl, 2-methylphenylacetyl, 2,3-dichlorobenzoyl,
2,3-dimethylbenzoyl, 2,4-dichlorophenylacetyl, 2,4-dichlorobenzoyl,
2,4-dimethylbenzoyl, 2-chloro-4-methylbenzoyl,
2-chloro-4-trifluoromethylbenzoyl, 3-fluorobenzoyl,
3-methylbenzoyl, 3-trifuoromethylbenzoyl, 3,4-dimethylbenzoyl,
3-fluoro-4-methylbenzoyl, 4-chloro-3-methylbenzoyl,
3,4-dimethylphenylacetyl, 3-chloro-4-methylbenzoyl,
4-chloro-3-fluorobenzoyl, 3-fluoro-4-trifluoromethylbenzoyl,
3-chloro-4-fluorobenzoyl, 4-methyl-3-trifluoromethylbenzoyl,
3-methyl-4-trifluoromethylbenzoyl,
3-methyl-5-trifluoromethylbenzoyl, 3,5-dimethylbenzoyl,
3,5-dichlorobenzoyl, 3,5-bis(trifluoromethyl) benzoyl,
3-fluoro-5-trifluoromethylbenzoyl, 3-chloro-5-methylbenzoyl,
3-chloro-5-fluorobenzoyl, 2,4,6-trimethylbenzoyl,
2,4,6-trichlorobenzoyl, 2-chloro-4-fluorobenzoyl,
4-chloro-2-fluorobenzoyl, 3,4,5-trifluoromethylbenzoyl,
4-chloro-2-trifluoromethylbenzoyl,
2-fluoro-4-trifluoromethylbenzoyl, 3-biphenylcarboxyl,
4-chlorobiphenyl-4-carboxyl, 3-phenylproponyl, 4-phenylbutanoyl,
2,4-dichlorophenylsulfonyl, 4-chloro-3-trifluoromethylbenzoyl,
4-isopropylbenzoyl, 4-chloro-3-fluorobenzoyl, .Iadd.and
.Iaddend.3-chloro-4-trifluoromethylbenzoyl; R.sup.3 represents a
side chain of an amino acid selected from leucine, phenylalanine,
norleucine, norvaline or t-butylglycine; R.sup.4 represents a side
chain of an amino acid selected from alanine, threonine, serine,
valine, t-butylglycine, 2-aminobutyric acid or 2-aminoisobutyric
acid; and k and q are individually selected from 1, 2, or 3; or
pharmaceutically acceptable salts thereof.
2. The compound according to claim 1, wherein R.sup.4 represents
the side chain of an amino acid selected from alanine, threonine,
serine, 2-aminobutyric acid, or 2-aminoisobutyric acid.
.Iadd.3. The compound of claim 2, wherein R.sup.3 represents the
side chain of D-leucine; R.sup.4 represents the side chain of
2-aminobutyric acid; R.sup.1 is 2,4-dichlorobenzoyl; k is 2; and q
is 1..Iaddend.
.Iadd.4. The compound of claim 1, selected from the group
consisting of: TABLE-US-00010 Compound R.sup.1 R.sup.3 R.sup.4 k q
29 4-BPC D-Leu Thr 2 2 30 PA D-Leu Thr 2 2 56 4-TFMB D-Leu Thr 2 1
57 3,4-DCB D-Leu Thr 2 1 61 3-CPA D-Leu Thr 2 1 62 2,4-DCPA D-Leu
Thr 2 1 70 2,4-DCPA D-Leu Abu 2 1 71 3,4-DCB D-Leu Abu 2 1 72 2-CB
D-Leu Abu 2 1 73 2-FB D-Leu Abu 2 1 74 4-TFMB D-Leu Abu 2 1 75 2-MB
D-Leu Abu 2 1 76 2-MPA D-Leu Abu 2 1 77 4-CPA D-Leu Abu 2 1 78 PA
D-Leu Abu 2 1 79 3-CPA D-Leu Abu 2 1 80 4-MPA D-Leu Abu 2 1 81
3,4-DCPA D-Leu Abu 2 1 82 2,4-DCB D-Leu Abu 2 1 83 3,4-DMB D-Leu
Abu 2 1 84 2-CPA D-Leu Abu 2 1 85 2-FPA D-Leu Abu 2 1 86 3-FB D-Leu
Abu 2 1 87 3-MB D-Leu Abu 2 1 88 3-CB D-Leu Abu 2 1 89 2,4-DMB
D-Leu Abu 2 1 90 2,3-DCB D-Leu Abu 2 1 91 2,3-DMB D-Leu Abu 2 1 92
2,4,6-TMB D-Leu Abu 2 1 93 3,5-DMB D-Leu Abu 2 1 94 4-CB D-Leu Abu
2 1 95 2,4,6-TCB D-Leu Abu 2 1 96 3,5-DCB D-Leu Abu 2 1 97
3,5-BTFMB D-Leu Abu 2 1 98 4-MB D-Leu Abu 2 1 99 4-IPB D-Leu Abu 2
1 100 4-EB D-Leu Abu 2 1 101 2-C-4-MB D-Leu Abu 2 1 102 3-F-4-MB
D-Leu Abu 2 1 103 3,4-DMPA D-Leu Abu 2 1 104 4-C-3-MB D-Leu Abu 2 1
105 3-C-4-MB D-Leu Abu 2 1 106 3-TFMB D-Leu Abu 2 1 107 4-C-3-Fb
D-Leu Abu 2 1 108 3-F-5-TFMB D-Leu Abu 2 1 109 2-C-4-TFMB D-Leu Abu
2 1 110 3-C-4-FB D-Leu Abu 2 1 111 3-F-4-TFMB D-Leu Abu 2 1 112
4-C-3-TFMB D-Leu Abu 2 1 113 4-M-3-TFMB D-Leu Abu 2 1 114 3-C-5-MB
D-Leu Abu 2 1 115 3-C-4-TFMB D-Leu Abu 2 1 116 3-C-5-FB D-Leu Abu 2
1 117 3,5-DCB D-Leu Ala 2 1 118 3,5-DCB D-Leu Thr 2 1 119
3-M-4-TFMB D-Leu Abu 2 1 120 3-M-5-TFMB D-Leu Abu 2 1 121 3-TFMB
D-Nle Abu 2 1 122 3-TFMB D-Phe Abu 2 1 123 3-TFMB D-Nlc Thr 2 1 124
3-TFMB D-Phe Thr 2 1 125 3-TFMB D-Nlc Ala 2 1 126 3-TFMB D-Phe Ala
2 1 127 4-TFMPA D-Leu Abu 2 1 134 4-C-2-FB D-Leu Abu 2 1 135
2-C-4-FB D-Leu Abu 2 1 136 4-C-2-TFMB D-Leu Abu 2 1 137 2-F-4-TFMB
D-Leu Abu 2 1 138 3-BPC D-Leu Abu 2 1 141 3-TFMB D-Leu Abu 3 1 142
4-BPC D-Leu Abu 2 1 144 4-Cl-BP-4-C D-Leu Ala 2 1 145 3-PP D-Leu
Abu 2 1 146 4-PB D-Leu Ala 2 1 147 2,4-DCB D-Leu Abu 2 3 148
2,4-DCPS D-Leu Abu 2 1,
or pharmaceutically acceptable salts thereof, wherein
4-BPC=4-biphenylcarboxyl, PA=phenylacetyl,
4-TFMPA=4-trifluoromethylphenylacetyl, 2-MB=2-methylbenzoyl,
3-MB=3-methylbenzoyl, 4-MB=4-methylbenzoyl,
3-F-4-MB=3-fluoro-4-methylbenzoyl,
4-C-3-MB=4-chloro-3-methylbenzoyl,
3-C-4-MB=3-chloro-4-methylbenzoyl,
3-C-5-MB=3-chloro-5-methylbenzoyl, 2-FPA=2-fluorophenylacetyl,
3-TFMB=3-trifuoromethylbenzoyl, 4-TFMB=4-trifluoromethylbenzoyl,
2-C-4-TFMB=2-chloro-4-trifluoromethylbenzoyl,
4-C-3-TFMB=4-chloro-3-trifluoromethylbenzoyl,
3-C-4-TFMB=3-chloro-4-trifluoromethylbenzoyl,
3-F-4-TFMB=3-fluoro-4-trifluoromethylbenzoyl,
3-F-5-TFMB=3-fluoro-5-trifluoromethylbenzoyl,
4-M-3-TFMB=4-methyl-3-trifluoromethylbenzoyl,
3-M-4-TFMB=3-methyl-4-trifluoromethylbenzoyl,
3-M-5-TFMB=3-methyl-5-trifluoromethylbenzoyl,
2-F-4-TFMB=2-fluoro-4-trifluoromethylbenzoyl,
4-C-2-TFMB=4-chloro-2-trifluoromethylbenzoyl,
3,5-BTFMB=3,5-bis(trifluoromethyl)benzoyl,
2,4,6-TMB=2,4,6-trimethylbenzoyl, 2,3-DMB=2,3-dimethylbenzoyl,
2,4-DMB=2,4-dimethylbenzoyl, 3,4-DMB=3,4-dimethylbenzoyl,
3,5-DMB=3,5-dimethylbenzoyl, 2-C-4-MB=2-chloro-4-methylbenzoyl,
4-EB=4-ethylbenzoyl, 4-IPB=4-Isopropylbenzoyl,
2,4-DCPA=2,4-dichlorophenylacetyl,
3,4-DCPA=3,4-dichlorophenylacetyl, 2-CPA=2-chlorophenylacetyl,
3-CPA=3-chlorophenylacetyl, 4-CPA=4-chlorophenylacetyl,
2-CB=2-chlorobenzoyl, 3-CB=3-chlorobenzoyl, 4-CB=4-chlorobenzoyl,
2,3-DCB=2,3-dichlorobenzoyl, 2,4-DCB=2,4-dichlorobenzoyl,
3,4-DCB=3,4-dichlorobenzoyl, 3,5-DCB=3,5-dichlorobenzoyl,
2,4,6-TCB=2,4,6-trichlorobenzoyl, 2-FB=2-fluorobenzoyl,
3-FB=3-fluorobenzoyl, 2-C-4-FB=2-chloro-4-fluorobenzoyl,
3-C-4-FB=3-Chloro-4-fluorobenzoyl,
3-C-5-FB=3-chloro-5-fluorobenzoyl,
4-C-2-FB=4-chloro-2-fluorobenzoyl,
4-C-3-FB=4-chloro-3-fluorobenzoyl, 2-MPA=2-methylphenylacetyl,
4-MPA=4-methylphenylacetyl, 3,4-DMPA=3,4-dimethylphenylacetyl,
3-BPC=3-biphenylcarboxyl, 4-C1-BP-4-C=4-chloro-biphenyl-4-carboxyl,
3-PP=3-phenylproponyl, 4-PB=4-phenylbutanoyl,
2,4-DCPS=2,4-dichlorophenylsulfonyl, Abu=2-aminobutyric acid,
Phe=phenylalanine, Thr=threonine, Ala=alanine, Leu=leucine, and
Nle=norleucine..Iaddend.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national stage application of the PCT
International Application No. PCT/AU2015/050149, filed Apr. 1, 2015
which claims the benefit of foreign priority of Australian
application 2014901182, filed on Apr. 1, 2014. The foregoing
applications are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
The present invention relates to antimicrobial compounds and their
uses, and in particular to peptide antibiotics which may be used in
the treatment of bacterial infections such as Gram-negative
bacterial infections, particularly those caused by
multidrug-resistant (MDR) pathogens.
BACKGROUND OF THE INVENTION
The world is facing an enormous and growing threat from the
emergence of bacteria that are resistant to almost all available
antibiotics. Whilst a small number of new antibiotics targeting
multidrug-resistant (MDR) Gram-positive bacteria have been approved
in the past two decades, there has been a marked decline in the
discovery of novel antibiotics for the treatment of Gram-negative
bacteria.
Representative genera of Gram-negative bacteria are: Acinetobacter;
Actinobacillus; Bartonella; Bordetella; Brucella; Burkholderia;
Campylobacter; Cyanobacteria; Enterobacter; Envinia; Escherichia;
Francisella; Helicobacter; Hemophilus; Klebsiella; Legionella;
Moraxella; Morganella; Neisseria; Pasteurella; Proteus;
Providencia; Pseudomonas; Salmonella; Serratia; Shigella;
Stenotrophomonas; Treponema; Vibrio; and Yersinia.
The Infectious Diseases Society of America (IDSA) has placed P.
aeruginosa, A. baumannii and K. pneumoniae on a `hit list` of the
six top-priority dangerous MDR microorganisms, the so-called
`superbugs`, in its recent `Bad Bugs Need Drugs` campaign. While
the recently approved tigecycline is active against a range of
clinically important Gram-negative pathogens, including
Acinetobacter baumannii, it is reported to not be effective against
Pseudomonas aeruginosa. Numerous hospitals worldwide have
experienced outbreaks of infections caused by P. aeruginosa, A.
baumannii or K. pneumoniae that are resistant to all commercially
available antibiotics, except for the last-line therapy
polymyxins.
Polymyxins belong to a class of peptides which was discovered more
than 60 years ago. They are produced by nonribosomal biosynthetic
enzymes from the secondary metabolic pathways in Paenibacillus
polymyxa. There are two polymyxins clinically available, colistin
(polymyxin E) and polymyxin B. Commercial preparations of polymyxin
B and colistin are mixtures of closely related peptides obtained
from fermentation (Orwa, J. A., et al. (2001) J. Chromatography A.
912, 369-373; Govaerts, C., et al. (2002) J. Chromatography A. 976,
65-78). The two major components found in polymyxin B preparations
are namely polymyxin B.sub.1 and B.sub.2, whilst commercial
preparations of colistin contain two major components labelled with
colistin A and B. The structures of these polymyxin B and colistin
components are shown below.
##STR00001##
Polymyxins are now being used as a last-line class of antibiotics
in patients where all other available antibiotics are inactive.
Despite the efficacy of polymyxins in treating certain
Gram-negative bacterial infections, it has been shown that
parenteral administration of colistin (as its inactive prodrug
colistin methanesulphonate) and polymyxin B can be potentially
nephrotoxic in up to 60% of patients, which limits them from being
used more routinely to treat MDR Gram-negative infections.
Furthermore, since nephrotoxicity is the major dose-limiting factor
for the currently available polymyxins, suboptimal dosing of
polymyxins can promote the emergence of polymyxin resistance.
Accordingly there exists a need to develop novel polymyxin
compounds that provide similar or better efficacy as the clinical
available polymyxins but without the nephrotoxic side effects.
SUMMARY OF THE INVENTION
It has now been found that certain polymyxin analogues have reduced
nephrotoxic side effects relative to polymyxin B or colistin,
whilst retaining or improving their efficacy against Gram-negative
bacteria, in particular, MDR Gram-negative bacteria.
Accordingly, in one aspect the present invention provides a method
of preventing or treating a multidrug-resistant (MDR) Gram-negative
bacterial infection comprising administering a therapeutically
effective amount of one or more compounds of the formula (I) or
formula (II) to a subject in need thereof:
##STR00002## wherein R.sup.1 is selected from
--C(O)C.sub.1-22alkyl, --C(O)C.sub.2-22alkenyl,
--C(O)C.sub.5-12aryl, --C(O)C.sub.1-22alkylC.sub.5-12aryl,
--C(O)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(O)C.sub.5-10arylC.sub.2-22alkenyl, --C(O)C.sub.4-12cycloalkyl,
--C(O)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(O)C.sub.3-12cycloalkylC.sub.2-22alkenyl, --C(S)C.sub.1-22alkyl,
--C(S)C.sub.2-22alkenyl, --C(S)C.sub.5-10aryl,
--C(S)C.sub.1-22alkylC.sub.5-12aryl,
--C(S)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(S)C.sub.4-12cycloalkyl, --C(S)C.sub.5-10arylC.sub.1-22alkyl,
--C(S)C.sub.5-10arylC.sub.2-22alkenyl,
--C(S)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(S)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--C(NH)C.sub.1-22alkyl, --C(NH)C.sub.2-22alkenyl,
--C(NH)C.sub.5-10aryl, --C(NH)C.sub.1-22alkylC.sub.5-12aryl,
--C(NH)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(NH)C.sub.4-12cycloalkyl, --C(NH)C.sub.5-10arylC.sub.1-22alkyl,
--C(NH)C.sub.5-10arylC.sub.2-22alkenyl,
--C(NH)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(NH)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.1-22alkyl, --S(O).sub.2C.sub.2-22alkenyl,
--S(O).sub.2C.sub.5-10aryl, --S(O).sub.2C.sub.4-12cycloalkyl,
--S(O).sub.2C.sub.5-10arylC.sub.1-22alkyl,
--S(O).sub.2C.sub.5-10arylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.3-12cycloalkylC.sub.1-22alkyl and
--S(O).sub.2C.sub.3-12cycloalkylC.sub.2-22alkenyl, each optionally
substituted with one or more C.sub.1-2alkyl, halo, or
trihaloC.sub.1-2alkyl; R.sup.2 represents a side chain of an amino
acid selected from serine or threonine; R.sup.3 represents a side
chain of an amino acid selected from leucine, phenylalanine,
norleucine, norvaline or t-butylglycine; R.sup.4 represents a side
chain of an amino acid selected from alanine, threonine, serine,
valine, t-butylglycine, 2-aminobutyric acid or 2-aminoisobutyric
acid; X is a residue of the side chain of an amino acid selected
from diaminobutyric acid, diaminopropionic acid, lysine or
ornithine; and k, m, n and p are individually selected from 1, 2,
or 3; or formula (II):
##STR00003## wherein R.sup.1, R.sup.3, R.sup.4, X, k, m, n and p
are as defined above for formula (I); and q is 1, 2 or 3; or
pharmaceutically acceptable salts thereof.
In another aspect, the present invention provides the use of one or
more compounds of formula (I) and/or formula (II) as hereinbefore
described, or pharmaceutically acceptable salts thereof, in the
manufacture of a medicament for the prevention or treatment of a
multidrug-resistant (MDR) Gram-negative bacterial infection.
In another aspect, the present invention provides one or more
compounds of formula (I) and/or formula (II) as hereinbefore
described, or pharmaceutically acceptable salts thereof, for use in
the prevention or treatment of a multidrug-resistant (MDR)
Gram-negative bacterial infection.
