KR102413027B1 - High-intensity focused ultrasound responsive nanostructure and nitric oxide delivery system using thereof - Google Patents

Abstract

The present invention relates to a micelle nanostructure carrying an N-heterocyclic carbene nitric oxide radical compound and a nitrogen monoxide release system using the same, and it can efficiently release nitrogen monoxide from a target site, so that it can be used for the treatment and diagnosis of various diseases including cancer. can be applied

Description

High-intensity focused ultrasound responsive nanostructure and nitric oxide delivery system using the same

The present invention relates to a nanostructure containing an N-heterocyclic carben nitric oxide radical compound, which is sensitive to high-intensity focused ultrasound (HIFU), and a nitrogen monoxide delivery system using the same. It is an invention about

According to recent studies, nitric oxide (NO) generated in various cells has been found to have anticancer, immunomodulatory, and antibacterial properties, so research on disease treatment using it is active.

For the treatment of diseases using nitric oxide, nitric oxide is directly administered to a target tissue or cell, or a compound capable of efficiently releasing nitric oxide is synthesized and used. As an example of a compound that releases nitrogen monoxide, Korean Patent Application Laid-Open No. 10-2016-0062697 discloses an N-heterocyclic carbene nitrogen monoxide radical compound that releases nitrogen monoxide at a specific temperature or higher. However, research on efficiently delivering nitric oxide to a desired target tissue or cell, including the Korean Patent Application Laid-Open No. 10-2016-0062697, is still in its infancy.

Korean Patent Publication No. 10-2016-0062697

The present invention is to develop a structure, method, and system capable of efficiently releasing nitrogen monoxide from an N-heterocyclic carbene nitric oxide radical compound at a target site, and to apply it to the treatment of various diseases including cancer.

HIFU (high-intensity focused ultrasound) sensitive micelles formed by self-assembly of an amphiphilic polymer including a hydrophobic polymer and a hydrophilic polymer, and loaded with an N-heterocyclic carbene nitric oxide radical compound represented by the following [Formula 1] Provided are a nanostructure, a nitric oxide delivery system using the same, and a composition for anticancer treatment comprising the same.

[In Formula 1, R ₁ and R ₂ are each independently (C ₁ -C ₃₀ )alkyl, (C ₃ -C ₃₀ )cycloalkyl, (C ₃ -C ₃₀ )heterocycloalkyl, (C ₆ - C ₃₀ )aryl or (C ₃ -C ₃₀ )heteroaryl, wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are each independently (C ₁ -C ₃₀ )alkyl, halo(C ₁ -C ₃₀ ) )alkyl, halogen, cyano, (C ₃ -C ₃₀ )cycloalkyl, (C ₃ -C ₃₀ )heterocycloalkyl, (C ₁ -C ₃₀ )alkoxy, (C ₆ -C ₃₀ )aryloxy, (C ₆ -C ₃₀ )aryl, (C ₆ -C ₃₀ )ar(C ₁ -C ₃₀ )alkyl, (C ₁ -C ₃₀ )alkyl(C ₆ -C ₃₀ )aryl, (C ₃ -C ₃₀ )heteroaryl , (C ₁ -C ₃₀ )alkyl substituted (C ₃ -C ₃₀ )heteroaryl, (C ₆ -C ₃₀ )aryl substituted (C ₃ -C ₃₀ )heteroaryl, mono or di(C ₁ - C ₃₀ )alkylamino, mono or di(C ₆ -C ₃₀ )arylamino, tri(C ₁ -C ₃₀ )alkylsilyl, di(C ₁ -C ₃₀ )alkyl(C ₆ -C ₃₀ )arylsilyl, tri (C ₆ -C ₃₀ ) May be further substituted with one or more selected from the group consisting of arylsilyl, nitro and hydroxy, wherein Z ₁ is -C(R ₃ )- or -N-, wherein R ₃ is hydrogen or (C ₁ -C ₃₀ )alkyl.]

The present invention is a step of 1) irradiating high-intensity focused ultrasound (HIFU) to the nanostructure on which the carbene nitric oxide radical compound is supported, 2) the micelles are dissociated by the HIFU irradiation of the 1) step, and the carbene nitric oxide radical compound is released and 3) provides a nitrogen monoxide release method comprising the step of irradiating HIFU to the carbene nitrogen monoxide radical compound released in step 2) to release nitrogen monoxide.

The nanostructure of the present invention can selectively release nitric oxide to an onset site in the body, such as cancer tissue, non-invasive treatment and diagnosis of disease, and furthermore, effective disease treatment is possible through combination with various drugs.

1 is a graph showing the results of X-ray diffraction analysis (XRD) measurement of the crystal structure of the compound prepared in Example 1 (IMesNO).
2 is a graph comparing and analyzing the electromagnetic resonance (EPR) spectra of the compounds prepared in Examples 1 and 2;
3 is a graph showing cyclic voltammograms of the compound prepared in Example 1. FIG.
Fig. 4 shows the apparatus used in Example 2.
5 and 6 show the NO release behavior and amount of IMesNO, which is an N-heterocyclic carbene nitric oxide compound by HIFU irradiation.
7 shows the manufacturing process of IMesNO@PEG-PCLmicelIe, a micellar nanostructure supported with IMesNO ((a)), the appearance and size of IMesNO@PEG-PCLmicelIe ((b)), and the loading efficiency and loading of IMesNO ( (c)).
FIG. 8 shows fluorescence at each organ and tumor site when IR820@PEG-PCLmicelIe, a micellar nanostructure carrying a fluorescent material, IR820, was injected intravenously into mice.
Figure 9 is a schematic diagram of the state in which NO is released when HIFU is irradiated to the IMesNO nanostructure.
Figure 10 shows an animal test procedure for determining whether NO is released from IMesNO@PEG-PCLmicelIe when irradiating HIFU.
11 shows the results of tumor site-specific NO release after intravenous (IV) injection of IMesNO@PEG-PCLmicelIe, a micellar nanostructure loaded with IMesNO.

