U.S. patent application number 10/925367 was filed with the patent office on 2005-03-10 for carboxylesterase inhibitors.
This patent application is currently assigned to St. Jude Children's Research Hospital. Invention is credited to Beroza, Paul P., Damoradan, Komath V., Hyatt, Janice L., Morton, Christopher L., Potter, Philip M..
Application Number | 20050054691 10/925367 |
Document ID | / |
Family ID | 34272725 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050054691 |
Kind Code |
A1 |
Potter, Philip M. ; et
al. |
March 10, 2005 |
Carboxylesterase inhibitors
Abstract
This disclosure relates to amides, aryl sulphonamides, aryl
ureas, and .alpha.,.beta.-diketones derivatives useful as
carboxylesterase esterase inhibitors. The disclosure is also
directed to the use of these compounds as selective human
intestinal carboxylesterase inhibitors and insect carboxylesterase
inhibitors. The disclosure is also directed to pharmaceutical
compositions and pesticide formulations containing these compounds,
and to methods for treating or ameliorating the toxic effects
following administration of drugs such as cancer therapy drugs,
treating or ameliorating the effects of a drug overdose, and to the
use of the compounds for increasing the effectiveness of
insecticides and pesticides.
Inventors: |
Potter, Philip M.; (Memphis,
TN) ; Hyatt, Janice L.; (Memphis, TN) ;
Morton, Christopher L.; (Memphis, TN) ; Beroza, Paul
P.; (Redwood City, CA) ; Damoradan, Komath V.;
(Cupertino, CA) |
Correspondence
Address: |
DARBY & DARBY
P.O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Assignee: |
St. Jude Children's Research
Hospital
Memphis
TN
|
Family ID: |
34272725 |
Appl. No.: |
10/925367 |
Filed: |
August 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60498778 |
Aug 29, 2003 |
|
|
|
Current U.S.
Class: |
514/357 ;
514/408; 514/617; 514/621 |
Current CPC
Class: |
A61K 31/165 20130101;
A61K 31/44 20130101; A61K 31/40 20130101 |
Class at
Publication: |
514/357 ;
514/408; 514/617; 514/621 |
International
Class: |
A61K 031/44; A61K
031/40; A61K 031/165 |
Goverment Interests
[0002] This invention was made with government support under core
grant P30-CA21765 awarded by the National Institutes of Health
Cancer Center. The U.S. government may have certain rights in this
invention.
Claims
What is claimed is:
1. A method of inhibiting an esterase in a patient in need thereof,
which method comprises administering an esterase inhibiting amount
of an esterase inhibitor selected from amides, aryl sulphonamides,
aryl ureas, .alpha.,.beta.-diketones, and mixtures thereof to the
patient.
2. The method of claim 1, wherein the amide is of Formula (I):
70wherein R.sup.1 to R.sup.12 are each independently selected from
hydrogen, C.sub.1-C.sub.6 linear alkyl, C.sub.1-C.sub.6 branched
alkyl, substituted alkyl, phenyl, substituted phenyl, hydroxy,
halogen, and C.sub.1-C.sub.6 alkoxy; and optionally R.sup.8 and
R.sup.9 are linked to form an optionally substituted aryl or
heteroaryl ring system.
3. The method of claim 1, wherein the amide is of Formula (II):
71wherein R.sup.11 to R.sup.12 are each independently selected from
hydrogen, C.sub.1-C.sub.6 linear alkyl, C.sub.1-C.sub.6 branched
alkyl, substituted alkyl, phenyl, substituted phenyl, alkylphenyl,
hydroxyl, halogen, and C.sub.1-C.sub.6 alkoxy; and Ar is optionally
substituted phenyl or naphthyl.
4. The method of claim 1, wherein the aryl sulphonamide is of
Formula (III): 72wherein R.sup.13, R.sup.14, R.sup.16 and R.sup.17
are each independently selected from hydrogen, halogen, and
C.sub.1-C.sub.6 linear or branched alkyl, or R.sup.16 and R.sup.17
are optionally linked to form an optionally substituted aryl or
heteroaryl ring system; R.sup.15 is selected from hydrogen,
C.sub.1-C.sub.6 alkoxy, optionally substituted phenoxy, and
NHSO.sub.2R.sup.19; wherein R.sup.19 is C.sub.1-C.sub.6 linear or
branched alkyl, phenyl, mono-, di- or tri-halosubstituted phenyl or
S.sub.2C.sub.6H.sub.5NHSO.sub.2CH.sub.3; and R.sup.18 is selected
from hydrogen, C.sub.1-C.sub.6 linear or branched alkyl, halogen,
phenyl, halosubstituted phenyl and [C.sub.6H.sub.2(CH.sub.3).sub-
.2]SO.sub.2NHC.sub.6H.sub.5.
5. The method of claim 1, wherein the aryl urea is of Formula (IV):
73wherein R.sup.20 to R.sup.29 are each independently selected from
hydrogen, halogen, C.sub.1C.sub.6 linear or branched alkyl and
NO.sub.2.
6. The method of claim 1, wherein the .alpha.,.beta.-diketone is of
Formula (V) 74wherein R.sup.31 and R.sup.32 are each independently
aryl or heteroaryl, wherein the aryl or heteroaryl is optionally
substituted with one or more hydrogen, halogen, hydroxy,
C.sub.1-C.sub.6 linear or branched alkyl, C.sub.1-C.sub.6 linear or
branched halo-alkyl, C.sub.1-C.sub.6 alkoxy, NR.sub.33R.sub.34,
COOH, or NO.sub.2; or R.sup.31 and R.sup.32 may optionally be
linked to form an optionally substituted polycyclic aryl or
heteroaryl ring system; and R33 and R are independently hydrogen or
C.sub.1-C.sub.6 linear or branched alkyl.
7. The method of claim 1, wherein the esterase is a
carboxylesterase.
8. The method of claim 7, wherein the carboxylesterase is human
intestinal carboxylesterase.
9. The method of claim 8, wherein the amide is selected from:
N-(5-chloro-2,4-dimethoxyphenyl)-3-hydroxy-2-naphthamide,
N-(4-chlorophenyl)-8-hydroxy-4aH-carbazole-7-carboxamide,
2-Benzoylamino-3-phenyl-propionic acid naphthalene-2-yl ester, and
2-naphthyl 2-(acetylamino)-4-methylpentanoate.
10. The method of claim 8, wherein the aryl sulfonamide is selected
from:
N-{2,3,5,6-tetrachloro-4-[(phenylsulfonyl)amino]phenyl}benzenesulfonamide-
,
4-chloro-N-(4{[(4-chlorophenyl)sulfonyl]amino}-2,3,4,6-tetrafluorophenyl-
) benzenesulfonamide,
4-chloro-N-(4-{[(4-chlorophenyl)sulfonyl]amino}pheny-
l)benzenesulfonamide,
4-bromo-N-(4-phenoxyphenyl)benzenesulfonamide,
4-chloro-N-(4-{[(4-chlorophenyl)sulfonyl]amino}-1-naphthyl)benzenesulfona-
mide, 4,6-dimethyl-N,N'-diphenylbenzene-1,3-disulfonamide,
N-{2-methyl-4-[(phenylsulfonyl)amino]phenyl}benzenesulfonamide,
N-[4-({4-[(methylsulfonyl)amino]phenyl}dithio)phenyl]methanesulfonamide,
N-{4-[(phenylsulfonyl)amino]phenyl}benzenesulfonamide, and
4-chloro-N-(4-ethoxyphenyl)benzenesulfonamide.
11. The method of claim 8, wherein the aryl urea is selected from:
N-(2-chloro-4-nitrophenyl)-N'-(4-chlorophenyl)urea,
N-(2,6-dimethylphenyl)-N'-(4-nitrophenyl)urea,
N-(3-fluorophenyl)-N'-(2-m- ethyl-4-nitrophenyl)urea, and
N-(2-methyl-4-nitrophenyl)-N'-phenylurea.
12. The method of claim 7, wherein the carboxylesterase is an
insect carboxylesterase.
13. A method of reducing the gastrointestinal toxicity following
administration of a drug to a patient in need thereof, which method
comprises administering an esterase inhibiting amount of an
esterase inhibitor selected from amides, aryl sulphonamides, aryl
ureas, .alpha.,.beta.-diketones, and mixtures thereof to the
patient.
14. The method of claim 13, wherein the amide is of the Formula
(I): 75wherein R.sup.1 to R.sup.12 are each independently selected
from hydrogen, C.sub.1-C.sub.6 linear alkyl, C.sub.1-C.sub.6
branched alkyl, substituted alkyl, phenyl, substituted phenyl,
hydroxyl, halogen, and C.sub.1-C.sub.6 alkoxy; and optionally
R.sup.8 and R.sup.9 are linked to form an optionally substituted
aryl or heteroaryl ring system.
15. The method of claim 13, wherein the amide is of the Formula
(II): 76wherein R.sup.11 to R.sup.12 are each independently
selected from hydrogen, C.sub.1-C.sub.6 linear alkyl,
C.sub.1-C.sub.6 branched alkyl, substituted alkyl, phenyl,
substituted phenyl, alkylphenyl, hydroxyl, halogen, and
C.sub.1-C.sub.6 alkoxy; and Ar is optionally substituted phenyl or
naphthyl.
16. The method of claim 13 wherein the aryl sulphonamide is of the
Formula (III): 77wherein R.sup.13, R.sup.14, R.sup.16 and R.sup.17
are each independently selected from hydrogen, halogen, and
C.sub.1-C.sub.6 linear or branched alkyl, or R.sup.16 and R.sup.17
are linked to form an optionally substituted aryl or heteroaryl
ring system; R.sup.15 is selected from hydrogen, C.sub.1-C.sub.6
alkoxy, optionally substituted phenoxy, and NHSO.sub.2R.sup.19;
wherein R.sup.19 is C.sub.1-C.sub.6 linear or branched alkyl,
phenyl, mono-, di- or tri-halosubstituted phenyl or
S.sub.2C.sub.6H.sub.5NHSO.sub.2CH.sub.3; and R.sup.18 is selected
from hydrogen, C.sub.1-C.sub.6 linear or branched alkyl, halogen,
phenyl, halosubstituted phenyl and [C.sub.6H.sub.2(CH.sub.3).sub-
.2]SO.sub.2NHC.sub.6H.sub.5.
