U.S. patent application number 16/353824 was filed with the patent office on 2019-07-11 for compounds and methods for treating bacterial infections.
The applicant listed for this patent is Washington University. Invention is credited to Corinne Cusumano, Zhenfu Han, Scott Hultgren, James W. Janetka, Jerome S. Pinkner.
Application Number | 20190211045 16/353824 |
Document ID | / |
Family ID | 46638932 |
Filed Date | 2019-07-11 |
![](/patent/app/20190211045/US20190211045A1-20190711-C00001.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00002.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00003.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00004.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00005.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00006.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00007.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00008.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00009.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00010.png)
![](/patent/app/20190211045/US20190211045A1-20190711-C00011.png)
View All Diagrams
United States Patent
Application |
20190211045 |
Kind Code |
A1 |
Janetka; James W. ; et
al. |
July 11, 2019 |
COMPOUNDS AND METHODS FOR TREATING BACTERIAL INFECTIONS
Abstract
The present invention encompasses compounds and methods for
treating urinary tract infections.
Inventors: |
Janetka; James W.; (St.
Louis, MO) ; Han; Zhenfu; (St. Louis, MO) ;
Hultgren; Scott; (St. Louis, MO) ; Pinkner; Jerome
S.; (St. Louis, MO) ; Cusumano; Corinne; (St.
Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Washington University |
St. Louis |
MO |
US |
|
|
Family ID: |
46638932 |
Appl. No.: |
16/353824 |
Filed: |
March 14, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15373260 |
Dec 8, 2016 |
10273260 |
|
|
16353824 |
|
|
|
|
14570322 |
Dec 15, 2014 |
9567362 |
|
|
15373260 |
|
|
|
|
13453991 |
Apr 23, 2012 |
8937167 |
|
|
14570322 |
|
|
|
|
PCT/US2010/053848 |
Oct 22, 2010 |
|
|
|
13453991 |
|
|
|
|
PCT/US2012/024169 |
Feb 7, 2012 |
|
|
|
13453991 |
|
|
|
|
61254135 |
Oct 22, 2009 |
|
|
|
61321738 |
Apr 7, 2010 |
|
|
|
61384535 |
Sep 20, 2010 |
|
|
|
61440260 |
Feb 7, 2011 |
|
|
|
61451455 |
Mar 10, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 55/08 20130101;
A01N 43/78 20130101; A61K 31/7048 20130101; C07D 309/10 20130101;
C07H 15/203 20130101; A61P 13/02 20180101; A01N 43/42 20130101;
C07H 19/056 20130101; A01N 43/54 20130101; A61K 31/706 20130101;
A01N 43/38 20130101; A61P 31/04 20180101; C07H 15/26 20130101; A61K
31/505 20130101; A01N 47/36 20130101; A01N 43/16 20130101; A01N
43/52 20130101; C07H 15/207 20130101; A61K 31/7056 20130101; A61K
31/7042 20130101; A01N 43/76 20130101; C07H 15/20 20130101; A01N
43/10 20130101; C07H 15/22 20130101; A61K 31/7034 20130101 |
International
Class: |
C07H 15/20 20060101
C07H015/20; C07H 15/26 20060101 C07H015/26; A01N 43/10 20060101
A01N043/10; A61K 31/706 20060101 A61K031/706; A61K 31/7056 20060101
A61K031/7056; A61K 31/7048 20060101 A61K031/7048; A61K 31/7042
20060101 A61K031/7042; A61K 31/7034 20060101 A61K031/7034; A61K
31/505 20060101 A61K031/505; C07D 309/10 20060101 C07D309/10; C07H
15/22 20060101 C07H015/22; C07H 15/207 20060101 C07H015/207; C07H
15/203 20060101 C07H015/203; A01N 55/08 20060101 A01N055/08; A01N
47/36 20060101 A01N047/36; A01N 43/78 20060101 A01N043/78; A01N
43/76 20060101 A01N043/76; A01N 43/54 20060101 A01N043/54; A01N
43/52 20060101 A01N043/52; A01N 43/42 20060101 A01N043/42; A01N
43/38 20060101 A01N043/38; A01N 43/16 20060101 A01N043/16; C07H
19/056 20060101 C07H019/056 |
Goverment Interests
GOVERNMENTAL RIGHTS
[0002] This invention was made with government support under grants
numbered 1 RC1 DK086378, RO1AI029549, P50DK064540 and
RO1BK051406-12 each of which were awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1-10. (canceled)
11. A compound of Formula (I): ##STR00120## wherein: X is selected
from the group consisting of hydrogen, OR.sup.2, SR.sup.2, and
NR.sup.z; Z is selected from the group consisting of O, S, CR.sup.3
and NR.sup.4; Y is selected from the group consisting of oxygen,
sulfur, CR.sup.3, NR.sup.4, --N(R.sup.5)CO--,
--CH.sub.2N(R.sup.5)--, --CH.sub.2N(R.sup.5)CO--, CO.sub.2,
SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO, --CON(R.sup.5)--,
--SO.sub.2N(R.sup.5)--, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, --N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--,
NR.sup.5, and an optionally substituted alkyl, alkene, alkyne, or
heterocycle; R.sup.2 is independently selected from the group
consisting of hydrogen, COR.sup.x, CONR.sup.x, hydrocarbyl, and
substituted hydrocarbyl; R.sup.3, R.sup.4, R.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; R.sup.z is independently
selected from the group consisting of hydrogen hydrocarbyl,
substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; R.sup.x
is independently selected from the group consisting of hydrogen,
NR.sup.4R.sup.5, and an optionally substituted alkyl, cycloalkyl,
heterocycle, or aryl; n is an integer from 1 to 10; and R.sup.1 is
Formula (IV): ##STR00121## wherein: A is independently selected
from the group consisting of C, CR.sup.6 and N; R.sup.6 is
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; R.sup.11 and R.sup.12 are
independently selected from the group consisting of OR.sup.15,
--NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.15,
--CONR.sup.16R.sup.17, --SO.sub.2NR.sup.18R.sup.19, alkenyl,
alkynyl, hydrocarbyl, and substituted hydrocarbyl; R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; and (a) of R.sup.11 is the
integer 1 and (a) of R.sup.12 is either the integer 1 or the
integer 2.
12. The compound of claim 1, wherein X is OR.sup.2 and Z is O.
13. The compound of claim 1, wherein X is OR.sup.2; Z is O;
R.sup.11 is selected from the group consisting of --OR.sup.15,
methyl, halogen, CO.sub.2Me, hydrocarbyl, and substituted
hydrocarbyl.
14. The compound of claim 14, wherein Formula (IV) is:
##STR00122##
15. The compound of claim 1, wherein X is OR.sup.2; Z is O; Y is
selected from the group consisting of oxygen, sulfur, CR.sup.3,
NR.sup.4, --N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--, --CH.sub.2O--,
--CON(R.sup.5)--, --CH.sub.2N(R.sup.5)CO--, --(CH.sub.2).sub.n--
and an optionally substituted alkyl.
16. The compound of claim 1, wherein X is OR.sup.2; Z is O; Y is
selected from the group consisting of oxygen, sulfur, NH,
--CH.sub.2O--, --CONH, --CH.sub.2NHCO--, --(CH.sub.2).sub.n-- and
an optionally substituted alkyl.
17. The compound of claim 15, wherein Y is selected from the group
consisting of oxygen, sulfur, NH, --(CH.sub.2).sub.n-- and an
optionally substituted alkyl; and R.sup.12 is selected from the
group consisting of --COOR.sup.15 and --CONR.sup.16R.sup.17.
18. The compound of claim 1, wherein X is OR.sup.2; Z is O; Y is
selected from the group consisting of oxygen and an optionally
substituted alkyl; Formula (IV) is: ##STR00123## R.sup.11 is
selected from the group consisting of --OR.sup.15, methyl, halogen,
CO.sub.2Me, and hydrocarbyl; and R.sup.12 is selected from the
group consisting of --COOR.sup.15 and --CONR.sup.16R.sup.17.
19. A method of treating a urinary tract infection, the method
comprising administering a compound of claim 1 to a subject in need
thereof.
20. The method of claim 19, wherein the subject is further
administered a bactericidal composition.
21. A method of reducing the resistance of a bacterium to a
bactericidal compound, the method comprising administering a
compound of claim 1 to a subject in need thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No. 14/570,322
filed Dec. 15, 2014, which is a divisional of U.S. Ser. No.
13/453,991 filed Apr. 23, 2012, now U.S. Pat. No. 8,937,167, which
is a continuation-in-part of PCT application number
PCT/US2010/053848 filed Oct. 22, 2010, which claims priority of
U.S. provisional application No. 61/254,135, filed Oct. 22, 2009;
U.S. provisional application No. 61/384,535, filed Sep. 20, 2010;
and U.S. provisional application No. 61/321,738, filed Apr. 7,
2010; and PCT application number PCT/US2012/024169 filed Feb. 7,
2012, which claims the priority of U.S. provisional application No.
61/440,260, filed Feb. 7, 2011; and U.S. provisional application
No. 61/451,455, filed Mar. 10, 2011, all of which are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention encompasses compounds and methods for
treating urinary tract infections.
BACKGROUND OF THE INVENTION
[0004] Urinary tract infection (UTI) caused by uropathogenic
Escherichia coli (UPEC) is one of the most common infectious
diseases in women. The morbidity and economic impact are enormous,
with over $2.5 billion spent annually on treatment. Further,
recurrent infections are a significant problem despite appropriate
antibiotic therapy of the index case. The high rates of recurrence,
and the large numbers of women that end up in urology clinics due
to their chronic recurrent UTIs highlights the need for a better
understanding of the pathogenic mechanisms involved in this disease
and the development of new and better therapies.
[0005] Gram-negative bacteria are the causative agents of a wide
variety of acute and chronic infectious diseases. Many of these
infections are initiated by a critical interaction between host
ligands (frequently polysaccharide moieties) and bacterial adhesins
(frequently expressed at the distal tip of polymeric pilus fibers
assembled by the chaperone/usher pathway). The mannose binding FimH
adhesin of type 1 pili is critical for the colonization and
invasion into the bladder epithelium. After invasion, UPEC are able
to rapidly multiply inside superficial umbrella cells of the
bladder forming biofilm-like intracellular bacterial communities
(IBCs). Upon maturation, bacteria disperse from the IBC, spread to
neighboring cells, and form next generation IBCs. This is the
mechanism by which UPEC rapidly amplify in numbers in the urinary
tract and cause disease.
[0006] The X-ray crystal structure of FimH bound to mannose showed
that mannose is bound in a negatively charged pocket on FimH. The
mannose binding site is highly conserved as it is invariant in 300
fimH genes sequenced from clinical UPEC strains. Thus, FimH is the
critical node of the entire UPEC pathogenic cascade.
[0007] Recurrence is a serious problem for many women. Women who
present with an initial episode of acute UTI have a 25-44% chance
of developing a second and a 3% chance of experiencing three
episodes within six months of the initial UTI. Recurrence occurs
despite appropriate antibiotic treatment and clearance of the
initial infection from the urine. A large percentage of recurrent
UTI are caused by the same strain of bacteria as the initial
infection. One study followed 58 women and found that 68% of
recurrences were caused by the same initial index strain of UPEC as
determined by restriction fragment length polymorphism (RFLP)
analysis. In a separate study, 50% of recurrent strains isolated
from female college students appeared genotypically identical to
the bacterial strain corresponding to the initial UTI. Another
long-term prospective study demonstrated that the same strain of
UPEC can cause a recurrent UTI up to 3 years later. The high
frequency of same-strain recurrences supports the notion that a
UPEC reservoir can exist in the affected individual. The inventors
have shown that a quiescent intracellular reservoir (QIR) can form
in the bladder tissue itself after acute infection and persist even
after antibiotic therapy and urine cultures become sterile. Thus,
reactivation of bacteria in QIRs may also be a contributing factor
in recurrent UTIs.
[0008] Therefore, there is a need for effective treatments that can
cure urinary tract infections and prevent the formation of
quiescent intracellular reservoir that are the source of so many
recurrent infections.
SUMMARY OF THE INVENTION
[0009] One aspect of the present invention encompasses a compound
comprising formula (I):
##STR00001##
wherein: [0010] X is selected from the group consisting of
hydrogen, OR.sup.2, SR.sup.2, and NR.sup.z; [0011] Z is selected
from the group consisting of O, S, CR.sup.3 and NR.sup.4; [0012] Y
is selected from the group consisting of oxygen, sulfur, CR.sup.3,
NR.sup.4, --N(R.sup.5)CO--, -- [0013] CH.sub.2N(R.sup.5)--,
--CH.sub.2N(R.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, NR.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0014] R.sup.2 is independently selected from the group consisting
of hydrogen, COR.sup.x, CONR.sup.x, hydrocarbyl, and substituted
hydrocarbyl; [0015] R.sup.3, R.sup.4, R.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0016] R.sup.z is independently selected
from the group consisting of hydrogen hydrocarbyl, substituted
hydrocarbyl, --COR.sup.x, --CONR.sup.xR.sup.xSO.sub.2R.sup.x, and
--CO.sub.2R.sup.x; [0017] R.sup.x is independently selected from
the group consisting of hydrogen, NR.sup.4R.sup.5, and an
optionally substituted alkyl, cycloalkyl, heterocycle, or aryl;
[0018] n is an integer from 1 to 10; and [0019] R.sup.1 is selected
from the group comprising Formulas (IIIA), (IIIB), (IV), (V), (VI),
(VII) and (VIII):
[0019] ##STR00002## [0020] wherein: [0021] A is independently
selected from the group consisting of CR.sup.6 and N; [0022] G is
selected from the group consisting of S, O, CR.sup.8, and NR.sup.9;
[0023] R.sup.6, R.sup.8 and R.sup.9 are independently selected from
the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl;
[0024] R.sup.10, R.sup.11, R.sup.12, R.sup.13 and R.sup.14 are
independently selected from the group consisting of hydrogen,
--OR.sup.16, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.16CO.sub.2R.sup.16, --NR.sup.16CONR.sup.16,
--NR.sup.16SO.sub.2R.sup.16, --COR.sup.16, --SO.sub.2R.sup.16,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.16,
--CONR.sup.16R.sup.17, --SO.sub.2NR.sup.18R.sup.19, alkenyl,
alkynyl, hydrocarbyl, and substituted hydrocarbyl; [0025] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; and [0026] a is either the
integer 1 or the integer 2.
[0027] Another aspect of the invention encompasses a compound
(II):
##STR00003## [0028] wherein [0029] X is selected from the group
consisting of hydrogen, OR.sup.2, SR.sup.2, and NR.sup.z; [0030] Z
is selected from the group consisting of O, S, CR.sup.3 and
NR.sup.4; [0031] Y is selected from the group consisting of oxygen,
sulfur, CR.sup.3, NR.sup.4, --N(R.sup.5)CO--,
--CH.sub.2N(R.sup.5)--, --CH.sub.2N(R.sup.5)CO--, CO.sub.2,
SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO, --CON(R.sup.5)--,
--SO.sub.2N(R.sup.5)--, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, --N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--,
NR.sup.5, and an optionally substituted alkyl, alkene, alkyne, or
heterocycle; [0032] L is selected from --O(CH.sub.2).sub.nO--,
--S(CH.sub.2).sub.nS--, --N(CH.sub.2).sub.nN--,
--(CH.sub.2).sub.n--,
--O[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xO--,
--N(CH.sub.2).sub.mO(CH.sub.2).sub.nN--, heterocycle, alkene,
alkyne, --CON[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xNCO--,
--SO.sub.2N[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xNO.sub.2S--,
and NCO[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xCON--, where L is
bound to a ring of R.sup.1 at a meta or para position; [0033]
R.sup.2 is independently selected from the group consisting of
hydrogen, COR.sup.x, CONR.sup.x, hydrocarbyl, and substituted
hydrocarbyl; [0034] R.sup.3, R.sup.4, R.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl, [0035] R.sup.z is independently selected
from the group consisting of hydrogen hydrocarbyl, substituted
hydrocarbyl, --COR.sup.x, --CONR.sup.xR.sup.xSO.sub.2R.sup.x, and
--CO.sub.2R.sup.x; [0036] R.sup.x is independently selected from
the group consisting of hydrogen, NR.sup.4R.sup.5, and an
optionally substituted alkyl, cycloalkyl, heterocycle, or aryl;
[0037] m, n, and x are integers from 1 to 10; and [0038] R.sup.1 is
selected from the group comprising Formulas (IIIA), (IIIB), (IV),
(V), (VI), (VII) and (VIII):
[0038] ##STR00004## [0039] wherein [0040] A is independently
selected from the group consisting of CR.sup.6 and N; [0041] G is
selected from the group consisting of S, O, CR.sup.8, and NR.sup.9;
[0042] R.sup.6, R.sup.8 and R.sup.9 are independently selected from
the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0043] R.sup.10, R.sup.11, R.sup.12, R.sup.13 and
R.sup.14 are independently selected from the group consisting of
hydrogen, --OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.15,
--CONR.sup.16R.sup.17, --SO.sub.2NR.sup.18R.sup.19, alkenyl,
alkynyl, hydrocarbyl, and substituted hydrocarbyl; [0044] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; and [0045] a is either the
integer 1 or the integer 2.
[0046] Yet another aspect of the present invention encompasses a
method of treating a urinary tract infection. The method comprises
administering a compound of the invention to a subject in need
thereof.
[0047] Still another aspect of the invention encompasses a method
of reducing the resistance of a bacterium to a bactericidal
compound. The method comprises administering a compound of the
invention to a subject in need thereof.
[0048] Other aspects and iterations of the invention are described
below.
Reference to Color Figures
[0049] The application file contains at least one photograph
executed in color. Copies of this patent application publication
with color photographs will be provided by the Office upon request
and payment of the necessary fee.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIG. 1 depicts images and diagrams detailing uropathogenic
E. coli pathogenesis.
[0051] FIG. 2 depicts chaperone/usher mediated pilus biogenesis as
shown for the pilus system.
[0052] FIG. 3 depicts a GRASP view of the FimH adhesion domain
bound to a D-mannose residue (shown in stick). Red=acidic residues.
Blue=basic residues.
[0053] FIG. 4 depicts a ribbon representation of the receptor
binding pocket in complex with the mannose. The D-mannose (pink),
the mannose interacting residues (green) and the residues of the
hydrophobic ridge around the pocket (white) are shown as
ball-and-stick model.
[0054] FIG. 5 depicts a graph showing the reduction of bacterial
load in the bladder upon incubation with heptyl mannose at 6 hours
post-infection. HM=heptyl mannose, MM=methyl mannose, p value
obtained by Mann-Whitney test.
[0055] FIG. 6 depicts FimH residues under positive selection. Green
represents the adhesin FimH and gray represents the chaperone FimC.
Residues in red are under positive selection. Residues in blue are
not under positive selection. All residues are located outside the
mannose binding pocket.
[0056] FIG. 7 depicts the alignment of FimH sequence from a panel
of clinical isolates previously examined. 18 clinical isolates
examined for IBC formation were used to assess the different FimH
sequences. The bold columns represent the residues identified as
under positive selection. The residues highlighted in aqua
represent residues that differ from UT189.
[0057] FIG. 8A,B depicts bacterial load and IBC formation of
isolate acute4 with its native FimH and UT 189's FimH. The FimH
sequence from acute4 was replaced with the fimH sequence of UT189
on the chromosome. (FIG. 8A) Depicts results obtained using
bacterial titers, and (FIG. 8B) depicts results obtained using IBC
formation.
[0058] FIG. 9 depicts the structure of FimH with the compound
number 15 in Table 1, illustrating interactions formed by compound
number 15 with FimH A that are not shared with monomannose.
Compound number 15 is shown in the final refined electron density
calculated to 2.55 .ANG. with 2Fo-Fc coefficients and contoured at
1 .sigma..
[0059] FIG. 10 depicts a graph showing that mannosides number 7 and
number 15 (Table 1) reduced intracellular bacteria at 1 hour
post-infection. A 1 hour gentamicin protection assay was used to
evaluate the amount of bacteria present in the luminal versus
intracellular fraction in the presence of mannosides number 7 and
number 15. A small decrease was seen in the luminal fraction in the
presence of mannoside number 7 (p<0.05) whereas a significant
decrease was seen in the intracellular fraction in the presence of
either mannoside (p<0.0001).
[0060] FIG. 11A,B depicts graphs showing that mannosides number 7
and number 15 reduce infection capacity in vivo. (FIG. 11A) 6 hour
LacZ reveals significantly reduced IBCs at 1 mM and 0.1 mM
mannoside for both mannosides number 7 and number 15 (p<0.0001).
(FIG. 11B) 6 hour bacterial load quantification reveals
significantly reduced colonization with 1 mM mannoside number 7 and
1 mM mannoside number 15 treated bacteria, p<0.001 and
p<0.01, respectively.
[0061] FIG. 12 depicts a diagram showing pilicide inhibit bacterial
pilus biogenesis. The schematic diagram shows the model of
chaperone-assisted pilus assembly and the mode of pilicide action.
During normal pilus biogenesis, newly synthesized pilus subunits
cross into the periplasm via the Sec secretion pathway. These pilus
subunits bind to their periplasmic chaperone proteins (orange
molecules) and fold into correct confirmations (1). In the absence
of the chaperones [A], pilus subunits are degraded [B].
Chaperone-subunit complexes (2) are transported to the outer
membrane usher proteins (blue cylinders) for assembly (3). Pilus
assembly follows an ordered process with the chaperone-adhesin
complexes as the initiatiors (4). As subunits are assembled into
the growing pilus, accompanying chaperones disassodate from the
membrane complexes (5), which could potentially be recycled to bind
newly synthesized subunits. Pilicides (yellow circles) can cross
freely through bacterial outer membranes and bind to periplasmic
chaperones (7). Pilicides block the interaction of
chaperone-subunit complexes with the outer membrane usher (8) and
prevent pilus assembly (9 & 10).
[0062] FIG. 13A-C Effect of pilicides on pilus function and
pilicide interaction with the PapD chaperone. (FIG. 13A) E. coli
NU14 was grown in the presence of pilicides 2d, 2a, 2b and 2c
(np048) (3.5 mM each). HA titers, adherence to 5637 bladder cells,
and the ability to form biofilms was determined. (FIG. 13B) New
pilicides EC220-EC282 exhibit significant improvement in biofilm
inhibitory activity compared to the original pilicide, np048. (FIG.
13C) PapD-np048 complex in overlay with the FimD1-125 N-terminal
usher domain in complex with the FimC-FimH158-279 chaperone/adhesin
complex. PapD and np048 are shown in light blue ribbon and
ball-and-stick representation, respectively. FimC, FimH158-279 and
FimD1-125 N-terminal domain are shown in magenta, green and yellow,
respectively.
[0063] FIG. 14A,B depicts a graph showing that UTI89 bacteria, when
inoculated in the presence of FN075, makes significantly less IBCs
in the bladder (FIG. 14A) and has a significantly reduced bacterial
load within the bladder at 6 hours post-infection (FIG. 14B).
p<0.0001.
[0064] FIG. 15 depicts a representation of an efficacy study in
mice using mannoside alone, antibiotic treatment alone, or dual
mannoside/antibiotic treatment.
[0065] FIG. 16 depicts a graph showing that dual treatment of mice
inoculated with bacteria showed significantly lower levels of
bacteria in the bladder than antibiotics alone.
[0066] FIG. 17 depicts a diagram illustrating two strategies to
inhibit bacterial binding to host cells. The schematic diagram
shows the model of chaperone-assisted pilus assembly and the mode
of mannoside (left panel) and pilicide (right panel) action. During
normal pilus biogenesis, newly synthesized pilus subunits cross
into the periplasm via the Sec secretion pathway. These pilus
subunits bind to their periplasmic chaperone proteins (orange
molecules) and fold into correct confirmations (1). In the absence
of the chaperones, pilus subunits are degraded (*).
Chaperone-subunit complexes (2) are transported to the outer
membrane usher proteins (blue cylinders) for assembly (3). Pilus
assembly follows an ordered process with the chaperone-adhesin
complexes as the initiators (4). As subunits are assembled into the
growing pilus, accompanying chaperones disassociate from the
membrane complexes (5), which could potentially be recycled to bind
newly synthesized subunits. Mannosides (teal triangles) interact
with the pilus adhesin and prevent binding to mannosylated residues
(light brown triangles) on the surface of epithelial cells.
Pilicides (yellow circles) can cross freely through bacterial outer
membranes and bind to periplasmic chaperones (B). Pilicides block
the interaction of chaperone-subunit complexes with the outer
membrane usher (C) and prevent pilus assembly (D).
[0067] FIG. 18A-C depicts diagrams illustrating chaperone-subunit
donor strand exchange (DSE) and donor strand complementation (DSC).
(FIG. 18A) Each subunit consists of six of the seven strands of a
standard immunoglobulin (Ig)-fold and an N-terminal extension
(Nte). (FIG. 18B) In order to form a stable structure prior to
incorporation into the growing fiber, the chaperone donates the
missing seventh strand of the subunit in a process called donor
strand complementation (DSC). (FIG. 18C) During pilus assembly, the
free Nte of one subunit displaces the chaperone bound to another
subunit and serves as the seventh strand of the Ig-like fold in a
process called donor strand exchange (DSE).
[0068] FIG. 19A-E depicts the inhibition and disruption of UTI89
biofilm by mannoside and phase switching of UTI89 in the presence
of mannoside. (FIG. 19A) Structure of mannosides. (FIG. 19B) The
median inhibitory concentration (IC50) of mannoside on UTI89
biofilm formation. (FIG. 19C) The IC50 of mannoside on UTI89
biofilm disruption. After treatment with mannoside, the amount of
UTI89 biofilm was measured. Bars show the mean value of the
experiments (n=3). (FIG. 19D and FIG. 19E) ZFH-2056 (Compound 50)
dispersed biofilm as measured by confocal microscopy of UTI89
biofilms grown for 24 h, then incubated for an additional 16 h in
the absence (FIG. 19D) or presence (FIG. 19E) of 0.3 .mu.M
ZFH-2056.
[0069] FIG. 20A-G depicts the mannoside effect on UTI89
colonization. (FIG. 20A) Pretreatment of UTI89 with mannosides
ZFH-2050 (Compound 49) and ZFH-2056 (Compound 50) resulted in
reduced IBC formation at 6 hpi. (FIG. 20B) Urine PK analysis of
ZFH-2056 (Compound 50) (n>3 mice) showing amounts in urine over
time for each dosing regimen indicated. Horizontal dashed line is
at IC50 (0.74 .mu.M) as determined by the biofilm inhibition assay,
(c and d) Confocal microscopy of bladders from untreated (FIG. 20C)
and ZFH-2056-treated (FIG. 20D) mice. Bacteria were stained with
SYTO9 (green) and the bladder luminal surface was stained with WGA
(red). The image in c shows a normal, robust IBC whereas the arrows
in d indicate luminal bacteria. (FIG. 20E) Total bacterial CPUs at
6 hpi from untreated mice or mice treated with ZFH-2056 either by
IP (5 mg/kg) or oral (100 mg/kg) dosing 30 min prior to inoculation
of UTI89. (FIG. 20F) IBC quantification at 6 hpi from untreated
mice or mice treated with ZFH-2056 either by IP (5 mg/kg) or oral
(100 mg/kg) dosing 30 min prior to inoculation of UTI89. (FIG. 20G)
6 hpi ex vivo gentamicin protection assay revealed both luminal and
intracellular bacteria are significantly reduced upon IP (5 mg/kg)
pretreatment of mice with ZFH-2056. Bars indicate geometric mean.
Statistical significance according to Mann-Whitney is at
**P<0.01, ***P<0.0001. ns, not significant; LCD, limit of
detection.
[0070] FIG. 21A,B depicts mannoside synergy with TMP-SMZ at
reducing UTI89 colonization. (FIG. 21A) Total bacterial CPUs were
quantitated 6 hpi. UTI89 colonization was reduced in mice treated
with ZFH-2056 (Compound 50), TMP-SMZ and TMP-SMZ+ZFH-2056. There
was further decreased colonization in TMP-SMZ+ZFH-2056-treated mice
over ZFH-2056 or TMP-SMZ alone. PBC-1 colonization was reduced in
mice treated with ZFH-2056 and TMP-SMZ+ZFH-2056, but not TMP-SMZ
alone. Enhanced efficacy as measured by bacterial CPUs was observed
upon treatment with TMP-SMZ+ZFH-2056 over ZFH-2056 or TMP-SMZ
treatment alone. Bars indicate geometric mean. Statistical
significance according to Mann-Whitney is at *P<0.05,
**P<0.01, ***P<0.0001. ns, not significant; LOD, limit of
detection. (FIG. 21B) Growth curve of PBC-1 with TMP-SMZ in the
absence (solid line) or presence (dashed line) of 100 .mu.M
ZFH-2056. SMZ concentration is 5.times.TMP concentration
listed.
[0071] FIG. 22 depicts X-ray structure of biphenyl mannoside
meta-methyl ester (Compound 29) with electron density (mesh)
calculated with 2Fo-Fc coefficients, contoured at 1 sigma.
Interaction with the Arg-98-Glu-50 salt bridge (dashes), pi-pi
stacking with Tyr-48 and hydrophobic interaction with Tyr-137 are
shown. Surface electrostatic potential of FimH, calculated with
APBS, is displayed such that pure blue and red would be +4 kT/e and
-4 kT/e respectively.
[0072] FIG. 23 depicts the correlation of HA Titer and Binding
Data.
[0073] FIG. 24 depicts mannoside esters and their amidated
counterparts with enhanced solubility.
[0074] FIG. 25A,B depicts results of hemagglutination (FIG. 25A)
and LacZ quantification of IBCs (FIG. 25B).
[0075] FIG. 26 depicts the pharmacokinetic results of ZFH56
(Compound 50) injected IP and PO into mice.
[0076] FIG. 27A-C depicts IBC numbers (FIG. 27A), bacterial titers
(FIG. 27B) and lacZ staining (FIG. 27C) in mice treated with ZFH56
(Compound 50).
[0077] FIG. 28A-C depicts fluorescent microscopy imaging of
infected bladders treated with ZFH56 (Compound 50).
[0078] FIG. 29 depicts efficacy studies of mannosides against
TMP-SMZ resistant strains.
[0079] FIG. 30A-C depicts efficacy of ZFH56 (Compound 50) under
various doses of inoculum.
[0080] FIG. 31 depicts efficacy studies of ZFH56 (Compound 50) with
compound dose boosting.
[0081] FIG. 32A-B depicts time course of infection and treatment
with mannosides (FIG. 32A) and inhibition of preformed biofilm by
ZFH56 (Compound 50) (FIG. 32B).
[0082] FIG. 33 depicts a graph showing that there is no defect in
UTI89 invasion following implantation. Graph represents bacterial
titers from homogenized bladders from non-implanted (o) or
implanted ( ) animals infected with UTI89 3 hpi following
gentamicin protection assay. Horizontal dashed lines represent the
limit of detection (lod) for viable bacteria (lnt=intracellular,
Ext=Extracellular). Each symbol represents a mouse from two
independent experiments with n=5/condition. The horizontal bars
represent the median of each dataset; p value by the Mann Whitney U
test.
[0083] FIG. 34A-C depicts microscope images and a graph showing
that uropathogenic E. coli produce IBCs in the superficial umbrella
cells of implanted bladders. (FIG. 34A) Representative images of
splayed bladders of female C57BI/6Ncr mice infected with UTI89 6
hpi in the absence (non-implanted) or presence (Implanted) of
implants following LacZ staining. Each black arrow indicates a
purple speck, indicative of an IBC. (FIG. 34B) Quantification of
IBC formation following LacZ staining at 6 hpi. Each symbol
represents IBC number from a single mouse from two independent
experiments with n=5/group. p value obtained from the Mann Whitney
U test. (FIG. 34C) Representative CLSM images of whole bladders
from non-implanted and implanted animals infected with UTI89
ectopically expressing GFP (Green), stained with DMA dye SYTO83
(Red) and Alexa-fluor 633-conjugate of WGA (Blue) reveal the
presence of IBC within umbrella cells. Scale bar=20 .mu.m.
[0084] FIG. 35A-F depicts micrographs showing that IBC and
filamentation occur following urinary catheterization.
Representative CLSM images of whole bladders from non-implanted and
implanted animals infected with UTI89 ectopically expressing GFP
(Green), stained with DMA dye SYTO83 (Red) and Alexa-fluor
633-conjugate of WGA (Blue) reveal the presence of multiple IBCs
within single umbrella cells (FIG. 35A-B), that unlike
non-implanted bladders (FIG. 35C), the underlying epithelium is
exposed following urinary catheterization (FIG. 35D-F), depict the
absence of bacterial colonization of the exposed underlying
epithelium in implanted animals (FIG. 35D-F), and the presence of
filamenting bacteria in umbrella cells (FIG. 35E-F). Scale bar=20
.mu.m
[0085] FIG. 36A-B depicts two graphs showing that UPEC reservoir
reactivation can lead to urinary implant and bladder colonization.
Graphs represent bacterial titers in log scale recovered from
implants, homogenized bladders and kidneys of non-bacteriuric
animals 14 days post infection with UTI89HK::GFP that were
non-implanted or implanted for 3 day (FIG. 36A) or 5 days (FIG.
36B). Horizontal dashed lines represent the limit of detection for
viable bacteria. Each symbol represents a mouse from at least two
independent experiments with n=10-20/group/experiment. The
horizontal bars represent the median of each dataset; p value by
the Mann Whitney U test.
[0086] FIG. 37A-C depicts two graphs showing that deletion of fimH
reduces biofilm formation and attenuates UPEC virulence. Graphs
represent crystal violet based quantification (FIG. 37A) and CFU
enumeration in logarithmic scale (log scale) (FIG. 37B) of 24 h old
UTI89 and UTI89.DELTA.fimH (.DELTA.fimH) biofilms under human urine
flow on silicone tubings at 37.degree. C. indicating that
.DELTA.fimH is defective in biofilm formation under these
conditions. Bars represent mean of three independent experiments,
error bars indicate standard error of the mean (SEM), p values from
Mann Whitney U test. (FIG. 37C) Graph represents bacterial titers
in log scale recovered from implants, homogenized bladders and
kidneys of non-implanted (open symbols) and implanted (closed
symbols) infected with either UTI89 (square) or A fimH (circle) for
24 h. Horizontal dashed lines represent the limit of detection for
viable bacteria. Each symbol represents a mouse from at least two
independent experiments with n=5/group. The horizontal bars
represent the median of each dataset; *p<0.05 and ***p<0.0005
by the Mann Whitney U test.
[0087] FIG. 38A-B depicts two graphs showing that S pili and curli
are not critical for UPEC virulence following urinary
catheterization. Graphs represent bacterial titers in log scale
recovered at 24 hpi from implants, homogenized bladders and kidneys
of (FIG. 38A) implanted animals infected with either UTI89 ( ) or
UTI89 mutant strains deficient in type 1 pili, .DELTA.fimH
(.box-solid.), S pili .DELTA.sfaA-H (.tangle-solidup.), both type 1
and S pili AsfaA-HAfimB-H (.diamond-solid.) and (FIG. 38B)
non-implanted (open symbols) or implanted (closed symbols) animals
infected with UTI89 (o, ), .DELTA.fimH (.quadrature.,.box-solid.)
or UTI89 mutant strains deficient in curli components .DELTA.csgA
(.DELTA.,.tangle-solidup.) and .DELTA.csgB.DELTA.csgG
(.diamond-solid.,.diamond.). Horizontal dashed lines represent the
limit of detection for viable bacteria. Each symbol represents a
mouse from at least two independent experiments with n=5/group. The
horizontal bars represent the median of each dataset; *p<0.05,
***p<0.005 **p<0.0005, ns corresponds to p>0.05 by the
Mann Whitney U test.
[0088] FIG. 39A-B depicts two graphs showing that methyl mannose
inhibits UPEC biofilm in human urine. Graphs represent crystal
violet based quantification (FIG. 39A) and CFU enumeration in
logarithmic scale (log scale) (FIG. 39B) of 24 h old UTI89 biofilms
in human urine with or without 1% methyl mannose under flow on
silicone tubings at 37.degree. C. indicating that methyl mannose
prevents UPEC biofilm formation. Bars represent mean of three
independent experiments, error bars indicate standard error of the
mean (SEM), p values from Mann Whitney U test.
[0089] FIG. 40A-B depicts two graphs showing that mannoside
treatment prevents IBC formation and UPEC virulence when used in
combination with TMP-SMZ. (FIG. 40A) Graph represents IBC
enumeration from LacZ staining of splayed bladders of female
C57BI/6Ncr mice treated i.p. with mannoside or saline prior to
transurethral implantation and inoculation with UTI89 6 hpi. Each
symbol represents IBC number from a single mouse from two
independent experiments with n=5/group. p value obtained from the
Mann Whitney U test. (FIG. 40B) Graph represents bacterial titers
in log scale recovered at 6 hpi from implants, homogenized bladders
and kidneys of animals treated with saline (o), mannoside
(.quadrature.), TMP-SMZ ( ) and TMP-SMZ+Mannoside (.box-solid.)
prior to urinary implantation and inoculation with UTI89.
Horizontal dashed lines represent the limit of detection for viable
bacteria. Each symbol represents a mouse from at least two
independent experiments with n=5/group. The horizontal bars
represent the median of each dataset; *p<0.05, **p<0.0005,
***p<0.0005, ns corresponds to p>0.05 by the Mann Whitney U
test.
[0090] FIG. 41 depicts a graph showing that treatment of chronic
mice with 5 doses of Mannoside 8 eliminates UPEC from the bladder.
Mannoside 8 treatment (.tangle-solidup.), PBS treatment ( ).
[0091] FIG. 42 depcist a graph showing that Mannoside 8 is
effective against the multidrug resistant UPEC isolate EC958.
Mannoside 8 treatment (.DELTA.), PBS treatment
(.tangle-solidup.).
[0092] FIG. 43A-E depicts inhibition, prevention and disruption of
UTI89 biofilm by mannoside. (FIG. 43A), Discovery of biphenyl
mannoside lead FimH inhibitors. Cellular HAI titers (EC.sub.>90)
are shown in parentheses. (FIG. 43B) The median inhibitory
concentration (IC.sub.50) of mannosides 1-3 and 6 on UTI89 biofilm
formation. Mannoside was added at the initiation of biofilm
formation. (FIG. 43C) The IC50 of 1-3 and 6 on UTI89 biofilm
prevention. Mannoside was added 24 h after biofilm growth was
initiated and % biofilm was calculated 16 h after addition of
mannoside. Bars show the mean value of the experiments (n=3). (FIG.
43D, E) 6 dispersed biofilm as measured by confocal microscopy of
UTI89 biofilms grown for 24 h (FIG. 43D), then incubated for an
additional 16 h in the presence of 0.3 .mu.M 6 (FIG. 43E).
[0093] FIG. 44A-G depicts mannoside effect on UTI89 colonization.
(FIG. 44A) Urine PK analysis of 6 (n.gtoreq.3 mice) showing amounts
in urine over time for each dosing regimen indicated. Horizontal
dashed line is at IC50 (0.74 .mu.M) as determined by the biofilm
inhibition assay. (FIG. 44B) Mannoside effectively treats UTI.
Chronically infected mice were treated with PBS or 6 (PO, 100 and
50 mg/kg). 6 hours post-treatment bacterial counts in the bladder
were enumerated. In the mannoside-treated groups, there was a
significant 3-log drop in bacterial load relative to PBS-treated
mice. (C, D) Confocal microscopy of bladders from PBS-treated (FIG.
44C) and 6-treated (FIG. 44D) mice. Bacteria were stained with
SYTO9 (green) and the bladder luminal surface was stained with WGA
(red). The image in C shows a normal, robust IBC whereas the arrows
in D indicate luminal bacteria. (FIG. 44E) Total bacterial CPUs at
6 hpi from mice treated with PBS or 6 either by IP (5 mg/kg) or
oral (100 mg/kg) dosing 30 min prior to inoculation of UTI89. (F)
IBC quantification at 6 hpi from mice treated with PBS or 6 either
by IP (5 mg/kg) or oral (100 mg/kg) dosing 30 min prior to
inoculation of UTI89. (FIG. 44G) 6 hpi ex vivo gentamicin
protection assay revealed both luminal and intracellular bacteria
are significantly reduced upon IP (5 mg/kg) pretreatment of mice
with 6. Bars indicate geometric mean. Statistical significance
according to Mann-Whitney is at *P<0.05, **P<0.01,
***P<0.0001. ns, not significant; LOD, limit of detection.
[0094] FIG. 45 depicts mannoside potentiates TMP-SMZ treatment.
Total bacterial CPUs were quantified 6 hpi. UTI89 colonization was
reduced in mice treated with 6, TMP-SMZ and TMP-SMZ+6. There was
further decreased colonization in TMP-SMZ+6-treated mice over 6 or
TMP-SMZ alone. PBC-1 colonization was reduced in mice treated with
6 and TMP-SMZ+6, but not TMP-SMZ alone. Enhanced efficacy as
measured by bacterial CPUs was observed upon treatment with
TMP-SMZ+6 over 6 or TMP-SMZ treatment alone. Bars indicate
geometric mean. Statistical significance according to Mann-Whitney
is at *P<0.05, **P<0.01, ***P<0.0001. ns, not significant;
LOD, limit of detection.
[0095] FIG. 46A-C depicts newly designed mannosides show enhanced
PK and potency at treating infection. (FIG. 46A) Optimized
ortho-methyl and trifluoromethyl substituted biphenyl mannosides.
Cellular HAI titers (EC.sub.>90) are shown in parentheses. (FIG.
46B) Mannosides show improved PK. Mannosides 8 and 10 dosed at 50
mg/kg had equivalent levels of compound in the urine 6 h
post-treatment relative to mannoside 6 dosed at 100 mg/kg. (FIG.
46C) Chronically infected mice were treated with PBS or mannoside
6, 10 or 8 (PO, 50 mg/kg). 6 hours post-treatment bacterial counts
in the bladder were enumerated. In the mannoside-treated groups,
there was a significant 3-log drop in bacterial load relative to
PBS-treated mice. The optimized mannoside 8 showed increased
efficacy over 6.
[0096] FIG. 47 depicts structures of substituted biphenyl
mannosides.
[0097] FIG. 48 depicts a graph showing curves of HAI, Octet and DSF
assay results.
[0098] FIG. 49 depicts a proposed model of mannoside 7 bound to
FimH calculated with APBS (Adaptive Poisson-Boltzmann Solver)
software using PDB code: 3MCY.
[0099] FIG. 50A-B depicts (FIG. 50A) Metabolic lability of the
mannoside 3 glycosidic bond from hydrolysis to mannose and
biphenol; (FIG. 50B) Elimination kinetics and clearance of
mannoside 3 and biphenol (R) hydrolysis product in mouse urine.
[0100] FIG. 51A depicts plasma pharmacokinetics of optimized
ortho-substituted mannosides 5a-c, 7, 8 and mannoside 3. (FIG. 51B)
depicts elimination clearance kinetics of optimized
ortho-substituted mannosides 5a-c, 7, 8 and mannoside 3 in
urine.
[0101] FIG. 52A-B depicts two graphs showing that UTI89.DELTA.qseC
has a defect in bladder invasion. (FIG. 52A) Bladder titers at 1
and 3 h.p.i showing decreased overall bacterial numbers recovered
from bladders infected with UTI89.DELTA.qseC compared to those
infected with wt UTI89. (FIG. 52B) Bacterial titers recovered from
gentamicin-treated bladders (intracellular population) after
washing 3 times with PBS to remove the extracellular, adherent
bacteria (luminal population). Fewer UTI89.DELTA.qseC bacteria are
internalized, indicating that the qseC mutant is less efficient in
invading the host bladder compared to wt UTI89. The average of 3
independent experiments is shown (*, P<0.02; **, P<0.003, by
two-tailed Mann-Whitney).
[0102] FIG. 53A-D depicts a diagram detailing and data showing that
restoring production of type 1 pili in UTI89.DELTA.qseC does not
influence other .DELTA.qseC-mediated defects. (FIG. 53A) Schematic
showing the strategy used to lock the phase-variable fim promoter
in the ON orientation. (FIG. 53B) Hemagglutination assays and (FIG.
53C) western blot analyses depicting restoration of type 1 pili
expression in UTI89.DELTA.qseC locked ON strain (.DELTA.qseC_LON).
(FIG. 53D) Assessment of curli production on YESCA-CR agar verifies
that, similarly to UTI89.DELTA.qseC, UTI89.DELTA.qseC_LON remains
defective for curli expression, exhibiting a white and smooth
morphotype. In contrast, wt UTI89 and UTI89_LON appear red, dry and
rough a phenotype indicative of curli expression.
[0103] FIG. 54 depicts a graph showing co-inhibition of type 1 pili
and QseC as a prophylactic measure for UTIs. Chart depicting the
bladder cfu obtained at 2 wks post infection from mice pre-treated
with mannoside and subsequently infected with either wt UTI89, or
UTI89.DELTA.qseC (used as a proxy for a QseC inhibitor).
Significantly fewer cfu (1.5-log reduction) were obtained from
pre-treated mice infected with UTI89.DELTA.qseC. The average of 3
independent experiments is shown (**, P<0.01; ***, P<0.001,
by two-tailed Mann-Whitney). UTI89_MAN, mice pre-treated with
mannoside and challenged with wt UTI89; .DELTA.qseC_MAN, mice
pre-treated with mannoside and challenged with UTI89.DELTA.qseC;
UTI89, mice pre-treated with PBS and challenged with wt UTI89;
.DELTA.qseC, mice pre-treated with PBS and challenged with
UTI89.DELTA.qseC.
DETAILED DESCRIPTION
[0104] Compounds that inhibit the function of type I pili of
bacteria have been developed. The compounds may be useful for the
treatment of urinary tract infections. Significantly, the compounds
may prevent bacterial colonization and invasion of the bladder
tissue to prevent infection and the establishment of reservoirs
that can serve as a source of recurrent infections. The invention
also encompasses methods of use of a compound of the invention.
I. Compounds
[0105] One aspect of the invention is a compound of Formula
(I):
##STR00005##
wherein: [0106] X is selected from the group consisting of
hydrogen, OR.sup.2, SR.sup.2, and NR.sup.z; [0107] Z is selected
from the group consisting of O, S, CR.sup.3 and NR.sup.4; [0108] Y
is selected from the group consisting of oxygen, sulfur, CR.sup.3,
NR.sup.4, --N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--,
--CH.sub.2N(R.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, NR.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0109] R.sup.2 is independently selected from the group consisting
of hydrogen, COR.sup.x, CONR.sup.x, hydrocarbyl, and substituted
hydrocarbyl; [0110] R.sup.3, R.sup.4, R.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0111] R.sup.z is independently selected
from the group consisting of hydrogen hydrocarbyl, substituted
hydrocarbyl, --COR.sup.x, --CONR.sup.xR.sup.xSO.sub.2R.sup.x, and
--CO.sub.2R*; [0112] R.sup.z is independently selected from the
group consisting of hydrogen, NR.sup.4R.sup.5, and an optionally
substituted alkyl, cycloalkyl, heterocycle, or aryl; [0113] n is an
integer from 1 to 10; and [0114] R.sup.1 is selected from the group
comprising Formulas (IIIA), (IIIB), (IV), (V), (VI), (VII) and
(VIII):
##STR00006##
[0114] wherein: [0115] A is independently selected from the group
consisting of CR.sup.6 and N; [0116] G is selected from the group
consisting of S, O, CR.sup.8, and NR.sup.9; [0117] R.sup.6, R.sup.8
and R.sup.9 are independently selected from the group consisting of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0118]
R.sup.10, R.sup.11, R.sup.12, R.sup.13 and R.sup.14 are
independently selected from the group consisting of hydrogen,
--OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.15,
--CONR.sup.16R.sup.17, --SO.sub.2NR.sup.18R.sup.19, alkenyl,
alkynyl, hydrocarbyl, and substituted hydrocarbyl; [0119] R.sup.15,
R.sup.16, R.sup.17. R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; and [0120] a is either the
integer 1 or the integer 2.
