KR101806485B1 - A composition comprising of Piper amide derivative for skin whitening - Google Patents

Abstract

The present invention relates to a composition for skin whitening comprising a piperamide derivative, wherein the compound inhibits the melanin synthesis and the expression of tyrosinase, TRP-1, TRP-2 and MITF protein which are involved in melanogenesis And thus can be effectively used as a skin whitening composition.

Description

FIELD OF THE INVENTION The present invention relates to a skin whitening composition comprising a piperamide derivative,

The present invention relates to a skin whitening composition comprising a piperamide derivative.

Melanin is a phenolic biopolymer found in skin, hair, retina, adrenal gland, inner ear and substantia nigra (Alaluf, S. et al., 2002). In the human body, melanin has two important functions. First, skin color is determined by the amount of melanin. The higher the amount of melanin, the darker skin color, and the different skin color is not due to the difference in the number of melanocytes, but because the size of melanocytes and the amount of melanin produced are different. In addition, melanin absorbs more than a certain amount of ultraviolet rays and blocks harmful ultraviolet rays from penetrating into the human body, thereby protecting the human body. The sunlight turns the skin brown, because the melanin cells present in the lower layers of the skin are stimulated by ultraviolet light to create melanin to protect the body, and the melanin that is made up to the skin is raised to prevent ultraviolet rays from penetrating deep into the skin Because. In the same logic, people who are exposed to the sun tend to have a lot of melanin to protect the skin from ultraviolet rays (Quevedo, W. C. et al., 1974; Slominski, A. et al., 2012).

In the melanin formation process, a melanoblast derived from a neural crest migrates to the epidermis and is differentiated into a melanocyte, a cell with a dendrite, where melanin is formed. Melanin is converted to tyrosine by tyrosinase, tyrosinase-related protein 1 (TRP-1), and DCT (DOPAchrome tautomerase) by the stepwise regulation of tyrosine by dopaquinone, Melanin (Jimbow, K. et al., 1994; Sulaimon, SS et al., 2003).

The most important transcription factor in the expression of tyrosinase-related protein (TYRP) genes is the microphtalmia-associated transcription factor (MITF) (Vance, KW et al., 2004), activation of cAMP-dependent protein kinase A And activation of CREB (cAMP response element-binding protein) by PKA induces MITF expression (Karin, M., 1994). In addition, the expression of TRPM1 (transient receptor potential cation channel subfamily M member 1) is regulated by MITF, and the expression of TRPM1 corresponds to the content of melanin in melanocytes (Oancea, E. et al., 2009). In a previous article [Devi, S. et al., 2009], knockdown of TRPM1 in melanocytes reduced intracellular Ca ²⁺ , resulting in decreased tyrosinase activity and melanin pigmentation.

Pigmentation can be caused by four steps: expression of a melanogenic enzyme, regulation of melanogenesis enzyme activity before or during melanin synthesis, melanosome transfer to keratinocytes, It is controlled through the deterioration and turnover of melanoma cotton. Among them, the melanogenesis process is the most important step for controlling the pigmentation.

The process of melanin formation is a naturally occurring phenomenon and does not result in overproduction of melanin in normal skin. However, when the skin responds to external stimuli such as ultraviolet light, environmental pollution or stress, melanin is overproduced and is not discharged out of the skin. It is transferred to keratinocyte and accumulates in the skin surface layer to accumulate spots, freckles and senile pigmentation (geriatric pigment spot), as well as promote skin aging and skin cancer.

Various skin protecting agents and whitening agents have been developed and used so far, but various problems have been raised. 4-hydroxyanisole and hydroquinone, which are whitening agents, are locally used for the treatment of spots, freckles, spots, and gestational hyperplasia. However, these compounds exhibit potent melanin inhibitory activity, Causing the denaturation or lethality of pigment cells and impairing the original function of the cells. In particular, the hydroquinone series exhibited a strong melanin biosynthesis inhibitory activity and was developed as a whitening cream. However, it has been approved for use in some countries because it causes skin irritation and skin diseases due to cytotoxicity. Kojic acid is currently used as an anti-browning agent for foods, cosmetics, and whitening agents for medicines along with arbutin (hydroquinone-β-D-glucopyranoside) and ascorbic acid (vitamin C) Antioxidants, antioxidants, and leading preservatives have also been reported. However, problems such as low inhibitory activity of kojic acid, discoloration during use, and instability of the substance itself have been raised. In addition, many substances such as 4-hydroxyindole and 4-hexylresorcinol exhibit tyrosinase-inhibiting activity, thereby lowering skin pigment synthesis. However, Mutation or the like, or exhibit strong toxicity, so that research on a whitening composition having low toxicity is required.

In addition, hyperpigmentation occurs due to ultraviolet damage and the protection of melanocytes to resist the onset of melanomas (Quevedo, WC et al., 1974; Slominski, A. et al., 2012) , Malignant melanoma can be formed by long-term exposure to ultraviolet light, resulting in direct damage and mutation in DNA. The mortality rate of metastatic melanotic melanoma is higher than other skin cancer, and its treatment is performed by surgery, chemotherapy, and radiation therapy. Conventional chemotherapy or radiotherapy has been reported to be ineffective, but more recently, biochemical therapy or adjuvant therapy has been shown to provide a palliative effect for cancer patients (Bhatia, S. et al., 2009 ). In particular, inhibition of melanogenesis or inhibition of tyrosinase expression (as a melanogenesis intermediate, L-DOPA is detected at high levels in melanoma patients) may be useful as an adjunct to skin metastatic or melanoma patients And aminobenzoic acid, a melanin formation inhibitor, can be used for the treatment of melanotic cancer and melanoma metastasis has been reported in International Publication WO 2005/070043. Therefore, as a new treatment against metastatic skin cancer, research on the resistance of hyperpigmentation is required.

Piper amide is predominantly composed of piper and is characterized by the presence of an α, β-unsaturated amide group. Piperamide natural products are known to exhibit a variety of physiological activities such as anti-inflammatory, antifungal, insecticidal, analgesic, antidepressant, and anti-cancer effects.

In general, the structure of the piperamide is characterized by having an α, β-unsaturated amide group as a parent, and the parent has been developed as a drug or a drug candidate. For example, AMG-9810 and SB-355791 are known as very potent vanilloid receptor TRPV1 antagonists, and tranilast is used as an anti-allergic agent in clinical practice. However, the possibility of its use as a skin whitening pharmaceutical composition or cosmetic composition through the activity of inhibiting the melanin formation of a piperamide derivative has not been disclosed yet.

Korean Patent Laid-Open Publication No. 2014-0109273, Piperamide-based compounds as Bombesin receptor subtype-3 regulator, published on Sep. 15, 2014. International Publication No. WO 2005/070043, Methods for treating non-melanoma cancers with paba, published Aug. 04, 2005. International Publication No. WO 2008/057300, TRPV1 antagonists and uses thereof, published May 15, 2008.

Alaluf, S. et al., The impact of epidermal melanin on objective measures of human skin color, Pigment Cell Res., 15 (2), 119-126, 2002. Bhatia, S. et al., Treatment of Metastatic Melanoma: An Overview, Oncology (Williston Park), 23 (6), 488-496, 2009. Busca, R. et al., Inhibition of the phosphatidylinositol 3-kinase / p70 (S6) -kinase pathway induces B16 melanoma cell differentiation, J Biol Chem., 271 (50), 31824-31830, 1996. Devi, S. et al., Calcium homeostasis in human melanocytes: role of transient receptor potential melastatin 1 (TRPM1) and its regulation by ultraviolet light, Am J Physiol Cell Physiol, 297 (3), C679-687, 2009. Hosoi, J. et al., Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid, Cancer research, 45, 1474-1478, 1985. Jimbow, K. et al., Molecular control of melanogenesis in malignant melanoma: a possible melanogenesis "chaperone", J Dermatol , 21 (11), 894-906, 1994. Karin, M., Signal transduction from the cell surface to the nucleus through the phosphorylation of transcription factors, Curr Opin Cell Biol., 6 (3), 415-424, 1994. Kim, D. S. et al., Delayed ERK activation by ceramide reduced melanin synthesis in human melanocytes, Cell Signal., 14 (9), 779-785, 2002. Kim, J. H. et al., Downregulation of Melanin Synthesis by Haginin A and Its Application to In Vivo Lightening Model, J Invest Dermatol., 128 (5), 1227-1235, 2008. Kim, S. M. et al., Column chromatography-free solution-phase synthesis of a natural piper-amide-like compound library, ACS Comb Sci., 15 (4), 208-215, 2013. Oancea, E. et al., TRPM1 forms ion channels associated with melanin content in melanocytes, Sci Signal., 2 (70), ra21, 2009. Park, K. T. et al., Inhibitory effect of mulberoside A and its derivatives on melanogenesis induced by ultraviolet B irradiation, Food Chem. Toxicol., 49, 3038-3045, 2011. Quevedo, W. C. et al., Light and skin color. In "Sunlight and Man ", Univ. of Tokyo Press, 165-194, 1974. Slominski, A. et al., Melanin pigmentation in mammalian skin and its hormonal regulation, Physiol Rev., 84 (4), 1155-1228, 2004. Slominski, A. et al., L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions, Pigment Cell Melanoma Res., 25 (1), 14-27, 2012. Sulaimon, S. S. et al., The biology of melanocytes, Vet Dermatol., 14 (2), 57-65, 2003. Vance, K. W. et al., The transcription network regulating melanocyte development and melanoma, Pigment Cell Res., 17 (4), 318-325, 2004. Xu, X. Z. et al., Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform, Proc Natl Acad Sci U S A., 98 (19), 10692-10697, 2001.

It is an object of the present invention to provide a skin whitening composition comprising a piperamide derivative.

The present invention relates to a skin whitening composition containing an?,? - unsaturated amide compound represented by the following formula (1).

[Chemical Formula 1]

In Formula 1,

R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, C 1 to C 10 alkyl, alkylphenyl, 2,3-dihydrobenzo [b] [1,4] dioxin (2,3-dihydrobenzo [b] [1,4 ] dioxine, 2-thiazole, benzyl, alkoxybenzyl, phenylalkyl, phenylalkyl or hydroxyphenylalkyl substituted with one or more alkoxy groups, or comprising nitrogen of said amide group to form a ring;

R ₃ is selected from the group consisting of C1 to C10 alkyl, C1 to C10 alkenyl, furanyl, phenyl, phenyl substituted with one or more alkyl groups, phenyl substituted with halogen, hydroxyphenyl, nitrophenyl, phenyl substituted with one or more alkoxy, Alkoxyalkylphenyl, halogenalkoxyphenyl or benzo [d] [1,3] dioxole;

R < ₄ > is hydrogen or C1 to C10 alkyl.

The term " alkyl " in the present invention refers to a straight or branched chain saturated hydrocarbon group of a single bond, for example, methyl, ethyl, propyl, n-butyl, isobutyl, tert-butyl and 1-methylpropyl.

The term " alkoxy " of the present invention refers to an oxygen group to which a single bond of a linear or branched saturated hydrocarbon is bonded, and includes, for example, methoxy, ethoxy, propoxy, n-butoxy, Propoxy and the like.

