U.S. patent application number 15/060179 was filed with the patent office on 2016-08-18 for phenoxy thiophene sulfonamides and other compounds for use as inhibitors of bacterial glucuronidase.
This patent application is currently assigned to NORTH CAROLINA CENTRAL UNIVERSITY. The applicant listed for this patent is NORTH CAROLINA CENTRAL UNIVERSITY, THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL. Invention is credited to MATTHEW R. REDINBO, JOHN E. SCOTT, ALFRED L. WILLIAMS, LI-AN YEH.
Application Number | 20160237058 15/060179 |
Document ID | / |
Family ID | 44563852 |
Filed Date | 2016-08-18 |
United States Patent
Application |
20160237058 |
Kind Code |
A1 |
WILLIAMS; ALFRED L. ; et
al. |
August 18, 2016 |
PHENOXY THIOPHENE SULFONAMIDES AND OTHER COMPOUNDS FOR USE AS
INHIBITORS OF BACTERIAL GLUCURONIDASE
Abstract
This invention relates generally to compounds that are
glucuronidase inhibitors. The glucuronidase inhibitors include
phenoxy thiophene sulfonamides, and other compounds such as
pyridine sulfonyls, benzene sulfonyls, thiophene sulfonyls,
thiazole sulfonyls, thiophene carbonyls, and thiazole carbonyls.
These compounds include nialamide, isocarboxazid, phenelzine,
amoxapine, loxapine and mefloquine. Also compositions including one
or more of such compounds for use in inhibiting glucuronidase and
methods of using one or more of such compounds for selective
inhibition of bacterial .beta.-glucoronidase. These compounds may
be used as a co-drug in combination with the anticancer drug
CPT-11. Also a method for screening compounds to determine their
usefulness in reducing diarrhea associated with irinotecan
chemotherapy.
Inventors: |
WILLIAMS; ALFRED L.;
(Durham, NC) ; SCOTT; JOHN E.; (Durham, NC)
; YEH; LI-AN; (Cary, NC) ; REDINBO; MATTHEW
R.; (Chapel Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NORTH CAROLINA CENTRAL UNIVERSITY
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL |
DURHAM
CHAPEL HILL |
NC
NC |
US
US |
|
|
Assignee: |
NORTH CAROLINA CENTRAL
UNIVERSITY
DURHAM
NC
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL
CHAPEL HILL
NC
|
Family ID: |
44563852 |
Appl. No.: |
15/060179 |
Filed: |
March 3, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13479590 |
May 24, 2012 |
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15060179 |
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PCT/US2011/027974 |
Mar 10, 2011 |
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13479590 |
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61312512 |
Mar 10, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/553 20130101;
C07D 295/135 20130101; A61K 31/4709 20130101; C07D 413/12 20130101;
A61P 35/00 20180101; A61K 31/4409 20130101; G01N 2500/02 20130101;
A61K 31/42 20130101; C07D 409/12 20130101; C07D 417/12 20130101;
A61K 31/137 20130101; C07D 333/34 20130101; C12Q 1/34 20130101;
C07D 409/06 20130101; G01N 2333/924 20130101 |
International
Class: |
C07D 333/34 20060101
C07D333/34; C12Q 1/34 20060101 C12Q001/34; C07D 417/12 20060101
C07D417/12; C07D 409/12 20060101 C07D409/12; C07D 413/12 20060101
C07D413/12 |
Goverment Interests
[0002] This invention was supported in part by funds from the U.S.
Government (National Cancer Institute 04-051311 and National
Institutes of Health grant 1SC2GM081129). The U.S. Government may
have certain rights in the invention.
Claims
1-18. (canceled)
19. A compound of formula (I) ##STR00098## or a pharmaceutically
acceptable salt thereof wherein: each of R, and R.sub.2 is the same
or different and is selected from H, naphthalene,
naphthalene-(C.sub.1-C.sub.4) alkyl, naphthalene-1-ylmethyl,
naphthalene-1-ylethyl, naphthalene-1-ylpropyl, 3-fluorobenzyl,
3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl,
3-(trifluoromethyl)benzyl, 3-(trichloromethyl)benzyl,
3-(tribromomethyl) benzyl, 3-(triiodomethyl)benzyl,
3-(C.sub.1-C.sub.4 alkyl)-benzyl, 3-methylbenzyl, 3-ethyl-benzyl,
3-propylbenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl,
3,5-dibromobenzyl, 3,5-diiodobenzyl, 3-chlorophenyl,
3-fluorophenyl, 3-bromophenyl, 3-iodophenyl, 3-(C.sub.1-C.sub.4
alkyoxy) phenyl, 3-methoxyphenyl, 3-ethoxyphenyl, 3-propoxyphenyl,
4-methoxyphenyl, 4-(C.sub.1-C.sub.4 alkyoxy) phenyl,
4-ethoxyphenyl, 4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl,
4-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl,
2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl,
3-iodobenzyl, 4-iodobenzyl, 3-(C.sub.1-C.sub.4 alkyoxy) benzyl;
3-methoxybenzyl, 4-methoxy-benzyl, 3-ethoxybenzyl, 4-ethoxybenzyl,
3-propoxybenzyl, 4-(C.sub.1-C.sub.4 alkyoxy)phenyl and 4
propoxybenzyl; each of R.sub.3 and R.sub.4 is the same or different
and is selected from H, F, Cl, Br, and I, and R.sub.5 is selected
from 3-(R-1-yl)phenyl, and 4-(R-1-yl)phenyl, wherein R is selected
from piperazin, and 4-(C.sub.1-C.sub.4 alkyl) piperazin,
4-methylpiperazin, 4-ethyl-piperazin, and 4-propylpiperazin, or a
pharmaceutically acceptable salt of the compound.
20. A method of treating a subject in need of a glucuronidase
inhibitor comprising administering to the subject a composition
comprising an amount of compound that is effective as an inhibitor
of glucuonidase activity, or a pharmaceutically acceptable salt of
the compound.
21. The method of claim 20, wherein the compound is selected from
one or more of phenoxy thiophene sulfonamides, pyridine sulfonyls,
benzene sulfonyls, thiophene sulfonyls, thiazole sulfonyls,
thiophene carbonyls, and thiozole carbonyls.
22. The method according to claim 20, wherein the compound of
formula (I) ##STR00099## or a pharmaceutically acceptable salt
thereof wherein: each of R.sub.1 and R.sub.2 is the same or
different and is selected from H, naphthalene,
naphthalene-(C.sub.1-C.sub.4) alkyl, naphthalene-1-ylmethyl,
naphthalene-1-ylethyl, naphthalene-1-ylpropyl, 3-fluorobenzyl,
3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl,
3-(trifluoromethyl)benzyl, 3-(trichloromethyl)benzyl,
3-(tribromomethyl) benzyl, 3-(triiodomethyl)benzyl,
3-(C.sub.1-C.sub.4 alkyl)-benzyl, 3-methylbenzyl, 3-ethyl-benzyl,
3-propylbenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl,
3,5-dibromobenzyl, 3,5-diiodobenzyl, 3-chlorophenyl,
3-fluorophenyl, 3-bromophenyl, 3-iodophenyl, 3-(C.sub.1-C.sub.4
alkyoxy) phenyl, 3-methoxyphenyl, 3-ethoxyphenyl, 3-propoxyphenyl,
4-methoxyphenyl, 4-(C.sub.1-C.sub.4alkyoxy) phenyl, 4-ethoxyphenyl,
4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl,
2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-bromobenzyl,
3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl,
4-iodobenzyl, 3-(C.sub.1-C.sub.4 alkyoxy) benzyl; 3-methoxybenzyl,
4-methoxybenzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl,
4-(C.sub.1-C.sub.4 alkyoxy)phenyl and 4 propoxybenzyl, each of
R.sub.3 and R.sub.4 is the same or different and is selected from
H, F, Cl, Br, and I, and R.sub.5 is selected from 3-(R-1-yl)phenyl
and 4-(R-1-yl)phenyl, wherein R is selected from piperazin,
4-(C.sub.1-C.sub.4 alkyl) piperazin, 4-methylpiperazin,
4-ethyl-piperazin, and 4-propylpiperazin.
23. A compound that is an inhibitor of glucuronidase, or a
pharmaceutically acceptable salt of the compound, wherein the
compound is a phenoxy thiophene sulfonamide.
24. A composition comprising a compound according to claim 19 and
one or more pharmaceutically acceptable carriers, diluents and
excipients.
25. A method for making a compound of formula (I): ##STR00100## or
a pharmaceutically acceptable salt thereof of claim 19, wherein:
each of R.sub.1 and R.sub.2 is the same or different and is
selected from H, naphthalene, naphthalene-(C.sub.1-C.sub.4) alkyl,
naphthalene-1-ylmethyl, naphthalene-1-ylethyl,
naphthalene-1-ylpropyl, 3-fluorobenzyl, 3-chlorobenzyl,
3-bromobenzyl, 3-iodobenzyl, 3-(trifluoromethyl)benzyl,
3-(trichloromethyl)benzyl, 3-(tribromomethyl) benzyl,
3-(triiodomethyl)benzyl, 3-(C.sub.1-C.sub.4 alkyl)-benzyl,
3-methylbenzyl, 3-ethyl-benzyl, 3-propylbenzyl, 3,5-dichlorobenzyl,
3,5-difluorobenzyl, 3,5-dibromobenzyl, 3,5-diiodobenzyl,
3-chlorophenyl, 3-fluorophenyl, 3-bromophenyl, 3-iodophenyl,
3-(C.sub.1-C.sub.4 alkyoxy) phenyl, 3-methoxyphenyl,
3-ethoxyphenyl, 3-propoxyphenyl, 4-methoxyphenyl,
4-(C.sub.1-C.sub.4 alkyoxy) phenyl, 4-ethoxyphenyl,
4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl,
2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-bromobenzyl,
3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl,
4-iodobenzyl, 3-(C.sub.1-C.sub.4 alkyoxy) benzyl; 3-methoxybenzyl,
4-methoxybenzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl,
4-(C.sub.1-C.sub.4 alkyoxy)phenyl and 4 propoxybenzyl; each of
R.sub.3 and R.sub.4 is the same or different and is selected from
H, F, Cl, Br, and I, and R.sub.5 is selected from 3-(R-1-yl)phenyl
and 4-(R-1-yl)phenyl, wherein R is selected from piperazin,
4-(C.sub.1-C.sub.4 alkyl) piperazin, 4-methylpiperazin,
4-ethyl-piperazin, and 4-propylpiperazin, comprising the steps of:
(a) reacting a halo thiophene-sulfonyl halo and
R.sub.1--N--H.sub.2, wherein R.sub.1 as defined above to form an
N-monoprotected thiophene sulfonamide having a first N-protecting
group comprising R.sub.1, (b) reacting the resultant
N-monoprotected thiophene sulfonamide with R.sub.2--N-halo wherein
R.sub.2 is as defined above and a catalyst in a base, forming a
resultant N,N-diprotected thiophene sulfonamide having a second
N-protecting group comprising R.sub.2, (c) reacting the
N,N-diprotected thiophene sulfonamide of step (b) with
Cs.sub.2CO.sub.3; and a phenol substituted by R, wherein R is
selected from piperazin, 4-(C.sub.1-C.sub.4 alkyl) piperazin,
4-methylpiperazin, 4-ethylpiperazin, and 4-propylpiperazin, in a
solvent, and then removing the solvent, to obtain N,N-diprotected
phenoxy thiophene sulfonamide, and (d) reacting the N,N-diprotected
phenoxy thiophene sulfonamide with a deprotecting agent that is
selective for deprotecting the second N-protecting group, removing
the second N-protecting group, and forming a N-monoprotected
phenoxy thiophene sulfonamide.
26. The method of claim 25 for making a compound of formula (I)
wherein: (a) the halothiophene sulfonyl halo is
dichlorothiophene-sulfonyl chloride and the group R.sub.1--N--H is
naphthylmethylamine, and the dichlorothiophene-sulfonyl chloride
and naphthylmethylamine, are mixed and cooled, thereby forming a
N-monoprotected thiophene sulfonamide, having a first N-protecting
group that comprises naphthylmethyl, (b) adding with mixing and
cooling to the resultant N-monoprotected thiophene sulfonamide,
methoxybenzyl bromide and a catalyst in a base that is sodium
hydride, thereby forming a N,N-diprotected thiophene sulfonamide
having also a second N-protecting group that comprises
methoxybenzyl, (c) adding with mixing and heating to the resultant
N,N-diprotected thiophene sulfonamide, and Cs.sub.2CO.sub.3 and
butyl (hydroxyphenyl) piperazine-carboxylate in a solvent, and then
removing the solvent, to obtain a resultant N,N-diprotected phenoxy
thiophene sulfonamide, and (d) mixing the resultant N,N-diprotected
phenoxy thiophene sulfonamide with a deprotecting agent that is
selective for deprotecting the second N-protecting group, thereby
removing the methoxy benzyl that is the second N-protecting group,
and forming a N-monoprotected phenoxy thiophene sulfonamide.
27. The method of claim 26, wherein (i) the
dichlorothiophene-sulfonyl chloride is
4,5-dichlorothiophene-2-sulfonyl chloride, (ii) the
naththylmethylamine is 1-naphthylmethylamine, (iii) the
methoxybenzyl bromide is 4-methoxybenzyl bromide, (iv) the catalyst
is tetrabutylammonium iodide, (v) the butyl (hydroxyphenyl)
piperazine-carboxylate is
tert-butyl-4-(3-hydroxyphenyl)piperazine-1-carboxylate, (vi) the
solvent is dimethyl formamide, or (vii) the selective deprotecting
agent comprises dichloromethane and triflouroacetic acid; or a
combination of two or more thereof.
