KR101340556B1

KR101340556B1 - Novel phytospingosine-1-phosphate derivatives, a process for the preparation thereof, and a composition for hair tonic or treating or preventing hair loss comprising the same

Info

Publication number: KR101340556B1
Application number: KR1020120015889A
Authority: KR
Inventors: 최명준
Original assignee: 주식회사 피토스
Priority date: 2012-02-16
Filing date: 2012-02-16
Publication date: 2013-12-11
Also published as: KR20130094547A

Abstract

The present invention relates to O-cyclic phytosphingosine-1-phosphate (O-C-P1P), N-cyclic phytosphingosine-1-phosphate (N-C-P1P), pharmaceutically acceptable salts thereof, or solvates thereof; Its production method; And it provides a cosmetic composition for preventing hair loss or hair growth, and a pharmaceutical composition for preventing, treating, or hair loss.

Description

Novel phytospingosine-1-phosphate derivatives, a process for the preparations, and a composition for hair tonic or novel phytosphingosine-1-phosphate derivatives, methods for preparing the same, and compositions for the prevention, treatment, or hair growth of hair loss comprising the same treating or preventing hair loss comprising the same}

The present invention relates to a novel phytosphingosine-1-phosphate derivative, a method for preparing the same, and a composition for the prevention, treatment, or hair growth comprising the same, and more specifically, a novel substance useful for the prevention, treatment, or hair growth of hair loss. A phytosphingosine-1-phosphate derivative, a method for producing the same, and a cosmetic composition for preventing hair loss or hair growth comprising the same, and a pharmaceutical composition for preventing, treating or hair loss.

Among sphingolipids, sphingosine-1-phosphate is a physiologically active autocrine that produces a strong biological response through binding to G-protein-coupled endothelial differentiation gene (EDG) receptors. ) As a molecule (Non Patent Literature 1), produced in vivo by sphingosine kinase, and degraded by sphingosine lyase or sphingosine phosphatase (Non Patent Literature 2).

The dynamic balance between the sphingolipid metabolites, ceramide and sphingosine 1-phosphate content, and the regulation of reverse signaling processes are important factors in determining cell life and death (Non-Patent Document 3). Increased intracellular ceramide inhibits proliferation in cancer cells, but in inflammatory cells exhibits both side effects that exacerbate the inflammatory response. Recently, it has been suggested that ceramide, as a function of new sphingolipids, is involved in neurites formation in the nervous system as well as cell death and cell proliferation inhibition, and the structure of sphingomyelin is important for intracellular transport. In other words, three-dimensional asymmetry in the inner and outer membranes of sphingolipids and non-uniformity in the horizontal structure can be considered to be involved in various cellular functions such as differentiation, proliferation and secretion. Glucosyl ceramide accumulates in the cells, and it has been suggested that such resistant cells have a mechanism for converting ceramide, an apoptosis-inducing factor, into another metabolite, glycosphingolipid, and excluding it (Non-Patent Document 4). Therefore, a change in the amount of sphingolipids is likely to be involved in conditions such as blood tumors and nervous system diseases (Non-Patent Document 5).

Accordingly, sphingosine-1-phosphate, an important intermediate in sphingosine metabolism, has been shown to exhibit various physiological activities. Accordingly, much research has been focused on the production, metabolism, action, and synthesis of the substance (Non-Patent Document 6). ). However, for phytosphingosine-1-phosphate, which plays a role comparable to sphingosine-1-phosphate, the amount of research is extremely limited, and the studies are mainly focused on the role in vivo. For the preparation of these two substances, sphingosine-1-phosphate and phytosphingosine-1-phosphate, enzymatic synthesis using kinase and chemical synthesis are known. Enzymatic synthesis methods (Non Patent Literatures 7, 8, 9) all have limited structural identification of the final material. While the chemical synthesis of D-sphingosine-1-phosphate has been relatively studied, the research on chemical synthesis of D-phytosphingosine-1-phosphate is very rare, and only three papers have been published (Non-Patent Documents 10, 11, 14). These two synthesis methods also apply the same method by substituting phytosphingosine for the starting material instead of sphingosine for the method already used in the synthesis method of D-sphingosine-1-phosphate (Non Patent Literatures 12 and 13).

The method according to the following Reaction Scheme 1 was published as the first chemical synthesis method of D-Phytosphingosine-1-phosphate (Non-Patent Document 10).

[Reaction Scheme 1]

The final yield of the entire process of the above synthesis method is reported to be 43% and data on high performance FAB-MS, ¹ H-NMR for the final material D-Phytosphingosine-1-phosphate are provided.

As another efficient chemical synthesis method of D-phytosphingosine-1-phosphate, a method according to Scheme 2 below has been reported (Non Patent Literature 14).

[Reaction Scheme 2]

The above method is a total of 6 steps, the yield of the whole process is reported to be 14.2%, and the spectroscopic data of all intermediates are described, but no specific spectroscopic data on the final target substance, D-Phytosphingosine-1-phosphate, are reported.

