U.S. patent application number 15/237258 was filed with the patent office on 2017-02-16 for antibacterials and/or modulators of biofilm formation and methods of using the same.
The applicant listed for this patent is INDIANA UNIVERSITY RESEARCH AND TECHNOLOGY CORPORATION. Invention is credited to Martin J. O'Donnell, Jack Geno Samaritoni, William L. Scott.
Application Number | 20170044097 15/237258 |
Document ID | / |
Family ID | 57994470 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170044097 |
Kind Code |
A1 |
Scott; William L. ; et
al. |
February 16, 2017 |
ANTIBACTERIALS AND/OR MODULATORS OF BIOFILM FORMATION AND METHODS
OF USING THE SAME
Abstract
Amides substituted with aromatic groups were synthesized and
some were purified to create enantiomer pure compounds. The
compounds were tested to determine their ability to inhibit the
growth of bacteria and the formation of biofilms created by
bacteria. Some of these compounds were found to be effective
antibacterials and to effectively inhibit the formation of
biofilms.
Inventors: |
Scott; William L.;
(Indianapolis, IN) ; O'Donnell; Martin J.;
(Indianapolis, IN) ; Samaritoni; Jack Geno; (Avon,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDIANA UNIVERSITY RESEARCH AND TECHNOLOGY CORPORATION |
Indianapolis |
IN |
US |
|
|
Family ID: |
57994470 |
Appl. No.: |
15/237258 |
Filed: |
August 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62205367 |
Aug 14, 2015 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 295/13 20130101;
C07C 229/34 20130101; C07D 213/40 20130101; C07C 237/20 20130101;
C07D 207/08 20130101; C07D 211/32 20130101 |
International
Class: |
C07C 237/20 20060101
C07C237/20; C07D 207/08 20060101 C07D207/08; C07D 211/32 20060101
C07D211/32; C07D 213/40 20060101 C07D213/40; C07D 295/13 20060101
C07D295/13 |
Goverment Interests
STATEMENT OF GOVERNMENTAL RIGHTS
[0002] This invention was made with government support under
GM028193 awarded by the National Institutes of Health and 1140602
awarded by the National Science Foundation. The government has
certain rights in the invention.
Claims
1. A compound, comprising formula: ##STR00051## wherein: R.sup.1 is
selected from the group consisting of: biphenyl,
--(CH.sub.2).sub.n-biphenyl, biphenyl ketone, naphthalene,
anthracene, benzyl optionally substituted with 1 to 3 halogens,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, --NH.sub.2,
--NO.sub.2, --CN, or --CF.sub.3, and phenyl optionally substituted
with 1 to 3 halogens, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, --NH.sub.2, --NO.sub.2, --CN, or --CF.sub.3; R.sup.2 is
selected from the group consisting of: --OH, --NH.sub.2,
--NHR.sup.3, and --NR.sup.3R.sup.4; R.sup.3 and R.sup.4 are
independently selected from the group consisting of: H, halogen,
--NH.sub.2, --NO.sub.2, C.sub.1-C.sub.6 alkyl being unbranched,
branched or cyclic, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
haloalkoxy, hydroxyl, acyl, acyl amides, carboxyl, tetrazolyl, and
--(CH.sub.2).sub.n--R.sup.5; alternatively, R.sup.3 and R.sup.4 are
taken together to form a pyridine, a piperidine, a pyrrolidine, or
a pyrrole ring; R.sup.5 is --OH, --CF.sub.3, morpholinyl,
pyridinyl, benzyl optionally substituted with 1 to 3 halogens,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, --NH.sub.2,
--NO.sub.2, --CN, or --CF.sub.3, or phenyl optionally substituted
with 1 to 3 halogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
--OH, --NH.sub.2, --NO.sub.2, --CN, or --CF.sub.3; and n is 1, 2,
3, or 4; or a pharmaceutically acceptable salt thereof, or a
metabolite thereof.
2. The compound according to claim 1, wherein: R.sup.1 is benzyl
substituted with 1 to 3 halogens, --NH.sub.2, --NO.sub.2, --CN, or
--CF.sub.3; and R.sup.2 is --OH or --NH.sub.2; or a
pharmaceutically acceptable salt thereof, or a metabolite
thereof.
3. The compound according to claim 1, wherein: R.sup.1 is benzyl
substituted with 1 to 3 halogens, --NH.sub.2, --NO.sub.2, --CN, or
--CF.sub.3; R.sup.2 is --NHR.sup.3; R.sup.3 is
--(CH.sub.2).sub.n--R.sup.5; R.sup.5 is --OH, --CF.sub.3,
morpholinyl, pyridinyl, or benzyl optionally substituted with 1 to
3 halogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, --OH,
--NH.sub.2, --NO.sub.2, --CN, or --CF.sub.3; and n is 1, 2, 3, or
4; or a pharmaceutically acceptable salt thereof, or a metabolite
thereof.
4. The compound according to claim 1, wherein the compound is at
least one enantiomer of at least one compound selected from the
group consisting of: ##STR00052## or a pharmaceutically acceptable
salt thereof, or a metabolite thereof.
5. The compound according to claim 1, wherein the compound is
selected from the group consisting of: ##STR00053## or a
pharmaceutically acceptable salt thereof, or a metabolite
thereof.
6. The compound according to claim 1, wherein the compound is
selected from the group consisting of: ##STR00054## ##STR00055## or
a pharmaceutically acceptable salt thereof, or a metabolite
thereof.
7. A method for reducing the growth of bacteria, comprising the
steps of: treating bacteria with at least one compound selected
from the compounds of claim 1.
8. The method according to claim 7, wherein the at least one
compound is selected from the group consisting of: ##STR00056##
##STR00057## or a pharmaceutically acceptable salt thereof, or a
metabolite thereof.
9. The method according to claim 7, wherein the bacteria is
gram-negative bacteria.
10. The method according to claim 7, wherein the bacteria is
Pseudomonas aeruginosa.
11. The method according to claim 7, further comprising the step
of: reducing biofilm formation of the bacteria.
12. The method according to claim 7, further comprising the step
of: treating an area that has been infected by the bacteria.
13. The method according to claim 12, wherein the area comprises
surfaces or hair of an animal, a human, or a plant.
14. A method of treating bacterial infections, comprising the steps
of: providing at least one therapeutically effective dose of at
least one compound selected from the compounds of claim 1 to a
patient.
15. The method according to claim 14, further comprising the step
of: diagnosing a patient with bacterial infections, wherein the
bacterial infections are caused by gram-negative bacteria.
16. The method according to claim 15, wherein the gram-negative
bacteria is Pseudomonas aeruginosa.
17. The method according to claim 14, wherein the at least one
compound is selected from the group consisting of: ##STR00058##
##STR00059## or a pharmaceutically acceptable salt thereof, or a
metabolite thereof.
18. The method according to claim 14, wherein the therapeutically
effective dose of the compound selected from the compounds of claim
1 is on the order of between about 1 mg/kg to about 7 mg/kg and the
dose of the compound is administered to the patient at least once
per day.
19. The method according to claim 18, wherein the therapeutically
effective dose of the compound selected from the compounds of claim
1 is on the order of between about 3 mg/kg to about 5 mg/kg and the
dose of the compound is administered to the patient at least once
per day.
20. The method according to claim 14, wherein the therapeutically
effective dose of the compound selected from the compounds of claim
1 is administered by intravenous or intramuscular injections.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of priority under 35
U.S.C. 119(e) of U.S. Provisional Application Ser. No. 62/205,367,
filed Aug. 14, 2015, the disclosure of which is hereby expressly
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates generally to compounds that can
inhibit the growth of bacterial and/or inhibit or reduce the
formation of biofilms formed by some types of bacteria and methods
of using these compounds to treat bacterial infection and outbreaks
and/or to reduce the formation of biofilms.