28. A method for screening compounds for their usefulness in
reducing diarrhea associated with irinotecan chemotherapy, the
method comprising: (a) assaying the compounds for activity in
inhibiting purified bacterial .beta.-glucoronidase; (b) assaying
the compounds for activity in inhibiting purified mammalian
.beta.-glucoronidase; and (c) selecting from the compounds assayed
in steps (a) and (b) a compound that inhibits the bacterial
.beta.-glucoronidase; in step (a) with a potency that is more than
2.50-fold greater than the compound inhibits the mammalian
.beta.-glucoronidase in step (b).
29. The method according to claim 28, wherein the assaying in step
(a) comprises assaying the compounds for activity in inhibiting
purified E. coli bacterial .beta.-glucoronidase.
30. The method according to claim 28, wherein the assaying in step
(a) comprises also assaying the compounds for activity in
inhibiting endogenous .beta.-glucoronidase activity in a culture
comprising intact bacterial cells.
31. The method according to claim 30, wherein the intact bacterial
cells are E. coli cells.
32. The method according to claim 30, wherein the assaying in step
(b) comprises assaying the compounds for inhibiting mammalian
.beta.-glucoronidase from B. taurus.
33. The method according to claim 28, further comprising
administering the selected compound to a patient to whom irinotecan
chemotherapy is being or will be administered.
34. The method according to claim 29, wherein the selected compound
generates an average IC.sub.50 value of 388 nM or less in the E.
coli .beta.-glucoronidase enzyme assay.
35. A method for inhibiting or reducing diarrhea in a patient being
treated with a drug that metabolizes to form a metabolite that is a
substrate for a bacterial .beta.-glucoronidase enzyme, the method
comprising administering to the patient a compound in an amount
effective to inhibit the bacterial .beta.-glucoronidase enzyme,
wherein the compound is selected from the group consisting of
nialamide, isocarboxazid, phenelzine, amoxapine, loxapine, and
mefloquine.
36. The method according to claim 35, wherein the drug is
irinotecan.
37. A composition comprising a compound according to claim 23 and
one or more pharmaceutically acceptable carriers, diluents and
excipients.
38. The compound according to claim 23, wherein the glucoronidase
is .beta.-glucoronidase.
39. The compound according to claim 1, wherein R is
4-(C.sub.1-C.sub.4 alkyl) piperazin, selected from
4-methylpiperazin, 4-ethyl-piperazin, and 4-propylpiperazin.
40. A compound of the formula ##STR00101## or a pharmaceutically
acceptable salt thereof.
41. A composition comprising a compound according to claim 40 and
one or more pharmaceutically acceptable carriers, diluents and
excipients.
42. The method of claim 20, wherein the compound is ##STR00102## or
a pharmaceutically acceptable salt thereof.
Description
RELATED APPLICATION INFORMATION
[0001] This application is a CONTINUATION of application Ser. No.
13/479,590 filed May 24, 2012 which is a CIP of International
Application PCT/US2011/027974 filed 10 Mar. 2011 entitled "Phenoxy
Thiophene Sulfonamides And Their Use As Inhibitors Of
Glucuronidase", which was published in the English language on 15
Sep. 2011, with International Publication Number WO 2011/112858 A1,
and which claims priority from U.S. Patent Application No.
61/312,512 filed on 10 Mar. 2010, the content of which is
incorporated herein by reference.
[0003] This invention relates generally to phenoxy thiophene
sulfonamides and other drugs that inhibit bacterial glucuronidase.
This invention also relates to compositions including one or more
of such compounds and methods of using one or more of such
compounds as a co-drug in combination with a camptothecin-derived
anticancer drug or other drug that, in a patient, is metabolized to
form a metabolite that is a substrate for a bacterial
.beta.-glucuronidase enzyme. The invention further relates to a
method of screening for such compounds. The invention also
encompasses a method for selectively inhibiting, in a patient to be
treated, bacterial .beta.-glucuronidase as compared with mammalian
.beta.-glucuronidase, wherein the method comprises administering to
the patient an effective amount of a compound selected from the
group consisting of nialamide, isocarboxazid, phenelzine,
amoxapine, loxapine and mefloquine.
BACKGROUND
[0004] Camptothecin, a plant alkaloid derived from the Chinese
Camptotheca acluminata tree, was added to the National Cancer
Institute's natural products screening set in 1966. It showed
strong anti-neoplastic activity but poor bioavailability and toxic
side effects. After thirty years of modifying the camptothecin
scaffold, two derivatives emerged and are now approved for clinical
use. Topotecan (Hycamptin.RTM. GlaxoSmithKline) is currently
employed to treat solid ovarian, lung and brain tumors. CPT-11
(also called Irinotecan, and Camptosar.RTM.; Pfizer) contains a
carbamate-linked dipiperidino moiety that significantly increases
bioavailability in mammals. This dipiperidino group is removed from
the CPT-11 prodrug in vivo by carboxylesterase enzymes that
hydrolyze the carbamate linkage to produce the drug's active
metabolite, SN-38. CPT-11 is currently used to treat solid colon,
lung, and brain tumors, along with refractory forms of leukemia and
lymphoma.
[0005] The sole target of the camptothecins is human topoisomerase
I. This enzyme relieves superhelical tension throughout the genome
and is essential for DNA metabolism, including DNA replication,
transcription, and homologous recombination. Topoisomerase I breaks
one strand in duplex DNA, forming a covalent 3'-phosphotyrosine
linkage, and guides the relaxation of DNA supercoils. It then
reseals the single-strand DNA break and releases a relaxed duplex
DNA molecule. The camptothecins bind to the covalent topoisomerase
I-DNA complex and prevent the religation of the broken single DNA
strand, effectively trapping the 91 kDa protein on the DNA. Such
immobilized macromolecular adducts act as roadblocks to the
progression of DNA replication and transcription complexes, causing
double-strand DNA breaks and apoptosis. Because cancer cells are
growing rapidly, the camptothecins impact neoplastic cells more
significantly than normal human tissues. Structural studies have
established that the camptothecins stack into the duplex DNA,
replacing the base pair adjacent to the covalent phosphotyrosine
linkage. Religation of the nicked DNA strand is prevented by
increasing the distance between the 5'-hydroxyl and the
3'-phosphotyrosine linkage to >11 .ANG..
[0006] CPT-11 efficacy is severely limited by delayed diarrhea that
accompanies treatment. While an early cholinergic syndrome that
generates diarrhea within hours can be successfully treated with
atropine, the diarrhea that appears .about.2-4 days later is
significantly more debilitating and difficult to control. CPT-11
undergoes a complex cycle of activation and metabolism that
directly contributes to drug-induced diarrhea. CPT-11 administered
by intravenous injection can traffic throughout the body, but
concentrates in the liver where it is activated to SN-38 by the
human liver carboxylesterase hCE1. The SN-38 generated in the liver
is conjugated in the liver to yield SN-38 glucuronide (SN-38G).
SN-38G is excreted from the liver via the bile duct and into the
intestines. Once in the intestines, however, SN-38G serves as a
substrate for bacterial glucuronidase enzymes in the intestinal
flora that remove the glucuronide moiety and produce the active
SN-38. SN-38 in the intestinal lumen produced in this manner
contributes to epithelial cell death and the severe diarrhea that
limits CPT-11 tolerance and efficacy. This effect has been
partially reversed in rats using the relatively weak (IC.sub.50=90
.mu.M) .beta.-glucuronidase inhibitor saccharic acid
1,4-lactone.
[0007] While broad-spectrum antibiotics have been used to eliminate
enteric bacteria from the gastrointestinal tract prior to CPT-11
treatment, this approach has several drawbacks. First, intestinal
flora play essential roles in carbohydrate metabolism, vitamin
production, and the processing of bile acids, sterols and
xenobiotics. Thus, the partial or complete removal of
gastrointestinal bacteria is non-ideal for patients already
challenged by neoplastic growths and chemotherapy. Second, it is
well established that the elimination of the symbiotic
gastrointestinal flora from even healthy patients significantly
increases the chances of infections by pathogenic bacteria,
including enterohemorrhagic E. coli and C. dificile. Third,
bacterial antibiotic resistance is a human health crisis, and the
unnecessary use of antimicrobials is a significant contributor to
this problem. For these reasons, we pursued the targeted inhibition
of gastrointestinal bacterial glucuronidases rather than the
broad-spectrum elimination of all enteric microflora.
[0008] Glucuronidases hydrolyze glucuronic acid sugar moieties in a
variety of compounds. The presence of glucuronidases in a range of
bacteria is exploited in commonly-used water purity tests, in which
the conversion of 4-methylumbelliferyl glucuronide (4-MUG) to
4-methylumbelliferone (4-MU) by glucuronidases is assayed to detect
bacterial contamination. Whereas relatively weak inhibitors of
glucuronidase have been reported, no potent and/or selective
inhibitors of the bacterial enzymes have been presented. Thus,
there is a need for selective inhibitors of bacterial glucuronidase
with a purpose of reducing the dose-limiting side effect and
improving the efficacy of the CPT-11 anticancer drug.
SUMMARY OF THE INVENTION
[0009] In a first aspect, the present invention relates to
compounds that are effective as inhibitors of bacterial
glucuronidase activity. In this respect, the inventors have found
that compounds that have GUS inhibitory activity can be used to
prevent dose-limiting diarrhea to the irinotecan therapy.
[0010] In another aspect, the present invention relates to a
compound for use with camptothecin-derived anticancer drugs. Use of
a compound of the invention with an camptothecin-derived anticancer
drug like CPT-11 for treating cancer reduces the dose-limiting side
effects and improves the efficacy of CPT-11. In an aspect of the
invention the compound is of formula (I) as described below, which
are phenoxy thiophene sulfonamides. In another aspect of the
invention, the compound may be a pyridine sulfonyl, benzene
sulfonyl, thiophene sulfonyl, thiazole sulfonyl, thiophene
carbonyl, and/or thiazole carbonyl. In still another aspect of the
invention, the compound of formula (I), or a compound that is a
pyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazole
sulfonyl, thiophene carbonyl, and/or thiazole carbonyl, is
administered prior to, at the same time as or following
administration of CPT-11. The present invention also relates to a
method for synthesizing compounds for inhibiting glucuronidases. In
an aspect of the invention the compound used of formula (I) as
described below, which is a phenoxy thiophene sulfonamide. In
another aspect of the invention the compound used may be a pyridine
sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazole sulfonyl,
thiophene carbonyl, and/or thiazole carbonyl.
[0011] In a further embodiment, the present invention relates to a
compound of the formula (I):
##STR00001##
or a pharmaceutically acceptable salt thereof wherein
[0012] each of R.sub.1 and R.sub.2 is the same or different and is
selected from H, naphthalene, naphthalene-(C.sub.1-C.sub.4) alkyl,
naphthalene-1-ylmethyl, naphthalene-1-ylethyl,
naphthalene-1-ylpropyl; 3-fluorobenzyl, 3-chlorobenzyl,
3-bromobenzyl, 3-iodobenzyl, 3-(trifluoromethyl)benzyl,
3-(trichloromethyl)benzyl, 3-(tribromomethyl) benzyl,
3-(triiodomethyl)benzyl, 3-(C.sub.1-C.sub.4 alkyl)-benzyl,
3-methylbenzyl, 3-ethyl-benzyl, 3-propylbenzyl, 3,5-dichlorobenzyl,
3,5-difluorobenzyl, 3,5-dibromobenzyl, 3,5-diiodobenzyl,
3-chlorophenyl, 3-fluorophenyl, 3-bromophenyl, 3-iodophenyl,
3-(C.sub.1-C.sub.4 alkyoxy) phenyl, 3-methoxyphenyl,
3-ethoxyphenyl, 3-propoxyphenyl, 4-methoxyphenyl,
4-(C.sub.1-C.sub.4 alkyoxy) phenyl, 4-ethoxyphenyl,
4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl,
2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-bromobenzyl,
3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl,
4-iodobenzyl, 3-(C.sub.1-C.sub.4 alkyoxy) benzyl; 3-methoxybenzyl,
4-methoxy-benzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl,
4-(C.sub.1-C.sub.4 alkyoxy)phenyl and 4 propoxybenzyl;
[0013] each of R.sub.3 and R.sub.4 is the same or different and is
selected from H, F, Cl, Br, and I, and
[0014] R.sub.5 is selected from 3-(R-1-yl)phenyl, and
4-(R-1-yl)phenyl, wherein R is selected from piperazin,
4-(C.sub.1-C.sub.4 alkyl) piperazin, 4-methylpiperazin,
4-ethyl-piperazin, and 4-propylpiperazin.
[0015] In yet another embodiment, the invention relates to a method
for inhibiting or reducing diarrhea in a patient being treated with
a drug that gets metabolized to form a metabolite that is a
substrate for a bacterial .beta.-glucuronidase enzyme. The method
comprises administering to the patient a compound in an amount
effective to inhibit the bacterial .beta.-glucuronidase enzyme,
wherein the compound is selected from the group consisting of
nialamide, isocarboxazid, phenelzine, amoxapine, and mefloquine. In
a preferred embodiment, the drug is irinotecan.
[0016] In another aspect, the present invention also relates to a
method for inhibiting bacterial .beta.-glucuronidase in a subject
in need thereof which comprises administering to the subject one or
more compounds that inhibit the glucuronidase. In an aspect of the
invention the compound is of formula (I) as described below, which
is a phenoxy thiophene sulfonamide. In another aspect of the
invention the compound may be a pyridine sulfonyl, benzene
sulfonyl, thiophene sulfonyl, thiazole sulfonyl, thiophene
carbonyl, and/or thiazole carbonyl.