[0121] One embodiment of formula (I), where R.sup.1 is formula
(IIIA), comprises formula (IX):
##STR00007##
wherein: [0122] X is selected from the group consisting of
hydrogen, OR.sup.2, SR.sup.2, NR.sup.z; [0123] Z is selected from
the group consisting of O, S, OR.sup.3 and NR.sup.4; [0124] Y is
selected from the group consisting of oxygen, sulfur, OR.sup.3,
NR.sup.4, --N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--,
--CH.sub.2N(R.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, NR.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0125] R.sup.2, R.sup.3, R.sup.4, R.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0126] n is an integer from 1 to 10;
[0127] A is independently selected from the group consisting of
CR.sup.6 and N; [0128] R.sup.6, R.sup.8 and R.sup.9 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0129] R.sup.10 is
independently selected from the group consisting of hydrogen,
--OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.15,
--CONR.sup.16R.sup.17, --SO.sub.2NR.sup.18R.sup.19, alkenyl,
alkynyl, hydrocarbyl, and substituted hydrocarbyl; [0130] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; [0131] R.sup.z is
independently selected from the group consisting of hydrogen
hydrocarbyl, substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; [0132]
R.sup.x is independently selected from the group consisting of
hydrogen, --NR.sup.4R.sup.5, and an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl; and [0133] a is either the
integer 1 or the integer 2.
[0134] In an exemplary embodiment of a compound of formula (IX),
R.sup.10 is aryl or heterocyle.
[0135] Another embodiment of formula (I), where R.sup.1 is formula
(NIB), comprises formula (X):
##STR00008##
wherein [0136] X is selected from the group consisting of hydrogen,
OR.sup.2, SR.sup.2, and NR.sup.z; [0137] Z is selected from the
group consisting of O, S, CR.sup.3 and NR.sup.4; [0138] Y is
selected from the group consisting of oxygen, sulfur, CR.sup.3,
NR.sup.4, --N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--,
--CH.sub.2N(R.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, NR.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0139] R.sup.2 is independently selected from the group consisting
of hydrogen, --COR.sup.x, and --CONR.sup.x; [0140] R.sup.3,
R.sup.4, R.sup.5 are independently selected from the group
consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl;
[0141] R.sup.z is independently selected from the group consisting
of hydrogen hydrocarbyl, substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; [0142]
R.sup.x is independently selected from the group consisting of
hydrogen, --NR.sup.4R.sup.5, and an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl; [0143] n is an integer from 1 to
10; [0144] A is independently selected from the group consisting of
CR.sup.6 and N; [0145] G is selected from the group consisting of
S, O, CR.sup.8, and NR.sup.9; [0146] R.sup.6, R.sup.8 and R.sup.9
are independently selected from the group consisting [0147] of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0148] R.sup.10
is independently selected from the group consisting of hydrogen,
OR.sup.15, NR.sup.15R.sup.16, NR.sup.15COR.sup.16,
NR.sup.15CO.sub.2R.sup.16, NR.sup.15CONR.sup.16,
NR.sup.15SO.sub.2R.sup.16, COR.sup.15, SO.sub.2R.sup.15, nitro,
cyano, halogen, aryl, heterocycle, COOR.sup.15,
CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19, alkenyl, alkynyl,
hydrocarbyl, and substituted hydrocarbyl; and [0149] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; and [0150] a is either the
integer 1 or the integer 2.
[0151] Yet another embodiment of formula (I), where R.sup.1 is
formula (IV), comprises formula (XI):
##STR00009##
wherein [0152] X is selected from the group consisting of hydrogen,
OR.sup.2, SR.sup.2, NR.sup.z; [0153] Z is selected from the group
consisting of O, S, CR.sup.3 and NR.sup.4; [0154] Y is selected
from the group consisting of oxygen, sulfur, CR.sup.3, NR.sup.4,
--N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--, --CH.sub.2N(R.sup.5)CO--,
CO.sub.2, SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO,
--CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, --N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--,
NR.sup.5, and an optionally substituted alkyl, alkene, alkyne, or
heterocycle; [0155] R.sup.2, R.sup.3, R.sup.4, R.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0156] n is an integer
from 1 to 10; [0157] A is independently selected from the group
consisting of CR.sup.6 and N; [0158] R.sup.6, R.sup.8 and R.sup.9
are independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0159] R.sup.11 and
R.sup.12 are independently selected from the group consisting of
hydrogen, --OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, [0160] --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, COOR.sup.15,
CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19, alkenyl, alkynyl,
hydrocarbyl, and substituted hydrocarbyl; [0161] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; [0162] R.sup.z is
independently selected from the group consisting of hydrogen
hydrocarbyl, substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; [0163]
R.sup.x is independently selected from the group consisting of
hydrogen, --NR.sup.4R.sup.5, and an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl; and a is either the integer 1 or
the integer 2.
[0164] Still another embodiment of formula (I), where R.sup.1 is
formula (V), comprises formula (XII):
##STR00010##
wherein [0165] X is selected from the group consisting of hydrogen,
OR.sup.2, SR.sup.2, NR.sup.z; [0166] Z is selected from the group
consisting of O, S, CR.sup.3 and NR.sup.4; [0167] Y is selected
from the group consisting of oxygen, sulfur, CR.sup.3, NR.sup.4,
--N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--, --CH.sub.2N(R.sup.5)CO--,
CO.sub.2, SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO,
--CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, --N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--,
NR.sup.5, and an optionally substituted alkyl, alkene, alkyne, or
heterocycle; [0168] R.sup.2, R.sup.3, R.sup.4, R.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0169] n is an integer
from 1 to 10 [0170] A is independently selected from the group
consisting of CR.sup.6 and N; [0171] G is selected from the group
consisting of S, O, CR.sup.8, and NR.sup.9; [0172] R.sup.6, R.sup.8
and R.sup.9 are independently selected from the group consisting of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0173] R.sup.13
and R.sup.14 are independently selected from the group consisting
of hydrogen, --OR.sup.15, --NR.sup.15R.sup.16,
--NR.sup.15COR.sup.16, --NR.sup.15CO.sub.2R.sup.16,
--NR.sup.15CONR.sup.16, --NR.sup.15SO.sub.2R.sup.16, --COR.sup.15,
--SO.sub.2R.sup.15, nitro, cyano, halogen, aryl, heterocycle,
COOR.sup.15, CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19,
alkenyl, alkynyl, hydrocarbyl, and substituted hydrocarbyl; [0174]
R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently
selected from the group consisting of hydrogen, hydrocarbyl,
substituted hydrocarbyl, aryl, and heterocycle, and R.sup.16 and
R.sup.17 together can optionally form a ring, and R.sup.18 and
R.sup.19 together can optionally form a ring; [0175] R.sup.z is
independently selected from the group consisting of hydrogen
hydrocarbyl, substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; [0176]
R.sup.x is independently selected from the group consisting of
hydrogen, --NR.sup.4R.sup.5, and an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl; and a is either the integer 1 or
the integer 2.
[0177] Yet still another embodiment of formula (I), where R.sup.1
is formula (VI), comprises formula (XIII):
##STR00011##
wherein [0178] X is selected from the group consisting of hydrogen,
OR.sup.2, SR.sup.2, NR.sup.z; [0179] Z is selected from the group
consisting of O, S, CR.sup.3 and NR.sup.4; [0180] Y is selected
from the group consisting of oxygen, sulfur, CR.sup.3, NR.sup.4,
--N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--, --CH.sub.2N(R.sup.5)CO--,
CO.sub.2, SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO,
--CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, --N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--,
NR.sup.5, and an optionally substituted alkyl, alkene, alkyne, or
heterocycle; [0181] R.sup.2, R.sup.3, R.sup.4, R.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0182] n is an integer
from 1 to 10; [0183] A is independently selected from the group
consisting of CR.sup.6 and N; [0184] R.sup.6 is independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0185] R.sup.13 and R.sup.14 are
independently selected from the group consisting of hydrogen,
--OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, COOR.sup.15,
CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19, alkenyl, alkynyl,
hydrocarbyl, and substituted hydrocarbyl; [0186] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; [0187] R.sup.z is
independently selected from the group consisting of hydrogen
hydrocarbyl, substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; [0188]
R.sup.x is independently selected from the group consisting of
hydrogen, --NR.sup.4R.sup.5, and an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl; and [0189] a is either the
integer 1 or the integer 2.
[0190] Still another embodiment of formula (I), where R.sup.1 is
formula (VII), comprises formula (XIV):
##STR00012##
wherein [0191] X is selected from the group consisting of hydrogen,
OR.sup.2, SR.sup.2, NR.sup.z; [0192] Z is selected from the group
consisting of O, S, CR.sup.3 and NR.sup.4; [0193] Y is selected
from the group consisting of oxygen, sulfur, CR.sup.3, NR.sup.4,
--N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--, --CH.sub.2N(R.sup.5)CO--,
CO.sub.2, SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO,
--CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, --N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--,
NR.sup.5, and an optionally substituted alkyl, alkene, alkyne, or
heterocycle; [0194] R.sup.2, R.sup.3, R.sup.4, R.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0195] n is an integer
from 1 to 10; [0196] A is independently selected from the group
consisting of CR.sup.6 and N; [0197] R.sup.6 is independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0198] R.sup.13 and R.sup.14 are
independently selected from the group consisting of hydrogen,
--OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, COOR.sup.15,
CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19, alkenyl, alkynyl,
hydrocarbyl, and substituted hydrocarbyl; [0199] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; [0200] R.sup.z is
independently selected from the group consisting of hydrogen
hydrocarbyl, substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; [0201]
R.sup.x is independently selected from the group consisting of
hydrogen, --NR.sup.4R.sup.5, and an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl; and [0202] a is either the
integer 1 or the integer 2.
[0203] A further embodiment of formula (I), where R.sup.1 is
formula (VIII), comprises formula (XV):
##STR00013##
wherein [0204] X is selected from the group consisting of hydrogen,
OR.sup.2, SR.sup.2, and NR.sup.z; [0205] Z is selected from the
group consisting of O, S, CR.sup.3 and NR.sup.4; [0206] Y is
selected from the group consisting of oxygen, sulfur, CR.sup.3,
NR.sup.4, --N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--,
--CH.sub.2N(R.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, NR.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0207] R.sup.2, R.sup.3, R.sup.4, R.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0208] n is an integer from 1 to 10;
[0209] A is independently selected from the group consisting of
CR.sup.6 and N; [0210] G is selected from the group consisting of
S, O, CR.sup.8, and NR.sup.9; [0211] R.sup.6, R.sup.8 and R.sup.9
are independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0212] R.sup.11 and
R.sup.12 are independently selected from the group consisting of
hydrogen, --OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, COOR.sup.15,
CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19, alkenyl, alkynyl,
hydrocarbyl, and substituted hydrocarbyl; [0213] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; [0214] R.sup.z is
independently selected from the group consisting of hydrogen
hydrocarbyl, substituted hydrocarbyl, --COR.sup.x,
--CONR.sup.xR.sup.xSO.sub.2R.sup.x, and --CO.sub.2R.sup.x; [0215]
R.sup.x is independently selected from the group consisting of
hydrogen, --NR.sup.4R.sup.5, and an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl; and [0216] a is either the
integer 1 or the integer 2.
[0217] A still further another embodiment of formula (I), where
R.sup.1 is formula (IV), comprises formula (XVI):
##STR00014##
wherein [0218] Z is selected from the group consisting of O, S,
CR.sup.3 and NR.sup.4; [0219] Y is selected from the group
consisting of O, S, CR.sup.3, and NR.sup.4; [0220] A is
independently selected from the group consisting of CR.sup.6 and N;
[0221] R.sup.3, R.sup.4, and R.sup.6 are independently selected
from the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0222] R.sup.11 is independently selected from the
group consisting of hydrogen, --NR.sup.5, --OR.sup.5, nitro, cyano,
chloro, bromo, iodo, fluoro, --COOR.sup.15, --CONR.sup.16R.sup.17,
--SO.sub.2R.sup.15, --SO.sub.2NR.sup.18R.sup.19, methyl,
hydrocarbyl, and substituted hydrocarbyl; [0223] R.sup.12 is
independently selected from the group consisting of hydrogen,
--OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.15,
--CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19, hydrocarbyl, and
substituted hydrocarbyl; and [0224] R.sup.15, R.sup.16, R.sup.17,
R.sup.18, R.sup.19 are independently selected from the group
consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,
and heterocycle, and R.sup.16 and R.sup.17 together can optionally
form a ring, and R.sup.18 and R.sup.19 together can optionally form
a ring; and [0225] a is either the integer 1 or the integer 2.
[0226] A still further another embodiment of formula (I), where
R.sup.1 is formula (IV), comprises formula (XVII):
##STR00015##
wherein [0227] Y is selected from the group consisting of O, S,
CR.sup.3, and NR.sup.4; [0228] A is independently selected from the
group consisting of CR.sup.6 and N; [0229] R.sup.3, R.sup.4, and
R.sup.6 are independently selected from the group consisting of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0230] R.sup.11
is selected from the group consisting of hydrogen, --NR.sup.5,
--OR.sup.5, nitro, cyano, chloro, bromo, iodo, fluoro,
--COOR.sup.15, --CONR.sup.16R.sup.17, --SO.sub.2R.sup.15,
--SO.sub.2NR.sup.18R.sup.19, methyl, hydrocarbyl, and substituted
hydrocarbyl; [0231] R.sup.12 is selected from the group consisting
of hydrogen, --OR.sup.15, --NR.sup.15R.sup.16,
--NR.sup.15COR.sup.16, --NR.sub.15CO.sub.2R.sup.16,
--NR.sup.15CONR.sup.16, --NR.sup.15SO.sub.2R.sup.16, --COR.sup.15,
--SO.sub.2R.sup.15, nitro, cyano, halogen, aryl, heterocycle,
--COOR.sup.15, --CONR.sup.16R.sup.17, --SO.sub.2NR.sup.18R.sup.19,
hydrocarbyl, and substituted hydrocarbyl; [0232] R selected from
the group consisting of --NR.sup.15SO.sub.2R.sup.16, --COOR.sup.15,
--NR.sup.15CONR.sup.16, --CONR.sup.16R.sup.17,
--SO.sub.2NR.sup.18R.sup.19; and [0233] R.sup.15, R.sup.16,
R.sup.17, R.sup.18, R.sup.19 are independently selected from the
group consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl,
aryl, and heterocycle, and R.sup.16 and R.sup.17 together can
optionally form a ring, and R.sup.18 and R.sup.19 together can
optionally form a ring.
[0234] In a preferred embodiment of formula (XVII), R.sup.11 is
selected from the group consisting of chloro, bromo, iodo, and
fluoro, R.sup.12 is selected from the group consisting of nitro,
cyano, --COOR.sup.15, --CONR.sup.16R.sup.17, and
--SO.sub.2NR.sup.18R.sup.19, and R selected from the group
consisting of --COOR.sup.15, --NR.sup.15CONR.sup.16,
--CONR.sup.16R.sup.17, and --SO.sub.2NR.sup.18R.sup.19.
[0235] In another preferred embodiment of formula (XVII), R.sup.11
is chloro, R.sup.12 is selected from the group consisting of nitro,
cyano, --COOR.sup.15, --CONR.sup.16R.sup.17, and
--SO.sub.2NR.sup.18R.sup.19, and R selected from the group
consisting of --COOR.sup.15, --NR.sup.15CONR.sup.16,
--CONR.sup.16R.sup.17, and --SO.sub.2NR.sup.18R.sup.19.
[0236] A yet further embodiment of formula (I), where R.sup.1 is
formula (VIII), comprises formula (XVIII):
##STR00016##
wherein [0237] Z is selected from the group consisting of O, S,
CR.sup.3 and NR.sup.4; [0238] Y is selected from the group
consisting of O, S, CR.sup.3, and NR.sup.4; [0239] A is
independently selected from the group consisting of CR.sup.6 and N;
[0240] G is selected from the group consisting of S, O, CR.sup.8,
and NR.sup.9; [0241] R.sup.3, R.sup.4, R.sup.6, R.sup.8, and
R.sup.9 are independently selected from the group consisting of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0242] R.sup.11
is independently selected from the group consisting of hydrogen,
--NR.sup.5, --OR.sup.5, nitro, cyano, chloro, bromo, iodo, fluoro,
--COOR.sup.15, --CONR.sup.16R.sup.17, --SO.sub.2R.sup.15,
--SO.sub.2NR.sup.18R.sup.19, methyl, hydrocarbyl, and substituted
hydrocarbyl; [0243] R.sup.12 is independently selected from the
group consisting of hydrogen, --OR.sup.15, --NR.sup.15R.sup.16,
--NR.sup.15COR.sup.16, --NR.sup.15CO.sub.2R.sup.16,
--NR.sup.15CONR.sup.16, --NR.sup.15SO.sub.2R.sup.16, --COR.sup.15,
--SO.sub.2R.sup.15, nitro, cyano, halogen, aryl, heterocycle,
--COOR.sup.15, --CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19,
hydrocarbyl, and substituted hydrocarbyl; and [0244] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; and [0245] a is either the
integer 1 or the integer 2.
[0246] In some embodiments of formula (I), where R.sup.1 is formula
(VIII), comprises formula (XIX):
##STR00017##
wherein [0247] Y is selected from the group consisting of O, S,
CR.sup.3, and NR.sup.4; [0248] A is independently selected from the
group consisting of CR.sup.6 and N; [0249] G is selected from the
group consisting of S, O, CR.sup.8, and NR.sup.9; [0250] R.sup.3,
R.sup.4, R.sup.6, R.sup.8, and R.sup.9 are independently selected
from the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0251] R.sup.11 is independently selected from the
group consisting of hydrogen, --NR.sup.5, --OR.sup.5, nitro, cyano,
chloro, bromo, iodo, fluoro, --COOR.sup.15, --CONR.sup.16R.sup.17,
--SO.sub.2R.sup.15, --SO.sub.2NR.sup.18R.sup.19, methyl,
hydrocarbyl, and substituted hydrocarbyl; [0252] R.sup.12 is
independently selected from the group consisting of hydrogen,
--OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.15,
--CONR.sup.16R.sup.17, SO.sub.2NR.sup.18R.sup.19, hydrocarbyl, and
substituted hydrocarbyl; and [0253] R.sup.15, R.sup.16, R.sup.17,
R.sup.18, R.sup.19 are independently selected from the group
consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,
and heterocycle, and R.sup.16 and R.sup.17 together can optionally
form a ring, and R.sup.18 and R.sup.19 together can optionally form
a ring.
[0254] In a preferred embodiment of formula (XIX), R.sup.11 is
selected from the group consisting of chloro, bromo, iodo, and
fluoro, R.sup.12 is selected from the group consisting of
--COOR.sup.15, and --CONR.sup.16R.sup.17, and R.sup.11 selected
from the group consisting of --COOR.sup.15, --NR.sup.15CONR.sup.16,
--CONR.sup.16R.sup.17, and --SO.sub.2NR.sup.18R.sup.19.
[0255] In another preferred embodiment of formula (XVII), R.sup.11
is chloro, R.sup.12 is selected from the group consisting of
--COOR.sup.15, and --CONR.sup.16R.sup.17, and R.sup.11 selected
from the group consisting of --COOR.sup.15, --NR.sup.15CONR.sup.16,
--CONR.sup.16R.sup.17, and --SO.sub.2NR.sup.18R.sup.19.
[0256] Another aspect of the present invention is a compound of
formula (II):
##STR00018##
wherein [0257] X is selected from the group consisting of hydrogen,
OR.sup.2, SR.sup.2, and NR.sup.z; [0258] Z is selected from the
group consisting of O, S, CR.sup.3 and NR.sup.4; [0259] Y is
selected from the group consisting of oxygen, sulfur, CR.sup.3,
NR.sup.4, --N(R.sup.5)CO--, --CH.sub.2N(R.sup.5)--,
--CH.sub.2N(R.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(R.sup.5)--, --SO.sub.2N(R.sup.5)--,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, NR.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0260] L is selected from --O(CH.sub.2).sub.nO--,
--S(CH.sub.2).sub.nS--, --N(CH.sub.2).sub.nN--,
--(CH.sub.2).sub.n--,
--O[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xO--,
--N(CH.sub.2).sub.mO(CH.sub.2).sub.nN--, heterocycle, alkene,
alkyne, --CON[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xNCO--,
--SO.sub.2N[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xNO.sub.2S--,
and NCO[(CH.sub.2).sub.mO(CH.sub.2).sub.n].sub.xCON--, where L is
bound to a ring of R.sup.1 at a meta or para position; [0261]
R.sup.2 is independently selected from the group consisting of
hydrogen, COR.sup.x, CONR.sup.x, hydrocarbyl, and substituted
hydrocarbyl; [0262] R.sup.3, R.sup.4, R.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl, [0263] R.sup.z is independently selected
from the group consisting of hydrogen hydrocarbyl, substituted
hydrocarbyl, --COR.sup.x, --CONR.sup.xR.sup.xSO.sub.2R.sup.x, and
--CO.sub.2R.sup.x; [0264] R.sup.x is independently selected from
the group consisting of hydrogen, NR.sup.4R.sup.5, and an
optionally substituted alkyl, cycloalkyl, heterocycle, or aryl;
[0265] m, n, and x are integers from 1 to 10; and [0266] R.sup.1 is
selected from the group comprising Formulas (IIIA), (IIIB), (IV),
(V), (VI), (VII) and (VIII):
##STR00019##
[0266] wherein [0267] A is independently selected from the group
consisting of CR.sup.6 and N; [0268] G is selected from the group
consisting of S, O, CR.sup.8, and NR.sup.9; [0269] R.sup.6, R.sup.8
and R.sup.9 are independently selected from the group consisting of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0270]
R.sup.10, R.sup.11, R.sup.12, R.sup.13 and R.sup.14 are
independently selected from the group consisting of hydrogen,
--OR.sup.15, --NR.sup.15R.sup.16, --NR.sup.15COR.sup.16,
--NR.sup.15CO.sub.2R.sup.16, --NR.sup.15CONR.sup.16,
--NR.sup.15SO.sub.2R.sup.16, --COR.sup.15, --SO.sub.2R.sup.15,
nitro, cyano, halogen, aryl, heterocycle, --COOR.sup.15,
--CONR.sup.16R.sup.17, --SO.sub.2NR.sup.18R.sup.19, alkenyl,
alkynyl, hydrocarbyl, and substituted hydrocarbyl; [0271] R.sup.15,
R.sup.16, R.sup.17, R.sup.18, R.sup.19 are independently selected
from the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle, and R.sup.16 and R.sup.17
together can optionally form a ring, and R.sup.18 and R.sup.19
together can optionally form a ring; and [0272] a is either the
integer 1 or the integer 2.
[0273] In an exemplary alternative of each of the foregoing
embodiments, a compound comprising formula (I) is a compound
comprising any of the Formulas in Table 15.
[0274] In a further exemplary alternative of each of the foregoing
embodiments, a compound comprising formula (I) is compound number
50, 75, 76, or 77.
[0275] Another aspect of the invention is a compound of Formula
(XXI):
##STR00020##
wherein [0276] X is selected from the group consisting of hydrogen,
OD.sup.2, SD.sup.2, ND.sup.z; [0277] Z is selected from the group
consisting of O, S, CD.sup.3 and ND.sup.4; [0278] Y is oxygen;
[0279] D.sup.2, D.sup.3, and D.sup.4 are independently selected
from the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0280] D.sup.5 is selected from the group consisting
of CF.sub.3, halogen, CH.sub.3, OMe, hydrocarbyl, and substituted
hydrocarbyl; [0281] D.sup.6, D.sup.7, D.sup.8, and D.sup.9 are
independently selected from the group consisting of hydrogen,
--COD.sup.10D.sup.11, --COND.sup.10D.sup.11, --COOD.sup.12, and
-ND.sup.12COND.sup.10, or D.sup.6 and D.sup.7 may optionally form a
cycloalkyl or heterocyclo ring, D.sup.7 and D.sup.8 may optionally
form a cycloalkyl or heterocyclo ring, and D.sup.8 and D.sup.9 may
optionally form a cycloalkyl or heterocyclo ring; and [0282]
D.sup.10, D.sup.11, and D.sup.12 are independently selected from
the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle; [0283] D.sup.18 and D.sup.19
are independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0284] D.sup.z is
independently selected from the group consisting of hydrogen,
hydrocarbyl, substituted hydrocarbyl, --COD.sup.x,
--COND.sup.xD.sup.xSO.sub.2D.sup.x, and --CO.sub.2D.sup.x; and
[0285] D.sup.x is independently selected from the group consisting
of hydrogen, --ND.sup.18D.sup.19, and an optionally substituted
alkyl, cycloalkyl, heterocycle, or aryl.
[0286] In one embodiment, a compound of the invention comprises
Formula (XXI), wherein [0287] X is selected from the group
consisting of OD.sup.2; [0288] Z is selected from the group
consisting of O, S, CD.sup.3 and ND.sup.4; [0289] Y is oxygen;
[0290] D.sup.2, D.sup.3, and D.sup.4 are independently selected
from the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0291] D.sup.5 is selected from the group consisting
of CF.sub.3, halogen, CH.sub.3, and OMe; [0292] D.sup.6, D.sup.7,
D.sup.8, and D.sup.9 are independently selected from the group
consisting of hydrogen, --COD.sup.10D.sup.11,
--COND.sup.10D.sup.11, --COOD.sup.12, and -ND.sup.12COND.sup.10, or
D.sup.6 and D.sup.7 may optionally form a cycloalkyl or heterocyclo
ring, D.sup.7 and D.sup.8 may optionally form a cycloalkyl or
heterocyclo ring, and D.sup.8 and D.sup.9 may optionally form a
cycloalkyl or heterocyclo ring; and [0293] D.sup.10, D.sup.11, and
D.sup.12 are independently selected from the group consisting of
hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl, and
heterocycle.
[0294] In another embodiment, a compound of the invention comprises
Formula (XXI), wherein [0295] X is selected from the group
consisting of OD.sup.2; [0296] Z is selected from the group
consisting of O, S, CD.sup.3 and ND.sup.4; [0297] Y is oxygen;
[0298] D.sup.2, D.sup.3, and D.sup.4 are independently selected
from the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0299] D.sup.5 is selected from the group consisting
of CF.sub.3, halogen, CH.sub.3, and OMe; [0300] D.sup.6 and D.sup.8
are hydrogen; [0301] D.sup.7 and D.sup.9 are independently selected
from the group consisting of hydrogen, --COD.sup.10D.sup.11,
--COND.sup.10D.sup.11, --COOD.sup.12, and -ND.sup.12COND.sup.10;
and [0302] D.sup.10, D.sup.11, and D.sup.12 are independently
selected from the group consisting of hydrogen, hydrocarbyl,
substituted hydrocarbyl, aryl, and heterocycle.
[0303] In yet another embodiment, a compound of the invention
comprises Formula (XXI), wherein [0304] X is OH; [0305] Z is O;
[0306] Y is oxygen; [0307] D.sup.2, D.sup.3, and D.sup.4 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0308] D.sup.5 is
selected from the group consisting of CF.sub.3, halogen, CH.sub.3,
and OMe; [0309] D.sup.6 and D.sup.8 are hydrogen; D.sup.7 and
D.sup.9 are independently selected from the group consisting of
hydrogen, --CONHCH.sub.3, --COOCH.sub.3, and --NHCONH.sub.2.
[0310] Another aspect of the invention is a compound of Formula
(XXII):
##STR00021##
wherein [0311] X is selected from the group consisting of hydrogen,
OD.sup.2, SD.sup.2, ND.sup.z; [0312] Z is selected from the group
consisting of O, S, CD.sup.3 and ND.sup.4; [0313] Y is selected
from the group consisting of sulfur, CD.sup.3, ND.sup.4, [0314]
N(D.sup.18)CO--, --CH.sub.2N(D.sup.18)-, --CH.sub.2N(D.sup.18)CO--,
CO.sub.2, SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO,
--CON(D.sup.18)-, --SO.sub.2N(D.sup.18)-, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, [0315] --N(CH.sub.2).sub.n--,
--(CH.sub.2).sub.n--, ND.sup.18, and an optionally substituted
alkyl, alkene, alkyne, or heterocycle; [0316] n is an integer from
1 to 10; [0317] D.sup.2, D.sup.3, and D.sup.4 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0318] D.sup.10 is selected from the group
consisting of hydrogen, CF.sub.3, halogen, CH.sub.3, OMe,
hydrocarbyl, and substituted hydrocarbyl; [0319] D.sup.11,
D.sup.12, D.sup.13, and D.sup.14 are independently selected from
the group consisting of hydrogen, --COD.sup.15D.sup.16,
--COND.sup.15D.sup.16, --COOD.sup.17, and -ND.sup.17COND.sup.15, or
D.sup.11 and D.sup.12 may optionally form a cycloalkyl or
heterocyclo ring, D.sup.12 and D.sup.13 may optionally form a
cycloalkyl or heterocyclo ring, and D.sup.13 and D.sup.14 may
optionally form a cycloalkyl or heterocyclo ring; [0320] D.sup.15,
D.sup.16, and D.sup.17 are independently selected from the group
consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,
and heterocycle; [0321] D.sup.18 and D.sup.19 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; and [0322] D.sup.z is independently
selected from the group consisting of hydrogen hydrocarbyl,
substituted hydrocarbyl, --COD.sup.x,
--COND.sup.xD.sup.xSO.sub.2D.sup.x, and --CO.sub.2D.sup.x; and
[0323] D.sup.x is independently selected from the group consisting
of hydrogen, --ND.sup.18D.sup.19, and an optionally substituted
alkyl, cycloalkyl, heterocycle, or aryl.
[0324] In one embodiment, a compound of the invention comprises
Formula (XXII), wherein [0325] X is OD.sup.2; [0326] Z is selected
from the group consisting of O, S, CD.sup.3 and ND.sup.4; [0327] Y
is selected from the group consisting of sulfur, CD.sup.3,
ND.sup.4, [0328] N(D.sup.18)CO--, --CH.sub.2N(D.sup.18)-,
--CH.sub.2N(D.sup.18)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(D.sup.18)-, --SO.sub.2N(D.sup.18)-,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--, [0329]
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, ND.sup.18, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0330] n is an integer from 1 to 10; [0331] D.sup.2, D.sup.3,
D.sup.4 and D.sup.18 are independently selected from the group
consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl;
[0332] D.sup.10 is selected from the group consisting of hydrogen,
CF.sub.3, halogen, CH.sub.3, and OMe; [0333] D.sup.11, D.sup.12,
D.sup.13, and D.sup.14 are independently selected from the group
consisting of hydrogen, --COD.sup.15D.sup.16,
--COND.sup.15D.sup.16, --COOD.sup.17, and -ND.sup.17COND.sup.15, or
D.sup.11 and D.sup.12 may optionally form a cycloalkyl or
heterocyclo ring, D.sup.12 and D.sup.13 may optionally form a
cycloalkyl or heterocyclo ring, and D.sup.13 and D.sup.14 may
optionally form a cycloalkyl or heterocyclo ring; and [0334]
D.sup.15, D.sup.16, and D.sup.17 are independently selected from
the group consisting of hydrogen, hydrocarbyl, substituted
hydrocarbyl, aryl, and heterocycle.
[0335] In another embodiment, a compound of the invention comprises
Formula (XXII), wherein [0336] X is OD.sup.2; [0337] Z is selected
from the group consisting of O, S, CD.sup.3 and ND.sup.4; [0338] Y
is selected from the group consisting of sulfur, CD.sup.3,
ND.sup.4, [0339] N(D.sup.18)CO--, --CH.sub.2N(D.sup.18)-,
--CH.sub.2N(D.sup.18)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(D.sup.18)-, --SO.sub.2N(D.sup.18)-,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--, [0340]
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, and ND.sup.18; [0341]
n is an integer from 1 to 10; [0342] D.sup.2, D.sup.3, D.sup.4 and
D.sup.18 are independently selected from the group consisting of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0343] D.sup.10
is selected from the group consisting of hydrogen, CF.sub.3,
halogen, CH.sub.3, and OMe; [0344] D.sup.11, D.sup.12, D.sup.13,
and D.sup.14 are independently selected from the group consisting
of hydrogen, --COD.sup.15D.sup.16, --COND.sup.15D.sup.16,
--COOD.sup.17, --ND.sup.17COND.sup.15; and [0345] D.sup.15,
D.sup.16, and D.sup.17 are independently selected from the group
consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,
and heterocycle.
[0346] In yet another embodiment, a compound of the invention
comprises Formula (XXII), wherein [0347] X is OH; [0348] Z is
selected from the group consisting of O, S, CD.sup.3 and ND.sup.4;
[0349] Y is selected from the group consisting of sulfur, CD.sup.3,
ND.sup.4, [0350] N(D.sup.18)CO--, and --CH.sub.2N(D.sup.18); [0351]
D.sup.3, D.sup.4 and D.sup.18 are independently selected from the
group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0352] D.sup.10 is selected from the group consisting
of hydrogen, CF.sub.3, halogen, CH.sub.3, and OMe; [0353] D.sup.11
and D.sup.13 are hydrogen; and [0354] D.sup.12 and D.sup.14 are
independently selected from the group consisting of hydrogen,
--CONHCH.sub.3, --COOCH.sub.3, and --NHCONH.sub.2.
[0355] In still yet another embodiment, a compound of the invention
comprises Formula (XXII), wherein [0356] X is OH; [0357] Z is
selected from the group consisting of O, S, CD.sup.3 and ND.sup.4;
[0358] Y is selected from the group consisting of CD.sup.3 and
--CH.sub.2N(D.sup.18); [0359] D.sup.3, D.sup.4 and D.sup.18 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0360] D.sup.10 is
selected from the group consisting of hydrogen, CF.sub.3, halogen,
CH.sub.3, and OMe; [0361] D.sup.11 and D.sup.13 are hydrogen; and
[0362] D.sup.12 and D.sup.14 are independently selected from the
group consisting of hydrogen, --CONHCH.sub.3, --COOCH.sub.3, and
--NHCONH.sub.2.
[0363] Yet another aspect of the invention is a compound of the
Formula (XXIII):
##STR00022##
wherein [0364] X is selected from the group consisting of hydrogen,
OD.sup.2, SD.sup.2, ND.sup.z; [0365] Z is selected from the group
consisting of O, S, CD.sup.3 and ND.sup.4; [0366] Y is selected
from the group consisting of oxygen, sulfur, CD.sup.3, ND.sup.4,
[0367] N(D.sup.5)CO--, --CH.sub.2N(D.sup.5)-,
--CH.sub.2N(D.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(D.sup.5)-, --SO.sub.2N(D.sup.5)-,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, ND.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0368] D.sup.2, D.sup.3, D.sup.4, D.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0369] n is an integer from 1 to 10;
[0370] A is independently selected from the group consisting of
CD.sup.6 and N; [0371] D.sup.6 is selected from the group
consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl;
[0372] D.sup.20 and D.sup.22 are selected from the group consisting
of hydrogen and --COOD.sup.15; [0373] D.sup.21 is selected from the
group consisting of hydrogen, a five membered cycloalkyl or
heterocyclo ring, and a halogen; [0374] D.sup.23 is selected from
the group consisting of hydrogen, halogen, hydrocarbyl, and
substituted hydrocarbyl; [0375] D.sup.15 is selected from the group
consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,
and heterocycle; [0376] D.sup.z is independently selected from the
group consisting of hydrogen hydrocarbyl, substituted hydrocarbyl,
--COD.sup.x, --COND.sup.xD.sup.xSO.sub.2D.sup.x, and
--CO.sub.2D.sup.x; and [0377] D.sup.x is independently selected
from the group consisting of hydrogen, --ND.sup.4D.sup.5, and an
optionally substituted alkyl, cycloalkyl, heterocycle, or aryl.
[0378] In one embodiment, a compound of the invention comprises
Formula (XXIII), wherein [0379] X is OD.sup.2; [0380] Z is selected
from the group consisting of O, S, CD.sup.3 and ND.sup.4; [0381] Y
is selected from the group consisting of oxygen, CD.sup.3,
--CH.sub.2N(D.sup.5)CO--; [0382] D.sup.2, D.sup.3, D.sup.4, D.sup.5
are independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0383] A is CD.sup.6;
[0384] D.sup.6 is selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0385] D.sup.20 and
D.sup.22 are selected from the group consisting of hydrogen and
--COOD.sup.15; [0386] D.sup.21 is selected from the group
consisting of hydrogen, halogen, and a five membered cycloalkyl or
heterocyclo ring; [0387] D.sup.23 is selected from the group
consisting of hydrogen, halogen, hydrocarbyl, and substituted
hydrocarbyl; and [0388] D.sup.15 is selected from the group
consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,
and heterocycle.
[0389] In another embodiment, a compound of the invention comprises
Formula (XXIII), wherein [0390] X is OH; [0391] Z is selected from
the group consisting of O, S, CD.sup.3 and ND.sup.4; [0392] Y is
selected from the group consisting of oxygen, CD.sup.3,
--CH.sub.2N(D.sup.5)CO--; [0393] D.sup.3, D.sup.4, and D.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0394] A is CD.sup.6;
[0395] D.sup.6 is selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0396] D.sup.20 and
D.sup.22 are selected from the group consisting of hydrogen and
--COOD.sup.15; [0397] D.sup.21 is selected from the group
consisting of hydrogen, halogen, a five membered cycloalkyl or
heterocyclo ring; [0398] D.sup.23 is selected from the group
consisting of hydrogen, halogen, hydrocarbyl, and substituted
hydrocarbyl; and [0399] D.sup.15 is selected from the group
consisting of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl,
and heterocycle.
[0400] In yet another embodiment, a compound of the invention
comprises Formula (XXIII), wherein [0401] X is OH; [0402] Z is
selected from the group consisting of O, S, CD.sup.3 and ND.sup.4;
[0403] Y is selected from the group consisting of oxygen, CD.sup.3,
--CH.sub.2N(D.sup.5)CO--; [0404] D.sup.3, D.sup.4, and D.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0405] A is CD.sup.6;
[0406] D.sup.6 is selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0407] D.sup.20 and
D.sup.22 are --COOD.sup.15; [0408] D.sup.21 and D.sup.23 and
hydrogen; and [0409] D.sup.15 is selected from the group consisting
of hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl, and
heterocycle.
[0410] In still yet another embodiment, a compound of the invention
comprises Formula (XXIII), wherein [0411] X is OH; [0412] Z is
selected from the group consisting of O, S, CD.sup.3 and ND.sup.4;
[0413] Y is selected from the group consisting of oxygen, CD.sup.3,
--CH.sub.2N(D.sup.5)CO--; [0414] D.sup.3, D.sup.4, D.sup.5,
D.sup.8, and D.sup.9 are independently selected from the group
consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl;
[0415] A is CD.sup.6; [0416] D.sup.6 is selected from the group
consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl;
[0417] D.sup.20 and D.sup.22 are hydrogen; [0418] D.sup.21 is
selected from the group consisting of hydrogen, halogen,
[0418] ##STR00023## [0419] wherein G is selected from the group
consisting of S, O, CD.sup.8, and ND.sup.9; and [0420] D.sup.23 is
selected from the group consisting of hydrogen, halogen,
hydrocarbyl, and substituted hydrocarbyl.
[0421] Still yet another aspect of the invention encompasses a
compound of Formula (XXIV):
##STR00024##
wherein [0422] X is selected from the group consisting of hydrogen,
OD.sup.2, SD.sup.2, and ND.sup.z; [0423] Z is selected from the
group consisting of O, S, CD.sup.3 and ND.sup.4; [0424] Y is
selected from the group consisting of oxygen, sulfur, CD.sup.3,
ND.sup.4, [0425] N(D.sup.5)CO--, --CH.sub.2N(D.sup.5)-,
--CH.sub.2N(D.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(D.sup.5)-, --SO.sub.2N(D.sup.5)-,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, ND.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0426] D.sup.2, D.sup.3, D.sup.4, D.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0427] n is an integer from 1 to 10;
[0428] A is independently selected from the group consisting of
CD.sup.6 and N; [0429] G is selected from the group consisting of
S, O, CD.sup.8, and ND.sup.9; [0430] D.sup.6, D.sup.8 and D.sup.9
are independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0431] D.sup.24 is
selected from the group consisting of hydrogen, halogen,
hydrocarbyl, and substituted hydrocarbyl; [0432] D.sup.25 and
D.sup.26 is selected from the group consisting of hydrogen,
--NHCONH.sub.2I--COOMe, and --CONHMe, and D.sup.25 and D.sup.26 can
optionally form a cycloalkyl or heterocyclo ring; [0433] D.sup.z is
independently selected from the group consisting of hydrogen
hydrocarbyl, substituted hydrocarbyl, --COD.sup.x,
--COND.sup.xD.sup.xSO.sub.2D.sup.x, and --CO.sub.2D.sup.x; and
[0434] D.sup.x is independently selected from the group consisting
of hydrogen, --ND.sup.4D.sup.5, or an optionally substituted alkyl,
cycloalkyl, heterocycle, or aryl.
[0435] In one embodiment, a compound of the invention encompasses
Formula (XXIV) wherein: [0436] X is selected from the group
consisting of hydrogen, OD.sup.2, and SD.sup.2; [0437] Z is
selected from the group consisting of O, S, CD.sup.3 and ND.sup.4;
[0438] Y is selected from the group consisting of oxygen, sulfur,
CD.sup.3, ND.sup.4, [0439] N(D.sup.5)CO--, --CH.sub.2N(D.sup.5)-,
--CH.sub.2N(D.sup.5)CO--, CO.sub.2, SO.sub.2, --CH.sub.2O--,
--CH.sub.2S--, CO, --CON(D.sup.5)-, --SO.sub.2N(D.sup.5)-,
--O(CH.sub.2).sub.n--, --S(CH.sub.2).sub.n--,
--N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--, ND.sup.5, and an
optionally substituted alkyl, alkene, alkyne, or heterocycle;
[0440] D.sup.2, D.sup.3, D.sup.4, D.sup.5 are independently
selected from the group consisting of hydrogen, hydrocarbyl, and
substituted hydrocarbyl; [0441] n is an integer from 1 to 10;
[0442] A is independently selected from the group consisting of
CD.sup.6 and N; [0443] G is selected from the group consisting of
S, O, CD.sup.8, and ND.sup.9; [0444] D.sup.6, D.sup.8 and D.sup.9
are independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0445] D.sup.24 is
selected from the group consisting of hydrogen, halogen,
hydrocarbyl, and substituted hydrocarbyl; [0446] D.sup.25 and
D.sup.26 is selected from the group consisting of hydrogen,
--NHCONH.sub.2, --COOMe, and --CONHMe, or D.sup.25 and D.sup.26 can
optionally form a cycloalkyl or heterocyclo ring.
[0447] In another embodiment, a compound of the invention
encompasses Formula (XXIV) wherein: [0448] X is OD.sup.2; [0449] Z
is selected from the group consisting of O, S, CD.sup.3 and
ND.sup.4; [0450] Y is selected from the group consisting of oxygen,
sulfur, CD.sup.3, ND.sup.4, [0451] N(D.sup.5)CO--,
--CH.sub.2N(D.sup.5)-, --CH.sub.2N(D.sup.5)CO--, CO.sub.2,
SO.sub.2, --CH.sub.2O--, --CH.sub.2S--, CO, --CON(D.sup.5)-,
--SO.sub.2N(D.sup.5)-, --O(CH.sub.2).sub.n--,
--S(CH.sub.2).sub.n--, --N(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--,
and ND.sup.5; [0452] D.sup.2, D.sup.3, D.sup.4, D.sup.5 are
independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0453] n is an integer
from 1 to 10; [0454] A is independently selected from the group
consisting of CD.sup.6 and N; [0455] G is selected from the group
consisting of S, O, CD.sup.8, and ND.sup.9; [0456] D.sup.6, D.sup.8
and D.sup.9 are independently selected from the group consisting of
hydrogen, hydrocarbyl, and substituted hydrocarbyl; [0457] D.sup.24
is selected from the group consisting of hydrogen, halogen,
hydrocarbyl, and substituted hydrocarbyl; [0458] D.sup.25 and
D.sup.26 are selected from the group consisting of hydrogen,
--NHCONH.sub.2I--COOMe, and --CONHMe, or D.sup.25 and D.sup.28 can
optionally form the structure:
##STR00025##
[0459] In yet another embodiment, a compound of the invention
encompasses a compound of Formula (XXIV) wherein: [0460] X is
OD.sup.2; [0461] Z is selected from the group consisting of O, S,
CD.sup.3 and ND.sup.4; [0462] Y is selected from the group
consisting of oxygen, CD.sup.3, ND.sup.4, [0463] and
--CH.sub.2N(D.sup.5)-; [0464] D.sup.2, D.sup.3, D.sup.4, D.sup.5
are independently selected from the group consisting of hydrogen,
hydrocarbyl, and substituted hydrocarbyl; [0465] A is independently
selected from the group consisting of CD.sup.6 and N; [0466] G is
selected from the group consisting of S, O, CD.sup.8, and ND.sup.9;
[0467] D.sup.6, D.sup.8 and D.sup.9 are independently selected from
the group consisting of hydrogen, hydrocarbyl, and substituted
hydrocarbyl; [0468] D.sup.24 is selected from the group consisting
of hydrogen, halogen, CH.sub.3, CF.sub.3, and OMe; [0469] D.sup.25
and D.sup.26 are selected from the group consisting of hydrogen,
--NHCONH.sub.2I--COOMe, and --CONHMe, or D.sup.25 and D.sup.26 can
optionally form the structure
##STR00026##
[0470] In an exemplary alternative of each of the foregoing
embodiments, a compound comprising Formula (XXI) is a compound
comprising any of the Formulas in Table 21 or 22.
[0471] In a further exemplary alternative of each of the foregoing
embodiments, a compound of the invention is 4ZFH284 or 4ZFH269 from
Table 22.
[0472] In certain embodiments, the sugar residue of the above
compounds may encompass any stereoisomer of mannose. In other
embodiments, the sugar residue of the above compounds may encompass
any stereoisomer of mannose other than glucose. In an exemplary
embodiment, the sugar residue of the above compounds is alpha D
mannose.
[0473] Exemplary methods of synthesizing a compound of the
invention are detailed in the Examples.
##STR00027##
[0474] A compound of the invention may also be an intermediate in
the synthesis of a compound of formula (I)-(XIX). For instance, in
one embodiment, a compound of the invention may be an ester
intermediate in the synthesis of a compound of formula (I)-(XIX).
In another embodiment, a compound of the invention may be a
boronate ester of a mannoside or a boronic acid ester of a
mannoside. In yet another embodiment, a compound of the invention
may have the formula (XX), wherein R' is selected from H, alkyl, or
both R' groups may together form a ring (for instance, see the
synthesis of compound 77 detailed in the Examples below). In still
another embodiment, a compound of the invention may be a compound
illustrated in Schemes 1, 2, 3, 1.sup.a, 2.sup.a, 3.sup.a, 4.sup.a,
or 5.sup.a in the Examples below.
