US20060128683A1

US20060128683A1 - Novel use of porphyrin derivatives

Info

Publication number: US20060128683A1
Application number: US11/255,405
Authority: US
Inventors: Nam-Tae Woo; Min-Suk Kang; Won-Young Lee; Chang-Hee Lee; Yong-Rok Kim; Dai-Woon Lee; Dong-Hoon Won; Si-Hwan Ko
Original assignee: TECHNO MART CO Ltd
Current assignee: TECHNO MART CO Ltd
Priority date: 2003-01-16
Filing date: 2005-10-20
Publication date: 2006-06-15
Also published as: JP2006514064A; US7019132B2; WO2004063200A1; US20040142917A1; EP1594875A4; CA2513133A1; AU2003272116A1; EP1594875A1

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.

Description

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in part of U.S. Ser. No. 10/718,734 filed on Nov. 20, 2003.

DESCRIPTION

1. Technical Field
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.
2. Background Art
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.
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.
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.
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.
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).
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.
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.
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.
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

Present invention provides novel use of porphyrin compounds and the pharmaceutically acceptable salts thereof useful as anti-cancer agent.
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.
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

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:

wherein
R₁, R₂is independently a straight or branched lower alkyl or alkoxy group having 1 to 6 carbon atoms, a polyethyleneglycol group or a sulfonyl group;
R₃is a hydrogen atom, an alkoxy group having 1 to 6 carbon atoms or a polyethyleneglycol group;
R₄is 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 may be chelating with transition metal ion comprising Ni metal ion.
A preferred embodiment comprises the compounds of the formula I where R₁, R₂is 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; R₃is 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; R₄is a hydrogen atom, a hydroxyl group or an methoxy group; and A is linked directly providing that R₁and R₂is the same group and R₂is different from R₁or R₃.
Exemplary preferable compound of the present invention comprises following compounds represented by general formula (II) to (VII):
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:

wherein
R₁, R₂is 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.
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:

wherein
R₁is a polyethyleneglycol group;
R₄is a hydrogen atom or a hydroxyl group.
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:

wherein
R₂is a bromopropyl group, or a polyethyleneglycol group;
R₄is a hydrogen atom or a hydroxyl group.
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:

wherein
R₁is a methyl, ethyl group, or an ethyleneglycol group.
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:

wherein
R₁, R₂is independently a polyethyleneglycol group.
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:
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

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 (R₁OH) 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).
As depicted in above Scheme 1, pheophytin a (1) is reacted with conventional alcohol (R₁OH) 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.
Detailed procedure described in scheme 1 will be explained in following Example 1 to 3.
As depicted in above Scheme 2, methyl pheophorbide a methyl ester (3) is reacted with alcohol (R₂OH) 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.
Detailed procedure described in scheme 2 will be explained in following Example 4 to 5.
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.
Detailed procedure described in scheme 3 will be explained in following Example 6.
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.
Detailed procedure described in scheme 4 will be explained in following Example 7 and 8.
As depicted in above Scheme 5, pheophytin a (9) is reacted with conventional alcohol (R₁OH) 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 KMnO₄, NaIO₄, 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.
Detailed procedure described in scheme 5 will be explained in following Example 9.
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.
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.
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.
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.
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.
Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.
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.
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.
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.
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.
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.
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

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;
FIG. 1 a shows UV spectrum of DH-1-180-3;
FIG. 2 shows the result of determination of singlet oxygen state at 508 nm (λexcitation);
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;
FIG. 4 presents the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated CT26 cell;
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.
FIG. 6 presents the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated TC-1 cell;
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;
FIG. 8 depicts the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated SNU-1 cell line;
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;
FIG. 10 depicts the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated HT-1197 cell line;
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;
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