Therefore, D-Phytosphingosine-1-phosphate has a very low yield of 43% or less according to the above synthesis method, and it can be said that the material is very inefficient in the synthesis.

Phytosphingosine-1-phosphate has also been patented for the prevention, treatment or hair growth use of phytosphingosine-1-phosphate derivatives in recognition of its hair growth and hair growth efficacy equivalent to that of minoxidil (Patent Document 1). The causes of such alopecia, in which the hair falls off the scalp, can be variously divided into male hormones, mental stress, lipid peroxide accumulation in the scalp, drug side effects, chronic diseases such as leukemia or tuberculosis, side effects from radiation therapy, and nutrition. Deficiency. In addition, hair loss, which has been known as a men's worry recently, is increasing the demand for prevention and treatment of hair loss in women and hair loss in young people.

Drugs currently used as hair growth and wool agents include large vasodilators to circulate blood sufficient to the scalp, female hormones to inhibit the action of male hormones, and 5α- to convert testosterone to 5-DHT (5-dihydrotesteone). There are testosterone activity inhibitors that inhibit reductase. Examples of the vasodilators include caproium chloride, minoxidil, and various plant extracts. Examples of female hormones include estrogen, estradiol, and progesterone. Examples of male hormone activity inhibitors include pinasteride and pentadecanoic acid.

However, there is a need for more effective and safe hair loss treatment or development of drugs for hair growth due to insufficient effects or side effects. Minoxidil, administered topically or orally, is irritating to the skin such as scalp erythema, inflammation, infection, irritation or pain, and has a lowering effect on blood pressure. Therefore, patients with hypertension, including patients with hypotensive agents, And the like. In addition, finasteride has a disadvantage of impaired erectile dysfunction due to the inhibitory effect of male hormone activity, and a decrease in sexual desire.

The phytosphingosine-1-phosphate derivative is a substance that has been recognized for use in the prevention, treatment or hair growth of hair loss due to the effect of promoting angiogenesis. In addition, the conventional hair loss treatment agent can be used without fear of side effects such as lowering blood pressure or decreased sexual function.

However, as described above, phytosphingosine-1-phosphate is a material having low yield in the synthesis. Therefore, there is a need for the development of a material that can be synthesized in high yield and effective in treating hair loss without fear of the side effects of the conventional hair loss treatment.

1. Korea Patent Registration 1003532

Lee, M.J. et al., Science, 279, pp 1552-1555, 1996 2. Pyne, S. and Pyne, N. J., Biochem. J., 349, pp 385-402, 2000 Cuvillier, O. et al., Nature, 381, pp 800-803, 1996 4. Lavie Y. et al., J. Biol. Chem., 272, pp1682-1687, 1997 5. Okazaki T. et al., Int. J. Hematolo., 65, p40, 1997 6.FASEB J, 10: 1388-1379 Hoppe-Seyler's Z. Physiol. Chem., 351, 635-642, 8. J. Biol. Chem., 273, 19437-19442, 9. Biochem. J., 332, 525-531 10. J. of Lipid Research, 40, 1999, 117-125 Bull. Korean Chem. Soc., 2003, 24, 267-268 12.J. of Lipid Research, 35, 1994, 2305-2311 13.Tetrahedron Letters, 2000, 41, 7821-7824 14. Bull. Korean Chem. Soc., 2003, 24, 267-268

Therefore, the present inventors have studied for the development of a new hair loss treatment active material that can solve the problems of the conventional hair loss treatment drug, a new phytosphingosine derivative material that can be synthesized in a high yield while being effective in treating hair loss and hair growth The present invention has been completed. In addition, the developed material was found to be effective in improving hair loss and hair growth as well as wrinkles.

Accordingly, it is an object of the present invention to provide new substances effective for the prevention, treatment, and hair growth of hair loss.

Another object of the present invention is to provide a method for producing a material according to the present invention.

Still another object of the present invention is to provide a cosmetic composition for preventing hair loss or hair growth comprising the substance according to the present invention.

Still another object of the present invention is to provide a pharmaceutical composition for preventing, treating, or hair loss comprising the substance according to the present invention.

Still another object of the present invention is to provide a cosmetic composition for preventing, ameliorating or treating wrinkles comprising the substance according to the present invention.

In order to achieve the above object, one aspect of the present invention is

O-cyclic phytosphingosine-1-phosphate of formula 1a (OC-P1P) or N-cyclic phytosphingosine-1-phosphate of formula 1b, pharmaceutically acceptable salts thereof, or solvates thereof to provide:

[Formula 1a]

[Chemical Formula 1b]

Another aspect of the invention provides a process for preparing a compound of formula 1a comprising reacting a compound of formula 4 with trifluoroacetic acid or concentrated hydrochloric acid to deprotect it:

[Formula 1a]

[Formula 4]

In Formula 4, R ¹ is a protecting group.

Another aspect of the present invention provides a method for preparing a compound of Formula 1b, comprising reacting a compound of Formula 5 with bromotrimethylsilane and then adding water:

[Chemical Formula 1b]

[Chemical Formula 5]

In Formula 5, R ² is a protecting group.