In addition, the compound of formula (1)

(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide;

(E) -3- (4-bromophenyl) -N- (4-hydroxyphenethyl) acrylamide;

(E) -3- (2-fluoro-4-methoxyphenyl) -N- (4-hydroxyphenethyl) acrylamide;

(E) -3- (2,5-dimethylphenyl) -N- (4-hydroxyphenethyl) acrylamide;

(E) -3- (4-chlorophenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide;

(E) -3- (benzo [d] [1,3] dioxol-5-yl) -N- (2,3- dihydrobenzo [b] [1,4] dioxin- Amide; And

(E) -N- (3-methoxyphenyl) -3- (4-methoxyphenyl) acrylamide.

The present invention also provides a pharmaceutical composition for skin whitening comprising the piperamide derivatives of Formula 1, wherein the pharmaceutical composition is a skin whitening composition comprising at least one selected from the group consisting of spots, freckles, liver, brown or black spots, pregnant brown spots, , Hyperpigmentation after drug use, scratches such as scars and burns, or hyperpigmentation due to melanin overproduction such as hyperpigmentation after inflammation due to dermatitis.

The pharmaceutical composition for skin whitening comprising the piperamide derivatives of Formula 1 may be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, May be formulated in the form of injection solutions. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition may be used as an external preparation for skin having one type of formulation selected from ointments, lotions, spray agents, patches, creams, gels and gels, and the external preparation for skin is applied to the skin And can be used as a composition for preventing or treating skin hypercholesterolemia, and can be used as a composition for increasing transdermal absorption, for example, in a non-limiting sense, especially dimethylsulfoxide, dimethylacetamide, dimethylformamide, Acetone, propylene glycol or polyethylene glycol. The frequency of application may vary considerably depending on the age, sex, weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber and the frequency of application is 10 times per month, Preferably four times a week to one day, more preferably three times per week to three times a day, more preferably once or twice a day.

Meanwhile, the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and the term " administration " of the present invention means introduction of a predetermined substance into a subject by an appropriate method. The administration route of the composition may be administered through any conventional route so long as it can reach the target tissue, for example, orally, rectally, intravenously, muscularly, subcutaneously, intraperitoneally or topically, Do not.

The term " individual " refers to a subject in need of treatment of a disease, and more particularly refers to a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, .

The term " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. Preferably, the piperamide derivatives of formula (I) of the present invention may be administered at a daily dose of 0.0001 to 100 mg / kg body weight, more preferably 0.001 to 100 mg / kg body weight, based on the solid content. The administration may be carried out once per day, or divided into several doses. Further, when administering the initial drug, administration may be started in an amount less than that required for the desired effect, and the dose may be gradually increased until the desired effect is obtained.

In another embodiment, the piperamide derivative of Formula 1 of the present invention may be used as a cosmetic composition having a skin whitening effect. In addition to the piperamide derivatives, the cosmetic composition may include all components commonly used in cosmetics . For example, it may contain common auxiliary components such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers and perfumes. The above-mentioned components can be selected within a range that does not impair the effect inherent in the cosmetic, depending on the purpose of formulation or use.

In addition, the cosmetic composition may be prepared in any form conventionally produced in the art, and examples thereof include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More preferably, it may have one type of formulation selected from softening longevity (skin), astringent lotion, nutritional lotion, nutritional cream, massage cream, essence, pack, skin sticking patch and skin sticking gel.

The cosmetic composition containing the piperamide derivative of Formula 1 may be used every day or may be used for an unspecified period of time and is preferably used depending on the age, skin condition or skin type of the user, or the concentration of the piperamide derivative You can control usage, frequency and duration.

The present invention relates to a composition for skin whitening comprising a piperamide derivative, wherein the compound inhibits the melanin synthesis and the expression of tyrosinase, TRP-1, TRP-2 and MITF protein which are involved in melanogenesis And thus can be effectively used as a skin whitening composition.

1 is a graph showing cell viability and melanin content in melan-A cells for Example 2-1 of the present invention.
FIG. 2 is a graph showing the results of measurement of tyrosinase activity in Example 2-1 of the present invention. FIG. 2A shows the results of measurement in melan-A cells, FIGS. 2B and 2C show the results of measurement of tyrosinase activity in melan- And the results are shown by the L-DOPA staining method.
FIG. 3 shows the results (FIG. 3A) of locally applied guinea pig skin induced hyperpigmentation and the Melanin Index (FIG. 3B) according to Example 2-1 of the present invention.
FIG. 4 shows the result of the application of Example 2-1 of the present invention to the skin of guinea pigs induced by hyperpigmentation, followed by the isolation of epidermal cells and the calculation of melanin content (FIG. 4A) and Fontana-Masson silver staining (FIG. 4B) .
FIG. 5 is a result of locally applying the Example 2-1 of the present invention to guinea pig skin induced hyperpigmentation, and then separating epidermal cells and measuring tyrosinase activity.
Fig. 6 shows mRNA expression of tyrosinase, TRP-1, TRP-2 and MITF in melan-A cells for Example 2-1 of the present invention.
Figure 7 shows the protein expression of tyrosinase, TRP-1, TRP-2 and MITF for Example 2-1 of the invention in melan-A cells (Figures 7A and 7B) and guinea pig epidermal cells (Figure 7C) And Western blot.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

< Example  One. Piper Amide  Synthesis of derivatives>

The piperamide derivative of the present invention can be obtained by following the procedure of Example 2 of Korean Laid-Open Patent Application No. 2014-0109273 and a prior art [Kim, SM et al., 2013] 1. &Lt; / RTI &gt;

[Reaction Scheme 1]

With reference to the scheme 1, the solubility key intermediate 1 (β-phosphono-N- hydroxysuccinimidyl ester) of 0.85 equivalents of the amine and the condensation reaction with 2 (β-phosphono amides) and N- hydroxysuccinimide soluble in water in water (N-hydroxysuccinimide). The remaining 1 was treated with a carbonyl compound scavenger, Girard's reagent T (3), to make 4 (hydrazine derivative) which was soluble in water and then removed by aqueous work-up. Aldehyde with 2 obtained by the above process is via an aldehyde and HWE reaction (Horner-Wadsworth-Emmons reaction) of 1.5 equivalent, to produce a soluble phosphate and 5 (α, β- unsaturated amide) in water, remains, like the removal of Girard's The reagent T (3) was removed by treatment.

[Reaction Scheme 2]

Referring to Scheme 2 above, Core Intermediate 1a ([beta] -phosphono-N-hydroxysuccinimidyl ester) can be readily isolated from the commercially available 6 ([beta] -phosphonoester) in two steps without column chromatography purification Were synthesized. In order to increase the (E) - selectivity in the HWE reaction, a sterically large isopropyl phosphonate group was selected. On the ethanol solvent conditions in 6 ml of 1N NaOH for hydrolysis to obtain the 7 (phosphoryl acetic acid). Without further purification aqueous work-up 7 methylene chloride (methylene chloride) EDCI (1- Ethyl-3- (3-dimethylaminopropyl) carbodiimide) and a good reactivity 1a processes the NHS (N-hydroxysuccinimide) in solvent conditions and obtained only . The aqueous work-up alone gave the compound of good purity and the compound obtained was used in the next reaction.

[Reaction Scheme 3]

Notes to Scheme 3 a Intermediate 1a obtained in the above procedure to, methylene chloride solvent conditions to condensation reaction with 0.85 equivalents of a variety of amine 8 {1} to {14}, 8 of 14 in 2 to obtain the amide of 14. The reaction of 1a with alkylamines 8 {1} to 8 {3} and 8 {6} to 8 {14} was terminated within 4 hours at room temperature and the reaction with arylamines 8 {4} and 8 {5} The reaction was terminated under triethylamine and DMAP (4-Dimethylaminopyridine) catalyst and terminated at high temperature (40 ° C) for 12 hours under the condition of no additives. After confirming that the amine 8 had disappeared with TLC, Girard's reagent T (3) was processed to make the remaining 1a as soluble derivative 4a . The condensation reaction of 1a with Girard's reagent T (3) was terminated at room temperature only for 12 hours, and β-phosphonamide 2a {1-14} of good yield and purity was obtained after aqueous work-up.

[Reaction Scheme 4]

Referring to Reaction Scheme 4, t-BuOK served as the most suitable base for the HWE reaction with 2a and 1.5 equivalents of aldehyde. Most of the aldehydes reacted with 2a treated with t-BuOK under tetrahydrofuran (THF) solvent conditions to yield 5 (α, β-unsaturated amide) in high yields and the products were characterized by high (E) Respectively. However, in the case of 9 {9} (3-hydroxy benzaldehyde), NaH functions as a more suitable base. Seventeen kinds of aldehydes 9 {1} to {17} were chemically synthesized by HWE reaction with 14 kinds of 2a {1-14} . After completion of the reaction, remaining aldehyde 9 was removed by aqueous work-up treatment with Girard's reagent T (3) , and 238 piferamide-based compound libraries with an average purity of 95% .

[Reaction Scheme 5]

Scheme 5 devises 1b (α-methyl β-phosphono-N-hydroxysuccinimidyl ester) with a substituent at the α position to give diversity to the piperamide skeleton. 1b was synthesized from 10 (? -Methyl? -Phosphono ester) in the same manner as the synthesis of 1a in scheme 2. Condensation reaction of 6 kinds of amines (8 {1}, 8 {3}, 8 {5}, 8 {6}, 8 {8} and 8 {10}) with 1b yielded 6 2b (β-phosphono Amide). The 2b and the 17 aldehydes 9 {1-17} were chemically HWE reacted to give 11 . In the above procedure, 11 was isolated only by aqueous work-up without purification by column chromatography. The α-methylpiperamide compound prepared in the above procedure exhibited a high yield and (E) -electivity in most cases.

<Example 2> Identification of physicochemical structure of the piperamide derivative>

The compounds of the following Examples 2-1 to 2-45 were able to be synthesized with reference to Reaction Formulas 1 to 5 of Example 1, and the physicochemical properties of the synthesized compounds are as follows.

Example 2-1. (E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide

(E) -3- (4- (t-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide;

light yellow solid;

m.p. 86-89 DEG C;

^{1 H NMR (300 MHz, CDCl} 3): δ 1.33 (s, 9H), 4.26 (s, 4H), 6.47 (1H, d, J = 15.3 Hz), 6.83 (1H, d, J = 8.7 Hz), 6.99 (1H, br d), 7.13 (1H, s), 7.41 (2H, d, J = 8.7 Hz), 7.48 (2H, d, J = 8.4 Hz), 7.72 (1H, d, J = 15.3 Hz) ;

^{13 C NMR (300 MHz, CDCl} 3): δ 31.2 (3C), 34.9, 64.3, 64.4, 109.9, 113.5, 117.2 (2C), 119.9, 125.8 (2C), 127.7 (2C), 131.9, 141.9, 141.8, 142.0, 143.5, 153.4;

FT-IR (neat): ν _max 2956, 1658, 1607, 1507, 1215 cm ^-1 ;

HRMS (FAB): m / z calc'd for C 21 H 24 NO 3; [M + H] &lt; ⁺ &gt;:338.1756; found: 338.1767.

