JP2011173960A - Polymer micelle type photostimulation-responsive nitrogen monoxide donor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound, a structure or a conjugate that has low toxicity and high dispersion stability, accumulates on a target portion in a body (for example, a cancer tissue), produces NO at the target portion only when irradiated with light without slowly releasing NO, and can demonstrate the effect of NO in situ. <P>SOLUTION: There is provided a polymer derivative which is produced by covalent bonding of a low molecular weight NO donor such as 3-trifluoromethyl-4-nitrophenoxy with a block copolymer that can form a polymer micelle in an aqueous medium (for example, a polymer of polyethylene glycol and p-chloromethylstyrene). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a polymer micelle-forming polymer derivative having a function of generating nitric oxide by light stimulation and use thereof, and more specifically, a low-molecular compound capable of generating nitric oxide and a polymer-forming property. The present invention relates to covalent derivatives of polymers and their use as anticancer agents.

  Nitric oxide (NO) is a free radical having unpaired electrons composed of nitrogen and oxygen, and is involved as a signal transmitter in many physiological phenomena such as vasodilation and nerve transmission in vivo. In addition, macrophages, which are part of the immune system, recognize tumor cells and pathogens and release large amounts of NO when they are detected, thereby inhibiting these external enemy mitochondrial electron transport systems and inducing DNA damage. (Non-patent document 1, Non-patent document 2). Furthermore, NO is known to have a concentration-dependent effect on cell growth, and low concentrations of NO promote cell growth, while high concentrations of NO inhibit cell growth or cause cell death (apoptosis). Cause (non-patent document 3). That is, if a high concentration of NO can be temporarily generated at a tumor site such as a cancer tissue, it can be expected to suppress the growth and eventually kill the tumor site.

  NO is a highly reactive gas at normal temperature and normal pressure, and its half-life in vivo is about 5 seconds. Therefore, it is practically difficult to dissolve NO in water and effectively act at a specific location in the living body. Therefore, NO donors (NO donors) that generate NO under physiological conditions have been developed for the purpose of supplying NO into the living body. For example, S-nitroso compounds, aromatic N-nitroso compounds, diazonium diolates (NONOates), (sub) nitrate compounds, metal nitrosyl, guanidines, oxatriazoliums, etc. are known as NO donors. However, most of them are gradually decomposed by heat, pH, the function of metal ions and protein functional groups contained in blood, or metabolism by enzymes in a living environment, and release NO gradually (non-patent literature). 4). When the antitumor effect of NO described above is expected, NO is allowed to act only at a necessary timing at a specific site in the living body, that is, spatiotemporal control of the NO function is required. The use of conventional NO donors that release slowly is not suitable.

  In order to achieve spatiotemporal control of NO function in vivo, NO donors utilizing photochemical reactions of nitrobenzene derivatives are disclosed by Miyata et al. (Non-patent Documents 5 and 6) and Sortino et al. (Non-patent Document 7). Reported by a research group. In these nitrobenzene derivatives, intramolecular atomic rearrangement is induced in the photoexcited state, and nitrogen and oxygen of the nitro group are released as NO. It is a stable compound that is not decomposed by functional groups in heat, pH, metal ions, or proteins in a biological environment, and can selectively generate NO only during light irradiation. However, these nitrobenzene derivatives are generally very poor in water solubility, and need to be dissolved in a mixed solution of an organic solvent such as dimethyl sulfoxide. Furthermore, since these nitrobenzene derivatives are low molecular weight compounds, even if they can be administered into blood, they are excreted by renal glomeruli and are rapidly excreted outside the body after administration. Sortino et al. Have proposed a NO carrier in which an appropriate amount of the above nitrobenzene derivative is introduced onto the surface of a platinum nanoparticle whose surface has a particle diameter of several nanometers stabilized with a carboxyl group (Non-patent Document 8, Non-patent Document). 8) Dispersion stability under the physiological conditions of particles dispersed only with a negative charge of a carboxyl group is expected to be extremely low, and there is a concern about long-term toxicity due to accumulation of platinum particles as a carrier in vivo. Therefore, it has a big problem in aiming at formulation at the time of using as a medicine.

