U.S. patent application number 11/255405 was filed with the patent office on 2006-06-15 for novel use of porphyrin derivatives.
Invention is credited to Min-Suk Kang, Yong-Rok Kim, Si-Hwan Ko, Chang-Hee Lee, Dai-Woon Lee, Won-Young Lee, Dong-Hoon Won, Nam-Tae Woo.
Application Number | 20060128683 11/255405 |
Document ID | / |
Family ID | 36383809 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060128683 |
Kind Code |
A1 |
Woo; Nam-Tae ; et
al. |
June 15, 2006 |
Novel use of porphyrin derivatives
Abstract
The present invention is related to novel use of photopyrin
compounds useful as an anticancer agent by way of reproducing
singlet state oxygen radical and the inventive compounds showed
potent inhibition effect on colon cancer, cervical cancer, gastric
cancer, cystic cancer or lung cancer. Accordingly, the porphyrin
compound of the present invention can be useful in treating or
preventing colon cancer, cervical cancer, gastric cancer, cystic
cancer or lung cancer in human or mammal.
Inventors: |
Woo; Nam-Tae; (Seoul,
KR) ; Kang; Min-Suk; (Ansan, KR) ; Lee;
Won-Young; (Seoul, KR) ; Lee; Chang-Hee;
(Chuncheon, KR) ; Kim; Yong-Rok; (Seoul, KR)
; Lee; Dai-Woon; (Sungnam, KR) ; Won;
Dong-Hoon; (Wonju, KR) ; Ko; Si-Hwan; (Goyang,
KR) |
Correspondence
Address: |
ANDERSON, KILL & OLICK, P.C.
1251 AVENUE OF THE AMERICAS
NEW YORK,
NY
10020-1182
US
|
Family ID: |
36383809 |
Appl. No.: |
11/255405 |
Filed: |
October 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10718734 |
Nov 20, 2003 |
7019132 |
|
|
11255405 |
Oct 20, 2005 |
|
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Current U.S.
Class: |
514/185 ;
514/410 |
Current CPC
Class: |
A61K 41/0071 20130101;
A61K 31/555 20130101; A61K 49/0036 20130101; C07D 487/22 20130101;
A61K 31/409 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/185 ;
514/410 |
International
Class: |
A61K 31/555 20060101
A61K031/555; A61K 31/409 20060101 A61K031/409 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 2003 |
KR |
10-2003-0002921 |
Jan 16, 2003 |
KR |
10-2003-0002922 |
Claims
1. A method of treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer in a mammal
said method comprises administering a therapeutically effective
amount of porphyrin derivatives represented by the following
formula (I), and the pharmaceutically acceptable salts thereof:
##STR13## wherein R.sub.1, R.sub.2 is independently a straight or
branched lower alkyl or alkoxy group having 1 to 6 carbon atoms, a
polyethyleneglycol group or a sulfonyl group; R.sub.3 is a hydrogen
atom, an alkoxy group having 1 to 6 carbon atoms or a
polyethyleneglycol group; R.sub.4 is a hydrogen atom, a hydroxyl
group or an alkoxy group having 1 to 6 carbon atom, A is linked
directly or bridged with oxygen atom, which can be chelating with
transition metal ion comprising Ni metal ion.
2. The method of claim 1, said porphyrin derivative represented by
the formula (I), comprise the compounds wherein R.sub.1, R.sub.2 is
selected from the group consisting of an ethyl group, a propyl
group, an ethyleneglycol group, diethyleneglycol group,
triethyleneglycol group, tetraethyleneglycol group,
hexaethyleneglycol group, heptaethyleneglycol group or a
methoxyethyleneglycol group; R.sub.3 is selected from the group
consisting of a hydrogen atom, an ethyl group, a propyl group, a
methoxy, an ethoxy group, an ethyleneglycol group,
triethyleneglycol group, hexaethylene group; R.sub.4 is a hydrogen
atom, a hydroxyl group or an methoxy group; and A is linked
directly providing that R.sub.1 and R.sub.2 is the same group and
R.sub.2 is different from R.sub.1 or R.sub.3.
3. A method of treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer in a mammal
said method comprises administering a therapeutically effective
amount of porphyrin derivatives represented by the following
formula (II), and the pharmaceutically acceptable salts thereof:
##STR14## wherein R.sub.1, R.sub.2 is independently a straight or
branched lower alkyl or alkoxy group having 1 to 6 carbon atoms, a
polyethyleneglycol group or a sulfonyl group, which can be
chelating with transition metal ion comprising Ni metal ion.
wherein X is oxygen atom; A is --CH.sub.2--; R.sub.1 is hydrogen
atom or aminoethyl group; R.sub.2 is an hydrogen or halogen atom or
an alkyl group having 1 to 6 carbon atoms.
4. A method of treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer in a mammal
said method comprises administering a therapeutically effective
amount of porphyrin derivatives represented by the following
general formula (III), and the pharmaceutically acceptable salts
thereof: ##STR15## wherein R.sub.1 is a polyethyleneglycol group;
R.sub.4 is a hydrogen atom or a hydroxyl group.
5. A method of treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer in a mammal
said method comprises administering a therapeutically effective
amount of porphyrin derivatives represented by the following
general formula (IV), and the pharmaceutically acceptable salts
thereof: ##STR16## wherein R.sub.2 is a bromopropyl group, or a
polyethyleneglycol group; R.sub.4 is a hydrogen atom or a hydroxyl
group.
6. A method of treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer in a mammal
said method comprises administering a therapeutically effective
amount of porphyrin derivatives represented by the following
general formula (V), the pharmaceutically acceptable salts thereof:
##STR17## wherein R.sub.1 is a methyl, ethyl group, or an
ethyleneglycol group.
7. A method of treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer in a mammal
said method comprises administering a therapeutically effective
amount of porphyrin derivatives represented by the following
general formula (VI), the pharmaceutically acceptable salts
thereof: ##STR18## wherein R.sub.1, R.sub.2 is independently a
polyethyleneglycol group.
8. A method of treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer in a mammal
said method comprises administering a therapeutically effective
amount of porphyrin derivatives represented by the following
general formula (VII), the pharmaceutically acceptable salts
thereof: ##STR19## wherein R.sub.1 is a polyethyleneglycol
group.
9. The method of claim 1, said porphyrin derivative represented by
the formula (VII), comprise the compounds wherein X is oxygen atom;
A is --NHCH.sub.2--; R.sub.1 is hydrogen atom or aminoethyl group;
R.sub.2 is an hydrogen or halogen atom or an alkyl group having 1
to 6 carbon atoms.
10. The method of claim 1, said porphyrin derivative represented by
the formula (I) comprise the compound which is one selected from
the group consisting of
N-[4-(3,4-dimethylphenyl)-2-(pivaloyloxymethyl)butyl]-N-[4-hydroxy-3-meth-
oxybenzyl]thiourea,
N-[4-t-bytulphenyl-2-(pivaloyloxymethyl)butyl]-N-[4-hydroxy-3-methoxybenz-
yl]thiourea,
N-[4-(3,4-dimethylphenyl)-2-(pivaloyloxymethyl)butyl]-N-[4-hydroxy-3-meth-
oxybenzyl]urea,
N--[4-t-dimethylphenyl-2-(pivaloyloxymethyl)butyl]-N-[4-hydroxy-3-methoxy-
benzyl]urea,
N-[4-(3,4-dimentylphenyl-2-(pivaloyloxymethyl)butyl)-2-[4-hydroxy-3-metho-
xyphenyl]acetamide,
N-[4-(4-t-butylphenyl)-2-(pivaloyloxymethyl)butyl]-2-[4-hydroxy-3-methoxy-
phenyl]acetamide,
N-[4-(3,4-dimethylphenyl)-2-(pivaloyloxymethyl)butyl]-2-[4-(2-aminoethoxy-
)-3-methoxyphenyl]acetamide,
N-[4-(4-t-butylphenyl)-2-(pivaloyloxymethyl)butyl]-2-[4-(2-aminethoxy)-3--
methoxyphenyl]acetamide.