Example  2-2. (E) -3- (4- Bromophenyl ) -N- (4- Hydroxyphenethyl ) Acrylamide

(E) -3- (4-bromophenyl) -N- (4-hydroxyphenethyl) acrylamide;

yield 66%;

yellow solid;

m.p. 179-180 DEG C;

^{1 H NMR (300 MHz, CD} 3 OD): δ H 2.75 (2H, t, J = 7.3 Hz, CH 2 Ph), 3.46 (2H, t, J = 7.3 Hz, NHCH 2), 6.57 (1H, d , J = 15.9 Hz, CH = CH), 6.71 (2H, d, J = 8.6 Hz, Ph), 7.05 (2H, d, J = 8.6Hz, Ph), 7.46 (2H, d, J = 8.4 Hz, Ph), 7.50 (1H, d, J = 16.3 Hz, CH = CH), 7.54 (2H, d, J = 8.6 Hz, Ph);

_{^{13 C NMR (125MHz, CD 3}} OD): δ C 35.7, 42.5, 116.3 (2C), 122.8, 124.6, 130.4 (2C), 130.7 (2C), 131.2, 133.1 (2C), 135.5, 140.2, 156.9, 168.3 ;

FT-IR (neat): ν _max 3289, 2452, 1649, 1609, 816 cm ^-1 ;

MS (FAB): m / z 346.44 (100) 136 (70);

HRMS (FAB): calc'd for C 17 H 17 BrNO 2; [M + H] &lt; ⁺ &gt;:346.0443; found: 346.0456.

Example  2-3. (E) -3- (2- Fluoro -4- Methoxyphenyl ) -N- (4- Hydroxyphenethyl ) Acrylamide

(E) -3- (2-fluoro-4-methoxyphenyl) -N- (4-hydroxyphenethyl) acrylamide;

yield 93%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.76 (2H, t, J = 7.0 Hz, CH 2 Ph), 3.57 (2H, q, J = 6.6 Hz, NHCH 2), 3.78 (s, 3H, _{OCH 3), 5.76 (br s} , 1H, NH), 6.33 (1H, d, J = 15.6 Hz, CH = CH), 6.58 (1H, dd, J = 2.6, 12.6 Hz, Ph), 6.64 (1H, dd, J = 2.6, 8.6Hz, Ph), 6.80 (2H, d, J = 8.4 Hz, Ph), 7.01 (2H, d, J = 8.4 Hz, Ph), 7.34 (1H, t, J = 8.6 Hz , Ph), 7.60 (1H, d, J = 15.8 Hz, CH = CH);

MS (FAB): m / z 316.44 (100) 136 (70).

Example  2-4. (E) -3- (2,5- Dimethylphenyl ) -N- (4- Hydroxyphenethyl ) Acrylamide

(E) -3- (2,5-dimethylphenyl) -N- (4-hydroxyphenethyl) acrylamide;

yield 82%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.29 (3H, s, PhCH 3), 2.35 (3H, s, PhCH 3), 2.80 (2H, t, J = 6.9 Hz, CH 2 Ph), 3.62 (2H, td, J = 6.0 , 7.2 Hz, CH 2 NH 2), 5.77 (1H, br t, J = 5.4 Hz, NH), 6.23 (1H, d, J = 15.6 Hz, CH = CH), 6.83 (2H, td, J = 2.1 , 8.7 Hz, Ph), 7.02-7.10 (4H, m, Ph), 7.29 (1H, s, Ph), 7.89 (1H, d, J = 15.6 Hz, CH = CH) ;

HRMS (FAB): calc'd for C 19 H 22 NO 2; [M + H] &lt; ⁺ &gt;:296.1651; found: 296.1653.

Example  2-5. (E) -3- (4- Chlorophenyl ) -N- (2,3- Dihydrobenzo [b] [1,4] dioxin -6-yl) acrylamide

(E) -3- (4-chlorophenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide;

yield 95%;

brown solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 4.25 (4H, s, CH 2), 6.50 (1H, d, J = 15.6 Hz, CH = CH), 6.82 (1H, d, J = 8.7 Hz, Ph), 7.01 (1H, d , J = 7.5 Hz, NH), 7.33 (2H, d, J = 8.4 Hz, Ph), 7.37 (1H, s, Ph), 7.43 (2H, d, J = 8.4 Hz , Ph), 7.67 (1H, d, J = 15.3 Hz, CH = CH);

HRMS (FAB): calc'd for C 17 H 15 ClNO 3; [M + H] &lt; ⁺ &gt;:316.0740; found: 316.0727.

Examples 2-6. (E) -3- (benzo [d] [1,3] dioxol-5-yl) -N- (2,3- dihydrobenzo [b] [1,4] dioxin- Amide

(E) -3- (benzo [d] [1,3] dioxol-5-yl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide;

yield 79%;

brown solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 4.20 (4H, s, CH 2), 5.96 (2H, s, CH 2), 6.38 (1H, d, J = 15.3 Hz, CH = CH), 6.74 (1H, d, J = 8.1 Hz, Ph), 6.78 (1H, d, J = 8.7 Hz, Ph), 6.90-6.93 , s, Ph), 7.59 (1H, d, J = 15.6 Hz, CH = CH), 7.83 (1H, s, Ph);

HRMS (FAB): calc'd for C 18 H 16 NO 5; [M + H] &lt; ⁺ &gt;:326.1028; found: 326.1039.

Example  2-7. (E) -N- (3- Methoxyphenyl ) -3- (4- Methoxyphenyl ) Acrylamide

(E) -N- (3-methoxyphenyl) -3- (4-methoxyphenyl) acrylamide;

yield 75%;

yellow oil;

^{1 H NMR (300 MHz, CDCl} 3): δ H 3.78 (3H, s, OCH 3), 3.81 (3H, s, OCH 3), 6.46 (1H, d, J = 15.3 Hz, CH = CH), 6.66 (1H, ddd, J = 0.9 , 2.4, 8.1 Hz, Ph), 6.85 (2H, td, J = 2.1, 8.7 Hz, Ph), 7.21 (1H, t, J = 8.1 Hz, Ph), 7.41 (2H (t, J = 2.7, 9.0 Hz, Ph), 7.45 (1H, s, Ph), 7.69 (1H, d, J = 15.6 Hz, CH = CH), 7.84 (1H, br s, NH);

HRMS (FAB): calc'd for C 17 H 18 NO 3; [M + H] &lt; ⁺ &gt;:284.1287; found: 284.1292.

Example  2-8. (E) -3- (2,3- Dimethoxyphenyl ) -N- Phenetyl acrylamide

(E) -3- (2,3-dimethoxyphenyl) -N-phenethylacrylamide;

yield 99%;

white solid;

^{1 H NMR (400 MHz, CDCl} 3): δ H 2.87 (2H, t, J = 6.9 Hz, CH 2 Ph), 3.63 (2H, dt, J = 6.3, 6.8 Hz, CH 2 NH 2), 3.81 ( _{3H, s, OCH 3),} 3.85 (3H, s, OCH 3), 5.67 (1H, br s, NH), 6.39 (1H, d, J = 15.9 Hz, CH = CH), 6.88 (1H, dd, J = 1.2, 8.0 Hz, Ph ), 7.00 (1H, t, J = 7.9 Hz, Ph), 7.20-7.24 (3H, m, Ph), 7.28-7.32 (2H, m, Ph), 7.83 (1H, d, J = 15.8 Hz, CH = CH);

HRMS (FAB): calc'd for C 19 H 22 NO 3; [M + H] &lt; ⁺ &gt;:312.1600; found: 312.1591.

Example  2-9. (E) -3- (4- Bromophenyl ) -N- Phenetyl acrylamide

(E) -3- (4-bromophenyl) -N-phenethylacrylamide;

yield 99%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.87 (2H, t, J = 6.8 Hz, CH 2 Ph), 3.63 (2H, dt, J = 6.0, 6.8 Hz, CH 2 NH 2), 5.70 ( 1H, br s, NH), 6.29 (1H, d, J = 15.6 Hz, CH = CH), 7.17-7.27 (3H, m, Ph), 7.32-7.41 (4H, m, Ph), 7.44-7.47 ( 2H, m, Ph), 7.53 (1H, d, J = 15.6 Hz, CH = CH);

HRMS (FAB): calc'd for C 17 H 17 BrNO; [M + H] &lt; ⁺ &gt;:330.0494; found: 330.0493.

Example  2-10. (E) -3- (4- Methoxy -2- Methylphenyl ) -N- Phenetyl acrylamide

(E) -3- (4-methoxy-2-methylphenyl) -N-phenethylacrylamide;

yield 100%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.40 (3H, s, CH 3), 2.89 (2H, t, J = 6.6 Hz, CH 2 Ph), 3.65 (2H, dt, J = 6.3, 6.6 _{Hz, CH 2 NH 2),} 3.79 (3H, s, OCH 3), 5.85 (1H, br s, NH), 6.17 (1H, d, J = 15.3 Hz, CH = CH), 6.67-6.70 (2H, m, Ph), 7.21-7.32 (5H, m, Ph), 7.43 (1H, d, J = 8.1 Hz, Ph), 7.85 (1H, d, J = 15.0 Hz, CH = CH);

HRMS (FAB): calc'd for C 19 H 22 NO 2; [M + H] &lt; ⁺ &gt;:296.1651; found: 296.1655.

Example  2-11. (E) -N- Penetil -3- (p- Tolyl ) Acrylamide

(E) -N-phenethyl-3- (p-tolyl) acrylamide;

yield 100%;

white solid;

^{1 H NMR (400 MHz, CDCl} 3): δ H 2.34 (3H, s, CH 3), 2.87 (2H, t, J = 6.9 Hz, CH 2 Ph), 3.65 (2H, q, J = 6.6 Hz, _{CH 2 NH 2), 5.52 (} 1H, br s, NH), 6.24 (1H, d, J = 15.5 Hz, CH = CH), 6.98-7.23 (5H, m, Ph), 7.29-7.37 (4H, m , &Lt; / RTI &gt; Ph), 7.57 (1H, d, J = 15.6 Hz, CH = CH);

HRMS (FAB): calc'd for C 18 H 20 NO; [M + H] &lt; ⁺ &gt;:266.1545; found: 266.1550.

Examples 2-12. (E) -3-phenyl-1- (piperidin-1-yl) prop-2-

(E) -3-phenyl-1- (piperidin-1-yl) prop-2-en-1-one;

yield 100%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 1.58-1.70 (6H, m, CH 2), 3.56-3.65 (4H, m, CH 2), 6.88 (1H, d, J = 15.6 Hz, CH = CH), 7.28-7.37 (3H, m, Ph), 7.46-7.51 (2H, m, Ph), 7.63 (1H, d, J = 15.4 Hz, CH = CH);

HRMS (FAB): calc'd for C 14 H 18 NO; [M + H] &lt; ⁺ &gt;:216.1388; found: 216.1382.

