KR101405403B1 - Chlorin derivatives and unsaturated fatty acids conjugate, a photosensitizer comprising the same, and a composition for treatment of cancer for using in photodynamic therapy comprising the same - Google Patents

Abstract

The present invention relates to a conjugate of a chlorine derivative and an unsaturated fatty acid, a photosensitizer containing the same, and a composition for treating cancer, which is used for photodynamic therapy comprising the same, wherein the chlorin derivative is a docosahexaenoic acid by providing a conjugate of a choline derivative and an unsaturated fatty acid having improved selectivity to the tumor and reduced cytotoxicity by binding to an unsaturated fatty acid such as an acid, DHA or oleic acid (OA) There is an effect that can be usefully used.

Description

TECHNICAL FIELD [0001] The present invention relates to a conjugate of a choline derivative and an unsaturated fatty acid, a photosensitizer containing the same, and a composition for treatment of cancer for use in photodynamic therapy including the same, of cancer using for photodynamic therapy comprising the same}

The present invention relates to a conjugate of a chlorine derivative and an unsaturated fatty acid, a photosensitizer containing the same, and a composition for treating cancer for use in photodynamic therapy comprising the same.

Photodynamic therapy (PDT) is a medical treatment using a combination of light and photosensitizers (PS). Its mechanism of action is largely dependent on the molecular mechanism of tumor selective accumulation of photosensitizers, photosensitizers and light Of the tumors. Each factor is not harmful by itself, but when combined with oxygen, they can produce a lethal cytotoxic agonist that inactivates tumor cells (Sternberg ED et al., Tetrahedron, 1998, 54: 4151-4202; Kadish KM et al., The Porphyrin Handbook. 2000, Vol 6: 158-161).

Photodynamic therapy exhibits dual selectivity, where photosensitizers are preferentially absorbed by diseased tissues, and photosensitizers are activated by irradiating light in specific areas. Photodynamic therapy kills cells through the production of singlet oxygen and other reactive oxygen species (ROS) that overwhelm numerous intracellular antioxidant defense mechanisms and cause oxidative damage to cellular macromolecules [Weishaupt KR et al., Cancer Res., 1976, 36: 2326-2329].

Photochemical reactions that generate cytotoxic agents, singlet oxygen and reactive oxygen species, that are formed during photodynamic therapy are represented by the modified Yablonski diagram (Fig. 1). In short, after absorption of the light, the photosensitizer is in a triplet state [S ₁ , (~ 10 ^-6 s)] electrically excited from the bottom one state (S ₀ ) through an excited singlet state [ T ₁ , (~ 10 ^-2 s)]. Of particular importance in photodynamic therapy is the excited singlet state photosensitizer, which has a short half-life, can perform the non-radioactive process of intersystem crossing (ISC). This is a spin-forbidden process because it requires spin inversion and thereby converts the photosensitizer to a relatively long half-life excited triplet state (T ₁ ) with electron spin parallelism. Any 'prohibited' pathway is less likely than the 'permissive' pathway, but superior photosensitizers perform 'prohibited' inter-crossover paths with very high efficiency. The excited triplet state photosensitizer can perform two types of reactions [Macdonald JI et al., J. Porphyrins Phthalocyanines, 2001, 5: 105-129].

First, it can participate in electron-transfer processes with biological substrates to form radicals and radical ions that can produce peroxide products such as superoxide ions, O ₂ ^- after interaction with oxygen [Type I reactions]. Alternatively, it can perform a photochemical process known as a type II reaction in which stable triple oxygen ( ³ O ₂ ) is converted to singlet oxygen ( ¹ O ₂ ), which has a short half life but is highly reactive.

Furthermore, the tumor cell lethal effects of photodynamic therapy are related to the depth of light penetration within the cancer mass. The effect of light in tissue decreases exponentially with distance [Moser JG. In Photodynamic Tumor Therapy - 2nd & 3rd Generation Photosensitizers. Harwood Academic Publishers, London, 1997, 3-8]. Tissue weakening is affected by optimal absorption, endogenous molecules, and scattering by the drug chromophore itself. The maximum transmittance of the skin tissue is in the region of 700-800 nm, and development of a photosensitizer exhibiting maximum absorption in this region is required. Effective penetration at 630 nm was between 1 and 3 mm, whereas at 700-850 nm at least 6 mm light penetration was observed. Thus, an ideal photosensitizer should exhibit strong absorption in the near infrared region.

Photosensitizers are defined as chemical species that induce chemical or physical modifications of other species under the absorption of light. Clinicians and chemists have different views on ideal photosensitizers [Kirchner C et al., Nano Lett., 2005, 5: 331]. For example, chemists can place more emphasis on high extinction and high quantum yield of singlet oxygen, while clinicians can emphasize low toxicity and high selectivity. Nevertheless, both clinical photodynamic therapy and ideal photosensitizers are clinically relevant and have been described by Allison et al. [Zheng H. Technology in Cancer Research & Treatment, 2005, 4: 283-293] and Castano et al. [Anna C et al ., Photochem Photobiol, 2006, 82: 617-625].

1. Strong absorption (> 650 nm) in the red portion of the visible spectrum,

2. High quantum yields of triplet formation with triplet energies greater than 94 kJ / mol ^-1 ,

3. High quantum yield of singlet oxygen production (excited state with a long half-life),

4. Low dark toxicity,

5. Tumor tissues versus healthy tissues, especially those showing concentration selectivity in the skin; General skin sensitization should be avoided; Certain therapeutic modalities require skin sensitization, where rapid sensitization and desensitization of the skin is desirable,

6. Simple formulation of drugs; The formulated drug should have a long shelf life,

7. A pharmacokinetic profile that is rapidly removed from the body,

8. An easy derivatization (side chain) option that allows for improvement of the above properties,

9. Easy synthesis from readily available starting materials, easy modification to multi-kilogram scale,

10. No self-aggregation in the body due to reduced ¹ O ₂ quantum yield.

In the field of photodynamic therapy, tetrapyrrole macrocycles are often used as photosensitizers. Strong absorption in the red region of the visible light spectrum is a highly desirable feature for effective photosensitizers because this allows for the treatment of thicker tumors [Johnson CK et al., Tetrahedron Lett., 1998, 39: 4619-4622 ]. For this reason, tetrapyrroles such as porphyrin, chlorine, bacterioclopene, foresin, phthalocyanine, naphthalocyanine, and extended porphyrin have been synthesized and their photodynamic therapeutic efficacy has been evaluated. Photosensitizers can be classified by their chemical structure and their origins. In general, they can be divided into three broad classes: (i) porphyrin-based (eg, photoprin, ALA / PpIX and BPDMA), (ii) chlorin-based (eg, perforin and bacterioclopins) iii) dyes (e.g. phthalocyanine, naphthalocyanine).

Among these, choline- and bacteriocin-based photosensitizers are monomeric compounds that effectively produce singlet oxygen (φ = 45-60%) and treat tumors that are large (wide) or deeply localized due to their long absorption wavelength Effective [Zheng, X. et al., J. Med. Chem. 2009, 52: 4306-4318.

However, most first- and second-generation photosensitizers have been studied for photodynamic therapy displays that prefer only malignant cells, often with increased sensitivity to the skin (photoprin), absorption into healthy cells and tissues [Sharman, WM et al., Advanced Drug Delivery Reviews, 2004, 56: 53-76; Allen, C. M. et al., Tumor Targeting in Cancer Therapy, Humana Press, New Jersey, 2002, 329-361]. To overcome this problem, the third generation of photosensitizers focuses on more actively targeting diseased tissues. These third-generation photosensitizers can be designed and synthesized to modify the photothermographic species through their physicochemical properties, or can be conjugated to species such as antibodies, polymers, protein scaffolds and carbohydrates to form targets [Brown, SB et al., The Lancet Oncology, 2004, 5: 497-508; Savellano, M. D. et al., Clin. Cancer Res., 2005, 11: 1658-1668].

Recently, polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) have been reported to have potential therapeutic effects on cancer and neurodegenerative disorders. Jaracz, S. et al., Bioorg. Med. Chem., 2005,13: 5043-5054; Youdim, K. A. et al., J. Devl Neuroscience, 2000, 18: 383-399; Yu, J. H. et al., Ann. N. Y. Acad. Sc., 2009, 1171: 359-364]. Some polyunsaturated fatty acids that are rapidly absorbed by tumor cells from arterial blood can be used as biochemical precursors and energy sources and can also be readily attached to the lipid bilayers of cells, (Sauer, LA et al., Cancer Res., 1986, 46: 3469-3475; Sauer, L. A. et al., Brit. J. Cancer, 1992, 66: 297-303; Takahashi, M. et al., J. M. Cancer Res., 1992, 52: 154; Grammatikos, S. I. et al., J. Cancer, 1994, 70: 219].

