U.S. patent application number 13/130159 was filed with the patent office on 2012-01-19 for chemical inhibitor of p53-snail binding and pharmaceutical composition for treating cancer disease containing same as its active ingredient.
This patent application is currently assigned to The Industry & Academic Cooperation in Chungnam National University. Invention is credited to Nam Chul Ha, Jee Hyun Lee, Sun Hye Lee, Bum Joon Park, Gyu Yong Song.
Application Number | 20120015960 13/130159 |
Document ID | / |
Family ID | 42281250 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120015960 |
Kind Code |
A1 |
Park; Bum Joon ; et
al. |
January 19, 2012 |
CHEMICAL INHIBITOR OF P53-SNAIL BINDING AND PHARMACEUTICAL
COMPOSITION FOR TREATING CANCER DISEASE CONTAINING SAME AS ITS
ACTIVE INGREDIENT
Abstract
Provided are compounds for inhibiting Snail-p53 binding and
therapeutic agents for cancer including the compounds as an
effective component. The Snail-p53 binding inhibitors induce
expression of p53 in K-Ras mutant cell lines, thereby enabling
effective treatment or prevention of K-Ras mutant cancer, such as,
pancreatic cancer, lung cancer, cholangioma, and colon cancer, of
which diagnosis or treatment is not easy.
Inventors: |
Park; Bum Joon; (Busan,
KR) ; Ha; Nam Chul; (Busan, KR) ; Lee; Sun
Hye; (Busan, KR) ; Song; Gyu Yong; (Daejeon,
KR) ; Lee; Jee Hyun; (Daejeon, KR) |
Assignee: |
The Industry & Academic
Cooperation in Chungnam National University
Daejeon
KR
Pusan National University Industry-University Cooperation
Foundation
Busan
KR
|
Family ID: |
42281250 |
Appl. No.: |
13/130159 |
Filed: |
November 23, 2009 |
PCT Filed: |
November 23, 2009 |
PCT NO: |
PCT/KR2009/006896 |
371 Date: |
May 19, 2011 |
Current U.S.
Class: |
514/255.01 ;
435/375; 435/7.1; 435/7.92; 514/319; 514/510; 514/569; 514/608;
514/618; 514/712; 544/391; 546/206; 554/109; 554/116; 562/427;
564/102; 568/43 |
Current CPC
Class: |
G01N 33/5748 20130101;
A61K 31/122 20130101; A61K 31/10 20130101; A61P 43/00 20180101;
C07C 2602/10 20170501; C07C 317/44 20130101; C07C 323/52 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
514/255.01 ;
544/391; 546/206; 564/102; 568/43; 562/427; 554/116; 554/109;
435/7.92; 435/375; 435/7.1; 514/319; 514/608; 514/712; 514/569;
514/510; 514/618 |
International
Class: |
A61K 31/495 20060101
A61K031/495; C07D 211/16 20060101 C07D211/16; C07C 313/24 20060101
C07C313/24; C07C 323/22 20060101 C07C323/22; C07C 323/62 20060101
C07C323/62; C07C 323/41 20060101 C07C323/41; G01N 33/566 20060101
G01N033/566; C12N 5/09 20100101 C12N005/09; A61K 31/451 20060101
A61K031/451; A61K 31/165 20060101 A61K031/165; A61K 31/10 20060101
A61K031/10; A61K 31/192 20060101 A61K031/192; A61K 31/216 20060101
A61K031/216; A61P 35/00 20060101 A61P035/00; C07D 295/192 20060101
C07D295/192 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 21, 2008 |
KR |
10-2008-0116343 |
Mar 5, 2009 |
KR |
10-2009-0018956 |
Aug 17, 2009 |
KR |
10-2009-0075529 |
Nov 18, 2009 |
KR |
10-2009-0111710 |
Claims
1. A compound represented by Formula 1 below or salt thereof:
##STR00007## wherein in Formula 1, m is an integer of 0 to 10, n
and p each are an integer of 0 or 1, Z is selected from the group
consisting of --NH(CH.sub.2).sub.qCH.sub.3, --OH, 4-phenylpiperidin
group, 4-phenylpiperazine group, isobutylamino group, and
isobutyloxy group, and q is an integer of 0 to 9.
2. The compound or salt thereof of claim 1, wherein the compound is
selected from the group consisting of
2-nonylamino-5,8-dimethoxy-1,4-naphtoquinone;
2-decylamino-5,8-dimethoxy-1,4-naphtoquinone;
3-(5,8-dimethoxy-1,4-dioxo-naphthalene-2-ylthio)propanoic acid;
11-(5,8-dimethoxy-1,4-dioxo-naphthalene-2-ylthio)undecanoic acid;
isobutyl-11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)-und-
ecanoate;
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)-N-i-
sobutyl undecanamide; and isobutyl
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylsulfinyl)undecanoa-
te.
3. A therapeutic agent for cancer, comprising a compound for
inhibiting Snail-p53 binding as an effective component.
4. The therapeutic agent of claim 3, wherein the compound is a
compound represented by Formula 1 below or salt thereof:
##STR00008## wherein in Formula 1, m is an integer of 0 to 10, n
and p each are an integer of 0 or 1, Z is selected from the group
consisting of --NH(CH.sub.2).sub.qCH.sub.3, --OH, 4-phenylpiperidin
group, 4-phenylpiperazine group, isobutylamino group, and
isobutyloxy group, and q is an integer of 0 to 9.
5. The therapeutic agent of claim 3, wherein the cancer is a K-Ras
mutant cancer.
6. The therapeutic agent of claim 5, wherein the K-Ras mutant
cancer is any one selected from the group consisting of pancreatic
cancer, lung cancer, cholangioma, and colon cancer.
7. A method of screening a therapeutic agent for K-Ras mutant
cancer, the method comprising: culturing Snail and a candidate drug
on a plate on which p53 is immobilized; and screening a candidate
drug for inhibiting Snail-p53 binding by using an ELISA leader.
8. A K-Ras mutant cells specific drug delivery method, comprising
delivering a target drug specifically to K-Ras mutant cells by
using endocytosis of a DNA binding domain of p53.
9. The K-Ras mutant cells specific drug delivery method of claim 8,
wherein the DNA binding domain comprises a sequence of 90-280 of a
human p53 amino acid sequence.
10. The K-Ras mutant cells specific drug delivery method of claim
8, the method comprising: treating a DNA binding domain of p53 and
a target drug into a cell; and delivering the target drug to
neighboring K-Ras mutant cells by endocytosis of the DNA binding
domain of p53.
11. A method of early diagnosing K-Ras mutant cancer, the method
comprising detecting expression of a Snail antibody.
12. The method of claim 11, wherein the expression of Snail
antibody is detected in a serum of a patient having K-Ras mutant
cancer.
13. The method of claim 11, wherein the K-Ras mutant cancer is any
one selected from the group consisting of pancreatic cancer, lung
cancer, cholangioma, and colon cancer.
14. The therapeutic agent of claim 4, wherein the cancer is a K-Ras
mutant cancer.
15. The K-Ras mutant cells specific drug delivery method of claim
9, the method comprising: treating a DNA binding domain of p53 and
a target drug into a cell; and delivering the target drug to
neighboring K-Ras mutant cells by endocytosis of the DNA binding
domain of p53.
16. The method of claim 12, wherein the K-Ras mutant cancer is any
one selected from the group consisting of pancreatic cancer, lung
cancer, cholangioma, and colon cancer.
Description
TECHNICAL FIELD
[0001] The present invention is directed to a compound that
inhibits Snail-p53 binding to induce expression of p53 so as to be
effectively used in treating a K-Ras mutant cancer, such as
pancreatic cancer, lung cancer, cholangioma, and colon cancer and a
therapeutic agent for cancer including the compound as an effective
component.
BACKGROUND ART
[0002] Improvement of anti-cancer drugs and diagnostic tools has
raised 5 years survival rate of overall cancer patients up to 50%.
However, some kinds of cancer including lung and pancreatic cancer
still show extremely low survival rate, less than 10%. Accordingly,
development of early diagnostic methods for these cancers is
urgently needed to increase survival rates of the cancer patients.
Interestingly, K-Ras is predominant event in such cancer, in
particular, in pancreatic cancer of which survival rate is 5% or
less.
[0003] Oncogenic Ras is known to induce senescence and apoptosis
through p53 activation, and formation of an oncogenic Ras mediated
tumor is assumed to occur under p53 deficient condition, and in
particular, H-Ras induced cancer cells are suppressed by rapidly
activated p53.
[0004] Currently available drugs for treating lung and pancreatic
cancer have relatively weak effect on extension of life span, and
cause various adverse effects. Accordingly, there is a need to
develop a drug for effectively treating or early diagnosing such
disease.
DETAILED DESCRIPTION OF THE INVENTION
TECHNICAL PROBLEM
[0005] The inventors of the present invention found that oncogenic
K-Ras suppresses p53 by inducing Snail, identified that a compound
can block an interaction between p53 and Snail, and found that the
compound induces p53 expression in K-Ras mutant cell lines, thereby
completing the present invention.
[0006] The present invention provides a method of screening a
therapeutic agent for K-Ras mutant cancer, in which the method
includes screening a candidate drug for inhibiting Snail-p53
binding.
[0007] The present invention also provides a compound for
inhibiting Snail-p53 binding and a therapeutic agent for cancer
including the compound as an effective component.
[0008] Also, the inventors of the present invention revealed that a
particular region of p53, for example, a DNA binding domain has
increasing permeation in K-Ras mutant cells, and thus, p53 can be
used as a carrier to deliver drug specifically to K-Ras mutant
cells. Also, they found that a K-Ras mutant cancer, such as
pancreatic, lung, cholangiocarcinoma, and colon cancer, can be
early diagnosed by detecting expression of Snail autoantibody,
thereby completing the present invention
[0009] Thus, the present invention also provides a drug delivery
method for delivering drug specifically to K-Ras mutant cells by
using endocytosis of a DNA binding domain of p53.
[0010] The present invention also provides a method of early
diagnosing K-Ras mutant cancer by detecting expression of Snail
autoantibody.
TECHNICAL SOLUTION
[0011] According to an aspect of the present invention, there is
provided a method of screening a therapeutic agent for K-Ras mutant
cancer, in which the method includes culturing Snail and a
candidate drug on a plate on which p53 is immobilized, and
screening a candidate drug that inhibits Snail-p53 binding by using
an ELISA leader.
[0012] According to an aspect of the present invention, there is
provided a compound represented by Formula 1 below or a salt
thereof:
##STR00001##
[0013] wherein in Formula 1,
[0014] m is an integer of 0 to 10, n and p each are 0 or 1,
[0015] Z is selected from the group consisting of
--NH(CH.sub.2).sub.qCH.sub.3, --OH, a 4-phenylpiperidin group, a
4-phenylpiperazine group, an isobutylamino group, and an
isobutyloxy group, and q is an integer of 0 to 9.