Stuhr et al. Exp. Med. , 1989, 169, 1011-1020. Hibbs et al. Science, 1987, 235, 473-476. Wink et al. Free Rad. Biol. Med. , 2008, 45, 18-31) The Chemical Society of Japan, “NO-Chemistry and Biology” Quarterly Chemical Review 1996 Fukuhara et al. Am. Chem. Soc. 2001, 123, 8662-8666. Suzuki et al. Am. Chem. Soc. 2005, 127, 11720-11726. Sortino et al. Chem. Commun. , 2001, 1226-1227 Caruso et al. Am. Chem. Soc. , 2007, 129, 480-481 Barone et al. Mater. Chem. , 2008, 18, 5531-5536

The problems of the prior art described above are summarized as follows.
1) When a NO donor compound is used when supplying NO into a living body, the NO donor is decomposed due to external factors in the living environment, and NO is gradually released. It is difficult to act on a specific site in the living body.
2) When a NO donor that generates NO by light irradiation is used, there is a general concern that the donor's low water solubility and, on the other hand, low molecular weight compounds cause in vitro filtration and excretion by renal glomeruli. The
3) A structure or conjugate in which an appropriate amount of the NO donor is introduced on the surface of the platinum nanoparticle carrier developed by supporting the photoresponsive NO donor on the carrier has low dispersion stability and target in the actual use (living) environment. Accumulation at the site cannot be expected.

  Therefore, it has low toxicity and high dispersion stability, accumulates in the target site (for example, cancer tissue) in the body, does not release NO gradually, generates NO only at the time of irradiation with light at the target site. If a compound or construct or conjugate capable of exerting the action effect of NO can be provided, it will be possible to provide a new stage in the art.

  In order to solve the above problems, the present inventors have studied to develop a novel derivative of the low molecular NO donor. As a result, polymer micelles derived from polymer derivatives in which the donor is covalently bonded to a block copolymer capable of forming polymer micelles in an aqueous medium have low molecular NO donors attached to the normal biological environment. It was confirmed that NO could be generated in situ by effectively responding to light stimulation while NO was maintained without being decomposed below and without sustained release.

More specifically, a block copolymer (PEG-b-PCMS) comprising a polyethylene glycol chain segment and a hydrophobic poly (4-chloromethylstyrene) chain segment that are biocompatible and excellent in water solubility. It was found that a block copolymer (PEG-b-PCNTP) having a photostimulation-responsive NO donor can be efficiently provided by introducing a photostimulation-responsive NO donor into the side chain via a covalent bond. Further, this PEG-b-PCCNT forms polymer micelles having a high dispersion stability with a particle size of about 40 nm by a simple dialysis method, and the obtained polymer micelles are selectively NO by irradiation with light in a living environment. Polymeric micelle-type photostimulation response that can generate NO at a desired timing in a desired in vivo region by generating PEG-b-PCMS and using a low molecular NO donor as a polymerized derivative It has been found that a sex NO donor can be provided very efficiently. Furthermore, although it may be less preferred, it has also been found that a block copolymer using a certain hydrophobic polymer chain instead of the PCMS chain can provide a polymer micelle-type photostimulation-responsive NO donor having similar characteristics. It was.

Thus, according to the invention, the general formula (I)

_{A- (CH 2 -CH 2 O)} m -L 1 - (NO-donor) -Z (I)

Represented by
Wherein A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent is a formyl group, the formula R ^a R ^b CH— (wherein R ¹ and R ² are independently _and, C 1 -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or -O _{(CH 2)} represents a 4 O-. )
L ₁ is a bond, — (CH ₂ ) _c S—, —CO (CH ₂ ) _c S—, — (CH ₂ ) _c NH—, — (CH ₂ ) _c CO— (where each c is 1 -5, preferably an integer of 1 or 2, and represents a linking group selected from the group consisting of -CO-, -COO- and -CONH-,
NO-donor represents a hydrophobic polymer chain in which a low molecular NO donor is covalently bonded to a side chain;
Z is hydrogen atom, hydroxy, C ₁ -C ₁₂ alkyloxy optionally substituted with one phenyl group or benzhydryl group at the non-bonding end, or substituted with one phenyl group or benzhydryl group at the non-bonding end. Represents a good C ₁ -C ₁₂ alkylcarbonyl,
m represents an integer of 20 to 5,000,
A polymeric derivative of the NO donor is provided.