BACKGROUND AND SUMMARY
[0004] Bacterial infections may cause severe suffering and even
death. While virtually all humans and animals are susceptible to
bacterial infections certain sub-population are especially
vulnerable to such infections. Vulnerable populations of human
include the very young, the very old, persons with poorly developed
or weakened immune systems or patients that already have underlying
health challenges such as Cystic Fibrosis that make them
susceptible to certain bacterial infections. Since the introduction
of penicillin in the 1940s, antibiotics have served as one of the
major treatments for pathogenic bacterial infections.
[0005] Unfortunately not all bacteria are susceptible to treatment
with antibiotics. Classes of bacteria that are especially
pathogenic include bacteria that readily form biofilms. Some of the
uses of these biofilms in the pathogenic setting include helping to
anchor bacteria to a portion of the body and to help the bacteria
existing in conjunction with the biofilm to evade the body's immune
system. Moreover, the widespread use of broad spectrum antibiotics
has helped to give rise to antibiotic resistant strains of
pathogenic bacteria which were once easily controlled with such
compounds. Accordingly, there exists a need for compounds that have
the ability to stop or at least slow the growth of pathogenic
bacteria.
[0006] Biofilms are present everywhere, on man-made structures, in
nature, and even in the mouth of an animal or a human. A biofilm is
a community of bacteria which adhere to a solid surface and other
bacteria. Biofilms allow bacteria to remain in a favorable niche,
act as a defense, and allow bacterial cells to live in close
association with each other (increasing genetic exchange).
[0007] The biofilm itself is composed of polysaccharides, proteins,
various signaling molecules, and amino acids, all produced by the
bacterial cells within. Bacteria begin forming the biofilms in the
presence of valuable nutrient resources and begin to rapidly grow
in size and population. Biofilms form in three stages: attachment,
colonization, and development. During attachment, a few cells
adhere to a solid surface. Adhering to the solid surface causes the
cell to express biofilm-specific genes. Next, the biofilm begins
colonization. In this stage, intercellular communication is
essential, along with bacterial multiplication and polysaccharide
secretion. Lastly, the biofilm matures into a large structure with
numerous bacteria encased in a matrix of polysaccharide, DNA, and
protein. Biofilms can trap nutrients for the microbial population
and prevent detachment of the population in flowing systems, such
as the saliva running across your teeth or water flowing down a
stream. As the biofilm ages, and nutrients become depleted,
dispersal of the bacterial cells within the biofilm allows them to
spread to other resource rich areas.
[0008] Although many biofilms are relatively harmless, some can be
very malicious and cause serious illnesses. They have been
associated with urinary tract infections, ear infections, and
colonization of implanted medical devices. For example, one culprit
which produces biofilms is Pseudomonas aeruginosa (PA). PA is
particularly dangerous to those suffering from cystic fibrosis.
Cystic fibrosis is a recessively inherited genetic disease caused
by a mutation in the gene which codes for the protein cystic
fibrosis transmembrane conductance regulator (CFTR). The
malfunction of CFTR leads to increased mucus accumulation in the
lungs and other organs, which is an ideal medium for P. aeruginosa
and other bacteria to colonize into biofilms.
[0009] The study of biofilms has afforded a new strategy in
combating virulent bacteria, such as P. aeruginosa. Instead of
formulating drugs to kill harmful bacteria, biofilms can be treated
with drugs which signal the bacteria to leave the biofilm and
disperse or reduce biofilm formation. Causing the bacteria to
disperse comes with the benefit of reducing them to a benign and
defenseless state, allowing the body's own immune system to destroy
the invaders without side effects from drugs and without inducing
antibiotic resistance in the bacteria. Accordingly, the ability of
a drug to inhibit or reduce formation of biofilms of bacteria
provides an excellent indication that the drug will likely inhibit
or reduce the growth of the bacteria.
[0010] For more information about biofilm composition and
development, see Flemming, H. C. et al., The Biofilm Matrix. NATURE
REVIEWS MICROBIOLOGY 2010, 8, 623-633, Hall-Stoodley, L. et al.,
Bacterial Biofilms: From the Natural Environment to Infectious
Diseases. NATURE REVIEWS MICROBIOLOGY 2004, 2, 95-108;
Kolodkin-Gal, I. et al., D-Amino Acids Trigger Biofilm Disassembly.
SCIENCE 2010 328, 627-629, disclosures of which are incorporated by
reference in its entirety to the extent that they are not
inconsistent with the explicit teachings of this specification.
[0011] Aspects of the instant invention include compounds that
inhibit or reduce the growth of bacteria including certain
pathogenic bacteria some of these compounds also reduce or inhibit
the development of biofilms that may contribute to the pathology of
certain strains of bacteria. These compounds include amide
derivatives of fluorinated phenyl groups, in some instances
specific enantiomers of such compounds are especially effective
anti-bacterial agents. One such compound is an amide derivative of
4-fluorophenylalanine; this and related compounds exhibit the
ability stop or at least slow the growth of Pseudomonas aeruginosa,
a pathogen known to be responsible for severe bacterial infection
in patient who has Cystic Fibrosis.
[0012] Some aspects of the invention include methods of
synthesizing such compounds and using the same to treat bacterial
infections and to eliminate or at least reduce the formation of
biofilms associated with the growth of certain types of
bacteria.
[0013] A first embodiments of the present disclosure includes at
least one compound of the following Formula or a pharmaceutically
acceptable salt thereof, or a metabolite thereof:
##STR00001##
[0014] wherein R.sup.1 is selected from the group consisting of:
biphenyl, --(CH.sub.2).sub.n-biphenyl, biphenyl ketone,
naphthalene, anthracene, benzyl, benzyl substituted with 1 to 3
halogens, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
--NH.sub.2, --NO.sub.2, --CN, or --CF.sub.3, phenyl, and phenyl
substituted with 1 to 3 halogens, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, --NH.sub.2, --NO.sub.2, --CN, or
--CF.sub.3;
[0015] R.sup.2 is selected from the group consisting of: --OH,
--NH.sub.2, --NHR.sup.3, and --NR.sup.3R.sup.4;
[0016] R.sup.3 and R.sup.4 are independently selected from the
group consisting of: H, halogen, --NH.sub.2, --NO.sub.2,
C.sub.1-C.sub.6 alkyl being unbranched, branched or cyclic,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 haloalkoxy, hydroxyl, acyl,
acyl amides, carboxyl, tetrazolyl, and
--(CH.sub.2).sub.n--R.sup.5;
[0017] Alternatively, R.sup.3 and R.sup.4 are taken together to
form a pyridine, a piperidine, a pyrrolidine, or a pyrrole
ring;
[0018] R.sup.5 is --OH, --CF.sub.3, morpholinyl, pyridinyl, benzyl,
benzyl substituted with 1 to 3 halogens, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, --NH.sub.2, --NO.sub.2, --CN, or
--CF.sub.3, phenyl, or phenyl optionally substituted with 1 to 3
halogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, --OH,
--NH.sub.2, --NO.sub.2, --CN, or --CF.sub.3; and
[0019] n is 1, 2, 3, or 4.
[0020] A second embodiment includes the compound of the first
embodiment, wherein: R.sup.1 is benzyl substituted with 1 to 3
halogens, --NH.sub.2, --NO.sub.2, --CN, or --CF.sub.3; and R.sup.2
is --OH or --NH.sub.2; or a pharmaceutically acceptable salt
thereof, or a metabolite thereof.
[0021] A third embodiment includes the compound of the first and
the second embodiments, wherein: R.sup.1 is benzyl substituted with
1 to 3 halogens, --NH.sub.2, --NO.sub.2, --CN, or --CF.sub.3;
R.sup.2 is --NHR.sup.3; R.sup.3 is --(CH.sub.2).sub.n--R.sup.5;
R.sup.5 is --OH, --CF.sub.3, morpholinyl, pyridinyl, or benzyl
optionally substituted with 1 to 3 halogen, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, --OH, --NH.sub.2, --NO.sub.2, --CN, or
--CF.sub.3; and n is 1, 2, 3, or 4; or a pharmaceutically
acceptable salt thereof, or a metabolite thereof.