17. The method of claim 13, wherein the aryl urea is of the Formula
(IV): 78wherein R.sup.20 to R.sup.29 are each independently
selected from hydrogen, halogen, C.sub.1-C.sub.6 linear or branched
alkyl and NO.sub.2.
18. The method of claim 13, wherein the .alpha.,.beta.-diketone is
of Formula (V): 79wherein R.sup.31 and R.sup.32 are each
independently aryl or heteroaryl, wherein the aryl or heteroaryl is
optionally substituted with one or more hydrogen, halogen, hydroxy,
C.sub.1-C.sub.6 linear or branched alkyl, C.sub.1-C.sub.6 linear or
branched halo-alkyl, C.sub.1-C.sub.6 alkoxy, NR.sub.33R.sub.34,
COOH, or NO.sub.2; or R.sup.31 and R.sup.32 may optionally be
linked to form an optionally substituted polycyclic aryl or
heteroaryl ring system; and R33 and R34 are independently hydrogen
or C.sub.1-C.sub.6 linear or branched alkyl.
19. The method of claim 13, wherein the gastrointestinal toxicity
is delayed diarrhea.
20. The method of claim 13, wherein the drug is an anti-cancer
drug.
21. The method of claim 20, wherein the anti-cancer drug is
CPT-11.
22. The method of claim 13, where the drug and the esterase
inhibitor are administered simultaneously or sequentially.
23. A method of treating or ameliorating the effects of an overdose
of a drug metabolized by a carboxylesterase in a patient in need
thereof, which method comprises administering an esterase
inhibiting amount of an esterase inhibitor selected from amides,
aryl sulphonamides, aryl ureas, .alpha.,.beta.-diketones, and
mixtures thereof to the patient.
24. The method of claim 23 wherein the amide is of Formula (I):
80wherein R.sup.1 to R.sup.12 are each independently selected from
hydrogen, C.sub.1-C.sub.6 linear alkyl, C.sub.1-C.sub.6 branched
alkyl, substituted alkyl, phenyl, substituted phenyl, hydroxyl,
halogen, and C.sub.1-C.sub.6 alkoxy; and optionally R.sup.8 and
R.sup.9 are linked to form an optionally substituted aryl or
heteroaryl ring system.
25. The method of claim 23, wherein the amide is of Formula (II):
81wherein R.sup.11 to R.sup.12 are each independently selected from
hydrogen, C.sub.1-C.sub.6 linear alkyl, C.sub.1-C.sub.6 branched
alkyl, substituted alkyl, phenyl, substituted phenyl, alkylphenyl,
hydroxyl, halogen, and C.sub.1-C.sub.6 alkoxy; and Ar is optionally
substituted phenyl or naphthyl.
26. The method of claim 23, wherein the aryl sulphonamide is of
Formula (III): 82wherein R.sup.13, R.sup.14, R.sup.16 and R.sup.17
are each independently selected from hydrogen, halogen, and
C.sub.1-C.sub.6 linear or branched alkyl, or R.sup.16 and R.sup.17
are linked to form an optionally substituted aryl or heteroaryl
ring system; R.sup.15 is selected from hydrogen, C.sub.1-C.sub.6
alkoxy, optionally substituted phenoxy, and NHSO.sub.2R.sup.19;
wherein R.sup.19 is C.sub.1-C.sub.6 linear or branched alkyl,
phenyl, mono-, di- or tri-halosubstituted phenyl or
S.sub.2C.sub.6H.sub.5NHSO.sub.2CH.sub.3; and R.sup.18 is selected
from hydrogen, C.sub.1-C.sub.6 linear or branched alkyl, halogen,
phenyl, halosubstituted phenyl and [C.sub.6H.sub.2(CH.sub.3).sub-
.2]SO.sub.2NHC.sub.6H.sub.5.
27. The method of claim 23, wherein the aryl urea is of Formula
(IV): 83wherein R.sup.20 to R.sup.29 are each independently
selected from hydrogen, halogen, C.sub.1-C.sub.6 linear or branched
alkyl and NO.sub.2.
28. The method of claim 23, wherein the .alpha.,.beta.-diketone is
of Formula (V): 84wherein R.sup.31 and R.sup.32 are each
independently aryl or heteroaryl, wherein the aryl or heteroaryl is
optionally substituted with one or more hydrogen, halogen, hydroxy,
C.sub.1-C.sub.6 linear or branched alkyl, C.sub.1-C.sub.6 linear or
branched halo-alkyl, C.sub.1-C.sub.6 alkoxy, NR.sub.33R.sub.34,
COOH, or NO.sub.2; or R.sup.31 and R.sup.32 may optionally be
linked to form an optionally substituted polycyclic aryl or
heteroaryl ring system; and R.sub.33 and R.sub.34 are independently
hydrogen or C.sub.1-C.sub.6 linear or branched alkyl.
29. The method of claim 23, wherein the drug is selected from
cocaine, heroin, meperidine, capecitabine and flumazenil.
30. A formulation comprising (i) a compound active as a pesticide;
and (ii) an insect esterase inhibiting amount of an esterase
inhibitor selected from amides, aryl sulphonamides, aryl ureas,
.alpha.,.beta.-diketones, and mixtures thereof.
31. The formulation of claim 30, wherein the amide is of Formula
(I): 85wherein R.sup.1 to R.sup.12 are each independently selected
from hydrogen, C.sub.1-C.sub.6 linear alkyl, C.sub.1-C.sub.6
branched alkyl, substituted alkyl, phenyl, substituted phenyl,
hydroxyl, halogen, and C.sub.1-C.sub.6 alkoxy; and optionally
R.sup.8 and R.sup.9 are linked to form an optionally substituted
aryl or heteroaryl ring system.
32. The formulation of claim 30, wherein the amide is of Formula
(II): 86wherein R.sup.11 to R.sup.12 are each independently
selected from hydrogen, C.sub.1-C.sub.6 linear alkyl,
C.sub.1-C.sub.6 branched alkyl, substituted alkyl, phenyl,
substituted phenyl, alkylphenyl, hydroxyl, halogen, and
C.sub.1-C.sub.6 alkoxy; and Ar is optionally substituted phenyl or
naphthyl.
33. The formulation of claim 30, wherein the aryl sulphonamide is
of Formula (III): 87wherein R.sup.13, R.sup.14, R.sup.16 and
R.sup.17 are each independently selected from hydrogen, halogen,
and C.sub.1-C.sub.6 linear or branched alkyl, or R.sup.16 and
R.sup.17 are linked to form an optionally substituted aryl or
heteroaryl ring system; R.sup.15 is selected from hydrogen,
C.sub.1-C.sub.6 alkoxy, optionally substituted phenoxy, and
NHSO.sub.2R.sup.19; wherein R.sup.19 is C.sub.1-C.sub.6 linear or
branched alkyl, phenyl, mono-, di- or tri-halosubstituted phenyl or
S.sub.2C.sub.6H.sub.5NHSO.sub.2CH.sub.3; and R.sup.18 is selected
from hydrogen, C.sub.1-C.sub.6 linear or branched alkyl, halogen,
phenyl, halosubstituted phenyl and [C.sub.6H.sub.2(CH.sub.3).sub-
.2]SO.sub.2NHC.sub.6H.sub.5.
34. The formulation of claim 30, wherein the aryl urea is of
Formula (IV): 88wherein R.sup.20 to R.sup.29 are each independently
selected from hydrogen, halogen, C.sub.1-C.sub.6 linear or branched
alkyl and NO.sub.2.
35. The formulation of claim 30, wherein the
.alpha.,.beta.-diketone is of Formula (V): 89wherein R.sub.31 and
R.sup.32 are each independently aryl or heteroaryl, wherein the
aryl or heteroaryl is optionally substituted with one or more
hydrogen, halogen, hydroxy, C.sub.1-C.sub.6 linear or branched
alkyl, C.sub.1-C.sub.6 linear or branched halo-alkyl,
C.sub.1-C.sub.6 alkoxy, NR.sub.33R.sub.34, COOH, or NO.sub.2; or
R.sup.31 and R.sup.32 may optionally be linked to form an
optionally substituted polycyclic aryl or heteroaryl ring system;
and R.sub.33 and R.sub.34 are independently hydrogen or
C.sub.1-C.sub.6 linear or branched alkyl.
36. A pharmaceutical composition comprising an esterase inhibiting
amount of an esterase inhibitor selected from amides, aryl
sulphonamides, aryl ureas, .alpha.,.beta.-diketones, and mixtures
thereof, and a pharmaceutically acceptable carrier or
excipient.
37. The composition of claim 36, wherein the amide is of Formula
(I): 90wherein R.sup.1 to R.sup.12 are each independently selected
from hydrogen, C.sub.1-C.sub.6 linear alkyl, C.sub.1-C.sub.6
branched alkyl, substituted alkyl, phenyl, substituted phenyl,
hydroxyl, halogen, and C.sub.1-C.sub.6 alkoxy; and optionally
R.sup.8 and R.sup.9 are linked to form an optionally substituted
aryl or heteroaryl ring system.
38. The composition of claim 36, wherein the amide is of Formula
(II): 91wherein R.sup.11 to R.sup.12 are each independently
selected from hydrogen, C.sub.1-C.sub.6 linear alkyl,
C.sub.1-C.sub.6 branched alkyl, substituted alkyl, phenyl,
substituted phenyl, alkylphenyl, hydroxyl, halogen, and
C.sub.1-C.sub.6 alkoxy; and Ar is optionally substituted phenyl or
naphthyl.
39. The composition of claim 36, wherein the aryl sulphonamide is
of Formula (III): 92wherein R.sup.13, R.sup.14, R.sup.16 and
R.sup.17 are each independently selected from hydrogen, halogen,
and C.sub.1-C.sub.6 linear or branched alkyl, or R.sup.16 and
R.sup.17 are linked to form an optionally substituted aryl or
heteroaryl ring system; R.sup.15 is selected from hydrogen,
C.sub.1-C.sub.6 alkoxy, optionally substituted phenoxy, and
NHSO.sub.2R.sup.19; wherein R.sup.19 is C.sub.1-C.sub.6 linear or
branched alkyl, phenyl, mono-, di- or tri-halosubstituted phenyl or
S.sub.2C.sub.6H.sub.5NHSO.sub.2CH.sub.3; and R.sup.18 is selected
from hydrogen, C.sub.1-C.sub.6 linear or branched alkyl, halogen,
phenyl, halosubstituted phenyl and [C.sub.6H.sub.2(CH.sub.3).sub-
.2]SO.sub.2NHC.sub.6H.sub.5.