  As a more specific and preferred embodiment of the present invention, the general formula (II)

In which A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent represents a formyl group or a group of formula R ¹ R ² CH—, wherein R ¹ and R ² is _{independently,} C 1 -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or -O _{(CH 2)} 4 O -
L ₁ represents a linking group selected from the group consisting of an atomized bond, — (CH ₂ ) _a S—, —CO (CH ₂ ) _a S—, wherein a is 1 to 5, preferably 2. An integer,
L ₂ represents a bond, —O—, —NH—, — (CH ₂ ) _b —O—, — (CH ₂ ) _b —NH—,
_{- (CH 2) b -OCO -} , - (CH 2) b -NHCO -, - (CH 2) b -COO- and _{- (CH} 2) b -CONH- become more linking group selected from the group (Note, Bond represents the direction in which the leading-bonds to the benzene ring), b is an integer of 0 to 3,
R is the formula

Represents the group of
R ^1-1 is at least one is bound in the ortho-position to the nitro _group, C 1 -C ₆ alkyl, one or more _C 1 -C ₆ alkyl substituted with halogen atoms, mono- or di -C ₁ -C _{6 alkyl,} C 1 -C ₆ alkyloxy, di - or tri _-C 1 -C ₆ alkyl silyl, phenyl, naphthyl, anthracenyl, pyrenyl, phenoxy, pyrrolyl, pyridyl, imidazole, morpholyl, thiophenyl, thiazole, amino , Nitro, cyano, hydroxyl, hydroxymethyl, aldehyde, carboxyl, carboxylic acid ester, carboxylic acid amide, sulfo, sulfonic acid ester, sulfonic acid amide and hydrazide,
p represents an integer of 1 to 4,
-X- is a _{bond, - (CH 2) d -} , and _{- (CH 2) d - (} CH = CH) e- (CH 2) d - is selected from the group consisting of, d is an integer from 1 to 6 E represents an integer of 0 to 3,
L ₂ represents a linking group selected from the group consisting of a bond, methylimino, methyliminomethyl, methyloxy, methyloxymethyl, methyl ester, and methyl ester methyl;
m is an integer of 20 to 5,000,
n is an integer from 3 to 1,000, and
R may be less than 50% of the total number n of R, preferably less than 30%, more preferably less than 10%, particularly preferably less than 5% are hydrogen atoms,
A polymeric derivative of the NO donor represented by:

The above polymerized derivative of the NO donor forms a polymer micelle in an aqueous medium.
a) Since it is nano-sized and has a water-soluble polymer PEG chain in its outer shell (shell), it has high biocompatibility and dispersion stability;
b) Since it has a particle size of several tens of nanometers, filtration and excretion by the glomeruli can be avoided during in vivo administration, and an accumulation effect in cancer tissue can be expected due to the enhanced permeation and retention (EPR) effect.

  In addition, the polymer micelle is predicted to have an NO donor site that generates NO for the first time by light stimulation in the core (core) of the micelle. In addition to the effect of accumulation in the cancer tissue (passive targeting) by the EPR effect Also, a double targeting effect that appears only when the NO donor accumulation site and the light irradiation site match can be expected.

  Therefore, according to the present invention, there is also provided an anticancer agent comprising the above-described polymerized derivative of NO donor as an active ingredient and, if necessary, a diluent or an excipient commonly used in pharmacy.

Detailed description of the invention

  Hereinafter, specific aspects of the present invention or terms defining the present invention will be described.

  The low molecular NO donor is a nitro mono- to poly-cyclic aromatic hydrocarbon including a nitrobenzene derivative, which can generate NO by a photochemical reaction. A low molecule is a conceptual compound facing a polymer, and when polymerized according to the present invention, the molecular size of the polymer micelle formed in an aqueous medium is on the order of several hundred nm or less. Refers to a compound.

