KR20190011207A - Tyrosinase antisense oligonucleotides - Google Patents

Abstract

Provided are peptide nucleic acid derivatives targeting a 3′ splice site of a human tyrosinase pre-mRNA. The peptide nucleic acid derivatives potently induce a splice variant of the human tyrosinase mRNA in cells, and are useful to safely treat dermatological indications or diseases involving a human tyrosinase protein upon topical administration. The present invention comprises the peptide nucleic acid derivatives or a pharmaceutically acceptable salt thereof.

Description

TYROSINASE ANTISENSE OLIGONUCLEOTIDES < RTI ID = 0.0 >

The present invention relates to a peptide nucleic acid derivative that specifically targets human tyrosinase pre-mRNA for improving skin pigmentation mediated by tyrosinase.

Melanin refers to the natural polymeric black pigment produced in living organisms. There are three types of melanin: eumelanin, peromelanin and neuromelanin. Eumelanin and peomelanin are synthesized in the skin. On the other hand, neuromelanin is a melanin found in the brain, and its physiological function has not yet been revealed.

Melanin in the skin effectively absorbs UV rays (ultraviolet rays) and protects the animals from UV rays (ultraviolet rays) in natural light. Thus, sun tanning is a natural process by which animals protect themselves from UV rays in sunlight.

Amino acid tyrosine is synthesized as eumelanin according to a series of complex reactions in the presence of tyrosinase. Eumelanin refers to a polymer of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Peomelanin is formed when tyrosine is polymerized by tyrosinase in the presence of cysteine or glutathione.

(Tyrosinase-related protein 1, that is, DHICA oxidase) and TYRP-2 (tyrosinase-related protein 2, that is, DOPA (tyrosinase-related protein 2)) among the biosynthesis of eumelanin and peomelanin, ≪ / RTI > chromotomeolactam ) ( Int . J. Mol . Sci . vol. 10, 4066-4087 ( 2009 )].

[Reaction Scheme 1]

Considering that tyrosinase (TYR) plays an important role in melanin biosynthesis in the skin, TYR inhibitors have been developed and used as a cosmetic ingredient to reduce excessive skin pigmentation, hyperpigmentation. TYR inhibitors have been reviewed for their effect on melanin formation in a number of articles [ Int . J. Cosmetic Sci . vol 33, 210-221 ( 2011 ); Int. J. Mol . Sci . vol 10, 2440-2475 ( 2009 )).

Hydroquinone (1,4-dihydroxybenzene) has been used for topical treatment of hyperpigmentation for decades [ JAMA vol 194 (9), 965-967 ( 1965 )]. Hydroquinone is known to inhibit TYR activity by binding to the active site of tyrosinase. In 2006, the US FDA withdrew prior approval for all OTC (non-prescription) products containing hydroquinone due to safety concerns, including potential carcinogenicity. Hydroquinone induces reactive oxygen species (ROS) that damage membrane lipids and proteins, including tyrosinase. Melanocytes are permanently removed by oxidative damage of the vaginal epithelium.

Arbutin (hydroquinone-O- [beta] -D-glucopyranoside) is a glycosylated hydroquinone extracted from a bearberry plant. Arbutin is also found in wheat and pear shells. Like hydroquinone, arbutin inhibits melanogenesis by competitively binding to the active site of the TYR [ J. Pharmacol . Exp . Ther . vol. 276 (2), 765-769 ( 1996 )]. Arbutin is less toxic to melanocytes than hydroquinone.

Deoxy arbutin is a synthetic derivative of arbutin, comparable to hydroquinone and arbutin in its TYR inhibitory activity. Deoxy arbutin is less cytotoxic than arbutin as well as hydroquinone [ J. Dermatol . Sci . vol 55 (3), 179-184 ( 2009 )). Deoxy arbutin markedly improved skin whitening in human subjects who were locally treated for 12 weeks ( Exp . Dermatol . vol 14 (8), 601-608 ( 2005 )).