[0475] A compound of the invention may also comprise an imaging
agent, such as a fluorescent moiety. For example, see compounds 98
and 99 of Table 15. In an exemplary embodiment, the imaging agent
is bound to the sugar portion of a compound of the invention,
either directly, or via a linker.
[0476] Compounds of the invention may block the function of FimH of
the type I pili of pathogenic bacteria and prevent bacterial
adherence and invasion and thus prevent bacterial amplification in
the IBC and subsequent spreading and repeated rounds of
amplification via new generation IBCs.
[0477] FimH functional assays used to measure activity of the
compounds are known to individuals skilled in the art. Non-limiting
examples of functional assays include hemmagglutination titer using
guinea pig red blood cells, affinity of binding to FimH, and the
ability of the compounds to prevent biofilm formation.
[0478] In some embodiments, activity of the compound is measured
using hemmagglutination titer of guinea pig red blood cells.
Hemagglutination of guinea pig red blood cells by type1 piliated
UPEC is dependent upon FimH mannose binding ability and serial
dilutions allow a quantitative analysis. Hemagglutination titer may
generally be defined as the amount of compound required for
decreasing hemagglutination by 75%. In some embodiments, the
hemmagglutination titer of the compound of the invention may be
less than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
.mu.M. In a preferred alternative of the embodiments, the
hemmagglutination titer of the compound of the invention may be
less than about 9 .mu.M. In another preferred alternative of the
embodiments, the hemmagglutiantion titer of the compound of the
invention may be less than about 7 .mu.M. In yet another preferred
alternative of the embodiments, the hemmagglutination titer of the
compound of the invention may less than about 1 .mu.M.
[0479] In yet other embodiments, activity of the compound may be
measured using the ability of the compound to prevent or disrupt
biofilm formation. In general, titration curves measuring the
ability of a compound inhibit biofilm formation may be performed to
determine the IC50. In some embodiments, the IC50 of the compound
may be less than about 700, 600, 500, 400, 300, 200 or 100 .mu.M.
In other embodiments, the IC50 of the compound may be less than
about 500, 400, 300, 200, 100, 50, 40, 30, 20 10, 9, 8, 7, 6, or 5
.mu.M. In preferred embodiments, the IC50 of the compound may be
less than about 20 .mu.M. In other preferred embodiments, the IC50
of the compound may be less than about 9 .mu.M.
II. Combinations
[0480] Another aspect of the present invention encompasses a
combination of a compound of the invention (described in Section I
above) with one or more bactericidal compounds. In some
embodiments, a compound of the invention may comprise a combination
with 1, 2, 3, 4, or 5 bactericidal compounds. In one embodiment,
the bactericidal compound is an antibiotic. Suitable antibiotics
are known in the art, and may include Amikacin, Gentamicin,
Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin,
Geldanamycin, Herbimycin, Carbacephem, Loracarbef, Ertapenem,
Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin,
Cefalotin, Cefalexin, Cephalosporins, Cefaclor, Cefamandole,
Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,
Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,
Ceftizoxime, Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin,
Vancomycin, Telavancin, Clindamycin, Lincomycin, Azithromycin,
Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,
Troleandomycin, Telithromycin, Spectinomycin, Aztreonam,
Furazolidone, Nitrofurantoin, Amoxicillin, Ampicillin, Azlocillin,
Carbenicillin, Cloxacillin, Didoxacillin, Fludoxacillin,
Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G,
Penicillin V, Piperacillin, Temocillin, Ticarcillin, Bacitracin,
Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin,
Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic add,
Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin,
Temafloxacin, Mafenide, Sulfonamidochrysoidine, Sulfacetamide,
Sulfadiazine, Silver sulfadiazine, Sulfamethizole, Sulfamethoxazole
(SMZ), Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim
(TMP), TrimethoprimSulfamethoxazole (such as Bactrim, Septra),
Demeclocycline, Doxycycline, Minocycline, Oxytetracycline,
Tetracycline, Clofazimine, Dapsone, Capreomycin, Cydoserine,
Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin,
Rifabutin, Rifapentine, Streptomycin, Arsphenamine,
Chloramphenicol, Fosfomycin, Fusidic add, Linezolid, Metronidazole,
Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Rifaximin,
Thiamphenicol, or Tinidazole. In an exemplary embodiment, the
antibiotic is TMP, SMZ, or a combination thereof.
III. Pharmaceutical Compositions
[0481] Yet another aspect of the invention encompasses a
pharmaceutical composition. A compound of the invention described
in section I above may exist in tautomeric, geometric or
stereoisomeric forms. The present invention contemplates all such
compounds, including cis- and trans-geometric isomers, E- and
Z-geometric isomers, R- and S-enantiomers, diastereomers,
d-isomers, l-isomers, the racemic mixtures thereof and other
mixtures thereof. Pharmaceutically acceptable salts of such
tautomeric, geometric or stereoisomeric forms are also included
within the invention. The terms "cis" and "trans", as used herein,
denote a form of geometric isomerism in which two carbon atoms
connected by a double bond will each have a hydrogen atom on the
same side of the double bond ("cis") or on opposite sides of the
double bond ("trans"). Some of the compounds described contain
alkenyl groups, and are meant to include both cis and trans or "E"
and "Z" geometric forms. Furthermore, some of the compounds
described contain one or more stereocenters and are meant to
include R, S, and mixtures of R and S forms for each stereocenter
present.
[0482] In a further embodiment, the inhibitors of the present
invention may be in the form of free bases or pharmaceutically
acceptable add addition salts thereof. The term
"pharmaceutically-acceptable salts" are salts commonly used to form
alkali metal salts and to form addition salts of free adds or free
bases. The nature of the salt may vary, provided that it is
pharmaceutically acceptable. Suitable pharmaceutically acceptable
acid addition salts of compounds for use in the present methods may
be prepared from an inorganic add or from an organic add. Examples
of such inorganic acids are hydrochloric, hydrobromic, hydroiodic,
nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic
acids may be selected from aliphatic, cydoaliphatic, aromatic,
araliphatic, heterocyclic, carboxylic and sulfonic classes of
organic adds, examples of which are formic, acetic, propionic,
succinic, glycolic, gluconic, lactic, malic, tartaric, citric,
ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic,
benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic,
mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,
benzenesulfonic, pantothenic, stearic, algenic, algenic,
hydroxybutyric, salicylic, galactaric and galacturonic add.
Suitable pharmaceutically-acceptable base addition salts of
compounds of use in the present methods include metallic salts made
from aluminum, calcium, lithium, magnesium, potassium, sodium and
zinc or organic salts made from N, N'-dibenzylethylenediamine,
chloroprocaine, choline, diethanolamine, ethylenediamine,
meglumine-(N-methylglucamine) and procaine. All of these salts may
be prepared by conventional means from the corresponding compound
by reacting, for example, the appropriate add or base with any of
the compounds of the invention.
[0483] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions, may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed, including synthetic mono- or diglycerides. In addition,
fatty adds such as oleic add are useful in the preparation of
injectables. Dimethyl acetamide, surfactants including ionic and
non-ionic detergents, and polyethylene glycols can be used.
Mixtures of solvents and wetting agents such as those discussed
above are also useful.
[0484] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, the compound is ordinarily combined with one or more
adjuvants appropriate to the indicated route of administration. If
administered per os, the compound can be admixed with lactose,
sucrose, starch powder, cellulose esters of alkanoic adds,
cellulose alkyl esters, talc, stearic add, magnesium stearate,
magnesium oxide, sodium and calcium salts of phosphoric and
sulfuric acids, gelatin, acacia gum, sodium alginate,
polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted
or encapsulated for convenient administration. Such capsules or
tablets can contain a controlled-release formulation as can be
provided in a dispersion of active compound in hydroxypropylmethyl
cellulose. In the case of capsules, tablets, and pills, the dosage
forms can also comprise buffering agents such as sodium citrate, or
magnesium or calcium carbonate or bicarbonate. Tablets and pills
can additionally be prepared with enteric coatings.
[0485] For therapeutic purposes, formulations for parenteral
administration may be in the form of aqueous or non-aqueous
isotonic sterile injection solutions or suspensions. These
solutions and suspensions may be prepared from sterile powders or
granules having one or more of the carriers or diluents mentioned
for use in the formulations for oral administration. The compounds
may be dissolved in water, polyethylene glycol, propylene glycol,
ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl
alcohol, sodium chloride, and/or various buffers. Other adjuvants
and modes of administration are well and widely known in the
pharmaceutical art. For instance, a compound of the invention may
be administered with a carrier. Non-limiting examples of such a
carrier include protein carriers and lipid carriers.
[0486] Liquid dosage forms for oral administration may include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs containing inert diluents commonly used in the
art, such as water. Such compositions may also comprise adjuvants,
such as wetting agents, emulsifying and suspending agents, and
sweetening, flavoring, and perfuming agents.
[0487] The amount of the compound of the invention that may be
combined with the carrier materials to produce a single dosage of
the composition will vary depending upon the subject and the
particular mode of administration. Those skilled in the art will
appreciate that dosages may also be determined with guidance from
Goodman & Goldman's The Pharmacological Basis of Therapeutics,
Ninth Edition (1996), Appendix II, pp. 1707-1711 and from Goodman
& Goldman's The Pharmacological Basis of Therapeutics, Tenth
Edition (2001), Appendix II, pp. 475-493.
[0488] A compound of the invention may also be formulated as a
prodrug. Such a prodrug formulation may increase the
bioavailability of a compound of the invention. In one embodiment,
the sugar portion of a compound of the invention may encompass a
prodrug. In another embodiment R.sup.1 may comprise a prodrug.
Non-limiting examples of a compound of the invention formulated as
a prodrug include the compounds below:
##STR00028##
IV. Methods of the Invention
[0489] Compounds of the invention may be used in methods of
treating a bacterial infection and methods of reducing resistance
to a bactericidal compound in a bacterium.
(a) Methods of Treating a Bacterial Infection
[0490] One embodiment of the invention encompasses a method for
treating bacterial infections. As used herein, "treating" refers to
preventing infection in a subject not currently infected, and
reducing or eliminating infection in a subject that is currently
infected. Generally, such a method comprises administering a
pharmaceutical composition comprising a compound of the invention
to a subject. As used herein, "subject" includes any mammal prone
to urinary tract infections by E. coli. In one embodiment, a
subject is prone to recurring UTIs. In some embodiments, a subject
may not have clinical symptoms of a UTI. In such embodiments, the
subject may have a latent infection. In other embodiments, a
subject may have clinical symptoms of a UTI.
[0491] In some embodiments, a compound of the invention may be
administered to a subject in combination with a bactericidal
compound as described in Section II above. When administered in a
combination, a compound of the invention may be administered
before, simultaneously, or after administration of a bactericidal
compound. When administered before or after a bactericidal
compound, the time between administration of a compound of the
invention and a bactericidal compound may be about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, or 60 min. In another embodiment, the time between
administration of a compound of the invention and a bactericidal
compound may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, or 72 hours.
[0492] A compound or pharmaceutical composition of the invention
may be administered by several different means that will deliver a
therapeutically effective dose. Such compositions may be
administered orally, parenterally, by inhalation spray, rectally,
intradermally, intracistemally, intraperitoneally, transdermally,
bucally, as an oral or nasal spray, topically (i.e. powders,
ointments or drops), or via a urinary cathetar in dosage unit
formulations containing conventional nontoxic pharmaceutically
acceptable carriers, adjuvants, and vehicles as desired. Topical
administration may also involve the use of transdermal
administration such as transdermal patches or iontophoresis
devices. The term parenteral as used herein includes subcutaneous,
intravenous, intramuscular, or intrasternal injection, or infusion
techniques. In an exemplary embodiment, the pharmaceutical
composition will be administered in an oral dosage form.
Formulation of drugs is discussed in, for example, Hoover, John E.,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa. (1975), and Liberman, H. A. and Lachman, L., Eds.,
Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.
(1980).
[0493] The amount of a compound of the invention that constitutes
an "effective amount" can and will vary. The amount will depend
upon a variety of factors, including whether the administration is
in single or multiple doses, and individual subject parameters
including age, physical condition, size, and weight. Those skilled
in the art will appreciate that dosages may also be determined with
guidance from Goodman & Goldman's The Pharmacological Basis of
Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and
from Goodman & Goldman's The Pharmacological Basis of
Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.
[0494] In order to selectively control the release of an inhibitor
to a particular region of the gastrointestinal tract for release,
the pharmaceutical compositions of the invention may be
manufactured into one or several dosage forms for the controlled,
sustained or timed release of one or more of the ingredients. In
this context, typically one or more of the ingredients forming the
pharmaceutical composition is microencapsulated or dry coated prior
to being formulated into one of the above forms. By varying the
amount and type of coating and its thickness, the timing and
location of release of a given ingredient or several ingredients
(in either the same dosage form, such as a multi-layered capsule,
or different dosage forms) may be varied.
[0495] In an exemplary embodiment, the coating may be an enteric
coating. The enteric coating generally will provide for controlled
release of the ingredient, such that drug release can be
accomplished at some generally predictable location in the lower
intestinal tract below the point at which drug release would occur
without the enteric coating. In certain embodiments, multiple
enteric coatings may be utilized. Multiple enteric coatings, in
certain embodiments, may be selected to release the ingredient or
combination of ingredients at various regions in the lower
gastrointestinal tract and at various times.
[0496] As will be appreciated by a skilled artisan, the
encapsulation or coating method can and will vary depending upon
the ingredients used to form the pharmaceutical composition and
coating, and the desired physical characteristics of the
microcapsules themselves. Additionally, more than one encapsulation
method may be employed so as to create a multi-layered
microcapsule, or the same encapsulation method may be employed
sequentially so as to create a multi-layered microcapsule. Suitable
methods of microencapsulation may include spray drying, spinning
disk encapsulation (also known as rotational suspension separation
encapsulation), supercritical fluid encapsulation, air suspension
microencapsulation, fluidized bed encapsulation, spray
cooling/chilling (including matrix encapsulation), extrusion
encapsulation, centrifugal extrusion, coacervation, alginate beads,
liposome encapsulation, inclusion encapsulation, colloidosome
encapsulation, sol-gel microencapsulation, and other methods of
microencapsulation known in the art. Detailed information
concerning materials, equipment and processes for preparing coated
dosage forms may be found in Pharmaceutical Dosage Forms: Tablets,
eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and in
Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery
Systems, 6th Ed. (Media, Pa.: Williams & Wilkins, 1995).
[0497] A bacterium may be contacted with a compound of the
invention in vivo, in vitro, in situ, or ex vivo. In some
embodiments, a bacterium may be directly contacted with the
compound of the invention. In other embodiments, an intracellular
bacterium may be contacted with a compound of the invention.
Suitable cells comprising one or more bacteria may be grown,
sub-cultured, stored and manipulated using standard techniques
known to individuals skilled in the art. Cell culture and
microbiological techniques for growing, culturing, storing, and
manipulating cells comprising one or more bacteria are commonly
known in the art.
(b) Methods of Reducing Bactericidal Resistance
[0498] Another method of the invention comprises reducing the
resistance of a bacterium to a bactericidal compound. Such a method
comprises contacting a bacterium resistant to a bactericidal
compound with a compound of the invention. For instance, a subject
infected with a bacterium resistant to a bactericidal compound may
be administered a compound of the invention, as described in
section IV(a) above. In an exemplary embodiment, a method comprises
contacting a bacterium resistant to an antibiotic with a compound
of the invention. In a further exemplary embodiment, a method
comprises contacting a bacterium resistant to TMP or SMZ with a
compound of the invention.
[0499] Methods of measuring resistance of a bacterium to an
antibiotic are known in the art. For more details, see the
examples.
(c) Methods of Treating Catheter-Associated Urinary Tract
Infections
[0500] In a further embodiment, a method of the invention
encompasses a method for treating catheter-associated urinary tract
infections. As used herein, "treating" refers to preventing
infection in a subject not currently infected, and reducing or
eliminating infection in a subject that is currently infected.
Generally, such a method comprises administering a pharmaceutical
composition comprising a compound of the invention to a subject.
For this embodiment, "subject" refers to any mammal with an
indwelling urinary catheter. In one embodiment, a subject with a
urinary catheter is prone to recurring UTIs. In some embodiments, a
subject with a urinary catheter may not have clinical symptoms of a
UTI. In such embodiments, the subject may have a latent infection.
In other embodiments, a subject with a urinary catheter may have
clinical symptoms of a UTI.
[0501] In some embodiments, a compound of the invention may be
administered to a subject in combination with a bactericidal
compound as described in Section II and IV(a) above.
V. Coatings
[0502] An additional aspect of the present invention encompasses
coatings comprising a compound of the invention. Such a coating may
be used on a medical device to prevent bacterial adherence or
infection of the host. Suitable means of coating medical devices
are known in the art. In one embodiment, a catheter may be coated
with a compound of the invention. In another embodiment, a urinary
catheter may be coated with a compound of the invention.
VI. Nutritional Supplement
[0503] An alternative aspect of the present invention encompasses a
nutritional supplement that comprises a compound of the invention.
Such a supplement may be used to treat a bacterial infection as
described in section IV above.
Definitions
[0504] The term "acyl," as used herein alone or as part of another
group, denotes the moiety formed by removal of the hydroxyl group
from the group --COOH of an organic carboxylic add, e.g., RC(O)--,
wherein R is R', R.sub.1O--, R'R.sub.2N--, or R.sub.1S--, R.sub.1
is hydrocarbyl, heterosubstituted hydrocarbyl, or heterocyclo and
R.sub.2 is hydrogen, hydrocarbyl or substituted hydrocarbyl.
[0505] The term "acyloxy," as used herein alone or as part of
another group, denotes an acyl group as described above bonded
through an oxygen linkage (--O--), e.g., RC(O)O-- wherein R is as
defined in connection with the term "acyl."
[0506] Unless otherwise indicated, the alkyl groups described
herein are preferably lower alkyl containing from one to eight
carbon atoms in the principal chain and up to 20 carbon atoms. They
may be straight or branched chain or cyclic and include methyl,
ethyl, propyl, isopropyl, butyl, hexyl and the like.
[0507] Unless otherwise indicated, the alkenyl groups described
herein are preferably lower alkenyl containing from two to eight
carbon atoms in the principal chain and up to 20 carbon atoms. They
may be straight or branched chain or cyclic and include ethenyl,
propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the
like.
[0508] Unless otherwise indicated, the alkynyl groups described
herein are preferably lower alkynyl containing from two to eight
carbon atoms in the principal chain and up to 20 carbon atoms. They
may be straight or branched chain and include ethynyl, propynyl,
butynyl, isobutynyl, hexynyl, and the like.
[0509] The terms "aryl" or "ar" as used herein alone or as part of
another group denote optionally substituted homocyclic aromatic
groups, preferably monocyclic or bicyclic groups containing from 6
to 12 carbons in the ring portion, such as phenyl, biphenyl,
naphthyl, substituted phenyl, substituted biphenyl or substituted
naphthyl. Phenyl and substituted phenyl are the more preferred
aryl.
[0510] As used herein, the term "functional group" includes a group
of atoms within a molecule that is responsible for certain
properties of the molecule and/or reactions in which it takes part.
Non-limiting examples of functional groups include, alkyl,
carboxyl, hydroxyl, amino, sulfonate, phosphate, phosphonate,
thiol, alkyne, azide, halogen, and the like.
[0511] The terms "halogen" or "halo" as used herein alone or as
part of another group refer to chlorine, bromine, fluorine, and
iodine.
[0512] The terms "heterocyclo" or "heterocyclic" as used herein
alone or as part of another group denote optionally substituted,
fully saturated or unsaturated, monocyclic or bicyclic, aromatic or
nonaromatic groups having at least one heteroatom in at least one
ring, and preferably 5 or 6 atoms in each ring. The heterocyclo
group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms,
and/or 1 to 4 nitrogen atoms in the ring, and may be bonded to the
remainder of the molecule through a carbon or heteroatom. Exemplary
heterocyclo include heteroaromatics such as furyl, thienyl,
pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl, or isoquinolinyl
and the like. Exemplary substituents include one or more of the
following groups: hydrocarbyl, substituted hydrocarbyl, keto,
hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy,
alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, thiol,
ketals, acetals, esters and ethers.
[0513] The term "heteroaromatic" as used herein alone or as part of
another group denote optionally substituted aromatic groups having
at least one heteroatom in at least one ring, and preferably 5 or 6
atoms in each ring. The heteroaromatic group preferably has 1 or 2
oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in
the ring, and may be bonded to the remainder of the molecule
through a carbon or heteroatom. Exemplary heteroaromatics include
furyl, thienyl, pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl,
or isoquinolinyl and the like. Exemplary substituents include one
or more of the following groups: hydrocarbyl, substituted
hydrocarbyl, keto, hydroxy, protected hydroxy, acyl, acyloxy,
alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino, nitro,
cyano, thiol, ketals, acetals, esters and ethers.
[0514] The terms "hydrocarbon" and "hydrocarbyl" as used herein
describe organic compounds or radicals consisting exclusively of
the elements carbon and hydrogen. These moieties include alkyl,
alkenyl, alkynyl, and aryl moieties. These moieties also include
alkyl, alkenyl, alkynyl, and aryl moieties substituted with other
aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenaryl
and alkynaryl. Unless otherwise indicated, these moieties
preferably comprise 1 to 20 carbon atoms.
[0515] The "substituted hydrocarbyl" moieties described herein are
hydrocarbyl moieties which are substituted with at least one atom
other than carbon, including moieties in which a carbon chain atom
is substituted with a hetero atom such as nitrogen, oxygen,
silicon, phosphorous, boron, sulfur, or a halogen atom. These
substituents include halogen, carbocycle, aryl, heterocyclo,
alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protected hydroxy,
keto, acyl, acyloxy, nitro, amino, amido, nitro, cyano, thiol,
ketals, acetals, esters and ethers.
[0516] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention. Those of skill in the art
should, however, in light of the present disclosure, appreciate
that many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention, therefore all
matter set forth or shown in the accompanying drawings is to be
interpreted as illustrative and not in a limiting sense.
EXAMPLES
[0517] The following examples illustrate various iterations of the
invention.
Example 1. Uropathogenic E. Coli (UPEC) Pathogenesis in the Urinary
Tract
[0518] Clinically, it has been presumed that UPEC infection
consists of a relatively simple extracellular colonization of the
luminal surface after inoculation of fecal flora into the bladder
via the urethra. In contrast, using a murine model of UPEC
infection of the UT, the inventors have detailed an unexpectedly
complex UPEC pathogenesis cycle that involves both intracellular
and extracellular niches (FIG. 1). Using genetic, biochemical and
cell biological approaches together with a variety of imaging
techniques including transmission, quick freeze-deep etch and
scanning electron microscopy, as well as confocal and time lapse
video microscopy, the inventors discovered that UPEC invade bladder
facet cells via a FimH-dependent mechanism (see below). After
invasion, cytoplasmic intracellular bacterial communities (IBCs)
are formed. Rapid replication of the initial invading bacteria
results in the formation of an early IBC of loosely-packed
rod-shaped bacteria. The bacteria continue to replicate and
progress to form a large densely packed mid-stage IBC of
morphologically coccoid bacteria, with biofilm-like characteristics
including positive periodic add-Schiff (PAS) staining and
differential gene expression throughout the community. After the
IBC matures, bacteria detach from the biomass, often become
filamentous, and spread to neighboring cells forming new generation
IBCs. Thus, the IBC pathway facilitates massive expansion of the
invading bacteria in a niche protected from host defenses.
Translational studies have shown that the majority of UPEC isolates
form IBCs when introduced into the murine bladder and that IBCs and
filamentous bacteria occur in the urine of human UTI patients.
Population dynamic studies conducted by the inventors using ex vivo
gentamicin protection assays demonstrated that .about.10.sup.4 UPEC
of an initial 10.sup.7 inoculum invaded the bladder tissue within
15 minutes after infection and that one percent of the invaded
bacteria went on to form IBCs, resulting in an average of 100 IBCs
per infected mouse bladder. If this is extrapolated to the human
situation, innate defenses in the bladder most likely prevent the
majority of bacterial inoculation events into the bladder from
leading to disease. However, the ramifications of the IBC cascade
are striking. Invasion of a single infecting bacterium can lead to
rapid expansion of the infection via IBC formation, replicating
within hours to 10.sup.4 bacteria and even higher numbers followed
by dispersal of the bacteria from the biomass and spreading to
neighboring cells to reinitiate the IBC cascade. This process
allows the bacteria to gain a critical foothold. Bacterial
descendants of the acute IBC cascade have been shown using a murine
model, to be able to form a quiescent intracellular reservoir (QIR)
that can persist, protected from antibiotics and seemingly
undetected by the host immune system even after the acute infection
is resolved and bacteria are no longer detectable in the urine.
Bacteria in the QIR can later seed a recurrent infection,
manifested by IBC formation, bacteruria and inflammation.
Example 2. FimH as a Therapeutic Target
[0519] There are several key implications from understanding UPEC
pathogenesis. Mannosides and pilicides that block FimH function
will prevent bacterial adherence and invasion and thus prevent
bacterial amplification in the IBC and subsequent spreading and
repeated rounds of amplification via new generation IBCs. These
compounds will have potent therapeutic activity by preventing
bacterial expansion which may also have the consequence of
eliminating or significantly reducing the QIR thus reducing
predisposition to recurrent infection.
Type 1 Pili/FimH are Critical for UPEC Pathogenesis in the UT.
[0520] Type 1 pili are essential cystitis virulence determinants.
Using scanning and high-resolution EM and the mouse cystitis model
developed by the inventors, it was shown that adhesive type 1
piliated bacteria are able to bind and invade host superficial
umbrella cells, while UPEC lacking type 1 pili are not.
Colonization and invasion of the bladder epithelium is dependent on
the FimH adhesion located at the distal end of the pilus that binds
mannose residues on bladder epithelial cells. High-resolution
freeze-dry/deep-etch EM revealed that FimH interacts directly with
receptors on the luminal surface of the bladder (FIG. 1). Standard
gentamicin protection assays of infected tissue culture cells and
ex vivo gentamicin treatment of infected bladders demonstrated that
fimH.sup.+ type 1 piliated clinical cystitis isolates, but not
fimH.sup.- mutants, could invade bladder epithelial cells. Using
immunohistochemistry and Pfim-gfp transcriptional fusions it was
demonstrated that type 1 pili are expressed within IBCs. Using
high-resolution EM, pilus-like fibers radiating from bacteria and
interacting with matrix material within the intracellular IBC were
also visualized (FIG. 1). These results combined with work showing
that type 1 pili are required for biofilm formation in in vitro
systems led to the hypothesis that type 1 pili promote IBC
formation and/or maintenance. Therefore, an anhydrotetracycline
(AHT) inducible fim strain was constructed which can be
"pre-piliate" UTI89 in vitro by growth in AHT before infecting
mouse bladders, allowing the initial invasion event to normally.
However, once inoculated into the mouse, AHT is no longer present,
fim transcription ceases and piliation is diluted upon each
bacterial division. Using this system, the earliest events of
colonization and invasion were identical between the wild type and
conditional strain. However, the inability of the conditional
strain to produce type 1 pili intracellularly abolished its ability
to form IBCs, as shown by confocal microscopy, and thus
dramatically attenuated virulence as determined by CFUs at later
time points. These results strongly suggest that type 1 pili are
required for the survival and proliferation of UPEC within
superficial facet cells. Additionally, this conditional mutant is
significantly impaired in its ability to form QIRs, arguing that
the bacteria in QIRs are descendents and thus dependent on the
acute IBC cascade.
Structural Studies of FimH and its Ligand.
[0521] Adhesive type 1 pili are prototypic structures of a family
of adhesive fibers produced by diverse Gram-negative bacteria via
the chaperone/usher assembly pathway. Using biochemistry,
mutational studies, nuclear magnetic resonance, and x-ray
crystallography, the molecular basis of pili assembled by the
chaperone/usher pathway in gram-negative bacteria, including type 1
pili of UPEC, were delineated (FIG. 2) The three dimensional
structure of FimH bound to its mannose receptor was solved in order
to gain a molecular snapshot of a critical initial event in UTI
pathogenesis (FIG. 3).
[0522] FimH is a two domain protein, with a receptor binding domain
linked to a typical pilin domain that joins the adhesin to the
pilus fiber. The structure of the complex of the FimC chaperone
bound to FimH (which was bound to D-mannopyranoside) was determined
to 2.8 .ANG. resolution. The mannose binding site of FimH is a deep
negatively charged pocket at the tip of its receptor-binding
domain. The FimH pocket engages in extensive hydrogen bonding to
mannose (FIG. 4), which are abundant in the oligosaccharide
moieties of uroplakins that coat the lumenal surface of the bladder
epithelium. A hydrophobic ridge surrounds the mannose binding
pocket in a manner that may facilitate polar interactions within
the FimH pocket. Mutational studies revealed that each residue is
critical in mannose binding and pathogenesis, emphasizing why the
pocket is invariant among UPEC isolates.
Development of Anti-Adhesives.
[0523] The FimH-mannose interaction was further investigated in an
effort to develop potential ligand-based antagonists of UTIs. The
chitobiose unit on oligomannose was found to bridge various mannose
derivatives to the asparagine in the Asn-X-Ser/Thr motif of FimH
resulting in higher affinity binding. Crystallization of FimH in
complex with oligomannose-3 revealed the mechanism of this higher
affinity binding. The non-reducing Man4 anchors into the
mannose-binding pocket while the GlcNAc folds over Thr51 allowing
specific interactions with a hydrophobic tyrosine gate. Heptyl
mannoside mimics the GlcNAc tail of oligomannose-3 and extends it
further to increase interactions outside the binding pocket
resulting in high affinity binding (Kd=5 nM). Based on the high
affinity of heptyl mannose for FimH, the ability of heptyl mannose
to reduce bacterial infection in our mouse model of UTI was tested.
First, biofilm formation as a surrogate for IBCs formed in the
bladder was evaluated. Heptyl mannose at 1 mM inhibited UPEC
biofilm formation in vitro, suggesting that the mannose binding
properties of the FimH adhesin is required for biofilm formation.
Thus, UPEC strain UTI89 was incubated with heptyl mannose prior to
inoculation into the bladders of mice. This resulted in a
significant attenuation of virulence at 6 hours post-infection at 5
mM heptyl mannose (FIG. 5). The ability of these compounds to
significantly attenuate virulence establishes mannosides as a
potential treatment for UTI. Therefore, more potent mannosides that
mimic the natural receptor for FimH but with increased affinity and
avidity in order to ultimately block bacterial colonization,
invasion, IBC formation and disease were developed as described
below.
Example 3. Genetic Analysis of UPEC Bacterial Isolates
[0524] Due to the necessity of FimH, it was tested to see if this
gene was under positive selection. Through sequencing of more than
200 isolates, the majority of which were UTI isolates, it was
discovered that residues on FimH are evolving under positive
selection, none of which were in the mannose binding pocket. These
residues are A48, A83 and V184 in UT189 (FIG. 6). Mutants were
constructed containing the other naturally occurring amino acids
commonly found at the positively selected positions. A mutation of
A83 to a serine resulted in a defect in binding in vitro, however
only a log drop in bacterial titers were observed in vivo. The
double mutant containing a valine at position 48 and an alanine at
position 184 resulted in a severe defect in vivo based on bacterial
titers and no defect in the ability to bind mannose in vitro.
Mutations in the non-positively selected amino adds had no
detectable effect. This study shows that residues outside the
binding pocket are also important to FimH's function in vivo
suggesting there is more to FimH than just its ability to bond to
the surface of the bladder through its mannose binding pocket.
[0525] Since FimH has been the only essential virulence factor
determined to date, it was investigated if the FimH from UT189
would result in establishing a more robust infection if it was put
into a clinical isolate containing an altered FimH. A panel of
clinical isolates was examined for their ability to form IBCs (FIG.
7). Isolates with a valine at position 48 seemed to perform worse
in vivo in regards to IBC formation.
Example 4. Role of FimH in Infection
[0526] To assess FimH's role in infection, the native FimH of the
clinical strain acute 4 (FIG. 7) was replaced with the FimH
sequence from UT189 under the control of the native promoter. This
was done through lambda red recombinase. The acute4 FimH was first
deleted and replaced with a chloramphenicol (Cm) cassette. The Cm
cassette was then replaced with UT189 FimH. The isolate was then
sequenced to confirm proper sequence.
[0527] Acute4 and acute4+UT189FimH were grown 2.times.24 hours to
obtain type 1 expression. C3H/HeN mice were then inoculated with
10.sup.7 of either strain. For bacterial titers (FIG. 8A), bladders
were harvested at 6 and 24 hours post-infection. At 6 hours
post-infection there was no significant difference in bacterial
load between the two stains and both strains were much lower that a
typical UT189 6 hour titer (10.sup.6). However, at 24 hours
post-infection, acute4+UT189FimH significantly colonized the murine
bladder more than actue 4 (p<0.001 by Mann-Whitney). 24 hour
titers in the acute4+UT 189FimH isolate were equivalent to what is
typically observed with UT 189. For IBC formation (FIG. 8B), LacZ
staining was performed at 6 hours post-infection. No IBCs were
observed in either isolate.
Example 5. Synthesis of Compounds
[0528] The compounds of the present invention may be prepared in a
number of ways well known to one skilled in the art of organic
synthesis. More specifically, the novel compounds of this invention
may be prepared using the reactions and techniques described
herein. In the description of the synthetic methods described
below, it is to be understood that all proposed reaction
conditions, including choice of solvent, reaction atmosphere,
reaction temperature duration of the experiment and workup
procedures, are chosen to be the conditions standard for that
reaction. It is understood by one skilled in the art of organic
synthesis that the functionality present on various portions of the
molecule must be compatible with the reagents and reactions
proposed. Such restrictions to the substituents, which are not
compatible with the reaction conditions, will be apparent to one
skilled in the art and alternate methods must then be used. Unless
otherwise stated, the starting materials for the examples contained
herein are either commercially available or are readily prepared by
standard methods from known materials. The compounds of Formula (I)
and (II) may be synthesized through standard organic chemistry
methodology and purification known to those trained in the skill
and art by using commercially available starting materials and
reagents. General procedures for synthesizing the compounds of the
invention are as follows:
[0529] The chemical synthesis of compounds with the general Formula
(I) and (II) is achieved using the routes described in Scheme 1
starting from commercially available .alpha.-D-mannose,
.alpha.-D-mannose pentaacetate (Route A), or .alpha.-methyl-D
mannose (Route B). Shown in Scheme 1A, Lewis-acid mediated
glycosidation of .alpha.-D-mannose pentaacetate followed by acetate
ester hydrolysis with sodium methoxide yields glycosides of Formula
(I). Further chemical transformation of the substituent R.sup.1 may
be accomplished using standard synthetic organic procedures such
as, but not limited to, metal-mediated aryl Suzuki coupling
reactions through aryl bromide or aryl boronate ester glycosides,
shown in Scheme 2A and 2B, respectively, to produce exemplified
compounds of Formula (I) described in Tables 1-9. All compounds are
also listed in Table 15.
##STR00029##
##STR00030##
[0530] The compounds of Formula (I) may be transformed into
compounds of Formula (II) as described in Scheme 3 by employing
various functional groups (R.sup.1) and linkers (L), such as, but
not limited to, alkyl ethers, esters, amines and amides.
##STR00031##
[0531] The invention is further described with reference to the
following illustrative examples in which, unless stated otherwise:
[0532] (i) temperatures are given in degrees Celsius (.degree. C.);
operations are carried out at room temperature or ambient
temperature, that is, in a range of 18-25.degree. C., unless
otherwise stated; [0533] (ii) solutions are dried over anhydrous
sodium sulphate or magnesium sulphate; evaporation of organic
solvent is carried out using a rotary evaporator under reduced
pressure (4.5-30 mmHg) with a bath temperature of up to 60.degree.
C.; [0534] (iii) chromatography means flash chromatography on
silica gel; thin layer chromatography (TLC) is carried out on
silica gel plates; [0535] (iv) in general, the course of reactions
are followed by TLC or liquid chromatography/mass spectroscopy
(LC/MS) and reaction times are given for illustration only; [0536]
(v) final products have satisfactory proton nuclear magnetic
resonance (NMR) spectra and/or mass spectra data; [0537] (vi)
yields are given for illustration only and are not necessarily
those which can be obtained by diligent process development;
preparations are repeated if more material is required; [0538]
(vii) when given, nuclear magnetic resonance (NMR) data is in the
form of delta (o) values for major diagnostic protons, given in
part per million (ppm) relative to tetra methylsilane (TMS) as an
internal standard, determined at 300 MHz ind.sub.3-MeOD unless
otherwise stated; [0539] (viii) chemical symbols have their usual
meanings; [0540] (ix) solvent ratio is given in volume: volume
(v/v) terms; [0541] (x) Purification of the compounds is carried
out using one or more of the following methods: [0542] a) flash
chromatography on regular silica gel; [0543] b) flash
chromatography on silica gel using an MPLC separation system
(Teledyne ISCO):prepackednormal phase flash column cartridge, flow
rate, 10-80 ml/min; [0544] c) Preparatory HPLC system using a
reverse-phase C18 column, 100.times.20 mm, 5 .mu.M (or larger) and
eluting with combinations of water (0.1% TFA) and MeCN (0.1% TFA)
as the mobile phase; [0545] (xi) the following abbreviations have
been used: [0546] Ac acetyl; [0547] CIV concentrated in vacuo;
[0548] RTandrt room temperature; [0549] BOC tert-butoxycarbonyl;
[0550] HATU 2-(7-Aza-1
H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; [0551] DIPEA N,N-diisopropylethylamine; [0552]
CBZ benzyloxycarbonyl; [0553] Bn benzyl; [0554] DCM
dichloromethane; [0555] DMF N,N-di methylformamide; [0556] DMSO
dimethylsulfoxide; [0557] NMP N-methyl-2-pyrrolidinone; [0558]
EtOAc ethyl acetate; [0559] ether diethyl ether; [0560] EtOH
ethanol; [0561] THF tetrahydrofuran; [0562] MeOH methanol; [0563]
MeCN acetontrile; [0564] TFA trifluoracetic add; and [0565] TEA
triethylamine.
1. General Procedures for the Preparation of Mannosides when
.alpha.-D-Mannose Pentaacetate is Used as the Starting Material:
Methyl
3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran--
2-yl]oxyphenyl]benzoate (29)
##STR00032##
[0566] 1.1 Methyl
3-[4-[(2R,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran--
2-yl]oxyphenyl]benzoate
[0567] Under nitrogen atmosphere, at 0.degree. C. boron trifluoride
diethyl etherate (0.128 g, 0.90 mmol) was added dropwise into the
solution of .alpha.-D-mannose pentaacetate (0.120 g, 0.3 mmol) and
methyl 3-(4-hydroxyphenyl)benzoate (0.140 g, 0.6 mmol) in 6 ml of
CH.sub.2Cl.sub.2. After a few mins the mixture was heated to reflux
and kept stirring for more than 36 hrs. The reaction was then
quenched with water and extracted with CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layer was collected, dried with Na.sub.2SO.sub.4,
concentrated. The resulting residue was purified by silica gel
chromatography with hexane/ethyl acetate combinations as eluent,
giving rise to methyl
3-[4-[(2R,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran--
2-yl]oxyphenyl]benzoate (0.136 g) in 81% yield. .sup.1H NMR (300
MHz, CDCl.sub.3) .sigma. ppm 8.23 (m, 1H), 7.99 (m, 1H), 7.74 (m,
1H), 7.57 (m, 2H), 7.50 (t, J=7.8 Hz, 1H), 7.18 (m, 2H), 5.59 (dd,
J=3.6, 9.9 Hz, 1H), 5.58 (d, J=1.5 Hz, 1H), 5.48 (dd, J=2.1, 3.3
Hz, 1H), 5.39 (t, J=10.2 Hz, 1H), 4.30 (dd, J=5.4, 12.3 Hz, 1H),
4.06.about.4.15 (m, 2H), 3.95 (s, 3H), 2.15 (s, 3H), 2.06 (s, 3H),
2.05 (s, 3H), 2.04 (s, 3H); MS (ESI): found: [M+H].sup.+,
559.1.
1.2 Methyl
3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrah-
ydropyran-2-yl]oxyphenyl]benzoate (29)
[0568] Methyl 3-[4-[(2R, 3S,4S,5R,
6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyphenyl]benz-
oate (0.120 g) was stirred in 6 mL of methanol with catalytic
amount of sodium methoxide (0.02 M) at room temperature overnight.
H.sup.+ exchange resin (DOWEX 50WX4-100) was added to neutralize
the mixture. The resin was filtered off and the filtrate was
concentrated, then dried in vacuo giving rise to pure product 29
(0.084 g) in quantitative yield. .sup.1H NMR (300 MHz, CD.sub.3OD)
.sigma. ppm 8.21 (m, 1H), 7.95 (m, 1H), 7.83 (m, 1H), 7.59 (m, 2H),
7.53 (m, 1H), 7.23 (m, 2H), 5.55 (d, J=1.8 Hz, 1H), 4.03 (dd,
J=1.8, 3.3 Hz, 1H), 3.91.about.3.96 (m, 4H), 3.70.about.3.82 (m,
3H), 3.59.about.3.65 (m, 1H); MS (ESI): found: [M+H].sup.+, 391.1.
(see Table 1).
2. Procedures for the preparation of mannosides when
2,3,4,6-tetra-O-benzyl-1-acetyl-.alpha.-D-man nopyranoseis used as
the starting
material:[3-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydropyran-2-yl]oxyphenyl] acetate (42)
##STR00033##
[0569] 2.1 [3-[(2R,3S,4S,5R,6R)-3,4,5-Tribenzyloxy-6
(benzyloxymethyl)tetrahydropyran-2-yl]oxyphenyl] acetate
[0570] Under nitrogen atmosphere, at 0.degree. C. boron trifluoride
diethyl etherate (0.051 g, 0.36 mmol) was added dropwise into the
solution of 2,3,4,6-tetra-O-benzyl-1-acetyl-.alpha.-D-mannopyranose
(0.106 g, 0.18 mmol) and resorcinol monoacetate (0.055 g, 0.36
mmol) in 7 ml of CH.sub.2Cl.sub.2. The mixture was stirred at
0.degree. C. and monitored by TLC. The reaction was then quenched
with water and extracted with CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layer was collected, dried with Na.sub.2SO.sub.4,
concentrated. The resulting residue was purified by silica gel
chromatography with hexane/ethyl acetate combinations as eluent to
give
[3-[(2R,3S,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyra-
n-2-yl]oxyphenyl] acetate (0.093 g) in 75% yield. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. ppm 7.15.about.7.40 (m, 21H),
6.88.about.6.91 (m, 1H), 6.81 (t, J=2.4 Hz, 1H), 6.73.about.6.76
(m, 1H), 5.58 (d, J=1.8 Hz, 1H), 4.90 (d, J=10.8 Hz, 1H), 4.78 (s,
2H), 4.64.about.4.69 (m, 3H), 4.52 (d, J=10.8 Hz, 1H), 4.45 (d,
J=12.3 Hz, 1H), 4.05.about.4.18 (m, 2H), 3.94 (t, J=2.4 Hz, 1H),
3.77.about.3.85 (m, 2H), 3.64.about.3.70 (m, 1H), 2.27 (s, 3H). MS
(ESI): found: [M+Na].sup.+, 697.2.
2.2
[3-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-
-2-yl]oxyphenyl] acetate (42)
[0571] Under hydrogen atmosphere
[3-[(2R,3S,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)
tetrahydropyran-2-yl]oxyphenyl] acetate (0.085 g, 0.13 mmol) was
stirred with Pd/C (10 wt. %) (0.132 g, 0.063 mmol) in ethanol (6
mL) and ethyl acetate (6 mL). The reaction was monitored by TLC
until it went into completion. The mixture was filtered through a
celite plug. The filtrate was concentrated then dried in vacuo
furnishing pure 42 (0.040 g) in quantitative yield. .sup.1H NMR
(300 MHz, CD.sub.3OD) .delta. ppm 7.30 (t, J=8.1 Hz, 1H), 7.00 (m,
1H), 6.90 (t, J=2.1 Hz, 1H), 6.76 (m, 1H), 5.47 (d, J=1.8 Hz, 1H),
4.00 (dd, J=1.8, 3.3 Hz, 1H), 3.88 (dd, J=3.3, 9.3 Hz, 1H),
3.68.about.3.79 (m, 3H), 3.54.about.3.61 (m, 1H). MS (ESI): found:
[M+H].sup.+, 314.9. (see Table 2).
3. Procedure for the Preparation of
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyran--
2-yl]oxyphenyl]benzene-1,3-dicarboxylic acid (45)
##STR00034##
[0573] 35 (0.025 g, 0.056 mmol) was added into 7 mL methanol. Then
0.20 M NaOH aqueous (3 mL) was added. The mixture was stirred at rt
overnight. H.sup.+ exchange resin (DOWEX 50WX4-100) was added to
neutralize the mixture. The resin was filtered off and the filtrate
was concentrated, then dried in vacuo giving rise to pure product
45 (0.023 g) in quantitative yield. .sup.1H NMR (300 MHz,
METHANOL-d.sub.4) .delta. ppm 3.59-3.67 (m, 1H) 3.70-3.84 (m, 3H)
3.94 (dd, J=3.30, 9.30 Hz, 1H) 4.05 (dd, J=3.30, 1.80 Hz, 1H) 5.56
(d, J=1.80 Hz, 1H) 7.26 (m, 2H) 7.63 (m, 2H) 8.41 (d, J=1.50 Hz,
2H) 8.57 (t, J=1.50 Hz, 1H). MS (ESI): found: [M+Na]*, 443.0. (see
Table 3).
4. Procedure for the preparation of
N1,N3-dimethyl-5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-2-yl]oxyphenyl]benzene-1,3-dicarboxamide (50)
##STR00035##
[0575] 35 (0.050 g, 0.11 mmol) was stirred with 15 mL of
MeNH.sub.2/EtOH (33 wt. %) at rt for 40 hrs. The solvent was
removed and the residue was dried in vacuo to afford pure 50 (0.050
g) in quantitative yield. .sup.1H NMR (300 MHz, METHANOL-d) 6 ppm
2.96 (s, 6H) 3.61-3.66 (m, 1H) 3.67-3.84 (m, 3H) 3.86-3.97 (m, 1H)
4.04 (dd, J=3.30, 1.92 Hz, 1H) 5.56 (d, J=1.92 Hz, 1H) 7.21-7.34
(m, 2H) 7.63-7.74 (m, 2H) 8.13-8.26 (m, 3H). MS (ESI): found:
[M+H].sup.+, 447.4. (see Table 4).