Usually, the photochemical reactivity of nitro mono- or polycyclic aromatic hydrocarbons is greatly influenced by the orientation of the nitro group. In the ground state, when the nitro group is oriented out of plane with respect to the aromatic ring (when the orientation is twisted with the plane formed by the aromatic ring), the oxygen p-orbital of the nitro group and the aromatic ring adjacent to the nitro group It overlaps with the p orbital of carbon. When this is in a photoexcited state, a rearrangement of nitrogen-oxygen atoms from a nitro (NO ₂ ) group to a nitrite (—O—NO) group (nitro-nitrite photorearrangement) is induced, and the nitrite group is Arise. This reaction is more likely to occur when the plane of the aromatic ring and the orientation of the nitro group are close to perpendicular (that is, the greater the twist). In the nitrite thus produced, the carbon-nitrogen bond is easily cleaved by light irradiation, and nitric oxide (NO) is produced. Although it is not limited in the present invention, the phrase “NO can be generated by photoreaction” means that NO can be generated by the reaction mechanism as described above. Therefore, typical low molecular NO donors or low molecular NO donor moieties include, for example, the compounds described in Non-Patent Documents 6 and 7, or residues obtained by removing hydrogen atoms from the compounds.

A hydrophobic polymer chain (NO-donor) segment in which the low molecular NO donor in the polymerized derivative of the NO donor according to the present invention obtained by polymerizing such a low molecular NO donor is covalently bonded to the side chain, Although not limited, a polymer chain having the following reactive side group and, if necessary, suitable functional groups (—OH, —NH ₂ , —NHNH) by a means known per se to a low molecular NO donor. ₂ , -COOH, etc.), and then reacted and covalently bonded.

  Examples of polymer chains with reactive side groups:

Here, p represents 1 or 2, R ₁ represents a C ₁ -C ₁₂ alkyl group optionally substituted by one phenyl group or benzhydryl group at the non-bonding end, and n represents 3 to 1,000. , Preferably 5 to 500, more preferably an integer of 10 to 300,
A polyamino acid ester chain segment of

Wherein, R ₁ represents one phenyl or benzhydryl optionally C ₁ -C ₁₂ alkyl group optionally substituted by a group in the non-binding end, R ₂ is a hydrogen atom or a C ₁ - ₅ alkyl group, preferably Represents a methyl group, and n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300,
A poly ((meth) acrylate) chain segment of

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
Of styrene-maleic anhydride copolymer chain segment;

Here, R ₁ represents a C ₁ -C ₁₂ alkyl group optionally substituted by one phenyl group or benzhydryl group at the non-bonding end, and n is 3 to 1,000, preferably 5 to 500, Preferably it represents an integer of 10 to 300,
A polymalate chain segment of

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
A polyamic acid chain segment of

Here, R ₁ represents a C ₁ -C ₁₂ alkyl group optionally substituted by one phenyl group or benzhydryl group at the non-bonding end, and n is 3 to 1,000, preferably 5 to 500, Preferably it represents an integer of 10 to 300,
A polymaleimide chain segment of

Here, L ₁ represents a chlorine, bromine or iodine atom, and n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
A poly (halomethylstyrene) chain segment of

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
Of poly (vinyl chloride) chain segments;

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
A poly (chloroprene) chain segment of

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
Of poly (glycidyl methacrylate) chain segments;

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
A poly (2-hydroxyethyl methacrylate) chain segment;

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
A polyepichlorohydrin chain segment of

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
A poly-3,3-bischloromethyloxetane chain segment of

Here, R ₁ represents a C ₁ -C ₁₂ alkyl group optionally substituted by one phenyl group or benzhydryl group at the non-bonding end, and n is 3 to 1,000, preferably 5 to 500, Preferably it represents an integer of 10 to 300,
A poly (oxazoline) chain segment of

Here, a and b independently represent an integer of 3 to 500.
Of polysiloxane chain segments;

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
A poly (vinylphenylboronic acid) chain segment of

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300,
A 1-nylon chain segment of

Here, n represents an integer of 3 to 1,000, preferably 5 to 500, more preferably 10 to 300.
Poly (vinylbenzaldehyde) chain segment.