[0016] For example, the compound may be selected from the group
consisting of 2-nonylamino-5,8-dimethoxy-1,4-naphtoquinone;
2-decylamino-5,8-dimethoxy-1,4-naphtoquinone;
3-(5,8-dimethoxy-1,4-dioxonaphthalene-2-ylthio)propanoic acid;
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)undecanoic
acid;
isobutyl-11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydroxynaphthalene-2-ylt-
hio)-undecanoate;
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)-N-isobutyl
undecanamide; and isobutyl
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylsulfinyl)undecanoa-
te, or a salt thereof.
[0017] The compound of Formula 1 enables the method of screening a
therapeutic agent for K-Ras mutant cancer to be used to selectively
screen a drug for inhibiting Snail-p53 binding, thereby effectively
treating or preventing K-Ras mutant cancer, such as pancreatic,
lung, and colon cancer, that are difficult to be diagnosed or
treated.
ADVANTAGEOUS EFFECTS
[0018] A method of screening a therapeutic agent for K-Ras mutant
cancer according to the present invention enables a drug for
inhibiting Snail-p53 binding to be specifically screened, thereby
effectively treating or preventing a K-Ras mutant cancer, such as
pancreatic, lung, cholangiocarcinoma, and colon cancer, of which
diagnosis or treatment is not easy.
[0019] Also, a DNA binding domain of p53 is used as a carrier to
deliver drug specifically to K-Ras mutant cells, which is very
useful for treatment of K-Ras mutant cancer. In addition, K-Ras
mutant cancer may be early diagnosed through identification of
expression of Snail autoantibody. Thus, it is possible to early
diagnose pancreatic cancer of which diagnosis is difficult, thereby
increasing a survival rate of caner patients or treatment
efficiency.
DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1 to 3, and 12 show molecular mechanisms of Snail
mediated p53 expression suppression.
[0021] FIG. 4 shows that oncogenic K-Ras mediated p53 suppression
is not blocked by a chemical inhibitor.
[0022] FIG. 5 shows that Snail is a critical mediator for oncogenic
K-Ras mediated p53 suppression.
[0023] FIG. 6 shows a direct interaction between Snail and p53.
[0024] FIG. 7 shows conditions required to activate ATR in
stabilizing K-Ras induced Snail.
[0025] FIG. 8 shows a direct binding between Snail and p53.
[0026] FIG. 9 shows identification of a Snail and p53 binding
inhibitor.
[0027] FIGS. 10 and 11 show that p53 function is induced in K-Ras
mutant cells by blocking Snail and p53 binding.
[0028] FIG. 13 shows results of an exporting mechanism of p53 from
a nucleus to a cytoplasm.
[0029] FIG. 14 shows that p53 is secreted through
vesicle-transport.
[0030] FIG. 15 shows results that p53 is removed by protease and
endocytosis, and Snail shows a resistance to protease and
endocytosis.
[0031] FIG. 16 shows re-absorption of His-p53 in K-Ras mutant
cells.
[0032] FIG. 17 shows secretion of p53 and Snail in cancer
tissues.
[0033] FIG. 18 shows analysis results of anti-Snail antibody in a
lung cancer serum.
[0034] FIG. 19 shows schematic secretion morphology of p53 and
Snail in oncogenic K-Ras.
[0035] FIG. 20 shows western blot assay results for identifying an
induction capability of compounds synthesized according to Examples
1 to 5.
[0036] FIG. 21 shows GST-full down assay results for identifying a
Snail-p53 binding inhibiting effect of Nutlin-3 and compounds 5o
and 7a.
[0037] FIG. 22 shows apoptosis rates when K-Ras mutant cancer cell
lines and K-Ras wild type cancer cell lines are treated with
compounds 5o and 7a and Nutlin-3.
[0038] FIG. 23 is a graph of western blot assay results that p21
activity is induced by treating compound 5o and Nutlin-3 into p53
mutant cell lines.
[0039] FIG. 24 shows a survival rate of a mouse treated with
compound 5o after A549 cells were injected into athymic mouse
through intraperitoneal injection.
[0040] FIG. 25 shows a tissue image of a tumor generated through
intraperitoneal injection.
[0041] FIG. 26 shows an overall anatomical abnormal finding
according to treatment with compound 5o in A549 cells.
BEST MODE
[0042] According to an embodiment, the compound of Formula 1 may be
present in a form of a salt. The salt may be a pharmaceutically
available salt of an inorganic acid, such as a hydrochloric acid or
a sulfuric acid, or an organic acid, such as p-toluene sulfonic
acid.
[0043] The compound of Formula 1 may be prepared through Reaction
Schemes 1 to 5.
##STR00002##
##STR00003##
##STR00004##
##STR00005##
##STR00006##
[0044] Reaction Scheme 2 will now be described in detail.
[0045] 1,4,5,8-tetramethoxynaphthalene (3) is synthesized from
1,5-dihydroxynaphthalene (2) as a starting material through a known
three-phase reaction, and then 1,4,5,8-tetramethoxynaphthalene (3)
is demethylated to produce 5,8-dimethoxy-1,4-naphtoquinone(4) as a
synthesis intermediate. A detailed synthesis method is disclosed in
cited references presented in the reaction schemes above, and a
solvent used herein may be a solvent that does not adversely affect
the reaction, and examples of such a solvent are sodium hydroxide,
acetonitrile, anhydrous methanol, N,N-dimethylformamide, and
chloroform. The initial methylation is performed in such a manner
that dimethyl sulfate is dropped to 1,5-dihydroxynaphthalene
dissolved in sodium hydroxide in the presence of a nitrogen gas for
1 hour and the reaction was performed for 2 hours. The reaction
product is re-crystallized with benzene to produce
1,5-dimethoxynaphthalene.
[0046] 1,4,5,8-tetramethoxynaphthalene (3) is prepared by
thermal-refluxing sodiummethoxide and iodine copper in
dimethylformamide and methanol under an anhydrous condition for 30
hours. The refluxing is continued at reaction temperature of
80.degree. C. or higher. Intermediate
5,8-dimethoxy-1,4-naphtoquinone is synthesized using nitric acid
ceriumdiammonium. That is, nitric acid ceriumdiammonium is dropped
thereto at room temperature for 30 hours and the reaction is
further performed for 30 minutes. In order to prepare compounds 5a
to 5p, intermediate 5,8-dimethoxy-1,4-naphtoquinone(4) is dissolved
in methanol, and desired amine or mercaptan or a mercaptan having
an end to which a carboxylic group or a hydroxyl group is bound is
added thereto and stirred at room temperature for 4 hours
overnight, and a reaction progress is identified by TLC and the
reaction is worked-up by using sulfuric acid and dichromate sodium
aqueous solution, and the reaction product is isolated by silicagel
column chromatography.
[0047] When a 4-phenylpiperidine or 4-phenylpiperazine derivative
is attached to a carboxylic group at site 2 of compound 5o in the
following step, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydro chloride (EDC), instead of 1,3-dicyclohexylcarbodiimide (DCC)
and N,N-dimethylaminopyridine (DMAP), is added thereto to
synthesize compounds 6a and 6b, and in this case, the reaction
yield is high and in a separation process, the reaction product is
clearly separated without urea. In the next step, when
isobutylalcohol and isobutylamine are attached by using compound
5p, compounds 7a and 7b may be easily obtained by using
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride (EDC).
When sulfoxide compound 8 is synthesized, compound 7a and MCPBA are
used and a reaction progress is identified by TLC. The reaction is
worked-up by using sodium bicarbonate, and the reaction product was
passed through a silicagel column to obtain purified compound
7a.
[0048] However, the methods according to Reaction Schemes 2 to 5
are just an example of a method of preparing the compound of
Formula 1. For example, reaction conditions, such as an amount of a
reaction solvent used, an amount of a base used, and an amount of a
reaction material used, are not limited thereto, and various other
synthesis methods that are known to one of ordinary skill in the
art, in addition to the methods according to Reaction Scheme 2 to
5, may also be used to prepare the compound of Formula 1.
[0049] Also, the present invention provides a therapeutic agent for
cancer that includes a compound for inhibiting Snail-p53 binding as
an effective component.
[0050] The compound may be a compound of Formula 1 or a salt
thereof, and preferably, a compound selected from the group
consisting of 2-nonylamino-5,8-dimethoxy-1,4-naphtoquinone;
2-decylamino-5,8-dimethoxy-1,4-naphtoquinone;
3-(5,8-dimethoxy-1,4-dioxo-naphthalene-2-ylthio)propanoic acid;
11-(5,8-dimethoxy-1,4-dioxo-naphthalene-2-ylthio)undecanoic acid;
isobutyl-11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)-und-
ecanoate;
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)-N-i-
sobutyl undecanamide; and isobutyl
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylsulfinyl)
undecanoate, or a salt thereof.
[0051] The cancer may be K-Ras mutant cancer. For example, the
cancer may be selected from the group consisting of pancreatic
cancer, lung cancer, cholangioma, and colon cancer.
[0052] The therapeutic agent for cancer according to the present
invention may further include a carrier, an excipient, or a
diluting agent, each of which is appropriate for use as a
therapeutic agent and is conventionally used in preparing a
pharmaceutical composition.
[0053] Examples of a carrier, an excipient, and a diluting agent
which are available for the present invention are lactose,
dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,
maltitol, amylum, acacia rubber, alginate, gelatin, calcium
phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinylidon, water,
methylhydroxybenzoate, propyl hydroxybenzoate, magnesium stearate,
and mineral oil.
[0054] The therapeutic agent for cancer may be prepared in an oral
formation, such as powder, a granule, a tablet, an capsule, a
suspension, an emulsion, a syrup, or an aerosol, an external
applicable formulation, an suppository formulation, or a sterile
injectable solution, according to a corresponding conventional
preparation method.
[0055] The preparation may be performed using a conventional
diluting agent or excipient, such as a filling agent, an extender,
a binder, a wetting agent, a disintegrant, or a surfactant.
Examples of a solid formulation for oral administration are a
tablet, a pill, powder, a granule, and a capsule, and such solid
formulations are prepared by mixing the compound as described above
with one or more excipients, for example, amylum, calcium
carbonate, sucrose or lactose, or gelatin.
[0056] Also, in addition to the excipients, a lubricant, such as
magnesium stearate, or talc, may additionally be used. Examples of
a liquid formulation for oral administration are a suspension, a
preparation dissolved in liquid, an emulsion, and syrup. The liquid
preparation may include, in addition to a conventional simple
diluting agent, such as water or liquid paraffin, various other
excipients including a wetting agent, a sweetener, an odorant, and
a preservative.
[0057] Examples of a formulation for parenteral administration are
a sterile aqueous solution, a non-aqueous solvent, a suspension, an
emulsion, a lyophillization formulation, and a suppository
formulation. Examples of a non-aqueous solvent and a suspension are
vegetable oil, such as propylene glycol, polyethylene glycol, or
olive oil, and an injectable ester such as ethylollate. As a
support for suppositories, witepsol, macrogol, tween 61, Cacao oil,
laurin oil, or glycerogeratin may be used.
[0058] A dose of the therapeutic agent for cancer may differ
according to the age, gender, or weight of a patient, and for
example, a dose of 0.1 to 100 mg/kg may be administered as a bolus
or divided into a few portions per day.
