U.S. patent application number 13/687207 was filed with the patent office on 2014-01-09 for composition of 5-nitrobenzoate derivatives as anti-metastatic agent that inhibits tumor cell-induced platelet aggregation.
This patent application is currently assigned to CHANG GUNG UNIVERSITY. The applicant listed for this patent is CHANG GUNG UNIVERSITY. Invention is credited to YAO-WEN CHANG, PEI-WEN HSIEH, CHING-PING TSENG.
Application Number | 20140011882 13/687207 |
Document ID | / |
Family ID | 49878996 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011882 |
Kind Code |
A1 |
TSENG; CHING-PING ; et
al. |
January 9, 2014 |
COMPOSITION OF 5-NITROBENZOATE DERIVATIVES AS ANTI-METASTATIC AGENT
THAT INHIBITS TUMOR CELL-INDUCED PLATELET AGGREGATION
Abstract
Disclosed are 5-nitrobenzoate derivatives of Formula I,
##STR00001## and the preparation method therefor, wherein R is
referred to hydrogen (H), unsubstituted, mono-substituted,
di-substituted or tri-substituted benzoyl moiety. 5-Nitrobenzoate
derivatives of Formula I do not affect the platelet aggregation,
possesses the inhibitory activity related to the tumor cell-induced
platelet aggregation (TCIPA), and further specifically inhibits
podoplanin-induced platelet aggregation. Therefore,
5-nitrobenzoates of the invention are applicable in its therapeutic
use as the novel therapeutic agent in preventing tumor
metastasis.
Inventors: |
TSENG; CHING-PING; (TAOYUAN
COUNTY, TW) ; HSIEH; PEI-WEN; (NEW TAIPEI CITY,
TW) ; CHANG; YAO-WEN; (PINGTUNG COUNTY, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHANG GUNG UNIVERSITY |
TAO-YUAN |
|
TW |
|
|
Assignee: |
CHANG GUNG UNIVERSITY
TAO-YUAN
TW
|
Family ID: |
49878996 |
Appl. No.: |
13/687207 |
Filed: |
November 28, 2012 |
Current U.S.
Class: |
514/616 ;
564/158 |
Current CPC
Class: |
C07C 235/60 20130101;
C07C 233/65 20130101; A61P 7/02 20180101; C07C 231/08 20130101;
A61K 31/166 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/616 ;
564/158 |
International
Class: |
C07C 233/65 20060101
C07C233/65 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 2012 |
TW |
101124690 |
Claims
1. A method of inhibiting metastasis of a tumor, the method
comprising a step of administrating to a subject in need thereof an
effective amount of a 5-nitrobenzoate derivative as represented by
formula I, ##STR00017## wherein R one is selected from a group
consisting of a hydrogen, an unsubstituted benzoyl moiety
##STR00018## a mono-substituted benzoyl moiety ##STR00019## a
di-substituted benzoyl moiety ##STR00020## and a tri-substituted
benzoyl moiety ##STR00021## and each of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 is one selected from a group
consisting of a fluoride, a chloride, a bromide, an iodide and a
methyl group.
2. The method according to claim 1, wherein the subject is one of a
human and a mammal excluding the human.
3. A method for blocking an interaction between a C-type
lectin-like receptor 2 and a podoplanin or blocking an effect
caused by the interaction therebetween, by comprising step of using
a 5-nitrobenzoate derivative as represented by formula I,
##STR00022## wherein R is one selected from a group consisting of a
hydrogen, an unsubstituted benzoyl moiety ##STR00023## a
mono-substituted benzoyl moiety ( ##STR00024## a di-substituted
benzoyl moiety ##STR00025## and a tri-substituted benzoyl moiety
##STR00026## and each of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5 and R.sub.6 is one selected from a group consisting of a
fluoride, a chloride, a bromide, an iodide and a methyl group.
4. A method for blocking a pathway of a tumor cell-induced platelet
aggregation, comprising a step of using a 5-nitrobenzoate
derivative as represented by formula I, ##STR00027## wherein R is
one selected from a group consisting of a hydrogen, an
unsubstituted benzoyl moiety ##STR00028## a mono-substituted
benzoyl moiety ( ##STR00029## a di-substituted benzoyl moiety
##STR00030## and a tri-substituted benzoyl moiety ##STR00031## and
each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is
one selected from a group consisting of a fluoride, a chloride, a
bromide, an iodide and a methyl group.