40. The composition of claim 36, wherein the aryl urea is of
Formula (IV): 93wherein R.sup.20 to R.sup.29 are each independently
selected from hydrogen, halogen, C.sub.1-C.sub.6 linear or branched
alkyl and NO.sub.2.
41. The composition of claim 36, wherein the
.alpha.,.beta.-diketone is of Formula (V): 94wherein R.sup.31 and
R.sup.32 are each independently aryl or heteroaryl, wherein the
aryl or heteroaryl is optionally substituted with one or more
hydrogen, halogen, hydroxy, C.sub.1-C.sub.6 linear or branched
alkyl, C.sub.1-C.sub.6 linear or branched halo-alkyl,
C.sub.1-C.sub.6 alkoxy, NR.sub.33R.sub.34, COOH, or NO.sub.2; or
R.sup.31 and R.sup.32 may optionally be linked to form an
optionally substituted polycyclic aryl or heteroaryl ring system;
and R.sub.33 and R.sub.34 are independently hydrogen or
C.sub.1-C.sub.6 linear or branched alkyl.
42. The composition of claim 36, wherein the amide is selected
from: N-(5-chloro-2,4-dimethoxyphenyl)-3-hydroxy-2-naphthamide,
N-(4-chlorophenyl)-8-hydroxy-4aH-carbazole-7-carboxamide,
2-Benzoylamino-3-phenyl-propionic acid naphthalene-2-yl ester, and
2-naphthyl 2-(acetylamino)4-methylpentanoate.
43. The composition of claim 36, wherein the aryl sulfonamide is
selected from:
N-{2,3,5,6-tetrachloro-4-[(phenylsulfonyl)amino]phenyl}benzenesulfo-
namide,
4-chloro-N-(4{[(4-chlorophenyl)sulfonyl]amino}-2,3,4,6-tetrafluoro-
phenyl) benzenesulfonamide,
4-chloro-N-(4-{[(4-chlorophenyl)sulfonyl]amino-
}phenyl)benzenesulfonamide,
4-bromo-N-(4-phenoxyphenyl)benzenesulfonamide,
4-chloro-N-(4-{[(4-chlorophenyl)sulfonyl]amino}-1-naphthyl)benzenesulfona-
mide, 4,6-dimethyl-N,N'-diphenylbenzene-1,3-disulfonamide,
N-{2-methyl-4-[(phenylsulfonyl)amino]phenyl}benzenesulfonamide,
N-[4-({4-[(methylsulfonyl)amino]phenyl}dithio)phenyl]methanesulfonamide,
N-{4-[(phenylsulfonyl)amino]phenyl}benzenesulfonamide, and
4-chloro-N-(4-ethoxyphenyl)benzenesulfonamide.
44. The composition of claim 36, wherein the aryl urea is selected
from: N-(2-chloro4-nitrophenyl)-N'-(4-chlorophenyl)urea,
N-(2,6-dimethylphenyl)-N'-(4-nitrophenyl)urea,
N-(3-fluorophenyl)-N'-(2-m- ethyl-4-nitrophenyl)urea, and
N-(2-methyl-4-nitrophenyl)-N'-phenylurea.
45. The composition of claim 36, further comprising a drug that is
metabolized by a carboxylesterase in a patient to whom the drug is
administered.
46. The composition of claim 45, wherein the drug is an anti-cancer
drug.
47. The composition of claim 46, wherein the anti-cancer drug is
CPT-11
48. The composition of claim 45, wherein the drug is metabolized by
a carboxylesterase to generate a topoisomerase I inhibitor.
Description
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Ser. No. 60/498,778,
filed Aug. 29, 2003, which is incorporated by reference herein in
its entirety.
FIELD OF THE INVENTION
[0003] This invention is directed to the use of amide, aryl
sulphonamide, aryl urea, and .alpha.,.beta.-diketone compounds as
carboxylesterase inhibitors, and particularly to the use of these
compounds as selective human intestinal carboxylesterase
inhibitors. The compounds are also useful as insect
carboxylesterase inhibitors. The invention is also directed to
pharmaceutical compositions and pesticide formulations containing
the amide, aryl sulphonamide, aryl urea, and
.alpha.,.beta.-diketone esterase inhibitors, and to methods for
treating or ameliorating the toxic effects of drugs, such as cancer
therapy drugs, treating or ameliorating the effects of a drug
overdose, and to the use of the compounds for increasing the
effectiveness of insecticides and pesticides.
BACKGROUND OF THE INVENTION
[0004] Carboxylesterase (CE) is one of a sub-class of enzymes known
collectively as hydrolases. Hydrolase enzymes catalyze the
hydrolysis of various bonds, with carboxylesterase specific for the
hydrolysis of both aliphatic and aromatic carboxylic esters,
thereby generating an alcohol and a carboxylic acid anion. CEs are
ubiquitous serine esterase enzymes that are thought to be involved
in the detoxification of xenobiotics, and are found in animal
tissues (primarily the liver, serum, lung, kidney, intestine, and
blood brain barrier), plants, molds, and yeast. The tissue
distribution of these enzymes correlates with their involvement in
xenobiotic detoxification.
[0005] As yet, no endogenous substrates for CEs have been
identified, although they are responsible for the metabolism of
many drugs, including CPT-11, cocaine, heroin, meperidine, and
capecitabine. These carboxylesterase enzymes are processed in the
endoplasmic reticulum of mammalian cells, and hence these proteins
can be secreted into the extracellular milieu. Recently, the x-ray
crystal structure of a rabbit-liver and a human-liver
carboxylesterase have been determined. These studies indicate that
the proteins demonstrate similar structures to other esterases
including acetylcholinesterases, lipases, etc.
[0006] Previously known inhibitors of esterases have included
organophosphorous compounds such as di-isopropyl fluorophosphate
(DFP), carbamates, piperidine derivatives, and acridine
derivatives. These compounds are regarded as highly toxic poisons.
The inhibition of chicken liver carboxylesterase by benzil has been
reported in a study of the enzymatic mechanism involved. However,
the only selective inhibitors of CEs that have been reported are
the cyclic organophosphate derivatives, Bomin-1, 2, and 3 (Latoxan,
France).
[0007]
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin
(Irinotecan, CPT-11) is a widely used anti-cancer drug that has
demonstrated remarkable promise in the treatment of solid tumors.
CPT-11 has demonstrated remarkable antitumor activity in both
preclinical models and patients with refractory disease and, as
such, has recently been approved for the treatment of colon cancer
in adults. When administered to patients, CPT-11 is activated by
human carboxylesterase to yield its active metabolite,
7-ethyl-10-hydroxycamptothecin (SN-38), which is a potent
topoisomerase I poison. Topoisomerases are the enzymes responsible
for unwinding and winding chromosomal DNA. In order to allow
transcription and translation, DNA must be unwound. SN-38 prevents
DNA unwinding, and thus inhibits critical cellular processes in
tumor cells, resulting in cell death.
[0008] The toxicities associated with this agent include a
cholinergic syndrome due to direct inhibition of
acetylcholinesterase, and delayed diarrhea due to gastrointestinal
toxicity. The gastrointestinal toxicity is thought to occur via two
independent mechanisms:
[0009] 1. SN-38 is conjugated in the liver to yield SN-38
glucuronide (SN-38G). Following deposition into the small intestine
via the bile, SN-38G can be cleaved by bacterial glucuronidases to
yield the toxic metabolite SN-38, resulting in local irritation and
toxicity to the gut. 2. CPT-11 is also eliminated via the bile, and
following entry into the gut, CEs present within the intestinal
epithelia can convert the drug to SN-38. Hence very high, local
concentrations of SN-38 will be produced, resulting in cytotoxicity
and hence diarrhea.
[0010] The delayed diarrhea associated with CPT-11 administration
can be life-threatening and is the dose limiting toxicity for this
agent. Potential solutions for ameliorating this toxicity include:
(i) the aggressive use of antidiarrheals, such as loperamide and
diphenoxylate/atropine, and (ii) the alkalinization of the gut
using bicarbonate.
[0011] The level of activation of CPT-11 by human plasma in vitro
is very low. In contrast, plasma derived from rats and mice is very
proficient at CPT-11 activation, with greater than 50% of the drug
converted to SN-38 within 1 hour of incubation. Either the levels
of the enzymes responsible for CPT-11 metabolism in humans are low,
or these human proteins have significantly diverged in structure
from their rodent counterparts. Hence, animal models designed to
predict tumor responses in humans may overestimate the efficacy of
the drug due to the increased plasma activation of CPT-11.
[0012] Recently, a rabbit liver carboxylesterase that could
efficiently convert CPT-11 to SN-38 was isolated. A human homolog
of this carboxylesterase (hCE1) is known. However, expression of
hCE1 in human tumor cells does not alter their sensitivity to
CPT-11. More recently, it has been demonstrated that both the human
and mouse small intestine expresses high levels of
carboxylesterases that can convert CPT-11 to SN-38. A cDNA encoding
a human small intestinal carboxylesterase (hiCE) has subsequently
been identified that is highly efficient at activating CPT-11.
Expression of this protein in mammalian cells sensitizes them to
the drug.
[0013] Therefore, it appears that the activation of CPT-11 in the
human intestine by hiCE results in local toxicity, and hence
produces the unwanted toxic side effects such as diarrhea.
Therefore, before CPT-11 and similar anti-cancer drugs can be
utilized to their full potential, there is a need to develop
methods for alleviating the toxicity problems associated with
administration of these drugs.
[0014] There is therefore a need to develop new compounds that are
not only useful as general esterase inhibitors, but further, to
develop new compounds that are specific for the inhibition of
selected carboxylesterases, such as the human small intestine
carboxylesterases (hiCEs) that activate drugs such as CPT-11.
