U.S. patent application number 14/528511 was filed with the patent office on 2015-07-30 for oxime-based compound, pharmaceutical composition containing the same and method for preparing the same.
The applicant listed for this patent is Chang Gung Univesity. Invention is credited to Pei-Wen Hsieh, Tsong-Long Hwang, Ting-Yi Wang, Wen-Hui Wang.
Application Number | 20150210636 14/528511 |
Document ID | / |
Family ID | 53491911 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150210636 |
Kind Code |
A1 |
Hsieh; Pei-Wen ; et
al. |
July 30, 2015 |
OXIME-BASED COMPOUND, PHARMACEUTICAL COMPOSITION CONTAINING THE
SAME AND METHOD FOR PREPARING THE SAME
Abstract
An oxime-based compound having the following formula (I) or a
pharmaceutically acceptable salt thereof: ##STR00001## wherein: Y
is a carbonyl group or a sulfonyl group; R.sup.1 is selected from
H, OH, a C.sub.1-C.sub.4 alkyl group, and a C.sub.1-C.sub.4 alkoxyl
group; R.sup.2 is selected from OH, a methoxyl group, --OR.sup.4OH,
and --OR.sup.4NH2, R.sup.4 being a C.sub.1-C.sub.3 alkyl group; and
R.sup.3 is H or a pivaloyloxybenzenesulfonyl group.
Inventors: |
Hsieh; Pei-Wen; (Tao-Yuan,
TW) ; Hwang; Tsong-Long; (Tao-Yuan, TW) ;
Wang; Wen-Hui; (Taichung City, TW) ; Wang;
Ting-Yi; (Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chang Gung Univesity |
Tao-Yuan |
|
TW |
|
|
Family ID: |
53491911 |
Appl. No.: |
14/528511 |
Filed: |
October 30, 2014 |
Current U.S.
Class: |
560/138 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 9/10 20180101; A61P 13/12 20180101; A61P 9/02 20180101; A61P
7/00 20180101; A61P 31/04 20180101; C07C 311/29 20130101; A61P
17/00 20180101; A61P 11/00 20180101; A61P 25/00 20180101; A61P
43/00 20180101; A61P 19/02 20180101 |
International
Class: |
C07C 311/29 20060101
C07C311/29; C07C 303/36 20060101 C07C303/36 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 2014 |
TW |
103102695 |
Claims
1. An oxime-based compound having the following formula (I) or a
pharmaceutically acceptable salt thereof: ##STR00021## wherein: Y
is a carbonyl group or a sulfonyl group; R.sup.1 is selected from
the group consisting of H, OH, a C.sub.1-C.sub.4 alkyl group, and a
C.sub.1-C.sub.4 alkoxyl group; R.sup.2 is selected from the group
consisting of OH, a methoxyl group, --OR.sup.4OH, and
--OR.sup.4NH2, R.sup.4 being a C.sub.1-C.sub.3 alkyl group; and
R.sup.3 is H or a pivaloyloxybenzenesulfonyl group.
2. The oxime-based compound of claim 1, wherein Y is a sulfonyl
group.
3. The oxime-based compound of claim 1, wherein R.sup.1 is selected
from the group consisting of H and a C.sub.1-C.sub.4 alkyl
group.
4. The oxime-based compound of claim 3, wherein R.sup.1 is selected
from the group consisting of H, methyl, ethyl, and n-propyl.
5. The oxime-based compound of claim 1, wherein R.sup.2 is selected
from the group consisting of OH, a methoxyl group, and
--OR.sup.4OH.
6. The oxime-based compound of claim 5, wherein --OR.sup.4OH of
R.sup.2 is --OC.sub.3H.sub.6OH.
7. The oxime-based compound of claim 1, selected from the group
consisting of:
(E)-4-(N-(2-(1-(hydroxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate,
(E)-4-(N-(2-(1-(hydroxyimino)propyl)phenyl)sulfamoyl)phenyl
pivalate,
(E)-4-(N-(2-(1-(hydroxyimino)butyl)phenyl)sulfamoyl)phenyl
pivalate, (E)-4-(N-(2-((hydroxyimino)methyl)phenyl)sulfamoyl)phenyl
pivalate,
(E)-4-(N-(2-(1-(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate,
(Z)-4-(N-(2-(1-(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate, and
(E)-4-(N-(2-(1-(3-hydroxypropoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate.
8. A pharmaceutical composition having inhibitory activity on
neutrophil elastase, comprising the oxime-based compound of claim 1
or the pharmaceutically acceptable salt thereof.
9. A pharmaceutical composition having inhibitory activity on
proteinase 3, comprising the oxime-based compound of claim 1 or the
pharmaceutically acceptable salt thereof.
10. A pharmaceutical composition for treatment of an inflammatory
disorder, comprising the oxime-based compound of claim 1 or the
pharmaceutically acceptable salt thereof.
11. The pharmaceutical composition of claim 10, wherein the
inflammatory disorder is selected from the group consisting of:
lung injury, chronic obstructive pulmonary disease, acute
respiratory distress syndrome, emphysema, cystic fibrosis, focal
cerebral ischemic, ischemic-reperfusion injury, glomerulonephritis,
rheumatoid arthritis, bullous pemphigoid, sepsis, and Wegener's
granulomatosis.
12. The pharmaceutical composition of claim 11, wherein the
inflammatory disorder is lung injury.
13. The pharmaceutical composition of claim 12, wherein the
inflammatory disorder is acute lung injury.
14. A method for preparing the oxime-based compound of claim 1,
comprising: reacting a compound of formula (A) with a compound of
formula (B) and a compound of R.sub.2NH.sub.2, ##STR00022## wherein
R.sup.1 and R.sup.3 in formula (A), Y in formula (B), and R.sup.2
in the compound of R.sub.2NH.sub.2 have the same definitions as
R.sup.1, R.sup.3, Y, and R.sup.2 in formula (I).
15. The method of claim 14, which is conducted by: (a) reacting the
compound of formula (A) with the compound of formula (B) so as to
obtain a compound of formula (II); ##STR00023## in which R.sup.1
and R.sup.3 in formula (II) have the same definitions as R.sup.1
and R.sup.3 in formula (I); and (b) reacting the compound of
formula (II) with the compound of R.sub.2NH.sub.2 so as to obtain
the compound of formula (I).
16. The method of claim 14, which is conducted by: (a) reacting the
compound of formula (A) with the compound of R.sub.2NH.sub.2 so as
to form a compound of formula (III) ##STR00024## in which R.sup.1,
R.sup.2, and R.sup.3 in formula (III) have the same definitions as
R.sup.1, R.sup.2, and R.sup.3 in formula (I); and (b) reacting the
compound of formula (III) with the compound of formula (B) so as to
obtain the compound of formula (I).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of Taiwanese Patent
Application No. 103102695, filed on Jan. 24, 2014.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to an oxime-based compound, more
particularly to a 2-aminobenzaldehyde oxime compound that exhibits
anti-inflammatory effect, a method for preparing the same, and a
pharmaceutical composition containing the same.
[0004] 2. Background Information
[0005] Neutrophils play a vital role in the defense of a human body
against infections. In response to inflammatory stimulus, activated
neutrophils secrete a series of cytotoxins, such as superoxide
anion (O.sub.2.sup..cndot.-), precursors of other reactive oxygen
species, serine proteases, and bioactive lipids.
[0006] The molecular weights of neutrophil elastase (NE, also known
as leukocyte elastase or lysosomal elastase) (EC 3.4.21.37) and
proteinase 3 (also known as leukocyte proteinase 3) (EC 3.4.21.76)
are 29-33 kDa and 29-32 kDa, respectively. Both neutrophil elastase
and proteinase 3 belong to chymotrypsin-like serine proteinase and
are usually stored in azurophil granules of the neutrophils.
[0007] The activities of neutrophil elastase and proteinase 3 are
modulated by endogenous inhibitor protein (such as
.alpha.1-protease inhibitor and .alpha.2-macrogloblin) in the body
to maintain homeostasis. The excessive proteases release may cause
tissue damage, and prolonged neutrophil accumulation has an
important role in the pathogenesis of inflammatory disorders.
[0008] The inflammatory disorders related to neutrophil elastase
and proteinase 3 include lung injury (such as acute lung injury),
chronic obstructive pulmonary disease, acute respiratory distress
syndrome, emphysema, cystic fibrosis, focal cerebral ischemic,
ischemic-reperfusion injury, glomerulonephritis, arthritis (such as
rheumatoid arthritis), bullous pemphigoid, sepsis and Wegener's
granulomatosis (see B. Korkmaz et al. (2008), Biochimie,
90:227-242; A. S. Cowburn et al. (2008), Chest, 134:606-612; Y.
Nakano et al. (2009), Journal of Surgical Research, 155:311-317; M.