5. A 5-nitrobenzoate derivative represented by formula I,
##STR00032## wherein R is one selected from a group consisting of a
hydrogen, an unsubstituted benzoyl moiety ##STR00033## a
mono-substituted benzoyl moiety ( ##STR00034## a di-substituted
benzoyl moiety ##STR00035## and a tri-substituted benzoyl moiety
##STR00036## each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5
and R.sub.6 is one selected from a group consisting of a fluoride,
a chloride, a bromide, an iodide and a methyl group.
6. The 5-nitrobenzoate derivative according to claim 5 being
prepared as a pharmaceutical composition.
7. The 5-nitrobenzoate derivative according to claim 5 being
prepared as a pharmacologically acceptable salt.
8. A method for preparing a 5-nitrobenzoate derivative as
represented by formula I, ##STR00037## the method comprising a step
of using an N-(2-aminoethyl)-2-hydroxy-5-nitrobenzamide as one of a
starting material and an intermediate, wherein R is one selected
from a group consisting of a hydrogen, an unsubstituted benzoyl
moiety ( ##STR00038## a mono-substituted benzoyl moiety
##STR00039## a di-substituted benzoyl moiety ##STR00040## and a
tri-substituted benzoyl moiety ##STR00041## and each of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is one selected from
a group consisting of a fluoride, a chloride, a bromide, an iodide
and a methyl group.
Description
CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY
[0001] The application claims the benefit of Taiwan Patent
Application No. 101124690, filed on Jul. 9, 2012, in the Taiwan
Intellectual Property Office, the disclosures of which are
incorporated herein in their entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a 5-nitrobenzoate
derivative which is used in the therapy for cancer metastasis via
the inhibition of tumor cell-induced platelet aggregation
(TCIPA).
BACKGROUND OF THE INVENTION
[0003] Tumor cells can stimulate platelet activation and form the
aggregation complex with platelets in the vascular circulation
system. This interaction is termed as tumor cell-induced platelet
aggregation (TCIPA). The ability of tumor cells to induce platelet
aggregation has been proven to highly correlate with the metastatic
capability of malignant tumor. At present, it is known that there
are many factors and mechanisms involved in TCIPA. For instance,
tumor cells activate coagulation cascade via thrombin generation
and induce platelet aggregation. Besides, adenosine diphosphate
(ADP) release is involved in MCF-7 tumor cells-induced platelet
aggregation that is relevant to the expression of platelet surface
P2Y12 receptor (Alonso-Escolano et al., Br. J. Pharmacol. 141:
241-252, 2004). Other factors including (1) proteinases: cathepsin
B and matrix metalloprotease (MMPs), (2) thromboxane A2 and
prostacyclin, (3) nitric oxide (NO), (4) platelet surface proteins
(e.g. GPIb-IX-V, GPIIb/IIIa and P-selectin, etc.) and so on are
involved in TCIPA (Jurasz et al., Br. J. Pharmacol. 143: 819-826,
2004). Based on these results, the detail mechanism of TCIPA seems
to very complicated and still be obscured. Nevertheless, these
results highlight TCIPA as a target for development of cancer
therapeutic strategies in translational medicine.
[0004] As so far, various approaches and anti-platelet agents have
been reported to inhibit TCIPA that potentially can be used as the
strategies for treatment of tumor metastasis. For example, the
anti-platelet/coagulation molecule heparin can reduce the
cancer-associated thromboembolism risk. In clinic, low molecular
weight heparin (LMWH) is administrated to cancer patients to
inhibit factor Xa and thrombin to block platelet aggregation,
despite that LMWH is not tumor-specific and LMWH overdose usually
increases the bleeding risk of cancer patients (Borsig, Progress in
Molecular Biology and Translational Science. 93: 335-349, 2010;
Lee, et al., N. Engl. J. Med. 349: 146-153, 2003). A common
salicylate acid drug, aspirin, inhibits cyclooxygenase (COX) and
subsequently blocks thromboxane A2 (TXA2) generation and platelet
aggregation. Nevertheless, high dose aspirin does not show
specificity and does not have effects on cancer metastasis and
patient protection in clinics (Jurasz et al., Br. J. Pharmacol.