[0015] The following patents and publications provide relevant
background to the present invention. All references cited below are
incorporated herein by reference in their entirety and to the same
extent as if each reference was individually incorporated by
reference. U.S. Pat. Nos. 5,762,314, 6,407,117; Published
International Application No. WO 99/42593; Khanna et al., Cancer
Research, 2000, 60: pp. 47254728; Wadkins et al., Molecular
Pharmacology, 2001, 60(2): pp. 355-362; Wierdl et al., Cancer
Research, 2001, 61: pp. 5078-5082; Tanizawa et al., J. Natl. Cancer
Inst., 1994, 86: pp. 836-42; Morton et al., Mol. Biotechnol., 2000,
16, pp. 193-202; Soares, E. R., Biochem. Genet., 1979, 17, pp.
577-583; and Berndt et al., Biochimica et Biophysica Acta., 1996,
1298, pp. 159-166.
SUMMARY OF THE INVENTION
[0016] This invention is directed to the use of amide, aryl
sulphonamide, aryl urea, and .alpha.,.beta.-diketone compounds as
esterase inhibitors.
[0017] In one embodiment, these compounds as useful as selective
human intestinal carboxylesterase inhibitors.
[0018] In a second embodiment, these compounds are useful as insect
carboxylesterase inhibitors.
[0019] A further embodiment is directed to pharmaceutical
compositions containing the amide, aryl sulphonamide, aryl urea,
and .alpha.,.beta.-diketone esterase inhibitors.
[0020] A further embodiment is directed to pesticide formulations
containing the amide, aryl sulphonamide, aryl urea, and
.alpha.,.beta.-diketone esterase inhibitors
[0021] Further embodiments are directed to a method for treating or
ameliorating the toxic effects of drugs (such as the cancer therapy
drug CPT-11) administered to a patient, to treating or ameliorating
the effects of a drug overdose, and to the use of the compounds for
increasing the effectiveness of insecticides and pesticides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows hyperbolic plots of the inhibition of o-NPA
metabolism by hiCE using sulphonamide inhibitor 44 at two different
concentrations (149 nM and 447 nM).
[0023] FIG. 2 shows the carboxylesterase activity in enzyme
preparations after preincubation with sulphonamide inhibitors 44-52
and 59.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention is directed to the novel use of amide,
aryl sulphonamide, aryl urea, and .alpha.,.beta.-diketone
derivatives as esterase inhibitors. In one embodiment, the amide,
aryl sulphonamide, aryl urea, and .alpha.,.beta.-diketone
derivatives are useful general carboxylesterase inhibitors. In
another embodiment, the amide, aryl sulphonamide, aryl urea, and
.alpha.,.beta.-diketone derivatives are useful selective
carboxylesterase inhibitors. In more specific embodiments, the
invention is directed to the use of amide, aryl sulphonamide, aryl
urea, and .alpha.,.beta.-diketone derivatives as selective human
intestinal carboxylesterase (hiCE) inhibitors or selective insect
carboxylesterase inhibitors.
[0025] The present invention is based, in part, on the discovery
that amide, aryl sulfonamide, aryl urea, and
.alpha.,.beta.-diketone derivatives of Formulas (I)-(V) have
esterase inhibitor activity. Previously, these classes of compounds
have not been shown to be esterase inhibitors. Furthermore, many of
these compounds are useful as selective inhibitors of human
intestinal carboxylesterase.
[0026] CPT-11 is an anti-cancer drug that is selectively hydrolyzed
to SN-38 by a human carboxylesterase. SN-38 is a potent
topoisomerase I inhibitor. One of the major problems associated
with CPT-11 administration is gastrointestinal toxicity, such as
delayed diarrhea, due to activation of CPT-11 by carboxylesterases
in the human intestine. By administering the selective hiCE
inhibitors of Formula (I)-(V), the conversion of CPT-11 to the
active metabolite SN-38 in the gut is minimized, and hence
CPT-11-induced gastrointestinal toxicity is ameliorated.
[0027] Additionally, these compounds, which do not readily cross
cell membranes, are useful as selective inhibitors of other
important carboxylesterases, such as insect carboxylesterases.
[0028] Definitions
[0029] Unless defined otherwise, all technical and scientific terms
used herein generally have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention
belongs.
[0030] The term "pharmaceutically acceptable salts" includes salts
of the active compounds which are prepared with relatively nontoxic
acids or bases, depending on the particular substituents found on
the compounds described herein. Suitable salts include, but are not
limited to, organic and inorganic salts, for example, ammonium,
acetate salt, citrate salt, halide salt, such as hydrochloride and
hydrobromide, hydroxide, sulfate, nitrate, phosphate, perchlorate,
tetrafluoroborate, carboxylate, mesylate, fumerate, malonate,
succinate, tartrate, acetate, gluconate, and maleate.
[0031] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviations, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, preferably up
to 10%, more preferably up to 5%, and more preferably still up to 1
% of a given value. Alternatively, particularly with respect to
biological systems or processes, the term can mean within an order
of magnitude, preferably within 5-fold, and more preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated the term "about"
meaning within an acceptable error range for the particular value
should be assumed.
[0032] The term "esterase inhibiting amount" means an amount of the
esterase inhibitor sufficient to inhibit esterase activity by a
measurable amount, e.g., by at least 50%, preferably by at least
75%, and more preferably by at least 90%.
[0033] The term "gastrointestinal toxicity" means a disorder of the
gastrointestinal tract, such as, but not limited to, delayed
diarrhea.
[0034] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain radical which may be fully saturated, mono- or
polyunsaturated and can include di- and multivalent radicals,
having the number of carbon atoms designated (e.g.,
C.sub.1-C.sub.10 means one to ten carbons). Examples of saturated
hydrocarbon radicals include, but are not limited to, groups such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,
sec-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. An unsaturated
alkyl group is one having one or more double bonds ("alkenyl") or
triple bonds ("alkynyl"). "Alkenyl" refers to a branched or
straight chain C.sub.2-C.sub.24 hydrocarbon (for example a
C.sub.2-C.sub.10 hydrocarbon, for further example a C.sub.2-C.sub.8
hydrocarbon, for even further example a C.sub.2-C.sub.6
hydrocarbon) which can comprise one or more carbon-carbon double
bonds. Exemplary alkenyl groups include propylenyl, buten-1-yl,
isobutenyl, penten-1-yl, 2,2-methylbuten-1-yl, 3-methylbuten-1-yl,
hexan-1-yl, hepten-1-yl, octen-1-yl, and the like. "Alkynyl" refers
to an unsaturated acyclic C.sub.2-C.sub.24 hydrocarbon for example
a C.sub.2-C.sub.10 hydrocarbon, for further example a C.sub.2-C8
hydrocarbon, for even further example a C.sub.2-C.sub.6
hydrocarbon) which can comprise one or more carbon-carbon triple
bonds. Exemplary alkynyl groups include ethynyl, propynyl,
butyn-1-yl, butyn-2-yl, pentyl-1-yl, pentyl-2-yl,
3-methylbutyn-1-yl, hexyl-1-yl, hexyl-2-yl, hexyl-3-yl,
3,3-dimethyl-butyn-1-yl, and the like. Typically, an alkyl,
alkenyl, or alkynyl group having 6 or fewer carbon atoms is
referred to as "lower alkyl", "lower alkenyl" , or "lower alkynyl",
respectively.
[0035] Cycloalkyl means a saturated or unsaturated cyclic
hydrocarbon radical, such as, but not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,
cyclohepta-1,3-dienyl, cyclooctadienyl, (cyclohexyl)methyl, and
cyclopropylmethyl.
[0036] Heterocycloalkyl means a cycloalkyl radical possessing one
or more heteroatoms selected from N, O, and S, wherein the
nitrogen, carbon and sulfur atoms are optionally oxidized, and the
nitrogen atom(s) are optionally quaternized. Additionally, for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to the remainder of the molecule. Examples
of heterocycloalkyl include, but are not limited to,
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
furan-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the
like.
[0037] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom.
[0038] The term "aryl" means, unless otherwise stated, a
substituted or unsubstituted polyunsaturated, aromatic, hydrocarbon
substituent which can be a single ring or multiple rings
(preferably from 1 to 3 rings), which are fused together or linked
covalently. The term "heteroaryl" refers to a category of aryl
groups (or rings) that contain from one to four heteroatoms
selected from N, O, and S, wherein the nitrogen, carbon and sulfur
atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. "Aryl", including "heteroaryl", also encompass
ring systems in which one or more non-aromatic ring systems are
fused, or otherwise bound, to an aryl or heteroaryl system.
[0039] Each of the above terms (e.g., "alkyl," "cycloalkyl", "aryl"
and "heteroaryl") include both substituted and unsubstituted forms
of the indicated radical. Preferred substituents for each type of
radical are provided below.
[0040] Substituents for the alkyl, and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generally referred to as "alkyl substituents" and "heteroalkyl
substituents," respectively, and they can be one or more of a
variety of groups selected from, but not limited to: --OR', .dbd.O,
.dbd.NR', .dbd.N--OR', --NR'R", --SR', -halogen, --SiR'R"R'",
--OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R", --OC(O)NR'R",
--NR"C(O)R', --NR'-C(O)NR"R'", --NR"C(O).sub.2R',
--NR--C(NR'R"R'").dbd.NR"", --NR--C(NR'R").dbd.NR'", --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R", --NRSO.sub.2R', --CN and
--NO.sub.2 in a number ranging from zero to (2m'+1), where m' is
the total number of carbon atoms in such radical. R', R", R'" and
R"" each preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R", R'" and R"" groups when more than one of these groups is
present. When R' and R" are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R" is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0041] Similar to the substituents described for the alkyl radical,
the aryl substituents and heteroaryl substituents are generally
referred to as "aryl substituents" and "heteroaryl substituents,"
respectively and are varied and selected from, for example:
halogen, --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R", --SR',
-halogen, --SiR'R"R'", --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R", --OC(O)NR'R", --NR"C(O)R', --NR'--C(O)NR"R'",
--NR"C(O).sub.2R', --NR--C(NR'R").dbd.NR'", --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R", --NRSO.sub.2R', --CN,
--NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R", R'" and R"" are
preferably independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1-C.sub.4)alkyl. When a compound of
the invention includes more than one R group, for example, each of
the R groups is independently selected as are each R', R", R'" and
R"" groups when more than one of these groups is present.