[0017] In another aspect of the invention, the compound is one
which selectively inhibits bacterial glucuronidase. In this
connection, nialamide, isocarboxazid, and amoxapine were identified
as potent inhibitors of bacterial GUS activity in purified enzyme
and whole bacteria cell-based assays, but do not inhibit mammalian
GUS. These drugs and their average IC.sub.50 values for inhibiting
GUS include the monoamine oxidase inhibitors nialamide (71 nM) and
isocarboxazid (128 nM), the tricyclic antidepressant amoxapine (388
nM) and the antimalarial drug mefloquine (1.2 .mu.M). These drugs
had no significant activity (75 .mu.M to >100 .mu.M IC.sub.50)
against purified mammalian GUS. Nialamide, isocarboxazid and
amoxapine also showed potent activity for inhibiting endogenous GUS
activity in whole E. coli cells with average IC.sub.50 values of
17, 336 and 119 nM, respectively. These drugs have potential to be
repurposed as therapeutic treatments to reduce diarrhea associated
with irinotecan chemotherapy.
[0018] The compounds of the invention are useful in eliminating or
reducing the diarrhea associated with CPT-11 use for the treatment
of cancer.
[0019] In yet another embodiment, the method involves screening
compounds for their usefulness in reducing diarrhea associated with
irinotecan chemotherapy. In one aspect, the method comprises: (a)
assaying the compounds for activity in inhibiting purified
bacterial .beta.-glucuronidase, (b) assaying the compounds for
activity in inhibiting purified mammalian .beta.-glucuronidase; and
(c) selecting from the compounds assayed in steps (a) and (b) a
compound that inhibits the bacterial .beta.-glucuronidase in step
(a) with a potency that is more than 250-fold greater than that
with which the compound inhibits the mammalian .beta.-glucuronidase
in step (b).
[0020] In a preferred embodiment, the assaying in step (a)
comprises assaying the compounds for activity in inhibiting
purified E. coli bacterial .beta.-glucuronidase. In another
preferred embodiment, the assaying in step (a) comprises also
assaying the compounds for activity in inhibiting endogenous
.beta.-glucuronidase activity in a culture comprising intact
bacterial cells. In yet another preferred embodiment, the intact
bacterial cells are E. coli cells. In still another preferred
embodiment, the assaying in step (b) comprises assaying the
compounds for inhibiting mammalian .beta.-glucuronidase from B.
taurus.
[0021] The method for screening can further comprise administering
the selected compound to a patient to whom irinotecan chemotherapy
is being or will be administered. In a preferred embodiment of the
screening method, the selected compound generates an average
IC.sub.50 value of 10 .mu.M or less in an E. coli
.beta.-glucuronidase enzyme assay.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 Flow scheme for discovery of drugs with GUS
inhibitory activity.
[0023] FIG. 2 Structures of studied drugs.
[0024] FIG. 3 Potency determinations using the E. coli GUS enzyme
assay. Concentration response data for compounds was normalized to
controls with and without enzyme and plotted as percent activity.
The drugs evaluated were nialamide ( ), isocarboxazid
(.tangle-solidup.), phenelzine (.quadrature.), amoxapine
(.box-solid.), loxapine (.largecircle.) and mefloquine
(.diamond-solid.). The average Hill slope values derived from the
IC.sub.50 curves of all active drugs ranged from 0.9-1.2. Data
points represent the average of three determinations per
concentration and error bars represent standard deviation. Data are
representative of three independent experiments.
[0025] FIG. 4 Activities in the E. coli cell-based GUS activity
assay. Concentration response data for compounds was normalized to
controls with and without whole E. coli cells and plotted as
percent activity. The drugs evaluated were nialamide ( ),
isocarboxazid (.tangle-solidup.), phenelzine (.quadrature.),
amoxapine (.box-solid.), loxapine (.largecircle.) and mefloquine
(.diamond-solid.). The average Hill slope values derived from the
IC.sub.50 curves of all active drugs ranged from 0.8-1.0. Data
points represent the average of three determinations per
concentration and error bars represent standard deviation. Data are
representative of three independent experiments.
[0026] FIG. 5 Bacterial cytotoxicity assessment of studied drugs.
E. coli bacteria were treated with compounds at 10 and 100 .mu.M,
as indicated, for two hours. Viability was assessed with MTS
viability reagent. Absorbance data was normalized to controls with
and without whole E. coli cells and plotted as percent viability,
with `no cells` considered as 0% viability and DMSO only treated
cells set at 1000% viable. Kanamycin, a known cytotoxic antibiotic
drug, was used as a control.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Definitions
[0027] The following abbreviations are used in this
specification:
[0028] Br=bromine
[0029] Cl=chlorine
[0030] CPT=camptothecin
[0031] DCM=Dichloromethane
[0032] DMEM=Dulbecco's Minimal Essential Media
[0033] DMF=Dimethylformamide
[0034] DMSO=Dimethylsulfoxide
[0035] DNA=deoxyribonucleic acid
[0036] F=fluorine
[0037] FPLC=fast performance liquid chromatography
[0038] H=hydrogen
[0039] HEPES=(4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
[0040] I=iodine
[0041] kDal=kilodalton
[0042] MHz=megahertz
[0043] mmol=millimole
[0044] .mu.Mol=micromolar
[0045] NMR=nuclear magnetic resonance
[0046] nm=nanometer
[0047] OD=optical density
[0048] PMB=p-methoxybenzyl
[0049] PMSF=phenylmethylsulfonyl fluoride
[0050] ppm=parts per million
[0051] SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel
electrophoresis
[0052] TBAI=tetrabutylammonium iodide
[0053] TFA=Trifluoroacetic acid
[0054] The term "pharmaceutically acceptable salts" refers to the
non-toxic, inorganic and organic acid addition salts and base
addition salts of compounds of the present invention.
[0055] Such conventional non-toxic salts include those derived from
inorganic acids such as hydrochloric, hydrobromic, sulfuric,
sulfamic, phosphoric, and nitric acid; and the salts prepared from
organic acids such as acetic, propionic, succinic, glycolic,
stearic, lactic, malic, tartaric, citric, ascorbic, palmoic,
maleic, hydroxy-maleic, phenylacetic, glutamic, benzoic, salicylic,
sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, and isethionic acid.
Pharmaceutically acceptable salts from amino acids may also be
used. Such as salts of arginine and lysine.
[0056] Pharmaceutically acceptable salts may be synthesized from
the parent compound which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts may be
prepared by reacting the free acid or base forms of these compounds
with a stoichiometric amount of the appropriate base or acid in
water or in an organic solvent, or in a mixture of the two.
[0057] As used herein, the terms "treatment" and "therapy" and the
like refer to alleviate, slow the progression, prophylaxis, or
attenuation of existing disease.
[0058] As used herein, the terms "inhibit." "inhibiting." and the
like means that the activity of glucuronidase is reduced.
[0059] As used herein, the term "subject" means an animal or
human.
[0060] The pharmaceutical compositions of this invention comprise
one or more compounds that inhibit glucuronidase and one or more
pharmaceutically acceptable carriers, diluents, and excipients.
[0061] Pharmaceutical compositions of the present invention may be
in a form suitable for use in this invention for examples
compositions may be formulated for i) oral use, for example,
aqueous or oily suspensions, dispersible powders or granules,
elixirs, emulsions, hard or soft capsules, lozenges, syrups,
tablets or trouches; ii) parenteral administration, for example,
sterile aqueous or oily solution for intravenous, subcutaneous,
intraperitoneal, or intramuscular; iii) delivered intracerebrally
or iv) topical administration, for example, a suppository or
ointment.
[0062] As used herein the term "pharmaceutically acceptable" is
meant that the carrier, diluent, excipients, and/or salt must be
compatible with the other ingredients of the formulation including
the active ingredient(s), and not deleterious to the recipient
thereof.
[0063] "Pharmaceutically acceptable" also means that the
compositions, or dosage forms are within the scope of sound medical
judgment, suitable for use for an animal or human without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0064] A compound can also be used in the manufacture of a
medicament. This medicament can be used for the purposes described
herein.
[0065] The compositions or medicaments normally contain about 1 to
99%, for example, about 5 to 70%, or from about 5 to about 30% by
weight of the compound or its pharmaceutically acceptable salt. The
amount of the compound or its pharmaceutically acceptable salt in
the composition will depend on the type of dosage form and the
pharmaceutically acceptable excipients used to prepare it.
[0066] The dose of the compounds of this invention, which is to be
administered, can cover a wide range. The dose to be administered
daily is to be selected to suit the desired effect. Actual dosage
levels of the active ingredients in the pharmaceutical compositions
of this invention may be varied so as to obtain an amount of the
active ingredient, which is effective to achieve the desired
therapeutic response for a particular patient, composition, and
mode of administration without causing undue side effects or being
toxic to the patient.
[0067] The selected dosage level will depend upon a variety of
factors, including the activity of the particular compound of the
present invention employed, the route of administration, the time
of administration, the rate of excretion of the particular compound
being employed, the duration of the treatment, other drugs,
compounds and/or materials used in combination with the particular
compounds employed, the age, sex, weight, condition, general health
and prior medical history of the patient being treated, and like
factors well known in the medical arts.
[0068] As used herein, "effective amount" and the like means the
amount of the compound or composition necessary to achieve a
therapeutic effect.
[0069] An effective amount of the therapeutic compound necessary to
achieve a therapeutic effect may vary according to factors such as
the age, sex, and weight of the subject. Dosage regimens can be
adjusted to provide the optimum therapeutic response. For example,
several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0070] Compounds of the invention can be formulated into
compositions that can be administered to a subject in need of a
glucuronidase inhibitor.
[0071] The compounds or compositions thereof are used for
inhibition of glucuronidase.
[0072] The compounds or compositions thereof are used in methods
for treating a subject in need of a glucuronidase inhibitor. The
compounds or compositions are administered in an amount that is
effective to inhibit the glucuronidase. In some embodiments of the
invention it is 3 glucuronidase or bacterial 3 glucoronidase that
is inhibited.
[0073] The compounds or compositions described herein can be
administered prior to, concurrently with or after administration of
a camptothecin-derived anticancer agent such as CPT-11.
Administration of the compounds or compositions may result in
certain benefits such as decreasing the dose of the anticancer
drug, increasing the tolerance of the anticancer drug and
alleviating side effects from the use of the anticancer drug. Side
effects include gastrointestinal side effects.
[0074] For purposes of the following detailed description, it is to
be understood that the invention may assume various alternative
variations and step sequences, except where expressly specified to
the contrary. Moreover, other than in any operating examples, or
where otherwise indicated, all numbers expressing, for example,
quantities of ingredients used in the specification and claims are
to be understood as being modified in all instances by the term
"about". Accordingly, unless indicated to the contrary, the
numerical parameters set forth in the following specification and
attached claims are approximations that may vary depending upon the
desired properties to be obtained by the present invention. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should at least be construed in light of the number of
reported significant digits and by applying ordinary rounding
techniques.
[0075] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard variation found in their respective testing
measurements.
[0076] Also, it should be understood that any numerical range
recited herein is intended to include all sub-ranges subsumed
therein. For example, a range of "1 to 10" is intended to include
all sub-ranges between (and including) the recited minimum value of
1 and the recited maximum value of 10, that is, having a minimum
value equal to or greater than 1 and a maximum value of equal to or
less than 10.
[0077] In this application, the use of the singular includes the
plural and plural encompasses singular, unless specifically stated
otherwise. In addition, in this application, the use of"or" means
"and/or" unless specifically stated otherwise, even though "and/or"
may be explicitly used in certain instances.
[0078] The references cited herein are hereby incorporated by
reference as fully as if set forth herein.
PREFERRED EMBODIMENTS
[0079] In a first aspect of the present invention, seventy-six (76)
phenoxythiophene sulfonamides from a 35,000 compound diversity set
library were tested for their ability to inhibit the bacterial
enzyme .beta.-glucuronidase. The structures and inhibitory activity
of the compounds are shown in Table 1.
[0080] Eighteen (18) analogs of BRITE-355252 were synthesized and
tested to initially explore the structural relationship these
compounds display towards inhibition of 3-glucuronidase. The
structures and inhibitory activity of the 18 analogs of
BRITE-355252 are shown in Table 2.
[0081] Compounds of formula (I)
##STR00002##
[0082] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
as defined above can be prepared by a process comprising the steps
of
[0083] (a) reacting a halo thiophene-sulfonyl halo and
R.sub.1--N--H.sub.2, to form a resultant N-monoprotected thiophene
sulfonamide having a first N-protecting group comprising
R.sub.1,
[0084] (b) reacting the resultant N-monoprotected amide with
R.sub.2--N-halo and a catalyst in a base, to form a resultant
N,N-diprotected thiophene sulfonamide having also a second
N-protecting group comprising R.sub.2,
[0085] (c) reacting the resultant N,N-diprotected thiophene
sulfonamide with Cs.sub.2CO.sub.3 and phenol group substituted by
R;
[0086] wherein R is selected from piperazin, 4-(C.sub.1-C.sub.4
alkyl) piperazin, 4-methylpiperazin, 4-ethyl-piperazin, and
4-propylpiperazin, in a solvent, and then removing the solvent, to
obtain a resultant N,N-diprotected phenoxy thiophene sulfonamide,
and
[0087] (d) reacting the resultant N,N-diprotected phenoxy thiophene
sulfonamide with a deprotecting agent that is selective for
deprotecting the second N-protecting group, thereby removing the
second N-protecting group, and forming a N-monoprotected phenoxy
thiophene sulfonamide.
[0088] The halogen atom of the halo thiophene-sulfonyl halo
compound is selected from bromine, chlorine, fluorine and
iodine.
[0089] Any base that will in combination with the N-monoprotected
amide with R.sub.2--N-halo and a catalyst result in a
N,N-diprotected thiophene sulfonamide can be used.
[0090] Non-limiting examples of bases that can be used are
Et.sub.3N, Na.sub.2CO.sub.3, K.sub.2CO.sub.3 and NaH and any base
described in the examples.