11. A use of porphyrin compound selected from the group consisting
of compounds of formula (I) to (VII) as set forth in claims 1 and 3
to 8 or pharmaceutical acceptable salts thereof as an anticancer
agent to treat or prevent colon cancer, cervical cancer, gastric
cancer, cystic cancer or lung cancer.
12. A use of porpyrin compound (I) to (VII) as set forth in claim 1
and 3 to 8 for the manufacture of medicament employed for treating
or preventing colon cancer, cervical cancer, gastric cancer, cystic
cancer or lung cancer in human or mammal.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in part of U.S. Ser. No.
10/718,734 filed on Nov. 20, 2003.
DESCRIPTION
[0002] 1. Technical Field
[0003] The present invention relates to a method of treating or
preventing colon cancer, cervical cancer, gastric cancer, or cystic
cancer in a mammal said method comprises administering a
therapeutically effective amount of porphyrin derivatives or their
pharmaceutically acceptable salts thereof.
[0004] 2. Background Art
[0005] Photodynamic tumor therapy is one of therapeutic techniques
to treat incurable diseases using by photosensitizer drugs having a
selectivity and photoenhancing activity to cancer cells or various
tumors without a surgical operation and complication occurring in
Chemotherapy.
[0006] The action mechanism of photosensitizer drugs is that for
example, the drug is administrated intravenously to a patient and
the optimum amount of light is irradiated thereto to form excited
state of photosensitizer. The drugs give rise to activating oxygen
molecule to transform to be excited singlet oxygen state, new
radical or new chemical species resulting in attacking and
demolishing cancer cells or various tumor cells selectively.
[0007] Representative photosensitizers are porphyrin compounds
which have been extracted from silk worm feces or mulberry leaf or
green algae and have appropriate spectrophotometric characteristics
to be used as photosensitizers. Their most important
characteristics are to give rise to electron transition due to
infrared light whose wavelength from 700 to 900 nm allowing
relative great cell penetrating activity and the production of
excited state of triplet oxygen thereby.
[0008] Porphyrin derivatives as photosensitizers can selectively
not only penetrate or be accumulated in tumor site but also emit
fluorescence or phosphorescence and therefore, can be useful as an
early stage diagnostic tool.
[0009] There have been lots of reports on several porphyrin
derivatives in prior art. For example, U.S. Pat. Nos. 5,633,275;
5,654,423; 5,675,001; 5,703,230; 5,705,622 and U.S. Pat. No.
4,882,234 disclose several photoprin II compounds. It has been
reported that one of those is on sale and some of those are on
clinical trials, however, those porphyrin II are the mixtures
consisting of several oligomers ether-linked with haematoporphyrin
(HpD).
[0010] PCT/WO 97/29915 (A) discloses BPDMA (verteporphin), a
benzoporphyrin derivative, known to show specific effect on skin
cancer, psoriasis and AMD. M-THPC disclosed in PCT/WO97/48393 and
known to be useful in treating trachea and lung cancer or
Monoaspitylchlorine disclosed in CA Registration No. 2121716 and
Japanese Patent Registration No. 09071531 and known to be useful to
photodynamic therapy as one of chlorine derivatives have been
reported together with related several patents i.e.,
PCT/WO97/19081, PCT/WO 97/32885; EP 569113; U.S. Pat. Nos.
5,587,394; 5,648,485 and 5,693,632; all of which are incorporated
herein by reference.
[0011] However, most of those porphyrin group compounds are
meso-tetraphenylporphyrin derivatives, chlorine group, chlorophyll
group, purpurine group, nerdine, Diels-Elder Reaction Adducts and
so on and 5-aminolevulanic acid, phthalocyanin and the like as
non-porphyrin group compounds.
[0012] Since the yield of producing singlet oxygen molecule is
correlated with cell cytotoxic activity directly, the yield is in
proportion with cell cytotoxic activity, which is most crucial
factor together with the retention time in human body in
photodynamic therapy and remains to be improved till now. However,
above described clinically using porphyrin compounds as
photosensitizer drugs have been reported to have several
disadvantages such as too long retention time in human body
delivering unfavorable photo-toxicity, which remains to be improved
till now.
[0013] Accordingly, present inventors have endeavored to find novel
use of porphyrin derivatives or their pharmaceutically acceptable
salt thereof which shows potent anti-cancer activity verified by
several experiments, i.e., in vitro cancer cell line test and in
vivo animal model test, and finally accomplished the present
invention.
SUMMARY OF THE INVENTION
[0014] Present invention provides novel use of porphyrin compounds
and the pharmaceutically acceptable salts thereof useful as
anti-cancer agent.
[0015] Present invention also provides a use of above described
porpyrin compounds for the preparation of for manufacture of
medicament employed for treating or preventing various cancers in
human or mammal.
[0016] Present invention also provides a method of treating or
preventing cancer in a mammal wherein said method comprises
administering a therapeutically effective amount of above described
compound or pharmaceutically acceptable salts thereof.
DISCLOSURE OF THE INVENTION
[0017] Thus, It is an object to provide a method of treating or
preventing colon cancer, cervical cancer, gastric cancer, cystic
cancer, lung cancer in a mammal said method comprises administering
a therapeutically effective amount of porphyrin derivatives
represented by the following formula (I), and the pharmaceutically
acceptable salts thereof: ##STR1## wherein
[0018] R.sub.1, R.sub.2 is independently a straight or branched
lower alkyl or alkoxy group having 1 to 6 carbon atoms, a
polyethyleneglycol group or a sulfonyl group;
[0019] R.sub.3 is a hydrogen atom, an alkoxy group having 1 to 6
carbon atoms or a polyethyleneglycol group;
[0020] R.sub.4 is a hydrogen atom, a hydroxyl group or an alkoxy
group having 1 to 6 carbon atom,
[0021] A is linked directly or bridged with oxygen atom, which may
be chelating with transition metal ion comprising Ni metal ion.
[0022] A preferred embodiment comprises the compounds of the
formula I where R.sub.1, R.sub.2 is selected from the group
consisting of an ethyl group, a propyl group, an ethyleneglycol
group, diethyleneglycol group, triethyleneglycol group,
tetraethyleneglycol group, hexaethyleneglycol group,
heptaethyleneglycol group or a methoxyethyleneglycol group; R.sub.3
is selected from the group consisting of a hydrogen atom, an ethyl
group, a propyl group, a methoxy, an ethoxy group, an
ethyleneglycol group, triethyleneglycol group, hexaethylene group;
R.sub.4 is a hydrogen atom, a hydroxyl group or an methoxy group;
and A is linked directly providing that R.sub.1 and R.sub.2 is the
same group and R.sub.2 is different from R.sub.1 or R.sub.3.
[0023] Exemplary preferable compound of the present invention
comprises following compounds represented by general formula (II)
to (VII):
[0024] Accordingly, it is a further object to provide a method of
treating or preventing colon cancer, cervical cancer, gastric
cancer, cystic cancer or lung cancer in a mammal said method
comprises administering a therapeutically effective amount of
porphyrin derivatives represented by the following formula (II),
and the pharmaceutically acceptable salts thereof: ##STR2##
wherein
[0025] R.sub.1, R.sub.2 is independently a straight or branched
lower alkyl or alkoxy group having 1 to 6 carbon atoms, a
polyethyleneglycol group or a sulfonyl group, which may be
chelating with transition metal ion comprising Ni metal ion.
[0026] Accordingly, it is a still further object to provide a
method of treating or preventing colon cancer, cervical cancer,
gastric cancer, cystic cancer or lung cancer in a mammal said
method comprises administering a therapeutically effective amount
of porphyrin derivatives represented by the following general
formula (III), and the pharmaceutically acceptable salts thereof:
##STR3## wherein
[0027] R.sub.1 is a polyethyleneglycol group;
[0028] R.sub.4 is a hydrogen atom or a hydroxyl group.
[0029] Accordingly, it is a still further object to provide a
method of treating or preventing colon cancer, cervical cancer,
gastric cancer, cystic cancer or lung cancer in a mammal said
method comprises administering a therapeutically effective amount
of porphyrin derivatives represented by the following general
formula (IV), and the pharmaceutically acceptable salts thereof:
##STR4## wherein
[0030] R.sub.2 is a bromopropyl group, or a polyethyleneglycol
group;
[0031] R.sub.4 is a hydrogen atom or a hydroxyl group.