[0042] As used herein, the term "heteroatom" includes oxygen (O),
nitrogen (N), sulfur (S) and silicon (Si).
[0043] The symbol "R" is a general abbreviation that represents a
substituent group that is selected from substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, 5
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, and substituted or unsubstituted heterocyclic
groups.
Esterase Inhibitor Compounds
[0044] Amide compounds that have been found to be esterase
inhibitors are of Formula (I) and (II): 1
[0045] wherein R.sup.1 to R.sup.2 are each independently hydrogen,
C.sub.1-C.sub.6 linear or branched alkyl, substituted alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl,
halogen, or C.sub.1-C.sub.6 alkoxy; and Ar is phenyl, substituted
phenyl, naphthyl or substituted naphthyl. Optionally, R.sup.8 and
R.sup.9 may be linked to form a substituted or unsubstituted aryl
or heteroaryl ring system.
[0046] In one embodiment of the compound of Formula (I), at least
one of R.sup.1 to R.sup.5 is chlorine and R.sup.6 is hydroxyl. In a
specific embodiment of the compound of Formula (II), Ar is
naphthyl, R.sup.11 is phenyl, and R.sup.12 is benzyl. In a second
specific embodiment of the compound of Formula (II), Ar is
naphthyl, R.sup.11 is methyl, and R.sup.12 is isopropyl. In a third
specific embodiment of the compound of Formula (II), Ar is
naphthyl, R.sup.11 is methyl, and R.sup.12 is isobutyl.
[0047] Aryl sulphonamide compounds that have been found to be
esterase inhibitors are of formula (III): 2
[0048] wherein R.sup.13, R.sup.14, R.sup.16 and R.sup.17 are each
independently hydrogen, halogen, C.sub.1-C.sub.6 linear or branched
alkyl, or R.sup.16 and R.sup.17 may optionally be linked to form an
aryl or heteroaryl ring system; R.sup.15 is hydrogen,
C.sub.1-C.sub.6 alkoxy, optionally substituted phenoxy, or
NHSO.sub.2R.sup.19; wherein R.sup.19 is C.sub.1-C.sub.6 linear or
branched alkyl, phenyl, mono-, di- or tri-halosubstituted phenyl or
S.sub.2C.sub.6H.sub.5NHSO.sub.2CH.sub.3; and R.sup.18 is hydrogen,
C.sub.1-C.sub.6 linear or branched alkyl, halogen, phenyl,
halosubstituted phenyl or [C.sub.6H.sub.2(CH.sub.3).sub.-
2]SO.sub.2NHC.sub.6H.sub.5.
[0049] Aryl urea compounds that have been found to be to be
esterase inhibitors are of formula (IV): 3
[0050] wherein R.sup.20 to R.sup.29 are each independently
hydrogen, halogen, C.sub.1-C.sub.6 linear or branched alkyl or
NO.sub.2. In one embodiment, R.sup.22 or R.sup.27 is NO.sub.2.
[0051] .alpha.,.beta.-diketone compounds that have been found to be
to be esterase inhibitors are of formula (V): 4
[0052] wherein R.sup.31 and R.sup.32 are each independently aryl or
heteroaryl, optionally substituted with one or more hydrogen,
halogen, hydroxy, C.sub.1-C.sub.6 linear or branched alkyl,
C.sub.1-C.sub.6 linear or branched halo-alkyl, C.sub.1-C.sub.6
alkoxy, NR.sub.33R.sub.34, COOH, or NO.sub.2, wherein R.sup.33 and
R.sup.34 are independently hydrogen or C.sub.1-C.sub.6 linear or
branched alkyl.
[0053] R.sup.31 and R.sup.32 may include, but are not limited to,
phenyl, pyridyl, furanyl, thienyl, and substituted derivatives
thereof. Alternatively, R.sup.31 and R.sup.32 may be linked to form
an optionally substituted polycyclic aryl or heteroaryl ring
system. In one embodiment of the compound of Formula (V), when
R.sup.31 is unsubstituted phenyl, R.sup.32 is not unsubstituted
phenyl (i.e., R.sup.31 and R.sup.32 are not both unsubstituted
phenyl).
[0054] Examples of .alpha.,.beta.-diketone compounds useful as
esterase inhibitors include, but are not limited to, benzil, furil,
thenil, pyridil, aceanthrenequinone, 9,10-phenanthrenequinone,
acenaphthenequinone, 1,2,-naphthoquinone, and substituted
derivatives thereof.
[0055] Additionally, since the structures of these compounds are
novel with respect to their ability to inhibit mammalian esterases,
certain esterase inhibitors of Formulae (I)-(V), or derivatives
thereof, are useful as selective inhibitors of specific mammalian
carboxylesterases. Hence inhibitors with these core structures with
selectivity toward other classes of carboxylesterases are
provided.
[0056] Many amide, aryl sulfonamide, aryl urea, and
.alpha.,.beta.-diketone compounds falling into the foregoing
classes are available from commercial sources, including Sigma
Aldrich (St. Louis, Mo.), ChemDiv (San Diego, Calif.), Asinex
(Moscow, Russia), and Maybridge (Cornwall, U.K.) The commercially
available compounds can be readily modified by routine synthetic
methods to generate derivative compounds.
[0057] Alternatively, any compound for use in the present invention
can be generated synthetically, by standard organic synthetic
methods readily known to one of ordinary skill in the art, e.g., as
set forth below.
Preparation of Compounds of Formulas (I) and (II)
[0058] Numerous synthetic methods are known to the art to form
amides (see "Comprehensive Organic Transformations", Chapter 9,
Larock, R. C. 1989, VCH Publishers, New York). Compounds of
Formulas (I) and (II) may be prepared in a similar manner to that
outlined below for the preparation of compound 42. 5
[0059] A protected naphthoic acid (PG=protecting group, see
"Protective Groups in Organic Synthesis", 2.sup.nd, Greene and
Wuts, 1991, Wiley, New York) is converted to the acyichioride using
oxalyichioride. The acid chloride is directly converted to the
amide by addition of the appropriate aniline dissolved in pyridine.
Deprotection of the alcohol followed by isolation and purification
affords Compound 42.
Preparation of Compounds of Formula (III)
[0060] Methods to prepare aryl and alkyl sulphonamides are well
known to the art (see "Advanced Organic Chemistry", 4.sup.th ed, pp
496-498, March, J., 1992, Wiley, New York). Compounds of Formula
(III) may be prepared in a similar manner to that outlined below
for the preparation of compound 50. 6
[0061] The 4-thiolaniline is oxidatively dimerized to form the
disulfide. Treatment of the disulfide with methanesulfonylchloride
in dichloromethane/pyridine yields compound 50.
[0062] Compound 59 was prepared according to the following
synthetic scheme. 7
Preparation of Compounds of Formula (IV)
[0063] Ureas and their preparation are well know to one skilled in
the art (see "Introduction to Organic Chemistry", 2.sup.nd ed., pp.
785-786, Streitweiser and Heathcock, 1981, MacMillan N.Y.). Diaryl
ureas may be prepared in a similar manner to that outlined below
for the preparation of compound 53. 8
[0064] The diaryl urea is directly formed by the reaction of an
isocyanate and the appropriate amine.
Preparation of Compounds of Formula (V)
[0065] Two potential synthetic schemes are possible for the
preparation of .alpha.,.beta.-diketone compounds of Formula (V).
For benzil, and derivatives thereof, the first uses the
condensation of the substituted benzaldehyde in the presence of
NaOH and thiamine to yield the benzoin derivative. Oxidation of the
benzoin derivative in the presence of Cu.sup.2+, acetic acid, and
ammonium nitrate, or concentrated nitric acid, produces the benzil
compound of Formula (V) virtually quantitative yield. This is shown
below for compound 17. 9
[0066] An alternate synthetic scheme for the preparation of benzil
compounds of Formula (V) involves the use of trimethylsilyl
cyanide. Following reaction of the substituted benzaldehyde with
trimethylsilyl cyanide in the presence of a zinc iodide catalyst,
the cyanohydrin derivative thus formed is reacted with the
substituted benzoyl acid chloride to yield the benzil compound of
Formula (V). 10
[0067] Thenil, pyridil, and furil analogs may be prepared in a
similar manner to that given above, using thiophenecarboxyaldehyde,
pyridinecarboxaldehyde, and furaldehyde, or substituted derivatives
thereof, as the starting material, respectively.
[0068] Suitable, but non limiting, examples of
.alpha.,.beta.-diketone derivatives useful as carboxylesterase
inhibitors in the present invention are set forth in Table 1.