[0091] In an embodiment of the invention the halo
thiophene-sulfonyl halo is dichlorothiophene-sulfonyl chloride and
R1-N--H.sub.2 is naphthylmethylamine. These groups are mixed and
cooled to form a N-monoprotected thiophene sulfonamide, having a
first N-protecting group that comprises naphthylmethyl.
[0092] In an embodiment of the invention the resultant
N-monoprotected thiophene sulfonamide, is mixed with methoxybenzyl
bromide and a catalyst that can be used in a Finkelstein reaction
in sodium hydride, and cooled thereby forming a N,N-diprotected
thiophene sulfonamide having also a second N-protecting group that
comprises methoxybenzyl, and the resultant N,N-monoprotected
thiophene sulfonamide, and Cs.sub.2CO.sub.3 and tert-butyl
(hydroxyphenyl)piperazine-carboxylate in a solvent, are mixed and
heated. The solvent is then removed to obtain a resultant
N,N-diprotected phenoxy thiophene sulfonamide.
[0093] In an embodiment of the invention the resultant
N,N-diprotected phenoxy thiophene sulfonamide is mixed with a
deprotecting agent that is selective for deprotecting the second
N-protecting group, thereby removing the methoxy benzyl that is the
second N-protecting group, and thereby forming a N-alkyl or N-aryl
phenoxy thiophene sulfonamide.
[0094] Examples of non-limiting embodiments of the invention are
where: the dichlorothiophene-sulfonyl chloride is
4,5-dichlorothiophene-2-sulfonyl chloride; the naththylmethylamine
is 1-naphthylmethylamine; the methoxybenzyl bromide is
4-methoxybenzyl bromide; the catalyst is tetrabutyl-ammonium
iodide; the butyl (hydroxyphenyl)piperazine-carboxylate is
tert-butyl-4-(3-hydroxyphenyl)piperazine-1-carboxylate; the solvent
is dimethyl formamide and/or the selective deprotecting agent
comprises dichloromethane and triflouroacetic acid; or a
combination thereof.
[0095] In addition to dimethyl formamide, non-limiting examples of
solvents that can be used are DMSO and dioxane and the solvents
described in the examples.
[0096] The following reaction Scheme 1 illustrates the preparation
of compounds within the scope of the present invention:
##STR00003##
[0097] Scheme 1 refers to the preparation of compounds of formula
I. Referring to Scheme 1, compounds of the formula I are prepared
by reacting commercially available 4,5-dichlorothiophene-2-sulfonyl
chloride 1 with an amine to generate dichlorothiophene sulfonamide
2. PMB (p-methoxybenzyl) protected 4,5-dichlorothiophene
sulfonamide 3 is generated by reacting compound 2 with NaH in DMF,
pmethoxybenzyl bromide and a catalytic amount of TBAI. Nucleophilic
displacement of the C-5 chlorine with a phenol in the presence of
Cs.sub.2CO.sub.3 produce N,N-diprotected
5-(3-phenoxy)-thiophene-2-sulfonamide 3. In the final step, the
protecting group is removed using TFA in DCM (1:1) to give the
desired compound.
[0098] In another aspect of the present invention, known compounds
were assayed for their ability to inhibit bacterial glucuronidase.
The Prestwick collection of FDA-approved drugs were screened with
the GUS enzyme assay to validate the GUS enzyme assay for HTS. This
screen of 1,120 compounds resulted in 40 actives having .gtoreq.50%
inhibition for a hit rate of 3.6% and all plates had Z'-factors of
.gtoreq.0.8 (average Z'-factor was 0.90). Since the collection was
screened at 10 .mu.M compound, a high concentration relative to in
vivo drug levels, a cut-off of 91% inhibition was applied as
criteria for selecting initial compounds for follow-up studies.
This requirement allowed us to focus on the more potent actives,
resulting in a short list of 7 compounds. Furthermore, antibiotics
and antiseptics were eliminated since the desire is to identify
drugs that do not disrupt the gut microbial flora, but instead only
inhibit bacterial GUS activity. This further triaging of actives
resulted in 4 compounds. We observed that two of these actives
belong to the monoamine oxidase inhibitor (MAOI) class of drugs,
though another MAOI while active (62% inhibition), did not quite
meet the 91% inhibition criteria. So to test more examples of this
class of inhibitors, we also included this compound (phenelzine) in
our studies. Thus, a total of five compounds were selected for
follow-up studies which included IC.sub.50 confirmation and E. coli
cell-based assays. A flow chart providing an overview of this
screening process is depicted in FIG. 1. The materials and method
we used to assay these compounds are described in Example 3,
infra.
[0099] The five compounds that remained after triage were
nialamide, isocarboxazid, phenelzine, amoxapine and mefloquine
(FIG. 1). Nialamide, isocarboxazid and phenelzine are all
irreversible hydrazine-class MAOI drugs, though nialamide is no
longer on the market. Amoxapine is a tricyclic antidepressant and
mefloquine is an antimalarial drug. Concentration response data for
each of the five compounds was generated using the purified E. coli
GUS enzyme assay from which IC.sub.50 values and Hill slopes were
calculated (FIG. 3). The average IC.sub.50 values and standard
deviations (SDs) for the MAOIs nialamide, isocarboxazid and
phenelzine were 71.+-.31, 128.+-.56, and 2,282.+-.1041 nM (see
Table 3 for all compound IC.sub.50 data).
[0100] Amoxapine generated an average IC.sub.50 value and SD of
388.+-.98 nM in the E. coli GUS enzyme assay. Loxapine is another
tricyclic antidepressant drug that has the identical structure as
amoxapine, except that loxapine has a methyl group, instead of
hydrogen, on the secondary amine of the piperazine ring (FIG. 1).
We tested loxapine as a specificity control and this compound
resulted in an average IC.sub.50 value of >100 .mu.M in the GUS
enzyme assay. Thus, the methyl group on the piperazine ring of
loxapine resulted in >250-fold loss in potency. The antimalarial
drug mefloquine hydrochloride generated an average IC.sub.50 value
of 1,212.+-.234 nM in this GUS enzyme assay.
[0101] Compound aggregation has been reported as a common
non-specific inhibitor mechanism for purified enzyme assays. The
Hill slopes calculated from concentration response data can be used
to eliminate many non-specific inhibitors in enzyme assays. For
single site binding, the Hill slope of an IC.sub.50 curve should be
1.0. IC.sub.50 curves with steep slopes, i.e. significantly greater
than 1.0, can be an indicator of non-specific mechanisms, including
compound aggregation. The IC.sub.50 curves for all the tested
compounds (with measurable IC.sub.50 values) had average Hill slope
values that ranged from 0.93 to 1.26 in the E. coli GUS enzyme
assay, which is close to the ideal value expected when measuring
inhibition of a single enzyme. To assess whether the compounds were
inhibiting signal by merely quenching fluorescence of the product
formed, GUS enzyme assays were done in which compound (100 .mu.M)
was added after the enzyme reaction was stopped and then
fluorescence was measured as usual. Adding the compounds at the end
of the assay resulted in no inhibition of signal for any of the
studied compounds (data not shown), indicating that the observed
activity is not due to fluorescence quenching, color quenching or
other assay artifact. Thus, these compounds produced data
consistent with specific binding to a single site on GUS and not
inhibition by non-specific mechanisms or assay artifact.
[0102] Tumor-derived mammalian GUS activity may be important for
optimal anti-tumor efficacy of irinotecan. Recent evidence suggests
that mammalian GUS may convert SN-38G back to SN-38 within the
tumor and thus increase the concentration of active drug (SN-38) in
the tumor. Therefore, any inhibitor of bacterial GUS used
therapeutically should not inhibit the mammalian GUS since this may
decrease the efficacy of irinotecan at the site of the tumor.
Therefore, we tested the three most potent drugs from the
screen--nialamide, isocarboxazid and amoxapine--in enzyme assays
identical to the E. coli GUS enzyme assay, except for the use of
mammalian GUS purified from Bovine taurus liver. Nialamide
generated an average IC.sub.50 of 74.8 .mu.M, while isocarboxazid
and amoxapine had IC.sub.50 values >100 .mu.M. Thus, nialamide
was over 1,000-fold more potent against E. coli GUS than mammalian
GUS, while the other two drugs where >250-fold more selective
for the E. coli GUS.
[0103] An E. coli cell based assay was developed in order to assess
the activity of these drugs against whole cells, instead of
purified enzyme. We took advantage of the well-known specificity
and sensitivity of the 4MUG substrate to detect GUS activity in E.
coli cells. This assay mimicked the enzyme assay in format, with
the GUS enzyme replaced by live log-phase E. coli cells and the
assay incubated for a longer time (2 hr) to detect GUS activity in
these un-modified cells. Four experimental results confirmed that
this cell-based assay was measuring GUS activity and no other E.
coli cell enzymes. First, The K.sub.m value for the substrate was
determined with this cell-based assay to be 151 .mu.M (data not
shown), which is similar to the 125 .mu.M K.sub.m value we
previously reported for the purified enzyme assay. Secondly, the
Hill slopes derived from concentration-response data for all 5
active drugs tested in this cell-based assay were in the 0.8-1.1
range, close to the expected value of 1.0 for inhibition of a
single enzyme. Thirdly, maximal inhibition was achieved by all
active compounds (FIG. 4), which would be expected if 100% of the
observed activity was coming from a single enzyme rather than a
mixture of enzymes. Finally, the overall rank order of potencies in
the cell based assay is similar to the E. coli GUS assay and
absolute IC.sub.50 values derived from the cell-based assay for the
test compounds are within 5-fold of the purified bacterial GUS
enzyme assay (see Table 3).
[0104] The potencies of the five hits from the Prestwick collection
and the one control compound were determined using the E. coli
cell-based assay (FIG. 4 and Table 3). In the irreversible MOAI
class of drugs, nialamide potently inhibited the cell-based assay
with an IC.sub.50 of 17.+-.2 nM, while isocarboxazid generated an
IC.sub.50 of 336.+-.120 nM. Thus, the potency of nialamide shifted
4-fold more potent in the cell-based assay compared to the E. coli
GUS enzyme assay, while isocarboxazid was about 3-fold less potent
in the cell-based assay. The other irreversible MOAI drug,
phenelzine, was also tested in the cell-based assay and resulted in
an IC.sub.50 value of 7,123 nM, which is 2.6-fold less potent
compared to the enzyme assay. Amoxapine was also tested in this
assay resulting in an IC.sub.50 of 119.+-.61 nM, which is 3.3-fold
more potent than the 388 nM IC.sub.50 value generated using the
enzyme assay. As a specificity control, loxapine was also tested,
but was completely inactive (>100 .mu.M) in this assay,
consistent with its inactivity in the enzyme assay. Mefloquine
generated an average IC.sub.50 value of 5,961.+-.1,526 nM and thus
shifted over 4.9-fold less potent in the cell-based assay compared
to the enzyme assay.
[0105] One possible explanation for the inhibitory activity of the
drugs in the cell-based assay is E. coli cell toxicity and/or
bacteriostatic activity resulting in reduced GUS activity.
Therefore, the viability of drug-treated E. coli cells was assessed
with a metabolic viability assay (MTS kit, Promega). Each compound
was tested at 100 and 10 .mu.M for 2 hrs and data normalized to
solvent (DMSO) controls with and without cells (FIG. 4). Kanamycin
was used as a control to demonstrate assay sensitivity to a known
bactericidal antibiotic. Kanamycin treatment (50 .mu.g/ml) reduced
activity in this assay by over 90%. Amoxapine, isocarboxazid,
loxapine, and nialamide showed no signs of toxicity in this assay
at up to 100 .mu.M drug. In contrast, the 100 .mu.M concentration
of mefloquine completely inhibited viability in this assay while
the 10 .mu.M concentration was at maximum control levels (100%
viability). Thus, the IC.sub.50 value of mefloquine in the
cell-based GUS assay may be a blend of GUS inhibitory activity and
bactericidal activity at higher concentrations. Phenelzine showed
some inhibitory activity (.about.10-20/o) at both the 100 and 10
.mu.M concentrations, but not enough to explain all of its
cell-based activity.
[0106] To summarize, we screened a collection of FDA-approved
drugs, the Prestwick collection, using our high throughput GUS
enzyme assay. The hit rate was high, with 40 actives displaying
.gtoreq.50% inhibition at the screening concentration of 10 .mu.M.
Raising the cut-off to 91% and elimination of
antiseptics/antibiotics resulted in a short list of 4 actives for
follow-up. We also included a compound (phenelzine) that did not
meet the activity cut-off. It was also chosen for follow-up since
it was in the same class as two on the short list and it had
>50% inhibition. Thus, the actives were nialamide,
isocarboxazid, phenelzine, amoxapine and mefloquine and all of
these compounds confirmed by IC.sub.50 determinations in the E.
coli GUS enzyme assay. The five hits can be categorized into three
drug classes: irreversible MAOI, tricyclic antidepressant and
antimalarial.
[0107] In the irreversible MAOI class, nialamide was a very potent
inhibitor of GUS activity with an IC.sub.50 of 71 nM in the GUS
enzyme assay. Surprisingly, this is more potent than its reported
in vitro IC.sub.50 values of 2.6-13 .mu.M for inhibiting monoamine
oxidase (in rat brain homogenates), its original intended target.