Hayakawa et al. (2010), Shock, 33:14-18; K. J. Kwon et al. (2013),
Neurosci. Lett., 548:67-72; B. Korkmaz et al. (2013), Semin.
Immunopathol., 35:411-421; B. Korkmaz et al. (2013), Int.
Immunopharmacol., doi: 10.1016/j.intimp.2013.07.003). Therefore,
inhibition of neutrophil elastase and proteinase 3 plays an
important role in the design of a drug for inflammatory disorder
treatment.
[0009] Sivelestat (marketed as Elaspol) is an inhibitor for
neutrophil elastase. However, the manufacturing process for
sivelestat is complex and hazardous, and sivelestat has a lesser
pharmacokinetic effect and is a potential toxin for organs, thereby
limiting its use in clinical applications. Sivelestat is currently
used for treatment of acute respiratory distress syndrome-related
respiratory failure in Japan and Korea (T. Stevens et al. (2011),
The Journal of Pharmacology and Experimental Therapeutics,
339:313-320).
[0010] EP 0347168 B1 discloses phenyl ester derivatives of pivalic
acid having a general formula (I) as follows:
##STR00002##
[0011] Each group of the phenyl ester derivatives of formula (I) is
defined as that in the disclosure of EP 0347168 B1. From Table 1 of
EP 0347168 B1, twenty compounds are proven to exhibit inhibitory
effect on the activity of neutrophil elastase. The following two
compounds are included in the twenty compounds:
Example 2(63)
[0012] N--[O-(p-pivaloyloxybenzene) sulfonylaminobenzoyl]glycine,
wherein R.sup.1 is
##STR00003##
R.sup.2 is H, R.sup.3 is H, m is 1, and Y is SO.sub.2; and
Example 5(3)
[0013] p-[N--(O-carboxyphenyl)sulfamoyl]phenyl ester of pivalic
acid, wherein R.sup.1 is
##STR00004##
R.sup.2 is H, R.sup.3 is H, m is 1, and Y is SO.sub.2,
[0014] Han-Hsiang Wang disclosed several anthranilate derivatives,
in which compounds WHH51, WHH52, and WHH53 having the following
general formula (II) were proven to be capable of effectively
inhibiting release of neutrophil elastase (see the thesis of "The
Structure-activity Relationships Study of Anti-inflammatory
Activity Anthranilate Derivatives," Graduate Institute of Natural
Products, Chang Gung University (2010)).
##STR00005##
[0015] In compound WHH51, ring A is benzene ring, R.sup.1 is
OCH.sub.3, R.sup.2 is 4-OCOC(CH.sub.3).sub.3, W is CH, X is NH, Y
is SO.sub.2, and Z is a single bond. In compound WHH52, ring A is
benzene ring, R.sup.1 is OCH.sub.2CH.sub.3, R.sup.2 is
4-OCOC(CH.sub.3).sub.3, W is CH, X is NH, Y is SO.sub.2, and Z is a
single bond. In compound WHH53, ring A is benzene ring, R.sup.1 is
NHCH.sub.2COOCH.sub.3, R.sup.2 is 4-OCOC(CH.sub.3).sub.3, W is CH,
X is NH, Y is SO.sub.2, and Z is a single bond.
[0016] The manufacture of the abovementioned derivatives having
formulas (I) and (II) is complex and hazardous due to the use of
hydrogen gas. Moreover, the effects thereof on inhibition of
proteinase 3 and on treatment of inflammation disorders in vivo
have not been proven. Therefore, there is a need in the art to
develop novel compounds that are effective in the treatment of
inflammatory disorders and that can be easily and safely
manufactured.
SUMMARY OF THE INVENTION
[0017] Accordingly, the present invention is to provide a compound
having superior effects on inhibition of proteinase 3 and
neutrophil elastase and on treatment of inflammation disorders.
[0018] Initially, the present invention provides an oxime-based
compound having the following formula (I) or a pharmaceutically
acceptable salt thereof:
##STR00006## [0019] wherein: [0020] Y is a carbonyl group or a
sulfonyl group; [0021] R.sup.1 is selected from the group
consisting of H, OH, a C.sub.1-C.sub.4 alkyl group, and a
C.sub.1-C.sub.4 alkoxyl group; [0022] R.sup.2 is selected from the
group consisting of OH, a methoxy group, --OR.sup.4OH, and
--OR.sup.4NH.sub.2, R.sup.4 being a C.sub.1-C.sub.3 alkyl group;
and [0023] R.sup.3 is H or a pivaloyloxybenzenesulfonyl group.
[0024] Secondly, the present invention also provides a method for
preparing the oxime-based compound of formula (I), including
reacting a compound of formula (A) with a compound of formula (B)
and a compound of R.sub.2NH.sub.2,
##STR00007## [0025] wherein R.sup.1 and R.sup.3 in formula (A), Y
in formula (B), and R.sup.2 in the compound of R.sub.2NH.sub.2 have
the same definitions as R.sup.1, R.sup.3, Y, and R.sup.2 in formula
(I).
[0026] Thirdly, this invention provides a pharmaceutical
composition having inhibitory activity on neutrophil elastase,
including the oxime-based compound of formula (I) or the
pharmaceutically acceptable salt thereof.
[0027] Fourthly, this invention provides a pharmaceutical
composition having inhibitory activity on proteinase 3, including
the oxime-based compound of formula (I) or the pharmaceutically
acceptable salt thereof.
[0028] Fifthly, the present invention provides a pharmaceutical
composition for treatment of inflammatory disorders, including the
oxime-based compound of formula (I) or the pharmaceutically
acceptable salt thereof.
[0029] Sixthly, this invention provides a method to inhibit
neutrophil elastase activity through administering the oxime-based
compound of formula (I) or the pharmaceutically acceptable salt
thereof.
[0030] Seventhly, this invention provides a method to inhibit
protease 3 activity through administering the oxime-based compound
of formula (I) or the pharmaceutically acceptable salt thereof.
[0031] Eighthly, the present invention provides a method to treat
an inflammatory disorder through administering the oxime-based
compound of formula (I) or the pharmaceutically acceptable salt
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Other features and advantages of the present invention will
be clarified not only via following detailed description of the
preferred embodiments of this invention, but also through reference
to the figures (FIG. 1-3), in which:
[0033] FIG. 1 shows the effect of compound (1) of an example of
this invention upon the change of foot thickness of mice from each
of a pathological control group (injected with human neutrophil
elastase only), a positive control group (injected with human
neutrophil elastase and Sivelestat) and experimental groups 1 and 2
(injected with human neutrophil elastase and compound (1)),
measured using a vernier caliper at 10.sup.th, 20.sup.th,
30.sup.th, 40.sup.th, 50.sup.th, 60.sup.th, 90.sup.th, 120.sup.th
and 240.sup.th minute after injection of human neutrophil elastase;
the change of foot thickness was determined as the difference in
foot thickness before and after injection of human neutrophil
elastase; "*" is indicative of p<0.05 when compared to the
pathological control group; "*" is indicative of p<0.01 when
compared to the pathological control group; and "***" is indicative
of p<0.001 when compared to the pathological control group;
[0034] FIG. 2 shows the effect of compound (1) on myeloperoxidase
(MPO) activity in lung tissues of mice from each of a normal
control group (injected with a saline solution but not administered
with LPS), a pathological control group (injected with a saline
solution and administered with LPS), a positive control group
(administered with LPS and injected with Sivelestat) and an
experimental group (administered with LPS and injected with
Compound (1)); "**" is indicative of p<0.01 when compared to the
pathological control group; and
[0035] FIG. 3 shows pictures of lung tissues from the mice in the
normal control group, the pathological control group, the positive
control group and the experimental group of FIG. 2, observed using
an optical microscope (IX81, Olympus) at 400.times. magnification
and captured using a digital camera (DP72, Olympus).
DETAILED DESCRIPTION
[0036] An oxime-based compound of the present invention has the
following formula (I):
##STR00008## [0037] wherein:
[0038] Y is a carbonyl group or a sulfonyl group;
[0039] R.sup.1 is selected from the group consisting of H, OH, a
C.sub.1-C.sub.4 alky group, and a C.sub.1-C.sub.4 alkoxyl
group;
[0040] R.sup.2 is selected from the group consisting of OH, a
methoxyl group, --OR.sup.4OH, and OR.sup.4NH2, R.sup.4 being a
C.sub.1-C.sub.3 alkyl group; and
[0041] R.sup.3 is H or a pivaloyloxybenzenesulfonyl group.