Examples 2-13. (E) -3- (furan-2-yl) -1- (piperidin- 1 -yl)

(E) -3- (furan-2-yl) -1- (piperidin-1-yl) prop-2-en-1-one;

yield 86%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 1.59-1.69 (6H, m, CH 2), 3.57-3.65 (4H, m, CH 2), 6.43 (1H, dd, J = 1.8, 3.3 Hz, furan), 6.52 (1H, d , J = 3.6 Hz, furan), 6.81 (1H, d, J = 15.3 Hz, CH = CH), 7.42 (1H, d, J = 1.8 Hz, furan), 7.44 (1H , d, J = 15.0 Hz, CH = CH);

HRMS (FAB): calc'd for C 12 H 16 NO 2; [M + H] &lt; ⁺ &gt;:206.1181; found: 206.1180.

Examples 2-14. (E) -3- (furan-2-yl) -N- (2-methylbutyl) acrylamide

(E) -3- (furan-2-yl) -N- (2-methylbutyl) acrylamide;

yield 89%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.88 (3H, s, CH 3), 0.90 (3H, s, CH 3), 1.07-1.19 (1H, m, CH 2), 1.22-1.45 (1H _{, m, CH 2), 1.47-1.63} (1H, m, CH), 3.11-3.20 (1H, m. CH 2), 3.25-3.33 (1H, m, CH 2), 5.78 (1H, br s, NH ), 6.30 (1H, d, J = 15.4 Hz, CH = CH), 6.41 (1H, dd, J = 1.8, 3.3 Hz, furan), 6.49 (1H, d, J = 3.3 Hz, furan), 7.37 ( 1H, d, J = 15.2 Hz, CH = CH), 7.39 (1H, d, J = 1.3 Hz, furan);

HRMS (FAB): calc'd for C 12 H 18 NO 2; [M + H] &lt; ⁺ &gt;:208.1338; found: 208.1341.

Example  2-15. (E) -3- (3,4- Dimethoxyphenyl ) -N- (4- Methoxyphenethyl ) Acrylamide

(E) -3- (3,4-dimethoxyphenyl) -N- (4-methoxyphenethyl) acrylamide;

yield 52%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.81 (2H, t, J = 6.8 Hz, CH 2 Ph), 3.61 (2H, td, J = 6.2, 6.6 Hz, NHCH 2), 3.78 (3H, _{s, OCH 3), 3.88 (} 6H, s, OCH 3), 5.53 (1H, br s, NH), 6.17 (1H, d, J = 15.6 Hz, CH = CH), 6.81-6.86 (3H, m, Ph), 6.98 (1H, d , J = 1.8 Hz, Ph), 7.05 (1H, dd, J = 1.8, 8.3 Hz, Ph), 7.13 (2H, d, J = 8.2 Hz, Ph), 7.54 (1H , d, J = 15.6 Hz, CH = CH);

HRMS (FAB): Calc'd for C ₂₀ H ₂₄ NO ₄ ; [M + H] &lt; ⁺ &gt;:342.1705; found: 342.1703.

Example  2-16. (E) -N- (4- Methoxyphenethyl ) -3- (p- Tolyl ) Acrylamide

(E) -N- (4-methoxyphenethyl) -3- (p-tolyl) acrylamide;

yield 58%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.34 (3H, s, CH 3), 2.81 (2H, t, J = 6.8 Hz, CH 2 Ph), 3.60 (2H, td, J = 6.2, 6.8 _{Hz, NHCH 2), 3.78 (} 3H, s, OCH 3), 5.64 (1H, br s, NH), 6.26 (1H, d, J = 15.5 Hz, CH = CH), 6.84 (2H, td, J = 1.7, 8.2 Hz, Ph), 7.12 (2H, d, J = 8.4 Hz, Ph), 7.14 (2H, d, J = 7.7 Hz, Ph), 7.36 (2H, d, J = 8.1 Hz, Ph), 7.57 (1H, doublet, J = 15.6 Hz, CH = CH);

HRMS (FAB): calc'd for C 19 H 22 NO 2; [M + H] &lt; ⁺ &gt;:296.1651; found: 296.1653.

Example  2-17. (E) -3- (4- Bromophenyl ) -N- (3,4- Dimethoxyphenethyl ) Acrylamide

(E) -3- (4-bromophenyl) -N- (3,4-dimethoxyphenethyl) acrylamide;

yield 100%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.80 (2H, t, J = 7.0 Hz, CH 2 Ph), 3.6 (2H, td, J = 6.2, 6.8 Hz, NHCH 2), 3.83 (6H, _{s, OCH 3), 5.75 (} 1H, br s, NH), 6.29 (1H, d, J = 15.5 Hz, CH = CH), 6.72 (1H, d, J = 7.5 Hz, Ph), 6.74 (1H, s, Ph), 6.79 (1H , d, J = 7.9 Hz, Ph), 7.30 (2H, d, J = 8.6 Hz, Ph), 7.44 (2H, d, J = 8.6 Hz, Ph), 7.52 (1H , d, J = 15.6 Hz, CH = CH);

HRMS (FAB): calc'd for C 19 H 21 BrNO 3; [M + H] &lt; ⁺ &gt;:390.0705; found: 390.0716.

Example  2-18. (E) -N- (3,4- Dimethoxyphenethyl ) -3- (2- Fluoro -4- Methoxyphenyl ) Acrylamide

(E) -N- (3,4-dimethoxyphenethyl) -3- (2-fluoro-4-methoxyphenyl) acrylamide;

yield 93%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.81 (2H, t, J = 6.8 Hz, CH 2 Ph), 3.6 (2H, td, J = 6.1, 6.8 Hz, NHCH 2), 3.79 (3H, _{s, OCH 3), 3.84 (} 6H, s, OCH 3), 5.67 (1H, br s, NH), 6.32 (1H, d, J = 15.8 Hz, CH = CH), 6.59 (1H, dd, J = 2.6, 12.6 Hz, Ph), 6.65 (1H, dd, J = 2.4, 8.5 Hz, Ph), 6.75-6.81 (3H, m, Ph), 7.35 (1H, t, J = 8.6 Hz, Ph), 7.60 (1H, d, J = 15.8 Hz, CH = CH);

HRMS (FAB): calc'd for C 20 H 23 FNO 4; [M + H] &lt; ⁺ &gt;:360.1611; found: 360.1621.

Example  2-19. (E) -N- Benzyldeca -2- Enamide

(E) -N-benzyldeca-2-enamide;

yield 95%;

white solid;

m.p. 68-70 캜;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.85 (3H, t, J = 6.7Hz, CH3), 1.15-1.33 (8H, m, CH 2), 1.35-1.44 (2H, m, CH 2) , 2.15 (2H, td, J = 7.2, 7.3 Hz, CH 2), 4.49 (2H, d, J = 5.7 Hz, CH2Ph), 5.65 (1H, br s, NH), 5.75 (1H, td, J = 1.5, 15.2 Hz, CH = CH), 6.87 (1H, td, J = 7.0, 15.2 Hz, CH = CH), 7.25-7.35 (5H, m, Ph);

^{13 C NMR (75 MHz, CDCl} 3): δ C = 14.1, 22.6, 28.2, 29.0, 29.1, 31.7, 32.0, 43.5, 123.2, 127.4, 127.8 (2C), 128.6 (2C), 138.3, 145.3, 165.9;

FT-IR (neat): ν _max 3291, 2921, 1669, 1622, 1552 cm ^-1 ;

MS (FAB): m / z 260 (100);

HRMS (FAB): calc'd for C17H26NO; [M + H] &lt; ⁺ &gt;:260.1970; found: 260.2019.

Example  2-20. (E) -N- Phenetyldeca -2- Enamide

(E) -N-phenethyldeca-2-enamide;

yield 97%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.85 (3H, t, J = 7.0 Hz, CH 3), 1.24 (8H, s, CH 2), 1.35-1.42 (2H, m, CH 2), 2.12 (2H, dq, J = 1.3, 7.0 Hz, CH 2), 2.82 (2H, t, J = 7.0 Hz, CH 2 Ph), 3.56 (2H, dt, J = 6.1, 7.0 Hz, NHCH 2), 5.46 (1H, br s, NH), 5.66 (1H, td, J = 1.5, 15.4 Hz, CH = CH), 6.80 (1H, td, J = 6.8, 15.4 Hz, CH = CH), 7.14-7.32 5H, m, Ph);

HRMS (FAB): calc'd for C 18 H 28 NO; [M + H] &lt; ⁺ &gt;:274.2171; found: 274.2167.

Example  2-21. (E) -N- (4- Methoxyphenethyl ) Deca -2- Enamide

(E) -N- (4-methoxyphenethyl) deca-2-enamide;

yield 96%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.85 (3H, t, J = 7.0 Hz, CH 3), 1.23 (8H, s, CH 2), 1.34-1.41 (2H, m, CH 2), 2.12 (2H, dq, J = 1.3, 7.0 Hz, CH 2), 2.75 (2H, t, J = 7.0 Hz, CH 2 Ph), 3.51 (2H, dt, J = 6.1, 7.0 Hz, NHCH 2), _{3.77 (3H, s, OCH 3} ), 5.47 (1H, br s, NH), 5.66 (1H, td, J = 1.5, 15.2 Hz, CH = CH), 6.81 (2H, d, J = 9.2 Hz, Ph ), 7.09 (2H, d, J = 8.4 Hz, Ph).

Example  2-22. (E) -N- (2- Methyl butyl ) Deca -2- Enamide

(E) -N- (2-methylbutyl) deca-2-enamide;

yield 98%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.83-0.90 (9H, m, CH 3), 1.20-1.31 (10H, m, CH 2), 1.29-1.45 (2H, m, CH 2), 1.49 -1.60 (1H, m, CH) , 2.14 (2H, dq, J = 1.5, 7.0 Hz, CH 2), 3.10-3.28 (2H, m, CH 2), 5.45 (1H, br s, NH), 5.73 (1H, td, J = 1.5, 15.2 Hz, CH = CH), 6.80 (1H, td, J = 7.0, 15.2 Hz, CH = CH);

HRMS (FAB): calc'd for C 15 H 30 NO; [M + H] &lt; ⁺ &gt;:240.2327; found: 240.2333.

Example  2-23. (E) -N- (3,4- Dimethoxyphenethyl ) Deca -2- Enamide

(E) -N- (3,4-dimethoxyphenethyl) deca-2-enamide;

yield 98%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.85 (3H, t, J = 7.0 Hz, CH 3), 1.21-1.31 (8H, m, CH 2), 1.37-1.42 (2H, m, CH 2 ), 2.12 (2H, dq, J = 1.5, 7.0 Hz, CH 2), 2.77 (2H, t, J = 6.8 Hz, CH 2 Ph), 3.53 (2H, dt, J = 6.0, 6.8 Hz, NHCH 2 ), 3.84 (s, 6H), 5.43 (1H, br s, NH), 5.66 (1H, td, J = 1.5, 15.4 Hz, CH = CH), 6.67-6.85 (4H, m, Ph);

MS (FAB): m / z 334 164 (100) 136 (70).

