U.S. patent application number 16/185017 was filed with the patent office on 2020-05-14 for neutrophil inflammation inhibitor and uses thereof.
This patent application is currently assigned to Chang Gung University of Science and Technology. The applicant listed for this patent is Chang Gung University of Science and Technology. Invention is credited to Yeh-Long CHEN, Tsong-Long HWANG, Chih-Hua TSENG, Cherng-Chyi TZENG.
Application Number | 20200148674 16/185017 |
Document ID | / |
Family ID | 70551695 |
Filed Date | 2020-05-14 |
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United States Patent
Application |
20200148674 |
Kind Code |
A1 |
HWANG; Tsong-Long ; et
al. |
May 14, 2020 |
NEUTROPHIL INFLAMMATION INHIBITOR AND USES THEREOF
Abstract
Disclosed herein are compounds of formula (I), and
pharmaceutical compositions comprising the same. The compounds of
formula (I) are neutrophilic inflammation inhibitors, thus, they
are useful for treatment and/or prophylaxis of inflammatory
diseases and/or disorders associated with abnormal activation of
neutrophils, such as ARDS, ALI, COPD, lung fibrosis, chronic
bronchitis, pulmonary emphysema, .alpha.-1 anti-trypsin deficiency,
cystic fibrosis, idiopathic pulmonary fibrosis, liver injury,
steatohepatitis, liver fibrosis, damages caused by ischemia and
reperfusion, myocardial infarction, shock, stroke, and organ
transplantation, ulcerative cholitis, vasculitis, SLE, sepsis,
SIRS, arthritis, psoriasis, atopic dermatitis, and inflammatory
skin diseases. ##STR00001##
Inventors: |
HWANG; Tsong-Long; (New
Taipei City, TW) ; CHEN; Yeh-Long; (Kaohsiung City,
TW) ; TZENG; Cherng-Chyi; (Kaohsiung City, TW)
; TSENG; Chih-Hua; (Tainan City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chang Gung University of Science and Technology |
Taoyuan City |
|
TW |
|
|
Assignee: |
Chang Gung University of Science
and Technology
Taoyuan City
TW
|
Family ID: |
70551695 |
Appl. No.: |
16/185017 |
Filed: |
November 9, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 11/00 20180101;
C07D 471/04 20130101; A61P 17/06 20180101; C07D 498/04
20130101 |
International
Class: |
C07D 471/04 20060101
C07D471/04; A61P 17/06 20060101 A61P017/06; C07D 498/04 20060101
C07D498/04; A61P 11/00 20060101 A61P011/00 |
Claims
1. A compound of formula (I), a salt, or a solvate thereof:
##STR00005## wherein, X is N or O; R.sub.1 is alkyl or nil, in
which R.sub.1 is nil when X is O, and R.sub.1 is alkyl when X is N;
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently H,
hydroxyl, sulfhydryl, halogen, alkyl, haloalkyl, --OR.sub.6,
--SR.sub.6, --(C.dbd.O)R.sub.6, or --COOH; and R.sub.6 is alkyl or
haloalkyl.
2. The compound of claim 1, wherein X is N; R.sub.1 is methyl;
R.sub.2, R.sub.3, and R.sub.5 are independently H; and R.sub.4 is
hydroxyl.
3. A pharmaceutical composition comprising a compound of formula
(I), a salt, or a solvent thereof, ##STR00006## wherein, X is N or
O; R.sub.1 is alkyl or nil, in which R.sub.1 is nil when X is O,
and R.sub.1 is alkyl when X is N; R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 are independently H, hydroxyl, sulfhydryl, halogen, alkyl,
haloalkyl, --OR.sub.6, --SR.sub.6, --(C.dbd.O)R.sub.6, or --COOH;
and R.sub.6 is alkyl or haloalkyl; and a pharmaceutically
acceptable excipient.
4. The pharmaceutic composition of claim 3, wherein in the compound
of formula (I), X is N; R.sub.1 is methyl; R.sub.2, R.sub.3, and
R.sub.5 are independently H; and R.sub.4 is hydroxyl.
5. A method for treating an inflammatory disease and/or disorder in
a subject comprising administering to the subject a therapeutically
effective amount of a compound of formula (I), a salt, or a solvate
thereof, ##STR00007## wherein, X is N or O; R.sub.1 is alkyl or
nil, in which R.sub.1 is nil when X is O, and R.sub.1 is alkyl when
X is N; R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently H,
hydroxyl, sulfhydryl, halogen, alkyl, --OR.sub.6, --SR.sub.6,
--(C.dbd.O)R.sub.6, or --COOH; and R.sub.6 is alkyl optionally
substituted with at least one halogen.
6. The method of claim 5, wherein in the compound of formula (I), X
is N; R.sub.1 is methyl; R.sub.2, R.sub.3, and R.sub.5 are
independently H; and R.sub.4 is hydroxyl.
7. The method of claim 5, wherein the inflammatory disease and/or
disorder is selected from the group consisting of acute respiratory
distress syndrome (ARDS), acute lung injury (ALI), chronic
Obstructive Pulmonary Disease (COPD), lung fibrosis, liver injury,
steatohepatitis, liver fibrosis, ischemia-reperfusion injury,
myocardial infarction, shock, stroke, and organ transplantation,
vasculitis, systemic lupus erythematosus (SLE), sepsis, systemic
inflammatory response syndrome (SIRS), arthritis, psoriasis, atopic
dermatitis, and inflammatory skin diseases.
8. The method of claim 7, wherein the inflammatory disease and/or
disorder is ALI.
9. The method of claim 7, wherein the inflammatory disease and/or
disorder is ARDS.
10. The method of claim 7, wherein the inflammatory disease and/or
disorder is psoriasis.
11. The method of claim 5, wherein the compound of formula (I) is
administered to the subject in the amount of 0.001-100 mg/Kg.
12. The method of claim 5, wherein the subject is a human.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] The present disclosure in general relates to the field of
inhibitors of neutrophilic inflammation, and their uses in the
treatment or prophylaxis of neutrophil associated diseases and/or
disorders.
2. Description of Related Art
[0002] Neutrophils are a class of white blood cells (leukocytes)
that comprise an essential component of the host defense system
against microbial invasion. In response to soluble inflammatory
mediators released by cells at the site of injury, neutrophils
emigrate into tissue from the bloodstream by crossing the blood
vessel wall. At the site of injury, activated neutrophils kill
foreign cells by phagocytosis and by the release of cytotoxic
compounds, such as oxidants, proteases and cytokines. Despite their
importance in fighting infection, neutrophils themselves can
promote tissue damage. During an abnormal inflammatory response,
neutrophils can cause significant tissue damage by releasing toxic
substances at the vascular wall or in uninjured tissue.
Alternatively, neutrophils that stick to the capillary wall may
produce tissue damage by ischemia. Such abnormal inflammatory
responses have been implicated in the pathogenesis of a variety of
clinical disorders including, but not limiting to, acute
respiratory distress syndrome (ARDS), acute lung injury (ALI),
chronic obstructive pulmonary disease (COPD), lung fibrosis, liver
injury, steatohepatitis, liver fibrosis, ischemia-reperfusion
injury, myocardial infarction, shock, stroke, and organ
transplantation, vasculitis, systemic lupus erythematosus (SLE),
sepsis, systemic inflammatory response syndrome (SIRS), arthritis,
psoriasis, atopic dermatitis, and inflammatory skin diseases.
[0003] Therefore, there is an unmet medical need for new
therapeutic agents that may suppress abnormal activation of
neutrophils, for the treatment and/or prophylaxis of inflammatory
diseases and/or disorders associated therewith.
SUMMARY
[0004] The present disclosure is based on unexpected discovery that
certain newly synthetic compounds are potent neutrophil inhibitors
and may antagonize the inflammatory process resulted from abnormal
activation of neutrophils, these compounds are thus useful as lead
compounds for the development of medicaments for treating
inflammatory diseases and/or disorders associated with abnormal
activation of neutrophils, such as ARDS, ALI, COPD, lung fibrosis,
chronic bronchitis, pulmonary emphysema, .alpha.-1 anti-trypsin
deficiency, cystic fibrosis, idiopathic pulmonary fibrosis, liver
injury, steatohepatitis, liver fibrosis, damages caused by ischemia
and reperfusion, myocardial infarction, shock, stroke, and organ
transplantation, ulcerative cholitis, vasculitis, SLE, sepsis,
SIRS, arthritis, psoriasis, atopic dermatitis, and inflammatory
skin diseases.
[0005] Accordingly, one aspect of the present disclosure is to
provide a novel compound having the structure of formula (I), a
salt, or a solvate thereof:
##STR00002##
wherein, [0006] X is N or O; [0007] R.sub.1 is H, alkyl or nil, in
which R.sub.1 is nil when X is O, and R.sub.1 is H or alkyl when X
is N; [0008] R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently H, hydroxyl, sulfhydryl, halogen, alkyl, haloalkyl,
--OR.sub.6, --SR.sub.6, --(C.dbd.O)R.sub.6, or --COOH; [0009]
R.sub.6 is alkyl or haloalkyl.
[0010] According to one preferred embodiment of the present
disclosure, in the formula (I), X is N; R.sub.1 is methyl; R.sub.2,
R.sub.3, and R.sub.5 are independently H; and R.sub.4 is
hydroxyl.
[0011] The present disclosure also encompasses a pharmaceutical
composition suitable for the treatment or prophylaxis of a disease
and/or disorder resulted from abnormal activation of neutrophils.
The pharmaceutical composition comprises the compound of formula
(I), a salt, or a solvate thereof; and a pharmaceutically
acceptable excipient.
[0012] Also encompasses in the present disclosure is a method for
the treatment or prophylaxis of a subject having or suspected of
having an inflammatory disease and/or disorder associated with
abnormal activation of neutrophils. The method comprises the step
of administering a therapeutically or prophylactically effective
amount of the compound of formula (I), a salt or a solvate thereof
to the subject, so as to ameliorate, mitigate and/or prevent the
symptoms of the inflammatory disease and/or disorder associated
with abnormal activation of neutrophils.
[0013] According to embodiments of the present disclosure, the
inflammatory disease and/or disorder associated with abnormal
activation of neutrophils is selected from the group consisting of
ARDS, ALI, COPD, lung fibrosis, chronic bronchitis, pulmonary
emphysema, .alpha.-1 anti-trypsin deficiency, cystic fibrosis,
idiopathic pulmonary fibrosis, liver injury, steatohepatitis, liver
fibrosis, damages caused by ischemia and reperfusion, myocardial
infarction, shock, stroke, and organ transplantation, ulcerative
cholitis, vasculitis, SLE, sepsis, SIRS, arthritis, psoriasis,
atopic dermatitis, and inflammatory skin diseases.
[0014] According to one preferred embodiments of the present
disclosure, the inflammatory disease and/or disorder is ARDS.
[0015] According to one preferred embodiments of the present
disclosure, the inflammatory disease and/or disorder is ALI
[0016] According to another preferred embodiments of the present
disclosure, the inflammatory disease and/or disorder is
psoriasis.
[0017] According to some embodiments of the present disclosure, the
compound of formula (I) is administered to the subject in the
amount of 0.001 to 100 mg/Kg.
[0018] According to preferred embodiments of the present
disclosure, the subject is a human.
[0019] The details of one or more embodiments of this disclosure
are set forth in the accompanying description below. Other features
and advantages of the invention will be apparent from the detail
descriptions, and from claims.
[0020] It is to be understood that both the foregoing general
description and the following detailed description are by examples,
and are intended to provide further explanation of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0022] The accompanying drawings, which are incorporated in and
constitute a part of the specification, illustrate various example
systems, methods and other exemplified embodiments of various
aspects of the invention. The present description will be better
understood from the following detailed description read in light of
the accompanying drawings, where,
[0023] FIG. 1 illustrate the effect of compound HKI-4971 on
imiquimod-induced psoriasis in mice. (A) Presentation of phenotype
of mice from IMQ group (left) and HKI4971 group (right). (B)
Microscopic presentation of skin from the IMQ group mouse. (C)
Microscopic presentation of skin from the HKI4971 group mouse.