[0059] In addition, the dose of the therapeutic agent for cancer
may be increased or decreased according to an administration
pathway, a progress degree of disease, a gender, a weight, or an
age. Accordingly, the dose may not limit the scope of the present
invention in any respects.
[0060] The therapeutic agent for cancer may be administered to a
mammal, such as rats, mice, livestock, or humans, through various
administration pathways. All administration methods may be obvious,
for example, oral administration, rectal administration, or
intravenous, intramuscular, hypodermic, intrauterine epidural, or
intracerebroventricular injection.
[0061] Also, the present invention provides a K-Ras mutant
cells-specific drug delivery method of delivering a target drug
specifically to K-Ras mutant cells by endocytosis of a DNA binding
domain of p53.
[0062] The DNA binding domain includes a sequence of 90-280 of
human p53 amino acid sequence (Genbank Accession No. P04637).
[0063] Preferably, the drug delivery method may include treating
cells with a DNA binding domain of p53 and a target drug; and
delivering the target drug to neighboring K-Ras mutant cells by
endocytosis of the DNA binding domain of p53.
[0064] Also, the present invention provides a method of early
diagnosing a K-Ras mutant cancer by detecting Snail antibody
expression.
[0065] The Snail antibody expression may be detected in a serum of
a patient having K-Ras mutant cancer, and the K-Ras mutant cancer
may be cancer selected from the group consisting of pancreatic
cancer, lung cancer, cholangioma, and colon cancer.
MODE OF THE INVENTION
[0066] Preferred embodiments of the present invention will now be
described in detail. However, the embodiments are presented only
for illustration purposes.
[0067] [Molecular Mechanism of Snail Mediated p53 Suppression]
EXAMPLE 1
[0068] 1. Isolation of Mouse Fibroblast and Immortalization
[0069] 6 month-old male mouse was scarified to collect fibroblast.
After isolation of lung, tissue was chopped and dissociated using a
culture-mess. After three-day of incubation in DMEM medium
containing 20% FBS, attached cells were seeded in culture dishes
and transfected with mutant H-Ras, N-Ras, and K-Ras using Jetpei
following a manufacturer's protocol. After 72 hours, transfected
cells were selected using 400 .mu.g/ml of G418 containing DMEM.
[0070] 2. Cell Culture and Reagents Preparation
[0071] Cell lines used herein were obtained from ATCC and
maintained in RPMI-1640 or DMEM containing 10% FBS. Antibodies used
herein were purchased from Santa Cruz or Cell Signaling (p53-R,
p-Erk). Ras expression vectors and Snail vectors were provided by
Dr. Chi SG and Hung M-C respectively. Chemicals used herein were
purchased from Calbiochem. Recombinant p53 was obtained from Assay
designs.
[0072] Cell fraction analysis was performed using a Subcell
fraction Kit (Merck) according to the manufacturer's protocol. For
analysis of media, a cell cultured media was collected and
concentrated by using Centricon (Millipore) or EtOH
precipitation.
[0073] 3. Immuno-Staining and Western Blotting
[0074] For cell staining, the cultured cells were washed and fixed
with 100% Me-OH and incubated with antibodies (First antibody:
1:200, overnight at 4.degree. C.; secondary antibody: 1:1000, 2
hours at RT). To detect secreted p53 and Snail, HCT116 p53.sup.-/-
cells were transfected with vectors for 24 hours in 1 ml PRMI 1640
medium and fixed by adding of 1 ml of 2% PFA without washing. After
fixation, cells were washed with PBS twice and incubated with
blocking buffer (PBS+anti-Human antibody (1:500)) to eliminate
non-specific binding. After washing with PBS, cells were incubated
with anti-p53 and anti-Snail antibodies and matched with the
secondary antibody. For protein analysis, protein was extracted
through RIPA buffer and the sample was applied to SDS-PAGE
according to a conventional western blot protocol.
[0075] Immuno-precipitation analysis was performed according to a
conventional protocol. That is, cell lysate was incubated first
with an antibody for 4 hours and then with protein-A/G-agarose for
2 hours The incubation product was centrifuged and washed 3 times.
The precipitated complex was subjected into SDS-PAGE/WB
analysis.
[0076] 4. Transfection and Reviewing of si-RNA Effect
[0077] For cell transfection, Jetpei was performed according to the
manufacturer's protocol. Cells were incubated with DNA/Jetpei
mixture for 24 hours in a complete media. For in- vitro gene knock
out, si-RNA against Snail and MDM2 were prepared. si-RNA was
transfected using Jetpei, and after 24 hours, the effect was
checked.
[0078] 5. Experimental Results
[0079] As shown in FIG. 1A, the transfection of N-Ras or H-Ras
induced apoptosis or senescence, but K-Ras transfected cells were
growing and maintained even over than 6 months from the
transfection of K-Ras.
[0080] Also, Forced expression of oncogenic K-Ras suppressed the
p53 expression in wild type p53-containg cell lines (see FIG. 2A),
and differentially from H-Ras or N-Ras, K-Ras evoked p53
suppression, which was not blocked by si-MDM2 (see FIG. 2B).
However, wild type K-Ras did not suppress the p53 expression (see
FIGS. 2C and 2D).
[0081] As shown in FIG. 1B, p53 was suppressed as in a K-Ras-dose
dependent manner. Blocking of Ras activity though DN-Ras increased
the p53 expression only in K-Ras mutated A549 but not in HepG2
(FIG. 2E).
[0082] This result suggests that endogenous oncogenic K-ras
suppresses p53 expression. However, as shown in FIG. 10, However,
DN-Ras did not show obvious and synergic effects on DNA
damage-mediated p53 suppression, implying that strong genotoxic
stress overcame the oncogenic K-Ras-mediated p53 suppression.
[0083] As shown in FIGS. 2F and 2G, K-Ras-mediated p53 suppression
was detected in point mutant. However, p53 S46D, active form of
p53, showed the resistance to K-Ras-mediated p53 suppression. This
result is consistent with the previous result that genotoxin
induced p53 activation overcame K-Ras-mediated suppression.
[0084] Since 22/23 mutant does not associate with MDM2, it was
confirm that K-ras-mediated p53 suppression is achieved through
MDM2 independent pathway. Also, since proteasome-inhibitors did not
block the K-Ras-mediated p53, this result shows the irrelevance of
MDM2 or p53 ubiquintin system (see FIGS. 1A and 2H).
[0085] Also, the effect of MAPK signaling inhibitors on
K-Ras-mediated p53 suppression was checked, and it was confirmed
that blocking of MAPK pathway did not abolish the effect on
K-Ras-mediated p53 suppression. These results implied that K-Ras
mediated p53 suppression would be achieved through novel
pathway.
[0086] Also, distribution of p53 by Snail in an insoluble fraction
was checked, and it was found that as shown in FIG. 12A, p53 was
reduced by Snail or oncogenic K-Ras, And cells were divided into 4
fractions, that is, nuclear, cytoplasmic, membrane/organells, and
insoluble fractions, and checked, and it was found that as shown in
FIG. 12B, p53, reduced by Snail, was not recovered in any kinds of
subcellular fractions.
[0087] Also, elimination of Snail from K-Ras mutant cells induced
p53 dominantly than si-MDM2 (FIG. 12C). And, as shown in FIGS. 12D
and 12E, differentially from MDM2-mediated suppression, reduction
of p53 by Snail was not recovered by proteasome inhibitors.
EXAMPLE 2
[0088] 1. Western Blot and In Vitro Kinase or Binding Assay
[0089] To address direct binding between Snail and p53, a membrane
was loaded with recombinant p53 or Snail or p53 transfected cell
lysate through typical SDS-PAGE and gel transfer method. After
blocking with 5% non-fat dry milk, the membrane was incubated with
p53 or Snail transfected p53.sup.-/- HCT116 cell lysate for 4 hours
at 4.degree. C. After washing, the membrane was subjected into a
typical WB procedure with p53 antibody or Snail antibody.
[0090] For in vitro binding, the recombinant p53 and GST-Snail were
alternately incubated for 1 hour at 4.degree. C. and performed the
IP with p53 antibody or GST antibody and WB with GST or p53
antibody. To examine the modification of Snail, 293 cells were used
for transfection. After fraction or lysis, lysates were incubated
with GST or GST-Snail for 1 hour at 25.degree. C. and subjected
into SDS-PAGE and WB analysis. Antibodies against p-MAPK substrate
and p-ATM/ATR substrate were obtained from cell signaling.
[0091] 2. Experimental Results
[0092] As shown in FIG. 3A, the effect of K-Ras on Snail expression
was checked and it was found that Snail was induced by K-Ras. Also,
the effect of Snail on p53 expression in cell lines was checked and
it was found that as shown in FIG. 3B, overexpression of Snail
suppressed p53 in A549 and HepG2 cell lines, whereas Snail knock
down induced p53 only in A549 (oncogenic K-Ras containing cell
line) but not HepG2.
[0093] In addition, si-Snail increased the sensitivity to a DNA
damage agent (see FIG. 4F). Thus overexpression of Snail promoted
cell proliferation and render the resistance to DNA-damage-induced
cell death (see FIG. 4G).
[0094] As show in FIG. 3C, Snail also suppressed the exo-p53 as
well as endo-p53, similarly to K-Ras (FIG. 2C), and when Snail and
p53 were co-transfected as shown in FIGS. 3C and 3D, they were
reduced together, in regardless of mutant p53. However, mRNA of
Snail and p53 were not reduced (FIGS. 3C and 3D). Also, the effect
of Snail on p53 transcript was checked and it was found that Snail
did not reduce p53 mRNA (FIG. 4H).
[0095] Accordingly, these results indicated that although p53 and
Snail were well-confirmed transcriptional regulators, their
reduction was irrelevance with transcriptional regulation. In
addition, elimination of Snail blocked the K-Ras-mediated p53
suppression as shown in FIGS. 3G and 3H. Also, the similar result
was obtained from exo-p53. These results indicate that
K-Ras-mediated p53 suppression is achieved through Snail
induction.
[0096] Also, as shown in FIG. 5C, oncogenic K-Ras induced p53
within 4 hours, whereas p53 was reduced after 6 hours. This result
indicated that p53 suppression was not achieved by
transfection-artifact but an effect of transfected proteins.
[0097] Also, as shown in FIG. 5E, apoptosis and cell cycle in
KRas/Snail transfected cells were checked, and it was found that
apoptosis and cell cycle inhibition was not obviously induced by
K-Ras/Snail.
[0098] Also, the reduction of p53 by K-Ras/Snail in Aphidicolin
treated cells was observed. This result suggested that reduction of
p53 was not linked to cell cycle.
[0099] Also, the effect of Snail on half-life of p53 through
CHX-pulse chase was checked, and it was found that as shown in FIG.
5F, Snail did not shorten p53 half-life. In addition, the effect of
Snail on the expression of p53 S46D was checked and it was found
that, as shown in FIG. 6A, differentially from K-Ras, Snail
suppressed p53 S46D expression.