[0022] A fourth embodiment includes the compound of any of the
first to the third embodiments, wherein the compound is at least
one enantiomer of at least one compound selected from the group
consisting of:
##STR00002##
or a pharmaceutically acceptable salt thereof, or a metabolite
thereof.
[0023] A fifth embodiment includes the compound of any of the first
to the third embodiments, wherein the compound is selected from the
group consisting of:
##STR00003##
or a pharmaceutically acceptable salt thereof, or a metabolite
thereof.
[0024] A sixth embodiment includes the compound of any of the first
to the third embodiments, wherein the compound is selected from the
group consisting of:
##STR00004## ##STR00005##
or a pharmaceutically acceptable salt thereof, or a metabolite
thereof.
[0025] A seventh embodiment includes a method for reducing the
growth of bacteria, comprising the steps of: treating bacteria with
at least one compound selected from the compounds of the first to
the third and the fifth to the sixth embodiments.
[0026] An eighth embodiment includes the method of the seventh
embodiment, wherein the at least one compound is selected from the
group consisting of:
##STR00006## ##STR00007##
or a pharmaceutically acceptable salt thereof, or a metabolite
thereof.
[0027] A ninth embodiment includes at least one method according to
any of the seventh and the eighth embodiments, wherein the bacteria
are gram-negative bacteria.
[0028] A tenth embodiment includes at least one method according to
any of the seventh to the ninth embodiments, wherein the bacteria
is Pseudomonas aeruginosa.
[0029] An eleventh embodiment includes at least one method
according to any of the seventh to the tenth embodiments, further
comprising the step of: reducing biofilm formation of the
bacteria.
[0030] A twelfth embodiment includes at least one method according
to any of the seventh to the eleventh embodiments, further
comprising the step of: treating an area that has been infected by
the bacteria.
[0031] A thirteenth embodiment includes at least one method
according to the twelfth embodiment, wherein the area comprises wet
or dry wet or dry surfaces including the skin, hair or fur of
animals and/or humans, or the outside of seeds or plants including
leaves, stems, shoots, roots, branches, blooms, fruits and the
like, or any inanimate object surfaces.
[0032] A fourteenth embodiment includes a method of treating
bacterial infections, comprising the steps of: providing at least
one therapeutically effective dose of at least one compound
selected from the compounds of the first to the third and/or the
compounds of the fifth to the sixth embodiments to a patient.
[0033] A fifteenth embodiment includes the method according to the
fourteenth embodiment, further comprising the step of: diagnosing a
patient with bacterial infections, wherein the bacterial infections
are caused by gram-negative bacteria.
[0034] A sixteenth embodiment includes the method according to the
fifteenth embodiment, wherein the gram-negative bacteria is
Pseudomonas aeruginosa.
[0035] A seventeenth embodiment includes at least one method
according to any one of the fourteenth to the sixteenth
embodiments, wherein the at least one compound is selected from the
group consisting of:
##STR00008## ##STR00009##
or a pharmaceutically acceptable salt thereof, or a metabolite
thereof.
[0036] An eighteenth embodiment includes at least one method
according to any one of the fourteenth to the seventeenth
embodiments, wherein the therapeutically effective dose of the
compound selected from the compounds of claim 1 is on the order of
between about 1 mg/kg to about 7 mg/kg and the dose of the compound
is administered to the patient at least once per day.
[0037] A nineteenth embodiment includes the eighteenth embodiment,
wherein the therapeutically effective dose of the compound selected
from the compounds of claim 1 is on the order of between about 3
mg/kg to about 5 mg/kg and the dose of the compound is administered
to the patient at least once per day.
[0038] A twentieth embodiment includes at least one method
according to any one of the fourteenth to the nineteenth
embodiments, wherein the therapeutically effective dose of the
compound selected from the compounds of the first to the third
and/or the compounds of the fifth to the sixth embodiments is
administered by intravenous or intramuscular injections.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1. Various compounds and their corresponding chemical
structures.
[0040] FIG. 2. A list showing a structure index for some 4-F-Phe
and 2-F-Phe analogs.
[0041] FIG. 3. Schematic diagram illustrating a reproducible P.
aeruginosa Biofilm Assay.
[0042] FIG. 4. Photographs showing the effects of some compounds on
the growth of P. aeruginosa as indicated by biofilm formation.
Gentamicin (Gent) and Tobramycin (Tob) were used as positive
controls.
[0043] FIG. 5. Photographs showing the effects of
4-fluorophenylalanine (4-F; red box) the growth of P. aeruginosa as
indicated by biofilm formation.
[0044] FIG. 6. Photographs showing the effects of some separately
synthesized crude lots of 4-fluorophenylalanine (1a-1d; red box) on
the growth of P. aeruginosa as indicated by biofilm formation.
[0045] FIG. 7. Photographs showing the effects of Gentamicin (Gent;
red box) and Tobramycin (Tob; red box) on the growth of P.
aeruginosa as indicated by biofilm formation.
[0046] FIG. 8. Photographs showing the effects of bacteria alone
(PA14; red box) on the growth of P. aeruginosa as indicated by
biofilm formation.
[0047] FIG. 9. Photographs showing the effects of media alone (M63;
red box) on the growth of P. aeruginosa as indicated by biofilm
formation.
[0048] FIG. 10. Photographs showing the effects of separate lots of
either crude 2-fluorophenylalanine (2-F, lots 02a, 02b, 02c, 02d,
and 02e; red box) or crude 3-fluorophenylalanine (3-F, lots 031,
03b, or 03c; red box) on the growth of P. aeruginosa as indicated
by biofilm formation.
[0049] FIG. 11. Photographs showing the effects of separate lots of
3,4-difluorophenylalanine (3,4-F2; red box), lot 08a of
3-bromophenylalanine (3-Br; red box), and lot 03a of
3-fluorophenylalanine (3-F; red box) on the growth of P. aeruginosa
as indicated by biofilm formation.
[0050] FIG. 12. Graph summarizing the effects of some compounds on
the growth of P. aeruginosa as indicated by biofilm formation.
[0051] FIG. 13. Graphs illustrating the effects of some compounds
on the growth of P. aeruginosa as indicated by biofilm formation
determine in microtiter plates.
[0052] FIG. 14. Graphs illustrating the effects of some compounds
on the growth of P. aeruginosa with biofilm formation determined in
microtiter plates.
[0053] FIG. 15. Graph illustrating the effects of some compounds on
the growth of P. aeruginosa with biofilm formation determine in
microtiter plates.
[0054] FIG. 16. Graph summarizing the effects of some compounds on
the growth of P. aeruginosa as indicated by biofilm formation.
[0055] FIG. 17. Graphs illustrating the effects of some compounds
on the growth of P. aeruginosa with biofilm formation determined in
microtiter plates.
[0056] FIG. 18. Graphs illustrating the effects of some compounds
on the growth of P. aeruginosa with biofilm formation determined in
microtiter plates.
[0057] FIG. 19. Graph illustrating the effects of some compounds on
the growth of P. aeruginosa with biofilm formation determined in
microtiter plates.
[0058] FIG. 20. Graphs illustrating the effects of some
mono-fluorinated lots, mono-brominated lots, mono-iodinated lots,
and mono-chlorinated lots on the growth of P. aeruginosa as
indicated by biofilm formation.
[0059] FIG. 21. Graphs illustrating the effects of some
di-fluorinated lots, tri-fluorinated lots, di-aryl lots on the
growth of P. aeruginosa as indicated by biofilm formation.
[0060] FIG. 22. Graph illustrating the dose dependent effects of
4-fluorophenylalanine (4-F), 3,4-difluorophenylalanine (3,4 diF),
and amide on the growth of P. aeruginosa as indicated by biofilm
formation.
[0061] FIG. 23. Graph summarizing the effects of some compounds on
the growth of P. aeruginosa as indicated by biofilm formation.
[0062] FIG. 24. Photographs of microtiter plates showing the
effects of 4-fluorophenylalanine and Tobramycin on the growth of P.
aeruginosa as indicated by biofilm formation, and a graph
illustrating the effects of 4-fluorophenylalanine and Tobramycin on
the growth of P. aeruginosa as indicated by biofilm formation.