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)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.29 (d, 1H, J=17.8 Hz, CH₂═CH), 6.28(s. 1H, CH), 6.18 (d, 1H, J=11.6 Hz CH₂═CH), 4.48-4.41 (m, 1H, CH), 4.24-4.22 (m. 1H. CH), 4.19-4.04 (m, 2H, OCH₂), 3.87 (s, 3H, OCH₃), 3.71-3.66 (m, 2H, CH₂), 3.68 (s, 3H, CH₃), 3.64-3.42 (m, 6H, CH₂OCH₂CH₂), 3.40 (s, 3H, CH₃), 3.22 (s, 3H, CH₃), 2.68-2.15 (m, 4H, CH₂CH₂), 1.82 (d, 3H, J=7.3 Hz, CH₃), 1.69 (t, 3H, J=7.6 Hz, CH₃), 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)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.26 (s. 1H, CH), 6.25 (d, 1H, J=17.8 Hz CH═CH₂), 6.14 (d, 1H, J=11.3 Hz CH═CH₂), 4.49-4.44 (m, 1H, CH), 4.22-4.20 (m. 1H. CH), 4.15-4.02 (m, 2H, OCH₂), 3.88 (s, 3H, OCH₃), 3.67 (s, 3H, CH₃), 3.60(q, 2H, CH₃—CH₂), 3.51-3.45 (m, 8H, CH₂OCH₂CH₂OCH₂), 3.41-3.37 (m, 2H, CH₂), 3.38 (s, 3H, CH₃), 3.25 (s, 3H, OCH₃), 3.16 (s, 3H, CH₃), 2.65-2.18 (m, 4H, CH₂CH₂), 1.82 (d, 3H, J=7.2 Hz, CH₃), 1.68-1.64 (m, 3H, CH₃), 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)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.31 (d, 1H, J=17.8 Hz, CH═CH₂), 6.20(d, 1H, J=11.6 Hz, CH═CH₂), 5.78 (s, 1H, OH), 4.52-4.47 (m, 1H, CH), 4.30-4.14 (m, 3H, CH and OCH₂), 3.74 (s, 3H, OCH₃), 3.74-3.70(m, 2H, CH₂), 3.63-3.57 (m, 8H, CH₂OCH₂CH₂OCH₂), 3.60 (s, 3H, CH₃), 3.47-3.46 (m, 2H, CH₂), 3.43 (s, 3H, CH₃), 3.29 (s, 3H, CH₃), 3.27 (s, 3H, OCH₃), 3.02-2.95, 2.64-2.57 and 2.35-2.21 (m, 4H, CH₂CH₂), 1.71 (t, 3H, J=7.5 Hz, CH₃), 1.60 (d, 3H, J=7.1 Hz, CH₃), 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)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.28 (d, 1H, J=19.3 Hz, CH═CH₂), 6.26(d, 1H, CH), J=11.6 Hz, CH═CH₂), 6.18 (d, 1H, J=11.6 Hz, CH═CH₂), 4.52-4.45 (m, 3H, CH and OCH₂), 4.24-4.22 (m, 1H, CH), 3.68 (s, 3H, CH₃), 3.67 (m, 2H, CH₂), 3.56 (s, 3H, OCH₃), 3.47-3.34 (m, 2H, CH₂), 3.40 (s, 3H, CH₃), 3.23 (s, 3H, CH₃), 2.68-2.17 (m, 6H, CH₂CH₂and CH₂), 1.83 (d, 3H, J=7.3 Hz, CH₃), 1.69 (t, 3H, J=7.6 Hz, CH₂—CH₃), 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)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.30 (d, 1H, J=18.1 Hz, CH═CH₂), 6.27 (s, 1H, CH), 6.19 (d, 1H, J=6.3, 12.8 Hz, CH═CH₂), 4.49-4.45 (m, 3H, CH and OCH₂), 4.26-4.24 (m, 1H, CH), 3.72-3.66 (m, 4H, CH₂and OCH₂), 3.69 (s, 3H, CH₃), 3.55 (s, 3H, OCH₃), 3.49-3.39(m, 4H, CH₂OCH₂), 3.41 (s, 3H, CH₃), 3.31 (t, 2H, J=4.6 Hz, CH₂), 3.26-3.23 (m, 5H, CH₂and CH₃), 2.66-2.21 (m, 5H, CH₂CH₂and OH), 1.82 (d, 3H, J=7.3 Hz, CH₃), 1.70 (t, 3H, J=7.6 Hz, CH₃), 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)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.34 (d, 1H, J=17.9 Hz, CH═CH₂), 6.18 (d, 1H, J=11.6 Hz, CH═CH₂), 6.09 (s, 1H, OH), 4.47-4.42 (m, 1H, CH), 4.07-4.05 (m, 1H, CH), 3.90 (s, 3H, OCH₃), 3.77 (s, 3H, CH₃), 3.74(q, 2H, CH₃—CH₂), 3.54 (s, 3H, OCH₃), 3.44 (s, 3H, CH₃), 3.26 (s, 3H, CH₃), 2.61-1.78 (m, 4H, CH₂CH₂), 1.71 (t, 3H, J=7.6 Hz, CH₂—CH₃), 1.60 (d, 3H, J=7.1 Hz, CH₃), -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)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.30 (d, 1H, J=17.9 Hz, CH═CH₂), 6.29 (d, 1H, CH), 6.19 (d, 1H, J=12.5 Hz, CH═CH₂), 4.49-4.44 (m, 3H, CH and OCH₂), 4.26-4.24 (m, 1H, CH), 4.15-4.07 (m, 2H, OCH₂), 3.74-3.65 (m, 4H, CH₂and OCH₂), 3.69 (s, 3H, OCH₃), 3.58-3.43 (m, 14H, OCH₂), 3.41 (s, 3H, CH₃), 3.35 (t, 2H, J=4.5 Hz, CH₂), 3.30-3.28 (m, 2H, CH₂), 3.24 (s, 3H, CH₃), 2.66-2.02 (m, 6H, CH₂CH₂and OH), 1.82 (d, 3H, J=7.3 Hz, CH₃), 1.70 (t, 3H, J=7.6 Hz, CH₃), 0.55 (br. s., 1H, N-H), -1.62 (br. s., 1H, N-H).