5. Procedures for the preparation of biphenyl mannoside derivatives
through Suzuki coupling reaction:
3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran--
2-yl]oxyphenyl]benzonitrile (62)
##STR00036##
[0576] 5.1
[(2R,3S,4S,5R,6R)-3,4,5-Triacetoxy-6-[4-(3-cyanophenyl)phenoxy]-
tetrahydropyran-2-yl]methyl acetate
[0577] Under nitrogen atmosphere, the mixture of acetyl-protected
4-bromophenyl .alpha.-D-mannoside (0.101 g, 0.2 mmol),
m-cyanophenylboronic acid (0.044 g, 0.3 mmol), cesium carbonate
(0.196 g, 0.6 mmol) and tetrakis(triphenylphosphine)palladium
(0.023 g, 0.02 mmol) in dioxane/water (5 mL/1 mL) was heated at
80.degree. C. with stirring for 1 h. The solvent was removed and
the resulting residue was purified by silica gel chromatography
with hexane/ethyl acetate combinations as eluent to give
[(2R,3S,4S,5R,6R)-3,4,5-triacetoxy-6-[4-(3-cyanophenyl)phenoxy]tetrahydro-
pyran-2-yl]methyl acetate (0.080 g) in 76% yield. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. ppm 7.82 (t, J=1.5 Hz, 1H), 7.76 (dt,
J=7.69, 1.65 Hz, 1H), 7.58.about.7.65 (m, 1H), 7.47.about.7.57 (m,
3H), 7.16.about.7.24 (m, 2H), 5.55.about.5.65 (m, 2H), 5.47 (dd,
J=3.57, 1.92 Hz, 1H), 5.39 (t, J=9.9 Hz, 1H), 4.25.about.4.34 (m,
1H), 4.04.about.4.17 (m, 2H), 2.22 (s, 3H), 2.06 (s, 3H), 2.05 (s,
3H), 2.04 (s, 3H). MS (ESI): found: [M+Na]*, 548.7.
5.2
3-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyr-
an-2-yl]oxyphenyl]benzonitrile (62)
[0578] [(2R,3S,4S,5R,6R)-3,4,5-Triacetoxy-6-[4-(3-cyanophenyl)
phenoxy]tetrahydropyran-2-yl]methyl acetate (0.075 g) was stirred
in 6 mL of methanol with catalytic amount of sodium methoxide (0.02
M) at room temperature overnight. H.sup.+ exchange resin (DOWEX
50WX4-100) was added to neutralize the mixture. The resin was
filtered off and the filtrate was concentrated, then dried in vacuo
giving rise to pure product 62 (0.045 g) in 88% yield. .sup.1H NMR
(300 MHz, CD.sub.3OD) .delta. ppm 3.55.about.3.65 (m, 1H),
3.67.about.3.83 (m, 3H), 3.84.about.3.99 (m, 1H), 4.03 (dd, J=3.43,
1.79 Hz, 1H), 5.55 (J=1.65 Hz, 1H), 7.16.about.7.33 (m, 2H),
7.54.about.7.75 (m, 4H), 7.83.about.8.01 (m, 2H). MS (ESI): found:
[M+H].sup.+, 358.3. (see Table 5)
6. Procedures for the preparation of biphenyl mannoside derivatives
through Suzuki coupling reaction: Methyl
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran--
2-yl]oxyphenyl]pyridine-3-carboxylate (77)
##STR00037##
[0579] 6.1
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-
-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl]
acetate
[0580] Under nitrogen atmosphere, the mixture of acetyl-protected
4-bromophenyl-D-mannoside (2.791 g, 5.55 mmol),
bis(pinacolato)diboron (1.690 g, 6.66 mmol), potassium acetate
(2.177 g, 22.18 mmol) and
(1.1'-bis(diphenylphosohino)ferrocene)dichloropalladium(II) (0.244
g, 0.33 mmol) in DMSO (50 ml) was heated at 80.degree. C. with
stirring for 2.5 h. The solvent was removed and the resulting
residue was purified by silica gel chromatography with hexane/ethyl
acetate combinations as eluent to give
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl] acetate
(2.48 g) in 81% yield. .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta.
ppm 1.33 (s, 12H) 1.98-2.12 (m, 9H) 2.20 (s, 3H) 3.93-4.19 (m, 2H)
4.21-4.36 (m, 1H) 5.32-5.42 (m, 1H) 5.45 (dd, J=3.57, 1.92 Hz, 1H)
5.51-5.62 (m, 2H) 7.00-7.15 (m, 2H) 7.67-7.84 (m, 2H). MS (ESI):
found: [M+Na].sup.+, 573.2.
6.2 Methyl
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrah-
ydropyran-2-yl]oxyphenyl]pyridine-3-carboxylate (77)
[0581] Under nitrogen atmosphere, the mixture of
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl] acetate
(0.132 g, 0.24 mmol), methyl 5-bromonicotinate (0.043 g, 0.2 mmol),
cesium carbonate (0.196 g, 0.6 mmol) and
tetrakis(triphenylphosphine)palladium (0.023 g, 0.02 mmol) in
dioxane/water (5 mL/1 mL) was heated at 80.degree. C. with stirring
for 1 h. After cooling down, the mixture was filtered through
silica gel column to remove the metal catalyst and salts with
hexane/ethyl acetate (2/1) containing 2% triethylamine as eluent.
The filtrate was concentrated, and then dried in vacuo. Into the
residue, 6 mL of methanol with catalytic amount of sodium methoxide
(0.02 M) was added and the mixture was stirred at room temperature
overnight. H.sup.+ exchange resin (DOWEX 50WX4-100) was added to
neutralize the mixture. The resin was filtered off and the filtrate
was concentrated. The resulting residue was purified by silica gel
chromatography with CH.sub.2Cl.sub.2/MeOH combinations containing
2% NH/H.sub.2O as eluent, giving rise to 77 (0.031 g) in 40% yield.
.sup.1H NMR (300 MHz, CD.sub.3OD) 5 ppm 3.53-3.65 (m, 1H) 3.67-3.83
(m, 3H) 3.89-3.96 (m, 1H) 3.99 (s, 3H) 4.04 (dd, J=3.43, 1.79 Hz,
1H) 5.57 (d, J=1.92 Hz, 1H) 7.22-7.37 (m, 2H) 7.58-7.73 (m, 2H)
8.54 (t, J=2.06 Hz, 1H) 8.97 (d, J=2.20 Hz, 1H) 9.04 (d, J=1.92 Hz,
1H). MS (ESI): found: [M+H].sup.+, 392.1. (see Table 6).
7. Procedure for the Preparation of Mannosides Via Amide Coupling
reaction
##STR00038##
[0582]
3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyphenyl]-N-[2-[2-[2-[[3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-
-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyphenyl]benzoyl]amino]ethoxy]etho-
xy]ethyl]benzamide (82)
[0583] Under nitrogen atmosphere, at 0.degree. C. anhydrous DMF (5
mL) was added into the RB flask containing 44 (0.062 g, 0.165 mmol)
and HATU (0.069 g, 0.182 mmol). After stirring for 10 min,
1,2-bis(2-aminoethoxy)ethane (0.0123 g, 0.083 mmol), then
N,N-isopropylethylamine (0.024 g, 0.182 mmol) were added. The
mixture was stirred overnight while being warmed to rt naturally.
The solvent was removed and the residue was purified by HPLC
(C18,15*150 mm column; eluent: acetonitrile/water (0.1% TFA)) to
give 82 (0.044 g) in 82% yield. .sup.1H NMR (300 MHz,
METHANOL-d.sub.4) 5 ppm 3.51-3.65 (m, 6H), 3.65-3.82 (m, 14H),
3.88-3.96 (m, 2H), 4.03 (dd, J=3.57, 1.92 Hz, 2H), 5.52 (d, J=1.65
Hz, 2H), 7.14-7.24 (m, 4H), 7.40-7.50 (m, 2H), 7.53-7.63 (m, 4H),
7.66-7.77 (m, 4H), 8.00 (t, SI 0.65 Hz, 2H). MS (ESI): found:
[M+H].sup.+, 865.9. (see Table 7).
8. Procedures for the Preparation of Fluorescent Mannoside (99 and
100)
##STR00039##
[0584] 8.1
[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(9Hflu ore
n-9-ylmethoxycarbonylamino)butoxy]tetrahydropyran-3-yl] acetate
[0585] Under nitrogen atmosphere, at 0.degree. C. boron trifluoride
diethyl etherate (0.115 g, 0.81 mmol) was added dropwise into the
solution of .alpha.-D-mannose pentaacetate (0.107 g, 0.27 mmol) and
4-(Fmoc-amino)-1-butanol (0.168 g, 0.54 mmol) in 5 ml of
CH.sub.2Cl.sub.2. After a few mins the mixture was heated to reflux
and kept stirring for more than 36 hrs. The reaction was then
quenched with water and extracted with CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layer was collected, then dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The resulting residue
was purified by silica gel chromatography with hexane/ethyl acetate
combinations as eluent to give
[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(9H-fluoren-9-ylme-
thoxycarbonylamino)butoxy]tetrahydropyran-3-yl] acetate (0.106 g)
in 60% yield. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm
1.48.about.1.79 (m, 4H), 2.01 (s, 3H), 2.05 (s, 3H), 2.11 (s, 3H),
2.17 (s, 3H), 3.25 (m, 2H), 3.50 (m, 1H), 3.73 (m, 1H),
3.91.about.4.04 (m, 1H), 4.05.about.4.18 (m, 1H), 4.18.about.4.37
(m, 2H), 4.41 (d, J=6.87 Hz, 2H), 4.74.about.4.94 (m, 2H),
5.17.about.5.41 (m, 3H), 7.29.about.7.37 (m, 2H), 7.37.about.7.47
(m, 2H), 7.61 (d, J=7.42 Hz, 2H), 7.78 (d, J=7.42 Hz, 2H). MS
(ESI): found: [M+H].sup.+, 642.2.
8.2
(2S,3S,4S,5S,6R)-2-(4-aminobutoxy)-6-(hydroxymethyl)tetrahydropyran-3,-
4,5-triol (98)
[0586]
[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(9H-fluoren--
9-ylmethoxycarbonylamino)butoxy]tetrahydropyran-3-yl] acetate
(0.106 g, 0.165 mmol) was stirred in 6 mL of methanol with
catalytic amount of sodium methoxide (0.02M) at rt overnight. The
solvent was removed and the residue was purified by silica gel
chromatography with combination of ammonia aqueous/methanol as
eluent to afford 98 (0.036 g) in 88% yield. .sup.1H NMR (300 MHz,
CD.sub.3OD) 5 ppm 1.46.about.1.78 (m, 4H), 2.61.about.2.88 (m, 2H),
3.41.about.3.55 (m, 2H), 3.59 (t, J=9.3 Hz, 1H), 3.65.about.3.87
(m, 5H), 4.75 (d, J=1.65 Hz, 1H). MS (ESI): found: [M+H].sup.+,
252.1.
8.3 Fluorescent Mannosides (99 and 100)
[0587] The mixture of 98 (0.018 g, 0.070 mmol), 5-(and
-6)-carboxyfluoresceinsuccinimidyl ester (0.022 g, 0.046 mmol) and
triethylamine (0.058 g, 0.57 mmol) in DMF (2 mL) was stirred
overnight. After removing the solvent, the residue was purified by
silica gel chromatography with dichloromethane/methanol combination
as eluent, giving rise to 99 and 100 (0.023 g) in 82% yield. MS
(ESI): found: [M+H].sup.+, 610.6. (see Table 8).
9. Procedures for the preparation of
(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-(4-phenyltriazol-1-yl)tetrahydropyra-
n-3,4,5-triol (101)
##STR00040##
[0588] 9.1
[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-phenyltr-
iazol-1-yl)tetrahydropyran-3-yl] acetate
[0589] Under nitrogen atmosphere, EtOH/H.sub.2O (4 mL/1 mL) was
added into the RB flask containing .alpha.-azido-D-mannose
tetraacetate (0.075 g, 0.2 mmol), phenyl acetylene (0.026 g, 0.24
mmol), copper (II) sulfate pentahydrate (0.01 g, 0.04 mmol) and
sodium ascorbate (0.016 g, 0.08 mmol). The mixture was stirred at
room temperature overnight. The solvent was removed and the residue
was purified by silica gel chromatography with hexane/ethyl acetate
combination as eluent, giving rise to
[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-phenyltriazol-1-yl-
)tetrahydropyran-3-yl] acetate (0.030 g) in 31% yield. .sup.1H NMR
(300 MHz, CHLOROFORM-d) .delta. ppm 2.05-2.12 (m, 9H) 2.20 (s, 3H)
3.89-3.99 (m, 1H) 4.08 (dd, J=2.50, 2.61 Hz, 1H) 4.40 (dd, J=2.50,
5.36 Hz, 1H) 5.40 (t, J=8.79 Hz, 1H) 5.95-6.04 (m, 2H) 6.07 (d,
J=2.75 Hz, 1H) 7.34-7.53 (m, 3H) 7.79-7.92 (m, 2H) 7.96 (s, 1H). MS
(ESI): found: [M+Na]*, 498.1.
9.2
(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-(4-phenyltriazol-1-yl)tetrahydrop-
yran-3,4,5-triol (101)
[0590]
[(2S,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-phenyltriazo-
l-1-yl)tetrahydropyran-3-yl] acetate (0.028 g, 0.059 mmol) was
stirred in 6 mL of methanol with catalytic amount of sodium
methoxide (0.02 M) at room temperature overnight. H.sup.+ exchange
resin (DOWEX 50WX4-100) was added to neutralize the mixture. The
resin was filtered off and the filtrate was concentrated, then
dried in vacuo giving rise to pure product 101 (0.015 g) in
quantitative yield. .sup.1H NMR (300 MHz, CD.sub.3OD) 5 ppm
3.34-3.44 (m, 1H) 3.70-3.92 (m, 3H) 4.12 (dd, J=8.52, 3.57 Hz, 1H)
4.71-4.80 (m, 1H) 6.08 (d, J=2.75 Hz, 1H) 7.30-7.41 (m, 1H)
7.41-7.51 (m, 2H) 7.77-7.90 (m, 2H) 8.51 (s, 1H). (ESI): found:
[M+Na].sup.+, 330.2. (see Table 9)
10. Procedures for the preparation of Methyl
3-[4-[[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-
-2-yl]amino]phenyl]benzoate (102)
##STR00041##
[0592] .alpha.-D-mannose (0.360 g, 2 mmol) and methyl
4'-aminobiphenyl-3-carboxylate (0.454 g, 2 mmol) in ethanol (5 mL)
was heated to 55.degree. C. for 17 h. After cooling down to rt, the
white precipitate formed was collected by filtration. The
precipitate was washed with ethanol (3 mL) two times, then dried in
vacuo to afford pure 102 in 77% yield. .sup.1H NMR (300 MHz,
ACETONITRILE-d.sub.3 and D.sub.2O) .delta. ppm 3.28-3.38 (m, 1H)
3.54-3.59 (m, 2H) 3.64-3.71 (m, 2H) 3.86 (s, 3H) 3.88-3.92 (m, 1H)
4.89 (d, J=1.10 Hz, 1H) 6.80-6.91 (m, 2H) 7.44-7.58 (m, 3H)
7.75-7.90 (m, 2H) 8.11-8.20 (m, 1H). (ESI): found: [M+H].sup.+,
390.1.
11. Procedures for the preparation of methyl
3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran--
2-yl]sulfanylphenyl]benzoate (103)
##STR00042##
[0593] 11.1
[(2R,3S,4S,5R,6R)-4,5-Diacetoxy-6-(acetoxymethyl)-2-(4-bromophenyl)sulfan-
yl-tetrahydropyran-3-yl] acetate
[0594] Linder nitrogen atmosphere, at 0.degree. C. boron
trifluoride diethyl etherate (0.427 g, 3.0 mmol) was added dropwise
into the solution of .alpha.-D-mannose pentaacetate (0.390 g, 1.0
mmol) and 4-bromobenzenethiol (0.378 g, 2.0 mmol) in 6 ml of
CH.sub.2Cl.sub.2. After a few mins the mixture was warmed to rt and
kept stirring for 48 hrs. The reaction was then quenched with water
and extracted with CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layer was
collected, dried with Na.sub.2SO.sub.4, filtered, and concentrated.
The resulting residue was purified by silica gel chromatography
with hexane/ethyl acetate combinations as eluent, giving rise to
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromophenyl)sulfan-
yl-tetrahydropyran-3-yl] acetate (0.40 g) in 77% yield. .sup.1H NMR
(300 MHz, CHLOROFORM-d) .delta. ppm 2.01-2.10 (m, 9H) 2.16 (s, 3H)
4.10 (dd, J=12.09, 2.47 Hz, 1H) 4.30 (dd, J=12.09, 6.04 Hz, 1H)
4.43-4.61 (m, 1H) 5.24-5.40 (m, 2H) 5.43-5.52 (m, 2H) 7.32-7.39 (m,
2H) 7.42-7.49 (m, 2H). MS (ESI): found: [2M+H].sup.+, 1039.1.
11.2 Methyl
3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran--
2-yl]sulfanylphenyl]benzoate (103)
[0595] Under nitrogen atmosphere, the mixture of
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromophenyl)sulfan-
yl-tetrahydropyran-3-yl] acetate (0.20 g, 0.39 mmol),
3-methoxycarbonylphenylboronic acid (0.106 g, 0.59 mmol), cesium
carbonate (0.381 g, 1.17 mmol) and
tetrakistriphenylphosphinepalladium (0.045 g, 0.04 mmol) in
dioxane/water (5 mL/1 mL) was heated at 80.degree. C. with stirring
for 1 h. After cooling down, the mixture was filtered through
silica gel column to remove the metal catalyst and salts with
hexane/ethyl acetate (2/1) as eluent. The filtrate was
concentrated, and then dried in vacuo. Into the residue, 6 mL of
methanol with catalytic amount of sodium methoxide (0.02 M) was
added and the mixture was stirred at room temperature overnight.
H.sup.+ exchange resin (DOWEX 50WX4-100) was added to neutralize
the mixture. The resin was filtered off and the filtrate was
concentrated. The resulting residue was purified by HPLC (C18,
15*150 mm column; eluent: acetonitrile/water (0.1% TFA)) to give
103 (0.095 g) in 63% yield. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. ppm 3.66-3.91 (m, 4H) 3.94 (s, 3H) 4.01-4.16 (m, 2H) 5.51
(d, J=1.37 Hz, 1H) 7.51-7.69 (m, 5H) 7.81-7.93 (m, 1H) 8.00 (dt,
J=7.83, 1.44 Hz, 1H) 8.24 (J=1.65 Hz, 1H). MS (ESI): fou: [M
nd+H].sup.+, 407.1.
12. Procedures for the preparation of methyl
3-[4-[[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-
-2-carbonyl]amino]phenyl]benzoate (104)
##STR00043##
[0597] The solution of .alpha.-cyano-D-mannose tetraacetate (0.107
g, 0.3 mmol) in 25% hydrochloric acid was heated at 50.degree. C.
for 48 h. The solvent was removed. Water (10 ml) and H.sup.+
exchange resin (DOWEX 50WX4-100) was added and kept stirring for 5
mins. The resin was filtered off and the filtrate was concentrated,
and then dried in vacuo. Into the resulting residue, HATU (0.274 g,
0.72 mmol) and anhydrous DMF (6 ml) were added at 0.degree. C.
After stirring for 10 min, methyl 4'-aminobiphenyl-3-carboxylate
(0.164 g, 0.72 mmol), then isopropylethylamine (0.233 g, 1.80 mmol)
were added. The mixture was stirred overnight while being warmed to
rt naturally. The solvent was removed and the residue was purified
by silica gel chromatography with CH.sub.2Cl.sub.2/MeOH
combinations as eluent to give 104 (0.066 g) in 52% yield. .sup.1H
NMR (300 MHz, CD.sub.3OD) 5 ppm 3.50 (dt, J=4.67, 2.33 Hz, 1H)
3.58-3.65 (m, 2H) 3.76 (dd, J=11.81, 7.42 Hz, 1H) 3.86-4.03 (m, 4H)
4.52 (t, J=2.61 Hz, 1H) 4.57 (d, J=2.75 Hz, 1H) 7.55 (t, J=7.69 Hz,
1H) 7.60-7.68 (m, 2H) 7.68-7.76 (m, 2H) 7.82-7.91 (m, 1H) 7.97 (dt,
J=7.69, 1.37 Hz, 1H) 8.21-8.28 (m, 1H). MS (ESI): found:
[M+H].sup.+, 418.1.
Example 6. Structure-Based Drug Design and Optimization of
Mannoside Bacterial FimH Antagonists
[0598] As shown in Scheme 1, .alpha.-D-mannoside derivatives were
prepared using traditional Lewis add mediated glycosidation.
Reaction of acylated .alpha.-D-(+)-mannose 1a with a variety of
phenols and BF.sub.3-OEt.sub.2 resulted in exclusive formation of
the .alpha.-isomer mannosides 2. Subsequent deacylation with NaOMe
in methanol gave the desired aryl mannosides 3-8 in good yield.
Biological activity against FimH was evaluated using a guinea pig
red blood cell-based hemagglutination (HA) assay (ref) in which the
concentration of the mannoside on reducing agglutination by 50% was
used as the primary endpoint (50% HA titer). This assay was
preferred to a simple FimH binding assay for screening and
developing SAR since it assesses the compound's ability to prevent
bacterially-mediated adhesion directly in a cellular assay.
##STR00044##
.sup.aReagents and conditions: (a) R'OH, BF.sub.3-OEt.sub.2,
CH.sub.2Cl.sub.2, reflux; (b) (i) NaOMe, MeOH; (ii) H.sup.+
exchange resin; (c) R'OH, BF.sub.3--OEt.sub.2. CH.sub.2Cl.sub.2,
0.degree. C. to 25.degree. C.; (d) H.sub.2, 10% Pd/C, MeOH.
[0599] As shown in Table 10, it was found that the general trend
that 2- and 3-substitution was optimal for potency relative to
4-substitution in most examples with the exception of the acyl
anilines 3q-s in which this trend was dramatically reversed.
Ortho-substituted chlorophenyl 3 g, cyanophenyl 3m, and
meta-substituted methyl ester 3j all showed an impressive greater
than 5-fold improvement in potency relative to parent phenyl
mannoside 3a. Interestingly, carboxylic acid 3l lost 10-fold
potency relative to matched pair methyl ester 3j showing a 50% HA
titer of only 60 .mu.M. Incorporation of an additional methyl ester
in the 5-position of 3j, as with compound 4, resulted in a
relatively large 3-fold enhancement in potency. Benzylic analogs 5a
and 5b (pictured below) which have different conformational space
and flexibility relative to direct phenyl substitution of the
anomeric oxygen, as with to matched pairs 3a and 3b, show a 2-fold
decrease (50% HA titer=60 .mu.M) in potency.
##STR00045##
TABLE-US-00001 TABLE 10 SAR of simple aryl substitution mannoside
library. ##STR00046## HA HA 50% 50% Titer Com- Titer Compound R
(.mu.M) pound R (.mu.M) 3a H 30 3m 2-CN 6 3b 4-NO.sub.2 31 3n 3-CN
23 3c 3-NO.sub.2 16 3o 4-CN 30 3d 4-NH.sub.2 32 3p 4-OMe 8 3e 3-Me
4 3q 2-NHAc 125 3f 4-Me 8 3r 3-NHAc 12 3g 2-Cl 4 35 4-NHAc 8 3h
3-Cl 6 3t 3-CONH.sub.2 16 31 4-Cl 32 3u 4-CONH.sub.2 15 3j
3-CO.sub.2Me 8 3w 4-CH.sub.2CO.sub.2Me 30 3l 3-CO.sub.2H 60 4
3,5-CO.sub.2Me 2
[0600] Upon examination of the X-ray structures of
.alpha.-D-butylmannoside and oligomannose-3 coupled with docking
studies of the monophenyl inhibitors bound to FimH, it was
encouraging to observe that additional improvements in binding
affinity could be achieved by addition of a second aryl or
aliphatic ring system in anticipation of introducing both
additional hydrophobic and n-n stacking interactions with Tyr-48
and Tyr-137 as seen from the directionality of the butyl side chain
in butyl mannoside and position of the second mannose residue in
oligomannose-3.
[0601] To this end, analogs with an additional ring system either
directly attached or fused ring to the parent aryl mannosides of
Table 10 were explored. It was discovered that a variety of ring
systems were tolerated (Table 11) relative to the previously
described coumarin analog 4-methylumbellferyl-.alpha.-D-mannoside
6. The most attractive inhibitor in this series of compounds was
the 4'-biphenyl derivative 8e bearing a methyl ester off the meta
position of the second aryl ring and showing a 2-fold improvement
relative to 6 displaying an HA titer 1 .mu.M. In order to determine
the source of this potency enhancement and aid in further
improvements, a high-resolution X-ray crystal structure of 8e bound
to FimH was obtained. Shown in FIG. 22, inhibitor 8e binds in a
very complementary "lock and key" fashion to the FimH binding
pocket. The mannose ring is making conserved interactions with the
mannose-binding pocket similar to those described previously while
the two aromatic rings of the biphenyl moiety exist in a non-planar
conformation allowing for a .pi.-stacking and hydrophobic
interactions with Tyr-48 and other residues encompassing the
exterior hydrophobic deft of FimH. The .pi.-stacking interaction
with Tyr 48 occurs on the opposite side of the phenyl ring than
that engaged by oligomannose-3, which inserts itself into the open
"tyrosine gate" formed by Tyr-48 and Tyr-137. Thus, 8e engages the
"tyrosine gate" in a way that results in alteration of the extended
FimH binding pocket through closure of the tyrosine gate upon
rotation of Tyr-48. This binding mode places the ester of 8e within
H-bonding distance to a salt bridge formed between Arg-98 and
Glu-50 (FIG. 22). It is noteworthy that the HA 50% titer for
Methylaman is >1 mM making compound 8e greater than 1000-fold
more potent from the addition of the biphenyl ester.
TABLE-US-00002 TABLE 11 SAR of multi-ring system analogs.
##STR00047## HA 50% titer EC.sub.90 Compound R (.mu.M) 6
##STR00048## 2 7a ##STR00049## 8 7b ##STR00050## 6 7c ##STR00051##
4 7d ##STR00052## 2 7e ##STR00053## 2 8a ##STR00054## 62 8b
##STR00055## 6 8c ##STR00056## 8 8d ##STR00057## 8 8e ##STR00058##
1 8f ##STR00059## 4 8g ##STR00060## 2 8h ##STR00061## 2
[0602] This observation lead to investigating replacing the mannose
with alternate sugars or mimics since the biphenyl portion alone
provides a significant contribution to biological activity
presumably from tighter binding to FimH. It was decided to start by
replacing the mannose portion of 8e with glucose since all chiral
centers are identical except for the 3-hydroxyl group adjacent to
the anomeric center, being of R stereochemistry in mannose and S in
glucose (Scheme 2). Standard Lewis acid mediated glycosidation gave
isomeric mixtures at the anomeric center favoring the undesired
.alpha.-isomer, conversely to the clean formation of the
.alpha.-isomer seen for the mannosides. Correspondingly, reaction
of benzoyl protected .alpha.-D-glucose 9 with phenol 10 using
BF.sub.3-OEt.sub.2 followed debenzoylation yielded both the
.alpha.-D glucoside 11a and .alpha.-D-glucoside 11b in a 3:7 ratio.
Unfortunately, neither glucoside isomer showed activity in the
hemagglutination assay even up to a concentration of 2.5 mM.
Therefore, modification of a single chiral hydroxyl group
stereocenter on the mannoside is suffident to significantly
decrease biological activity, presumably resulting from the
inability to tightly bind FimH. Upon analysis of the specific
interactions realized from .alpha.-D-mannose bound to FimH,.sup.4
this hydroxyl group is making an H-bonding interaction with the
N-terminal residue of FimH and a water molecule contained inside
the binding pocket. In any event, this finding demonstrates the
exquisite specificity of FimH for mannose epitopes which has
enabled UPEC bacteria to exclusively recognize mannose-presenting
cells.
##STR00062##
[0603] Next, the biaryl portion of the mannoside was optimized by
undertaking an extensive SAR evaluation of the second ring through
a modified Toplisstype evaluation of substituents. This focused set
of compounds was designed with some bias toward improving
interactions with Arg-98 and Glu-50 but also with interest in
elucidating the importance of ring electronic properties as a means
to improve the stacking interaction with Tyr-48. A few initial
analogs were prepared using the synthetic route outlined previously
in Scheme 1 buta more convergent synthesis was developed based on
Suzuki coupling of arylbromide intermediate 12 as shown in Scheme
3.
##STR00063##
[0604] Employing standard Suzuki conditions by reaction of aryl
boronic adds or esters with Pd(Ph.sub.3P).sub.4 and
Cs.sub.2CO.sub.3, 12 was converted to protected biphenyl mannosides
13 in good yield. The acylated biaryl mannosides 13 were then
deprotected as before to generate the final target compounds 14 or
15a displayed in Table 12. Di-ester 15a was further functionalized
to di-methyl amide 15b by reaction with dimethylamine or converted
to di-add 15c by basic hydrolysis in methanol. Upon evaluation of
HA titers, the matched pair meta-substituted methyl amide 14a was
equipotent to ester 8e while reverse amide 14b and free acid 14c
displayed moderately lower potencies of 2 .mu.M and 4 .mu.M
respectively. The latter result was surprising since these
modifications were designed to introduce an electrostatic
interaction with Arg-98 and disrupt the Arg-98/Glu-50 salt bridge.
On the other hand, sulfonamide 14r has equivalent potency to
carboxamide 14a providing further evidence that a hydrogen bond
acceptor is required for optimal potency presumably from
interaction with Arg-98. From analysis of ortho, meta, and para
matched pairs, it was obvious that para-substitution was least
preferred for activity while meta substitution was preferred to
ortho substitution. This preference for meta substitution was most
pronounced with the methyl alcohols 14 g and 14 h where the ortho
analog 14 g was over 5-fold less potent with an HA titer of only 16
.mu.M. It was surmised that this large substituent results in an
increased rotation of the two rings out of the plane causing a
non-productive alignment for interactions with Tyr-48. Although a
majority of the analogs tested contained electron withdrawing
groups, in general electron donating groups such as the methyl
alcohols 14 g-i and phenols 14j-l were less active in the HA titer
assay. The most potent of the mono-substituted mannosides is meta
nitro derivative 14m with a 50% HA titer of 0.5 .mu.M. It is
possible that the partial negative charge on the nitro oxygen atoms
allow for an optimized H-bond acceptor-donor interaction with
Arg-98 coupled with the increased electron withdrawing ability of
the nitro group possibly enhancing stacking interactions with
Tyr-48.
TABLE-US-00003 TABLE 12 SAR of substituted biphenyl mannosides.
##STR00064## HA 50% titer EC.sub.90 Compound R.sub.1 (.mu.M) 14a
3-CONHMe 1 14b 3-NHAc 2 14c 3-CO.sub.2H 4 14d 2-CN 2 14e 3-CN 1 14f
4-CN 8 14g 2-CH.sub.2OH 16 14h 3-CH.sub.2OH 3 14i 4-CH.sub.2OH 6
14j 2-OH 8 14k 3-OH 4 14l 4-OH 6 14m 2-OMe 1 14n 3-OMe 1.5 14o
2-NHSO.sub.2Me 12 14p 3-NHSO.sub.2Me 2 14q 2-SO.sub.2NHMe 4 14r
3-SO.sub.2NHme 1 14s 3-NO.sub.2 0.5 14t 4-NO.sub.2 2 14u
2-CONH.sub.2 6 14v 3-CONH.sub.2 2 14w 2-CF.sub.3 8 14x 3-CF.sub.3 2
14y 3-F 6 15a 3,5-CO.sub.2Me 0.15 15b 3,5-CONHMe 0.37 15c
3,5-CO.sub.2H 2
[0605] Perhaps the most unexpected finding from this study was that
addition of another ester or amide substituted in the other meta
position such as di-ester 15a and di-methyl amide 15b resulted in
the two most potent mannoside inhibitors of FimH with activities of
150 nM and 370 nM, respectively. With respect to di-ester 15a this
constitutes a 7-fold improvement in activity relative to mono ester
8e. The di-amide 15b, while slightly less potent, has much improved
solubility relative to di-ester 15a. It was hypothesized that the
addition of the second ester or amide serves a two-fold purpose for
improving activity: First, the electron withdrawing group results
in less electron density of the aryl ring thus improving .pi.-.pi.
stacking interactions with Tyr48. Second, the mono-ester (or amide)
analog can presumably access conformations in which the aryl ring
is not in close proximity to Arg-98 whereas the meta di-ester (or
di-amide) analog likely exists only in conformations where the
ester or amide resides in dose proximity to Arg-98 resulting in
less entropic loss upon binding and a lower energy bound
conformation resulting in increased binding affinity and
potency.
[0606] In order to more accurately determine the relative
contributions of both binding affinity to FimH versus other
compound properties to the potency seen in the cellular HA titer
assay, a high-throughput fluorescence polarization (FP) (or
anisotropy) competitive binding assay was developed using a
fluorescent-labeled mannoside ligand shown in Scheme 4. Synthesis
was achieved as before using Lewis add mediated glycosylation with
protected aminoalcohol 17 followed by dual Fmoc and acyl
deprotection of intermediate 18 to give free amine 19. Subsequent
reaction of 5(6)FAM-OSu with 19 using triethylamine in DMF gave the
desired fluorescently tagged FAM-mannoside 20.
##STR00065##
[0607] The K.sub.D for FAM mannoside 20 was measured to be 0.17
.mu.M while the 50% HA titer was only 125 .mu.M. Twenty mannoside
ligands with structural diversity and activities were evaluated in
the HA titer cell assay ranging from 150 nM to 125 .mu.M for their
ability to competitively inhibit binding of FAM 20. Interestingly,
it was found that all potently competed for FimH binding having
EC.sub.50's less than 0.25 .mu.M but having no clear correlation of
EC.sub.50 with the activity found in the HA cell assay (Table 13).
Although there was over a 60-fold range in cellular potency, there
was only about a 5-fold range in binding affinity (FIG. 23). In
addition, there was also no linear correlation of data with the two
assays. Simple alkyl mannosides have the highest drop in cell
activity as demonstrated with butyl mannoside having the most
dramatic difference with a 510-fold drop in cell activity relative
to binding.
TABLE-US-00004 TABLE 13 Binding affinity of selected mannosides in
fluorescence polarization assay. FP FP Binding HA 50% Binding HA
50% EC.sub.50 titer EC.sub.50 titer Compound (.mu.M) (.mu.M)
Compound (.mu.M) (.mu.M) 6 0.044 2 Heptylman 0.089 15 (MeUmbman) 8e
0.052 1 4 0.091 2 8f 0.059 4 14p 0.097 2 3k 0.064 8 14c 0.099 4 14b
0.07 2 3a 0.114 30 14a 0.075 1 3s 0.122 8 15a 0.077 0.15 3r 0.160
12 14v 0.078 2 3n 0.162 23 3j 0.084 6 5a 0.187 60 15b 0.087 0.37
Butylman 0.245 125
[0608] Interestingly, the most potent biphenyl mannoside series
shows a modest correlation of binding affinity having a much
smaller drop in cell activity relative to binding. The di-ester 15a
and di-amide 15b show the best correlation with only a 2-fold and
4-fold difference in cell potency vs. binding. It is important to
note that even though these two analogs are the most potent in the
HA titer cell assay, they have about 2-fold less binding affinity
in the FP assay than the tightest binding mannosides. While it is
unclear why there is no correlation of binding to activity in the
cell, there is a moderate correlation when compounds with binned
affinities are compared. However, the latter observation cannot
explain the non-linear increase in cell activity of 15a and 15b
relative to the mono-substituted biphenyl mannosides. Perhaps
differential binding kinetics including varied on rates and off
rates of mannoside analogs to FimH can provide a plausible
explanation for the latter. It is worth mentioning that the
previously reported K.sub.D values obtained through surface plasmon
resonance (SPR) for 6 (MeUmb.alpha.man), butyl mannoside, and
Heptyl.alpha.man are 20 nM, 151 nM, and 5 nM respectively. While
the result for butyl mannoside is similar, the FP assay suggests
MeUmbaman is the most potent (44 nM) being 2-fold more potent than
Heptyl.alpha.man (89 nM). Considering both the lack of correlation
between binding affinity and biological activity in the cell plus
the variability seen comparing two separate binding assays, the HA
titer assay coupled with the use of structural information is a
preferred route for medicinal chemistry optimization of mannoside
FimH ligands. Nonetheless, based on the HA titer cellular data,
mannoside 15a is the most potent FimH antagonist reported to date
and represents an excellent lead candidate for further optimization
of the monovalent mannosides toward a novel preclinical candidate
with tremendous therapeutic potential for treating urinary tract
infections.
[0609] The development of multivalent and dendrimeric mannosides
has been the major focus of previous work on FimH antagonists since
simple mannosides, while having respectable binding affinity to
FimH, show poor cellular activity in the HA assay. It has been
demonstrated that multivalent mannosides are effective at
increasing avidity through a phenomenon termed "duster effect"
resulting in an overall increased binding affinity when calculated
per mannoside monomer. Therefore, it was of interest to determine
if the improved monovalent mannosides 15a and 15b would produce a
similar increase in avidity or duster effect previously reported
for dendrimeric mannosides. For synthetic simplicity, a model
system based on monoamide 14a was chosen. A symmetrical divalent
mannoside was designed by connecting two monomers via an amide
based linker to the two biphenyl rings (Scheme 5). Accordingly,
dimeric inhibitors based on 14a were synthesized by coupling
carboxylic add 13c to either diamine 2-(2-aminoethoxy)ethanamine or
2-[2-(2-aminoethoxy)ethoxy]ethanamine yielding diamides 21 and 22
respectively using standard HATU coupling conditions with Hunig's
base in DMF. The shorter chain analog 21 showed a 50% HA titer of
0.75 .mu.M thus showing no noticeable improvement relative to 14a,
while the longer ethylene glycol linked diamide 22 displayed an
almost 8-fold increase in activity with an impressive HA titer of
130 nM. This constitutes a 4-fold increase in avidity relative to
the expected potency of 500 nM based on two monomeric units of 14a.
In addition to increased potency, 22 has much improved solubility
relative to 21 making it an ideal starting point for further
optimization. Although these analogs have less potential to be
effective as oral agents relative to the monovalent mannosides, the
divalent mannosides are very useful chemical research tools and can
potentially be developed into topical or intravenously dosed
antibacterial agents.
##STR00066##
Example 7. In Vitro Analysis of Mannoside Inhibitors
[0610] Three FimH function assays were used to screen mannosides
for inhibitory function, and are described below. Selected results
for the SAR and testing of these compounds are presented in
Examples 5 and 6, and show that some synthesized and tested
compounds are more potent inhibitors of HA titer and biofilm
formation than anything commercially available and previously
tested, some by as much as 2 logs.
[0611] i. Hemagglutination of guinea pig red blood cells by type1
piliated UPEC is dependent upon FimH mannose binding ability.
Serial dilutions allow a quantitative analysis using this
assay.
[0612] ii. Compounds that decrease hemagglutination by 75% were
then tested in a type 1 dependent biofilm assay. First, the ability
of mannoside derivatives to prevent UPEC type 1 dependent biofilm
formation in the published Kolter assay was measured as optimized
for the prototypic UTI89 strain used in these assays. Mannoside
derivatives that allow growth but inhibit biofilm formation at 50%
or greater were verified by repeating the biofilm assay and
performing titration curves to determine the IC50. A more rigorous
assay tested the ability of the strongest inhibitory mannoside
derivatives to reverse hemagglutination of guinea pig red blood
cells and to disrupt preformed type 1 dependent biofilms.
[0613] iii. A fluorescent polarization assay was developed and
miniaturized to monitor the direct binding of a synthesized
fluorescently labeled butyl mannose derivative to the FimH receptor
as well as competitive displacement by other synthetic mannosides.
This fluorescent polarization assay provides a rapid method for
monitoring improvements to receptor affinity during the synthetic
process, as well as providing Kd binding affinity measurements.
Example 8. Crystallography
[0614] In order to further rationally design better mannoside
derivatives to inhibit FimH binding the most efficient inhibitors,
such as ZFH253, with the FimH adhesin domain (FimH.sub.A) were
crystallized to detail their interactions with FimH (FIG. 9).
[0615] The adhesin domain of FimH (residues 1-175 with a C-terminal
6-histidine tag, hereafter FimH.sub.A) was cloned into a pTRC99
plasmid and expressed in E. coli. FimH.sub.A was purified from
bacterial periplasm by passage over cobalt affinity (Talon;
Clontech) and Q Sepharose (GE Healthcare) columns. Protein was
subsequently dialyzed against 10 mM MES at pH 6.5 and concentrated
to 15 mg/ml for crystallization trials. FimH.sub.A crystallized in
20% ethanol, 100 mM imidazole pH 8.0, and 200 mM MgCl2. Tetragonal
bipyramidal crystals measuring 150.times.150 .mu.m formed in two
days and did not diffract unless slowly dehydrated over the course
of at least 12 hours. The FimH.sub.A-ZFH253 complex was therefore
formed by soaking individual crystals in a stabilizing solution
initially containing 15% ethanol, 100 mM imidazole pH 8.0, 200 mM
MgCl2, 10% PEG200, 5% glycerol and 1 mM ZFH253. Individual crystals
were placed in 150 pl stabilizing solution and allowed to dehydrate
to one-third their original volume over the course of 24 hours.
Crystals were then harvested without further cryopretection and
plunged into liquid nitrogen. Crystals diffracted to 2.55 .ANG. at
beamline 4.2.2 at the Advanced Light Source. The structure of
FimH.sub.A-ZFH253 was solved by molecular replacement with the
program PHASER using a previously-solved model of FimH.sub.A with
its methylmannose ligand removed as the search model (PDB ID 1
UWF).
[0616] An initial map calculated with Fo-Fc coefficients showed
unambiguous difference density for ZFH253 occupying the mannose
binding pocket of all four copies of FimH.sub.A present in the
asymmetric unit. These studies showed that the mannose portion of
ZFH253 engages in conserved interactions with FimH.sub.A. ZFH253
buries 293 .ANG..sup.2 (calculated with PISA). This places ZFH253
between monomannose, which buries approximately 185 .ANG..sup.2,
and oligomannose-3, which buries 388 .ANG..sup.2. Relative to
monomannose, most of the additional surface area is buried by the
two phenyl groups of ZFH253, which do not engage the "tyrosine
gate" of Tyr48 and Tyr137 but orient toward Tyr48 such that the
phenyl group most distal to the mannose moiety forms a close
pi-stacking interaction with Tyr48 (FIG. 9). This interaction
mimics the aromatic-to-saccharide stacking between Tyr48 and Man3
of oligomannose-3 that contributes to the high affinity of
high-mannose epitopes to FimH in vivo. Additionally, the terminal
carboxyl group of ZFH253 forms two hydrogen bonds with FimH (FIG.
9), offering a structural basis for its 16-fold increase in potency
in the HA titer assay over ZFH296, which contains only two
unsubstituted phenyl groups. Further, the FimH.sub.A/ZFH253 crystal
structure suggested several avenues for rational improvement of
mannoside derivatives. First, there is an ordered water at 2.7
.ANG. from the carboxyl group of the first carbon of the mannose
ring that forms close hydrogen bonds with the N-terminus, the
main-chain carbonyl of Phe1, and the epsilon oxygen of Gln133.
Extension of this carboxyl group by two bond lengths would allow
the mannoside to engage these interactions. There is a second
ordered water 4.7 .ANG. from an ortho carbon of the first phenyl
group of the mannoside that could be coordinated by a hydrogen bond
donor. Importantly, the FimH.sub.A/ZFH253 crystal structure serves
as proof of concept that computational docking may serve as a
useful tool in rational design of related mannosides. The
conformation of ZFH253 seen in the crystal structure is essentially
identical to that of a lowest-energy conformer generated by OMEGA2
and docked into a previous crystal structure of FimH by FRED
(OpenEye, Inc.). This approach will speed the design of further
compounds, with additional crystallography for the exploration of
scaffolds other than the biphenyl group of ZFH253.
Example 9. Inhibition of In Vivo Functions of FimH
[0617] Based on the extensive elucidation of the UPEC pathogenic
cascade, critical nodes in pathogenesis that mannosides would
target and thereby have powerful and potent therapeutic
ramifications were identified. The first step in the UPEC pathway
involves FimH-mediated colonization of the bladder epithelium via
the recognition of mannose receptors. Bacterial invasion into
superficial bladder umbrella cells can then ensue. By preventing
bacterial attachment, mannosides will also inhibit the subsequent
invasion of bacteria into the bladder epithelial cells. To test
this, a well-established gentamicin protection assay that allows
the determination of the luminally bound fraction versus the
intracellular fraction was utilized (FIG. 10). In the presence of
mannosides ZFH177 and ZFH253 there was a significant reduction in
intracellular bacteria at 1 hour post-infection arguing that the
mannosides are disrupting specific type 1-mediated binding required
for subsequent invasion. It is not clear why a reduction was not
observed in the luminal UPEC population. This may be due to
contamination from bacteria in the urine or bacteria that are
non-specifically associated with the epithelium. In any case,
blocking invasion blocks the ability of the bacteria to rapidly
expand in numbers. This is due to the ability of the mannosides to
block the IBC mechanism.
[0618] After invasion, UPEC are able to rapidly replicate to form
IBCs, thus dramatically expanding in numbers. After maturation of
the IBC, bacteria are able to disperse from the biomass and spread
to neighboring cells to expand the buildup in bacterial numbers via
next generation IBC. The ability of mannosides to block IBC
formation was evaluated based on LacZ staining of whole mount
bladders, a protocol developed to enumerate IBCs. Whole bladder
titers were also determined by quantifying colony forming units
(CFUs) at 6 hours post-infection and later time points as test
compounds were further developed. In the presence of mannosides
ZFH177 and ZFH253 there was a significant decrease in IBCs observed
6 hours postinfection at concentrations as low as 0.1 mM and almost
no IBCs observed at 1 mM Mannoside (FIG. 11A). A significant
decrease was also seen in CFUs at 6 hours post-infection with 1 mM
ZFH177 and ZFH253 (FIG. 11B). These results strongly argue that the
mannosides are able to inhibit first round IBC formation by
preventing FimH-mediated binding and invasion into the bladder
epithelial cells. The success of these experiments suggested that
these compounds would have dramatic potential as potent
therapeutics for treating UTI, the most common bacterial infection
in highly industrialized countries.
Example 10. Pharmokinetics/Bioavailability/Toxicity
[0619] Pharmokinetic, bioavailability and evaluation of toxicity
studies will be performed to elucidate the effectiveness of
synthesized mannosides in treating UTI. Using these studies we will
identify: i. the kinetics by which the mannoside is excreted in the
urine, ii. the dosage administered to the mouse versus the amount
that reaches the urine to obtain 1 mM and 0.1 mM concentrations
(determined effective through above results), iii. the length of
time the mannoside remains in the urine after one dose and iv. the
presence of mannoside within the tissue, namely the bladder. The
most effective way of delivering the mannoside (oral, IP or tail
vein) with no toxicity to the animal will be determined. Toxicity
will be evaluated based on animal survival and weight loss/gain of
treated animals relative to non-treated animals. Urine was
evaluated for mannoside presence and concentration using LC-MS. The
delivery method, dose and timing, which gave the highest levels in
the urine were then used in treatment trials.
Example 11. Mannosides as Inhibitors of UTI
[0620] Once the proper delivery method, dose and timing will be
determined, female mice will be pre-treated with mannoside (two
doses) prior to infection (using our well-developed mouse model of
UTI) and treated with mannoside throughout the course of infection
to maintain efficacious levels within the urine. The potency of the
mannoside in preventing UTI based on CFUs in the bladder will be
assessed at 6 hours, 48 hours and 2 weeks post-infection and IBC
formation at 6 hours post-infection compared to untreated mice.