[0063] FIG. 25. Graphs illustrating the existence of individual
4-fluorophenylalanine (4-F-Phe) enantiomer and the effects of
enantiomer A and B on the growth of P. aeruginosa as indicated by
biofilm formation.
[0064] FIG. 26. Graph illustrating the dose dependent effects of
4-fluorophenylalanine (4-F-Phe) and 3-fluorophenylalanine (3-F-Phe)
on the growth of P. aeruginosa as indicated by biofilm
formation.
[0065] FIG. 27. Graph illustrating the dose dependent effects of
2-fluorophenylalanine (2-F-Phe) and 3,4-difluorophenylalanine
(3,4-diF-Phe) on the growth of P. aeruginosa as indicated by
biofilm formation.
[0066] FIG. 28. Graph summarizing the dose dependent effects of
4-fluorophenylalanine (4-F-Phe), 3-fluorophenylalanine (3-F-Phe),
2-fluorophenylalanine (2-F-Phe), 3,4-difluorophenylalanine
(3,4-diF-Phe), 4-fluorophenylalanine amide on the growth of P.
aeruginosa as indicated by biofilm formation.
[0067] FIG. 29. Graph illustrating separation of D and L forms of
racemic 4-fluoropheylalamine (4-F-Phe). Mass of D form of racemic
4-fluoropheylalamine (D-4-F-Phe): 33.1 .mu.g (amount injected: 34.5
.mu.g); Mass of L form of racemic 4-fluoropheylalamine (L-4-F-Phe):
30.4 .mu.g (amount injected: 34.5 .mu.g).
[0068] FIG. 30. Graph illustrating an enantiopure D-4-F-Phe.
[0069] FIG. 31. Graph illustrating an enantiopure L-4-F-Phe.
[0070] FIG. 32. Exemplary structures of some primary amides and
N-methyl amides.
[0071] FIG. 33. Exemplary structures of some primary amides and
N-methyl amides.
[0072] FIG. 34. Graph summarizing the effects of 2-, 3-, 4-, and
3,4-substituted phenylalanine primary amides on the growth of P.
aeruginosa as indicated by biofilm formation. Results were
expressed as % biofilm formation (control=bacteria alone=100%
biofilm formation).
[0073] FIG. 35. Graph summarizing the effects of some 2-, 3-, 4-,
and 3,4-substituted phenylalanine N-methyl amides on the growth of
P. aeruginosa as indicated by biofilm formation. Results were
expressed as % biofilm formation (control=bacteria alone=100%
biofilm formation).
[0074] FIG. 36. Graph summarizing the dose dependent effects of
some secondary amides on the growth of P. aeruginosa as indicated
by biofilm formation. Results are expressed as % biofilm formation
(control=bacteria alone=100% biofilm formation).
[0075] FIG. 37. Graph summarizing the dose dependent effects of
some secondary amides on the growth of P. aeruginosa as indicated
by biofilm formation. Results were expressed as % biofilm formation
(control=bacteria alone=100% biofilm formation).
[0076] FIG. 38. Graph summarizing the dose dependent effects of
some secondary and tertiary amides on the growth of P. aeruginosa
as indicated by biofilm formation. Results were expressed as %
biofilm formation (control=bacteria alone=100% biofilm
formation).
DESCRIPTION
[0077] For the purposes of promoting an understanding of the
principles of the novel technology, reference will now be made to
the preferred embodiments thereof, and specific language will be
used to describe the same. It will nevertheless be understood that
no limitation of the scope of the novel technology is thereby
intended, such alterations, modifications, and further applications
of the principles of the novel technology being contemplated as
would normally occur to one skilled in the art to which the novel
technology relates are within the scope of this disclosure and the
claims.
[0078] As used herein, unless explicitly stated otherwise or
clearly implied otherwise the term `about` refers to a range of
values plus or minus 10 percent, e.g. about 1.0 encompasses values
from 0.9 to 1.1.
[0079] As used herein, unless explicitly stated otherwise or
clearly implied otherwise the meaning of the terms "treatment" or
"treating" used in conjunction with various compounds and methods
disclosed and claimed herein include, but are not limited to,
applying certain compounds to various either wet or dry surfaces
including, but not limited to the skin, hair or fur of animals or
the outside of seeds or plants including leaves, stems, shoots,
roots, branches, blooms, fruits and the like, or any inanimate
object either directly or indirectly. The terms "treatment" or
"treating" also includes adding compounds to liquids, either
aqueous or non-aqueous or mixtures thereof including simple
mixtures of such or emulsions. The terms "treatment" or "treating"
used in also include administering compounds to plants, cells,
animals and humans. The terms "treatment" or "treating" include but
are not limited to contacting bacteria or the biofilms of bacteria,
directly or indirectly and may affect the growth of bacteria, and
either directly or indirectly the formation of bacteria biofilms.
The terms "treatment" or "treating" as used herein may include
administering a `therapeutically effective dose or doses of
compounds.
[0080] As used herein, unless explicitly stated otherwise or
clearly implied otherwise the terms `therapeutically effective
dose,` `therapeutically effective amounts,` and the like, refers to
a portion of a compound that has a net positive effect on the
health and wellbeing of a human or other animal. Therapeutic
effects may include an improvement in longevity, quality of life
and the like these effects also may also include a reduced
susceptibility to developing disease or deteriorating health or
well being. The effects may be immediately realized after a single
dose and/or treatment or they may be cumulatively realized after a
series of doses and/or treatments.
[0081] Pharmaceutically acceptable salts include salts of compounds
of the invention that are safe and effective for use in mammals and
that possess a desired therapeutic activity. Pharmaceutically
acceptable salts include salts of acidic or basic groups present in
compounds of the invention. Pharmaceutically acceptable acid
addition salts include, but are not limited to, hydrochloride,
hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate,
acid phosphate, isonicotinate, acetate, lactate, salicylate,
citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate,
maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,
formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzensulfonate, p-toluenesulfonate, trifluoroacetic acid, and
pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts.
Certain compounds of the invention may form pharmaceutically
acceptable salts with various amino acids. Suitable base salts
include, but are not limited to, aluminum, calcium, lithium,
magnesium, potassium, sodium, zinc, and diethanolamine salts. For
addition information on some pharmaceutically acceptable salts that
can be used to practice the invention please reviews such as Berge,
et al., 66 J. PHARM. SCI. 1-19 (1977), and Haynes, et at, J.
Pharma. Sci., Vol. 94, No. 10, October 2005, pgs. 2111-2120.
[0082] Bacteria are known to communicate via small molecules.
Through this communication they are able to create complex,
highly-organized communities responsible for biofilm formation,
antibiotic resistance, and other important processes. These
biofilms are involved in many disease states, including cystic
fibrosis, which is a genetically inherited disease affecting
approximately 70,000 people worldwide. Dr. Richard Losick and
colleagues at Harvard have reported that certain D-amino acids (1,
R.sup.1=naturally occurring amino acid side chains in the D
configuration) are a trigger for the disassembly of bacterial
biofilms (Science, 2010, 328, 627-629). D-Tyrosine was reported to
be particularly active:
##STR00010##
[0083] Using the synthetic procedure discussed herein compounds
such as 1 and 2 were
##STR00011##
wherein R.sup.1 is selected from the group consisting of a benzyl
group optionally substituted with a halogen, methyl group, or a
nitro group, a bi-phenyl, a naphthalene, and an anthracene.
Experiments and Results
##STR00012##
[0085] One aspect of this approach uses D3-6 (Scheme 1) developed
for our Distributed Drug Discovery (D3) project to synthesize
analogs of compound 1. This approach employs solid-phase chemistry,
Bill-Board equipment, and alkylating agents R.sup.1X to synthesize
a small "library" of unnatural amino acids 1.
[0086] A straightforward biological assay was developed to provide
for the study these important bacterial biofilm communities. This
assay also provided an efficient means for screening for compounds
that have an effect on bacterial growth and/or biofilm
formation.