Hereinafter, the present invention will be described in detail. Unless otherwise defined, terms used in this specification should be interpreted as content commonly understood by those of ordinary skill in the art. The drawings and embodiments of the present specification are for those skilled in the art to easily understand and practice the present invention, and content that may obscure the gist of the present invention may be omitted from the drawings and embodiments, and the present invention is limited to the drawings and embodiments it's not going to be

The present invention relates to a HIFU (high-intensity focused ultrasound)-sensitive micelle nanostructure formed by self-assembly of an amphiphilic polymer and carrying an N-heterocyclic carbene nitrogen monoxide radical compound, and the delivery of nitrogen monoxide using the same.

The amphiphilic polymer is a polymer including a hydrophobic polymer and a hydrophilic polymer, and can form a micelle nanostructure having hydrophobicity on the inside and hydrophilicity on the outside by self-assembly. Various compounds may be supported inside the micellar nanostructure, and in the present invention, an N-heterocyclic carbene nitric oxide radical compound is supported. Since the N-heterocyclic carbene nitric oxide radical compound has hydrophobic properties, it must be provided with hydrophilic properties or supported on another carrier having hydrophilic properties in order to be delivered well in an in vivo or ex vivo environment. For example, in order to be efficiently delivered in a living body, it must dissolve well in the blood, but in the case of a hydrophobic material, it is difficult to efficiently transfer it because it is not easily soluble in the blood. According to an embodiment of the present invention, an N-heterocyclic carbene nitric oxide radical compound can be supported on micelles using an amphiphilic polymer to effectively deliver it in vivo.

A hydrophobic polymer forming an amphiphilic polymer, such as caprolactone, hydrophobic amino acids, glycolic acid, lactic acid, maleic acid, vinyl acetate, vinyl propionate, vinyl butyrate, styrene, alkylstyrene, chlorostyrene, alkyl acrylate, acrylonitrile And a polymer of one or two or more monomers selected from the group consisting of hydroxy butyrate may be used, but the present invention is not limited thereto.

A polymer of one or more monomers selected from the group consisting of ethylene glycol, ethyleneimine, alkylene oxide, acrylamide, vinyl alcohol, hydrophilic amino acids and sugars can be used as the hydrophilic polymer forming the amphiphilic polymer, but is limited thereto not.

As a preferred amphiphilic polymer, a polyethylene glycol-based multi-copolymer (block or random copolymer) having polyethylene glycol may be used. For example, polyethyleneglycol based diblock copolymer is PEG-PLA double copolymer (Poly(ethylene glycol) methyl ether-block-poly(D, L lactide)), PEG-PLGA double copolymer ( Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide)), PEG-PE double copolymer (Poly(ethylene glycol)-block-polyethylene) and PEG-PS double copolymer (Poly(ethylene glycol) -block-poly(styrene)) may be one or more selected from, but is not limited thereto. And, as a polyethyleneglycol based triblock copolymer, a PEG-PPG-PEG triple copolymer (Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) may be used, As another triple copolymer, PEO-PPO-PEO (Poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide)) may be used, but is not limited thereto.

In addition, as the amphipathic polymer, a polysaccharide-based amphiphilic polymer formed by polymerization of polysaccharides may be used. For example, at least one of dextran, heparin, alginate, and hyaluronic acid is a hydrophobic polymer polycaprolactone, polylactic acid, and poly It may be an amphiphilic polymer (polysaccharide based amphiphilic polymer) formed by forming a polymer with at least one of (lactic-co-glycolate) (PLGA), but is not limited thereto.

According to an embodiment of the present invention, PEG-PLA double copolymer (Poly (ethylene glycol), an amphiphilic polymer formed by polymerization of polyethylene glycol with polycaprolactone hydrophilic polymer as an amphiphilic polymer for forming micelle nanostructures as a hydrophobic polymer ) methyl ether-block-poly(D, L lactide)) is particularly preferred, but is not limited thereto. The inner core of the micellar nanostructure is formed by polycaprolactone among the amphiphilic polymers, and the N-heterocyclic carbene nitric oxide compound having hydrophobic properties is supported on the inner core, so that solubility can be increased.

In the present invention, high-intensity focused ultrasound (hereinafter referred to as 'HIFU') irradiation is used. HIFU is a non-invasive, non-radiative ultrasound that can be irradiated to the inside of a living body, and is intensively irradiated on a specific site in the living body. When this happens, heat is generated rapidly or cavitation occurs, which can coagulate the tissue or burn the tissue to remove it. However, this method of direct treatment by irradiating HIFU on the tissue requires a high level of technology and requires attention because it deforms or physically damages the living tissue. According to the present invention, this difficulty can be solved by using a micelle nanostructure in which an N-heterocyclic carbene nitric oxide radical compound is supported.

The micelle nanostructure loaded with the N-heterocyclic carbene nitric oxide radical compound of the present invention is characterized in that it is sensitive to high-intensity focused ultrasound (hereinafter 'HIFU'). This feature uses HIFU irradiation (stimulation), but because micelles loaded with N-heterocyclic carbene nitric oxide radical compounds sensitive to HIFU are used, it is administered through blood vessels so that it can be delivered to the desired site (systemic) It makes it possible to treat diseases through delivery). When HIFU is irradiated to micelles that have moved to the target site through blood vessels, the micelles are decomposed (dissociated) in response to HIUF, so the N-heterocyclic carbene nitric oxide radical compound supported inside is released to the outside. Subsequently, HIFU is irradiated to the released N-heterocyclic carbene nitric oxide radical compound to release nitric oxide, so intensive treatment using HIFU and nitric oxide is possible.