When tested for inhibitory potency against purified mammalian GUS,
nialamide had an IC.sub.50 of approximately 75 .mu.M. Thus,
nialamide displayed a dramatic 1,000-fold selectivity for
inhibiting E. coli GUS over mammalian GUS. Furthermore, nialamide
had more potent activity for inhibiting endogenous GUS in the E.
coli cell-based assay, generating an IC.sub.50 of 17 nM. This
activity was not due to acute toxicity of nialamide. This unusual
increased potency in a cell-based assay (also observed with
amoxapine) may be due to a unique mechanism of action or due to
compound concentration inside the bacterial cell. This same
phenomenon was observed previously for some, but not all, GUS
inhibitor compounds. The other compound in this same class,
isocarboxazid, was also relatively potent with an IC.sub.50 of 128
nM, which is more potent than its reported potency of 4.8 .mu.M
IC.sub.50 for MAO in rat brain homogenates. Isocarboxazid was
>780-fold more selective for inhibiting bacterial GUS compared
to its activity against mammalian GUS, which was not measurable
(>100 .mu.M IC.sub.50). This drug also inhibited in the
cell-based assay with an IC.sub.50 of 336 nM, 2.6-fold less potent
compared to the purified enzyme assay. Phenelzine is also an MAOI
that is structurally similar to nialamide and isocarboxazid in that
it contains a hydrazine group and is irreversible against its
original target. Phenelzine was a much weaker inhibitor of GUS at
2.2 .mu.M IC.sub.50, in contrast to its IC.sub.50 for MAO that was
reported to be 70-900 nM (depending on subclass of MAO-A or MAO-B,
or total activity). Thus, the phenelzine results indicated that
inhibition by the MAOIs was not solely due to the presence of a
hydrazine group or the irreversible nature of these drugs.
Phenelzine showed some toxicity to E. coli at 10 and 100 .mu.M, but
not enough to account for all of its GUS inhibitory activity.
[0108] The tricyclic antidepressant amoxapine potently inhibited
purified GUS with an IC.sub.50 of 388 nM. In comparison, amoxapine
had no measurable IC.sub.50 against mammalian GUS (>100 .mu.M)
thus resulting in a >250-fold selectivity for inhibiting
bacterial GUS over mammalian GUS. Furthermore, amoxapine had more
potent activity in the cell-based assay with an IC.sub.50 of 119
nM. Loxapine was used as a control compound for this class since it
has an identical structure to amoxapine except that loxapine has a
methyl group on the nitrogen of the piperazine group. Despite this
very small structural difference, loxapine had no measurable
IC.sub.50 value (>100 .mu.M) for both the GUS enzyme assay and
the cell-based assay. Thus, the amoxapine/loxapine pair served to
illustrate the exquisite structural selectivity for inhibiting
signal in these assays and demonstrated that a free amine in the
piperazine group is critical for inhibiting GUS.
[0109] Finally, mefloquine is an antimalarial drug that was also
identified in our screen. This drug had only weak activity for
inhibiting purified GUS (IC.sub.50=1.2 .mu.M) and its potency
worsened by 5-fold when tested in the cell-based assay (IC.sub.50=6
.mu.M). Since this compound resulted in complete inhibition in the
toxicity assay at 100 .mu.M, though none evident at 10 .mu.M, it is
possible that some of the cell-based activity is due to toxicity.
Nialamide, the most potent of these drugs for inhibiting bacterial
GUS, has a number of issues with respect to its use as a
therapeutic. First, nialamide is no longer on the market. Nialamide
was withdrawn from the market in 1963 due to interactions with food
products containing high levels of tyramine. Ingestion of certain
foods high in tyramine (e.g. aged cheese) resulted in sometimes
severe tyramine toxicity in patients taking nialamide, a general
problem with all the non-selective irreversible MAOIs. Therefore,
toxicity of nialamide is a major concern, even if it were available
on the market again. However, given the high potency of this drug
for inhibiting GUS, may be possible to use lower, and thus safer,
doses of nialamide that would have acceptable side effects. Special
diets, especially avoiding intake of tyramine-enriched foods, help
reduce food toxicity side effects of MAOIs. It is also conceivable
that nialamide could be re-formulated for low-dose time release in
the intestine. The food-induced toxicity reported for nialamide is
assumed to be due to inhibition of MAO in the intestine. Thus, we
believe that dosing with nialamide may result in sufficient
concentrations of nialamide in the GI tract to effectively inhibit
GUS. In contrast to nialamide, isocarboxazid is still on the market
for treatment of major depression. Like all drugs in the MAOI
class, isocarboxazid has toxicity/side effect concerns and can be
problematic in combination with many other medicines due to
drug-drug interactions. Phenelzine had relatively weak activity in
our assays and so it is not clear if effective in vivo
concentrations could be achieved. It should be recognized that any
GUS inhibitor would only be needed short term (weeks) and perhaps
even intermittently. Thus, we believe that the long term toxicity
of nialamide or isocarboxazid is avoidable with strategic, short
term dosing regimens to minimize long term drug exposure with the
accompanying toxicity/side effects.
[0110] Amoxapine is a marketed member of an older class of
antidepressant drugs with significantly fewer toxicity concerns
compared to the MAOI drug class. It also has a safer side effect
profile and far fewer drug-drug interactions than the MAOI class of
drugs in general. Antidepressant use in general is common in cancer
patients and thus amoxapine could also treat cancer-induced
depression. According to a recent report, amoxapine and loxapine
have been discovered to be potent non-competitive inhibitors of
P-glycoprotein, a transporter responsible for multidrug resistance
displayed by some cancer cells. Thus, the use of amoxapine as a GUS
inhibitor could also have the added benefit of enhancing the
sensitivity of multidrug resistant cancer cells to irinotecan
and/or other chemotherapeutic drugs given in combination with
irinotecan. Tricyclic antidepressants typically take about three
weeks to reach peak efficacy for treatment of depression. Unlike
the long term, slow acting mechanism of amoxapine for depression,
amoxapine as a GUS inhibitor will only be needed short term and
should act immediately to prevent re-activation of SN-38G. Thus,
some of the side effects encountered with chronic use of amoxapine
may be minimized with strategic intermittent dosing. The
combination of its potency for inhibiting GUS and its safer profile
suggests that amoxapine is the preferred drug as a therapeutic
treatment of irinotecan induced diarrhea. Moreover, based on the
potency of amoxapine for inhibiting GUS, compounds that get
metabolized in vivo to form amoxapine as a metabolite would
similarly be potent GUS inhibitors. In particular, loxapine
undergoes metabolism that includes some of the drug being
de-methylated at the piperazine ring--essentially generating
amoxapine and amoxapine-like molecules in vivo. Thus, loxapine,
though it lacked activity in our in vitro assays, would similarly
be expected to have GUS inhibitory activity in vivo due to its
metabolites.
[0111] In short, we have identified five known drugs that inhibit
E. coli GUS activity in enzyme assays: nialamide, isocarboxazid,
phenelzine, amoxapine and mefloquine. These compounds displayed
IC.sub.50 values ranging from 71 nM to 2.3 .mu.M against purified
E. coli GUS. Furthermore, nialamide, isocarboxazid and amoxapine
had no significant activity against purified mammalian GUS. All
five compounds also had activity in an E. coli cell-based assay
with IC.sub.50 values for inhibiting endogenous GUS ranging from 17
nM to 7.1 .mu.M.
[0112] Each of these drugs, or drugs that get metabolized in vivo
to form these drugs as metabolites, can be administered to a
patient selectively to inhibit bacterial .beta.-glucoronidase (as
compared with mammalian .beta.-glucoronidase) in the patient
whereby to reduce diarrhea associated with, for example, irinotecan
chemotherapy. Each of these drugs can be administered to the
patient in an amount effective to inhibit the bacterial
.beta.-glucoronidase with the dosages being controlled within the
following limits:
[0113] Amoxapine: less than or equal to 400 mg/day (or 600 mg/day
for a hospitalized patient);
[0114] Isocarboxazid: less than or equal to 60 mg/day;
[0115] Nialamide: less than or equal to 3.3 mg/kg (from FDA web
site);
[0116] Loxapine: less than or equal to 250 mg/day;
[0117] Mefloquine: less than or equal to 1250 mg/day; and
[0118] Phenelzine: less than or equal to 90 mg/day.
EXAMPLES
[0119] The invention is further understood by reference to the
following Examples, which are intended to be purely exemplary of
the invention. The present invention is not limited in scope by the
exemplified embodiments, which are intended as illustrations of
single aspects of the invention only. Any methods that are
functionally equivalent to those described in the Examples are
within the scope of the invention. Various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing
description. Such modifications fall within the scope of the
appended claims.
Example 1
.beta.-Glucuronidase Activity Assay
[0120] Expression and Purification of E. coli .beta.-Glucuronidase.
The full-length E. coli .beta.-glucuronidase gene was obtained from
bacterial genomic DNA and was cloned into the pET-28a expression
plasmid (Novagen) with an N-terminal 6.times.-Histidine tag.
BL21-DE3 competent cells were transformed with the expression
plasmid and grown in the presence of kanamycin (25 ug/ml) in LB
medium with vigorous shaking at 37.degree. C. until an OD.sub.600
of 0.6 was attained. The expression was induced with the addition
of 0.3 mM isopropyl-1-thio-D-galactopyranoside (IPTG) and further
incubated at 37.degree. C. for 4 hours. Cells were collected by
centrifugation at 4500.times.g for 20 min at 4.degree. C. Cell
pellets were resuspended in Buffer A (20 mM Potassium Phosphate, pH
7.4, 25 mM Imidazole, 500 mM NaCl), along with PMSF (2 .mu.L/mL
from 100 mM stock) and 0.05 .mu.L/mL of protease inhibitors
containing 1 mg/mL of aprotinin and leupeptin. Resuspended cells
were sonicated and centrifuged at 14,500.times.g for 30 min to
clarify the lysate. The cell lysate was flowed over a pre-formed
Ni-NTA His-Trap gravity column and washed with Buffer A. The
Ni-bound protein was eluted with Buffer B (20 mM Potassium
Phosphate, pH 7.4, 500 mM Imidazole, 500 mM NaCl). Collected
fractions were then tested for initial purity by SDS-PAGE.
Relatively pure (.about.85%) fractions were combined and loaded
into the Aktaxpress FPLC system and passed over a HiLoad.TM. 16/60
Superdex.TM. 200 gel filtration column. The protein was eluted into
20 mM HEPES, pH 7.4, and 50 mM NaCl for crystallization and
activity assays. Two milliliter fractions were collected based on
highest ultraviolet absorbance at 280 nm. Fractions were analyzed
by SDS-PAGE (which indicated >95% purity), combined, and
concentrated to 10 mg/mL for long-term storage at -80.degree. C. In
addition, some experiments were performed with purified E. coli
.beta.-glucuronidase enzyme purchased from Sigma-Aldrich.
[0121] High Throughput Screening .beta.-Glucuronidase Assay
[0122] The .beta.-glucuronidase assay was performed by the addition
of 0.5 .mu.l of compound (or DMSO) to the well of a black 384-well
plate followed by the addition of 30 .mu.l of diluted
.beta.-glucuronidase enzyme. The enzyme was diluted in assay buffer
(50 mM HEPES, pH 7.4) plus 0.0166% Triton X-100 for a final enzyme
concentration of 50 .mu.M and final detergent concentration of
0.01%. After a 15 minute incubation at room temperature (23.degree.
C.), 20 ul of substrate, 4-Methylumbelliferyl 3-D-glucuronide
hydrate (4MUG) diluted into assay buffer, was added to the reaction
for a final concentration of 125 uM. .beta.-glucuronidase
hydrolyzes the non-fluorescent 4MUG resulting in a fluorescent
product, 4-methylumbelliferyl. After a 30 minute incubation at room
temperature, the reaction was stopped by the addition of 20 ul 1 M
Na.sub.2CO.sub.3. The fluorescence (in relative fluorescence units,
RFU) was measured using a 355 nm excitation filter and 460 nm
emission filter using a Victor V (Perkin Elmer) plate reader.
Minimum (min) controls were performed using reactions with no
enzyme. Maximum (max) controls were performed using no compound. 1%
DMSO was maintained in all reactions. Percent inhibition was
calculated using RFU data by the following formula: [1-(assay
readout-average of min)/(Average of Max-Average of Min)].times.100.
The known .beta.-glucuronidase inhibitor, D-Glucaric
acid-1,4-lactone monohydrate, was used to validate the assay and
serve as a positive control. IC.sub.50 value was defined as the
concentration of inhibitor calculated to inhibit 50% of the assay
signal based on a serial dilution of compound. Values were
calculated using either a four or three-parameter dose response
(variable slope) equation in GraphPad Prism.TM. or
ActivityBase.TM.. For the IC.sub.50 determinations, serial
dilutions of compounds were performed in 100% DMSO with a two-fold
dilution scheme resulting in 10 concentrations of compound. These
results are shown in Tables 1 and 2.
Example 2
Preparation of Analogs of BRITE-355252
[0123] General procedures for the preparation of analogs of
BRITE-355252 All solvents and reagents were obtained from
commercial sources and used without further purification unless
otherwise stated. All reactions were performed in oven-dried
glassware (either in RB flasks or 20 ml vials equipped with septa)
under an atmosphere of nitrogen and the progress of reactions was
monitored by thin-layer chromatography and LC-MS. Analytical
thin-layer chromatography was performed on precoated 250 .mu.m
layer thickness silica gel 60 F.sub.254 plates (EMD Chemicals
Inc.). Visualization was performed by ultraviolet light and/or by
staining with phosphomolybdic acid (PMA) orp-anisaldehyde. All the
silica gel chromatography purifications were carried out by using
Combiflash.RTM. Rf (Teledyne Isco) and CombiFlash.RTM.
Companion.RTM. (Teledyne Isco) either with EtOAc/hexane or
MeOH/CH.sub.2Cl.sub.2 mixtures as the eluants. Melting points were
measured on a MEL-TEMP.RTM. capillary melting point apparatus and
are uncorrected. Proton nuclear magnetic resonance (.sup.1H NMR)
spectra and carbon nuclear magnetic resonance (.sup.13C NMR)
spectra were recorded on a Varian VNMRS-500 (500 MHz) spectrometer.
Chemical shifts (.delta.) for proton are reported in parts per
million (ppm) downfield from tetramethylsilane and are referenced
to it (TMS 0.0 ppm). Coupling constants (J) are reported in Hertz.
