WO2023176796A1 - Scar formation inhibitor, fibroblast proliferation inhibitor, and method for producing nanoparticles - Google Patents

Abstract

The problem to be solved by the present invention is to provide a novel scar formation inhibitor having, as an active ingredient, a compound that has not been conventionally known to have an effect for inhibiting scar formation. The present invention provides a scar formation inhibitor containing a compound represented by general formula (I) or a salt thereof [see description, etc., for R1, R2, R3, R4, and Y].

Description

Scar formation inhibitor, fibroblast proliferation inhibitor, and method for producing nanoparticles

[Cross reference to related applications]
This application claims priority based on Japanese Patent Application No. 2022-040442, filed on March 15, 2022, the entire disclosure of which is incorporated herein by reference. The present invention relates to a scar formation inhibitor, a fibroblast proliferation inhibitor, and a method for producing nanoparticles.

Scars, which are scars that remain after cuts, burns, ulcers, etc., have healed, are not only a problem in appearance, but can also be accompanied by pain, itching, etc., so it is desirable to suppress their formation. Another problem is that scarring that occurs after glaucoma surgery may impair the effectiveness of the surgery. Specifically, glaucoma is a disease in which the outlet of the aqueous humor that fills the eye is narrowed and intraocular pressure increases, and filtration surgery is performed to drain the aqueous humor outside the eye. However, the problem is that the new aqueous humor drainage channel is blocked as the filtering bleb becomes scarred, causing the intraocular pressure to gradually rise again. It has been found that mitomycin C (antimetabolite) suppresses scarring, but continuous use causes severe ocular tissue damage, and the permeability of water-soluble molecules to the corneal and conjunctival membranes is extremely low. Therefore, when mitomycin C is used as an eye drop, it hardly reaches the surgical scar and is not expected to be effective, so its application is limited to intraoperative application. Therefore, the challenge is to find a drug and its drug form that can easily and long-term suppress scarring even after surgery.

Patent No. 6479485

An object of the present invention is to provide a novel scar formation inhibitor containing as an active ingredient a compound whose effect on inhibiting scar formation has not been known so far.

Under such circumstances, the present inventors continued their intensive research and found that the above problems could be solved by using a compound represented by general formula (I) or a salt thereof, which will be described later. The present invention is based on this new knowledge. Accordingly, the present invention provides the following items:

Item 1. A scar formation inhibitor containing a compound represented by the following general formula (I) or a salt thereof:

[In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different, hydrogen, optionally substituted C1-18 alkyl, optionally substituted C1-18 alkoxy, substituted C7-18 aralkyloxy or optionally substituted phenoxy.
Two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form an optionally substituted ring together with the carbon atom to which these groups are bonded.
Y is optionally substituted C1-18 alkyl, optionally substituted C7-18 aralkyl, optionally substituted phenyl, optionally substituted C1-18 alkoxy, optionally substituted C7-18 aralkyloxy, optionally substituted phenoxy, optionally substituted mono C1-18 alkylamino, optionally substituted mono C7-18 aralkylamino, or optionally substituted monophenylamino show].

Item 2. Fibroblast proliferation inhibitor containing a compound represented by the following general formula (I) or a salt thereof:

[In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different, hydrogen, optionally substituted C1-18 alkyl, optionally substituted C1-18 alkoxy, substituted C7-18 aralkyloxy or optionally substituted phenoxy.
Two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form an optionally substituted ring together with the carbon atom to which these groups are bonded.
Y is optionally substituted C1-18 alkyl, optionally substituted C7-18 aralkyl, optionally substituted phenyl, optionally substituted C1-18 alkoxy, optionally substituted C7-18 aralkyloxy, optionally substituted phenoxy, optionally substituted mono C1-18 alkylamino, optionally substituted mono C7-18 aralkylamino, or optionally substituted monophenylamino show].

Item 3. In general formula (I), R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents hydrogen, optionally substituted C1-18 alkyl, or optionally substituted C7-18 aralkyloxy. show,
Y represents optionally substituted C1-18 alkyl,
Item 1. The scar formation inhibitor according to Item 1 or the fibroblast proliferation inhibitor according to Item 2.

Section 4. The scar formation inhibitor according to Item 1, the fibroblast proliferation inhibitor according to Item 2, or the scar formation inhibitor according to Item 3, which contains nanoparticles containing the compound represented by general formula (I) or a salt thereof. agent or fibroblast proliferation inhibitor.

Item 5. A method for producing nanoparticles, comprising a step of injecting a water-miscible organic solvent solution of a compound represented by the following general formula (I) or a salt thereof into water:

[In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different, hydrogen, optionally substituted C1-18 alkyl, optionally substituted C1-18 alkoxy, substituted C7-18 aralkyloxy or optionally substituted phenoxy.
Two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form an optionally substituted ring together with the carbon atom to which these groups are bonded.
Y is optionally substituted C1-18 alkyl, optionally substituted C7-18 aralkyl, optionally substituted phenyl, optionally substituted C1-18 alkoxy, optionally substituted C7-18 aralkyloxy, optionally substituted phenoxy, optionally substituted mono C1-18 alkylamino, optionally substituted mono C7-18 aralkylamino, or optionally substituted monophenylamino show].

Item 6. In general formula (I), R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents hydrogen, optionally substituted C1-18 alkyl, or optionally substituted C7-18 aralkyloxy. show,
Y represents optionally substituted C1-18 alkyl,
The method described in Section 5.

According to the present invention, it is possible to provide a novel scar formation inhibitor containing as an active ingredient a compound whose effect on inhibiting scar formation has not been known so far.

FIG. 1 shows a SEM image (left side of FIG. 1) of the nanoparticle dispersion prepared in Production Example (1) and a graph (left side of FIG. 1) of changes over time in the particle size distribution of nanoparticles. FIG. 2 shows a SEM image (left side of FIG. 2) of a highly concentrated nanoparticle dispersion prepared in Production Example (2) and a graph (left side of FIG. 2) of changes over time in particle size distribution of nanoparticles. 1 shows the results of cell proliferation inhibition evaluation using nanoparticles in Example 1. The results of HE staining of mouse eyeball samples in Example 2 are shown. FIG. 4 shows the results of Picrosirius red staining of the mouse eyeball sample in Example 2. 2 shows an SEM image and a graph of particle size distribution of nanoparticles of Compound 5a in Production Example (14). 2 shows an SEM image and a graph of particle size distribution of nanoparticles of Compound 5b in Production Example (15). 2 shows a SEM image and a graph of particle size distribution of nanoparticles of Compound 5a in Production Example (16). 2 shows a SEM image and a graph of particle size distribution of nanoparticles of Compound 5b in Production Example (17). FIG. 10(A): Shows a graph of the Alamar Blue assay in Example 3. Figure 10(B): Shows a micrograph of HTF. FIG. 10(C): Shows a graph showing the temporal effect of 0.5 μM N-Col on the viability of HTF. FIG. 10(D): A graph showing the relationship between HTF proliferation and N-Col treatment time. FIG. 10(E): A graph showing the relationship between TGF-β1-induced HTF proliferation and N-Col treatment time. FIG. 11(A): Shows a photograph of HTF in Example 3. FIG. 11(B): Shows a graph showing the relationship of wound area to incubation time. FIG. 11(C): Shows a micrograph in transwell assay. FIGS. 11(D) and (E): Graphs showing the results of statistical analysis of invasive cells. FIG. 12(A): A photograph of the collagen gel culture kit in Example 5 is shown. FIG. 12(B): A graph showing the results of statistical analysis of the remaining gel area. Figure 12(C): Shows the results of statistical analysis of α-SMA expression detected by Western blot and banding. FIG. 12(D): Shows the results of α-SMA immunostaining. Figure 12(E): Shows the results of F-actin immunostaining. FIG. 13(A): Shows the results of Ki67 antibody staining in Example 6. Figure 13(B): Shows the analysis results of Ki67 staining. FIG. 13(C): Shows the results of indirect TUNEL method. Figure 13(D): Shows the results of quantitative analysis of TUNEL. FIG. 14(A): Shows the results of Western blotting in Example 7. 14(B) to (F): Shows the quantitative results of Western blot in Example 7. FIG. 14(G): Shows the results of immunostaining for caspase-3. 10 shows a graph of the results of the alamar blue assay in Example 8.

Scar formation inhibitor In one embodiment, the present invention provides a scar formation inhibitor comprising a compound represented by the following general formula (I) or a salt thereof:

[In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different, hydrogen, optionally substituted C1-18 alkyl, optionally substituted C1-18 alkoxy, substituted C7-18 aralkyloxy or optionally substituted phenoxy.
Two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form an optionally substituted ring together with the carbon atom to which these groups are bonded.
Y is optionally substituted C1-18 alkyl, optionally substituted C7-18 aralkyl, optionally substituted phenyl, optionally substituted C1-18 alkoxy, optionally substituted C7-18 aralkyloxy, optionally substituted phenoxy, optionally substituted mono C1-18 alkylamino, optionally substituted mono C7-18 aralkylamino, or optionally substituted monophenylamino show]. In the present invention, the compound represented by general formula (I) may be simply referred to as compound (I).