That is, as a method of delivering the micelle nanostructure of the present invention to a target site, a method of directly administering and delivering to a desired site (local delivery) and a method of administering through a blood vessel so that it can be delivered to a desired site (systemic delivery) method All are available. Among them, the method of delivery through blood vessels is one of the non-invasive methods, and has the advantage of being painless and capable of delivering a large amount of material, but it is difficult to relatively increase the treatment efficiency intensively on the target site, and it is a method of delivering directly to the desired site. The disadvantage is that pain is accompanied at the target site and the administered substance is easily diffused to the surrounding area. However, according to an embodiment of the present invention, the micelle nanostructure of the present invention is hardly delivered to other normal organs even when administered through blood vessels, and can be very effectively delivered to disease-causing sites such as tumor (cancer) sites. Only the advantages of local delivery and systemic delivery can be maximized.

The N-heterocyclic carben nitric oxide radical (N-heterocyclic carben nitric oxide radical) supported on the micellar nanostructure of the present invention may be represented by the following [Formula 1].

) 또는 -N-(

)이고, 상기 R₃는 수소 또는 (C₁-C₃₀)알킬이다.] 본 발명의 일 실시예에 따르면, N-헤테로고리 카벤 일산화질소 라디칼 화합물의 상기 Z1은 -N-일 수 있다. 본 발명의 다른 일 실시예에 따른 N-헤테로고리 카벤 일산화질소 라디칼 화합물의 상기 R₁ 및 R₂는 각각 독립적으로 페닐, 나프틸, 비페닐, 터페닐, 안트릴, 인데닐(indenyl), 플루오레닐, 페난트릴, 트라이페닐레닐, 피렌일, 페릴렌일, 크라이세닐, 나프타세닐 및 플루오란텐일 등에서 선택되는 아릴기이며, 아릴기들은 각각 독립적으로 (C₁-C₁₀)알킬, 할로(C₁-C₁₀)알킬, 할로겐, 시아노, (C₁-C₁₀)알콕시, 나이트로 및 하이드록시 등으로 이루어진 군으로부터 선택되는 하나 이상으로 더 치환될 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 다른 일 실시예에 따른 N-헤테로고리 카벤 일산화질소 라디칼 화합물은 바람직하게 상기 Z₁이 N일 수 있으나 이에 한정되는 것은 아니다. [In Formula 1, R ₁ and R ₂ are each independently (C ₁ -C ₃₀ )alkyl, (C ₃ -C ₃₀ )cycloalkyl, (C ₃ -C ₃₀ )heterocycloalkyl, (C ₆ -C ₃₀ )aryl or (C ₃ -C ₃₀ )heteroaryl, wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are each independently (C ₁ -C ₃₀ )alkyl, halo(C ₁ -C ₃₀ ) alkyl, halogen, cyano, (C ₃ -C ₃₀ )cycloalkyl, (C ₃ -C ₃₀ )heterocycloalkyl, (C ₁ -C ₃₀ )alkoxy, (C ₆ -C ₃₀ )aryloxy, (C ₆ -C ₃₀ )aryl, (C ₆ -C ₃₀ )ar(C ₁ -C ₃₀ )alkyl, (C ₁ -C ₃₀ )alkyl(C ₆ -C ₃₀ )aryl, (C ₃ -C ₃₀ )heteroaryl, (C ₁ -C ₃₀ )alkyl substituted (C ₃ -C ₃₀ )heteroaryl, (C ₆ -C ₃₀ )aryl substituted (C ₃ -C ₃₀ )heteroaryl, mono or di(C ₁ -C ₃₀ )alkylamino, mono or di(C ₆ -C ₃₀ )arylamino, tri(C ₁ -C ₃₀ )alkylsilyl, di(C ₁ -C ₃₀ )alkyl(C ₆ -C ₃₀ )arylsilyl, tri( C ₆ -C ₃₀ ) May be further substituted with one or more selected from the group consisting of arylsilyl, nitro and hydroxy, wherein Z ₁ is -C(R ₃ )-(

) or -N-(

), and R ₃ is hydrogen or (C ₁ -C ₃₀ )alkyl.] According to an embodiment of the present invention, Z1 of the N-heterocyclic carben monoxide radical compound may be —N—. In the N-heterocyclic carben monoxide radical compound according to another embodiment of the present invention, R ₁ and R ₂ are each independently phenyl, naphthyl, biphenyl, terphenyl, anthryl, indenyl, flu orenyl, phenanthryl, triphenylenyl, pyrenyl, peryleneyl, chrysenyl, naphthacenyl, and an aryl group selected from fluoranthenyl, and the aryl groups are each independently (C ₁ -C ₁₀ )alkyl, halo (C ₁ -C ₁₀ ) It may be further substituted with one or more selected from the group consisting of alkyl, halogen, cyano, (C ₁ -C ₁₀ )alkoxy, nitro and hydroxy, but is not limited thereto. In the N-heterocyclic carbene nitrogen monoxide radical compound according to another embodiment of the present invention, Z ₁ may be N, but is not limited thereto.

The N-heterocyclic carbene nitrogen monoxide radical compound represented by [Formula 1] of the present invention may generate nitrogen monoxide by thermal decomposition. According to an embodiment of the present invention, the thermal decomposition may be performed at 80 to 200 ° C. In terms of energy efficiency, preferably 80 to 150 ° C, more preferably 80 to 120 ° C., but is not limited thereto. .