Scale bar=1 mm.
[0024] FIG. 2 illustrate the H&E staining and
immunohistochemistry staining of the skin taken from psoriasis mice
treated with compound HKI-4971 in according to one embodiment of
the present disclosure. Scale bars=100 .mu.m.
[0025] FIG. 3 illustrates the effect of HK-4971 on liver and kidney
functions in imiquimod-induced mice. (A) Alanine aminotransferase,
(B) aspartate transaminase, (C) blood urine nitrogen, and (D)
creatinine were measured. Data are expressed as the mean.+-.S.E.M
(n=3).
[0026] FIG. 4 illustrates the effect of HK-4971 on acute lung
injury (ALI) induced by lipopolysaccharide (LPS) in mice. (A)
Myeloperoxidase (MPO) activity of lung tissues. (B) Upper panel:
Representative photos of lungs. Down panel: Photomicrograph of
H&E and immunohistochemistry stained lung sections by light
microscopy. Data are expressed as mean.+-.S.E.M. (n=3). Scale
bar=100 .mu.m. ***P<0.01
DETAILED DESCRIPTION OF THE INVENTION
[0027] The detailed description provided below in connection with
the appended drawings is intended as a description of the present
disclosure and is not intended to represent the only forms in which
the present disclosure may be constructed or utilized.
[0028] 1. Definitions
[0029] The term "alkyl" includes substituted and unsubstituted
straight, branched and cyclic alkyl groups. Unless otherwise
specified, each instance of an alkyl group is independently
optionally substituted, i.e., unsubstituted (an "unsubstituted
alkyl") or substituted (a "substituted alkyl") with one or more
substituents. The term "substituted," when used to describe a
chemical structure or moiety, refers to a derivative of that
structure or moiety wherein one or more of its hydrogen atoms is
substituted with one or more of: halo, haloalkyl, or hydroxyl. In
certain embodiments, the alkyl group is unsubstituted alkyl,
particularly, alkyl having 1-6 carbon atoms (or lower alkyl), such
as methyl, ethyl, propyl, butyl, and the like. In certain
embodiments, the alkyl group is substituted with at least one
substituents (e.g., haloalkyl).
[0030] The term "halo" or "halogen" refers to fluorine (fluoro,
--F), chlorine (chloro, --Cl), bromine (bromo, --Br), or iodine
(iodo, --I).
[0031] The term "haloalkyl" denotes an alkyl group, as defined
above, having one, two, three, four, five, six, or more halogen
atoms attached thereto. Haloalkyl includes fluoroalkyl,
chloroalkyl, bromoalkyl, and iodoalkyl. "Fluoroalkyl" refers to an
alkyl group having one, two, three, four, five, six, or more
fluorine atoms attached thereto. A haloalkyl group may include more
than one type of halogen atoms. For example, fluoroalkyl includes
an alkyl group having one or more fluorine atoms and one or more
chlorine atoms. A haloalkyl group may be substituted with one or
more substituents that are not halogen atoms.
[0032] The term "alkoxy" refers to a moiety of the formula: --OR',
wherein R' is an optionally substituted alkyl group described
herein.
[0033] The present disclosure is not intended to be limited in any
manner by the above exemplary listing of substituents.
[0034] The term "salt" refers to a pharmaceutically acceptable salt
which is, within the scope of sound medical judgment, suitable for
use in contact with the tissues of humans and lower animals without
undue toxicity, irritation, allergic response and the like, and is
commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well known in the art. Pharmaceutically
acceptable salts of the compounds of this invention include those
derived from suitable inorganic and organic acids and bases.
Examples of pharmaceutically acceptable, nontoxic acid addition
salts are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid, and perchloric acid or with organic acids such as acetic
acid, oxalic acid, maleic acid, tartaric acid, citric acid,
succinic acid, or malonic acid or by using other methods known in
the art such as ion exchange. Other pharmaceutically acceptable
salts include adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4.sup.-
salts. Representative alkali or alkaline earth metal salts include
sodium, lithium, potassium, calcium, magnesium, and the like.
Further pharmaceutically acceptable salts include, when
appropriate, nontoxic ammonium, quaternary ammonium, and amine
cations formed using counterions such as halide, hydroxide,
carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and
aryl sulfonate.
[0035] The term "solvate" refers to forms of the compound that are
associated with a solvent, usually by a solvolysis reaction. This
physical association may include hydrogen bonding. Conventional
solvents include water, methanol, ethanol, acetic acid, dimethyl
sulfoxide (DMSO), tetrahydrofuran (THF), diethyl ether, and the
like. The compounds described herein may be prepared, e.g., in
crystalline form, and may be solvated. Suitable solvates include
pharmaceutically acceptable solvates and further include both
stoichiometric solvates and non-stoichiometric solvates. In certain
instances, the solvate will be capable of isolation, for example,
when one or more solvent molecules are incorporated in the crystal
lattice of a crystalline solid. "Solvate" encompasses both
solution-phase and isolatable solvates. Representative solvates
include hydrates, ethanolates, and methanolates.
[0036] The term "hydrate" refers to a compound which is associated
with water. Typically, the number of the water molecules contained
in a hydrate of a compound is in a definite ratio to the number of
the compound molecules in the hydrate. Therefore, a hydrate of a
compound may be represented, for example, by the general formula
RxH.sub.2O, wherein R is the compound, and x is a number greater
than 0. A given compound may form more than one type of hydrate,
including, e.g., monohydrates (x is 1), lower hydrates (x is a
number greater than 0 and smaller than 1, e.g., hemihydrates
(R0.5H.sub.2O)), and polyhydrates (x is a number greater than 1,
e.g., dihydrates (R2H.sub.2O) and hexahydrates (R6H.sub.2O)).
[0037] The term "pharmaceutically acceptable excipient" refers to
compounds that are compatible with other ingredients in a
pharmaceutical composition and would not endanger the subject after
administering.
[0038] Unless otherwise indicated, the term "effective amount" as
referred to herein designate the quantity of a compound which is
sufficient to yield a desired response. For therapeutic purposes,
the effective amount is also one in which any toxic or detrimental
effects of the compound are outweighed by the therapeutically
beneficial effects. The specific effective or sufficient amount
will vary with such factors as the particular condition being
treated, the physical condition of the patient (e.g., the patient's
body mass, age, or gender), the type of mammal or animal being
treated, the duration of the treatment, the nature of concurrent
therapy (if any), and the specific formulations employed and the
structure of the compounds or its derivatives. Effective amount may
be expressed, for example, in grams, milligrams or micrograms or as
milligrams per kilogram of body weight (mg/Kg). Alternatively, the
effective amount can be expressed in the concentration of the
active component (e.g., the compound of formula (I) the present
disclosure), such as molar concentration, mass concentration,
volume concentration, molality, mole fraction, mass fraction and
mixing ratio. Further, persons having ordinary skills in the
related art could calculate the human equivalent dose (HED) for the
medicament (such as the compound of the present disclosure) based
on the doses determined from animal models. For example, one may
follow the guidance for industry published by US Food and Drug
Administration (FDA) entitled "Estimating the Maximum Safe Starting
Dose in Initial Clinical Trials for Therapeutics in Adult Healthy
Volunteers" in estimating a maximum safe dosage for use in human
subjects.
[0039] Unless otherwise indicated, a "therapeutically effective
amount" of a compound is an amount sufficient to provide a
therapeutic benefit in the treatment or management of a disease or
condition, or to delay or minimize one or more symptoms associated
with the disease or condition. A therapeutically effective amount
of a compound is an amount of therapeutic agent, alone or in
combination with other therapies, which provides a therapeutic
benefit in the treatment or management of the disease or condition.
The term "therapeutically effective amount" can encompass an amount
that improves overall therapy, reduces or avoids symptoms or causes
of a disease or condition, or enhances the therapeutic efficacy of
another therapeutic agent.
[0040] Unless otherwise indicated, a "prophylactically effective
amount" of a compound is an amount sufficient to prevent a disease
or condition, or one or more symptoms associated with the disease
or condition, or prevent its recurrence. A prophylactically
effective amount of a compound means an amount of therapeutic
agent, alone or in combination with other agents, which provides a
prophylactic benefit in the prevention of the disease. The term
"prophylactically effective amount" can encompass an amount that
improves overall prophylaxis or enhances the prophylactic efficacy
of another prophylactic agent.
[0041] Unless otherwise indicated, the terms "treat," "treating"
and "treatment" contemplate an action that occurs while a patient
is suffering from the specified disease or disorder, which reduces
the severity of the disease or disorder, or one or more of its
symptoms, or retards or slows the progression of the disease or
disorder.
[0042] The term "subject" or "patient" is used interchangeably
herein and is intended to mean a mammal including the human species
that is treatable by the compound of the present invention. The
term "mammal" refers to all members of the class Mammalia,
including humans, primates, domestic and farm animals, such as
rabbit, pig, sheep, and cattle; as well as zoo, sports or pet
animals; and rodents, such as mouse and rat. Further, the term
"subject" or "patient" intended to refer to both the male and
female gender unless one gender is specifically indicated.
Accordingly, the term "subject" or "patient" comprises any mammal
which may benefit from the treatment method of the present
disclosure. Examples of a "subject" or "patient" include, but are
not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat,
cow, horse, dog, cat, bird and fowl. In a preferred embodiment, the
subject is a human.
[0043] It should also be noted that if the stereochemistry of a
structure or a portion of a structure is not indicated with, for
example, bold or dashed lines, the structure or the portion of the
structure is to be interpreted as encompassing all stereoisomers of
it. Similarly, names of compounds having one or more chiral centers
that do not specify the stereochemistry of those centers encompass
pure stereoisomers and mixtures thereof. Moreover, any atom shown
in a drawing with unsatisfied valences is assumed to be attached to
enough hydrogen atoms to satisfy the valences.
[0044] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in the respective testing measurements.
Also, as used herein, the term "about" generally means within 10%,
5%, 1%, or 0.5% of a given value or range. Alternatively, the term
"about" means within an acceptable standard error of the mean when
considered by one of ordinary skill in the art. Other than in the
operating/working examples, or unless otherwise expressly
specified, all of the numerical ranges, amounts, values and
percentages such as those for quantities of materials, durations of
times, temperatures, operating conditions, ratios of amounts, and
the likes thereof disclosed herein should be understood as modified
in all instances by the term "about." Accordingly, unless indicated
to the contrary, the numerical parameters set forth in the present
disclosure and attached claims are approximations that can vary as
desired. At the very least, each numerical parameter should at
least be construed in light of the number of reported significant
digits and by applying ordinary rounding techniques.
[0045] The singular forms "a", "and", and "the" are used herein to
include plural referents unless the context clearly dictates
otherwise.
[0046] 2. Neutrophil Inhibitors
[0047] Inventors of the present disclosure unexpectedly identify
certain synthetic compounds are potent inhibitors of neutrophilic
inflammation. It was found that the compounds of the present
disclosure may antagonize the inflammatory process caused by
abnormal activation of neutrophils, these compounds are thus useful
as lead compounds for the development of medicaments for treating
inflammatory diseases and/or disorders associated therewith.
[0048] The compounds as described herein can have the structure of
formula (I), which is described herein, and pharmaceutically
acceptable salts or solvates thereof.
##STR00003##
[0049] In some embodiments, they can be salts, solvates, or
hydrates of formula (I).
[0050] In formula (I), X can be N or O. Alternatively or in
addition, R.sub.1 is H, alkyl or nil, in which R.sub.1 is nil when
X is O, and R.sub.1 is H or alkyl when X is N. Further, R.sub.1 can
be substituted or unsubstituted. In certain embodiments, R.sub.1 is
methyl.
[0051] Alternatively or in addition, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 are independently H, hydroxyl, sulfhydryl, halogen, alkyl,
haloalkyl, --OR.sub.6, --SR.sub.6, --(C.dbd.O)R.sub.6, or --COOH;
and R.sub.6 is alkyl or haloalkyl.