In another aspect the present invention provides compound of the
formula (Ia):
##STR00004## wherein R.sup.1 is selected from
--C(O)C.sub.1-22alkyl, --C(O)C.sub.2-22alkenyl,
--C(O)C.sub.5-12aryl, --C(O)C.sub.1-22alkylC.sub.5-12aryl,
--C(O)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(O)C.sub.5-10arylC.sub.2-22alkenyl, --C(O)C.sub.4-12cycloalkyl,
--C(O)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(O)C.sub.3-12cycloalkylC.sub.2-22alkenyl, --C(S)C.sub.1-22alkyl,
--C(S)C.sub.2-22alkenyl, --C(S)C.sub.5-10aryl,
--C(S)C.sub.1-22alkylC.sub.5-12aryl,
--C(S)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(S)C.sub.4-12cycloalkyl, --C(S)C.sub.5-10arylC.sub.1-22alkyl,
--C(S)C.sub.5-10arylC.sub.2-22alkenyl,
--C(S)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(S)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--C(NH)C.sub.1-22alkyl, --C(NH)C.sub.2-22alkenyl,
--C(NH)C.sub.5-10aryl, --C(NH)C.sub.1-22alkylC.sub.5-12aryl,
--C(NH)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(NH)C.sub.4-12cycloalkyl, --C(NH)C.sub.5-10arylC.sub.1-22alkyl,
--C(NH)C.sub.5-10arylC.sub.2-12alkenyl,
--C(NH)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(NH)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.1-22alkyl, --S(O).sub.2C.sub.2-22alkenyl,
--S(O).sub.2C.sub.5-10aryl, --S(O).sub.2C.sub.4-12cycloalkyl,
--S(O).sub.2C.sub.5-10arylC.sub.1-22alkyl,
--S(O).sub.2C.sub.5-10arylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.3-12cycloalkylC.sub.1-22alkyl and
--S(O).sub.2C.sub.3-12cycloalkylC.sub.2-22alkenyl, each optionally
substituted with one or more C.sub.1-2alkyl, halo, or
trihaloC.sub.1-2alkyl; R.sup.2 represents a side chain of an amino
acid selected from serine or threonine; R.sup.3 represents a side
chain of an amino acid selected from leucine, phenylalanine,
norleucine, norvaline or t-butylglycine; R.sup.4 represents a side
chain of an amino acid selected from alanine, threonine, serine,
valine, t-butylglycine, 2-aminobutyric acid or 2-aminoisobutyric
acid; X is a residue of the side chain of an amino acid selected
from diaminobutyric acid, diaminopropionic acid, lysine or
ornithine; and k, m, n and p are individually selected from 1, 2,
or 3; with the proviso that when R.sup.3 is the side chain residue
of leucine or phenylalanine, R.sup.4 is the side chain residue of
threonine and k, m, n and p are 2, R.sup.1 is not
S-6-methyloctanoyl or 6-methylheptanoyl; or pharmaceutically
acceptable salts thereof
In a further aspect, the present invention provides a compound of
the formula (IIa):
##STR00005## wherein R.sup.1 is selected from
--C(O)C.sub.1-22alkyl, --C(O)C.sub.2-22alkenyl,
--C(O)C.sub.5-12aryl, --C(O)C.sub.1-22alkylC.sub.5-12aryl,
--C(O)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(O)C.sub.5-10arylC.sub.2-22alkenyl, --C(O)C.sub.4-12cycloalkyl,
--C(O)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(O)C.sub.3-12cycloalkylC.sub.2-22alkenyl, --C(S)C.sub.1-22alkyl,
--C(S)C.sub.2-22alkenyl, --C(S)C.sub.5-10aryl,
--C(S)C.sub.1-22alkylC.sub.5-12aryl,
--C(S)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(S)C.sub.4-12cycloalkyl, --C(S)C.sub.5-10arylC.sub.1-22alkyl,
--C(S)C.sub.5-10arylC.sub.2-22alkenyl,
--C(S)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(S)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--C(NH)C.sub.1-22alkyl, --C(NH)C.sub.2-22alkenyl,
--C(NH)C.sub.5-10aryl, --C(NH)C.sub.1-22alkylC.sub.5-12aryl,
--C(NH)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(NH)C.sub.4-12cycloalkyl, --C(NH)C.sub.5-10arylC.sub.1-22alkyl,
--C(NH)C.sub.5-10arylC.sub.2-22alkenyl,
--C(NH)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(NH)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.1-22alkyl, --S(O).sub.2C.sub.2-22alkenyl,
--S(O).sub.2C.sub.5-10aryl, --S(O).sub.2C.sub.4-12cycloalkyl,
--S(O).sub.2C.sub.5-10arylC.sub.1-22alkyl,
--S(O).sub.2C.sub.5-10arylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.3-12cycloalkylC.sub.1-22alkyl and
--S(O).sub.2C.sub.3-12cycloalkylC.sub.2-22alkenyl, each optionally
substituted with one or more C.sub.1-2alkyl, halo, or
trihaloC.sub.1-2alkyl; R.sup.3 represents a side chain of an amino
acid selected from leucine, phenylalanine, norleucine, norvaline or
t-butylglycine; R.sup.4 represents a side chain of an amino acid
selected from alanine, threonine, serine, valine, t-butylglycine,
2-aminobutyric acid or 2-aminoisobutyric acid; X is a residue of
the side chain of an amino acid selected from diaminobutyric acid,
diaminopropionic acid, lysine or ornithine; and k, m, n, p and q
are individually selected from 1, 2, or 3; with the proviso that
when R.sup.3 is the side chain residue of leucine, R.sup.4 is the
side chain residue of threonine and k, m, n, p and q are 2, R.sup.1
is not S-6-methyloctanoyl or 6-methylheptanoyl, or pharmaceutically
acceptable salts thereof.
In another aspect the invention provides a pharmaceutical
composition comprising a therapeutically effective amount of one or
more compounds as hereinbefore defined, or pharmaceutically
acceptable salts thereof, together with at least one
pharmaceutically acceptable carrier or diluent.
In another aspect the invention provides a method of preventing or
treating a Gram-negative bacterial infection comprising the step of
administering a therapeutically effective amount of one or more
compounds of the formula (Ia) and/or (IIa) as hereinbefore
described, or pharmaceutically acceptable salts thereof, to a
subject in need thereof.
In another aspect the invention provides the use of one or more
compounds of the formula (Ia) and/or (IIa) as hereinbefore
described, or pharmaceutically acceptable salts thereof, in the
manufacture of a medicament for the prevention or treatment of a
Gram-negative bacterial infection.
In another aspect the invention provides one or more compounds of
the formula (Ia) and/or (IIa) as hereinbefore described, or
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of a Gram-negative bacterial infection.
These and other aspects of the present invention will become more
apparent to the skilled addressee upon reading the following
detailed description in connection with the accompanying examples
and claims.
DETAILED DESCRIPTION OF THE INVENTION
The initial cellular target of polymyxins in Gram-negative bacteria
is the lipopolysaccharide (LPS) component of the outer membrane
(OM). It is believed that the LPS target is generally conserved
across most, if not all, Gram-negative bacteria.
In general, LPS is composed of three domains, a conserved inner
core 2-keto-3-deoxyoctanoic acid bound to lipid A and a variable
O-antigen composed of repeating units of various polysaccharides.
The consensus structure of lipid A consists of a .beta.-1'-6-linked
D-glucosamine disaccharide that is phosphorylated at the 1- and
4'-positions. An example of the structure of lipid A from P.
aeruginosa is shown below:
##STR00006##
Lipid A usually contains six acyl chains. Four .beta.-hydroxy acyl
chains (usually C.sub.10 to C.sub.14 in length) are attached
directly to the glucosamine sugars, while a secondary acyl chain is
often attached to the .beta.-hydroxy group on each of two of the
chains. Lipid A acts as a hydrophobic anchor with the tight packing
of the fatty acyl chains helping to stabilise the overall outer
membrane structure.
It is believed that there is an initial polar interaction between
the cationic polymyxin peptide (particularly the charged
.alpha.,.gamma.-diaminobutyric acid (Dab) residues) and the lipid A
component of LPS in the outer membrane, thereby displacing divalent
cations (Ca.sup.2+ and Mg.sup.2+) from the negatively charged
phosphate groups of lipid A. This initial interaction is followed
by uptake across the outer membrane and interaction with the
cytoplasmic membrane.
Polymyxin B and colistin (polymyxin E) first became available for
clinical use as antibiotics in the 1950s. Shortly after, their use
fell out of favour because of concerns about nephrotoxic side
effects. These observed nephrotoxic side effects for colistin
resulted in the peptide rarely being used as an antibiotic during
the period of 1980-2000. More recently it has found use again as a
last-line antibiotic, predominantly due to necessity, in patients
where all other antibiotics are found to be ineffective.
Furthermore, since nephrotoxicity is the major dose-limiting factor
for the current polymyxins, compounds having an improved
nephrotoxicity profile would allow higher doses to be administered
to more effectively treat infections and suppress the emergence of
polymyxin resistance.
It has now surprisingly been found that the compounds of the
present invention are effective against Gram-negative bacteria
whilst displaying an improved nephrotoxicity profile relative to
polymyxin B or colistin. The present inventors have discovered that
certain amino acid residues at three key locations within the
polymyxin structure, in combination with specific N-terminal fatty
acyl groups, can significantly reduce the level of nephrotoxicity
of the compound whilst maintaining or improving the compound's
antibacterial efficacy.
In this specification a number of terms are used which are well
known to a skilled addressee. Nevertheless, for the purposes of
clarity a number of terms will be defined. As used herein, the term
"alkyl", used either alone or in compound words, denotes straight
chain or branched alkyl. Preferably the alkyl group is a straight
chain alkyl group. Prefixes such as "C.sub.1-22" are used to denote
the number of carbon atoms within the alkyl group (from 1 to 22 in
this case). Examples of straight chain and branched alkyl include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl,
n-pentyl, hexyl, heptyl, 5-methylheptyl, 5-methylhexyl, octyl,
nonyl, decyl, undecyl, dodecyl and docosyl (C.sub.22).
As used herein, the term "alkenyl", used either alone or in
compound words, denotes straight chain or branched hydrocarbon
residues containing at least one carbon to carbon double bond
including ethylenically mono-, di- or polyunsaturated alkyl groups
as previously defined. Preferably the alkenyl group is a straight
chain alkenyl group. Prefixes such as "C.sub.2-22" are used to
denote the number of carbon atoms within the alkenyl group (from 2
to 22 in this case). Examples of alkenyl include vinyl, allyl,
1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl,
1-pentenyl, 1-hexenyl, 3-hexenyl, 1-heptenyl, 3-heptenyl,
1-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl,
1,3-butadienyl, 1,4-pentadienyl, 1,3-hexadienyl, 1,4-hexadienyl and
5-docosenyl (C.sub.22).
As used herein, the term "cycloalkyl", used either alone or in
compound words, denotes a cyclic alkyl group. Prefixes such as
"C.sub.3-12" are used to denote the number of carbon atoms within
the cyclic portion of the alkyl group (from 3 to 12 in this case).
Examples of cyclic alkyl include mono- or polycyclic alkyl groups
such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and
cyclododecyl.
As used herein, the term "aryl" denotes any single- or polynuclear,
conjugated or fused residues of aromatic hydrocarbon ring systems.
Prefixes such as "C.sub.6-16" are used to denote the number of
carbon atoms within the cyclic portion of the aryl group (from 6 to
16 in this case). Examples of aryl include phenyl (single nuclear),
naphthyl (fused polynuclear), biphenyl (conjugated polynuclear) and
tetrahydronaphthyl (fused polynuclear).
The term "halo" used herein refers to fluoro, chloro, bromo or
iodo.
As used herein, reference to an amino acid "side chain" takes its
standard meaning in the art. Examples of side chains of amino acids
are shown below:
##STR00007##
As used herein, non-naturally occurring amino acids include any
compound with both amino and carboxyl functionality, derivatives
thereof, or derivatives of a naturally occurring amino acid. These
amino acids form part of the peptide chain through bonding via
their amino and carboxyl groups. Alternatively, these derivatives
may bond with other natural or non-naturally occurring amino acids
to form a non-peptidyl linkage.
In addition to the negatively charged side chains shown above, it
will be appreciated that a number of the side chains may also be
protonated and so become positively charged, such as the side chain
of lysine. The present invention contemplates within its scope
these protonated side chains as well.
It will be understood that the compounds of the present invention
may exist in one or more stereoisomeric forms (e.g. diastereomers).
The present invention includes within its scope all of these
stereoisomeric forms either isolated (in, for example, enantiomeric
isolation), or in combination (including racemic mixtures and
diastereomic mixtures). The present invention contemplates the use
of amino acids in both L and D forms, including the use of amino
acids independently selected from L and D forms, for example, where
the peptide comprises two Dab residues, each Dab residue may have
the same, or opposite, absolute stereochemistry. Unless stated
otherwise, the amino acid is taken to be in the
L-configuration.
The invention thus also relates to compounds in substantially pure
stereoisomeric form with respect to the asymmetric centres of the
amino acid residues, e.g., greater than about 90% de, such as about
95% to 97% de, or greater than 99% de, as well as mixtures,
including racemic mixtures, thereof. Such diastereomers may be
prepared by asymmetric synthesis, for example, using chiral
intermediates, or mixtures may be resolved by conventional methods,
e.g., chromatography, or use of a resolving agent.
In some preferred embodiments of the invention, and with reference
to the general formulae (Ia) and (IIa), one or more of the
following preferred embodiments apply:
a) R.sup.1 is selected from --C(O)C.sub.1-22alkyl,
--C(O)C.sub.2-22alkenyl, --C(O)C.sub.5-12aryl,
--C(O)C.sub.1-22alkylC.sub.5-12aryl,
--C(O)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(O)C.sub.5-10arylC.sub.2-22alkenyl, --C(O)C.sub.4-12cycloalkyl,
--C(O)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(O)C.sub.3-12cycloalkylC.sub.2-22alkenyl, --C(S)C.sub.1-22alkyl,
--C(S)C.sub.2-22alkenyl, --C(S)C.sub.5-10aryl,
--C(S)C.sub.1-22alkylC.sub.5-12aryl,
--C(S)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(S)C.sub.4-12cycloalkyl, --C(S)C.sub.5-10arylC.sub.1-22alkyl,
--C(S)C.sub.5-10arylC.sub.2-22alkenyl,
--C(S)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(S)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--C(NH)C.sub.1-22alkyl, --C(NH)C.sub.2-22alkenyl,
--C(NH)C.sub.5-10aryl, --C(NH)C.sub.1-22alkylC.sub.5-12aryl,
--C(NH)C.sub.1-22alkylC.sub.3-12cycloalkyl,
--C(NH)C.sub.4-12cycloalkyl, --C(NH)C.sub.5-10arylC.sub.1-22alkyl,
--C(NH)C.sub.5-10arylC.sub.2-22alkenyl,
--C(NH)C.sub.3-12cycloalkylC.sub.1-22alkyl,
--C(NH)C.sub.3-12cycloalkylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.1-22alkyl, --S(O).sub.2C.sub.2-22alkenyl,
--S(O).sub.2C.sub.5-10aryl, --S(O).sub.2C.sub.4-12cycloalkyl,
--S(O).sub.2C.sub.5-10arylC.sub.1-22alkyl,
--S(O).sub.2C.sub.5-10arylC.sub.2-22alkenyl,
--S(O).sub.2C.sub.3-12cycloalkylC.sub.1-22alkyl and
--S(O).sub.2C.sub.3-12cycloalkylC.sub.2-22alkenyl, each optionally
substituted with one or more C.sub.1-2alkyl, halo, or
trihaloC.sub.1-2alkyl. b) R.sup.1 is selected from hexanoyl,
hepatanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl,
S,R-6-methyloctanoyl (racemic mixture), R-6-methyloctanoyl,
7-methyl octanoyl, S-5-methyl heptanoyl, R-5-methyl heptanoyl,
S,R-5-methyl heptanoyl (racemic mixture), 4-biphenylcarboxyl,
4-trifluoromethylbenzoyl, 4-ethylbenzoyl, 3,4-dichlorobenzoyl,
4-chlorobenzoyl, 3-chlorobenzoyl, pentafluorobenzoyl,
4-methylbenzoyl, 4-ethylphenylacetyl, phenylacetyl,
4-methylphenylacetyl, 4-trifluoromethylphenylacetyl,
pentafluorophenyl acetyl, 3,4-dichlorophenylacetyl, 4-chlorophenyl
acetyl, 3-chlorophenyl acetyl, 2-chlorobenzoyl, 2-fluorobenzoyl,
2-methyl benzoyl, 2-chlorophenylacetyl, 2-methylphenyl acetyl,
2-fluorophenylacetyl, 2,3-dichlorobenzoyl, 2,3-dimethylbenzoyl,
2,4-dichlorophenylacetyl, 2,4-dichlorobenzoyl, 2,4-dimethylbenzoyl,
2-chloro-4-methylbenzoyl, 2-chloro-4-trifluoromethylbenzoyl,
3-fluorobenzoyl, 3-methylbenzoyl, 3-trifuoromethylbenzoyl,
3,4-dimethylbenzoyl, 3-fluoro-4-methylbenzoyl,
4-chloro-3-methylbenzoyl, 3,4-dimethylphenylacetyl,
3-chloro-4-methylbenzoyl, 4-chloro-3-fluorobenzoyl,
3-fluoro-4-trifluoromethylbenzoyl, 3-chloro-4-fluorobenzoyl,
4-methyl-3-trifluoromethylbenzoyl,
3-methyl-4-trifluoromethylbenzoyl,
3-methyl-5-trifluoromethylbenzoyl, 3,5-dimethylbenzoyl,
3,5-dichlorobenzoyl, 3,5-bis(trifluoromethyl)benzoyl,
3-fluoro-5-trifluoromethylbenzoyl, 3-chloro-5-methylbenzoyl,
3-chloro-5-fluorobenzoyl, benzoyl, 2,4,6-tri methylbenzoyl,
2,4,6-tri chlorobenzoyl, 2-chloro-4-fluorobenzoyl,
4-chloro-2-fluorobenzoyl, 3,4,5-trifluoromethylbenzoyl,
4-chloro-2-trifluoromethylbenzoyl,
2-fluoro-4-trifluoromethylbenzoyl, 3-biphenylcarboxyl,
4-chlorobiphenyl-4-carboxyl, 3-phenylproponyl, 4-phenylbutanoyl,
2,4-di chlorophenyl sulfonyl, 4-chloro-3-tri fluoromethyl benzoyl,
4-isopropylbenzoyl, 4-chloro-3-fluorobenzoyl,
3-chloro-4-trifluoromethylbenzoyl. c) R.sup.1 is selected from
hexanoyl, hepatanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl,
S,R-6-methyloctanoyl (racemic mixture), R-6-methyloctanoyl,
7-methyl octanoyl, S-5-methylheptanoyl, R-5-methylheptanoyl,
S,R-5-methylheptanoyl (racemic mixture), 4-biphenylcarboxyl,
4-trifluoromethylbenzoyl, 4-ethylbenzoyl, 3,4-dichlorobenzoyl,
4-chlorobenzoyl, 3-chlorobenzoyl, pentafluorobenzoyl,
4-methylbenzoyl, 4-ethylphenylacetyl, phenylacetyl,
4-methylphenylacetyl, 4-trifluoromethylphenylacetyl,
pentafluorophenyl acetyl, 3,4-dichlorophenylacetyl, 4-chlorophenyl
acetyl, 3-chlorophenyl acetyl, 2-chlorobenzoyl, 2-fluorobenzoyl,
2-methylbenzoyl, 2-chlorophenyl acetyl, 2-fluorophenylacetyl,
2-methylphenylacetyl, 2,3-dichlorobenzoyl, 2,3-dimethylbenzoyl,
2,4-dichlorophenylacetyl, 2,4-dichlorobenzoyl, 2,4-dimethylbenzoyl,
2-chloro-4-methylbenzoyl, 2-chloro-4-trifluoromethylbenzoyl,
3-fluorobenzoyl, 3-methylbenzoyl, 3-trifuoromethylbenzoyl,
3,4-dimethylbenzoyl, 3-fluoro-4-methylbenzoyl,
4-chloro-3-methylbenzoyl, 3,4-dimethylphenyl acetyl,
3-chloro-4-methylbenzoyl, 4-chloro-3-fluorobenzoyl,
3-fluoro-4-trifluoromethylbenzoyl, 3-chloro-4-fluorobenzoyl,
4-methyl-3-trifluoromethylbenzoyl,
3-methyl-4-trifluoromethylbenzoyl,
3-methyl-5-trifluoromethylbenzoyl, 3,5-dimethylbenzoyl,
3,5-dichlorobenzoyl, 3,5-bis(trifluoromethyl)benzoyl,
3-fluoro-5-trifluoromethylbenzoyl, 3-chloro-5-methylbenzoyl,
3-chloro-5-fluorobenzoyl, 2,4,6-trimethylbenzoyl, 2,4,6-tri
chlorobenzoyl, 2-chloro-4-fluorobenzoyl, 4-chloro-2-fluorobenzoyl,
3,4,5-trifluoromethylbenzoyl, 4-chloro-2-trifluoromethylbenzoyl,
2-fluoro-4-trifluoromethylbenzoyl, 3-biphenylcarboxyl,
4-chloro-biphenyl-4-carboxyl, 3-phenylproponyl, 4-phenylbutanoyl,
2,4-di chlorophenyl sulfonyl, 4-chloro-3-trifluoromethylbenzoyl,
4-isopropylbenzoyl, 4-chloro-3-fluorobenzoyl,
3-chloro-4-trifluoromethylbenzoyl. d) R.sup.1 is selected from
hexanoyl, hepatanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl,
S,R-6-methyloctanoyl (racemic mixture), R-6-methyloctanoyl,
7-methyl octanoyl, S-5-methylheptanoyl, R-5-methylheptanoyl,
S,R-5-methylheptanoyl (racemic mixture), 4-biphenylcarboxyl,
4-trifluoromethylbenzoyl, 4-ethylbenzoyl, 3,4-di chlorobenzoyl,
4-chlorobenzoyl, 3-chlorobenzoyl, pentafluorobenzoyl,
4-methylbenzoyl, 4-ethylphenyl acetyl, phenylacetyl, 4-methylphenyl
acetyl, 4-trifluoromethyl phenyl acetyl, pentafluorophenylacetyl,
3,4-dichlorophenylacetyl, 4-chlorophenylacetyl and
3-chlorophenylacetyl. e) R.sup.2 represents a side chain of an
amino acid selected from serine or threonine. f) R.sup.3 represents
a side chain of an amino acid selected from leucine, phenylalanine,
norleucine, norvaline or t-butylglycine. g) R.sup.4 represents a
side chain of an amino acid selected from alanine, threonine,
serine, valine, t-butylglycine, 2-aminobutyric acid or
2-aminoisobutyric acid. h) R.sup.4 represents a side chain of an
amino acid selected from alanine, threonine, serine, 2-aminobutyric
acid, or 2-aminoisobutyric acid. i) X is a residue of the side
chain of an amino acid selected from diaminobutyric acid,
diaminopropionic acid, lysine or ornithine. j) X is a residue of
the side chain of diaminobutyric acid. k) m, n and p are each 2
In a preferred embodiment X is the side chain residue of
diaminobutyric acid and m, n and p are 2.