Bradley et al. First discovered a paclitaxel-DHA conjugate, and the paclitaxel-DH conjugate is more effective than paclitaxel in cancer treatment by enhancing its anticancer activity (Bradley, M.O. et al., Clinical Cancer Research, 2001, 7: 3229-3238; Bradley, M.O. et al., Journal of Controlled Release, 2001, 74: 233-236). Siddiqui et al. Found that docosahexaenoic acid (DHA), a type of unsaturated fatty acid in long chains, is very easy to oxidize, and this oxidation can occur in both enzymatic and non-enzymatic environments. Through a variety of non-enzymatic mechanisms, docosahexaenoic acid (DHA) can be transformed into a number of oxidized materials that appear to play a major role in mediating DHA-induced apoptosis, which accounts for its anti-cancer properties [Siddiqui, RA et al., Chemistry and Physics of Lipids, 2008, 153: 47-56].

Under these circumstances, the present inventors conjugated chlorophyll derivatives with unsaturated fatty acids (UFAs) such as docosahexaenoic acid or oleic acid in order to obtain a more selective and affinity strong photosensitizer for cancer cells. As a result, the present inventors completed the present invention by demonstrating the fact that the conjugate has an excellent effect of targeting cancer cells and exhibiting a synergistic effect.

It is an object of the present invention to provide a method for producing a cholinergic derivative which is more selective for tumor cells and exhibits excellent photosensitizing effect by conjugating a chlorine derivative with an unsaturated fatty acid such as docosahexaenoic acid (DHA) or oleic acid (OA) Fatty acid conjugate.

Another object of the present invention is to provide a method for producing a conjugate of a chlorin derivative and an unsaturated fatty acid which is more selective for tumor cells and exhibits a photosensitizing effect by binding a chlorin derivative to an unsaturated fatty acid such as docosahexaenoic acid or oleic acid .

In one embodiment, the present invention provides a conjugate of a choline derivative and an unsaturated fatty acid represented by the following general formula (I).

[Formula I]

또는Wherein R is

or

이다.

to be.

In another aspect, the present invention provides a conjugate of a chlorine derivative and an unsaturated fatty acid represented by the following general formula (II).

[Formula II]

또는Wherein R is

or

이다.

to be.

In another embodiment, the present invention provides a process for producing a choline derivative represented by the following formula 4 and the following compound 8; And a step of binding the chlorine derivative with an unsaturated fatty acid. The present invention also provides a method for producing a chlorinated derivative and an unsaturated fatty acid conjugate.

[Compound 4]

[Compound 8]

In the present invention, the unsaturated fatty acid is preferably docosahexaenoic acid (DHA) or oleic acid (OA), but is not limited thereto.

In the present invention, the chlorine derivative is preferably methyl pheopoboid-a, pyropheopoboid-a, or methyl pyropheopoide-a, but is not limited thereto. Spirulina maxima ) algae.

In another aspect, the present invention provides a photosensitizer containing a conjugate of a chlorine derivative and an unsaturated fatty acid.

In the present invention, the photosensitizer is characterized in that it exhibits photosensitizing activity for light rays in the range of 650 nm to 800 nm.

In another aspect, the present invention provides a composition for treating cancer, which comprises a photosensitizer containing a conjugate of a chlorine derivative and an unsaturated fatty acid as an active ingredient for photodynamic therapy.

In the composition for treating cancer, a conjugate of a chlorine derivative and an unsaturated fatty acid is characterized in that it is optically activated in vitro or in vivo for light rays in the range of 650 nm to 800 nm.

In the present invention, the cancer may be selected from the group consisting of skin, digestive, urinary, reproductive, respiratory, circulatory, brain and nervous system cancers.

More particularly, the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma , Cancer of the uterine cervix, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute (CNS) tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, and pituitary adenomas. The present invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis of leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, But is not limited thereto.

The compositions for treating cancer of the present invention can be administered orally or parenterally by intravenous injection, intraperitoneal injection, intramuscular injection, intracranial injection, intratumoral injection, intraepithelial injection, skin penetration delivery, esophageal administration, abdominal administration, intraarterial injection, ≪ / RTI > and the like.

The composition according to the present invention may be formulated into a formulation for parenteral administration in the form of a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion or an emulsion according to a conventional method. When formulating, it may be prepared using a diluent or an excipient such as a surfactant usually used. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.

The preferred dosage of the conjugate according to the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the conjugate of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, once or several times a day.

The conjugate of the choline derivative and the unsaturated fatty acid of the present invention not only reduces toxicity as a photosensitizer but also promotes selectivity, thereby effectively suppressing the proliferation of cancer cells while minimizing adverse effects on normal cells.

In another aspect, the present invention relates to a composition comprising as an active ingredient a photosensitizing agent containing a conjugate of a chlorine derivative and an unsaturated fatty acid; And

There is provided a kit for cancer treatment for use in photodynamic therapy comprising a light source for irradiating a light beam having a wavelength in the range of 650 nm to 800 nm.

Hereinafter, the configuration of the present invention will be described in detail.

Photodynamic therapy (PDT) is a combination of photosensitizers (PS), light and oxygen. When the photosensitizer is administered to the human body, the photosensitizer accumulates in the tumor, ie, the tumor tissue. When the light is irradiated thereafter, the tumor is selectively destroyed by maximizing the production of singlet oxygen.

Chlorine is a compound having a structure represented by the following formula (1), and is a large heterocyclic aromatic ring composed of pyrrole and pyrroline connected by four methine linkages at the center. Magnesium-containing chlorine is called chlorophyll and is a central light sensitizing dye in the chloroplast.

[Chemical Formula 1]

In the present invention, a chlorine derivative means a compound having the above-mentioned chlorine as a basic skeleton structure. Chlorine and choline derivatives are effectively used as photosensitizers in photodynamic therapy because of their photosensitizing properties. In particular, chlorine and chlorine derivatives are more useful in synthesizing photosensitizers, on the one hand having good spectral characteristics and low toxicity, and on the other hand having many reaction center points which enable various chemical conversions to be performed Can be used.

Among the chlorin derivatives that can be used in the present invention, there are methyl pheopoboid-a, pyropheopoboid-a, and methyl pyropheopoide-a which can be obtained from chlorophyll, but the present invention is not limited thereto. The methylpeoplebide-a, pyropheopoboid-a, and methylpyropopobide-a can be activated by red light rays that are much longer at ~ 670 nm than photoprenrine II and can produce less long-term normal tissue phototoxicity It is therefore used as a kind of potential new compound. The chlorine derivative used in the present invention has an advantage of high commercial yield due to its high yield in the extraction process. However, the higher the concentration, the darker the toxicity.

In the present invention, the Spirulina maxima as chlorin derivative a type of bird to produce a mixture of natural chlorine (Spirulina The peoh Poby de -a (pheophorbide-a, MPa) , fatigue peoh Poby de -a and methyl fatigue peoh Poby de -a (methyl pyropheophorbide-a, MPPa ) obtained from the Maxima) may be used.

(Methyl pyropheophorbide-d-OH) to confirm the tumor-selectivity and photosensitizing effect on a conjugate of a chlorinated derivative and polyunsaturated fatty acids (PUFAs) or fatigue peoh Poby provides de -a-17 ⁵ -N- hexanol (pyropheophorbide-a-175-N -hexanol) conjugate connecting the acid in Toko hexahydro chlorin derivatives such as.

In order to compare the effect of polyunsaturated fatty acids and monounsaturated acids (MUFAs) on tumor cells, the present invention relates to a method for producing polyunsaturated fatty acids by reacting oleic acid, which is a monounsaturated fatty acid, with methyl pyrophosphobde- RTI ID = 0.0 > ^5- N-hexanol. ≪ / RTI >

The conjugate thus formed has an effect of improving the selectivity to the tumor and decreasing the cytotoxicity, so that it can be usefully used for photodynamic cancer treatment.

In particular, FIG. 31 shows that when a 10 uM choline derivative alone (Free-Ps) was used, the cell showed a side effect of dark toxicity because it exhibited cell growth inhibition effect of 90% or more even in the state without laser . In the case of 1 uM, which is a cytotoxic concentration, there is no cytotoxicity but only about 50% inhibition of cell growth in the irradiation with laser. Therefore, when the chlorine derivative alone is used, the concentration must be increased to obtain a cell growth inhibiting effect of 90% or more, but the concentration can not be used due to toxicity. However, in the case of the choline derivative and the unsaturated fatty acid (DHA) conjugate (Ps-DHA), the cell growth inhibitory effect (photodynamic treatment effect) at a low concentration in the presence of the laser is lower than that of the chlorin derivative alone, Is not observed even at an excellent 10 uM, there is an advantage that a cell growth inhibitory effect of 90% or more can be obtained. 32, when the choline derivative alone was used, there was almost no cell due to toxicity, and the shape of the cell was changed. However, the shape of the cells treated with the choline derivative and the unsaturated fatty acid (DHA) conjugate is healthy and the absorption rate of fluorescence is not different from that of the choline derivative alone.