In the present invention, when a group included in general formula (I) has a substituent, unless otherwise specified, the substituent includes an alkyl group, a dialkylamino group, a monoalkylamino group, an alkoxy group. , an alkoxycarbonyl group, an alkoxycarbonyloxy group, a halogen group, and the like. When a group included in general formula (I) has a substituent, the number of substituents per said group is, for example, 1 to 3, 1 to 2, unless it is specified otherwise. , 1, etc.

In the present invention, "C1-18 alkyl", "C7-18 aralkyl", "phenyl", "C1-18 alkoxy", "C7-18 aralkyloxy", "phenoxy", "mono C1 "-18 alkylamino", "mono C7-18 aralkylamino" or "monophenylamino" may be substituted, and such substituents include dialkylamino group, monoalkylamino group, alkoxy group, alkoxycarbonyl group, alkoxy Examples include carbonyloxy group and halogen group.

As mentioned above, in general formula (I), two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form a ring together with the carbon atom to which these groups are bonded. Examples of such a ring include a 5- to 7-membered ring (9- to 11-membered ring if the number of carbon atoms in the benzene ring to which R ¹ , R ² , R ³ and R ⁴ are bonded is included). Such a ring may be substituted, and examples of such substituents include an alkyl group, a dialkylamino group, a monoalkylamino group, an alkoxy group, an alkoxycarbonyl group, an alkoxycarbonyloxy group, and a halogen group.

In the present invention, the term "alkyl group" refers to a linear or branched saturated hydrocarbon group, such as a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-undecyl group, dodecyl group , C1-C18 alkyl groups such as tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, etc., preferably C1-C10 alkyl group, more preferably C1-C8 alkyl group etc. More preferred are C1-C6 alkyl groups, particularly preferred are C1-C4 alkyl groups.

In the present invention, the "alkoxy group" includes an alkoxy group in which the alkyl moiety is the alkyl group described above. More specifically, examples of the alkoxy group include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group. group, isopentyloxy group, n-hexyloxy group, n-heptyloxy group, n-octyloxy group, n-nonyloxy group, n-decyloxy group, n-undecyloxy group, dodecyloxy group, tridecyloxy group , a C1-C18 alkoxy group such as a tetradecyloxy group, a pentadecyloxy group, a hexadecyloxy group, a heptadecyloxy group, an octadecyloxy group, etc., preferably a C1-C10 alkoxy group, and more preferably Examples include a C1-C8 alkoxy group, more preferably a C1-C6 alkoxy group, particularly preferably a C1-C4 alkoxy group.

In the present invention, the "alkoxycarbonyl group" includes an alkoxycarbonyl group in which the alkoxy moiety is the aforementioned alkoxy group. More specifically, examples of the alkoxycarbonyl group include methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, isopropoxycarbonyl group, n-butoxycarbonyl group, isobutoxycarbonyl group, sec-butoxycarbonyl group, tert-butoxycarbonyl group, n-pentyloxycarbonyl group, isopentyloxycarbonyl group, n-hexyloxycarbonyl group, n-heptyloxycarbonyl group, n-octyloxycarbonyl group, n-nonyloxycarbonyl group, n-decyl Oxycarbonyl group, n-undecyloxycarbonyl group, dodecyloxycarbonyl group, tridecyloxycarbonyl group, tetradecyloxycarbonyl group, pentadecyloxycarbonyl group, hexadecyloxycarbonyl group, heptadecyloxycarbonyl group, octadecyloxycarbonyl group C1-C18 alkoxycarbonyl groups such as groups, preferably C1-C10 alkoxycarbonyl groups, more preferably C1-C8 alkoxycarbonyl groups, still more preferably C1-C6 alkoxycarbonyl groups, etc. Among them, C1-C4 alkoxycarbonyl group is particularly preferred.

In the present invention, the "alkoxycarbonyloxy group" includes an alkoxycarbonyloxy group in which the alkoxy moiety is the aforementioned alkoxy group. More specifically, examples of the alkoxycarbonyloxy group include methoxycarbonyloxy group, ethoxycarbonyloxy group, n-propoxycarbonyloxy group, isopropoxycarbonyloxy group, n-butoxycarbonyloxy group, and isobutoxycarbonyloxy group. , sec-butoxycarbonyloxy group, tert-butoxycarbonyloxy group, n-pentyloxycarbonyloxy group, isopentyloxycarbonyloxy group, n-hexyloxycarbonyloxy group, n-heptyloxycarbonyloxy group, n-octyloxy group Carbonyloxy group, n-nonyloxycarbonyloxy group, n-decyloxycarbonyloxy group, n-undecyloxycarbonyloxy group, dodecyloxycarbonyloxy group, tridecyloxycarbonyloxy group, tetradecyloxycarbonyloxy group, penta Examples include C1-C18 alkoxycarbonyloxy groups such as decyloxycarbonyloxy group, hexadecyloxycarbonyloxy group, heptadecyloxycarbonyloxy group, and octadecyloxycarbonyloxy group, and preferably C1-C10 alkoxycarbonyloxy group. More preferred are C1-C8 alkoxycarbonyloxy groups, still more preferred are C1-C6 alkoxycarbonyloxy groups, and particularly preferred are C1-C4 alkoxycarbonyloxy groups.

In the present invention, the "aryl group" refers to an aromatic hydrocarbon ring group, such as a C6-C14 aryl group such as a phenyl group, a naphthyl group, an anthracenyl group, and preferably a C6-C10 aryl group. , more preferably a phenyl group.

In the present invention, the "aralkyl group" includes, for example, an aralkyl group in which the aryl part is the above-mentioned aryl group and the alkyl part is the above-mentioned alkyl group. More specifically, for example, the aralkyl group includes a benzyl group, 2-phenylethyl group, 3-phenylpropyl group, 1-phenyl-propan-2-yl group, 4-phenyl-butyl group, 4-phenylbutane group. -2-yl group, 2-methyl-3-phenylpropyl group, 2-methyl-1-phenylpropan-2-yl group, 5-phenylpentyl group, 6-phenylhexyl group, 7-phenylheptyl group, 8- C7-18 aralkyl such as phenyloctyl group, 9-phenylnonyl group, 10-phenyldecyl group, 11-phenylundecyl group, 12-phenyldodecyl group, naphthylmethyl group, 2-naphthylethyl group, anthracenylmethyl group, etc. Examples include C7-C10 aralkyl groups, and more preferably C7-C8 aralkyl groups.

In the present invention, the "aralkyloxy group" includes, for example, an aralkyloxy group in which the aralkyl moiety is the aralkyl group described above. More specifically, for example, as the aralkyl group, benzyloxy group, 2-phenylethyloxy group, 3-phenylpropyloxy group, 1-phenyl-propan-2-yloxy group, 4-phenyl-butyloxy group, 4-phenyl-butyloxy group, -phenylbutan-2-yloxy group, 2-methyl-3-phenylpropyloxy group, 2-methyl-1-phenylpropan-2-yloxy group, 5-phenylpentyloxy group, 6-phenylhexyloxy group, 7- Phenylheptyloxy group, 8-phenyloctyloxy group, 9-phenylnonyloxy group, 10-phenyldecyloxy group, 11-phenylundecyloxy group, 12-phenyldodecyloxy group, naphthylmethyloxy group, 2-naphthylethyl Examples include C7-18 aralkyloxy groups such as oxy and anthracenylmethyloxy groups, preferably C7-C10 aralkyloxy groups, and more preferably C7-C8 aralkyloxy groups.

In the present invention, the "monoalkylamino group" includes, for example, the above-mentioned amino group having one alkyl group. More specifically, the monoalkylamino group includes, for example, N-methylamino group, N-ethylamino group, N-n-propylamino group, N-isopropylamino group, N-n-butylamino group, N- -isobutylamino group, N-sec-butylamino group, N-tert-butylamino group, N-n-pentylamino group, N-isopentylamino group, N-n-hexylamino group, N-n-heptylamino group group, N-n-octylamino group, N-n-nonylamino group, N-n-decylamino group, N-n-undecylamino group, N-dodecylamino group, N-tridecylamino group, N-tetradecyl Examples include mono-C1-C18 alkylamino groups such as amino group, N-pentadecylamino group, N-hexadecylamino group, N-heptadecylamino group, and N-octadecyl group, preferably mono-C1-C10 alkylamino group. More preferred are mono C1-C8 alkylamino groups, even more preferred are mono C1-C6 alkylamino groups, and particularly preferred are mono C1-C4 alkylamino groups.

In the present invention, the "dialkylamino group" includes, for example, the above-mentioned amino group having two alkyl groups. The two alkyl groups contained in the dialkylamino group may be the same or different. More specifically, the dialkylamino group includes, for example, monomethylamino group, N,N-diethylamino group, N-methyl-N-ethylamino group, N,N-di-n-propylamino group, N-methyl -Nn-propylamino group, N,N-diisopropylamino group, N,N-di-n-butylamino group, N,N-diisobutylamino group, N,N-di-sec-butylamino group, N , N-di-tert-butylamino group, N,N-di-n-pentylamino group, N,N-diisopentylamino group, N,N-di-n-hexylamino group, N,N-di-n-hexylamino group, -n-heptylamino group, N,N-di-n-octylamino group, N,N-di-n-nonylamino group, N,N-di-n-decylamino group, N,N-di-n-un Decylamino group, N,N-didodecylamino group, N,N-ditridecylamino group, N,N-ditetradecylamino group, N,N-dipentadecylamino group, N,N-dihexadecylamino group group, di-C1-C18 alkylamino groups such as N,N-diheptadecylamino group, N,N-dioctadecyl group, etc., preferably di-C1-C10 alkylamino group, more preferably di-C1-C10 alkylamino group. Examples include -C8 alkylamino group, more preferably di-C1-C6 alkylamino group, particularly preferably di-C1-C4 alkylamino group.