[0087] The racemic amino acid derivatives 1 (synthesized in row A
of each Bill-Board) are then screened for biological activity
(antibacterial activity in a Kirby-Bauer disc plate assay) and/or
K357 laboratory (assay for biofilm formation by Pseudomonas
aeruginosa).
##STR00013##
[0088] Fmoc-Gly-Wang resin (15.725 g, 12.108 mmol) was swelled for
30 minutes with 100 mL of NMP in a 250-mL solid-phase peptide
synthesis vessel. The vessel was drained and the resin was treated
with 70 mL of 20% piperidine in NMP for 2 minutes. The vessel was
drained and the resin was treated with 120 mL of 20% piperidine and
was rocked for 45 minutes on an orbital shaker. The vessel was
drained, the resin was washed with 6.times.75 mL.times.2 min NMP.
The deprotected resin was then treated with 20.32 mL of
benzophenone imine in 110 mL of NMP followed by 6.02 mL of acetic
acid. The vessel was rocked overnight at room temperature. After 22
h the vessel was drained and the resin was washed with 4.times.75
mL.times.2 min NMP and dichloromethane each. The resin 1 was dried
under a slow stream of dry nitrogen gas for approximately 30 h and
was then stored at 2.degree. C.
[0089] 50 .mu.mols of resin 1 was treated with 100 .mu.mols of 0.20
M BTPP in NMP followed by 100 .mu.mols of 0.20 M fluorinated benzyl
bromide R.sup.1X in NMP. After two days the reaction mixture was
filtered and the resulting resin 2 was washed once with 3 mL of
THF. To the resin was then added 2.5 mL of 1.0 N HCl in THF (1:2).
After 20 minutes the resin was filtered and was washed with 3 mL of
THF followed by 2.times.2.5 mL of 0.20 M diisopropylethylamine in
NMP, 2.times.2.5 mL of NMP, 3.times.2 mL of THF and 3.times.3 mL of
dichloromethane to give resin 3. Treatment of resin 3 with 2 mL of
35:60:5 TFA/DCM/H.sub.2O for 30 minutes was followed by filtering
and washing the resin with 2 mL of 35:60:5 TFA/DCM/H.sub.2O and 2
mL of DCM. The combined filtrates were evaporated to a residue
which was chromatographed on silica gel using iPrOH/MeOH/NH.sub.4OH
mobile phases to elute the free bases 4.
##STR00014##
2-Amino-3-(4-fluorophenyl)propanoic acid (4a)
[0090] 6.9 mg (75%), .sup.1HNMR (D.sub.2O) .delta. 3.04 (dd, 1H,
J=14.7 and 7.8 Hz), 3.18 (dd, 1H, J=14.6 and 5.3 Hz), 3.89 (dd, 1H,
J=7.8 and 5.3 Hz), 7.06 (t, 2H, J=8.9 Hz), 7.21 (m, 2H).
##STR00015##
2-Amino-3-(2-fluorophenyl)propanoic acid (4b)
[0091] 9.1 mg (99%), .sup.1HNMR (D.sub.2O) .delta. 3.08 (dd, 1H,
J=14.6 and 7.9 Hz), 3.30 (dd, 1H, J=14.6 and 5.4 Hz), 3.94 (t, 1H,
J=6.6 Hz), 7.11-7.17 (m, 2H), 7.26-7.36 (m, 2H).
##STR00016##
2-Amino-3-(3-fluorophenyl)propanoic acid (4c)
[0092] 7.5 mg (82%), .sup.1HNMR (D.sub.2O) .delta. 3.03 (dd, 1H,
J=14.5 and 7.9 Hz), 3.18 (dd, 1H, J=14.6 and 5.3 Hz), 3.89 (dd, 1H,
J=7.9 and 5.4 Hz), 6.96-7.00 (m, 2H), 7.02 (d, 1H, J=7.5 Hz),
7.28-7.32 (m, 1H).
##STR00017##
2-Amino-3-(3,4-difluorophenyl)propanoic acid (4d)
[0093] 4.0 mg (40%), .sup.1HNMR (D.sub.2O) .delta. 3.03 (dd, 1H,
J=14.6 and 7.7 Hz), 3.16 (dd, 1H, J=14.4 and 5.1 Hz), 3.88 (t, 1H,
J=6.5 Hz), 6.99-7.01 (br m, 1H), 7.11-7.21 (m, 2H).
##STR00018##
[0094] A 50-mL round-bottom flask sealed with a rubber septum and
charged with 501 mg of 10 in 5 mL of dichloromethane under dry
argon gas was treated dropwise via syringe 1.08 mL of
3,4-difluorobenzyl bromide. The contents were cooled to -78.degree.
C. and 0.734 mL of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphor-
ine (BEMP) was added dropwise via syringe over 10 min. The solution
was stirred at -78.degree. C. for 8 h and was then stored at
-20.degree. C. overnight. The solution was concentrated to an oil
which was chromatographed on silica gel using hexanes to remove the
excess benzyl bromide and 95/5 hexanes/ethyl acetate to elute 11d
(575 mg, 80%). Imine 11d (572 mg) was heated under reflux in 8 mL
of 6N HCl for 6 h and was allowed to cool. The solution was washed
with 2.times.5 mL of diethyl ether (106 mg of an insoluble solid
was collected and later identified as the hydrochloride salt of the
desired product). The solution was then transferred to a 200-mL rb
flask and was concentrated to dryness in vacuo. Approximately 5 mL
of boiling ethanol was used to transfer the residue to a 25-mL
Erlenmeyer flask as a solution. Upon cooling the solution was
treated with 400 uL of propylene oxide and was allowed to stand.
The precipitate was collected by filtration and dried in vacuo to
afford 104 mg (38%) of 4d.
##STR00019##
[0095] A 50-mL round-bottom flask was charged with 500.4 mg of
protected amino acid 6 and 10 mL of THF. To the solution was added
204.1 mg of N-hydroxysuccinimide. The solution was chilled in an
ice bath and 367 mg of dicyclohexylcarbodiimide was added. The
mixture was stirred at 0-5.degree. C. for 4 h and then was allowed
to warm to room temperature and stand overnight. The mixture was
chilled in an ice bath, was filtered, and the filtrate was
concentrated to give 7 quantitatively as a white solid. Compound 7
(20-30 mg) was dissolved in 1 mL of THF and was treated with 1.2
equivalents of the benzylamine (X=H, Cl, or OMe) and the mixture
was allowed to stand overnight. The reaction mixture was then
filtered and the filtrate was evaporated to dryness. The residue
was then chromatographed on 500 mg of silica gel using a step
gradient as follows to give 8; dichloromethane, 99/1, 98/2, 97/3,
96/4, and 95/5 DCM/MeOH. Compound 8 was then treated with 500-700
uL of 1.0 M HCl in acetic acid and 50-70 uL of anisole and the
solution was stirred for 1.5-2 h. The solution was added dropwise
to 5 mL of cold, saturated potassium carbonate (pH9) and was
extracted two times with 15 mL of diethyl ether. The combined
extracts were washed once with 5 mL of potassium carbonate and once
with brine and were dried over sodium sulfate. Concentration gave
crude amides 9 which were chromatographed on 500 mg of silica gel
using a step gradient as follows; dichloromethane, 99/1, 98/2,
97/3, 96/4, and 95/5 DCM/MeOH.
##STR00020##
2-Amino-N-(4-chlorobenzyl)-3-(4-fluorophenyl)propanamide (9a)
[0096] 5.7 mg (53%), .sup.1HNMR (CDCl.sub.3) .delta. 2.79 (dd, 1H,
J=13.8 and 8.7 Hz), 3.21 (dd, 1H, J=13.9 and 4.2 Hz), 3.64 (dd, 1H,
J=8.3 and 4.0 Hz), 4.38 (dd, 1H, J=14.7 and 6.1 Hz), 4.42 (dd, 1H,
J=14.9 and 6.1 Hz), 6.69 (t, 2H, J=8.6 Hz), 7.15-7.18 (m, 4H), 7.29
(d, 2H, J=8.4 Hz), 7.59 (br s, 1H).