Another aspect of the present invention provides a hair loss preventing or hair growth cosmetic composition comprising the compound of Formula 1a or 1b, a pharmaceutically acceptable salt thereof, or a solvate thereof.

Another aspect of the present invention provides a pharmaceutical composition for the prevention, treatment, or hair growth of hair loss comprising the compound of Formula 1a or 1b, a pharmaceutically acceptable salt thereof, or solvate thereof.

Another aspect of the present invention provides a cosmetic composition for preventing, ameliorating or treating wrinkles comprising the compound of Formula 1a or 1b, a pharmaceutically acceptable salt thereof, or a solvate thereof.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications cited herein are hereby incorporated by reference in their entirety.

The present inventors have studied for the development of an effective material for the treatment of hair loss that can be synthesized in high yield and can be safely used without fear of the side effects of the conventional hair loss treatment, as a result of the novel phytosphingosine-1-phosphate known as a conventional safe material One derivative, the compounds of Formula 1a and Formula 1b, has been developed.

Accordingly, the present invention in one aspect, O-cyclic phytosphingosine-1-phosphate of formula 1a (OC-P1P) or N-cyclic phytotosphingosine-1-phosphate of formula 1b (NC-P1P), Or a pharmaceutically acceptable salt thereof, or solvate thereof:

[Formula 1a]

[Chemical Formula 1b]

The pharmaceutically acceptable salt may be present as an acid addition salt wherein the compound of Formula 1a or 1b forms a salt with the free acid. The compounds of Formula 1a or 1b may form pharmaceutically acceptable acid addition salts according to conventional methods known in the art. The free acid may be an organic acid or an inorganic acid, and the inorganic acid may be hydrochloric acid, bromic acid, sulfuric acid, or phosphoric acid, and the organic acid may be citric acid, acetic acid, lactic acid, tartaric acid, or tartariac acid. ), Maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embon acid, Glutamic acid or aspartic acid, etc. can be used. In particular, the compound of Formula 1a may exist in the form of hydrochloride.

The pharmaceutically acceptable salt may be present as an inorganic salt of the compound of Formula 1a or 1b. The compound of Formula 1a or 1b may form a pharmaceutically acceptable inorganic salt according to conventional methods known in the art. The inorganic salts include, but are not limited to, salts with aluminum, ammonium, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium, or zinc, and ammonium, calcium, magnesium, potassium, or sodium salts are preferred. .

In addition, the compound of Formula 1a or 1b according to the present invention may include not only pharmaceutically acceptable salts, but also all solvates including all salts and hydrates that may be prepared by conventional methods.

In another aspect, the present invention provides a process for preparing a compound of formula 1a comprising reacting a compound of formula 4 with trifluoroacetic acid or hydrochloric acid gas to deprotect it:

[Formula 1a]

[Formula 4]

In Formula 4, R ¹ is a protecting group.

R ¹ of Chemical Formula 4 may be any protecting group that may be commonly used for protecting an amino group as a protecting group, for example, t-butyloxycarbonyl (t-Boc), p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group or allyloxycarbonyl group, and the like, but is not limited thereto.

As a solvent for carrying out the reaction for deprotecting the compound of Formula 4 by reacting with trifluoroacetic acid or hydrochloric acid gas, any solvent that does not inhibit the reaction may be used, and preferably, upon reaction with trifluoroacetic acid. Ethyl acetate may be used for the reaction with methylene chloride and HCl gas. Specific reaction conditions of the deprotection reaction may vary depending on the type of the protecting group and the reactants. According to one embodiment, the reaction may be performed at room temperature during the reaction with the trifluoroacetic acid, and during the reaction with the hydrochloric acid gas. The reaction may be cooled to about 0 ° C.

The compound of Formula 4 may be prepared by reacting a compound of Formula 3 with POCl ₃ :

(3)

In Formula 3, R ¹ is a protecting group.

As a solvent for carrying out the reaction of phosphorylating the compound of Formula 3 with POCl ₃ , any solvent that does not inhibit the reaction may be used, but preferably a pyridine solvent is used. Preferably, according to one embodiment, the reaction may be carried out by first dissolving the compound of Chemical Formula 3 in a pyridine solvent, cooling to about −20 ° C., and then adding a solution of POCl ₃ in pyridine solvent. After the reaction is completed, a compound such as Formula 4 may be formed by adjusting the pH to about 2 using a strong acid such as hydrochloric acid. The obtained compound of formula 4 can be used to prepare the compound of formula 1a without any special purification process.

The compound of Chemical Formula 3 may be prepared by introducing a protecting group to the amino group of D-phytosphingosine of Chemical Formula 2 below.

(2)

The reaction of introducing a protecting group to the amino group of the D-phytosphingosine may be carried out according to any method known in the art, for example, may be performed according to the method of Non-Patent Document 14.

One embodiment of the method for preparing a compound of Formula 1a described above may be represented by the following Scheme 3.

Scheme 3

In another aspect, the present invention provides a process for preparing a compound of Formula 1b comprising reacting a compound of Formula 5 with bromotrimethylsilane and then adding water:

[Chemical Formula 1b]

[Chemical Formula 5]

In Formula 5, R ² is a protecting group.