[0100] Also, the effect of p53 S46D on Snail expression was checked
and it was found that, as shown in FIG. 6B, S46D suppressed Snail
expression at transcription and translation levels. Accordingly,
differentially from wild type p53 in which si-Snail restored the
p53 suppression, as shown in FIG. 6C, si-Snail did not induce p53
expression when S46D was transfected.
[0101] These results indicate that under certain stress condition,
activated p53 by modification at serine 46 residue may overcome
K-Ras mediated suppression.
[0102] Also, induction of Snail was achieved through ATR.
[0103] To address how K-ras induce Snail, the engagement of AKT was
examined. It was known that Ras activates AKT to suppress
GSK-3-mediated Snail destabilization.
[0104] But AKT-KD did not block the Snail or K-Ras-induced p53
suppression as shown in FIG. 7A. In contrast, suppression of ATR
through si-RNA blocked the p53 suppression as shown in FIG. 7B.
Indeed, Snail was increased by ATR but not ATM and nccodazole
treatment as shown in FIGS. 7C and 7D. In vitro kinase assay showed
that Snail was phosphorylated by ATR as shown in FIG. 6D. K-Ras,
which has been known to activate ATR, also increased p-Snail as in
ATR-dependent manner as shown in FIG. 7E and extended half-life of
Snail.
EXAMPLE 3
[0105] 1. Recombinant Proteins and GST-Pull Down Analysis
[0106] Three human snail fragments (residues 1-90, 91-112, and
113-264) and p53 fragments (1-93 and 93-292) were expressed in
Escherichia coli (E. coli) as a GST-fusion protein. Each of the
fragments was loaded on to GSH-agarose, washed, and then eluted
using a buffer containing 20 mM reduced glutathione. The eluted
fractions were further purified using an anionexchange
chromatography (HitrapQ). The recombinant human p53 protein
(residues 94 292) was expressed in E. coli using a vector pET28A
which contains a hexa-histidine tag at C terminus.
[0107] The p53 protein was purified using Ni--NTA affinity and size
exclusion chromatography (Superdex 200). To identify a direct
binding between p53 and Snail, agarose bead conjugated GST or
GST-Snail was incubated with cell lysate or His-p53 in RIPA for 45
min at 4.degree. C. After washing with PBS and RIPA, precipitated
protein was subjected into SDS PAGE and WB.
[0108] 2. Experimental Results
[0109] Since Snail is nuclear protein, p53 were disappeared when
they were co-transfected (see FIGS. 3D to 3F). As shown in FIG. 8A,
it was confirmed from endo-IP that these proteins are associated
with each other. As shown in FIGS. 8B and 8C, far-western blot
analysis and GST-Pull down assay indicated that Snail and p53 were
directly interacted with each other. Also, a DNA binding domain of
p53 and a middle region of Snail performed as a binding domain (see
FIGS. 8D to 8F, 6E, and 6F.)
EXAMPLE 4
[0110] 1. Preparation of ELISA System for Chemical Screening
[0111] To isolate Snail-p53 binding inhibitor, ELISA system was
prepared. His-p53 (93-292) was immobilized on 96 well plates using
0.5% PFA. After drying and washing, the 96 well plates were
incubated with GST Snail with 0.1 .mu.M of chemicals (final
concentration). After 1 hour incubation, the 96 well plates were
washed with TBST and incubated with anti-GST-antibody (1:10000, 45
min) and anti-mouse-IgG-HRP (1; 50000, 30 min). After washing
twice, plates were incubated with a TMB solution and a stop
solution. Measurement was performed using an ELISA reader.
[0112] 2. Experimental Results
[0113] As shown in FIGS. 9A and 9B, p53 and Snail binding
inhibition was checked by ELISA system. As shown in FIG. 9C, from
about 150 chemicals, 3 kinds of chemicals were identified as an
inhibitor of Snail and p53 binding. As shown in FIGS. 10A and 11A,
these chemicals showed dose-dependent inhibition of Snail and p53
binding.
[0114] Through the GST-pull down assay, the expression of p53 and
its targets after treatment of these chemicals was measured, and it
was found that all of them blocked the interaction of p53 and Snail
and induced p53 expression (see FIGS. 8B and 11B).
[0115] Also, induction of PUMA and p21 by treatment of these
chemicals was observed. In particular, induction of p53 was
detected only in K-Ras mutated cells but not wild type K-Ras
harboring cells (see FIG. 10D). Similar structure of quercetin and
morin suggested that the screening system was reliable (see FIG.
10E).
[0116] Also, the effect of the chemicals on Snail-mediated p53
suppression was checked. As shown in FIGS. 10E and 10F, p53
reduction by co-transfection of Snail was blocked by treatment of
chemicals by #3 and #9. These results suggested that blocking of
p53-Snail interaction was restored the p53 expression.
[0117] Also, the effect of these chemicals on cell proliferation
was examined using tryphan blue staining. It was found that these
chemicals obviously suppressed cell proliferation in A549, whereas
they did not show anti-proliferating effect on MKN45 (see FIG.
11C).
[0118] Also, ferulic acid evoked cell death in K-Ras mutated cells
(see FIG. 11D). Moreover, Quercetin (#2) was identified as an
inhibitor of Snail-p53 interaction.
[0119] [K-Ras Mutant Cells Specific Drug Delivery Method Using
Endocytosis of DNA Binding Domain of p53]
EXAMPLE 5
[0120] 1. Exporting Mechanism of p53 from a Nucleus to a
Cytoplasm
[0121] To perform a GST-pull down assay, first, a human Snail and
p53 recombinant protein was prepared using a known method
(Neoplasia 11: 1-10, 2009). To identify a direct binding between
p53 and Snail in media and whole cell lysate, agarose bead
conjugated GST or GST-Snail was incubated with cell lysate or
culture media at 4.degree. C. for 2 hours. After washing with PBS
and RIPA, precipitated protein was subjected into SDS PAGE and WB
by using the same method as described above.
[0122] 2. Experimental Results
[0123] The effect of Snail on p53 NES that is mutated in a p53
nuclear exporting sequence from a nucleus to a cytoplasm was
checked, and it was found that Snail reduced p53 NES expression
(see FIG. 13A) and leptomycin B(LMB; nuclear exporting blocker from
a nucleus to a cytoplasm) did not block the Snail-mediated p53
reduction (see FIGS. 13B and 13C). The reduction of p53 by Snail
was confirmed in real time (see FIG. 13D). Expression of p53 in
culture-media was checked and within 2 hours, the expression of p53
in media was detected (see FIG. 13E). Moreover, p53 was detected in
cytosol as a vesicle-like-structure in Snail or K-Ras transfected
cells, and finally detected in an extracellular region (see FIG.
13F). Meanwhile, Snail was located in vesicle of cytosol with p53
(see FIG. 17A).
[0124] To confirm secretion of p53 in K-Ras mutated cells, the
GST-Pull down assay was performed using Snail-GST in culture media
and cell lysates. It was confirmed that although Snail-associated
p53 was detected in all cell lysates, the median p53 was observed
only in K-Ras mutated pancreatic cancer cell lines (see FIG. 13G).
Also, the median p53 was identified in K-Ras mutated cell's culture
media, without Snail-pull down (see FIG. 13H).
[0125] Since p53 is reduced by vesicle-like transport, p53
expression was checked by disrupting cytoskeleton-network by
Nocodazole (Noc). It was found that the Noc treatment blocked the
Snail or K-Ras mediated p53 reduction (see FIG. 14A). Despite of
cytoplasmic vesicle-like staining of p53 in Noc-treated cells,
cellular morphology was changed differentially from control cells
(see FIG. 14B). Noc blocked the p53 reduction (see FIG. 14C).
Median p53 was disappeared by Noc-treatment, whereas p53 was
accumulated in cytoplasm as vesicle (see FIGS. 14D and 14E).
Aph/Noc blocked the reduction of p53, which was not achieved by LMB
(FIGS. 14F and 14G).
EXAMPLE 6
[0126] 1. Behaviors of Extracellular p53 and Snail
[0127] 1) Tissue Analysis
[0128] Normal and tumor paired cholangioma and liver tissues were
obtained from Shunchunhyang Medical Center. Tissues were rapidly
frozen in the deep freezer until use. Frozen tissues were sliced
and 0.5 mg of tissues was incubated in 0.25 ml serum free medium
for 30 min. at 37.degree. C. to allow the release of tissue fluid.
After incubation, the culture medium was collected and precipitated
with 0.5 ml 100% Et-OH. Precipitated materials were dissolved using
RIPA and used for SDS-PAGE and WB analysis. We also obtained same
culture medium through the same method and used the culture medium
to detect p53 antibody.
[0129] 2) p53 ELISA Assay
[0130] To examine the p53, we performed the ELISA following
manufacture's protocol (Assay Design). In brief, 0.2 ml tissue
cultured media was added to wells and incubated with detection
antibody. After washing with a wash buffer, 0.2 ml of a substrate
sol and 0.05 ml of a stop solution were added thereto.
[0131] 3) Snail Antibody Detection in Blood Samples
[0132] Human blood samples were obtained from Shunchunhyang
University (pancreatic cancer and gall stone patients), and the
medical center of Pusan National University (lung cancer). Normal
blood samples were collected from volunteers or non-cancer
patients. Serum was collected by centrifugation and kept at
-70.degree. C. until use. 3 .mu.l of serum was incubated with
agarose-conjugated GST-Snail-N after pre-clearing with GST-protein.
Precipitated GST-Snail-antibody complex was dissolved with RIPA and
SDS sample buffer, and subjected into SDS-PAGE in the same method
as described above. After transfer to PVDF membrane, protein was
incubated with anti-human antibody and anti-GST antibody.
[0133] 2. Experimental Results
[0134] First, A549 and MKN 45 cells were treated with recombinant
p53, and their locations were identified. In comparison with
control protein (His-lamin A) recovered from media, His-p53 was
expressed in whole cell lysate of A549 (FIG. 15A). In addition,
His-p53 was completely removed from MKN45 cells and culture media
thereof (FIG. 15A).
[0135] To get more detail, in the present invention, recombinant
p53 was cultured using fresh media, A549-culture media, PC3, and
HCT116. In comparison with His-laminA recovered from media, p53-His
was detected in whole cell lysate of HCT116. In addition, p53 was
not recovered neither from A549 culture media nor PC3. These
results show that p53 was digested by protease that was secreted
from cultured cells and also resorpted by K-Ras mutated cells.
[0136] Also, protease inhibitor (PMSF) and endocytosis inhibitor
(Brefeldin A; BFA) were treated into A549 and Capan-1. Despite of
non-effect on intrecellular p53 expression, both chemicals
increased expression of median p53 (see FIG. 15B). BFA blocked the
location of recombinant p53 in A549 whole cell lysate (FIG.
15C).
[0137] To know the behavior of secreted snail, treatment with
recombinant Snail was performed and the p53 destination was
compared. p53 middle region was recovered from WCL of A549, which
was suppressed by BFA. But recombinant p53, treated in MKN 45, was
disappeared in media and in WCL. These results suggested that
secreted p53 would be digested by not only serine protease but also
other kinds of proteases such as MMP. Meanwhile, Snail was
recovered from media, and the recombinant Snail was recovered
without protease inhibitor. These results suggested that Snail
would be resistant to endocytosis as well as protease-mediated
digestion.