Accordingly, in a further embodiment, the present invention
provides compounds of the formula (Ia) represented by the formula
(Ib):
##STR00008## wherein R.sup.1 is selected from hexanoyl, hepatanoyl,
octanoyl, nonanoyl, decanoyl, dodecanoyl, S,R-6-methyloctanoyl
(racemic mixture), R-6-methyloctanoyl, 7-methyloctanoyl,
S-5-methylheptanoyl, R-5-methylheptanoyl, S,R-5-methylheptanoyl
(racemic mixture), 4-biphenylcarboxyl, 4-trifluoromethylbenzoyl,
4-ethylbenzoyl, 3,4-dichlorobenzoyl, 4-chlorobenzoyl,
3-chlorobenzoyl, pentafluorobenzoyl, 4-methylbenzoyl, 4-ethylphenyl
acetyl, phenyl acetyl, 4-methylphenyl acetyl,
4-trifluoromethylphenyl acetyl, pentafluorophenyl acetyl,
3,4-dichlorophenylacetyl, 4-chlorophenyl acetyl, 3-chlorophenyl
acetyl, 2-chlorobenzoyl, 2-fluorobenzoyl, 2-methylbenzoyl,
2-chlorophenyl acetyl, 2-fluorophenylacetyl, 2-methylphenylacetyl,
2,3-dichlorobenzoyl, 2,3-dimethylbenzoyl, 2,4-dichlorophenylacetyl,
2,4-dichlorobenzoyl, 2,4-dimethylbenzoyl, 2-chloro-4-methylbenzoyl,
2-chloro-4-trifluoromethylbenzoyl, 3-fluorobenzoyl,
3-methylbenzoyl, 3-trifuoromethylbenzoyl, 3,4-dimethylbenzoyl,
3-fluoro-4-methylbenzoyl, 4-chloro-3-methylbenzoyl,
3,4-dimethylphenyl acetyl, 3-chloro-4-methylbenzoyl,
4-chloro-3-fluorobenzoyl, 3-fluoro-4-trifluoromethylbenzoyl,
3-chloro-4-fluorobenzoyl, 4-methyl-3-trifluoromethylbenzoyl,
3-methyl-4-trifluoromethylbenzoyl,
3-methyl-5-trifluoromethylbenzoyl, 3,5-dimethylbenzoyl,
3,5-dichlorobenzoyl, 3,5-bis(trifluoromethyl)benzoyl,
3-fluoro-5-trifluoromethylbenzoyl, 3-chloro-5-methylbenzoyl,
3-chloro-5-fluorobenzoyl, 2,4,6-trimethylbenzoyl, 2,4,6-tri
chlorobenzoyl, 2-chloro-4-fluorobenzoyl, 4-chloro-2-fluorobenzoyl,
3,4,5-trifluoromethylbenzoyl, 4-chloro-2-trifluoromethylbenzoyl,
2-fluoro-4-trifluoromethylbenzoyl, 3-biphenylcarboxyl,
4-chloro-biphenyl-4-carboxyl, 3-phenylproponyl, 4-phenylbutanoyl,
2,4-di chlorophenyl sulfonyl, 4-chloro-3-trifluoromethylbenzoyl,
4-isopropylbenzoyl, 4-chloro-3-fluorobenzoyl,
3-chloro-4-trifluoromethylbenzoyl; R.sup.2 represents a side chain
of an amino acid selected from serine or threonine; R.sup.3
represents a side chain of an amino acid selected from leucine,
phenylalanine, norleucine, norvaline or t-butylglycine; R.sup.4
represents a side chain of an amino acid selected from alanine,
threonine, serine, valine, t-butylglycine, 2-aminobutyric acid or
2-aminoisobutyric acid; and k is 1, 2 or 3; or pharmaceutically
acceptable salts thereof.
In a further embodiment the present invention provides compounds of
the formula (Ila) represented by the formula (IIb):
##STR00009## wherein R.sup.1 is selected from hexanoyl, hepatanoyl,
octanoyl, nonanoyl, decanoyl, dodecanoyl, S,R-6-methyloctanoyl
(racemic mixture), R-6-methyloctanoyl, 7-methyloctanoyl,
S-5-methylheptanoyl, R-5-methylheptanoyl, S,R-5-methylheptanoyl
(racemic mixture), 4-biphenylcarboxyl, 4-trifluoromethylbenzoyl,
4-ethylbenzoyl, 3,4-dichlorobenzoyl, 4-chlorobenzoyl,
3-chlorobenzoyl, pentafluorobenzoyl, 4-methylbenzoyl, 4-ethylphenyl
acetyl, phenyl acetyl, 4-methylphenyl acetyl,
4-trifluoromethylphenyl acetyl, pentafluorophenyl acetyl,
3,4-dichlorophenylacetyl, 4-chlorophenyl acetyl, 3-chlorophenyl
acetyl, 2-chlorobenzoyl, 2-fluorobenzoyl, 2-methylbenzoyl,
2-chlorophenyl acetyl, 2-fluorophenylacetyl, 2-methylphenylacetyl,
2,3-dichlorobenzoyl, 2,3-dimethylbenzoyl, 2,4-dichlorophenylacetyl,
2,4-dichlorobenzoyl, 2,4-dimethylbenzoyl, 2-chloro-4-methylbenzoyl,
2-chloro-4-trifluoromethylbenzoyl, 3-fluorobenzoyl,
3-methylbenzoyl, 3-trifuoromethylbenzoyl, 3,4-dimethylbenzoyl,
3-fluoro-4-methylbenzoyl, 4-chloro-3-methylbenzoyl,
3,4-dimethylphenyl acetyl, 3-chloro-4-methylbenzoyl,
4-chloro-3-fluorobenzoyl, 3-fluoro-4-trifluoromethylbenzoyl,
3-chloro-4-fluorobenzoyl, 4-methyl-3-trifluoromethylbenzoyl,
3-methyl-4-trifluoromethylbenzoyl,
3-methyl-5-trifluoromethylbenzoyl, 3,5-dimethylbenzoyl,
3,5-dichlorobenzoyl, 3,5-bis(trifluoromethyl)benzoyl,
3-fluoro-5-trifluoromethylbenzoyl, 3-chloro-5-methylbenzoyl,
3-chloro-5-fluorobenzoyl, 2,4,6-trimethylbenzoyl, 2,4,6-tri
chlorobenzoyl, 2-chloro-4-fluorobenzoyl, 4-chloro-2-fluorobenzoyl,
3,4,5-trifluoromethylbenzoyl, 4-chloro-2-trifluoromethylbenzoyl,
2-fluoro-4-trifluoromethylbenzoyl, 3-biphenylcarboxyl,
4-chloro-biphenyl-4-carboxyl, 3-phenylproponyl, 4-phenylbutanoyl,
2,4-dichlorophenylsulfonyl, 4-chloro-3-trifluoromethylbenzoyl,
4-isopropylbenzoyl, 4-chloro-3-fluorobenzoyl,
3-chloro-4-trifluoromethylbenzoyl; R.sup.3 represents a side chain
of an amino acid selected from leucine, phenylalanine, norleucine,
norvaline or t-butylglycine; R.sup.4 represents a side chain of an
amino acid selected from alanine, threonine, serine, valine,
t-butylglycine, 2-aminobutyric acid or 2-aminoisobutyric acid; and
k and q are individually selected from 1, 2, or 3; or
pharmaceutically acceptable salts thereof.
In another embodiment compounds of the formula (Ia) are selected
from those compounds listed in Table 1.
TABLE-US-00001 TABLE 1 Compounds of formula (Ia): (Ia) ##STR00010##
Compound R.sup.1 R.sup.2 R.sup.3 R.sup.4 X k m n p 1 Octanoyl
D-Ser.sup.a D-Leu.sup.a Thr.sup.a Dab.sup.a 2 2 2 2 2 Octanoyl
D-Ser D-Leu Ala Dab 2 2 2 2 3 Octanoyl D-Ser D-Phe Thr Dab 2 2 2 2
4 Octanoyl Ser D-Leu Thr Dab 2 2 2 2 5 Octanoyl D-Ser D-Leu Val Dab
2 2 2 2 6 Octanoyl D-Ser D-Leu Ser Dab 2 2 2 2 7 Octanoyl D-Ser
D-Nle Thr Dab 2 2 2 2 8 Hexanoyl D-Ser D-Leu Thr Dab 2 2 2 2 9
Decanoyl D-Ser D-Leu Thr Dab 2 2 2 2 10 Dodec.sup.a D-Ser D-Leu Thr
Dab 2 2 2 2 11 4-BPC D-Ser D-Leu Thr Dab 2 2 2 2 12 PA D-Ser D-Leu
Thr Dab 2 2 2 2 13 Octanoyl D-Ser D-Leu Thr Dab 1 2 2 2 14 Octanoyl
D-Ser D-Leu Thr Dab 3 2 2 2 15 Octanoyl D-Thr D-Leu Thr Dab 2 2 2 2
16 Heptanoyl D-Ser D-Leu Thr Dab 2 2 2 2 17 Nonanoyl D-Ser D-Leu
Thr Dab 2 2 2 2 18 3-TFMB D-Ser D-Leu Abu Dab 2 2 2 2 19 3-TFMB
D-Ser D-Leu Thr Dab 2 2 2 2 .sup.afor R.sup.2, R.sup.3, R.sup.4 and
X, the amino acid shown in these columns is indicative of the side
chain and stereochemistry at these positions; Dodec = dodecanoyl,
4-BPC = 4-biphenylcarboxyl, PA = phenylacetyl, 3-TFMB =
3-trifluoromethylbenzoyl, Dab = diaminobutyric acid, Nle =
norleucine, Abu = 2-aminobutyric acid, Phe = phenylalanine, Thr =
threonine, Ala = alanine, Ser = serine, Val = valine, D- indicates
D-amino acids.
In another embodiment compounds of the formula (IIa) are selected
from those compounds listed in Table 2.
TABLE-US-00002 TABLE 2 Compounds of formula (IIa): (IIa)
##STR00011## Compound R.sup.1 R.sup.3 R.sup.4 X k q m n p 20
Octanoyl D-Leu.sup.a Thr.sup.a Dab.sup.a 2 2 2 2 2 21 Octanoyl
D-Leu Ala Dab 2 2 2 2 2 22 Octanoyl D-Phe Thr Dab 2 2 2 2 2 23
Octanoyl D-Leu Val Dab 2 2 2 2 2 24 Octanoyl D-Nle Thr Dab 2 2 2 2
2 25 Octanoyl D-Leu Ser Dab 2 2 2 2 2 26 Hexanoyl D-Leu Thr Dab 2 2
2 2 2 27 Decanoyl D-Leu Thr Dab 2 2 2 2 2 28 Dodec.sup.a D-Leu Thr
Dab 2 2 2 2 2 29 4-BPC D-Leu Thr Dab 2 2 2 2 2 30 PA D-Leu Thr Dab
2 2 2 2 2 31 Octanoyl D-Leu Thr Dab 1 2 2 2 2 32 Octanoyl D-Leu Thr
Dab 3 2 2 2 2 33 Octanoyl D-Leu Thr Dab 2 1 2 2 2 34 Octanoyl D-Leu
Thr Dab 2 3 2 2 2 35 Octanoyl D-Leu Ala Dab 2 1 2 2 2 36 Octanoyl
D-Leu Val Dab 2 1 2 2 2 37 Decanoyl D-Leu Thr Dab 2 1 2 2 2 38
Decanoyl D-Leu Ala Dab 2 1 2 2 2 39 Decanoyl D-Leu Val Dab 2 1 2 2
2 40 Decanoyl D-Leu Ala Dab 2 2 2 2 2 41 Decanoyl D-Leu Val Dab 2 2
2 2 2 42 Heptanoyl D-Leu Thr Dab 2 2 2 2 2 43 Nonanoyl D-Leu Thr
Dab 2 2 2 2 2 44 Heptanoyl D-Leu Thr Dab 2 1 2 2 2 45 Octanoyl
D-Leu Aib Dab 2 1 2 2 2 46 Octanoyl D-Leu Abu Dab 2 1 2 2 2 47
Octanoyl D-Leu Tle Dab 2 1 2 2 2 48 Octanoyl D-Leu Thr Dab 1 1 2 2
2 49 Octanoyl D-Nva Thr Dab 2 1 2 2 2 50 Octanoyl D-Leu Thr Dab 2 1
2 2 1 51 Octanoyl D-Leu Thr Dab 2 1 2 1 2 52 Octanoyl D-Leu Thr Dab
2 1 1 2 2 53 Octanoyl D-Leu Thr Dab 2 1 2 2 3 54 Octanoyl D-Leu Thr
Dab 2 1 2 3 2 55 Octanoyl D-Leu Thr Dab 2 1 3 2 2 56 4-TFMB D-Leu
Thr Dab 2 1 2 2 2 57 3,4-DCB D-Leu Thr Dab 2 1 2 2 2 58 Nonanoyl
D-Leu Thr Dab 2 1 2 2 2 59 Nonanoyl D-Leu Abu Dab 2 1 2 2 2 60
Octanoyl D-Leu Abu Dab 2 2 2 2 2 61 3-CPA D-Leu Thr Dab 2 1 2 2 2
62 2,4-DCPA D-Leu Thr Dab 2 1 2 2 2 63 Heptanoyl D-Leu Abu Ala 2 1
2 2 2 64 Heptanoyl D-Leu Abu Dab 2 1 2 2 2 65 6-MH D-Leu Ala Dab 2
1 2 2 2 66 6-MH D-Leu Abu Dab 2 1 2 2 2 67 Hexanoyl D-Leu Ala Dab 2
1 2 2 2 68 Hexanoyl D-Leu Abu Dab 2 1 2 2 2 69 Octanoyl D-Nva Abu
Dab 2 1 2 2 2 70 2,4-DCPA D-Leu Abu Dab 2 1 2 2 2 71 3,4-DCB D-Leu
Abu Dab 2 1 2 2 2 72 2-CB D-Leu Abu Dab 2 1 2 2 2 73 2-FB D-Leu Abu
Dab 2 1 2 2 2 74 4-TFMB D-Leu Abu Dab 2 1 2 2 2 75 2-MB D-Leu Abu
Dab 2 1 2 2 2 76 2-MPA D-Leu Abu Dab 2 1 2 2 2 77 4-CPA D-Leu Abu
Dab 2 1 2 2 2 78 PA D-Leu Abu Dab 2 1 2 2 2 79 3-CPA D-Leu Abu Dab
2 1 2 2 2 80 4-MPA D-Leu Abu Dab 2 1 2 2 2 81 3,4-DCPA D-Leu Abu
Dab 2 1 2 2 2 82 2,4-DCB D-Leu Abu Dab 2 1 2 2 2 83 3,4-DMB D-Leu
Abu Dab 2 1 2 2 2 84 2-CPA D-Leu Abu Dab 2 1 2 2 2 85 2-FPA D-Leu
Abu Dab 2 1 2 2 2 86 3-FB D-Leu Abu Dab 2 1 2 2 2 87 3-MB D-Leu Abu
Dab 2 1 2 2 2 88 3-CB D-Leu Abu Dab 2 1 2 2 2 89 2,4-DMB D-Leu Abu
Dab 2 1 2 2 2 90 2,3-DCB D-Leu Abu Dab 2 1 2 2 2 91 2,3-DMB D-Leu
Abu Dab 2 1 2 2 2 92 2,4,6-TMB D-Leu Abu Dab 2 1 2 2 2 93 3,5-DMB
D-Leu Abu Dab 2 1 2 2 2 94 4-CB D-Leu Abu Dab 2 1 2 2 2 95
2,4,6-TCB D-Leu Abu Dab 2 1 2 2 2 96 3,5-DCB D-Leu Abu Dab 2 1 2 2
2 97 3,5-BTFMB D-Leu Abu Dab 2 1 2 2 2 98 4-MB D-Leu Abu Dab 2 1 2
2 2 99 4-IPB D-Leu Abu Dab 2 1 2 2 2 100 4-EB D-Leu Abu Dab 2 1 2 2
2 101 2-C-4-MB D-Leu Abu Dab 2 1 2 2 2 102 3-F-4-MB D-Leu Abu Dab 2
1 2 2 2 103 3,4-DMPA D-Leu Abu Dab 2 1 2 2 2 104 4-C-3-MB D-Leu Abu
Dab 2 1 2 2 2 105 3-C-4-MB D-Leu Abu Dab 2 1 2 2 2 106 3-TFMB D-Leu
Abu Dab 2 1 2 2 2 107 4-C-3-FB D-Leu Abu Dab 2 1 2 2 2 108
3-F-5-TFMB D-Leu Abu Dab 2 1 2 2 2 109 2-C-4-TFMB D-Leu Abu Dab 2 1
2 2 2 110 3-C-4-FB D-Leu Abu Dab 2 1 2 2 2 111 3-F-4-TFMB D-Leu Abu
Dab 2 1 2 2 2 112 4-C-3-TFMB D-Leu Abu Dab 2 1 2 2 2 113 4-M-3-TFMB
D-Leu Abu Dab 2 1 2 2 2 114 3-C-5-MB D-Leu Abu Dab 2 1 2 2 2 115
3-C-4-TFMB D-Leu Abu Dab 2 1 2 2 2 116 3-C-5-FB D-Leu Abu Dab 2 1 2
2 2 117 3,5-DCB D-Leu Ala Dab 2 1 2 2 2 118 3,5-DCB D-Leu Thr Dab 2
1 2 2 2 119 3-M-4-TFMB D-Leu Abu Dab 2 1 2 2 2 120 3-M-5-TFMB D-Leu
Abu Dab 2 1 2 2 2 121 3-TFMB D-Nle Abu Dab 2 1 2 2 2 122 3-TFMB
D-Phe Abu Dab 2 1 2 2 2 123 3-TFMB D-Nle Thr Dab 2 1 2 2 2 124
3-TFMB D-Phe Thr Dab 2 1 2 2 2 125 3-TFMB D-Nle Ala Dab 2 1 2 2 2
126 3-TFMB D-Phe Ala Dab 2 1 2 2 2 127 4-TFMPA D-Leu Abu Dab 2 1 2
2 2 128 Octanoyl D-Phe Ala Dab 2 1 2 2 2 129 Octanoyl D-Phe Thr Dab
2 1 2 2 2 130 Octanoyl D-Phe Abu Dab 2 1 2 2 2 131 Heptanoyl D-Phe
Abu Dab 2 1 2 2 2 132 Nonanoyl D-Leu Ala Dab 2 1 2 2 2 133
3,4,5-TFB D-Leu Abu Dab 2 1 2 2 2 134 4-C-2-FB D-Leu Abu Dab 2 1 2
2 2 135 2-C-4-FB D-Leu Abu Dab 2 1 2 2 2 136 4-C-2-TFMB D-Leu Abu
Dab 2 1 2 2 2 137 2-F-4-TFMB D-Leu Abu Dab 2 1 2 2 2 138 3-BPC
D-Leu Abu Dab 2 1 2 2 2 139 (S,R)-6-MO D-Leu Abu Dab 2 1 2 2 2 140
Octanoyl D-Phe Aib Dab 2 1 2 2 2 141 3-TFMB D-Leu Abu Dab 3 1 2 2 2
142 4-BPC D-Leu Abu Dab 2 1 2 2 2 143 Nonanoyl D-Phe Ser Dab 2 1 2
2 2 144 4-Cl-BP-4-C D-Leu Ala Dab 2 1 2 2 2 145 3-PP D-Leu Abu Dab
2 1 2 2 2 146 4-PB D-Leu Ala Dab 2 1 2 2 2 147 2,4-DCB D-Leu Abu
Dab 2 3 2 2 2 148 2,4-DCPS D-Leu Abu Dab 2 1 2 2 2 149 Octanoyl
D-Leu Thr Orn 2 2 2 2 2 .sup.afor R.sup.3, R.sup.4 and X, the amino
acid shown in these columns is indicative of the side chain and
stereochemistry at these positions; Dodec = dodecanoyl, 4-BPC =
4-biphenylcarboxyl, PA = phenylacetyl, 6-MH = 6-methylheptanoyl,
4-TFMPA = 4-trifluoromethylphenylacetyl, 2- MB = 2-methylbenzoyl,
3-MB = 3-methylbenzoyl, 4-MB = 4-methylbenzoyl, 3-F-4-MB =
3-fluoro-4-methylbenzoyl, 4-C-3-MB = 4-chloro-3-methylbenzoyl,
3-C-4-MB = 3-chloro-4-methylbenzoyl, 3-C-5-MB =
3-chloro-5-methylbenzoyl, 2-FPA = 2-fluorophenylacetyl, 3-TFMB =
3-trifluoromethylbenzoyl, 4-TFMB = 4-trifluoromethylbenzoyl,
2-C-4-TFMB = 2-chloro-4-trifluoromethylbenzoyl, 4-C-3-TFMB =
4-chloro-3-trifluoromethylbenzoyl, 3-C-4-TFMB =
3-chloro-4-trifluoromethylbenzoyl, 3-F-4-TFMB =
3-fluoro-4-trifluoromethylbenzoyl, 3-F-5- TFMB =
3-fluoro-5-trifluoromethylbenzoyl, 4-M-3-TFMB =
4-methyl-3-trifluoromethylbenzoyl, 3-M-4-TFMB =
3-methyl-4-trifluoromethylbenzoyl, 3-M-5-TFMB =
3-methyl-5-trifluoromethylbenzoyl, 2-F-4-TFMB =
2-fluoro-4-trifluoromethylbenzoyl, 3,4,5-TFMB =
3,4,5-trifluoromethylbenzoyl, 4-C-2-TFMB =
4-chloro-2-trifluoromethylbenzoyl, 3,5-BTFMB =
3,5-bis(trifluoromethyl)benzoyl, 2,4,6-TMB =
2,4,6-trimethylbenzoyl, 2,3-DMB = 2,3-dimethylbenzoyl, 2,4-DMB =
2,4-dimethylbenzoyl, 3,4-DMB = 3,4-dimethylbenzoyl, 3,5-DMB =
3,5-dimethylbenzoyl, 2-C-4-MB = 2-chloro-4-methylbenzoyl, 4-EB =
4-ethylbenzoyl, 4-IPB = 4-Isopropylbenzoyl, 2,4-DCPA =
2,4-dichlorophenylacetyl, 3,4-DCPA = 3,4-dichlorophenylacetyl,
2-CPA = 2-chlorophenylacetyl, 3-CPA = 3-chlorophenylacetyl, 4-CPA =
4-chlorophenylacetyl, 2-CB = 2-chlorobenzoyl, 3-CB =
3-chlorobenzoyl, 4-CB = 4-chlorobenzoyl, 2,3-DCB =
2,3-dichlorobenzoyl, 2,4- DCB = 2,4-dichlorobenzoyl, 3,4-DCB =
3,4-dichlorobenzoyl, 3,5-DCB = 3,5-dichlorobenzoyl, 2,4,6-TCB =
2,4,6-trichlorobenzoyl, 2-FB = 2-fluorobenzoyl, 3-FB =
3-fluorobenzoyl, 2-C-4-FB = 2-chloro-4-fluorobenzoyl, 3-C-4-FB =
3-Chloro-4-fluorobenzoyl, 3-C-5-FB = 3-chloro-5-fluorobenzoyl,
4-C-2-FB = 4-chloro-2-fluorobenzoyl, 4-C-3-FB =
4-chloro-3-fluorobenzoyl, 2-MPA = 2-methylphenylacetyl, 4-MPA =
4-methylphenylacetyl, 3,4-DMPA = 3,4-dimethylphenylacetyl,
(S,R)-6-MO = (S,R)-6-methyloctanoyl, 3-BPC = 3-biphenylcarboxyl,
4-Cl-BP-4-C = 4-chloro-biphenyl-4-carboxyl, 3-PP =
3-phenylproponyl, 4-PB = 4-phenylbutanoyl, 2,4-DCPS =
2,4-dichlorophenylsulfonyl, Dab = diaminobutyric acid, Tle =
t-butylglycine, Aib = aminoisobutyric acid, Abu = 2-aminobutyric
acid, Phe = phenylalanine, Thr = threonine, Ala = alanine, Ser =
serine, Val = valine, Nva = norvaline, Nle = norleucine, D-
indicates D-amino acids.
In another preferred embodiment there is provided methods
preventing or treating a MDR Gram-negative bacterial infection
comprising administering a therapeutically effective amount of one
or more compounds of the formula (I) and/or formula (II) as herein
defined.
Accordingly, in a further preferred embodiment there is provided