R ² of Chemical Formula 5 may be any protecting group that may be conventionally used for protecting an amino group, for example, t-butyloxycarbonyl (t-Boc), p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group or allyloxycarbonyl group, and the like, but is not limited thereto.

As a solvent for carrying out the reaction of the compound of Formula 5 with bromotrimethylsilane, any solvent that does not inhibit the reaction may be used, and methylene chloride may be preferably used. Specific reaction conditions of the deprotection reaction may vary depending on the type of the protecting group. According to one embodiment, the compound of Formula 5 is dissolved in a solvent, cooled to about −20 ° C., and then reacted by adding bromotrimethylsilane. Can be. Then, by adding water and further reacting at about 0 ° C., a compound of Formula 1b may be formed.

The compound of Formula 5 may be prepared according to any method known in the art, for example, may be prepared according to the method of Non-Patent Document 14.

One embodiment of the method for preparing the compound of Formula 1b described above may be represented by the following Scheme 4.

[Reaction Scheme 4]

In another aspect, the present invention provides a hair loss preventing or hair growth cosmetic composition comprising the compound of Formula 1a or 1b, a pharmaceutically acceptable salt thereof, or a solvate thereof.

In another aspect, the present invention provides a pharmaceutical composition for preventing, treating, or raising hair, comprising the compound of Formula 1a or Formula 1b, a pharmaceutically acceptable salt thereof, or a solvate thereof.

In another aspect, the present invention provides a cosmetic composition for preventing, ameliorating, or treating wrinkles, comprising the compound of Formula 1a or Formula 1b, a pharmaceutically acceptable salt thereof, or a solvate thereof. .

Hereinafter, the cosmetic composition and the pharmaceutical composition are collectively referred to as "composition according to the present invention".

It is demonstrated in the following examples that the compounds of Formula 1a (O-C-P1P) and Formula 1b (N-C-P1P) are effective in treating, preventing and hair loss. Specifically, the hair growth test using C3H mice was performed on the compounds of the present invention. As a result, the compounds of the present invention showed a remarkably superior hair growth effect compared to the minoxyl 3% (minoxidil 3% liquid, Hyundai Pharmaceutical) currently sold as a hair loss treatment from the FDA. In addition, as a result of the hair growth test using the C3H mouse at a concentration of 0.005% to 0.02% of the compound of the present invention, it showed a remarkably superior effect as compared to the negative control, and there was almost no difference in the hair growth effect by the concentration. It showed an effect similar to that of minoxidil 3%, and was found to have effective hair growth at a concentration 300 times lower than the conventional hair loss treatment. In addition, as a result of the hair growth test using C3H mice in the form of HCl salt and disodium salt, the compound of the present invention showed a remarkably superior effect regardless of the type of salt compared to the negative control. In addition, as a result of clinical trials conducted on bald patients with the compounds of the present invention, hair loss prevention and hair growth effects were confirmed. Thus, the compounds according to the invention have been found to be effective in treating, preventing and hair loss.

Hair loss prevention or hair growth cosmetic composition according to the present invention and hair loss prevention, treatment or hair growth pharmaceutical composition may be used as a complex preparation further comprising the prevention, treatment, or hair growth drugs or supplements. Other drugs or adjuvants include retinoic acid, minoxidil, finasteride, zinc peptide, zinc oxide, biotin, genistein, onion extract, pumpkin seed oil, emu oil, Green tea extract, willow bark extract, centella extract, nettle extract, iris extract, rosemary extract, chamomile extract, and the like, but is not limited thereto.

Hair loss prevention or hair growth cosmetic composition according to the present invention and hair loss prevention, treatment or hair growth pharmaceutical composition, including, but not limited to, oral administration, injections, suppositories, transdermal administration, and non-administration It may be formulated and administered in a dosage form, but may preferably be formulated into a formulation suitable for topical application to the skin area covered with the scalp or hair. Such topical formulations include, but are not limited to, liposomes, nanoemulsions, shampoos, hair conditioners, or hair lotions. Preferably, in order to promote the effect by increasing transdermal absorption, it is preferable to formulate into liposomes or nanoemulsions. In formulating the composition, it may be prepared using a carrier, diluent, or additive suitable for the preparation of each formulation known in the cosmetic or pharmaceutical arts.

Hair loss prevention or hair growth cosmetic composition according to the present invention and hair loss prevention, treatment or hair growth pharmaceutical composition is about 0.001% to 1% by weight, preferably 0.005% by weight of the compound of Formula 1a or 1b It may be contained in the range of% to 0.1% by weight, and may be increased or decreased depending on the type of active ingredient. In the case of the pharmaceutical composition according to the present invention, the topical administration may be administered by applying the compound of Formula 1a or the compound of Formula 1b once or twice a day to a site for the prevention, treatment, or hair growth of hair loss. When the active ingredient is 1% by weight, the daily application amount is about 0.5-3 mg, and may be increased or decreased depending on the area of the application site. Such dosages and frequency can be used appropriately depending on the age, sex, and degree of hair loss of the patient.