[0138] The effect of K-Ras on p53-endocytosis was measured and it
was found that recombinant p53 was selectively eliminated by K-ras
transfected cells. If p53 middle region could be re-entered by
K-Ras mutated cells selectively, this property would be useful for
chemical delivery to K-Ras mutated cells.
[0139] To confirm this, PI (propidium iodine; red dye, 50 .mu.g/mL)
and His-p53(2 .mu.g/mL) were treated into A549 and MKN 45. PI alone
was not accumulated into cells in both cell lines. When, however,
PI and p53 were co-treated, PI, but not MKN 45, was accumulated in
inner cells of A549 (FIGS. 15E, and 16A to 16B). These results
suggested that p53 could be useful for K-Ras specific drug delivery
system.
[0140] To check whether autoantibody of p53 is produced by
Snail-mediated secreted p53, p53 and Snail expression in
tissue-fluid was identified. p53 and Snail were detected in
cholangioma, but not in hepatocellular carcinoma (HCC) and
non-cancer tissue fluid (see FIGS. 17A and 17B). As shown in FIG.
17C, the presence of p53 in tissue fluid was examined using ELISA.
It was found that anti-p53 antibody and anti-Snail antibody were
detected in cholangioma, but not in HCC (see FIGS. 17D and 17E). In
addition, autoantibody against p53 and Snail in pancreatic or Bile
duct cancer patient's blood serum was examined, and it was
confirmed that anti-p53 antibody did not show relevance with
cancer. These results suggested that p53 autoantibody did not show
relevance with cancer status, and secreted p53 was rapidly removed
by protease and endocytosis (see FIG. 15A).
TABLE-US-00001 TABLE 1 sample serum serum anti-p53 Anti- NO Age Sex
Stage Diagnosis p53 Snail Ab Snail Ab 1 45 F IV CBD
cancer/adenocarcinoma Y Y Y Y 2 78 F III CBD cancer/adenocarcinoma
Y Y Y Y 3 58 IIB adenocarcinoma (intrahepatic Y Y Y Y
cholagiocarcinoma) 4 74 M IIA CBD cancer/adenocarcinoma Y Y Y Y 5
62 M IIB Gall Bladder adenocarcinoma Y Y Y Y 6 38 M IIA CBD
cancer/adenocarcinoma Y Y Y Y 7 56 F IIB adenocarcinoma
(intrahepatic Y Y Y Y cholagiocarcinoma) 8 59 M IIA CBD
cancer/adenocarcinoma Y Y Y Y 9 68 M IB Amulla of Vater Y N N Y
adenocarcinoma (weak) 10 54 F IIB CBD cancer/adenocarcinoma Y Y N N
HCC 1 58 M II HCC N N N N 2 43 M I HCC N N N Y(weak) 3 66 M III HCC
N N N Y(weak) 4 63 F F HCC N N N N
[0141] In contrast, Snail was resistant to protease and endocytosis
(FIG. 15D). Thus, Snail autoantibody in serum was checked.
Expression of Snail antibody was detected in pancreatic cancer
patient's serum and gallstone patient's serum (see FIG. 15F). In
contrast, Snail antibody was not detected in normal healthy
population (see FIG. 15G). Snail antibody was detected in lung
cancer patient's serum (FIG. 18). Accordingly, it was deemed that
presence of Snail antibody would be very useful as a cancer
diagnostic marker.
TABLE-US-00002 TABLE 2 No sex/age cell type TNM Stage Meta Snail Ab
1 M/64 ADC T2N0M0 IB x 2 M/65 ADC T4N3M1 IV brain lost to f/u
positive 3 M/73 SQC T4N3M1 IV Lung 4 M/68 SQC T4N2Mx IIIB ?
positive 5 M/63 SQC T4N2Mx IIIB ? 6 M/50 ADC T4N3M1 IV brain, bone
7 M/72 SQC T4N2M1 IV brain positive 8 M/66 SQC T4N3Mx IIIB ? 9 M/65
SQC T2N2Mx IIIA ? NA positive 10 M/56 SOC T2N1Mx IIB ? NA positive
11 M/72 SQC T4N2M1 IV bone lost to f/u 12 M/58 SQC T2N3M0 IIIB x
expired 13 M/72 ADC T1N2M0 IIIA x 14 M/80 ADC T4N3M1 IV bone 15
M/71 SQC T3N2M1 IV lung expired positive 16 M/74 SCLC -- extensive
lung expired 17 M/62 SQC T4N3M1 IV lung 18 M/48 non-cancer 19 M/73
SQC T2N1Mx IIIB ? NA positive 20 M/63 SQC T4N3M0 IIIB x expired
positive 21 F/64 ADC T4N3M1 IV lung NA positive 22 M/69 SQC T4N3Mx
IIIB ? positive 23 M/80 SQC T4N1Mx IIIB ? 24 F/47 ADC T4N2M1 IV
lung lost to f/u 25 M/61 SQC T2N3M0 IIIB x expired positive 26 M/63
SQC T2N3Mx IIIB ? positive 27 F/65 SCLC -- limited x positive 28
M/62 SQC T3N3Mx IIIB ? NA positive 29 M/67 ADC T4N3Mx IIIB ?
positive 30 F/53 ADC T1N0M0 IA -- positive
[0142] [Snail-p53 Binding Inhibitor Identification]
EXAMPLE 1
1-1. Synthesis of 2-methylthio-1,4-naphthoquinone (1a)
[0143] 0.617 mM 1,4-naphtoquinone was dissolved in 30 ml of
methanol in 100 ml one-neck round flask, and 1.54 mM sodium
thiomethoxide was added thereto and stirred overnight. 50 ml of
saturated sodium chloride solution was added to the reaction
mixture, followed by extraction three times with 50 ml of
chloroform, and an organic layer was dehydrated with an anhydrous
sodium sulfate and filtered. The filtrate was concentrated under
reduced pressure and the obtained residue was re-crystallized with
methanol to produce 2-methylthio-1,4-naphthoquinone that was yellow
crystal.
[0144] Yield: 14.0%, melting point: 185-186.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.07(m, 2H), 7.78-7.70(m, 2H),
6.58(s, 1H), 2.40(s, 3H), m/z 205.1 (M+H).sup.+.
1-2. Synthesis of 2-ethylthio-1,4-naphthoquinone (1b)
[0145] 2-ethylthio-1,4-naphthoquinone that was yellow crystal was
prepared in the same manner as in Example 1-1, except that
ethylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0146] Yield: 40.7%, melting point: 135-136.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.08(m, 2H), 7.78-7.69(m, 2H),
6.62(s, 1H), 2.87(q, J=7.2 Hz, 2H), 1.44(t, J=7.2 Hz, 3H), m/z
219.1 (M+H).sup.+.
1-3. Synthesis of 2-propylthylthio-1,4-naphthoquinone (1c)
[0147] 2-propylthylthio-1,4-naphthoquinone that was yellow crystal
was prepared in the same manner as in Example 1-1, except that
propylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0148] Yield: 33.9%, melting point: 118-119.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.07(m, 2H), 7.77-7.68(m, 2H),
6.61(s, 1H), 2.82(t, J=7.6 Hz, 2H), 1.86-1.77(m, 2H), 1.11(t, J=7.2
Hz, 3H), m/z 233.0 (M+H).sup.+.
1-4. Synthesis of 2-butylthio-1,4-naphthoquinone (1d)
[0149] 2-butylthio-1,4-naphthoquinone (1d) that was yellow crystal
was prepared in the same manner as in Example 1-1, except that
butylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0150] Yield: 33.9%, melting point: 97-98.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.07(m, 2H), 7.77-7.68(m, 2H),
6.61(s, 1H), 2.84(t, J=7.2 Hz, 2H), 1.80-1.72(m, 2H), 1.57-1.48(m,
2H), 0.98(t, J=7.6 Hz, 3H), m/z247.1 (M+H).sup.+.
1-5. Synthesis of 2-pentylthio-1,4-naphthoquinone (1e)
[0151] 2-pentylthio-1,4-naphthoquinone that was yellow crystal was
prepared in the same manner as in Example 1-1, except that
pentylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0152] Yield: 15.3%, melting point: 111-112.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.11-8.06(m, 2H), 7.77-7.68(m, 2H),
6.60(s, 1H), 2.83(t, J=7.6 Hz, 2H), 2.17-1.74(m, 2H), 1.51-1.33(m,
4H), 0.93(t, J=7.2 Hz, 3H), m/z261.2 (M+H).sup.+.
1-6. Synthesis of 2-hexylthio-1,4-naphthoquinone (1e)
[0153] 2-hexylthio-1,4-naphthoquinone that was yellow crystal was
prepared in the same manner as in Example 1-1, except that
hexylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0154] Yield: 15.0%, melting point: 101-102.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.13-8.08(m, 2H), 7.77-7.69(m, 2H),
6.61(s, 1H), 2.83(t, J=7.6 Hz, 2H), 1.81-1.73(m, 2H), 1.56-1.46(m,
2H), 1.35-1.31(m, 4H), 0.91(t, J=6.8 Hz, 3H), m/z 275.3
(M+H).sup.+.
1-7. Synthesis of 2-heptylthio-1,4-naphthoquinone (1g)
[0155] 2-heptylthio-1,4-naphthoquinone that was yellow crystal was
prepared in the same manner as in Example 1-1, except that
heptylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0156] Yield: 46.4%, melting point: 114-115.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.07(m, 2H), 7.77-7.68(m, 2H),
6.61(s, 1H), 2.83(t, J=14.8 Hz, 2H), 1.77(quint, J=7.6 Hz, 2H),
1.52-1.45(m, 2H), 1.35-1.29(m, 6H), 0.90(t, J=6.8 Hz, 3H), m/z
289.2 (M+H).sup.+.
1-8. Synthesis of 2-octylthio-1,4-naphthoquinone (1h)
[0157] 2-octylthio-1,4-naphthoquinone that was yellow crystal was
prepared in the same manner as in Example 1-1, except that
octylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0158] Yield: 76.8%, melting point: 114-115.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.10(m, 2H), 7.78-7.70(m, 2H),
6.61(s, 1H), 2.84(t, J=7.6 Hz, 2H), 1.77(quint, J=7.6 Hz, 2H),
1.49-1.47(m, 2H), 1.35-1.29(m, 8H), 0.89(t, J=6.8 Hz, 3H), m/z
304.5 (M+H).sup.+.
1-9. Synthesis of 2-nonylthio-1,4-naphthoquinone (1i)
[0159] 2-nonylthio-1,4-naphthoquinone that was yellow crystal was
prepared in the same manner as in Example 1-1, except that
nonylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0160] Yield: 87.4%, melting point: 105-106.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.07(m, 2H), 7.77-7.68(m, 2H),
6.61(s, 1H), 2.83(t, J=7.6 Hz, 2H), 1.77(quint, J=7.6 Hz, 2H),
1.51-1.45(m, 2H), 1.33-1.29(m, 10H), 0.89(t, J=6.4 Hz, 3H), m/z
317.5 (M+H).sup.+.