143: 819-826, 2004). Recently, utilizing antibody to obstruct the
interaction between platelets and tumor cells serves as a promising
approach to block metastasis. For example, anti-aggrus/podoplanin
antibody has been used to inhibit the interaction of TCIPA between
the transmembrane protein ("podoplanin") of cancer cell and C-type
lectin-like receptor 2 (CLEC-2) of platelet. Antibody therapy is
usually expensive and patients might suffer from the risk of
autoantibody generation (Nakazawa et al., Cancer Sci. 102:
2051-2057, 2011). On the whole, the problems in the prior art lie
in that the functions of platelet aggregation are still influenced
such that tumor cells cannot be specifically inhibited and TCIPA is
not efficiently inhibited.
[0005] It is therefore attempted by the applicant to deal with the
above situation encountered in the prior art.
SUMMARY OF THE INVENTION
[0006] For overcoming the drawbacks in the prior art, a series of
5-nitrobenzoate derivatives are obtained via the synthesis in the
invention, do not affect the platelet aggregation, and can
effectively inhibit TCIPA and specifically inhibit the platelet
aggregation induced by surface protein, podoplanin, of tumor cells.
Therefore, 5-nitrobenzoate derivatives of the invention can be
applicable as the targeted therapy drug for inhibiting tumor
cells.
[0007] The present invention provides a 5-nitrobenzoate derivative
represented by formula I,
##STR00002##
wherein R may be hydrogen (H), unsubstituted benzoyl moiety
##STR00003##
mono-substituted benzoyl moiety
##STR00004##
di-substituted benzoyl moiety
##STR00005##
or tri-substituted benzoyl moiety
##STR00006##
each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 may
be but not limited to fluoride (F), chloride (Cl), bromide (Br),
iodide (I) or methyl (--CH.sub.3) group. R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 respectively are referred to bind to
the para-, meta- or ortho-position of the benzene structure.
[0008] The 5-nitrobenzoate derivative can be prepared as a
pharmaceutical composition or a pharmacologically acceptable
salt.
[0009] The present invention further provides a method for
preparing N-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide,
including steps of: (a) reacting
1-chloro-4-nitro-2-(trifluoromethyl)benzene dissolved in dimethyl
sulfoxide (DMSO) with sodium hydroxide (NaOH) to obtain
4-nitro-2-(trifluoromethyl)phenol (compound 1); (b) reacting
compound 2 with tert-butyl 2-aminoethylcarbamate in a solution
containing NaOH and 1,4-dioxane to obtain tert-butyl
2-(2-hydroxy-5-nitrobenzamido)ethylcarbamate (compound 2); (c)
reacting compound 3 with a dichloromethane (CH.sub.2Cl.sub.2)
solution containing trifluoric acid (TFA) to obtain
N-(2-aminoethyl)-2-hydroxy-5-nitrobenzamide (compound 3); and (d)
reacting compound 3 with benzoyl chloride to obtain
N-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide (compound 4).
[0010] Preferably, compound 1 is obtained by an extraction with
CH.sub.2Cl.sub.2, and compound 2 is obtained by further an
extraction with CH.sub.2Cl.sub.2.
[0011] The present invention further provides a method for
preparing a 5-nitrobenzoate derivative of formula I. The method
includes: reacting compound 3 with benzoyl chloride
##STR00007##
mono-substituted benzoyl chloride
##STR00008##
di-substituted benzoyl chloride
##STR00009##
or tri-substituted benzoyl chloride
##STR00010##
when R is correspondingly the unsubstituted benzoyl moiety,
mono-substituted benzoyl moiety, di-substituted benzoyl moiety or
tri-substituted benzoyl moiety, as mentioned above. Each of
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 may be
fluoride (F), chloride (Cl), bromide (Br), iodide (I) or methyl
(--CH.sub.3) group.
[0012] The present invention further provide a method for preparing
a 5-nitrobenzoate derivative of formula I by using
1-chloro-4-nitro-2-(trifluoromethyl)benzene as a starting material
or an intermediate, wherein the substituted groups R, R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 of formula I are
described above.