one or more compounds of formula (I) and/or formula (II) as herein
defined for use in the prevention or treatment of a MDR
Gram-negative bacterial infection.
It will be appreciated that for Gram-negative bacteria to be
multidrug-resistant the bacteria will be non-susceptible to at
least one agent in three or more antibacterial categories.
Gram-negative bacteria that are non-susceptible to at least one
agent in all but two or fewer antibacterial categories are
classified as extensively, or extremely, drug resistant (XDR).
Gram-negative bacteria that are non-susceptible to all agents in
all antibacterial categories are classified as "pandrug-resistant"
(PDR) (Magiorakos, A. P. et al. (2011) European Society of Clinical
Microbiology and Infectious Diseases, Clin Microbiol Infect, 18,
268-281). Table 3 provides a list of antibacterial agents falling
within each of the antibacterial categories.
TABLE-US-00003 TABLE 3 Antibacterial categories and agents
Antibacterial Category Antibacterial Agent Aminoglycosides
Gentamicin Tobramycin Amikacin Netilmicin Antipseudomonal
carbapenems Imipenem Meropenem Doripenem Antipseudomonal
cephalosporins Ceftazidime Cefepime Antipseudomonal
fluoroquinolones Ciprofloxacin Levofloxacin Antipseudomonal
penicillins + Ticarcillin-clavulanic acid .beta.-lactamase
inhibitors Piperacillin-tazobactum Monobactams Aztreonam Phosphonic
acids Fosfomycin Polymyxins Colistin Polymyxin B
It will be appreciated that in order to treat a Gram-negative
bacterial infection in a subject in need thereof, it may be
beneficial to administer to the subject one or more compounds of
the formula (I) as herein described or one or more compounds of the
formula (II) as herein described. It is envisaged that in one
embodiment, treatment of a Gram-negative bacterial infection will
comprise administering to a subject in need thereof a compound of
the formula (I). It is also envisaged that treatment of a
Gram-negative bacterial infection will comprise administration of a
compound of the formula (II) to a subject in need thereof.
It will be appreciated that in order to minimise the nephrotoxic
side effects associated with the polymyxin analogues in current
clinical use and to maintain or improve upon the efficacy of the
compounds against a broad spectrum of Gram-negative bacteria, it
may be beneficial to administer to the subject in need thereof a
combination of two or more compounds of the present invention. It
is envisaged that in one embodiment, treatment of a Gram-negative
bacterial infection will comprise administration of two or more
compounds of the formula (I) to a subject in need thereof. It is
also envisaged that treatment of a Gram-negative bacterial
infection will comprise administration of two or more compounds of
the formula (II) to a subject in need thereof. In other embodiments
it is envisaged that treatment of a Gram-negative bacterial
infection will comprise administration of one or more compounds of
the formula (I) together with one or more compounds of the formula
(II) to a subject in need thereof. In further embodiments it is
envisaged that treatment of a Gram-negative bacterial infection
will comprise administration of one or both of the naturally
occurring polymyxin analogues polymyxin D.sub.1/D.sub.2 to a
subject in need thereof (compounds 150 and 151, respectively). In
another embodiment it is envisaged that treatment of a
Gram-negative bacterial infection will comprise administration of
one or both of the naturally occurring polymyxin analogues
polymyxin M.sub.1/M.sub.2 to a subject in need thereof (compounds
152 and 153, respectively).
In a preferred embodiment there is provided the use of one or more
compounds of the formula (Ia) and/or (IIa) as hereinbefore defined
in the manufacture of a medicament for preventing or treating a
Gram-negative bacterial infection.
In a further preferred embodiment there is provided one or more
compounds of the formula (Ia) and/or (IIa) as hereinbefore defined
for use in the prevention or treatment a Gram-negative bacterial
infection.
Polymyxin D and polymyxin M, like polymyxin B and colistin, are
mixtures of closely related peptides obtained from fermentation
(Table 4). However, unlike polymyxin B and colistin, the components
of the polymyxin D and M mixtures obtained from fermentation have
not been well characterised. To date, the components that have been
identified in polymyxin D preparations are polymyxin D.sub.1 and
D.sub.2, whilst for polymyxin M the components are polymyxin
M.sub.1 and M.sub.2 (Kimura, Y, et al. (1981), J. Chromatography,
206, 563-572; Orwa, J. A., et al. (2001) J. Chromatography A. 912,
369-373; Govaerts, C., et al. (2002) J. Chromatography A. 976,
65-78). Because of their perceived nephrotoxic side effects,
polymyxin D and M mixtures, or their individual components, have
not found use in the clinical treatment of Gram-negative bacterial
infections, in particular, MDR Gram-negative bacterial infections
(Bryer, M. S., et al. (1949) Ann. N. Y. Acad. Sci., 51, 935-943;
Brownlee, G., et. Al. (1949) Ann. N. Y. Acad. Sci., 51, 952-957;
Filippos'yan, S. T. Antibiotiki, (1969) 14, 5, 459-463).
TABLE-US-00004 TABLE 4 The chemical structures of the polymyxin B,
E (Colistin), D and M lipopeptides. ##STR00012## Polymyxin
Fatty-acyl group Pos 3 Pos 6 Pos 7 B.sub.1 (S)-6-methyloctanoyl
L-Dab D-Phe L-Leu B.sub.2 6-methylheptanoyl L-Dab D-Phe L-Leu
E.sub.1 (S)-6-methyloctanoyl L-Dab D-Leu L-Leu (Colistin A) E.sub.2
6-methylheptanoyl L-Dab D-Leu L-Leu (Colistin B) D.sub.1 (150)
(S)-6-methyloctanoyl D-Ser D-Leu L-Thr D.sub.2 (151)
6-methylheptanoyl D-Ser D-Leu L-Thr M.sub.1 (152)
(S)-6-methyloctanoyl L-Dab D-Leu L-Thr M.sub.2 (153)
6-methylheptanoyl L-Dab D-Leu L-Thr D-Dab = D-diaminobutyric acid,
L-Dab = L-diaminobutyric acid, D-Phe = D-phenylalanine, L-Leu =
L-Leucine, D-Ser = D-Serine, L-Thr = L-Threonine
However, the present inventors have discovered that the perceived
nephrotoxicity associated with the individual components polymyxin
D.sub.1/D.sub.2 and polymyxin M.sub.1/M.sub.2 is unwarranted.
Without wishing to be limited by theory, it is believed that this
may be attributed to the fact that earlier toxicity tests were
conducted on samples of polymyxin D mixtures and polymyxin M
mixtures that were not well characterised in terms of chemical
composition and purity, and not on pure samples of the individual
components obtained by total organic synthesis or pure samples
obtained by extensively purifying fermentation products (Bell. P.
H. and Bone J. F., (1949) Ann. N. Y. Acad. Sci., 51, 897-908;
Bryer, M. S., et al. (1949) Ann. N. Y. Acad. Sci., 51, 935-943;
Brownlee, G., et. Al. (1949) Ann. N. Y. Acad. Sci., 51, 952-957).
The present inventors have discovered that pure isolates of the
individual components polymyxin D.sub.2 and polymyxin M.sub.2
exhibit no significant nephrotoxicity in the in vivo nephrotoxicity
model tested. Pure isolates of the individual components polymyxin
D.sub.1 and polymyxin M.sub.1 exhibit some nephrotoxicity, but have
improved nephrotoxicity profiles compared to the clinically
available polymyxin B and colistin.
It has now been found that certain combinations of amino acid
residues at the 3.sup.rd, 6.sup.th and 7.sup.th positions of the
polymyxin core, together with select N-terminal fatty acyl groups,
can reduce the nephrotoxicity of the resultant compounds relative
to polymyxin B or colistin, whilst maintaining or improving the
compound's antibacterial efficacy. Without wishing to be limited by
theory, it is believed that replacement of one or both of the
6.sup.th and 7.sup.th residues in the polymyxin compound with less
hydrophobic residues can reduce the level of nephrotoxicity. It is
also believed that selection of certain amino acid residues at the
3.sup.rd position and certain N-terminal fatty acyl group reduces
the nephrotoxicity of the resultant compound due to the effect
these groups have on the overall conformation of the compound. It
is believed that the change in conformation interferes with the
compounds ability to form key interactions with molecular targets
that trigger physiological events that lead to nephrotoxicity.
In general, techniques for preparing the compounds of the invention
are well known in the art for example see:
a) Alewood, P.; Alewood, D.; Miranda, L.; Love, S.; Meutermans, W.;
Wilson, D. (1997) Meth. Enzymol., 289, 14-28;
b) Merrifield, R. B. (1964) J. Am. Chem. Soc., 85, 2149;
c) Bodanzsky, "Principles of Peptide Synthesis", 2nd Ed.,
Springer-Verlag (1993); and
d) Houghten, (1985) Proc. Natl. Acad. Sci. USA, 82, 5131.
Of particular relevance to the synthesis polymyxin type compounds
are: Sharma, S. K., et al. (1999) J. Pept. Res. 53, 501-506; Kline,
T., Holub, D., Therrien, J. et al. (2001) J. Pept. Res. 57,
175-187; de Visser, P. C., et al. (1999) J. Pept. Res. 61, 298-306;
Sukura, N., et al. (2004) Bull. Chem. Soc. Jpn. 77, 1915-1924; and
Vaara, M., Fox, J., Loidl, G., Siikanen, O. et al. (2008)
Antimicrob. Agents Chemother. 52(9), 3229-3236. The entire contents
of these documents are incorporated herein by reference.
Known solid or solution phase techniques may be used in the
synthesis of the compounds of the present invention, such as
coupling of the N- or C-terminus to a solid support (typically a
resin) followed by step-wise synthesis of the linear peptide. An
orthogonal protecting group strategy may be used to facilitate
selective deprotection and cyclization to form the cyclic
heptapeptide core of the compound. Protecting group chemistries for
the protection of amino acid residues, including side chains, are
well known in the art and may be found, for example, in: Theodora
W. Greene and Peter G. M. Wuts, Protecting Groups in Organic
Synthesis (Third Edition, John Wiley & Sons, Inc, 1999), the
entire contents of which is incorporated herein by reference.
As a general strategy, the synthesis of the compounds of the
present invention may be performed in four stages. In the first
stage, amino acids may be protected for incorporation into the
compound, such as the protection of isoleucine as Fmoc-isoleucine.
Second, a partially protected linear peptide which selectively
exposes only the functional groups required for cyclisation may be
synthesised using solid phase techniques. Third the cyclisation
reaction may be performed in solution to produce the protected
cyclic lipopeptide. Fourth the remaining side chain protecting
groups may be deprotected to furnish the compound.
Where the compounds of the present invention require purification,
chromatographic techniques such as high-performance liquid
chromatography (HPLC) and reversed-phase HPLC may be used. The
peptides may be characterised by mass spectrometry and/or other
appropriate methods.
Where the compound comprises one or more functional groups that may
be protonated or deprotonated (for example at physiological pH) the
compound may be prepared and/or isolated as a pharmaceutically
acceptable salt. It will be appreciated that the compound may be
zwitterionic at a given pH. As used herein the expression
"pharmaceutically acceptable salt" refers to the salt of a given
compound, wherein the salt is suitable for administration as a
pharmaceutical. Such salts may be formed, for example, by the
reaction of an acid or a base with an amine or a carboxylic acid
group respectively.
Pharmaceutically acceptable acid addition salts may be prepared
from inorganic and organic acids. Examples of inorganic acids
include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid and the like. Examples of organic acids
include acetic acid, propionic acid, glycolic acid, pyruvic acid,
oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,
fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid and the like.
Pharmaceutically acceptable base addition salts may be prepared
from inorganic and organic bases. Corresponding counter ions
derived from inorganic bases include the sodium, potassium,
lithium, ammonium, calcium and magnesium salts. Organic bases
include primary, secondary and tertiary amines, substituted amines
including naturally-occurring substituted amines, and cyclic
amines, including isopropylamine, trimethyl amine, diethylamine,
tri ethyl amine, tripropylamine, ethanolamine,
2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine,
caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, N-alkylglucamines, theobromine, purines, piperazine,
piperidine, and N-ethylpiperidine.
Acid/base addition salts tend to be more soluble in aqueous
solvents than the corresponding free acid/base forms.
The compounds of the invention may be in crystalline form or as
solvates (e.g. hydrates) and it is intended that both forms are
within the scope of the present invention. The term "solvate" is a
complex of variable stoichiometry formed by a solute (in this
invention, a peptide of the invention) and a solvent. Such solvents
should not interfere with the biological activity of the solute.
Solvents may be, by way of example, water, ethanol or acetic acid.
Methods of solvation are generally known within the art.
The compounds of the invention may be in the form of a pro-drug.
The term "pro-drug" is used in its broadest sense and encompasses
those derivatives that are converted in vivo to the peptides of the
invention. Such derivatives would readily occur to those skilled in
the art and include, for example, compounds where a free hydroxy
group is converted into an ester derivative or a ring nitrogen atom
is converted to an N-oxide. Examples of ester derivatives include
alkyl esters (for example acetates, lactates and glutamines),
phosphate esters and those formed from amino acids (for example
valine). Any compound that is a prodrug of a compound of the
invention is within the scope and spirit of the invention.
Conventional procedures for the preparation of suitable prodrugs
according to the invention are described in text books, such as
"Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985, the entire
contents of which is incorporated herein by reference.