1-10. Synthesis of 2-decylthio-1,4-naphthoquinone (1-j)
[0161] 2-decylthio-1,4-naphthoquinone that was yellow crystal was
prepared in the same manner as in Example 1-1, except that
decylmercaptan was used instead of sodium thiomethoxide. The yield
and properties of the synthesized compound are as follows.
[0162] Yield: 87.4%, melting point: 101-102.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 8.12-8.08(m, 2H), 7.77-7.68(m, 2H),
6.61(s, 1H), 2.83(t, J=7.2 Hz, 2H), 1.77(quint, J=7.6 Hz, 2H),
1.50-1.42(m, 2H), 1.33-1.29(m, 12H), 0.88(t, J=6.4 Hz, 3H), m/z
331.1 (M+H).sup.+.
EXAMPLE 2
2-1. Synthesis of 2-methylamino-5,8-dimethoxy-1,4-naphthoquinone
(5a)
[0163] 0.45 mM 5,8-dimethoxy-1,4-naphthoquinone (4) which had been
prepared above was dissolved in 30 ml of methanol in 100 ml
one-neck round flask and then, 0.687 mmol methylamine was added
thereto and stirred at room temperature for 3 hours 0.64 mM sodium
dichromate and 0.18 mM sulfuric acid dissolved in water were slowly
dropped to the reaction mixture and stirred at room temperature for
3 minutes. Then, 50 ml of saturated sodium chloride was added to
the reaction mixture, followed by extraction three times with 50 ml
of chloroform and obtained organic layers were gathered and
dehydrated with anhydrous sodium sulfate and filtered. The filtrate
was concentrated under reduced pressure and the residue was
subjected to silicagel column chromatography, thereby producing
2-methylamino-5,8-dimethoxy-1,4-naphthoquinone that was reddish
brown. The yield and properties of the synthesized compound are as
follows.
[0164] Yield: 56.7%, melting point: 203-204.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, J=9.6 MHz), 7.19(d, J=9.2
MHz, 1H), 5.75(BR, 1H), 5.60(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
2.87(d, J=5.2 MHz, 3H), m/z 248(M+H).sup.+.
2-2. Synthesis of 2-ethylamino-5,8-dimethoxy-1,4-naphthoquinone
(5b)
[0165] 2-ethylamino-5,8-dimethoxy-1,4-naphthoquinone (5b) was
prepared in the same manner as in Example 2-1, except that
ethylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0166] Yield: 23.6%, melting point: 172-173.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, J=9.2 Hz, 1H), 7.19(d, J=9.6
Hz, 1H), 5.63(BR, 1H), 5.61(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.09(q, 2H), 1.29(t, J=7.2 Hz, 3H), m/z 262.1(M+H).sup.+.
2-3. Synthesis of 2-propylamino-5,8-dimethoxy-1,4-naphthoquinone
(5c)
[0167] 2-propylamino-5,8-dimethoxy-1,4-naphthoquinone (5c) was
prepared in the same manner as in Example 2-1, except that
propylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0168] Yield: 46.5%, melting point: 175-176.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, J=9.2 Hz, 1H), 7.19(d, J=9.2
Hz, 1H), 5.72(BR, 1H), 5.61(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.09(q, 2H), 1.68(J=6.8 Hz, 2H), 0.99(t, J=7.6 Hz, 3H), m/z
276(M+H).sup.+.
2-4. Synthesis of 2-butylamino-5,8-dimethoxy-1,4-naphthoquinone
(5d)
[0169] 2-butylamino-5,8-dimethoxy-1,4-naphthoquinone (5d) was
prepared in the same manner as in Example 2-1, except that
butylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0170] Yield: 46.2%, melting point: 104-105.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, J=9.6 Hz, 1H), 7.19(d, J=9.6
Hz, 1H), 5.70(BR, 1H), 5.61(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.11(q, 2H), 1.64(p, 2H), 1.46-1.38(m, 2H), 0.95(t, J=7.2 Hz, 3H),
m/z 290(M+H).sup.+.
2-5. Synthesis of 2-pentylamino-5 8-dimethoxy-1,4-naphthoquinone
(5e)
[0171] 2-pentylamino-5,8-dimethoxy-1,4-naphthoquinone (5e) was
prepared in the same manner as in Example 2-1, except that
pentylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0172] Yield: 55.9%, melting point: 102-103.degree. C. .sup.1H-NMR
(CDCl.sub.3, 400): .delta. 7.34(d, j=9.2 MHz, 1H), 7.19(d, J=9.2
Hz, 1H), 5.70(BR, 1H), 5.61(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.11(q, 2H), 1.29(t, J=7.2 Hz, 3H), m/z 303.6(M+H).sup.+.
2-6. Synthesis of 2-hexylamino-5,8-dimethoxy-1,4-naphthoquinone
(5f)
[0173] 2-hexylamino-5,8-dimethoxy-1,4-naphthoquinone (5f) was
prepared in the same manner as in Example 2-1, except that
hexylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0174] Yield: 47.3%, melting point: 83-84.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, J=9.6 Hz, 1H), 7.18(d, J=9.6
Hz, 1H), 5.69(BR, 1H), 5.61(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.11(q, 2H), 1.66-1.58(m, 4H), 1.32-1.30(m, 4H), 0.89(t, J=6.8 Hz,
3H), m/z 318(M+H).sup.+.
2.7 Synthesis of 2-heptylamino-5,8- dimethoxy-1,4-naphthoquinone
(5g)
[0175] 2-heptylamino-5,8-dimethoxy-1,4-naphthoquinone (5g) was
prepared in the same manner as in Example 2-1, except that
heptylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0176] Yield: 41.8%, melting point: 74-75.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, J=9.6 Hz, 1H), 7.33(d, j=9.6
Hz, 1H), 7.18(d, J=9.6 Hz, 1H), 5.69(BR, 1H), 5.60(s, 1H), 3.96(s,
3H), 3.94(s, 3H), 3.11(q, 2H), 1.66-1.61(m, 2H), 1.35-1.29(m, 8H),
0.89(t, J=6.4 Hz, 3H), m/z 332(M+H).sup.+.
2-8. Synthesis of 2-octylamino-5,8- dimethoxy-1,4-naphthoquinone
(5h)
[0177] 2-octylamino-5,8- dimethoxy-1,4-naphthoquinone (5h) was
prepared in the same manner as in Example 2-1, except that
octylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0178] Yield: 45.1%, melting point: 81-82.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, j=9.6 Hz, 1H), 7.18(d, J=9.6
Hz, 1H), 5.69(br, 1H), 5.61(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.11(q, 2H), 1.68-1.61(m, 2H), 1.41-1.20(m, 10H), 0.88(t, J=6.4 Hz,
3H), m/z 346(M+H).sup.+.
2-9. Synthesis of 2-nonylamino-5,8- dimethoxy-1,4-naphthoquinone
(5i)
[0179] 2-nonylamino-5,8-dimethoxy-1,4-naphthoquinone (5i) was
prepared in the same manner as in Example 2-1, except that
nonylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0180] Yield: 44.0%, melting point: 85-86.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.34(d, j=9.6 Hz, 1H), 7.18(d, J=9.6
Hz, 1H), 5.69(br, 1H), 5.61(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.11(m, 2H), 1.66-1.61(m, 2H), 1.41-1.20(m, 12H), 0.88(t, J=6.4 Hz,
3H), m/z 360(M+H).sup.+.
2-10. Synthesis of 2-decylamino-5,8- dimethoxy-1,4-naphthoquinone
(5j)
[0181] 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (5j) was
prepared in the same manner as in Example 2-1, except that
decylamine was used instead of methylamine in the round flask of
Example 2-1. The yield and properties of the synthesized compound
are as follows.
[0182] Yield: 17.6%, melting point: 86-87.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.7.33(d, j=9.2 Hz, 1H), 7.18(d, J=9.2
Hz, 1H), 5.69(br, 1H), 5.60(s, 1H), 3.96(s, 3H), 3.94(s, 3H),
3.11(q, 2H), 1.66-1.61(m, 2H), 1.40-1.20(m, 14H), 0.88(t, J=6.4 Hz,
3H), m/z 374(M+H).sup.+.
2-11. Synthesis of
2-(2-hydroxyethylthio)-5,8-dimethoxy-1,4-naphthoquinone (5k)
[0183] 2-(2-Hydroxyethylthio)-5,8-dimethoxy-1,4-naphthoquinone (5k)
was prepared in the same manner as in Example 2-1, except that
2-mercaptoethanol was used instead of methylamine in the round
flask of Example 2-1. The yield and properties of the synthesized
compound are as follows.
[0184] Yield: 59.5% melting point: 117-118.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.7.34(d, J=9.6 Hz, 1H), 7.28(d, J=9.6
Hz, 1H), 6.61(s, 1H), 3.96(s, 6H), 3.93(t, J=6.4 Hz, 2H), 3.05(t,
J=6.4 Hz, 2H), m/z 316.9(M+Na).sup.+.
2-12. Synthesis of
2-(3-hydroxypropylthio)-5,8-dimethoxy-1,4-naphthoquinone (5l)
[0185] 2-(3-Hydroxypropylthio)-5,8-dimethoxy-1,4-naphthoquinone
(5l) was prepared in the same manner as in Example 2-1, except that
3-mercaptopropanol was used instead of methylamine in the round
flask of Example 2-1. The yield and properties of the synthesized
compound are as follows.
[0186] Yield: 69.9%, melting point: 125.about.126.degree. C.,
.sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.7.33(d, J=9.6 Hz, 1H),
7.27(d, J=9.6 Hz, 1H), 6.51(s, 1H), 3.96(s, 6H), 3.81(t, J=6.4 Hz,
2H), 2.91(t, J=7.2 Hz, 2H), 1.99(m, 2H), m/z 331.1
(M+Na).sup.+.
2-13. Synthesis of
2-(4-hydroxybutylthio)-5,8-dimethoxy-1,4-naphthoquinone (5m)
[0187] 2-(4-Hydroxybutylthio)-5,8-dimethoxy-1,4-naphthoquinone (5m)
was prepared in the same manner as in Example 2-1, except that
4-mercaptobutanol was used instead of methylamine in the round
flask of Example 2-1. The yield and properties of the synthesized
compound are as follows.
[0188] Yield: 64.0%, melting point: 122-123.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.33(d, J=9.6 Hz, 1H), 7.29(d,
J=10.0 Hz, 1H), 6.46(s, 1H), 3.96(s, 3H), 3.95(s, 3H), 3.71(t,
J=6.4 Hz, 2H), 1.87-1.81(m, 2H), 1.78-1.50 (m, 2H), m/z 345.1
(M+Na).sup.+.
2-14. Synthesis of
2-(6-hydroxyhexylthio)-5,8-dimethoxy-1,4-naphthoquinone (5n)
[0189] 2-(6-hydroxyhexylthio)-5,8-dimethoxy-1,4-naphthoquinone (5n)
was prepared in the same manner as in Example 2-1, except that
6-mercaptohexanol was used instead of methylamine in the round
flask of Example 2-1. The yield and properties of the synthesized
compound are as follows.