[0013] The present invention further provide a method for preparing
a 5-nitrobenzoate derivative of formula I by using compound 3 as a
starting material or an intermediate
[0014] The present invention further provides a method of
inhibiting tumor growth and/or platelet aggregation by
administrating to a subject in need thereof an effective amount of
a 5-nitrobenzoate derivative of formula I. The subject may include
but not limited to a human and a mammal excluding the human.
[0015] The present invention further provides a method for blocking
an interaction between C-type lectin-like receptor 2 (CLEC-2) and
podoplanin or blocking an effect caused by the interaction
therebetween by using a 5-nitrobenzoate derivative of formula
I.
[0016] The present invention further provides a method for blocking
a pathway of tumor cell-induced platelet aggregation (TCIPA) by
using a 5-nitrobenzoate derivative of formula I.
[0017] The term "derivative" herein is referred to that one
hydrogen atom or substituted group of a molecule is replaced to
form another molecule. The term "compound" herein is referred to
that two or more than two molecules are chemically bound to form
another molecule at a certain molar ratio (or weight ratio) under
an adequate reaction condition.
[0018] The term "compound" herein can be made by the preparation
method disclosed in the embodiments. The substituted group of a
specific compound can be replaced by other substituted groups, and
thus other derivatives can be prepared under this spirit.
Therefore, the terms "5-nitrobenzoate derivative", "compound" and
"derivative" herein can be alternately used.
[0019] The term "platelet activation stimulator" herein is widely
referred to a reagent capable of activating platelets, including
but not limited to adenosine diphosphate (ADP), collegen,
thromboxane A2 analogs
(9,11-dideoxy-11.alpha.,9.alpha.-epoxymethanoprostaglandin
F2.alpha. (U46619), Sigma, U.S.) and thrombin for promoting
platelet aggregation, or A23187 (calcium ion carrier, also named as
calcimycin or calcium ionophore, AG Scientific, U.S.) for
increasing cationic ions (e.g. Ca.sup.2+) concentration in
cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The above objectives and advantages of the present invention
will become more readily apparent to those ordinarily skilled in
the art after reviewing the following detailed descriptions and
accompanying drawings.
[0021] FIGS. 1(a), 1(b), 1(c), 1(d) and 1(e) respectively depict
the effect of compound 4 or the control compound 5 on (a) ADP-, (b)
collagen-, (c) U46619-, (d) thrombin- and (e) A23187-induced wash
platelet aggregation tests.
[0022] FIG. 2(a) depicts the effect of compound 4 and control
compounds 5, 6 and 7 on ADP-induced platelet-rich plasma
aggregation test, wherein group 1 is ADP (10 .mu.M, control), group
2 is compound 4 (20 .mu.M) pre-treatment+ADP (10 .mu.M), group 3 is
compound 5 (20 .mu.M) pre-treatment+ADP (10 .mu.M), group 4 is
compound 6 (20 .mu.M) pre-treatment+ADP (10 .mu.M), and group 5 is
compound 7 (20 .mu.M) pre-treatment+ADP (10 .mu.M).
[0023] FIG. 2(b) depicts the effect of compound 4 and control
compounds 5, 6 and 7 on collagen-induced platelet-rich plasma
aggregation test, wherein group 1 is collagen (4 .mu.g/ml,
control), group 2 is compound 4 (20 .mu.M) pre-treatment+collagen
(4 .mu.g/ml), group 3 is compound 5 (20 .mu.M)
pre-treatment+collagen (4 .mu.g/ml), group 4 is compound 6 (20
.mu.M) pre-treatment+collagen (4 .mu.g/ml), and group 5 is compound
7 (20 .mu.M) pre-treatment+collagen (4 .mu.g/ml).
[0024] FIG. 3(a) depicts the immunoblotting pattern showing the
level of podoplanin of C6 tumor cell lines (including C6-LG,
C6-Blood and C6-Lung).
[0025] FIG. 3(b) depicts the effect of compound 4 (20 .mu.M) on
C6-Lung tumor cell-induced platelet aggregation.