Furthermore, in the above formula (I),
Z is hydrogen atom, hydroxy, C ₁ -C ₁₂ alkyloxy optionally substituted with one phenyl group or benzhydryl group at the non-bonding end, or substituted with one phenyl group or benzhydryl group at the non-bonding end. Mention may be made, for example, of diblock copolymers representing C ₁ -C ₁₂ alkylcarbonyl.

  However, in particular, the hydrophobic chain segment carrying the reactive group of the block copolymer has the formula

Here, a block copolymer which is a poly (halomethylstyrene) chain segment in which L ₁ represents a chlorine, bromine or iodine atom and n represents an integer of 3 to 1,000 can be preferably used.

  Some of such block copolymers are known, for example, those in which the hydrophobic segment represents a polyamino acid ester chain segment are disclosed in Japanese Patent No. 2690276 (or US Pat. No. 5,449,513), etc. In addition, those representing poly ((meth) acrylic ester chain segments are known from Patent Document 3. Other block copolymers are the above-described block copolymer production methods or production examples described later in this specification. It can be easily obtained by those skilled in the art with reference to 1. It should be noted that when the linking group or repeating unit in the general formula defining the block copolymer used in the present invention is represented by a formula, it is described. It is intended to be incorporated into the general formula in a certain direction.

  A preferred embodiment of the polymerized derivative of the NO donor according to the present invention comprising a hydrophobic polymer chain (NO-donor) segment in which a low molecular NO donor is covalently bonded to the side chain can be provided in the above general formula (II). It is a compound represented. In the general formula (II), the above formula

Preferred examples of the group include, but are not limited to, based on the phenyl group bonded to X,
(I) 3- (C ₁ -C ₆ alkyl) -4-nitrophenyl, 3- (C ₁ -C ₆ branched alkyl) -4-nitrophenyl, 3,5-di- (C ₁ -C ₆ alkyl) -4-nitrophenyl,
(Ii) 3- [C ₁ -C ₆ alkyl substituted with one or more halogen atoms (fluorine, chlorine, bromine or iodine)]-4-nitrophenyl, 3,5-di- [one or more halogens atom (fluorine, chlorine, bromine or iodine) _C 1 -C ₆ alkyl substituted with] -4-nitrophenyl,
(Iii) 3- (mono- - or di _-C 1 -C ₆ alkyl) amino-4-nitrophenyl, 3,5-di - (mono - or di _-C 1 -C ₆ alkyl) amino-4-nitrophenyl ,
(Iv) A phenyl group selected from 3- (C ₁ -C ₆ alkyl) oxy-4-nitrophenyl and 3,5-di- (C ₁ -C ₆ alkyl) oxy-4-nitrophenyl bonded to X Things can be mentioned.

For each group or part of each group that identifies the above compounds, for example, the alkyl moiety in C ₁ -C ₁₂ alkoxy or C ₁ -C ₆ alkyloxy is a straight or branched alkyl, such as methyl, ethyl N-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-octyl, n-dodecyl and the like. Accordingly, C ₁ -C ₆ alkyl includes, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl and the like.

A halogen atom referred to in C ₁ -C ₆ alkyl substituted with one or more halogen atoms is fluorine, chlorine, bromine or iodine. Examples of such an alkyl group substituted with a halogen atom include, but are not limited to, trifluoromethyl, trichloromethyl, tribromomethyl, dichloromatyl, dibromomethyl, tetrafluoroethyl, tetrachloroethyl, and tetrabromoethyl. Etc.

  In accordance with the present invention, polymerized derivatives of NO donors can be used in aqueous media such as water, phosphorylated saline, or water-miscible organic solvents (N, N-dimethylformamide, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, acetone, dimethyl). Polymer micelles can be formed in an aqueous solution containing acetamide, methanol, etc.).