Example  2-24. (E) -3- ( Benzo [d] [1,3] dioxole -5-yl) -N- (4- Methoxyphenethyl ) Acrylamide

(E) -3- (benzo [d] [1,3] dioxol-5-yl) -N- (4-methoxyphenethyl) acrylamide;

yield 75%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.80 (2H, t, J = 6.8 Hz, CH 2 Ph), 3.89 (2H, td, J = 6.0, 6.8 Hz, NHCH 2), 3.77 (3H, _{s, OCH 3), 5.60 (} 1H, br s, NH), 5.96 (2H, s, CH 2), 6.12 (1H, d, J = 15.4 Hz, CH = CH), 6.76 (1H, dd, J = 1.3, 7.2 Hz, Ph), 6.84 (2H, td, J = 2.0, 8.6 Hz, Ph), 6.94 (2H, dd, J = 1.8, 7.9 Hz, Ph), 7.11 (2H, td, J = 1.8, 8.4 Hz, Ph), 7.50 (1H, d, J = 15.6 Hz, CH = CH);

MS (FAB): m / z 326 164 (100) 136 (70).

Examples 2-25. (E) -3- (benzo [d] [1,3] dioxol-5-yl) -N- (2-methylbutyl) acrylamide

(E) -3- (benzo [d] [1,3] dioxol-5-yl) -N- (2-methylbutyl) acrylamide;

yield 99%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.92 (3H, s, CH 3), 0.94 (3H, s, CH 3), 1.16-1.23 (2H, m, CH 2 CH 3), 1.39-1.62 _{(1H, m, CHCH 2)} , 3.15-3.37 (2H, m. CH 2), 5.64 (1H, br s, NH), 5.98 (2H, s, CH 2), 6.22 (1H, d, J = 15.6 Hz, CH = CH), 6.79 (1H, d, J = 7.8 Hz, Ph), 6.98 (2H, dd, J = 1.2, 9.6 Hz, Ph), 7.53 (1H, d, J = 15.6 Hz, CH = CH);

HRMS (FAB): calc'd for C 15 H 20 NO 3; [M + H] &lt; ⁺ &gt;:262.1443; found: 262.1451.

Example  2-26. N- Phenethyltin acid amide

N-phenethylcinnamamide;

yield 93%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.89 (2H, t, J = 6.9 Hz, CH 2 Ph), 3.66 (2H, td, J = 6.0, 6.7 Hz, NHCH 2), 5.79 (1H, (1H, d, J = 15.6 Hz, CH = CH), 7.21-7.49 (10H, m, Ph), 7.62 (1H, d, J = 15.6 Hz, CH = CH).

HRMS (FAB): calc'd for C 17 H 18 NO; [M + H] &lt; ⁺ &gt;:252.1388; found: 252.1390.

Example  2-27. (E) -3- (3- Hydroxyphenyl ) -N- (4- Methoxyphenethyl ) Acrylamide

(E) -3- (3-hydroxyphenyl) -N- (4-methoxyphenethyl) acrylamide;

yield 76%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.81 (2H, t, J = 6.8 Hz, CH 2 Ph), 3.60 (2H, td, J = 6.2, 6.8 Hz, NHCH 2), 3.77 (3H, _{s, OCH 3), 5.68 (} 1H, br s, NH), 6.26 (1H, d, J = 15.4 Hz, CH = CH), 6.84 (3H, d, J = 8.6 Hz, Ph), 7.02 (2H, d, J = 7.9 Hz, Ph ), 7.12 (2H, d, J = 8.6 Hz, Ph), 7.20 (1H, t, J = 8.0 Hz, Ph), 7.58 (1H, d, J = 15.4 Hz, CH = CH).

Example 2-28. (2E, 4E) -N- (p-tolyl) deca-2,4-diene amide

(2E, 4E) -N- (p-tolyl) deca-2,4-dienamide;

yield 88%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.87 (3H, t, J = 6.8 Hz, CH 3), 1.20-1.43 (6H, m, CH 2), 2.14 (2H, q, J = 6.8 Hz CH ₂ ), 2.29 (3H, s, CH ₃ ), 5.88 (1H, d, J = 14.8 Hz, CH = CH), 6.03-6.19 (2H, m, CH = CH), 7.08-7.14 m, Ph), 7.28-7.45 (2H, m, Ph);

MS (FAB): m / z 258 154 (100) 107 (70).

Examples 2-29. (2E, 4E) -N- (4-methoxybenzyl) deca-2,4-diene amide

(2E, 4E) -N- (4-methoxybenzyl) deca-2,4-dienamide;

yield 94%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.86 (3H, t, J = 6.6 Hz, CH 3), 1.20-1.44 (6H, m, CH 2), 2.11 (2H, td, J = 6.0, _{6.8 Hz, CH 2), 3.76} (3H, s, OCH 3), 4.41 (2H, d, J = 5.7 Hz, CH = CH), 5.72 (1H, d, J = 15.0 Hz, CH = CH), 5.77 (1H, br t, NH), 5.99-6.14 (1H, m, CH = CH), 6.83 (2H, td, J = 2.0, 8.6 Hz, Ph), 7.16-7.23 (2H, m, Ph);

HRMS (FAB): calc'd for C 18 H 26 NO 2; [M + H] &lt; ⁺ &gt;:288.1964; found: 288.1973.

Example 2-30. (2E, 4E) -N-isobutyldeca-2,4-diene amide

(2E, 4E) -N-isobutyldeca-2,4-dienamide;

yield 99%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.87 (3H, t, J = 6.6 Hz, CH 3), 0.91 (3H, d, J = 6.6 Hz, CH 3), 0.92 (3H, s, CH _{3), 1.25-1.45 (6H, m} , CH 2), 1.74-1.83 (1H, m, CH), 2.13 (2H, td, J = 6.3, 7.2 Hz, CH 2), 3.15 (2H, t, J = 6.6 Hz, CH ₂ ), 5.64 (1H, br s, NH), 5.76 (1H, d, J = 15.0 Hz, CH = CH), 5.99-6.16 , dd, J = 6.6, 15.0 Hz, CH = CH);

HRMS (FAB): calc'd for C 14 H 26 NO; [M + H] &lt; ⁺ &gt;:224.2014; found: 224.2016.

Example  2-31. (2E, 4E) -N- (4- Methoxyphenethyl ) Deca -2,4- Dienamide

(2E, 4E) -N- (4-methoxyphenethyl) deca-2,4-dienamide;

yield 98%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.88 (3H, t, J = 6.9 Hz, CH 3), 1.21-1.45 (6H, m, CH 2), 2.04-2.22 (2H, m, CH 2 ), 2.78 (2H, t, J = 6.9 Hz, CH 2), 3.55 (2H, td, J = 6.6, 6.9 Hz, CH 2), 3.78 (3H, s, OCH 3), 5.56 (1H, br s , NH), 5.68 (1H, d, J = 15.0 Hz, CH = CH), 6.05-6.15 (2H, m, CH = CH), 6.82-6.85 (2H, m, Ph), 7.08-7.26 (3H, m, Ph);

HRMS (FAB): calc'd for C 19 H 28 NO 2; [M + H] &lt; ⁺ &gt;:302.2120; found: 302.2127.

Example 2-32. (2E, 4E) -N- (2-methylbutyl) deca-2,4-diene amide

(2E, 4E) -N- (2-methylbutyl) deca-2,4-dienamide;

yield 98%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.86-0.92 (6H, m, CH 3), 1.08-1.48 (6H, m, CH 2), 1.52-1.60 (1H, m, CH), 2.14 ( 2H, q, J = 6.6 Hz , CH 2), 3.10-3.32 (2H, m, CH 2), 5.49 (1H, br s, NH), 5.75 (1H, d, J = 15.0 Hz, CH = CH) , 6.01-6.17 (2H, m, CH = CH), 7.19 (1H, dd, J = 15.0 Hz, CH = CH);

HRMS (FAB): calc'd for C 15 H 28 NO; [M + H] &lt; ⁺ &gt;:238.2171; found: 238.2175.

Example  2-33. (2E, 4E) -N- (3,4- Dimethoxyphenethyl ) Deca -2,4- Dienamide

(2E, 4E) -N- (3,4-dimethoxyphenethyl) deca-2,4-dienamide;

yield 91%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.87 (3H, t, J = 6.9 Hz, CH 3), 1.24-1.45 (6H, m, CH 2), 2.13 (2H, td, J = 5.4, _{7.2 Hz, CH 2), 2.79} (2H, t, J = 6.9 Hz, CH 2 Ph), 3.56 (2H, td, J = 6.0, 6.6 Hz, NHCH 2), 3.85 (6H, s, OCH 3), 5.57 (1H, br s, NH ), 5.68 (1H, d, J = 15.0 Hz, CH = CH), 6.00-6.15 (2H, m, CH = CH), 6.71-6.82 (3H, m, Ph), 7.18 (1H, dd, J = 1.8, 15.0 Hz, CH = CH);

HRMS (FAB): calc'd for C 20 H 30 NO 3; [M + H] &lt; ⁺ &gt;:332.2226; found: 332.2223.

Example 2-34. (2E, 4E) -1- (piperidin-1-yl) deca-2,4-dien-

(2E, 4E) -1- (piperidin-1-yl) deca-2,4-dien-1-one;

yield 78%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.88 (3H, t, J = 6.9 Hz, CH 3), 1.24-1.46 (6H, m, CH 2), 1.52-1.68 (6H, m, CH 2 ), 2.14 (2H, q, J = 7.2 Hz, CH 2), 3.48-3.61 (4H, m, CH 2), 5.99-6.09 (1H, m, CH = CH), 6.17 (1H, ddd, J = CH = CH), 6.25 (1H, d, J = 14.7 Hz, CH = CH), 7.21 (1H, dd, J = 10.5, 14.7 Hz, CH = CH);

HRMS (FAB): calc'd for C 15 H 26 NO; [M + H] &lt; ⁺ &gt;:236.2014; found: 236.2015.

Example  2-35. N- (4- Hydroxyphenethyl ) Cinnamic acid amide

N- (4-hydroxyphenethyl) cinnamamide;

yield 57%;

yellow solid;

^{1 H NMR (300 MHz, CD} 3 OD): δ H 2.75 (2H, t, J = 7.2 Hz, CH 2 Ph), 3.47 (2H, t, J = 6.9 Hz, NHCH 2), 6.57 (1H, d , J = 15.6 Hz, CH = CH), 6.71 (2H, td, J = 2.1, 8.4 Hz, Ph), 7.05 (2H, td, J = 2.1, 8.7 Hz, Ph), 7.34-7.39 (3H, m , Ph), 7.51 (1H, d, J = 15.9 Hz, CH = CH), 7.52-7.55 (2H, m, Ph);

HRMS (FAB): calc'd for C 17 H 18 NO 2; [M + H] &lt; ⁺ &gt;:268.1338; found: 268.1342.