Kojisan is a chelating agent that is produced during fungal fermentation for the production of soy sauce and sake, that is, Japanese sake. Kojisan was used to preserve or change color in food and cosmetics. Koji acid chelates copper ions in the active sites of TYR to inhibit melanin formation. It also inhibits the homomerization of DOPA chromium to 5,6-dihydroxyindole-2-carboxylic acid [ J. Pharm . Pharmacol . vol 46 (12), 982-985 ( 1994 )]. Kojisan has been widely used for the treatment of spots, but it can cause contact dermatitis, sensitization and erythema. J. Drugs Dermatol . vol 6 (1), 32-39 ( 2007 )]

While there are a variety of TYR inhibitors, their IC ₅₀ values for TYR are in most cases micromolar (μM) [ J. Enzyme Inhibition Med . Chem . vol 32 (1), 403-425 ( 2017 )]. Such IC ₅₀ values are considered insufficient when considering their molecular size and cause cross-reactivity with other molecular target (s). Since insufficient inhibitory activity is interpreted as a large amount of transdermal dosage, significant improvement in inhibitory efficacy is required to meet the desired transdermal activity and also safety for skin pigmentation.

free - mRNA :

Genetic information is on DNA (2-deoxyribose nucleic acid). DNA is transcribed to generate pre-mRNA (free-messenger ribonucleic acid) in the nucleus. Mammalian pre-mRNAs usually consist of exons and introns, and exons and introns are mutually linked to each other as provided schematically below. Exons and introns are numbered as illustrated in the following figure.

free - mRNA  Graphical representation of structure

(: Fit after splicing): - pre-splicing of pre-mRNA is -mRNA collectively as "splicing (from match)" in accordance with the deletion of the introns by a complex series of reactions referred to is converted to mRNA ( It is schematically summarized in the picture) [ Ann. Rev. Biochem . 72 (1), 291-336 ( 2003 ); Nature Rev. Mol . Cell Biol . 6 (5), 386-398 ( 2005 ); Nature Rev. Mol . Cell Biol . 15 (2), 108-121 ( 2014 )].

Splicing is initiated by the formation of a " spliceosome E complex " (i.e., an initial spliceosomal complex) between the pre-mRNA and the splice adapter element. In the "splicing ohsom E complex", U1 is coupled to the junction of exons and introns N and N, U2AF ³⁵ is coupled to the junction of the intron and exon N (N + 1). Thus, exon / intron or intron / exon junctions are important for the formation of the initial spliceosome complex. "Splice Osom E complex" evolves into a "Splice Osom A complex" when forming additional complexes with U2. The " spliceomeome A complex " undergoes a series of complicated reactions to delete or join the intron and splice out neighboring exons.

Protein synthesis of ribosomes: Proteins are encoded by DNA (2-deoxyribose nucleic acid). DNA responds to cell stimulation or is spontaneously transcribed to produce pre-mRNA (pre-messenger ribonucleic acid) in the nucleus. The introns of the pre-mRNA are joined by an enzyme to remove mRNA (messenger ribonucleic acid), and then the mRNA is transferred to the cytoplasm. In the cytoplasm, a complex of translational machinery, referred to as ribosomes, binds to mRNA and the complex performs protein synthesis by scanning the encoded genetic information along the mRNA ( Biochemistry vol 41, 4503-4510 ( 2002 ); Cancer Res . vol 48, 2659-2668 ( 1988 )],

Antisense oligonucleotides ( ASO ): Oligonucleotides that bind in a sequence-specific manner (i.e., complementarily) to nucleic acids including DNA, mRNA and pre-mRNA are referred to as antisense oligonucleotides (ASO).

When ASO binds to, for example, mRNA in the cytoplasm, the ASO can inhibit protein synthesis from the mRNA by ribosomes. ASO must be present in the cytoplasm to inhibit ribosomal protein synthesis of its target protein.

Antisense Splicing inhibition: If the ASO is strongly bonded to the free -mRNA in the nucleus, the ASO can inhibit or modulate the spliced to the pre-mRNA -mRNA. ASO must be present in the nucleus to inhibit or regulate splicing of the pre-mRNA into mRNA. Such splice inhibition of the antisense produces mRNA or mRNA without exon targeted by the ASO. Such mRNA (s) are referred to as " splice variant (s) " that encode a protein (s) smaller than the protein encoded by the full length mRNA.