[0026] FIG. 4 depicts the activation analysis of compound 4 (20
.mu.M) on recombinant fusion protein (i.e. recombinant
podoplanin/Fc)-induced platelet aggregation test.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0027] The present invention will now be described more
specifically with reference to the following Embodiments. It is to
be noted that the following descriptions of preferred Embodiments
of this invention are presented herein for purpose of illustration
and description only; it is not intended to be exhaustive or to be
limited to the precise form disclosed.
Embodiment 1: Preparation of
N-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide
[0028] The preparation procedure of
N-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide is represented by
the following formula II.
##STR00011##
[0029] 1-Chloro-4-nitro-2-(trifluoromethyl)benzene (2.0 mL, 13.3
mmole) was dissolved in dimethyl sulfoxide (DMSO, 12 mL), NaOH (1.6
g) was batchwise added at a temperature lower than 25.degree. C.,
and the reaction solution was reacted at room temperature (RT) for
8 hours. After the reaction was terminated, the pH of the reaction
solution was adjusted to 1.0 using concentrated HCl, and then the
reaction solution was poured into the separatory funnel and
extracted with CH.sub.2Cl.sub.2 for five times (each for 20 ml).
The obtained CH.sub.2Cl.sub.2 solution was hydrated over MgSO.sub.4
and concentrated under vacuum. The obtained concentrate was
subjected to the purification of silica gel column (50 g) and
eluted with the system of CHCl.sub.3/n-hexane (2:1) to afford
compound 1 (1.85 g), yield of about 67%.
[0030] Compound 1: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.48
(1H, d, J=2.4 Hz), 8.32 (1H, dd, J=8.8, 2.4 Hz), 7.14 (1H, d, J=8.8
Hz).
[0031] Compound 1 was nominated as
4-nitro-2-(trifluoromethyl)phenol.
[0032] Next, compound 1 (500 mg, 2.4 mmole) and tert-butyl
2-aminoethylcarbamate (769 mg, 4.8 mmole) were transferred in a
reaction bottle, 1 M aqueous NaOH solution (7.2 mmole) and dioxane
(10 mL) were added, and then the mixture solution were heated to
100.degree. C. and reacted for 24 hours. After the reaction was
terminated, the pH of the reaction solution was adjusted to 1.0
using 1 N HCl solution, and then the reaction solution was poured
into the separatory funnel and extracted with CH.sub.2Cl.sub.2 for
five times (each for 20 ml). The obtained CH.sub.2Cl.sub.2 solution
was hydrated over MgSO.sub.4 and concentrated under vacuum. The
obtained concentrate was subjected to the purification of silica
gel column (50 g) and eluted with the system of CHCl.sub.3/n-hexane
(19:1) to afford compound 2 (600.0 mg), yield of about 77%.
[0033] Compound 2: .sup.1H NMR (400 MHz, Acetone-d6): .delta. 8.96
(1H, s), 8.74 (1H, d, J=2.4 Hz), 8.30 (1H, dd, J=8.8, 2.4 Hz), 7.10
(1H, d, J=8.8, 2.4 Hz), 6.32 (1H, s), 3.58 (2H, m), 3.39 (2H,
m).
[0034] Compound 2 was nominated as tert-butyl
2-(2-hydroxy-5-nitrobenzamido)ethylcarbamate.
[0035] Next, compound 2 (600 mg) was installed in the reaction
bottle, the CH.sub.2Cl.sub.2 solution containing 20% TFA was added,
and the reaction solution was reacted at RT for 2 hours to form the
reaction mixture. After the reaction was terminated, the reaction
mixture was concentrated under vacuum. The obtained concentrate was
subjected to the purification on silica gel column (45 g) and
eluted using the system of CHCl.sub.3/n-hexane (4:1) to afford
compound 3 (390 mg), yield of about 95%.
[0036] Compound 3: .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 8.79
(1H, d, J=1.6 Hz), 8.21 (1H, dd, J=9.2, 1.6 Hz), 7.01 (1H, d, J=9.2
Hz), 3.72 (2H, m), 3.21 (2H, m).
[0037] Compound 3 was nominated as
N-(2-aminoethyl)-2-hydroxy-5-nitrobenzamide.