Example  2-36. (E) -3- (3,4- Dimethoxyphenyl ) -N- (4- Hydroxyphenethyl ) Acrylamide

(E) -3- (3,4-dimethoxyphenyl) -N- (4-hydroxyphenethyl) acrylamide;

yield 60%;

white solid;

m.p. 161-162 占 폚;

^{1 H NMR (600 MHz, CDCl} 3): δ H 2.79 (2H, t, J = 6.9 Hz, CH 2), 3.60 (2H, q, J = 6.6 Hz, CH 2), 3.87 (3H, s, OCH _{3), 3.88 (3H, s} , OCH 3), 5.55 (1H, br s, OH), 5.57 (1H, br s, NH), 6.17 (1H, d, J = 15.5 Hz, CH), 6.79 (2H (t, J = 2.5, 9.0 Hz, Ph), 6.82 (1H, d, J = 8.2 Hz, Ph), 6.98 (1H, d, J = 1.8 Hz, Ph), 7.03-7.06 ), 7.54 (1H, d, J = 15.6 Hz, CH);

_{^{13 C NMR (150MHz, CDCl 3}} ): δ C 34.8, 40.9, 55.9, 56.0, 109.7, 111.1, 115.6 (2C), 118.3, 122.0, 127.7, 129.9 (2C), 130.6, 141.1, 149.1, 150.6, 154.6, 166.3;

FT-IR (neat) :? _Max 3271, 3015, 1654, 1595, 1260 and 1140 cm ^-1 ;

HRMS (FAB): calc'd for C 19 H 22 NO 4; [M + H] &lt; ⁺ &gt;:328.1549; found: 328.1557.

Example  2-37. (E) -3- ( Furan Yl) -N- (4- Hydroxyphenethyl ) Acrylamide

(E) -3- (furan-2-yl) -N- (4-hydroxyphenethyl) acrylamide;

yield 62%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 2.80 (2H, t, J = 6.9 Hz, CH 2 Ph), 3.60 (2H, td, J = 6.0, 7.2 Hz, NHCH 2), 5.56 (1H, br s, NH), 6.21 ( 1H, d, J = 15.3 Hz, CH = CH), 6.44 (1H, dd, J = 1.8, 3.3 Hz, furan), 6.54 (1H, d, J = 3.3 Hz, furan ), 6.80 (2H, td, J = 2.1,8.4 Hz, Ph), 7.07 (2H, d, J = 8.4 Hz, Ph), 7.37-7.42 (2H, m, furan);

HRMS (FAB): calc'd for C 15 H 16 NO 3; [M + H] &lt; ⁺ &gt;:258.1130; found: 258.1136.

Example  2-38. (E) -N- (4- Methoxyphenethyl ) -3- Methylocte -2- Enamide

(E) -N- (4-methoxyphenethyl) -3-methyloct-2-enamide;

yield 76%;

yellow oil;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.87 (3H, t, J = 6.9 Hz, CH 3), 1.23-1.34 (4H, m, CH 2), 1.39-1.44 (2H, m, CH 2 ), 2.04 (2H, t, J = 6.6 Hz, CH 2), 2.11 (3H, d, J = 1.2 Hz, CH 3), 2.76 (2H, t, J = 6.9 Hz, CH 2 Ph), 3.49 ( 2H, td, J = 6.6 Hz , NHCH 2), 3.78 (3H, s, OCH 3), 5.45 (1H, d, J = 1.2 Hz, CH = CH), 6.84 (2H, td, J = 2.1, 8.4 Hz, Ph), 7.11 (2H, td, J = 1.8, 8.7 Hz, Ph);

HRMS (FAB): calc'd for C 18 H 28 NO 2; [M + H] &lt; ⁺ &gt;:290.2120; found: 290.2125.

Example 2-39. (E) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) -3- (3-hydroxyphenyl) acrylamide

(E) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) -3- (3-hydroxyphenyl) acrylamide;

yield 58%;

white solid;

^{1 H NMR (300 MHz, CD} 3 OD): δ H 4.17-4.23 (4H, m, CH 2), 6.67 (1H, d, J = 15.6 Hz, CH = CH), 6.75-6.82 (2H, m, Ph), 6.98-7.05 (3H, m , Ph), 7.20 (1H, t, J = 8.1 Hz, Ph), 7.29 (1H, d, J = 2.4 Hz, Ph), 7.53 (1H, d, J = 15.6 Hz, CH = CH);

HRMS (FAB): calc'd for C 17 H 16 NO 4; [M + H] &lt; ⁺ &gt;:298.1079; found: 298.1090.

Example  2-40. (E) -N- (2,3- Dihydrobenzo [b] [1,4] dioxin Yl) Deca -2- Enamide

(E) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) deca-2-enamide;

yield 89%;

yellow oil;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.88 (3H, t, J = 6.9 Hz, CH 3), 1.27-1.29 (8H, m, CH 2), 1.43-1.45 (2H, m, CH 2 ), 2.20 (2H, td, J = 6.6, 7.5 Hz, CH 2), 4.23 (4H, s, CH 2), 5.88 (1H, d, J = 15.3 Hz, CH = CH), 6.78 (1H, d , J = 8.4 Hz, Ph), 6.90-6.99 (2H, m, Ph), 7.21 (2H, br s, CH = CH);

HRMS (FAB): calc'd for C 18 H 26 NO 3; [M + H] &lt; ⁺ &gt;:304.1913; found: 304.1907.

Example 2-41. (2E, 4E) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) deca-2,4-

(2E, 4E) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) deca-2,4-dienamide;

yield 90%;

yellow oil;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.89 (3H, t, J = 6.9 Hz, CH 3), 1.23-1.47 (6H, m, CH 2), 2.12-2.19 (2H, m, CH 2 ), 4.23 (4H, s, CH 2), 5.86 (1H, d, J = 15.0 Hz, CH = CH), 6.05-6.21 (2H, m, CH = CH), 6.79 (1H, d, J = 8.4 Hz, Ph), 6.95 (1H, d, J = 8.1 Hz, Ph), 7.21 (1H, br s, NH), 7.25-7.33 (2H, m, Ph);

HRMS (FAB): Calc'd for C ₁₈ H ₂₄ NO ₃ ; [M + H] &lt; ⁺ &gt;:302.1756; found: 302.1766.

Examples 2-42. (2E, 4E) -1- (pyrrolidin-1-yl) deca-2,4-dien-

(2E, 4E) -1- (pyrrolidin-1-yl) deca-2,4-dien-1-one;

yield 98%;

yellow oil;

^{1 H NMR (300 MHz, CDCl} 3): δ H 0.88 (3H, t, J = 6.6 Hz, CH 3), 1.25-1.46 (6H, m, CH 2), 1.81-2.00 (4H, m, CH 2 ), 2.14 (2H, q, J = 6.9 Hz, CH 2), 3.52 (4H, q, J = 6.9 Hz, CH 2), 6.05-6.22 (3H, m, CH = CH), 7.22-7.31 (1H , m, CH = CH);

HRMS (FAB): calc'd for C 14 H 24 NO; [M + H] &lt; ⁺ &gt;:222.1858; found: 222.1865.

Example 2-43. N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl)

N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) cinnamamide;

yield 100%;

white solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 4.23 (4H, s, CH 2), 6.54 (1H, d, J = 15.6 Hz, CH = CH), 6.81 (1H, d, J = 9.0 Hz, Ph), 7.02 (1H, d, J = 7.8 Hz, Ph), 7.28 1H, br s, NH), 7.72 (1H, d, J = 15.6 Hz, CH = CH);

HRMS (FAB): calc'd for C 17 H 16 NO 3; [M + H] &lt; ⁺ &gt;:282.1130; found: 282.1138.

Example 2-44. (E) -3- (4- (tert-butyl) phenyl) -N- (3-methoxyphenyl) acrylamide

(E) -3- (4- (t-butyl) phenyl) -N- (3-methoxyphenyl) acrylamide;

yield 91%;

yellow oil;

^{1 H NMR (300 MHz, CDCl} 3): δ H 1.32 (9H, s, CH 3), 3.80 (3H, s, OCH 3), 6.58 (1H, d, J = 15.6 Hz, CH = CH), 6.67 (1H, dd, J = 2.1 , 8.4 Hz, Ph), 7.10 (1H, d, J = 8.4 Hz, Ph), 7.21 (1H, t, J = 8.4 Hz, Ph), 7.36 (2H, td, J = 2.1, 8.4 Hz, Ph), 7.43-7.46 (3H, m, Ph), 7.73 (1H, d, J = 15.6 Hz, CH = CH), 7.84 (1H, br s, NH);

HRMS (FAB): calc'd for C 20 H 24 NO 2; [M + H] &lt; ⁺ &gt;:310.1807; found: 310.1820.

Examples 2-45. (E) -3- (4- (tert-butyl) phenyl) -N- (thiazol-2-yl)

(E) -3- (4- (t-butyl) phenyl) -N- (thiazol-2-yl) acrylamide;

yield 96%;

yellow solid;

^{1 H NMR (300 MHz, CDCl} 3): δ H 1.34 (9H, s, CH 3), 6.71 (1H, d, J = 15.6 Hz, CH = CH), 7.07 (1H, d, J = 3.6 Hz, Ph), 7.45 (2H, d , J = 8.4 Hz, Ph), 7.52 (2H, d, J = 8.7 Hz, Ph), 7.55 (1H, d, J = 3.6 Hz, Ph), 7.88 (1H, d , J = 15.9 Hz, CH = CH);

HRMS (FAB): Calc'd for C ₁₆ H ₁₉ N ₂ OS; [M + H] &lt; ⁺ &gt;:287.1218; found: 287.1223.

&Lt; Example 3: Cell culture >

The cell culture conditions used to measure the whitening activity of the piperamide derivative of the present invention are as follows.

First, the murine melanocyte melan-A cells were obtained from AmorePacific Skin Research Institute (Yongin, Korea) and the melan-A cells were cultured in DMEM supplemented with 10% [v / v] FBS 1640 medium (Gibco, Carlsbad, CA, USA) containing 1% [v / v] PS (penicillin-streptomycin) and 200 nM TPA (phorbol 12-myristate 13-acetate) Respectively.

NHEM (normal human epidermal melanocytes) cells were purchased from ATCC and cultured in melanocyte growth medium (PromoCell, Heidelberg, Germany). The cells were cultured in a humidified incubator at 37 ° C, 5% CO ₂ for 4 days to monolayer the cells. The cells cultured in the monolayer were resuspended with trypsin-EDTA, and were inoculated in a 24-well plate at 10 ⁵ cells / well and cultured for 24 hours.

Then, the cultured melan-A or NHEM cells were treated with the piperamide derivative of the present invention at a concentration, and 990 μl of a fresh culture solution and 10 μl of a piperamide derivative (concentration-dependent compound dissolved in DMSO) were added daily for 3 days (Puma et al., 1999). As a positive control, a melanin-forming inhibitor, PTU (1-phenyl-2-thiourea, Sigma Chemical Co. USA) was used.

&Lt; Example 4: Measurement of cell viability &

(3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to measure the cell viability of the piperamide derivative of the present invention.

First, 100 μl of a 0.5 mg / ml MTT reagent was added to 600 μl of the melan-A cell culture solution treated with the piperamide derivative of Example 3. The cells were mixed with 5% CO ₂ , 95% O ₂ , And cultured at 37 ° C for 2 hours. Subsequently, the medium containing the substrate was removed, 500 쨉 l of DMSO (dimethyl sulfoxide) was dissolved and dissolved in the formazan crystal of each well, the plate was stirred at room temperature for 30 minutes, Absorbance was measured at 570 nm with a reader (microplate reader, Molecular Devices E09090, USA) and is shown in Table 1 below.