Additionally, it was demonstrated in the examples below that the compounds of Formula 1a (O-C-P1P) and Formula 1b (N-C-P1P) are effective in preventing, improving, or treating wrinkles. Specifically, after treating the compounds of the present invention by concentration, the proliferative effect of human fibroblasts was measured by MTT assay, and the amount of collagen in fibroblasts was measured. As a result, as the concentration of the compound of the present invention increases, the survival rate of the fibroblasts was increased, and the amount of collagen was also increased, which was confirmed to have an effective wrinkle prevention, improvement and treatment effect.

The cosmetic composition for preventing, improving and treating wrinkles according to the present invention may further be used as a complex preparation including further substances for preventing, improving and treating wrinkles.

The cosmetic composition for preventing, improving and treating wrinkles according to the present invention may be formulated into a formulation suitable for topical application to wrinkled or susceptible skin areas. Such topical formulations include, but are not limited to, liposomes or nanoemulsions. In formulating the composition, it may be prepared using a carrier, diluent, or additive suitable for the preparation of each formulation known in the cosmetic art.

The cosmetic composition for preventing, improving and treating wrinkles according to the present invention contains the compound of Formula 1a or 1b in the range of about 0.001% to 1% by weight, preferably 0.005% to 0.1% by weight based on the total composition. It may be increased or decreased depending on the type of active ingredient. In the case of the pharmaceutical composition according to the present invention, the topical administration may be administered by applying the compound of Formula 1a or the compound of Formula 1b to the site for wrinkle prevention, improvement and treatment once or twice a day. When the active ingredient is 1% by weight, the daily application amount is about 0.5-3 mg, and may be increased or decreased depending on the area of the application site. Such dosages and frequency may be appropriately increased or decreased depending on the age, sex, and extent of wrinkles of the user.

As described above, the compound of Formula 1a or Formula 1b, a pharmaceutically acceptable salt thereof, or solvate thereof according to the present invention may be used for the treatment, prevention, or hair growth, and the prevention, improvement, or treatment of hair loss. In addition to being excellent in effect, they can be easily prepared synthetically and are economical and can be easily delivered to hair follicles when formulated into liposomes, such as liposomes, nanoemulsions. In addition, compared to minoxidil and finasteride which are conventionally used as hair loss treatment agents, there is no skin irritation and there is no side effect and it is highly desirable.

1 is a ¹ H NMR spectrum of a compound of Formula 1a (O-cyclic phytosphingosine- ¹ -phosphate) prepared according to one embodiment of the present invention.
2 is a ¹ H NMR spectrum of the compound of Formula 1b (N-cyclic phytosphingosine- ¹ -phosphate) prepared according to one embodiment of the present invention.
Figure 3 shows the hairs of C3H mice and then the compound OC-P1P, NC-P1P solution according to the present invention was applied once a day, and then positive control (3% minoxidil solution on day 11, 14 and 18) ) And pictures taken in comparison with the negative control.
Figure 4 shows the hair of a C3H mouse and then OC-P1P liposome prepared according to an embodiment of the present invention once a day, and then compared to the positive and negative controls on day 14 and day 18 It is a photograph of English.
FIG. 5 is a photograph taken in comparison to the negative control on the 13th and 17th days after depilating the flanks of C3H mice and applying the OC-P1P solution prepared according to one embodiment of the present invention once a day by concentration. .
FIG. 6 is a daily application of a solution of OC-P1P free base, OC-P1P hydrochloride, and OC-P1P disodium salt, which is prepared according to an embodiment of the present invention after depilating the side of a C3H mouse The photographs were taken on days 14 and 18 compared to the negative control.
7A and 7B are OC-P1P liposomes prepared in accordance with an embodiment of the present invention, after applying to the applicant for the hair loss 1-2 times a day, and then photographed the head over time.
Figure 8 is a double-blind method of 0.002% by weight (Group A), 0.01% by weight (Group B), 0.05% by weight (Group C) OC-P1P liposome prepared in accordance with an embodiment of the present invention to volunteers After the clinical trials, the heads were taken before and 16 weeks after application for each concentration group.

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

Example 1 Preparation of N-Boc-D-Phytosphingosine

Bull D-Phytosphingosine . Korean Chem. According to the method of Soc., 2003 , 24 , 267-268, N-Boc-D-phytosphingosine was prepared by reacting with (Boc) ₂ O in t-butanol to introduce a protecting group (Boc) to an amino group.

Example 2: Preparation of N-Boc-D-Phytosphingosine-3-O, P-cyclophosphate

1.05 equivalents of POCl ₃ was slowly added dropwise to a pyridine solvent (1/3 of the total volume) cooled to about −30 ° C. to prepare a POCl ₃ / pyridine solution. Example 1 a N-Boc-D- phytosphingosine pyridine solvent (1 equivalent), prepared in a prepared in advance and was dissolved in (2/3 of total volume) was cooled to -20 ℃ and then maintaining the temperature POCl ₃ / Pyridine solution was added. When the reaction temperature rose to room temperature, the solvent pyridine was removed under reduced pressure, adjusted to pH 2 with 6N HCl, and extracted with ethyl acetate. Then, the ethyl acetate extract was removed with water of MgSO ₄ and the solvent was concentrated under reduced pressure to quantitatively obtain the desired substance N-Boc-D-phytosphingosine-3-O, P-cyclophosphate. Used as starting material in Examples 3 and 4 without further purification.