As used herein, "cycloalkyl" means a fully saturated and partially unsaturated hydrocarbon ring of 3 to 9 carbon atoms, and includes cases in which aryl or heteroaryl is fused. In addition, the "aryl" described in the present invention is an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, and is preferably a single or fused group containing 4 to 7, preferably 5 or 6 ring atoms in each ring. It includes a ring system, and may include a form in which a plurality of aryls are connected by a single bond. Specific examples include, but are not limited to, phenyl, naphthyl, biphenyl, anthryl, indenyl, fluorenyl, and the like. In addition, the "heteroaryl" described in the present invention is an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, and includes one or more heteroatoms selected from B, N, O, S, P(=O), Si and P may be a monocyclic or polycyclic aromatic hydrocarbon radical containing 3 to 8 ring atoms, and a single or fused ring system comprising suitably 3 to 7, preferably 5 or 6 ring atoms in each ring It includes, and may include a form in which a plurality of heteroaryls are connected by a single bond.

In the present invention, C ₁ -C _n is C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ ... Like C _n , it may include each of 1 to n carbon atoms.

The substituents including "alkyl", "alkoxy" and other "alkyl" moieties described in the present invention may include both straight-chain or pulverized forms.

The N-heterocyclic carbene nitric oxide radical compound according to the present invention can not only be used as a bioavailable source of nitrogen monoxide, but also can provide nitrogen monoxide in high yield from an external heat source, resulting in a sufficient level of nitrogen monoxide (NO) It can be used for diseases that require In addition, the compound according to the present invention can emit nitrogen monoxide with high efficiency by minimizing the generation of harmful active gases such as nitrous oxide (N ₂ O) during thermal decomposition.

The N-heterocyclic carbene nitrogen monoxide radical compound of the present invention may be preferably represented by the following [Formula 2].

The N-heterocyclic carbene nitrogen monoxide radical compound of the present invention may be preferably represented by the following [Formula 2] and may be classified according to a substituent.

In [Formula 2] , Ar ₁ and Ar ₂ are each independently phenyl, naphthyl, biphenyl, terphenyl, anthryl, indenyl, fluorenyl, phenanthryl, triphenylenyl, pyrenyl, peryleneyl, a substituted or unsubstituted aryl group selected from chrysenyl, naphthacenyl and fluorantenyl; R ₁₁ and R ₁₂ are each independently hydrogen, (C ₁ -C ₁₀ )alkyl, halo(C ₁ -C ₁₀ )alkyl, halogen, cyano, (C ₁ -C ₁₀ )alkoxy, nitro and hydroxy is selected, and p and q are each independently selected from an integer of 0 to 3.]

[In Formula 2, Ar ₁ and Ar ₂ are each independently phenylene, naphthylene, biphenylene, terphenylene, anthrylene, indenylene, fluorenylene, phenanthrylene, triphenylenylene, pyrenylene , an arylene group selected from peryleneylene, chrysenylene, naphthacenylene and fluorantenylene; R ₁₁ and R ₁₂ are each independently hydrogen, (C ₁ -C ₁₀ )alkyl, halo(C ₁ -C ₁₀ )alkyl, halogen, cyano, (C ₁ -C ₁₀ )alkoxy, nitro and hydroxy is selected, and at least one of R ₁₁ and R ₁₂ has a substituent other than hydrogen at the ortho-position of the arylene group based on -N- of imidazole; p and q are each independently selected from an integer of 1 to 3.]

The N-heterocyclic carbene nitrogen monoxide radical compound of the present invention may be preferably represented by the following [Formula 3].

[In Formula 3, R ₁₃ to R ₁₈ are each independently hydrogen, (C ₁ -C ₇ )alkyl, halo (C ₁ -C ₇ )alkyl, halogen, cyano, (C ₁ -C ₇ )alkoxy, nite and hydroxy, and at least one of R ₁₃ , R ₁₅ , R ₁₆ and R ₁₈ has a substituent other than hydrogen.]

More preferably, the (C ₁ -C ₇ )alkyl is a straight-chain alkyl of methyl, ethyl, propyl, butyl, butyl, pentyl, heptyl, or hexyl.

The N-heterocyclic carbene nitrogen monoxide radical compound of the present invention can be prepared through a simple and economical method using the N-heterocyclic carbene compound.

According to an embodiment of the present invention, the N-heterocyclic carbene compound represented by the following [Formula 4] and nitrogen oxide are reacted with the N-heterocyclic carbene compound represented by the following [Formula 1] for the preparation of the nitrogen oxide radical compound. -preparing a heterocyclic carbene nitric oxide radical compound.

[In Formulas 1 and 4, R ₁ and R ₂ are each independently (C ₁ -C ₃₀ )alkyl, (C ₃ -C ₃₀ )cycloalkyl, (C ₃ -C ₃₀ )heterocycloalkyl, ( C ₆ -C ₃₀ )aryl or (C ₃ -C ₃₀ )heteroaryl, wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are each independently (C ₁ -C ₃₀ )alkyl, halo(C ₁ -C ₃₀ )alkyl, halogen, cyano, (C ₃ -C ₃₀ )cycloalkyl, (C ₃ -C ₃₀ )heterocycloalkyl, (C ₁ -C ₃₀ )alkoxy, (C ₆ -C ₃₀ )aryloxy , (C ₆ -C ₃₀ )aryl, (C ₆ -C ₃₀ )ar(C ₁ -C ₃₀ )alkyl, (C ₁ -C ₃₀ )alkyl(C ₆ -C ₃₀ )aryl, (C ₃ -C ₃₀ ) )heteroaryl, (C ₁ -C ₃₀ )alkyl substituted (C ₃ -C ₃₀ )heteroaryl, (C ₆ -C ₃₀ )aryl substituted (C ₃ -C ₃₀ )heteroaryl, mono or di( C ₁ -C ₃₀ )alkylamino, mono or di(C ₆ -C ₃₀ )arylamino, tri(C ₁ -C ₃₀ )alkylsilyl, di(C ₁ -C ₃₀ )alkyl(C ₆ -C ₃₀ )aryl It may be further substituted with one or more selected from the group consisting of silyl, tri(C ₆ -C ₃₀ )arylsilyl, nitro and hydroxy; Z ₁ is -C(R ₃ )- or -N-, and R ₃ is hydrogen or (C ₁ -C ₃₀ )alkyl.] In the manufacturing method according to an embodiment of the present invention, a higher reactivity In terms of realizing a high yield within a short time, Z ₁ may be -N-. In addition, in the manufacturing method according to an embodiment of the present invention, nitrogen monoxide can be effectively generated by thermal decomposition, and in terms of minimizing the generation of by-products, preferably, R ₁ and R ₂ are each independently phenyl , an aryl group selected from naphthyl, biphenyl, terphenyl, anthryl, indenyl, fluorenyl, phenanthryl, triphenylenyl, pyrenyl, peryleneyl, chrysenyl, naphthacenyl and fluoranthenyl and the aryl groups are each independently composed of (C ₁ -C ₁₀ )alkyl, halo (C ₁ -C ₁₀ )alkyl, halogen, cyano, (C ₁ -C ₁₀ )alkoxy, nitro and hydroxy. It may be further substituted with one or more selected from the group, but is not limited thereto.