[0190] Yield: 38.2% Melting point: 87.about.88.degree. C.,
.sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta. 7.33(d, J=9.6 Hz, 1H),
7.27(d, J=9.6 Hz, 1H), 6.45(s, 1H), 3.96(s, 6H), 3.66(t, J=6.4 Hz,
2H), 2.76(t, J=7.6 Hz, 2H), 1.78-1.12(m, 2H), 1.61-1.25(m, 6H), m/z
372.9 (M+Na).sup.+.
2-15. Synthesis of
3-(5,8-dimethoxy-1,4-dioxo-naphthalen-2-ylthio)propanoic acid
(5o)
[0191] 3-(5,8-dimethoxy-1,4-dioxo-naphthalen-2-ylthio)propanoic
acid (5o) was prepared in the same manner as in Example 2-1, except
that 3-mercaptopropionic acid was used instead of methylamine in
the round flask of Example 2-1. The yield and properties of the
synthesized compound are as follows.
[0192] Yield: 80.6%, melting point: 208.about.209.degree. C.,
.sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta. 7.35(d, J=9.2 Hz, 1H),
7.28(d, J=13.6 Hz, 1H), 6.51(s, 1H), 3.97(s, 3H), 3.96(s, 3H),
3.07(t, J=7.2 Hz, 2H), 2.81(t, J=7.2 Hz, 2H), m/z 348.4
(M+Na).sup.+.
2-16. Synthesis of
11-(5,8-dimethoxy-1,4-dioxo-naphthalen-2-ylthio)undecanoic acid
(5p)
[0193] 11-(5,8-dimethoxy-1,4-dioxo-naphthalen-2-ylthio)undecanoic
acid (5p) was prepared in the same manner as in Example 2-1, except
that 11-mercaptoundecanoic acid was used instead of methylamine in
the round flask of Example 2-1. The yield and properties of the
synthesized compound are as follows.
[0194] Yield: 77.9%, melting point: 146-147.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.33(d, J=9.6 Hz, 1H), 7.27(d, J=9.6
Hz, 1H), 6.46(s, 1H), 3.96(s, 3H), 3.95(s, 3H), 3.34(t, J=7.2 Hz,
2H), 2.75(t, J=2.7 Hz, 2H), 2.41.about.2.32(m, 5H),
2.06.about.2.00(m, 2H), 1.76.about.1.56(m, 5H), 1.48.about.1.39(m,
2H), 0.97.about.0.88(m, 2H), m/z 435 (M+H).sup.+
EXAMPLE 3
3-1. Synthesis of
5,8-dimethoxy-2-(3-oxo-3-(4-phenylpiperazin-1-yl)propylthio)naphthalene-1-
,4-dione (6a)
[0195] 0.163 mM
3-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)propanoic
acid that had been prepared above was dissolved in 40 n of
chloroform in 100 ml one-neck round flask, and then 0.26 mM
N-(3-dimethylaminopropyl)-N'-ethylcarbodimide hydro chloride (EDC)
and 0.26 mM 4-phenylpiperidine were added thereto and stirred
overnight. 1N hydrochloric acid was added to the reaction mixture
and stirred at room temperature for 3 minutes. Then, 50 ml of
saturated sodium chloride solution was added thereto, followed by
extraction three times with 50 ml of chloroform, and obtained
organic layers were gathered and dehydrated with anhydrous sodium
sulfate and filtered. The filtrate was concentrated under reduced
pressure and the residue was subjected to silicagel column
chromatography, thereby producing
5,8-dimethoxy-2-(3-oxo-3-(4-phenylpiperazin-1-yl)propylthio)naphthalene-1-
,4-dione (6a) that was reddish brown. The yield and properties of
the synthesized compound are as follows.
[0196] Yield: 73.5%, melting point: 94.about.95.degree. C., H-NMR
(CDCl.sub.3, 400 MHz): .delta.7.34.about.7.28(m, 5H), 7.19(d, J=7.6
Hz), 6.26(s, 1H), 3.95(s, 3H), 3.94(s, 3H), 314(t, 4H),
2.79.about.2.74(q, 4H), 2.67(t, J=12 Hz, 1H), 1.89(t, J=12.4 Hz,
2H), 1.65(t, J=12.4 Hz, 2H),m/z 466.3(M+H).sup.+.
EXAMPLE 4
4-1. Synthesis of
isobutyl-11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)unde-
canoate (7a)
[0197] 1.15 mM
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)undecanoic
acid (5p) that had been prepared above was dissolved in 60 ml of
chloroform in 100 ml one-neck round flask and then, 1.38 mM
N-(3-dimethylaminopropyl)-N'-ethylcarbodimide hydro chloride (EDC)
and 1.38 mM isobutylalcohol were added thereto and stirred
overnight. 1N hydrochloric acid was added to the reaction mixture
and stirred at room temperature for 3 minutes. Then, 50 ml of
saturated sodium chloride solution was added thereto, followed by
extraction three times with 50 ml of chloroform, and obtained
organic layers were gathered and dehydrated with anhydrous sodium
sulfate and filtered. The filtrate was concentrated under reduced
pressure and the residue was subjected to silicagel column
chromatography, thereby producing
isobutyl-11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)unde-
canoate (7a) that was reddish brown. The yield and properties of
the synthesized compound are as follows.
[0198] Yield: 52.4%, melting point: 58-59.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.32(d, J=9.6 Hz, 1H), 7.26(d, J=9.6
Hz, 1H), 6.44(s, 1H), 3.96(s, 3H), 3.95(s, 3H), 3.85(d, J=6.8 Hz,
2H), 2.75(t, J=7.6 Hz, 2H), 2.31(t, 7.6 Hz, 2H), 1.96-1.89(m, 1H),
1.72-1.68(m, 4H), 1.29-1.25(m, 12H), 0.94(s, 3H), 0.92(s, 3H), m/z
491 (M+H).sup.+
4-2. Synthesis of
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)-N-isobutyl
undecanamide (7b)
[0199]
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydronaphthalene-2-ylthio)-N-isob-
utyl undecanamide (7b) was prepared in the same manner as in
Example 4-1, except that isobutylamine was used instead of
isobutylalcohol in the round flask of Example 4-1. The yield and
properties of the synthesized compound are as follows.
[0200] Yield: 77.9%, melting point: 74-75.degree. C., .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta. 7.33(d, J=9.6 Hz, 1H), 7.27(d, J=9.2
Hz), 6.44(s, 1H), 5.55(s, 1H), 3.96(s, 3H), 3.95(s, 3H), 3.10(t,
J=6.6 Hz, 2H), 2.75(t, J=7.6 Hz, 2H), 2.17(t, J=7.4 Hz, 2H),
1.78-1.7(m, 2H), 1.63(m, 2H), 1.45(m, 1H), 0.92(s, 3H), 0.90(s,
3H), m/z 490.0 (M+H).sup.+.
EXAMPLE 5
5-1. Synthesis of isobutyl
11-(5,8-dimethoxyl-1,4-dioxo-1,4-dihydronaphthalene-2-yl
sulfinyl)undecanoate (8)
[0201] 0.163 mM isobutyl
11-(5,8-dimethoxy-1,4-dioxo-1,4-dihydroxynaphthalene-2-ylthio)-undecanoat-
e (7a) that had been prepared above was dissolved in 30 ml of
dichloromethane in 100 ml one-neck round flask and then 0.196 mM
3-chloroperoxybbenzoic acid was added thereto and stirred for 2
hours A sodium bicarbonate was added to the reaction mixture and
stirred at room temperature for 3 minutes. Then, 50 ml of saturated
sodium chloride solution was added thereto, followed by extraction
three times with 50 ml of chloroform, and obtained organic layers
were gathered and dehydrated with anhydrous sodium sulfate and
filtered. The filtrate was concentrated under reduced pressure and
the residue was subjected to silicagel column chromatography,
thereby producing isobutyl
11-(5,8-dimethoxyl-1,4-dioxo-1,4-dihydronaphthalene-2-yl
sulfinyl)undecanoate (8) that was red. The yield and properties of
the synthesized compound are as follows.
[0202] Yield: 45.8%, melting point: 92.about.93.degree. C.,
.sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.7.41(d, J=9.6 Hz, 1H),
7.36(d, J=9.6 Hz, 1H), 7.31(s, 1H), 3.99(s, 3H), 3.98(s, 3H),
3.85(d, J=6.8 Hz, 1H), 3.28.about.3.21(m, 1H), 2.96.about.1.89(m,
2H), 1.69.about.1.59(m, 4H), 1.41.about.1.23(m, 12H), 0.93(d, J=6.8
MHz, 6H), m/z 507(M+H).sup.+
[0203] Effects of the compounds synthesized as described above on
Snail-p53 binding inhibition were confirmed in the following
experiments.
EXPERIMENTAL EXAMPLE 1
Confirmation of p53 Activity Recovery and Target Gene Induction
[0204] To confirm whether the compounds prepared according to
Examples 1 to 5 are effective for inhibiting Snail-p53 binding at
cell level, the compound was treated into K-Ras mutant cancer cell
lines HCT116 and results were analyzed by western blotting.
[0205] Oncogenic K-ras suppresses the p53 activity through Snail.
In the cell line HCT116, Snail is always expressed by K-Ras
mutation and bound to p53 to suppress normal activity of p53. If
the K-Ras mutant cancer cell line HCT116 is treated with a compound
for suppressing Snail-p53 binding, p53 is normally activated and
more expressed, and target genes of p53 are induced.
[0206] First, western blotting was performed to detect expression
of p53 and p21, which is a target gene of p53. All cell lines used
in the present invention were obtained from ATCC, and maintained in
10% FBS-containing RPMI-1640 or DMEM. Protein was extracted with
RIPA buffer solution and a membrane loaded with cell lysate through
typical SDS-PAGE and gel transfer method was prepared. After
blocking with 5% non-fat dry milk, the membrane was subjected into
a typical western blot procedure with an antibody corresponding to
each gene. Antibodies used herein were obtained from Cell
signaling, SantaCruz. p53 activity was confirmed by expression of
p21, which is a target gene of p53. FIG. 1 shows images and graphs
showing western blot results of HCT116 cells, K-Ras mutant cancer
cell lines, using some of the compounds of Examples 1 to 5, and
numeral values of the results are shown in Table 3 below.
TABLE-US-00003 TABLE 3 Control Protein expression ratio of compound
to control Gene DMSO control 5i 5j 5k 5l 5n 5m 5o 5p 8 7b 7a 6a 6b
p53 1.0 1.6 1.3 0.3 0.6 1.1 0.8 2.6 1.6 2.3 4.6 4.2 1.9 4.0 p21 1.0
4.6 5.0 0.2 0.1 0.2 0.1 2.5 0.3 1.7 7.0 7.2 2.8 8.7
[0207] Referring the treatment results of the compounds at a
concentration of 10 .mu.M into K-Ras mutant HCT116 cancer cell
lines, one of the compounds showed p53 expression 2 to 5 times
stronger than that of a DMSO control and another compound showed
p21 expression 2 to 8 times stronger than that of the DMSO control
(see FIG. 20 and Table 3). The enhanced expression of p53 and p21
as a target gene of p53 by the compounds indicates that functions
of Snail are suppressed by the compounds in K-Ras mutant HCT116
cell lines in which Snail is always activated and Snail-p53 binding
inhibition is maintained.