[0042] The term "alkyl group" referred to herein is a saturated
monovalent hydrocarbon group having straight chain or branched
chain moieties. Thus, the term "C.sub.1-C.sub.4 alkyl group"
referred to herein is a straight or branched saturated monovalent
hydrocarbon group having 1 to 4 carbon atoms, and the term
"C.sub.1-C.sub.3 alkyl group" referred to herein is a straight or
branched saturated monovalent hydrocarbon group having 1 to 3
carbon atoms. Examples of the C.sub.1-C.sub.4 alkyl group include,
but are not limited to, methyl, ethyl, n-propyl, isopropyl,
n-butyl, iso-butyl, sec-butyl, tert-butyl, and branched chain
isomers thereof. Examples of the C.sub.1-C.sub.3 alkyl group
include, but are not limited to, methyl, ethyl, propyl, and
isopropyl.
[0043] The term "C.sub.1-C.sub.4 alkoxyl group" referred to herein
is a group with chemical formula --OR', in which R' is a
C.sub.1-C.sub.4 alkyl group as defined above. Examples of the
C.sub.1-C.sub.4 alkoxy group include, but are not limited to,
methoxyl, ethoxyl, n-propoxyl, iso-propoxyl, n-butoxyl,
iso-butoxyl, sec-butoxyl, and tert-butoxyl.
[0044] Preferably, Y is a sulfonyl group.
[0045] Preferably, R.sup.1 is H or a C.sub.1-C.sub.4 alkyl
group.
[0046] In examples of this invention, R.sup.1 is H, methyl, ethyl,
or n-propyl.
[0047] Preferably, R.sup.2 is OH, a methoxyl group, or
--OR.sup.4OH.
[0048] In examples of this invention, R.sup.2 is OH, a methoxyl
group, or --OC.sub.3H.sub.6OH.
[0049] The oxime-based compound of this invention is preferably
selected from: [0050]
(E)-4-(N-(2-(1-(hydroxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate, [0051]
(E)-4-(N-(2-(1-(hydroxyimino)propyl)phenyl)sulfamoyl)phenyl
pivalate, [0052]
(E)-4-(N-(2-(1-(hydroxyimino)butyl)phenyl)sulfamoyl)phenyl
pivalate, [0053]
(E)-4-(N-(2-((hydroxyimino)methyl)phenyl)sulfamoyl)phenyl pivalate,
[0054] (E)-4-(N-(2-(1-(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate, [0055]
(Z)-4-(N-(2-(1-(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate, and [0056]
(E)-4-(N-(2-(1-(3-hydroxypropoxyimino)ethyl)phenyl)sulfamoyl)pheny-
l pivalate.
[0057] The oxime-based compound of the present invention can exist
in free form, or where appropriate, as a pharmaceutically
acceptable salt, solvate, hydrate, or stereoisomers thereof.
[0058] The pharmaceutically acceptable salt of the oxime-based
compound may be, but is not limited to, a salt with an inorganic
acid, such as HCl, HBr, H.sub.2SO.sub.4, and H.sub.2PO.sub.4, a
salt with an organic acid, such as acetate, maleate, tartrate, and
methanesulfonate, or a salt with an amino acid, such as arginine,
aspartic acid, and glutamic acid.
[0059] A method for preparing the oxime-based compound of formula
(I) includes: [0060] reacting a compound of formula (A) with a
compound of formula (B) and a compound of R.sub.2NH.sub.2,
[0060] ##STR00009## [0061] wherein R.sup.1 and R.sup.3 in formula
(A), Y in formula (B), and R.sup.2 in the compound of
R.sub.2NH.sub.2 have the same definitions as R.sup.1, R.sup.3, Y,
and R.sup.2 in formula (I).
[0062] Preferably, the method is conducted by: [0063] (a) reacting
the compound of formula (A) with the compound of formula (B) so as
to obtain a compound of formula (II);
[0063] ##STR00010## [0064] in which R.sup.1 and R.sup.3 in formula
(II) have the same definitions as R.sup.1 and R.sup.3 in formula
(I); and [0065] (b) reacting the compound of formula (II) with the
compound of R.sub.2NH.sub.2 so as to obtain the compound of formula
(I).
[0066] Preferably, the method is conducted by: [0067] (a) reacting
the compound of formula (A) with the compound of R.sub.2NH.sub.2 so
as to form a compound of formula (III)
[0067] ##STR00011## [0068] is in which R.sup.1, R.sup.2, and
R.sup.3 in formula (III) have the same definitions as R.sup.1,
R.sup.2, and R.sup.3 in formula (I); and [0069] (b) reacting the
compound of formula (III) with the compound of formula (B) so as to
obtain the compound of formula (I).
[0070] The oxime-based compound of formula (I) has been proven to
be able to inhibit release of human neutrophil elastase and to
exhibit inhibitory activity on human neutrophil elastase and
protease-3 in vitro. Moreover, the oxime-based compound of formula
(I) could effectively treat lipopolysaccharide (LPS)-induced acute
lung injury and foot edema induced by human neutrophil elastase in
vivo. The oxime-based compound of formula (I) is thus predicted to
be able to treat an inflammatory disorder.
[0071] Therefore, this invention provides a pharmaceutical
composition having inhibitory activity on neutrophil elastase,
including the oxime-based compound of formula (I) or the
pharmaceutically acceptable salt thereof. This invention also
provides a pharmaceutical composition having inhibitory activity on
proteinase 3, including the oxime-based compound of formula (I) or
the pharmaceutically acceptable salt thereof. This invention
further provides a pharmaceutical composition for treatment of an
inflammatory disorder, including the oxime-based compound of
formula (I) or the pharmaceutically acceptable salt thereof.
[0072] As used herein, the terms "treat," "treatment," and
"treating," refer to preventing, reducing, alleviating,
ameliorating, relieving, or controlling at least one of clinical
signs of a disease or a disorder, or lowering, stopping, and
reversing severity or progression of a condition or a symptom.
[0073] The inflammatory disorder of this invention, includes, but
is not limited to, lung injury, chronic obstructive pulmonary
disease, acute respiratory distress syndrome, emphysema, cystic
fibrosis, focal cerebral ischemic, ischemic-reperfusion injury,
glomerulonephritis, rheumatoid arthritis, bullous pemphigoid,
sepsis, and Wegener's granulomatosis. (B. Korkmaz et al. (2008),
Biochimie, 90:227-242; A. S. Cowburn et al, (2008), Chest,
134:606-612; Y. Nakano et al. (2009), Journal of Surgical Research,
155:311-317; M. Hayakawa et al. (2010), Shock, 33:14-18; K. J. Kwon
et al. (2013), Neurosci. Lett., 548:67-72; B. Korkmaz et al.
(2013), Semin. Immunopathol., 35:411-421; B. Korkmaz et al. (2013),
Int. Immunopharmacol., doi: 10.1016/j.intimp.2013.07.003).
Preferably, the inflammatory disorder is lung injury, more
preferably, acute lung injury.
[0074] The pharmaceutical composition of this invention may be
parenterally, orally, or topically administrable and can be
formulated into a dosage form, such as injection (e.g., sterile
aqueous solutions, dispersions, etc.), sterile powders, tablets,
troches, pills, capsules, external preparation, etc.
[0075] The pharmaceutical composition of this invention may be
administered by a parenteral route, such as intraperitoneal,
subcutaneous, intramuscular, or intravenous injection. In this
embodiment, the pharmaceutical composition of this invention is
administrated by intraperitoneal rejection.
[0076] The pharmaceutical composition of this invention further
includes a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier includes at least one of the
following agents: a solvent, a buffer, an emulsifier, a suspending
agent, a decomposer, a disintegrating agent, a dispersing agent, a
binding agent, an excipient, a stabilizing agent, a chelating
agent, a diluent, a gelling agent, a preservative, a wetting agent,
a lubricant, an absorption delaying agent, a liposome, etc.
Examples of the pharmaceutically acceptable carrier include, but
are not limited to, water, normal saline, phosphate buffered saline
(PBS), a sugar-containing solution, and an aqueous solution
containing alcohol.
[0077] This invention provides a method to inhibit neutrophil
elastase activity or protease 3 activity in a subject, including
administering the oxime-based compound of formula (I) or the
pharmaceutically acceptable salt thereof to the subject. This
invention also provides a method to treat an inflammatory disorder
in a subject, including applying the oxime-based compound of
formula (I) or the pharmaceutically acceptable salt thereof to the
subject.
EXAMPLES
Preparation Examples
General Method
[0078] 1. Silica gel column chromatography was conducted using a
silica gel 60 (sieve mesh 230-400, available from Silicycle).
[0079] 2. .sup.1H-NMR and .sup.13C-NMR were obtained using Brucker
AVANCe-400 MHz FT-NMR, a nuclear magnetic resonance spectrometer.
CDCl.sub.3 with 7.265 ppm of .delta. and CDCl.sub.3 with 77.0 ppm
of .delta. were used as internal standards to determine chemical
shift. Coupling constant is referred to as J, and the unit is Hz.