[0052] In certain embodiment, the compound of formula (I) can be
compound 3, in which X is N, R.sub.1 is methyl, and R.sub.2,
R.sub.3, R.sub.4 and R.sub.5 are independently H.
[0053] In certain embodiment, the compound of formula (I) can be
compound 4, in which X is N, R.sub.1 is methyl, and R.sub.2,
R.sub.3 and R.sub.5 are independently H, and R.sub.4 is a
fluoro.
[0054] In certain embodiment, the compound of formula (I) can be
compound 7, in which X is N, R.sub.1 is methyl, R.sub.4 is
--OR.sub.6, with R.sub.6 being methyl; and R.sub.2, R.sub.3 and
R.sub.5 are independently H.
[0055] In certain embodiment, the compound of formula (I) can be
compound 8, in which X is N, R.sub.1 is methyl, R.sub.2 is H, and
R.sub.3, R.sub.4, and R.sub.5 are independently --OR.sub.6, with
R.sub.6 being methyl.
[0056] In certain embodiment, the compound of formula (I) can be
compound 10, in which X is N, R.sub.1 is methyl, R.sub.4 is
--(C.dbd.O)R.sub.6, with R.sub.6 being methyl, and R.sub.2,
R.sub.3, and R.sub.5 are independently H.
[0057] In certain embodiment, the compound of formula (I) can be
compound 11, in which X is N, R.sub.1 is methyl, R.sub.2 is
hydroxyl, and R.sub.3, R.sub.4 and R.sub.5 are independently H.
[0058] In certain embodiment, the compound of formula (I) can be
compound 12, in which X is N, R.sub.1 is methyl, R.sub.3 is
hydroxyl; while R.sub.2, R.sub.4 and R.sub.5 are independently
H.
[0059] In certain embodiment, the compound of formula (I) can be
compound 13, in which X is N, R.sub.1 is methyl, R.sub.4 is
hydroxyl, and R.sub.2, R.sub.3 and R.sub.5 are independently H.
[0060] In certain embodiment, the compound of formula (I) can be
compound 15, in which X is N, R.sub.1 is methyl, R.sub.2, R.sub.3,
and R.sub.5 are independently H, and R.sub.4 is carboxyl.
[0061] In certain embodiment, the compound of formula (I) can be
compound 16, in which X is N, R.sub.1 is methyl, R.sub.2 is
sulfhydryl, and R.sub.3, R.sub.4 and R.sub.5 are independently
H.
[0062] In certain embodiment, the compound of formula (I) can be
compound 17, in which X is N, R.sub.1 is methyl, R.sub.4 is a
chloro, and R.sub.2, R.sub.3 and R.sub.5 are independently H.
[0063] In certain embodiment, the compound of formula (I) can be
compound 28, in which X is O, R.sub.2 is hydroxy, and R.sub.3,
R.sub.4 and R.sub.5 are independently H.
[0064] In certain embodiment, the compound of formula (I) can be
compound 30, in which X is O, R.sub.4 is alkyl substituted with
three fluoro (i.e., trifluoromethyl (--CF.sub.3)), and R.sub.2,
R.sub.3 and R.sub.5 are independently H.
[0065] The compounds of the present disclosure may selectively
inhibit the activities of neutrophils, which include inhibition of
one or more of the following activities by neutrophils: release of
superoxide anion, release of reactive oxygen species, release of
myeloperoxidase, release of elastase, neutrophil aggregation,
adhesion to plastic surfaces, adhesion to vascular endothelial
cells, chemotaxis, and transmigration across a monolayer of
endothelial cells. In some embodiments, the compounds of the
present disclosure may inhibit superoxide anion generated from
formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF)-activated
neutrophils. In other embodiments, the compounds of the present
disclosure may inhibit release of elastase from fMLF-activated
neutrophils.
[0066] The inhibition of the superoxide anion generation by a
compound of the present disclosure may be measured by the half
maximal inhibitory concentrations (IC.sub.50) value of a compound
of the present disclosure when the compound of formula (I), or a
salt or a solvate thereof, is contacted, directly or indirectly,
with the fMLF-activated neutrophils. In certain embodiments, the
IC.sub.50 value of a compound of the present disclosure is at most
about 4 nM, at most about 0.5 .mu.M, at most about 5 .mu.M, at most
about 8 .mu.M, at most about 10 .mu.M for the suppression of
superoxide anion generation in fMLF-activated neutrophils. In
certain embodiments, the IC.sub.50 value of a compound of the
present disclosure is at least about 30 nM, at least about 2 .mu.M,
at least about 5 .mu.M, at least about 10 .mu.M for the suppression
of elastase release fMLF-activated neutrophils.
[0067] 3. Method of Preparing the Compounds
[0068] Any of the compounds described herein can be prepared by
routine methods known in the art or by methods described in working
examples.
[0069] In general, a compound as described herein (e.g., a compound
of Formula (I), can be prepared by treating (e.g., contacting) a
pyrazolo[4,3-c]quinoline (i.e., compound 2a or 2b of Example 1),
with a substituted aniline (e.g., 2-methoxy aniline,
4-fluoroaniline and etc.). The reaction (e.g., the step of
contacting) can be conducted in an aprotic solvent, such as
dimethyl sulfoxide (DMSO), dimethylformamide (DMF),
N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM),
tetrahydrofuran (THF), acetone, ethanol, propanol and the like. The
reaction is monitored by thin layer chromatography (TLC) or high
performance liquid chromatography (HPLC), and the product is
identified by MS and NMR spectra.
[0070] 4. Pharmaceutical Composition and Kits
[0071] This disclosure also encompasses pharmaceutical compositions
for the treatment or prophylaxis of an inflammatory disease and/or
disorder, particularly the one that is associated with abnormal
activation of neutrophils. The pharmaceutical composition comprises
a therapeutically or prophylactic effective amount of the compound
of formula (I) of the present disclosure (e.g., compound 13), a
slat or a solvate thereof; and a pharmaceutical acceptable
excipient.
[0072] The compound of formula (I) is present at a level of about
0.1% to 99% by weight, based on the total weight of the
pharmaceutical composition. In some embodiments, the compound of
formula (I) is present at a level of at least 1% by weight, based
on the total weight of the pharmaceutical composition. In certain
embodiments, the compound of formula (I) is present at a level of
at least 5% by weight, based on the total weight of the
pharmaceutical composition. In still other embodiments, the
compound of formula (I) is present at a level of at least 10% by
weight, based on the total weight of the pharmaceutical
composition. In still yet other embodiments, the compound of
formula (I) is present at a level of at least 25% by weight, based
on the total weight of the pharmaceutical composition.
[0073] Certain pharmaceutical compositions are single unit dosage
forms suitable for oral, mucosal (e.g., nasal, sublingual, vaginal,
buccal, or rectal), parenteral (e.g., subcutaneous, intravenous,
bolus injection, intramuscular, or intra-arterial), or transdermal
administration to a patient. Examples of dosage forms include, but
are not limited to: tablets; caplets; capsules, such as soft
elastic gelatin capsules; cachets; lozenges; dispersions;
suppositories; ointments; cataplasms (poultices); pastes; powders;
dressings; creams; plasters; solutions; patches; aerosols (e.g.,
nasal sprays or inhalers); gels; liquid dosage forms suitable for
oral or mucosal administration to a patient, including suspensions
(e.g., aqueous or non-aqueous liquid suspensions, oil-in-water
emulsions, or a water-in-oil liquid emulsions), solutions, and
elixirs; liquid dosage forms suitable for parenteral administration
to a patient; and sterile solids (e.g., crystalline or amorphous
solids) that can be reconstituted to provide liquid dosage forms
suitable for parenteral administration to a patient.
[0074] The formulation should suit the mode of administration. For
example, oral administration requires enteric coatings to protect
the compounds of this invention from degradation within the
gastrointestinal tract. Similarly, a formulation may contain
ingredients that facilitate delivery of the active ingredient(s) to
the site of action. For example, compounds may be administered in
liposomal formulations, in order to protect them from degradative
enzymes, facilitate transport in circulatory system, and effect
delivery across cell membranes to intracellular sites.
[0075] Similarly, poorly soluble compounds may be incorporated into
liquid dosage forms (and dosage forms suitable for reconstitution)
with the aid of solubilizing agents, emulsifiers and surfactants
such as, but not limited to, cyclodextrins (e.g.,
.alpha.-cyclodextrin or .beta.-cyclodextrin), and non-aqueous
solvents, such as, but not limited to, ethyl alcohol, isopropyl
alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate, propylene glycol, 1,3-butylene glycol, dimethyl
formamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g.,
cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),
glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty
acid esters of sorbitan, and mixtures thereof (e.g., DMSO:corn
oil).
[0076] The composition, shape, and type of a dosage form will vary
depending on its use. For example, a dosage form used in the acute
treatment of a disease may contain larger amounts of one or more of
the active ingredients it comprises than a dosage form used in the
chronic treatment of the same disease. Similarly, a parenteral
dosage form may contain smaller amounts of one or more of the
active ingredients it comprises than an oral dosage form used to
treat the same disease. These and other ways in which specific
dosage forms encompassed by this invention will vary from one
another will be readily apparent to those skilled in the art. See,
e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing, Easton, Pa. (1990).
[0077] 4.1 Oral Dosage Forms
[0078] Pharmaceutical compositions of the present invention
suitable for oral administration can be presented as discrete
dosage forms, such as, but are not limited to, tablets (e.g.,
chewable tablets), caplets, capsules, and liquids (e.g., flavored
syrups). Such dosage forms contain predetermined amounts of active
ingredients, and may be prepared by methods of pharmacy well known
to those skilled in the art. See, e.g., Remington's Pharmaceutical
Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990).
[0079] Typical oral dosage forms are prepared by combining the
active ingredient(s) in an intimate admixture with at least one
excipient according to conventional pharmaceutical compounding
techniques. Excipients can take a wide variety of forms depending
on the form of preparation desired for administration.
[0080] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit forms. If
desired, tablets can be coated by standard aqueous or non-aqueous
techniques. In general, pharmaceutical compositions and dosage
forms are prepared by uniformly and intimately admixing the active
ingredients with liquid carriers, finely divided solid carriers, or
both, and then shaping the product into the desired presentation if
necessary. Dis-integrants may be incorporated in solid dosage forms
to facility rapid dissolution. Lubricants may also be incorporated
to facilitate the manufacture of dosage forms (e.g., tablets).
[0081] 4.2 Parenteral Dosage Forms
[0082] Parenteral dosage forms can be administered to patients by
various routes including, but not limited to, subcutaneous,
intravenous (including bolus injection), intramuscular, and
intra-arterial. Because their administration typically bypasses
patients' natural defenses against contaminants, parenteral dosage
forms are specifically sterile or capable of being sterilized prior
to administration to a patient. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
and emulsions.
[0083] Suitable vehicles that can be used to provide parenteral
dosage forms of the invention are well known to those skilled in
the art. Examples include, but are not limited to: water; aqueous
vehicles such as, but not limited to, sodium chloride solution,
Ringer's solution, and Dextrose; water-miscible vehicles such as,
but not limited to, ethyl alcohol, polyethylene glycol, and
polypropylene glycol; and non-aqueous vehicles such as, but not
limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl
oleate, isopropyl myristate, and benzyl benzoate.
[0084] 4.3 Transdermal, Topical and Mucosal Dosage Forms
[0085] Transdermal, topical, and mucosal dosage forms include, but
are not limited to, ophthalmic solutions, sprays, aerosols, creams,
lotions, ointments, gels, solutions, emulsions, suspensions, or
other forms known to one of skill in the art. See, e.g.,
Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing,
Easton, Pa. (1990). Transdermal dosage forms include "reservoir
type" or "matrix type" patches, which can be applied to the skin
and worn for a specific period of time to permit the penetration of
a desired amount of active ingredients.
[0086] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide transdermal, topical, and
mucosal dosage forms are well known to those skilled in the
pharmaceutical arts, and depend on the particular tissue to which a
given pharmaceutical composition or dosage form will be
applied.