Condition Cell survival rate (%) Condition Cell survival rate (%) Example 2-1 97.81 Examples 2-24 88.07 Example 2-2 98.95 Examples 2-25 100.72 Example 2-3 102.99 Example 2-26 100.38 Examples 2-4 95.89 Example 2-27 101.94 Example 2-5 99.99 Example 2-28 101.94 Examples 2-6 101.75 Example 2-29 94.01 Examples 2-7 89.73 Example 2-30 100.22 Examples 2-8 101.03 Example 2-31 98.19 Examples 2-9 101.56 Example 2-32 101.28 Examples 2-10 93.32 Example 2-33 105.65 Examples 2-11 90.62 Example 2-34 100.21 Examples 2-12 98.72 Example 2-35 100.76 Examples 2-13 100.93 Example 2-36 97.90 Examples 2-14 92.64 Example 2-37 101.28 Examples 2-15 97.84 Example 2-38 100.19 Examples 2-16 99.78 Example 2-39 101.52 Examples 2-17 104.67 Example 2-40 96.37 Examples 2-18 97.34 Example 2-41 101.60 Example 2-19 95.50 Examples 2-42 104.96 Examples 2-20 101.21 Examples 2-43 84.94 Examples 2-21 102.20 Examples 2-44 81.96 Examples 2-22 99.42 Example 2-45 91.51 Examples 2-23 101.99 Positive control (PTU) 100.12

Referring to Table 1, the piperamide derivative of the present invention does not show cytotoxicity in melan-A cells, and thus can be usefully used as a skin whitening composition.

&Lt; Example 5: Measurement of melanin content and tyrosinase activity >

In order to evaluate the whitening activity against the piperamide derivatives, the melanin content and tyrosinase activity were analyzed in vitro and in vivo.

Example 5-1. Measurement of melanin content-in vitro

In order to measure the melanin content in vitro, the method reported by the prior art [Hosoi et al., 1985] was modified and applied.

First, the melamine-A cells of Example 3 were treated with the piperamide derivatives of Examples 2-1 to 2-7 at a concentration of 10 μM for 3 days, the cultured medium was removed, and the wells were washed with PBS buffer saline). Then, to dissolve melanin, 400 1 of 1N NaOH was added to each well, and melanin was measured at an absorption wavelength of 405 nm with a microplate reader, and is shown in Table 2 and Fig.

Condition Melanin content (%) Example 2-1 33.90 Example 2-2 49.90 Example 2-3 74.77 Examples 2-4 67.38 Example 2-5 58.53 Examples 2-6 45.28 Examples 2-7 63.48 Positive control (PTU) 10 uM: 51.95 100 uM: 27.85

Table 2 shows the melanin content of the piperamide derivative of the present invention in comparison with the melanin content of the present invention. In particular, in the case of Example 2-1, melanin content was the lowest. The results are shown in Fig. 1. As shown in Fig. 1, the content of melanin was determined to be 58% at a concentration of 5 μM and 66% at a concentration of 10 μM in a concentration-dependent manner And it showed similar tendency to PTU which is a positive control group, and it can be confirmed that the activity to inhibit melanin formation is excellent.

Example 5-2. Measurement of tyrosinase activity - in vitro

Melan-A cells were used to measure tyrosinase activity in vitro, and tyrosinase activity was analyzed for melan-A and NHEM cells by L-DOPA staining as another method. The in situ tyrosinase activity by the L-DOPA staining provides a more sensitive change to the melanin formation, so that it can be used for analysis by referring to the method reported in the prior art [Richard et al., 2007] Respectively.

First, in order to measure tyrosinase activity in melan-A cells, the cells were inoculated with ~5 × 10 ⁵ cells per plate (100 mm culture dish) and cultured. After 24 hours, the cultured cells were treated with positive control kojic acid (kojic acid, tyrosinase inhibitor) or Example 2-1 at a concentration of 1 μM, 5 μM and 10 μM, respectively, and cultured for 3 days while changing the medium. After 3 days, the medium was removed, the cells were washed with PBS, the cells were separated and resuspended using trypsin-EDTA, and centrifuged at 7500 rpm for 5 minutes. Cells were disrupted using an ultrasonicator for 2 minutes by adding tyrosinase buffer (80 mM phosphate buffer, 1% Triton-X100 and 100 μg / ml PMSF) to the cell pellet, . Then, the supernatant was obtained by centrifuging the cells (12500 rpm, 15 minutes, 4 ° C). The protein concentration of the supernatant was determined by the Bradford method (Bio-Rad, USA) using bovine serum albumin . 150 μg of the protein measured by the Bradford method and 120 μl of L-DOPA (8.3 mM in 80 mM phosphate buffer, pH 6.8) were mixed and reacted at 37 ° C. for 20 minutes in a dark room. Thereafter, the absorbance at 490 nm was measured with a microplate reader to show tyrosinase activity (expressed as% relative to the control group), and is shown in Table 3 and FIG. 2A.

In order to perform L-DOPA staining as another method for measuring tyrosinase activity, melan-A cells cultured in Example 3 and 4% [w / v] paraformaldehyde dissolved in PBS in NHEM cells (paraformaldehyde) at 37 &lt; 0 &gt; C for 10 min. Thereafter, the cells were permeabilized with 0.1% [v / v] Triton X-100 (Sigma Chemical Co., USA) dissolved in PBS for 2 minutes, washed with PBS, v] L-DOPA (Sigma Chemical Co. USA) was added and reacted at 37 ° C for 2 hours. After 2 hours, the cells were washed with PBS and the activity of tyrosinase was measured with a microscope (Olympus Corp., Tokyo, Japan) and shown in Fig. 2B and Fig. 2C.

Condition Concentration (μM) Tyrosinase activity (%) Example 2-1 One 90.2 ± 3.3 10 31.6 ± 1.3 Positive control group
(kojic acid) One 92.8 ± 6.8 10 78.3 ± 4.4 100 50.5 ± 0.2

Referring to Table 3 and FIG. 2A, it can be confirmed that Example 2-1 of the present invention decreases the tyrosinase activity in a concentration-dependent manner in melan-A cells, which indicates that melan-A cells by L-DOPA staining (FIG. 2B) and NHEM cells (FIG. 2C). In addition, since the effect of reducing tyrosinase activity is more excellent than that of the positive control group, it can be usefully used as a skin whitening composition.

Example 5-3. Induction of hyperpigmentation by UVB - topical application

After inducing hyperpigmentation by UVB in the skin of guinea pigs, the whipped effect was measured by topical application of the piperamide derivative of the present invention.

The hyperpigmentation was induced by Brown guinea pig (~ 350 g, Central Lab Animals, Inc. Seoul, Korea), which was modified by the method reported in the previous paper [Candan et al., 2012] (IA1300229) was approved by the Korea Conformity Laboratories' Institutional Animal Care and Use Committee.

After adjusting for 6 weeks, the skin was shaved and exposed to UVB (G8T5E lamps, Sankyo Denki, Hiratsuka, Japan) at an illuminance of 310 nm. The bulb was located 15 cm above and the lamp was measured with the IL1700 Research Radiometer (International Light, Newburyport, MA, USA) equipped with a UVB sensor.

The guinea pigs were exposed to UVB only during the first week, followed by UVB at 350 mJ / cm 2 (4 min, 7 sec) three times a week for 2 weeks (total 2100 mJ / cm 2) during the first week. Thereafter, guinea pigs were treated with 1% arbutin, tyrosinase inhibitor, 0.1% Example 2-1, 1% Example 2-1 and negative control (propylene glycol: ethanol, 7: 3, v / v) Is applied topically for 5 weeks, 5 times a week for 2 weeks, and the result for skin such as guinea pig after 5 weeks is shown in Fig. 3A. In addition, the melanin index was measured at the end of 3, 4 and 5 weeks using a skin colorimeter (DermaLab ^® Combo, Cortex Technology, Denmark) and is shown in FIG. 3B.

3A and 3B, toxic symptoms such as red spots do not appear when topical application of the piperamide derivative of the present invention is applied to the skin of guinea pigs. When treating Example 2-1 of the present invention, Similar to the positive control, the melanin index decreased by 3%, 17% and 20% in a time-dependent manner.

Example 5-4. Measurement of melanin content-in vivo

In order to measure melanin content in vivo, guinea pigs induced hypervigilance by UVB in Example 5-3 were evaluated by locally applying Example 2-1 of the present invention. The melanin content was measured by two methods. The first method was to calculate the melanin content using a microplate reader, and the second method was Fontana-Masson silver staining.

To calculate the melanin content using a microplate reader, 2M NaBr was added to the locally applied guinea pig dermis, followed by reaction at 37 ° C for 5 hours to isolate the epidermis. The separated epidermis was dissolved in 1 N NaOH solution containing 10% [v / v] DMSO at 80 ° C for 1 hour and centrifuged at 16,000 x g for 30 minutes to take the supernatant. The protein concentration of the supernatant was measured by a Bradford method using BSA as a standard substance at 405 nm using a microplate reader and the melanin content was expressed as [expressed melanin (μg) / total expressed protein (μg)] 4A.

Next, in order to measure the content of melanin by Fontana-Masson silver staining, we refer to the previous paper [Park et al., 2011]. The melanin production was calculated on an area of 435 x 325 mu m and its density measurement was shown in Fig. 4B using NIS element imaging software (ver. 4.0, Nikon, Japan).

Referring to the results of FIG. 4A, when the Example 2-1 of the present invention was treated at a concentration of 0.1% or 1%, the melanin content was reduced to 25% and 26%, respectively. 4B, dark spots were increased in the case of UVB irradiation compared to the non-treatment group (steady state), but the positive control group after UVB irradiation or the Example 2-1 of the present invention When treated with 0.1% or 1%, black spots were reduced by 66%, 67%, and 77%, respectively, compared with the UVB irradiated group.

In addition, although not shown in FIG. 4, it was confirmed that H & E (Hematoxylin and eosin) staining, which is commonly used for observation of cells and tissue structure, did not affect the morphological change to the skin tissue. It was confirmed that the compound had an effect of reducing only the melanin content without affecting the morphological changes of the skin tissue.

Examples 5-5. Determination of tyrosinase activity-in vivo

In order to measure tyrosinase activity in vivo, the epidermis was separated from the dermis of guinea pigs and used for tyrosinase activity measurement in the same manner as in Example 5-4.

To measure tyrosinase activity, the epidermis separated from the dermis was homogenized with 1% Triton X-100 solution (MP fast prep, MP Biomedicals, Solon, USA) containing 0.1 mM PMSF, Min at 4 ° C to obtain a supernatant. The extracted protein (30 μg) was resuspended in 0.2 ml of PBS and 2 mg / ml of L-DOPA, and the dopachrome formation was carried out using a microplate reader at 490 nm and 37 ° C for 10 minutes As shown in Fig.

Referring to FIG. 5, when Example 2-1 of the present invention was treated at a concentration of 0.1% or 1%, tyrosinase activity was reduced to 34% and 38%, respectively. This is similar to FIG. 4A, and it can be confirmed that the compound of the present invention can be easily applied as a composition for skin whitening.