[0080] 3. Electrospray ionization mass spectra (ESI-MS) and
high-resolution electrospray ionization mass spectra (HRESI-MS)
were obtained using TSQ Quantum Triple Quadrupole mass spectrometer
and Orbitrap mass spectrometer respectively, both available from
Thermo Finnigan LLC. [0081] 4. Preparation of oxime-based compounds
of formula (I) Compounds of formula (I) can be manufactured using
one of the following Schemes 1 to 3.
[0082] In Scheme 1, 2-aminobenzaldehyde or 2-aminophenyl ketone
(Compound (a1)) is reacted with p-pivaloyloxybenzenesulfonyl
chloride to obtain 4-(N-(2-acylphenyl)sulfamoyl)phenyl pivalate
(Compound (a2)). Compound (a2) is then reacted with hydroxylamine
hydrochloride to obtain the compound of formula (I). The following
Compounds (1), (2), (3), and (4) in Preparation Examples 1 to 4
were prepared using Scheme 1.
##STR00012##
[0083] In Scheme 2, Compound (a1) is reacted with methoxylamine
hydrochloride to obtain 1-(2-aminophenyl)ethanone O-methyl oxime
(Compound (a3)). Compound (a3) is then reacted with
p-pivaloyloxybenzenesulfonyl chloride to obtain the oxime-based
compound of formula (I). The following Compounds (5) and (6), which
are geometrical isomers, in Preparation Examples 5 were prepared
using Scheme 2.
##STR00013##
[0084] In Scheme 3, Compound (a1) is reacted with hydroxylamine
hydrochloride to obtain (E)-1-(2-aminophenyl)ethanone oxime
(Compound (a4)). Compound (a4) is reacted with 3-chloropropan-1-ol
to obtain (E)-1-(2-aminophenyl)ethanone O-3-hydroxypropyl oxime
(Compound (a5)). Compound (a5) is then reacted with
p-pivaloyloxybenzenesulfonyl chloride to obtain the compound of
formula (I). The following Compound (7) in Preparation Examples 6
was prepared using Scheme 3.
##STR00014##
[0085] It should be noted that Compounds (1), (2), (3), and (4) can
be prepared through Scheme 2, in which hydroxylamine hydrochloride
is used instead of methoxylamine hydrochloride. That is, Compound
(a1) is first reacted with hydroxylamine hydrochloride, followed by
reacting with p-pivaloyloxybenzenesulfonyl chloride. Compound (7)
also can be prepared through Scheme 2, in which
O-3-hydroxypropylhydroxyamine hydrochloride is used instead of
methoxylamine hydrochloride.
Preparation Example 1
Preparation of Compound (1)
((E)-4-(N-(2-(1(hydroxyimino)ethyl)phenyl)sulfamoyl) phenyl
pivalate) having the following formula
##STR00015##
[0086] A. Preparation of 4-(N-(2-acetylphenyl)sulfamoyl)phenyl
pivalate
[0087] 2-Aminoacetophenone (1 mmol) and
p-pivaloyloxybenzenesulfonyl chloride (1.5 equivalent weights) were
added into pyridine (5 mL) to obtain a mixture, followed by
subjecting to reaction at room temperature for 4 hours and then
removing pyridine under vacuum to obtain a reaction product. The
reaction product was purified using a silica gel column
(n-hexane/acetone=4:1), thereby obtaining
4-(N-(2-acetylphenyl)sulfamoyl)phenyl pivalate (331 mg, yield
89%).
B. Preparation of Compound (1)
[0088] 4-(N-(2-acetylphenyl)sulfamoyl)phenyl pivalate (0.2 mmol)
and hydroxylamine hydrochloride (1.5 equivalent weights) were added
into ethanol (5 mL) to obtain a mixture, followed by heating the
mixture under reflux for 14 hours and then removing ethanol by
vacuum concentration to obtain a reaction product. The reaction
product was purified using a silica gel column
(n-hexane/acetone=4:1), thereby obtaining Compound (1) as a white
powder (105 mg, yield 27%).
Structure Identification
[0089] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 10.86 (1H, s,
NH), 8.51 (1H, s, OH), 7.73 (2H, d, J=8.8 Hz), 7.65 (1H, dd, J=1.2,
8.0 Hz), 7.33 (1H, dd, J=1.2, 8.0 Hz), 7.28 (1H, td, J=1.2, 8.0
Hz), 7.11 (1H, td, J=1.2, 8.0 Hz), 7.09 (2H, d, J=8.8 Hz), 2.05
(3H, s, CH.sub.3), 1.33 (9H, s, (CH.sub.3).sub.3); .sup.13C-NMR
(CDCl.sub.3, 100 MHz) .delta.: 176.9 (s, --OCO--), 157.3 (s,
C.dbd.N), 154.9 (s, C-4'), 136.5 (s, C-1'), 135.7 (s, C-2), 130.0
(d, C-4), 129.2 (d, C-2', 6'), 129.0 (d, C-6), 125.9 (s, C-1),
125.1 (d, C-5), 122.9 (d, C-3), 122.5 (d, C-3', 5'), 39.6 (s,
C(CH.sub.3).sub.3), 27.4 (q, (CH.sub.3).sub.3), 12.8 (q, CH.sub.3);
ESI-MS: m/z 390.9 ([M+H].sup.+); HRESI-MS: m/z 391.1322
([M+H].sup.+) (calcd. for C.sub.19H.sub.23O.sub.5N.sub.2S,
391.1330).
Preparation Example 2
Preparation of Compound (2)
((E)-4-(N-(2-(1-(hydroxyimino)propyl)phenyl)sulfamoyl)phenyl
pivalate) having the following formula
##STR00016##
[0090] A. Preparation of 1-(2-aminophenyl)propan-1-one
[0091] 2-Aminobenzonitrile (1 mmol) was disposed in anhydrous
tetrahydrofuran (5 ml) followed by adding ethylmagnesium bromide
(10 equivalent weights) at 0.degree. C. under nitrogen gas
atmosphere and reacting for 4 hours to obtain a reaction product.
The pH of the reaction product was adjusted to be in the range of 3
to 4 using 1N HCl, followed by partitioning using ethyl acetate and
saturated NaHCO.sub.3 for three times. The ethyl acetate layer was
collected and purified using a silica gel column
(n-hexane/acetone=4:1), thereby obtaining
[1-(2-aminophenyl)propan-1-one] (83.5 mg, yield 56%).
B. Preparation of 4-(N-(2-propionylphenyl)sulfamoyl)phenyl
pivalate
[0092] The procedure for preparing
4-(N-(2-propionylphenyl)sulfamoyl)phenyl pivalate is similar to
procedure A in Preparation Example 1. The difference resides in
that the 1-(2-aminophenyl)propan-1-one (0.5 mmol) was used to
replace 2-aminoacetophenone (1 mmol).
4-(N-(2-propionylphenyl)sulfamoyl)phenyl pivalate was purified
using a silica gel column (n-hexane/acetone=4:1) (263 mg, yield
68%).
C. Preparation of Compound (2)
[0093] The procedure for preparing Compound (2) is similar to
procedure B in Preparation Example 1. The difference resides in
that 4-(N-(2-propionylphenyl)sulfamoyl)phenyl pivalate (0.5 mmol)
was used to replace 4-(N-(2-acetylphenyl)sulfamoyl)phenyl pivalate]
(0.2 mmol). A silica gel column (n-hexane/acetone=4:1) was used to
afford compound (2) of a white powder (153 mg, yield 37.8%).
Structure Identification
[0094] .sup.1H NMR (400 MHz, CDC.sub.13) .delta.: 11.04 (1H, s,
NH), 8.40 (1H, s, OH), 7.76 (2H, d, J=8.8 Hz), 7.67 (1H, dd, J=1.2,
8.0 Hz), 7.37 (1H, dd, J=1.2, 8.0 Hz), 7.27 (1H, td, J=1.2, 8.0
Hz), 7.10 (1H, td, J=1.2, 8.0 Hz), 7.09 (2H, d, J=8.8 Hz), 2.64
(2H, q, J=7.6 Hz, CH2), 1.32 (9H, s, (CH.sub.3).sub.3), 0.99 (3H,
t, J=7.6 Hz, CH.sub.3); .sup.13C-NMR (CDCl.sub.3, 100 MHz) .delta.:
176.4 (s, --OCO--), 161.8 (s, C.dbd.N), 154.4 (s, C-4'), 136.3 (s,
C-1'), 135.9 (s, C-2), 129.6 (d, C-4), 128.8 (d, C-2', 6'), 128.3
(d, C-6), 124.4 (s, C-1), 123.6 (d, C-5), 122.0 (d, C-3, 3', 5'),
39.2 (s, C(CH.sub.3).sub.3), 27.0 (q, (CH.sub.3).sub.3), 19.5 (t,
CH.sub.2CH.sub.3), 10.8 (q, CH.sub.2CH.sub.3); ESI-MS: m/z 405.2
([M+H].sup.+), 427.2 ([M+Na].sup.+), 830.8 ([M+M+Na].sup.+);
HRESI-MS: m/z 405.1500 ([M+H].sup.+) (calcd. for
C.sub.20H.sub.25O.sub.5N.sub.2S, 405.1500).