[0032] Accordingly, it is a still further object to provide a
method of treating or preventing colon cancer, cervical cancer,
gastric cancer, cystic cancer or lung cancer in a mammal said
method comprises administering a therapeutically effective amount
of porphyrin derivatives represented by the following general
formula (V), the pharmaceutically acceptable salts thereof:
##STR5## wherein
[0033] R.sub.1 is a methyl, ethyl group, or an ethyleneglycol
group.
[0034] It is a still further object to provide a method of treating
or preventing colon cancer, cervical cancer, gastric cancer, cystic
cancer or lung cancer in a mammal said method comprises
administering a therapeutically effective amount of porphyrin
derivatives represented by the following general formula (VI), the
pharmaceutically acceptable salts thereof: ##STR6## wherein
[0035] R.sub.1, R.sub.2 is independently a polyethyleneglycol
group.
[0036] It is a still further object to provide a method of treating
or preventing colon cancer, cervical cancer, gastric cancer, cystic
cancer or lung cancer in a mammal said method comprises
administering a therapeutically effective amount of porphyrin
derivatives represented by the following chemical formula (VII),
the pharmaceutically acceptable salts thereof: ##STR7##
[0037] The compounds of the present invention may be chemically
synthesized by the methods in the reaction schemes hereinafter,
which are merely exemplary and in no way limit the invention. The
reaction schemes show the steps for preparing the representative
compounds of the present invention, and other compounds also may be
produced by following the steps with appropriate modifications of
reagents and starting materials, which are envisaged by those
skilled in the art.
General Synthetic Procedures
[0038] For example, the porphyrin compounds represented by general
formula (I) the pharmaceutically acceptable salt thereof may be
prepared by the following steps: pheophytin a or
10-hydroxypheophytin a is obtained by extracting dried silk worm
feces or green algae with water or organic solvent such as alcohol,
acetone or chloroform etc to obtain porphyrin containing extract;
and the extract is subjected to repeated column chromatography and
Thin layer chromatography to isolate pheophytin a (1) or
10-hydroxypheophytin a; and then the isolated compound is reacted
with alcohol (R.sub.1OH) in the presence of acid or base at room
temperature or reflux condition to obtain pheophorbide a alkylester
(2) or 10-hydroxy pheophorbide a methylester which is used as
starting material for the preparation of the compounds represented
by formula (II) to (VII). ##STR8##
[0039] As depicted in above Scheme 1, pheophytin a (1) is reacted
with conventional alcohol (R.sub.1OH) such as 3-bromo-1-propanol or
PEG such as ethylenglycol, triethyleneglycol, in the presence of
acid preferably, sulfuric acid at nitrogen gas atmosphere in
solvent such as toluene, oxazine, dichloromethane for 1 hr to 3
days, preferably 24 hrs, washed with appropriate washing solution
and then remaining solvent is removed by evaporator in vaccuo to
obtain pheophorbide a ester (2) as a final product, which is
further purified and isolated with column chromatography or TLC
well-known in the art.
[0040] Detailed procedure described in scheme 1 will be explained
in following Example 1 to 3. ##STR9##
[0041] As depicted in above Scheme 2, methyl pheophorbide a methyl
ester (3) is reacted with alcohol (R.sub.2OH) such as
3-bromo-1-propanol or PEG such as ethylenglycol, triethyleneglycol,
in the presence of acid preferably, sulfuric acid or base
preferably, pyridine, at nitrogen gas atmosphere in inert solvent
such as toluene, oxazine, dichloromethane for 1 hr to 3 days,
preferably 24 hrs, washed with appropriate washing solution and
remaining solvent is removed by evaporator in vaccuo to obtain
pheophorbide a methylester, final product (4), which is further
purified and isolated with column chromatography or TLC well-known
in the art.
[0042] Detailed procedure described in scheme 2 will be explained
in following Example 4 to 5. ##STR10##
[0043] As depicted in above Scheme 3, methyl pheophorbide a methyl
ester (5) is reacted with oxazine in the presence of base
preferably, pyridine, at nitrogen gas atmosphere in inert solvent
such as toluene, oxazine, dichloromethane for 1 hr to 3 days,
preferably 24 hrs, washed with appropriate washing solution such as
ammonium sulfate and then remaining solvent is removed by
evaporator in vaccuo to obtain oxazine type pheophorbide a
methylester, final product (6) which is further purified and
isolated with column chromatography or TLC well-known in the
art.
[0044] Detailed procedure described in scheme 3 will be explained
in following Example 6. ##STR11##
[0045] As depicted in above Scheme 4, methyl pheophorbide a methyl
ester (7) is reacted with triethyleneglycol in the presence of acid
preferably, sulfuric acid, at nitrogen gas atmosphere in inert
solvent such as toluene, oxazine, dichloromethane for 1 hr to 3
days, preferably 24 hrs, washed with appropriate washing solution
such as sodium bicarbonate and remaining solvent is removed by
evaporator in vaccuo to obtain pheophorbide a methylester, final
product (8), which is further purified and isolated with column
chromatography or TLC well-known in the art.
[0046] Detailed procedure described in scheme 4 will be explained
in following Example 7 and 8. ##STR12##
[0047] As depicted in above Scheme 5, pheophytin a (9) is reacted
with conventional alcohol (R.sub.1OH) such as 3-bromo-1-propanol or
PEG such as ethylenglycol, triethyleneglycol, in the presence of
acid preferably, sulfuric acid at nitrogen gas atmosphere in
solvent such as toluene, oxazine, dichloromethane for 1 hr to 3
days, preferably 24 hrs, washed with appropriate washing solution
and remaining solvent is removed by evaporator in vaccuo. The
compound is further subjected to oxidation with oxidizing agent
such as KMnO.sub.4, NaIO.sub.4, in the presence of under basic
condition at nitrogen gas atmosphere in protic solvents such as
water to obtain pheophorbide a alcohol as a final product (10),
which is further purified and isolated with column chromatography
or TLC well-known in the art.
[0048] Detailed procedure described in scheme 5 will be explained
in following Example 9.
[0049] The compound of the present invention has potent anticancer
activity and therefore, the pharmaceutical composition of the
present invention thus may be employed to treat or prevent various
cancers such as colon cancer, cervical cancer, gastric cancer, or
cystic cancer by way of reproducing singlet state oxygen radical
and superior cell cytotoxic activity.
[0050] The present invention also provides an use of compound
selected from the group consisting of compounds of formula (I) to
(VII) or pharmaceutical acceptable salts thereof as anti-cancer
agent useful in treating or preventing colon cancer, cervical
cancer, gastric cancer, cystic cancer or lung cancer.
[0051] In accordance with another aspect of the present invention,
there is also provided an use of the compound (I) to (VII) for
manufacture of medicament employed for treating or preventing colon
cancer, cervical cancer, gastric cancer, cystic cancer or lung
cancer in human or mammal.
[0052] The present invention also provides a pharmaceutical
composition comprising a compound of formula (I) to (VII) or a
pharmaceutically acceptable salt thereof as an active
ingredient.
[0053] The compound of formula (I) to (VII) according to the
present invention can be provided as a pharmaceutical composition
containing pharmaceutically acceptable carriers, adjuvants or
diluents. For example, the compounds of the present invention can
be dissolved in oils, propylene glycol or other solvents which are
commonly used to produce an injection. Suitable examples of the
carriers include physiological saline, polyethylene glycol,
ethanol, vegetable oils, isopropyl myristate, etc., but are not
limited to them. For topical administration, the compounds of the
present invention can be formulated in the form of ointments and
creams.
[0054] Hereinafter, the following formulation methods and
excipients are merely exemplary and in no way limit the
invention.
[0055] The compounds of the present invention in pharmaceutical
dosage forms may be used in the form of their pharmaceutically
acceptable salts, and also may be used alone or in appropriate
association, as well as in combination with other pharmaceutically
active compounds.