Example 8

Preparation of methyl pheophorbide a diethyleneglycol ester (9)

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):
¹H-NMR (500 MHz, CDCl₃) δ: 9.26 (s, 1H, meso-H), 8.34 (s, 1H, meso-H), 7.92 (dd, 1H, J=6.0, 11.8 Hz, CH₂═CH), 6.33 (s, 1H, OH), 6.25 (d, 1H, J=17.8 Hz, CH═CH₂), 6.17 (d, 1H, J=11.6 Hz, CH═CH₂), 4.88(br. s, 2H. OH), 4.76-4.60 (m, 4H, CH₂CH₂), 4.41-4.33 (m, 3H, CH and CH₂), 4.27-4.23 (m, 2H, CH and OCH₂), 3.96-3.94 (m, 2H, CH₂), 3.88-3.80 (m, 6H, CH₂), 3.75 (s, 3H, OCH₃), 3.75-3.69 (m, 2H, CH₂), 3.58 (s, 3H, CH₃), 3.31 (s, 3H, CH₃), 3.18 (s, 3H, CH₃), 2.75-2.00 (m, 4H, CH₂CH₂), 2.03 (d, 3H, J=7.0 Hz, CH₃), 1.64 (t, 3H, J=7.3 Hz, CH₃), 0.87 (br. s., 1H, N-H), -1.85 (br. s., 1H, N-H).