Multiplicities are reported using the following abbreviations:
br=broad; s=singlet; d=doublet; t=triplet; q=quartet; m=multiplet.
Chemical shifts (6) for carbon are reported in parts per million
(ppm) downfield from tetramethylsilane and are referenced to
residual solvent peaks: carbon (CDCl.sub.3 77.0 ppm). Mass spectra
were recorded on an Agilent 1200 Series LC/MS instrument equipped
with a XTerra MS (C-18, 3.5 m) 3.0.times.100 mm column.
Representative Procedure for the Preparation of
4,5-dichloro-N(aryl/alkyl)thiophene-2-sulfonamides
[0124] To a solution of 4,5-dichlorothiophene-2-sulfonyl chloride
(1.000 g, 4.002 mmol) in anhydrous CH.sub.2Cl.sub.2 (20 mL) was
added 1-naphthylmethylamine (0.630 g, 4.007 mmol) followed by
Et.sub.3N (0.84 mL, 6.027 mmol) and stirred at room temperature for
2 h. The reaction mixture was diluted with water (20 mL) and
extracted with CH.sub.2Cl.sub.2 (100 mL), washed with brine, dried
(Na.sub.2SO.sub.4) and concentrated under vacuo. The residue was
purified by recrystallization from CH.sub.2Cl.sub.2-hexane to
afford the pure
4,5-dichloro-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide
(1.350 g, 91%) as a white crystalline product.
Representative Procedure for the PMB Protection of 4,5-dichloro-N
(aryl/alkyl)thiophene-2-sulfonamides
[0125] Sodium hydride (0.081 g, 3.375 mmol) was slowly added in
portions to a solution of
4,5-dichloro-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide
(1.250 g, 3.358 mmol) in anhydrous DMF (10 mL) at 0.degree. C. and
stirred for 15 min. Then, 4-methoxybenzyl bromide (PMBBr) (0.675 g,
3.357 mmol), and a catalytic amount of TBAI (0.030 g, 0.081 mmol)
were added at 0.degree. C., and allowed to stir at room temperature
for 2 h. After completion of the reaction, it was quenched by slow
addition of water (5 mL) and extracted with EtOAc (100 mL), washed
with water and brine, dried (Na.sub.2SO.sub.4), concentrated under
vacuo and the residue purified by flash silica gel column
chromatography (Combiflash.RTM. Rf) using EtOAc-hexane (1:9) as
eluant to afford
4,5-dichloro-N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)
thiophene-2-sulfonamide (1.500 g, 91%) as a white solid.
Representative Procedure for the Coupling of Phenols with PMB
Protected 4,5-dichloro-N-(aryl/alkyl)thiophene-2-sulfonamides
[0126] A mixture of
4,5-dichloro-N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)thiophene-2-sul-
fonamide (0.100 g, 0.203 mmol), tert-butyl
4-(3-hydroxyphenyl)piperazine-1-carboxylate (0.068 g, 0.244 mmol)
and Cs.sub.2CO.sub.3 (0.099 g, 0.304 mmol) in anhydrous DMF (2 mL)
was heated at 80.degree. C. for 2.5 h. The solvent was removed
under vacuo and the residue was purified by Combiflash.RTM. Rf
(Isco) using EtOAc-hexanes (1:9) to obtain a white solid (0.140 g,
94%).
Representative Procedure for the Deprotection PMB Group
[0127] To a solution of tert-butyl
4-(3-(3-chloro-5-(N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)sulfamoyl)-
thio-phen-2-yloxy)phenyl)piperazine-1-carboxylate (0.085 g, 0.116
mmol) in anhydrous CH.sub.2Cl.sub.2 (2 mL) was added TFA (2 mL) and
stirred at room temperature for 3 h. The solvent mixture was
removed under vacuo and the residue was re-dissolved in
CH.sub.2Cl.sub.2 (20 mL) washed with aqueous sat. NaHCO.sub.3
followed by brine, dried (Na.sub.2SO.sub.4), and concentrated under
vacuo.
BRITE-355252
4-Chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene--
2-sulfonamide
[0128] The crude product was purified by flash silica gel column
chromatography using MeOH--CH.sub.2Cl.sub.2 (1:9) to afford a light
orange solid (0.055 g, 92%). .sup.1H NMR (500 MHz, DMSO-ds):
.delta. (ppm): 2.81 (t, 4H, J=5.0 Hz), 3.09 (t, 4H, J=5.0 Hz), 4.56
(s, 2H), 6.43 (dd, 1H, J=2.0, 8.0 Hz), 6.75 (t, 1H, J=2.5 Hz), 6.83
(dd, 1H, J=2.5, 8.0 Hz), 7.26 (t, 1H, J=8.0 Hz), 7.43-7.48 (m, 3H),
7.54-7.58 (m, 2H), 7.87 (dd, 1H, J=1.5, 7.5 Hz), 7.93-7.96 (m, 1H),
8.06-8.09 (m, 1H). APCI/ESI MS: mz 513.9 [M+H].sup.+
BRITE-492796
4-Chloro-N-methyl-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)t-
hiophene-2-sulfonamide
[0129] The product was prepared in 89%% yield; White solid, mp:
144-146.degree. C.;
[0130] .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta. (ppm): 2.55 (s,
3H), 2.86 (t, 4H, J=4.5 Hz), 3.14 (t, 41H, J=4.5 Hz), 4.63 (s, 2H),
6.62-6.66 (m, 1H), 6.84-6.88 (m, 2H), 7.30 (t, 1H, J=8.0 Hz),
7.48-7.62 (m, 4H), 7.91 (s, 1H), 7.94 (d, 1H, J=8.0 Hz), 7.98 (d,
1H, J=9.0 Hz), 8.29 (d, 1H, J=8.0 Hz). APCI/ESI MS m/z 527.9
[M+H].sup.+
BRITE-492794
4-Chloro-5-(3-(4-methylpiperazin-1-yl)phenoxy)-N-(naphthalen-1-yl
methyl)thiophene-2-sulfonamide
[0131] The product was prepared in 89%% yield; White solid, mp:
156-158.degree. C.; .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta.
(ppm): 2.21 (s, 3H), 2.43 (t, 4H, J=5.0 Hz), 3.17 (t, 4H, J=5.0
Hz), 4.55 (d, 2H, J=4.5 Hz), 6.44 (dd, 1H, J=2.0, 8.0 Hz), 6.78 (t,
1H, J=2.0 Hz), 6.84 (dd, 1H, J=2.0, 8.0 Hz), 7.27 (t, 1H, J=8.0
Hz), 7.43-7.48 (m, 3H), 7.53-7.58 (m, 2H), 7.88 (dd, 1H, J=1.5, 7.5
Hz), 7.93-7.97 (m, 1H), 8.05-8.09 (m, 1H), 8.52 (t, 1H, J=4.5 Hz,
NH). APCI/ESI MS Im/z 527.9 [M+H].sup.+
BRITE-492809
4-Chloro-N-(3-fluorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfo-
namide
[0132] The product was prepared in 75% yield; White solid, mp:
86-88.degree. C. (decomposed); .sup.1H NMR (500 MHz, DMSO-d.sub.6):
.delta. (ppm): 3.06 (t, 4H, J=5.0 Hz), 3.20 (t, 4H, J=5.0 Hz), 4.23
(s, 2H), 6.54 (dd, 1H, J=2.5, 8.0 Hz), 6.67 (t, 1H, J=2.5 Hz), 6.77
(dd, 1H, J=2.5, 8.0 Hz), 6.95-7.02 (m, 2H), 7.04 (d, 1H, J=7.0 Hz),
7.23-7.32 (m, 2H), 7.33 (s, 1H).
[0133] APCI/ESI MS m/z 481.9 [M+H].sup.+
BRITE-354873
4-Chloro-5-(3-(piperazin-1-yl)phenoxy)-N-(3-(trifluoromethyl)benzyl)thioph-
ene-2-sulfonamide
[0134] The product was prepared in 71% yield; White solid, mp:
58-60.degree. C. (decomposed): .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta. (ppm): 3.03 (t, 4H, J=5.0 Hz), 3.17 (t, 4H, J=5.0 Hz), 4.29
(s, 2H), 6.53 (dd, 1H, J=2.5, 8.0 Hz), 6.66 (t, 1H, J=2.5 Hz), 6.76
(dd, 1H, J=2.5, 8.0 Hz), 7.22-7.25 (m, 1H), 7.31 (s, 1H), 7.43-7.50
(m, 3H), 7.56 (d, 1H, J=7.0 Hz).
[0135] APCI/ESI MS m/z 531.9 [M+H].sup.+
BRITE-492808
4-Chloro-N-(3-methylbenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfo-
namide
[0136] The product was prepared in 71% yield; White solid, mp:
122-124.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 2.33 (s, 3H), 3.03 (t, 4H, J=5.0 Hz), 3.17 (t, 4H, J=5.0
Hz), 4.20 (s, 2H), 6.52 (dd, 1H, J=2.5, 8.0 Hz), 6.66 (t, 1H, J=2.5
Hz), 6.76 (dd, 1H, J=2.5, 8.0 Hz), 7.02 (d, 1H, J=8.0 Hz), 7.04 (s,
1H), 7.11 (d, 1H, J=7.5 Hz), 7.20 (d, 1H, J=8.0 Hz), 7.23 (d, 1H,
J=8.0 Hz), 7.32 (s, 1H). APCI ESI-MS m/z 477.9 [M+H].sup.+
BRITE-492807
4-Chloro-N-(3,5-dichlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-s-
ulfonamide
[0137] The product was prepared in 89% yield; Yellowish syrup:
.sup.1H NMR (500 MHz, CDCl.sub.3): .delta. (ppm): 3.06 (t, 4H,
J=5.0 Hz), 3.20 (t, 4H, J=5.0 Hz), 4.19 (s, 2H), 6.56 (dd, 1H,
J=1.5, 8.0 Hz), 6.68 (s, 1H), 6.77 (dd, 1H, J=1.5, 8.0 Hz), 7.14
(d, 2H, J=0.5 Hz), 7.24-7.29 (m, 2H), 7.31 (s, 1H). APCI/ESI MS m/z
531.8 [M+H]
BRITE-354909
4-Chloro-N-(4-methoxyphenyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulf-
onamide
[0138] The product was prepared in 89% yield; White solid, mp:
118-120.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3+CD.sub.3OD):
.delta. (ppm): 3.02 (t, 4H, J=5.0 Hz), 3.16 (t, 4H, J=5.0 Hz), 3.79
(s, 3H), 6.48 (dd, 1H, J=1.5, 8.0 Hz), 6.60 (t, 1H, J=1.5 Hz), 6.74
(dd, 1H, J=2.0, 8.5 Hz), 6.81-6.85 (m, 2H), 7.05-7.09 (m, 2H), 7.18
(s, 1H), 7.22 (t, 1H, J=8.0 Hz). APCI/ESI MS m/z 479.9 [M+H]
BRITE-492806
4-Chloro-N-(naphthalen-1-ylmethyl)-5-(4-(piperazin-1-yl)phenoxy)thiophene--
2-sulfonamide
[0139] The product was prepared in 97% yield; Light orange solid,
mp: 156-158.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 3.04-3.07 (m, 4H), 3.13-3.16 (m, 4H), 4.65 (s, 2H),
6.89-6.92 (m, 2H), 7.02-7.05 (m, 2H), 7.32 (s, 1H), 7.37-7.42 (m,
2H), 7.52-7.55 (m, 2H), 7.83 (dd, 1H, J=2.0, 7.0 Hz), 7.86-7.89 (m,
1H), 7.92-7.94 (m, 1H).
[0140] APCI/ESI MS m/z 514.0 [M+H].sup.+
BRITE-492805
4-Chloro-N-(2-chlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfo-
namide
[0141] The product was prepared in 83% yield: Light orange solid,
mp: 99-101.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 3.02-3.04 (m, 4H), 3.15-3.18 (m, 4H), 4.35 (s, 2H), 6.49
(dd, 1H, J=2.0, 8.0 Hz), 6.64 (t, 1H, J=2.0 Hz), 6.75 (dd, 1H,
J=2.5, 8.5 Hz), 7.20 (s, 1H), 7.22 (d, 1H, J=0.5 Hz), 7.24 (s, 1H),
7.30 (s, 1H), 7.32-7.35 (m, 2H).
[0142] APCI/ESI MS m/z 497.9 [M+H]
BRITE-355123
4-Chloro-N-(3-chlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfo-
namide
[0143] The product was prepared in 85% yield; White solid, mp:
123-125.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 3.01-3.04 (m, 4H), 3.15-3.18 (m, 4H), 4.21 (s, 2H), 6.54
(dd, 1H, J=2.5, 8.0 Hz), 6.67 (t, 1H, J=2.5 Hz), 6.77 (dd, 1H,
J=2.5, 8.0 Hz), 7.13-7.16 (m, 1H), 7.21-7.30 (m, 4H), 7.32 (s, 1H).
APCI/ESI MS m/z 497.9 [M+H].sup.+
BRITE-492802
4-Chloro-N-(4-chlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfo-
namide
[0144] The product was prepared in 78% yield; Light orange solid,
mp: 118-120.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 3.01-3.04 (m, 4H), 3.15-3.18 (m, 4H), 4.20 (s, 2H), 6.53
(ddd, 1H, J=0.5, 2.0, 8.0 Hz), 6.67 (t, 1H, J=2.0 Hz), 6.77 (dd,
1H, J=2.0, 8.0 Hz), 7.18-7.21 (m, 2H), 7.22 (s, 1H), 7.29-7.32 (m,
2H), 7.34 (s, 1H).