The process for preparing the methyl pyrophosphobde-d-OH and docosahexaenoic acid conjugate represented by compound 5 of the present invention comprises the following steps (Fig. 2):

Synthesizing methyl pheophorbide-a (MPa) from chlorophyll a;

Synthesizing methyl pyrophosphobde-a (MPPa) from the methyl pheopoboid-a (MPa);

Synthesizing methyl pyropheophorbide-d (MPPd) from the methyl pyropheopoebide-a (MPPa);

Synthesizing methyl pyrophosphobde-d-OH from the methyl pyrophosphobde-d; And

And coupling docohexanoic acid to the methyl pyrophosphobde-d-OH.

The process for preparing the methyl pyrophosphobde-d-OH and oleic acid conjugate represented by compound 6 of the present invention comprises the following steps (Fig. 3):

Synthesizing methyl pheopoboid-a (MPa) from chlorophyll-a;

Synthesizing methyl pyrophosphobde-a (MPPa) from the methyl pheopoboid-a (MPa);

Synthesizing methyl pyropheophorbide-d (MPPd) from the methyl pyropheopoebide-a (MPPa);

Synthesizing methyl pyrophosphobde-d-OH from the methyl pyrophosphobde-d; And

Linking oleic acid to the methyl pyrophosphobde-d-OH.

The process for preparing the pyropheopoboid-a-17 ⁵ -N-hexanol and docosahexaenoic acid conjugate represented by the present invention compound 9 comprises the following steps (Fig. 4):

Synthesizing methyl pheopoboid-a (MPa) from chlorophyll-a;

Synthesizing methyl pyrophosphobde-a (MPPa) from the methyl pheopoboid-a (MPa);

Synthesizing pyrophosphopoda-a (PPa) from the methyl pyrophosphopeide-a (MPPa);

Synthesizing pyropheopoboid-a-17 ⁵ -N -hexanol from the pyropeoplebide-a (PPa); And

Binding the docohexanoic acid to the pyrophosphopoda-a-17 ⁵ -N-hexanol.

The process for preparing the pyropheopoboid-a-17 ^5- N-hexanol and oleic acid conjugate of compound 10 of the present invention comprises the following steps (Figure 5):

Synthesizing methyl pheopoboid-a (MPa) from chlorophyll-a;

Synthesizing methyl pyrophosphobde-a (MPPa) from the methyl pheopoboid-a (MPa);

Synthesizing pyrophosphopoda-a (PPa) from the methyl pyrophosphopeide-a (MPPa);

Synthesizing pyropheopoboid-a-17 ⁵ -N -hexanol from the pyropeoplebide-a (PPa); And

Bonding oleic acid to the pyrophosphopoda-a-17 ⁵ -N -hexanol.

Hereinafter, a method for producing a conjugate of a chlorin derivative and an unsaturated fatty acid of the present invention will be described in detail.

A method for producing a conjugate of a chlorine derivative and an unsaturated fatty acid represented by the general formula I will be described in detail.

First, a method for producing methyl pyrophosphobde-d-OH and docosahexaenoic acid conjugate represented by compound 5 will be described in detail.

Spirulina maxima is treated with acetone to extract chlorophyll-a, which is treated with methanol under acidic conditions to obtain methyl perphosphite-a (MPa). This process is schematically shown in the following reaction formula 1.

[Reaction Scheme 1]

In the present invention, chlorophyll-a may be obtained separately from Spirulina maxima or commercially available one.

Then, methylpheopoboid-a (MPa) is refluxed in the collidine to obtain methyl pyrophosphobde-a (MPPa). This process is schematically shown in the following Reaction Scheme 2.

[Reaction Scheme 2]

The methyl pyrophosphobde-a (MPPa) obtained according to the above method was dissolved in acetic acid and oxidized with osmium tetroxide (OsO ₄ ) - and sodium periodate (NaIO ₄ ) to obtain methyl pyrophosphate To obtain povidone-d (MPPd). This process is schematically shown in the following Reaction Scheme 3.

[Reaction Scheme 3]

Then, methyl pyrophosphobde-d (MPPd) is added with tBuNH ₂ BH ₃ and reduced to obtain methyl pyrophosphobde-d-OH. This process is schematically shown in the following Reaction Scheme 4.

[Reaction Scheme 4]

(DHA), which is one type of unsaturated fatty acid, is bound to methyl pyrophosphobde-d-OH, and then carboimide-mediated reaction is carried out under 4-dimethylaminopyridine (DMPA) The esterification reaction is carried out using a coupling reagent (DCC or EDC-HCl).

More specifically, methyl pyrophosphobde-d-OH, dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMPA) were dissolved in dichloromethane, and docosahexaenoic acid (DHA) Is added and reacted to obtain Compound (5). This process is briefly shown in the following reaction formula (5).

[Reaction Scheme 5]

Secondly, a method for producing methyl pyrophosphobde-d-OH and an oleic acid conjugate represented by compound 6 will be described in detail.

(MPa) was obtained by extracting chlorophyll-a from the spirulina maxima algae and treating it with methanol under acidic conditions to obtain methylpeopoboid-a (MPa), refluxing in methylene pyrophosphate-a (MPPa) was obtained by oxidizing methyl pyrophosphobde-a (MPPa) with sodium osmium tetraoxide and sodium iodate to obtain methyl pyropheopbide-d (MPPd) -d-OH and docosahexaenoic acid conjugate represented by the above-mentioned compound 5 were prepared until the step of reducing methylpyrrolidone-d (MPPd) to tBuNH ₂ BH ₃ to obtain methyl pyrophosphobde-d-OH .

Thereafter, oleic acid (OA), which is one type of unsaturated fatty acid, is bound to methyl pyrophosphobde-d-OH. (OA) was added to the reaction mixture to dissolve methyl pyrophosphobde-d-OH, dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMPA) in dichloromethane, . This process is schematically shown in the following reaction formula (6).

[Reaction Scheme 6]

A method for producing a conjugate of a chlorine derivative and an unsaturated fatty acid represented by the general formula II will be described in detail.

First, a method for producing pyropheopoboid-a-17 ⁵ -N -hexanol and docosahexaenoic acid conjugate represented by Compound 9 will be described in detail.

(MPa) was obtained by extracting chlorophyll-a from the spirulina maxima algae and treating it with methanol under acidic conditions to obtain methylpeopoboid-a (MPa), refluxing in methylene pyrophosphate-a Up to the step of obtaining povidone-a (MPPa) is the same as the method for producing a conjugate of a chlorine derivative and an unsaturated fatty acid represented by the above general formula I.

Then, methyl pyrophosphobde-a (MPa) is treated with hydrochloric acid (HCl) in tetrahydrofuran (THF) to obtain pyropefovoid-a (PPa). This process is schematically shown in the following Reaction Scheme 7.

[Reaction Scheme 7]

Subsequently, pyrropefovoid-a (PPa), N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were dissolved in dichloromethane and triethanolamine , TEA) is added to react. Then, hexanolamine is added thereto and stirred to obtain pyropheopoboid-a-17 ⁵ -N-hexanol. This process is schematically shown in the following reaction formula 8.

[Reaction Scheme 8]

Then, it attaches docohexanoic acid (DHA), which is one kind of unsaturated fatty acid, to pyropheopoboid-a-17 ⁵ -N -hexanol. Fatigue peoh Poby de -a-17 ⁵ -N- hexanol, dicyclohexyl carbodiimide an imide (DCC) and 4-dimethylaminopyridine (DMPA) was dissolved in dichloromethane, the docosahexaenoic acid herein ( DHA) is added and reacted to obtain Compound (9). This process is shown briefly in the following reaction equation (9).

[Reaction Scheme 9]

Secondly, a method for preparing the pyropheopoboid-a-17 ⁵ -N -hexanol and oleic acid conjugate represented by Compound 10 is described in detail.

(MPa) was obtained by extracting chlorophyll-a from the spirulina maxima algae and treating it with methanol under acidic conditions to obtain methylpeopoboid-a (MPa), refluxing in methylene pyrophosphate-a (PPA) was obtained by treatment with hydrochloric acid (HCl) in terahydrofuran (THF) to give pivalo-a-pivalo-a (MPPa), and then dicyclohexylcarbodiimide (DCC ), N-hydroxysuccinimide (NHS), triethylamine (TEA) and hexanolamine to obtain pyropheopoboid-a-17 ⁵ -N- The procedure is the same as the method for producing pyropefovoid-a-17 ^5- N-hexanol and docosahexaenoic acid conjugate.