The present invention also provides a pharmaceutical composition
comprising a therapeutically effective amount of a compound as
hereinbefore defined, or a pharmaceutically acceptable salt
thereof, together with at least one pharmaceutically acceptable
carrier or diluent.
The term "composition" is intended to include the formulation of an
active ingredient with encapsulating material as carrier, to give a
capsule in which the active ingredient (with or without other
carrier) is surrounded by carriers.
While the compounds as hereinbefore described, or pharmaceutically
acceptable salts thereof, may be the sole active ingredient
administered to the subject, the administration of other active
ingredient(s) with the compound is within the scope of the
invention. In one or more embodiments it is envisaged that a
combination of two or more of the compounds of the invention will
be administered to the subject. It is envisaged that the
compound(s) could also be administered with one or more additional
therapeutic agents in combination. The combination may allow for
separate, sequential or simultaneous administration of the
compound(s) as hereinbefore described with the other active
ingredient(s). The combination may be provided in the form of a
pharmaceutical composition.
The term "combination", as used herein refers to a composition or
kit of parts where the combination partners as defined above can be
dosed dependently or independently or by use of different fixed
combinations with distinguished amounts of the combination
partners, i.e., simultaneously or at different time points. The
combination partners can then, e.g., be administered simultaneously
or chronologically staggered, that is at different time points and
with equal or different time intervals for any part of the kit of
parts. The ratio of the total amounts of the combination partners
to be administered in the combination can be varied, e.g. in order
to cope with the needs of a patient sub-population to be treated or
the needs of the single patient which different needs can be due to
age, sex, body weight, etc. of the patients.
As will be readily appreciated by those skilled in the art, the
route of administration and the nature of the pharmaceutically
acceptable carrier will depend on the nature of the condition and
the mammal to be treated. It is believed that the choice of a
particular carrier or delivery system, and route of administration
could be readily determined by a person skilled in the art. In the
preparation of any formulation containing the active compound care
should be taken to ensure that the activity of the compound is not
destroyed in the process and that the compound is able to reach its
site of action without being destroyed. In some circumstances it
may be necessary to protect the compound by means known in the art,
such as, for example, micro encapsulation. Similarly the route of
administration chosen should be such that the compound reaches its
site of action.
Those skilled in the art may readily determine appropriate
formulations for the compounds of the present invention using
conventional approaches. Identification of preferred pH ranges and
suitable excipients, for example antioxidants, is routine in the
art. Buffer systems are routinely used to provide pH values of a
desired range and include carboxylic acid buffers for example
acetate, citrate, lactate and succinate. A variety of antioxidants
are available for such formulations including phenolic compounds
such as BHT or vitamin E, reducing agents such as methionine or
sulphite, and metal chelators such as EDTA.
The compounds as hereinbefore described, or pharmaceutically
acceptable salts thereof, may be prepared in parenteral dosage
forms, including those suitable for intravenous, intrathecal, and
intracerebral or epidural delivery. The pharmaceutical forms
suitable for injectable use include sterile injectable solutions or
dispersions, and sterile powders for the extemporaneous preparation
of sterile injectable solutions. They should be stable under the
conditions of manufacture and storage and may be preserved against
reduction or oxidation and the contaminating action of
microorganisms such as bacteria or fungi.
The solvent or dispersion medium for the injectable solution or
dispersion may contain any of the conventional solvent or carrier
systems for the active compound, and may contain, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about where necessary by the
inclusion of various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal
and the like. In many cases, it will be preferable to include
agents to adjust osmolarity, for example, sugars or sodium
chloride. Preferably, the formulation for injection will be
isotonic with blood. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminium monostearate and
gelatin. Pharmaceutical forms suitable for injectable use may be
delivered by any appropriate route including intravenous,
intramuscular, intracerebral, intrathecal, epidural injection or
infusion.
Sterile injectable solutions are prepared by incorporating the
compounds of the invention in the required amount in the
appropriate solvent with various of the other ingredients such as
those enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the various sterilised active ingredient into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
preferred methods of preparation are vacuum drying or freeze-drying
of a previously sterile-filtered solution of the active ingredient
plus any additional desired ingredients.
Other pharmaceutical forms include oral and enteral formulations of
the present invention, in which the active compound may be
formulated with an inert diluent or with an assimilable edible
carrier, or it may be enclosed in hard or soft shell gelatin
capsule, or it may be compressed into tablets, or it may be
incorporated directly with the food of the diet. For oral
therapeutic administration, the active compound may be incorporated
with excipients and used in the form of ingestible tablets, buccal
or sublingual tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. The amount of active compound in such
therapeutically useful compositions is such that a suitable dosage
will be obtained.
The tablets, troches, pills, capsules and the like may also contain
the components as listed hereafter: a binder such as gum, acacia,
corn starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such a sucrose, lactose or saccharin may be added
or a flavouring agent such as peppermint, oil of wintergreen, or
cherry flavouring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup or elixir may contain the active compound,
sucrose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavouring such as cherry or orange
flavour. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the compounds of the invention
may be incorporated into sustained-release preparations and
formulations, including those that allow specific delivery of the
active peptide to specific regions of the gut.
Liquid formulations may also be administered enterally via a
stomach or oesophageal tube. Enteral formulations may be prepared
in the form of suppositories by mixing with appropriate bases, such
as emulsifying bases or water-soluble bases. It is also possible,
but not necessary, for the compounds of the present invention to be
administered topically, intranasally, intravaginally, intraocularly
and the like.
The compounds of the present invention may be administered by
inhalation in the form of an aerosol spray from a pressurised
dispenser or container, which contains a propellant such as carbon
dioxide gas, dichlorodifluoromethane, nitrogen, propane or other
suitable gas or combination of gases. The compounds may also be
administered using a nebuliser.
It will be appreciated that the compounds of the present invention,
having improved nephrotoxicity profiles, are particularly useful
when the compounds are administered enterally or parentarally, for
example, orally, intravenously or intramuscularly.
Pharmaceutically acceptable vehicles and/or diluents include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the
like. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, use thereof in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
It is especially advantageous to formulate the compositions in
dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit containing a predetermined quantity of
active material calculated to produce the desired therapeutic
effect in association with the required pharmaceutically acceptable
vehicle. The specification for the novel dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique
characteristics of the active material and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding active materials for the treatment of
disease in living subjects having a diseased condition in which
bodily health is impaired as herein disclosed in detail.
As mentioned above the principal active ingredient may be
compounded for convenient and effective administration in
therapeutically effective amounts with a suitable pharmaceutically
acceptable vehicle in dosage unit form. A unit dosage form can, for
example, contain the principal active compound in amounts ranging
from 0.25 .mu.g to about 2000 mg. Expressed in proportions, the
active compound may be present in from about 0.25 .mu.g to about
2000 mg/mL of carrier. In the case of compositions containing
supplementary active ingredients, the dosages are determined by
reference to the usual dose and manner of administration of the
said ingredients.
As used herein, the term "effective amount" refers to an amount of
compound which, when administered according to a desired dosing
regimen, provides the desired therapeutic activity. Dosing may
occur once, or at intervals of minutes or hours, or continuously
over any one of these periods. Suitable dosages may lie within the
range of about 0.1 .mu.g per kg of body weight to 1 g per kg of
body weight per dosage. A typical dosage is in the range of 1 .mu.g
to 1 g per kg of body weight per dosage, such as is in the range of
1 mg to 1 g per kg of body weight per dosage. In one embodiment,
the dosage may be in the range of 1 mg to 500 mg per kg of body
weight per dosage. In another embodiment, the dosage may be in the
range of 1 mg to 250 mg per kg of body weight per dosage. In yet
another embodiment, the dosage may be in the range of 1 mg to 100
mg per kg of body weight per dosage, such as up to 50 mg per body
weight per dosage.
The terms "treatment" and "treating" as used herein cover any
treatment of a condition or disease in an animal, preferably a
mammal, more preferably a human, and includes: (i) inhibiting the
bacterial infection, e.g. arresting its proliferation; (ii)
relieving the infection, e.g. causing a reduction in the severity
of the infection; or (iii) relieving the conditions caused by the
infection, e.g. symptoms of the infection. The terms "prevention"
and "preventing" as used herein cover the prevention or prophylaxis
of a condition or disease in an animal, preferably a mammal, more
preferably a human and includes preventing the bacterial infection
from occurring in a subject which may be predisposed to infection
but has not yet been diagnosed as being infected.
In some embodiments the Gram-negative bacterial infection may be
caused by one or more species selected from one or more of the
genera: Acinetobacter; Actinobacillus; Bartonella; Bordetella;
Brucella; Burkholderia; Campylobacter; Cyanobacteria; Enterobacter;
Envinia; Escherichia; Francisella; Helicobacter; Hemophilus;
Klebsiella; Legionella; Moraxella; Morganella; Neisseria;
Pasteurella; Proteus; Providencia; Pseudomonas; Salmonella;
Serratia; Shigella; Stenotrophomonas; Treponema; Vibrio; and
Yersinia. Specific examples of species are Pseudomonas aeruginosa,
Acinetobacter baumannii, Klebsiella pneumoniae, Stenotrophomonas
maltophilia, Enterobacter cloacae, Escherichia coli and Salmonella
enterica.
The invention will now be described with reference to the following
non-limiting examples:
Example 1: Methods for Preparing Compounds of the General Formulae
(I) and (II)
The following example is representative of the present invention,
and provides detailed methods for preparing exemplary compounds of
the present invention.
Synthesis of Compound 1:
##STR00013##
Synthesis of the protected linear peptide (residues 1-10 and the
N-terminal cap) was conducted on a Protein Technologies Prelude
automated peptide synthesizer using standard Fmoc solid-phase
peptide chemistry.
Specifically, synthesis was undertaken using TCP-Resin, pre-loaded
with Fmoc-Thr(tBu)-OH, 0.1 mmol scale. Coupling of the Fmoc-amino
acids was performed using the default instrument protocol: 3 molar
equivalents (relative to resin loading) of Fmoc amino acid and HCTU
in DMF with activation in situ, using 6 molar equivalents of DIPEA.
This was carried out for 50 min at room temperature. Fmoc
deprotection was conducted using the default instrument protocol:
20% piperidine in dimethylformamide (1.times.5 min, 1.times.10 min)
at room temperature. The resin was washed with DMF then treated
with 3% hydrazine in DMF (4.times.15 min) to remove the ivDde
group.
The protected linear peptide was cleaved from the resin by treating
the resin with 10-20% hexafluoroisopropanol (HFIP) in DCM
(1.times.30 min, 1.times.5 min). The resulting solution was
concentrated in vacuo and the resulting residue (crude protected
linear peptide) dissolved in DMF (10 mL) to which DPPA, (0.3 mmol,
0.65 .mu.L, 3 molar equivalents relative to the loading of the
resin) and DIPEA (0.6 mmol, 104 .mu.L, 6 molar equivalents relative
to the loading of the resin) were added. This solution was stirred
at room temperature overnight. The reaction solution was then
concentrated under vacuum overnight. The resulting residue was
taken up in a solution of 2.5% EDT, 5% TIPS in TFA and stirred at
room temperature for 2 h. To this solution 40 mL of diethyl ether
was added. The resulting precipitate was collected by
centrifugation and washed twice more with diethyl ether (40 mL)
then air-dried in a fume food to give the crude cyclic peptide as a
white solid. The resulting solid was taken up in Milli-Q water (5
mL) and de-salted using a Vari-Pure WE SAX column.
The crude cyclic peptide was purified by reversed-phase HPLC
(RP-HPLC) on an Agilent 1200 quaternary pump system with a
photodiode array detector (214 nm) using a Phenomenex Axia column
(Luna C.sub.8(2), 50.times.21.3 mm ID). A gradient of 60%
acetonitrile in 0.1% aqueous TFA over 60 min were employed at a
flow rate of 5 mL/min. Fractions collected were analysed using a
Shimadzu 2020 LCMS system, incorporating a photodiode array
detector (214 nm) coupled directly to an electrospray ionization
source and a single quadrupole mass analyser. RP-HPLC was carried
out employing a Phenomenex column (Luna C8(2), 100.times.2.0 mm ID)
eluting with a gradient of 80% acetonitrile in 0.05% aqueous TFA,
over 10 min at a flow rate of 0.2 mL/min. Mass spectra were
acquired in the positive ion mode with a scan range of 200-2,000
m/z. The combined fractions were freeze-dried for two days to give
compound 1 as a white TFA salt in a yield of 42.9 mg. The purity
was 99.7% as estimated by RP-HPLC at 214 nm. The compound was
confirmed as having the correct molecular weight (1130.2) by ESI-MS
analysis: m/z (monoisotopic): [M+2H].sup.2+ 566.15.
It will be understood that this representative synthesis may be
applied to the synthesis of a range of compounds described herein.
For example, the representative synthesis may be applied to the
synthesis of compounds 2 to 153 as herein described and listed in
Tables 5 and 6 below.
TABLE-US-00005 TABLE 5 Characterisation data for compounds of the
invention represented by formula (Ic): (Ic) ##STR00014## No R.sup.1
R.sup.2 R.sup.3 R.sup.4 X k Compound data 2 Octanoyl D-Ser D-Leu
Ala Dab 2 Yield: 43.0 mg, Purity: (99.4%) MS Data: [M + 2H].sup.2+
= 551.5 3 Octanoyl D-Ser D-Phe Thr Dab 2 Yield: 59.0 mg, Purity:
(98.4%) MS Data: [M + 2H].sup.2+ = 583.4 4 Ocatanoyl Ser D-Leu Thr
Dab 2 Yield: 58.7 mg, Purity: (99.7%) MS Data: [M + 2H].sup.2+ =
566.3 5 Octanoyl D-Ser D-Leu Val Dab 2 Yield: 65.5 mg, Purity:
(98.9%) MS Data: [M + 2H].sup.2+ = 565.4 6 Octanoyl D-Ser D-Leu Ser
Dab 2 Yield: 53.1 mg, Purity: (99.3%) MS Data: [M + 2H].sup.2+ =
559.4 7 Octanoyl D-Ser D-Nle Thr Dab 2 Yield: 40.0 mg, Purity:
(99.2%) MS Data: [M + 2H].sup.2+ = 566.3 8 Hexanoyl D-Ser D-Leu Thr
Dab 2 Yield: 61.0 mg, Purity: (99.2%) MS Data: [M + 2H].sup.2+ =
552.25 9 Decanoyl D-Ser D-Leu Thr Dab 2 Yield: 63.7 mg, Purity:
(99.6%) MS Data: [M + 2H].sup.2+ = 580.3 10 Dodec D-Ser D-Leu Thr
Dab 2 Yield: 39.0 mg, Purity: (98.6%) MS Data: [M + 2H].sup.2+ =
594.30 11 4-BPC D-Ser D-Leu Thr Dab 2 Yield: 68.5 mg, Purity:
(99.4%) MS Data [M + 2H].sup.2+ = 593.25 12 PA D-Ser D-Leu Thr Dab
2 Yield: mg, Purity: (99.1%) MS Data: [M + 2H].sup.2+ = 562.20 13
Octanoyl D-Ser D-Leu Thr Dab 1 Yield: 63.8 mg, Purity: (98.3%) MS
Data: [M + 2H].sup.2+ = 559.25 14 Octanoyl D-Ser D-Leu Thr Dab 3
Yield: 69.3 mg, Purity: (99.5%) MS Data: [M + 2H].sup.2+ = 573.30
15 Octanoyl D-Thr D-Leu Thr Dab 2 Yield: 60.3 mg, Purity: (98.6%)
MS Data: [M + 2H].sup.2+ = 573.3 16 Heptanoyl D-Ser D-Leu Thr Dab 2
Yield: 50.0 mg, Purity: (99.3%) MS Data: [M + 2H].sup.2+ = 559.25
17 Nonanoyl D-Ser D-Leu Thr Dab 2 Yield: 56.4 mg, Purity: (99.0%)
MS Data: [M + 2H].sup.2+ = 573.25 18 3-TFMB D-Ser D-Leu Abu Dab 2
Yield: 15.5 mg, Purity: (97.3%) MS Data: [M + 2H].sup.2+ = 582.1 19
3-TFMB D-Ser D-Leu Thr Dab 2 Yield: 12.6 mg, Purity: (97.3%) MS
Data: [M + 2H].sup.2+ = 590.05 150 (S)-6-MO D-Ser D-Leu Thr Dab 2
Yield: 56.6 mg, Purity: (99.2%) (polymyxin D.sub.1) MS Data: [M +
2H].sup.2+ = 573.3 151 6-MH D-Ser D-Leu Thr Dab 2 Yield: 47.7 mg,
Purity: (96.8%) (polymyxin D.sub.2) MS Data: [M + 2H].sup.2+ =
566.3 a) for R.sup.2, R.sup.3 R.sup.4 and X, the amino acid shown
in these columns is indicative of the side chain and
stereochemistry at these positions; Dodec = dodecanoyl, 3-TFMB =
3-trifluoromethylbenzoyl, (S)-6-MO = (S)-6-methyloctanoyl, 6-MH =
6-methylheptanoyl, Dab = di- aminobutyric acid, Phe =
phenylalanine, Thr = threonine, Ala = alanine, Ser = serine, Val =
valine, Nle = norleucine, Abu = 2-aminobutyric acid, D- indicates
D-amino acids.
TABLE-US-00006 TABLE 6 Characterisation data for compounds of the
invention represented by formula IIc: (IIc) ##STR00015## No.