Example 9

Preparation of methyl pheophorbide a triethyleneglycol ester (10)

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
¹H-NMR (500 MHz, CDCl₃) δ: 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, CH₂═CH), 6.30 (d, 1H, J=18.1 Hz, CH═CH₂), 6.27 (s, 1H. CH), 6.19 (d, 1H, J=12.8 Hz, CH═CH₂), 4.49-4.45 (m, 3H, CH and OCH₂), 4.26-4.24 (m, 1H, CH), 3.72-3.66 (m, 4H, CH₂and CH₂), 3.69 (s, 3H, OCH₃), 3.55 (s, 3H, OCH₃), 3.49-3.39 (m, 4H, CH₂OCH₂), 3.41 (s, 3H, CH₃), 3.31(t, 2H, J=4.6 Hz, CH₂), 3.26-3.23 (m, 5H, CH₂and CH₃), 2.66-2.21 (m, 5H, CH₂CH₂and OH), 1.82 (d, 3H, J=7.3 Hz, CH₃), 1.70 (t, 3H, J=7.6 Hz, CH₃), 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

The transforming activity of the target compounds into singlet oxygen state was measured by following methods.
Methods
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 2.1×10⁻³M at 21° C.
Result
At the result of determination of singlet oxygen state at 508 nm (λexcitation), 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

The anticancer activity of the target compounds on colon cancer was determined by following experiments.
In Vitro Assay
DH-1-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
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° C. in 5% CO₂.
2) CT26 cell lines were inoculated into a 96-well, flat-bottomed microplate at a volume of 100 ul (1×10⁵cells/well), respectively. 24 hrs later, the medium was removed and the cultures were washed three times in PBS.
3) Various concentrations of DH-I-180-3 (final conc.: 0.1˜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/cm²using by halogen lamp), and then the cells were further incubated at 37° C. for 24 hrs in humidified incubator.
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.
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
1) DH-I-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
2) CT26 cell lines were (2×10⁵cells/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.
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/cm²using by halogen lamp).
Conventional photosensitizer, i.e., photogem®, 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
FIG. 3 represents the result of photosensitizing effect of DH-I-180-3 with light against CT26 cells.
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.
FIG. 4 depicts the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated CT26.
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®) treated group.

Experimental Example 3

Anticancer Effect on Cervical Cancer

The anticancer activity of the target compounds on cervical cancer was determined by following experiments.
In Vitro Assay
DH-1-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
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° C. in 5% CO₂.
2) TC-1 cell lines were inoculated into a 96-well, flat-bottomed microplate at a volume of 100 ul (1×10⁵cells/well), respectively. 24 hrs later, the medium was removed and the cultures were washed three times in PBS.
3) Various concentrations of DH-I-180-3 (final conc.: 0.1˜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/cm²using by halogen lamp), and then the cells were further incubated at 37° C. for 24 hrs in humidified incubator.
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.
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
1) DH-I-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
2) TC-1 cell lines were (3×10 ⁵cells/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.
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/cm²using by halogen lamp).
Result
FIG. 5 represents the result of photosensitizing effect of DH-I-180-3 with light against TC-1 cell lines.
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.
FIG. 6 depicts the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated TC-1 cell lines.
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®) treated group.

Experimental Example 4

Anticancer Effect on Gastric Cancer

The anticancer activity of the target compounds on gastric cancer was determined by following experiments.
In Vitro Assay
DH-1-180-3 as a test sample of the present invention and photoprin (photogem®) as a control group were used in test.
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° C. in 5% CO₂.
2) SNU-1 cell lines were inoculated into a 96-well, flat-bottomed microplate at a volume of 100 ul (1×10⁵cells/well), respectively. 24 hrs later, the medium was removed and the cultures were washed three times in PBS.
3) Various concentrations of DH-I-180-3 (final conc.: 0.1˜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/cm²using by halogen lamp), and then the cells were further incubated at 37° C. for 24 hrs in humidified incubator.
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.
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
1) DH-I-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
2) SNU-1 cell lines were (3×10⁵cells/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.
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/cm²using by halogen lamp).
Result
FIG. 7 represents the result of photosensitizing effect of DH-I-180-3 with light against SNU-1 cell lines.
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.
FIG. 8 depicts the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated SNU-1 cell lines.
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®) treated group.