Preparation Example 3
Preparation of Compound (3)
((E)-4-(N-(2-(1-(hydroxyimino)butyl)phenyl)sulfamoyl)phenyl
pivalate) having the following formula
##STR00017##
[0095] A. Preparation of 1-(2-aminophenyl)butan-1-one
[0096] The procedure for preparing 1-(2-aminophenyl)butan-1-one is
similar to procedure A in Preparation Example 2. The difference
resides in that propylmagnesium chloride was used to replace
ethylmagnesium bromide. 1-(2-aminophenyl)butan-1-one was purified
using a silica gel column (n-hexane/acetone=4:1) (91 mg, yield
56%).
B. Preparation of 4-(N-(2-butyrylphenyl)sulfamoyl)phenyl
pivalate
[0097] The procedure for preparing
4-(N-(2-butyrylphenyl)sulfamoyl)phenyl pivalate is similar to
procedure A in Preparation Example 1. The difference resides in
that 1-(2-aminophenyl)butan-1-one (0.5 mmol) was used to replace
2-amino acetophenone (1 mmol).
4-(N-(2-butyrylphenyl)sulfamoyl)phenyl pivalate was purified using
a silica gel column (n-hexane/acetone=5:1) (293 mg, yield 73%).
C. Preparation of Compound (3)
[0098] The procedure for preparing Compound (3) is similar to
procedure B in Preparation Example 1. The differences reside in
that 4-(N-(2-butyrylphenyl)sulfamoyl)phenyl pivalate (0.5 mmol) was
used to replace 4-(N-(2-acetylphenyl)sulfamoyl)phenyl pivalate (0.2
mmol). Compound (3) was purified using a silica gel column
(n-hexane/acetone=5:1), thereby obtaining Compound (3) of a white
powder (292 mg, yield 70%).
Structure Identification
[0099] .sup.1H NMR (400 MHz, CDCl3) .delta.: 11.07 (1H, s, NH),
7.77 (2H, d, J=8, 8 Hz), 7.68 (1H, dd, J=1.2, 8.0 Hz), 7.36 (1H,
dd, J=1.2, 8.0 Hz), 7.27 (1H, td, J=1.2, 8.0 Hz), 7.10 (1H, td,
J=1.2, 8.0 Hz), 7.09 (2H, d, J=8.8 Hz), 2.61 (2H, t, J=7.6 Hz,
CH.sub.2), 1.35 (2H, sext., J=7.6 Hz, CH.sub.2), 1.32 (9H, s,
(CH.sub.3).sub.3), 0.89 (3H, t, J=7.6 Hz, CH.sub.3); .sup.13C-NMR
(CDCl3, 100 MHz) .delta.: 176.3 (s, --OCO--), 160.8 (s, C.dbd.N),
154.4 (s, C-4'), 136.4 (s, C-1'), 136.0 (s, C-2), 129.6 (d, C-4),
128.7 (d, C-2', 6'), 128.4 (d, C-6), 124.3 (s, C-1), 123.7 (d,
C-5), 122.0 (d, C-3', 5'), 121.8 (d, C-3), 39.2 (s,
C(CH.sub.3).sub.3), 27.9 (t, CH.sub.2CH.sub.2CH.sub.3), 27.0 (q,
(CH.sub.3).sub.3), 19.9 (t, CH.sub.2CH.sub.2CH.sub.3), 14.2 (q,
CH.sub.2CH.sub.2CH.sub.3); ESI-MS: m/z 419.2 ([M+H].sup.+), 441.2
([M+Na].sup.+); HRESI-MS: m/z 419.1635 ([M+H].sup.+) (calcd. for
C.sub.21H.sub.27O.sub.5N.sub.2S, 419.1643).
Preparation Example 4
Preparation of Compound (4)
((E)-4-(N-(2-((hydroxyimino)methyl)phenyl)sulfamoyl)phenyl
pivalate) having the following formula
##STR00018##
[0100] A. Preparation of 4-(N-(2-formylphenyl)sulfamoyl)phenyl
pivalate
[0101] The procedure for preparing
4-(N-(2-formylphenyl)sulfamoyl)phenyl pivalate is similar to
procedure A in Preparation Example 1. The difference resides in
that 2-aminobenzaldehyde was used to replace 2-aminoacetophenone.
4-(N-(2-formylphenyl)sulfamoyl)phenyl pivalate was purified using a
silica gel column (n-hexane/acetone=5:1) (230 mg, yield 64%).
B. Preparation of Compound (4)
[0102] The procedure for preparing Compound (4) is similar to
procedure B in Preparation Example 1. The difference resides in
that 4-(N-(2-formylphenyl)sulfamoyl)phenyl pivalate (0.5 mmol) was
used to replace 4-(N-(2-acetylphenyl)sulfamoyl)phenyl pivalate]
(0.2 mmol). Compound (4) was purified using a silica gel column
(n-hexane/acetone=5:1), thereby obtaining Compound (4) of a white
powder (214 mg, yield 57%).
Structure Identification
[0103] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 10.52 (1H, s,
NH), 8.05 (1H, s, H), 7.87 (2H, d, J=8.8 Hz), 7.64 (1H, br. dd,
J=1.2, 8.0 Hz), 7.26 (1H, td, J=1.2, 8.0 Hz), 7.14 (2H, d, J=8.8
Hz), 7.13 (1H, dd, J=1.2, 8.0 Hz), 7.05 (1H, td, J=1.2, 8.0 Hz),
1.33 (9H, s, (CH.sub.3).sub.3); .sup.13C-NMR (CDCl.sub.3, 100 MHz)
.delta.: 176.4 (s, --OCO--), 154.6 (s, C.dbd.N), 152.1 (s, C-4'),
136.3 (s, C-1', 2), 132.1 (d, C-4), 130.5 (d, C-6), 128.9 (d, C-2',
6'), 123.7 (d, C-5), 122.2 (d, C-3', 5'), 119.5 (s, C-1), 119.1 (d,
C-3), 39.2 (s, C(CH.sub.3).sub.3), 27.0 (q, (CH.sub.3).sub.3);
ESI-MS: 399.1 ([M+Na].sup.+); HRESI-MS: m/z 399.0985 ([M+Na].sup.+)
(calcd. for C.sub.18H.sub.20N.sub.2O.sub.5SNa, 399.0983).
Preparation Example 5
Preparation of Compound (5)
((E)-4-(N-(2-(1-(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate) and Compound (6)
((Z)-4-(N-(2-(1-(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate) having the following formulas
##STR00019##
[0104] A. Preparation of 1-(2-aminophenyl)ethanone O-methyl
oxime
[0105] 2-Aminoacetophenone (1 mmol) was added into ethanol (10 mL),
followed by adding methoxylamine hydrochloride (2.5 equivalent
weights) therein to obtain a mixture. The mixture was heated under
reflux for 12 hours at 80.degree. C., followed by removal of the
ethanol by vacuum concentration. The reaction product was then
partitioned using water and dichloromethane (DCM) for three times.
The organic layer was collected and dried with anhydrous
MgSO.sub.4, followed by filtration and then removal of the solvent
by vacuum concentration, thereby obtaining
1-(2-aminophenyl)ethanone O-methyl oxime (154 mg, yield 94%).
B. Preparation of Compounds (5) and (6)
[0106] The procedure for preparing Compounds (5) and (6) is similar
to procedure A in Preparation Example 1. The difference resides in
that 1-(2-aminophenyl)ethanone O-methyl oxime (0.5 mmol) was used
to replace 2-aminoacetophenone (1 mmol). Compounds (5) and (6) were
purified using a silica gel column (n-hexane/acetone=4:1), thereby
obtaining Compound (5) of a white powder (242 mg, yield 60%) and
Compound (6) of a white powder (10 mg, yield 2.5%).