[0056] The compounds of the present invention may be formulated
into preparations for injections by dissolving, suspending, or
emulsifying them in aqueous solvents such as normal saline, 5%
Dextrose, or non-aqueous solvent such as vegetable oil, synthetic
aliphatic acid glycerides, esters of higher aliphatic acids or
propylene glycol. The formulation may include conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0057] The desirable dose of the inventive compounds varies
depending on the condition and the weight of the subject, severity,
drug form, route and period of administration, and may be chosen by
those skilled in the art. However, in order to obtain desirable
effects, it is generally recommended to administer at the amount
ranging 0.0001-100 mg/kg, preferably 0.001-100 mg/kg by weight/day
of the inventive compounds of the present invention. The dose may
be administered in single or divided into several times per day. In
terms of composition, the compounds should be present between
0.0001 to 10% by weight, preferably 0.0001 to 1% by weight based on
the total weight of the composition.
[0058] The pharmaceutical composition of present invention can be
administered to a subject animal such as mammals (rat, mouse,
domestic animals or human) via various routes. All modes of
administration are contemplated, for example, administration can be
made orally, rectally or by intravenous, intramuscular,
subcutaneous, intrathecal, epidural or intracerebroventricular
injection.
[0059] It will be apparent to those skilled in the art that various
modifications and variations can be made in the compositions, use
and preparations of the present invention without departing from
the spirit or scope of the invention.
[0060] The present invention is more specifically explained by the
following examples. However, it should be understood that the
present invention is not limited to these examples in any
manner.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] The above and other objects, features and other advantages
of the present invention will more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which;
[0062] FIG. 1a shows UV spectrum of DH-1-180-3;
[0063] FIG. 2 shows the result of determination of singlet oxygen
state at 508 nm (.lamda.excitation);
[0064] FIG. 3 represents the result of photosensitizing effect of
DH-I-180-3 with light against CT26 cell line determined by MTT
assay, of which control group (control), light only irradiation
group (light), the group treated with solvent (DMF), the group
treated with photosensitizer samples without light (Ps(2) only) and
the group treated with present photosensitizing substance
(DH-1-180-3) denote respectively;
[0065] FIG. 4 presents the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated CT26 cell;
[0066] FIG. 5 presents the result of photosensitizing effect of
DH-I-180-3 with light against TC-1 cell line determined by MTT
assay, of which control group (control), light only irradiation
group (light), the group treated with solvent (DMF), the group
treated with photosensitizer samples without light (Ps(2) only) and
the group treated with present photosensitizing substance
(DH-1-180-3) denote respectively.
[0067] FIG. 6 presents the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated TC-1 cell;
[0068] FIG. 7 presents the result of photosensitizing effect of
DH-I-180-3 with light against SNU-1 cell line determined by MTT
assay, of which control group (control), light only irradiation
group (light), the group treated with solvent (DMF), the group
treated with photosensitizer samples without light (Ps(2) only) and
the group treated with present photosensitizing substance
(DH-1-180-3) denote respectively;
[0069] FIG. 8 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated SNU-1 cell line;
[0070] FIG. 9 presents the result of photosensitizing effect of
DH-I-180-3 with light against HT-1197 cell line determined by MTT
assay, of which control group (control), light only irradiation
group (light), the group treated with solvent (DMF), the group
treated with photosensitizer samples without light (Ps(2) only) and
the group treated with present photosensitizing substance
(DH-1-180-3) denote respectively;
[0071] FIG. 10 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated HT-1197 cell line;
[0072] FIG. 11 presents the result of photosensitizing effect of
DH-I-180-3 with light against LLC1 cell line determined by MTT
assay, of which control group (control), light only irradiation
group (light), the group treated with solvent (DMF), the group
treated with photosensitizer samples without light (Ps(2) only) and
the group treated with present photosensitizing substance
(DH-1-180-3) denote respectively;
[0073] FIG. 12 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated LLC1 cell line.
BEST MODE FOR CARRYING OUT THE INVENTION
[0074] The present invention is more specifically explained by the
following examples. However, it should be understood that the
present invention is not limited to these examples in any
manner.
EXAMPLES
Example 1
Preparation of (13-diethylene glycol-oxycarbonyl)-pheophorbide a,
methyl ester (2)
[0075] A solution of phephytin a 60 mg in dichloromethane (3 ml)
was poured in 50 ml of flask and treated with diethyleneglycol (20
ml) with stirring. 1 ml of sulfuric acid was added thereto, stirred
for 3 hrs and then sodium bicarbonate water solution was added
thereto. The solution was extracted with chloroform and the
collected chloroform layer was concentrated by removing organic
solvent. Remaining residue was purified by column chromatography to
isolate 39 mg of (13-diethylene glycol-oxycarbonyl)-pheophorbide a,
methyl ester (2):
[0076] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.51 (s, 1H,
meso-H), 9.37(s, 1H, meso-H), 8.56(s, 1H, meso-H), 7.99(dd, 1H,
J=6.2, 11.6 Hz, CH.sub.2.dbd.CH), 6.29 (d, 1H, J=17.8 Hz,
CH.sub.2.dbd.CH), 6.28(s. 1H, CH), 6.18 (d, 1H, J=11.6 Hz
CH.sub.2.dbd.CH), 4.48-4.41 (m, 1H, CH), 4.24-4.22 (m. 1H. CH),
4.19-4.04 (m, 2H, OCH.sub.2), 3.87 (s, 3H, OCH.sub.3), 3.71-3.66
(m, 2H, CH.sub.2), 3.68 (s, 3H, CH.sub.3), 3.64-3.42 (m, 6H,
CH.sub.2OCH.sub.2CH.sub.2), 3.40 (s, 3H, CH.sub.3), 3.22 (s, 3H,
CH.sub.3), 2.68-2.15 (m, 4H, CH.sub.2CH.sub.2), 1.82 (d, 3H, J=7.3
Hz, CH.sub.3), 1.69 (t, 3H, J=7.6 Hz, CH.sub.3), 0.56 (br. S., 1H,
N-H), -1.61 (br. s., 1H, N-H).
Example 2
Preparation of (13- methoxytriethylene glycol-oxycarbonyl)
pheophorbide a methyl ester (3)
[0077] 29 mg of (13- methoxytriethylene glycol -oxycarbonyl)
pheophorbide a methyl ester (3) was prepared by the same procedure
with that described in above Example 1 except using phephytin (60
mg) and methoxytriethyleneglycol (30 ml):
[0078] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.45 (s, 1H,
meso-H), 9.30(s, 1H, meso-H), 8.55(s, 1H, meso-H), 7.93(dd, 1H,
J=6.2, 11.5 Hz, CH.sub.2.dbd.CH), 6.26 (s. 1H, CH), 6.25 (d, 1H,
J=17.8 Hz CH.dbd.CH.sub.2), 6.14 (d, 1H, J=11.3 Hz
CH.dbd.CH.sub.2), 4.49-4.44 (m, 1H, CH), 4.22-4.20 (m. 1H. CH),
4.15-4.02 (m, 2H, OCH.sub.2), 3.88 (s, 3H, OCH.sub.3), 3.67 (s, 3H,
CH.sub.3), 3.60(q, 2H, CH.sub.3--CH.sub.2), 3.51-3.45 (m, 8H,
CH.sub.2 OCH.sub.2 CH.sub.2 OCH.sub.2), 3.41-3.37 (m, 2H,
CH.sub.2), 3.38 (s, 3H, CH.sub.3), 3.25 (s, 3H, OCH.sub.3), 3.16
(s, 3H, CH.sub.3), 2.65-2.18 (m, 4H, CH.sub.2CH.sub.2), 1.82 (d,
3H, J=7.2 Hz, CH.sub.3), 1.68-1.64 (m, 3H, CH.sub.3), 0.51 (br. s.,
1H, N-H), -1.61 (br. s., 1H, N-H).