Example 6 Analysis of Proteins Associated with Melanogenesis-Tyrosinase, TRP-1, TRP-2 and MITF [

In order to evaluate the whitening activity of the piperamide derivatives of the present invention, tyrosinase, TRP-1, TRP-2 and MITF proteins related to melanogenesis were analyzed in vitro and in vivo.

Example 6-1. mRNA expression analysis-in vitro

MRNA was extracted and expressed in vitro to investigate the effects of tyrosinase, TRP-1, TRP-2 and MITF, proteins involved in melanogenesis.

RNA was prepared by reference to the protocol of TRIzol reagent (Invitrogen, USA). First, the cultured melan-A cells were washed twice with PBS, and then centrifuged to obtain pellets. The TRIzol reagent was added to the pellet, and the cells were dissolved by pipetting and left on ice for 5 minutes. Then, chloroform was added thereto, and the mixture was centrifuged at 12,000 rpm, 15 minutes, and 4 ° C. The aqueous phase was transferred to a new tube, and isopropanol was added. Then, the supernatant was allowed to stand at 4 DEG C for 10 minutes, and centrifuged at 12,000 rpm, 10 minutes, and 4 DEG C. The RNA pellet was added with a 75% [v / v] aqueous ethanol solution and then resuspended with dried and distilled water treated with DEPC (diethylpyrocarbonate). Total RNA was identified by electrophoresis, and the amount of RNA was analyzed by measuring OD (optical density) at 260 nm. CDNA was obtained from reverse transcriptase and total RNA (1 μg). The primers used for cDNA amplification and RT-PCR (reverse transcription-PCR) conditions were as follows.

* Primer sequence used

tyrosinase upstream: 5'-GGC CAG CTT TCA GGC AGA GGT-3 '

tyrosinase downstream: 5'-TGG TGC TTC ATG GGC AAA ATC-3 '

TRP-1 upstream: 5'-GCT GCA GGA GCC TTC TTT CTC-3 '

TRP-1 downstream: 5'-AAG ACG CTG G TCT-3 '

TRP-2 upstream: 5'-TAC CAT CTG TTG TGG CTG GA-3 '

TRP-2 downstream: 5'-TGG CAC TGC TG GTC ATC TTG TCT TGC TG-3 '

MITF upstream: 5'-CTA GAG CGC ATG GAC TTT CC-3 '

MITF downstream: 5'-AAG TTG GAG CCC ATC TTC CT-3 '

GAPDH upstream: 5'-CGT CTT CAC CAT GGA GA-3 '

GAPDH downstream: 5'-CGG CCA TCA CGC AGT TT-3 '.

RT-PCR conditions: 35 cycles at 94 ° C for 150 seconds, 1 cycle at 60 ° C for 30 seconds, and 1 cycle at 72 ° C for 60 seconds.

The PCR products obtained under the above conditions were confirmed by electrophoresis (2% [w / v] agarose gel), where GAPDH was used as a control. Each experiment was repeated three or more times. Densitometry analysis of the bands during electrophoresis was performed using ImageMaster 2D Elite software (version 3.1, Amersham Pharmacia Biotech, USA) and is shown in FIG.

6, when Example 2-1 of the present invention was treated with melan-A cells, mRNA expression levels of tyrosinase, TRP-1, TRP-2 and MITF were measured after 6 hours Were significantly reduced to 79%, 91%, 87%, and 93%, respectively, confirming that Example 2-1 of the present invention has an effect of reducing the mRNA expression level in a time-dependent manner.

Example  6-2. Western Blot  Analysis-in vitro and in vivo

In order to investigate the effects of tyrosinase, TRP-1, TRP-2 and MITF, proteins involved in melanin formation in vitro and in vivo, Western blot for melanin-A cells and skin proteins of guinea pigs Respectively.

First, in order to evaluate protein expression of tyrosinase, TRP-1, TRP-2 and MITF on melan-A cells, Example 2-1 of the present invention was treated with Melan-A cells for 3 days by concentration Similarly, in order to evaluate protein expression of tyrosinase, TRP-1, TRP-2 and MITF on skin cells of guinea pigs, epidermal cells were separated from the skin of guinea pigs, and Example 2-1 was treated with 1, 5 and 10 μM Respectively.

The epidermal cells of the melan-A cells and the guinea pigs were washed with cold lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 0.5% sodium deoxycholate, 100 μg / , 1 ㎍ / ㎖ aprotinin and phosphatase inhibitors). Protein concentration was measured by Bradford method using BSA as a standard and BSA as a standard. Then, 40 μg of the protein was subjected to SDS-PAGE (10% [w / v] acrylamide gels) and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The membranes were blocked with 5% [w / v] skimmed milk dissolved in TBS-T (Tris-buffered saline-Tween buffer) and primary antibodies (tyrosinase, TRP-1, TRP- tubulin, Santa Cruz Biotech, USA and Amersham Pharmacia Biotech, UK) was added and reacted at 4 ° C for 16 hours. The antibody-treated membrane was washed three times with TBS-T and then incubated with a secondary antibody (goat anti-rabbit IgG, Amersham Pharmacia Biotech, UK; donkey anti-goat IgG, Santa Cruz Biotechnology, USA) Lt; / RTI &gt; Protein-transferred membranes were then analyzed by adding ECL reagent (Amersham Pharmacia Biotech, UK) and density analysis of bands was performed using ImageMaster 2D Elite software (ver.3.1, Amersham Pharmacia Biotech, UK) 7A to 7C.

FIGS. 7A and 7B show protein expression of tyrosinase, TRP-1, TRP-2 and MITF on melan-A cells. As a result, when Example 2-1 of the present invention was treated, Is decreased. In particular, FIG. 7A shows that protein expression decreases in a concentration-dependent manner, and FIG. 7B shows that protein expression decreases in a time-dependent manner. Fig. 7C shows the protein expression of tyrosinase, TRP-1, TRP-2 and MITF on the epidermal cells of guinea pigs. In the case of the group irradiated only with UVB compared to the untreated group, The expression levels of the proteins were increased to 158%, 230%, 133%, and 133%, respectively. However, when the present invention was applied to Example 2-1 at a concentration of 1% To 51%, 34%, 21% and 66%, respectively. This shows that the effect of reducing the expression of the protein involved in melanin formation is excellent.

< Formulation example  1. Pharmaceutical preparations>

Formulation example  1-1. Preparation of hydrophilic ointment

(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl ) Acrylamide was used to prepare a hydrophilic ointment agent by a conventional method according to the composition shown in Table 4 below.

Raw material name Content (% by weight) Example 2-1 8.0 White vaseline 35.0 Stearyl alcohol 30.0 Ethyl (or methyl) p-oxybenzoate a very small amount Propylene glycol 20.0 Sodium lauryl sulfate 3.4 Propyl p-oxybenzoate a very small amount

Formulation Example 1-2. Manufacture of tablets

(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl ) Acrylamide was used to mix the components listed in Table 5, followed by tableting according to a conventional method for producing tablets.

Raw material name Weight (mg) Example 2-1 100 mg Corn starch 100 mg Lactose 100 mg Magnesium stearate 2 mg

&Lt; Preparation Example 2: Preparation of cosmetic &

Formulation Example 2-1. Production of flexible lotion

(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl ) Acrylamide, a flexible lotion was prepared by a conventional method according to the composition shown in Table 6 below.

Raw material name Content (% by weight) Example 2-1 4.0 Butylene glycol 3.5 glycerin 2.5 Polyoxyethylene hardened castor oil 0.1 ethanol 2.5 Betaine 1.0 Citric acid 0.01 Sodium citrate 0.03 antiseptic Suitable amount Spices Suitable amount Purified water to 100

Formulation example  2-2. Manufacture of nutrition essences

(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl ) Acrylamide was used to prepare nutritional essences according to the compositions shown in Table 7 below.

Raw material name Content (% by weight) Example 2-1 7.0 Cetostearyl alcohol 1.0 Self emulsifying monostearic acid 1.0 Wax 0.5 Squalane 5.0 Isocetyl octanoate 3.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 Propylene glycol 0.2 Carboxy polymer 0.22 Triethanolamine 0.25 antiseptic Suitable amount Spices Suitable amount flash Suitable amount Purified water to 100

Formulation example  2-3. Manufacture of cream

(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl ) Acrylamide, creams were prepared according to the compositions shown in Table 8 below by a conventional method.

Raw material name Content (% by weight) Example 2-1 7.0 Cetostearyl alcohol 3.0 Self emulsifying monostearic acid 1.5 Chin type monostearate 1.5 Wax 0.5 Liquid paraffin 8.0 Squalane 7.0 Isocetyl octanoate 4.0 Refined jojoba oil 4.0 Dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Suitable amount flash Suitable amount Purified water to 100

Formulation Example 2-4. Manufacture of packs

(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl ) &Lt; / RTI &gt; acrylamide, packs were prepared according to the compositions listed in Table 9 below in a conventional manner.

Raw material name Content (% by weight) Example 2-1 8.0 glycerin 7.0 1,3-butylene glycol 3.0 Squalene 3.0 Dimethicone 3.0 Sodium hydrulonate 0.1 Glycine 2.0 Polyacrylamide 5.0 Triethanolamine 0.1 EDTA 0.1 Purified water to 100

Claims (6)

A skin whitening composition comprising an?,? - unsaturated amide compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

In Formula 1,
R &lt; ₁ &gt; is hydrogen,
R ₂ is selected from the group consisting of hydrogen, 2,3-dihydrobenzo [b] [1,4] dioxine, 2,3-dihydrobenzo [b] [1,4] dioxine, 2-thiazole, benzyl, Benzyl, phenylalkyl, phenylalkyl substituted with one or more alkoxy groups, or hydroxyphenylalkyl;
R ₃ is selected from the group consisting of C1 to C10 alkyl, C1 to C10 alkenyl, furanyl, phenyl, phenyl substituted with one or more alkyl groups, phenyl substituted with halogen, hydroxyphenyl, nitrophenyl, phenyl substituted with one or more alkoxy, Alkoxyalkylphenyl, halogenalkoxyphenyl or benzo [d] [1,3] dioxole;
R &lt; ₄ &gt; is hydrogen or C1 to C10 alkyl. The method according to claim 1,
The compound of formula (1)
(E) -3- (4- (tert-butyl) phenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide;
(E) -3- (4-chlorophenyl) -N- (2,3-dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide;
(E) -3- (benzo [d] [1,3] dioxol-5-yl) -N- (2,3- dihydrobenzo [b] [1,4] dioxin- Amide; And
(E) -N- (3-methoxyphenyl) -3- (4-methoxyphenyl) acrylamide. A pharmaceutical composition for skin whitening comprising the whitening composition of claim 1 or 2 as an active ingredient. The method of claim 3,
Wherein the pharmaceutical composition has one type of formulation selected from ointments, lotions, sprays, patches, creams, gels and gels. A cosmetic composition for skin whitening comprising the whitening composition of claim 1 or 2 as an active ingredient. 6. The method of claim 5,
The cosmetic composition according to claim 1, wherein the cosmetic composition has one type of formulation selected from the group consisting of a softening agent, a convergent lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, a pack, .

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