A preferred N-heterocyclic carbene compound in the preparation method of the present invention may be represented by the following [Formula 5], but is not limited thereto.

[In Formula 5, Ar ₁ and Ar ₂ are each independently phenylene, naphthylene, biphenylene, terphenylene, anthrylene, indenylene, fluorenylene, phenanthrylene, triphenylenylene, pyrenylene , an arylene group selected from peryleneylene, chrysenylene, naphthacenylene and fluorantenylene; R ₁₁ and R ₁₂ are each independently hydrogen, (C ₁ -C ₁₀ )alkyl, halo(C ₁ -C ₁₀ )alkyl, halogen, cyano, (C ₁ -C ₁₀ )alkoxy, nitro and hydroxy is selected, and at least one of R ₁₁ and R ₁₂ has a substituent other than hydrogen at the ortho-position of the arylene group based on -N- of imidazole; p and q are each independently selected from an integer of 1 to 3.]

In the present invention, the N-heterocyclic carbene compound can be stably and tumor-specifically delivered in micelles, and can selectively release nitric oxide to a target site in response to HIFU. In addition, according to the type of the N-heterocyclic carbene compound, micelle loading efficiency, tumor specificity, or nitric oxide release efficiency may be different. This difference is because steric properties, steric effects, or electronic effects may differ depending on the type of the N-heterocyclic carbene compound. Among the N-heterocyclic carbene compounds, compounds having a similar three-dimensional structure or electron effect may have similar stability in micelles or a release degree of nitric oxide. For example, when the steric hindrance caused by the substituent in the N-heterocyclic carbene compound is large, stability in micelles, loading efficiency, nitrogen monoxide release efficiency, etc. may be rapidly reduced. According to an embodiment of the present invention, when the steric hindrance caused by the substituent of aryl or arylene connected to nitrogen of the imidazole moiety of the N-heterocyclic carbene compound is large in the N-heterocyclic carbene compound, stability, loading efficiency, and monoxide in micelles of the N-heterocyclic carbene compound The nitrogen release efficiency may not be good.

In the preparation method of the present invention, the reaction of the N-heterocyclic carbene compound and nitrogen oxide may be carried out at 80 to 40° C., preferably at 80 to 0° C., after mixing each reactant, and then at room temperature (23° C.) to 40 The reaction may be carried out at ℃, but is not limited thereto. The reaction time may be carried out for 10 minutes to 5 hours, and when Z ₁ is -N-, high purity is maintained and reactivity is excellent, and the reaction is performed for 10 minutes to 1 hour to obtain the desired N-heterocyclic carben monoxide Nitrogen radical compounds can be obtained.

When heat is applied to the N-heterocyclic carbene nitrogen monoxide radical compound of the present invention, the compound may decompose and release nitrogen monoxide. According to an embodiment of the present invention, nitrogen monoxide may be generated when heat of 80 ° C or higher is applied, and if the temperature is 80 ° C or higher, it is not particularly limited, but 80 to 200 ° C, preferably 80 to 150 ° C in terms of energy efficiency, More preferably, it is good to be carried out at 80 to 120 ℃.

The N-heterocyclic carbene nitrogen monoxide radical compounds of the present invention can release nitrogen monoxide while decomposing in response to HIFU like a micellar nanostructure. According to an embodiment of the present invention, the amount of nitrogen monoxide emitted tends to increase in proportion to the HIFU irradiation intensity and time. And, even when HIFU is irradiated with an intensity that does not damage the tissue, nitrogen monoxide can be effectively released, so it can be used for non-invasive treatment.

The irradiation time of HIFU in the present invention is 25 to 100 seconds, 25 to 75 seconds, 25 to 50 seconds, 1 to 100 seconds, 1 to 90 seconds, 1 to 75 seconds, 1 to 60 seconds, 1 to 50 seconds, 1 to 40 seconds, 1 to 30 seconds, 1 to 25 seconds, 15 to 100 seconds, 15 to 75 seconds, 15 to 60 seconds, 15 to 45 seconds, 15 to 30 seconds, preferably 20 to 30 seconds or limited thereto not.

In the present invention, the irradiation intensity of HIFU is 25 to 100W, 25 to 75W, 25 to 50W, 1 to 100W, 1 to 90W, 1 to 75W, 1 to 60W, 1 to 50W, 1 to 40W, 1 to 30W, 1 to 25W, 15 to 100W, 15 to 75W, 15 to 60W, 15 to 45W, 15 to 30W, preferably 40 to 60W, but is not limited thereto.

In the present invention, the N-heterocyclic carbene nitric oxide radical compounds, which can be represented by [Formula 1] to [Formula 5], can release nitrogen monoxide, so they have good room for biomedical applications such as disease treatment, but various solvents such as water It is a stable compound that is not readily soluble in sodium, so it is difficult to easily use it for the treatment of diseases. However, according to the present invention, the N-heterocyclic carbene nitric oxide radical compound can be effectively delivered to the site of disease by utilizing the excellent solubility and biocompatibility characteristics of the nanostructure, which can be effectively used for disease treatment.