1TABLE 1 .alpha.,.beta.-Diketone Derivatives Useful as
Carboxylesterase Inhibitors Compound Chemical Structure Name of
Compound Code 11 4-[oxo(phenyl)acetyl]benzoic acid 1 12 Benzil
Diphenylethane-1,2-dione 2 13 1-[4-bromomethyl)phenyl]-2-
phenylethane-1,2-dione 3 14 1,2-bis(4-bromo-3-
nitrophenyl)ethane-1,2-dione 4 15 1-(3,4-dimethylphenyl)-- 2-
phenylethane-1,2-dione 5 16 1-(4-methoxyphenyl)-2-
2phenylethane-1,2-dione 6 17 1-(4-methyl-3-nitrophenyl)-2- -
phenylethane-1,2-dione 7 18 1-(2-chlorophenyl)-2-(3,4-
dimethoxyphenyl)ethane-1,2-dione 8 19 1,2-bis(5-bromo-2-
hydroxyphenyl)ethane-1,2-dione 9 20 1,2-bis(2,4-
dihydroxyphenyl)ethane-1,2-dione 10 21 1-(2,4-dinitrophenyl)-2-
phenylethane-1,2-dione 11 22 1-(pentachlorophenyl)-2-
(pentafluorophenyl)ethane-1,2- dione 12 23 1,2-
bis[4(dimethylamino)phenyl]ethane- 1,2-dione 13 24
1-(4-nitrophenyl)-2-phenylethane- 1,2-dione 14 25 4-4'
dibromobenzil ((1,2-bis(4-bromophenyl)ethane- 1,2-dione 15 26 4-4'
difluorobenzil (1,2-bis(4-fluorophenyl)ethane-1,2- dione) 16 27
1,2-bis(4-methylphenyl)ethane-1,2- dione 17 28 4-4' dichlorobenzil
1,2-bis(4-chlorophenyl)ethane-1,2- dione 18 29
1,2-bis(3,5-difluorophenyl)ethane- 1,2-dione 19 30 1,2-bis(3,4,5-
trifluorophenyl)ethane-1,2-dione 20 31
1,2-bis(4-methoxyphenyl)ethane- 1,2-dione 21 32
1-(4-chlorophenyl)-2-(4- methylphenyl)ethane-1,2-dione 22 33 Thenil
1,2-dithien-2-yl-ethane-1,2-dione 23 34 Furil
1,2-di-2-furylethane-1,2-dione 24 35 Pyridil
1,2-dipyridin-2-ylethane-1,2-dione 25 36 Aceanthrenequinone
Aceanthrylene-1,2-dione 26 37 9,10-Phenanthrenequinone
Phenanthrene-9,10-dione 27 38 Acenaphthenequinone
Acenaphthylene-1,2-dione 28 39 1,2-Napthoquinone
Naplithalene-1,2-dione 29 40 2-2' dichlorobenzil
1,2-bis(2-chlorophenyl)ethane-1,2- dione 30 41
1-(4-chlorophenyl)-2-phenylethane- 1,2-dione 31 42
1-(4-methylphenyl)-2-phenylethane- 1,2-dione 32 43
1,2-bis(3-methoxyphenyl)ethane- 1,2-dione 33 44
1,2-bis(3-nitrophenyl)ethane-1,2- dione 34 45
1,2-bis(4-hydroxyphenyl)ethane- 1,2-dione 35 46
1,2-bis(4-hydroxy-3- nitrophenylethane-1,2-dione 36 47
1,2-bis(4-methoxy-3- nitrophenyl)ethane-1,2-dione 37 48 Bisbenzil
1-{4-[oxo(phenyl)acetyl]phenyl}-2- phenylethane-1,2-dione 38
[0069] Compounds 26-29, and 33 are commercially available from
Sigma Aldrich (St. Louis, Mo.) Compound 30 is commercially
available from VWR/Lancaster (Swedesboro, N.J.). Compound 31 is
commercially available from Alfa Aesa (Ward Hill, Mass.). Compound
32 is commercially available from Toronto Research Chemicals
(Toronto, Canada). Compounds 34-37 are commercially available from
Industrial Research Limited (Auckland, New Zealand). Compound 38 is
commercially available from TCI America (Portland, Oreg.).
Uses of the Esterase Inhibitors
General Inhibition of Carboxylesterases
[0070] One embodiment of the present invention is the use of amide,
aryl sulphonamide, aryl urea, and benzil compounds of Formulas
(I)-(V), or derivatives thereof, as 5 general carboxylesterase
inhibitors. Specific, but none limiting, examples of the compounds
of Formula (V) that have been found to be general carboxylesterase
inhibitors include compounds 1, 2, 3, 5, 6, 7, 14, 16, 17, 19, 20,
22, and 23.
[0071] In addition, other compounds that have been found to be
general carboxylesterase inhibitors are set forth in Table 2.
2TABLE 2 General carboxylesterase Inhibitors Compound Chemical
Structure Class and Name of Compound Code 49 Sulfone
4-{[4-(benzyloxy)phenyl]sulfonyl}phenol 39 50 Sulfonamide
N-(2-bromo-4{[4- methylphenyl)sulfonyl]ami- no} phenyl)-4-
methylbenzenesulfonamide 40 51 Triazene
1-{4-[(1-bromo-2-naphthyl)methyl]-3-
chlorophenyl}-6,6-dimethyl-1,6-dihyd- ro-
1,3,5-triazine-2,4-diamine 41
Inhibition of Human Intestinal Carboxylesterase (hiCE)
[0072] A further embodiment of the present invention is the use of
amide, aryl sulphonamide aryl urea, and .alpha.,.beta.-diketone
compounds of Formulas (I)-(V), or derivatives thereof, as selective
human intestinal carboxylesterase (hiCE) inhibitors. Specific, but
non-limiting, examples of the compounds of Formulas (I)-(V) that
have been found to be selective inhibitors of human small
intestinal carboxylesterase are given in Table 3.
3TABLE 3 Structures of Selective Human Small Intestinal
Carboxylesterase Inhibitors Compound Chemical Structure Class and
Name of Compound Code 52 Amide N-(5-chloro-2,4-dimethoxyphenyl)-3-
hydroxy-2-naphthamide 42 53 Amide N-(4-chlorophenyl)-8-hydroxy-4aH-
carbazole-7-carboxamide 43 54 Sulphonamide
N-{2,3,5,6-tetrachloro-4- [(phenylsulfonyl)amino]phenyl}benzene
sulfonamide 44 55 Sulphonamide 4-chloro-N-(4-{[(4-
chlorophenyl)sulfonyl]amino}phenyl) benzenesulfonamide 45 56
Sulphonamide 4-bromo-N-(4- phenoxyphenyl)benzenesulfonamide 46 57
Sulphonamide 4-chloro-N-(4-{[(4- chlorophenyl)sulfonyl]amino}-- 1-
naphthyl)benzenesulfonamide 47 58 Sulphonamide
4,6-dimethyl-N,N'-diphenylbenzene-1,3- disulfonamide 48 59
Sulphonamide N-{2-methyl-4- [(phenylsulfonyl)amino]phenyl}benzene
sulfonamide 49 60 Sulphonamide N-[4-({4-
[(methylsulfonyl)amino]phenyl}dithio) phenyl]methanesulfonamide 50
61 Sulphonamide N-{4- [(phenylsulfonyl)amino]phenyl}benzene
sulfonamide 51 62 Sulphonamide 4-chloro-N-(4-
ethoxyphenyl)benzenesulfonamide 52 63 Urea
N-(2-chloro-4-nitrophenyl)-N'-(4- chlorophenyl)urea 53 64 Urea
N-(2,6-dimethylphenyl)-N'-(4- nitrophenyl)urea 54 65 Urea
N-(3-fluorophenyl)-N'-(2-methyl-4- nitrophenyl)urea 55 66 Urea
N-(2-methyl-4-nitrophenyl)-N'-phenylurea 56 67 Amide
2-Benzoylamino-3-phenyl-propionic acid naphthalene-2-yl ester 57 68
Amide 2-naphthyl 2-(acetylamino)-4- methylpentanoate 58 69
Sulfonamide 4-chloro-N-(4{[(4-chlorophenyl) sulfonyl]amino}
2,3,4,6- tetrafluorophenyl) benzenesulfonamide 59
[0073] The compounds identified in Table 3 were obtained from the
following sources, Table 4:
4TABLE 4 Commercial Sources of the Compounds Identified in Table 3
Compound Alternate Code Source Catalog # Source Catalog # 42
ChemDiv 1125-0434 Asinex BAS 3819227 43 ChemDiv 000A-0340 Asinex
BAS 1057195 44 Asinex BAS 0126340 45 ChemDiv 0896-7238 Asinex BAS
0126335 46 ChemDiv 4049-0210 ChemDiv 3365-0568 47 Asinex BAS
0459805 48 ChemDiv 0262-0298 Asinex BAS 0116665 49 Sigma S739391
Aldrich 50 Synthesis described herein 51 ChemDiv 0896-7239 52
ChemDiv 4063-0024 Asinex BAS 1358536 53 Sigma S607258 Aldrich 54
Sigma S713805 Aldrich 55 Sigma S844829 Aldrich 56 Sigma S610461
Aldrich 57 Sigma S44975 Aldrich 58 Sigma S776955 Aldrich 59
Synthesis described herein
[0074] Inhibition of acetylcholinesterase (AcChE) would be an
undesirable property of the compounds of the selective human
intestinal carboxylesterase inhibitors identified in Table 3, and
make their use clinically impractical. Since AcChE is the target of
many nerve gases, compounds that inhibit this enzyme would be
highly toxic and would have very little use in humans. None of the
selective carboxylesterases identified in Table 3 were found to
inhibit AcChE.
[0075] The selective human intestinal carboxylesterase inhibitors
identified in Table 3 have been found not to cross cell membranes,
i.e., not to inhibit carboxylesterase activity intracellularly.
Thus the compounds identified in Table 3 cannot easily translocate
from the intestine to the bloodstream.
[0076] The sulphonamide human intestinal carboxylesterase
inhibitors identified in Table 3 have also been found to be
partially competitive inhibitors of hiCE, and to inhibit hiCE in a
reversible manner. As seen in FIG. 1, when hiCE is incubated in the
presence of 149 nM or 447 nM of compound 44,
(N-{2,3,5,6-tetrachloro4-[(phenylsulfonyl)amino]phenyl} benzene
sulfonamide), no change in the apparent Vmax (the velocity of the
enzyme at infinite substrate concentration) is observed, but an
increase in the Km values for hiCE occurs from 0.391 nM to 0.584 nM
and 1.132 nM, respectively. FIG. 2 demonstrates that the
sulfonamide inhibitors 44-52 and 59 do not result in irreversible
inhibition of hiCE. After incubation with either 10 .mu.M of the
compounds, or a concentration equivalent to five times the Ki
value, for 1 hour on ice, residual carboxylesterase activity was
determined. Bis-(4-nitrophenyl) phosphine (BNPP), an irreversible
esterase inhibitor, was used as a control. Since the enzyme and
inhibitor were diluted at least 250-fold in the CE assay, loss of
activity can only occur from direct inactivation of the protein by
the sulfonamide analog during the preincubation period. As can be
seen from FIG. 2, none of the sulfonamide compounds resulted in
irreversible inhibition of the hiCE protein.
Inhibition of Insect Carboxylesterases
[0077] Insects detoxify pesticides with carboxylesterases, and
insecticide resistance to pesticide compounds can occur via a
single acid point mutation in the carboxylesterase protein that
renders the enzyme more efficient at detoxification. Development of
pesticide resistance can lead to ineffective pesticide application
and widespread crop damage, or in the case of mosquitoes carrying
diseases like West Nile virus, public health threats. The
development of pesticide resistance may result in heavier
application of pesticides, which can harm the environment,
including fish and animals, and ultimately harm people,
particularly due to pesticide residues in foods. Therefore, a
further embodiment of the present invention is the use of the
amide, aryl sulphonamide, aryl urea, and .alpha.,.beta.-diketone
compounds of Formulas (I)-(V), or derivatives thereof, as selective
insect carboxylesterase inhibitors, thereby allowing for an
increase in the efficacy of currently available pesticides.