Example 3
Preparation of 13-hydroxy-(13-methoxytriethylene glycoloxy
carbonyl) pheophorbide a methyl ester (4)
[0079] 22 mg of 13-hydroxy-(13- methoxytriethylene glycoloxy
carbonyl) pheophorbide a methyl ester (4) was prepared by the same
procedure with that described in above Example 1 except using
10-hydroxyphephytin a (60 mg) and methoxytriethyleneglycol (20
ml):
[0080] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.62 (s, 1H,
meso-H), 9.49(s, 1H, meso-H), 8.65(s, 1H, meso-H), 8.03(dd, 1H,
J=6.3, 11.4 Hz, CH.sub.2.dbd.CH), 6.31 (d, 1H, J=17.8 Hz,
CH.dbd.CH.sub.2), 6.20(d, 1H, J=11.6 Hz, CH.dbd.CH.sub.2), 5.78 (s,
1H, OH), 4.52-4.47 (m, 1H, CH), 4.30-4.14 (m, 3H, CH and
OCH.sub.2), 3.74 (s, 3H, OCH.sub.3), 3.74-3.70(m, 2H, CH.sub.2),
3.63-3.57 (m, 8H, CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2), 3.60 (s, 3H,
CH.sub.3), 3.47-3.46 (m, 2H, CH.sub.2), 3.43 (s, 3H, CH.sub.3),
3.29 (s, 3H, CH.sub.3), 3.27 (s, 3H, OCH.sub.3), 3.02-2.95,
2.64-2.57 and 2.35-2.21 (m, 4H, CH.sub.2CH.sub.2), 1.71 (t, 3H,
J=7.5 Hz, CH.sub.3), 1.60 (d, 3H, J=7.1 Hz, CH.sub.3), 0.30 (br.
s., 1H, N-H), -1.83 (br. s., 1H, N-H).
Example 4
Preparation of [13-(3-bromo-1-propyloxycarbonyl)]-pheophorbide a
methy ester (5)
[0081] A solution of methyl pheophorbide a methyl ester (4) (20 mg)
in pyridine (4 ml) and toluene (8 ml), was poured in 50 ml of flask
and treated with 3-bromo-1-propanol (0.003 ml) with stirring and
heated for 5 hrs. And then the solution was washed with ammonium
chloride water solution and extracted with methylene chloride. The
collected methylene chloride layer was concentrated by removing
organic solvent and remaining residue was purified by column
chromatography to isolate 11 mg of
[13-(3-bromo-1-propyloxycarbonyl)]-pheophorbide a methy ester
(5):
[0082] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.52 (s, 1H,
meso-H), 9.38(s, 1H, meso-H), 8.56(s, 1H, meso-H), 7.99(dd, 1H,
J=6.2, 11.7 Hz, CH.sub.2.dbd.CH), 6.28 (d, 1H, J=19.3 Hz,
CH.dbd.CH.sub.2), 6.26(d, 1H, CH), J=11.6 Hz, CH.dbd.CH.sub.2),
6.18 (d, 1H, J=11.6 Hz, CH.dbd.CH.sub.2), 4.52-4.45 (m, 3H, CH and
OCH.sub.2), 4.24-4.22 (m, 1H, CH), 3.68 (s, 3H, CH.sub.3), 3.67 (m,
2H, CH.sub.2), 3.56 (s, 3H, OCH.sub.3), 3.47-3.34 (m, 2H,
CH.sub.2), 3.40 (s, 3H, CH.sub.3), 3.23 (s, 3H, CH.sub.3),
2.68-2.17 (m, 6H, CH.sub.2CH.sub.2 and CH.sub.2), 1.83 (d, 3H,
J=7.3 Hz, CH.sub.3), 1.69 (t, 3H, J=7.6 Hz, CH.sub.2--CH.sub.3),
0.54 (br. s., 1H, N-H), -1.62 (br. s., 1H, N-H).
Example 5
Preparation of (13- triethylene glycoloxy carbonyl)pheophorbide a
methy ester (6)
[0083] In similar method in Example 4, A solution of methyl
pheophorbide a methyl ester (4) (100 mg) in pyridine (16 ml) and
toluene (15 ml), was poured in 50 ml of flask and treated with
triethyleneglycol (0.033 ml) at nitrogen gas atmosphere and heated
for 16 hrs. And then the solution was washed with ammonium chloride
water solution and extracted with methylene chloride. The collected
methylene chloride layer was concentrated by removing organic
solvent and remaining residue was purified by column chromatography
to isolate 73 mg of (13- triethylene glycoloxy
carbonyl)pheophorbide a methy ester (6):
[0084] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.53 (s, 1H,
meso-H), 9.40(s, 1H, meso-H), 8.57(s, 1H, meso-H), 8.00(dd, 1H,
J=6.3, 11.5 Hz, CH.sub.2.dbd.CH), 6.30 (d, 1H, J=18.1 Hz,
CH.dbd.CH.sub.2), 6.27 (s, 1H, CH), 6.19 (d, 1H, J=6.3, 12.8 Hz,
CH.dbd.CH.sub.2), 4.49-4.45 (m, 3H, CH and OCH.sub.2), 4.26-4.24
(m, 1H, CH), 3.72-3.66 (m, 4H, CH.sub.2 and OCH.sub.2), 3.69 (s,
3H, CH.sub.3), 3.55 (s, 3H, OCH.sub.3), 3.49-3.39(m, 4H, CH.sub.2
OCH.sub.2), 3.41 (s, 3H, CH.sub.3), 3.31 (t, 2H, J=4.6 Hz,
CH.sub.2), 3.26-3.23 (m, 5H, CH.sub.2 and CH.sub.3), 2.66-2.21 (m,
5H, CH.sub.2CH.sub.2 and OH), 1.82 (d, 3H, J=7.3 Hz, CH.sub.3),
1.70 (t, 3H, J=7.6 Hz, CH.sub.3), 0.54 (br. s., 1H, N-H), -1.62
(br. s., 1H, N-H).
Example 6
Preparation of [13-hydroxy-(13- triethylene glycoloxy
carbonyl)]-pheophorbide a methyl ester (7)
[0085] A solution of methyl pheophorbide a methyl ester (4) (50 mg)
in pyridine (8 ml) and oxazine (23 ml), was dissolved in 10 ml of
toluene and heated for 5 hrs. And then the solution was washed with
ammonium chloride water solution and extracted with methylene
chloride. The collected methylene chloride layer was concentrated
by removing organic solvent and remaining residue was purified by
column chromatography to isolate 21 mg of
[13-hydroxy-(13-triethylene glycoloxy carbonyl)]-pheophorbide a
methyl ester (7):
[0086] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.76 (s, 1H,
meso-H), 9.54 (s, 1H, meso-H), 8.71 (s, 1H, meso-H), 8.01 (dd, 1H,
J=6.2, 11.6 Hz, CH.sub.2.dbd.CH), 6.34 (d, 1H, J=17.9 Hz,
CH.dbd.CH.sub.2), 6.18 (d, 1H, J=11.6 Hz, CH.dbd.CH.sub.2), 6.09
(s, 1H, OH), 4.47-4.42 (m, 1H, CH), 4.07-4.05 (m, 1H, CH), 3.90 (s,
3H, OCH.sub.3), 3.77 (s, 3H, CH.sub.3), 3.74(q, 2H,
CH.sub.3--CH.sub.2), 3.54 (s, 3H, OCH.sub.3), 3.44 (s, 3H,
CH.sub.3), 3.26 (s, 3H, CH.sub.3), 2.61-1.78 (m, 4H,
CH.sub.2CH.sub.2), 1.71 (t, 3H, J=7.6 Hz, CH.sub.2--CH.sub.3), 1.60
(d, 3H, J=7.1 Hz, CH.sub.3), -1.09 (br. s., 1H, N-H), -1.41 (br.
s., 1H, N-H).
Example 7
Preparation of pheophorbide a triethylene glycol methyl ester
(8)
[0087] A solution of methyl pheophorbide a methyl ester (30 mg) was
dissolved in 20 ml of triethyleneglycol and stirred with adding 1
ml of sulfuric acid thereto. And then the solution was washed with
sodium bicarbonate water solution and extracted with ethyl acetate.