[0621] The ability of mannosides to treat established infections
will be also tested. Mice will be treated with mannoside at 24
hours post-infection and the effect on bacterial clearance will be
assessed by determining CFUs in the bladder 24 and 48 hours and two
weeks post-mannoside treatment. The last set of experiments mimic a
potential situation within the clinic.
[0622] These experiments will show that inhibition of binding of
the FimH adhesin to the bladder epithelium can eliminate infection.
The above experiments also establish that the mannosides block the
formation of QIRs, which proves their efficacy in reducing the
predisposition of the host to suffer a recurrent infection. Due to
increasing antibiotic resistance and high rates of recurrence,
these compounds introduce an entirely new means of treating
microbial infections, using an anti-virulence agent instead of an
antimicrobial.
Example 12. Investigation of Dual Therapy with Adhesion and Pilus
Assembly Inhibitors and/or Antibiotics
[0623] Due to the critical nature of type 1 pili in UPEC
pathogenesis, in addition to the work to develop high affinity
inhibitors of FimH binding described above, the extensive
structural and functional knowledge of the mechanism of pili
assembly by the chaperone/usher pathway (see biosketch) was used to
develop efficient inhibitors of pilus biogenesis ("pilicides")
(FIG. 12). The most efficient pilicides show greater than 90%
inhibition of hemagglutination and type 1 dependent biofilm at
concentrations in the low micromolar range (FIG. 13). Further, the
structural basis by which pilicide block pilus assembly were
identified. These compounds were rationally designed based on a
2-pyridone scaffold that has affinity for beta-sheet structures.
One of the best pilicides to date, FN075, was able to achieve
significant attenuation of virulence as measured by CFUs and IBC
formation (FIG. 14).
[0624] Using pilicides and mannosides in combination would
potentially give a "double hit" to this critical type 1 pilus
system, potentially resulting in synergy by inhibiting pre-existing
type 1 pilus function while eliminating the ability to produce new
pili. Combination therapy using the most effective mannoside
inhibitors and pilicides with antibiotics were tested. In one
experiment, groups of mice were treated with ZFH56 mannoside
treatment only, with antibiotic treatment only, or with a dual
treatment (mannoside+antibiotic). One group of untreated mice was
used as a control (FIG. 15).
[0625] For mannoside treatment, bacteria were incubated with 1 mM
ZFH56 prior to inoculation. Every 24 h for 3 days, mice were
transurethrally inoculated with 1 mM ZFH56. For antibiotic
treatment, mice were inoculated with UTI89 and immediately given
antibiotics in the drinking water for 3 days. Antibiotics used were
trimethoprim-sulfamethoxazole (SXT) at 54 and 270 mg/ml,
respectively. For dual treatment, bacteria were incubated with 1 mM
ZFH56 prior to inoculation. Every 24 h for 3 days, mice were
transurethrally inoculated with 1 mM ZFH56. Additionally, mice were
immediately given antibiotics in the drinking water for 3 days as
in the antibiotic only treatment. Urine was collected every day for
13 days. At 2 weeks, mice were sacrificed and bladders were
titered.
[0626] Dual treatment showed significantly lower levels of bacteria
in the bladder than antibiotics alone (FIG. 16).
Example 13. Bacterial Adhesion--A Source of Alternate Antibiotic
Targets
Introduction
[0627] More than a century ago, the discovery of Penicillin marked
a significant, albeit not immediately recognized, advance in the
field of medicine. By the middle of the 20.sup.th century this
naturally occurring fungal antibiotic had single-handedly
vanquished the biggest wartime killer--infected wounds. Just four
years after the mass production of penicillin began in 1943,
resistant microbes started to appear beginning with Staphylococcus
aureus, followed shortly thereafter by Streptococcus pneumoniae and
Neiserria gonorrheae. Today this list includes antibiotic resistant
Enterococcus, Salmonella, Mycobacterium tuberculosis and
Escherichia coli, to name just a few. The bacterial infections
which contribute most to human disease are also those in which
emerging and microbial resistance is most evident: diarrheal
diseases, respiratory tract infections, urinary tract infections,
meningitis, sexually transmitted infections, and hospital acquired
infections. Thus, there is dire need for new therapeutics to save
the lives and ease the suffering of millions of people.
Rising Antibiotic Resistance
[0628] The antibiotics that are available today are primarily
variations on a single theme-bacterial eradication based on
inhibition of essential molecular processes required for bacterial
growth (bacteriostatic) or cellular maintenance (bactericidal).
Some target cell-wall biosynthesis, whereas others inhibit protein
synthesis or DNA replication. These life or death treatments put
selective pressure on the bacteria to adapt or die, selecting for
antibiotic resistance. Second generation drugs were developed to
combat the resistances that arose, however, they employed the same
general mechanism of action. In addition, while some last resort
drugs have minimal resistance thus far, they are much more
expensive, often are more toxic and typically unavailable to many
countries around the world. Consequences of treatment failure due
to antibiotic resistance are high with greater morbidity, mortality
and transmission of the resistant organism. Even if pharmaceutical
companies increased efforts to develop next generation replacement
drugs, bacterial evolution is outpacing drug development for these
classes of antibiotics. We are nearing an end to the seemingly
endless flow of antimicrobial drugs. We are now faced with a long
list of microbes that have found ways to circumvent different
structural classes of drugs and are no longer susceptible to most,
if not all, therapeutic regimens. New tactics and weapons are
needed to combat bacteria that are, owing to evolution and
selection, moving targets.
Alternative Antibiotic Targets
[0629] Targeting bacterial virulence is an alternative approach to
the development of new classes of antimicrobials that can be used
to specifically target and disarm pathogens in the host, while
leaving commensal bacteria unperturbed. Prevention of the activity
of virulence factors will render the bacteria less able to colonize
and give beneficial symbionts and the host immune system time to
eradicate the disease causing pathogen prior to colonization or
facilitate clearing of an established infection. Furthermore,
stripping microorganisms of their virulence properties without
threatening their existence may offer a reduced selective pressure
for drug-resistant mutations and provide increased potency. Upon
exposure to the host, bacteria respond by the production of an
arsenal of virulence factors to combat host immune responses and
facilitate persistence in their desired niche. There are many
examples of virulence factors that represent potential therapeutic
targets. Polysaccharide capsules prevent phagocytosis. Siderophores
facilitate iron acquistion. Flagella promote motility and
chemotaxis. Some bacterial species secrete toxins that alter and
disrupt the eukaryotic host cell resulting in disease. Yet another
strategy employed by bacteria is the production of a molecular
syringe, known as a type three secretion system, that injects
effectors into the host cell causing disease. Another fundamental
virulence property of nearly all microbes is their ability to
adhere to host cells. This step is typically crucial in
pathogenesis and without it bacteria are more likely to be
eradicated by the host. Bacterial adhesins typically mediate
attachment to a specific receptor with stereochemical specificity,
a process that is thought to determine the host and tissue tropisms
of a pathogen. While many virulence factors may represent
attractive therapeutic targets, this review will focus on two
strategies to develop anti-virulence therapeutics based on
targeting adhesive pili using uropathogenic E. coli(UPEC) as the
model system. The first strategy depends on competitively
inhibiting bacterial binding to host cells through addition of
exogenous carbohydrates that mimic host ligands. The second
strategy targets the mechanism by which adhesive pili are assembled
thus inhibiting bacterial adhesion to host cells. Thus, both
strategies could be used in synergy: the first compound prevents
bacterial attachment and colonization by occupying the binding
pocket of the bacterial adhesin while the second compound enters
the cell and prevents future pilus production. Such a two-pronged
approach could eliminate adherence to host cells and promote
clearance of the bacteria.
Pili in Bacteria
[0630] Pathogens are capable of presenting multiple adhesins that
can be expressed differentially to permit binding in specific sites
and at particular times over the course of a complex infectious
cycle. To achieve this, many bacterial species possess long
filamentous structures known as pili or fimbriae extending from
their surfaces. Pili are extracellular polymers that also have been
shown to play a role in invasion, biofilm formation, cell motility,
and protein and DNA transport across membranes. Pilus formation is
common to many pathogenic bacteria, both Gram-negative and
Gram-positive pathogens. Despite the diversity in pilus structure
and biogenesis, assembly mechanisms among Gram-negative and
Gram-positive bacteria are conserved within each group.
Gram-positive pili are formed by covalent polymerization of pilin
subunits in a process that requires a dedicated sortase enzyme. In
contrast, Gram-negative pili are typically formed by non-covalent
homopolymerization of major pilus subunit proteins. This review
will focus on a major family of adhesive pili in Gram-negative
bacteria that are classified by their mechanism of assembly; the
so-called chaperone/usher (CU) pili (Table 14).
[0631] In Gram-negative bacteria, one of the best-characterized
pilus assembly systems is the CU pathway. Hundreds of operons
encoding CU systems have been reported in a plethora of pathogenic
organisms with as many as twelve encoded by a single organism.
Examples of CU-assembled adhesive virulence fibers include: Type 1
and P pili predominately on UPEC that cause urinary tract infection
(UTI), S pili of E. coli strains that cause sepsis, meningitis and
UTI and Hif pili of Haemophilus influenza which causes otitis
media. The causative agents of whooping cough (Bordetella
pertussis), gastroenteritis (Salmonella typhimurium), and pneumonia
(Klebsiella pneumoniae) also assemble fibers through the CU
pathway. Additionally there are `non-pilus adhesins` assembled by
the CU pathway that are generally homopolymers composed of a single
protein subunit, like the Afa/Dr family of adhesins of E. coli and
the polymeric F1 capsular antigen of Yersinia pestis. The
ubiquitous nature of this pathway paves the way for a potentially
broad-spectrum anti-virulence therapeutic that disrupts the
assembly of the pilus fibers thus eliminating bacterial
colonization.
[0632] Pili in UTI
[0633] Of the CU pathway assembled pili, P and type 1 pili are the
most extensively characterized. Type 1 pili are expressed
throughout the Enterobacteriaceae family but have been shown to be
essential for UPEC in colonization of the urinary epithelium. UPEC
is the leading causative agent for greater than 80% of UTIs which
is one of the most common bacterial infections in industrialized
countries. UTIs are a significant burden, resulting in more than 8
million outpatient visits per year in the United States and
expended costs of US $3.5 billion annually for evaluation and
treatment. Women are the most frequent sufferers--a female has a
60% chance of suffering from at least one UTI in her lifetime.
Additionally, up to 44% of women who present with an initial
episode of UTI will experience recurrence. While the increased
likelihood of recurrence it not entirely understood, data suggests
that bacteria can persist within bladder tissue despite antibiotic
treatments, and may serve as bacterial reservoirs for recurrent
infections.
[0634] Type 1 pili are expressed by virtually all UPEC isolates.
The FimH adhesin at the tip of type 1 pili achieves specific
binding to host cells within the urinary tract. FimH specifically
binds mannose groups that abundantly decorate uroplakins on the
luminal surface of the Madder. A deep, negatively charged pocket in
the N-terminal receptor-binding domain of FimH accommodates the
mannose with stereochemical specificity. Once bound, a pathogenic
cascade is initiated that involves several distinct phases as
examined in the murine cystitis model and human UTIs. Numerous
innate defenses are activated early in an attempt to stem off the
infection. These defenses include, cytokine induction followed by
inflammation, iron sequestering, exfoliation of the colonized
epithelial cells and sheer forces elicited by urine flow. Even if
UPEC successfully invade into a bladder epithelial cell, the
bladder cell has been shown to be able to expel UPEC, potentially
serving as an additional innate defense. However, if UPEC are able
to escape into the cytoplasm of superficial umbrella cells, they
are able to rapidly replicate to form tightly packed intracellular
bacterial communties (IBCs) with biofilm-like qualities. Within the
urothelial cells, the bacteria are protected from the flow of urine
and host defenses. Eventually UPEC detach from the IBC, disperse
and initiate new rounds of IBC formation. This represents a
mechanism by which a single invasion event dependent on type 1 pili
adhesion can lead to rapid bacterial expansion and colonization of
the urinary tract. IBC formation occurs in the acute states of
infection. One long-term outcome of infection is that UPEC are also
able to form a quiescent intracellular reservoir (QIR) within the
bladder. Bacteria in the QIR do not rapidly replicate, but remain
dormant, hidden from the immune system and antibiotics. They are
then able to reestablish an infection at later timepoints
post-treatment resulting in a recurrence. Due to this alternative
pathway of continuous colonization, UTIs are more frequently being
identified as chronic infections. Detailed analysis of the UPEC
pathogenic cascade has identified several potential targets to
inhibit virulence, such as siderophores, capsule and flagella.
However, type 1 pili represent a particularly attractive drug
target because they are ubiquitous among UPEC, and are required to
initiate a pathogenic cascade that evades the host immune system
and can lead to acute, chronic, persistent or asymptomatic
infection. Disruption of type 1 pili function could break the cycle
of chronic infection.
New Drug Development Strategies to Inhibit Pilus Mediated
Function
[0635] Carbohydrates of various forms are abundantly expressed on
the animal cell surface. Microbes have taken advantage of this
property and evolved with the ability to adhere to sugar receptors
in an organ-specific manner for colonization and infection. Sugars
are commonly involved in cell recognition and communication playing
important roles in microbial adherence and uptake, colonization and
biofilm formation, and in virulence. Since bacterial adhesion to
host cells is extremely specific, exogenous sugars can block
binding to carbohydrate receptors and competitively displace or
inhibit bacteria from attachment to cells. The ability to impair
bacterial adhesion represents an ideal strategy to combat bacterial
pathogenesis because it targets an important early step in the
infectious process, and would also be suitable for use as a
prophylactic to prevent infection. In the case of UPEC, knowledge
of the structural basis of how FimH recognizes the mannose
receptor, has lead to the development of a ligand-based antagonist,
termed mannoside, that mimics the natural receptor for FimH but
with increased affinity and avidity. Mannosides effectively block
the adhesive properties of type 1 pili by inhibiting bacterial
colonization, invasion, IBC formation and disease. Furthermore, the
sequence of approximately 300 fimH genes of clinical UPEC isolates
showed that the mannose-binding pocket is invariant. Thus
mannosides are a powerful new class of therapeutics that could have
a potent therapeutic effect by preventing UPEC pathogenesis and
disease. This approach can be readily extended to other adherent
organisms by tailoring the anti-adhesive compounds to antagonize
their specific receptor-ligand interactions (FIG. 17). Indeed,
sugars designed to target adhesive structures of organisms such a
Pseudomonas aeruginosa, Stapyhlococcus and Streptococcus have
already proved efficacious at inhibiting adherence in vitro.
[0636] The goal of the second strategy is to interrupt pilus
assembly, which, like the first strategy, blocks pilus-mediated
adhesion and subsequent disease. To disrupt pilus assembly, one
must first understand the mechanisms employed by bacteria to form
fibers. Chaperone-usher mediated assembly prevents premature
subunit aggregation and facilitates the ordered secretion, folding
and assembly of hundreds of thousands of subunits into fibers on
the surface of a bacterium.
[0637] Components of the pilus are secreted through the general
secretory pathway into the periplasm. Once in the periplasmic
space, a specific chaperone binds each pilus subunit to facilitate
proper folding and prevent premature assembly of subunits. Each
subunit consists of six of the seven strands of a standard
immunoglobulin (Ig)-fold and an N-terminal extension (Nte) (FIG.
18A). In order to form a stable structure prior to incorporation
into the growing fiber, the chaperone donates structural
information to each subunit, its G1 .beta. strand, to compensate
for the missing seventh strand of the subunit in a process called
donor strand complementation (DSC) (FIG. 18B). DSC prevents
aggregation of the subunits prior to fiber formation. The
pilin/chaperone complex is then delivered to the usher, which is a
pore in the outer membrane and serves as a platform for pilus
assembly. During pilus assembly, the free Nte of one subunit
displaces the chaperone bound to another subunit and serves as the
seventh strand of the Ig-like fold in a process called donor strand
exchange (DSE) (FIG. 18C). DSE leads to the polymerization of the
fiber extending from the bacterium. Since chaperone-usher systems
are necessary for the assembly of extracellular adhesive organelles
in a wide range of pathogens, inhibitors may serve as broad-range
anti-virulence therapeutics, an attractive feature that would
enhance the marketability of an effective drug.
[0638] Pilicides are a class of pilus inhibitors that target
chaperone function and inhibit pilus biogenesis. A recent study
(Pinkner J S et al: PNAS (2006) 103(47):17897-17902) identified a
new class of pilicides based on a bi-cyclic 2-pyridone chemical
scaffold that inhibit pilus assembly by binding to the chaperone
and disrupting the chaperone-usher interaction. As a result,
pilicides inhibit pilus dependent activities such as colonization,
invasion and biofilm formation. Biofilms are structured communities
of microorganisms encapsulated within a self-developed polymeric
matrix that defends them against antibiotics and the host immune
system. They are of great medical importance, accounting for over
80% of microbial infections in the body. Pilicides target regions
on the chaperone that are highly conserved among chaperone-usher
pathways and are thus able to inhibit assembly of multiple pilus
systems (type 1 and P pili of E. coli) (FIG. 17). Pyridinones also
avoid the severe drawbacks observed for peptide-based drugs; poor
absorption after oral administration, rapid enzymatic degradation,
and quick excretion.
[0639] Another distinct mechanism of adhesive fiber assembly among
Gram-negative bacteria is fibers assembled by the extracellular
nucleation/precipitation pathway (curli). Curii are proteinaceous
fibers found on enteric bacteria such as E. coli and Salmonella
spp. Despite curli's unclear role in pathogenesis, their
biochemical and structural properties resemble eucaryotic amyloid
fibers found in neurodegenerative diseases, such as Alzheimer's,
Parkinson's, and prion diseases making them an ideal model system
to study amyloid biogenesis. Curli are heteropolymers that consist
of a major subunit, CsgA, and minor subunit, CsgB. CsgA and CsgB
are secreted into the extracellular milieu via the outer membrane
pore, CsgG. With the help of two chaperone-like proteins, CsgB and
CsgF, CsgA is nucleated into a fiber by CsgB. Curli fibers have
been implicated in biofilm formation, environmental survival and
resistance to sanitizing agents. A recent study (Cegelski L et aL:
submitted) showed that derivatives of the 2-pyridone compounds
mentioned above were able to inhibit curli assembly giving them the
name curlicides. The lack of curli fibers prevented curlidependent
biofilm formation and reduced infection load in vivo as measured by
the murine model of UTI. Pilicides and curlicides selectively
disrupt a protein-protein interaction required for the biogenesis
of a bacterial virulence factor instead of targeting a process
essential for survival.
[0640] Employing both anti-virulence strategies, inhibition of
receptor binding and disruption of pilus assembly, could result in
very potent anti-adhesive therapeutics. For example, synergistic
effects could be obtained by inhibiting preexisting type 1 pilus
function through mannosides while also eliminating the ability to
produce new pili through pilicides. Furthermore, if infection is
not entirely controlled by the two-pronged approach, antibiotics
can be added, however, at much lower concentration to reduce the
opportunity to develop resistance. Type 1 pili are an essential
virulence factor for colonization of the urinary tract and are the
most extensively studied chaperone-usher fimbrial system. Thus,
they have been described in this review as an excellent target
resulting in the development of new prototypic therapeutics to
treat UTIs. This new drug class could translate into a multitude of
other therapies for Gram-negative infections.
Conclusion
[0641] While this review focuses on the development of therapeutics
to inhibit adhesion, there are many other virulence factors
employed by bacteria to colonize and persist within the host. The
general strategy of inhibiting virulence factors rather than
essential growth factors changes the paradigm of drug development
and reduces the evolutionary pressure for bacteria to develop
resistance. Bacteria can survive in the presence anti-virulence
drugs, but will be eliminated by the host immune system prior to
establishing a foothold. Furthermore, since a complex virulence
mechanism is targeted, if mutants arise, they will most likely be
avirulent. If the host immune system cannot resolve any residual
infection, it is possible to couple anti-virulence therapeutics
with traditional antibiotics although at lower doses thus reducing
resistance potential. A commitment to developing therapeutics that
target virulence requires a serious change in our perspective for
treating infectious diseases and increased efforts should be
focused on bringing these new therapeutics from bench to
bedside.
Example 14. Orally Active Mannosides Subvert Antibiotic Resistance
of E. Coli in Bladder Infection
[0642] In this example, therapeutic utility of mannosides that are
competitive, high-affinity inhibitors of FimH mannose binding
activity is analyzed, as measured by biofilm formation, type 1
pilus expression and bladder colonization, invasion and IBC
formation. The mannose binding site of FimH consists of a deep
acidic pocket surrounded by a ridge of hydrophobic residues. The
acidic amino add residues of the binding pocket are invariant in
all UPEC and mutational studies showed that altering any of these
residues abolishes binding to mannose and attenuates virulence.
From the rational design and optimization of mannosides, several
potent inhibitors of FimH-dependent hemagglutination were
identified, including ZFH-1101, ZFH-1177, ZFH-1253, ZFH-2050 and
ZFH-2056 (FIG. 19a). These mannosides were evaluated first for
their ability to inhibit biofilm formation or disrupt preformed
biofilms. Biofilm inhibition was evaluated on FimH-dependent UTI89
biofilms grown on PVC in LB at room temperature. The median
inhibitory concentration (IC50) values for each of the compounds
were found to be in the low micromolar range. Compounds ZFH-1253
and ZFH-2056 were the most potent biofilm inhibitors with IC50
values of 0.94 and 0.74 .mu.M, respectively (FIG. 19b). Biofilm
disruption was evaluated by the addition of mannosides to preformed
24 h biofilms followed by growth for an additional 16 h. All five
mannosides were shown to facilitate dispersal of the preformed
biofilm. The IC50 values for dispersal were higher than those
concentrations needed to prevent biofilm formation, however the
efficacy trend remained the same (FIG. 19c). Visualization of
mannoside-treated PVC biofilms by confocal microscopy showed that
ZFH-2056 treated biofilm lacked continuity as demonstrated by holes
in the biofilm, and it lacked the tall mushroom-like structures
observed in untreated biofilm (FIG. 19 d,e). Biofilm causes many
antibiotic treatment failures since antibiotics are unable to
penetrate its dense matrix. These results demonstrate that
mannoside can prevent biofilm formation and dissolve preformed
biofilms arguing that mannosides may augment antibiotic efficacy by
altering bacterial community behavior.
[0643] To test the efficacy of the mannosides in vivo, UTI89
expressing type 1 pili were pre-incubated with mannosides,
inoculated into mice and IBCs were quantitated at 6 hours
post-infection (hpi). The efficacy of compounds ZFH-1177, ZFH-1253,
ZFH-2050 and ZFH-2056 in inhibiting IBC formation correlated with
their potency observed in the in vitro assays. The most significant
reduction in IBCs occurred with as little as 10 .mu.M ZFH-2050 or
ZFH-2056 (FIG. 20a). These studies identified ZFH-2056 as the most
potent compound tested in preventing colonization.
[0644] Pharmacokinetic (PK) studies with ZFH-2056 evaluated in vivo
dosing conditions after intraperitoneal (IP) injection or oral (PO)
gavage. ZFH-2056 was dosed IP and compound levels in the urine were
evaluated at several timepoints by HPLC using mass spectrometry
(MS) detection. Doses of 5 mg/kg and 10 mg/kg resulted in
concentrations of nearly 5 mM in the urine 30 min after treatment
(FIG. 20b). Eight hours after dosing, ZFH-2056 remained at levels
equal to or slightly below the IC50 (0.74 .mu.M) as determined by
biofilm inhibition. Since oral dosing is the desired route of
delivery, we next evaluated the concentration of drug after 100
mg/kg PO of ZFH-2056. The latter resulted in a 10 fold lower
initial concentration of ZFH-2056 versus IP but, significantly,
urine levels were 10-fold higher at 8 hours post-dosing. It is
noteworthy that >95% of drug cleared to the urine is intact with
the remaining <5% being the hydrolysis products, D-mannose and
phenol.
[0645] The efficacy of in vivo mannoside treatment was evaluated
after dosing animals with mannoside either IP or PO 30 min prior to
infecting with UTI89. At 6 hpi the bladders were removed and total
bacterial CPUs were quantitated. In both the IP and PO treated
cohort, there was a significant drop in bacterial counts
demonstrating the efficacy of mannoside in reducing overall
colonization of the bladder (FIG. 20e). Furthermore, IBC
quantification showed a significant reduction in IBCs in the mice
that were pretreated with mannoside by either the IP or oral route
(FIG. 20f). To test whether mannoside reduced IBC formation by
blocking UPEC invasion into the bladder tissue, gentamicin
treatment assays were performed. Gentamicin efficiently kills
extracellular UPEC but is unable to penetrate tissue and thus
intracellular bacteria survive gentamicin treatment 18. In the
ZFH-2056-treated mice, gentamicin treatment of the bladders
eliminated all CPUs (FIG. 20g). In bladders from untreated mice,
10.sup.3-10.sup.4 CPUs remained after gentamicin treatment. These
results argued that mannoside effectively prevented UPEC
penetration into the bladder tissue. Confocal microscopy confirmed
these results. Bladders of the untreated cohort showed normal,
robust IBC formation (FIG. 20c) whereas IBCs were rarely seen in
the mannoside treated mouse bladders but bacteria were observed on
the bladder epithelial surface (FIG. 20d). These results
demonstrate that mannosides prevent bacterial invasion into the
bladder tissue and significantly reduce infection in the
bladder.
[0646] The first-line treatment of choice for UTI has long been a
3-day course of TMP-SMZ. However, resistance to TMP-SMZ is on the
rise. Furthermore, humans have a 38% carriage rate of TMP-SMZ
resistant (TMP-SMZR) strains, and these numbers are even higher in
low resource countries with a 94% carriage rate. TMP-SMZ is known
to concentrate in the urine. Therefore, it was reasoned that by
preventing bacterial invasion into the bladder tissue, mannoside
may have an anti-virulence synergism with TMP-SMZ and may
circumvent the TMP-SMZ resistance problem. This was tested in mice
given TMP-SMZ for 3 days prior to inoculation with either UTI89 or
TMP-SMZR strain, PBC-1. Mice were either untreated or IP treated
with mannoside ZFH-2056 30 min prior to inoculation with bacteria.
After inoculation with UTI89 or PBC-1, bacterial CPUs were
quantitated 6 hpi. Upon treatment with only ZFH-2056 there was a
significant drop in bacterial load of both strains in the bladder.
As expected, treatment with TMP-SMZ alone resulted in a significant
drop in bacterial load in the UTI89-infected mice but had no effect
on PBC-1, since it is resistant to TMP-SMZ. In the dual treatment
group there was a significant drop in bacterial CFUs compared to
mannoside alone or TMP-SMZ alone for both strains which was most
pronounced for PBC-1 (FIG. 21 a). The observation that TMP-SMZR
strain PBC-1 was able to succumb to antibiotic treatment in the
presence of mannoside suggested that either mannoside increases the
efficacy of TMP-SMZ killing or that it exposes the bacteria to
concentrations at or above the MIC necessary for killing. Mannoside
was not found to increase the efficacy of TMP-SMZ killing when
bacteria were grown in varying concentrations of TMP-SMZ with and
without 100 .mu.M of ZFH-2056 (FIG. 21 b). This analysis revealed
that the TMP-SMZ resistant strain had a MIC of 256 and 1280
.mu.g/ml, respectively. Furthermore the HA titer of PBC-1 after
growth in varying concentrations of TMP-SMZ showed that type 1 pili
function was not affected by antibiotic (data not shown). It has
long been known that TMP concentrates in the urine, and this
serendipitous feature is one reason TMP-SMZ has been the antibiotic
of choice for UTI over the last several decades. Using MS, the
concentration of TMP-SMZ was measured in the urine of mice after 3
days of treatment with 54 .mu.g/ml and 270 .mu.g/ml TMP and SMZ,
respectively. TMP concentrations were determined to be 9.95+/-4.36
mg/ml. SMZ was present at 67.17+/-32.51 .mu.g/ml. These results
show that by preventing bacterial invasion, mannoside keeps UPEC
extracellular thus exposing them to TMP concentrations in the urine
that are well above the MIC of the UPEC strain. This enables the
killing of bacteria that clinically are resistant to TMP-SMZ. Thus,
the invasion of the TMP-SMZR UPEC strain PBC-1 into bladder tissue
protected it from the exposure of TMP-SMZ concentrations of
antibiotic necessary for killing. However, mannoside prevented
access of the bacteria to the intracellular compartment and thus
kept the bacteria extracellular where TMP-SMZ concentrates to high
levels in the urine, resulting in their succumbing to killing by
the antibiotic. These results highlight the importance of the
intracellular pathway in bacterial persistence. Not only do the
bacteria escape the immune system in their intracellular niche,
they are also able to escape exposure to effective antibiotics.
Methods for Example 13
[0647] UTI89 biofilm was grown in LB+/-mannoside for 24 h at
22.degree. C. in PVC plates and quantitated using crystal violet.
UTI89 biofilm for confocal microscopy was grown in LB for 24 h at
22.degree. C. on PVC coverslips. Mannoside was then added and
biofilm was grown for an additional 16 h. Coverslips were then
washed in PBS, fixed in 2% paraformaldehyde and stained with SYTO9
prior to confocal microscopy. For all animal experiments UTI89 or
PBC-1 was grown 2.times.24 h statically in LB at 37.degree. C. and
inoculated at a dose of 1.times.10.sup.7 bacteria in 50 .mu.L. All
mice used were female C3H/HeN (Jackson). For the pretreatment
studies, bacteria were incubated with mannoside for 20 min prior to
inoculation into the mouse. For IP dosing, 50 .mu.l of 2 mg/ml (5
mg/kg) or 4 mg/ml (10 mg/kg) ZFH-2056 in PBS was injected into the
mouse 30 min prior to inoculation of bacteria. For oral dosing, 100
.mu.l of 20 mg/ml (100 mg/kg) ZFH-2056 in 8% DMSO was inoculated
with a gavage needle 30 min prior to inoculation of bacteria. Mass
spectrometry was used to quantify urinary mannoside or TMP-SMZ
concentrations. For CFU counts, bladders were harvested at 6 hpi
and placed in 1 mL PBS. Bladders were then homogenized, diluted and
plated on LB. After growth at 37.degree. C. overnight, bacterial
counts were determined. LacZ staining and gentamicin protection
assays were performed at 6 hpi. For antibiotic experiments, mice
were given TMP-SMZ in the drinking water at a concentration of 54
.mu.g/ml and 270 .mu.g/ml, respectively. Water was changed daily
with fresh antibiotics. Standard growth curve assays and
hemagglutination assays were performed with TMP-SMZ. All
statistical analysis performed was a two-tailed Mann-Whitney U
test.
[0648] Bacterial strains. UTI89 is a prototypical cystitis isolate
of serotype 018:K1:H7. PBC-1 is a TMP-SMZR strain of serotype
OX13:K1:H10 isolated from a 59 year old asymptomatic female with a
history of recurrent UTI and diagnosis of primary biliary
cirrhosis.
[0649] Synthesis of mannosides. Following the procedure outlined in
Han et. al. 2010 (J Med Chem 53 (12), 4779-4792), ZFH-2056 was
synthesized on a multi-gram scale with >98% purity by HPLC and
NMR.
[0650] Biofilm assay. UTI89 was grown in LB broth in wells of PVC
microtiter plates at 23.degree. C. in the presence of individual
mannosides at varying concentrations. After 48 h of growth, wells
were rinsed with water and stained with crystal violet for
quantification as described. For biofilm disruption activity in PVC
plates, UTI89 was grown in LB broth in wells of PVC microtiter
plates at 23.degree. C. After 24 h of growth, mannoside was added
and biofilms were grown for an additional 16 h. Wells were then
rinsed, stained with crystal violet and quantified. For biofilm
disruption activity on PVC coverslips, UTI89 was grown in LB broth
in 50 mL conicals containing PBC coverslips at 23.degree. C. After
24 h of growth, 0.3 nM ZFH-2056 was added and biofilm was grown for
an additional 16 h. Coverslips were then rinsed, fixed with 2%
paraformaldehyde (v/v), stained with SYTO9 (1:1000 in PBS;
Molecular Probes) and observed with a Zeiss LSM410 confocal laser
scanning microscope under a 63.times. objective.
[0651] Animal infections. Bacteria were grown under type 1
pili-including conditions (2.times.24 h at 37.degree. C. statically
in LB). The bacteria were harvested and resuspended to an
OD.sub.600 Of 0.5 in PBS. Eight-week-old C3H/HeN (Harlan) female
mice were anesthetized by inhalation of isoflurane and infected via
transurethral catheterization with 50 .mu.l of the bacterial
suspension, resulting in 1-2.times.10.sup.7 inoculum. At 6 hpi,
mice were sacrificed by cervical dislocation under anesthesia and
the bladders were immediately harvested and processed as described
below. All animal studies using mice were approved by the Animal
Studies Committee of Washington University (Animal Protocol Number
20100002).
[0652] Enumeration of bladder IBCs. For bacterial pretreatment
experiments, mannoside was added to the bacterial suspension and
incubated for 20 min. For animal pretreatment experiments,
mannoside ZFH-2056 was administered either IP (5 mg/kg) or orally
(100 mg/kg) 30 min prior to inoculation with UTI89. To accurately
count the number of IBCs, mice were sacrificed 6 hpi and bladders
were aseptically removed, bisected, splayed on silicone plates and
fixed in 2% paraformaldehyde (v/v). IBCs, readily discernable as
punctate violet spots, were quantified by LacZ staining of whole
bladders.
[0653] Pharamacokinetic analysis. For intraperitoneal dosing, 50
.mu.l of a 2 mg/ml (5 mg/kg) or 4 mg/ml (10 mg/kg) solution of
ZFH-2056 in PBS was injected into the peritoneal cavity of the
mouse. For oral dosing, 100 .mu.l of a 20 mg/ml (100 mg/kg)
solution of ZFH-2056 in 8% DMSO was inoculated with a gavage needle
into the mouse stomach. Urine was collected at 30 min, 1, 2, 3, 4,
6, and 8 h post-treatment. An equal volume of 10 internal standard
(ZFH-2050) was added to the urine. Mannosides were extracted from
the urine by loading on C18 columns (100 mg, Waters), washing with
30% methanol, and eluting with 60% methanol. Vacuum-concentrated
eluates were analyzed using liquid chromatography-mass spectrometry
system 30 with a lower heated capillary temperature of 190.degree.
C. and a gradient as follows: Solvent B (80% acetonitrile in 0.1%
formic add) was held constant at 5% for 5 minutes, increased to 44%
B by 45 minutes, and then to a 95% B by 65 minutes. SRM mode
quantification was performed with collision gas energy of 30% for
the following MS/MS transitions (precursor m/z/product m/z):
compound ZFH-2056, 447/285; compound ZFH-2050, 390/228. Absolute
quantification was achieved by comparison to a calibration
curve.
[0654] Bladder tissue bacterial titer determination. Mannoside
ZFH-2056 was administered either IP (5 mg/kg) or orally (100 mg/kg)
30 min prior to inoculation with UTI89. To enumerate the bacteria
present, mice were sacrificed at 6 hpi and bladders were
aseptically removed and homogenized in 1 ml PBS, serially diluted
and plated onto LB agar plates. CFU was enumerated after 16 h of
growth at 37.degree. C.
[0655] Confocal microscopy. Mannoside ZFH-2056 was administered IP
(5 mg/kg) 30 min prior to inoculation with UTI89. To visualize
bacterial behavior within the bladder, mice were sacrificed at 6
hpi and bladders were aseptically removed, bisected, splayed on
silicone plates revealing the luminal surface and fixed in 2%
paraformaldehyde (v/v). The splayed bladders were then incubated
for 20 min at room temperature with (i) SYTO9 (1:1000 in PBS;
Molecular Probes) to stain bacteria and (ii) Alexa Fluor
594-conjugated wheat germ agglutinin (WGA; 1:1000 in PBS; Molecular
Probes) to stain the bladder luminal surface. Bladders were rinsed
with PBS, mounted using Prolong Gold antifade reagent (Invitrogen)
and examined with a Zeiss LSM510 confocal laser scanning microscope
under a 63.times. objective. SYTO9 and WGA were excited at 488 and
594 nm, respectively.
[0656] Gentamicin protection assay. To enumerate bacteria present
in the intracellular versus extracellular compartments, bladders
were aseptically harvested at 6 hpi. The bladders were then
bisected twice and washed three times in 500 .mu.l of PBS each. The
wash fractions were pooled, lightly spun at 500 rpm for 5 min to
pellet exfoliated bladder cells, serially diluted, and plated onto
LB agar to obtain the luminal fraction. The Madders were treated
with 100 .mu.g of gentamicin/ml for 90 min at 37.degree. C. After
treatment, the bladders were washed twice with PBS to eliminate
residual gentamicin, homogenized in 1 ml of PBS, serially diluted,
and plated onto LB agar to enumberate the CPUs in the intracellular
fraction.
[0657] Antibiotic treatment. Mice were given TMP-SMZ in the
drinking water at a concentration of 54 ng/ml and 270 .mu.g/ml,
respectively. Water was changed daily for 3 days prior to
inoculation with UTI89. Mice remained on TMP-SMZ during the
infection. To determine TMP-SMZ concentration in the urine, urine
was collected after 3 days of TMP-SMZ treatment and quantified by
LC-MS following addition of sulfisoxazole as an internal
standard.
[0658] Growth curve. An overnight culture of PBC-1 was diluted
1:1000 in LB in the absence or presence of TMP-SMZ and/or mannoside
ZFH-2056. The highest concentration of TMP-SMZ used was 512 ng/ml
and 2560 .mu.g/ml, respectively. Two-fold dilutions of TMP-SMZ were
performed. Mannoside ZFH-2056 was added at 100 .mu.M. Growth curves
were performed in a 96-well plate at 37.degree. C. with A600
readings taken every 30 min for 8 h.
[0659] Hemagglutination assay. PBC-1 was grown statically in LB in
the absence or presence of TMP-SMZ for 2.times.24 h at 37.degree.
C. The highest concentration of TMP-SMZ used was 256 .mu.g/ml and
1280 .mu.g/ml, respectively. Two-fold dilutions of TMP-SMZ were
performed. Hemagglutination assays for mannose-sensitive
agglutination of guinea pig red blood cells were performed as
previously described.
[0660] Statistical analysis. Observed differences in bacterial
titers and IBC numbers were analyzed for significance using the
nonparametric Mann-Whitney U test (Prizm; GraphPad Software).
Example 15. Improving Solubility and Potency of Mannosides
[0661] Amide groups were added to the insoluble 1ZFH253 and 1ZFH296
mannosides to produce mannoside compounds with increased solubility
as shown in the FIG. 24. A divalent compound was also produced
using 2ZFH50. The efficacy of these compounds along with 1ZFH177
was tested in an in vitro inhibition of hemagglutination and
biofilm assays (FIG. 25). These results demonstrate improved in
vitro potency of mannosides comprising enhanced solubility.
[0662] ZFH56 was injected IP into mice at various doses and levels
of the compound were measured in the animal (FIG. 26). 10 mg/kg, 5
mg/kg or 5 mg/kg followed by a 4 hour boost were injected into
animals.
[0663] IBC numbers were also determined in animals pretreated with
ZFH56 (FIG. 27). ZFH56 also altered the pathogenic pathway as
measured by confocal microscopy (FIG. 28). ZFH56 also showed
increased efficacy against TMP-SMZ resistant strains (FIG. 29).
Example 16. Testing Against Various Inoculum Levels
[0664] An inoculum of 10.sup.7 is likely well above the
physiological dose typically found in a human. It is likely that
only a few bacteria are inoculated into the urinary tract for a
successful infection. Thus, a lower dose of bacteria was introduced
to the mouse and determined whether this increased the efficacy of
mannoside ZFH56 (FIG. 30). In order to obtain a consistent
infection in the mouse model of UTI the dose could only be lowered
by 10 fold. With an inoculum of UTI89 of 10.sup.6, there was a
significant decrease (p<0.0001) in bacterial titers in the ZFH56
treated mice compared to the untreated mice. However, the level of
infection in the mannoside treated mice at both the 10.sup.7 and
10.sup.6 inoculum was approximately equivalent suggesting that the
lower dose did not further enhance efficacy of the mannoside. Using
IBC counts in the bladder at the lower dose there were no IBCs
observed in the ZFH56 treated mice at the 10.RTM. inoculum. However
the overall number of IBCs in the untreated mice were also much
lower. Since there were no IBCs at 6 h in the 10.RTM. inoculum, it
was hypothesized that the level of infection at 2 weeks
post-infection would be significantly lower. Evaluation of 2 week
titers revealed a significant decrease in bacterial load in the
mannoside treated bladders with the 10.sup.6 inoculum. This data
suggests that upon lowering the inoculum, mannoside is able to
significantly reduce colonization at 2 wpi with a single dose of 5
mg/kg ZFH56 30 min prior to inoculation of UTI89.
[0665] PK studies with IP dosing revealed that after injection,
mannoside ZFH56 is rapidly eliminated in the urine and is mostly
gone by 6-8 h post injection. Thus the mice were boosted with 5
mg/kg mannoside ZFH56 every 8 h to maintain a constant presence of
mannoside in the mouse. This experiment comprised 4 groups: (1)
untreated, (2) pretreat by IP injection of 5 mg/kg ZFH56 30 min
prior to inoculation with UTI89, (3) pretreat by IP injection of 5
mg/kg ZFH56 30 min prior to inoculation plus 2 additional
treatments every 8 h, and (4) inoculation with UTI89 followed by IP
treatment with 5 mg/kg ZFH56 1 h post-infection and 2 additional
treatments every 8 h. Mice were harvested at 24 hpi. This was done
with the 10.sup.7 and 10.sup.6 inoculum of UTI89. There was a
significant drop in bacterial titers with the single pretreatment
of ZFH56 with both inoculum doses. In the mice inoculated with
10.sup.7 UTI89 there was further efficacy of ZFH56 upon boosting.
However, this same effect was not observed with the 10e inoculum.
With both inoculums there was no efficacy observed upon
post-infection treatment with ZFH56. These data suggest that
mannoside is currently most efficacious when used prophylactically
(FIG. 31).
[0666] Next, it was determined if a 10 fold increase in ZFH56 would
increase efficacy and if the divalent ZFH308 was as efficacious as
ZFH56 at 5 mg/kg (FIG. 32). Mice were treated with 5 mg/kg ZFH56,
50 mg/kg ZFH56 or 5 mg/kg ZFH308 30 min prior to infection with
10.sup.7 UTI89. At 6 and 24 hpi bladders were harvested and
titered. At 6 hpi, 5 mg/kg ZFH56 and 50 mg/kg ZFH56 both
significantly inhibited bacterial infection. However, 50 mg/kg
ZFH56 did not significantly enhance efficacy over the 5 mg/kg ZFH56
dose. 5 mg/kg ZFH308 did not significantly reduce bacterial
infection at 6 hpi. At 24 hpi, both 5 mg/kg and 50 mg/kg ZFH56
significantly reduced bacterial titers. 50 mg/kg ZFH56 showed
slight enhancement over 5 mg/kg ZFH56 relative to untreated mice.
Again ZFH308 did not show efficacy in reducing infection. Thus, 5
mg/kg ZFH56 seems to be the optimal dose for inhibiting bacterial
infection and the divalent ZFH308 does not show enhanced efficacy
over the monovalent compound.
Example 17. ZFH56 Disrupts Preformed Biofilm
[0667] UTI89 was grown in LB in PVC plates and allowed to form
biofilm at RT for 24 or 48 h. At 24 or 48 h, varying concentrations
of mannoside ZFH56 was added. Biofilm was then allowed to grow for
another 24 h at RT. Biofilm formation was then quantitated using
the standard crystal violet assay. Results showed that ZFH56 was
able to disrupt preformed biofilm (FIG. 32). At 24 h the IC50 for
biofilm disruption was 6 .mu.M and at 48 h the IC50 was 25 .mu.M.
This shows that not only can mannoside inhibit biofilm formation it
can dissolve biofilm that has already formed.
Introduction for Examples 18-22
[0668] Catheter-associated urinary tract infections (CAUTIs) often
arise from multidrug resistant Gram-positive and Gram-negative
bacterial colonization and biofilm aggregation on the surface of
indwelling urologic devices such as urinary catheters, rendering
treatment very difficult. Uropathogenic Escherichia coli (UPEC),
the primary cause of community-acquired UTI, account for 50% of
nosocomial UTIs, including CAUTIs. Yet, very little is known about
its pathogenesis following urinary catheterization, which results
in the disruption of the normal mechanical and antimicrobial
defenses of the bladder. Previous reports using human biopsies and
rodent models of infections have shown that the catheterized
bladder is edematous and highly inflamed with immune cell
infiltration and pro-inflammatory cytokine production, an
environment quite different from that which UPEC encounters in a
non-catheterized bladder. We hypothesized that these profound
catheter-related changes may affect UPEC pathogenesis.
[0669] The UPEC pathogenic cascade has been extensively
characterized in a non-catheterized murine model of cystitis. UPEC
elaborate on their surface adhesive type 1 pili which mediate
binding to and invasion of superficial umbrella cells lining the
bladder epithelium. Once intracellular UPEC can escape into the
cytoplasm, replicate rapidly and undergo morphological
differentiation within bladder epithelial cells to produce mature
intracellular bacterial communities (IBCs) of 10.sup.4-10.sup.5
bacteria with biofilm-like properties. UPEC then flux out from
infected cells and can reinvade neighboring cells and start the
process de novo. This acute phase of UPEC infection can lead to the
development of chronic cystitis, pyelonephritis, and the formation
of quiescent intracellular reservoirs (QIRs) with absence of
bacteriuria. Detailed understanding of the critical steps of this
pathogenic cascade has led to the development of small molecule
inhibitors called mannosides. Mannosides specifically target the
bacterial type 1 pili tip adhesin, FimH, which binds to
mannosylated residues present on the surface of the bladder
epithelium. Rationally designed to interfere and prevent FimH
interaction with these residues, mannosides inhibit UPEC binding
and invasion of the superficial umbrella cells during urinary tract
infections (UTIs). Mannosides, in combination with existing
antibiotic-based UTI therapy, have recently been shown to be
effective in preventing and treating UPEC infections in
non-catheterized infection models. The present study investigates
whether this therapeutic approach could be beneficial in the
prevention and treatment of CAUTIs.
[0670] In Examples 18-22, the optimized murine model of foreign
body-associated UTI that closely mimics CAUTI was used to
investigate the consequences of urinary catheterization on the
pathophysiology of UPEC infection. For these studies, several UPEC
virulence parameters, including the contribution of type 1 pili,
IBC formation, and QIR reactivation, were assessed. The results
obtained indicate that urinary catheterization provides UPEC with
the opportunity to exploit the extracellular milieu of the bladder
via type 1 pili-mediated biofilm formation on the surface of the
foreign body, which results in a shift in the niche population.
Administration of mannosides in combination with
trimethoprim/sulfamethoxazole prior to urinary catheterization
prevents UPEC colonization of the urinary tract. These Examples
provide important insights into the mechanisms underlying
UPEC-mediated CAUTI, and informs efforts to design better
therapeutic approaches to prevent and potentially treat these
infections.
Materials and Methods for Examples 18-22
Bacterial Strains and Growth Conditions.
[0671] All strains used in this study and their characteristics are
listed in Table 16. Unless otherwise specified, a single colony of
E. coli grown on Luria Bertani (LB, Becton Dickinson) agar plate
supplemented with appropriate antibiotics was inoculated into LB
broth and grown statically at 37.degree. C. for 18 h.