In theory, splicing can be prevented by inhibiting the formation of " spliceome E-complex ". When ASO binds strongly to the junction of the (5 '→ 3') exon-intron, ie, the "5 'splice site", the ASO prevents complex formation between the pre-mRNA and the factor U1 and thus "spliceosome E Complex " Similarly, a "spliceome E-complex" can not be formed when the ASO is strongly bound to the (5 '→ 3') intron-exon junction, ie, the "3 'splice site".

The 3 'splice location and 5' splice location are schematically illustrated in the figure provided below.

Non-natural oligonucleotides: DNA or RNA oligonucleotides are sensitive to degradation by endogenous nuclease, limiting their therapeutic usefulness. To date, many types of non-natural (non-naturally occurring) oligonucleotides have been developed and intensively studied ( Clin . Exp . Pharmacol . Physiol . vol 33, 533-540 ( 2006 )). Some of them exhibit prolonged metabolic stability compared to DNA and RNA. The chemical structure of some typical non-native oligonucleotides is provided below. These oligonucleotides bind to complementary nucleic acids such as DNA or RNA as expected.

Phosphorothioate oligonucleotides: Phosphorothioate oligonucleotides (PTO) are DNA analogues in which one of the main chain phosphate oxygen atoms per monomer is replaced by one sulfur atom. Such small structural changes have made PTO relatively resistant to degradation by nuclease [ Ann. Rev. Biochem . vol 54, 367-402 ( 1985 )].

In view of the structural similarity in the backbone of PTO and DNA, they both insufficiently permeabilize the cell membrane in most mammalian cell types. However, for some types of cells that abundantly express transporter (s) of DNA, DNA and PTO exhibit good cellular permeability. Systemically administered PTOs are known to be readily distributed in the liver and kidney [ Nucleic Acids Res . vol 25, 3290-3296 ( 1997 )).

In order to facilitate PTO cell penetration in vitro, lipofection was widely practiced. However, lipofection physically alters the cell membrane, induces cytotoxicity, and thus may not be ideal for long-term in vivo treatment.

Over the past 30 years, variants of antisense PTO and PTO have been clinically evaluated for the treatment of cancer, immunological diseases, metabolic diseases, etc. [ Biochemistry vol 41, 4503-4510 ( 2002 ); Clin . Exp . Pharmacol . Physiol . vol 33, 533-540 ( 2006 )). Many of these antisense drug candidates have not been successfully developed in part due to insufficient cellular permeability of the PTO. To overcome this insufficient cellular permeability, the PTO needs to be administered in high doses for therapeutic activity. However, PTO has been implicated in immune stimulation including increased blood coagulation time, complement activation, tubular nephropathy, Kupffer cell activation, and splenomegaly, lymphoid hyperplasia, Lt ; RTI ID = 0.0 & gt ; ( Clin . Exp . Pharmacol . Physiol . vol 33, 533-540 ( 2006 )).

It has been found that a large number of antisense PTOs exhibit clinical activity commensurate with a disease predominantly originating from the liver or kidney. Mipomersen is a PTO analog that inhibits the synthesis of apoB-100, a protein involved in LDL cholesterol transport. Miformeen has shown clinical activity in patients with atherosclerosis, which appears to be due to its preferential distribution to the liver [ Circulation vol 118 (7), 743-753 ( 2008 )]. ISIS-113715 is a PTO antisense analog that inhibits the synthesis of protein tyrosine phosphatase 1B (PTP1B) and has been shown to exhibit therapeutic activity in patients with type II diabetes [ Curr . Opin . Mol . Ther . vol 6, 331-336 ( 2004 )).

Locked Nucleic Acid: In Locked Nucleic Acid (LNA), the backbone ribose ring of RNA is structurally bound, thereby increasing the binding affinity for RNA or DNA. Thus, LNA can be regarded as a high affinity DNA or RNA analogue [ Biochemistry vol 45, 7347-7355 ( 2006 )].