EXPERIMENTAL EXAMPLE 2
Confirmation of Snail-p53 Binding Inhibiting Effect
[0208] Subsequently, among compounds that induced p53 and p21 and
showed strong expression, compounds 5o and 7a were selected to
identify relationships among K-Ras, Snail, and p53. To prove the
Snail-p53 binding inhibition, GST pull down assay was performed
using compounds 5o and 7a.
[0209] GST pull down assay is a method for identifying a binding
degree of two proteins. GST-fused Snail protein and p53 recombinant
protein were prepared and treated with the compound 5o or 7a to
identify a binding degree of GST-Snail protein and p53 protein.
Nutlin-3 was used as a control for the compounds. Nutlin-3 is known
as a protein that blocks binding between p53 and MDM2, which is
used as a negative control factor of p53, and is over-expressed in
various tumor cells. If MDM2 is over-expressed, proteolysis of p53
is induced to suppress apoptosis occurring through target genes of
p53, and an anti-proliferation effect of cells is induced. Although
MDM2 is a representative control factor of p53, MDM2 is not applied
to K-Ras mutant disease or mutant p53 containing disease.
[0210] To perform GST pull down assay, three human Snail fragments
(residues 1-90, 91-112, and 113-264) and p53 fragments (1-93 and
93-292) were expressed in E. coli as GST-fusion protein. Each of
the fragments was loaded on to GSH-agarose, washed, and then eluted
using a buffer containing 20 mM reduced glutathione. The eluted
fractions were purified using an anionexchange chromatography
(HitrapQ). The recombinant human p53 protein (residues 94-292) was
expressed in E. coli using the vector pET28A which contains a
hexa-histidine tag at the C terminus. The p53 protein including the
hexa-histidine tag was purified using Ni--NTA affinity and a size
exclusion chromatography (Superdex 200). To confirm the direct
binding between p53 and Snail, agarose-bead conjugated GST or
GST-Snail was incubated with His-p539 (histidine-p53) in PBS for 45
minutes at 4.degree. C. After washing with PBS, the precipitated
protein was subjected into SDS-PAGE and western blotting.
[0211] As shown in FIG. 21, GST pull down assay results show that
in the case of control (c), the binding between GST-Snail and p53
was strong. Since Nutlin-3 also controls MDM2 and induces p53
activity, it did not inhibit Snail-p53 binding and showed the
similar binding degree to that of the control. In contrast, in
fractions using the compounds 5o and 7a, the binding between
GST-Snail protein and p53 was substantially weak. This result shows
that the compounds inhibited Snail-p53 binding.
EXPERIMENTAL EXAMPLE 3
Confirmation of K-Ras Dependent p53 Induction Capability
[0212] To confirm that the compounds did not affect normal cells,
and selectively affected only K-Ras mutant cancer cells to induce
apoptosis, cytotoxicity and apoptosis effects of the compounds 5o
and 7a were confirmed through cell vitality, by counting the number
of cells using a tryphan blue solution. To confirm that the p53
induction of the compounds is related to K-Ras, A549 and HCT116,
which are K-Ras mutant cancer cell lines, and MKN45, which is K-Ras
wild type cancer cell line, were used.
[0213] Table 4 shows numeral values of death rates of the
respective cell lines, and FIG. 22 is a graph showing the numeral
values of death rates.
TABLE-US-00004 TABLE 4 Control (%) Cancer cell death rate of
compounds with respect to control (%) Type of Control (DMSO) 5o 7a
Nutlin-3 cancer cell 0 .mu.M 5 .mu.M 10 .mu.M 20 .mu.M 5 .mu.M 10
.mu.M 20 .mu.M 5 .mu.M 10 .mu.M 20 .mu.M A546 (k-Ras 1 .+-. 1.3 30
.+-. 51 .+-. 62 .+-. 23 .+-. 34 .+-. 45 .+-. 11 .+-. 19 .+-. 25
.+-. mutant) 4.5 1.1 1.7 3.4 2.3 2.3 4.0 4.5 4.7 HCT116 1 .+-. 1.12
35 .+-. 57 .+-. 67 .+-. 32 .+-. 44 .+-. 57 .+-. 21 .+-. 32 .+-. 40
.+-. (k-Ras 3.2 1.6 2.5 2.1 2.5 2.8 2.4 2.9 3.1 mutant) MKN45 1
.+-. 1.1 10 .+-. 15 .+-. 18 .+-. 1 .+-. 1 .+-. 3 .+-. 28 .+-. 38
.+-. 47 .+-. (k-Ras wild 2.9 2.4 1.9 1.0 1.0 2.4 2.2 2.9 2.2
type)
[0214] Referring to the results obtained through cell death rates,
in A549 and HCT116, which are K-Ras mutant cancer cell lines, the
compound 5o showed, at the same concentration of about 10 .mu.M,
death rates of about 51% and about 57%, respectively, and the
compound 7a showed, at the same concentration of about 10 .mu.M,
death rates of about 34% and about 44%, respectively (see Table 4
and FIG. 22). In contrast, differentially from in the K-Ras mutant
cancer cell lines, in MKN45, which is K-Ras wild type cancer cell
line, at the same concentration of about 10 .mu.M, the compound 5o
showed a death rate of about 15%, and the compound 7a showed a
death rate of about 1%. These results show that the compounds
selectively affect only cancer cell lines in which K-Ras is not
normally activated and show high death rates. This result implies
that the compounds selectively affect only K-Ras damaged cancer
cells and are thus useful for patients who develop cancer by K-Ras
damage. On the other hand, results of Nutlin-3 did not have
consistency. That is, Nutlin-3 showed death rates of 19% and 32%
respectively in A549 and HCT116, which are K-Ras mutant cancer cell
lines, at the same concentration of 10 .mu.M, and showed a cell
death rate of 38% in MKN45, which is K-Ras wild type cancer cell
line, at the same concentration of 10 .mu.M. As described above, it
was confirmed that Nutlin-3 did not show a significant reactivity
regardless of normal state or mutant state of K-Ras and did not
show a selective reactivity to K-Ras. Thus, it was confirmed that
differentially from Nutlin-3, the compounds 5o and 7a selectively
affect K-Ras.
EXPERIMENTAL EXAMPLE 4
Induction of Target Genes of p53 Activity
[0215] Then, the effect of compound 5o on mutant p53 was
checked.
[0216] The compound 5o was treated into mutant p53-type MT/WT-p53
gene-containing MDA-MB 468, a human breast cancer cell line, and
western blotting was performed. FIG. 23 shows a graph showing
western blot results when the compound 5o was treated into MDA-MB,
and Table 5 shows numeral values of the graph.
TABLE-US-00005 TABLE 5 Control Expression ratio of Control
Expression ratio of (DMSO gene to control with (DMSO gene to
control with control) respect to compound 5o contorl) respect to
Nutlin-3 Gene 0 .mu.M 5 .mu.M 10 .mu.M 20 .mu.M 0 .mu.M 5 .mu.M 10
.mu.M 20 .mu.M p21 1.0 13.5 25.9 31.3 1.0 1.4 0.9 1.2
[0217] When the compound 5o and Nutlin-3 were treated into p53
mutant-type cancer cell lines MDA-MB 468, expression of p21 was
strong only when treated with the compound 5o (see FIG. 23 and
Table 5). The result shows that Nutlin-3 did not affect p21 as a
target gene in the presence of mutant p53, and differentially from
the Nutlin-3, the compound 5o induced activity of p21.
EXPERIMENTAL EXAMPLE 5
Xenograft In Vivo
[0218] 1. Experimental Method
[0219] Athymic mouse was obtained from Daehan Biolink Co. Ltd, and
raised under temperature and light conditions (20-23.degree. C.,
cycle of 12 hours light/12 hours darkness) and fed with sterile
diet and water freely. After 2 weeks, 1.times.10.sup.7 A549 cells
were inoculated into the athymic mouse (n=21) by intraperitoneal
injection. After 2 weeks, each group was divided into three
subgroups and PBS, and 10 mg/kg or 20 mg/kg of the compound 5o were
intraperitoneally injected once per week for 10 weeks, and vitality
thereof was measured. This animal test was approved by the Animal
Protection Committee of Pusan National University, and performed
according to a guideline presented by the same.
[0220] 2. Experimental Results
[0221] As shown in FIG. 24, when 10 mg/kg and 20 mg/kg of the
compound 5o was treated, tumor-caused death was prevented. On the
other hand, vitality of the PBS treatment group after 10 weeks was
equal to or lower than 50%, and as shown in FIG. 25, the group
treated with the compound 5o showed almost no tumor. In addition,
as shown in FIG. 26, an overall anatomical abnormal finding
according to weight loss or injection of the compound was not
observed. Table 6 shows tumor development sites and morphological
characteristic thereof.
TABLE-US-00006 TABLE 6 Tumor site Control 10 mg/kg 20 mg/kg Liver 1
(death) 0 0 Lung 1 (death) 0 0 Pancreas 2 (1/2 death) 0 0 A. C 2 3
(regression) 2 (regression)
[Sequence List Pre Text]
[0222] SEQ ID NO: 1 shows an amino acid sequence of human p53
cellular tumor antibody.
Sequence CWU 1
1
11393PRTHomo sapiens 1Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu
Pro Pro Leu Ser Gln1 5 10 15Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu
Pro Glu Asn Asn Val Leu 20 25 30Ser Pro Leu Pro Ser Gln Ala Met Asp
Asp Leu Met Leu Ser Pro Asp 35 40 45Asp Ile Glu Gln Trp Phe Thr Glu
Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60Arg Met Pro Glu Ala Ala Pro
Arg Val Ala Pro Ala Pro Ala Ala Pro65 70 75 80Thr Pro Ala Ala Pro
Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95Val Pro Ser Gln
Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110Phe Leu
His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120
125Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln
130 135 140Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg
Ala Met145 150 155 160Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu
Val Val Arg Arg Cys 165 170 175Pro His His Glu Arg Cys Ser Asp Ser
Asp Gly Leu Ala Pro Pro Gln 180 185 190His Leu Ile Arg Val Glu Gly
Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200 205Arg Asn Thr Phe Arg
His Ser Val Val Val Pro Tyr Glu Pro Pro Glu 210 215 220Val Gly Ser
Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser225 230 235
240Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr
245 250 255Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe
Glu Val 260 265 270Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr
Glu Glu Glu Asn 275 280 285Leu Arg Lys Lys Gly Glu Pro His His Glu
Leu Pro Pro Gly Ser Thr 290 295 300Lys Arg Ala Leu Pro Asn Asn Thr
Ser Ser Ser Pro Gln Pro Lys Lys305 310 315 320Lys Pro Leu Asp Gly
Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335Arg Phe Glu
Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350Ala
Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360
365Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met
370 375 380Phe Lys Thr Glu Gly Pro Asp Ser Asp385 390
* * * * *