##STR00021##
2-Amino-3-(4-fluorophenyl)-N-(4-methoxybenzyl)propanamide (9b)
[0097] 12.7 mg (67%), .sup.1HNMR (CDCl.sub.3) .delta. 2.78 (dd, 1H,
J=13.9 and 8.8 Hz), 3.21 (dd, 1H, J=13.9 and 4.2 Hz), 3.61 (dd, 1H,
J=8.6 and 4.2 Hz), 3.80 (s, 3H), 4.33 (dd, 1H, J=14.5 and 5.8 Hz),
4.40 (dd, 1H, J=14.5 and 5.9 Hz), 6.85 (d, 2H, J=8.7 Hz), 6.98 (t,
2H, J=8.6 Hz), 7.14-7.18 (m, 4H), 7.16 br s, 1H).
##STR00022##
2-Amino-N-benzyl-3-(4-fluorophenyl)propanamide (9c)
[0098] 10.3 mg, (44%), .sup.1HNMR (CDCl.sub.3) .delta. 2.80 (dd,
1H, J=13.8 and 8.7 Hz), 3.22 (dd, 1H, J=13.8 and 4.1 Hz), 3.64 (br
s, 1H), 4.41 (dd, 1H, J=14.8 and 5.8 Hz), 4.46 (dd, 1H, J=14.8 and
6.0 Hz), 6.98 (t, 2H, J=8.7 Hz), 7.16-7.18 (m, 2H), 7.22 (d, 2H,
J=7.0 Hz), 7.27 (t, 1H, J=7.2 Hz), 7.32 (t, 2H, J=6.9 Hz), 7.54 (br
s, 1H).
##STR00023##
[0099] Compound 7 (20-30 mg) was dissolved in 1 mL of THF and was
treated with 3.0 equivalents of isopropylamine or isobutylamine and
the mixture was allowed to stand overnight. The reaction mixture
was evaporated to dryness. The residue was then chromatographed on
500 mg of silica gel using a step gradient as follows;
dichloromethane, 99/1, 98/2, 97/3, 96/4, and 95/5 DCM/MeOH.
Compounds 14 were then treated with 500-700 uL of 1.0 M HCl in
acetic acid and 50-70 uL of anisole and the solution was stirred
for 1.5-2 h. The solution was added dropwise to 5 mL of cold,
saturated potassium carbonate (pH9) and was extracted two times
with 15 mL of diethyl ether. The combined extracts were washed once
with 5 mL of potassium carbonate and once with brine and were dried
over sodium sulfate. Concentration gave crude amides of 5g/5h which
were chromatographed on 500 mg of silica gel using a step gradient
as follows; dichloromethane, 99/1, 98/2, 97/3, 96/4, and 95/5
DCM/MeOH.
##STR00024##
2-Amino-3-(4-fluorophenyl)-N-isopropylpropanamide (5g)
[0100] 7.0 mg (38%), .sup.1HNMR (CDCl.sub.3) .delta. 1.12 (d, 3H,
J=6.1 Hz), 1.13 (d, 3H, J=6.1 Hz), 1.50 (br s, 2H), 2.74 (dd, 1H,
J=13.8 and 8.7 Hz), 3.17 (dd, 1H, J=13.8 and 4.3 Hz), 3.54 (dd, 1H,
J=8.3 and 4.0 Hz), 4.05 (octet, 1H, J=7.1 Hz), 6.96-7.02 (m, 3H),
7.17-7.19 (m, 2H).
##STR00025##
2-Amino-3-(4-fluorophenyl)-N-isobutylpropanamide (5h)
[0101] 6.7 mg (38%), .sup.1HNMR (CDCl.sub.3) .delta. 0.89 (d, 3H,
J=6.7 Hz), 0.94 (d, 3H, J=6.7 Hz), 1.75 (nonet, 1H, J=6.7 Hz), 2.74
(dd, 1H, J=13.8 and 8.9 Hz), 3.08 (nonet, 2H, J=6.5 Hz), 3.21 (dd,
1H, J=13.9 and 4.0 Hz), 3.59 (br s, 1H), 7.00 (t, 2H, J=8.7 Hz),
7.17-7.20 (m, 2H), 7.31 (br s, 1H).
##STR00026##
2-Amino-3-(4-fluorophenyl)-N-(pyridin-3-ylmethyl)propanamide
(5i)
[0102] Using the procedure above for 5g and 5h gave 2-3 mg of crude
product. The aqueous phase was concentrated to dryness and was then
continuously extracted for six hours in a Soxhlet extractor using
dichloromethane affording 13 mg of material which was
chromatographed as above for 5g/5h to give 7.0 mg (41%), .sup.1HNMR
(CDCl.sub.3) .delta. 2.79 (dd, 1H, J=13.9 and 8.7 Hz), 3.22 (dd,
1H, J=13.9 and 4.1 Hz), 3.65 (dd, 1H, J=8.7 and 4.2 Hz), 4.43 (dd,
1H, J=15.0 and 6.0 Hz), 4.48 (dd, 1H, J=15.0 and 6.1 Hz), 6.99 (t,
2H, J=8.7 Hz), 7.15-7.18 (m, 2H), 7.26 (dd, 1H, J=7.8 and 4.9 Hz),
7.57 (d, 1H, J=7.8 Hz), 7.70 (br s, 1H), 8.52 (br s, 2H).
##STR00027##
2-amino-3-(4-fluorophenyl)-N-(pyridin-4-ylmethyl)propanamide 2,2,2
trifluoroacetate (5j)
[0103] Using the procedure above for 5g and 5h gave intermediate
14j. The Boc group was removed by treatment with 25%
trifluoroacetic acid in dichloromethane over three hours. The
reaction mixture was evaporated to 32.9 mg of a residue which was
chromatographed of silica gel using 18:2:1
isopropanol:methanol:ammonium hydroxide and proceeding to 9:2:1 to
give 4.2 mg (14%) and 19.3 mg of contaminated target material,
.sup.1HNMR (CD.sub.3OD) .delta. 3.13 (dd, 1H, J=13.9 and 8.7 Hz),
3.20 (dd, 1H, J=13.8 and 7.9 Hz), 4.13 (t, 1H, J=7.5 Hz), 4.37 (d,
1H, J=16.0 Hz), 4.48 (d, 1H, J=16.0 Hz), 7.08 (t, 2H, J=8.7 Hz),
7.27-7.29 (m, 4H), 8.55 (br s, 2H).
##STR00028##
2-Amino-3-(4-fluorophenyl)-N-(2,2,2-trifluoroethyl)propanamide
(5k)
[0104] 8.8 mg (42%), .sup.1HNMR (CDCl.sub.3) .delta. 2.79 (dd, 1H,
J=14.0 and 8.8 Hz), 3.21 (dd, 1H, J=13.9 and 3.9 Hz), 3.67 (br s,
1H), 3.82-4.00 (m, 2H), 7.01 (t, 2H, J=8.6 Hz), 7.16-7.18 (m, 2H),
7.79 (br s, 1H).
##STR00029##
2-Amino-3-(4-fluorophenyl)-N-phenethylpropanamide (5l)
[0105] 14.4 mg (64%); .sup.1HNMR (CDCl.sub.3) .delta. 2.69 (dd, 1H,
J=13.9 and 8.8 Hz), 2.79 (t, 2H, J=7.0 Hz), 3.17 (dd, 1H, J=13.9
and 4.2 Hz), 3.46-3.57 (m, 3H), 6.99 (t, 2H, J=8.6 Hz), 7.14-7.16
(m, 4H), 7.20-7.25 (m, 2H), 7.26-7.30 (m, 2H).