[0087] Depending on the specific tissue to be treated, additional
components may be used prior to, in conjunction with, or subsequent
to treatment with active ingredients of the invention. For example,
penetration enhancers may be used to assist in delivering active
ingredients to the tissue.
[0088] The pH of a pharmaceutical composition or dosage form, or of
the tissue to which the pharmaceutical composition or dosage form
is applied, may also be adjusted to improve delivery of one or more
active ingredients. Similarly, the polarity of a solvent carrier,
its ionic strength, or tonicity can be adjusted to improve
delivery. Compounds such as stearates may also be added to
pharmaceutical compositions or dosage forms to advantageously alter
the hydrophilicity or lipophilicity of one or more active
ingredients so as to improve delivery. In this regard, stearates
can serve as a lipid vehicle for the formulation, as an emulsifying
agent or surfactant, and as a delivery-enhancing or
penetration-enhancing agent. Different salts, hydrates or solvates
of the active ingredients can be used to further adjust the
properties of the resulting composition
[0089] 4.4 Kits
[0090] Also encompasses within the present disclosure is an article
of manufacture or "kit," containing materials useful for the
treatment or prophylaxis of a disease and/or disorder associated
with neutrophils in a subject.
[0091] In one embodiment, the kit comprises a container comprising
the compound of the present disclosure. The kit is suitable for the
treatment or prophylaxis of an inflammatory disease and/or
disorder, particularly the one that is associated with abnormal
activation of neutrophils, such as chronic obstructive pulmonary
disease (COPD), chronic bronchitis (CB), pulmonary emphysema,
.alpha.-1 anti-trypsin deficiency, cystic fibrosis, idiopathic
pulmonary fibrosis, adult respiratory distress syndrome, psoriasis,
rheumatoid arthritis, ulcerative cholitis, sepsis, and damages
caused by ischemia and reperfusion. Suitable containers include,
for example, bottles, vials, syringes, blister pack, and etc. The
container may be formed from a variety of materials such as glass,
or plastic. The container may hold a compound of the present
disclosure or a pharmaceutical formulation thereof, in an amount
effective for the treatment or prophylaxis of the inflammatory
disease and/or disorder associated with abnormal activation of
neutrophils, and may have a sterile access port, for example, the
container may be an intravenous solution bag or a vail having a
stopper pierceable by a hypodermic injection needle). The kit may
further comprise a label or package insert on or associated with
the container. The label or package insert indicates that the
composition is used for treating condition of choice. Alternatively
or additionally, the kit may further comprise a second container
comprising a pharmaceutically acceptable buffer, such as a
phosphate-buffered saline, Ringer's solution or dextrose solution.
It may further include other materials desirable from a commercial
and user standpoint, including other buffers, diluents, filters,
needles, and syringes.
[0092] The kit may further include directions for the
administration of the compound of the present invention and, if
present, the second formulation for treating or preventing the
inflammatory disease and/or disorder associated with abnormal
activation of neutrophils. For example, if the kit comprises a
first composition comprising the compound of the present
disclosure, and a second pharmaceutical formulation, the kit may
further include directions for the simultaneous, sequential, or
separate administration of the first and second pharmaceutical
compositions to a patient in need thereof.
[0093] In another embodiment, the kits are suitable for the
delivery of solid oral forms of a compound of the present
disclosure, such a kit includes, for example, a number of unit
dosages. Such kits include card having the dosages oriented in the
order of their intended use. An example of such kit is a "blister
pack." Blister packs are well known in the packaging industry and
are widely used for packaging pharmaceutical unit dosage forms. If
desired, an aid may be provided, for example, in the form of
numbers, letters, or other markings or with a calendar insert,
designating the days in the treatment schedule in which the dosage
can be administered.
[0094] According to one embodiment, the Kit may include, at least,
(a) a first container containing the present compound of formula
(I); and optionally, (b) a second container containing a second
therapeutic agent that is any of a known anti-inflammatory agent,
an anti-biotic, an anti-neoplasm agent, or a cardiovascular drug;
and (c) a legend associated with the kit for instructing a user how
to use the kit. The legend may be in a form of pamphlet, tape, CD,
VCD or DVD.
[0095] 5. Methods of Treatment
[0096] As the compounds of the present disclosure may antagonize
the activation of neutrophils signaling, thus they are useful as
lead compounds for the development of medicaments for treating
inflammatory diseases and/or disorders, particularly those
associated with abnormal activation of neutrophils. The present
invention thus is directed to the therapeutic use of the compound
of formula (I) for the treatment or prophylaxis of neutrophils
associated diseases and/or disorders, such as ARDS, ALI, COPD, lung
fibrosis, chronic bronchitis, pulmonary emphysema, .alpha.-1
anti-trypsin deficiency, cystic fibrosis, idiopathic pulmonary
fibrosis, liver injury, steatohepatitis, liver fibrosis, damages
caused by ischemia and reperfusion, myocardial infarction, shock,
stroke, and organ transplantation, ulcerative cholitis, vasculitis,
SLE, sepsis, SIRS, arthritis, psoriasis, atopic dermatitis, and
inflammatory skin diseases.
[0097] The present invention thus encompasses a method for the
treatment or prophylaxis of a subject having or suspected of having
an inflammatory disease and/or disorder associated with abnormal
activation of neutrophils. The method comprises the step of
administering a therapeutically or prophylactically effective
amount of the compound of formula (I), a salt or a solvate thereof
to the subject, so as to ameliorates, mitigates and/or prevents the
symptoms of the inflammatory disease and/or disorder associated
with the abnormal activation of neutrophils.
[0098] The compound of formula (I) of the present disclosure may be
synthesized in accordance with process set forth in the working
examples of the present disclosure.
[0099] According to embodiments of the present disclosure, the
inflammatory disease and/or disorder associated with abnormal
activation of neutrophils may be ARDS, ALI, COPD, lung fibrosis,
chronic bronchitis, pulmonary emphysema, .alpha.-1 anti-trypsin
deficiency, cystic fibrosis, idiopathic pulmonary fibrosis, liver
injury, steatohepatitis, liver fibrosis, damages caused by ischemia
and reperfusion, myocardial infarction, shock, stroke, and organ
transplantation, ulcerative cholitis, vasculitis, SLE, sepsis,
SIRS, arthritis, psoriasis, atopic dermatitis, or inflammatory skin
diseases.
[0100] Other existing treatments of such disease and/or disorders
may also be used along with the present treatment. Accordingly, the
present invention also encompasses administering another
therapeutic agent conventionally used to treat inflammatory
diseases and/or disorders associated with the activation of
neutrophils, before, together with, or after the administration of
the compound of formula (I). The therapeutic agent conventionally
used to treat inflammatory diseases and/or disorders may be a known
an anti-inflammatory agent (e.g., steroid), an anti-biotic (e.g.,
penicillin), an anti-neoplasm agent, or a cardiovascular drug. For
example, in the case of treating ARDS, in addition to administering
the compound of formula (I), a salt or solvate thereof to the
asthma patient, the present method may further include
administering any agent known to attenuate, reduce or alleviate the
level of bronchoconstriction or bronchospasm associated with
ARDS.
[0101] According to embodiments of the present disclosure, the
compound of formula (I) may be administered to the subject in the
amount of about 0.001-100 mg/Kg, such as 0.001, 0.002, 0.003,
0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.05, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0,
7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 mg/Kg; preferably in
the amount of about 0.01-90 mg/Kg, such as 0.01, 0.02, 0.03, 0.04,
0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 mg/Kg; more preferably
in the amount of about 0.11-80 mg/Kg, such as 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0,
9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, and 80 mg/Kg.
[0102] The amount, route of administration and dosing schedule of
the compound of formula (I), a salt, a solvate thereof, will depend
upon factors such as the specific indication to be treated,
prevented, or managed, and the age, sex and condition of the
patient. The roles played by such factors are well known in the
art, and may be accommodated by routine experimentation.
[0103] The present invention will now be described more
specifically with reference to the following embodiments, which are
provided for the purpose of demonstration rather than limitation.
While they are typically of those that might be used, other
procedures, methodologies, or techniques known to those skilled in
the art may alternatively be used.
EXAMPLES
[0104] Materials and Methods
[0105] Neutrophil Preparation
[0106] Human neutrophils were obtained in lipopolysaccharide-free
conditions by means of dextran sedimentation and Ficoll
centrifugation.
[0107] Animals
[0108] Male BALB/c mice aged 7-8 weeks were purchased from BioLASCO
(Taipei, Taiwan) and maintained in a room with 12 hr light/dark
cycles. Standard laboratory chow and water were available ad
libitum. All animal experimental procedures were performed in
accordance with guidelines and approved by the Institutional Animal
Care and Use Committee of Chang Gung University.
[0109] Measurement of O.sub.2.sup.108 - Production
[0110] O.sub.2.sup. - production was determined based on the
SOD-inhibitable reduction of ferricytochrome c. Briefly,
neutrophils (6.times.10.sup.5/mL) were treated with a candidate
compound at 37.degree. C. at various concentrations (0.001 to 10
.mu.M) for 5 min after being supplementing with 0.5 mg/mL
ferricytochrome c and 1 mM Ca.sup.2+. Cells were then activated
with fMLF (0.1 .mu.M) for 10 min in the pre-process of cytochalasin
B (CB, 1 .mu.g/mL) for 3 min. Changes in absorbance with the
reduction of ferricytochrome c at 550 nm were continuously
monitored in a double-beam, six-cell positioner spectrophotometer
with constant stirring (Hitachi U-3010, Tokyo, Japan). Calculations
were based on the differences in the reactions with and without SOD
(100 U ml.sup.-1) divided by the extinction coefficient for the
reduction of ferricytochrome c (.epsilon.=21.1 mM.sup.-1/10
mm).
[0111] Measurement of Elastase Release
[0112] Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide was employed
as the elastase substrate for the determination of elastase
release. Briefly, after supplementation with
Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (100 .mu.M),
neutrophils (6.times.10.sup.5/mL) were equilibrated at 37.degree.
C. for 2 min and incubated with a candidate compound at various
concentrations for 5 min. Cells were then activated with fMLF (0.1
.mu.M) for 10 min in the pre-process of CB (0.5 .mu.g/mL) for 3
min, and changes in absorbance at 405 nm were continuously
monitored to evaluate elastase release. Results are expressed as
the percentage of elastase release in the drug-free control group,
DMSO.
[0113] Cell Viability Test
[0114] Cytotoxicity of a candidate compound towards neutrophils was
evaluated by measuring the level of lactate dehydrogenase (LDH)
using a commercial kit (Promega). LDH is a cytosolic enzyme that is
released only when cell membrane is disrupted (i.e., cell death),
thus may be used as an indicator of cell viability. The kit
measures a color change at (.lamda.max=450 nm) when LDH reduces NAD
to NADH. Cytotoxicity was expressed as the percent LDH activity
obtained in cell-free medium compared to the total LDH activity.
Total LDH activity was determined by lysing cells with 0.1% Triton
X-100 for 30 min at 37.degree. C.
[0115] LPS-Induced ALI Model
[0116] Mice were pre-treated with a candidate compound (e.g.,
HKI-4971), which was dissolved in 50 .mu.l saline solution
containing 10% DMSO and 20% Kolliphor EL (Sigma-Aldrich, USA), by
intravenous injection. One hour after the administration, mice were
anaesthetized with Zoletil 50 (30 mg/kg) and xylazine (6 mg/kg) and
challenged with an intratracheal aerosol spray of 50 .mu.g of LPS
(Escherichia coli 0111:B4; Sigma-Aldrich, USA) dissolved in 40
.mu.l normal saline. Mice without LPS spray were injected with
saline as the controls. At 5 h after inoculation, mice were
sacrificed by cervical dislocation and lungs were removed. The left
lobe of lung was fixed in 10% buffered neutral formalin for
histological examination and the right four lobes were assayed for
MPO activity.