The present invention provides a system capable of delivering nitrogen monoxide to a target site based on the HIFU sensitivity of a micelle nanostructure loaded with an N-heterocyclic carbene nitric oxide radical compound.

The micelle nanostructure carrying the N-heterocyclic carbene nitric oxide radical compound of the present invention can be effectively delivered to a target site where a disease or the like has occurred in the body. The micellar nanostructure delivered to the target site is decomposed by irradiation with high-intensity ultra-focused ultrasound (HIFU) to release the N-heterocyclic carbene nitric oxide radical compound supported therein. When HIFU is irradiated to the released N-heterocyclic carbene nitric oxide radical compound, nitrogen monoxide is released from the N-heterocyclic carbene nitric oxide radical compound, and the nitrogen monoxide is finally delivered to the target site.

In addition, by utilizing the micelle nanostructure of the present invention, there is provided a method capable of releasing nitrogen monoxide when necessary at a desired site. For example, 1) after administering the N-heterocyclic carbene nitric oxide radical compound-supported nanostructure, irradiating HIFU (high-intensity focused ultrasound) to the target site to which the nanostructure or nanostructure is to be delivered; 2) a step in which micelles are dissociated by HIFU irradiation in step 1) to release a carbene nitric oxide radical compound and 3) a step in which carbene nitric oxide radical compound released in step 2) is irradiated with HIFU to release nitrogen monoxide Nitric oxide may be released.

The micellar nanostructures carrying the N-heterocyclic carbene nitric oxide radical compound of the present invention can be delivered more specifically to the tumor site, and by utilizing this, a composition for anticancer treatment including the micellar nanostructures can be provided. .

The micellar nanostructure of the present invention can be utilized for more effective disease treatment by supporting other drugs together with the N-heterocyclic carbene nitric oxide radical compound. And, in addition to the method of directly supporting the drugs on the micelle nanostructure, a method of physically or chemically binding the drugs can also be used for disease treatment.

The micellar nanostructure carrying the N-heterocyclic carbene nitrogen monoxide radical compound of the present invention is prepared by combining a hydrophilic polymer and a hydrophobic polymer to prepare an amphiphilic polymer, and an amphiphilic polymer and an N-heterocyclic carbene nitrogen monoxide radical compound It can be prepared according to the steps of dissolving in a solvent by mixing, evaporating the solvent and then forming the micellar nanostructures through sonication. According to an embodiment of the present invention, the solvent for dissolving the amphiphilic polymer and the N-heterocyclic carbenyl nitrogen oxide radical compound is preferably chloroform, but is not limited thereto.

The method for manufacturing a micelle nanostructure of the present invention is simple and very economical, and the N-heterocyclic carbene nitric oxide radical compound can be efficiently supported on the micelle nanostructure. According to an embodiment of the present invention, it may have a loading efficiency of 65%, 66%, 67%, 68%, 69%, 70% or more.

The size of the micelle nanostructure on which the N-heterocyclic carbene nitric oxide radical compound of the present invention is supported has an average particle diameter of 50 to 200 nm, 50 to 150 nm, 50 to 100 nm, 50 to 80 nm, 60 to 200 nm, 60 to 150 nm, 60 to 100 nm, 60 to 80 nm, 65 to 85 nm, 65 to 75 nm, preferably 60 to 70 nm, more preferably 65 to 70 nm. Since the size of the micellar nanostructure loaded with the N-heterocyclic carbene nitric oxide radical compound of the present invention is suitable for selectively accumulating through the blood vessels of the tissue in which the tumor (cancer) is formed, it may hardly accumulate in normal tissues. .

The N-heterocyclic carbene nitric oxide radical compound supported on the micellar nanostructure of the present invention is 2 to 10% by weight, 2 to 9% by weight, 2 to 8% by weight, 2 to 7% by weight, 3 to 10 based on the total weight wt%, 3-9 wt%, 3-8 wt%, 3-7 wt%, 4-10 wt%, 4-9 wt%, 4-8 wt%, 4-7 wt%, 5-10 wt% , 5-9 wt%, 5-8 wt%, 5-7 wt%, 6-10 wt%, 6-9 wt%, 6-8 wt% may be supported.

In the present invention, the tumor (cancer) is one selected from breast cancer, ovarian cancer, bladder cancer, lung cancer, kidney cancer, uterine cancer, liver cancer, prostate cancer, pancreatic cancer, stomach cancer, bone cancer, skin cancer, epithelial cancer and malignant tumor, but is not limited thereto.

Examples for carrying out the present invention will be described below. The examples correspond to one example for carrying out the present invention, and the present invention should not be construed as being limited by the examples.

Hereinafter, Examples 1 and 2 of the present invention were carried out in a glove box maintaining a nitrogen atmosphere, and solvents such as benzene, diethyl ether, toluene, and tetrahydrofuran (THF) were purified by distillation.

The chemical structure of the target compound was confirmed by NMR (model: Bruker DRX 500 spectrometer), electron-impact GC/MS spectrum, and electron paramagnetic resonance (Bruker X-band A200 spectrometer). Electrochemical performance was confirmed by measuring cyclic voltammograms (Princeton Applied Research (PAR) 273A potentiostat).