##STR00030##
2-Amino-3-(4-fluorophenyl)-N-(2-hydroxyethyl)propanamide
2,2,2-trifluoroacetate (5m)
[0106] Using the procedure above for 5g and 5h gave intermediate
14m. The Boc group was removed by treatment with 25%
trifluoroacetic acid in dichloromethane over three hours. The
reaction mixture was evaporated to a residue which was
chromatographed of silica gel using 95:5 and 9:1
dichloromethane:methanol to give 18.7 mg (69%) of 5m; .sup.1HNMR
(D.sub.2O) .delta. 3.11 (m, 2H), 3.22 (m, 2H), 3.44 (m, 1H), 3.50
(m, 1H), 4.08 (t, 1H, J=7.4 Hz), 7.07 (t, 2H, J=8.9 Hz), 7.20-7.22
(m, 2H); .sup.1HNMR (CD.sub.3OD) .delta. 3.06 (dd, 1H, J=13.8 and
7.3 Hz), 3.19 (dd, 1H, J=12.8 and 6.5 Hz), 3.27-3.37 (m, 2H),
3.52-3.62 (m, 2H), 4.00 (br t, 1H, J=5.9 Hz), 7.11 (t, 2H, J=8.7
Hz), 7.29-7.32 (m, 2H).
##STR00031##
2-Amino-3-(4-fluorophenyl)-N-(2-morpholinoethyl)propanamide
(5n)
[0107] Appreciable water-solubility required three extractions of
the basic aqueous phase with dichloromethane to give 10.9 mg (47%);
.sup.1HNMR (CDCl.sub.3) .delta. 2.41-2.46 (m, 6H), 2.76 (dd, 1H,
J=13.8 and 8.6 Hz), 3.17 (dd, 1H, J=13.8 and 4.6 Hz), 3.35 (q, 2H,
J=5.9 Hz), 3.57 (dd, 1H, J=8.4 and 4.6 Hz), 3.67 (t, 4H, J=4.6 Hz),
7.00 (t, 2H, J=8.6 Hz), 7.17-7.20 (m, 2H), 7.35 (br s, 1H).
##STR00032##
2-Amino-3-(4-fluorophenyl)-1-(piperidin-1-yl)propan-1-one (5o)
[0108] 10.1 mg (59%); .sup.1HNMR (CDCl.sub.3) .delta. 1.14-1.22 (m,
1H), 1.40-1.63 (m, 5H), 2.74 (dd, 1H, J=13.4 and 7.2 Hz), 2.91 (dd,
1H, J=13.4 and 6.7 Hz), 3.12-3.16 (m, 1H), 3.27-3.32 (m, 1H),
3.49-3.58 (m, 2H), 3.93 (br s, 1H), 6.98 (t, 2H, J=8.6 Hz),
7.15-7.18 (m, 2H).
##STR00033##
2-Amino-3-(4-fluorophenyl)-1-(pyrrolidin-1-yl)propan-1-one (5p)
[0109] Appreciable water-solubility required three extractions of
the basic aqueous phase with dichloromethane to give 10.8 mg (58%);
.sup.1HNMR (CDCl.sub.3) .delta. 1.67-1.86 (m, 4H), 2.77 (dd, 1H,
J=13.3 and 7.0 Hz), 2.82-2.86 (m, 1H), 2.92 (dd, 1H, J=13.2 and 7.4
Hz), 3.35-3.40 (m, 2H), 3.45-3.50 (m, 1H), 3.69 (t, 1H, J=7.1 Hz),
6.97 (t, 2H, J=8.6 Hz), 7.15-7.18 (m, 2H).
##STR00034##
The procedure in Scheme 2 for the preparation of resins 3 was used.
Resins 3 were then treated with 7N methanolic ammonia or 33%
methylamine in ethanol or isoamylamine in ethanol for four to
twelve days. The vessels were drained and the resins washed with
THF. The filtrates were then evaporated to dryness to afford crude
amides 5a-f, and i.
##STR00035##
2-Amino-3-(2-fluorophenyl)propanamide (5a)
[0110] 7.1 mg (78%), .sup.1HNMR (DMSO-d.sub.6) .delta. 2.65 (dd,
1H, J=13.3 and 8.3 Hz), 2.94 (dd, 1H, J=13.3 and 5.2 Hz), 3.36 (dd,
1H, J=8.3 and 5.8 Hz), 6.96 (br s, 1H), 7.10-7.14 (m, 2H),
7.23-7.27 (m, 1H), 7.29-7.32 (m, 1H), 7.34 (br s, 1H).
##STR00036##
2-Amino-3-(3-fluorophenyl)propanamide (5b)
[0111] 9.0 mg (99%), .sup.1HNMR (DMSO-d.sub.6) .delta. 2.65 (dd,
1H, J=13.2 and 8.2 Hz) 2.93 (dd, 1H, J=13.3 and 4.9 Hz), 3.39 (dd,
1H, J=7.9 and 5.2 Hz), 7.01-7.04 (m, 2H), 7.06 (d, 2H, J=7.9 Hz),
7.29-7.33 (m, 1H), 7.36 (br s, 1H).
##STR00037##
2-Amino-3-(4-fluorophenyl)propanamide (5c)
[0112] 15.2 mg (38%), .sup.1HNMR (D.sub.2O) .delta. 2.82 (dd, 1H,
J=13.7 and 6.9 Hz), 2.87 (dd, 1H, J=13.6 and 6.7 Hz), 3.57 (t, 1H,
J=6.8 Hz), 7.02 (t, 2H, J=8.9 Hz), 7.18 (m, 2H).
##STR00038##
2-Amino-3-(3,4-difluorophenyl)propanamide (5d)
[0113] 4.0 mg, (40%), .sup.1HNMR (5% CD.sub.3OD in CDCl.sub.3)
.delta. 2.75 (dd, 1H, J=13.5 and 8.7 Hz), 3.14 (br d, 1H, J=13.1
Hz), 3.61 (br s, 1H), 6.00 (br s, 1H), 6.94-6.96 (br m, 1H),
7.05-7.14 (m, 2H), 7.18 (br s, 1H).
##STR00039##
2-Amino-3-(4-fluorophenyl)-N-methylpropanamide (5e)
[0114] 3.9 mg (40%), .sup.1HNMR (CD.sub.3OD) 2.60 (s, 3H), 2.73
(dd, 1H, J=13.5 and 7.0 Hz), 2.88 (dd, 1H, J=13.5 and 6.7 Hz), 3.40
(t, 1H, J=6.6 Hz), 6.94 (t, 2H, J=8.8 Hz), 7.12-7.15 (m, 2H).
##STR00040##
2-Amino-3-(3,4-difluorophenyl)-N-methylpropanamide (5i)
[0115] 4.4 mg (41%), .sup.1HNMR (CDCl.sub.3) 2.73 (dd, 1H, J=13.9
and 8.9 Hz), 2.82 (d, 3H, J=4.9 Hz), 3.20 (dd, 1H, J=13.9 and 4.1
Hz), 3.58 (dd, 1H, J=8.8 and 4.1 Hz), 6.91-6.94 (br m, 1H), 7.04
(ddd, 1H, J=10.6, 7.5, and 1.9 Hz), 7.07-7.13 (m, 1H), 7.21 (br s,
1H).
##STR00041##
2-Amino-3-(4-fluorophenyl)-N-isopentylpropanamide (5f)
[0116] 5.6 mg (44%), .sup.1HNMR (CDCl.sub.3) .delta. 0.91 (d, 6H,
J=6.7 Hz), 1.37 (q, 2H, J=7.3 Hz), 1.58 (heptet, 1H, J=6.7 Hz),
2.73 (dd, 1H, J=13.9 and 8.9 Hz), 3.20 (dd, 1H, J=13.9 and 4.2 Hz),
3.26 (ddd, 2H, J=9.1, 7.1, 1.3 Hz), 3.56 (dd, 1H, J=8.8 and 4.2
Hz), 7.00 (t, 2H, J=8.6 Hz), 7.14 (br s, 1H), 7.14-7.19 (m,
2H).