TABLE-US-00005 TABLE 16 Strains used in this study Relevant
Antibiotic Species and Strains resistance Characteristics UTI89
Parental UPEC UTI89 strain, cystitis isolate UTI89.DELTA.fimH UTI89
with an in-frame deletion of fimH, type 1 pili defective
UTI89.DELTA.sfaA-H UTI89 with an in-frame deletion of sfa operon, S
pili defective UTI89.DELTA.sfaA-H.DELTA.fimB-H Kan.sup.R Cm.sup.R
UTI89 with in-frame deletion of the sfa operon and the fim operon
from fimB to fimH, S and type1 pili defective UTI89.DELTA.csgA
UTI89 with an in-frame deletion of csgA, curli deficient
UTI89.DELTA.csgB.DELTA.csgG UTI89 with in-frame deletions of csgB
and csgG, curli deficient UTI89HK::GFP Kan.sup.R UTI89 with an
insertion of kanamycin cassette and GFP at the HK site UTI89pCOMGFP
Kan.sup.R UTI89 ectopically expressing GFP from pCOM plasmid
In Vitro Cultivation and Quantification of Biofilms.
[0672] Biofilms were grown as described by Ferrieres et al. (2007;
FEMS Immun. Med. Micro 51212) on All Silicone Foley catheters (Bard
Medical, GA) or silicone tubing (Thermo Fisher Scientific Inc., PA)
and modified as follows. All tubing and connectors in the system
were autoclaved and ethanol sterilized prior to use. The system was
assembled similar to the previously described flow-chamber system.
Priming of the catheter or the silicone tubing occurred at
37.degree. C. for 20 min by flowing pre-warmed pooled human urine.
Urine was collected from healthy volunteers as approved by the
Institutional Review Board of Washington University in St. Louis.
Pooled samples were spun at 10000.times.g for 15 min, filtered
through 33 .mu.M filters, and, if necessary, stored at 4.degree. C.
for no more than 3 days. Three milliliters of stationary-phase E.
coli from overnight cultures were diluted to 1-2.times.10.sup.6
CFU/ml in human urine and injected into the catheter or silicone
tubing using a 30 cc gauge needle. The bacteria were allowed to
attach to the substratum for 1 h before urine flow via
Watson-Marlow peristaltic pump 205S was resumed at 0.5 ml
min.sup.-1. When indicated, urine was supplemented with 1% methyl
mannose (Sigma, MO) prior to the experiment. After 24 hours, the
remaining medium was exchanged for sterile ddH.sub.2O that was
allowed to flow at 0.5 ml min.sup.-1 to remove residual urine and
non-adherent bacteria in the system. The liquid from catheter or
silicone tubing was then removed by capillary action onto absorbent
paper. The tubing was cut into pieces for CFU enumeration or
crystal violet staining, respectively. For CFU enumeration, at
least three pieces (1 cm in length) of incubated tubing were
separately further cut into smaller pieces and placed into 1 ml
PBS. Adherent cells were detached by sonication (10 min) and
vigorous vortexing (3 min). Viable bacterial counts were assessed
by serial dilution on LB plate with appropriate antibiotics.
Crystal violet staining was used to determine biofilm biomass. At
least 3 pieces of incubated tubing (3 cm in length) were filled
with 0.5% crystal violet at room temperature for 10 min. Excess dye
was removed by washing three times with ddH.sub.2O and dried by
capillary action on absorbent paper. The bound crystal violet was
then dissolved in 200 .mu.l of 33% acetic add and absorbance
measured at 595 nm. The amount of biofilm was expressed as CFU/ml
per cm.sup.2 and A595/cm.sup.2. The experiment was repeated at
least three times with different urine samples.
Animal Implantation and Infections.
[0673] Six to seven week-old female wild-type C57BL/6Ncr mice
purchased from the National Cancer Institute (NCI) were used in
this study. Experiments were performed following one week
adaptation in the animal facility after arrival from NCI. Animals
were implanted and infected with the indicated bacterial strain as
previously described. Briefly, seven to eight week-old female mice
were anesthetized by inhalation of isoflurane and implanted with
platinum-cured silicone tubing (4-5 mm in length) (Implanted).
Immediately following implantation, 50 .mu.l of 1-2.times.10.sup.7
CFU bacteria in 1.times.PBS were introduced in the bladder lumen by
transurethral inoculation. Non-implanted animals were inoculated in
the same manner. Animals were sacrificed at indicated time points
by cervical dislocation under anesthesia inhalation. Bladders and
kidneys were aseptically harvested. Subsequently, the silicone
implant was retrieved from the bladder when present, placed in PBS,
sonicated for 10 min and then vortexed at maximum speed for 3 min.
Bladder and kidneys from each mouse were homogenized in PBS.
Samples were serially diluted and plated on LB agar plates
supplemented with appropriate antibiotics. CFU were enumerated
after 24 h incubation at 37*C. In all cases, experiments were
performed at least twice with n=5 mice/strain/condition. All
studies and procedures were approved by the Animal Studies
Committee at Washington University School of Medicine.
Mannoside and Antibiotic Treatment.
[0674] SdfFor pretreatment experiments, 50 .mu.l mannoside
(mannoside 6; 5 mg/kg mouse body weight) or PBS was administered
intraperitoneally 30 min prior to implantation as previously
described. As indicated for pre-infection treatment,
trimethoprim/sulfamethoxazole (TMP-SMZ) was added to the drinking
water for three days prior to infection at 54 and 270 .mu.g/ml,
respectively. The drinking water was changed every 24 h. To assess
the effects of mannoside and/or TMP-SMZ on established infections,
animals were implanted and infected for 24 h. At 24 hpi, TMP-SMZ
was added to the drinking at the concentrations indicated above and
mannoside 6 or PBS was administered i.p. 6 h prior to sacrifice.
Animals were sacrificed 48 hpi. UPEC reservoir reactivation.
[0675] Non-implanted animals were infected with UTI89HK::GFP as
described above. At fourteen days post infection, urine was
collected, serially diluted and plated for CPU and a subset of
animals implanted as described above. Animals determined to be
bacteriuric (bacterial loads greater than or equal to 10.sup.4
CFU/ml in urine) as counted on titer plates the next day were
eliminated from further study. QIR reactivation post-implantation
was assessed by CFU enumeration of bacteria on implants and in the
organs 3 or 5 days post-implantation (17 or 19 dpi). UTI89HK::GFP
titers greater than 10.sup.4 CFU/ml on implants or bladders were
considered reactivation events. Measures of reactivation events of
animals, which were non-bacteruric at 14 dpi but non-implanted
served as controls.
[0676] IBC enumeration and visualization. Implanted and
non-implanted animals were infected with UTI89 for 6 h. When
indicated, mannoside 6 (5 mg/kg) or PBS was administered i.p. at 30
min prior to implantation. At 6 hpi, bladders were harvested,
bisected, splayed on silicone plates and fixed in 2%
paraformaldehyde. LacZ staining of whole bladders was performed as
previously described. Punctate violet spots characteristic of IBCs
were enumerated by light microscopy.
[0677] For IBC visualization, animals were infected with UTI89
constitutively expressing GFP (UTI89pCom-GFP). At the indicated
time point, bladders were removed, bisected, splayed, and fixed as
described above. The splayed bladders were then incubated for 20
min at room temperature with Alexa Fluor 633-conjugated wheat germ
agglutinin (WGA; 1:1000 in PBS; Molecular Probes) for staining of
the bladder surface and, when indicated, SYTO83 (1:1000 in PBS;
Molecular Probes) to stain bacteria. Bladders were rinsed with PBS,
mounted using Prolong Gold antifade reagent (Invitrogen) and
examined with a Zeiss LSM510 confocal laser scanning microscope
under a 63.times. objective. SYTO83 and WGA were excited at 543 and
633 nm, respectively.
Gentamicin Protection Assay.
[0678] To quantify intracellular and extracellular bacteria,
bladders were aseptically harvested at 3 and 6 hpi. Bladders were
cut in 4 parts and washed three times in 500 .mu.l PBS. The wash
fractions were pooled, centrifuged at 500 rpm for 5 min to pellet
exfoliated bladder cells. The supernatants were then serially
diluted and plated on LB agar supplemented with appropriate
antibiotics, which were incubated at 37.degree. C. for 24 hrs to
obtain extracellular bacterial CPU counts. Rinsed bladders were
then treated with 100 .mu.g/ml gentamicin for 90 min at 37.degree.
C. Following gentamicin treatment, the bladder tissue was washed
twice with PBS to eliminate residual antibiotics, homogenized in 1
ml PBS, and bacterial CPU counts of determined as above to
determine the levels of intracellular bacteria (protected from
gentamicin killing). Statistical methods.
[0679] Comparisons between groups were conducted by nonparametric
Mann-Whitney U test using GraphPad Prism (GraphPad software,
version 5). Values below the limit of detection for in vivo
experiments (20 CFU for organs, 40 CFU for implants) were assigned
the appropriate LOD value for statistical analyses. All tests were
two tailed, and a p-value less than 0.05 was considered
significant. Colonization and infection was defined as
organs/implants with bacterial titers above the limit of
detection.
Example 18: UPEC Adherence, Invasion, and IBC Morphology are
Unaltered in Catheterized Bladders
[0680] IBC formation occurs in the pathogenesis of UPEC in
non-catheterized patients and has been shown in mouse models to be
critical for infection. To assess the effects of urinary
catheterization on IBC formation, 4-5 mm platinum-cured silicone
tubing sections were implanted in the bladders of C57BI/6Ncr female
mice, which were then immediately infected with 1-2.times.10.sup.7
CFU of the well-studied virulent UPEC strain UTI89 by transurethral
catheterization. Gentamicin protection assays performed at 3 hpi
revealed no statistical difference in either the extracellular or
intracellular UPEC populations in the presence or absence of
implants (FIG. 33), indicating no gross defect in bacterial
invasion in implanted animals. IBC formation within both implanted
and non-implanted bladders was assessed by LacZ staining and
confocal scanning laser microscopy (CSLM) at 6 hpi (FIG. 34A).
Inoculation of UPEC into implanted animals resulted in
significantly fewer IBCs with a median of 8 IBCs/bladder (p=0.0044;
FIG. 34B) compared to non-implanted animals in which IBC numbers
ranged up to >250 IBCs/bladder with a median of 55 IBCs/bladder.
However, bacterial CFU in implanted bladders were similar to those
in non-implanted animals (data not shown). To account for this
observation, we postulated that IBC morphology might be different
in implanted animals. However, IBCs formed in implanted animals
were observed to be overall similar in size and shape as those
produced in non-implanted bladders (FIG. 34C). UPEC were also seen
to produce multiple IBCs within one umbrella cell and filamentous
bacterial clumps in both catheter implanted and non-implanted
bladders at 6 hpi (FIG. 35). UPEC colonization at 6 hpi was
selectively localized in the remaining umbrella cells and not
observed in the exposed underlying epithelium in implanted bladders
(FIG. 35). Together, these findings indicate that urinary
catheterization negatively impacts IBC formation by UPEC, possibly
due to a correlated increase in exfoliation.
Example 19: Bacteria Originating from Existing UPEC Reservoirs can
Seed Urinary Implant Colonization
[0681] One troubling possible outcome of the UPEC pathogenic
cascade is the establishment of quiescent intracellular reservoirs
(QIR) in the underlying epithelial layers, which have been shown
can be a source of recurrent UTIs (rUTIs). QIRs were shown to be
reactivated following treatment with protamine sulfate, a chemical
that leads to exfoliation of the superficial umbrella cells of the
uroepithelium. Like protamine sulfate, we have previously shown
that urinary catheterization causes severe damage to the protective
uroepithelial layer. Thus, we hypothesized that urinary
catheterization might also reactivate existing UPEC reservoirs,
resulting in bacteruria, catheter colonization and further
dissemination. To test this hypothesis, mice were infected with
1-2.times.10.sup.7 CFU UTI89HK::GFP and infection allowed to
resolve over the course of 2 weeks. On day 14 post-infection, urine
was collected from each animal to assess infection state prior to
urinary implantation of a subset of these animals. Those animals in
which titering of urines 14 dpi indicating bacteriuria
(.gtoreq.10.sup.4 CFU/ml) were considered to have active
(non-resolved and or recurrent) infection and were removed from
further analysis. The remaining mice were presumed to either have
completely cleared the infection or to have established QIRs with
bacteria loads between 10.sup.1 and 10.sup.4 CFU/ml. 3 or 5 days
post implantation, reservoir reactivation was assessed by bacterial
colonization of implants and bladder. On day 3 post implantation,
UPEC UTI89HK::GFP was recovered from implants in 3 of 26 implanted
mice (11.5%). One mouse with implant had bladder colonization
greater than 10.sup.4 CFU/ml compared to none of the 22 similarly
infected but non-implanted animals (FIG. 36A). There was no
significant difference between groups at 3 dpi. For mice assessed
at 5 days post-implantation, UTI89HK::GFP was recovered from
implants of 4 out of 32 animals (13%) with two of them having
bladder titers greater than 10.sup.4 CFU/ml (FIG. 36B) compared to
0 out of 23 in non-implanted animals. Interestingly, there were
overall significantly fewer bacteria recovered from the bladders of
implanted animals compared to non-implanted animals at 5 dpi
(p=0.0010), suggesting that either reactivated reservoirs are
cleared by the immune response prior to day 5 following
implantation or increased exfoliation prevents establishment of
persistent infections. Together, these data indicate that urinary
catheter colonization can occur from previous urinary infections
even if those infections appear by bacteriuria counts to have been
resolved.
Example 20: FimH is Required for Biofilm Formation and UPEC
Colonization of the Urinary Tract Following Catheter
Implantation
[0682] Biofilm formation is a critical component of CAUTI
pathophysiology. We have previously shown that UPEC is able to
produce biofilms on the surface of the foreign body and is
recovered for the catheterized murine bladder at very high titers.
Type 1 pili are major UPEC virulence factors that have been shown
to be critical for biofilm aggregation, IBC formation, and other
aspects of UPEC uropathogenesis. Thus, we assessed the contribution
of these extracellular pili as well as other UPEC fibers, including
curli which contribute to biofilm formation or S pili associated
with E. coli clinical isolates producing strong biofilms, to
biofilm formation in filtered human urine under flow conditions and
UPEC-mediated CAUTI in vivo. Deletion of the gene for the tip
adhesin of type 1 pili, FimH, in UTI89 resulted in significantly
(p<0.0001) lower biomass (FIG. 37A) and an approximate 2-fold
reduction in adherent viable bacteria (FIG. 37B) in biofilms formed
in human urine in vitro. These data indicate that type 1 pili are a
major contributor to UPEC biofilm formation in urine. The biofilm
defect was specifically associated with the fimH mutant under these
conditions, and was not observed following deletions of the sfa
operon to prevent S pili formation, csgA required for curli fiber
formation, or a component of the flagellar system fliC (data not
shown).
[0683] In vivo, similar to findings in a murine model of cystitis,
UTI89.DELTA.fimH is severely attenuated in the murine model of
foreign body-associated UTI (FIG. 37C). UTI89.DELTA.fimH displayed
>3 log fewer CFU in the bladder and was unable to ascend to the
kidneys at 24 hpi. Further, deletion of fimH resulted in
significant reduction in implant colonization (p<0.0001).
Similar to in vitro experiments, S-pili are not required for CAUTI
since UTI89.DELTA.sfaA-His as virulent as wildtype UTI89 and a
double deletion of both sfaA-H and fimB-H recapitulated the
UTI89.DELTA.fimH phenotype (FIG. 38A). Furthermore, components of
the curli system important for biofilm formation in vitro under
certain conditions, but not in human urine (FIG. 37A-B), were also
dispensable during CAUTI (FIG. 38B). The residual binding to
implants and bladders in implanted animals could therefore be
attributed to other pili or biofilm determinants. Together, these
data strongly suggest that the tip adhesin FimH of type I pili is a
critical determinant of UPEC virulence in mediating biofilm
formation and virulence during CAUTI.
Example 21: Mannoside Treatment Reduces IBC Formation
[0684] Having established that FimH is required for UPEC virulence
in implanted bladders, we investigated this as a potential
therapeutic target for CAUTI using small molecules inhibitors
designed to interfere with FimH binding to mannosylated residues.
This family of small molecules, called mannosides, has recently
been shown to prevent acute and chronic UPEC infections and
potentiated the effectiveness of antibiotics in combinatorial
treatment.
[0685] To investigate the potential therapeutic effects of
mannosides on CAUTI, we first assessed the inhibitory effects of
methyl-.alpha.-D-mannopyranoside (methyl mannose), on UTI89 biofilm
formation in urine under flow. Similar to the deletion of fimH
(FIG. 37A), UTI89 biofilms grown in presence of 1% methyl mannose
had significantly reduced biomass (p=0.0022) and biofilm-adherent
cells (p=0.0012), compared to untreated controls (FIG. 39). Since
methyl mannose is a FimH antagonist, these data confirm the
critical role of type 1 pili to biofilm formation in urine as was
previously described for biofilms formed in LB media.
[0686] The effects of mannoside treatment were then assessed in
vivo by using IBC formation as well as implant and urinary tract
colonization as benchmarks of disease progression. Mice were
treated intraperitoneally (i.p.) with saline or 5 mg/kg of
mannoside 6, which is more potent than methyl mannose in vitro and
in vivo, in PBS 30 min prior to urinary implantation. Catheter
implantation was immediately followed by transurethral inoculation
of UTI89. IBC formation and bacterial colonization were assayed by
LacZ staining and CFU enumeration of implants, bladders, and
kidneys at 6 hpi and 24 hpi, respectively. As shown in FIGS. 40A
and 40B, mannoside treatment further reduced IBC formation
(p=0.0051) and bladder colonization (p=0.0114) in implanted animals
at 6 hpi, suggesting that this treatment prevents intracellular
infection. While eliminated from their intracellular niche, data
further indicated that UPEC were able to persist in the
extracellular milieu where they can colonize the surface of the
implants to relatively similar levels as saline-treated animals
(p=0.0547) (FIG. 40B). No statistical difference was observed in
kidney colonization in the presence or absence of mannosides (FIG.
40B). By 24 hpi, a time point at which the mannosides have been
eliminated from the bladder, similar bacterial loads were recovered
from implants, bladders, and kidneys in implanted animals in the
presence or absence of mannoside treatment (data not shown).
Example 22: Mannoside Treatment Increases the Efficiency of TMP-SMZ
in Preventing UPEC Colonization
[0687] In order to examine whether mannosides could prevent
establishment of CAUTI when used in combination with antibiotics,
animals were treated with 54 and 270 .mu.g/ml of TMP-SMZ,
respectively, in their drinking water for three days and then
treated with saline or mannoside (5 mg/kg) i.p. 30 min prior to
implantation and bacterial inoculation. At 6 hpi, UPEC colonized
the implants and bladders at significantly lower levels in animals
that only received antibiotics compared to those who received water
or were only administered mannoside (FIG. 40B). Interestingly,
mannoside treatment in addition to TMP-SMZ further decreased UPEC
colonization of implants, bladders, and kidneys compared to
treatment with antibiotic alone (p<0.0005 in all cases).
Furthermore, treatment with mannosides alone did not reduce
bacterial titers from a 24 h old UPEC infection and in combination
with TMP-SMZ showed no additive effects on established UPEC CAUTI
24 hpi (data not shown). Together, these findings indicate that
virulence-targeted therapies in combination with established
antibiotic treatment can help prevent or delay the onset of CAUTI
and that further research is warranted for enhancing mannosides
potential as therapeutics against CAUTIs.
Discussion for Examples 18-22
[0688] UPEC is the major etiological agents of CAUTI. Yet, the
molecular mechanisms of urinary catheter and bladder colonization
following urinary catheterization have not been elucidated. Studies
in an optimized murine model of foreign body-associated UTI10 show
that urinary catheterization favors UPEC exploitation of the
bladder extracellular milieu. This occurs via type 1 pili-dependent
biofilm formation on the surface of silicone implants in the murine
bladder. The data further indicate that of the biofilm determinants
tested, type 1 pili are necessary for implant, bladder, and kidney
colonization during CAUTI; providing definitive experimental
evidence for previous reports postulating that type 1 pili may be
required for UPEC persistence during CAUTIs. Interestingly,
fimH-deficient UPEC strains have the ability to adhere to some
extent to the surface of the foreign body, probably using other
biofilm determinants such as other chaperone-usher pili systems,
curli, or surface adhesins.
[0689] In addition to colonizing the foreign body in the bladder
lumen, UPEC is able to exploit intracellular niches in implanted
animals, albeit to a lesser degree than in non-implanted animals,
by invading and producing IBCs in the early stages of infection.
Reduced IBC formation in implanted animals may be a result of loss
of the host superficial facet cells in which IBCs form due to
increased exfoliation or damage to the uroepithelium following
urinary catheterization. Nonetheless, this finding is of particular
interest for treatment strategies against UPEC-mediated CAUTI. In
humans, removal of the contaminated urinary catheter is the
preferred method for treatment of these infections; however, the
presence of bacteria in an intracellular compartment protected from
host immune defenses and antibiotic treatment requires more
comprehensive approaches as intracellular UPEC can lead to
re-infection of a novel catheter or serve as a nidus for future
UTI.
[0690] Quiescent intracellular reservoirs are an important outcome
of UPEC pathogenic cascade because they are proposed to be a
mechanism of recurrent UTI following damage to the uroepithelium.
Findings from the current study indicate that urinary implantation
of animals with a history of UTI can lead to bladder infection and
implant colonization with the UPEC causing the first infection.
This finding suggests that in addition to introduction of
extracellular and periurethral bacteria, urinary catheter
colonization can occur from bacteria originating from pre-existing
reservoirs or other niches within the urinary tract not appreciated
by assessment of bacteriuria. Interestingly, by day 5 following
implantation in animals with a history of UTI, there is overall a
significant reduction in the number of QIRs compared to that in
non-implanted animals as assessed by bladder CFU. This reduction in
bacterial load could be a result of enhanced immune-mediated
clearance of infected cells or to exfoliation of infected cells in
implanted animals. These hypotheses are currently being
evaluated.
[0691] The identification of FimH as a critical virulence factor
during UPEC CAUTI provides an interesting avenue for the
development of novel preventative measures against these
infections. In fact, there has recently been an upsurge in
recommendations and guidelines for management of CAUTIs and rUTIs
in catheterized patients, with a particular focus on preventative
measures including the limited use of catheters and even the
prophylatic use of antibiotics prior and following catheter
removal. However, the inappropriate use of antibiotics can worsen
the problems of increasing antibiotic resistance. Accordingly, the
present study suggests that the use of small molecules inhibitors,
such as mannosides, in combination with existing UTI treatment
regimes, can lead to prevention or delay of UPEC colonization.
Mannosides, were rationally designed to interfere with
FimH-mediated binding to mannosylated proteins on the surface of
the uroepithelium. Recent studies show that the use of mannosides
in combination with antibiotics is highly effective in preventing
IBC formation and acute stages of UPEC infection as well as
treating chronic cystitis. Similarly, pretreatment with mannosides
further prevents IBC formation following UPEC infection of
implanted bladders and reduces UPEC binding to the uroepithelium.
However, the presence of the abiotic implant surface provided a
favorable environment for adherence of extracellular bacteria. The
inability of mannoside treatment to eliminate UPEC from the
implant, given that the experimental evidence that mannose inhibits
UPEC biofilm on catheter material in urine in vitro, may be due to
the lack of urine flow in this implanted murine model. It is
possible that if it were possible to truly catheterize mice in a
manner analogous to clinical catheterization of humans in which
urine flows through the catheter that mannoside may have a more
efficacious effect on implant clearance. Nonetheless, by preventing
invasion and shifting UPEC's niche to the extracellular milieu,
mannosides enhanced the bacteriocidal efficacy of antibiotics, such
as TMP-SMZ, which does not cross host cell membranes. Mannosides
could also be used in combination with bacterial interference
strategy being offered as alternatives in the prevention of CAUTI.
Previous reports have shown that pre-colonization of urinary
catheters with an avirulent E. coli strain 83972 delays the onset
of CAUTI in catheterized patients. Thus, it is quite possible that
if used in combination with mannosides, UPEC will be kept from the
intracellular niche as well as from binding to the catheter during
bacterial interference with the avirulent strain. An attractive
avirulent strain should be able to colonize the catheter in a type
1-independent manner to prevent invasion into urothelial cell,
however E. coli strain 83972 enhanced binding to silicone catheter
requires ectopic expression of fim. However, the therapeutic
effects of mannoside 6 could not be recapitulated in our model of
CAUTI (data not shown), possibly because the small molecules are
ineffective at disrupting established biofilms in vivo or that UPEC
may employ additional type 1 pili-independent mechanisms for
maintaining biofilms. It is a well-established fact that the
extracellular matrix of bacterial biofilms is impermeable to
antimicrobial and antibiotics, providing a safe haven for the
microbes within. Further research is thus needed for better
therapeutics against CAUTIs. Nonetheless, the prophylatic use of
mannosides prior to urinary catheterization can help reduce the
rate of recurrent UTIs from intracellular bacterial reservoirs
following the removal of contaminated catheters.
[0692] Urinary catheterization is a necessary medical procedure
that causes major damage to the urinary tract. Pathogens, such as
UPEC, take advantage of this compromised environment to exploit new
and existing niches and establish severe infections. This report
uncovers important molecular mechanisms underlying UPEC
pathogenesis following urinary catheterization. It raises important
questions regarding the deleterious consequences of urinary
catheterization and the origins of urinary catheter colonization.
These novel aspects of CAUTI pathophysiology, especially the
presence of intracellular bacterial niches even in presence of
urinary catheters, should thus be taken into consideration for
better diagnosis and the development of anti-virulence based
preventative and therapeutic approaches against these
infections.
Example 23. Treatment of Chronic Mice with 5 Doses of Mannoside 8
Eliminates UPEC from the Bladder
[0693] Mice were infected with the uropathogenic E. coli (UPEC)
strain UTI89. Animals that established chronic infection were
treated at day 12 post infection with 5 doses of 50 mg/kg of
Mannoside 8 administered 8 h apart (mice treated with PBS in the
same way were included as controls). Mice were sacrificed 48 h post
initial dose and bladder titers were enumerated.
[0694] The results clearly show a greatly reduced titer of UPEC in
mice treated with Mannoside 8 versus control mice treated with PBS
(FIG. 41).
Example 24. Mannoside 8 is Effective Against the Multidrug
Resistant UPEC Isolate EC958
[0695] Mice were infected with the multidrug resistant (MDR) UPEC
strain EC958. Animals that established chronic infection were
treated at day 12 post infection with 1 dose of 50 mg/kg of
Mannoside 8 (mice treated with PBS in the same way were included as
controls). Mice were sacrificed 6 h post initial dose and bladder
titers were enumerated.
[0696] The results show that titers of MDR UPEC in mice treated
with Mannoside 8 had significantly lower titers of the MDR UPEC
compared to MDR UPEC titers in control mice treated with PBS (FIG.
42).
Introduction for Examples 25-29
[0697] The development of antibiotics to broadly treat infections
has led to significant advances in human health. Increasing
bacterial resistance to traditional antibiotics and the dearth of
programs to develop innovative antibiotics threatens to reverse
these advances. This has been described as an impending "public
health crisis". For example, over 15 million women suffer from
urinary tract infections (UTI) annually in the U.S. with an
estimated cost exceeding $2.5 billion. Uropathogenic E. coli (UPEC)
account for up to 85% of all UTIs, which are exacerbated by
increasing antimicrobial resistance. Resistance of UPEC to the
antibiotic cocktail, trimethoprim-sulfamethoxazole (TMP-SMZ) has
increased significantly in the past decade and thus therapy has
increasingly required the use of last-line antibiotics such as
fluoroquinolones, leading to increased treatment costs and an
associated rise in multidrug resistance. These developments make
UTI one of the most visible manifestations of increasing
gram-negative antibiotic resistance. Recurrent UTI in healthy women
is a major problem despite the fact that standard antibiotic
treatment typically results in clearance of bacteriuria.
[0698] The two relevant niches during acute UTI are tissue and
urine. The ability of bacteria to associate within the tissue niche
is critical for establishing infection. Extracellular adhesive
fibers known as pili are promising targets of anti-virulence
therapeutics as they are critical factors for colonizing and
invading host tissues and forming biofilms. UPEC use the
chaperone/usher pathway to assemble type 1 pili tipped with the
FimH adhesin. FimH mediates binding to mannosylated receptors
present on the luminal surface of mammalian bladder epithelial
cells, which facilitates bacterial invasion into these cells.
Subversion of innate expulsion mechanisms requires entry into the
cytoplasm where a single UPEC can replicate rapidly into
10.sup.4-10.sup.5 bacteria, which aggregate in a type 1 dependent
manner into a biofilm-like intracellular bacterial community (IBC)
that protects UPEC from host defenses and antibiotics. Bacteria
later disperse from the IBC to spread to neighboring cells.
FimH-dependent IBCs and the filaments that emerge from them
commonly exist in women with recurrent cystitis as revealed in a
clinical study of 100 women as IBC formation is thought to
represent a mechanism that allows UPEC to rapidly expand in
numbers. Virulence factors that increase the fitness of UPEC in the
urinary tract are predicted to be under positive selection and the
fimH gene is under positive selection in clinical isolates of UPEC,
consistent with its role in human disease. FimH is found in
virtually all UPEC, the mannose binding pocket is invariant and
mutations which disrupt mannose binding are attenuated. Previous
studies support the utility of mannosides, to disrupt FimH
function, as an effective therapeutic strategy to treat UTI our
refs. The rational X-ray structure based design and synthesis of
novel biphenyl mannosides FimH inhibitors were recently described,
starting from phenyl mannoside 1 (FIG. 43A). In the following
examples, it is shown that lead compound 6 (2ZFH56) and newly
developed orally bioavailable mannosides are potent and fast acting
antibacterial compounds. It is demonstrated that these mannosides
are effective at treating and preventing UTI in mice as well as
potentiating the antimicrobial effects of TMP-SMZ.
Example 25. Mannosides Disrupt and Inhibit the Formation of
Biofilms
[0699] Functional activity of mannoside FimH inhibitors 1-6 (FIG.
43A) was first assessed in vitro using a hemagglutination and UPEC
biofilm assays. The relative ability of compounds to block biofilm
formation and disrupt preformed biofilms was used to prioritize and
select compounds for further in vivo evaluation. Biofilm formation
is complex and the multiple determinants that contribute to their
development and maintenance may vary depending on growth
conditions, medium and substrates. E. coli biofilm formation in LB
at room temperature on polyvinyl chloride (PVC) is dependent on
type 1 pili and therefore these conditions were used to determine
the efficacy of the mannosides. The median inhibitory concentration
(IC50) values for compounds 1-3 and 6 were all low micromolar but
compound 6 showed the best activity with an IC50 of 0.74 .mu.M
(FIG. 43B). In addition to preventing the formation of biofilms, it
was also found that the mannosides inhibited the buildup up of
preformed biofilms (FIG. 43C). Confocal microscopy of preformed
biofilms treated with mannoside 6 showed effective disruption of a
preformed biofilm, likely explaining in part, the activity seen in
FIG. 43C. Furthermore, preformed biofilms treated with 6 lacked
continuity as seen by gaping holes and lack of the tall
mushroom-like structures observed in untreated biofilms (FIG. 43D,
E). The propensity of E. coli to form biofilms contributes to
antibiotic treatment failures since antibiotics are unable to
penetrate the dense biofilm matrix providing compelling evidence
that biofilm inhibitors can potentiate the effects of
antibiotics.
Example 26. Mannoside is Efficacious at Clearing Severe
Infection
[0700] From the in vitro studies in Example 25, mannosides 4 and 6
showed similar activity in the hemagglutination inhibition (HAI)
assay which is predictive of relative activity in biofilm
inhibition. However, the ester group in compound 4 was unstable for
oral dosing and it was found that its hydrolysis product 5 was
13-fold less potent. Mannoside 6 which contains an amide in place
of the ester, was not only equipotent to 4 but was also more stable
plus had increased solubility and thus was selected as our lead
compound for initial in vivo evaluation. Pharmacokinetic (PK)
studies with lead compound 6 in mice was performed using
intraperitoneal (IP) injection and oral (PO) gavage. Following IP
dosing, 6 concentrations in the urine were quantified at several
time points using HPLC and mass spectrometry (MS). Doses of 5 mg/kg
and 10 mg/kg resulted in concentrations of 1 mM in the urine 30 min
after treatment (FIG. 44A). Eight hours after administration, 6
levels remained near the IC50 (0.74 .mu.M) of biofilm inhibition.
The mouse PK of 6 dosed orally was next evaluated at several
concentrations up to 200 mg/kg. A 100 mg/kg dose of 6 resulted in
3-fold higher concentrations relative to IP (10 mg/kg) eight hours
post-dosing demonstrating some oral bioavailability of 6. It is
also noteworthy that >95% of drug was excreted in the urine
unchanged and no apparent toxicity was observed up to a 200 mg/kg
dose as measured by survival and weight gain/loss.
[0701] In order to determine the therapeutic potential of 6 for
treating chronic UTIs, a unique preclinical murine model was
adopted. In humans, the ultimate outcome of UPEC infection of the
urinary tract ranges from asymptomatic bacteriuria to acute
self-limiting infection to chronic/recurrent UTI. Similarly, the
outcome of UTI in C3H/HeN mice ranges from self-limiting to
long-lasting, chronic cystitis characterized by persistent, high
titer bacteriuria (>10.sup.4 colony forming units (CFU)/ml),
high titer bacterial bladder burdens at sacrifice >2 weeks
post-infection (wpi), chronic inflammation, and urothelial
necrosis. The acute host response, within the first 24 hours, to
tissue-associated UPEC has been shown to determine disease outcome
including predisposition to chronic/recurrent UTI. Thus, C3H/HeN
mice were infected with 1.times.10.sup.7 CPU of UTI89 and mice
developing chronic cystitis, as determined by persistent urine
titers of >10.sup.6 through 2 weeks post infection (wpi), were
PO treated at 2 wpi with 6 at a single dose of 100 or 50 mg/kg to
evaluate the ability of mannoside to treat UTI. 6 hours
post-treatment bacterial counts in the bladder were enumerated. A
dramatic 3-log drop in bacterial titers was observed suggesting
mannoside is efficacious at clearing severe infection within 6
hours of oral delivery of a chronic long lasting infection (FIG.
44B).
Example 27. Prophylactic Use of Mannoside
[0702] Since 6 successfully treated chronic cystitis, it was of
interest to elucidate if mannosides could also prevent a UTI as a
prospective prophylactic therapy. To mimic this clinical scenario,
the efficacy of 6 in in vivo treatment was evaluated by dosing mice
either IP or PO 30 min prior to infecting with UTI89. At 6 hours
post infection (hpi), bladders were removed and total bacterial
CPUs were quantified. In both the IP and PO treated cohorts a
significant drop in bacterial counts was observed, demonstrating
the efficacy of 6 in reducing overall UPEC colonization of the
bladder (FIG. 44E). Furthermore, there was also a significant
reduction of IBCs in the mice that were pretreated with mannoside
(FIG. 44F). To demonstrate 6 reduced IBC formation by blocking UPEC
invasion into the bladder tissue, gentamicin treatment assays were
performed. Gentamicin kills extracellular UPEC but is unable to
penetrate tissue and thus intracellular bacteria survive treatment.
It was found that in the 6-treated mice, gentamicin treatment of
the bladders eliminated all CPUs (FIG. 44G). In bladders from
untreated mice, 10.sup.3-10.sup.4 CPUs remained after gentamicin
treatment, reflecting the bacteria present that had invaded the
bladder epithelium and thus circumvented the treatment. Confocal
microscopy of bladders in the untreated cohort showed normal,
robust IBC formation (FIG. 44C) whereas IBCs were rarely seen in
the mannoside treated mouse bladders however bacteria were observed
in the bladder luminal compartment (FIG. 44D). These results
demonstrate that a mannoside FimH inhibitor prevents bacterial
invasion into the bladder tissue and significantly reduces
infection in the bladder. This novel class of orally active
biphenyl mannosides has potential utility for the treatment of
women suffering from chronic/recurrent UTIs as an alternative to
prophylactic antibiotic treatment and would significantly benefit
women with increased incidence of UTI due to sexual
intercourse.
Example 28. Mannosides Inhibit the Invasion of UPEC into the
Bladder Tissue and Potentiate the Efficacy of TMP-SMZ
[0703] The first-line treatment of choice for UTI has traditionally
been a 3-day course of TMP-SMZ. Women suffering from
chronic/recurrent UTIs are often given TMP-SMZ prophylactically to
prevent recurrence. However, resistance to this TMP-SMZ regimen is
rapidly expanding. It was hypothesized that by preventing bacterial
invasion into the bladder tissue, a FimH inhibitor may result in
anti-virulence synergism with TMP-SMZ and may curtail or circumvent
the problem of TMP-SMZ resistance. This theory was evaluated in a
preclinical animal model where mice given TMP-SMZ for 3 days were
infected with either UTI89 or the TMP-SMZR strain, PBC-1. Mice were
IP treated with 6 30 min prior to inoculation with bacteria and
compared to a control group of untreated animals. After inoculation
with UTI89 or PBC-1, bacterial CPUs were quantified at 6 hpi. As
expected, treatment with TMP-SMZ alone resulted in a significant
drop in bacterial load in the UTI89-infected mice but had no effect
on PBC-1, since it is resistant to TMP-SMZ. Upon treatment with 6
alone there was a significant drop in bacterial load of both
strains in the bladder. In the dual treatment group there was also
a significant drop in bacterial CPUs compared to mannoside alone or
TMP-SMZ alone for both strains which was most pronounced for PBC-1
(FIG. 45). It was determined that the presence of mannoside had no
effect on growth or killing efficiency of either strain during
growth in vitro in the presence or absence of TMPSMZ. Therefore,
the observation that in combination with 6, the TMP-SMZR strain
PBC-1 succumbed to antibiotic treatment suggested that the
mannoside potentiates the efficacy of TMP-SMZ by a unique
mechanism. Based on growth curves in TMP-SMZ, PBC-1 was calculated
to have a Minimum Inhibition Concentration (MIC) of 256 and 1280
.mu.g/ml for TMP and SMZ, respectively and UTI89 was calculated to
have an MIC of 0.05 .mu.g/ml TMP and 0.25 .mu.g/ml SMZ. The
presence of mannoside had no effect on growth or killing efficiency
of either strain. It is well established that TMP concentrates in
the urine and this serendipitous feature is a major reason TMP-SMZ
has been the preferred antibiotic for UTI over the last several
decades. Using quantitative HPLC-MS, the concentration of TMP-SMZ
was measured in the urine of mice after 3 days of treatment with 54
.mu.g/ml and 270 .mu.g/ml TMP and SMZ, respectively. TMP
concentrations were determined to be 9.95+/-4.36 mg/ml and SMZ at
67.17+/-32.51 .mu.g/ml. These results indicate that by preventing
bacterial invasion, 6 compartmentalizes the microbes to the bladder
lumen thus exposing them to TMP-SMZ concentrations above the MIC of
PBC-1, resulting in augmentation of bacterial cell killing.
Presumably TMP-SMZ concentrations reach tissue concentrations above
the MIC needed for UTI89 killing but fail to reach tissue levels
needed for killing PBC-1. These results clearly highlight the
importance of the intracellular pathway in bacterial persistence.
In addition to escaping the immune system in their intracellular
niche, bacteria are also able to evade exposure to antibiotics as
highlighted by the clinically TMP-SMZ resistant strain. In summary,
mannosides could benefit those women on suppressive antibiotic
therapy by inhibiting the invasion of UPEC into the bladder tissue
and potentiating the efficacy of TMP-SMZ creating a cost-effective
treatment, which is predicted to lower the rate of treatment
failures.
Example 29. Mannoside Optimization
[0704] While 6 shows good efficacy in vivo, it was sought to
identify optimized mannosides with further improved
pharmacokinetics in particular those encompassing increased cell
permeability and thus better oral bioavailability and bladder
tissue penetration. The inherent polarity of mannosides and other
sugar-derived compounds often limits their cellular permeability
and increasing their hydrophobicity (Log P/Log D) is predicted to
improve the latter. Computational modeling of mannosides bound to
FimH suggested that the ortho-position of the biphenyl ring
attached to mannose is aimed at Tyr137 and improved hydrophobic
contact could be achieved by substitution. The increased
hydrophobicity was not only predicted to improve FimH binding
affinity but also the oral bioavailability and bladder tissue
penetration relative to starting mannoside 6. Furthermore, these
derivatives will likely display increased metabolic stability
through protection of the glycosidic bond from hydrolysis both in
the gut and by .alpha.-mannosidases. Thus, a matched pair analysis
was performed of monoamide 3 compared to ortho-substituted analogs
bearing methyl, trifluoromethyl, and chloro groups. The compounds
were evaluated for their potency in the hemagglutination inhibition
(HAI) assay and it was discovered that all biphenyl ring
substitutions yielded more potent inhibitors (FIG. 46A).
Ortho-chloro mannoside 7 inhibited hemagglutination with a potency
of 125 nM which is 10-fold better than matched pair 3 while the
ortho-methyl analog 8 was even 2-fold more active (HAI=62 nM).
Substitution with trifluoromethyl gave the most potent analog 9
with an HAI=32 nM. This data suggests that our prediction for
increased hydrophobic contact with Tyr137 might explain this
enhanced potency since the trifluoromethyl analog 9 has the largest
hydrophobic surface area and also shows the highest activity.
However, it is also possible that the orientation of the phenyl
rings are altered slightly and conformationally restricted in a
more productive form conducive to improved FimH binding. In any
case, the outcome of this preliminary study directed us to develop
ortho-trifluoromethyl diamide 10 which is exponentially more potent
than any previously reported mannoside FimH inhibitor with an HAI=8
nM. This unprecedented level of potency corresponds to a
15,000-fold improvement over butyl-.alpha.-D-mannoside (125 .mu.M)
and is 50-fold better than lead 6. Based on their improved in vitro
properties, we tested the optimized mannosides for mouse oral PK
(FIG. 46B) and found at 50 mg/kg compounds 8 and 10 yielded the
highest concentrations in urine at 6 hours post dosing with 8
displaying equivalent concentrations to 100 mg/kg of mannoside 6.
Due to their enhanced potency and optimal PK, mannosides 8 and 10
were selected for further testing in our chronic infection mouse
model. C3H/HeN mice having chronic cystitis at 2 weeks post
infection were treated with 6, 8 or 10. Treatment with 8 and 10
resulted in a significant and dramatic 4 log reduction in bacterial
counts in the bladder 6 hours posttreatment (FIG. 4c). Although
ortho-trifluoromethyl diamide 10 is 8-fold more potent in vitro
than ortho-methyl monoamide 8, it was found that when dosed orally
at 50 mg/kg 8 showed better efficacy in vivo. 8 reduced bacterial
CPUs in the bladder almost 2 Log units better than 6 at the
identical 50 mg/kg dose and was still more efficacious than a 100
mg/kg dose of 6. These results can be explained by increased FimH
inhibition combined with improved PK of the ortho-substituted
mannosides relative to 6. Mannoside 8 represents a very promising
lead preclinical candidate for the oral treatment and prevention of
recurrent urinary tract infections.
Discussion for Examples 25-29
[0705] The efficacy of anti-virulence compounds in vivo has not
been extensively characterized. Herein, the most potent orally
active small molecule FimH antagonists described are reported to
date. This is also the first demonstration that a mannoside FimH
inhibitor shows therapeutic potential for treating an established
chronic urinary tract infection in vivo. These innovative compounds
also show excellent efficacy in vivo when used prophylactically.
Furthermore, the mechanism of action displayed by FimH inhibitors
keeps bacteria extracellular and sensitizes a TMP-SMZ resistant
strain by prolonging exposure to antibiotic levels above its MIC.
This enhanced susceptibility may help overcome the rising problem
of TMP-SMZ resistance amongst E. coli. Not only are these
anti-virulence FimH antagonists effective as a treatment against
UTI, but their oral availability represents a major step toward
effective preclinical optimization and drug development.
Alternative management strategies are needed for patients suffering
from chronic/recurrent UTIs. Prophylactic administration of
mannoside alone or in combination with TMP-SMZ could potentially
reduce the incidence of treatment failure and shorten the time span
of currently administered suppressive therapy. A shift to reduce
the use of fluoroquinolones would provide both a more
cost-effective treatment option as well as slow the spread of
resistance to this class of antibiotics. Given that resistance to
antibiotic therapy is rapidly increasing, it is time to reconsider
standard UTI therapy in order to preserve the effectiveness of
current antibiotics. The use of FimH inhibitors as a targeted
therapeutic strategy benefits from the fact that it is not broad
spectrum and will specifically target those bacteria expressing
type 1 pili, ubiquitous amongst UPEC. Given the unique welldefined
clinical population of patients with UTI, of which nearly 85% of
the cases are caused by UPEC, the narrow spectrum afforded by
mannosides provides an advantage over alternative therapies and
other broad spectrum anti-virulence drugs currently in clinical
trials. It is predicted that mannosides either dosed alone or in
combination with commonly used antibiotics can treat almost all
initial and chronic UTIs.
[0706] Outside of UTI, cell-cell adherence to host tissues is the
first step in most infectious diseases. Thus, due to the
commonality of adhesion mechanisms in bacterial and viral
pathogenesis, similar but unique anti-virulence compounds can be
tailored to treat a multitude of infectious diseases. The results
in Examples 25-29 validate the utility of employing a rational
approach to study the molecular mechanisms of pathogenesis and
resulted in the discovery and development of potential
anti-virulence therapeutics, which specifically target mechanisms
essential in UPEC pathogenesis. In conclusion, these studies have
the potential to revolutionize current approaches to both
antimicrobial and anti-viral treatment and provides not only new
opportunities for effectively treating a broad range of infectious
diseases, but also the potential to curtail the ever expanding
medical crisis of pathogen resistance to traditional
antibiotics.
Methods for Example 25-29
[0707] UTI89 biofilm was grown in LB+/-mannoside for 24 h at
22.degree. C. in PVC plates and quantified using crystal violet.
UTI89 biofilm for confocal microscopy was grown in LB for 24 h at
22.degree. C. on PVC coverslips followed by mannoside treatment for
16 h. For all animal experiments UTI89 or PBC-1 was grown
2.times.24 h statically in LB at 37.degree. C. and inoculated at a
dose of 1.times.107 bacteria in 50 .mu.l. All mice used were female
C3H/HeN (Harlan). For the chronic UTI model, mice were infected for
2 weeks prior to treatment with mannoside. For IP dosing, 50 .mu.l
of 2 mg/ml (5 mg/kg) or 4 mg/ml (10 mg/kg) 6 in PBS was injected
into the mouse 30 min prior to inoculation of bacteria. For oral
dosing, 100 .mu.l of 10 mg/ml (50 mg/kg) or 20 mg/ml (100 mg/kg)
mannoside in 8% DMSO was inoculated with a gavage needle 30 min
prior to inoculation of bacteria. Mass spectrometry was used to
quantify urinary mannoside or TMP-SMZ concentrations. For CFU
counts, bladders were harvested at 6 hpi and placed in 1 mL PBS.