[0117] Myeloperoxidase (MPO) Activity Assay
[0118] Lung tissues were homogenized in PBS (50 Mm, pH 6.0)
containing 0.5% (w/v) hexadecyltrimethylammonium bromide (HETAB)
(Sigma-Aldrich, USA) by using the MagNA Lyser Instrument (Roche,
Germany), and was further centrifuged at 12,000.times.g for 20 min
at 4.degree. C. to remove the insoluble material. The peroxidase
activity was measured as the oxidation of o-Dianisidine
dihydrochloride (Sigma-Aldrich, USA). Briefly, the homogenate were
added with 0.2 mg/ml o-Dianisidine dihydrochloride containing
0.001% H.sub.2O.sub.2 and then incubated at room temperature for 10
min. The absorption was determined spectrophotometrically at 405
nm. MPO activity was normalized to protein concentration based on
the Bradford method (Bio-Rad, USA).
[0119] Imiquimod-Induced Psoriasis-Like Skin Inflammation in
Mice
[0120] Male BALB/c mice aged 7-8 weeks were used for the
development of the imiquimod-induced psoriatic-like plaque model.
The dorsal skin of the mice were shaved and received a daily
topical dose of 62.5 mg of 5% imiquimod (IMQ) cream (Aldara, MEDA
Pharma, Sweden) with or without a test compound (e.g., HKI-4971)
treatment by intraperitoneal injection for 7 consecutive days.
[0121] Histopathology and Immunohistochemistry
[0122] Tissue samples were fixed in 10% neutral buffered formalin
and embedding in paraffin blocks. Sections of the samples were
prepared at 5 .mu.m thickness and stained with hematoxylin and
eosin (H&E). For immunohistochemistry staining, the sections
were incubated with primary antibodies against Ly6g, MPO, and ki67
then the secondary antibody was applied for the detection of the
primary antibody according to the manufacturer's instructions of
the SuperPicture Polymer Detection Kit (Thermo Fisher, USA).
Finally, the section slides were subsequently stained with DAB
chromogen and counterstained with hematoxylin.
[0123] Statistical Analysis
[0124] Results are expressed as the mean.+-.standard error of the
mean (SEM), and comparisons were made using Student's t-test. A
probability of 0.05 or less was considered significant. The
software SigmaPlot was used for the statistical analysis.
Example 1
Production and Characterization of Compound of Formula (I)
[0125] 1.1 Production of Compound of Formula (I)
[0126] In general, the compound of formula (I) was synthesized in
accordance with the procedures described in Scheme I.
##STR00004##
[0127] The Compound of Formula (I)
TABLE-US-00001 Compound # and name X R.sub.1 R.sub.2 R.sub.3
R.sub.4 R.sub.5 3 HKI-4961 N CH.sub.3 H H H H 4 HKI-4962 N CH.sub.3
H H F H 5 HKI-4966 N CH.sub.3 OCH.sub.3 H H H 6 HKI-4965 N CH.sub.3
H OCH.sub.3 H H 7 HKI-4964 N CH.sub.3 H H OCH.sub.3 H 8 HKI-4963 N
CH.sub.3 H OCH.sub.3 OCH.sub.3 OCH.sub.3 9 HKI-4967 N CH.sub.3 H H
SCH.sub.3 H 10 HKI-4968 N CH.sub.3 H H --(C.dbd.O)CH.sub.3 H 11
HKI-4973 N CH.sub.3 OH H H H 12 HKI-4972 N CH.sub.3 H OH H H 13
HKI-4971 N CH.sub.3 H H OH H 14 HKI-4974 N CH.sub.3 H H CF.sub.3 H
15 HKI-4970 N CH.sub.3 H H COOH H 16 HKI-4975 N CH.sub.3 SH H H H
17 TCH-30o N CH.sub.3 H H Cl H 18 TCH-30p N CH.sub.3 H H CH.sub.3 H
19 HKI-4978 O Nil H H H H 20 HKI-4979 O Nil H H F H 21 HKI-4983 O
Nil OCH.sub.3 H H H 22 HKI-4982 O Nil H OCH.sub.3 H H 23 HKI-4981 O
Nil H H OCH.sub.3 H 24 HKI-4980 O Nil H OCH.sub.3 OCH.sub.3
OCH.sub.3 25 HKI-4984 O Nil H H SCH.sub.3 H 26 HKI-4986 O Nil H
--(C.dbd.O)CH.sub.3 H H 27 HKI-4987 O Nil H H --(C.dbd.O)CH.sub.3 H
28 HKI-4990 O Nil OH H H H 29 HKI-4988 O Nil H H OH H 30 HKI-4991 O
Nil H H CF.sub.3 H 31 HKI-4985 O Nil H H COOH H 32 TCH-30q O Nil H
H Cl H 33 TCH-30r O Nil H H CH.sub.3 H
[0128] 4-Chloro-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(compound 2a; X=N, R.sub.1=CH.sub.3)
[0129] A mixture of 2,4-dichloro-3-cyanoquinoline (5.48 g, 24.6
mmol) and methylhydrazine (2.21 g, 48.0 mmol) in EtOH (100 mL) was
refluxed for 2 h (TLC monitoring). The reaction mixture was
concentrated in vacuo and the precipitate was collected by
filtration, washed with EtOH, H.sub.2O and dried to yield pale
yellow solid. The crude product was recrystallized with EtOH to
give compound 2a (4.23 g, 74%).
[0130] .sup.1H-NMR (200 MHz, DMSO-d.sub.6): .delta. 7.65-7.78 (m,
2H, Ar--H), 7.92-7.96 (m, 1H, Ar--H), 7.39-7.44 (m, 1H, Ar--H).
[0131] 4-Chloroisoxazolo[4,5-c]quinolin-3-amine (compound 2b; X=O,
R.sub.1=H)
[0132] A mixture of 2,4-dichloro-3-cyanoquinoline (1.12 g, 5 mmol),
hydroxylamine hydrochloride (1.03 g, 15 mmol), and K.sub.2CO.sub.3
(2.07 g, 15 mmol) in EtOH (30 mL) was refluxed for 1 hr (TLC
monitoring). The reaction mixture was concentrated in vacuo
followed by the addition of H.sub.2O (100 mL). Precipitate was
collected by filtration, washed with H.sub.2O and dried to yield
yellow solid. The crude product was recrystallized with MeOH to
give compound 2b (0.71 g, 65%).
[0133] Mp.: 194-196.degree. C.
[0134] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): 7.52-7.60 (m, 1H,
Ar--H), 7.73-7.78 (m, 2H, Ar--H), 8.06 (d, 1H, J=7.6 Hz, Ar--H),
8.72 (br s, 2H, NH.sub.2).
[0135] General Procedure for the synthesis of
4-substituted-anilino-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(compounds 3-18) and
4-substituted-anilinoisoxazolo[4,5-c]quinolin-3-amine (Compounds
19-33)
[0136] A mixture of 2a or 2b (2 mmol) and appropriate
substituted-aniline (4 mmol) in DMF (25 mL) was refluxed with
stirring for 2-5 hrs (by TLC monitoring). The mixture was then
cooled and evaporated in vacuo to give a residue which was treated
with H.sub.2O (50 mL). The precipitate thus collected was washed
with H.sub.2O and then dried to give a crude solid, which was
recrystallized from EtOH.
[0137] Each compound was identified by: thin layer chromatography
(TLC) (pre-coated (0.2 mm) silica gel 60 F.sub.254 plates from EM
Laboratories, Inc) detected by UV light (254 nm); melting point
(Electrothermal IA9100 digital melting-point apparatus;
uncorrected); and .sup.1H and .sup.13C NMR spectra
(Varian-Unity-400 spectrometer at 400 and 100 MHz or
Varian-Gemini-200 spectrometer at 200 and 50 MHz, chemical shifts
.delta. in ppm with SiMe.sub.4 as an internal standard (=0 ppm),
coupling constants J in Hz). Elemental analyses were carried out on
a Heraeus CHN-O-Rapid elemental analyzer, and results were within
.+-.0.4% of calculated values.
[0138] 4-Anilino-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(compound 3, HKI-4961)
[0139] Yield 40%. M.p.: 186-187.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 4.19 (s, 3H, NCH.sub.3), 5.58 (br s,
2H, NH.sub.2), 6.99-7.03 (m, 1H, Ar--H), 7.33-7.37 (m, 3H, Ar--H),
7.52-7.56 (m, 1H, Ar--H), 7.67-7.70 (m, 1H, Ar--H), 7.95-7.97 (m,
2H, Ar--H), 8.22-8.24 (m, 1H, Ar--H), 8.25 (br s, 1H, NH).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 39.18, 99.13, 114.49,
119.93 (2C), 121.71, 121.96, 122.46, 126.96, 128.45 (2C), 128.51,
140.52, 140.67, 146.07, 147.29, 148.95. Anal. calcd for
C.sub.17H.sub.15N.sub.5: C 70.57, H 5.23, N 24.21; found: C 70.20,
H 5.24, N 24.14.
[0140]
4-(4-Fluoroanilino)-1-methyl-1H-1-pyrazolo[4,3-c]quinolin-3-amine
(Compound 4, HKI-4962)
[0141] Yield 62%. M.p.: 218-219.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 4.19 (s, 3H, NCH.sub.3), 5.90 (br s,
2H, NH.sub.2), 7.24-7.28 (m, 2H, Ar--H), 7.37-7.41 (m, 1H, Ar--H),
7.55-7.59 (m, 1H, Ar--H), 7.72-7.74 (m, 1H, Ar--H), 7.84-7.87 (m,
2H, Ar--H), 8.24-8.26 (m, 1H, Ar--H), 9.26 (br s, 1H, NH).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 39.35, 97.75, 113.74,
115.39, 115.61, 112.36 (2C), 123.37, 123.84, 129.14, 135.390,
140.05, 142.33, 148.59, 157.37, 159.74. Anal. calcd for
C.sub.17H.sub.14FN.sub.5 0.3HCl: C 64.14, H 4.53, N 22.00; found: C
63.87, H 4.73, N 21.77.
[0142]
4-(2-Methoxyanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 5, HKI-4966)
[0143] Yield 74%. M.p.: 229-230.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 3.90 (s, 3H, OCH.sub.3), 4.21 (s, 3H,
NCH.sub.3), 5.21 (br s, 2H, NH.sub.2), 6.98-7.08 (m, 3H, Ar--H),
7.32-7.36 (m, 1H, Ar--H), 7.53-7.57 (m, 1H, Ar--H), 7.73-7.75 (m,
1H, Ar--H), 8.22-8.24 (m, 1H, Ar--H), 8.69 (br s, 1H, NH),
8.99-8.02 (m, 1H, Ar--H). .sup.13C-NMR (100 MHz, DMSO-d.sub.6):
.delta. 39.11, 56.04, 100.52, 110.55, 114.44, 119.16, 120.50,
121.46, 121.96, 122.43, 127.12, 128.50, 129.78, 140.20, 146.35,
147.28, 148.05, 149.12. Anal. calcd for C.sub.18H.sub.17FN.sub.5O:
C 67.70, H 5.37, N 21.93; found: C 67.37, H 5.39, N 21.85.
[0144]
4-(3-Methoxyanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 6, HKI-4965)
[0145] Yield 79%. M.p.: 284-286.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 3.80 (s, 3H, OCH.sub.3), 4.19 (s, 3H,
NCH.sub.3), 5.58 (br s, 2H, NH.sub.2), 6.58-6.60 (m, 1H, Ar--H),
7.22-7.26 (m, 1H, Ar--H), 7.32-7.42 (m, 2H, Ar--H), 7.53-7.75 (m,
1H, Ar--H), 7.69-7.71 (m, 1H, Ar--H), 7.88(br s, 1H, NH), 8.23-8.26
(m, 2H, Ar--H). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta.
39.17, 54.93, 99.19, 105.43, 107.30, 112.12, 114.45, 121.97,
122.57, 126.97, 128.56, 129.11, 140.48, 141.86, 145.95, 147.24,
148.88, 159.49. Anal. calcd for C.sub.18H.sub.17N.sub.5O 0.4HCl: C
64.72, H 5.26, N 20.97; found: C 64.96, H 5.29, N 21.08.