In the present invention, the "monoaralkyl amino group" includes, for example, the above-mentioned amino group having one aralkyl group. More specifically, the monoaralkyl amino group includes, for example, N-benzylamino group, N-2-phenylethylamino group, N-3-phenylpropylamino group, and N-1-phenyl-propan-2-ylamino group. group, N-4-phenyl-butylamino group, N-4-phenylbutan-2-ylamino group, N-2-methyl-3-phenylpropylamino group, N-2-methyl-1-phenylpropan-2- ylamino group, N-5-phenylpentylamino group, N-6-phenylhexylamino group, N-7-phenylheptylamino group, N-8-phenyloctylamino group, N-9-phenylnonylamino group, N- 10-phenyldecylamino group, N-11-phenylundecylamino group, N-12-phenyldodecylamino group, N-naphthylmethylamino group, N-2-naphthylethylamino group, N-anthracenylmethylamino group Examples include C7-18 aralkylamino groups such as, preferably C7-C10 aralkylamino groups, and more preferably C7-C8 aralkylamino groups.

In the present invention, examples of the "halogen group" include chlorine, fluorine, bromine, and iodine.

In the present invention, in general formula (I), R ¹ , R ² , R ³ and R ⁴ are the same or different and are hydrogen, optionally substituted C1-18 alkyl, or optionally substituted C7- Preferably it represents 18aralkyloxy. It is preferable that 1 to 2 of R ¹ , R ² , R ³ and R ⁴ represent "optionally substituted C7-18 aralkyloxy", and one represents "optionally substituted C7-18 aralkyloxy". It is more preferable to represent "aralkyloxy". When one or two of R ¹ , R ² , R ³ and R ⁴ represent "optionally substituted C7-18 aralkyloxy", R ³ represents "optionally substituted C7-18 aralkyloxy" ” or a total of two of R ³ and one of R ¹ , R ² and R ⁴ preferably represent “optionally substituted C7-18 aralkyloxy”. Furthermore, it is preferable that 1 to 3 of R ¹ , R ² , R ³ and R ⁴ represent "optionally substituted C1-18 alkyl", and 2 represent "optionally substituted C1-18 alkyl". -18 alkyl” is preferred. When 2 to 3 of R ¹ , R ² , R ³ and R ⁴ represent "optionally substituted C1-18 alkyl", R ² and R ⁴ represent "optionally substituted C1-18 alkyl" 18 alkyl" or a total of three of R ² and R ⁴ and one of R ¹ and R ³ preferably represents "optionally substituted C1-18 alkyl."

In general formula (I), Y represents "optionally substituted C1-18 alkoxy", "optionally substituted C1-18 alkyl", or "optionally substituted mono-C1-18 alkyl". It is preferable to indicate "amino". When Y represents "optionally substituted C1-18 alkoxy", the "C1-18 alkoxy" moiety is preferably C1-8 alkoxy, more preferably C2-7 alkoxy, and more preferably C3-6 alkoxy. . When Y represents "optionally substituted C1-18 alkyl", the "C1-18 alkyl" moiety is preferably C1-9 alkyl, more preferably C2-8 alkoxy, and more preferably C3-7 alkoxy. , C4-6 alkoxy are more preferred. When Y represents "optionally substituted mono C1-18 alkylamino", the "C1-18 alkyl" moiety is preferably C1-8 alkyl, more preferably C2-7 alkoxy, and C3-6 alkoxy is More preferred.

In the present invention, the term "salt" refers to a pharmaceutically acceptable salt. Further, the salt of compound (I) includes an acid addition salt and a base salt. Specific examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, phosphate, oxalate, malonate, and succinic acid. Salt, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethane Examples include organic acid salts such as sulfonates, and acidic amino acid salts such as glutamate and aspartate. Specific examples of salts with bases include alkali metal or alkaline earth metal salts such as sodium salts, potassium salts or calcium salts, salts with organic bases such as pyridine salts and triethylamine salts, and bases such as lysine and arginine. Examples include salts with natural amino acids.

Compound (I) or a salt thereof may exist in the form of a hydrate or solvate, and therefore, these hydrates and solvates are also included in compound (I), which is an active ingredient of the present invention. be done. Furthermore, when compound (I) has isomers such as geometric isomers, stereoisomers, optical isomers, etc., these isomers are included in the compounds of the present invention unless otherwise specified.

Examples of solvents that form solvates include water, alcohols such as ethanol and propanol, organic acids such as acetic acid, esters such as ethyl acetate, ethers such as tetrahydrofuran and diethyl ether, ketones such as acetone, DMSO, etc. Illustrated.

Compound (I) can be produced by various methods, for example, by the method shown in the reaction formula below.

[Reaction formula-1]

[In the formula, R ¹ , R ² , R ³ , R ⁴ and Y are as described above]

In Reaction Formula-1, Compound 2 can be obtained by first oxidizing Compound 1. Examples of the oxidizing agent used include pyridinium chlorochromate, pyridinium dichromate, dimethyl sulfoxide/oxalyl chloride, tetrapropylammonium perruthenate, Dess-Martin periodinane, 2,2,6,6-tetramethylpiperidine 1- Examples include oxyl free radicals. The ratio of Compound 1 and the oxidizing agent to be used is not particularly limited, but for example, the latter can be used in an amount of 1 to 2 moles per 1 mole of the former. The reaction can be carried out in the presence of additives such as triethylamine and molecular sieves. Moreover, the reaction temperature of the above reaction is not particularly limited, and the reaction is usually performed at room temperature, cooling, or heating. Preferably, the temperature is 0 to 25°C. The reaction time for the above reaction is also not particularly limited, but the reaction can be carried out for 1 to 24 hours, for example.

Next, Compound 2 can be further oxidized to obtain Compound 3. Examples of the oxidizing agent used include potassium permanganate, hypochlorous acid, and chromium trioxide. The ratio of Compound 1 and the oxidizing agent to be used is not particularly limited, but for example, the latter can be used in an amount of 1 to 2 moles per 1 mole of the former. The reaction can be carried out in the presence of additives such as sodium dihydrogen phosphate, 2-methyl-2-butene, and sulfuric acid. Moreover, the reaction temperature of the above reaction is not particularly limited, and the reaction is usually performed at room temperature, cooling, or heating. Preferably, the temperature is 0 to 25°C. The reaction time for the above reaction is also not particularly limited, but the reaction can be carried out for 1 to 24 hours, for example.

Next, compound (I) can be obtained by reacting compound 3 with deacetylcolchicine 4. The ratio of the compound 3 and deacetylcolchicine 4 to be used is not particularly limited, but for example, the latter can be used in a range of 1 to 1.5 mol per 1 mol of the former. Moreover, the reaction temperature of the above reaction is not particularly limited, and the reaction is usually performed at room temperature, cooling, or heating. Preferably, the temperature is 0 to 25°C. The reaction time for the above reaction is also not particularly limited, but the reaction can be carried out for 1 to 24 hours, for example.

These reactions are usually carried out using conventional solvents that do not adversely affect the reaction, such as dichloromethane, triethylamine, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, diethyl ether diglyme, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, etc. It is carried out in a mixed solvent of

Method for producing nanoparticles The present invention provides nanoparticles containing compound (I) or a salt thereof. In the present invention, nanoparticles refer to particles having an average particle diameter of less than 1 μm. In typical embodiments, nanoparticles of the invention are generally spherical. In the present invention, the average particle diameter can be measured by scanning electron microscopy (SEM) or dynamic light scattering (DLS). The average particle diameter of the nanoparticles of the present invention is preferably 10 to 500 nm, more preferably 10 to 100 nm. The nanoparticles of the present invention can typically be prepared by injecting a water-miscible organic solvent solution of compound (I) or its salt into water and dispersing it by the method described below. Therefore, in a typical embodiment, the nanoparticles of the present invention are organic nanocrystals of compound (I) or a salt thereof, and therefore consist essentially of compound (I) or a salt thereof. However, to the extent that the effects of the present invention can be obtained, the nanoparticles of the present invention may contain components other than compound (I) or its salt (for example, additives to the water-miscible organic solvent used in the nanoparticle manufacturing method). May contain. For example, the content of compound (I) or a salt thereof in the nanoparticles of the present invention is preferably 95% by mass or more, more preferably 99% by mass or more, and even more preferably 99.9% by mass or more. By making Compound (I) or its salt into nanoparticles, it has excellent permeability through the ocular surface (corneoconjunctiva) compared to other drug forms, and even after surgery, the drug can be administered through the conjunctiva by instillation. Delivery is expected to suppress scarring at the surgical site in the long term.