Experimental Example 5

Anticancer Effect on Cystic Cancer

The anticancer activity of the target compounds on cystic cancer was determined by following experiments.
In Vitro Assay
DH-1-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
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° C. in 5% CO₂.
2) HT-1197 cell lines were inoculated into a 96-well, flat-bottomed microplate at a volume of 100 ul (1×10⁵cells/well), respectively. 24 hrs later, the medium was removed and the cultures were washed three times in PBS.
3) Various concentrations of DH-I-180-3 (final conc.: 0.1˜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/cm²using by halogen lamp), and then the cells were further incubated at 37° C. for 24 hrs in humidified incubator.
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.
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
1) DH-I-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
2) HT-1197 cell lines were (3×10 ⁵cells/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.
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/cm²using by halogen lamp).
Result
FIG. 9 represents the result of photosensitizing effect of DH-I-180-3 with light against HT-1197 cell lines.
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.
FIG. 10 depicts the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated HT-1197 cell lines.
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®) treated group.

Experimental Example 6

Anticancer Effect on Lung Cancer

The anticancer activity of the target compounds on lung cancer was determined by following experiments.
In Vitro Assay
DH-1-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
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° C. in 5% CO₂.
2) LLC1 cell lines were inoculated into a 96-well, flat-bottomed microplate at a volume of 100 ul (1×10⁵cells/well), respectively. 24 hrs later, the medium was removed and the cultures were washed three times in PBS.
3) Various concentrations of DH-I-180-3 (final conc.: 0.1˜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/cm²using by halogen lamp), and then the cells were further incubated at 37° C. for 24 hrs in humidified incubator.
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.
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
1) DH-I-180-3 as a test sample of the present invention and photofrin (photogem®) as a control group were used in test.
2) LLC1 cell lines were (2×10⁵cells/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.
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/cm²using by halogen lamp).
Conventional photosensitizer, i.e., photogem®, 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
FIG. 11 represents the result of photosensitizing effect of DH-I-180-3 with light against LLC1 cells.
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.
FIG. 12 depicts the inhibition of tumor of BALB/c mouse caused by DH-1-180-3 treated LLC 1.
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®) treated group.

Experimental Example 7

Toxicity Test

Methods
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
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.
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

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.

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:

wherein

R₁, R₂is independently a straight or branched lower alkyl or alkoxy group having 1 to 6 carbon atoms, a polyethyleneglycol group or a sulfonyl group;

R₃is a hydrogen atom, an alkoxy group having 1 to 6 carbon atoms or a polyethyleneglycol group;

R₄is 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 R₁, R₂is 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; R₃is 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; R₄is a hydrogen atom, a hydroxyl group or an methoxy group; and A is linked directly providing that R₁and R₂is the same group and R₂is different from R₁or R₃.

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:

wherein

R₁, R₂is 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 —CH₂—; R₁is hydrogen atom or aminoethyl group; R₂is 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:

wherein

R₁is a polyethyleneglycol group;

R₄is 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:

wherein

R₂is a bromopropyl group, or a polyethyleneglycol group;

R₄is 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:

wherein

R₁is 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:

wherein

R₁, R₂is 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:

wherein

R₁is 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 —NHCH₂—; R₁is hydrogen atom or aminoethyl group; R₂is 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-methoxybenzyl]thiourea,

N-[4-t-bytulphenyl-2-(pivaloyloxymethyl)butyl]-N-[4-hydroxy-3-methoxybenzyl]thiourea,

N-[4-(3,4-dimethylphenyl)-2-(pivaloyloxymethyl)butyl]-N-[4-hydroxy-3-methoxybenzyl]urea,

N--[4-t-dimethylphenyl-2-(pivaloyloxymethyl)butyl]-N-[4-hydroxy-3-methoxybenzyl]urea,

N-[4-(3,4-dimentylphenyl-2-(pivaloyloxymethyl)butyl)-2-[4-hydroxy-3-methoxyphenyl]acetamide,

N-[4-(4-t-butylphenyl)-2-(pivaloyloxymethyl)butyl]-2-[4-hydroxy-3-methoxyphenyl]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.