R.sup.1 R.sup.3 R.sup.4 X k q m n p Compound Data 20 Octanoyl D-Leu
Thr Dab 2 2 2 2 2 Yield: 54.7 mg, Purity: (98.4%) MS Data: [M +
2H].sup.2+ = 572.80 21 Octanoyl D-Leu Ala Dab 2 2 2 2 2 Yield: 57.0
mg, Purity: (98.2%) MS Data: [M + 2H].sup.2+ = 557.65 22 Octanoyl
D-Phe Thr Dab 2 2 2 2 2 Yield: 54.8 mg, Purity: (99.3%) MS Data: [M
+ 2H].sup.2+ = 589.8 23 Octanoyl D-Leu Val Dab 2 2 2 2 2 Yield:
41.2 mg, Purity: (99.3%) MS Data: [M + 2H].sup.2+ = 571.80 24
Octanoyl D-Nle Thr Dab 2 2 2 2 2 Yield: 61.2 mg, Purity: (98.9%) MS
Data: [M + 2H].sup.2+ = 572.80 25 Octanoyl D-Leu Ser Dab 2 2 2 2 2
Yield: 49.8 mg, Purity: (99.2%) MS Data: [M + 2H].sup.2+ = 565.75
26 Hexanoyl D-Leu Thr Dab 2 2 2 2 2 Yield: 73.3 mg, Purity: (99.6%)
MS Data: [M + 2H].sup.2+ = 558.75 27 Decanoyl D-Leu Thr Dab 2 2 2 2
2 Yield: 64.1 mg, Purity: (98.9%) MS Data: [M + 2H].sup.2+ = 586.8
28 Dodec D-Leu Thr Dab 2 2 2 2 2 Yield: 51.0 mg, Purity: (99.6%) MS
Data: [M + 2H].sup.2+ = 600.85 29 4-BPC D-Leu Thr Dab 2 2 2 2 2
Yield: 69.0 mg, Purity: (98.8%) MS Data: [M + 2H].sup.2+ = 599.75
30 PA D-Leu Thr Dab 2 2 2 2 2 Yield: 69.3 mg, Purity: (99.1%) MS
Data: [M + 2H].sup.2+ = 568.75 31 Octanoyl D-Leu Thr Dab 1 2 2 2 2
Yield: 68.9 mg, Purity: (98.9%) MS Data: [M + 2H].sup.2+ = 565.80
32 Octanoyl D-Leu Thr Dab 3 2 2 2 2 Yield: 77.4 mg, Purity: (98.5%)
MS Data: [M + 2H].sup.2+ = 579.80 33 Octanoyl D-Leu Thr Dab 2 1 2 2
2 Yield: 72.2 mg, Purity: (98.8%) MS Data: [M + 2H].sup.2+ = 565.80
34 Octanoyl D-Leu Thr Dab 2 3 2 2 2 Yield: 57.9 mg, Purity: (98.4%)
MS Data: [M + 2H].sup.2+ = 579.85 35 Octanoyl D-Leu Ala Dab 2 1 2 2
2 Yield: 59.3 mg, Purity: (98.7%) MS Data: [M + 2H].sup.2+ = 550.75
36 Octanoyl D-Leu Val Dab 2 1 2 2 2 Yield: 47.8 mg, Purity: (98.6%)
MS Data: [M + 2H].sup.2+ = 564.80 37 Decanoyl D-Leu Thr Dab 2 1 2 2
2 Yield: 63.1 mg, Purity: (99.14%) MS Data: [M + 2H].sup.2+ =
579.75 38 Decanoyl D-Leu Ala Dab 2 1 2 2 2 Yield: 48.6 mg, Purity:
(99.3%) MS Data: [M + 2H].sup.2+ = 564.8 39 Decanoyl D-Leu Val Dab
2 1 2 2 2 Yield: 52.9 mg, Purity: (99.3%) MS Data: [M + 2H].sup.2+
= 578.85 40 Decanoyl D-Leu Ala Dab 2 2 2 2 2 Yield: 28.5 mg,
Purity: (99.6%) MS Data: [M + 2H].sup.2+ = 571.5 41 Decanoyl D-Leu
Val Dab 2 2 2 2 2 Yield: 59.3 mg, Purity: (99.6%) MS Data: [M +
2H].sup.2+ = 585.80 42 Heptanoyl D-Leu Thr Dab 2 2 2 2 2 Yield:
67.0 mg, Purity: (98.9%) MS Data: [M + 2H].sup.2+ = 568.80 43
Nonanoyl D-Leu Thr Dab 2 2 2 2 2 Yield: 35.0 mg, Purity: (98.9%) MS
Data: [M + 2H].sup.2+ = 579.75 44 Heptanoyl D-Leu Thr Dab 2 1 2 2 2
Yield: 59.8 mg, Purity: (99.1%) MS Data: [M + 2H].sup.2+ = 558.70
45 Octanoyl D-Leu Aib Dab 2 1 2 2 2 Yield: 67.5 mg, Purity: (97.7%)
MS Data: [M + 2H].sup.2+ = 557.75 46 Octanoyl D-Leu Abu Dab 2 1 2 2
2 Yield: 52.5 mg, Purity: (99.0%) MS Data: [M + 2H].sup.2+ = 557.75
47 Octanoyl D-Leu Tle Dab 2 1 2 2 2 Yield: 45.3 mg, Purity: (97.4%)
MS Data: [M + 2H].sup.2+ = 571.80 48 Octanoyl D-Leu Thr Dab 1 1 2 2
2 Yield: 57.5 mg, Purity: (98.9%) MS Data: [M + 2H].sup.2+ = 558.75
49 Octanoyl D-Nva Thr Dab 2 1 2 2 2 Yield: 53.0 mg, Purity: (99.3%)
MS Data: [M + 2H].sup.2+ = 558.75 50 Octanoyl D-Leu Thr Dab 2 1 2 2
1 Yield: 63.8 mg, Purity: (99.1%) MS Data: [M + 2H].sup.2+ = 558.75
51 Octanoyl D-Leu Thr Dab 2 1 2 1 2 Yield: 64.9 mg, Purity: (98.8%)
MS Data: [M + 2H].sup.2+ = 558.75 52 Octanoyl D-Leu Thr Dab 2 1 1 2
2 Yield: 43.8 mg, Purity: (99.0%) MS Data: [M + 2H].sup.2+ = 558.75
53 Octanoyl D-Leu Thr Dab 2 1 2 2 3 Yield: 52.0 mg, Purity: (96.7%)
MS Data: [M + 2H].sup.2+ = 572.75 54 Octanoyl D-Leu Thr Dab 2 1 2 3
2 Yield: 45.1 mg, Purity: (99.3%) MS Data: [M + 2H].sup.2+ = 572.75
55 Octanoyl D-Leu Thr Dab 2 1 3 2 2 Yield: 60.9 mg, Purity: (99.6%)
MS Data: [M + 2H].sup.2+ = 572.75 56 4-TFMB D-Leu Thr Dab 2 1 2 2 2
Yield: 52.5 mg, Purity: (99.1%) MS Data: [M + 2H].sup.2+ = 588.75
57 3,4-DCB D-Leu Thr Dab 2 1 2 2 2 Yield: 52.0 mg, Purity: (99.1%)
MS Data: [M + H].sup.+ = 596.75 58 Nonanoyl D-Leu Thr Dab 2 1 2 2 2
Yield: 54.0 mg, Purity: (99.3%) MS Data: [M + 2H].sup.2+ = 572.7 59
Nonanoyl D-Leu Abu Dab 2 1 2 2 2 Yield: 66.0 mg, Purity: (98.3%) MS
Data: [M + 2H].sup.2+ = 564.85 60 Octanoyl D-Leu Abu Dab 2 2 2 2 2
Yield: 23.0 mg, Purity: (99.6%) MS Data: [M + 2H].sup.2+ = 564.75
61 3-CPA D-Leu Thr Dab 2 1 2 2 2 Yield: 71.9 mg, Purity: (98.5%) MS
Data: [M + 2H].sup.2+ = 578.9 62 2,4-DCPA D-Leu Thr Dab 2 1 2 2 2
Yield: 74.9 mg, Purity: (98.9%) MS Data: [M + 2H].sup.2+ = 595.8 63
Heptanoyl D-Leu Ala Dab 2 1 2 2 2 Yield: 65.4 mg, Purity: (97.9%)
MS Data: [M + 2H].sup.2+ = 543.80 64 Heptanoyl D-Leu Abu Dab 2 1 2
2 2 Yield: 59.8 mg, Purity: (99.1%) MS Data: [M + 2H].sup.2+ =
550.80 65 6-MH D-Leu Ala Dab 2 1 2 2 2 Yield: 67.4 mg, Purity:
(98.7%) MS Data: [M + 2H].sup.2+ = 550.75 66 6-MH D-Leu Abu Dab 2 1
2 2 2 Yield: 68.0 mg, Purity: (97.9%) MS Data: [M + 2H].sup.2+ =
557.75 67 Hexanoyl D-Leu Ala Dab 2 1 2 2 2 Yield: 65.9 mg, Purity:
(99.2%) MS Data: [M + 2H].sup.2+ = 536.75 68 Hexanoyl D-Leu Abu Dab
2 1 2 2 2 Yield: 69.9 mg, Purity: (99.4%) MS Data: [M + 2H].sup.2+
= 543.80 69 Octanoyl D-Nva Abu Dab 2 1 2 2 2 Yield: 57.9 mg,
Purity: (98.9%) MS Data: [M + 2H].sup.2+ = 550.8 70 2,4-DCPA D-Leu
Abu Dab 2 1 2 2 2 Yield: 70.7 mg, Purity: (98.5%) MS Data: [M +
2H].sup.2+ = 596.85 71 3,4-DCB D-Leu Abu Dab 2 1 2 2 2 Yield: 60.7
mg, Purity: (98.2%) MS Data: [M + 2H].sup.2+ = 581.65 72 2-CB D-Leu
Abu Dab 2 1 2 2 2 Yield: 61.0 mg, Purity: (99.2%) MS Data: [M +
2H].sup.2+ = 563.95 73 2-FB D-Leu Abu Dab 2 1 2 2 2 Yield: 61.4 mg,
Purity: (97.8%) MS Data: [M + 2H].sup.2+ = 555.70 74 4-TFMB D-Leu
Abu Dab 2 1 2 2 2 Yield: 67.3 mg, Purity: (97.7%) MS Data: [M +
2H].sup.2+ = 58.75 75 2-MB D-Leu Abu Dab 2 1 2 2 2 Yield: 62.9 mg,
Purity: (98.1%) MS Data: [M + 2H].sup.2+ = 553.75 76 2-MPA D-Leu
Abu Dab 2 1 2 2 2 Yield: 67.2 mg, Purity: (98.8%) MS Data: [M +
2H].sup.2+ = 560.75 77 4-CPA D-Leu Abu Dab 2 1 2 2 2 Yield: 67.6
mg, Purity: (99.2%) MS Data: [M + 2H].sup.2+ = 570.90 78 PA D-Leu
Abu Dab 2 1 2 2 2 Yield: 43.9 mg, Purity: (98.3%) MS Data: [M +
2H].sup.2+ = 553.75 79 3-CPA D-Leu Abu Dab 2 1 2 2 2 Yield: 76.2
mg, Purity: (97.9%) MS Data: [M + 2H].sup.2+ = 570.60 80 4-MPA
D-Leu Abu Dab 2 1 2 2 2 Yield: 65.2 mg, Purity: (98.9%)
MS Data: [M + 2H].sup.2+ = 560.80 81 3,4-DCPA D-Leu Abu Dab 2 1 2 2
2 Yield: 60.7 mg, Purity: (99.1%) MS Data: [M + 2H].sup.2+ = 588.55
82 2,4-DCB D-Leu Abu Dab 2 1 2 2 2 Yield: 64.3 mg, Purity: (98.9%)
MS Data: [M + 2H].sup.2+ = 581.65 83 3,4-DMB D-Leu Abu Dab 2 1 2 2
2 Yield: 63.5 mg, Purity: (98.7%) MS Data: [M + 2H].sup.2+ = 560.75
84 2-CPA D-Leu Abu Dab 2 1 2 2 2 Yield: 61.4 mg, Purity: (99.4%) MS
Data: [M + 2H].sup.2+ = 570.60 85 2-FPA D-Leu Abu Dab 2 1 2 2 2
Yield: 60.4 mg, Purity: (99.5%) MS Data: [M + 2H].sup.2+ = 562.75
86 3-FB D-Leu Abu Dab 2 1 2 2 2 Yield: 62.3 mg, Purity: (98.2%) MS
Data: [M + 2H].sup.2+ = 555.70 87 3-MB D-Leu Abu Dab 2 1 2 2 2
Yield: 64.8 mg, Purity: (98.1%) MS Data: [M + 2H].sup.2+ = 553.75
88 3-CB D-Leu Abu Dab 2 1 2 2 2 Yield: 59.3 mg, Purity: (97.3%) MS
Data: [M + 2H].sup.2+ = 564.0 89 2,4-DMB D-Leu Abu Dab 2 1 2 2 2
Yield: 63.0 mg, Purity: (98.8%) MS Data: [M + 2H].sup.2+ = 560.80
90 2,3-DCB D-Leu Abu Dab 2 1 2 2 2 Yield: 67.9 mg, Purity: (99.1%)
MS Data: [M + 2H].sup.2+ = 581.60 91 2,3-DMB D-Leu Abu Dab 2 1 2 2
2 Yield: 60.6 mg, Purity: (98.5%) MS Data: [M + 2H].sup.2+ = 560.80
92 2,4,6-TMB D-Leu Abu Dab 2 1 2 2 2 Yield: 22.0 mg, Purity:
(98.2%) MS Data: [M + 2H].sup.2+ = 567.80 93 3,5-DMB D-Leu Abu Dab
2 1 2 2 2 Yield: 57.1 mg, Purity: (99.0%) MS Data: [M + 2H].sup.2+
= 560.80 94 4-CB D-Leu Abu Dab 2 1 2 2 2 Yield: 62.1 mg, Purity:
(98.4%) MS Data: [M + 2H].sup.2+ = 563.95 95 2,4,6-TCB D-Leu Abu
Dab 2 1 2 2 2 Yield: 58.0 mg, Purity: (98.7%) MS Data: [M +
2H].sup.2+ = 598.70 96 3,5-DCB D-Leu Abu Dab 2 1 2 2 2 Yield: 63.8
mg, Purity: (98.6%) MS Data: [M + 2H].sup.2+ = 581.65 97 3,5-BTFMB
D-Leu Abu Dab 2 1 2 2 2 Yield: 57.0 mg, Purity: (98.9%) MS Data: [M
+ 2H].sup.2+ = 614.75 98 4-MB D-Leu Abu Dab 2 1 2 2 2 Yield: 60.0
mg, Purity: (98.1%) MS Data: [M + 2H].sup.2+ = 553.80 99 4-IPB
D-Leu Abu Dab 2 1 2 2 2 Yield: 51.8 mg, Purity: (97.9%) MS Data: [M
+ 2H].sup.2+ = 567.80 100 4-EB D-Leu Abu Dab 2 1 2 2 2 Yield: 54.8
mg, Purity: (99.5%) MS Data: [M + 2H].sup.2+ = 560.75 101 2-C-4-MB
D-Leu Abu Dab 2 1 2 2 2 Yield: 37.0 mg, Purity: (98.6%) MS Data: [M
+ 2H].sup.2+ = 570.65 102 3-F-4-MB D-Leu Abu Dab 2 1 2 2 2 Yield:
61.5 mg, Purity: (98.7%) MS Data: [M + 2H].sup.2+ = 562.75 103
3,4-DMPA D-Leu Abu Dab 2 1 2 2 2 Yield: 64.7 mg, Purity: (97.2%) MS
Data: [M + 2H].sup.2+ = 567.80 104 4-C-3-MB D-Leu Abu Dab 2 1 2 2 2
Yield: 62.0 mg, Purity: (97.6%) MS Data: [M + 2H].sup.2+ = 570.90
105 3-C-4-MB D-Leu Abu Dab 2 1 2 2 2 Yield: 64.0 mg, Purity:
(98.7%) MS Data: [M + 2H].sup.2+ = 570.95 106 3-TFMB D-Leu Abu Dab
2 1 2 2 2 Yield: 60.0 mg, Purity: (97.6%) MS Data: [M + 2H].sup.2+
= 580.75 107 4-C-3-FB D-Leu Abu Dab 2 1 2 2 2 Yield: 62.7 mg,
Purity: (97.5%) MS Data: [M + 2H].sup.2+ = 572.90 108 3-F-5-TFMB
D-Leu Abu Dab 2 1 2 2 2 Yield: 66.0 mg, Purity: (98.5%) MS Data: [M
+ 2H].sup.2+ = 589.75 109 2-C-4-TFMB D-Leu Abu Dab 2 1 2 2 2 Yield:
67.5 mg, Purity: (97.3%) MS Data: [M + 2H].sup.2+ = 597.60 110
3-C-4-FB D-Leu Abu Dab 2 1 2 2 2 Yield: 65.4 mg, Purity: (98.1%) MS
Data: [M + 2H].sup.2+ = 572.90 111 3-F-4-TFMB D-Leu Abu Dab 2 1 2 2
2 Yield: 61.3 mg, Purity: (97.5%) MS Data: [M + 2H].sup.2+ = 589.75
112 4-C-3-TFMB D-Leu Abu Dab 2 1 2 2 2 Yield: 60.0 mg, Purity:
(96.9%) MS Data: [M + 2H].sup.2+ = 598.00 113 4-M-3-TFMB D-Leu Abu
Dab 2 1 2 2 2 Yield: 65.2 mg, Purity: (96.5%) MS Data: [M +
2H].sup.2+ = 587.75 114 3-C-5-MB D-Leu Abu Dab 2 1 2 2 2 Yield:
25.4 mg, Purity: (96.9%) MS Data: [M + 2H].sup.2+ = 570.95 115
3-C-4-TFMB D-Leu Abu Dab 2 1 2 2 2 Yield: 19.2 mg, Purity: (97.0%)
MS Data: [M + 2H].sup.2+ = 597.95 116 3-C-5-FB D-Leu Abu Dab 2 1 2
2 2 Yield: 67.7 mg, Purity: (97.4%) MS Data: [M + 2H].sup.2+ =
572.95 117 3,5-DCB D-Leu Ala Dab 2 1 2 2 2 Yield: 17.6 mg, Purity:
(95.9%) MS Data: [M + 2H].sup.2+ = 574.55 118 3,5-DCB D-Leu Thr Dab
2 1 2 2 2 Yield: 11.7 mg, Purity: (97.1%) MS Data: [M + 2H].sup.2+
= 589.50 119 3-M-4-TFMB D-Leu Abu Dab 2 1 2 2 2 Yield: 60.2 mg,
Purity: (97.4%) MS Data: [M + 2H].sup.2+ = 587.85 120 3-M-5-TFMB
D-Leu Abu Dab 2 1 2 2 2 Yield: 65.2 mg, Purity: (97.1%) MS Data: [M
+ 2H].sup.2+ = 587.75 121 3-TFMB D-Nle Abu Dab 2 1 2 2 2 Yield:
62.4 mg, Purity: (97.5%) MS Data: [M + 2H].sup.2+ = 580.75 122
3-TFMB D-Phe Abu Dab 2 1 2 2 2 Yield: 65.6 mg, Purity: (97.2%) MS
Data: [M + 2H].sup.2+ = 597.75 123 3-TFMB D-Nle Thr Dab 2 1 2 2 2
Yield: 52.4 mg, Purity: (97.5%) MS Data: [M + 2H].sup.2+ = 588.70
124 3-TFMB D-Phe Thr Dab 2 1 2 2 2 Yield: 63.6 mg, Purity: (97.2%)
MS Data: [M + 2H].sup.2+ = 605.70 125 3-TFMB D-Nle Ala Dab 2 1 2 2
2 Yield: 62.3 mg, Purity: (97.8%) MS Data: [M + 2H].sup.2+ = 573.70
126 3-TFMB D-Phe Ala Dab 2 1 2 2 2 Yield: 61.6 mg, Purity: (97.9%)
MS Data: [M + 2H].sup.2+ = 590.70 127 4-TFMPA D-Leu Abu Dab 2 1 2 2
2 Yield: 84.6 mg, Purity: (98.6%) MS Data: [M + 2H].sup.2+ = 595.70
128 Octanoyl D-Phe Ala Dab 2 1 2 2 2 Yield: 62.6 mg, Purity: 97.1%)
MS Data: [M + 2H].sup.2+ = 567.80 129 Octanoyl D-Phe Thr Dab 2 1 2
2 2 Yield: 56.8 mg, Purity: (97.7%) MS Data: [M + 2H].sup.2+ =
582.80 130 Octanoyl D-Phe Abu Dab 2 1 2 2 2 Yield: 66.3 mg, Purity:
(97.3%) MS Data: [M + 2H].sup.2+ = 574.75 131 Heptanoyl D-Phe Abu
Dab 2 1 2 2 2 Yield: 66.4 mg, Purity: (98.8%) MS Data: [M +
2H].sup.2+ = 567.75 132 Nonanoyl D-Leu Ala Dab 2 1 2 2 2 Yield:
62.2 mg, Purity: (97.7%) MS Data: [M + 2H].sup.2+ = 557.80 133
3,4,5-TFB D-Leu Abu Dab 2 1 2 2 2 Yield: 63.3 mg, Purity: (97.8%)
MS Data: [M + 2H].sup.2+ = 573.70 134 4-C-2-FB D-Leu Abu Dab 2 1 2
2 2 Yield: 70.3 mg, Purity: (97.7%) MS Data: [M + 2H].sup.2+ =
572.60 135 2-C-4-FB D-Leu Abu Dab 2 1 2 2 2 Yield: 66.2 mg, Purity:
(98.0%) MS Data: [M + 2H].sup.2+ = 572.90 136 4-C-2-TFMB D-Leu Abu
Dab 2 1 2 2 2 Yield: 74.2 mg, Purity: (98.1%) MS Data: [M +
2H].sup.2+ = 597.60 137 2-F-4-TFMB D-Leu Abu Dab 2 1 2 2 2 Yield:
56.7 mg, Purity: (98.2%) MS Data: [M + 2H].sup.2+ = 589.75 138
3-BPC D-Leu Abu Dab 2 1 2 2 2 Yield: 62.0 mg, Purity: (97.5%) MS
Data: [M + 2H].sup.2+ = 584.80 139 (S,R)-6-MO D-Leu Abu Dab 2 1 2 2