Phosphorodiamidate Morpholino oligonucleotides: In phosphorodiamidate morpholino oligonucleotides (PMO), the backbone phosphate of DNA and 2-deoxyribose are substituted with phosphoamidate and morpholine, respectively [ Appl . Microbiol . Biotechnol. vol 71, 575-586 ( 2006 )). The DNA backbone is negatively charged while the PMO backbone is not charged. Therefore, the binding between PMO and mRNA is not electrostatic repulsion between the main chains and tends to be stronger than the binding between DNA and mRNA. Because PMO is very structurally different from DNA, the PMO will not be recognized by the liver transporter (s) that recognize DNA or RNA. Nevertheless, PMOs do not readily penetrate cell membranes.

Peptide Nucleic Acid: Peptide nucleic acid (PNA) is a polypeptide with N- (2-aminoethyl) glycine as the unit backbone and was discovered by Dr. Nielsen and colleagues [ Science vol 254, 1497-1500 ( 1991 ). The chemical structure and abbreviation nomenclature of PNAs are illustrated in the figure provided below. Like DNA and RNA, PNAs also bind selectively to complementary nucleic acids [ Nature (London) vol. 365, 566-568 ( 1992 )]. In binding to a complementary nucleic acid, the N-terminus of the PNA is considered equivalent to the "5'-end" of DNA or RNA and the C-terminus of the PNA is considered equivalent to the & .

Like the PMO, the PNA backbone is not charged. Thus, the binding between PNA and RNA tends to be stronger than the binding between DNA and RNA. Since PNA is significantly different from DNA in its chemical structure, PNA will not be recognized by DNA-recognizing liver transporter (s) and will exhibit a tissue distribution profile that is different from DNA or PTO. However, PNA also permeabilizes mammalian cell membranes insufficiently ( Adv . Drug Delivery Rev. vol 55, 267-280, 2003 ).

The modified nucleobase : PNA modified to improve the membrane permeability of PNA became highly permeable to the mammalian cell membrane by the introduction of a modified nucleobase in which the cationic lipid or its equivalent was covalently attached. The chemical structure of such a modified nucleobase is provided below. These modified nucleobases of cytosine, adenine and guanine have been found to hybridize predictably and complementarily with guanine, thymine and cytosine, respectively [PCT Appl. No. PCT / KR2009 / 001256; EP2268607; US8680253].

PNA is similar to the situation of lipofection by the integration of such modified nucleobases. By lipofection, the oligonucleotide molecule with the phosphate backbone is surrounded by a cationic lipid molecule, such as lipofectamine, and the lipofectamine / oligonucleotide complex as described above is somewhat less than the naked oligonucleotide molecule The film tends to easily permeate through the film.

In addition to good membrane permeability, the PNA derivatives have been found to have a very-strong affinity for complementary nucleic acids. For example, the introduction of four to five modified nucleobases on 11- to 13-mer PNA derivatives produced a T _m gain of 20 ° C or higher in duplex formation with complementary DNA. Such PNA derivatives are very sensitive to single base mismatches. Single base mismatches produced a T _m loss of 11-22 ° C, depending on the type of base as well as the PNA sequence.

Small interfering RNA ( siRNA ) Small interfering RNA (siRNA) refers to 20-25 base pairs of double stranded RNA [ Microbiol . Mol . Biol . Rev. vol 67 (4), 657-685 ( 2003 )]. The antisense strand of the siRNA interacts with a portion of the protein to form an " RNA-induced silencing complex " (RISC). The RISC then binds to any part of the mRNA that is complementary to the antisense strand of the siRNA. The mRNA complexed with the RISC undergoes cleavage. Thus, siRNA catalytically induces cleavage of its target mRNA and consequently inhibits protein expression by the mRNA. The RISC does not always bind to the entire complementarity sequence in its target mRNA, which raises concerns associated with the target-external effects of siRNA therapy. Like other classes of oligonucleotides with DNA or RNA backbones, siRNAs have insufficient cellular permeability and thus lack inadequate in vitro or in vivo therapeutic activity unless properly formulated or chemically modified to have good membrane permeability .

Tyrosinase siRNA : TYR siRNA targeting the 19-mer RNA sequence in human TYR mRNA was found to inhibit the expression of TYR mRNA and protein in human epidermal melanocytes (HEM) at 20 nM concentration and lipofection conditions. siRNA downregulated the TYR mRNA and protein but did not affect the expression of tyrosinase related proteins 1 and 2. The siRNA inhibited the formation of melanin induced by UV irradiation ( BMB Reports , vol 42 (3), 178-183 ( 2009 )).