Structure Identification
Compound (5):
[0107] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 10.77 (1H, s,
NH), 7.69 (2H, d, J=8, 8 Hz), 7.65 (1H, br. dd, J=1.2, 8.4 Hz),
7.30 (1H, br, td, J=1.2, 8.4 Hz), 7.27 (1H, d, J=8.4 Hz), 7.11 (1H,
td, J=1.2, 8.4 Hz), 7.08 (2H, d, J=8.8 Hz), 4.06 (3H, s,
OCH.sub.3), 2.02 (3H, s, CH.sub.3), 1.33 (9H, s, (CH.sub.3).sub.3);
.sup.13C-NMR (CDCl.sub.3, 100 MHz) .delta.: 176.2 (s, --OCO--),
156.1 (s, C.dbd.N), 154.4 (s, C-4'), 136.4 (s, C-1'), 135.4 (s,
C-2), 129.7 (d, C-6), 128.7 (d, C-2', 6'), 128.5 (d, C-4), 125.0
(s, C-1), 124.5 (d, C-5), 122.3 (d, C-3', 5'), 121.9 (d, C-3), 62.6
(q, OCH.sub.3), 39.2 (s, C(CH.sub.3).sub.3), 27.0 (q,
(CH.sub.3).sub.3), 13.2 (q, CH.sub.3); ESI-MS: 403.4 ([M-H].sup.-);
HRESI-MS: m/z 403.1322 ([M-H].sup.-) (calcd. for
C.sub.20H.sub.23O.sub.5N.sub.2S, 403.1331).
Compound (6):
[0108] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 7.66 (2H, d,
J=8.8 Hz), 7.64 (1H, br. dd, J=1.2, 7.6 Hz), 7.40 (1H, td, J=1.2,
7.6 Hz), 7.23 (1H, td, J=1.2, 7.6 Hz), 7.10 (2H, d, J=8.8 Hz), 7.05
(1H, d, J=1.2, 7.6 Hz), 3.91 (3H, s, OCH.sub.3), 1.57 (3H, s,
CH.sub.3), 1.36 (9H, s, (CH.sub.3).sub.3); .sup.13C-NMR
(CDCl.sub.3, 100 MHz) .delta.: 176.3 (s, --OCO--), 154.6 (s,
C.dbd.N), 153.6 (s, C-4'), 136.7 (s, C-1'), 132.7 (s, C-2), 130.3
(d, C-1), 130.1 (s, C-6), 128.8 (d, C-2', 6'), 127.3 (d, C-4),
126.5 (d, C-3), 126.3 (d, C-3), 122.3 (d, C-3', 5'), 62.1 (q,
OCH.sub.3), 39.2 (s, C(CH.sub.3).sub.3), 27.0 (q,
(CH.sub.3).sub.3), 21.3 (q, CH.sub.3); ESI-MS: 427.1
([M+Na].sup.+); HRESI-MS: m/z 427.1298 ([M+Na].sup.+) (calcd. for
C.sub.18H.sub.20N.sub.2O.sub.5SNa, 427.1294).
Preparation Example 6
Preparation of Compound (7)
((E)-4-(N-(2-(1-(3-hydroxypropoxyimino)ethyl)phenyl)sulfamoyl)phenyl
pivalate) having the following formula
##STR00020##
[0109] A. Preparation of (E)-1-(2-aminophenyl)ethanone oxime
[0110] 2-Aminoacetophenone (1.5 mmol) and hydroxylamine
hydrochloride (3.75 equivalent weights) were added into ethanol (5
mL) to obtain a mixture. The mixture was heated under reflux for 16
hours at 70.degree. C., followed by removal of ethanol by vacuum
concentration and partition using water and ethyl acetate for three
times. The organic layer was collected and dried with anhydrous
MgSO.sub.4, followed by filtration and then removal of the solvent
by vacuum concentration, thereby obtaining
(E)-1-(2-aminophenyl)ethanone oxime (215 mg, yield 95%).
B. Preparation of (E)-1-(2-aminophenyl)ethanone O-3-hydroxypropyl
oxime
[0111] (E)-1-(2-aminophenyl)ethanone oxime (0.2 mmol) was added
into anhydrous acetonitrile (MeCN) (10 mL), followed by adding
K.sub.2CO.sub.3 (1.1 equivalent weights) and 3-chloropropan-1-ol (5
equivalent weights) therein to obtain a mixture. The mixture was
heated under reflux for 36 hours and was purified using a silica
gel column (n-hexane/acetone=2:1), thereby obtaining
(E)-1-(2-aminophenyl)ethanone O-3-hydroxypropyl oxime (168 mg,
yield 81%).
C. Preparation of Compound (7)
[0112] (E)-1-(2-aminophenyl)ethanone O-3-hydroxypropyl oxime (0.2
mmol) was added into dichloromethane (DCM) (5 mL), followed by
adding pyridine (10 equivalent weights) and
4-(chlorosulfonyl)phenyl pivalate (2 equivalent weights) therein to
obtain a mixture. The mixture was heated under reflux for 16 hours
and was purified using a silica gel column
(n-hexane/acetone=.delta.: 1), thereby obtaining Compound (7) of a
white powder (43 mg, yield 49%).
Structure Identification
[0113] .sup.1H-NMR (CDCl.sub.3, 400 MHz) .delta.: 10.82 (1H, s,
NH), 7.71 (2H, br. dd, J=8.8 Hz, H-3', 5'), 7.63 (1H, dd, J=1.2,
8.0 Hz, H-3), 7.33 (1H, dd, J=1.2, 8.0 Hz, H-6), 7.28 (1H, td,
J=1.2, 8.0 Hz, H-5), 7.09 (2H, d, J=8.8 Hz, H-2', 6'), 7.10 (1H,
td, J=1.2, 8.0 Hz, H-4), 4.39 (2H, t, J=6.4 Hz, .dbd.NOCH.sub.2--),
3.82 (2H, t, J=6.0 Hz, --CH.sub.2OH), 3.82 (2H, t, J=6.0 Hz,
--CH.sub.2OH), 2.06 (2H, m, CH.sub.2), 2.06 (3H, s, CH.sub.3), 1.33
(9H, s, (CH.sub.3).sub.3); .sup.13C-NMR (CDCl.sub.3, 100 MHz)
.delta.: 176.3 (s, --OCO--), 156.2 (s, C.dbd.N), 154.4 (s, C-4'),
136.4 (s, C-2), 135.4 (s, C-1'), 129.7 (d, C-4), 128.6 (d, C-6),
128.6 (d, C-2', 6'), 124.8 (d, C-5), 124.4 (s, C-1), 122.0 (d,
C-3', 5'), 121.7 (d, C-3), 72.1 (t, .dbd.NOCH.sub.2--), 59.8
(--CH.sub.2OH), 39.2 (s, C(CH.sub.3).sub.3), 32.2 (t, CH.sub.2),
27.0 (q, (CH.sub.3).sub.3), 13.3 (q, CH.sub.3); ESI-MS: 449.2
([M+H].sup.+), 471.2 ([M+Na].sup.+); HRESI-MS: m/z 449.1761
([M+H].sup.+) (calcd. for C.sub.22H.sub.29N.sub.2O.sub.6S,
449.1741).
[0114] Compounds (1) to (7) prepared by Preparation Examples 1 to 6
are listed in Table 1.
TABLE-US-00001 TABLE 1 Comp. IUPAC Naming R.sup.1 1 (E)-4-(N-(2-(1-
--CH.sub.3 (hydroxyimino)ethyl)phenyl)sulfamoyl)phenyl pivalate 2
(E)-4-(N-(2-(1- --CH.sub.2CH.sub.3
(hydroxyimino)propyl)phenyl)sulfamoyl)phenyl pivalate 3
(E)-4-(N-(2-(1- --CH.sub.2CH.sub.2CH.sub.3
(hydroxyimino)butyl)phenyl)sulfamoyl)phenyl pivalate 4
(E)-4-(N-(2-((hydroxyimino)methyl)phenyl)sulfamoyl)phenyl H
pivalate 5 (E)-4-(N-(2-(1- --CH.sub.3
(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl pivalate] 6
(Z)-4-(N-(2-(1- --CH.sub.3
(methoxyimino)ethyl)phenyl)sulfamoyl)phenyl pivalate 7
(E)-4-(N-(2-(1-(3-hydroxypropoxyimino)ethyl)phenyl)sulfamoyl)phenyl
--CH.sub.3 pivalate
Pharmacological Examples
[0115] The following analyses were performed in order to determine
the biological activities of Compounds (1) to (7).
Experimental Materials
1. Ca.sup.2+-Free Hank's Balanced Salt Solution:
[0116] Ca.sup.2+-free Hank's balanced salt solution having pH 7.4
includes ingredients shown in Table 2. The ingredients were
dissolved in deionized water.
TABLE-US-00002 TABLE 2 Ingredient Conc. (mg/L) NaCl 8000 KCl 400
KH.sub.2PO.sub.4 60 glucose 1000 Na.sub.2HPO.sub.4 48
MgCl.sub.2.cndot.6H.sub.2O 203
2. Hank's Balanced Salt Solution (HBSS)
[0117] HBSS having pH 7.4 includes ingredients shown in Table 3.
The ingredients were dissolved in deionized water.