[0146]
4-(4-Methoxyanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 7, HKI-4964)
[0147] Yield 62%. M.p.: 193-194.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. .82 (s, 3H, OCH.sub.3), 4.20 (s, 3H,
NCH.sub.3), 6.30 (br s, 2H, NH.sub.2), 7.06-7.08 (m, 2H, Ar--H),
7.43-7.47 (m, 1H, Ar--H), 7.59-7.63 (m, 3H, Ar--H), 7.81-7.83 (m,
1H, Ar--H), 8.26-8.28 (m, 1H, Ar--H), 9.74 (br s, 1H, NH).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 39.58, 55.37, 96.37,
112.90, 114.71 (2C), 121.28, 122.79, 124.23, 125.78, 129.59,
129.82, 138.43, 139.55, 148.50, 149.88, 157.43. Anal. calcd for
C.sub.18H.sub.17N.sub.5O 0.5HCl: C 64.02, H 5.23, N 20.74; found: C
64.13, H 5.42, N 20.69.
[0148]
4-(3,4,5-Trimethoxyanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-a-
mine (Compound 8, HKI-4963)
[0149] Yield 63%. M.p.: 219-220.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 3.68 (s, 3H, OCH.sub.3), 3.82 (s, 6H,
OCH.sub.3), 4.19 (s, 3H, NCH.sub.3), 5.75 (br s, 2H, NH.sub.2),
7.38 (m, 3H, Ar--H), 7.56-7.60 (m, 1H, Ar--H), 7.72-7.76 (m, 1H,
Ar--H), 8.24-8.26 (m, 1H, Ar--H). .sup.13C-NMR (100 MHz,
DMSO-d.sub.6): .delta. 39.29, 55.79 (2C), 60.12, 98.54, 113.92,
122.24, 122.97, 123.13, 127.10, 129.00, 140.11, 148.59, 152.85.
Anal. calcd for C.sub.20H.sub.21N.sub.5O.sub.3 0.5HCl: C 60.39, H
5.46, N 17.61; found: C 60.08, H 5.57, N 17.36.
[0150]
1-Methyl-4-(4-methylthioanilino)-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 9, HKI-4967)
[0151] Yield 84%. M.p.: 233-235.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 2.48 (s, 3H, SCH.sub.3), 4.18 (s, 3H,
NCH.sub.3), 5.71 (br s, 2H, NH.sub.2), 7.29-7.37 (m, 3H, Ar--H),
7.53-7.57 (m, 1H, Ar--H), 7.69-7.71 (m, 1H, Ar--H), 7.86-7.88 (m,
2H, Ar--H), 8.22-8.24 (m, 1H, Ar--H). .sup.13C-NMR (100 MHz,
DMSO-d.sub.6): .delta. 15.88, 39.22, 98.58, 114.15, 121.42, 122.10
(2C), 122.77, 125.80, 127.37 (2C), 128.74, 130.67, 137.67, 140.28,
144.66, 147.78, 148.65. Anal. calcd for C.sub.18H.sub.17N.sub.5S
0.2HCl: C 63.08, H 5.06, N 20.43, S 9.36; found: C 63.01, H 5.19, N
20.43, S 9.39.
[0152]
4-(4-Acetoylanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 10, HKI-4968)
[0153] Yield 60%. Mp.: 272-274.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6+TFA-d): .delta. 2.65 (s, 3H, COCH.sub.3), 4.28
(s, 3H, OCH.sub.3), 7.62-7.74 (m, 4H, Ar--H), 7.93-7.95 (m, 1H,
Ar--H), 8.14-8.17 (m, 2H, Ar--H), 8.39-8.41 (m, 1H, Ar--H).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6+TFA-d): .delta. 26.89, 40.06,
96.94, 113.17, 119.71, 123.61, 123.98 (2C), 126.17, 130.46 (2C),
131.04, 135.22, 135.53, 140.11, 140.97, 147.68, 151.36, 197.17.
Anal. calcd for C.sub.19H.sub.17N.sub.5O: C 68.87, H 5.17, N 21.13;
found: C 68.67, H 5.18, N 20.95
[0154]
4-(2-Hydroxyanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 11, HKI-4973)
[0155] Yield 64%. Mp.: 233-235.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 4.21 (s, 3H, NCH.sub.3), 5.98 (br s,
2H, NH.sub.2), 6.91-6.95 (m, 1H, Ar--H), 7.03-7.04 (m, 1H, Ar--H),
7.13-7.14 (m, 1H, Ar--H), 7.24-7.45 (m, 1H, Ar--H), 7.58-7.62 (m,
1H, Ar--H), 7.81-7.83 (m, 2H, Ar--H), 8.26-8.28 (m, 1H, Ar--H).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 39.28, 97.06, 113.06,
116.81 (2C), 119.44 (2C), 122.25, 122.76, 123.97, 124.94, 126.56,
129.68, 139.58, 148.58, 149.38, 150.37. Anal. calcd for
C.sub.17H.sub.15N.sub.5O 1.0HCl: C 59.72, H 4.72, N 20.49; found: C
59.62, H 5.06, N 20.30.
[0156]
4-(3-Hydroxyanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 12, HKI-4972)
[0157] Yield 70%. Mp.: 190-191.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 4.21 (s, 3H, NCH.sub.3), 6.23 (br s,
2H, NH.sub.2), 6.66-6.69 (m, 1H, Ar--H), 7.07-7.09 (m, 1H, Ar--H),
7.22-7.26 (m, 2H, Ar--H), 7.45-7.4 (m, 1H, Ar--H), 7.61-7.65 (m,
1H, Ar--H), 7.83-7.85 (m, 2H, Ar--H), 8.27-8.29 (m, 1H, Ar--H),
9.75 (br s, 1H, OH). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta.
39.58, 97.14, 110.25, 113.13, 113.59, 122.81, 124.34, 129.83,
130.13, 138.38, 139.77, 148.14, 149.81, 158.31. Anal. calcd for
C.sub.17H.sub.15N.sub.5O 0.9HCl: C 60.36, H 4.77, N 20.71; found: C
59.99, H 5.06, N 20.31.
[0158]
4-(4-Hydroxyanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 13, HKI-4971)
[0159] Yield 79%. Mp.: 320-322.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 4.20 (s, 3H, NCH.sub.3), 6.47 (br s,
2H, NH.sub.2), 6.93-6.97 (m, 2H, Ar--H), 7.34-7.37 (m, 2H, Ar--H),
7.48-7.52 (m, 1H, Ar--H), 7.62-7.66 (m, 1H, Ar--H), 7.89-7.91 (m,
1H, Ar--H), 8.27-8.29 (m, 1H, Ar--H), 9.94 (br s, 1H, OH), 11.62
(br s, 1H, NH). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta.
39.29, 95.29, 112.33, 116.47 (2C), 119.27, 123.10, 125.00, 125.97,
127.36, 130.31, 135.59, 139.17, 148.35, 150.77, 157.17. Anal. calcd
for C.sub.17H.sub.15N.sub.5O 1.0HCl: C 59.72, H 4.72, N 20.49;
found: C 59.64, H 4.85, N 20.41.
[0160]
4-(4-Trifluoromethylanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3--
amine (Compound 14, HKI-4974)
[0161] Yield 59%. Mp.: 219-220.degree. C. (EtOH). .sup.1H-NMR (400
MHz, TFA-d): .delta. 4.28 (s, 3H, NCH.sub.3), 7.60-7.63 (m, 1H,
Ar--H), 7.72-7.81 (m, 3H, Ar--H), 7.90-7.95 (m, 3H, Ar--H),
8.38-8.40 (m, 1H, Ar--H). Anal. calcd for
C.sub.18H.sub.14F.sub.3N.sub.5 1.3HCl: C 53.40, H 3.81, N 17.30;
found: C 53.00, H 4.12, N 17.20.
[0162]
4-[(3-Amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-4-yl)amino]benzoic
acid (Compound 15, HKI-4970)
[0163] Yield 68%. Mp.: 325-326.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 4.24 (s, 3H, NCH.sub.3), 7.59-7.59 (m,
1H, Ar--H), 7.69-7.79 (m, 3H, Ar--H), 7.87-7.89 (m, 1H, Ar--H),
8.04-8.06 (m, 1H, Ar--H), 8.34-8.37 (m, 1H, Ar--H), 8.27-8.29 (m,
1H, Ar--H). .sup.13C-NMR (100 MHz, SO-d.sub.6): .delta. 39.29,
97.33, 112.94, 120.22, 122.67, 123.15, 125.46, 127.67, 130.45,
130.91 (2C), 131.16, 136.34, 139.79, 141.47, 147.39, 150.81,
166.82. Anal. calcd for C.sub.18H.sub.15N.sub.5O.sub.2 1.0HCl: C
58.44, H 4.36, N 18.93; found: C 58.04, H 4.50, N 18.79.
[0164]
2-[(3-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-4-yl)amino]benzenet-
hiol (Compound 16, HKI-4975)
[0165] Yield 42%. Mp.: 235-236.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 3.14 (s, 3H, NCH.sub.3), 5.03 (br s,
1H, SH), 6.05 (br s, 1H, NH.sub.2), 6.93-6.97 (m, 1H, Ar--H),
6.85-6.87 (m, 1H, Ar--H), 7.02-7.04 (m, 1H, Ar--H), 7.20-7.30 (m,
2H, Ar--H), 7.36-7.41 (m, 1H, Ar--H), 7.81-7.86 (m, 1H, Ar--H).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 35.75, 100.80,
111.46, 115.40, 116.44, 120.82, 121.33, 123.74, 125.82, 131.11,
131.31, 132.51, 140.24, 147.51, 152.11, 153.59, 161.85. Anal. calcd
for C.sub.17H.sub.15N.sub.5S 0.1HCl: C 62.82, H 4.68, N 21.55, S
9.86; found: C 63.03, H 4.72, N 21.55, S 9.88.
[0166]
4-(4-Chloroanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 17, TCH-30o)
[0167] Yield 82%. Mp.: 281-282.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 4.23 (s, 3H, NCH.sub.3), 7.53-7.61 (m,
3H, Ar--H), 7.66-7.71 (m, 3H, Ar--H), 7.88-7.90 (m, 1H, Ar--H),
8.33-8.34 (m, 1H, Ar--H), 12.19 (br s, 1H, NH). .sup.13C-NMR (100
MHz, DMSO-d.sub.6): .delta. 39.77, 96.36, 112.62, 119.49, 123.18,
125.36, 126.43 (2C), 129.74 (2C), 130.46, 130.99, 135.31, 135.70,
139.49, 147.76, 150.97. Anal. calcd for C.sub.17H.sub.14ClN.sub.5
1.5HCl: C 53.93, H 4.13, N 18.50; found: C 54.09, H 4.44, N
18.53.
[0168]
4-(4-Methylanilino)-1-methyl-1H-pyrazolo[4,3-c]quinolin-3-amine
(Compound 18, TCH-30p)
[0169] Yield 85%. Mp.: 253-255.degree. C. (EtOH). .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta. 2.36 (s, 3H, CH.sub.3), 4.20 (s, 3H,
NCH.sub.3), 6.22 (br s, 2H, NH.sub.2), 7.27-7.29 (m, 2H, Ar--H),
7.43-7.47 (m, 1H, Ar--H), 7.559-7.63 (m, 3H, Ar--H), 7.79-7.81 (m,
1H, Ar--H), 8.26-8.28 (m, 1H, Ar--H), 9.72 (br s, 1H, NH).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 20.63, 39.50, 97.08,
113.19, 122.14, 122.67 (2C), 123.14, 124.05, 129.65, 129.78 (2C),
134.30, 135.09, 139.44, 139.76, 148.34, 149.57. Anal. calcd for
C.sub.18H.sub.17N.sub.5 1.1HCl: C 62.93, H 5.32, N 20.39; found: C
62.80, H 5.73, N 20.44.