Bladders were then homogenized, diluted and plated on LB. After
growth at 37.degree. C. overnight, bacterial counts were
determined. LacZ staining and gentamicin protection assays were
performed at 6 hpi. For antibiotic experiments, mice were given
TMP-SMZ in the drinking water at a concentration of 54 .mu.g/ml and
270 .mu.g/ml, respectively. Water was changed daily with fresh
antibiotics. Standard growth curve and hemagglutination assays were
performed. All statistical analysis performed was a two-tailed
Mann-Whitney U test. Compounds 1-6 were prepared as outlined in Han
et. al. (2010; J. Med Chem. 53:4779). Compounds 7-10 were prepared
using slightly modified procedures. All compounds are >95% pure
as determined by HPLC/MS and .sup.1H NMR.
Bacterial Strains.
[0708] UTI89 is a prototypical cystitis isolate of serotype
018:K1:H7. PBC-1 is a TMP-SMZ.sup.R strain of serotype OX13:K1:H10
isolated from a 59 year old asymptomatic female with a history of
recurrent UTI and diagnosis of primary biliary cirrhosis.
Synthesis of Mannosides.
[0709] 1. General Synthesis, Purification, and Analytical Chemistry
Procedures.
[0710] Starting materials, reagents, and solvents were purchased
from commercial vendors unless otherwise noted. .sup.1H NMR spectra
were measured on a Varian 300 MHz NMR instrument. The chemical
shifts were reported as .delta. ppm relative to TMS using residual
solvent peak as the reference unless otherwise noted. The following
abbreviations were used to express the multiplicities: s=singlet;
d=doublet; t=triplet; q=quartet; m=multiplet; br=broad.
High-performed liquid chromatography (HPLC) was carried out on
GILSON GX-281 using Waters C18 5 .mu.M, 4.6*50 mm and Waters Prep
C18 5 .mu.M, 19*150 mm reverse phase columns, eluted with a
gradient system of 5:95 to 95:5 acetonitrile:water with a buffer
consisting of 0.05% TFA. Mass spectra (MS) were performed on
HPLC/MSD using electrospray ionization (ESI) for detection. All
reactions were monitored by thin layer chromatography (TLC) carried
out on Merck silica gel plates (0.25 mm thick, 60F254), visualized
by using UV (254 nm) or dyes such as KMnO.sub.4, p-Anisaldehyde and
CAM. Silica gel chromatography was carried out on a Teledyne ISCO
Combi Flash purification system using pre-packed silica gel columns
(12 g.about.330 g sizes). All compounds used for biological assays
are greater than 95% purity based on NMR and HPLC by absorbance at
220 nm and 254 nm wavelengths.
2. Experimental Procedure for the Preparation of Mannoside 8
2.1
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxyinethyl)-2-(4-bromo-2-methyl-
-phenoxy)tetrahydropyran-3-yl] acetate
##STR00067##
[0712] Linder nitrogen atmosphere and at room temperature, boron
trifluoride diethyl etherate (3.41 g, 24 mmol) was added dropwise
into the solution of .alpha.-D-mannose pentaacetate (3.12 g, 8
mmol) and 4-bromo-2-methylphenol (2.99 g, 16 mmol) in 100 ml of
anhydrous CH.sub.2Cl.sub.2. After a few mins the mixture was heated
to reflux and kept stirring for 45 hrs. The reaction was then
quenched with water and extracted with CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layer was collected dried with Na.sub.2SO.sub.4,
concentrated. The resulting residue was purified by silica gel
chromatography with hexane/ethyl acetate combinations as eluent,
giving the title compound (3.22 g) in 77% yield. .sup.1H NMR (300
MHz, CHLOROFORM-d) .delta. 7.18-7.38 (m, 2H), 6.97 (d, J=8.79 Hz,
1H), 5.50-5.59 (m, 1H), 5.43-5.50 (m, 2H), 5.32-5.42 (m, 1H), 4.28
(dd, J=5.63, 12.50 Hz, 1H), 3.99-4.15 (m, 2H), 2.27 (s, 3H), 2.20
(s, 3H), 2.02-2.11 (three singlets, 9H); MS (ESI): found: [M+Na]+,
539.0.
2.2
N-methyl-3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxyme-
thyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide (8)
##STR00068##
[0714] Under nitrogen atmosphere, the mixture of
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phe-
noxy)tetrahydropyran-3-yl]acetate (0.517 g, 1 mmol),
3-(N-Methylaminocarbonyl)phenylboronic add pinacol ester (0.392 g,
1.5 mmol), cesium carbonate (0.977 g, 3 mmol) and
tetrakis(triphenylphosphine)palladium (0.116 g, 0.1 mmol) in
dioxane/water (15 mL/3 mL) was heated at 80.degree. C. with
stirring for 1 h under a nitrogen atmosphere. After cooling to RT,
the mixture was filtered through silica gel column to remove the
metal catalyst and salts with hexane/ethyl acetate combinations as
eluent. The filtrate was concentrated, and then dried in vacuo. The
residue was diluted with 15 mL of methanol containing a catalytic
amount of sodium methoxide (0.02 M) and the mixture was stirred at
RT overnight. H+ exchange resin (DOWEX 50WX4-100) was added to
neutralize the mixture. The resin was filtered off and the filtrate
was concentrated. The resulting residue was purified by silica gel
chromatography with CH.sub.2Cl.sub.2/MeOH combinations as eluent,
giving the title compound (0.260 g) in 64% yield for two steps.
.sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. 7.94 (t, J=1.65 Hz,
1H), 7.57-7.72 (m, 2H), 7.33-7.50 (m, 3H), 7.23 (d, J=8.52 Hz, 1H),
5.48 (d, J=1.92 Hz, 1H), 4.00 (dd, J=1.79, 3.43 Hz, 1H), 3.83-3.94
(m, 1H), 3.60-3.76 (m, 3H), 3.46-3.58 (m, 1H), 2.87 (s, 3H), 2.24
(s, 3H). MS (ESI): found: [M+H]+, 404.2.
[0715] Mannosides 7 and 9 were prepared following a similar
procedure to the synthesis of 8.
3. Experimental Procedure for the Preparation of Mannoside 10
3.1
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-trifluo-
romethylphenoxy)tetrahydropyran-3-yl] acetate
##STR00069##
[0717] Using the procedure outlined in 2.1 using
4-bromo-2-trifluoromethylphenol, the title compound was obtained
(2.5 g) in 54% yield. .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta.
7.75 (d, J=2.20 Hz, 1H), 7.61 (dd, J=2.47, 8.79 Hz, 1H), 7.15 (d,
J=8.79 Hz, 1H), 5.61 (d, J=1.65 Hz, 1H), 5.32-5.58 (m, 3H), 4.28
(dd, J=5.22, 12.36 Hz, 1H), 3.95-4.22 (m, 2H), 2.21 (s, 3H), 2.07
(s, 3H), 2.06 (s, 3H), 2.04 (s, 3H); MS (ESI): found: [M+Na]+,
593.0.
3.2
N1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene--
1,3-dicarboxamide
##STR00070##
[0719] Dimethyl 5-bromobenzene-1,3-dicarboxylate (10.6 g, 36.8
mmol) was dissolved in a 33 wt % solution of methylamine in EtOH
(30 mL) and stirred for 6 h at RT. The precipitate that formed
during the reaction was filtered to give 5.3 g (53%) of the
intermediate 5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide as a
white solid. Concentration of the remaining filtrate yielded an
additional 4.6 g (46%) of product.
5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide (5.3 g, 19.5
mmol), Pd(dppf).sub.2Cl.sub.2 (0.87 g, 1.2 mmol), di-pinacolborane
(6.1 g, 24 mmol), and potassium acetate (7.8 g, 80 mmol) were
dissolved in DMSO (100 mL). The solution was stirred under vacuum
and then repressurized with nitrogen. This process was repeated 3
times and then the resultant mixture was stirred at 80.degree. C.
for 5 h under a nitrogen atmosphere. After removal of the solvent
under high vacuum, the crude material was purified by silica gel
chromatography to give the title compound as a light tan solid (2.2
g, 35%). .sup.1H NMR (300 MHz, DMSO-d6 .delta. 8.63 (m, 2H), 8.41
(t, J=1.51 Hz, 1H), 8.23 (d, J=1.65 Hz, 2H), 2.80 (s, 3H), 2.78 (s,
3H), 1.33 (s, 12H). MS (ESI): found: [M+H]+, 319.2.
3.3
N1,N3-dimethyl-5-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihyd-
roxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarbox-
amide (10)
##STR00071##
[0721] Using the procedure outlined in 2.2 with
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-trifluorom-
ethyl-phenoxy)tetrahydropyran-3-yl] acetate (0.57 g) and
N1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-
-dicarboxamide (0.48 g), the title compound was obtained (0.340 g)
in 69% yield for the two steps. .sup.1H NMR (300 MHz, METHANOL-d4)
.delta. 8.17-8.24 (m, 1H), 8.14 (d, J=1.65 Hz, 2H), 7.92 (d, J=1.92
Hz, 1H), 7.87 (dd, J=2.20, 8.79 Hz, 1H), 7.57 (d, J=8.79 Hz, 1H),
5.64 (d, J=1.65 Hz, 1H), 4.04 (dd, J=1.65, 3.30 Hz, 1H), 3.87-3.96
(dd, 1H), 3.64-3.83 (m, 3H), 3.48-3.63 (m, 1H), 2.93 (s, 6H). MS
(ESI): found: [M+H]+, 515.1.
Biofilm Assay.
[0722] UTI89 was grown in LB broth in wells of PVC microtiter
plates at 23.degree. C. in the presence of individual mannosides at
varying concentrations. After 48 h of growth, wells were rinsed
with water and stained with crystal violet for quantification as
described. For biofilm disruption activity in PVC plates, UTI89 was
grown in LB broth in wells of PVC microtiter plates at 23.degree.
C. After 24 h of growth, mannoside was added and biofilms were
grown for an additional 16 h. Wells were then rinsed, stained with
crystal violet and quantified. For biofilm disruption activity on
PVC coverslips, UTI89 was grown in LB broth in 50 mL conicals
containing PBC coverslips at 23.degree. C. After 24 h of growth,
0.3 .mu.M ZFH-2056 was added and biofilm was grown for an
additional 16 h. Coverslips were then rinsed, fixed with 2%
paraformaldehyde (v/v), stained with SYTO9 (1:1000 in PBS;
Molecular Probes) and observed with a Zeiss LSM410 confocal laser
scanning microscope under a 63.times. objective.
Animal Infections.
[0723] Bacteria were grown under type 1 pili-including conditions
(2.times.24 h at 37.degree. C. statically in LB). The bacteria were
harvested and resuspended to an OD.sub.600 of 0.5 in PBS.
Eight-week-old C3H/HeN (Harlan) female mice were anesthetized by
inhalation of isoflurane and infected via transurethral
catheterization with 50 .mu.l of the bacterial suspension,
resulting in 1-2.times.10.sup.7 inoculum. At 6 hpi, mice were
sacrificed by cervical dislocation under anesthesia and the
bladders were immediately harvested and processed as described
below. All animal studies using mice were approved by the Animal
Studies Committee of Washington University (Animal Protocol Number
20100002).
Chronic Infection.
[0724] Mice were infected with UTI89 and the infection was allowed
to continue for 2 weeks. At 12 days post-infection, urine was
collected and titered to determine mice chronically infected (urine
titers >10.sup.6). At 2 wpi, chronically infected mice were
treated PO with 50 mg/kg or 100 mg/kg of mannoside. 6 h
posttreatment, mice were sacrificed and bladders were aseptically
removed and homogenized to determine tissue titers. Enumeration of
bladder IBCs.
[0725] For animal pretreatment experiments, mannoside ZFH-2056 was
administered either IP (5 mg/kg) or orally (100 mg/kg) 30 min prior
to inoculation with UTI89. To accurately count the number of IBCs,
mice were sacrificed 6 hpi and bladders were aseptically removed,
bisected, splayed on silicone plates and fixed in 2%
paraformaldehyde (v/v). IBCs, readily discernable as punctate
violet spots, were quantified by LacZ staining of whole
bladders.
Pharamacokinetic Analysis.
[0726] For intraperitoneal dosing, 50 .mu.l of a 2 mg/ml (5 mg/kg)
or 4 mg/ml (10 mg/kg) solution of 6 in PBS was injected into the
peritoneal cavity of the mouse. For oral dosing, 100 .mu.l of a 20
mg/ml (100 mg/kg) solution of ZFH-2056 in 8% DMSO was inoculated
with a gavage needle into the mouse stomach. Urine was collected at
30 min, 1, 2, 3, 4, 6, and 8 h post-treatment. An equal volume of
10 .mu.M internal standard (ZFH-2050) was added to the urine.
Mannosides were extracted from the urine by loading on C18 columns
(100 mg, Waters), washing with 30% methanol, and eluting with 60%
methanol. Vacuum-concentrated eluates were analyzed using liquid
chromatography-mass spectrometry system with a lower heated
capillary temperature of 190.degree. C. and a gradient as follows:
Solvent B (80% acetonitrile in 0.1% formic acid) was held constant
at 5% for 5 minutes, increased to 44% B by 45 minutes, and then to
a 95% B by 65 minutes. SRM mode quantification was performed with
collision gas energy of 30% for the following MS/MS transitions
(precursor m/z/product m/z): compound 6, 447/285; compound 3,
390/228. Absolute quantification was achieved by comparison to a
calibration curve.
Bladder Tissue Bacterial Titer Determination.
[0727] Mannoside ZFH-2056 was administered either IP (5 mg/kg) or
orally (100 mg/kg) 30 min prior to inoculation with UTI89. To
enumerate the bacteria present, mice were sacrificed at 6 hpi and
bladders were aseptically removed and homogenized in 1 ml PBS,
serially diluted and plated onto LB agar plates. CPU was enumerated
after 16 h of growth at 37.degree. C.
Confocal Microscopy.
[0728] Mannoside 6 was administered IP (5 mg/kg) 30 min prior to
inoculation with UTI89. To visualize bacterial behavior within the
bladder, mice were sacrificed at 6 hpi and bladders were
aseptically removed, bisected, splayed on silicone plates revealing
the luminal surface and fixed in 2% paraformaldehyde (v/v). The
splayed bladders were then incubated for 20 min at room temperature
with (i) SYTO9 (1:1000 in PBS; Molecular Probes) to stain bacteria
and (ii) Alexa Fluor 594-conjugated wheat germ agglutinin (WGA;
1:1000 in PBS; Molecular Probes) to stain the bladder luminal
surface. Bladders were rinsed with PBS, mounted using Prolong Gold
antifade reagent (Invitrogen) and examined with a Zeiss LSM510
confocal laser scanning microscope under a 63.times. objective.
SYTO9 and WGA were excited at 488 and 594 nm, respectively.
Gentamicin protection assay.
[0729] To enumerate bacteria present in the intracellular versus
extracellular compartments, bladders were aseptically harvested at
6 hpi. The bladders were then bisected twice and washed three times
in 500 .mu.l of PBS each. The wash fractions were pooled, lightly
spun at 500 rpm for 5 min to pellet exfoliated bladder cells,
serially diluted, and plated onto LB agar to obtain the luminal
fraction. The bladders were treated with 100 .mu.g of gentamicin/ml
for 90 min at 37.degree. C. After treatment, the bladders were
washed twice with PBS to eliminate residual gentamicin, homogenized
in 1 ml of PBS, serially diluted, and plated onto LB agar to
enumberate the CPUs in the intracellular fraction.
Antibiotic Treatment.
[0730] Mice were given TMP-SMZ in the drinking water at a
concentration of 54 .mu.g/ml and 270 .mu.g/ml, respectively. Water
was changed daily for 3 days prior to inoculation with UTI89. Mice
remained on TMP-SMZ during the infection. To determine TMP-SMZ
concentration in the urine, urine was collected after 3 days of
TMP-SMZ treatment and quantified by LC-MS following addition of
sulfisoxazole as an internal standard.
Growth Curve.
[0731] An overnight culture of PBC-1 was diluted 1:1000 in LB in
the absence or presence of TMP-SMZ and/or mannoside 6. The highest
concentration of TMP-SMZ used was 512 .mu.g/ml and 2560 .mu.g/ml,
respectively. Two-fold dilutions of TMP-SMZ were performed.
Mannoside 6 was added at 100 .mu.M. Growth curves were performed in
a 96-well plate at 37.degree. C. with A.sub.600 readings taken
every 30 min for 8 h. Minimum inhibitory concentration (MIC) was
calculated as the lowest concentration of antibiotic that prevented
growth of the bacterial strain.
Statistical Analysis.
[0732] Observed differences in bacterial titers and IBC numbers
were analyzed for significance using the nonparametric Mann-Whitney
U test (Prizm; GraphPad Software)
Introduction for Examples 30-36
[0733] FimH is a mannose-specific bacterial lectin located at the
tip of type 1 pili, an adhesive fiber produced by uropathogenic E.
coli (UPEC). FimH is known to bind to mannosylated human uroplakins
that coat the luminal surface of the bladder and has also been
shown to be involved in invasion of human bladder cells and mast
cells, triggering apoptosis and exfoliation and inducing elevated
levels of cAMP. Furthermore, FimH recognizes N-linked
oligosaccharides on beta1 and alpha3 integrins, which are expressed
throughout the urothelium. Murine uroplakin is highly homologous to
human and FimH has been shown to facilitate bacterial colonization
and invasion of the bladder epithelium in murine models.
Internalized UPEC are exocytosed in a TLR-4 dependent process;
however, bacteria can escape into the host cell cytoplasm, where
they are able to subvert expulsion and innate defenses by
aggregating into biofilm-like intracellular bacterial communities
(IBCs) in a FimH dependent process. Subsequently, UPEC disperse
from the IBC, escape into the bladder lumen, and re-initiate the
process by binding and invading naive epithelial cells where they
are able to establish quiescent intracellular reservoirs that can
persist in a dormant state, tolerant to antibiotic therapy and
subsequently serve as seeds for recurrent infection. In humans, the
severity of UTI was increased and the immunological response was
greater in children with infections caused by type 1 piliated UPEC
strains and type 1 pilus expression has been shown to be essential
for UTI in mouse models. In addition, a recent study concluded that
type 1 pili play an important role in human cystitis and it has
been reported that type 1 pili fulfill "Molecular Koch's postulates
of microbial pathogenesis. In agreement with these findings and in
support of a role for FimH in humans, it has been shown that the
fimH gene is under positive selection in human clinical isolates of
UPEC. Aspects of the UPEC pathogenic cascade extensively
characterized in a murine model of infection have been documented
in samples from human clinical studies such as filamentation and
IBC formation. Targeted inhibitors of FimH adhesion which block
both E. coli invasion and biofilm formation thus hold promising
therapeutic potential as new antibacterials for the treatment of
UTI and the prevention of recurrence. The discovery of simple
D-mannose derivatives as inhibitors of bacterial adherence was
first reported almost three decades ago but early mannosides showed
only weak inhibition of bacterial adhesion. Consequently, the vast
majority of research in this area has been focused on multivalent
mannosides, which have been pursued in an effort to improve binding
avidity to type 1 pili, which can be present in large numbers on a
single bacterium (up to hundreds). While substantial progress has
been made with this approach, these high molecular weight
structures are not suitable for in vivo evaluation or clinical
development as oral drugs. The recent X-ray crystal structures of
D-mannose, butyl mannoside, and mannotriose bound to FimH have
enabled the rational structure-based design of tighter binding
alkyl-, phenyl- and biphenyl-mannoside FimH inhibitors. The urgency
for developing new orally bioavailable FimH inhibitors as a
targeted strategy for the treatment of UTI alternative to broad
spectrum antibiotics is reinforced by the rate of recurrence seen
in these type of infections as well as increasing clinical
resistance of UPEC to first line antibiotic treatments.
Example 30. Tight Binding Ortho-Substituted Biphenyl Mannosides
[0734] A matched pair analysis was performed of monoester 1
compared to ortho-substituted analogs bearing halogen and small
alkyl groups shown in FIG. 47. The compounds were evaluated for
their potency in the hemagglutination inhibition (HAI) assay and it
was discovered that all biphenyl ring substitutions yielded more
potent inhibitors (Table 17). Ortho-Cl mannoside 4b had a HAI Titer
EC.sub.90 of 30 nM which is more than 30-fold better than matched
pair 1 while the Me analog 4c was 8-fold more active (HAI Titer
EC.sub.90=120 nM). Substitution with CF3 gave the most potent
analog 4d with an HAI Titer EC.sub.90 of 30 nM whereas the OMe (4e)
and F (4a) analogs showed smaller improvements in activity
following the trend CF3>Cl=Me>OMe>F. This data suggests
that increased hydrophobic contact with the tyrosine gate and
Ile52, or with Ile13 at the opposite ridge of the mannose binding
pocket could explain this enhanced potency since better activity
correlates well with increased hydrophobicity as evidenced by the
fact that fluoro analog 4a shows no improvement in activity
relative to unsubstituted matched pair 1 and the trifluoromethyl
analog 4c which has the largest hydrophobic surface area shows the
highest activity. However, it is possible that the orientation of
both phenyl rings are altered slightly and are restricted to a
conformation more conducive to improved FimH binding with Tyr137,
Tyr48, Ile52, MeI 3 and/or Arg98 residues.
TABLE-US-00006 TABLE 17 Potency enhancement and PAMPA data from
ortho- substitution of biphenyl mannosides. Biofilm HAI Preven-
PAMPA Titer tion MW LogP.sub.e EC.sub.>90 IC.sub.50 (g/
(cm.sup.2/ Compound (.mu.M) (.mu.M) mol) PSA CLogD.sub.7.4 sec) 1
(ester) 1.00 0.94 390.4 126 1.17 -5.42 4a (F) 0.75 408.4 126 1.00
4b (Cl) 0.03 0.26 424.8 126 1.64 -4.29 4c (Me) 0.12 0.33 404.4 126
1.82 4d (CF.sub.3) 0.03 0.17 458.4 126 1.62 -3.91 4e (OMe) 0.19
0.89 420.4 135 0.47 6 (m-CO.sub.2Me) 1.0 448.4 152 0.93 -4.08 2
(amide) 0.50 1.35 389.4 128 0.28 5a (Cl) 0.12 0.52 423.8 128 0.75
5b (Me) 0.06 0.16 403.4 128 0.93 -3.89 5c (CF.sub.3) 0.03 0.13
457.4 128 0.73 3 (di-amide) 0.37 0.74 446.4 158 0.71 -4.51 7
(CF.sub.3) 0.01 0.043 514.5 158 1.15 -6.27 8 (Me) 0.02 0.073 460.5
158 1.35 -8.46 1 (ester) 1.00 0.94 390.4 126 1.17 -5.42 4a (F) 0.75
408.4 126 1.00 4b (Cl) 0.03 0.26 424.8 126 1.64 -4.29 4c (Me) 0.12
0.33 404.4 126 1.82 4d (CF.sub.3) 0.03 0.17 458.4 126 1.62 -3.91 4e
(OMe) 0.19 0.89 420.4 135 0.47 6 (m-CO.sub.2Me) 1.0 448.4 152 0.93
-4.08 2 (amide) 0.50 1.35 389.4 128 0.28 5a (Cl) 0.12 0.52 423.8
128 0.75 5b (Me) 0.06 0.16 403.4 128 0.93 -3.89 5c (CF.sub.3) 0.03
0.13 457.4 128 0.73 3 (di-amide) 0.37 0.74 446.4 158 0.71 -4.51 7
(CF.sub.3) 0.01 0.043 514.5 158 1.15 -6.27 8 (Me) 0.02 0.073 460.5
158 1.35 -8.46
[0735] The outcome of this preliminary study directed the
investigation of analogs which were more metabolically stable and
soluble than esters such as amides 5a-c (FIG. 47 and Table 17). A
similar trend was observed (CF3>Me>Cl) with CF3 amide 5c
having the best activity but in this case the Me analog 5b showed
better activity than Cl analog 5a. Analogs were also explored with
substitution on the meta position, exemplified by ester 6, which
retains potency relative to 1 but did not lead to any enhancement.
Ortho-CF3 7 and Me 8 diamide matched pairs to original lead
compound 3 were next developed which were exponentially more potent
than any previously reported mannoside FimH inhibitors with an HAI
Titer EC.sub.90 of 8 nM and 16 nM, respectively. This unprecedented
level of cellular activity corresponds to a 200,000-fold
improvement over .alpha.-D-mannose and a 15,000-fold improvement
over an early reported inhibitor butyl-.alpha.-D-mannoside (HAI
Titer EC90=125 .mu.M) and 50-fold better than previous lead
compound 3.
[0736] The biofilm inhibition assay was utilized to test these
mannosides' ability to prevent bacteria from forming IBCs, a
critical pathogenic process in the development of UTIs. As shown in
Table 17, the Biofilm Prevention IC.sub.50S correlate quite well
with the potencies determined by the HAI assay. Introduction of an
ortho-substituent (e.g. methyl) to the biphenyl mannoside improved
the biofilm activity by 8-fold from mannoside 2 (IC50=1.35 .mu.M)
to mannoside 5b (IC50=0.16 .mu.M). This data confirmed the
mannoside's functional effect and activity on UPEC derived from
FimH inhibition with a secondary assay. Biofilm IC50S were utilized
in conjunction with pharmacokinetic parameters as a key measure of
the predicted lowest effective mannoside concentration in the urine
required for efficacy in vivo to be discussed vide infra.
[0737] Mannosides were synthesized by traditional Lewis acid
mediated glycosylation of mannose penta-acetate by reaction with
2-substituted 4-bromophenols using BF3 etherate (Scheme 1). Suzuki
cross-coupling with commercially available 3-substituted phenyl
boronic add derivatives gave protected ortho-substituted
4'-biphenyl mannosides in excellent yields and subsequent
deprotection with NaOMe gave mannosides 4-5. 6 was prepared
following the procedure previously described. Synthesis of
di-amides 7-8 followed a similar procedure but required the
synthesis of 3,5-di-(N-methyl aminocarbonylphenyl boronic acid
pinacol ester 9. Shown in Scheme 1, amidation of dimethyl
5-bromoisophthalate by reaction of methylamine in ethanol proceeded
in quantitative yield to give N,N-dimethyl 5-bromoisophthalamide.
Installation of the boronate ester was accomplished by Pd-mediated
coupling with bis(pinacolato)diboron to give 9. Suzuki coupling and
deprotection as before yielded compounds 7-8.
##STR00072## ##STR00073##
Example 31. Mannosides with Increased pKa Functionality
[0738] To help improve tissue penetration and exposure of
mannosides in the bladder, amide derivatives were explored
containing functional groups with higher pKa (Table 18). Higher pKa
compounds containing basic moieties such as amines tend to have
increased tissue penetration. Therefore, mannosides 10a-f were
prepared via standard HATU-mediated coupling reaction of
4'-(.alpha.-D-mannopyranosyloxy)biphenyl-3-carboxylic add with
various amines. Unexpectedly, aminoethyl amide 10b showed a
disappointing 6-fold drop in activity relative to methyl amide 2.
However, hydroxyethyl amide 10a was equipotent to 2. It was also
found that more hydrophobic tertiary amide piperazine analogs 10c
and 10d had largely decreased potency (HAI EC.sub.90=4 .mu.M).
Interestingly, pyridyl amides 10e and 10f showed slightly improved
activity most pronounced with 4-pyridyl derivative 10e. While it is
unclear to the reason for decreased potency of higher pKa
substituents it is plausible that this effect could originate from
charge-charge repulsion with Arg98 side chain at the edge of FimH
binding pocket.
TABLE-US-00007 TABLE 18 Exploration of amide substitution with
increased pKa ##STR00074## Compound R HAI Titer EC.sub.>90
(.mu.M) 10a ##STR00075## 0.50 10b ##STR00076## 3.0 10c ##STR00077##
4.0 10d ##STR00078## 4.0 10e ##STR00079## 0.25 10f ##STR00080##
0.37
Example 32. Mannosides with Heterocyclic Replacements
[0739] Heterocyclic replacements of the terminal biphenyl ring were
then persued as an alternate approach to improve the drug-like
properties of lead mannosides. In target compounds the key H-bond
donor to Arg98 of FimH was retained so we could directly compare
the effects of heterocyclic ring replacement. In order to
synthesize a library of heterocycles in a divergent fashion, a new
Suzuki synthesis was developed using a 4-mannopyranosyloxyphenyl
boronate intermediate 11 in place of
4-bromophenyl-.alpha.-D-mannoside. Only a limited number of
heteroaryl boronates are commercially available and this new
methodology allows for the use of more readily obtainable
heteroaryl bromides as coupling partners. All heteroaryl bromides
used in Table 19 were commercially available with reasonable prices
except bromothiophene derivatives 16 and 17 (Scheme 2). 16 was
prepared via Curtius reaction by first treatment of
5-bromothiophene-3-carboxylic acid with diphenylphosphoryl azide
(DPPA) to form isocyanate intermediate, then quenching with
ammonia. 17 was synthesized according to previous method. As shown
in Scheme 2, Starting from 4-bromophenyl-.alpha.-D-mannoside,
bis(pinacolato)diboron and Pd(dppf)Cl.sub.2 in DMSO, intermediate
11 was prepared in good yield. Suzuki cross coupling of 11 with
various aryl bromides, followed by acetyl deprotection gave target
compounds. Compounds synthesized, shown in Table 19, include
pyridyl esters 12a and 12b which showed excellent activity in the
HAI titer with improvement over phenyl ester 1. Furthermore, ring
replacement with a thiophene urea carboxylate led to dramatic
advancements in FimH activity as exemplified by compound 13a which
has an HAI EC.sub.90=16 nM. In order to ascertain whether the
carboxylate ester group or urea were responsible for this large
increase in potency, ester 13b and urea 13c were synthesized to
discover that the enhancement results from a combined effect of
both functional since 13b or 13c have much decreased activity. It
is unknown why there is a synergistic effect from the compound with
both urea and carboxylate but from previous work on thiophene
carboxamide ureas it was shown that an intramolecular H-bond exists
between the internal NH of the urea and the carbonyl of the ester.
This conformational restriction might enhance binding entropically
to FimH likely from the urea carbonyl. Several fused rings were
also persued such as isoquinoline derivatives 14 and 15 to examine
the effects of isosteric replacement for the aryl carbonyl H-bond
acceptor where the heterocyclic ring nitrogen is designed to accept
a H-bond from the FimH Arg98 side chain. The promising HAI assay
results for 14a-b to 15a-b dearly provides much evidence that the
orientation of the C.dbd.N in 15a or C.dbd.O in 15b is likely the
same as the conformation of C.dbd.O of mannoside 1 in crystal
structure of FimH-mannoside I.sup.25 bringing the potency up by as
much as 10-fold over mannoside 1.
##STR00081##
TABLE-US-00008 TABLE 19 Heterocyclic modifications to biphenyl ring
for improving drug-like properties HAI Titer Compound R
EC.sub.>90 (.mu.M) 12a ##STR00082## 0.50 12b ##STR00083## 0.19
13a ##STR00084## 0.02 13b ##STR00085## 1.0 13c ##STR00086## 1.0 13d
##STR00087## 2.0 14a ##STR00088## 0.75 14b ##STR00089## 0.38 15a
##STR00090## 0.25 15b ##STR00091## 0.10
Example 33. Direct Binding of FimH-Mannoside
[0740] In order to better understand how the excellent potency in
cell-based HAI and biofilm assays is correlated with FimH binding
by biaryl mannosides and to more precisely select the best lead
compounds for in vivo pharmacokinetic (PK) studies, a biolayer
interferometry method was developed to directly measure the binding
affinities (K<j) of FimH inhibitors. As shown in Table 20,
earlier mannosides 19 33 showing moderate potency in the HAI assay
had K.sub.d values as low as 1.about.10 nM. Strikingly, for
compounds 1 and 2, K.sub.d values were in the picomolar range. For
mannosides 5, 7, and 8, however, K.sub.d could not be calculated
because the off-rates were too low to measure. In order to overcome
this obstacle, differential scanning fluorimetry (DSF) was utilized
to rank the high-affinity mannosides. DSF measures the melting
temperature change of protein when binding to small molecules.
Melting temperature shifts are proportional to the free energy of
binding, and melting temperatures increase even as ligand
concentration exceeds the K<j. As illustrated in Table 20 and
FIG. 48, the melting temperature of FimH without mannoside was
about 60.degree. C. and rose to between 68.degree. C. and
74.degree. C. when binding to mannosides 19 33 of moderate potency.
With tight binding mannosides 5b, 7, 8 the melting temperature of
FimH ranged from 74.degree. C. to 76.degree. C., suggesting that
improved mannosides likely bind FimH with low picomolar affinity.
FIG. 48 illustrates that DSF ranks mannosides in a similar fashion
to the HAI assay except for 5c and 25, demonstrating that this is a
general and reliable method to qualitatively rank FimH-mannoside
binding when K.sub.dS span many orders of magnitude. Thus, these
direct FimH binding studies solidified that the high potencies
stemming from mannosides 5b, 7, 8 derives directly from extremely
tight binding to the FimH lectin and not other non-specific effects
from the cell assays.
TABLE-US-00009 TABLE 20 Results of Octet assay and DSF assay
Compound Structure of ##STR00092## HAI Titer EC.sub.>90 (.mu.M)
Octet K.sub.d (nM) DSF Melting Temp. (.degree. C.) 7 ##STR00093##
0.008 N.D.* 76.15 8 ##STR00094## 0.016 N.D.* 75.76 5c ##STR00095##
0.032 N.D.* 72.29 5b ##STR00096## 0.060 N.D.* 74.46 18 ##STR00097##
0.150 N.D.* 72.53 10f ##STR00098## 0.375 N.D.* 72.53 3 ##STR00099##
0.375 0.14 74.39 12a ##STR00100## 0.500 N.D.* 73.68 2 ##STR00101##
0.500 0.01 73.38 19 ##STR00102## 1.000 0.49 72.05 1 ##STR00103##
1.000 0.08 73.39 20 ##STR00104## 1.500 1.25 71.77 21 ##STR00105##
2.000 N.D.* 70.72 22 ##STR00106## 2.000 14.5 72.39 23 ##STR00107##
2.000 3.45 73.25 24 ##STR00108## 2.000 2.00 70.90 25 ##STR00109##
2.000 1.56 68.32 26 ##STR00110## 2.000 1.14 72.30 27 ##STR00111##
2.000 1.00 71.69 28 ##STR00112## 3.000 2.61 71.16 29 ##STR00113##
4.000 3.99 68.69 30 ##STR00114## 4.000 2.21 70.93 31 ##STR00115##
6.000 2.72 70.75 32 ##STR00116## 6.000 2.72 70.75 33 ##STR00117##
8.000 3.00 71.17 Phenyl-.alpha.- D-mannoside ##STR00118## 30.000
N.D. 68.93 34 ##STR00119## 60.000 43.66 68.11 * N.D. = not
determined
[0741] An electrostatic surface was generated with the most potent
mannoside 7 docked to FimH shown in FIG. 49. The large boost in
binding affinity to FimH can be attributed to the fact that in the
model the ortho trifluorometyl group orients directly at Ile13
resulting in a very strong hydrophobic interaction with FimH. The
added hydrophobicity encompassed by the fluorine atoms in mannoside
7 results in further augmented binding to FimH relative to ortho
methyl mannoside 8.
Example 34. Stability and Elimination Kinetics of Mannoside
[0742] Mannoside 3 shows efficacy in vivo in the treatment and
prevention of established UTIs in mice when dosed orally but the
compound displayed some metabolic instability from hydrolysis of
the glycosidic bond (FIG. 50A) and very rapid elimination kinetics
to the urine partly due to its low CLogD value. While renal
clearance is an attractive feature for UTI therapy, improved
mannosides were desirable encompassing lower clearance rates as
well as increased oral bioavailability and bladder tissue
permeability. In order to have a general idea of the elimination
rate of mannoside 3, pharmacokinetic (PK) studies of its urinary
clearance were conducted in mice (FIG. 50B). These experiments
demonstrated that the lower oral dosing of 20 mg/kg was unlikely to
maintain effective concentrations (as determined by Biofilm IC50)
due to rapid clearance. Maintenance of mannoside 3 levels above the
minimal effective level of 0.74 .mu.M during in an eight-hour
period required a larger, 100 mg/kg dose. During the PK study, a
small amount of a phenolic biphenyl metabolite (R) (FIG. 50A) was
detected in the urine indicating some glycoside bond hydrolysis
takes place upon oral dosing. Urine levels of the R group were
unchanged from the 100 and 200 mg/kg doses, suggesting that
metabolism by glycoside bond hydrolysis is saturated between the 20
and 100 mg/kg doses (FIG. 50B).
Example 35. Parallel Artificial Membrane Permeability Assay
(PAMPA)
[0743] The low Log D of polyhydroxylated sugar-based mannosides and
other carbohydrate-derived compounds can limit their ability to
cross cell membranes in the absence of active transport mechanisms
and so increasing their hydrophobicity is one strategy to help
improve cell permeability and oral bioavailability of this class of
molecules. The ortho-substituted mannosides described above were
designed for increased hydrophobic contact with FimH but also in
part to increase the Log D and were predicted to improve the
solubility, oral bioavailability and bladder tissue penetration
relative to starting mannosides 1-3. It was anticipated that the
newly designed inhibitors would also have increased metabolic
stability via protection of the glycosidic bond from acidic
hydrolysis in the gut and enzymatic hydrolysis by
.alpha.-mannosidases in blood and tissues. In order to test these
hypotheses experimentally, the Parallel Artificial Membrane
Permeability Assay (PAMPA) was used. PAMPA is commonly used as an
in vitro model of passive, transcellular permeability to predict
oral bioavailability for drug candidates. The most potent biphenyl
mannosides was tested in this model for prioritizing compounds to
evaluate further in animal PK and efficacy studies. Shown in Table
20, Compound 5b with Log P.sub.e of -3.89 cm.sup.2/sec proved to
have the highest oral absorption, while the most potent mannosides
(determined by HAI assay) 7 and 8 with Log P.sub.e of -6.27 and
-8.46 cm.sup.2/sec exhibited significantly lower oral
bioavailability.
Example 36. Pharmacokinetic Studies for Selected Mannoside
Inhibitors
[0744] Based on these results, oral PK studies were performed in
mice to assess any improvements in the PK of these
ortho-substituted mannosides. Compounds were dosed at 50 mg/kg and
plasma and urine samples were taken at 30 min and 1, 2, 3, 4, 6
hours after dosing. As demonstrated in FIG. 51, a generally 10-fold
higher mannoside concentration was observed in urine (FIG. 51B)
compared to plasma (FIG. 51A), indicating a high clearance rate for
these mannosides, which in this case aids in clearing uropathogens
on the bladder surface. It was found that compounds 8 and 5b
consistently maintain a high level of concentration in both urine
and plasma, which is well above the predicted minimum effective
concentration within a 6-hour period. While 100 mg/kg dosing of
mannoside 3 was required to achieve effective mannoside
concentrations, only 50 mg/kg dosing of mannoside 5b was required,
permitting a much larger therapeutic window for treatment. Taken
together with PAMPA results, high oral bioavailability and in vivo
efficacy in recently reported animal studies support mannoside 5b
as the most promising therapeutic candidate for UTI
treatment/prevention.
Conclusions for Examples 30-36
[0745] Using a combination of traditional ligand-based and X-ray
structure-guided approaches with structure-activity relationship
(SAR) driven by cell-based hemagglutination and biofilm assays in
combination with direct FimH binding assays, a diverse array of
biaryl mannoside FimH inhibitors that exhibit affinities into the
picomolar range were identified. While it was found the most potent
mannoside 7 with respect to FimH binding affinity and activity in
cell assays contains an ortho-trifluoromethyl group off the phenyl
ring adjacent to the mannose ring group, the most promising
inhibitor from in vivo studies is the ortho-methyl analog 8 showing
prolonged compound exposure in plasma and urine PK studies. A
variety of heterocyclic biaryl mannosides that either retain or
improve FimH binding activity were also discovered. The novel
inhibitors of UPEC type 1 mediated bacterial adhesion reported
herein show unprecedented activity in hemagglutination and biofilm
in vitro assays in addition to desirable pharmacokinetic properties
in vivo. Further optimization of lead mannosides is currently being
focused on the identification of mannose modifications with reduced
sugar-like character. Biaryl mannosides have high potential as
innovative therapeutics for the clinical treatment and prevention
of urinary tract infections. The unique mechanism of action of
targeting the pilus tip adhesin, FimH, circumvents the conventional
requirement for drug penetration of the outer membrane and the
potential for development of resistance by porin mutations, efflux
or degradative enzymes, all mechanisms that promote resistance to
antibiotics. Current efforts are directed at the selection of one
or more clinical candidate drugs through rigorous preclinical
evaluation in several models of recurrent UTI with antibiotic
resistant forms of UPEC. These preclinical models will facilitate
further optimization of current lead compounds.
Experimental Section for Examples 30-36
General Synthesis, Purification, and Analytical Chemistry
Procedures.
[0746] Starting materials, reagents, and solvents were purchased
from commercial vendors unless otherwise noted. .sup.1H NMR spectra
were measured on a Varian 300 MHz NMR instrument. The chemical
shifts were reported as .delta. ppm relative to TMS using residual
solvent peak as the reference unless otherwise noted. The following
abbreviations were used to express the multiplicities: s=singlet;
d=doublet; t=triplet; q=quartet; m=multiplet; br=broad.
High-performed liquid chromatography (HPLC) was carried out on
GILSON GX-281 using Waters C18 5 .mu.M, 4.6*50 mm and Waters Prep
C18 5 .mu.M, 19*150 mm reverse phase columns, eluted with a
gradient system of 5:95 to 95:5 acetonitrile:water with a buffer
consisting of 0.05% TFA. Mass spectra (MS) were performed on
HPLC/MSD using electrospray ionization (ESI) for detection. All
reactions were monitored by thin layer chromatography (TLC) carried
out on Merck silica gel plates (0.25 mm thick, 60F254), visualized
by using UV (254 nm) or dyes such as KMnO.sub.4, P-Anisaldehyde and
CAM. Silica gel chromatography was carried out on a Teledyne ISCO
Combi Flash purification system using pre-packed silica gel columns
(12 g330 g sizes). All compounds used for biological assays are
greater than 95% purity based on NMR and HPLC by absorbance at 220
nm and 254 nm wavelengths.
[0747] Procedures for the Preparation of Biphenyl Mannoside
Derivatives Through Suzuki Coupling Reaction with Bromophenyl
Mannoside Derivatives as Intermediate
N-methyl-3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl-
)tetrahydropyran-2-yl]oxy-phenyl]benzamide (5b).
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxyinethyl)-2-(4-bromo-2-methyl-ph-
enoxy)tetrahydropyran-3-yl] acetate
[0748] Under nitrogen atmosphere and at room temperature, boron
trifluoride diethyl etherate (3.41 g, 24 mmol) was added dropwise
into the solution of .alpha.-D-mannose pentaacetate (3.12 g, 8
mmol) and 4-bromo-2-methylphenol (2.99 g, 16 mmol) in 100 ml of
anhydrous CH.sub.2Cl.sub.2. After a few mins the mixture was heated
to reflux and kept stirring for 45 hrs. The reaction was then
quenched with water and extracted with CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layer was collected dried with Na.sub.2SO.sub.4,
concentrated. The resulting residue was purified by silica gel
chromatography with hexane/ethyl acetate combinations as eluent,
giving
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phe-
noxy)tetrahydropyran-3-yl] acetate (3.22 g) in 77% yield. .sup.1H
NMR (300 MHz, CHLOROFORM-d) .delta. ppm 7.18-7.38 (m, 2H), 6.97 (d,
J=8.79 Hz, 1H), 5.50-5.59 (m, 1H), 5.43-5.50 (m, 2H), 5.32-5.42 (m,
1H), 4.28 (dd, J=5.63, 12.50 Hz, 1H), 3.99-4.15 (m, 2H), 2.27 (s,
3H), 2.20 (s, 3H), 2.02-2.11 (three singlets, 9H). MS (ESI): found:
[M+Na].sup.+, 539.0.
N-methyl-3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl-
)tetrahydropyran-2-yl]oxy-phenyl]benzamide (5b)
[0749] Under nitrogen atmosphere, the mixture of
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phe-
noxy)tetrahydropyran-3-yl] acetate (0.517 g, 1 mmol),
3-(N-methylaminocarbonyl)phenylboronic add pinacol ester (0.392 g,
1.5 mmol), cesium carbonate (0.977 g, 3 mmol) and
tetrakistriphenylphosphinepalladium (0.116 g, 0.1 mmol) in
dioxane/water (15 mL/3 mL) was heated at 80.degree. C. with
stirring for 1 h under a nitrogen atmosphere. After cooling to RT,
the mixture was filtered through silica gel column to remove the
metal catalyst and salts with hexane/ethyl acetate combinations as
eluent. The filtrate was concentrated, and then dried in vacuo. The
residue was diluted with 15 mL of methanol containing a catalytic
amount of sodium methoxide (0.02 M) and the mixture was stirred at
RT overnight. H.sup.+ exchange resin (DOWEX 50WX4-100) was added to
neutralize the mixture. The resin was filtered off and the filtrate
was concentrated. The resulting residue was purified by silica gel
chromatography with CH.sub.2Cl.sub.2/MeOH combinations as eluent,
giving the title compound (0.260 g) in 64% yield for two steps.
.sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 7.94 (t, J=1.65
Hz, 1H), 7.57-7.72 (m, 2H), 7.33-7.50 (m, 3H), 7.23 (d, J=8.52 Hz,
1H), 5.48 (d, J=1.92 Hz, 1H), 4.00 (dd, J=1.79, 3.43 Hz, 1H),
3.83-3.94 (m, 1H), 3.60-3.76 (m, 3H), 3.46-3.58 (m, 1H), 2.87 (s,
3H), 2.24 (s, 3H). MS (ESI): found: [M+H].sup.+, 404.2.
Methyl
3-[3-fluoro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-2-yl]oxy-phenyl]benzoate (4a).
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-fluoro-phe-
noxy)tetrahydropyran-3-yl] acetate
[0750] it was prepared using the same procedure as for
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phe-
noxy)tetrahydropyran-3-yl] acetate in the synthesis of 5b. Yield:
25%. .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. ppm 7.30 (dd,
J=2.34, 10.03 Hz, 1H), 7.21 (td, J=1.79, 8.79 Hz, 1H), 7.08 (t,
J=8.52 Hz, 1H), 5.48-5.58 (m, 2H), 5.46 (d, J=1.65 Hz, 1H),
5.31-5.41 (m, 1H), 4.23-4.31 (m, 1H), 4.13-4.22 (m, 1H), 4.05-4.13
(m, 1H), 2.20 (s, 3H), 2.02-2.08 (three singlets, 9H). MS (ESI):
found: [M+Na].sup.+, 543.0.
Methyl
3-[3-fluoro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-2-yl]oxy-phenyl]benzoate (4a)
[0751] 4a was prepared using the same procedure as for 5b with
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-fluoro-phe-
noxy)tetrahydropyran-3-yl] acetate and 3-methoxycarbonylphenyl
boronic add as the reactants. Yield: 66%. .sup.1H NMR (300 MHz,
METHANOL-d.sub.4) .delta. ppm 8.21 (t, J=1.65 Hz, 1H), 7.99 (td,
J=1.44, 7.83 Hz, 1H), 7.84 (ddd, J=1.10, 1.92, 7.69 Hz, 1H),
7.35-7.62 (m, 4H), 5.55 (d, J=1.92 Hz, 1H), 4.10 (dd, J=1.79, 3.43
Hz, 1H), 3.94 (s, 3H), 3.86-4.00 (m, 1H), 3.59-3.86 (m, 4H). MS
(ESI): found [M+Na].sup.+, 431.1.