##STR00042##
[0117] Compound 4a (prepared as described above for 4d), 209.1 mg,
was treated with 711 .mu.L of t-butanol, 142.8 .mu.L of water, and
570.2 .mu.L of 2.0 N NaOH. To this solution was added 268.5 .mu.L
of di-t-butyl-dicarbonate. The solution was stirred overnight and
was then transferred to a separatory funnel containing 10 mL of
diethylether and mixed. The layers were separated and the aqueous
phase was washed with 5 mL of diethyl ether and was adjusted to
pH2-3 with 1N HCl. The mixture was extracted with 2.times.10 mL of
ethyl acetate. The combined extracts were washed with 2 mL of water
and were dried (MgSO.sub.4). Concentration gave 242.4 mg of 12a.
Compound 12a (117 mg) under dry argon gas was dissolved in 874
.mu.L of DMF and the solution was chilled to -9.degree. C. To the
solution was then added 45.6 .mu.L of N-methylmorpholine followed
by 56.6 .mu.L of isobutyl chloroformate. After 5-10 min, 600 .mu.L
of 7N ammonia in methanol was added. The mixture was stirred at -15
to 2.degree. C. over a 4.5 h period and was then added to 20-25 mL
of crushed ice and saturated sodium bicarbonate. The mixture was
extracted with 2.times.15 mL of ethyl acetate. The combined
extracts were washed once with 5 mL of water, once with 5 mL of 10%
citric acid, and once with water and were dried (MgSO.sub.4).
Concentration gave 123.8 mg of 13a as a white solid. To 121.8 mg of
13a was added a solution of 782 .mu.L of 1.0 M HCl in acetic acid
and 77.2 .mu.L of anisole. After one hour, 5 mL of diethyl ether
was added. The precipitate was collected and was dried in vacuo to
afford 70 mg of 5c as its hydrochloride salt. A portion (35.3 mg)
was stirred in 3 mL of saturated potassium carbonate for 3 h.
Collection afforded 15.2 mg of 5c. For additional information, see
U.S. Pat. No. 4,265,808, disclosure of which is incorporated by
reference in its entirety to the extent that it is not inconsistent
with the explicit teachings of this specification.
[0118] Referring now to FIG. 1, compounds were synthesized and
tested for the chemical activity. Compounds in red (i.e., 2-F, 3-F,
4-F, and 4-I) showed activity in preliminary screens.
[0119] Referring now to FIG. 2, twenty-one "unnatural" amino acids
were synthesized.
Effect of Some Exemplary Compounds on Biofilm Formation
[0120] Referring now to FIG. 3, reproducible P. aeruginosa Biofilm
Assays were carried out using newly synthesized twenty-one
"unnatural" amino acids. See George A. O'Toole, Microtiter Dish
Biofilm Formation Assay, J. VIS EXP. 2011; (47): 2437, disclosures
of which are incorporated by reference in its entirety to the
extent that they are not inconsistent with the explicit teachings
of this specification.
[0121] Referring now to FIG. 23, four compounds (2-F, 3-F, 4-F, and
3,4-F2) showed activity in the Biofilm Assay. Subsequent assay of a
purified sample of these compounds confirmed this activity, and the
compounds were active in a dose dependent manner (FIGS. 26-28).
Referring now to FIG. 24, in the biofilm assay 4-F was shown to be
as potent as tobramycin (the current treatment for P. aeruginosa
infections in patients with Cystic Fibrosis).
Exemplary Separation of Enantiomers
[0122] Enantiomers were separated by chiral chromatography using a
Regis Technologies (Morton Grove, Ill.) Chirosil SCA (-)
4.6.times.150 mm, 5-micron column using 7/3 MeOH/H.sub.2O with 10
mM AcOH at a flow rate of 1.0 mL/min and detection at 214, 230, and
254 nm. A typical injection volume was 30 .mu.L (34.5 .mu.g) of a
1.15 mg/mL solution of purified, racemic compound in 7/3
MeOH/H.sub.2O. Each enantiomer was collected manually in separate
test tubes. The process was repeated a second time. Combined
solutions of individual enantiomers were evaporated to dryness and
the residues were dissolved in 200 .mu.L of 7/3 MeOH/H.sub.2O.
These solutions were then each analyzed on the chiral column (5
.mu.L injections) to verify enantiopurity and then on an Agilent
Technologies (Santa Clara, Calif.) Zorbax SB C18, 4.6.times.150 mm,
5-micron column (30% 1:1 MeOH:MeCN with 5 mM NH.sub.4OAc--70%
H.sub.2O with 5 mM NH.sub.4OAc, 1.0 mL/min, 210, 214, 254 nm) to
determine mass quantities from peak areas using the calibration
equation.
[0123] Initially, un-purified samples of two of these compounds, of
structures 4-fluorophenylalanine (4-F) and 2-fluorophenylalanine
(2-F), were found to be active in the K357 biofilm assay.
Subsequent assay of a purified sample of 4-F confirmed this
activity. It was then demonstrated that in the biofilm assay 4-F is
as potent as tobramycin (the current treatment for P. aeruginosa
infections in patients with Cystic Fibrosis).
##STR00043##
[0124] Expanding on these initial results an enantiomeric
purification was carried out and the resolved enantiomers were
tested in the bioassay. Using these preparations it was established
that one of the enantiomers of 4-F is primarily responsible for the
direct or indirect potent inhibition of biofilm formation by P.
aeruginosa. The other enantiomer of 4-F appears to have some
activity but at a significantly higher concentration. A Structure
Activity Relationship (SAR) was developed to provide a means for
improving the utility of these compounds in modulating biofilm
formation, and/or inhibiting growth. Critical findings reported in
these studies are the potent activity, in addition to 4-F and 2-F,
of molecules 3-fluorophenylalanine (3-F) and
3,4-difluorophenylalanine (3,4-F). Also determined using a similar
experimental approach is that the amide derivative
(4-fluorophenylalanine amide) of 4-F is very potent inhibitor of
biofilm development.
##STR00044##
[0125] Referring now to FIG. 25, one of the enantiomers ("Isomer
B") was found to be primarily responsible for the direct or
indirect potent inhibition of biofilm formation by P. aeruginosa.
The other enantiomer ("Isomer A") appears to have some activity but
at a significantly higher concentration.
[0126] Referring now to FIGS. 34-35, newly synthesized compounds
were prepared according to the schemes described above. These
compounds include, but not limited to, compounds having the
formula:
##STR00045##
[0127] Referring to FIGS. 34-35, all newly synthesized compounds
prepared according to the schemes described above were assayed for
biofilm formation in multiplicate 24 well plates at a screening
dose of 20 .mu.g/mL using crystal violet optical density (OD)
readings as the readout. Results were expressed as % biofilm
formation relative to a control (control=bacteria alone=100%
biofilm formation). The procedure for the biofilm assay was the
same as that used by students in the K357 biofilm laboratory.
[0128] Referring now to FIGS. 36-38, all compounds assayed in
either 96 well or 24 well plates at a screening dose of either 20
or 40 .mu.g/mL, using a biofilm assay with crystal violet optical
density (OD) readings as the readout. Results were expressed as %
biofilm formation (control=bacteria alone=100% biofilm formation).
The procedure for the biofilm assay was the same as that used by
students in the K357 biofilm laboratory.
[0129] Newly synthesized compounds used in FIGS. 36-38 include:
##STR00046## ##STR00047## ##STR00048##
[0130] Referring now to FIGS. 34-38, the following compounds, for
example, exhibited 50% or greater inhibition of biofilm formation
at 20 .mu.g/mL:
##STR00049## ##STR00050##
[0131] While the novel technology has been illustrated and
described in detail in the figures and foregoing description, the
same is to be considered as illustrative and not restrictive in
character, it being understood that only the preferred embodiments
have been shown and described and that all changes and
modifications that come within the spirit of the novel technology
are desired to be protected. As well, while the novel technology
was illustrated using specific examples, theoretical arguments,
accounts, and illustrations, these illustrations and the
accompanying discussion should by no means be interpreted as
limiting the technology. All patents, patent applications, and
references to texts, scientific treatises, publications, and the
like referenced in this application are incorporated herein by
reference in their entirety.
* * * * *