2 Yield: 54.8 mg, Purity: (98.6%) MS Data: [M + 2H].sup.2+ = 564.85
140 Octanoyl D-Phe Aib Dab 2 1 2 2 2 Yield: 68.7 mg, Purity:
(97.6%) MS Data: [M + 2H].sup.2+ = 574.80 141 3-TFMB D-Leu Abu Dab
3 1 2 2 2 Yield: 74.0 mg, Purity: (97.1%) MS Data: [M + 2H].sup.2+
= 587.70 142 4-BPC D-Leu Abu Dab 2 1 2 2 2 Yield: 69.3 mg, Purity:
(97.3%) MS Data: [M + 2H].sup.2+ = 584.80 143 Nonanoyl D-Phe Ser
Dab 2 1 2 2 2 Yield: 36.2 mg, Purity: (97.3%) MS Data: [M +
2H].sup.2+ = 582.85 144 4-Cl-BP-4-C D-Leu Ala Dab 2 1 2 2 2 Yield:
56.4 mg, Purity: (96.2%) MS Data: [M + 2H].sup.2+ = 595.00 145 3-PP
D-Leu Abu Dab 2 1 2 2 2 Yield: 55.2 mg, Purity: (97.3%) MS Data: [M
+ 2H].sup.2+ = 560.80 146 4-PB D-Leu Ala Dab 2 1 2 2 2 Yield: 69.8
mg, Purity: (97.9%) MS Data: [M + 2H].sup.2+ = 560.80 147 2,4-DCB
D-Leu Abu Dab 2 3 2 2 2 Yield: 67.8 mg, Purity: (97.2%) MS Data: [M
+ 2H].sup.2+ = 595.65 148 2,4-DCPS D-Leu Abu Dab 2 1 2 2 2 Yield:
10.1 mg, Purity: (94.0%) MS Data: [M + 2H].sup.2+ = 599.6 149
Octanoyl D-Leu Thr Orn 2 2 2 2 2 Yield: 54.0 mg, Purity: (98.5%) MS
Data: [M + 2H].sup.2+ = 579.75 152 (S)-6-MO D-Leu Thr Dab 2 2 2 2 2
Yield: 40.0 mg, Purity: (polymyxin M.sub.1) (97.4%) MS Data: [M +
2H].sup.2+ = 579.65 153 6-MH D-Leu Thr Dab 2 2 2 2 2 Yield: 47.7
mg, Purity: (Polymyxin M.sub.2) (96.9%) MS Data: [M + 2H].sup.2+ =
572.65 for R.sup.3, R.sup.4 and X, the amino acid shown in these
columns is indicative of the side chain and stereochemistry at
these positions; Dodec = dodecanoyl, 4-BPC = 4-biphenylcarboxyl, PA
= phenylacetyl, 6-MH = 6-methylheptanoyl, 4-TFMPA =
4-trifluoromethylphenylacetyl, 2- MB = 2-methylbenzoyl, 3-MB =
3-methylbenzoyl, 4-MB = 4-methylbenzoyl, 3-F-4-MB =
3-fluoro-4-methylbenzoyl, 4-C-3-MB = 4-chloro-3-methylbenzoyl,
3-C-4-MB = 3-chloro-4-methylbenzoyl, 3-C-5-MB =
3-chloro-5-methylbenzoyl, 2-FPA = 2-fluorophenylacetyl, 3-TFMB =
3-trifluoromethylbenzoyl, 4-TFMB = 4-trifluoromethylbenzoyl,
2-C-4-TFMB = 2-chloro-4-trifluoromethylbenzoyl,
4-C-3- TFMB = 4-chloro-3-trifluoromethylbenzoyl, 3-C-4-TFMB =
3-chloro-4-trifluoromethylbenzoyl, 3-F-4-TFMB =
3-fluoro-4-trifluoromethylbenzoyl, 3-F-5-TFMB =
3-fluoro-5-trifluoromethylbenzoyl, 4-M-3-TFMB =
4-methyl-3-trifluoromethylbenzoyl, 3-M-4-TFMB =
3-methyl-4-trifluoromethylbenzoyl, 3-M-5-TFMB =
3-methyl-5-trifluoromethylbenzoyl, 2-F-4-TFMB =
2-fluoro-4-trifluoromethylbenzoyl, 3,4,5-TFMB =
3,4,5-trifluoromethylbenzoyl, 4-C-2-TFMB =
4-chloro-2-trifluoromethylbenzoyl, 3,5-BTFMB =
3,5-bis(trifluoromethyl)benzoyl, 2,4,6-TMB =
2,4,6-trimethylbenzoyl, 2,3-DMB = 2,3-dimethylbenzoyl, 2,4-DMB =
2,4-dimethylbenzoyl, 3,4-DMB = 3,4-dimethylbenzoyl, 3,5-DMB =
3,5-dimethylbenzoyl, 2-C-4-MB = 2-chloro-4-methylbenzoyl, 4-EB =
4-ethylbenzoyl, 4-IPB = 4-Isoprop- ylbenzoyl, 2,4-DCPA =
2,4-dichlorophenylacetyl, 3,4-DCPA = 3,4-dichlorophenylacetyl,
2-CPA = 2-chlorophenylacetyl, 3-CPA = 3- chlorophenylacetyl, 4-CPA
= 4-chlorophenylacetyl, 2-CB = 2-chlorobenzoyl, 3-CB =
3-chlorobenzoyl, 4-CB = 4-chlorobenzoyl, 2,3- DCB =
2,3-dichlorobenzoyl, 2,4-DCB = 2,4-dichlorobenzoyl, 3,4-DCB =
3,4-dichlorobenzoyl, 3,5-DCB = 3,5-dichlorobenzoyl, 2,4,6- TCB =
2,4,6-trichlorobenzoyl, 2-FB = 2-fluorobenzoyl, 3-FB =
3-fluorobenzoyl, 2-C-4-FB = 2-chloro-4-fluorobenzoyl, 3-C-4-FB =
3-Chloro-4-fluorobenzoyl, 3-C-5-FB = 3-chloro-5-fluorobenzoyl,
4-C-2-FB = 4-chloro-2-fluorobenzoyl, 4-C-3-FB =
4-chloro-3-fluorobenzoyl, 2-MPA = 2-methylphenylacetyl, 4-MPA =
4-methylphenylacetyl, 3,4-DMPA = 3,4-dimethylphenylacetyl,
(S,R)-6-MO = (S,R)-6-methyloctanoyl, (S)-6-MO =
(S)-6-methyloctanoyl, 3-BPC = 3-biphenylcarboxyl, 4-Cl-BP-4-C =
4-chloro-biphenyl-4-carboxyl, 3-PP = 3-phenylproponyl, 4-PB =
4-phenylbutanoyl, 2,4-DCPS = 2,4-dichlorophenylsulfonyl, Dab =
diaminobutyric acid, , Tle = t-butylglycine, Aib = aminoisobutyric
acid, Abu = 2-aminobutyric acid, Phe = phenylalanine, Thr =
threonine, Ala = alanine, Ser = serine, Val = valine, Nva =
norvaline, Nle = norleucine, Orn = ornithine, D- indicates D-amino
acids.
Example 2. Measurements of Minimum Inhibitory Concentrations
(MICs)
MICs of the lipopeptides (trifluoroacetic acid salt, TFA) were
determined by broth microdilution in cation-adjusted Mueller-Hinton
broth (CAMHB) (Oxoid Australia, Thebarton, SA, Australia) according
to Clinical and Laboratory Standards Institute standards (Clinical
and Laboratory Standards Institute. Performance standards for
antimicrobial susceptibility testing; eighteenth informational
supplement M100-S18. Wayne, Pa., 2008). Polymyxin B (sulphate) was
employed as control. Gram-negative bacteria were examined for
compounds 1-153 as well as for a 1:1 combination of compounds 1 and
20: for (1) Pseudomonas aeruginosa, 3 polymyxin-susceptible
isolates; (2) Acinetobacter baumannii, 3 polymyxin-susceptible
isolates; (3) Klebsiella pneumoniae, 2 polymyxin-susceptible
isolates; (4) Enterobacter cloacae, 3 polymyxin-susceptible
isolates. The results are illustrated in Table 7.
TABLE-US-00007 TABLE 7 Minimum inhibitory concentrations (mg/L) for
compounds 1-153 Pa Ab Kp Ec ATCC FADDI- FADDI- ATCC FADDI- A ATCC
ATCC FADDI- FADDI- FADDI- FADDI- Compound 27853 PA022 PA025 19606
AB034 17978 13883 KP032 EC006 EC001 EC003- Colistin 1 1 2 1 0.5 0.5
1 1 <0.125 0.25 <0.125 Polymyxin B 1 1 1 1 0.5 1 1 <0.125
0.5 0.25 0.5 1 4 4 >32 1 0.25 0.25 1 <0.125 0.5 <0.125
<0.125 2 8 8 >32 0.5 0.5 0.5 1 0.5 0.5 <0.125 <0.125 3
4 4 >32 0.25 <0.125 <0.125 2 <0.125 0.25 0.5 <0.125
4 4 8 >32 8 4 8 32 1 1 0.5 0.25 5 2 4 16 <0.125 0.25
<0.125 1 <0.125 0.25 0.25 <0.125 6 >32 4 >32 1 0.5 1
>32 0.5 0.25 0.25 0.25 7 2 4 >32 0.25 <0.125 <0.125 32
<0.125 0.5 0.25 0.25 8 4 8 >32 2 1 2 32 1 0.25 0.25 0.5 9 4 8
32 0.25 0.5 1 16 1 1 0.5 1 10 2 4 4 1 1 0.5 2 0.5 1 1 0.5 11 4 4 32
2 1 0.5 32 0.25 0.5 0.5 0.5 12 >32 8 >32 4 4 4 >32 2 1
0.25 0.5 13 4 4 >32 1 0.5 1 32 0.25 0.5 0.25 0.25 14 >32 8
>32 16 4 8 >32 1 4 0.25 0.5 15 16 16 >32 2 1 0.5 8 1 1 1 2
16 4 4 >32 2 2 0.5 >32 0.25 0.25 0.5 0.25 17 2 4 16 <0.125
0.25 <0.125 <0.125 0.25 0.5 0.25 0.25 18 8 8 2 0.5 0.5 0.5 8
0.25 0.5 1 0.5 19 32 16 >32 2 4 0.5 32 0.5 0.5 1 0.5 20 0.5 0.5
>32 4 2 4 0.25 0.25 <0.125 <0.125 0.5 21 0.5 0.5 >32 1
0.5 2 >32 0.25 0.125 0.125 0.125 22 1 1 16 1 0.5 1 >32 0.5
0.25 <0.125 <0.125 23 1 1 4 0.5 0.25 0.5 2 <0.125 0.25
<0.125 <0.125 24 0.5 0.5 16 1 1 1 >32 <0.125 0.25
<0.125 <0.125 25 >32 0.5 >32 8 4 4 >32 0.25 0.5
<0.125 0.25 26 0.5 2 >32 16 16 8 >32 2 1 0.5 0.5 27 1 1 16
1 1 1 1 0.5 0.25 <0.125 <0.125 28 2 4 4 2 1 2 0.5 2 0.5 0.5
0.5 29 1 4 32 8 4 2 1 1 0.5 0.5 1 30 1 1 >32 32 32 32 >32 1 4
2 2 31 0.5 0.5 16 4 2 4 >32 <0.125 0.5 <0.125 0.25 32
>32 1 >32 >32 >32 >32 >32 1 >32 1 1 33 0.5 0.5
8 0.5 0.25 0.25 <0.125 <0.125 1 <0.125 <0.125 34 1 0.5
>32 2 2 2 >32 <0.125 4 <0.125 <0.125 35 0.5 0.5 16
0.5 0.25 0.5 1 <0.125 <0.125 <0.125 0.25 36 1 1 4 0.25 0.5
0.5 0.5 0.25 0.25 0.25 <0.125 37 1 0.5 2 0.25 0.25 0.5 0.5 0.25
0.5 0.5 4 38 1 2 2 0.5 1 0.5 8 0.25 1 0.5 0.5 39 1 2 2 0.5 1 2 2
0.5 0.5 0.5 0.5 40 0.5 2 4 0.5 0.5 0.5 8 0.25 0.25 0.25 0.25 41 1 4
2 1 0.5 1 >32 0.5 0.5 1 0.5 42 0.5 2 >32 4 2 4 4 <0.125
0.25 <0.125 <0.125 43 0.5 1 16 0.5 1 0.5 0.5 <0.125 0.5
0.25 <0.125 44 0.5 1 16 1 1 1 32 <0.125 <0.125 0.25
<0.125 45 0.5 1 16 8 2 4 >32 0.5 0.5 0.5 0.25 46 0.5 0.5 2
0.25 0.25 0.25 <0.125 <0.125 <0.125 <0.125 0.25 47 1 1
8 8 1 2 16 <0.125 0.25 0.5 0.5 48 0.5 2 4 1 1 1 32 0.25
<0.125 0.25 <0.125 49 0.5 0.25 16 1 0.5 0.5 >32 <0.125
0.25 0.5 0.5 50 1 0.5 >32 0.5 1 0.5 32 <0.125 <0.125
<0.125 <0.125 51 1 0.5 >32 4 8 4 >32 0.25 1 0.25 0.25
52 0.5 1 >32 1 0.5 0.5 32 <0.125 0.5 0.25 0.25 53 8 2 >32
16 8 8 >32 0.25 8 0.5 2 54 4 2 16 0.5 0.25 0.5 0.5 0.25 0.25
<0.125 <0.125 55 >32 >32 >32 >32 >32 >32
>32 2 >32 1 4 56 0.5 2 4 2 1 1 16 0.25 0.25 <0.125
<0.125 57 0.5 0.5 4 2 0.5 1 0.5 <0.125 1 1 1 58 0.5 2 2 0.25
1 0.5 0.25 0.25 0.5 0.25 0.25 59 1 2 2 2 0.5 1 0.25 <0.125 0.5 1
1 60 0.5 0.5 8 1 0.5 0.5 1 <0.125 0.5 <0.125 <0.125 61 0.5
0.5 >32 4 1 2 >32 0.25 0.25 0.25 0.25 62 0.5 1 1 2 1 1 >32
0.25 0.25 <0.125 <0.125 63 0.5 2 >32 2 1 1 32 0.25 0.5 0.5
<0.125 64 1 2 8 0.5 1 0.5 16 <0.125 0.25 0.5 <0.125 65 1 2
16 0.5 1 0.25 4 <0.125 0.25 0.5 <0.125 66 1 2 4 0.25 0.5 0.25
4 <0.125 0.25 0.5 <0.125 67 0.5 0.5 2 4 4 2 >32 1 0.5
<0.125 0.25 68 0.5 0.5 2 2 1 1 32 0.25 <0.125 0.25 <0.125
69 0.5 0.5 8 0.5 0.25 0.5 16 <0.125 <0.125 0.25 <0.125 70
1 1 2 0.5 1 0.5 16 0.25 0.5 0.5 0.25 71 0.5 4 1 0.5 0.25 0.25 16
0.5 1 1 0.25 72 0.5 0.5 4 8 4 4 16 1 0.5 0.5 0.25 73 0.5 1 4 8 4 4
1 1 1 0.25 0.25 74 0.5 0.5 2 0.5 1 0.5 .25 0.25 2 <0.125 0.25 75
0.5 1 2 1 1 1 2 0.5 0.5 0.25 0.25 76 0.5 1 4 2 1 1 0.5 0.5 0.5 0.25
0.25 77 0.25 0.5 4 4 1 0.5 0.25 <0.125 0.25 0.5 <0.125 78 1
0.5 4 4 2 2 1 0.5 0.5 0.25 0.25 79 0.5 1 1 1 1 0.5 8 <0.125 2
0.25 <0.125 80 0.5 1 2 4 2 1 16 0.5 4 <0.125 0.25 81 2 4 1 1
0.25 0.5 4 <0.125 0.25 0.25 <0.125 82 0.5 0.5 1 0.5 0.25 0.5
1 <0.125 <0.125 <0.125 <0.125 83 1 1 2 2 0.5 1 >32
0.25 0.25 0.25 0.25 84 0.5 2 2 8 2 2 >32 0.5 0.5 <0.125 0.25
85 0.5 2 2 4 2 2 32 1 1 0.5 1 86 0.5 1 2 4 1 2 4 1 1 1 1 87 0.5 1 2
2 2 1 1 1 2 2 1 88 0.5 1 2 2 0.5 0.5 8 0.5 0.5 1 0.25 89 0.5 1 4 4
1 1 <0.125 0.25 2 <0.125 <0.125 90 0.5 2 2 2 8 1 <0.125
0.5 0.25 0.5 8 91 0.5 2 4 4 2 1 2 0.5 0.5 <0.125 <0.125 92
0.5 0.5 4 4 4 0.5 0.25 0.25 2 <0.125 0.25 93 2 4 2 1 0.5 0.5 2
0.5 4 0.25 1 94 0.5 0.5 2 1 0.25 0.5 1 0.25 0.5 0.25 0.25 95 2 4 2
0.25 0.25 0.25 16 <0.125 1 <0.125 0.25 96 0.25 0.25 0.5 0.5
0.25 0.25 <0.125 <0.125 0.5 1 1 97 0.5 0.5 1 0.5 0.25
<0.125 <0.125 <0.125 0.5 0.5 0.25 98 0.5 0.5 2 2 2 2
>32 1 2 1 0.25 99 1 0.5 2 1 0.25 0.25 >32 0.5 0.5 0.25 0.25
100 0.5 0.5 2 2 1 1 >32 0.5 0.5 0.25 0.5 101 0.5 2 2 0.5 1 0.5 8
0.25 0.25 <0.125 <0.125 102 0.5 0.5 2 2 1 1 >32 0.5 4 1
0.5 103 0.5 0.5 2 0.5 0.5 0.5 16 <0.125 0.5 <0.125 <0.125
104 0.5 0.5 2 1 0.25 0.5 8 1 0.5 0.5 0.25 105 0.5 1 2 0.5 0.25 0.5
>32 0.25 1 1 0.5 106 0.5 0.5 2 0.5 0.25 0.5 0.5 0.25 0.25 0.25
0.5 107 0.5 0.5 2 2 0.25 0.5 8 0.25 0.25 0.25 0.25 108 0.5 0.5 2
0.5 0.5 0.5 0.5 0.25 0.5 1 0.5 109 0.5 0.5 2 0.25 1 1 4 0.25 0.25 1
<0.125 110 0.5 0.5 1 2 0.5 0.5 16 0.25 0.5 1 1 111 0.5 1 1 0.5
0.25 0.5 16 0.25 0.25 0.5 0.25 112 0.5 0.5 1 0.25 0.25 <0.125
0.25 0.5 0.5 0.5 0.25 113 1 0.5 2 0.5 0.25 0.25 <0.125 0.25 1 1
0.25 114 0.5 1 1 2 0.5 0.5 32 0.25 0.25 0.25 0.25 115 1 0.5 1 0.5
0.5 0.5 0.25 <0.125 0.25 0.25 0.25 116 0.5 0.5 1 0.5 0.25 0.5
0.25 0.5 0.25 0.25 0.25 117 0.25 2 2 1 1 0.25 4 0.25 1 0.25 0.25
118 0.5 2 2 2 1 >32 32 0.25 0.5 1 0.25 119 0.5 0.5 2 1 0.25 0.5
1 <0.125 0.25 <0.125 <0.125 120 1 1 2 1 0.5 0.5 1 0.5 0.25
0.25 0.25 121 0.5 1 2 0.5 0.5 0.5 0.5 0.25 0.25 0.25 0.25 122 2 2 2
0.5 0.5 0.25 0.5 0.5 0.25 0.5 0.5 123 0.5 0.5 2 1 0.5 1 32 0.5 1
0.5 0.5 124 0.25 0.5 2 1 0.25 1 1 0.25 0.5 0.5 <0.125 125 0.25
0.5 2 1 0.5 1 0.5 0.25 1 0.5 0.5 126 0.25 1 2 1 1 1 0.25 0.5 0.5
0.5 0.5 127 0.5 0.5 16 4 0.5 1 16 0.5 0.5 <0.125 0.25 128 0.5 1
4 0.25 0.25 0.25 <0.125 0.25 0.5 <0.125 0.25 129 0.5 1 2 0.5
0.25 0.25 2 <0.125 0.5 0.5 <0.125 130 0.5 2 2 0.25 0.25 0.25
16 0.5 1 0.25 0.25 131 0.5 2 4 0.25 0.25 0.25 32 <0.125
<0.125 <0.125 <0.125 132 0.5 1 4 0.5 0.25 0.5 32 0.25
<0.125 0.25 <0.125 133 0.5 0.5 2 2 0.25 0.5 >32 0.25 0.25
0.5 0.25 134 0.5 1 1 2 0.5 2 >32 <0.125 <0.125 0.25
<0.125 135 0.5 1 2 4 1 1 4 0.25 <0.125 <0.125 <0.125
136 0.5 2 1 0.5 0.25 0.5 4 <0.125 0.25 <0.125 <0.125 137
0.5 1 4 0.5 0.25 0.25 1 <0.125 <0.125 <0.125 <0.125 138
1 2 1 1 0.5 0.5 2 0.5 0.5 0.5 1 139 1 2 1 0.5 0.25 0.25 <0.125
<0.125 1 0.5 0.25 140 1 1 4 2 1 0.5 0.25 0.25 1 0.5 0.25 141 1 1
2 16 4 4 >32 0.5 4 2 1 142 1 1 1 1 0.25 0.5 0.5 0.25 0.5 0.25 1
143 0.5 0.5 16 1 0.5 1 0.25 0.25 0.5 1 0.5 144 1 2 1 1 0.5 1 0.5 1
1 2 1 145 0.5 0.5 1 1 0.5 0.5 1 <0.125 <0.125 0.5 <0.125
146 0.5 0.5 >32 4 2 1 1 0.5 0.5 0.5 0.25 147 1 1 >32 4 2 4 4
0.5 1 <0.125 1 148 1 1 16 2 1 0.5 1 0.5 1 2 4 149 >32 >32
>32 >32 >32 >32 >32 16 >32 >32 >- 32
1/20.sup.# 0.5 0.5 >32 0.5 0.5 0.5 0.25 <0.125 0.25 <0.125
1 150 4 4 >32 0.25 0.25 0.25 8 0.5 0.5 0.5 0.5 151 4 4 >32
0.5 0.5 0.25 16 0.5 0.25 0.25 0.25 152 1 1 32 1 1 1 >32 0.5 0.25
0.5 0.5 153 0.5 4 >32 2 1 2 >32 0.25 0.5 0.25 0.25 Pa =
Pseudomonas aeruginosa, Ab = Acinetobacter baumannii, Kp =
Klebsiella pneumonia, Ec = Enterobacter cloacae, .sup.#1:1 ratio of
compounds 1 and 20.