Thereafter, oleic acid (OA), which is one kind of unsaturated fatty acid, is bound to pyrophosphopoda-a-17 ⁵ -N -hexanol. Fatigue peoh Poby de -a-17 ⁵ -N- hexanol, a dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMPA) was dissolved in dichloromethane, was added oleic acid (OA) here To obtain Compound (10). This process can be briefly described as the following reaction formula (10).

[Reaction Scheme 10]

The present invention relates to a method for preparing a choline derivative by conjugating a chlorine derivative with an unsaturated fatty acid such as docosahexaenoic acid (DHA) or oleic acid (OA) to improve the selectivity to the tumor and reduce the cytotoxicity of the choline derivative and the unsaturated fatty acid By providing a conjugate, the conjugate can be advantageously used for photodynamic cancer treatment.

Figure 1 shows a modified Yablonski diagram. Here, 1 means absorption, 2 means non-radioactive decay, 3 means fluorescence, 4 means inter-crossover, 5 means phosphorescence, 6 means energy transfer.
Figure 2 is a simplified representation of the process for preparing methyl pyrophosphobde-d-OH and docosahexaenoic acid conjugate, represented by compound 5 of the present invention.
Fig. 3 is a schematic view showing a process for preparing methyl pyrophosphobde-d-OH and oleic acid conjugate represented by the compound 6 of the present invention.
4 is a simplified representation of the process for preparing pyropheopoboid-a-17 ⁵ -N-hexanol and docosahexaenoic acid conjugate, represented by compound 9 of the present invention.
Figure 5 is a simplified representation of the process for preparing pyropheopoboid-a-17 ⁵ -N -hexanol and oleic acid conjugate, represented by compound 10 of the present invention.
6 is the UV spectrum of methyl perphosphide-a (MPa).
7 is a ¹ H-NMR spectrum of methyl perphosphide-a (MPa).
Figure 8 is the UV spectrum of methyl pyrophosphopaide-a (MPPa).
9 is a ¹ H-NMR spectrum of methyl pyrophosphobde-a (MPPa).
10 is a mass spectrometry spectrum of methyl pyrophosphobde-a (MPPa).
11 is the UV spectrum of methyl pyrophosphobde-d-OH.
12 is a ¹ H-NMR spectrum of methyl pyrophosphobde-d-OH.
13 is the UV spectrum of methyl pyrophosphobde-d-OH and docosahexaenoic acid conjugate.
14 is a ¹ H-NMR spectrum of methyl pyrophosphobde-d-OH and docosahexaenoic acid conjugate.
15 is a mass spectrometry spectrum of methyl pyrophosphobde-d-OH and docosahexaenoic acid conjugate.
16 is a UV spectrum of methyl pyrophosphobde-d-OH and oleic acid conjugate.
17 is a ¹ H-NMR spectrum of methyl pyrophosphobde-d-OH and an oleic acid conjugate.
18 is a mass spectrometry spectrum of methyl pyrophosphobde-d-OH and an oleic acid conjugate.
19 is a UV spectrum of pyropefovoid-a (PPa).
20 is a ¹ H-NMR spectrum of pyrophosphopoda-a (PPa).
21 is a mass spectrometry spectrum of pyrophosphopoda-a (PPa).
22 is a UV spectrum of pyropefovoid-a-17 ⁵ -N-hexanol.
23 is a ¹ H-NMR spectrum of pyropheopoboid-a-17 ⁵ -N-hexanol.
24 is a mass spectrometry spectrum of pyropheopoboid-a-17 ⁵ -N -hexanol.
25 is a UV spectrum of pyropheopoboid-a-17 ⁵ -N-hexanol and docosahexaenoic acid conjugate.
26 is a ¹ H-NMR spectrum of pyropheopoboid-a-17 ⁵ -N -hexanol and docosahexaenoic acid conjugate.
27 is a mass spectrometry spectrum of pyropheopoboid-a-17 ^5- N-hexanol and docosahexaenoic acid conjugate.
Figure 28 is the UV spectrum of pyropefovoid-a-17 ^5- N-hexanol and oleic acid conjugate.
29 is a ¹ H-NMR spectrum of pyropheopoboid-a-17 ⁵ -N -hexanol and oleic acid conjugate.
30 is a mass spectrometry spectrum of pyropheopoboid-a-17 ^5- N-hexanol and oleic acid conjugate.
Fig. 31 shows the results of comparing the cell growth inhibitory effect of the chlorin derivative alone, the chlorin derivative of the present invention and the unsaturated fatty acid conjugate at 10 uM, 1 uM, and 0.1 uM when and without photodynamic therapy, respectively.
Fig. 32 shows the results of showing the shape of the cells and the absorption rate of fluorescence when the chlorine derivative alone, the chlorine derivative of the present invention and the unsaturated fatty acid conjugate were used.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

All reagents were purchased from Sigma, Aldrich, Fluka, Alfa Aesar and Daihan, and if necessary, purified by distillation according to standard procedures. Other commercially available reagents were used without purification.

Column chromatography used silica gel 60 (63-200 and 200-400 mesh, Merck).

The progress of the reaction was monitored by TLC (silica gel F ₂₅₄ , 0.2 mm thick, Merck) and TLC was monitored at wavelengths in the range of UV lamp 265 nm or 365 nm during monitoring.

¹ H-NMR spectra were measured with a 500 MHz Unity Inova 500 (UI 500, Varian Inc, USA) spectrometer in NICRF at Seoul National University and chemical shifts were calculated using TMS (0.00) Relative to ppm.

MALDI mass spectra were measured by a yager-DETM STR Biospectrometry Workstation spectrometer in Seoul National University's Basic Science Institute (NICRF).

[Example 1]

methyl Peofovid -a ( methyl pheophorbide -a, MPa , Compound 1)

The dried Spirulina maxima (Spirulina maxima ) algae were refluxed with 2 l of acetone under a nitrogen atmosphere for 2 hours. The supernatant was then filtered on Wattman filter paper on a Buchner funnel when hot and excess acetone was added to the remaining solids above. The extraction and the filtration were repeated three times according to the same procedure. The green filtrate was evaporated and the residue redissolved in 300 mL of acetone, cooled in a refrigerator and filtered to remove red solid impurities. The filtrate containing pheophytin-a was evaporated and treated with a methanol solution of 5% sulfuric acid (500 ml) in a dark place at room temperature for 12 hours and 30 minutes. The solution was diluted with dichloromethane (~ 500 mL), washed with water (~ 500 mL), washed with 10% aqueous sodium bicarbonate solution (~ 500 mL) and then washed three times with water. The organic layer was separated, dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by column chromatography on silica gel 60 (230-400 mesh) eluting with 2% acetone in dichloromethane. The product was recrystallized from dichloromethane / methanol. The result of measuring the UV spectrum of the product was as shown in Fig. 6, two base peaks of the chlorine ring were observed at? 412.5 (1.52) nm and? 667.6 (0.655) nm, and other small peaks at? 507.9 nm,? 537.7 nm? Respectively. On the other hand, the 500 MHz ¹ H-NMR spectrum of the product (MPa) was measured in chloroform, and the result is shown in FIG. The signals determined using δ and J _{H -H} values were marked in the 1D proton spectrum. Signals in the delta-2 to 10 domains corresponding to a total of 39 protons were observed as 11 single, 4 multiple, 3 double and 1 quadruple signals from the NMR spectrum results, from which methylpeofovide-a ( MPa) was formed. The two NH 3 protons appeared as a wide signal at approximately -4 ppm due to rapid exchange.

Yield: 0.4%.

_{R f: 0.4 (CHCl 2:} CH 3 COCH 3 = 98: 2).

UV-vis (CH ₂ Cl ₂ ):? _Max , nm (log?) 667.6 (0.655), 610.4 (0.124), 537.7 (0.145), 507.9 (0.158), 412.5 (1.52).

^{1 H-NMR (300 MHz,} CDCl 3, TMS int): δ H, ppm 9.50 (1H, s, 5-meso-H), 9.35 (1H, s, 10-meso-H), 8.55 (1H, s , 20-meso-H), 7.97 (1H, m, 3 1 -CH), 6.30 and ^{6.19 (2H, dd, 3 2} -CH 2), 6.25 (1H, s, 13 2 -CH), 4.46 (1H 3H, s, 13 ⁴ -OCH ₃ ), 3.65 (2H, q, 8 ¹ -CH ₂ ), 3.68 (3H, s, _{^{, 17 4 -OCH 3), 3.57}} (3H, s, 12 1 -CH 3), 3.39 (3H, s, 2 1 -CH 3), 3.21 (3H, s, 7 1 -CH 3), 2.63-2.17 (4H, m, 17 ¹ and _{^{17 2 - 2 × CH 2)}} , 1.81 (3H, d, 18 1 -CH 3), 1.68 (3H, t, 8 2 -CH 3), 0.53 and -1.63 (2H, Respectively s, br, 2 x NH).