[0038] Subsequently, compound 3 (390 mg) was dissolved in 2 N NaOH
solution (10 ml) and reacted with benzoyl chloride at RT for 16
hours, and then the mixture concentrated under vacuum after the
reaction was terminated. The residue was subjected to the
purification on silica gel column (50 g) and eluted using the
system of CHCl.sub.3/n-hexane (30:1) to afford compound 4 (325 mg),
yield of about 57%.
[0039] Compound 4: .sup.1H NMR (400 MHz, C.sub.5D.sub.5N): .delta.
12.65 (1H, s), 10.14 (1H, s), 9.48 (1H, s), 9.09 (1H, s), 8.11 (3H,
m), 7.40 (3H, m), 7.05 (1H, J=8.8 Hz), 3.95 (4H, m). ESI-MS m/z 330
(100) [M+H].sup.+, 352 (32) [M+Na].sup.+. HRESI-MS m/z 352.0911
(calc.: 352.0909; C.sub.16H.sub.15N.sub.3O.sub.5Na).
[0040] Compound 4 was nominated as
N-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide.
Embodiment 2: Preparation of Other 5-nitrobenzoate Derivatives
[0041] For affording other 5-nitrobenzoate derivatives, compound 3
may be reacted with benzoyl chlorides bound with a various of
substituted groups, such as mono-substituted benzoyl chloride,
di-substituted benzoyl chloride or tri-substituted benzoyl
chloride
##STR00012##
respectively), and each of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5 and R.sub.6 may be fluoride, chloride, bromide, iodide or
methyl group, and R.sub.1 to R.sub.6 may be bound to the para-,
meta- or ortho-position of the benzoyl moiety. That is, the benzoyl
moiety of the prepared compound 4 may be substituted as
mono-substituted benzoyl moiety, di-substituted benzoyl moiety or
tri-substituted benzoyl moeity
##STR00013##
respectively).
Embodiment 3: Preparation of
4-O-benzoyl-3-methoxy-.beta.-nitrostyrene (compound 5)
[0042] trans-4-Hydroxyl 3-methoxyl-.beta.-nitrostyrene and benzoyl
chloride, were dissolved in a mixture solution of pyridine (1 ml)
and CH.sub.2Cl.sub.2 (10 ml), and reacted at RT for 24 hours. After
the removal of solvent, the obtained concentrate was subjected to
the purification of silica gel column (90 g) and eluted with the
system of n-hexane/acetone (3:1) to afford
4-O-benzoyl-3-methoxy-.beta.-nitrostyrene (compound 5; as
represented by formula III).
##STR00014##
[0043] Compound 5: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.25
(1H, s), 8.24 (1H, s), 8.02 (1H, d, J=13.6 Hz), 7.68(1H, d, J=7.6
Hz), 7.61(1H, d, J=13.6 Hz), 7.56(1H, d, J=7.6 Hz), 7.55(1H, d,
J=7.6 Hz), 7.28(1H, d, J=8.2 Hz), 7.24(1H, d, J=8.2 Hz), 7.17 (1H,
d , J=1.2 Hz), 3.88 (3H, s). ESI-MS m/z 322 (100) [M+Na].sup.+.
[0044] Compound 5 was nominated as
4-O-benzoyl-3-methoxy-.beta.-nitrostyrene).
Embodiment 4: Preparation of
4-O-nicotinoyl-3-methoxy-.beta.-nitrostyrene (compound 6)
[0045] trans-4-Hydroxyl 3-methoxyl-.beta.-nitrostyrene and
nicotinoyl chloride hydrochloride, were dissolved in a mixture
solution of pyridine (1 ml) and CH.sub.2Cl.sub.2 (10 ml), and
reacted at RT for 16 hours. After the removal of solvent, the
residue was subjected to the purification of silica gel column (60
g) and eluted with the system of n-hexane/CHCl.sub.3 (1:3) to
afford 4-O-nicotinoyl-3-methoxy-.beta.-nitrostyrene (compound 6; as
represented by formula IV).
##STR00015##
[0046] Compound 6: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.40
(1H, br.s), 8.87 (1H, d, J=4.8 Hz), 8.45 (1H, d, J=8.4 Hz), 8.00
(1H, d, J=13.6 Hz), 7.59 (1H, d, J=8.0, 2.0 Hz), 7.49 (1H, dd,
J=4.8, 8.0 Hz), 7.24 (2H, m), 7.15(1H, s), 3.88 (3H, s).