As is evident from the above data, the exemplified compounds have
comparable or improved antibacterial efficacy against one or more
of the above Gram-negative bacterial isolates.
Example 3. In Vivo Efficacy in Mouse Blood Infection Model
P. aeruginosa ATCC 27853, A. baumannii ATCC 19606 and K. pneumoniae
FADDI-KP032 were subcultured on nutrient agar plates. One colony of
each bacterial strain was dispersed in 10-mL CAMHB and incubated
overnight. On day 2, an aliquot (0.2 mL) of each overnight culture
suspension was dispersed in 20-mL CAMHB and incubated for 1.5-2.5 h
for production of early log-phase growth bacterial culture. The
bacteria in the early log-phase growth suspension were concentrated
by centrifugation (3,220 g for 10 min) and re-suspended in sterile
0.9% saline for inoculation into mice. The bacterial cell
concentration (colony forming unit [CFU]/mL) in saline was
estimated by determining the optical density (OD) of the suspension
at 600 nm, and confirmed by plating the suspension on nutrient agar
plates. Swiss mice (22 to 28 g) were rendered neutropenic by
injecting two doses of cyclophosphamide intraperitoneally, -4 day
(150 mg/kg) and -1 day (100 mg/kg) prior to inoculation.
Bloodstream infection was established by injecting intravenously 50
.mu.L of early log-phase bacterial suspension (10.sup.8-10.sup.9
CFU/mL). The exact injection volume for each bacterial suspension
was calculated based upon the OD value of the bacterial suspension
and the desired inoculum for each isolate.
Solutions for administration of colistin, polymyxin B or the
compounds were prepared at a concentration of 1 mg (free base) per
mL in sterile 0.9% saline. At 2 h after inoculation, a mouse in the
treatment groups was injected intravenously with one of the above
solutions at 4 .mu.L/g body weight (BW) (i.e. free base 4 mg/kg
BW), while the same volume of saline was injected into the control
mice. At 0 h or at 4 h after the administration of antibacterial
drug or saline (control), animals were euthanised by inhalation of
overdose isoflurane. The skin on the chest and fore-paws of each
animal was thoroughly cleansed with 70% ethanol and Betadine.RTM..
The blood was collected via cardiac puncture using a 1-mL syringe
rinsed with heparin (5,000 IU/mL), diluted serially in sterile 0.9%
saline and plated on nutrient agar plates using a spiral
plater.
The agar plates were incubated at 37.degree. C. overnight. The
bacterial colonies on the plate were counted and CFU/mL of the
blood was calculated. The log.sub.10 CFU/mL of blood in each mouse
was calculated. The in vivo activity of the compounds against the
bacteria was calculated as the difference of the log.sub.10 CFU/mL
values between the treated mice and the control mice at 4 h
(.DELTA. log=log.sub.10 (treated)CFU/mL-log.sub.10
(control)CFU/mL). The results obtained are documented in Table
8.
Any compound showing a decrease in bacterial loading (.DELTA. log)
of .gtoreq.2 at 4 h is considered to have good in vivo efficacy in
this initial screening model. As is evident from the Table below,
the compounds of the invention have comparable or improved in vivo
antibacterial efficacy compared to the clinically available
polymyxin B (Reduction of bacterial loading for the polymyxin B
control used in the corresponding experiment is shown in brackets
next to the reduction in bacterial loading for the compound).
TABLE-US-00008 TABLE 8 In vivo efficacy in mouse blood infection
model P. aeruginosa A. baumannii K. pneumoniae ATCC 27853 ATCC
19606 FADDI-KP032 MIC .DELTA. log (Treated- MIC .DELTA. log
(Treated- MIC .DELTA. log (Treated- Compound (mg/L) Control at 4
h){circumflex over ( )} (mg/L) Control at 4 h){circumflex over ( )}
(mg/L) Control at 4 h).sup. 1 4 0.40 (-2.44) <0.125 -2.13
(-2.32) 0.5 -3.49 (-3.66) 2 8 -- 0.5 -2.70 (-2.58) 0.5 -3.44
(-3.15) 3 4 -- 0.25 -2.02 (-2.58) <0.125 -3.45 (-3.15) 4 4 -- 8
-- 1 -1.84 (-3.15) 5 2 -0.54 (-2.21) <0.125 -2.60 (-2.58)
<0.125 -3.74 (-3.15) 6 >32 -- 1 -2.35 (-2.58) 0.5 -- 7 -0.61
(-2.21) 0.25 -2.87 (-2.58) <0.125 -3.39 (-3.15) 8 4 -0.05
(-3.45) 2 -- 1 -- 18 8 -- 0.5 -1.57 (-1.79) 0.25 -- 19 32 -- 2
-1.58 (-1.79) 0.5 -- 20 0.5 -2.13 (-2.21) 4 -1.86 (-1.90) 0.25
-3.50 (-3.15) 21 0.5 -2.26 (-2.21) 1 -2.43 (-2.58) 0.25 -- 22 1
-3.10 (-2.21) 1 -1.50 (-2.58) 0.5 -3.11 (-3.15) 23 1 -2.48 (-2.21)
0.5 -2.82 (-2.58) <0.125 -3.88 (-3.15) 24 0.5 -2.31 (-2.21) 1
-2.16 (-2.58) <0.125 -3.21 (-3.15) 25 >32 8 0.27 (-2.58) 0.25
-2.65 (-3.15) 26 0.5 -0.04 (-3.45) 16 -- 2 -- 27 1 -3.67 (-3.45) 1
-- 0.5 -- 29 1 -2.80 (-3.45) 8 -- 1 -- 30 1 -0.39 (-3.45) 32 -- 1
-- 33 0.5 -1.55 (-2.83) 0.5 -1.40 (-2.08) <0.125 -2.80 (-3.00)
35 0.5 -3.36 (-3.74) 0.5 -1.60 (-1.57) <0.125 -3.12 (-3.00) 46
0.5 -2.53 (-3.91) 0.25 -1.56 (-1.57) <0.125 -2.93 (-2.64) 58 0.5
-3.00 (-2.83) 0.25 -2.15 (-1.86) 0.25 -3.21 (-3.13) 59 1 -2.82
(-2.87) 2 -- <0.125 -- 61 0.5 -4.75 (-3.91) 4 -- 0.25 -- 62 0.5
-2.69 (3.35) 2 -- 0.25 -- 70 1 -3.11 (-3.91) 0.5 -- 0.25 -- 71 0.5
-4.04 (-3.91) 0.5 -1.82 (-1.86) 0.5 -2.98 (-3.13) 74 0.5 -3.10
(-3.83) 0.5 -2.34 (-1.86) 0.25 -2.94 (-3.13) 77 0.25 -2.28 (-3.35)
4 -- <0.125 -- 79 0.5 -2.28 (-3.35) 1 -- <0.125 -- 82 0.5
-3.44 (-3.35) 0.5 -1.44 (-1.79) <0.125 -3.33 (-3.13) 83 1 -3.63
(-3.35) 2 -- 0.25 -- 88 0.5 -3.33 (-3.83) 2 -2.46 (-1.86) 0.5 -- 94
0.5 -3.23 (-3.83) 1 -1.26 (1.86) 0.25 -- 96 0.25 -3.45 (-3.83) 0.5
-2.42 (-1.86) <0.125 -2.75 (-3.13) 99 1 -3.21 (-3.83) 1 -- 0.5
-- 100 0.5 -3.83 (-3.83) 2 -- 0.5 -- 104 0.5 -3.08 (-2.99) 1 -1.35
(-1.86) 1 -3.23 (-3.13) 105 0.5 -2.54 (-2.99) 0.5 -1.31 (-1.86)
0.25 -- 106 0.5 -2.33 (-2.99) 0.5 -- 0.25 -2.77 (-3.13) 107 0.5
-2.98 (-2.99) 2 -- 0.25 -- 108 0.5 -2.32 (-2.99) 0.5 -1.82 (-1.79)
0.25 -2.84 (-3.13) 109 0.5 -2.68 (-2.99) 0.25 -1.54 (-1.86) 0.25
-3.47 (-3.13) 110 0.5 -3.30 (-2.99) 2 -1.57 (-1.79) 0.25 -2.50
(-3.13) 111 1 -3.65 (-2.99) 0.5 -1.66 (-1.79) 0.25 -- 112 0.5 -3.15
(-2.99) 0.25 -- 0.5 -- 113 0.5 -2.61 (-2.87) 0.5 -1.60 (-1.79) 0.25
-- 114 0.5 -2.57 (-2.87) 2 -- 0.25 -- 115 1 -2.71 (-2.87) 0.5 --
0.125 -- 116 0.5 -2.81 (-2.87) 0.5 -1.51 (-.179) 0.5 -- 117 0.25
-2.99 (-3.14) 1 -- 0.25 -- 118 0.5 -3.53 (-3.14) 2 -- 0.25 -- 119
0.5 -3.40 (-3.14) 1 -- <0.125 -- 120 1 -3.20 (-3.14) 1 -- 0.5 --
121 0.5 -2.81 (-3.14) 0.5 -- 0.25 -- 122 2 -3.70 (-3.14) 0.5 -1.58
(-1.79) 0.5 -- 123 0.5 -2.96 (-3.14) 1 -1.53 (-1.79) 0.5 -- 124
0.25 -3.36 (-3.14) 1 -- 0.25 -- 125 0.25 -3.66 (-3.14) 1 -- 0.25 --
126 0.25 -3.97 (-3.14) 1 -- 0.5 -2.68 (-3.13) 127 0.5 -3.51 (-3.59)
4 -- 0.5 -- 129 0.5 -3.45 (-3.59) 0.5 -1.42 (-1.26) <0.125 -3.64
(-3.24) 130 0.5 -3.66 (-3.59) 0.25 -- 0.5 -- 131 0.5 -3.80 (-3.59)
0.25 -1.75 (-1.26) <0.125 -3.44 (-3.24) 132 0.5 -4.39 (-3.59)
0.5 -1.58 (-1.26) 0.25 -3.16 (-3.24) 133 0.5 -3.23 (-3.59) 2 --
0.25 -- 138 1 -2.98 (-3.23) 1 -- 0.5 -- 139 1 -3.19 (-3.23) 0.5 --
<0.125 -- 140 1 -3.27 (-3.23) 2 -- 0.25 -- 142 1 -3.89 (-3.23) 1
-- 0.25 -- 143 0.5 -3.47 (-3.23) 1 -- 0.25 -- 144 1 -2.98 (-3.23) 1
-- 1 -- 146 0.5 -3.63 (-3.23) 4 -- 0.5 -- 1/20* -1.52 (-2.83) -1.86
(-2.08) -2.82 (-3.00) 150 4 -0.29 (-3.18) 0.25 -1.46 (-1.17) 0.5
-3.77 (-2.99) 151 4 -0.40 (-2.20) 0.5 -1.59 (-2.53) 0.5 -4.71
(-4.06) 152 1 -3.24 (-3.18) 1 -1.61 (-1.17) 0.5 -3.86 (-2.99) 153
0.5 -2.73 (-3.18) 2 -1.26 (-1.17) 0.25 -3.59 (-2.99) -- Not
determined .sup. The .DELTA. log (Treated-Control at 4 h) for the
polymyxin B control used in the corresponding experiment is shown
in brackets next to the .DELTA. log (Treated-Control at 4 h) for
each compound. *1:1 ratio of compound 1 and 20.
Example 4. Nephrotoxicity in a Mouse Model
PMB sulphate (Batch 20120204) and colistin sulphate (Batch
20120719) were supplied by Betapharma (Shanghai Co., Ltd, China).
Stock solutions of compounds in saline (5 mg base/mL) were stored
at 4.degree. C. before use. The mice were subcutaneously
administered with the drug/compound at 12 mg base/kg, 6 doses in
one day every 2 h. At .about.20 h after the last dose, mice were
euthanised by inhalation of an overdose of isoflurane. Immediately
after blood sampling, the right kidney from each mouse was
collected immediately and placed in 10% formalin in 5-mL plastic
tubes separately, and the left kidney placed in a pre-weighed in
14-mL plastic tubes, weighed again and stored at -20.degree. C.
pending for homogenization and analysis of polymyxin and colistin.
The frozen kidney samples were thawed, homogenized in 2 mL of
Milli-Q water and stored in a -20.degree. C. freezer. The
formalin-fixed kidneys were then sent to the Australian Phenomics
Network-Histopathology and Organ Pathology (The University of
Melbourne, Parkville, VIC, Australia) for histological examination.
The samples were examined by a pathologist who was blind to the
treatment groups.
Lesions were rated as follows: mild acute tubular damage with
tubular dilation, prominent nuclei and a few pale 45 tubular casts
(Grade 1); severe acute tubular damage with necrosis of tubular
epithelial cells and numerous tubular casts (Grade 2); acute
cortical necrosis/infarction of tubules and glomeruli with or
without papillary necrosis (Grade 3). The grades were given the
following scores: grade 1=1, grade 2=4, and grade 3=10. The
percentages of the kidney slices affected were scored as follows:
<1%=0, 1 to <5%=1, 5 to <10%=2, 10 to <20%=3, 20 to
<30%=4, 30 to <40%=5, and >40%=6. The overall kidney
histology score was calculated as the product of percentage score
and grade score. These scores were then expressed as a
semiquantitative score on a scale of 0 to +5 for renal histological
changes. These scores were assigned as follows: SQS 0=no
significant change (overall score, <1); SQS+1=mild damage
(overall score, 1 to <15); SQS+2=mild to moderate damage
(overall score, 15 to <30); SQS+3=moderate damage (overall
score, 30 to <45); SQS+4=moderate to severe damage (overall
score, 45 to <60); and SQS+5=severe damage (overall score,
>60) (Yousef, J., Chen, G., Hill, P., Nation, R., Li, J., 2011,
Antimicrobial Agents And Chemotherapy [P], vol 55, issue 9,
American Society for Microbiology, USA., pp. 4044-4049).
The results obtained are documented in Table 9. Any compound with a
kidney histology score of .ltoreq.+1.0 is considered to have a low
nephrotoxicity in this model.
TABLE-US-00009 TABLE 9 In vivo nephrotoxicity in a mouse model Max
Overall Kidney Max Kidney Compound* Histology Score Histology Score
Polymyxin B 60.0 +5 Colistin 60.0 +5 1 5.0 +1 2 3.0 +1 3 3.0 +1 5
6.0 +1 6 0.0 0 7 6.0 +1 9 0.0 0 11 0.1 0 16 0.0 0 17 6.0 +1 19 5.0
+1 20 0.0 0 21 0.0 0 22 0.2 0 23 0.2 0 24 3.0 +1 27 0.1 0 33 0.0 0
35 2.0 +1 36 0.1 0 37 0.1 0 42 0.0 0 43 0.1 0 44 0.0 0 46 4.0 +1 58
0.1 0 59 4.0 +1 60 1.0 +1 62 0.1 0 63 0.0 0 64 0.0 0 65 0.0 0 66
4.0 +1 70 2.0 +1 71 0.0 0 75 0.0 0 77 2.0 +1 78 0.0 0 79 5.0 +1 82
0.0 0 83 0.0 0 88 0.0 0 89 0.1 0 90 0.0 0 91 0.0 0 92 0.0 0 93 0.0
0 94 0.0 0 95 0.0 0 96 2.0 +1 100 0.0 0 103 0.1 0 104 0.0 0 105 2.0
+1 106 0.0 0 107 0.0 0 108 6.0 +1 109 0.0 0 110 0.0 0 111 6.0 +1
113 6.0 +1 116 0.0 0 120 6.0 +1 121 4.0 +1 123 0.0 0 124 3.0 +1 125
0.0 0 126 0.0 0 128 3.0 +1 129 4.0 +1 132 0.0 0 133 0.0 0 134 0.0 0
135 0.0 0 1/20.sup.# 0.0 0 150 24.0 +2 151 0.1 0 152 2.0 +1 153 0.0
0 .sup.#1:1 ration of compound 1 and 20.
As can be observed from the above data, colistin and polymyxin B
display severe nephrotoxicity in this model. On the other hand, the
compounds of the present invention displayed no significant
nephrotoxicity.
Throughout this specification and claims which follow, unless the
context requires otherwise, the word "comprise", and variations
such as "comprises" or "comprising", will be understood to imply
the inclusion of a stated integer or group of integers or steps but
not the exclusion of any other integer or group of integers.
The reference in this specification to any prior publication (or
information derived from it), or to any matter which is known, is
not, and should not be taken as an acknowledgment or admission or
any form of suggestion that that prior publication (or information
derived from it) or known matter forms part of the common general
knowledge in the field of endeavour to which this specification
relates.
* * * * *