Methyl
3-[3-chloro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-2-yl]oxy-phenyl]benzoate (4b).
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxyinethyl)-2-(4-bromo-2-chloro-ph-
enoxy)tetrahydropyran-3-yl] acetate
[0752] it was prepared using the same procedure as for
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phe-
noxy)tetrahydropyran-3-yl] acetate in the synthesis of 5b. Yield:
46%. .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. ppm 7.55 (d,
J=2.47 Hz, 1H), 7.33 (dd, J=2.47, 8.79 Hz, 1H), 7.06 (d, J=8.79 Hz,
1H), 5.58 (dd, J=3.02, 10.16 Hz, 1H), 5.52 (s, 1H), 5.49-5.54 (m,
1H), 5.33-5.42 (m, 1H), 4.22-4.32 (m, 1H), 4.04-4.17 (m, 2H), 2.21
(s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H). MS (ESI): found
[M+Na].sup.+, 561.0.
Methyl
3-[3-chloro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-2-yl]oxy-phenyl]benzoate (4b)
[0753] 4b was prepared using the same procedure as for 5b with
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-chloro-phe-
noxy)tetrahydropyran-3-yl] acetate and 3-methoxycarbonylphenyl
boronic add as the reactants. It was further purified by HPLC (C18,
15*150 mm column; eluent: acetonitrile/water (0.1% TFA)). Yield:
43%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 3.59-3.71
(m, 1H) 3.71-3.85 (m, 3H) 3.94 (s, 3H) 4.01 (dd, J=9.34, 3.30 Hz,
1H) 4.12 (dd, J=3.30, 1.92 Hz, 1H) 5.61 (d, J=1.65 Hz, 1H)
7.40-7.49 (m, 1H) 7.49-7.62 (m, 2H) 7.68 (d, J=2.20 Hz, 1H) 7.82
(ddd, J=7.76, 1.85, 1.10 Hz, 1H) 7.98 (dt, J=7.83, 1.30 Hz, 1H)
8.14-8.25 (m, 1H). MS (ESI): found [M+H].sup.+, 425.1.
Methyl
3-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-2-yl]oxy-phenyl]benzoate (4c)
[0754] 4c was prepared using the same procedure as for 5b with
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phe-
noxy)tetrahydropyran-3-yl] acetate and 3-methoxycarbonylphenyl
boronic add as the reactants. Yield: 54%. .sup.1H NMR (300 MHz,
METHANOL-d.sub.4) .delta. ppm 8.20 (t, J=1.51 Hz, 1H), 7.94 (td,
J=1.41, 7.90 Hz, 1H), 7.77-7.87 (m, 1H), 7.52 (t, J=7.55 Hz, 1H),
7.39-7.48 (m, 2H), 7.27-7.38 (m, 1H), 5.56 (d, J=1.65 Hz, 1H), 4.08
(dd, J=1.92, 3.30 Hz, 1H), 3.94-4.01 (m, 1H), 3.90-3.94 (m, 3H),
3.68-3.83 (m, 3H), 3.55-3.65 (m, 1H), 2.31 (s, 3H). MS (ESI): found
[M+Na].sup.+, 427.1.
[0755] Methyl
3-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymet-
hyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate (4d).
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-bromo-2-(trifluoro-
methyl)phenoxy]tetrahydropyran-3-yl] acetate: it was prepared using
the same procedure as for
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phe-
noxy)tetrahydropyran-3-yl]acetate in the synthesis of 5b. Yield:
54%. .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. ppm 7.74 (d,
J=2.20 Hz, 1H), 7.61 (dd, J=2.33, 8.93 Hz, 1H), 7.15 (d, J=8.79 Hz,
1H), 5.61 (d, J=1.92 Hz, 1H), 5.48-5.56 (m, 1H), 5.33-5.48 (m, 2H),
4.21-4.34 (m, 1H), 3.97-4.14 (m, 2H), 2.21 (s, 3H), 1.99-2.12
(three singlets, 9H). MS (ESI): found [M+Na].sup.+, 593.0.
[0756] Methyl
3-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymet-
hyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate (4d). 4d was prepared
using the same procedure as for 5b with
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-bromo-2-(trifluoro-
methyl)phenoxy]tetrahydropyran-3-yl] acetate and
3-methoxycarbonylphenyl boronic add as the reactants. Yield: 53%.
.sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 8.23 (t, J=1.51
Hz, 1H), 8.01 (td, J=1.37, 7.69 Hz, 1H), 7.80-7.93 (m, 3H),
7.52-7.67 (m, 2H), 5.66 (d, J=1.65 Hz, 1H), 4.07 (dd, J=1.79, 3.43
Hz, 1H), 3.95 (s, 3H), 3.90-4.00 (m, 1H), 3.66-3.87 (m, 3H),
3.52-3.66 (m, 1H). MS (ESI): found [M+Na].sup.+, 481.0.
Methyl
3-[3-methoxy-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydropyran-2-yl]oxy-phenyl]benzoate (4e)
[0757] 4e was prepared using the same procedure as for 5b. In the
first step of glycosidation reaction 4-bromo-2-methoxyphenol was
used as glycosidation acceptor and in the second step of Suzuki
coupling reaction 3-methoxycarbonylphenyl boronic add was used
instead. All intermediates were directly taken to the next step
reaction without further purification. 4e was further purified by
HPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.1%
TFA)). Yield: 3%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta.
ppm 8.21 (t, J=1.51 Hz, 1H), 7.96 (td, J=1.44, 7.83 Hz, 1H), 7.84
(ddd, J=1.24, 1.92, 7.83 Hz, 1H), 7.49-7.61 (m, 1H), 7.30 (d,
J=8.24 Hz, 1H), 7.24 (d, J=1.92 Hz, 1H), 7.13-7.21 (m, 1H), 5.47
(d, J=1.92 Hz, 1H), 4.11 (dd, J=1.79, 3.43 Hz, 1H), 3.94 (s, 3H),
3.92 (s, 3H), 3.86-4.03 (m, 1H), 3.67-3.86 (m, 4H). MS (ESI): found
[M+Na].sup.+, 443.1.
3-[3-Chloro-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahyd-
ropyran-2-yl]oxy-phenyl]-M-methyl-benzamide (5a)
[0758] 5a was prepared using the same procedure as for 5b. Yield:
59%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 8.03 (t,
J=1.51 Hz, 1H), 7.69-7.83 (m, 3H), 7.32-7.67 (m, 3H), 5.60 (d,
J=1.65 Hz, 1H), 4.12 (dd, J=1.79, 3.43 Hz, 1H), 4.01 (dd, J=3.57,
9.34 Hz, 1H), 3.69-3.84 (m, 3H), 3.61-3.69 (m, 1H), 2.95 (s, 3H).
MS (ESI): found [M+Na].sup.+, 424.1.
N-Methyl-3-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hy-
droxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide (5c)
[0759] 5c was prepared using the same procedure as for 5b. Yield:
65%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 7.98 (t,
J=1.65 Hz, 1H), 7.77-7.87 (m, 2H), 7.65-7.77 (m, 2H), 7.42-7.60 (m,
2H), 5.58 (d, J=1.65 Hz, 1H), 3.99 (dd, J=1.79, 3.43 Hz, 1H),
3.81-3.91 (m, 1H), 3.59-3.80 (m, 3H), 3.45-3.58 (m, 1H), 2.87 (s,
3H). MS (ESI): found [M+H].sup.+, 458.1.
Methyl
2-(3-methoxycarbonylphenyl)-5-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6--
(hydroxymethyl)tetrahydropyran-2-yl]oxy-benzoate (6). Methyl
5-hydroxy-2-(3-methoxycarbonylphenyl)benzoate
[0760] The reactants of methyl 2-bromo-5-hydroxybenzoate (0.231 g,
1 mmol), 3-methoxycarbonylphenyl boronic add (0.214 g, 1.2 mmol),
palladium acetate (0.022 g, 0.1 mmol), potassium carbonate (0.346
g, 2.5 mmol) and tetrabutylammonium bromide (0.322 g, 1 mmol) in
1.2 ml of water was heated with stirring at 70.degree. C. for 1 h
and 20 mins in a sealed vial by microwave. Then the mixture was
partitioned between AcOEt and 1 N HCl aqueous solution. The organic
layer was collected, dried with Na.sub.2SO.sub.4, then
concentrated. The resulting residue was purified by silica gel
chromatography with AcOEt/Hex combinations as eluent, giving the
title compound (0.240 g) in 84% yield. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. ppm 10.02 (s, 1H), 7.90 (td, J=2.03, 6.66 Hz,
1H), 7.72-7.81 (m, 1H), 7.45-7.60 (m, 2H), 7.28 (d, J=8.52 Hz, 1H),
7.16 (d, J=2.47 Hz, 1H), 7.03 (dd, J=2.61, 8.38 Hz, 1H), 3.86 (s,
3H), 3.56 (s, 3H). MS (ESI): found [M+Na].sup.+, 309.2.
Methyl
2-(3-methoxycarbonylphenyl)-5-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6--
(hydroxymethyl)tetrahydropyran-2-yl]oxy-benzoate (6)
[0761] 6 was prepared via glycosidation between .alpha.-D-mannose
pentaacetate and methyl
5-hydroxy-2-(3-methoxycarbonylphenyl)benzoate following the
procedure previously described..sup.25 Yield: 73%. .sup.1H NMR (300
MHz, METHANOL-d.sub.4) .delta. ppm 3.56-3.68 (m, 4H) 3.68-3.84 (m,
3H) 3.86-3.98 (m, 4H) 4.06 (dd, J=3.30, 1.92 Hz, 1H) 5.60 (d,
J=1.92 Hz, 1H) 7.29-7.43 (m, 2H) 7.45-7.53 (m, 2H) 7.56 (d, J=2.47
Hz, 1H) 7.84-7.92 (m, 1H) 7.93-8.04 (m, 1H). MS (ESI): found
[M+H].sup.+, 449.0.
N1,N3-Dimethyl-5-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-
-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarboxamid-
e (7).
N1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benze-
ne-1,3-dicarboxamide (9)
[0762] dimethyl 5-bromobenzene-1,3-dicarboxylate (10.6 g, 36.8
mmol) was dissolved in a 33 wt % solution of methylamine in EtOH
(30 mL) and stirred for 6 h at RT. The precipitate that formed
during the reaction was filtered to give 5.3 g (53%) of the
intermediate 5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide as a
white solid. Concentration of the remaining filtrate yielded an
additional 4.6 g (46%) of product.
5-bromo-N1,N3-dimethyl-benzene-1,3-dicarboxamide (5.3 g, 19.5
mmol), Pd(dppf)Cl.sub.2 (0.87 g, 1.2 mmol), bis(pinacolato)diboron
(6.1 g, 24 mmol), and potassium acetate (7.8 g, 80 mmol) were
dissolved in DMSO (100 mL). The solution was stirred under vacuum
and then repressurized with nitrogen. This process was repeated 3
times and then the resultant mixture was stirred at 80.degree. C.
for 5 h under a nitrogen atmosphere. After removal of the solvent
under high vacuum, the crude material was purified by silica gel
chromatography to give 9 as a light tan solid (2.2 g, 35%). .sup.1H
NMR (300 MHz, DMSO-d6).quadrature. 5 ppm 8.63 (m, 2H), 8.41 (t,
J=1.51 Hz, 1H), 8.23 (d, J=1.65 Hz, 2H), 2.79 (d, J=4.40 Hz, 6H),
1.33 (s, 12H). MS (ESI): found: [M+H].sup.+, 319.2.
N1,N3-Dimethyl-5-[3-(trifluoromethyl)-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-
-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarboxamid-
e (7)
[0763] Using the procedure outlined in the synthesis of 5b with
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-trifluorom-
ethyl-phenoxy)tetrahydropyran-3-yl] acetate (0.57 g) and
N1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-
-dicarboxamide (9) (0.48 g), the title compound was obtained (0.340
g) in 69% yield for the two steps. .sup.1H NMR (300 MHz,
METHANOL-d.sub.4) .delta. ppm 8.17-8.24 (m, 1H), 8.14 (d, J=1.65
Hz, 2H), 7.92 (d, J=1.92 Hz, 1H), 7.87 (dd, J=2.20, 8.79 Hz, 1H),
7.57 (d, J=8.79 Hz, 1H), 5.64 (d, J=1.65 Hz, 1H), 4.04 (dd, J=1.65,
3.30 Hz, 1H), 3.87-3.96 (dd, 1H), 3.64-3.83 (m, 3H), 3.48-3.63 (m,
1H), 2.93 (s, 6H). MS (ESI): found: [M+H].sup.+, 515.1.
N1,N3-Dimethyl-5-[3-methyl-4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxy-
methyl)tetrahydropyran-2-yl]oxy-phenyl]benzene-1,3-dicarboxamide
(8)
[0764] 8 was prepared using the same procedure as for 5b with
N1,N3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,3-
-dicarboxamide as the Suzuki coupling partner. Yield: 56%. .sup.1H
NMR (300 MHz, METHANOL-d.sub.4) .delta. .quadrature.ppm 8.09-8.23
(m, 3H), 7.46-7.59 (m, 2H), 7.33 (d, J=8.52 Hz, 1H), 5.57 (d,
J=1.92 Hz, 1H), 4.08 (dd, J=1.92, 3.30 Hz, 1H), 3.97 (dd, J=3.43,
9.48 Hz, 1H), 3.67-3.85 (m, 3H), 3.60 (ddd, J=2.47, 5.01, 7.35 Hz,
1H), 2.96 (s, 6H), 2.32 (s, 3H). MS (ESI): found: [M+H].sup.+,
461.2.
Procedure for the Preparation of Mannosides Via Amide Coupling
Reaction:
N-(2-Hydroxyethyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethy-
l)tetrahydropyran-2-yl]oxyphenyl]benzamide (10a)
[0765] Under nitrogen atmosphere, at 0.degree. C. anhydrous DMF (5
mL) was added into the RB flask containing
4'-(.alpha.-D-mannopyranosyloxy)biphenyl-3-carboxylic acid.sup.25
(0.038 g, 0.1 mmol) and HATU (0.046 g, 0.12 mmol). After stirring
for 10 min, aminoethanol (0.007 g, 0.12 mmol), then
N,N-isopropylethylamine (0.039 g, 0.3 mmol) were added. The mixture
was stirred overnight while being warmed to rt naturally. The
solvent was removed and the residue was purified by HPLC (C18,
15*150 mm column; eluent: acetonitrile/water (0.1% TFA)) to give
the title compound (0.032 g) in 76% yield. .sup.1H NMR (300 MHz,
DEUTERIUM OXIDE) 5 ppm 3.53-3.63 (m, 2H) 3.68-3.94 (m, 6H) 4.11
(dd, J=9.20, 3.43 Hz, 1H) 4.22 (dd, J=3.30, 1.92 Hz, 1H) 5.66 (d,
J=1.65 Hz, 1H) 7.21 (d, J=8.79 Hz, 2H) 7.47-7.62 (m, 3H) 7.66-7.78
(m, 2H) 7.87 (t, J=1.65 Hz, 1H). MS (ESI): found [M+H].sup.+,
420.1.
N-(2-Aminoethyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydropyran-2-yl]oxyphenyl]benzamide (10b)
[0766] 10b was prepared using the same procedure as for 10a. Yield:
60%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 3.14-3.26
(m, 2H) 3.57-3.66 (m, 1H) 3.66-3.83 (m, 5H) 3.87-4.00 (m, 1H) 4.03
(dd, J=3.30, 1.92 Hz, 1H) 5.49-5.62 (m, 1H) 7.19-7.31 (m, 2H)
7.49-7.59 (m, 1H) 7.59-7.72 (m, 2H) 7.75-7.89 (m, 2H) 8.03-8.21 (m,
1H). MS (ESI): found [M+H].sup.+, 419.2.
Piperazin-1-yl-[3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-2-yl]oxyphenyl]phenyl]methanone (10c)
[0767] 10c was prepared using the same procedure as for 10a. Yield:
65%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 3.54-3.67
(m, 1H) 3.67-3.85 (m, 4H) 3.85-3.99 (m, 4H) 4.03 (dd, J=3.30, 1.92
Hz, 1H) 5.54 (d, J=1.65 Hz, 1H) 7.19-7.29 (m, 2H) 7.42 (dt, J=7.69,
1.24 Hz, 1H) 7.50-7.64 (m, 3H) 7.67-7.79 (m, 2H). MS (ESI): found
[M+H].sup.+, 445.3.
(4-Methylpiperazin-1-yl)-[3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydro-
xymethyl)tetrahydropyran-2-yl]oxyphenyl]phenyl]methanone (10d)
[0768] 10d was prepared using the same procedure as for 10a. Yield:
87%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 2.96 (s,
3H) 3.05.about.3.65 (m, 9H) 3.68-3.85 (m, 3H) 3.87-3.97 (m, 1H)
4.03 (dd, J=3.30, 1.92 Hz, 1H) 5.54 (d, J=1.65 Hz, 1H) 7.17-7.30
(m, 2H) 7.42 (dt, J=7.62, 1.27 Hz, 1H) 7.50-7.66 (m, 3H) 7.67-7.80
(m, 2H). MS (ESI): found [M+H].sup.+, 459.0.
N-(4-Pyridyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tet-
rahydropyran-2-yl]oxyphenyl]benzamide (10e)
[0769] 10e was prepared using the same procedure as for 10a. Yield:
93%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 3.57-3.69
(m, 1H) 3.69-3.84 (m, 3H) 3.93 (dd, J=9.34, 3.30 Hz, 1H) 4.04 (dd,
J=3.30, 1.92 Hz, 1H) 5.56 (d, J=1.65 Hz, 1H) 7.18-7.37 (m, 2H)
7.57-7.78 (m, 3H) 7.82-8.06 (m, 2H) 8.24 (t, J=1.65 Hz, 1H)
8.36-8.51 (m, 2H) 8.68 (d, J=7.42 Hz, 2H). MS (ESI): found
[M+H].sup.+, 453.1.
N-(3-Pyridyl)-3-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tet-
rahydropyran-2-yl]oxyphenyl]benzamide (10f)
[0770] 10f was prepared using the same procedure as for 10a. Yield:
75%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. ppm 3.55-3.68
(m, 1H) 3.68-3.85 (m, 3H) 3.88-3.98 (m, 1H) 4.04 (dd, J=3.43, 1.79
Hz, 1H) 5.56 (d, J=1.92 Hz, 1H) 7.20-7.32 (m, 2H) 7.52-7.73 (m, 3H)
7.80-7.91 (m, 1H) 7.91-7.99 (m, 1H) 8.04 (dd, J=8.65, 5.63 Hz, 1H)
8.23 (t, J=1.65 Hz, 1H) 8.59 (d, J=5.49 Hz, 1H) 8.67-8.79 (m, 1H)
9.55 (s, 1H). MS (ESI): found [M+H].sup.+, 453.2.
Procedure for the Preparation of Biphenyl Mannoside Derivatives
Through Suzuki coupling reaction with
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaboroian-2-yl)phenoxy]tetrahydropyran-3-yl] acetate
(9) as intermediates
methyl
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyphenyl]pyridine-3-carboxylate (12b).
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl] acetate
(11)
[0771] Under nitrogen atmosphere, the mixture of 4-bromophenyl
2,3,4,6-tetra-O-acetyl-.alpha.-D-mannopyranoside (2.791 g, 5.55
mmol), bis(pinacolato)diboron (1.690 g, 6.66 mmol), potassium
acetate (2.177 g, 22.18 mmol) and
(1.1'-bis(diphenylphosohino)ferrocene)dichloropalladium(II) (0.244
g, 0.33 mmol) in DMSO (50 ml) was heated at 80.degree. C. with
stirring for 2.5 h. The solvent was removed and the resulting
residue was purified by silica gel chromography with hexane/ethyl
acetate combinations as eluent to give 11 (2.48 g) in 81% yield.
.sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. ppm 1.33 (s, 12H)
1.98-2.12 (m, 9H) 2.20 (s, 3H) 3.93-4.19 (m, 2H) 4.21-4.36 (m, 1H)
5.32-5.42 (m, 1H) 5.45 (dd, J=3.57, 1.92 Hz, 1H) 5.51-5.62 (m, 2H)
7.00-7.15 (m, 2H) 7.67-7.84 (m, 2H). MS (ESI): found: [M+Na].sup.+,
573.2.
Methyl
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyphenyl]pyridine-3-carboxylate (12b)
[0772] Under nitrogen atmosphere, the mixture of
[(2R,3S,4S,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-2-[4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl] acetate
(0.132 g, 0.24 mmol), methyl 5-bromonicotinate (0.043 g, 0.2 mmol),
cesium carbonate (0.196 g, 0.6 mmol) and
tetrakis(triphenylphosphine)palladium (0.023 g, 0.02 mmol) in
dioxane/water (5 mL/1 mL) was heated at 80.degree. C. with stirring
for 1 h. After cooling down, the mixture was filtered through
silica gel column to remove the metal catalyst and salts with
hexane/ethyl acetate (2/1) containing 2% triethylamine as eluent.
The filtrate was concentrated, then dried in vacuo. Into the
residue, 6 mL of methanol with catalytic amount of sodium methoxide
(0.02 M) was added and the mixture was stirred at room temperature
overnight. The solvent was removed. The resulting residue was
purified by silica gel chromography with CH.sub.2Cl.sub.2/MeOH
combinations containing 2% NH.sub.3/H.sub.2O as eluent, giving rise
to 12b (0.031 g) in 40% yield. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. ppm 3.53-3.65 (m, 1H) 3.67-3.83 (m, 3H) 3.89-3.96 (m, 1H)
3.99 (s, 3H) 4.04 (dd, J=3.43, 1.79 Hz, 1H) 5.57 (d, J=1.92 Hz, 1H)
7.22-7.37 (m, 2H) 7.58-7.73 (m, 2H) 8.54 (t, J=2.06 Hz, 1H) 8.97
(d, J=2.20 Hz, 1H) 9.04 (d, J=1.92 Hz, 1H). MS (ESI): found:
[M+H].sup.+, 392.1.
Methyl
4-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyphenyl]pyridine-2-carboxylate (12a)
[0773] 12a was prepared using the same procedure as for 12b and was
purified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water
(0.1% TFA)). Yield: 15%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4)
.delta. ppm 3.51-3.63 (m, 1H) 3.65-3.84 (m, 3H) 3.88-3.95 (m, 1H)
4.00-4.13 (m, 4H) 5.62 (d, J=1.65 Hz, 1H) 7.28-7.40 (m, 2H)
7.82-7.95 (m, 2H) 8.13 (dd, J=5.49, 1.92 Hz, 1H) 8.55 (d, J=1.65
Hz, 1H) 8.73 (d, J=5.49 Hz, 1H). MS (ESI): found [M+H].sup.+,
392.2.
Methyl
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyphenyl]-3-ureido-thiophene-2-carboxylate (13a)
[0774] 13a was prepared using the same procedure as for 12b and was
purified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water
(0.1% TFA)). Yield: 10%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4)
.delta. ppm 3.58 (ddd, J=7.21, 4.88, 2.47 Hz, 1H) 3.66-3.83 (m, 3H)
3.83-3.96 (m, 4H) 4.02 (dd, J=3.30, 1.92 Hz, 1H) 5.54 (d, J=1.65
Hz, 1H) 7.12-7.27 (m, 2H) 7.56-7.69 (m, 2H) 8.12 (s, 1H). MS (ESI):
found [M+H].sup.+, 455.1.
Methyl
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyphenyl]thiophene-2-carboxylate (13b)
[0775] 13b was prepared using the same procedure as for 12b and was
purified by silica gel chromography with CH.sub.2Cl.sub.2/MeOH
combinations. Yield: 23%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4)
.delta. ppm 3.53-3.62 (m, 1H) 3.68-3.81 (m, 3H) 3.85-3.94 (m, 4H)
4.02 (dd, J=3.60, 2.1 Hz, 1H) 5.54 (d, J=1.8 Hz, 1H) 7.19 (m, 2H)
7.34 (d, J=3.9 Hz, 1H) 7.64 (m, 2H) 7.75 (d, J=3.9 Hz, 1H). MS
(ESI): found [M+Na].sup.+, 419.1.
(5-bromo-3-thienyl)urea (16)
[0776] Under nitrogen atmosphere N,N-diisopropylethylamine (0.390
g, 3 mmol) was added to the solution of
5-bromothiophene-3-carboxylic add (0.207 g, 1 mmol) and
diphenylphosphoryl azide (DPPA) (0.330 g, 1.2 mmol) in dioxane (5
ml) at rt. After stirring for 30 min, the mixture was heated at 85
for 1.5 h. After the mixture cooled down to rt, 0.5 M of ammonia
solution in dioxane (12 ml) was added. 30 min's later, the solvents
was removed and the resulting residue was purified by silica gel
chromography with CH.sub.2Cl.sub.2/MeOH combinations to give
(5-bromo-3-thienyl)urea (0.072 g) in 32% yield. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. ppm 8.80 (s, 1H), 7.09 (s, 2H), 5.87 (s,
2H). MS (ESI): found [M+H].sup.+, 223.0.
[5-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyran--
2-yl]oxyphenyl]-3-thienyl]urea (13c)
[0777] 13c was prepared using the same procedure as for 12b and was
purified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water
(0.1% TFA)). Yield: 80%. .sup.1H NMR (300 MHz,
METHANOL-d.sub.4/ACETONITRILE-d.sub.3(3/1)) .delta. 7.50-7.62 (m,
2H), 7.11-7.21 (m, 3H), 7.08 (d, J=1.37 Hz, 1H), 5.53 (d, J=1.65
Hz, 1H), 4.02 (dd, J=1.92, 3.30 Hz, 1H), 3.82-3.95 (m, 1H),
3.66-3.81 (m, 3H), 3.51-3.64 (m, 1H). MS (ESI): found [M+H].sup.+,
397.1.
Methyl
5-[4-[(2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyphenyl]thiophene-3-carboxylate (13d)
[0778] 13d was prepared using the same procedure as for 12b and was
purified by silica gel chromography with CH.sub.2Cl.sub.2/MeOH
combinations. Yield: 33%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4)
.delta. ppm 3.55-3.63 (m, 1H) 3.68-3.81 (m, 3H) 3.84-3.94 (m, 4H)
4.02 (dd, J=3.30, 1.8 Hz, 1H) 5.53 (d, J=1.8 Hz, 1H) 7.18 (m, 2H)
7.59 (m, 2H) 7.64 (d, J=1.5 Hz, 1H) 8.11 (d, J=1.5 Hz, 1H). MS
(ESI): found [M+Na].sup.+, 419.1.
(2R,3S,4S,5S,6R)-2-(Hydroxymethyl)-6-[4-(7-isoquinolyl)phenoxy]tetrahydrop-
yran-3,4,5-triol (14a)
[0779] 14a was prepared using the same procedure as for 12b and was
purified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water
(0.1% TFA)). Yield: 73%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4)
.delta. 9.73 (s, 1H), 8.59-8.71 (m, 1H), 8.47-8.58 (m, 2H), 8.42
(d, J=6.59 Hz, 1H), 8.33 (d, J=8.79 Hz, 1H), 7.76-7.89 (m, 2H),
7.25-7.41 (m, 2H), 5.60 (d, J=1.92 Hz, 1H), 4.06 (dd, J=1.92, 3.30
Hz, 1H), 3.87-4.00 (m, 1H), 3.68-3.87 (m, 3H), 3.55-3.68 (m, 1H).
MS (ESI): found [M+H].sup.+, 384.2.
(2R,3S,4S,5S,6R)-2-(Hydroxymethyl)-6-(4-quinazolin-6-ylphenoxy)tetrahydrop-
yran-3,4,5-triol (14b)
[0780] 14b was prepared using the same procedure as for 12b. Yield:
28%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4) .delta. 9.51 (s, 1H),
9.15 (s, 1H), 8.21-8.35 (m, 2H), 8.02 (d, J=8.52 Hz, 1H), 7.63-7.80
(m, J=8.79 Hz, 2H), 7.14-7.32 (m, J=8.79 Hz, 2H), 5.50 (d, J=1.37
Hz, 1H), 3.94-4.03 (m, 1H), 3.80-3.94 (m, 1H), 3.62-3.80 (m, 3H),
3.48-3.62 (m, 1H). MS (ESI): found [M+H].sup.+, 385.1.
(2R,3S,4S,5S,6#?)-2-(Hydroxymethyl)-6-[4-(5-isoquinolyl)phenoxy]tetrahydro-
pyran-3,4,5-triol (15a)
[0781] 15a was prepared using the same procedure as for 12b and was
purified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water
(0.1% TFA)). Yield: 90%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4)
.delta. 9.80 (s, 1H), 8.45-8.60 (m, 2H), 8.35 (d, J=6.59 Hz, 1H),
8.01-8.21 (m, 2H), 7.45-7.55 (m, 2H), 7.31-7.42 (m, 2H), 5.62 (d,
J=1.92 Hz, 1H), 4.07 (dd, J=1.92, 3.30 Hz, 1H), 3.89-3.99 (m, 1H),
3.70-3.86 (m, 3H), 3.60-3.69 (m, 1H). MS (ESI): found [M+H].sup.+,
384.2.
7-[4-[(2R,3S,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-
-yl]oxyphenyl]-2H-isoquinolin-1-one (15b)
[0782] 15b was prepared using the same procedure as for 12b and was
purified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water
(0.1% TFA)). Yield: 75%. .sup.1H NMR (300 MHz, METHANOL-d.sub.4)
.delta. 8.51 (d, J=2.20 Hz, 1H), 7.93-8.05 (m, 1H), 7.62-7.77 (m,
3H), 7.21-7.31 (m, 2H), 7.18 (d, J=7.14 Hz, 1H), 6.71 (d, J=7.14
Hz, 1H), 5.56 (d, J=1.92 Hz, 1H), 4.04 (dd, J=1.92, 3.57 Hz, 1H),
3.88-4.00 (m, 1H), 3.69-3.87 (m, 3H), 3.57-3.69 (m, 1H). MS (ESI):
found [M+H].sup.+, 400.2.
Affinity Measurement by Bio-Layer Interferometry.
[0783] Samples or buffer (200 .mu.L per well) were dispensed into
96-well microtiter plates (Greiner Bio-One, Monroe N.C.) and
maintained at 30.degree. C. with 1000 RPM shaking. Pre-manufactured
pins for individual assays were made by biotinylating FimH lectin
domain.sup.25 at a 1:1 molar ratio with NHS-PEO4-Biotin (Thermo
Fisher, Rockford Ill.), diluting it to 50 .mu.g/ml in 20 mM HEPES
pH 7.5, 150 mM NaCl (HBS), immobilizing it on high-binding
streptavidin-coated biosensor tips (Super Streptavidin, ForteBio,
Inc., Menlo Park, Calif.) for 10 minutes at 30.degree. C., blocking
the pins with 10 .mu.g/ml biocytin for 2 minutes, washing in HBS
for 1 hour, and rinsing in 15% sucrose in HBS. Pins were then air
dried for 30 minutes and stored in their original packaging with a
dessicant packet. Assays were performed by re-wetting premade pins
with HBS for 15 minutes, then storing them in fresh HBS until use.
Individual affinity assays were performed on an Octet Red
instrument (ForteBio, Inc., Menlo Park, Calif.) and consisted of a
short baseline measurement followed by incubation of pins for 10
minutes with 7 twofold dilutions (in HBS) of compound in a
concentration range experimentally determined to give well-measured
association and dissociation kinetics, then a 30-minute
dissociation phase in HBS. Each experimental pin was referenced to
a biocytin-blocked pin to control for instrument drift and a second
biocytin-blocked pin that was passed through a duplicate experiment
in the same 96-well plate to control for nonspecific binding of the
compound to the pin. Kinetics data and affinity constants were
generated automatically by the global fitting protocol in ForteBio
Data Analysis version 6.3. Typical signal for compound binding was
approximately 0.2 "nm shift" units, while the noise level of the
instrument is around 0.0025 nm.
Differential Scanning Fluorimetry (DSF) Method.
[0784] FimH lectin domain (residues 1-158 of UPEC J96 FimH, without
an affinity tag) was purified as described previously. Five
micrograms FimH in 5 .mu.l HBS were mixed with HBS to yield a final
volume of 50 .mu.l containing compound at a final concentration of
100 .mu.M and a "5.times." final concentration of SYPRO Orange
(sold as a "5000.times." stock in DMSO and mixed with HBS to a
working stock of SOX immediately before use: Life Technologies
Inc.; Grand Island, N.Y.). Compounds were diluted to 100 .mu.M from
stocks in DMSO and compared to matched control wells with FimH
alone plus 0.2% DMSO. 50 .mu.l reactions were placed in 96-well
clear-bottom PCR plates and subjected to a melt curve from
20-90.degree. C. in 0.5.degree. C. increments of 15 seconds each
followed by a fluorescence read of the "HEX" channel in a Bio-Rad
CFX96 thermocycler (Bio-Rad, Hercules, Calif.). Melt curves were
fitted to the Boltzmann equation to determine the melting
temperature (T.sub.m) (y=A2+(A1-A2)/(1+exp((x-x0)/dx)) where x0 is
the T.sub.m) using Origin Pro 8 (OriginLab, Northampton Mass.).
Pharmacokinetic Studies.
[0785] Compound levels in mouse urine and plasma were made using an
AB Sciex API-4000 QTrap (AB Sciex, Foster City, Calif.) as
previously described..sup.36 Selected reaction monitoring (SRM)
mode quantification was performed with using the following MS/MS
transitions [precursor mass/charge ratio (m/z)/product m/z]:
compound 3, 447/285; compound 5a, 424/262; compound 5b, 404/242;
compound 5c, 548/296; compound 7, 515/353; compound 8, 461/299;
compound 3 R group, 285/254.
Parallel Artificial Membrane Permeability Assay (PAMPA).
[0786] Materials. The assay was carried out using Multiscreen PVDF
96-well plates (Millipore, Billerica, Mass.) using the company's
Transporter Receiver Plate. The lipid (dioleyoylphosphotidylcholine
(DOPC):Stearic Acid (8020, wt %) in dodecane was obtained from
Avanti Polar lipids (Alabaster, Ala.). Hank's Buffered Salt
Solution (HBSS), pH 7.4 was obtained from MediaTech (Manassas,
Va.).
[0787] Methods. Each of the test compounds was diluted to 2.5 mM in
DMSO (Sigma, St Louis, Mo.) and further diluted prior to testing to
2.5 .mu.M in HBSS, pH 7.4. The assay was performed using a
Millipore 96-well Multiscreen-IP PAMPA plate, 5 .mu.l of the lipid
suspension was directly added to the PVDF membrane of the filter
plate. Immediately following the addition of the lipid to the
membrane, 200 .mu.l of HBSS solution containing the test compound
was added to the donor (upper) chamber. HBSS (300 .mu.l) is also
added to the receiver plate and the filter and receiver plates
assembled and incubated overnight at room temperature in a
moistened sealed bag to prevent evaporation. The concentration in
the receiver plate as well as an equilibrium plate, that represents
a theoretical, partition-free sample, was determined by HPLC-tandem
mass spectrometry. Analysis of each compound was performed in
triplicate.
[0788] Analysis. Analysis of both the acceptor and equilibrium
samples were performed using an AB 3200 triple quadripole mass
spectrometer linked to a Shimadzu DGU-20A HPLC with a Prevail C18
column (3 .mu.m, 2.1.times.10 mm) with a flow rate of 0.35 ml/min.
The mobile phase used was A: 0.1% Formic Acid in water, B: 0.1%
Formic Acid in methanol. The elation gradient method is described
in the table. Data acquisition and peak height determination was
performed using Analyst v.1.4.2.
TABLE-US-00010 Time, min A B 0.01 95 5 0.50 95 5 1.00 5 95 2.00 5
95 2.01 95 5 6.01 Stop
[0789] Calculations.
[0790] The log.sub.10 of the effective permeability (Log P.sub.e)
was calculated using the following equation
log P e = log { C ln ( 1 - [ drug ] acceptor [ drug ] equilibrium )
} where C = ( V D V A ( V D + V A ) Area Time ) ##EQU00001##
Where the drug concentration is the peak areas for the analyte and
V.sub.D and V.sub.A is the volume of the donor and acceptor
compartment respectively. The area is the surface area of the PVDF
membrane (0.11 cm.sup.2) and time is the incubation in seconds
(64,800 sec). Each value is the mean of triplicates performed on
the same day.
Introduction for Examples 37-39
[0791] Type 1 pili constitute the major UPEC virulence factor,
being critical for attachment to and invasion of the bladder
epithelium as well as for IBC formation. Type 1 pili belong to a
class of extracellular fibers assembled by the chaperone-usher
pathway. They are encoded by the fim gene cluster, and their
expression is directed by a phase-variable promoter (fimS), which
facilitates a switch between piliated and nonpiliated bacterial
states. UPEC populations are heterogeneous consisting of bacteria
that are bald, low-, moderately- and highly piliated. The ratio of
each piliated fraction shifts depending on the environment; studies
investigating the expression of type 1 pili revealed that UPEC
associated with epithelial cells are highly piliated, consistent
with the critical role of type 1 pili in urothelium colonization,
while bacteria recovered from urine samples of patients have fimS
primarily in the OFF phase and are likely non-piliated. Regulation
of fimS phase-variation is controlled by the FimB, FimE and FimX
recombinases, the expression of which is in turn influenced by
numerous regulatory factors. We recently identified the QseC sensor
kinase as one of the factors implicated in regulation of type 1
pili expression. In the qseC mutant, the fim promoter is found
primarily in the OFF orientation, resulting in reduced type 1 pili
expression, an effect that stems from uncontrolled activation of
the QseB response regulator in this mutant. In addition to
influencing type 1 pili expression in the absence of qseC,
over-active QseB aberrantly regulates conserved cellular pathways
and impacts several virulence-associated genes, resulting in UPEC
attenuation.
[0792] Numerous investigations highlighted the importance of type 1
pili in pathogenesis and spurred the onset of research towards the
development of anti-adhesion agents, known as mannosides, aimed at
inhibiting FimH-host receptor interactions. Given that disruption
of QseC simultaneously affects fim expression and a number of other
cellular processes, type 1 pili were disengaged from QseC control
to determine the infection stages at which other QseC-mediated
defects become important. By locking the invertible fim promoter
element in the ON orientation, type 1 pili expression was restored
in the qseC mutant and it was shown that the resulting mutant is
competent in infection initiation and IBC formation. However, even
upon expression of type 1 pili the qseC mutant is rapidly
outcompeted by the parent strain during co-infection studies and
cannot survive long-term in the urinary tract in mono-infections.
These findings indicate that although type 1 pili mask the qseC
deletion phenotypes at the onset of infection, additional
QseC-mediated processes are required for persistence. Therefore,
the examples below explore whether co-inhibition of QseC and type 1
pili would have a synergistic effect on clearing UPEC from the
urinary tract. Given the lack of available QseC inhibitors a
prophylactic approach was taken, using the qseC mutant as a proxy
to QseC-inhibition. The data showed that mice pre-treated with
mannoside (to block FimH-mediated adhesion) and subsequently
challenged with the qseC mutant were more effectively protected
against chronic UTI compared to mice challenged with the parent
strain. These examples indicate that targeting of QseC can be
exploited as a potential preventative strategy, alone or in
combination with other anti-virulence agents.
Example 37. Deletion of QseC Impairs the Ability of UPEC to Invade
the Bladder
[0793] It was previously shown that deletion of qseC in UTI89 leads
to reduced bladder bacterial titers during the acute stages of
infection (6 and 16 h post infection) in a cystitis murine model.
Given that type 1 pili are critical for bladder adherence and
invasion and are significantly reduced in the absence of QseC, it
was rationalized that the in vivo attenuation of UTI89.DELTA.qseC
could be, at least partly, attributed to reduced bacterial
internalization. Thus, the ability of UTI89.DELTA.qseC to adhere to
and invade the bladder epithelium was assessed and compared to the
parent strain. Female C3H/HeN mice were transurethrally inoculated
with 10.sup.7 wild-type (wt) UTI89 or UTI89.DELTA.qseC and
bacterial invasion was assessed at 1 and 3 h post infection
(h.p.i), time-points previously shown to be sufficient for
completion of the invasion events. Enumeration of colony forming
units (cfu) recovered from infected bladders indicated that
compared to wt UTI89, UTI89.DELTA.qseC had significantly lower
overall bacterial titers (intracellular and luminal) at both
time-points (FIG. 52A). Treatment of infected bladders with
gentamicin to eradicate the luminal bacterial population but leave
the intracellular population unharmed revealed decreased
intracellular numbers for UTI89.DELTA.qseC (FIG. 52B). Thus, these
data indicate that deletion of qseC affects the ability of UTI89 to
initiate infection by compromising its ability to colonize and
invade the host bladder.
Example 38. Expression of Type 1 Pill Rescues the Adherence
Properties of UTI89.DELTA.gseC In Vitro, but does not Influence
Other .DELTA.gseC-Related Defects
[0794] Attenuation of UTI89.DELTA.gseC is linked to reduced
bacterial internalization due to decreased type 1 pili production.
Therefore, it was investigated whether restoration of type 1 pili
expression alone would be sufficient to overcome the in vivo
defects of UTI89.DELTA.gseC. It was previously reported that in the
absence of QseC the phase-variable fim promoter driving type 1 pili
expression, fimS, is primarily switched to the OFF orientation.
Therefore, fimS was locked in the ON orientation in the chromosome
of wt UTI89 and UTI89.DELTA.gseC to attain expression of type 1
pili independently of QseC (FIG. 53A). Electron microscopy revealed
that the resulting strains, UTI89_LON and UTI89.DELTA.gseC_LON,
were not hyper-piliated, but rather a larger population of piliated
versus non-piliated bacteria was observed compared to wt UTI89
(data not shown). UTI89_LON and UTI89.DELTA.gseC_LON exhibited
comparable mannose-sensitive hemagglutination (HA) properties,
which were higher than wt UTI89 since inversion of the promoter to
the OFF state can no longer occur (FIG. 53B). Consistently, western
blot analyses probing for FimA, the major type 1 pilus subunit,
verified that type 1 pili production in UTI89AgseC_LON is
comparable to UTI89_LON, but again, higher than wt UTI89 (FIG.
53C).
Example 39. Dual Inhibition of Type 1 Pili and QseC Potentiates
UPEC Elimination from the Bladder
[0795] Type 1 pili are sufficient for establishment of acute
infection whereas QseC is a critical determinant for persistence in
the bladder; thus targeting both factors could constitute an
excellent means of potentiating UPEC clearance from the urinary
tract by interfering with both the acute and chronic aspects of
infection. Mannosides, compounds that bind with high affinity the
mannose-binding pocket of the FimH adhesin at the tip of type 1
pili, are potent inhibitors of type 1 pili function. In particular,
mannosides 6 and 8, the most potent mannosides tested, drastically
reduce bacterial titers within 6 hours of oral delivery when used
prophylactically or as a therapeutic strategy. These studies are a
significant step towards a new anti-bacterial therapeutic; however,
a fraction of the bacterial population persists after mannoside
treatment and could lead to relapse, suggesting that mannoside
effectiveness can be further optimized. It was therefore
rationalized that co-inhibition of QseC may enhance mannoside
efficacy. However, no QseC inhibitors are currently available.
Although previous work has identified LED209 as an inhibitor of the
kinase activity of QseC, It has been established that it is the
disruption of the QseC phosphatase property that is responsible for
the attenuation of a qseC mutant. To circumvent the lack of
available QseC-specific phosphatase inhibitors, UTI89.DELTA.qseC
was used as a proxy to QseC inhibition and a prophylactic strategy
was employed to investigate whether targeting both factors (type 1
pili and QseC) could effectively protect the host against UTI. Mice
were pretreated with mannoside 6, and infected 30 min post
treatment with wt UTI89 or UTI89.DELTA.qseC. Mice treated with PBS
prior to infection were included as controls. Bladder bacterial
titers were enumerated at 2 weeks post infection as a measure of
chronic infection. Compared to non-treated mice, there was a 2-log
reduction in mannoside-treated mice infected with wt UTI89 (FIG.
54, UTI89 vs. UTI89_MAN) that was similar to the reduction observed
in non-treated mice infected with UTI89.DELTA.qseC (FIG. 54,
UTI89_MAN vs. UTI89.DELTA.qseC). Notably, in mannoside-treated
animals infected with UTI89.DELTA.qseC, bacterial reduction was
further enhanced, as indicated by an additional 1.5-log drop
compared to mannoside-treated animals infected with wt UTI89 (FIG.
54, UTI89_MAN vs. UTI89.DELTA.qseC_MAN). These data strongly
indicate that dual targeting of type 1 pili and QseC could
potentiate UPEC elimination from the host bladder.
Materials and Methods for Examples 37-39
Strains, Constructs and Growth Conditions
[0796] UTI89_LON and UTI89AgseC_LON were created using .lamda. Red
Recombinase, so as to mutate 7 out of 9 nucleotides in the left
invertible repeat of the fim promoter in the chromosome. Bacteria
were incubated in Luria Bertani (LB) media at 37.degree. C. for 4 h
under shaking conditions, sub-cultured (1:1000) in fresh LB media
and incubated statically at 37.degree. C. for 18 h.
Mouse Infections
[0797] Female C3H/HeN mice (Harlan), 7-9 weeks old, were used for
all studies described below and in each case mice were infected
with 10.sup.7 bacteria.
[0798] Short-term infections: Mice were transurethrally infected
with bacteria carrying the GFP-expressing plasmid pCom-GFP, as
previously described, and sacrificed at 1 and 3 h.p.i. Bladders
were aseptically removed, homogenized and plated for total
bacterial enumeration or gentamicin-treated to determine
intracellular bacterial titers. Experiment was repeated 3
times.
[0799] Ex vivo gentamicin assay: Bladders were bisected and washed
3 times in 500 .mu.l PBS. The washes were collected and plated for
cfu enumeration, to determine luminal bacteria. Washed bladders
were incubated for 90 min at 37.degree. C. with 100 .mu.g/ml
gentamicin to kill adherent extracellular bacteria. Following wash
(3.times.) wit PBS, bladders were homogenized in 1 ml PBS and
plated to determine intracellular bacterial titers. Two-tailed
Mann-Whitney (P<0.05) was used for statistical analyses.
[0800] Acute infection studies: Mice were transurethrally infected
with 10.sup.7 bacteria carrying the plasmid pCom-GFP as previously
described. Experiments were repeated three times and statistically
analyzed using two-tailed Mann-Whitney (P<0.05, considered
significant).
[0801] Long-term infection studies: Mice were infected with
chromosomally marked strains of wt UTI89, UTI89_LON,
UTI89.DELTA.qseC or UTI89.DELTA.qseC_LON. Mice were sacrificed at 2
weeks post inoculation and organs were processed for cfu
enumeration on LB and LB/Kanamycin agar plates. Experiment was
repeated 2 times. Cumulative data from all experiments are
presented.
Immunoblots, HA and Phase Assays
[0802] Bacteria were grown statically in LB for 18 h at 37.degree.
C. Immunoblots (using anti-type 1 pili antibody), HA and phase
assays were performed on normalized cells (OD.sub.600=1) as
previously described.
qPCR Analyses
[0803] RNA extraction, DNase treatment and reverse transcription
were prerformed using reagents and methods as reported by
Kostakioti et al. Relative transcript abundance was determined by
qPCR as previously described using aceB- or qseB-primers.
* * * * *