The nanoparticles of the present invention can be produced by a method that includes a step of injecting a solution of compound (I) or a salt thereof in a water-miscible organic solvent into water. In the method of the present invention, first, a solution of compound (I) or a salt thereof in a water-miscible organic solvent is injected into water using a syringe. The water-miscible organic solvent is not particularly limited as long as it is a good solvent for compound (I) or its salt, and examples thereof include tetrahydrofuran, acetone, dioxane, acetonitrile, methanol, ethanol, propanol, N-methylpyrrolidone, dimethylsulfoxide, etc. From the viewpoint of solubility and safety, acetone, tetrahydrofuran, ethanol, dimethyl sulfoxide and the like are preferred. These solvents can be used alone or in combination. The content of compound (I) or its salt in a water-miscible organic solvent solution of compound (I) or its salt is not particularly limited, but is, for example, 0.1 to 15% by mass, preferably 0.1 to 10% by mass. It can be set appropriately within the range of %. In addition to compound (I) or its salt, polysorbate 80 (PS80), polyvinylpyrrolidone (PVP), etc. may be added to the water-miscible organic solvent solution. The amount of the water-miscible organic solvent solution of compound (I) or its salt to be injected into water is also not particularly limited, but for example, 0.1 to 1 ml of the water-miscible organic solvent solution to 10 ml of water, preferably 0. .1 to 0.2 ml can be added. Although the injection time is not particularly limited, it is preferable to inject in 0.1 to 1 second, and more preferably in 0.1 to 0.2 seconds. The temperature of the reaction system in this step is also not particularly limited, but can be appropriately set, for example, in the range of 0 to 30°C, preferably 10 to 20°C. In one embodiment of the invention, it is preferred to stir the water-miscible organic solvent solution after pouring it into water. The stirring speed is not particularly limited, but can be appropriately set, for example, in the range of 1000 to 1500 rpm, preferably 1200 to 1500 rpm. The temperature in the stirring step is the same as in the injection step. The stirring time is not particularly limited, but can be appropriately set, for example, in the range of 1 to 10 seconds, preferably 1 to 3 seconds. Injection of the water-miscible organic solvent solution into water and optionally stirring results in crystallization or particleization of compound (I) or its salt, resulting in an aqueous dispersion of nanoparticles of compound (I) or its salt. can be obtained. In one embodiment of the present invention, the obtained nanoparticles can be used as a dispersion liquid while being dispersed in water. When used as a dispersion, the water-miscible organic solvent used for dissolving compound (I) or its salt and adding it to water is preferably removed before use from the viewpoint of safety. The method for removing the organic solvent is not particularly limited, and any known method can be used, such as distillation under reduced pressure (or normal pressure), dialysis, or the like. In another embodiment of the present invention, the obtained nanoparticles can be isolated and used as a fine powder by performing a solid-liquid separation operation such as filtration on the dispersion. When using the above manufacturing method, for example, except for using compound (I) or a salt thereof as a raw material, the method can be performed with reference to the description in Patent Document 1. The compound of the present invention or a salt thereof is preferable because when nanoparticles are formed as described above, a highly concentrated dispersion (eg, 0.1 to 10 mM, preferably 1 to 10 mM dispersion) can be obtained.

In the present invention, even if compound (I) or a salt thereof itself is used as a scar formation inhibitor, various pharmaceutically acceptable carriers (such as isotonic agents, chelating agents, stabilizers, pH adjusting agents, etc.) may be used. , preservatives, antioxidants, solubilizing agents, thickening agents, etc.) as anti-scarring compositions.

The scarring inhibitor of the present invention can be used as a medicament for the treatment of diseases or conditions that can be treated by inhibiting scarring. In such embodiments, diseases or conditions that can be treated by inhibiting scarring include, but are not limited to, scarring of the filtering vesicles after glaucoma filtering surgery, interstitial lung disease, renal fibrosis, and liver fibrosis. For example,

Examples of tonicity agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and sodium chloride, potassium chloride, and calcium chloride. Examples include inorganic salts.

Examples of the chelating agent include edetate salts such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salt, hexamethalin. Examples include acid soda and citric acid.

Examples of the stabilizer include sodium hydrogen sulfite.

Examples of the pH adjuster include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate, or hydrogen carbonate. Examples include salts, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like.

Examples of preservatives include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorhexidine gluconate, benzalkonium chloride, and chloride. Examples include quaternary ammonium salts such as benzethonium and cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.

Examples of antioxidants include sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, and the like.

Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, and the like.
Examples of the thickening agent include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, and the like.

Furthermore, the above composition may further contain, in addition to compound (I) or a salt thereof, a compound known to have a scar formation inhibiting effect. Examples of compounds known to have an inhibitory effect on scar formation include mitomycin, tranilast, and the like.

In the embodiment of the composition, the content of compound (I) or its salt in the composition is not particularly limited, and is, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more in terms of the content of compound (I). It can be set as appropriate from conditions such as at least 30% by mass, at least 10% by mass, at least 5% by mass, and at least 1% by mass.

The formulation form is not particularly limited; for example, oral preparations such as tablets, pills, capsules, powders, granules, syrups, and sublingual preparations; eye drops, injections (intravenous injection, intramuscular injection, local injection, etc.) , gargles, drops, external preparations (ointments, creams, patches, inhalants), and parenteral preparations such as suppositories. Among the above formulations, preferred examples include eye drops.

The content of the compound (I) of the present invention in the preparation varies depending on the route of administration, age, weight, symptoms, etc. of the patient and cannot be unconditionally defined, but the daily dose of the compound (I) is usually about 10 to 5000 mg, More preferably, the amount may be about 100 to 1000 mg. When administering once a day, this amount may be contained in one preparation, and when administering three times a day, one third of this amount may be contained in one preparation.

The medicament of the present invention is administered to patients such as mammals. Mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep, and the like.

Fibroblast proliferation inhibitor In the present invention, compound (I) or a salt thereof has a fibroblast proliferation inhibitory effect and can inhibit scar formation. Therefore, in another embodiment, the present invention provides a fibroblast proliferation inhibitor comprising Compound (I) or a salt thereof. The active ingredient, formulation form, amount used, etc. of the fibroblast proliferation inhibitor can be selected in the same manner as the preventive or therapeutic agent of the present invention. Furthermore, the fibroblast proliferation inhibitor of the present invention can also suppress the proliferation of fibroblasts by adding it to fibroblasts in vitro. Furthermore, the fibroblasts may be isolated cells, or may be in the form of cell clusters, tissue pieces, or the like. Examples of these fibroblasts include those derived from humans, mice, rats, guinea pigs, monkeys, dogs, cats, Xenopus, and the like. In vitro proliferation of fibroblasts can be inhibited, for example, by adding compound (I) or a salt thereof to fibroblasts. The amount added is not particularly limited, but can be set so that, for example, the final concentration in the reaction system is in the range of 0.1 to 100 μM, preferably 1 to 10 μM.

In the method of the present invention, fibroblasts may be used while placed in a solution such as a medium. As the medium, any medium commonly used for culturing animal cells can be used as appropriate. Such a medium includes, for example, Dulbecco's modified Eagle's medium. Further, serum, buffer, antibiotics, etc. may be added to the medium as appropriate.

Specific embodiments of the present invention will be described below in detail by way of examples, but the present invention is not limited to these examples.

Production example (1) (S)-4-(benzyloxy)-3,5-dimethyl-2-(2-methyl-4-oxo-4-((1,2,3,10-tetramethoxy-9- Oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)amino)butan-2-yl)phenylisobutyl carbonate ((S)-4-(benzyloxy)-3,5-dimethyl-2 -(2-methyl-4-oxo-4-((1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)amino)butan- Production of 2-yl)phenyl isobutyl carbonate)

Deacetylcolchicine 4 (173.2 mg, 0.485 mmol), compound 3' (206.0 mg, 0.481 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in an eggplant flask equipped with a stirrer bar. Salt (EDC·HCl) (182.1 mg, 0.950 mmol) and 4-dimethylaminopyridine (DMAP) (10.7 mg, 0.0466 mmol) were added and dissolved in 5.0 mL of dichloromethane. After stirring at room temperature for 2 hours, the residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography (chloroform/methanol = 100/0-50/1) to give the title compound (319.7 mg, 86%). Obtained as a pale yellow solid. Hereinafter, the title compound may be referred to as compound 5.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 1.04-1.06 (6H, m), 1.35-1.42 (1H, m), 1.58 (3H, overlaps with peak of water ), 1.65 (3H, s), 2.04-2.17 (1H, m), 2.24-2.39 (9H, m), 2.69 (1H, d, J=13.4Hz ), 3.60 (3H, s), 3.73 (3H, s), 3.90 (3H, s), 3.94 (3H, s), 4.12-4.16 (1H, m) , 4.19-4.23 (1H, m), 4.30-4.37 (1H, m), 4.60 (1H, d, J=10.8Hz), 4.69 (1H, d, J = 10.8Hz), 6.14 (1H, d, J = 7.1Hz), 6.40 (1H, s), 6.71 (2H, d, J = 11.0Hz), 6.77 ( 1H, s), 7.16-7.19 (2H, m), 7.34-7.47 (5H, m)

Production example (2) Method for producing nanoparticles of compound 5 of the present invention 100 μL of an acetone solution of compound 5 adjusted to 11.1 mM was injected into 9 mL of water at room temperature using a syringe, and stirred at 1500 rpm for 2 seconds. , an aqueous dispersion of nanoparticles was obtained. When preparing the acetone solution, 10 wt% of the compound polysorbate 80 was added. After removing acetone, the volume was determined to be 10 mL, and mixed with a 9% aqueous sodium chloride solution at a ratio of 9:1 to prepare. The final concentration of the aqueous dispersion was 0.1 mM. SEM and DLS revealed a particle size of approximately 200 nm. The SEM image of the nanoparticle dispersion of Compound 5 obtained above is shown in the right photograph of FIG. The graph on the left of Figure 1 shows the change in particle size distribution of nanoparticles over time.