[Example 2]

methyl Pyropeofovid -a ( methyl pyropheophorbide -a, MPPa , Compound 2)

Methylperaphobde-a (1 g, 1.65 mmol) was dissolved in cholidine (100 mL, re-distilled, stored on KOH) and refluxed for 2 h 30 min. After cooling, the solution was diluted with dichloromethane, washed with 2N HCl (5 x 200 mL) and then twice with water. The combined organic phases were dried over anhydrous sodium sulfate and the solvent was removed with a vacuum rotator. The residue was purified over silica gel 60 (230-400 mesh) eluting with 2% acetone in dichloromethane and recrystallized from dichloromethane / hexanes. The result of measurement of the UV spectrum of the product was as shown in Fig. 8, two base peaks of the chlorine ring were observed at? 414.1 nm and? 667.7 nm, and other small peaks were observed at? 508.5 nm,? 539.2 nm and? 610.5 nm. On the other hand, the 500 MHz ¹ H-NMR spectrum of the product (MPPa) was measured in chloroform, and the result is shown in FIG. The signals determined using δ and J _{H -H} values were marked in the 1D proton spectrum. Signals in the range of δ -2 to 10 corresponding to a total of 37 protons were observed as ten single, four multiple, three double and two quadruple signals from the NMR spectrum results, from which the previous compound and two other protons It was confirmed that methyl pyrophosphobde-a was formed. The two NH 3 protons appeared as a wide signal at approximately -4 ppm due to rapid exchange. In addition, the mass spectrum of the product (MPPa) is shown in Fig. The molecular weight of the product was calculated as 548.7 g / mol and 549 (M ⁺ , 100%) was detected through EI-mass spectrometry analysis.

Yield: 85.15%.

_{R f: 0.28 (CHCl 2:} Ace = 98: 2).

UV-vis (in CH ₂ Cl ₂ ):? _Max , nm (log?) 667.7 (0.24), 610.5 (0.036), 539.2 (0.043), 508.5 (0.049), 414.1 (0.558).

^{1 H-NMR (300 MHz,} CDCl 3, TMS int): δ H, ppm 9.46 (1H, s, 5-meso-H), 9.35 (1H, s, 10-meso-H), 8.56 (1H, s , 20-meso-H), 8.04 (1H, m, 3 1 -CH), 6.31 and 6.19 (each _{^{1H, dd, 3 2 -CH 2}} ), 5.31 and 5.15 (2H, q, J = 20.1 Hz, 13 _{^{2 -CH 2), 4.51 (1H}} , m, 18-CH), 4.29 (1H, m, 17-CH), 3.66 (5H, s, 17 4 -OCH 3 , and 8 ¹ -CH ₂ different as overlapping), 3.63 (3H, s, 12 1 -CH 3), 3.41 (3H, s, 2 1 -CH 3), 3.22 (3H, s, 7 1 -CH 3), ^{2.70-2.58 (2H, m, 17 1} -CH 2), 2.33-2.31 (2H, m, 17 2 -CH 2), 1.82 (3H, d, 18 1 -CH 3), 1.69 (3H, t, 8 ² -CH ₃ ), 0.44 and -1.70 (1H, br, s, 2 x NH, respectively).

EI-MS: m / z; 549 (M ^< ^{+ &} gt ^; ).

[Example 3]

methyl Pyropeofovid -d ( methyl pyropheophorbide -d, MPPd , Compound 3)

Methyl pyrophosphoride-a (1 mmol) and osmium tetraoxide (OsO ₄ , 10 mg) were dissolved in tetrahydrofuran (100 ml, re-distilled), and this was dissolved in a peristaltic pump Was added to an aqueous solution (7.5 ml) in which sodium periodate (NaIO ₄ , 1.19 g) and acetic acid (0.5 ml) were dissolved and allowed to react overnight in a nitrogen atmosphere. The reaction mixture was added to distilled water and dichloromethane, stirred for 30 minutes, and then the aqueous layer was extracted with dichloromethane several times until the aqueous layer became discolored. The organic layer was washed with a saturated aqueous solution of sodium carbonate (NaHCO ₃ ) and water, dried over anhydrous sodium sulfate, and then the organic layer was concentrated. The concentrated organic material was purified by column chromatography on silica gel using 2% acetone in dichloromethane as an eluent to obtain the compound (MPPd). The obtained compound was confirmed by TLC and the following reaction proceeded. The obtained compound was confirmed by UV-vis, ¹ H-NMR and MS.

Yield: 60%.

_{UV-vis (CH 2 Cl 2} ): λ max, ㎚ (log ε) 695 (0.78), 634 (0.09), 555 (0.17), 522 (0.15), 427 (1.00). 388 (0.86).

_{^{1 H-NMR (CDCl 3)}} : δ H, ppm 11.55 (s, CHO), 10.30, 9.61 and 8.64 (3s, CH-5, CH-10 and CH-20, respectively), 5.35, 5.19 (2d, J = 20 Hz, CH ₂ -13 ² ), 4.58 (dq, J = 2 + 7 Hz, CH-18), 4.39 Hz, CH ₂ -8 ¹ ), 3.78, 3.72, 3.32, 3.62 (4s, CH ₃ -2 ¹ , CH ₃ -12 ¹ , CH ₃ -7 ¹ , CO ₂ CH ₃ ), 2.54-2.80, 2.25-2.38 _{(2m, 17-CH 2 CH} 2), 1.85 (d, J = 7 Hz, CH 3 -18 1), 1.72 (t, J = 8 Hz, CH 3 -8 2), -0.13, -2.06 (2 S, NH).

MS (Cl): m / z; 551 (MH ^< ⁺ & gt ^; ).

[Example 4]

methyl Pyropeofovid -d- OH ( methyl pyropheophorbide -d- OH , Compound 4)

Methyl pyrophosphobde-d (0.2 mmol) was dissolved in dichloromethane (100 ml), tBuNH ₂ BH ₃ (20 mg) was added thereto, and the reaction was allowed to proceed overnight under an argon atmosphere. The reaction mixture was added to a 2% aqueous HCl solution (100 ml) and stirred for 20 minutes in an ice bath. The aqueous layer was extracted with dichloromethane several times until the aqueous layer became discolored. The organic layer was washed with a 2% aqueous solution of HCl (500 ml), distilled water (500 ml), saturated sodium bicarbonate (NaHCO ₃ ) aqueous solution (100 ml) and an aqueous sodium chloride solution (500 ml), and the organic layer was washed with anhydrous sodium sulfate After drying, the organic layer was concentrated. The concentrated organic material was purified by column chromatography on silica gel using 10% acetone in dichloromethane as an eluent to obtain the compound (methyl pyrophosphobde-d-OH). The obtained compound was confirmed by UV-vis, ¹ H-NMR and MS, and UV-vis and ¹ H-NMR were shown in Fig. 11 and Fig. 12, respectively.

Yield: 90%

UV-vis (in CH ₂ Cl ₂ ): λ _max , nm (log ε) 664.8 (1.13), 606.8 (0.31), 537.3 (0.35), 504.0 (0.31), 472.5 (0.20), 411.3 (0.64).

_{^{1 H-NMR (CDCl 3)}} : δ H, ppm 9.46, 9.43, 8.55 (3H, 3s, CH-10, CH-5, CH-20), 5.89 (1H, s, CH 2 -3 1), 5.21 , 5.07 (2H, 2d, J = 20 Hz, CH 2 -13 2), 4.45-4.47 (1H, m, CH-18), 4.24-4.26 (1H, m, CH-17), 3.68 (2H, q , J = 7.5 Hz, CH ₂ -8 ¹ ), 3.63, 3.61, 3.41, 3.25 (4H, 4s, CH ₃ -12 ¹ , CH ₃ -2 ¹ , CH ₃ -7 ¹ , CO ₂ CH ₃ ) -2.67, 2.53-2.58, 2.24-2.30 (4H, 4m, 17-CH 2 CH 2), 2.15 (1H, s, br, OH), 1.79 (3H, d, J = 7.3 Hz, CH 3 -18 1 ), 1.69 (3H, t, J = 7.5 Hz, CH 3 -8 2), 0.24, -1.77 (2H, 2s, NH).

MS (Cl): m / z; 553 (MH ⁺ ), 537 ([(M-OH + H) + H] < ⁺ >).