[0170] 4-Anilinoisoxazolo[4,5-c]quinolin-3-amine (Compound 19,
HKI-4978)
[0171] Yield 40%. Mp.: 266-267.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 7.42-7.45 (m, 2H, Ar--H),
7.54-7.71 (m, 6H, Ar--H), 7.96-7.99 (m, 1H, Ar--H), 10.04 (br s,
2H, NH.sub.2), 11.47 (br s, 1H, 2H, NH, HC1). .sup.13C-NMR (100
MHz, DMSO-d.sub.6): .delta. 82.82, 111.73, 123.07, 125.37, 125.61
(2C), 127.63, 129.80 (2C), 132.22, 135.07, 136.29, 148.52, 155.35,
168.52. Anal. calcd for C.sub.16H.sub.12N.sub.4O 1.1HCl: C 60.71, H
4.18, N 17.70; found: C 60.94, H 4.45, N 17.45.
[0172] 4-(4-Fluoroanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 20, HKI-4979)
[0173] Yield 67%. Mp.: 271-272.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 7.39-7.44 (m, 3H, Ar--H),
7.60-7.70 (m, 4H, Ar--H), 7.96-7.98 (m, 1H, Ar--H), 7.96-7.99 (m,
1H, Ar--H), 10.02 (br s, 2H, NH.sub.2), 11.46 (br s, 1H, 2H, NH,
HCl). .sup.13C-NMR (100 MHz, DMSO-d6): .delta. 82.69, 111.71,
116.70 (2C, J=22.8 Hz), 118.97, 123.11, 125.39, 128.45 (2C, J=8.3
Hz), 131.16, 132.26, 136.33, 148.87, 155.33, 161.34 (J=242.6 Hz),
168.52. Anal. calcd for C.sub.16H.sub.11FN.sub.4O 1.0HCl: C 58.08,
H 3.66, N 16.94; found: C 57.97, H 3.95, N 16.85.
[0174] 4-(2-Methoxyanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 21, HKI-4983)
[0175] Yield 40%. Mp.: 261-262.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 3.79 (s, 3H, OCH.sub.3), 7.11-7.15
(m, 1H, Ar--H), 7.26-7.28 (m, 1H, Ar--H), 7.40-7.52 (m, 3H, Ar--H),
7.61-7.65 (m, 1H, Ar--H), 7.74-7.76 (m, 1H, Ar--H), 7.96-7.98 (m,
1H, Ar--H), 9.91 (br s, 2H, NH.sub.2), 10.99 (br s, 1H, 2H, NH,
HCl). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 55.95, 82.11,
111.55, 113.24, 118.81, 121.19, 122.12, 123.25, 125.54, 128.55,
130.19, 132.50, 136.21, 148.49, 154.43, 155.19, 168.64. Anal. calcd
for C.sub.17H.sub.14N.sub.4O.sub.2 1.1HCl: C 58.92, H 4.40, N
16.17; found: C 59.01, H 4.58, N 15.92.
[0176] 4-(3-Methoxyanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 22, HKI-4982)
[0177] Yield 50%. Mp.: 262-263.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 3.80 (s, 3H, OCH.sub.3), 6.99-7.02
(m, 1H, Ar--H), 7.26-7.28 (m, 2H, Ar--H), 7.40-7.48 (m, 2H, Ar--H),
7.62-7.66 (m, 1H, Ar--H), 7.71-7.73 (m, 1H, Ar--H), 7.97-7.98 (m,
1H, Ar--H), 10.02 (br s, 2H, NH.sub.2), 11.44 (br s, 2H, NH, HCl).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 55.38, 82.50, 111.27,
111.74, 113.43, 117.60, 119.07, 123.05, 125.36, 130.57, 132.23,
136.08, 136.35, 148.48, 155.36, 160.20, 168.51. Anal. calcd for
C.sub.17H.sub.14N.sub.4O.sub.2 1.1HCl: C 58.92, H 4.40, N 16.17;
found: C 58.92, H 4.56, N 15.85.
[0178] 4-(4-Methoxyanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 23, HKI-4981)
[0179] Yield 43%. Mp.: 257-259.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 3.84 (s, 3H, OCH.sub.3), 7.10-7.13
(m, 2H, Ar--H), 7.38-7.48 (m, 3H, Ar--H), 7.60-7.63 (m, 1H, Ar--H),
7.70-7.72 (m, 1H, Ar--H), 7.95-7.96 (m, 1H, Ar--H), 9.98 (br s, 2H,
NH.sub.2), 11.17 (br s, 1H, NH), 11.32 (br s, 1H, HCl).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 55.45, 82.38, 111.61,
115.06 (2C), 118.97, 123.05, 125.19, 127.13, 127.66 (2C), 132.17,
136.42, 148.83, 155.31, 158.81, 168.42. Anal. calcd for
C.sub.17H.sub.14N.sub.4O.sub.2 1.1HCl: C 58.92, H 4.40, N 16.17;
found: C 58.83, H 4.49, N 16.01.
[0180] 4-(3,4,5-Trimethoxyanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 24, HKI-4980)
[0181] Yield 63%. Mp.: 264-266.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 3.74 (s, 3H, OCH.sub.3), 3.79 (m,
6H, OCH.sub.3), 6.95 (s, 2H, Ar--H), 7.42-7.44 (m, 1H, Ar--H),
7.62-7.67 (m, 1H, Ar--H), 7.76-7.78 (m, 1H, Ar--H), 7.96-7.99 (m,
1H, Ar--H), 10.02 (br s, 2H, NH.sub.2), 11.34 (br s, 1H, NH), 11.44
(br s, 1H, HCl). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta.
56.12 (2C), 60.01, 82.50, 103.75 (2C), 111.62, 119.01, 123.03,
125.33, 130.14, 132.26, 136.27, 136.87, 148.50, 153.55 (2C),
155.31, 168.47. Anal. calcd for C.sub.19H.sub.18N.sub.4O.sub.4
1.2HCl: C 55.64, H 4.72, N 13.66; found: C 55.65, H 5.01, N
13.51.
[0182] 4-(4-Methylthioanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 25, HKI-4984)
[0183] Yield 42%. Mp.: 246-247.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 2.55 (s, 3H, SCH.sub.3), 7.39-7.52
(m, 5H, Ar--H), 7.61-7.69 (m, 3H, Ar--H), 7.95-7.97 (m, 1H, Ar--H),
10.02 (br s, 2H, NH.sub.2), 11.39 (br s, 2H, NH, HCl). .sup.13C-NMR
(100 MHz, DMSO-d.sub.6): .delta. 14.67, 82.70, 111.69, 119.03,
123.04, 125.25, 126.35 (2C), 126.87 (2C), 131.69, 132.16, 136.44,
137.79, 148.55, 155.33, 168.43. Anal. calcd for
C.sub.17H.sub.14N.sub.4OS 1.1HCl: C 56.33, H 4.20, N 15.46, S 8.85;
found: C 56.21, H 4.22, N 15.34, S 8.88.
[0184] 4-(3-Acetoylanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 26, HKI-4986)
[0185] Yield 40%. Mp.: 249-250.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 2.62 (s, 3H, COCH.sub.3), 7.36 (m,
1H, Ar--H), 7.60-7.65 (m, 3H, Ar--H), 7.89-7.96 (m, 3H, Ar--H),
8.25 (m, 1H, Ar--H), 9.61 (br s, 2H, NH.sub.2), 10.94 (br s, 1H,
NH), 11.27 (br s, 1H, HCl). .sup.13C-NMR (100 MHz, DMSO-d.sub.6):
.delta. 26.89, 112.30, 122.96 (2C), 124.10, 124.63, 125.77, 128.83,
129.84, 131.92, 137.90, 148.66, 156,13, 167.86, 197.60. Anal. calcd
for C.sub.18H.sub.14N.sub.4O.sub.2 1.2HCl: C 59.69, H 4.23, N
15.47; found: C 59.50, H 4.62, N 15.19.
[0186] 4-(4-Acetoylanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 27, HKI-4987)
[0187] Yield 50%. Mp.: 255-257.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 2.60 (s, 3H, COCH.sub.3), 7.36 (m,
1H, Ar--H), 7.60 (m, 2H, Ar--H), 7.79-7.80 (m, 2H, Ar--H),
7.94-7.96 (m, 1H, Ar--H), 8.04-8.06 (m, 1H, Ar--H), 9.45 (br s, 2H,
NH.sub.2), 10.48 (br s, 1H, NH), 11.83 (br s, 1H, HCl).
.sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 26.67, 84.16, 112.45,
121.48, 122.95 (2C), 123.33, 124.81, 125.15, 129.63 (2C), 130.57,
131.87, 133.32, 148.15, 156.25, 167.76, 196.85. Anal. calcd for
C.sub.18H.sub.14N.sub.4O.sub.2 1.3HCl: C 59.09, H 4.22, N 15.31;
found: C 59.20, H 4.58, N 14.96.
[0188] 4-(2-Hydroxyanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 28, HKI-4990)
[0189] Yield 42%. Mp.: 240-241.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 6.87-6.96 (m, 2H, Ar--H), 7.10 (m,
1H, Ar--H), 7.21 (m, 1H, Ar--H), 7.34-7.36 (m, 2H, Ar--H), 7.50 (m,
1H, Ar--H), 7.88 (m, 1H, Ar--H), 8.46 (br s, 1H, NH), 8.78 (br s,
2H, NH.sub.2), 11.42 (br s, 1H, OH). .sup.13C-NMR (100 MHz,
DMSO-d.sub.6): .delta. 83.79, 112.90, 119.13, 122.78, 123.03,
124.31, 124.92, 126.09, 128.00, 131.36 (2C), 145.30, 149.84,
150.29, 157.64, 166.34. Anal. calcd for
C.sub.16H.sub.12N.sub.4O.sub.2 0.7HCl: C 60.45, H 4.03, N 17.62;
found: C 60.34, H 4.45, N 17.52.
[0190] 4-(4-Hydroxyanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 29, HKI-4988)
[0191] Yield 45%. Mp.: 236-237.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 6.80-6.85 (m, 2H, Ar--H),
7.19-7.23 (m, 1H, Ar--H), 7.44-7.52 (m, 4H, Ar--H), 7.86-7.88 (m,
1H, Ar--H), 8.88 (br s, 2H, NH.sub.2), 9.43 (br s, 1H, NH), 10.84
(br s, 1H, OH). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta.
83.55, 112.63, 113.98, 115.39 (2C), 116.15, 122.67 (2C), 122.98,
125.34, 127.16, 131.27, 149.09, 154.57, 157.10, 166.70. Anal. calcd
for C.sub.16H.sub.12N.sub.4O.sub.2 1.9HCl: C 58.84, H 4.88, N
17.16; found: C 58.74, H 4.59, N 17.01.
[0192] 4-(4-Trifluoromethylanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 30, HKI-4991)
[0193] Yield 42%. Mp.: 227-229.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 7.34 (m, 1H, Ar--H), 7.57 (m, 2H,
Ar--H), 7.81-7.93 (m, 3H, Ar--H), 7.93-7.94 (m, 2H, Ar--H), 9.26
(br s, 2H, NH.sub.2), 10.16 (br s, 1H, NH), 11.65 (br s, 1H, OH).
Anal. calcd for C.sub.17H.sub.11F.sub.3N.sub.4O 0.4HCl: C 56.88, H
3.20, N 15.61; found: C 56.59, H 3.34, N 15.35.
[0194] 4-[(3-aminoisoxazolo[4,5-c]quinolin-4-yl)amino]benzoic acid
(Compound 31, HKI-4985)
[0195] Yield 58%. Mp.: 308-310.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 7.44-7.46 (m, 1H, Ar--H),
7.61-7.72 (m, 4H, Ar--H), 7.98-8.09 (m, 3H, Ar--H), 10.05 (br s,
2H, NH.sub.2), 11.62 (br s, 1H, NH), 11.80 (br s, 1H, HCl), 13.01
(br s, 1H, COOH). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta.