Production Example (3) Method for producing high-concentration nanoparticles of Compound 5 100 μL of an acetone solution of Compound 5 adjusted to 72.2 mM was injected into 9 mL of water at room temperature using a syringe, and stirred at 1500 rpm for 2 seconds. , an aqueous dispersion of nanoparticles was obtained. When preparing the acetone solution, 10 wt% of the compound polysorbate 80 was added. After removing acetone, the volume was determined to be 10 mL, and mixed with a 9% aqueous sodium chloride solution at a ratio of 9:1 to prepare. The final concentration of the aqueous dispersion was 0.65mM. SEM and DLS revealed a particle size of approximately 200 nm. The SEM image of the highly concentrated nanoparticles of Compound 5 obtained above is shown in the left photograph of FIG. The graph on the right side of Figure 2 shows the change in particle size distribution of nanoparticles over time.

As shown in the above results, by using Compound 5, it was possible to form nanoparticles that were stable for a long period of time. The ocular surface layer consists of a hydrophobic corneal epithelium, a hydrophilic corneal stroma, and a tight junction in the outermost layer, which has low permeability to common eye drops. On the other hand, nanoparticles prepared from Compound 5 are thought to pass through tight junctions due to their small size, and are thought to pass through hydrophobic corneal epithelium because they are an aggregate of hydrophobic compounds. It also has hydrophilic properties and is expected to penetrate the hydrophilic corneal stroma.

Example 1
Human fibroblasts were seeded in a 96-well plate, and 24 hours later, deacetylcolchicine and nanoparticles of Compound 5 prepared in Production Example (2) above were added to a final concentration of 10-1000 nM. After a further 24 hours, alamar blue reagent was added, and absorbance was measured 2 hours later. The results are shown in Figure 3. As shown in FIG. 3, the nanoparticles of Compound 5 showed approximately the same growth-inhibiting activity as deacetylcolchicine.

Example 2
A filtering bleb model was created in both eyes of an 8-week-old male C57BL/6J mouse using the following procedure. A mixed solution of ketamine (180 mg/kg), xylazine (90 mg/kg), and physiological saline was administered intraperitoneally for anesthesia.
Approximately one-fourth of the upper conjunctiva was removed using spring scissors to expose the sclera.
An anterior chamber puncture was performed from the sclera in the limbus using a 30-gauge needle to create a leakage path for the aqueous humor.
The exposed sclera was covered with the peeled conjunctiva, and the conjunctiva was sutured at two locations with 10-0 nylon at the limbus.
Immediately after surgery, ofloxacin ointment was applied to prevent infection.
From the day after surgery, 10 μl of high-concentration colchicine nano eye drops or physiological saline (control) eye drops were instilled into the eyes of two animals each four times a day (9:00, 1:00 p.m., 5:00 p.m., and 8:00 p.m.). In the above step, an aqueous dispersion of high-concentration nanoparticles of Compound 5 prepared in Production Example (3) above was used as the high-concentration colchicine nano-eye drop.
On the 7th and 14th days after surgery, one mouse each received high-concentration colchicine nano-eye drops and physiological saline eye drops.The mouse was euthanized by cervical dislocation, the eyeball was enucleated, and the mouse was fixed in 4% paraformaldehyde. Each sample obtained was subjected to HE staining or Picrosirius red staining (collagen staining). The results of HE staining are shown in FIG. 4, and the results of Picrosirius red staining are shown in FIG. As shown in FIGS. 4 and 5, when physiological saline was instilled, the filtering bleb was blocked by scarring, but when highly concentrated colchicine derivative nanoparticles were instilled into the eye, the filtering bleb structure was maintained.

Production Example (4) Production of 4-(benzyloxy)-2-(4-((tert-butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5-dimethylphenol (compound 7)

Compound 6 (10.7 g, 34.0 mmol) and 4-dimethylaminopyridine (DMAP) (6.32 g, 51.7 mmol) were placed in an eggplant flask equipped with a stirrer bar, and added to 50.0 mL of tetrahydrofuran (THF) at 0°C. It was dissolved in After adding 50.0 mL of a THF solution of tert-butyldimethylsilyl chloride (TBSCl) (6.44 g, 42.7 mmol) to the obtained melt, the obtained melt was reacted at room temperature for 15 hours. The resulting reaction product was purified by silica gel column chromatography (hexane/ethyl acetate = 100/0-1/1) to obtain Compound 7 (13.3 g, 91%) as a pale yellow solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 0.021 (6H, s), 0.872 (9H, s), 1.60 (6H, s), 2.07 (2H, t, J = 6.6Hz), 2.20 (3H, s), 2.41 (3H, s), 3.63 (2H, t, J=6.6Hz), 4.70 (2H, s), 5.75 (1H, s), 6.47 (1H, s), 7.32-7.36 (1H, m), 7.38-7.42 (2H, m), 7.46-7.49 (2H , m).

Production example (5) 4-(benzyloxy)-2-(4-((tert-butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5-dimethylphenyl 4-methylpentanoate (compound 8a) manufacturing stirrer bar Compound 7 (117.5 mg, 0.274 mmol) and 4- _{dimethylaminopyridine} (DMAP) (19.6 mg, 0.160 mmol) were placed in a _round bottom flask equipped with It was dissolved in After adding 4-methylvaleryl chloride (0.035 mL, 0.255 mmol) and triethylamine (TEA) (0.040 mL, 0.287 mmol) to the resulting lysate, the resulting lysate was incubated at room temperature for 24 hours. Made it react. The obtained reaction product was purified by silica gel column chromatography (chloroform) to obtain Compound 8a (115.0 mg, 80%) as a yellow oil.

^1H -NMR (CDCl ₃ , 400MHz): δ = -0.019 (6H, s), 0.850 (9H, s), 0.965 (6H, d, J = 6.4Hz), 1.47 (6H, s), 1.64-1.67 (3H, m), 2.06 (2H, t, J=7.6Hz), 2.25 (3H, s), 2.47 (3H, s ), 2.54 (2H, t, J=7.7Hz), 3.51 (2H, t, J=7.6Hz), 4.73 (2H, s), 6.56 (1H, s), 7.33-7.37 (1H, m), 7.39-7.43 (2H, m), 7.47-7.49 (2H, m).

Production Example (6) Production of 4-(benzyloxy)-2-(4-hydroxy-2-methylbutan-2-yl)-3,5-dimethylphenyl 4-methylpentanoate (Compound 9a) The compound was placed in an eggplant flask equipped with a stirrer bar. 8a (84.1 mg, 0.160 mmol) was added and dissolved in 4.8 mL of tetrahydrofuran (THF) at room temperature. After adding 1.6 mL of acetic acid (AcOH) and 1.6 mL of water (H ₂ O) to the obtained melt, the obtained melt was reacted at room temperature for 12 hours. The obtained reaction product was purified by silica gel column chromatography (chloroform) to obtain Compound 9a (53.4 mg, 84%) as a pale yellow oil.

^1H -NMR (CDCl ₃ , 400MHz): δ = 0.968 (6H, d, J = 6.4Hz), 1.27 (1H, t, J = 5.7Hz), 1.47 (6H, s ), 1.64-1.68 (3H, m), 2.09 (2H, t, J=7.2Hz), 2.26 (3H, s), 2.47 (3H, s), 2. 54 (2H, t, J=7.7Hz), 3.56 (2H, q, J=6.7Hz), 4.75 (2H, s), 6.57 (1H, s), 7.33- 7.37 (1H, m), 7.38-7.42 (2H, m), 7.46-7.48 (2H, m).

Production Example (7) Production of 4-(benzyloxy)-3,5-dimethyl-2-(2-methyl-4-oxobutan-2-yl)phenyl 4-methylpentanoate (compound 2a) In an eggplant flask equipped with a stirrer bar. Compound 9a (393.4 mg, 0.992 mmol) was added and dissolved in 12.0 mL of dichloromethane (CH ₂ Cl ₂ ) at 0°C. Dimethyl sulfoxide (DMSO) (1.00 mL, 14.1 mmol), triethylamine (TEA) (0.570 mL, 4.09 mmol), and sulfur trioxide-pyridine complex (Py.SO ₃ ) (339. After adding 2 mg, 2.13 mmol), the resulting lysate was reacted for 12 hours at room temperature. The obtained reaction product was purified by silica gel column chromatography (chloroform) to obtain Compound 2a (324.8 mg, 80%) as a colorless oil.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 0.964 (6H, d, J = 6.4Hz), 1.58 (6H, s), 1.64-1.67 (3H, m), 2.26 (3H, s), 2.47 (3H, s), 2.54 (2H, d, J=7.7Hz), 2.83 (2H, d, J=2.6Hz), 4. 76 (2H, s), 6.61 (1H, s), 7.32-7.37 (1H, m), 7.38-7.42 (2H, m), 7.45-7.48 ( 2H, m), 9.56 (1H, t, J=2.7Hz).