[Example 5]

methyl Pyropeofovid -d- OH  And Docosahexaenoic acid  Junction methyl  pyropheophorbide-d-OH and docosahexaenoic acid conjugate , Compound 5)

(30 mg, 0.054 mmol), dicyclohexylcarbodiimide (22.4 mg, 0.108 mmol) and 4-dimethylaminopyridine (9.9 mg, 0.081 mmol) were dissolved in dichloromethane And the mixture was added with docosahexaenoic acid (17.8 mg, 0.054 mmol). The reaction was carried out at room temperature under a nitrogen atmosphere for 2 hours. To the reaction mixture was added 10 ml of water and stirred, and it was extracted with 20 ml of dichloromethane. The organic layer obtained as 100 ml of water was washed twice, and the organic layer was dried over anhydrous sodium sulfate, and then the organic layer was concentrated. The concentrated organics were purified by column chromatography on silica gel using 5% methanol in dichloromethane as eluent to give the compound (Compound 5), which was dried at 50 < 0 > C in a thermostat. The obtained compound was identified by UV-vis, ¹ H-NMR and MS, and these are shown in Fig. 13, Fig. 14 and Fig.

Yield: 39.78 mg (85%).

R _f : 0.55 (CH ₂ Cl ₂ : MeOH = 98: 2).

_{UV-vis (in CHCl 3)} : λ max, ㎚ (Abs) 666.4 (0.79), 608.2 (0.25), 537.9 (0.28), 503.7 (0.24), 412 (1.35), 320 (0.46).

^{1 H-NMR (500 MHz,} CDCl 3, TMS int): δ H, ppm 9.53 (1H, s, 5-meso-H), 9.42 (1H, s, 10-meso-H), 8.6 (1H, s , 20-meso-H), 6,38 (2H, s, 3 1 -CH), 5.30 (13H, m, 3 5,6,8,9,11,12,14,15,17,18,20 ^{, 21,} 13 ² -CH ^a, fold deflection) and 5.11 (1H, d, J = 19.5 Hz, 13 2 -CH b), 4.5 (1H, q, J = 0.045 Hz, 18-CH), 4.31 (2H , d, J = 8.5 Hz, 17-CH), 3.70 (2H, q, J = 0.16 Hz, 8 1 -CH 2), 3.68 (3H, s, 17 4 -CH 3), 3.6 (3H, s, _{^{12 1 -CH 3), 3.44 (}} 3H, s, 2 1 -CH 3), 3.26 (3H, s, 7 1 -CH 3), 2.74 (10H, m, 3 7,10,13,16,19 - _{CH 2), 2.71-2.52 (2H,} m, 17 1 -CH 2), 2.50 (2H, m, 3 4 -CH 2), 2.46 (2H, t, J = 0.78 Hz 3 3 -CH 2), 2.29 ^{(2H, m, 17 2 -CH} 2), 2.03 (2H, p, J = 0.08 Hz, 3 22 -CH 2), 1.81 (3H, d, J = 7.3 Hz, 18 1 -CH 3), 1.70 ( 3H, t, J = 7.6 Hz, 8 ² -CH ₃ ), 0.93 (3H, t, 3 ²³ -CH ₃ ), 0.31 and -1.70 respectively (1H, br, s, 21, 23-NH).

MALDI-MS: m / z of C ₅₅ H ₆₆ N ₄ O ₅ ⁺ (M + H) < ⁺ >; Calculated value 863.1363, measured value 863.4110.

[Example 6]

methyl Pyropeofovid -d- OH  And oleic acid conjugate ( methyl pyropheophorbide -d- OH  and oleic acid conjugate , Compound 6)

(25 mg, 0.045 mmol), dicyclohexylcarbodiimide (22 mg, 0.09 mmol) and 4-dimethylaminopyridine (0.05 mmol) were stirred in dichloromethane to give To the mixture was added oleic acid (12.77 mg, 0.045 mmol). The reaction was carried out at room temperature under a nitrogen atmosphere for 2 hours. To the reaction mixture was added 10 ml of water and stirred, and it was extracted with 20 ml of dichloromethane. The organic layer obtained as 100 ml of water was washed twice, and the organic layer was dried over anhydrous sodium sulfate, and then the organic layer was concentrated. The concentrated organics were purified by column chromatography on silica gel using 5% methanol in dichloromethane as eluent to give the compound (Compound 6), which was dried at 50 < 0 > C in a thermostat. The obtained compound was confirmed by UV-vis, ¹ H-NMR and MS, and these are shown in Fig. 16, Fig. 17 and Fig. 18 in order.

Yield: 30.9 mg (84%).

_{R f: 0.42 (CH 2 Cl} 2: MeOH = 98: 2).

_{UV-vis (in CHCl 3)} : λ max, ㎚ (Abs) 666.1 (1.37), 608.2 (0.35), 537.9 (0.39), 505 (0.36), 411.5 (2.37), 319 (0.71).

^{1 H-NMR (500 MHz,} CDCl 3, TM Sint): δ H, ppm 9.44 (1H, s, 5-meso-H), 9.42 (1H, s, 10-meso-H), 8.61 (1H, s , 20-meso-H), 6.38 (1H, s, 3 1 -CH), 5.28 and 5.13 (2H, each d, J = 19.5 Hz, J = 19.5 Hz, 13 2 -CH 2), 5.18 (2H, ^{m, 3 10, 11 -CH)} , 4.50 (1H, q, J = 0.045 Hz, 18-CH), 4.31 (1H, d, J = 8.5 Hz, 17-CH), 3.7 (2H, q, J = _{^{0.46 Hz, 8 1 -CH 2)}} , 3.68 (3H, s, 17 4 -CH 3), 3.60 (3H, s, 12 1 -CH 3), 3.44 (3H, s, 2 1 -CH 3), 3.27 ^{(3H, s, 7 1 -CH} 3), 2.7-2.56 (2H, m, 17 1 -CH 2), 2.43 (2H, t, J = 0.02 Hz, 3 3 -CH 2), 2.29 (2H, m _{^{, 17 2 -CH 2), 1.88}} (4H, m, 3 9, 12), 1.80 (3H, d, J = 7.29 Hz, 18 1 -CH 3), 1.70 (3H, t, J = 0.07 Hz, 8 _{^{2 -CH 3), 1.67 (2H}} , m, 3 4 -CH 2), 1.13 (20H, m, 3 5,6,7,8,13,14,15,16,17,18), 0.83 (3H , t, J = 2.8 Hz, 3 19 -CH 3), 0.31 and -1.79 (each 1H, br, s, 21, 23-NH).

MALDI-MS: m / z of C ₅₁ H ₆₈ N ₄ O ₅ ⁺ (M + H) < ⁺ >; The calculated value is 817.1094, the measured value is 816.3974.

[Example 7]

Pyropeofovid -a ( pyropheophorbide -a, PPa , Compound 7)

Methyl pyrophosphoride-a (1.166 g, 2.125 mmol) was dissolved in tetrahydrofuran (230 mL). A 4N aqueous hydrochloric acid solution (580 mL) was added to the mixture. The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. Dichloromethane (150 mL) was added to the reaction mixture and the aqueous layer was separated and the organic layer was washed several times with water to remove the acid. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was recrystallized from dichloromethane / hexane. The residue was purified on silica gel 60 (230-400 mesh) eluting with 5% methanol in dichloromethane. The result of measuring the UV spectrum of the product was as shown in Fig. 19, two base peaks of the chlorine ring were observed at? 419.9 nm and? 667.5 nm, and other small peaks were observed at? 508.9 nm,? 539 nm, and? 609.8 nm. On the other hand, the 500 MHz ¹ H-NMR spectrum of the product (PPa) was measured in chloroform, and the result is shown in FIG. The signals determined using δ and J _{H -H} values were marked in the 1D proton spectrum. Signals of the delta-2 to 10 domains corresponding to a total of 34 (1) protons were observed as eight single, five multiple, three double and two quadruple signals from the NMR spectrum results, It was confirmed that pyropeobiode-a was formed by two protons. The proton of the carboxyl group did not appear because the proton was exchanged by deuterium. The two NH 3 protons appeared as a wide signal at approximately -4 ppm due to rapid exchange. The mass spectrum of the product (PPa) is shown in FIG. The molecular weight of the product was calculated to be 534.7 g / mol and 535 (M ⁺ , 100%) was detected through EI-mass spectrometry analysis.

Yield: 1.0 g (88.5%).

_{R f: 0.25 (CHCl 2:} MeOH = 95: 5).