Example 3: Preparation of O-cyclic Phytosphingosine-1-phosphate (Formula 1a) (1)

N-Boc-D-Pytosphingosine-3-O, P-cyclophosphate prepared in Example 2 was dissolved in CH ₂ Cl ₂ , and then 1/3 of the volume of the CH ₂ Cl ₂ solvent using trifluoroacetic acid were added at room temperature. Stirred at room temperature for 1 hour. Then, the solvent was removed under reduced pressure and vigorously stirred by adding Conc-HCl solvent to obtain the white solid produced by filtration, washed with water, washed with acetone and dried to dry the final target O-cyclic phytosphingosine-1-phosphate (OC -P1P) was obtained in the form of hydrochloride (total yield: 90%).

Example 4 Preparation of O-cyclic Phytosphingosine-1-Phosphate (Formula 1a) (2)

The N-Boc-D-phytosphingosine-3-O, P-cyclophosphate prepared in Example 2 was dissolved in an ethyl acetate solvent, cooled to 0 ° C., and HCl gas was injected for 1 hour. The resulting white solid was filtered, washed with ethyl acetate and dried to give the final product, O-cyclic phytosphingosine-1-phosphate (O-C-P1P), in the form of hydrochloride (total yield: 93%).

¹ H NMR (300 MHz, CD ₃ OD: CD ₃ COOD = 1: 1) data of the finally obtained material is shown in FIG. 1.

The obtained hydrochloride was treated with the same equivalent KOH ethanol solution to obtain O-cyclic phytosphingosine-1-phosphate from which the hydrochloride was removed.

Further, O-cyclic phytosphingosine-1-phosphate obtained above was treated with 3 equivalents of an aqueous NaOH solution to prepare an O-cyclic phytosphingosine-1-phosphate disodium salt.

Example 5: Preparation of N-cyclic phytosphingosine-1-phosphate (Formula 1b)

Bull. Korean Chem. N-Boc-D-Pytosphingosine-1-phosphoric acid dimethyl ester was prepared according to the method described in Soc., 2003 , 24 , 267-268. After dissolving in the prepared N-Boc-D- phytosphingosine-1-phosphoric acid dimethyl ester CH ₂ Cl ₂ and cooled to -20 ℃ 5 equivalents of bromodimethylsilane was added and stirred for about 1 hour. Then, water was added, and acetone was added to the residue obtained by heating and concentration. The resulting white solid was filtered to give the title compound (NC-P1P) (total yield: 13%).

¹ H NMR (300 MHz, CD ₃ OD: CD ₃ COOD = 1: 1) data of the finally obtained material is shown in FIG. 2.

Example 6: Preparation of O-C-P1P-containing Liposomes

First, 1 g of 75% soy phosphatidylcholine (Lipoid), 0.01 g of OC-P1P obtained in Example 4, and 0.3 g of vitamin E acetate were dissolved in a mixed solution of 20 g of ethanol and 2 g of ethoxydiglycol to prepare an OC-P1P ethanol solution. It was. Sonication was performed for 5 minutes for dissolution. Then, an aqueous solution of 0.5 g of menthol, 0.5 g of niacinamide, 3 g of natural organic sulfur, and 0.1 g of hesperidin in 71.59 g of distilled water was slowly added to the O-C-P1P ethanol solution. At the time of addition, it was stirred vigorously. After all the distilled water was added, the stirring was continued for 30 minutes. Thereafter, ultrasonic treatment was performed for 30 minutes using a bath-type sonicator to prepare the O-C-P1P-containing liposome by making the particle size around 100 nm.

Separately, when preparing liposomes in the preparation method, skin permeation promoting liposomes were prepared by adding poloxamer + polysorbate 80 (Tween 80) as a skin permeation promoter.

Example 7: Preparation of O-C-P1P-containing Liposomes (2)

In preparing the aqueous solution in Example 6, 1 g of willow bark extract, 1 g of green tea extract, 3 g of Centella extract, 0.5 g of nettle extract, 0.5 g of iris extract, 0.5 g of rosemary extract, 0.5 g of chamomile extract, and soybean fermentation Liposomes containing OC-P1P were prepared in the same manner as in Example 6, except that 1 g of water was additionally added to prepare an aqueous solution. However, liposomes were prepared using less distilled water as the amount of various extracts.

Example 8 Preparation of N-C-P1P-containing Liposomes (1)

Liposomes containing 0.01% NC-P1P were prepared in the same manner as in Example 6, except that NC-P1P prepared in Example 5 was used instead of OC-P1P in Example 6.