  The polymer micelles thus formed are also referred to herein as polymer micelle-type photostimulation-responsive NO donors, but these are usually polymer derivatives having an average diameter of 100 nm or less and constituting the same. Those in which about 10 to about 20 NO generating sites are introduced per (block copolymer) can be preferably used as an active ingredient of a pharmaceutical preparation, for example. For pharmaceutical preparations, if necessary, diluents, purified water, diluents such as buffered aqueous solutions with buffers commonly used under physiological conditions, or monosaccharides, disaccharides, oligosaccharides, polyethylene glycols, etc. Excipients commonly used in the art can also be included.

The measurement result of ¹ H-NMR of PEG-b-PCMS obtained in Example 1 (a) and the corresponding structural formula are shown. The measurement result of ¹ H-NMR of PEG-b-PCNTP obtained in Example 1 (b) and the corresponding structural formula are shown. It is the spectrum of FT-IR of each of PEG-b-PCMS and PEG-b-PCNTP obtained in (a) and (b) of Example 1. The dynamic light-scattering measurement result of the polymer micelle of PEG-b-PCNTP obtained by Example 2 is shown. It is an ESR spectrum in Example 3. The result of the grease test in Example 4 is represented. The result of the cytotoxicity test in Example 5 is represented.

  In the following, specific examples are described in order to more specifically explain the present invention, but this is for the purpose of simplifying the description of the invention and does not mean that the present invention is limited to these embodiments. .

Production of block copolymer having photo-stimulatory NO donor (a) 1 g of polyethylene glycol having a thiol group at one end (PEG-SH, M _n = 5000), 2,2′-azobisisobutyronitrile 17 mg Was added to the reaction vessel, and nitrogen substitution in the reaction vessel was repeated three times. Further, 1.5 g (1.4 ml) of p-chloromethylstyrene and 12 ml of toluene were added while blowing nitrogen into the reaction vessel. The sealed reaction vessel was heated to 60 ° C. and stirred for 24 hours. The reaction mixture was added dropwise to 600 ml of hexane, and the resulting white precipitate was collected by filtration. The recovered material was washed three times with diethyl ether and then freeze-dried with benzene. The yield was 1.2 g, and the yield was 48.2%. The composition of the obtained polyethylene glycol-polychloromethylstyrene block copolymer (PEG-b-PCMS) is that the molecular weight of the PEG segment is 5000 g / mol from the measurement result of ¹ H-NMR (see FIG. 1). The molecular weight was determined to be about 1830 g / mol (polymerization degree n = 12).

(B) PEG-b-PCMS 150 mg, 4-nitro- (3-trifluoromethyl) phenol (NTP) 128 mg, and potassium carbonate 256 mg were respectively weighed and dissolved in 5 ml of dimethylformamide, and then subjected to 90 ° C. under light-shielding conditions. And stirred overnight. After insoluble matter was removed by filtration, the filtrate was put into a dialysis membrane having a molecular weight cut off of 3500 and dialyzed against methanol. The dialysis membrane solution was recovered and the residue after evaporation was dissolved in benzene, lyophilized and further introduced with a 3-trifluoromethyl-4-nitrophenoxy-methyl group on the side chain of PEG-b-PCMS. A yellow powder of a polymerized derivative (PEG-b-PCNTP) was obtained. The recovered amount was 161 mg, and the recovery rate was 82.5%. The structure of PEG-b-PCCNTP was confirmed by FT-IR (see FIG. 3) and ¹ H-NMR, and it was confirmed that NTP was introduced into all chloromethyl groups of PEG-b-PCMS (FIG. 2).

Production of Polymeric Micelle Type Photostimulus-responsive NO Donor 10 mg of PEG-b-PCNTP was weighed and dissolved in 2 ml of dimethylformamide. This was put in a dialysis membrane having a molecular weight cut off of 3500 and dialyzed against pure water at room temperature in the dark. The dialysis membrane solution was recovered and used as the micelle solution. The concentration of the micelle solution was determined by lyophilizing a predetermined amount of the micelle solution and measuring the weight of the polymer contained therein. From the dynamic light scattering measurement of the obtained micelle solution, formation of a micelle-like aggregate having a monomodal particle size distribution with an average particle size of about 40 nm was confirmed (see FIG. 4).