[0047] Compound 6 was nominated as
4-O-nicotinoyl-3-methoxy-.beta.-nitrostyrene.
Embodiment 5: Preparation of
4-O-(2,4-dichlorobenzoyl)-3-methoxy-.beta.-nitrostyrene (compound
7)
[0048] trans-4-Hydroxyl 3-methoxyl-.beta.-nitrostyrene and
2,4-dichlorobenzoyl chloride, were dissolved in a mixture solution
of pyridine (1 ml) and CH.sub.2Cl.sub.2 (10 ml), and reacted at RT
for 24 hours. After the removal of solvent, the residue was
subjected to the purification of (a) silica gel column (100 g) and
eluted with the system of n-hexane/CHCl.sub.3 (1:2) and (b) silica
gel column (60 g) and eluted with the system of n-hexane/acetone
(4:1) twice to afford
4-O-(2,4-dichlorobenzoyl)-3-methoxy-.beta.-nitrostyrene (compound
7; as represented by formula V).
##STR00016##
[0049] Compound 7: .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.07
(1H, d, J=8.0 Hz), 7.98 (1H, d, J=13.6 Hz), 7.57 (1H, d, J=13.6
Hz), 7.55 (1H, d, J=2.0 Hz), 7.39 (1H, dd, J=8.0, 2.0 Hz), 7.25
(1H, d, J=8.0 Hz), 7.20 (1H, dd, J=8.0, 1.2 Hz), 7.14 (1H, d, J=1.2
Hz), 3.88 (3H, s).
[0050] Compound 7 was nominated as
4-O-(2,4-dichlorobenzoyl)-3-methoxy-.beta.-nitrostyrene.
[0051] Experiment 1: Preparation of Human Platelets
[0052] The venous blood was collected from 18 to 35 year-old
healthy volunteer donors (who didn't take any anti-platelet
medicine or other anti-inflammation medicine within two weeks
before blood draw), sufficiently mixed with anticoagulant (venous
blood:anticoagulant=9:1), and then centrifuged at 200 g at RT for
15 minutes. The upper layered platelet-rich plasma (PRP) was
collected, and centrifuged at 1000 g for 10 minutes after mixing
with anticoagulant (the final concentration: 0.5 .mu.M prostacyclin
and 10 U/ml heparin). The supernatant was removed, and the platelet
pellets were resuspended in Tyrode's solution and further
centrifuged at 1000 g for 10 minutes. Finally, the wash platelets
without plasma proteins were resuspended in the Tyrode's solution
containing calcium and magnesium ions (this sample is wash
platelets). The number of platelets were calculated using the
coulter counter before use, and the density of platelets was
adjusted to 3.times.10.sup.8 cells/ml and stored at RT for use.
[0053] Experiment 2: Human Platelet Aggregation test
[0054] Experiment 2 was performed to determine the variations of
light transmission upon the aggregation of platelets (the
platelet-rich plasma sample and the wash platelet sample) by using
platelet aggregometer (Model 570VS, Chrono-log Corp., U.S.).
Firstly, the platelets (3.times.10.sup.8 cells/ml) prepared in
Experiment 1 was pre-heated with stir at 900 rpm at 37.degree. C.
for 1 minute, and the prepared 5-nitrobenzoate derivative (compound
4 or other control compounds 5, 6 and 7) was added to react for 3
minutes. The separate platelet activation stimulator (includes but
not limit to ADP, collagen, U46619, thrombin and A23187) was added
to observe the effect of 5-nitrobenzoate derivative on the platelet
aggregation activation.
[0055] Please refer to FIGS. 1(a), 1(b), 1(c), 1(d) and 1(e), which
respectively depict the effect of compound 4 or control compound 5
on (a) ADP-, (b) collagen-, (c) U46619-, (d) thrombin- and (e)
A23187-induced wash platelet aggregation test. Compound 4 did not
inhibit or interfere ADP-, collagen-, U46619-, thrombin- or
A23187-induced platelet aggregation along with the increased dosage
of compound 4.