Example 9 Preparation of N-C-P1P-containing Liposomes (2)

Liposomes containing 0.01% NC-P1P were prepared in the same manner as in Example 7, except that NC-P1P prepared in Example 5 was used instead of OC-P1P in Example 7.

The composition of the liposomes prepared in Example 6-9 is shown in Table 1 below.

Example
Furtherance Example 6 Example 7 Example 8 Example 9 O-C-P1P 0.01 0.01 0 0 N-C-P1P 0 0 0.01 0.01 lecithin One One One One ethanol 20 20 20 20 Ethoxydiglycol 2 2 2 2 Pollock Sommer 0.5 0.5 0.5 0.5 Twin 80 0.5 0.5 0.5 0.5 Niacinamide 0.5 0.5 0.5 0.5 menthol 0.5 0.5 0.5 0.5 Natural Organic Sulfur 3 3 3 3 Hesperidin 0.1 0.1 0.1 0.1 Green tea extract 0 One 0 One Willow Bark Extract 0 One 0 One Soybean Fermentation 0 One 0 One Centipede extract 0 3 0 3 Nettle extract 0 0.5 0 0.5 Iris extract 0 0.5 0 0.5 Rosemary Extract 0 0.5 0 0.5 Chamomile Extract 0 0.5 0 0.5 Distilled water 71.89 63.89 71.89 63.89

Experimental Example 1 Hair Growth Test Using C3H Mouse (1)

A 6-week-old C3H mouse was purchased to remove some of the hair on the back, and then the epilation cream was applied to completely remove the existing hair. The mice that were not completely depilated were excluded from the experiment. After depilation, 5 mice per mouse cage were randomly bred and a total of 5 cages were used.

After depilation, the OC-P1P synthesized in Example 4 and the NC-P1P synthesized in Example 5 were mixed with ethanol and ethoxydiglycol in a mixed solution (9: 1, v / v) of 0.01 as a test compound. Dissolved in a concentration of weight% was applied once a day to the epilation site. A group coated with only a mixture of ethanol and ethoxydiglycol without the test compound was used as a negative control, and a 3% minoxidil formulation (minoxyl, Hyundai) was used as a positive control. Fig. 3 shows photographs taken of the epilation site at 14 and 18 days after the sample was applied.

According to the results of FIG. 3, in the non-treated group (negative control group), almost no hair grows and the skin is exposed to the outside, whereas in the OC-P1P-treated group and NC-P1P-treated group, hair growth is achieved very quickly. there was. The hair growth effect of OC-P1P was faster than that of the positive control, whereas the NC-P1P group was slightly lower than the positive control, but a significant hair growth effect was observed compared to the negative control. It is therefore clear from these results that the compounds according to the invention promote hair growth.

Experimental Example 2: Hair Growth Test Using C3H Mouse (2)

The O-C-P1P-containing liposomes prepared in Examples 6 and 7 were subjected to a hair growth test in the same manner as in Experimental Example 1. However, once a day the application was continued for three weeks, and then photographed the area of epilation. The results are shown in Fig.

According to Figure 4, OC-P1P liposome treatment group showed a significant hair growth effect compared to the negative control, it was shown to have a similar hair growth effect to the positive control.

Experimental Example 3: Hair growth test according to the concentration of O-C-P1P (3)

Samples were prepared by dissolving O-C-P1P prepared in Example 4 in a mixed solution (9: 1, v / v) of ethanol and ethoxydiglycol at three concentrations of 0.005% by weight, 0.01% by weight, and 0.02% by weight. The hair growth test was performed on the sample in the same manner as in Experimental Example 1.

5 shows photographs taken of the depilated areas on the 13th and 17th days after the sample is applied.

According to the results of FIG. 5, the hair growth effect was remarkably superior to the negative control at the concentration of 0.005-0.02 wt%, but there was no significant difference in hair growth effect among the three concentration groups of 0.005 wt%, 0.01 wt%, and 0.02 wt%. Since OC-P1P shows a similar effect to the 3% by weight of 3% minoxidil formulation administration group, even at a concentration of 300 times lower than minoxidil it can be seen that the same hair growth effect.

Experimental Example 4: Hair growth test according to the type of salt of O-C-P1P

Hair growth test was carried out in the same manner as in Experiment 1 for the OC-P1P hydrochloride synthesized in Example 4, OC-P1P prepared in Example 4, OC-P1P disodium salt prepared in Example 4. .

Fig. 6 shows photographs taken of the depilated areas on the 14th and 18th days after the sample was applied.

According to the results of Figure 6, regardless of the type of salt was found to have a significant hair growth effect compared to the negative control.

Experimental Example 5: hair growth efficacy clinical trial (1)

The O-C-P1P liposomes prepared in Example 7 were directly administered to volunteers whose hair loss progressed to compare hair growth efficacy. Two volunteers were applied 1-2 times a day, sprayed 5-7 times at a time, and then rubbed with hands after application to prevent the liposome solution from dripping.

7A and 7B show photographs of the head before and after application of the sample.

According to the results of FIGS. 7a and 7b, it was found that hair loss prevention and hair regrowth efficacy were very excellent over time compared to before application. Therefore, excellent hair loss prevention and hair growth effects were confirmed when applied directly to the human body.