The collected ethyl acetate layer was concentrated by removing
organic solvent and remaining residue was purified by column
chromatography to isolate 32 mg of pheophorbide a triethylene
glycol methyl ester (8):
[0088] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.53 (s, 1H,
meso-H), 9.40 (s, 1H, meso-H), 8.57 (s, 1H, meso-H), 8.00 (dd, 1H,
J=6.4, 11.5 Hz, CH.sub.2.dbd.CH), 6.30 (d, 1H, J=17.9 Hz,
CH.dbd.CH.sub.2), 6.29 (d, 1H, CH), 6.19 (d, 1H, J=12.5 Hz,
CH.dbd.CH.sub.2), 4.49-4.44 (m, 3H, CH and OCH.sub.2), 4.26-4.24
(m, 1H, CH), 4.15-4.07 (m, 2H, OCH.sub.2), 3.74-3.65 (m, 4H,
CH.sub.2 and OCH.sub.2), 3.69 (s, 3H, OCH.sub.3), 3.58-3.43 (m,
14H, OCH.sub.2), 3.41 (s, 3H, CH.sub.3), 3.35 (t, 2H, J=4.5 Hz,
CH.sub.2), 3.30-3.28 (m, 2H, CH.sub.2), 3.24 (s, 3H, CH.sub.3),
2.66-2.02 (m, 6H, CH.sub.2CH.sub.2 and OH), 1.82 (d, 3H, J=7.3 Hz,
CH.sub.3), 1.70 (t, 3H, J=7.6 Hz, CH.sub.3), 0.55 (br. s., 1H,
N-H), -1.62 (br. s., 1H, N-H).
Example 8
Preparation of methyl pheophorbide a diethyleneglycol ester (9)
[0089] A solution of pheophytin a (60 mg) was dissolved in small
amount of dichloromethane. 20 ml of diethyleneglycol and 1 ml of
sulfuric acid were added thereto and stirred for 23 hrs. And then
the solution was washed with saturated sodium bicarbonate water
solution and extracted with chloroform. The collected chloroform
layer was concentrated by removing organic solvent and remaining
residue was purified by column chromatography to isolate 2 mg of
methyl pheophorbide a diethylene glycol ester (9):
[0090] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.26 (s, 1H,
meso-H), 8.34 (s, 1H, meso-H), 7.92 (dd, 1H, J=6.0, 11.8 Hz,
CH.sub.2.dbd.CH), 6.33 (s, 1H, OH), 6.25 (d, 1H, J=17.8 Hz,
CH.dbd.CH.sub.2), 6.17 (d, 1H, J=11.6 Hz, CH.dbd.CH.sub.2),
4.88(br. s, 2H. OH), 4.76-4.60 (m, 4H, CH.sub.2 CH.sub.2),
4.41-4.33 (m, 3H, CH and CH.sub.2), 4.27-4.23 (m, 2H, CH and
OCH.sub.2), 3.96-3.94 (m, 2H, CH.sub.2), 3.88-3.80 (m, 6H,
CH.sub.2), 3.75 (s, 3H, OCH.sub.3), 3.75-3.69 (m, 2H, CH.sub.2),
3.58 (s, 3H, CH.sub.3), 3.31 (s, 3H, CH.sub.3), 3.18 (s, 3H,
CH.sub.3), 2.75-2.00 (m, 4H, CH.sub.2CH.sub.2), 2.03 (d, 3H, J=7.0
Hz, CH.sub.3), 1.64 (t, 3H, J=7.3 Hz, CH.sub.3), 0.87 (br. s., 1H,
N-H), -1.85 (br. s., 1H, N-H).
Example 9
Preparation of methyl pheophorbide a triethyleneglycol ester
(10)
[0091] A solution of methyl pheophorbide a methyl ester (100 mg)
dissolved in 16 ml of pyridine was added to 15 ml of toluene. 0.033
ml of triethyleneglycol was added thereto and heated for 16 hrs at
nitrogen gas atmosphere. And then the solution was extracted with
methylene chloride. The collected methylene chloride layer was
concentrated by removing organic solvent and remaining residue was
purified by column chromatography to isolate 73 mg of methyl
pheophorbide a triethylene glycol ester (10) designated as
DH-1-180-3 hereinafter:
UV Spectrum: See FIG. 1
[0092] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 9.53 (s, 1H,
meso-H), 9.40 (s, 1H, meso-H), 8.57 (s, 1H, meso-H), 8.00 (dd, 1H,
J=6.3, 11.5 Hz, CH.sub.2.dbd.CH), 6.30 (d, 1H, J=18.1 Hz,
CH.dbd.CH.sub.2), 6.27 (s, 1H. CH), 6.19 (d, 1H, J=12.8 Hz,
CH.dbd.CH.sub.2), 4.49-4.45 (m, 3H, CH and OCH.sub.2), 4.26-4.24
(m, 1H, CH), 3.72-3.66 (m, 4H, CH.sub.2 and CH.sub.2), 3.69 (s, 3H,
OCH.sub.3), 3.55 (s, 3H, OCH.sub.3), 3.49-3.39 (m, 4H,
CH.sub.2OCH.sub.2), 3.41 (s, 3H, CH.sub.3), 3.31(t, 2H, J=4.6 Hz,
CH.sub.2), 3.26-3.23 (m, 5H, CH.sub.2 and CH.sub.3), 2.66-2.21 (m,
5H, CH.sub.2CH.sub.2 and OH), 1.82 (d, 3H, J=7.3 Hz, CH.sub.3),
1.70 (t, 3H, J=7.6 Hz, CH.sub.3), 0.54 (br. s., 1H, N-H), -1.62
(br. s., 1H, N-H).
Experimental Example 1
Determination of Transforming Activity of Present Compounds Into
Singlet Oxygen State
[0093] The transforming activity of the target compounds into
singlet oxygen state was measured by following methods.
Methods
[0094] The experimental assay was performed under air-saturated
condition (99.999% ultra-purified gas) using toluene (Merck Co.
HPLC grade) as a solvent, in the oxygen concentration in the
solution of 2.1.times.10.sup.-3 M at 21.degree. C.
Result
[0095] At the result of determination of singlet oxygen state at
508 nm (.lamda.excitation), the transformed photon yield was
0.60(5%) as can be seen in FIG. 2. Therefore, it is confirmed that
the transforming activity of DH-1-180-3 into singlet oxygen state
is excellent and the physical stability thereof is also good.
Experimental Example 2
Anticancer Effect on Colon Cancer
[0096] The anticancer activity of the target compounds on colon
cancer was determined by following experiments.
In Vitro Assay
[0097] DH-1-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0098] 1) CT26 cell line (mouse colon cancer cell line) was
cultured in culture medium (DMEM, 10% FBS, 100 units of penicillin,
100 ug streptomycin, L-glutamine, 2.2 mg/ml sodium bicarbonate) at
37.degree. C. in 5% CO.sub.2.
[0099] 2) CT26 cell lines were inoculated into a 96-well,
flat-bottomed microplate at a volume of 100 ul (1.times.10.sup.5
cells/well), respectively. 24 hrs later, the medium was removed and
the cultures were washed three times in PBS.
[0100] 3) Various concentrations of DH-I-180-3 (final conc.:
0.1.about.2 ug/ml) dissolved in DMF were added at a volume of 100
ul/well for 1 hr. The concentration of DMF solvent didn't exceed
0.5% to exclude the effect of DMF. Following incubation, the cells
were exposed to light (1.2 J/cm.sup.2 using by halogen lamp), and
then the cells were further incubated at 37.degree. C. for 24 hrs
in humidified incubator.
[0101] 4) For the MTT assay, 20 ul of MTT reagent (5 mg/ml) was
added to each cell culture well and cultured for 4 hrs. The
supernatant was discarded and 200 ul of DMSO was added to the
culture. Shaken for 10 min and the absorbance measured with an
ELISA-reader at 570 nm.
[0102] 5) The control, light and DMF groups represent cell only,
light only and DMF only. Respectively. The photoesensitizer only
group was subtracted from this result due to having not influence
on tumor cells by itself.
In Vivo Assay
[0103] 1) DH-I-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0104] 2) CT26 cell lines were (2.times.10.sup.5 cells/mouse in 100
ul of PBS) inoculated to Balb/C mice (ten per group) and 7-10 days
later, test sample diluted with 1% Tween 80 and control drug
diluted with injection water were administrated to each mouse at
the dose of 0.4, 0.8 and 2 mg/kg of photofrin in control group.