Evaluation of NO generation ability of polymer micelle type photo-stimulated NO donor by spin trap method Ferrous sulfate 22mg and N- (dithiocarbamoyl) -N-methyl-D-glucamine sodium salt (MGD) 5.5mg Were mixed in pure water sufficiently deoxygenated to prepare an aqueous solution of Fe (II) (MGD) ₂ complex. The sample solution was prepared by mixing so that the molar ratio of the NTP site and the Fe (II) (MGD) ₂ complex in the polymer micelle-type photostimulation-responsive NO donor prepared in Example 2 was 1:20. The sample solution was put in a quartz cell, irradiated with ultraviolet rays (54 mW / cm ² at 365 nm) using a high-pressure mercury lamp, and then the ESR spectrum of this solution was measured. As a result, nitrosated Fe (II) (MGD ) A signal derived from the ₂ complex was confirmed, and it was confirmed that NO was generated from the polymer micelle-type photostimulation-responsive NO donor with light irradiation (see FIG. 5).

Evaluation of polymer micelle-type photostimulation-responsive NO donor by grease test method The concentration of the polymer micelle-type photostimulation-responsive NO donor solution produced in Example 2 was adjusted to 1 mM in terms of NTP units. Then, it was transferred to a quartz cell and irradiated with ultraviolet rays using a high-pressure mercury lamp. The solution was sampled 5, 10, 15 and 30 minutes after the start of irradiation, and the NO concentration in the solution was colorimetrically determined by the grease test method. The grease test is based on quantifying NO ²⁻ and NO ³⁻ in a solution by utilizing the fact that NO generated in water becomes NO ²⁻ and NO ³⁻ through reaction with water and dissolved oxygen. The NO concentration is converted. The grease test was conducted using Dojindo's NO ₂ / NO ₃ Assay Kit-CII (Colorimetric) “Gries Reagent Kit” according to the company's recommended protocol. The amount of NO produced in the solution varied with the irradiation time and irradiation light intensity. For example, when ultraviolet irradiation of 54 mW / cm ² was performed at 365 nm, it was confirmed that NO was generated at about 20 M in 10 minutes and about 60 M in 30 minutes after the start (see FIG. 6).

Cytotoxicity Evaluation of Polymeric Micelle Type Photostimulus-responsive NO Donor Mouse-derived colon cancer cells (colon 26) were seeded on a 96-well plate at 5 × 10 ³ cells / well and cultured for 24 hours. The polymer micelle-type photostimulation-responsive NO donor solution produced in Example 2 was added thereto, and after contacted for 24 hours, the medium was replaced with a new medium, and ultraviolet light (300-390 nm, 34 mW) was used using a mercury lamp. / Cm ² ) for a certain time (maximum 10 minutes). Thereafter, the culture was further continued, and the number of viable cells after 24 hours was evaluated using Cell Counting Kit-8 manufactured by Dojindo. As a result, the NO donor showed no cytotoxicity even at a concentration of 0.4 mg / ml unless irradiated with ultraviolet light, whereas it was irradiated with ultraviolet light after contacting with colon 26 under the same conditions. It was confirmed that the number of living cells was significantly reduced. Since no cytotoxicity was observed even when the same ultraviolet rays were irradiated to colon 26 which was not brought into contact with the micelles, the cytotoxicity observed here was the colon 26 cells incorporating the NO donor. It is considered that a large amount of NO is generated in the cells by irradiating the cells with ultraviolet rays, and the proliferation of cancer cells is suppressed or apoptosis is induced (see FIG. 7).

  As described above, when the NO generating ability of the NO donor according to the present invention was evaluated, NO was not generated at all in the absence of light (in the dark), whereas NO was only applied when irradiated with ultraviolet light of 300 nm or more. It was clarified that the amount depends on the irradiation wavelength, intensity, and time.

  Furthermore, when the photostimulation-responsive NO donor was brought into contact with cancer cells for a certain period of time and then irradiated with ultraviolet light, the effect was evaluated. As a result, a remarkable anticancer effect due to NO generated from the micelles was confirmed. It was.