Production Example (8) Production of 3-(3-(benzyloxy)-2,4-dimethyl-6-((4-methylpentanoyl)oxy)phenyl)-3-methylbutanoic acid (compound 3a) Eggplant flask equipped with stirrer bar Compound 2a (248.4 mg, 0.605 mmol) was added to the solution and dissolved in 4.0 mL of tetrahydrofuran 2 (THF) and 6.0 mL of tert-butyl alcohol (t-BuOH) at room temperature. 2-Methyl-2-butene (0.650 mL, 6.13 mmol), sodium chlorite (NaClO ₂ ) (107.7 mg, 1.19 mmol), and sodium dihydrogen phosphate (NaH ₂ PO After adding 6.0 mL of an aqueous solution of ₄ ) (153.4 mg, 1.28 mmol), the resulting solution was reacted at room temperature for 12 hours. The obtained reaction product was purified by silica gel column chromatography (chloroform) to obtain Compound 3a (247.4 g, 96%) as a white solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 0.956 (6H, d, J = 6.4Hz), 1.61 (6H, s), 1.63-1.69 (3H, m), 2.24 (3H, s), 2.48 (3H, s), 2.57 (2H, t, J=7.7Hz), 2.87 (2H, s), 4.75 (2H, s) , 6.59 (1H, s), 7.32-7.36 (1H, m), 7.38-7.42 (2H, m), 7.46-7.48 (2H, m).

Production example (9) (S)-4-(benzyloxy)-3,5-dimethyl-2-(2-methyl-4-oxo-4-((1,2,3,10-tetramethoxy-9-oxo- Production of 5,6,7,9-tetrahydrobenzo "a" heptalen-7-yl)amino)butan-2-yl)phenyl 4-methylpentanoate (compound 5a) Deacetylcolchicine 4 (26 .0mg, 72.7μmol), Compound 3a (26.5mg, 62.1μmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) (25.3mg, 0.132mmol) , 4-dimethylaminopyridine (DMAP) (1.50 mg, 12.3 μmol) was dissolved in 3.1 mL of dichloromethane (CH ₂ Cl ₂ ). After stirring at room temperature for 24 hours, the residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography (chloroform/methanol = 100/0-10/1) to obtain compound 5a (54.4 mg, 98%). Obtained as a yellow solid.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 0.986-1.01 (6H, m), 1.35-1.43 (1H, m), 1.52 (1H, overlaps with peak of water ), 1.59 (3H, s), 1.63 (3H, s), 1.70-1.74 (3H, m), 2.22-2.37 (9H, m) 2.60-2 .66 (3H, m), 3.60 (3H, s), 3.70 (3H, s), 3.89 (3H, s), 3.95 (3H, s), 4.24-4. 30 (1H, m), 4.58 (1H, d, J=10.8Hz), 4.71 (1H, d, J=10.8Hz), 6.00 (1H, d, J=6.6Hz ), 6.39 (1H, s), 6.63 (1H, s), 6.72 (1H, d, J=11.1Hz), 7.17-7.19 (2H, m), 7. 34-7.38 (1H, m), 7.40-7.43 (2H, m), 7.45-7.47 (2H, m).

Production Example (10) Production of 4-(benzyloxy)-2-(4-hydroxy-2-methylbutan-2-yl)-3,5-dimethylphenyl isobutylcarbamate (compound 9b) Carbonic acid was added to a two-necked eggplant flask equipped with a stirrer bar. A solution containing bis(trichloromethyl) (29.7 mg, 0.10 mmol) and dissolved in 2.0 mL of super-dehydrated dichloromethane (CH ₂ Cl ₂ ) under an argon atmosphere was cooled to 0° C. and stirred for 15 minutes. Then, pyridine (0.019 mL, 0.24 mmol) was added and stirred for an additional 25 minutes. A solution of compound 7 (85.7 mg, 0.20 mmol) dissolved in ₂ mL of super dry di _- CH2Cl2 was added dropwise to the mixture at 0<0>C. After stirring for 2 hours, pyridine (0.024 mL, 0.30 mmol) was further added, and the mixture was stirred at 0°C for 10 minutes. Subsequently, isobutylamine (0.024 mL, 0.24 mmol) was added dropwise at the same temperature. After stirring for 20 minutes, the temperature was raised to room temperature. After stirring at room temperature for 1.5 hours, the solution was extracted with CH ₂ Cl ₂ and the CH ₂ Cl ₂ extract was washed with water. The extracted solution was dried over magnesium sulfate and the solvent was removed by evaporation under reduced pressure. 1.0 mL of tetrahydrofuran (THF) was put into the eggplant flask containing the obtained residue, and after dissolving it, 0.5 mL of water and 1.5 mL of acetic acid (AcOH) were added. After stirring at room temperature for 11 hours, the solution was extracted with ethyl acetate, and the ethyl acetate extract was washed with water. After dehydrating the extracted solution with magnesium sulfate, the solvent was removed by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform/methanol = 100/1-50/1) to obtain Compound 9b (82.3 mg, 77%) as a pale yellow oil. Furthermore, two types of ¹ H-NMR spectra (Major: Minor = 100:16) were observed for Compound 9b due to rotamers.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 0.96 (6H, d, J = 6.7 Hz), 0.99 (0.96 H, d, J = 6.8 Hz), 1.47-1 .55 (6.96H, m), 1.69-1.91 (2.32H, m), 2.08 (2.32H, t, J=6.8Hz), 2.25 (3.48H, s), 2.47 (3.48H, s), 3.11 (2H, t, J=6.5Hz), 3.17 (0.32H, t, J=6.4Hz), 3.58( 2.32H, t, J=6.8Hz), 4.74(2.32H, s), 4.80-4.89(0.16H, m), 5.14(1H, t, J=6 .0Hz), 6.63 (0.16H, s), 6.66 (1H, s), 7.31-7.37 (1.16H, m), 7.37-7.44 (2.32H , m), 7.44-7.51 (2.32H, m).

Production Example (11) Production of 4-(benzyloxy)-3,5-dimethyl-2-(2-methyl-4-oxobutan-2-yl)phenyl isobutylcarbamate (compound 2b) Compound 9b was placed in an eggplant flask equipped with a stirrer bar. (27.3 mg, 0.066 mmol) was added and dissolved in 1.3 mL of dichloromethane (CH ₂ Cl ₂ ) at 0°C. Dimethyl sulfoxide (DMSO) (0.065 mL, 0.92 mmol), triethylamine (TEA) (0.037 mL, 0.26 mmol), sulfur trioxide-pyridine complex (Py SO ₃ ) (21 mg, After adding 0.13 mmol), the resulting lysate was reacted at room temperature for 13 hours. The resulting reaction product was purified by silica gel column chromatography (hexane/ethyl acetate = 5/1) to obtain Compound 2b (19.6 mg, 72%) as a colorless oil. Furthermore, two types of ¹ H-NMR spectra (Major: Minor = 100:20) were observed for Compound 2b due to rotamers.

^1H -NMR (CDCl ₃ , 400MHz): δ = 0.97 (6H, d, J = 6.7Hz), 0.99 (1.2H, d, J = 7.0Hz), 1.60 (7 .2H, s), 1.73-1.93 (1.2H, m), 2.26 (3.6H, s), 2.48 (3.6H, s), 2.80 (2H, d , J=2.6Hz), 2.85 (0.4H, d, J=2.4Hz), 3.11 (2H, t, J=6.5Hz), 3.16 (0.4H, t, J=6.5Hz), 4.74 (2.4H, s), 4.77-4.85 (0.2H, m), 5.06 (1H, t, J=6.0Hz), 6. 68 (0.2H, s), 6.70 (1H, s), 7.31-7.37 (1.2H, m), 7.37-7.44 (2.4H, m), 7. 44-7.50 (2.4H, m).

Production Example (12) Production of 3-(3-(benzyloxy)-6-((isobutylcarbamoyl)oxy)-2,4-dimethylphenyl)-3-methylbutanoic acid (compound 3b) Compound 2b was added to an eggplant flask equipped with a stirrer bar. (19.6 mg, 0.048 mmol) was dissolved in 1.0 mL of tetrahydrofuran (THF) and 0.5 mL of tert-butyl alcohol (t-BuOH) at room temperature. 2-Methyl-2-butene (0.051 mL, 0.48 mmol), sodium chlorite (NaClO ₂ ) (8.7 mg, 0.096 mmol), and sodium dihydrogen phosphate (NaH ₂ PO After adding 1.0 mL of an aqueous solution of ₄ ) (17.3 mg, 0.14 mmol), the resulting solution was reacted at room temperature for 12 hours. The resulting reaction product was purified by silica gel column chromatography (chloroform/methanol = 30/1) to obtain Compound 3b (19.1 mg, 93%) as a colorless oil. Furthermore, two types of ¹ H-NMR spectra (Major: Minor = 100:15) were observed for Compound 3b due to rotamers.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 0.92-1.01 (6.9 H, m), 1.62 (6.9 H, s), 1.83 (1.15 H, sep, J =6.7Hz), 2.23 (3.45H, s), 2.48 (3H, s), 2.49 (0.45H, s), 2.81 (2.3H, s), 3. 10 (2H, t, J=6.5Hz), 3.18 (0.3H, t, J=6.2Hz), 4.73 (2.3H, s), 5.26 (1.15H, t , J=6.1Hz), 6.63 (0.15H, s), 6.67 (1H, s), 7.31-7.37 (1.15H, m), 7.37-7.43 (2.3H, m), 7.43-7.50 (2.3H, m).