[0105] 3) Tumor bearing mice were given the intravenous (i.v.)
injection of DH-I-180-3 and Photofrin. At 4 hrs after
administration, the tumors were then given light treatment (1.2
J/cm.sup.2 using by halogen lamp).
[0106] Conventional photosensitizer, i.e., photogem.RTM., was used
as a comparative control group and several factors such as the
concentration of test samples, the irradiation strength of light
and the absorption time of samples etc were modified in the present
experiment.
Result
[0107] FIG. 3 represents the result of photosensitizing effect of
DH-I-180-3 with light against CT26 cells.
[0108] As can be seen in FIG. 3, the light was down to
approximately 10% at a dose of 2 ug/ml DH-I-180-3 compared with
that of control. Tumor cell killing by DH-I-180-3 mediated PDT was
clearly influenced by the time assayed after PDT as well as its
concentration treated to the cells. Light dependency in
cytotoxicity with DH-I-180-3 mediated PDT was demonstrated by
observing survival of DH-I-180-3 treated CT 26 cells when it was
protected from light.
[0109] FIG. 4 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated CT26.
[0110] As can be seen in FIG. 4, the tumor growth of the mice in
groups treated with DH-I-180-3/PDT was further delayed compared
with the photofrin/PDT group. It is confirmed that the growing
ratio of colon cancer in PDT treated group was remarkably decreased
with the treatment time comparing with those in control group and
photofrin (photogem.RTM.) treated group.
Experimental Example 3
Anticancer Effect on Cervical Cancer
[0111] The anticancer activity of the target compounds on cervical
cancer was determined by following experiments.
In Vitro Assay
[0112] DH-1-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0113] TC-1 cell line (mouse lung cancer cell line carrying HPV 16
E7) was cultured in culture medium (RPMI-1640, 10% FBS, 100 units
of penicillin, 100 ug streptomycin, L-glutamine, 2.2 mg/ml sodium
bicarbonate, 0.4 mg/ml G418 disulfate) at 37.degree. C. in 5%
CO.sub.2.
[0114] 2) TC-1 cell lines were inoculated into a 96-well,
flat-bottomed microplate at a volume of 100 ul (1.times.10.sup.5
cells/well), respectively. 24 hrs later, the medium was removed and
the cultures were washed three times in PBS.
[0115] 3) Various concentrations of DH-I-180-3 (final conc.:
0.1.about.2 ug/ml) dissolved in DMF were added at a volume of 100
ul/well for 1 hr. The concentration of DMF solvent didn't exceed
0.5% to exclude the effect of DMF. Following incubation, the cells
were exposed to light (1.2 J/cm.sup.2 using by halogen lamp), and
then the cells were further incubated at 37.degree. C. for 24 hrs
in humidified incubator.
[0116] 4) For the MTT assay, 20 ul of MTT reagent (5 mg/ml) was
added to each cell culture well and cultured for 4 hrs. The
supernatant was discarded and 200 ul of DMSO was added to the
culture. Shaken for 10 min and the absorbance measured with an
ELISA-reader at 570 nm.
[0117] 5) The control, light and DMF groups represent cell only,
light only and DMF only, respectively. The photosensitizer only
group was subtracted from this result due to having not influence
on tumor cells by itself
In Vivo Assay
[0118] 1) DH-I-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0119] 2) TC-1 cell lines were (3.times.10.sup.5 cells/mouse in 100
ul of PBS) inoculated to C57BL/6 mice (ten per group) and 7-10 days
later, test sample diluted with 1% Tween 80 and control drug
diluted with injection water were administrated to each mouse at
the dose of 0.4, 0.8 and 2 mg/kg of photofrin in control group.
[0120] 3) Tumor bearing mice were given the intravenous (i.v.)
injection of DH-I-180-3 and Photofrin. At 4 hrs after
administration, the tumors were then given light treatment (1.2
J/cm.sup.2 using by halogen lamp).
Result
[0121] FIG. 5 represents the result of photosensitizing effect of
DH-I-180-3 with light against TC-1 cell lines.
[0122] As can be seen in FIG. 5, the light was down to
approximately 10% at a dose of 2 ug/ml DH-I-180-3 compared with
that of control. Tumor cell killing by DH-I-180-3 mediated PDT was
clearly influenced by the time assayed after PDT as well as its
concentration treated to the cells. Light dependency in
cytotoxicity with DH-I-180-3 mediated PDT was demonstrated by
observing survival of DH-I-180-3 treated TC-1 cell lines when it
was protected from light.
[0123] FIG. 6 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated TC-1 cell lines.
[0124] As can be seen in FIG. 6, the tumor growth of the mice in
groups treated with DH-I-180-3/PDT was further delayed compared
with the photofrin/PDT group. It is confirmed that the growing
ratio of cervical cancer in PDT treated group was remarkably
decreased with the treatment time comparing with those in control
group and photofrin (photogem.RTM.) treated group.
Experimental Example 4
Anticancer Effect on Gastric Cancer
[0125] The anticancer activity of the target compounds on gastric
cancer was determined by following experiments.
In Vitro Assay
[0126] DH-1-180-3 as a test sample of the present invention and
photoprin (photogem.RTM.) as a control group were used in test.
[0127] 1) SNU-1 cell line was cultured in culture medium
(RPMI-1640, 10% FBS, 100 units of penicillin, 100 ug streptomycin,
L-glutamine, 2.2 mg/ml sodium bicarbonate) at 37.degree. C. in 5%
CO.sub.2.
[0128] 2) SNU-1 cell lines were inoculated into a 96-well,
flat-bottomed microplate at a volume of 100 ul (1.times.10.sup.5
cells/well), respectively. 24 hrs later, the medium was removed and
the cultures were washed three times in PBS.
[0129] 3) Various concentrations of DH-I-180-3 (final conc.:
0.1.about.2 ug/ml) dissolved in DMF were added at a volume of 100
ul/well for 1 hr. The concentration of DMF solvent didn't exceed
0.5% to exclude the effect of DMF. Following incubation, the cells
were exposed to light (1.2 J/cm.sup.2 using by halogen lamp), and
then the cells were further incubated at 37.degree. C. for 24 hrs
in humidified incubator.
[0130] 4) For the MTT assay, 20 ul of MTT reagent (5 mg/ml) was
added to each cell culture well and cultured for 4 hrs. The
supernatant was discarded and 200 ul of DMSO was added to the
culture. Shaken for 10 min and the absorbance measured with an
ELISA-reader at 570 nm.
[0131] 5) The control, light and DMF groups represent cell only,
light only and DMF only, respectively. The photoesensitizer only
group was subtracted from this result due to having not influence
on tumor cells by itself
In Vivo Assay
[0132] 1) DH-I-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0133] 2) SNU-1 cell lines were (3.times.10.sup.5 cells/mouse in
100 ul of PBS) inoculated to C57BL/6 mice (ten per group) and 7-10
days later, test sample diluted with 1% Tween 80 and control drug
diluted with injection water were administrated to each mouse at
the dose of 0.4, 0.8 and 2 mg/kg of photofrin in control group.
[0134] 3) Tumor bearing mice were given the intravenous (i.v.)
injection of DH-I-180-3 and Photofrin. At 4 hrs after
administration, the tumors were then given light treatment (1.2
J/cm.sup.2 using by halogen lamp).
Result
[0135] FIG. 7 represents the result of photosensitizing effect of
DH-I-180-3 with light against SNU-1 cell lines.
[0136] As can be seen in FIG. 7, the light was down to
approximately 46% at a dose of 2 ug/ml DH-I-180-3 compared with
that of control. Tumor cell killing by DH-I-180-3 mediated PDT was
clearly influenced by the time assayed after PDT as well as its
concentration treated to the cells. Light dependency in
cytotoxicity with DH-I-180-3 mediated PDT was demonstrated by
observing survival of DH-I-180-3 treated TC-1 cell lines when it
was protected from light.
[0137] FIG. 8 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated SNU-1 cell lines.
[0138] As can be seen in FIG. 6, the tumor growth of the mice in
groups treated with DH-I-180-3/PDT was further delayed compared
with the photofrin/PDT group. It is confirmed that the growing
ratio of gastric cancer in PDT treated group was remarkably
decreased with the treatment time comparing with those in control
group and photofrin (photogem.RTM.) treated group.