[0145] APCI/ESI MS m/z 497.9 [M+H]y
BRITE-492803
4-Chloro-N-(4-methoxybenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulf-
onamide
[0146] The product was prepared in 48% yield; White solid, mp:
106-108.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 2.97 (t, 4H, J=5.0 Hz), 3.12 (t, 4H, J=5.0 Hz), 3.77 (s,
3H), 4.14 (s, 2H), 6.51 (dd, 1H, J=2.0, 8.0 Hz), 6.64 (t, 1H, J=2.0
Hz), 6.73 (dd, 1H, J=2.0, 8.0 Hz), 6.82 (d, 2H, J=8.5 Hz), 7.14 (d,
2H, J=8.5 Hz), 7.23 (t, 1H, J=8.5 Hz), 7.28 (s, 1H). APCI/ESI MS mz
494.0 [M+H].sup.+
BRITE-355227
4-Chloro-N-(3-methoxybenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulf-
onamide
[0147] The product was prepared in 31% yield; White solid, mp:
60-62.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. (ppm):
2.97 (t, 4H, J=5.0 Hz), 3.12 (t, 4H, J=5.0 Hz), 3.76 (s, 3H), 4.18
(s, 2H), 6.52 (dd, 1H, J=2.0, 8.0 Hz), 6.64 (t, 1H, J=2.0 Hz), 6.73
(dd, 1H, J=2.0, 8.5 Hz), 6.76 (s, 1H), 6.78-6.83 (m, 2H), 7.22
(ABq, 2H, J=8.5 Hz), 7.28 (s, 1H). APCI/ESI MS m/z 494.1
[M+H].sup.+
BRITE-492800
4-Chloro-N-(3-chlorophenyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfo-
namide
[0148] The product was prepared in 78% yield, White solid, mp:
154-156.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 3.17 (t, 4H, J=5.0 Hz), 3.28 (t, 4H, J=5.0 Hz), 6.49 (dd,
1H, J=2.0, 8.0 Hz), 6.74 (s, 1H), 6.78 (d, 1H, J=7.5 Hz), 6.83 (dd,
1H, J=2.0, 8.0 Hz), 6.86 (dd, 1H, J=1.0, 7.5 Hz), 6.96 (s, 1H),
7.10 (t, 1H, J=8.0 Hz), 7.23 (s, 1H), 7.26 (t, 1H, J=8.0 Hz), 8.34
(br s, 1H, NH). APCI/ESIMS mz 484.0 [M+H].sup.+
BRITE-492799
4-Chloro-N-(3-methoxyphenyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulf-
onamide
[0149] The product was prepared in 78% yield; White solid, mp:
180-182.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 3.03 (t, 4H, J=5.0 Hz), 3.14 (t, 4H, J=5.0 Hz), 3.78 (s,
3H), 6.47 (dd, 1H, J=2.0, 8.0 Hz), 6.59 (t, 1H, J=2.0 Hz),
6.65-6.68 (m, 1H), 6.71-6.76 (m, 3H), 7.18-7.23 (m, 2H), 7.27 (s,
1H). APCI/ESI MS m/z 480.0 [M+H].sup.+
BRITE-492798
4-Chloro-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide
[0150] To a solution of tert-butyl
4-(3-(5-(N,N-bis(4-methoxybenzyl)
sulfamoyl)-3-chlorothiophen-2-yloxy)phenyl)piperazine-1-carboxylate
(0.430 g, 0.602 mmol) in anhydrous CH.sub.2Cl.sub.2 (0.5 mL) was
added TFA (4.5 mL) and stirred at room temperature for 4 h. The
solvent mixture was removed under vacuo and the residue was
re-dissolved in CH.sub.2Cl.sub.2 (30 mL) washed with aqueous sat.
NaHCO.sub.3 followed by brine, dried (Na.sub.2SO.sub.4), and
concentrated under vacuo. The residue was purified by
Combiflash.RTM. Rf (Isco) using MeOH--CH.sub.2Cl.sub.2 (1:5) to
give a white solid (0.180 g, 80%). .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta. (ppm): 3.02-3.05 (m, 4H), 3.19-3.22 (m, 4H),
6.56 (dd, 1H, J=2.0, 8.0 Hz), 6.73 (t, 1H, J=2.0 Hz), 6.84 (dd, 1H,
J=2.0, 8.5 Hz), 7.27 (t, 1H, J=8.5 Hz), 7.40 (s, 1H).
[0151] APCI/ESI MS m/z 374.0 [M+H].sup.+
BRITE-492797
N-(Naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfona-
mide
[0152] The product was prepared in 87% yield, Light orange solid,
mp: 65-67.degree. C.: .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
(ppm): 2.92 (t, 4H, I=5.0 Hz), 3.08 (t, 4H, J=5.0 Hz), 4.62 (s,
2H), 6.43 (d, 1H, J=4.5 Hz), 6.56-6.60 (m, 1H), 6.65 (t, 1H, J=2.5
Hz), 6.71 (dd, 1H, J=2.0, 8.5 Hz), 7.23 (t, 1H, J=8.5 Hz), 7.37 (d,
2H, J=4.5 Hz), 7.39 (d, 1H, J=4.0 Hz), 7.48-7.54 (m, 2H), 7.79 (t,
1H, J=4.5 Hz), 7.82-7.86 (m, 1H), 7.95 (dd, 1H, J=1.0, 7.5 Hz).
[0153] APCI/ESI MS m/z 480.1 [M+H].sup.+
Example 3
Materials And Methods
[0154] All common reagents such as HEPES, Triton X-100,
carbenicillin, and dimethyl sulfoxide (DMSO) were reagent-grade
quality and obtained from Thermo Fisher Scientific (Waltham, Mass.)
or Sigma-Aldrich (St. Louis, Mo.). 4-methylumbelliferyl glucuronide
(4MUG) was obtained from Sigma-Aldrich (St. Louis, Mo.). The solid
black 96-well plates (cat#3915) for the assay and 96 well clear
plates (cat#9017) for cytotoxicity assay were from Corning
Incorporated (Corning, N.Y.). Falcon polypropylene plates
(cat#1190) used for serial dilution of compounds were obtained from
Becton Dickinson (Franklin Lake, N.J.). Amoxapine, nialamide,
isocarboxazid and other compounds for follow-up studies were
obtained from Sigma-Aldrich. The Prestwick Chemical Collection was
obtained from Prestwick Chemical Company (Washington D.C.). E. coli
DH5a (Zymo Research, Irvine, Calif.) was used for the cell-based
assay. The expression and purification of GUS enzyme from E. coli
carrying an expression plasmid containing the full-length E. coli
GUS gene has been previously described (26). Bovine taurus GUS
enzyme was purchased from Sigma-Aldrich. In addition, some
experiments were performed with purified E. coli
.beta.-glucuronidase enzyme purchased from Sigma-Aldrich.
[0155] GUS Enzyme Assay--Manual Version
[0156] The semi-automated GUS high throughput enzyme assay was
performed as previously described [25] and was used to screen the
Prestwick Chemical Collection. The follow-up studies were performed
manually in a similar manner with the exception of plate type and
volumes, as briefly outlined here. Compound stock solutions were
made in 100% DMSO. Serial dilutions of compounds for IC.sub.50
determinations were initially performed in 100% DMSO in 96 well
polypropylene plates, then each compound concentration diluted into
assay buffer (50 mM HEPES, pH 7.4 and 0.017% Triton X-100),
producing a constant 50%/DMSO in all wells. Subsequently, 20.mu.l
of this aqueous diluted compound (or just 5% DMSO for controls) was
added to the wells of a solid black 96-well plate followed by 40
.mu.l of GUS enzyme (83 .mu.M GUS) diluted in assay buffer. After
addition of enzyme, the reaction was initiated by addition of 40
.mu.l of 4MUG substrate (312.5 .mu.M 4MUG) diluted in 50 mM HEPES,
pH 7.4. 4MUG stock solutions were prepared in the same buffer.
Final concentrations in the assembled assay were 50 mM HEPES, pH
7.4, 0.01% Triton X-100, 1% DMSO, 125 .mu.M 4MUG and 33 .mu.M GUS.
The enzyme reaction was allowed to proceed for 20 minutes at
23.degree. C. and was terminated by the addition of 40 .mu.l of a
1M sodium carbonate solution. Fluorescence at 460 nm was determined
using 355 nm excitation wavelength with a 0.1 s/well read time in a
BMG PheraStar (BMG LABTECH, Cary, N.C.). Fluorescence data,
expressed in relative fluorescence units (RFU), were normalized to
DMSO (100% activity) and "no enzyme" (0% activity) controls as
maximum and minimum responses, respectively. The Bovine taurus GUS
enzyme assay was performed in an identical manner except Bovine
taurus GUS enzyme (1 nM) was used instead of bacterial GUS. The
IC.sub.50 values and Hill slopes were calculated from concentration
response data using GraphPad Prism software (GraphPad Software
Inc., La Jolla, Calif.) employing either four-parameter or a three
parameter (fixed bottom) curve fit.
[0157] GUS Cell Based Assay
[0158] Cultures of E. coli (DH5a) carrying the empty expression
vector pCMV5 were grown over night in LB containing carbenicillin
(50 .mu.M) and then used to initiate fresh LB/carbenicillin
cultures adjusted to an initial OD of 0.1. These cultures were
allowed to reach an OD of 0.6 and then washed twice with 50 mM
HEPES, pH 7.4 containing carbenicillin 50 .mu.M and concentrated by
centrifugation to an OD of 1 for use in the assay. The GUS cell
based assay was performed in an identical manner as the enzyme
assay except the Triton X-100 was left out of the assay buffer, the
E. coli cells replaced the enzyme and the reaction was allowed to
proceed for 2 hrs at 37.degree. C. The resulting data was analyzed
as outlined for the enzyme assay.
[0159] Toxicity Assay
[0160] Compounds were tested for cytotoxicity in E. coli cells. The
cells were grown and prepared for assay as described above, and
plated in clear 96-well plates. Cells were treated with 100 .mu.M
and 10 .mu.M concentrations (1% DMSO) of test compounds and
incubated for 2 hours at 37.degree. C. Subsequently, 25 uL of MTS
viability reagent (CellTiter 96 Aqueous Non-Radioactive Cell
Proliferation Assay Kit, Promega Corp., Madison, Wis.) was added to
the wells and incubation continued for 5 minutes. The plates were
then analyzed for absorbance at 490 nm on a SpectraMax Plus 384
(Molecular Devices, Sunnyvale, Calif.). Controls included DMSO only
(considered 100% viability), "no cells" (representing 0% viable
cells), and the cytotoxic positive control compound kanamycin at 50
.mu.g/ml.
REFERENCES
[0161] Pommier, Y. (2006) Topoisomerase I inhibitors: camptothecins
and beyond, Nat Rev Cancer 6, 789-802 [0162] Pizzolato, J. F., and
Saltz, L. B. (2003) The camptothecins, Lancet 361, 2235-2242.
[0163] Smith, N. F., Figg, W. D., and Sparreboom, A. (2006)
Pharmacogenetics of irinotecan metabolism and transport: an update,
Toxicol In Vitro 20, 163-175. [0164] Ma, M. K., and McLeod, H. L.
(2003) Lessons learned from the irinotecan metabolic pathway, Curr
Med Chem 10, 41-49. [0165] Mathijssen, R. H. J., van Alphen, R. J.,
Verweij, J., Loos, W. J., Nooter, K., Stoter, G., and Sparreboom,
A. (2001) Clinical Pharmacokinetics and Metabolism of Irinotecan
(CPT-11), Clin Cancer Res 7, 2182-2194. [0166] Miley, M. J.,
Zielinska, A. K., Keenan, J. E., Bratton, S. M., Radominska-Pandya,
A., and Redinbo, M. R. (2007) Crystal structure of the
cofactor-binding domain of the human phase II drug-metabolism
enzyme UDP-glucuronosyltransferase 2B7, J Mol Biol 369, 498-511.
[0167] Nagar, S., and Blanchard, R. L. (2006) Pharmacogenetics of
uridine diphosphoglucuronosyltransferase (UGT) IA family members
and its role in patient response to irinotecan, Drug Metab Rev 38,
393-409. [0168] Stein, A., Voigt, W., and Jordan, K. Review:
Chemotherapy-induced diarrhea: pathophysiology, frequency and
guideline-based management, Therapeutic Advances in Medical
Oncology, pp. 51-63 vol. 2. [0169] Tobin, P. J., Dodds, H. M.,
Clarke, S., Schnitzler, M., and Rivory, L. P. (2003) The relative
contributions of carboxylesterase and beta-glucuronidase in the
formation of SN-38 in human colorectal tumours. Oncology reports
10, 1977-1979. [0170] Hu, Z. P., Yang, X. X., Chan, S. Y., Xu, A.
L., Duan, W., Zhu, Y. Z., Sheu, F. S., Boelsterli, U. A., Chan, E.,
Zhang, Q., Wang, J. C., Ee, P. L., Koh, H. L., Huang, M., and Zhou,
S. F. (2006) St. John's wort attenuates irinotecan-induced diarrhea
via down-regulation of intestinal pro-inflammatory cytokines and
inhibition of intestinal epithelial apoptosis, Toxicology and
applied pharmacology 216, 225-237. [0171] Kurita, A., Kado, S.,
Matsumoto, T., Asakawa, N., Kaneda, N., Kato, I., Uchida, K.,
Onoue, M., and Yokokura, T. Streptomycin alleviates
irinotecan-induced delayed-onset diarrhea in rats by a mechanism
other than inhibition of beta-glucuronidase activity in intestinal
lumen, Cancer chemotherapy and pharmacology. [0172] Basinska, A.,
and Florianczyk, B. (2003) Beta-glucuronidase in physiology and
disease, Annales Universitatis Mariae Curie-Sklodowska 58, 386-389.