Formulations comprising a compound active as a pesticide and an
amide, aryl sulphonamide, aryl urea, or .alpha.,.beta.-diketone
insect carboxylesterase inhibitor of Formulas (I)-(V), or
derivatives thereof, or combinations thereof, are also envisaged.
Suitable compounds active as pesticides include, but are not
limited to, malathion, parathion, pirimicarb, and chlorpyrifos.
Modulation of Drug Metabolism
[0078] In a further embodiment of the present invention, amide,
aryl sulphonamide, aryl urea, and .alpha.,.beta.-diketone compounds
of Formulas (I)-(V), or derivatives thereof, can be used to
modulate drug and metabolite levels in humans. Carboxylesterases
are involved in the metabolism of a wide variety of drugs in the
human body, including, but not limited to, cocaine, heroin,
meperidine, capecitabine and flumazenil. Therefore, the esterase
inhibitors of Formulas (I)-(V) identified in the present invention
may be used to modulate drug and metabolite levels in the human
body. One embodiment is the use of amide, aryl sulphonamide, aryl
urea, and .alpha.,.beta.-diketone compounds for the amelioration of
toxicity following a drug overdose in a human patient.
Specifically, the compounds of Formulas (I)-(V) may be used to
prevent drug metabolism and prove useful in the treatment of
individuals who have overdosed on drugs such as, but not limited
to, cocaine and/or heroin.
Therapeutic Compositions and Regimens
[0079] According to the present invention, a therapeutic compound
can be formulated in a pharmaceutical composition of the invention
to be introduced parenterally, transmucosally, e.g., orally,
nasally, or rectally, or transdermally. Parenteral administration
includes, but is not limited to, intravenous, intra-arteriole,
intramuscular, intradermal, subcutaneous, intraperitoneal,
intraventricular, and intracranial administration, e.g., by
injection.
[0080] When formulated in a pharmaceutical composition, a
therapeutic compound can be admixed with a pharmaceutically
acceptable carrier or excipient. The phrase "pharmaceutically
acceptable" refers to molecular entities and compositions that are
"generally regarded as safe", e.g., that are physiologically
tolerable and do not typically produce an allergic or similar
untoward reaction, such as gastric upset, dizziness and the like,
when administered to a human. Preferably, as used herein, the term
"pharmaceutically acceptable" means approved by a regulatory agency
of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers
to a diluent, adjuvant, excipient, or vehicle with which the
compound is administered. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water or
aqueous solution saline solutions and aqueous dextrose and glycerol
solutions are preferably employed as carriers, particularly for
injectable solutions. Alternatively, the carrier can be a solid
dosage form carrier, including but not limited to one or more of a
binder (for compressed pills), a glidant, an encapsulating agent, a
flavorant, and a colorant. Suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W.
Martin.
[0081] A preferred mode of administration of the specific
inhibitors of the human intestinal carboxylesterase, identified in
Table 1, is oral administration. When administered orally, these
poorly bioavailable molecules are expected to either remain in the
gut or only enter the epithelia of the lining of the intestine, and
hence inactivate any carboxylesterase in these tissues, thus
preventing subsequent activation of CPT-11 that is deposited in the
duodenum from the bile.
[0082] A preferred mode of administration of the esterase inhibits
of Formula (I)-(V), for the inhibition of drug metabolism in the
blood stream or highly perfused organs like the liver, is
intravenous administration. This should lead to complete inhibition
of CE activity and hence prevent esterase-mediated drug
catalysis.
[0083] The compounds of Formula (I)-(V) may be administered either
before or after (sequentially), or at the same time
(simultaneously) as the drug that is metabolized by a
carboxylesterase.
[0084] Formulations comprising a compound of Formula (I)-(V) and a
drug, for example, CPT-11, which is metabolized by a
carboxylesterase to generate, for example, a topoisomerase I
inhibitor, are also envisaged.
[0085] For the potentiation of insecticides, it is necessary to
have a readily bioavailable molecule that can be co-administered
with the active agent. A formulation of the insect esterase
inhibitor and the insecticide may be prepared. The composition of
the formulation largely depends on the solubility of the compounds
and their intended use e.g. as fast acting liquid forms, or slow
release pellets, etc.
[0086] A constant supply of the therapeutic compound can be ensured
by providing a therapeutically effective dose (i.e., a dose
effective to induce metabolic changes in a subject) at the
necessary intervals, e.g., daily, every 12 hours, etc. These
parameters will depend on the severity of the disease condition
being treated, the regimen of any other drugs being administered
(such as CPT-11, for example), other actions, such as diet
modification, that are implemented, the weight, age, and sex of the
subject, and other criteria, which can be readily determined
according to standard good medical practice by those of skill in
the art.
[0087] A subject or patient in whom administration of the
therapeutic compound is an effective therapeutic regimen for a
disease or disorder is preferably a human, but can be any animal,
including a laboratory animal in the context of a clinical trial or
screening or activity experiment. Thus, as can be readily
appreciated by one of ordinary skill in the art, the methods and
compositions of the present invention are particularly suited to
administration to any animal, particularly a mammal, and including,
but by no means limited to, domestic animals, such as feline or
canine subjects, farm animals, such as but not limited to bovine,
equine, caprine, ovine, and porcine subjects, wild animals (whether
in the wild or in a zoological garden), research animals, such as
mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian
species, such as chickens, turkeys, songbirds, etc., i.e., for
veterinary medical use.
[0088] For the esterase inhibitor compounds of the present
invention, as further studies are conducted, information will
emerge regarding appropriate dosage levels for treatment of various
conditions in various subjects, and the ordinary skilled worker,
considering the therapeutic context, age, and general health of the
recipient, will be able to ascertain proper dosing. The selected
dosage depends upon the desired therapeutic effect, on the route of
administration, and on the duration of the treatment desired.
Generally dosage levels of between 0.001 to 10 mg/kg of body weight
daily are administered to mammals. Generally, for intravenous
injection or infusion dosage may be lower. The dosing schedule may
vary, depending on the circulation half-life, and the formulation
used.
[0089] The esterase inhibitors of the present invention (or their
derivatives) may be administered in conjunction with one or more
additional active ingredients or pharmaceutical compositions.
EXAMPLES
[0090] The following Examples illustrate the invention, but are not
limiting.
Example 1
Carboxylesterase Inhibition Assay
[0091] (a) ortho-nitrophenyl Acetate (o-NPA) as the Substrate
[0092] Carboxylesterase (CE) inhibition was determined by a
spectroscopic assay using o-nitrophenyl acetate (o-NPA) as a
substrate. Recombinant CEs produced from expression in Spodoptera
frugiperda Sf9 insect cells via baculovirus were purified from
serum-free culture media for the enzyme inhibition studies. Enzymes
were incubated in 200 .mu.l of 50 mM Hepes (pH 7.4) containing 3 mM
o-NPA. Conversion of the o-NPA to nitrophenol was monitored by
measuring the change in the absorbance at 420 nm. Absorbance
readings were taken every 15 seconds for 2 minutes, and data
transferred to a computer data spreadsheet. Inhibitors were
dissolved in dimethylsulfoxide (DMSO) and inhibition of CE activity
determined by the addition of the inhibitor solution to the o-NPA
reaction mixture at a concentration of 100 .mu.M. The final DMSO
concentration was always 1% or less. The Ki value (i.e., the
concentration of inhibitor that will bind to half of the binding
sites in the enzyme at equilibrium) was calculated for those
compounds that produced a greater then 50% reduction in the rate of
change of absorbance. The above procedure was then repeated using
the same assay, with inhibitor concentrations ranging from 1 nM to
100 .mu.M. Results were then transferred to Prism software
(GraphPad Software, Inc., San Diego, Calif.) and Ki values
determined using sigmoidal curve fits of the data. Routinely, all
analyses were performed in duplicate, and Ki values determined
using at least 8 inhibitor concentrations.
[0093] Ki values, determined for human small intestine
carboxylesterase (hiCE), human liver carboxylesterase (hCE1),
rabbit liver carboxylesterase (rCE), human acetylcholinesterase
(hAcCHE), and human butyrylcholinesterase (hBuChE) for the
selective human intestinal (hiCE) carboxylesterase inhibitors
identified in Table 3 are set forth in Table 5:
5TABLE 5 Ki Values for the Compounds Identified in Table 3, with
hiCE, hCE1, hAcChE, and rCE, using o = NPA as the Substrate
Compound Ki hiCE Ki hCE1 Ki hAcChE Ki hBuChE Ki rCE Ki (hCE1)/ Code
(nM) (nM) (nM) (nM) (nM) Ki (hiCE) 42 159 >100,000 >100,000
43 850 >100,000 >100,000 44 451 .+-. 39 >100,000
>100,000 >100,000 >100,000 >220 45 53.3 .+-. 5.5 13,700
.+-. 4,870 >100,000 >100,000 1,200 .+-. 230 >250 46 165
.+-. 33 >100,000 >100,000 >100,000 319 .+-. 37 >600 47
194 .+-. 23 >100,000 >100,000 >100,000 >100,000 >500
48 218 .+-. 45 >100,000 >100,000 >100,000 >100,000
>450 49 365 .+-. 87 >100,000 >100,000 >100,000 3,230
.+-. 439 >250 50 767 .+-. 285 >100,000 >100,000
>100,000 739 .+-. 384 >125 51 1,060 .+-. 133 >100,000
>100,000 >100,000 1,550 .+-. 350 >90 52 1,310 .+-. 176
>100,000 >100,000 >100,000 2,000 .+-. 755 >75 53 150
>100,000 >100,000 54 320 >100,000 >100,000 55 1280
>100,000 >100,000 56 1524 >100,000 >100,000 57 61
>100,000 >100,000 58 331 >100,000 >100,000 59 41.5 .+-.