Experimental Example 6: Hair growth efficacy clinical trial (2)

As described in Example 7, 0.002% by weight (group A), 0.01% by weight (group B), 0.05% by weight (group C) of OC-P1P liposomes were prepared, and administered directly to volunteers with hair loss, and had a hair growth effect. Was compared. The subject and the experimenter performed the experiment by double blinding so that the concentration was unknown.

After application, the effect of O-C-P1P concentration on the hair density and thickness was observed during 4, 8, 12, and 16 weeks. The results of observing density change and thickness change of hair at each concentration are summarized in Tables 2 and 3 below. The evaluation criteria were as follows (unit: evaluation unit of scalp density and thickness, 0: no change, 1: perceived improvement, 2: weakly improved, 3: improved, 4: significantly improved, 5: very improved). . The results of comparing the photographs taken before the application and after 16 weeks for each concentration group are shown in FIG. 8.

Hair density change over 16 weeks Average 0 weeks 8 weeks 12 weeks 16 weeks Group A 0 0.17 1.08 1.92 * Group B 0 0.22 2.00 * 2.78 * Group C 0 1.11 1.78 * 2.78 *

* Shows statistically significant results

Hair thickness change over 16 weeks Average 0 weeks 8 weeks 12 weeks 16 weeks Group A 0 0.00 0.17 0.42 Group B 0 0.11 0.78 1.33 * Group C 0 0.11 0.78 1.56 *

* Shows statistically significant results

As shown in Tables 2 and 3, statistically significant hair density and thickness change were observed at 12 and 16 weeks in groups B and C. Also, according to FIG. You can see the increase.

Experimental Example 7: Promote cell proliferation and collagen synthesis

In order to measure the anti-wrinkle effect of NC-P1P and OC-P1P, the proliferative effect of human fibroblasts was investigated by MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide). In order to examine collagen synthesis, procollagen type I produced in human fibroblasts (dermis cells) was measured.

To measure cell proliferation, human fibroblasts were incubated for 24 hours in DMEM (Dulbecco's modified eagle medium) medium containing 10% FBS and 100 unit / mL penicillin / streptomycin. Cultured in a CO ₂ incubator. After removing the medium, and cultured by adding NC-P1P and OC-P1P by concentration, the results of measuring the cell proliferation effect through MTT assay are summarized in Table 4.

To measure collagen synthesis, human fibroblasts (dermis cells) were incubated for 24 hours under the above conditions, medium was removed, and NC-P1P and OC-P1P (1uM and 10uM) diluted with fresh serum free medium were added. After culturing again for 24 hours, the result of measuring the amount of collagen with the procollagen type I C-peptide EIA kit with the culture medium is shown in Table 5 below.

According to the results of Tables 4 and 5, N-C-P1P and O-C-P1P not only promote cell proliferation but also promote collagen synthesis. Therefore, N-C-P1P and O-C-P1P have been found to be applicable to wrinkle improvement and aging products that promote the proliferation of human fibroblasts and collagen synthesis.

Claims

O-cyclic phytosphingosine-1-phosphate of formula 1a (OC-P1P) or N-cyclic phytosphingosine-1-phosphate of formula 1b, or a pharmaceutically acceptable salt thereof:
[Formula 1a]

[Chemical Formula 1b]

A method for preparing a compound of Formula 1a comprising reacting a compound of Formula 4 with trifluoroacetic acid or a hydrochloric acid gas to deprotect it:
[Formula 1a]

[Chemical Formula 4]

In Formula 4, R ¹ is a protecting group.

The method of claim 2, wherein the compound of Formula 4 is prepared by reacting a compound of Formula 3 with POCl ₃
(3)

In Formula 3, R ¹ is a protecting group.

The method of claim 3, wherein the compound of Formula 3 is prepared by introducing a protecting group to an amino group of D-phytosphingosine of Formula 2 below:
(2)

A method for preparing a compound of Formula 1b comprising reacting a compound of Formula 5 with bromotrimethylsilane and then reacting with water:
[Chemical Formula 1b]

[Chemical Formula 5]

In Formula 5, R ² is a protecting group.

A hair loss preventing or hair growth cosmetic composition comprising a compound of formula 1a or 1b according to claim 1 or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition for the prevention, treatment or hair growth of hair loss comprising a compound of formula 1a or 1b according to claim 1 or a pharmaceutically acceptable salt thereof.

8. A composition according to claim 6 or 7, wherein said composition is an agent for topical application to a skin area covered with scalp or hair.

The composition of claim 8, wherein the composition has a formulation of liposomes, nanoemulsions, shampoos, hair conditioners, or hair lotions.

A cosmetic composition for preventing, ameliorating or treating wrinkles, comprising the compound of formula 1a or 1b according to claim 1 or a pharmaceutically acceptable salt thereof.

The composition of claim 10, wherein the composition is a formulation for topical application to skin areas that are wrinkled or susceptible to wrinkles.

12. The composition of claim 11, wherein said composition has a formulation of liposomes or nanoemulsions.