[0056] Please refer to FIG. 2(a), which depicts the effect of
compound 4 and control compounds 5 to 7 on the ADP-induced
platelet-rich plasma aggregation test. The measured light
transmission of platelet aggregation was enhanced depending on the
increased reaction time (after 300 seconds) of compound 4 (group
2), indicating that compound 4 did not inhibit or interfere
ADP-induced platelet-rich plasma aggregation. Please refer to FIG.
2(b), similarly, the measure light transmission of platelet-rich
plasma aggregation test was enhanced depending on the increased
reaction time (after 300 seconds) of compound 4 (group 2),
indicating that compound 4 did not inhibit or interfere
collagen-induced platelet aggregation.
[0057] Experiment 3: Tumor Cell-Induced Platelet Aggregation
(TCIPA)
[0058] The purified platelets (1.times.10.sup.9 cells/ml) was
preheated with stir at 900 rpm at 37.degree. C. for 1 minute, and
5-nitrobenzoate derivative of the invention was added. After a
3-minute reaction, C6 tumor cells "C6-Lung" and "C6-LG"
(1.times.10.sup.6 cells/ml, respectively) with different levels of
podoplanin was added to react with platelets for 15 minutes, and
the variations of light transmission upon the platelet aggregation
were measured by using platelet aggregometer, to analyze the TCIPA
effect.
[0059] Please refer to the immunoblotting pattern in FIG. 3(a),
which depicts that C6-Lung tumor cells had the higher expression
level of podoplanin relative to C6-LG or C6-Blood cells.
.beta.-Actin is the control for immunoblotting test.
[0060] Please refer to FIG. 3(b), which depicts that compound 4 (20
.mu.M) can effectively inhibit C6-Lung tumor cell (with high
expression level of podoplanin)-induced platelet aggregation along
with the increased reaction time.
[0061] Experiment 4: Platelet Aggregation Induced by the
Recombinant Podoplanin/Fc Fusion Protein
[0062] The purified wash platelets (1.times.10.sup.9 cells/ml) were
preheated with stir at 1000 rpm at 37.degree. C. for 1 minute, and
5-nitrobenzoate derivative of formula I of the invention was added.
After a 3-minute reaction, the genetically engineering recombinant
podoplanin/Fc fusion protein (abbreviated hereinafter "PDPN/Fc", 2
.mu.g, Sino Biological Inc., Beijing, People's Republic of China)
was added to react with platelets for 15 minutes, and the
variations of light transmission upon the platelet aggregation were
measured by using platelet aggregometer, to analyze the effect of
5-nitrobenzoate derivative on the recombinant PDPN/Fc-induced
platelet aggregation.
[0063] Please refer to FIG. 4, which depicts that compound 4 can
effectively inhibit the PDPN/Fc-induced platelet aggregation along
with the increased reaction time. The recombinant Fc is the
genetically engineering antibody Fc fragment and acts as the
control.
[0064] In concluding the above experimental results,
5-nitrobenzoate derivatives or compounds of Formula I with
mono-substituted benzoyl chloride, di-substituted benzoyl chloride
or tri-substituted benzoyl chloride, of the invention do not
influence platelet aggregation, can efficiently inhibit tumor
cell-induced platelet aggregation (TCIPA) and the TCIPA pathway,
can specifically inhibit podoplanin-induced platelet aggregation
and its pathway, in particular inhibit the recombinant
podoplanin/fc fusion protein-induced platelet aggregation
[0065] Since podoplanin of tumor cells would be combined with
CLEC-2 of platelets and 5-nitrobenzoate derivatives of the
invention would inhibit TCIPA induced by podoplanin-expressing
tumor cells, 5-nitrobenzoate derivatives of the invention can be
used to block the interaction between CLEC-2 and podoplanin and can
be applied as the targeted therapy medicine for inhibiting
metastasis of tumor cells.
[0066] While the invention has been described in terms of what is
presently considered to be the most practical and preferred
Embodiments, it is to be understood that the invention needs not be
limited to the disclosed Embodiments. On the contrary, it is
intended to cover various modifications and similar arrangements
included within the spirit and scope of the appended claims, which
are to be accorded with the broadest interpretation so as to
encompass all such modifications and similar structures.
* * * * *