6.5 >100,000 >100,000 >100,000 522 .+-. 144 >2,400
[0094] As can be seen from Table 5, Ki values of greater than
100,000 nM observed human liver carboxylesterase (hCE1), human
acetylcholinesterase (hAcChE) and human cholinesterase (hBuChE)
indicate no inhibition of these enzymes using the selective
ylesterase inhibitors identified in Table 3. These inhibitors
demonstrate inhibition of intestinal carboxylesterase hiCE,
confirming the selectivity of these molecules for ion of the hiCE
enzyme over hCE1, hAcCHE and hBuChE.
[0095] Ki values, determined for human small intestine
carboxylesterase (hiCE), human arboxylesterase (hCE1), rabbit liver
carboxylesterase (rCE), human acetylcholinesterase (hAcCHE), and
human butyrylcholinesterase (hBuChE) for other carboxylesterase
inhibitors of the present invention are set forth in Table 6:
6TABLE 6 Ki Values for other carboxylesterase inhibitors of the
present invention, with hiCE, hCE1, and rCE, using o-NPA as the
Substrate. Acetylthiocholine and butyrylthiocholine were used as
substrates for hAcChE and hBuChE, respectively. Compound hiCE
K.sub.i .+-. SE hCE1 K.sub.i .+-. SE rCE K.sub.i .+-. SE hAcChE
hBuChE K.sub.i code (nM) (nM) (nM) K.sub.i (nM) (nM) 1 71.5 .+-. 10
524 .+-. 46 64 .+-. 6.2 >100,000 >100,000 2 14.7 .+-. 1.9
45.1 .+-. 3.2 103 .+-. 19 >100,000 >100,000 3 21.3 .+-. 1.4
77.6 .+-. 5.5 15 .+-. 1.6 >100,000 >100,000 4 >100,000
>100,000 8.7 .+-. 1.3 >100,000 >100,000 5 4.1 .+-. 0.4
99.1 .+-. 10 108 .+-. 8.8 >100,000 >100,000 6 10.3 .+-. 0.6
175 .+-. 8.5 200 .+-. 60 >100,000 >100,000 7 7.9 .+-. 1.0 295
.+-. 9.6 11.9 .+-. 1.1 >100,000 >100,000 8 8.9 .+-. 0.9 3,300
.+-. 558 36 .+-. 12 >100,000 >100,000 9 72.7 .+-. 8.9
>100,000 53.5 .+-. 9.4 >100,000 >100,000 10 1,730 .+-. 245
>100,000 >100,000 >100,000 >100,000 11 209 .+-. 50
>100,000 520 .+-. 31 >100,000 >100,000 12 380 .+-. 113
1,690 .+-. 236 823 .+-. 215 >100,000 >100,000 13 >100,000
>100,000 2,400 >100,000 >100,000 14 30.6 .+-. 5.0 215 .+-.
24 21.7 .+-. 2.5 >100,000 >100,000 15 >100,000 >100,000
4.1 .+-. 0.5 >100,000 >100,000 16 167 .+-. 12 231 .+-. 12 398
.+-. 59 >100,000 >100,000 17 60.4 .+-. 6.0 532 .+-. 35 49.6
.+-. 6.8 >100,000 >100,000 18 >100,000 >100,000 14.1
.+-. 2.5 >100,000 >100,000 19 23.4 .+-. 3.6 73.5 .+-. 8.4
17.5 .+-. 2.7 >100,000 >100,000 20 259 .+-. 53 372 .+-. 99
47.9 .+-. 18 >100,000 >100,000 21 70.2 .+-. 1.1 3,410 .+-.
547 580 .+-. 157 >100,000 >100,000 22 22.8 .+-. 3.9 160 .+-.
32 16.2 .+-. 1.7 >100,000 >100,000 30 353 .+-. 62 1,650 .+-.
300 220 .+-. 37 >100,000 >100,000 31 18.2 .+-. 3.6 47.4 .+-.
6.8 23.9 .+-. 2.9 >100,000 >100,000 32 33.3 .+-. 6.0 125 .+-.
15 119 .+-. 20 >100,000 >100,000 33 139 .+-. 20 1,540 .+-.
316 72.1 .+-. 9.1 >100,000 >100,000 34 397 .+-. 66 18,100
.+-. 5,850 267 .+-. 30 >100,000 >100,000 35 1,200 .+-. 180
>100,000 >100,000 >100,000 >100,000 36 >100,000
>100,000 >100,000 >100,000 >100,000 37 >100,000
>100,000 >100,000 >100,000 >100,000 38 5.6 .+-. 0.4 8.0
.+-. 0.9 6.1 .+-. 0.6 >100,000 >100,000
[0096] Compound 29, whilst demonstrating inhibition of hiCE, hCE1,
and rCE, also inhibited hAcChE
[0097] (b) Irinotecan (CPT-11) as the Substrate
[0098] Since the sullphonamide compounds identified herein are
competitive inhibitors of hiCE, their activity is dependant upon
the substrate used. To access the ability of these compounds to
inhibit the hiCE-mediated activation of CPT-11, Ki values were
determined using CPT-11 as the substrate.
[0099] CPT-11 concenttrations were fixed at 100 .mu.m, and
inhibitor concentrations varied from 1 nM to 10 .mu.M. Following
incubation for an hour at 37.degree. C., the levels of SN-38 in the
reaction mixture was determined by High Performance Liquid
Chromatography (HPLC). Ki values for inhibition of the metabolism
of CPT-11 by the sulphonamide compounds identified in Table 3, are
set for the in Table 7. the Sulphonamide Compounds Identified in
Table 3,
7TABLE 7 Ki hiCE Values for the Sulphonamide Compounds Identified
in Table 3, using CPT-11 as the Substrate Compound Ki hiCE Code
(nM) 44 >100,000 45 141 .+-. 64 46 >100,000 47 >100,000 48
>100,000 49 238 .+-. 29 50 >100,000 51 892 .+-. 67 52 3,220
.+-. 950 59 110 .+-. 23
[0100] As can be seen from a comparison of the date set forth in
Tables 5 and 7, Ki hiCE values are approximately ten-fold higher
for the inhibition of metabolism of CPT-11, than the corresponding
values obtained using o-NPA as the substrate. The observed values
are in the high nM range, indicating that these compounds would be
effective for in vivo applications.
Example 2
Inhibition of Acetylcholinesterase and Butyrylcholinesterase
[0101] The activity of the hiCE selective carboxylesterase
inhibitors identified in Table 3 toward acetylcholinesterase
(AcChE) and butyrylcholinesterase metabolism was investigated.
Purified AcChE was purchased from Sigma Biochemicals (St. Louis,
Mo.) and substrate metabolism was monitored using a
spectrophotometric assay. 1 mM acetylthiocholine was mixed with 0.5
mM of 5,5'-dithio-bis-(2-nitrobenzoi- c acid) in 50 mM Hepes (pH
7.4) in the presence of the inhibitor (100 .mu.M). The reaction was
initiated by the addition of 0.22 U/ml AcChE (where 1 U is the
amount of enzyme that hydrolyzes 1 .mu.mol of acetylthiocholine
iodide per min at pH 7.4 at 37.degree. C.), and the reaction
monitored by measuring the change of the absorbance at 405 nM every
15 seconds 2 minutes. Data was transferred to GraphPad Prism
software, and Ki values were calculated as described in the Example
above.
[0102] Purified BuChE was purchased from Sigma Biochemicals (St.
Louis, Mo.) and substrate metabolism was monitored using a
spectrophotometric assay. 1 nM butyrylthiocholine was mixed with
0.5 mM of 5,5'-dithio-bis-(2-nitrobenzoic acid) in 50 mM Hepes (pH
7.4) in the presence of the inhibitor (100 .mu.M). The reaction was
initiated by the addition of 0.05 U/ml BuChE (where 1 U is the
amount of enzyme that hydrolyzes 1 .mu.mol of butyrylcholine iodide
per min at pH 8.0 at 37.degree. C.), and the reaction monitored by
measuring the change of the absorbance at 405 nM every 15 seconds 2
minutes. Data was transferred to GraphPad Prism software, and Ki
values were calculated as described in the Example above.
[0103] Table 5 sets forth the Ki values for human AcChE using the
compounds described in Table 3. As can clearly be seen, Ki values
greater than 100,000 nM indicate no inhibition of hAcChE or hBuChE
by the selective human intestinal carboxylesterase inhibitors
identified in Table 3.
Example 3
Ability of the Selective hiCE Inhibitors to Cross Cell
Membranes
[0104] To monitor the ability of the compounds to cross the cell
membrane and inhibit carboxylesterase activity intracellularly, an
assay using human tumor cells expressing hiCE was devised.
U373MGhiCE (a human glioma cell line transfected with a plasmid
expressing hiCE) were plated in T25 flasks and allowed to grow to
confluency (approximately 2-5.times.10.sup.5 cells). Inhibitors
were then added at a final concentration of 10 .mu.m, and
incubation allowed to continue for 1 hour. At this time, the cells
were washed extensively with 5 ml of complete media and 2.times.5
ml of Hanks balanced salt solution, and cell extracts were
prepared. Carboxylesterase activity was then determined using o-NPA
as a substrate.
[0105] Table 8 sets forth the level of intracellular enzyme
inhibition following incubation of human glioblastoma tumor cells
expressing human intestinal carboxylesterase with 10 .mu.M of each
compound identified in Table 3.
8TABLE 8 Inhibition of intracellular human intestinal
carboxylesterase following incubation with 10 .mu.M of the compound
identified in Table 3. Inhibition of intracellular Compound Code
carboxylesterase (%) 42 0 43 ND 44 0.6 45 7.5 46 0 47 9.2 48 8.9 49
7.6 50 14.6 51 5.5 52 9.6 53 3.9 54 0 55 ND 56 0 57 24.2 58 11.5 59
8.3 ND--not determined
[0106] As can be seen from Table 8, none of the selective hiCE
inhibitors identified in Table 3 significantly cross cell-membranes
and inhibit carboxylesterase activity intracellularly.
[0107] The present invention is not to be limited in scope by the
specific embodiments described herein. 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 are intended to fall within the
scope of the appended claims.
[0108] All patents, applications, publications, test methods,
literature, and other materials cited herein are hereby
incorporated by reference.
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