83.57, 111.94, 119.23, 123.12, 124.74, 125.61, 129.03, 130.84 (2C),
131.16, 132.29, 136.40, 139.78, 148.40, 155.43, 166.75, 168.60.
Anal. calcd for C.sub.17H.sub.12N.sub.4O.sub.3 1.4HCl: C 54.96, H
3.64, N 15.08; found: C 54.97, H 3.95, N 15.303.
[0196] 4-(4-Chloroanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 32, TCH-30q)
[0197] Yield 75%. Mp.: 268-269.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 7.41-7.46 (m, 1H, Ar--H),
7.59-7.69 (m, 6H, Ar--H), 7.97-7.99 (m, 1H, Ar--H), 9.94 (br s, 2H,
NH.sub.2), 11.31 (br s, 2H, NH, HCl). .sup.13C-NMR (100 MHz,
DMSO-d.sub.6): .delta. 83.09, 111.86, 119.17, 123.25, 125.67,
127.79 (2C), 129.99 (2C), 132.22, 132.45, 134.15, 136.45, 148.82,
155.48, 168.68. Anal. calcd for C.sub.16H.sub.11ClN.sub.4O 1.0HCl:
C 55.33, H 3.49, N 16.140; found: C 55.37, H 3.45, N 16.24.
[0198] 4-(4-Methylanilino)isoxazolo[4,5-c]quinolin-3-amine
(Compound 33, TCH-30r)
[0199] Yield 80%. Mp.: 173-175.degree. C. (Hexane). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta. 2.40 (s, 3H, CH.sub.3), 7.37-7.45
(m, 5H, Ar--H), 7.61-7.65 (m, 1H, Ar--H), 7.70-7.72 (m, 1H, Ar--H),
7.95-7.98 (m, 1H, Ar--H), 9.92 (br s, 2H, NH.sub.2), 11.20 (br s,
2H, NH, HCl). .sup.13C-NMR (100 MHz, DMSO-d.sub.6): .delta. 20.88,
82.71, 111.75, 119.14, 123.20, 125.50, 125.92 (2C), 130.52 (2C),
132.20, 132.40, 136.42, 137.57, 148.78, 155.44, 168.63. Anal. calcd
for C.sub.17H.sub.14N.sub.4O 1.1HCl: C 61.78, H 4.61, N 16.96;
found: C 61.63, H 4.62, N 16.97.
[0200] 1.2 Characterization of the Compound of Example 1
[0201] 1.2.1 Compound of formula (I) suppresses O.sub.2.sup. -
production and elastase release in human neutrophils
[0202] In this example, inhibitory effects of compound of Example 1
(i.e., compound of formula (I)) on superoxide anion production and
elastase release in human neutrophils were investigated, and
results are summarized in Table 2.
TABLE-US-00002 TABLE 2 Effects of compound of example 1 on
superoxide anion generation and elastase release in FMLP/CB-induced
human neutrophils. Superoxide Anion Elastase Release Compound
IC.sub.50 (.mu.M).sup.a Inhibition % IC.sub.50 (.mu.M).sup.a
Inhibition % 3 HKI-4961 2.39 .+-. 0.63 93.92 .+-. 6.32*** 4.09 .+-.
1.02 80.45 .+-. 2.57*** 4 HKI-4962 1.44 .+-. 0.22 91.16 .+-.
4.43*** 5.50 .+-. 1.50 68.04 .+-. 6.80*** 5 HKI-4966 1.37 .+-. 0.08
90.13 .+-. 3.38*** 4.65 .+-. 2.57 66.02 .+-. 6.51*** 6 HKI-4965
>10 27.54 .+-. 5.47** >10 33.36 .+-. 6.04** 7 HKI-4964 0.51
.+-. 0.17 99.26 .+-. 1.51*** 3.77 .+-. 0.71 96.67 .+-. 6.56*** 8
HKI-4963 0.80 .+-. 0.09 98.19 .+-. 2.41*** 3.25 .+-. 0.08 89.77
.+-. 6.51*** 9 HKI-4967 >10 47.72 .+-. 3.32*** >10 20.84 .+-.
3.75** 10 HKI-4968 1.58 .+-. 0.16 85.10 .+-. 1.66*** 4.63 .+-. 1.10
57.17 .+-. 7.34*** 11 HKI-4973 0.73 .+-. 0.23 93.51 .+-. 1.75***
2.24 .+-. 0.91 101.39 .+-. 4.50*** 12 HKI-4972 1.89 .+-. 0.69 93.17
.+-. 3.66*** 4.62 .+-. 1.74 94.36 .+-. 8.34*** 13 HKI-4971 0.00369
.+-. 0.00174 88.13 .+-. 1.19*** 0.03210 .+-. 0.01740 104.56 .+-.
2.53*** 14 HKI-4974 4.56 .+-. 0.83 63.09 .+-. 2.51*** >10 37.61
.+-. 6.22** 15 HKI-4970 1.83 .+-. 0.17 82.93 .+-. 2.86*** 3.68 .+-.
0.42 79.42 .+-. 1.91*** 16 HKI-4975 >10 4.05 .+-. 2.95 >10
-3.34 .+-. 1.78 17 TCH-30p 3.49 .+-. 1.25 82.78 .+-. 5.36*** 5.28
.+-. 0.66 75.78 .+-. 6.64*** 18 TCH-30o 5.21 .+-. 1.90 69.71 .+-.
7.05*** >10 39.66 .+-. 3.83*** 19 HKI-4978 >10 43.73 .+-.
6.43*** >10 5.81 .+-. 5.22 20 HKI-4979 6.16 .+-. 1.18 63.22 .+-.
7.76* >10 10.69 .+-. 4.00* 21 HKI-4983 >10 32.68 .+-. 4.92**
>10 -8.32 .+-. 2.23* 22 HKI-4982 6.53 .+-. 0.90 69.18 .+-.
3.14*** >10 -3.61 .+-. 0.98* 23 HKI-4981 8.59 .+-. 0.29 59.08
.+-. 2.03*** >10 -9.67 .+-. 3.44* 24 HKI-4980 >10 17.10 .+-.
3.77** >10 -10.55 .+-. 3.81* 25 HKI-4984 -- enhanced >10
18.10 .+-. 3.97** 26 HKI-4986 2.88 .+-. 0.27 76.17 .+-. 4.68***
>10 -4.55 .+-. 5.83 27 HKI-4987 >10 37.41 .+-. 5.43*** >10
-9.53 .+-. 5.26 28 HKI-4990 4.54 .+-. 1.18 91.04 .+-. 1.69*** 5.77
.+-. 1.14 77.57 .+-. 4.52*** 29 HKI-4988 0.65 .+-. 0.20 91.10 .+-.
0.38*** >10 29.85 .+-. 6.66** 30 HKI-4991 -- enhanced 6.23 .+-.
0.52 81.50 .+-. 6.97*** 31 HKI-4985 >10 7.44 .+-. 4.23 >10
-1.40 .+-. 3.53 32 TCH-30q >10 38.65 .+-. 2.30*** >10 3.69
.+-. 3.42 33 TCH-30r enhanced 7.24 .+-. 0.72 67.96 .+-. 5.23***
Percentage of inhibition (Inh %) at 10 .mu.M concentration. Results
are presented as mean .+-. S.E.M. (n = 3-5). *P < 0.05, **P <
0.01, ***P < 0.001 compared with the control value.
.sup.aConcentration necessary for 50% inhibition (IC.sub.50).
[0203] As evident from Table 2, most compounds, except compounds 6,
9, 16, 19, 21, 24, 25, 27, 30, 31, 32, and 33, were capable of
suppressing the superoxide anion generation (IC.sub.50 ranged from
about 4 nM to 8.6 .mu.M) and elastase released (IC.sub.50 ranged
from about 32 nM to 7.2 .mu.M) from the neutrophils in response to
fMLF. Among them, compound 13 (HKI-4971) appeared to be most
potent, with IC.sub.50 being about 3.7.+-.1.7 nM for suppressing
the superoxide anion generation, and 32 .+-.17 nM for suppressing
elastase release. Accordingly, HKI-4971 was chosen for further
evaluation on its biological activities in Examples 2 and 3.
Example 2
HKI-4971 Suppresses the Development of Imiquimod-Induced
Psoriasis
[0204] In this example, the effect of HKI-4971 (or compound 13) on
the development of psoriasis was investigated using the
well-established psoriasis animal model, in which imiquimod (IMQ)
was used to induce psoriasis-like skin inflammation. Briefly,
BALB/c mice are pre-treated by intraperitoneal injection of
HKI-4971 (20 mg/kg) for 30 min before 5% IMQ cream was topically
administered onto the back of the mice every day for 7 days. Then,
skin samples taken from these animals were subjected to microscopy
examination and immunohistochemistry staining, while blood samples
were subjected to analysis for liver and kidney functions. Results
are depicted in FIGS. 1 to 3.
[0205] FIG. 1(A) is a photograph taken from one representative
mouse treated with IMQ (left), and IMQ+HKI-4971 (right); while
photographs in FIGS. 1(B) and 1(C) respectively illustrating
microscopic presentation of the skins taken from the control
IMQ-treated mouse, and the mouse treated with IMQ+HKI-4971.
Immunostaining of skin samples taken from these animals confirmed
that the markers for cell proliferation (i.e., ki-67) and for
neutrophils (i.e., Ly6g and MPO) were reduced by HKI-4971 (FIG. 2).
Thus, it is clear that HKI-4971 may reduce the severity or
progression of IMQ-induced psoriasis in the tested animals.
[0206] Blood samples taken from IMQ- or IMQ+HKI-4971 treated
animals were analyzed for the levels of alanine aminotransferase
and aspartate transaminase, which were used as indicators for
assessing liver function; and the levels of blood urine nitrogen
(BUN) and creatinine (CRE), which were used as indicators for
assessing the kidney function. Results are illustrated in FIG. 3.
As evident from the data presented in FIG. 3, treatment of IMQ or
HKI-4971, either alone or in combination, did not result in
significant damage to the functions of liver or kidney.
[0207] Taken together, the data confirmed that HK-4971 manifested a
protective effect on IMQ-induced psoriasis, thus it may be used as
a medicament to treat psoriasis or skin-inflammatory diseases,
which often develops after a variety of insults, including but not
limiting to, bacterial infection, trauma, autoimmune disorder, and
allergy.
Example 3
HKI-497 Modulates LPS-Induced ALI
[0208] In this example, the Gram-negative bacterial endotoxin LPS
induced ALI in mice was employed to mimic ARDS in human, and the
effect of HKI-4971 was evaluated. Briefly, BALB/c mice were
pre-treated by intravenous injection of compound HKI-4971 (20
mg/Kg) for 1 hr, and then challenged with intratracheal injection
of LPS (2 mg/Kg) for 5 hrs. The level of MPO activity and the
markers for neutrophils (Ly6g and MPO) in lung tissues were taken
as indications of lung injury. Results are depicted in FIG. 4.
[0209] As FIG. 4(A) depicted, LPS evoked a surge in MPO activity in
lung tissues, an indication of inflammatory cells in lung tissue,
which was suppressed by the administration of HKI-4971. FIG. 4(B)
are representative photographs of lung sections staining by H&E
and the markers for neutrophils viewed under light microscopy. It
is evident that HKI-4971 significantly alleviated LPS-induced
neutrophil infiltration and lung injury.
[0210] Taken together, the data confirmed that HK-4971 manifested a
protective effect on LPS-induced lung injury, thus it may be used
as a medicament to treat ALI or ARDS, which often develops after a
variety of insults, including but not limiting to, sepsis, trauma,
and pneumonia.
[0211] It will be understood that the above description of
embodiments is given by way of example only and that various
modifications may be made by those with ordinary skill in the art.
The above specification, examples and data provide a complete
description of the structure and use of exemplary embodiments of
the invention. Although various embodiments of the invention have
been described above with a certain degree of particularity, or
with reference to one or more individual embodiments, those with
ordinary skill in the art could make numerous alterations to the
disclosed embodiments without departing from the spirit or scope of
the present disclosure.
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