Production example (13) (S)-4-(benzyloxy)-3,5-dimethyl-2-(2-methyl-4-oxo-4-((1,2,3,10-tetramethoxy-9-oxo- Production of 5,6,7,9-tetrahydrobenzo "a" heptalen-7-yl) amino) butan-2-yl) phenyl isobutylcarbamate (compound 5b) Deacetylcolchicine 4 (16.1 mg) was placed in an eggplant flask equipped with a stirrer bar. , 45.0 μmol), Compound 3b (19.1 mg, 45.0 μmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) (17.2 mg, 90.0 μmol), 4 -Dimethylaminopyridine (DMAP) (0.54 mg, 4.48 μmol) was added and dissolved in 1.0 mL of dichloromethane (CH ₂ Cl ₂ ). After stirring at room temperature for 8.5 hours, the residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography (chloroform/acetone/ethyl acetate = 6/1/1) to obtain compound 5b (32.2 mg, 93 %) as a white solid. Furthermore, two types of ¹ H-NMR spectra (Major: Minor = 100:12) were observed for Compound 5b due to rotamers.

¹ H-NMR (CDCl ₃ , 400 MHz): δ = 0.97-1.07 (6.72 H, m), 1.60-1.66 (7.84 H, m), 1.95 (1.12 H , sep, J=6.7Hz), 2.19-2.37 (5.96H, m), 2.40 (3H, s), 2.54 (1H, d, J=13.8Hz), 2 .64 (1.12H, d, J=13.8Hz), 2.69 (0.12H, d, J=12.8Hz), 3.14-3.33 (2.24H, m), 3. 59 (0.36H, s), 3.60 (3H, s), 3.67 (0.36H, s), 3.74 (3H, s), 3.90 (3.36H, s), 3 .93 (0.36H, s), 3.95 (3H, s), 4.28 (1.12H, ddd, J=11.7, 6.5, 6.4Hz), 4.57 (0. 12H, d, J=10.9Hz), 4.60-4.68 (1.12H, m), 4.71 (1H, d, J=10.8Hz), 6.27 (1.12H, d , J=6.5Hz), 6.36 (0.12H, s), 6.39 (1H, s), 6.48 (1.12H, t, J=6.1Hz), 6.65-6 .80 (2.24H, m), 7.20 (1H, d, J=10.6Hz), 7.32-7.51 (6.72H, m).

Production Example (14) Method for Preparing Nanoparticles of Compound 5a 100 μL of an acetone solution of Compound 5a adjusted to 10 mM was injected into 10 mL of water at room temperature using a syringe, and stirred at 1500 rpm for 2 seconds to form nanoparticles in water. A dispersion was obtained. When preparing the acetone solution, 10 wt% of the compound polysorbate 80 was added. The final concentration of the aqueous dispersion was 0.1 mM. SEM and DLS revealed the particle size to be approximately 130 nm. The photograph of FIG. 6 shows the results of the SEM image of the nanoparticle dispersion of compound 5a obtained above. The particle size distribution of the nanoparticles is shown in the graph of FIG.
Production Example (15) Method for Preparing Nanoparticles of Compound 5b 100 μL of an acetone solution of Compound 5b adjusted to 10 mM was injected into 10 mL of water at room temperature using a syringe, and stirred at 1500 rpm for 2 seconds to form nanoparticles in water. A dispersion was obtained. When preparing the acetone solution, 10 wt% of the compound polysorbate 80 was added. The final concentration of the aqueous dispersion was 0.1 mM. SEM and DLS revealed the particle size to be approximately 100 nm. The photograph of FIG. 7 shows the results of the SEM image of the nanoparticle dispersion of compound 5b obtained above. The particle size distribution of the nanoparticles is shown in the graph of FIG.

Production Example (16) Method for producing high-concentration nanoparticles of Compound 5a 100 μL of an acetone solution of Compound 5a adjusted to 72.2 mM was injected into 9 mL of water at room temperature using a syringe, and stirred at 1500 rpm for 2 seconds. , an aqueous dispersion of nanoparticles was obtained. When preparing the acetone solution, 10 wt% of the compound polysorbate 80 was added. After removing acetone, the volume was determined to be 10 mL, and mixed with a 9 w/v % sodium chloride aqueous solution at a ratio of 9:1 to prepare. The final concentration of the aqueous dispersion was 0.65mM. SEM and DLS revealed the particle size to be approximately 170 nm. The photograph of FIG. 8 shows the result of the SEM image of the nanoparticles with a high concentration of compound 5a obtained above. The particle size distribution of the nanoparticles is shown in the graph of FIG.

Production Example (17) Method for producing high-concentration nanoparticles of Compound 5b 100 μL of an acetone solution of Compound 5b adjusted to 72.2 mM was injected into 9 mL of water at room temperature using a syringe, and stirred at 1500 rpm for 2 seconds. , an aqueous dispersion of nanoparticles was obtained. When preparing the acetone solution, 10 wt% of the compound polysorbate 80 was added. After removing acetone, the volume was determined to be 10 mL, and mixed with a 9 w/v % sodium chloride aqueous solution at a ratio of 9:1 to prepare. The final concentration of the aqueous dispersion was 0.65mM. SEM and DLS revealed the particle size to be approximately 130 nm. The photograph of FIG. 9 shows the result of the SEM image of the nanoparticles with high concentration of compound 5b obtained above. The particle size distribution of the nanoparticles is shown in the graph of FIG.

Example 3
Figure 10. Evaluation of N-Col treatment on HTF viability by Alamar Blue assay and cell proliferation by BrdU assay. Human fibroblasts were seeded in 96-well plates, and after 24 hours, Compound 5 nanoparticles were added to a final concentration of 10- It was added at a concentration of 1000 nM. After a further 24 hours, alamar blue reagent was added, and absorbance was measured 2 hours later. Further, human fibroblast cells were seeded in a 96-well plate, and 24 hours later, compound 5 nanoparticles were added to a final concentration of 10-500 nM. After a further 24 hours, the BrdU reagent was added, and the absorbance was measured 24 hours and 48 hours later. The results are shown in FIGS. 10(A) to (E).

Figure 10(A) HTF (human tenon fibroblast) was treated with different concentrations of N-Col (compound 5 (0.1, 0.25, 0.5, 0.75, 1.0 μM) for 24 hours. At 0.1 μM and 0.25 μM, the statistical survival rate was Although no decrease was observed, a statistical decrease was shown from 0.5 μM to 1.0 μM compared to the solvent control. Figure 10(B) HTF with N-Col treatment (0.1, 0.5, 1.0 μM) was observed under a microscope for 24 hours. Cells contracted (black arrow) while HTF was co-treated with TGF-β1 and N-Col. Scale bar = 200 μm. Figure 10(C) 10 ng/mL TGF- Figure 10 shows the temporal effect of 0.5 μM N-Col on the survival rate of HTF in the presence or absence of β1. TGF-β1 increased the survival rate of HTF, but this activation was markedly suppressed by N-Col treatment. (D) Shows the temporal effects of N-Col treatment (0, 0.1, 0.25, and 0.5 μM, respectively) on the proliferation of normal HTF without TGF-β1 induction. Although no decrease in BrdU absorbance was observed at 24 h. , a decrease in BrdU absorbance was observed at 48 h while cells were treated with 0.25 and 0.5 μM N-Col. N-Col; nanocolchicine; ns; no significance, compared with untreated control, ^# p < 0.05, compared with TGF-β1 group, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Example 4
Figure 11 Evaluation of N-Col treatment on HTF motility by scratch assay and transwell assay.
Human fibroblasts were seeded in a 6-well plate, and after confirming proliferation, a wound was created and compound 5 nanoparticles were added at a final concentration of 100-500 nM. Images were taken with a microscope every 12 hours and the remaining area was measured. In addition, human fibroblast cells were seeded in transwells with a diameter of 8 μm, and cell migration and invasion were evaluated by crystal violet staining. The results are shown in FIGS. 11(A) to (E).
FIG. 11(A) HTF was treated with N-Col (compound 5) for 48 hours with or without TGF-β1 induction, and photographs were taken every 12 hours.

The decrease in wound area in the control group and TGF-β1 group was significantly faster than that in the two 0.5 μM N-Col groups. Scale bar = 1 mm. Figure 11 (B) Wound area was measured at 0, 12, 24, 36, and 48 hours. N-Col treatment was shown to significantly suppress wound healing induced by TGF-β1. Figure 11(C) HTF migration and invasion was assessed by transwell assay. N-Col treatment could significantly inhibit cell migration and invasion induced by TGF-β1, but the effect was less in normal THF. Scale bar = 2 mm Figure 11 (DE) Migrated or invasive cells were calculated under the microscope and statistical analysis was performed.
N-Col; nanocolchicine, ns; no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001 compared to untreated control.