Experimental Example 5
Anticancer Effect on Cystic Cancer
[0139] The anticancer activity of the target compounds on cystic
cancer was determined by following experiments.
In Vitro Assay
[0140] DH-1-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0141] 1) HT-1197 cell line was cultured in culture medium (MEM,
10% FBS, 100 units of penicillin, 100 ug streptomycin, L-glutamine,
2.2 mg/ml sodium bicarbonate) at 37.degree. C. in 5% CO.sub.2.
[0142] 2) HT-1197 cell lines were inoculated into a 96-well,
flat-bottomed microplate at a volume of 100 ul (1.times.10.sup.5
cells/well), respectively. 24 hrs later, the medium was removed and
the cultures were washed three times in PBS.
[0143] 3) Various concentrations of DH-I-180-3 (final conc.:
0.1.about.2 ug/ml) dissolved in DMF were added at a volume of 100
ul/well for 1 hr. The concentration of DMF solvent didn't exceed
0.5% to exclude the effect of DMF. Following incubation, the cells
were exposed to light (1.2 J/cm.sup.2 using by halogen lamp), and
then the cells were further incubated at 37.degree. C. for 24 hrs
in humidified incubator.
[0144] 4) For the MTT assay, 20 ul of MTT reagent (5 mg/ml) was
added to each cell culture well and cultured for 4 hrs. The
supernatant was discarded and 200 ul of DMSO was added to the
culture. Shaken for 10 min and the absorbance measured with an
ELISA-reader at 570 nm.
[0145] 5) The control, light and DMF groups represent cell only,
light only and DMF only, respectively. The photoesensitizer only
group was subtracted from this result due to having not influence
on tumor cells by itself
In Vivo Assay
[0146] 1) DH-I-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0147] 2) HT-1197 cell lines were (3.times.10.sup.5 cells/mouse in
100 ul of PBS) inoculated to C57BL/6 mice (ten per group) and 7-10
days later, test sample diluted with 1% Tween 80 and control drug
diluted with injection water were administrated to each mouse at
the dose of 0.4, 0.8 and 2 mg/kg of photofrin in control group.
[0148] 3) Tumor bearing mice were given the intravenous (i.v.)
injection of DH-I-180-3 and Photofrin. At 4 hrs after
administration, the tumors were then given light treatment (1.2
J/cm.sup.2 using by halogen lamp).
Result
[0149] FIG. 9 represents the result of photosensitizing effect of
DH-I-180-3 with light against HT-1197 cell lines.
[0150] As can be seen in FIG. 9, the light was down to
approximately 26% at a dose of 2 ug/ml DH-I-180-3 compared with
that of control. Tumor cell killing by DH-I-180-3 mediated PDT was
clearly influenced by the time assayed after PDT as well as its
concentration treated to the cells. Light dependency in
cytotoxicity with DH-I-180-3 mediated PDT was demonstrated by
observing survival of DH-I-180-3 treated TC-1 cell lines when it
was protected from light.
[0151] FIG. 10 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated HT-1197 cell lines.
[0152] As can be seen in FIG. 10, the tumor growth of the mice in
groups treated with DH-i-180-3/PDT was further delayed compared
with the photofrin/PDT group. It is confirmed that the growing
ratio of cystic cancer in PDT treated group was remarkably
decreased with the treatment time comparing with those in control
group and photofrin (photogem.RTM.) treated group.
Experimental Example 6
Anticancer Effect on Lung Cancer
[0153] The anticancer activity of the target compounds on lung
cancer was determined by following experiments.
In Vitro Assay
[0154] DH-1-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0155] 1) LLC1 cell line (mouse lung cancer cell line) was cultured
in culture medium (DMEM, 10% FBS, 100 units of penicillin, 100 ug
streptomycin, L-glutamine, 2.2 mg/ml sodium bicarbonate) at
37.degree. C. in 5% CO.sub.2.
[0156] 2) LLC1 cell lines were inoculated into a 96-well,
flat-bottomed microplate at a volume of 100 ul (1.times.10.sup.5
cells/well), respectively. 24 hrs later, the medium was removed and
the cultures were washed three times in PBS.
[0157] 3) Various concentrations of DH-I-180-3 (final conc.:
0.1.about.2 ug/ml) dissolved in DMF were added at a volume of 100
ul/well for 1 hr. The concentration of DMF solvent didn't exceed
0.5% to exclude the effect of DMF. Following incubation, the cells
were exposed to light (1.2 J/cm.sup.2 using by halogen lamp), and
then the cells were further incubated at 37.degree. C. for 24 hrs
in humidified incubator.
[0158] 4) For the MTT assay, 20 ul of MTT reagent (5 mg/ml) was
added to each cell culture well and cultured for 4 hrs. The
supernatant was discarded and 200 ul of DMSO was added to the
culture. Shaken for 10 min and the absorbance measured with an
ELISA-reader at 570 nm.
[0159] 5) The control, light and DMF groups represent cell only,
light only and DMF only, respectively. The photoesensitizer only
group was subtracted from this result due to having not influence
on tumor cells by itself.
In Vivo Assay
[0160] 1) DH-I-180-3 as a test sample of the present invention and
photofrin (photogem.RTM.) as a control group were used in test.
[0161] 2) LLC1 cell lines were (2.times.10.sup.5 cells/mouse in 100
ul of PBS) inoculated to Balb/C mice (ten per group) and 7-10 days
later, test sample diluted with 1% Tween 80 and control drug
diluted with injection water were administrated to each mouse at
the dose of 0.4, 0.8 and 2 mg/kg of photofrin in control group.
[0162] 3) Tumor bearing mice were given the intravenous (i.v.)
injection of DH-I-180-3 and Photofrin. At 4 hrs after
administration, the tumors were then given light treatment (1.2
J/cm.sup.2 using by halogen lamp).
[0163] Conventional photosensitizer, i.e., photogem.RTM., was used
as a comparative control group and several factors such as the
concentration of test samples, the irradiation strength of light
and the absorption time of samples etc were modified in the present
experiment.
Result
[0164] FIG. 11 represents the result of photosensitizing effect of
DH-I-180-3 with light against LLC1 cells.
[0165] As can be seen in FIG. 11, the light was down to
approximately 10% at a dose of 2 ug/ml DH-I-180-3 compared with
that of control. Tumor cell killing by DH-I-180-3 mediated PDT was
clearly influenced by the time assayed after PDT as well as its
concentration treated to the cells. Light dependency in
cytotoxicity with DH-I-180-3 mediated PDT was demonstrated by
observing survival of DH-I-180-3 treated LLC1 cells when it was
protected from light.
[0166] FIG. 12 depicts the inhibition of tumor of BALB/c mouse
caused by DH-1-180-3 treated LLC 1.
[0167] As can be seen in FIG. 12, the tumor growth of the mice in
groups treated with DH-I-180-3/PDT was further delayed compared
with the photofrin/PDT group. It is confirmed that the growing
ratio of lung cancer in PDT treated group was remarkably decreased
with the treatment time comparing with those in control group and
photofrin (photogem.RTM.) treated group.
Experimental Example 7
Toxicity Test
Methods
[0168] The acute toxicity tests on ICR mice (mean body weight
25.+-.5 g) and Sprague-Dawley rats (235.+-.10 g) were performed
using the compounds 1 and 10. Each group consisting of 3 mice or
rats was administrated intraperitoneally with 20 mg/kg, 10 mg/kg
and 1 mg/kg of test compounds or solvents (0.2 ml, i.p.),
respectively and observed for 24 hrs.
Results
[0169] There were no treatment-related effects on mortality,
clinical signs, body weight changes and gross findings in any group
or either gender. These results suggested that the compounds
prepared in the present invention were potent and safe.
[0170] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the present
invention, and all such modifications as would be obvious to one
skilled in the art are intended to be included within the scope of
the following claims.
INDUSTRIAL APPLICABILITY
[0171] The compounds according to the present invention are useful
in the prevention, or treatment of various cancer diseases and have
superior advantages such as excellent photon yield to produce
singlet oxygen, good physical stability and potent cell
cytotoxicity to conventional photosensitizers.
* * * * *