  ADVANTAGE OF THE INVENTION According to this invention, the polymeric micelle type | mold NO donor which produces | generates NO only when it accumulate | stores in the cancer structure | tissue in a biological body and light is irradiated to an accumulation | aggregation location can be provided. Such a polymeric micelle-type NO donor can be expected to develop cancer treatment by a new photodynamic therapy with few side effects on normal tissues. Therefore, the present invention can be used in the pharmaceutical manufacturing industry.

Claims (3)

Formula (I)

_{A- (CH 2 -CH 2 O)} m -L 1 - (NO-donor) -Z (I)

In the above formula, A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent is a formyl group, the formula R ^a R ^b CH— (where R ¹ and R ² are _{independently,} C 1 -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or -O _{(CH 2)} represents a 4 O- )) Group,
L ₁ is a bond, — (CH ₂ ) _c S—, —CO (CH ₂ ) _c S—, — (CH ₂ ) _c NH—, — (CH ₂ ) _c CO— (where each c is 1 -5, preferably an integer of 1 or 2, and represents a linking group selected from the group consisting of -CO-, -COO- and -CONH-,
NO-donor represents a hydrophobic polymer chain in which a low molecular NO donor is covalently bonded to a side chain;
Z is hydrogen atom, hydroxy, C ₁ -C ₁₂ alkyloxy optionally substituted with one phenyl group or benzhydryl group at the non-bonding end, or substituted with one phenyl group or benzhydryl group at the non-bonding end. Represents a good C ₁ -C ₁₂ alkylcarbonyl,
m represents an integer of 20 to 5,000,
A polymerized derivative of an NO donor represented by: Formula (II)
In the above formula, A represents unsubstituted or substituted C ₁ -C ₁₂ alkoxy, and when substituted, the substituent represents a formyl group or a group of the formula R ¹ R ² CH—, where R ¹ and ^{R 2} are _{independently,} C 1 -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or -O _{(CH 2) 4} Represents O-
L ₁ represents a linking group selected from the group consisting of an atomized bond, — (CH ₂ ) _a S—, —CO (CH ₂ ) _a S—, wherein a is 1 to 5, preferably 2. An integer,
L ₂ represents a bond, —O—, —NH—, — (CH ₂ ) _b —O—, — (CH ₂ ) _b —NH—, — (CH ₂ ) _b —OCO—, — (CH ₂ ) _b. A linking group selected from the group consisting of —NHCO—, — (CH ₂ ) _b —COO—, and — (CH ₂ ) _b —CONH— (note that the bond has a direction in which the leading-is bonded to the benzene ring). B is an integer from 0 to 3,
R is the formula
Represents the group of
At least one R ^1-1 is bonded to the ortho position with respect to the nitro group, and C ₁ -C ₆
Alkyl, C ₁ -C ₆ alkyl substituted with one or more halogen atoms, mono- or di-C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyloxy, di- or tri-C ₁ -C ₆ alkylsilyl, Phenyl, naphthyl, anthracenyl, pyrenyl, phenoxy, pyrrolyl, pyridyl, imidazole, morpholyl, thiophenyl, thiazole, amino, nitro, cyano, hydroxyl, hydroxymethyl, aldehyde, carboxyl, carboxylic acid ester, carboxylic acid amide, sulfo, sulfonic acid ester , Selected from the group consisting of sulfonic acid amides and hydrazides,
p represents an integer of 1 to 4,
-X- is a _{bond, - (CH 2) d -} , and _{- (CH 2) d - (} CH = CH) e- (CH 2) d - is selected from the group consisting of, d is an integer from 1 to 6 E represents an integer of 0 to 3,
L ₂ represents a linking group selected from the group consisting of methylimino, methyliminomethyl, methyloxy, methyloxymethyl, methyl ester and methyl ester methyl,
m is an integer of 20 to 5,000,
n is an integer from 3 to 1,000, and
R may be less than 50% of the total number n of Rs being hydrogen atoms,
A polymerized derivative of an NO donor represented by:   An anticancer agent comprising the polymerized derivative of the NO donor according to claim 1 or 2 as an active ingredient.