TABLE-US-00003 TABLE 3 Ingredient Conc. (mg/L)
CaCl.sub.2.cndot.2H.sub.2O 294 NaCl 8000 KCl 400 KH.sub.2PO.sub.4
60 glucose 1000 Na.sub.2HPO.sub.4 48 MgCl.sub.2.cndot.6H.sub.2O
203
3. Preparation of Human Neutrophil:
[0118] Healthy human volunteers (20 to 34 years old) were recruited
through a certified procedure approved by Chang Gung Medical
Foundation Institutional Review Board.
[0119] Blood obtained from the healthy human volunteers by
venipuncture was centrifuged at 650 g for 10 minutes, and a
leukocyte-rich lower layer was obtained by removing the upper layer
containing platelets. 3% dextran T500 solution was well mixed with
the leukocyte-rich lower layer at a volume ratio of 1:1 and was
allowed to stand at room temperature. After standing for 25
minutes, the clear supernatant containing neutrophils was slowly
transferred to a centrifuge tube of Ficoll-Pague.TM. Plus
(14-1440-03, GE Healthcare, Sweden) and was subjected to
density-gradient centrifugation at 400 g and 4.degree. C. for 40
minutes. The precipitate thus obtained was treated with a hypotonic
solution, i.e., 0.2% NaCl, to lyse erythrocytes, followed by
removal of the lysed erythrocytes using centrifugation at 200 g and
4.degree. C. for 8 minutes so as to obtain the neutrophils. The
purified neutrophils having >98% viable cells determined using a
trypan blue exclusion method was suspended in Ca.sup.2+-free Hank's
balanced salt solution (pH 7.4), thereby obtaining a neutrophil
suspension having a concentration of 1.times.10.sup.7 cell/ml. The
neutrophil suspension was stored at 4.degree. C. before use.
4. Experimental Animals:
[0120] Male C57BL/6 mice (6 to 8 weeks of age, a body weight
ranging from 20 to 25 g) were purchased from BioLasco Taiwan Co.,
Ltd. All mice were raised in an air-conditioned room with the
following conditions: a light-dark cycle of 12 hours of
illumination and 12 hours of darkness, 21-24.degree. C., relative
humidity of 40-70%, and ventilation rate at 75 to 100%.
Furthermore, food and water were provided ad libitum for all of the
experimental animals. All experimental procedures involving the
experimental animals were approved by Laboratory Animal Center of
Chang Gung University and were performed in accordance with the NIH
(National Institutes of Health) Guide for the Care and Use of
Laboratory Animals.
General Procedure
Statistical Analysis:
[0121] Each of the following pharmacological experiments was
repeated 3 times. The experimental data are presented by
"mean.+-.standard error of the mean (SEM)." All of the data were
analyzed by Student's t-test or one-way analysis of variance
(one-way ANOVA), followed by turkey's test to assess the
differences between groups in each pharmacological experiment. If
the analytical result shows p<0.05, it is of statistical
significance.
Pharmacological Experiment 1: Effect of Compounds (1) to (7) on
Anti-Inflammation Activity
A. Effect of Compounds (1) to (7) on Inhibition of Elastase
Activity:
[0122] Each of Compounds (1) to (7) was added into 50 .mu.L buffer
A (containing 20 mM Tris-HCl (pH 7.4), 0.1% NaN.sub.3 and 5 mM
CaCl.sub.2) to obtain compound solutions with concentrations of
0.02, 0.2, 2, or 20 .mu.M for each of Compounds (1) to (7),
followed by adding 50 .mu.L of the compound solutions into wells of
a 96-well plate. The buffer A and Sivelestat (under the same
solvent condition and concentrations as those of Compounds (1) to
(7)) with the same volume were respectively used as a control group
and a positive control group. Each well of the 96-well plate was
further added with 25 .mu.L of human neutrophil elastase (Enzo, 200
nM, in buffer B containing 20 mM Tris-HCl (pH 7.4) and 0.1% NaN3)
and 25 .mu.L MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (500 .mu.M,
prepared in the buffer B), followed by reacting in an incubator
(30.degree. C.) for 30 minutes. An ELISA reader (ThermoLabsystem,
USA) was used to detect the change of optical absorption at 405 nm
(OD.sub.405).
[0123] IC.sub.50 value, indicating the concentration of the
oxime-based compound that reduces 50% of absorbance at OD.sub.405
as compared with the control group, was recorded. The results are
shown in Table 4.
TABLE-US-00004 TABLE 4 Compound IC.sub.50 (.mu.M).sup.a 1 51.26
.+-. 7.88 2 679.37 .+-. 29.72 3 367.5 .+-. 16.69 4 394.44 .+-.
22.54 5 323.89 .+-. 38.46 6 367.18 .+-. 27.35 7 158.8 .+-. 15.50
Sivelestat 65.04 .+-. 4.26 .sup.aIC.sub.50 is presented by mean
.+-. SEM (n = 3).
[0124] It is revealed in Table 4, Compounds (1) to (7) could
effectively inhibit the activity of human neutrophil elastase.
Especially, Compound (1) has better inhibitory effect than
Sivelestat.
B. Effect of Compounds 1 to 6 on Release of Human Neutrophil
Elastase (NE):
[0125] Each of Compounds (1) to (6) was dissolved in 100% DMSO so
as to obtain stock compound solutions with concentrations of 0.01,
0.03, 0.1, 0.3, 1, 3 or 10 mM) for each of Compounds (1) to (6).
7500 of the neutrophil suspension obtained in "3. Preparation of
human neutrophil" under the section of "Experimental materials" was
added with 200 .mu.M of MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide
(454454, Calbiochem Limited, dissolved in 750 .mu.l HBSS and
functioned as a substrate for human neutrophils) at a volume ratio
of 1:1 (the final volume was 1.5 ml), stirred at 37.degree. C. for
2 minutes, followed by addition with 1.5 .mu.l of the aforesaid
compound solution and reaction at 37.degree. C. for 2 minutes. In a
control group, 100% DMSO was used to substitute the compound
solution. Moreover, Sivelestat (with concentrations of 0.01, 0.03,
0.1, 0.3, 1, 3 or 10 mM in DMSO) was used to substitute the
compound solution as a positive control group. The reacted mixture
was added with 1.5 .mu.l of cytochalasin B (CB, 0.5 mg/ml) and was
incubated for 3 minutes, followed by addition with 1.5 .mu.l of
formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 100 .mu.M) and
reaction for 10 minutes to activate neutrophils. Change in
absorbance for the final mixture at 405 nm was measured using a
spectrophotometer (U-3010, Hitachi). IC.sub.50 value, indicating
the concentration of the compound that reduces 50% of absorbance of
the final mixture as compared with the control group, was recorded.
The results are shown in Table 5.
TABLE-US-00005 TABLE 5 Compound IC.sub.50 (.mu.M).sup.a 1 0.03 .+-.
0.01 2 0.11 .+-. 0.03 3 0.13 .+-. 0.03 4 0.08 .+-. 0.02 5 0.17 .+-.
0.02 6 0.18 .+-. 0.02 Sivelestat 0.05 .+-. 0.001 .sup.aIC.sub.50 is
presented by mean .+-. SEM (n = 3).
[0126] It is found from Table 5 that Compounds (1) to (6) could
effectively inhibit release of human neutrophil elastase.
Especially, Compound (1) has better effect than Sivelestat.
[0127] According to the aforesaid results, since Compounds (1) to
(7) could inhibit activity of human neutrophil elastase and
suppress release thereof, the oxime-based compound of formula (I)
is predicted to exhibit anti-inflammation activity.
Pharmacological Experiment 2: Effect of Compound (1) on Inhibition
of Proteinase 3 Activity
[0128] Compound (1) was mixed with 50 .mu.L of buffer C [containing
100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES,
pH 7.5), 500 mM NaCl, 10% DMSO and 170 mM
5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB)], thereby obtaining
compound solutions with concentrations of 0.02, 0.2, 2, or 20 nM).
50 .mu.L of the compound solution was then added to each well of a
96-well plate. Sivelestat (under the same solvent condition and
concentrations as those of Compound (I)) with the same volume were
respectively used as a control group and a positive control. 25
.mu.L proteinase 3 (Merck, 200 nM, prepared in buffer C) was added
to each well of the 96-well plate, followed by adding 25 .mu.L
Boc-Ala-Ala-Nve-SBzl (400 .mu.M, prepared in buffer C). The 96-well
plate was disposed in an incubator at 30.degree. C. for 180
minutes. The change in optical absorption at 405 nm (OD.sub.405)
was measured by ELISA reader.
[0129] IC.sub.50 value, indicating the concentration of the
compound that reduces 50% of absorbance of the final mixture as
compared with the control group, was recorded. The results are
shown in Table 6.
TABLE-US-00006 TABLE 6 Compound 1 Sivelestat IC.sub.50
(.mu.M).sup.a 221.56 .+-. 17.31 338.22 .+-. 28.35 .sup.aIC.sub.50
is presented by mean .+-. SEM (n = 3).