[0173] Farnleitner, A. H., Hocke, L., Beiwl, C., Kavka, G. C., and
Mach, R. L. (2002) Hydrolysis of
4-methylumbelliferyl-beta-D-glucuronide in differing sample
fractions of river waters and its implication for the detection of
fecal pollution., Water Res. 36, 975-981. [0174] Wallace, B. D.,
Wang, H., Lane, K. T., Scott, J. E., Orans, J., Koo, J. S.,
Venkatesh, M., Jobin, C., Yeh, L. A., Mani, S., and Redinbo, M. R.
(2010) Alleviating cancer drug toxicity by inhibiting a bacterial
enzyme, Science 330, 831-835. [0175] Ahmad, S., Hughes, M. A.,
Lane, K. T., Redinbo, M. R., Yeh, L. A., and Scott, J. E. (2011) A
High Throughput Assay for Discovery of Bacterial beta-Glucuronidase
Inhibitors, Curr Chem Genomics 5, 13-20. [0176] McGovern, S. L.,
Caselli, E., Grigorieff, N., and Shoichet, B. K. (2002) A common
mechanism underlying promiscuous inhibitors from virtual and
high-throughput screening, J Med Chem 45, 1712-1722. [0177] Feng,
B. Y., Simeonov, A., Jadhav, A., Babaoglu, K., Inglese, J.,
Shoichet, B. K., and Austin, C. P. (2007) A high-throughput screen
for aggregation-based inhibition in a large compound library, J Med
Chem 50, 2385-2390. [0178] Tobin, P., Clarke, S., Seale, J. P.,
Lee, S., Solomon, M., Aulds, S., Crawford, M., Gallagher, J.,
Eyers, T., and Rivory, L. (2006) The in vitro metabolism of
irinotecan (CPT-11) by carboxylesterase and beta-glucuronidase in
human colorectal tumours, Br J Clin Pharmacol 62, 122-129. [0179]
Prijovich, Z. M., Chen, K. C., and Roffler, S. R. (2009) Local
enzymatic hydrolysis of an endogenously generated metabolite can
enhance CPT-11 anticancer efficacy, Mol Cancer Ther 8, 940-946.
[0180] Huang, P. T., Chen, K. C., Prijovich, Z. M., Cheng, T. L.,
Leu, Y. L., and Roffler, S. R. (2011) Enhancement of CPT-11
antitumor activity by adenovirus-mediated expression of
beta-glucuronidase in tumors, Cancer Gene Ther 18, 381-389. [0181]
Ulus, I. H., Maher, T. J., and Wurtman, R. J. (2000)
Characterization of phentermine and related compounds as monoamine
oxidase (MAO) inhibitors, Biochem Pharmacol 59, 1611-1621. [0182]
Maxwell, D. R., Gray, W. R., and Taylor, E. M. (1961) Relative
activity of some inhibitors of mono-amine oxidase in potentiating
the action of tryptamine in vitro and in vivo, Br J Pharmacol
Chemother 17, 310-320. [0183] Lexchin, J. (2005) Drug withdrawals
from the Canadian market for safety reasons, 1963-2004, CMAJ 172,
765-767. [0184] Wimbiscus, M., Kostenko, O., and Malone, D. (2010)
MAO inhibitors: risks, benefits, and lore, Cleve Clin J Med 77,
859-882. [0185] Fisch, M. (2004) Treatment of depression in cancer,
JNatl Cancer Inst Monogr, 105-111. [0186] Palmeira, A., Rodrigues,
F., Sousa, E., Pinto, M., Vasconcelos, M. H., and Fernandes, M. X.
(2011) New uses for old drugs: pharmacophore-based screening for
the discovery of P-glycoprotein inhibitors, Chem Biol Drug Des 78,
57-72. [0187] Leonard, B. E. (1984) Pharmacology of new
antidepressants, Prog Neuropsychopharmacol Biol Psychiatry 8,
97-108. [0188] Badway, M. A., and Dugas, J. E. (1984) Loxapine
yields amoxapine, J Clin Psychopharmacol 4, 363-364. [0189] Cheung,
S. W., Tang, S. W., and Remington, G. (1991) Simultaneous
quantitation of loxapine, amoxapine and their 7- and 8-hydroxy
metabolites in plasma by high-performance liquid chromatography, J
Chromatogr 564, 213-221. [0190] Hsiang, Y. H., Hertzberg, R.,
Hecht, S. & Liu, L. F. Camptothecin induces protein-linked DNA
breaks via mammalian DNA topoisomerase I. J Biol Chem 260, 14873-8
(1985). [0191] Redinbo, M. R., Champoux, J. J. & Hol, W. G.
Structural insights into the function of type IB topoisomerases.
Curr Opin Struct Biol 9, 29-36 (1999). [0192] Redinbo, M. R.,
Stewart, L., Kuhn, P., Champoux, J. J. & Hol, W. G. Crystal
structures of human topoisomerase I in covalent and noncovalent
complexes with DNA. Science 279, 1504-13 (1998). [0193] Stewart,
L., Redinbo, M. R., Qiu, X., Hol, W. G. & Champoux, J. J. A
model for the mechanism of human topoisomerase I. Science 279,
1534-41 (1998). [0194] Chrencik, J. E. et al. Mechanisms of
camptothecin resistance by human topoisomerase I mutations. J Mol
Biol 339, 773-84 (2004). [0195] Staker, B. L. et al. The mechanism
of topoisomerase I poisoning by a camptothecin analog. Proc Natl
Acad Sci USA 99, 15387-92 (2002). [0196] Fittkau, M., Voigt, W.,
Holzhausen, H. J. & Schmoll, H. J. Saccharic acid 1,4-lactone
protects against CPT-11-induced mucosa damage in rats. J Cancer Res
Clin Oncol 130, 388-94 (2004). [0197] Flieger, D. et al. Phase II
clinical trial for prevention of delayed diarrhea with
cholestyramine/levofloxacin in the second-line treatment with
irinotecan biweekly in patients with metastatic colorectal
carcinoma. Oncology 72, 10-6 (2007). [0198] Cummings, J. H. &
Macfarlane, G. T. Role of intestinal bacteria in nutrient
metabolism. JPEN J Parenter Enteral Nutr 21, 357-65 (1997). [0199]
Guarner, F. & Malagelada, J. R. Gut flora in health and
disease. Lancet 361, 512-519 (2003). [0200] Job, M. L. &
Jacobs, N. F., Jr. Drug-induced Clostridium difficile-associated
disease. Drug Saf 17, 37-46 (1997). [0201] Levy, S. B. &
Marshall, B. Antibacterial resistance worldwide: causes, challenges
and responses. Nat Med 10, S 122-9 (2004). [0202] Nord, C. E.,
Kager, L. & Heimdahl, A. Impact of antimicrobial agents on the
gastrointestinal microflora and the risk of infections. Am J Med
76, 99-106 (1984). [0203] Settle, C. D. & Wilcox, M. H. Review
article: antibiotic-induced Clostridium difficile infection.
Aliment Pharmacol Ther 10, 835-41 (1996). [0204] Sears, S.,
McNally, P., Bachinski, M. S. & Avery, R. Irinotecan (CPT-11)
induced colitis: report of a case and review of Food and Drug
Administration MEDWATCH reporting. Gastrointest Endosc 50, 841-4
(1999). [0205] Stamp, D. Antibiotic therapy may induce cancers in
the colon and breasts through a mechanism involving bile acids and
colonic bacteria. Med Hypotheses 63, 555-6 (2004). [0206] Yang, L.
& Pei, Z. Bacteria, inflammation, and colon cancer. World J
Gastroenterol 12, 6741-6 (2006). [0207] Russell, W. M. &
Klaenhammer, T. R. Identification and cloning of gusA, encoding a
new beta-glucuronidase from Lactobacillus gasseri ADH. Appl Environ
Microbiol 67, 1253-61 (2001).
TABLE-US-00001 [0207] TABLE 1 .beta.-Glucuronidase Inhibitory
activity of phenoxythiophene sulfonamides Compound ID Structure
IC.sub.50 (.mu.m) BRITE-354972 ##STR00004## 0.000 BRITE-355123
##STR00005## 0.090 BRITE-355252 ##STR00006## 0.090 BRITE-354975
##STR00007## 0.120 BRITE-354989 ##STR00008## 0.150 BRITE-354909
##STR00009## 0.170 BRITE-354725 ##STR00010## 0.190 BRITE-354969
##STR00011## 0.200 BRITE-355417 ##STR00012## 0.210 BRITE-355006
##STR00013## 0.210 BRITE-355017 ##STR00014## 0.220 BRITE-354979
##STR00015## 0.230 BRITE-355004 ##STR00016## 0.240 BRITE-354667
##STR00017## 0.270 BRITE-354966 ##STR00018## 0.270 BRITE-355262
##STR00019## 0.280 BRITE-354965 ##STR00020## 0.290 BRITE-354873
##STR00021## 0.290 BRITE-354615 ##STR00022## 0.310 BRITE-355016
##STR00023## 0.320 BRITE-354517 ##STR00024## 0.320 BRITE-354958
##STR00025## 0.330 BRITE-355360 ##STR00026## 0.340 BRITE-355227
##STR00027## 0.350 BRITE-354948 ##STR00028## 0.390 BRITE-355336
##STR00029## 0.410 BRITE-355423 ##STR00030## 0.430 BRITE-355468
##STR00031## 0.500 BRITE-355202 ##STR00032## 0.510 BRITE-355003
##STR00033## 0.510 BRITE-354946 ##STR00034## 0.560 BRITE-354983
##STR00035## 0.570 BRITE-355014 ##STR00036## 0.600 BRITE-355015
##STR00037## 0.630 BRITE-354627 ##STR00038## 0.710 BRITE-354764
##STR00039## 0.730 BRITE-354993 ##STR00040## 0.730 BRITE-354956
##STR00041## 0.750 BRITE-354984 ##STR00042## 0.750 BRITE-354974
##STR00043## 0.760 BRITE-354947 ##STR00044## 0.770 BRITE-354392
##STR00045## 0.790 BRITE-355045 ##STR00046## 0.880 BRITE-354994
##STR00047## 0.900 BRITE-354957 ##STR00048## 0.920 BRITE-355074
##STR00049## 0.970 BRITE-354565 ##STR00050## 1.000 BRITE-354998
##STR00051## 1.000 BRITE-354955 ##STR00052## 1.310 BRITE-355296
##STR00053## 1.370 BRITE-355008 ##STR00054## 1.390 BRITE-355005
##STR00055## 6.700 BRITE-355192 ##STR00056## 16.140 BRITE-354839
##STR00057## 20.030 BRITE-354428 ##STR00058## 20.560 BRITE-355224
##STR00059## 22.910 BRITE-355018 ##STR00060## 23.330 BRITE-355240
##STR00061## 28.570 BRITE-355329 ##STR00062## 31.510 BRITE-355250
##STR00063## 50.060 BRITE-355339 ##STR00064## 76.560 BRITE-355319
##STR00065## 84.560 BRITE-355243 ##STR00066## 92.780 BRITE-355180
##STR00067## 101.980 BRITE-355244 ##STR00068## 123.470 BRITE-355214
##STR00069## 134.850 BRITE-355030 ##STR00070## 149.400 BRITE-355234
##STR00071## 151.550 BRITE-355169 ##STR00072## 152.600 BRITE-354458
##STR00073## 175.430 BRITE-335211 ##STR00074## 192.490 BRITE-355201
##STR00075## 201.630 BRITE-355233 ##STR00076## 375.580 BRITE-355221
##STR00077## 401.950 BRITE-355253 ##STR00078## 443.900 BRITE-354502
##STR00079## 965.440
TABLE-US-00002 TABLE 2 Structure and .beta.-Glucuronidase
Inhibitory Activity of BRITE-355252 analogs Compound ID Structure
IC50 (.mu.M) BRITE-354873 ##STR00080## 0.030 BRITE-354909
##STR00081## 0.060 BRITE-355123 ##STR00082## 0.020 BRITE-355227
##STR00083## 0.050 BRITE-355252 ##STR00084## 0.020 BRITE-492794
##STR00085## 10.170 BRITE-492796 ##STR00086## 0.120 BRITE-492797
##STR00087## 0.090 BRITE-492798 ##STR00088## 0.330 BRITE-492799
##STR00089## 0.070 BRITE-492800 ##STR00090## 0.070 BRITE-492802
##STR00091## 0.030 BRITE-492803 ##STR00092## 0.100 BRITE-492805
##STR00093## 0.040 BRITE-492806 ##STR00094## 0.300 BRITE-492807
##STR00095## 0.130 BRITE-492808 ##STR00096## 0.030 BRITE-492809
##STR00097## 0.040
TABLE-US-00003 TABLE 3 Summary of GUS Inhibitory Activity of
Studied Drugs E. coli GUS B. taurus GUS E. coli Currently Enzyme
Assaya Enzyme Assay.sup.a Cell-Based Assay.sup.a Drug Drug Class
Marketed IC.sub.50 .+-. SD (nM) IC.sub.50 .+-. SD (nM) IC.sub.50
.+-. SD (nM) Nialamide Irreversible MAOI No 71 .+-. 32 74,813 .+-.
1,841 17 .+-. 2 Isocarboxazid Irreversible MAOI Yes 128 .+-. 56
>100,000 336 .+-. 120 Phenelzine Irreversible MAOI Yes 2,282
.+-. 1041 ND.sup.b 7,123 .+-. 1650 Amoxapine Tricyclic
Antidepressant Yes 388 .+-. 98 >100,000 119 .+-. 61
Loxapine.sup.c Tricyclic Antidepressant Yes >100,000 ND
>100,000 Mefloquine Antimalarial Yes 1,212 .+-. 234 ND 5,961
.+-. 1526 .sup.aIC.sub.50 value determinations were performed at
least three times, with average IC.sub.50 values and standard
deviations (SD) shown. The range of average Hill slopes for all
measurable IC.sub.50 curves (where at least 50% inhibition was
obtained) was 0.84 to 1.26. .sup.bND = not determined; .sup.cThis
drug was included as a study control
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