UV-vis (CH ₂ Cl ₂ ):? _Max , nm (log?) 667.5 (0.52), 609.8 (0.08), 539.0 (0.094), 508.9 (0.107), 413.9 (1.24). ¹ H-NMR (300 MHz, CDCl ₃ , TMS _int ):? _H , ppm 9.45 (1H, s, 5-meso-H), 9.34 , 20-meso-H), 8.02 (1H, m, 3 1 -CH), 6.29 and ^{6.17 (2H, dd, 3 2} -CH 2), 5.29 and ^{5.13 (2H, dd, 13 2} -CH 2), (2H, q, 8 ¹ -CH ₂ ), 3.63 (3H, s, 12 ¹ -CH ₃ ), 3.38 (1H, m, _{^{3H, s, 2 1 -CH 3}} ), 3.20 (3H, s, 7 1 -CH 3), 2.72-2.58 (2H, m, 17 1 -CH 2), 2.38-2.25 (2H, m, 17 2 - _{CH 2), 1.82 (3H,} d, J = 7.2 Hz, 18 1 -CH 3), 1.67 (3H, t, J = 7.8 Hz, 8 2 -CH 3), 0.48 and -1.70 (1H, br, s , NH).

EI-MS: m / z; 535 (M ^< ^{+ &} gt ^; ).

[Example 8]

Pyropeofovid -a-17 ⁵ -N-hexanol ( pyropheophorbide -a-17 ⁵ -N- hexanol , Compound 8)

N, N'-dicyclohexylcarbodiimide (77 mg, 0.37 mmol) and N-hydroxysuccinimide (26 mg, 0.226 mmol) were dissolved in anhydrous di After dissolving in 3 ml of chloromethane and adding 3 drops of trimethylamine to the mixture, the mixture was stirred for 4 hours at room temperature under an argon atmosphere. And hexanolamine (22 mg, 0.188 mmol) in methanol (2 ml) was added to the reaction and stirred for 1 hour 30 minutes.

10 ml of water was added to the reaction and stirred for 10 minutes, which was extracted with 30 ml of dichloromethane. The organic layer obtained as 100 ml of water was washed twice, and the organic layer was dried over anhydrous sodium sulfate, and then the organic layer was concentrated. To remove the by-products remaining in the concentrated organic material, the concentrated organic material was dissolved in a small amount of dichloromethane and cooled to cool. The organic matter from which the byproduct was removed was purified by silica gel column chromatography using 5% methanol in dichloromethane as an eluent to obtain a compound (Compound 8). The obtained compound was confirmed by UV-vis, ¹ H-NMR and MS, and these are shown in Fig. 22, Fig. 23 and Fig.

Yield: 140.2 mg (71%).

_Rf : 0.31 (CH ₂ Cl ₂ : MeOH = 95: 5).

_{UV-vis (in CHCl 3)} : λ max, ㎚ (Abs) 668.9 (2.03), 611.4 (0.51), 540.8 (0.58), 525.8 (0.381), 510 (0.57), 407.2 (3), 323.2 (1.16) , 269.7 (0.97).

^{1 H-NMR (500 MHz,} DMSO-d 6, TM Sint): δ H, ppm 9.60 (1H, s, 5-meso-H), 9.34 (1H, s, 10-meso-H), 8.86 (1H , s, 20-meso-H ), 8.15 (1H, dd, J = 11.51, J = 11.6, 31-CH), 7.70 (1H, t, J = 0.08 Hz, 17 3 -NH), 6.35 and 6.18 ( 2H, each d, J = 11.9 Hz, J = 11.76 Hz, 3 2 -CH 2), 5.22 and 5.08 (2H, each d, J = 19.8 Hz, J = 19.7 Hz, 13 2 -CH 2), 4.55 ( 1H, q, J = 1.54 Hz , 18-CH), 4.27 (1H, s, 17 9 -OH), 4.26 (2H, d, J = 10.1 Hz, 17-CH), 3.61 (2H, q, J = _{^{0.17 Hz, 8 1 -CH 2)}} , 3.57 (3H, s, 12 1 -CH 3), 3.41 (3H, s, 2 1 -CH 3), 3.30 (2H, q, J = 0.02 Hz, 17 9 - _{CH 2), 3.14 (3H,} s, 7 1 -CH 3), 2.95 (2H, m, 17 4 -CH 2), 2.60-2.32 (2H, m, 17 1 -CH 2), 2.16-2.06 (2H _{^{, m, 17 2 -CH 2)}} , 1.79 (3H, d, J = 7.29 Hz, 18 1 -CH 3), 1.58 (3H, t, J = 0.11 Hz, 8 2 -CH 3), 1.30 (2H, p, J = 0.22 Hz, 17 8 -CH 2), 1.22 (2H, p, J = 0.67 Hz, 17 5 -CH 2), 1.15 (4H, m, 17 6 and 17 ^7), 0.14 and -1.67 ( Respectively, 1H, br, s, 21, 23-NH).

MALDI-MS: m / z of C ₃₉ H ₄₇ N ₅ O ₃ ⁺ (M + H) ⁺ ; Calculated value 633.8222, measured value 634.2467.

[Example 9]

Pyropeofovid -a-17 ⁵ -N- Hexanol  And Docosahexaenoic acid  Junction pyropheophorbide -a-17 ⁵ -N-hexanol and docosahexaenoic acid conjugate , Compound 9)

Fatigue peoh Poby de -a-17 ⁵ -N- hexanol (30 ㎎, 0.047 mmol), dicyclohexyl carbodiimide (19.5, 0.094 mmol) and 4-dimethylaminopyridine (6.9 ㎎, 0.056 mmol) of dimethyl Was stirred in chloromethane and docoshexaenoic acid (15.5 mg, 0.047 mmol) was added to the mixture. The reaction was carried out at room temperature under a nitrogen atmosphere for 2 hours. To the reaction mixture was added 10 ml of water and stirred, and it was extracted with 20 ml of dichloromethane. The organic layer obtained as 100 ml of water was washed twice, and the organic layer was dried over anhydrous sodium sulfate, and then the organic layer was concentrated. The concentrated organics were purified by column chromatography over silica gel using 5% methanol in dichloromethane as eluent to give the compound (Compound 9), which was dried at 50 < 0 > C in a thermostat. The obtained compound was confirmed by UV-vis, ¹ H-NMR and MS, and these are shown in Fig. 25, Fig. 26 and Fig. 27 in order.

Yield: 33.5 mg (75%).

_Rf : 0.17 (CH ₂ Cl ₂ : MeOH = 98: 2).

_{UV-vis (in CHCl 3)} : λ max, ㎚ (Abs) 669.2 (0.81), 611 (0.27), 540.5 (0.31), 525.9 (0.24), 511.1 (0.28), 415.3 (1.63), 3.23 (0.56) .

¹ H-NMR (500 MHz, CDCl ₃ , TM _Sint ):? _H , ppm 9.5 (1H, s, 5-meso-H), 9.41 , 20-meso-H), 8.03 (1H, dd, J = 11.51, J = 11.6, 3 1 -CH), 6.29 and 6.18 (2H, dd, J = 17.9 Hz, J = 10.7 Hz, 3 2 -CH ₂ ), 5.34 (12H, m, 17 ^{13,14,16,17,19,20,22,23,24,25,27,28} ), 5.18 (2H, dd, J = 19.5 Hz, _{^{, 13 2 -CH 2), 4.77}} (1H, m, 17 3 -NH), 4.51 (1H, m, 18-CH), 4.36 (2H, d, J = 6.9 Hz, 17-CH), 3.93 (2H , t, J = 0.1 Hz, 17 9 -CH 2), 3.70 (2H, q, J = 0.16 Hz, 8 1 -CH 2), 3.66 (3H, s, 12 1 -CH 3), 3.41 (3H, _{^{s, 2 1 -CH 3),}} 3.25 (3H, s, 7 1 -CH 3), 2.93 (2H, m, 17 4 -CH 2), 2.79 (10H, m, 17 15,18,21,23, _{^{26 -CH 2), 2.7-2.4 (2H}} , m, 17 1 -CH 2), 2.30 (2H, m, 17 12 -CH 2), 2.06-1.82 (2H, m, 17 2 -CH 2), 2.04 (2H, p, J = 8.1 Hz, 17 30 -CH 2), 1.80 (3H, d, J = 7.29 Hz, 18 1 -CH 3), 1.70 (3H, t, J = 0.11 Hz, 8 2 -CH _{3), 1.61 (2H, m} , 17 11 -CH 2), 1.42 (2H, p, J = 3.1 Hz, 17 8 -CH 2), 1.05 (6H, m, 17 5,6,7 -CH 2) , 0.95 (3H, t, 17 31 -CH 3), 0.47 and -1.68 (each 1H, br, s, 21, 23-NH).

MALDI-MS: m / z of C ₆₁ H ₇₇ N ₅ O ₄ ⁺ (M) ⁺ ; Calculated value 944.2952, measured value 944.4903.

[Example 10]

Pyropeofovid -a-17 ⁵ -N- Hexanol  And oleic acid conjugate ( pyropheophorbide -a-17 ⁵ -N-hexanol and oleic acid conjugate , Compound 10)

Fatigue peoh Poby de -a-17 ⁵ -N- hexanol (30 ㎎, 0.047 mmol), dicyclohexyl carbodiimide (19.5, 0.094 mmol) and 4-dimethylaminopyridine (6.9 ㎎, 0.056 mmol) 3 ml dichloromethane and oleic acid (13.4 mg, 0.047 mmol) was added to the reaction mixture.