Example One- 1.N -heterogory Caven nitric oxide compound IMesNO's Produce

In a glovebox maintaining a nitrogen atmosphere, 1,3-bis-(2,4,6-trimethylphenyl)imidazolylidene ( I Mes (1b), 1.00 g, 2.57 mmol, 1.00 equiv in 25 mL toluene in a 40 mL vial. ) was added, and the solution was heated to 78°C. After nitric oxide (1.00 equiv) was supplied to the solution several times in the form of a gas, it was confirmed that the colorless solution changed to dark brown. The solution was heated to 23 °C and reacted for 1 hour. After the solvent was removed in vacuo, the obtained solid was suspended in 5 mL of ether (Et2O), filtered, and washed with ether (3×1 mL). It was then vacuum dried to give a light orange solid (560 mg). The solid was left at 30 °C for 48 hours to obtain a brown solid, which was washed with ether (Et2O) (3 x 0.5 mL), and the target compound (2b) as a yellow solid was prepared (760 mg, yield = 72%) ).

¹ H NMR (500 MHz, C ⁶ D ⁶ , 23° C., δ): 1.60 (s, br).

Anal. Calcd. for C21H24N3O: C, 75.42; H, 7.23; N, 12.56; found: C, 75.22; H, 6.96; N, 12.42.

In addition, the crystal structure of the target compound (2b) was shown in FIG. 1 by X-ray diffraction analysis (XRD), and 0.1 mg of the target compound (2b) was dissolved in 1.0 mL benzene in a glove box maintaining a nitrogen atmosphere. The ERP signal and its intensity were confirmed at 23 °C (electron paramagnetic resonance spectra were measured) (FIG. 2). Cyclic voltammetry of the target compound (2a) in acetonitrile containing 1.2 mM target compound (2a) in 0.1 M NBu ⁴ BF ⁴ Scan rate with respect to the reference electrode Ag/AgCl (KCl 3.5M) By scanning the electron potential of the electrode from 1.2 to 0.8 at 0.1 V/sec, the potential of the electrode was measured (the cyclic voltammetry characteristic) (FIG. 3).

Example 1-2. N-heterocyclic Caven nitric oxide compound IPrNO's Produce

In a glovebox maintaining a nitrogen atmosphere, 1,3-bis-(2,6-diisopropylphenyl)imidazolylidene (IPr (1a), 1.00 g, 2.57 mmol, 1.00 equiv) dissolved in 25 mL of toluene was added to a 40 mL vial. and the solution was heated to 78°C. After nitric oxide (1.00 equiv) was supplied to the solution several times in the form of a gas, it was confirmed that the colorless solution changed to dark brown. The solution was heated to 23 °C and reacted for 1 hour. After the solvent was removed in vacuo, the obtained solid was suspended in 5 mL of ether (Et2O), filtered, and washed with ether (3×1 mL). It was then vacuum dried to give a light orange solid (560 mg). The solid was left at 30° C. for 48 hours to obtain a brown solid, which was washed with ether (Et 2 O) (3 × 0.5 mL) and dried under vacuum to prepare the target compound (2a). (172 mg, yield = 68%).

¹ H NMR (500 MHz, C6D6, 23° C., δ): 1.60 (s, br).

Anal. Calcd. for C27H36N3O (a light orange solid): C, 77.47; H, 8.67; N, 10.04; found: C, 77.62; H, 8.51; N, 9.60.

Anal. Calcd. for C27H36N3O (a brown solid): C, 77.47; H, 8.67; N, 10.04; found: C, 77.30; H, 8.34; N, 9.58.

The crystal structure of the target compound (2a) was confirmed by X-ray diffraction analysis (XRD), electron paramagnetic resonance spectra were measured, and cyclic voltammetry characteristics were measured.

N-heterocyclic Caven nitric oxide compound IMesNO's NO Emission Confirmation

In a glove box maintaining a nitrogen atmosphere, the apparatus shown in FIG. 4 was installed, and 2a (5.0 mg, 12.1 μmol) prepared in Example 1 was put into the first tube. 1b (0.50 mg, 1.6 μmol) dissolved in 0.5 mL of benzene was added to the second tube. At this time, the apparatus was maintained in a vacuum state, and the second tube was maintained at 78 °C. Then, while the first tube was heat-treated at 120 °C for 1 hour, the second tube was maintained at 23 °C. Electron paramagnetic resonance spectra of the solution in the second tube were measured (FIG. 2). From this, it was found that nitrogen monoxide could be effectively released during thermal decomposition of the N-heterocyclic carbene nitrogen monoxide radical compound according to the present invention (FIG. 2). In addition, 2b causes an irreversible oxidation-reduction reaction (FIG. 3).

HIFU (High-intensity focused ultrasound) IMesNO's NO Emission Confirmation

By irradiating the compound (IMesNO) prepared in Example 1 with high-intensity focused ultrasound (HIFU), the degree of NO emission was analyzed using NO Analyzer (NO Analyzer, NOA 280i chemiluminescence) to analyze the NO emission behavior and emission amount did

Specifically, a mixture of 1 mg of IMesNO in 40 ml of DPBS (Dulbecco's Phosphate buffered saline) was irradiated with high-intensity focused ultrasound (VIFU 2000, Alpinion Medical Systems, Repubilc of Korea), and the unit of NO released according to the intensity of high-intensity focused ultrasound Emission amount per surface area ([NO] _t b), time (t _d ^c ), maximum concentration [NO] _m ^d , its required time (t _m ^e ), half-life (t _1/2 ^f ), etc. were measured (FIG. 5) and Fig. 6). As a result, NO was detected when the temperature was higher than a certain temperature, and as the temperature increased, a large amount of NO was released within a short time, but it was confirmed that the emission time decreased. In addition, when the intensity of high-intensity focused ultrasound was 20W, NO was released when irradiated for 100 seconds, and when the intensity was 50W and 100W, the longer the irradiation time, the more NO was released and the time was longer, but the half-life was was found to be similar. In addition, when irradiating high-intensity focused ultrasound in vivo , it was confirmed that the intensity of high-intensity focused ultrasound is preferably 100 W or less, and the irradiation time is 50 seconds or less, considering the condition that the tissue is not subjected to thermal shock due to high energy. .