Example 5
Figure 12. Evaluation of N-Col effects on TGF-β1-induced HTF morphology and function.
Human fibroblasts were included in a collagen gel, nanoparticles of compound 5 were added, and the cells were seeded in a 24-well plate and cultured. After that, photographs were taken with a microscope over time until 15 days later, and the size of the collagen gel was measured. Furthermore, human fibroblasts were cultured for 48 hours in the presence of compound 5 nanoparticles, and α-SMA expression was evaluated by Western blotting. Furthermore, human fibroblasts were cultured for 24 hours in the presence of compound 5 nanoparticles, and cell morphology was evaluated by F-actin immunostaining. The results are shown in FIGS. 12(A) to (E). Figure 12(A) Cell contractility was tested using a collagen gel culture kit. TGF-β1-induced HTF was treated with 0.1 or 0.5 μM N-Col (compound 5) for 15 days and photographs were taken daily. Significant differences in gel shrinkage area among all groups occurred on day 5 and reached a peak on day 9. In the TGF-β1 group, the gel area decreased and 5 μM N-Col was completely retained. Figure 12(B) The remaining gel area was measured and statistically analyzed. 0.5 μM N-Col significantly inhibited TGF-β1-induced gel contraction, which returned to untreated levels. Figure 12 (C) α-SMA expression detected by Western blot and banding was statistically analyzed. 0.5 μM N-Col inhibited α-SMA expression. The intensity of each band was normalized with GAPDH. FIG. 12(D) Immunostaining of α-SMA was performed. It was shown that 0.5 μM N-Col suppressed TGF-β1-induced α-SMA expression, reducing α-SMA fluorescence to untreated control levels. Scale bar = 200 μm.
(E) F-actin immunostaining with rhodamine phalloidin (red) was performed on HTFs subjected to different treatments. In the TGF-β1 group, the cell size was large, the fluorescence intensity indicating the cytoskeleton was strong, and morphological changes such as polygonal shapes were observed. After treatment with 0.5 μM N-Col, the cells shrank and became rod-shaped. Scale bar is shown in the figure. N-Col; nanocolchicine, ns; no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001 compared to untreated control.

Example 6
Figure 13. Effect of N-Col treatment on TGF-β1-induced HTF cell cycle and apoptosis.
Human fibroblasts were cultured for 24 hours in the presence of compound 5 nanoparticles, and the cell cycle was detected by Ki67 immunostaining, and apoptotic cells were detected by the TUNEL method. Furthermore, the results are shown in FIGS. 13(A) to (D). Figure 13(A) HTFs after different treatments were stained with Ki67 antibody (white arrowheads). It was revealed that 0.5 μM N-Col (compound 5) suppressed Ki67 expression activated by TGF-β1. Figure 13(B) Quantification of Ki67 staining showed that N-Col significantly reduced Ki67-positive cells in activated HTFs, but not in normal HTFs. FIG. 13(C) Apoptosis of HTF was detected by different treatments using the indirect TUNEL method (white arrow), which is an anti-digoxigenin antibody using rhodamine fluorescent dye. N-Col exerted a significant proapoptotic effect on TGF-β1-induced HTF, but this effect was weaker on HTF without TGF-β1 treatment. Figure 13(D) TUNEL quantification showed that N-Col significantly increased apoptotic cells in activated HTF and slightly increased in normal HTF. Scale bar = 200 μm. N-Col; nanocolchicine, ns; no significant difference, compared to untreated control, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Example 7
Figure 14. Human fibroblasts were cultured for 48 hours with the addition of compound 5 nanoparticles related to fibrosis markers and apoptosis-related proteins in normal and activated HTFs after N-Col treatment. The expression of apoptosis-related factors was evaluated by Western blotting. In addition, activation of caspase-3 was evaluated by culturing human fibroblasts for 24 hours in the presence of compound 5 nanoparticles and staining with an antibody that specifically recognizes cleaved caspase-3. The results are shown in FIGS. 14(A) to (G). Figure 14 (A) HTFs were treated with N-Col (compound 5) doses (0.1, 0.25, 0.5 μM, respectively) with or without TGF-β1 induction. Protein levels were measured by Western blot. Relative expression levels were determined by density analysis. Figure 14 (BF) Band quantification showed that 0.5 μM N-Col did not change the expression of Col-I, fibronectin, α-SMA, or the ratio of phosphorylated ERK1/2/total ERK1/2. We showed that normal HTF without TGF-β1 pretreatment slightly increased the Bax/Bcl-2 ratio. On the other hand, in activated HTF by addition of TGF-β1, 0.5 μMN-Col reduced the expression of Col-I and α-SMA, while decreasing phosphorylated ERK1/2/total ERK1/2 and increasing Bax/Bcl-2. significantly decreased. FIG. 14(G) Immunostaining of cleaved caspase-3 was performed. 0.5 μM N-Col increased apoptosis-positive cells in activated HTF, but only a slight increase in normal fibroblasts. Scale bar = 200 μm. N-Col; nanocolchicine, ns; no significant difference, compared to untreated control, *: p < 0.05, **: p < 0.01, ***: p < 0.001, TGF- ^# p < 0.05 compared to β1 group

Example 8
Figure 15. Alamar Blue assay was performed in the same manner as in Example 3 (Figure 10A) except that Compound 5a or Compound 5b was used instead of N-Col (Compound 5). The results are shown in FIG. HTF was treated with different concentrations of N-Col (0.1, 0.25, 0.5, 0.75, 1.0 μM) for 24 h. A decrease in cell viability was shown at 1.0 μM compared to the solvent control.

Claims (6)

1. A scar formation inhibitor containing a compound represented by the following general formula (I) or a salt thereof:
   

   

   [In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different, hydrogen, optionally substituted C1-18 alkyl, optionally substituted C1-18 alkoxy, substituted C7-18 aralkyloxy or optionally substituted phenoxy.
   Two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form an optionally substituted ring together with the carbon atom to which these groups are bonded.
   Y is optionally substituted C1-18 alkyl, optionally substituted C7-18 aralkyl, optionally substituted phenyl, optionally substituted C1-18 alkoxy, optionally substituted C7-18 aralkyloxy, optionally substituted phenoxy, optionally substituted mono C1-18 alkylamino, optionally substituted mono C7-18 aralkylamino, or optionally substituted monophenylamino show].
2. Fibroblast proliferation inhibitor containing a compound represented by the following general formula (I) or a salt thereof:
   

   

   [In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different, hydrogen, optionally substituted C1-18 alkyl, optionally substituted C1-18 alkoxy, substituted C7-18 aralkyloxy or optionally substituted phenoxy.
   Two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form an optionally substituted ring together with the carbon atom to which these groups are bonded.
   Y is optionally substituted C1-18 alkyl, optionally substituted C7-18 aralkyl, optionally substituted phenyl, optionally substituted C1-18 alkoxy, optionally substituted C7-18 aralkyloxy, optionally substituted phenoxy, optionally substituted mono C1-18 alkylamino, optionally substituted mono C7-18 aralkylamino, or optionally substituted monophenylamino show].
3. In general formula (I), R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents hydrogen, optionally substituted C1-18 alkyl, or optionally substituted C7-18 aralkyloxy. show,
   Y represents optionally substituted C1-18 alkoxy, optionally substituted C1-18 alkyl, or optionally substituted mono C1-18 alkylamino,
   The scar formation inhibitor according to claim 1 or the fibroblast proliferation inhibitor according to claim 2.
4. The scar formation inhibitor according to claim 1, the fibroblast proliferation inhibitor according to claim 2, or the fibroblast proliferation inhibitor according to claim 3, which comprises nanoparticles containing the compound represented by general formula (I) or a salt thereof. Scar formation inhibitors or fibroblast proliferation inhibitors.
5. A method for producing nanoparticles, comprising a step of injecting a water-miscible organic solvent solution of a compound represented by the following general formula (I) or a salt thereof into water:
   

   

   [In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different, hydrogen, optionally substituted C1-18 alkyl, optionally substituted C1-18 alkoxy, substituted C7-18 aralkyloxy or optionally substituted phenoxy.
   Two adjacent ones of R ¹ , R ² , R ³ and R ⁴ may form an optionally substituted ring together with the carbon atom to which these groups are bonded.
   Y is optionally substituted C1-18 alkyl, optionally substituted C7-18 aralkyl, optionally substituted phenyl, optionally substituted C1-18 alkoxy, optionally substituted C7-18 aralkyloxy, optionally substituted phenoxy, optionally substituted mono C1-18 alkylamino, optionally substituted mono C7-18 aralkylamino, or optionally substituted monophenylamino show].
6. In general formula (I), R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents hydrogen, optionally substituted C1-18 alkyl, or optionally substituted C7-18 aralkyloxy. show,
   Y represents optionally substituted C1-18 alkoxy, optionally substituted C1-18 alkyl, or optionally substituted mono C1-18 alkylamino,
   The method according to claim 5.

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