[0130] It is revealed From Table 6 that Compounds 1 could
effectively inhibit the activity of proteinase 3, and is better in
activity than Sivelestat.
[0131] According to the above results, it indicates that the
oxime-based compound of formula (I) could inhibit the activities of
neutrophil elastase and proteinase 3, and thus, might exhibit
anti-inflammation effect and could be used to treat inflammatory
disorders.
Pharmacological Experiment 3: Effect of Compounds (1) on Treatment
of Foot Edema Induced by Human Neutrophil Elastase
[0132] To further prove the effect of the compound (1) on treatment
of inflammatory disorders, compound (1) was further subjected to an
animal test. Specifically, the effect of compound (1) on treatment
of foot edema of a mouse induced by human neutrophil elastase was
measured.
[0133] Male C57BL/6 mice were randomly divided into four groups
(n=6 for each group) including a pathological control group, a
positive control group and experimental groups 1 and 2. The mice
were anesthetized with pentobarbital (50 mg/kg) by intraperitoneal
injection. The mice in the pathological control group and the
positive control group were respectively intraperitoneally injected
with 25 .mu.L saline solution and Sivelestat (prepared in a
DMSO/saline solution (v/v=7:3), 100 mg/kg dosage). The mice in
experimental groups 1 and 2 were respectively and intraperitoneally
injected with 25 .mu.L of 50 mg/kg and 100 mg/kg of Compound (1)
(prepared in a DMSO/saline solution (v/v=7:3)). After 60 minutes of
administration, 25 .mu.L of human neutrophil elastase (0.0075U, 5
.mu.g/mL, prepared in a saline solution) was injected into the rear
right foot of each of the mice to induce edema in the foot.
[0134] A vernier caliper (SL-A, Insize) was used to measure the
thicknesses of the rear right foot of each of the mice from each
group before injection of human neutrophil elastase and at
10.sup.th, 20.sup.th, 30.sup.th, 40.sup.th, 50.sup.th, 60.sup.th,
90.sup.th, 120.sup.th, and 240.sup.th minute after injection of
human neutrophil elastase. The change in foot thickness
(hereinafter referred to as edema degree) was determined by
subtracting the thickness of the foot before the injection from
that after the injection.
Results:
[0135] FIG. 1 shows the edema degree measured at 10.sup.th,
20.sup.th, 30.sup.th, 40.sup.th, 50.sup.th, 60.sup.th, 90.sup.th,
120.sup.th, and 240.sup.th minute after the injection of human
neutrophil elastase. As shown in FIG. 1, the mice in experimental
groups 1 and 2 have relatively lower edema degree as compared to
that in pathological control group. More particularly, compound (1)
at 100 mg/kg exhibits superior effect in reduction of edema degree
to Sivelestat. The experimental data show that compound (1) could
effectively ameliorate the foot edema induced by human neutrophil
elastase. Thus, the oxime-based compound of formula (I) of this
invention may have in vivo anti-inflammation activity by virtue of
inhibiting the activity of human neutrophil elastase, thereby
achieving the treating effect on inflammatory disorders.
Pharmacological Experiment 4: Effect of Compound (1) on Treatment
of Acute Lung Injury Induced by Lipopolysaccharides (LPS)
A. Induction of Acute Lung Injury
[0136] Male C57BL/6 mice were randomly divided into four groups
(n=6 for each group) including a pathological control group, a
positive control group, a normal control group and an experimental
group. The mice were anesthetized with pentobarbital (50 mg/kg) by
intraperitoneal injection. The mice from the pathological control
group and the normal control group were intraperitoneally injected
with 50 .mu.L saline solution. The mice from the positive control
group were intraperitoneally injected with Sivelestat [prepared in
DMSO/saline solution (v/v=7:3), 100 mg/kg dosage]. The mice in the
experimental group were intraperitoneally injected with compound
(1) [prepared in DMSO/saline solution (v/v=7:3), 100 mg/kg
dosage].
[0137] Sixty minutes after injection, each of the mice from each
group was subjected to tracheostomy to form an opening in the
trachea, and a PE10 catheter was inserted into the opening. Each of
the mice in the normal control group was injected with 50 .mu.L of
a saline solution through the PE10 catheter. 50 .mu.L of LPS
(prepared in saline solution, 800 .mu.L/mouse) was administered
through the PE10 catheter to each of the mice in the pathological
control group, the positive control group and the experimental
group, thereby causing acute lung injury.
[0138] After 6 hours of administering LPS or saline solution
through the PE10 catheter, the mice were sacrificed. The chest of
each of the mice was opened, and the left side of the lung was
obtained by clamping the left lung hilum. 0.5 g of the retrieved
left lung tissue was analyzed using the following MPO activity
analysis. The rest of the left lung tissue was subjected to the
following tissue pathological examination.
B. Analysis of Myeloperoxidase (MPO) Activity
[0139] 0.5 g of the left lung tissue thus obtained was suspended in
2.5 mL of homogenization buffer (containing 0.5%
hexadecyltrimethylammonium bromide, 0.25% protease inhibitor
(P2714, Sigma) and 50 mM phosphate buffer (pH 6.0)), followed by
vibrating using an ultrasonicator at 4.degree. C. for 3 times, each
time being conducted for 30 seconds, and allowing to cool on ice.
The suspension was then centrifuged at 12,000 rpm and 4.degree. C.
for 10 minutes. A supernatant was collected, and the total protein
concentration (mg/mL) in the supernatant was measured using a
Bio-Rad assay kit.
[0140] 290 .mu.L of phosphate buffer (50 mM), 3 .mu.L of
o-dianisidine hydrochloride solution (20 g/L, as a substrate for
MPO), and 3 .mu.L of H.sub.2O.sub.2 (20 mM) was added into each
well of a 96-well plate. 10 .mu.L of the supernatant was added into
each well to start reaction for 5 minutes. The reaction was stopped
using 3 .mu.L of 30% sodium azide. Absorbance (OD.sub.460) for the
reaction mixture was measured at 460 nm using an ELISA Reader.
Absorbance (OD.sub.460) was converted to MPO concentration based on
a standard curve. The standard curve was obtained by plotting MPO
standard solution commercially available from Sigma, St. Louis, Mo.
at concentrations of 5 U/mL, 2.5 U/mL, 1.25 U/mL, 0.625 U/mL,
0.3125 U/mL, 0.15625 U/mL, and 0.078125 U/mL with the respective
OD.sub.460. The activity was determined based on the following
equation:
MPO Activity(U/mg)=MPO concentration(U/ml)/total protein
concentration (mg/ml)
C. Tissue Pathological Examination
[0141] The left lung tissue of the mice from each group was rinsed
with PBS and was fixed using a fixating solution (10%
paraformaldehyde prepared in PBS) for 24 hours, followed by
subjecting to ethanol dehydration treatment. The dehydrated tissue
was then embedded in paraffin and sliced, thereby obtaining a
sample with 4 to 6 .mu.m thickness. The paraffin was then removed
from the sample. The tissue was then stained with hematoxylin and
eosin, was observed using an optical microscope (IX81, Olympus) at
400.times. magnification, and an image thereof was captured using a
digital camera (DP72, Olympus).
Results:
A. Analysis of MPO Activity
[0142] Referring to FIG. 2, it is revealed that, compared to the
normal control group, the MPO activity in the pathological control
group is increased, indicating that LPS successfully induces the
acute lung injury in the mice. The data also show that, compared
with the pathological control group, administration of compound (1)
to the mice in the experimental group significantly reduced MPO
activity, and compound (1) has an effect comparable to that of
Sivelestat.
B. Tissue Pathological Examination
[0143] FIG. 3 shows pictures of the lung tissues in the
pathological control group, the positive control group, the normal
control group and the experimental group. It is revealed that, in
the pathological control group, alveolar edema, interstitial lung
edema, and leukocyte infiltration were observed, showing that LPS
successfully induced acute lung injury in the mice. Compared with
the pathological control group, the lung tissue of the experimental
group shows minor alveolar edema, interstitial lung edema, and
leukocyte infiltration.
[0144] According to the aforesaid experimental results, it is
evident that compound (1) could effectively improve the LPS
induced-acute lung injury.
[0145] In conclusion, the oxime-based compound of formula (I) has
superior inhibitory activities on neutrophil elastase and
proteinase 3, and exhibits ability to ameliorate LPS-induced actue
lung injury and foot edema induced by human neutrophil elastase in
vivo, indicating that the oxime-based compound of formula (I) may
be a potential drug for the treatment of inflammatory disorders by
regulation of the activity of neutrophil elastase and proteinase
3.
[0146] While the present invention has been described in connection
with what are considered the most practical and preferred
embodiments, it is understood that this invention is not limited to
the disclosed embodiments but is intended to cover various
arrangements included within the spirit and scope of the broadest
interpretation and equivalent arrangements.
* * * * *