IMesNO supported micelles Nanostructure manufacturing

Polyethylene glycol having a molecular weight of 5000 (Sigma-Aldrich Co. (St. Louis, MO)) 1.0 g and polycaprolactone (Sigma-Aldrich Co. (St. Louis, MO)) 0.685 g and Sn(Oct) ₂ (Sigma- Aldrich Co. (St. Louis, MO)) 0.008 g as a catalyst is combined through a ring opening polymerization method to make an amphiphilic polymer. Briefly, these three compounds are dissolved in anhydrous toluene, purged with nitrogen, and then polymerized under high temperature of 100 for 24 hours. After that, after cooling to room temperature, the precipitate is collected using diethyl ether. The precipitated compound is filtered and dried under vacuum for 48 hours to obtain the final polymer. After dissolving the synthesized polymer and IMesNO prepared in Example 1 in 1 ml chloroform, the solvent was rapidly evaporated to form a film, and then about 3 ml of DPBS (Dulbecco's Phosphate buffered saline) was slowly dropped thereinto, followed by ultrasonication for 3 hours. (Sonication) treatment to prepare IMesNO-supported micellar nanostructures (hereinafter 'IMesNO@PEG-PCLmicelIe') (FIG. 7 (a)). As a result of confirming IMesNO@PEG-PCLmicelIe using dynamic light scattering and transmission electron microscopy, it has an average size of about 70 nm (67.89±19 nm), and IMesNO has an efficiency of about 69% (69.1%) through ultraviolet-visible spectroscopy. It was supported on the micellar nanostructures, and it was confirmed that the loading amount was about 7 wt% (6.91 wt%) (FIG. 7 (c)).

IMesNO supported micelles Confirmation of delivery to the target site of the nanostructure and release of NO

Example 5-1. Confirmation of delivery of nanostructures to the tumor site

In vivo ( in vivo ), it was confirmed whether the micellar nanostructure could be delivered to the target site through blood vessels (systemic delivery). Since IMesNO itself is not a fluorescence material, an IR820 probe (Sigma-Aldrich (St. Louis, USA)) that fluoresces in the near-infrared light was used instead of IMesNO in the nanostructure. The IR820 probe is similar to the method in which IMesNO was previously loaded onto PEG-PCLmicelIe. In the last step, when the ultrasonic treatment was performed, it was processed for 1 hour to prepare a micellar nanostructure on which IR820 was supported. Experimental mice were xenografted with breast cancer cells 4T1 (1 X 10 ⁶ ) into BALB/C females between 6 and 8 weeks of age. When the size of the cancer is about 10 to 13 mm ³ , micelles loaded with IR820 are intravenously injected into mice for 3 hours, 6 hours, and 24 hours using an In Vivo Imaging System (IVIS) (Califer). Lifescience, Hopkinton, MA) was used to confirm that the nanostructure was delivered to the target site. As a result, even in the case of a relatively short time of 3 hours, the material was delivered well, and in particular, the intensity of the fluorescence signal in the cancer tissue was the highest compared to other organs (FIG. 8). That is, it was confirmed that the micellar nanostructures (PEG-PLCmicelle) had excellent systemic delivery properties in vivo through blood vessels.

Example 5-2. delivered to the tumor IMesNO supported micelles in nanostructures HIFU Confirmation of NO emission according to irradiation

It was expected to have selective NO release properties when high-intensity focused ultrasound was irradiated to the micellar nanostructures (IMesNO@PEG-PCLmicelIe) loaded with IMesNO delivered in vivo (FIG. 9). To confirm this, a probe (4,5-DiaminofIuorescein diacetate, DAF-2DA) (Sigma-Aldrich Co. (St. Louis, MO)) that fluoresces at a wavelength of 540 nm when NO is present was used. Fluorescence was confirmed using a small animal fluorescence imaging system (IVIS) (Califer Lifescience, Hopkinton, MA). 100 μl (1 mg/ml) of IMesNO@PEG-PCLmicelIe prepared in Example 4 was first injected into an intravenous injection (IV) of an experimental mouse. After 24 hours of accumulating micelles as cancer targets, 50 μl of DAF-2DA is injected directly into the tumor site (local injection), and high-intensity focused ultrasound is applied to the target site cancer tissue for 10 seconds at 50 W or 25 seconds at 25 W for 10 seconds. or irradiated for 25 seconds. As a result, fluorescence by DAF-2DA was observed only in cancer tissue, the target site irradiated with high-intensity focused ultrasound, through which IMesNO@PEG-PCLmicelIe was specifically delivered to cancer tissue, and NO was released in response to HIFU. could check And when HIFU was irradiated with high intensity (50W) for a long time (25s), it was found that the fluorescence by DAF-2DA was high, and it was confirmed that the NO emission was the largest (FIG. 11).

As the specific parts of the present invention have been described in detail above, it will be clear to those of ordinary skill in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

HIFU (high-intensity focused ultrasound) sensitive micellar nanostructure formed by self-assembly of polyethylene glycol and polycaprolactone, and carrying N-heterocyclic carbene nitric oxide radical compound represented by the following formula:

.
delete delete delete According to claim 1,
A micelle nanostructure having an average particle diameter of 50 to 200 nm.
According to claim 1,
The N-heterocyclic carbene nitric oxide radical compound is supported in an amount of 2 to 10% by weight based on the total weight of the micelle nanostructure.
Nitrogen monoxide delivery based on HIFU (high-intensity focused ultrasound) sensitivity of micellar nanostructure formed by self-assembly of polyethylene glycol and polycaprolactone and carrying N-heterocyclic carbene nitrogen monoxide radical compound represented by the following formula system:

.
[Claim 7] A composition for treating a tumor comprising the nanostructure according to any one of claims 1, 5 and 6.

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