The reaction was carried out at room temperature under a nitrogen atmosphere for 2 hours. To the reaction mixture was added 10 ml of water and stirred, and it was extracted with 20 ml of dichloromethane. The organic layer obtained as 100 ml of water was washed twice, and the organic layer was dried over anhydrous sodium sulfate, and then the organic layer was concentrated. The concentrated organics were purified by column chromatography over silica gel using 5% methanol in dichloromethane as the eluent to give the compound (Compound 10), which was dried at 50 < 0 > C in a thermostat. The obtained compound was confirmed by UV-vis, ¹ H-NMR and MS, which are shown in Fig. 28, Fig. 29 and Fig. 30, respectively.

Yield: 34 mg (80%).

_{R f: 0.13 (CH 2 Cl} 2: MeOH = 98: 2).

_{UV-vis (CHCl 3):} λ max, ㎚ (Abs) 669.2 (1.45), 611.3 (0.38), 540.6 (0.43), 525.8 (0.31), 510.3 (0.42), 4.75.5 (0.25), 414 (2.72 ), 323.2 (0.83).

^{1 H-NMR (500 MHz,} CDCl 3, TM Sint): δH, ppm 9.49 (1H, s, 5-meso-H), 9.4 (1H, s, 10-meso-H), 8.56 (1H, s, 20-meso-H), 8.02 (1H, dd, J = 11.5, J = 11.5, 3 1 -CH), 6.29 and 6.18 (2H, each d, J = 17.9 Hz, J = 10.7 Hz, 3 2 -CH _{2), 5.3 (2H, m} , 17 18,19 -CH), 5.24 and 5.11 (2H, each d, J = 19.5 Hz J = 19.5 Hz, 13 2 -CH 2), 4.8 (1H, m, 17 3 -NH), 4.51 (1H, m , 18-CH), 4.36 (1H, m, 17-CH), 3.91 (2H, t, J = 0.02 Hz, 17 9 -CH 2), 3.7 (2H, q, ^{J = 0.16 Hz, 8 1 -CH} 2), 3.64 (3H, s, 12 1 -CH 3), 3.41 (3H, s, 2 1 -CH 3), 3.25 (3H, s, 7 1 -CH 3) , 2.89 (2H, m, 17 4 -CH 2), 2.65 and 2.45 (2H, each _{^{m, 17 1 -CH 2),}} 2.18 (2H, t, J = 0.085 Hz, 17 11 -CH 2), 2.15 and 1.86 (2H, each _{^{m, 17 2 -CH 2),}} 1.97 (4H, m, 17 17,20), 1.80 (3H, d, J = 7.29 Hz, 18 1 -CH 3), 1.70 (3H, t, ^{J = 0.11 Hz, 8 2 -CH} 3), 1.51 (2H, m, 17 12 -CH 2), 1.41 (2H, p, J = 0.25 Hz, 17 8 -CH 2), 1.24 (20H, m, 17 ^{13,14,15,16,21,22,23,24,25,26), 1.05 (6H, m} , 17 5,6,7 -CH 2), 0.86 (3H, t, J = 0.18 Hz, 17 ²⁷ -CH _3), 0.47 and -1.69 (each 1H, br, s, 21, 23-NH).

MALDI-MS: m / z of C ₅₇ H ₇₉ N ₅ O ₄ ⁺ (M) ⁺ ; Calculated value, measured value 898.4696.

[Experimental Example 1]

Chlorine  Inhibition of Cell Growth by Conjugates of Derivatives and Unsaturated Fatty Acids

To confirm the effect of inhibiting the growth of cells by pyripheopoboid-a-17 ⁵ -N -hexanol and docosahexaenoic acid conjugate (Compound 9) which is a conjugate of the chlorine derivative and the unsaturated fatty acid prepared in Example 9 Inhibition of cell growth was investigated in TC-1 cell line expressing human HPV 16 E6 / E7. (Gibco BRL), 0.22% sodium bicarbonate (Sigma-Aldrich, St. Louis, Mo., USA) was added to RPMI-1640 (Gibco BRL, Rockville, , 400 ㎎ / L of G418 (Sigma-Aldrich), and streptomycin / penicillin (Gibco BRL) were added and cultured at 37 ° C in a 5% CO ₂ incubator.

The TC-1 cell line was divided into 3 × 10 ³ cells / well in a 96-well plate, and cultured for 24 hours to treat a conjugate of chlorine derivative, chlorine derivative and unsaturated fatty acid of 0.1, 1 and 10 μM for 24 hours. Then, PDT was performed at 1.56 J / ㎠ using 662nm ± 3nm laser, and PDT was not performed in the control. Subsequently, 20 ㎕ of 5 ㎎ / ㎖ of MTT solution (Sigma-Aldrich) was added to each well of the control and treatment and cultured at 37 캜 for 4 hours. After removing the supernatant, dimethylsulfoxide (DMSO, Sigma- Aldrich) was added to 100 μl / well and shaken in a shaker for 10 seconds. Absorbance was measured at 570 nm with an ELISA reader (spectra max 250, Molecular Devices, Sunnyvale, CA, USA).

The results are shown in Fig. As can be seen from FIG. 31, when a 10 uM choline derivative alone (Free-Ps) was used, the side effect of dark toxicity was exhibited due to the effect of inhibiting cell growth by 90% . In the case of 1 uM, which is a cytotoxic concentration, there is no cytotoxicity but only about 50% inhibition of cell growth in the irradiation with laser. Therefore, when the chlorine derivative alone is used, the concentration must be increased to obtain a cell growth inhibiting effect of 90% or more, but the concentration can not be used due to toxicity. However, in the case of the choline derivative and the unsaturated fatty acid (DHA) conjugate (Ps-DHA), the cell growth inhibitory effect (photodynamic treatment effect) at a low concentration in the presence of the laser is lower than that of the chlorin derivative alone, Is not observed even at an excellent 10 uM, there is an advantage that a cell growth inhibitory effect of 90% or more can be obtained.

[Experimental Example 2]

Chlorine  Of a conjugate of a derivative and an unsaturated fatty acid Confocal  Microscope analysis

To examine the cytotoxicity and tumor selectivity of pyripheopoboid-a-17 ⁵ -N -hexanol and docosahexaenoic acid conjugate (Compound 9), which is a conjugate of the chlorin derivative and the unsaturated fatty acid prepared in Example 9, Confocal microscopy was performed on the TC-1 cell line expressing HPV 16 E6 / E7.

(Gibco BRL), 0.22% sodium bicarbonate (Sigma-Aldrich, St. Louis, Mo., USA) was added to RPMI-1640 (Gibco BRL, Rockville, , 400 ㎎ / L of G418 (Sigma-Aldrich), and streptomycin / penicillin (Gibco BRL) were added and cultured at 37 ° C in a 5% CO ₂ incubator. The TC-1 cell line was dispensed at 3 × 10 ³ cells / well in a 6-well plate containing sterilized cover glasses and cultured for 24 hours to treat the conjugate of the choline derivative and the choline derivative with the unsaturated fatty acid for 12 hours. After the culture, the medium was removed, washed twice with 1 × PBS buffer, and treated with 1 ml of 1% paraformaldehyde for 15 minutes to fix the cells. After fixation, the supernatant was removed, washed once with 1 × PBS buffer, dried on a dropping glass slide with mounting solution, and analyzed using a confocal microscope (TCS SP2, Leica, Wetzlar, Germany) Respectively. The excitation wavelength used in the analysis was 600 nm and the emission wavelength was 545 nm.

The results of the analysis are shown in Fig. 32, when the chlorin derivative alone was used, there was almost no cell due to toxicity, and the shape of the cell was also changed. However, the shape of the cells treated with the choline derivative and the unsaturated fatty acid (DHA) conjugate is healthy and the absorption rate of fluorescence is not different from that of the choline derivative alone.

Claims (14)

A conjugate of a choline derivative and an unsaturated fatty acid represented by the following general formula (I).

[Formula I]

Wherein R is

or

to be.
delete A photosensitizer containing a conjugate of a chlorine derivative and an unsaturated fatty acid according to claim 1.
4. The photosensitizer of claim 3, wherein the photosensitizer exhibits photosensitizing activity against light rays in the range of 650 nm to 800 nm.
delete delete delete delete delete delete delete delete delete A method for producing a chlorinated derivative and an unsaturated fatty acid conjugate comprising the step of conjugating a chlorine derivative represented by the following formula 4 with docosahexaenoic acid (DHA) or oleic acid (OA).

[Compound 4]

Images