SiRNAs designed to target mouse TYR mRNA were screened for their ability to inhibit the expression of the TYR mRNA and protein and the formation of melanin in mouse B16 melanoma cells at 40 nM concentration and lipofection conditions. The most effective siRNA was injected into the eye of C57BL / 6 mice and found to inhibit up to 60% of the TYR mRNA in the retina [ Mol . Biol . International , vol 2010, Article ID 240472 ( 2010 )].

There is an additional example of a TYR siRNA in a shared work. For example, one prior art disclosed siRNAs targeting human TYR mRNA for treatment of hyperpigmentation [US 7504385]. However, the practicality of such siRNAs is doubtful for applications for hyperpigmentation. siRNAs are too costly to manufacture and tend to limit their cosmetic use. Moreover, it needs to be delivered to the skin by transdermal administration for good user compliance. Transdermal delivery should be addressed with a major technical challenge in the oligonucleotide field.

The present invention provides a peptide nucleic acid derivative or a pharmaceutically acceptable salt thereof,

(I)

In this formula,

n is an integer between 10 and 21;

3') UGUACAGAUUGUCU]의 14-머 프리-mRNA 서열과 적어도 10-머 상보성 오버랩을 갖고;The compound of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-

Wherein the entire compound of Formula I is fully complementary to the human TYR pre-mRNA or is partially complementary to the human TYR pre-mRNA with one or two incompatibilities of the entire compound;

, S_{n -1}, S_n, T₁, T₂,

, T_{n -1}, 및 T_n 은 독립적으로 듀테리도(deuterido), 하이드리도(hydrido), 치환되거나 또는 비치환된 알킬, 또는 치환되거나 또는 비치환된 아릴 라디칼을 나타내고;S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n Are independently selected from the group consisting of deuterido, hydrido, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl radicals;

X and Y are independently dew Terry also, hydrido [H], formyl [HC (= O) -] , aminocarbonyl _{[NH 2 -C (= O)} -], amino thiocarbonyl [NH ₂ -C (= S) -], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl acyl, substituted or unsubstituted aryl acyl, substituted or unsubstituted alkyl Substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, Substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, substituted or unsubstituted alkyloxytithiocarbonyl, substituted or unsubstituted aryloxytithiocarbonyl, substituted or unsubstituted alkylsulfonyl, Substituted arylsulfonyl, substituted or unsubstituted aryl Or a substituted or unsubstituted arylphosphonyl radical;

Z is selected from the group consisting of hydrido, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amino, substituted or unsubstituted alkyl, or substituted or unsubstituted An aryl radical;

, B_{n -1}, 및 B_n은 아데닌, 티민, 구아닌, 시토신 및 우라실을 포함한 천연 핵염기, 및 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;

, B_{n -1}, 및 B_n 중 적어도 4개는 핵염기 부분에 공유 결합된 치환되거나 또는 비치환된 아미노 라디칼을 갖는 비천연 핵염기 중에서 독립적으로 선택된다.B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among non-natural nucleobases having a substituted or unsubstituted amino radical covalently bonded to the nucleobase moiety.

The compound of formula I induces skipping of " exon 2 " in human TYR pre-mRNA and produces a splice variant (s) of human TYR mRNA splice without " exon 2 " , Thus being useful for inhibiting the functional activity of the gene transcribing the human TYR pre-mRNA.

The phrase " n is an integer between 10 and 21 " means literally that n is an integer that can be selected from integer groups of 11, 12, 13, 14, 15, 16, 17, 18,

The chemical structures of the natural or non-natural nucleobases in the PNA derivatives of the above formula (I) are illustrated in Figures 1A-1C. The native (i.e., naturally occurring) or non-native (non-naturally occurring) nucleobases of the present invention include, but are not limited to, the nucleobases provided in Figures 1A-1C. The provision of such non-natural nucleobases is intended to illustrate the variety of acceptable nucleobases and should not be construed as limiting the scope of the invention.

The substituents employed to describe the PNA derivatives of formula (I) above are illustrated in Figures 2A-2E. Figure 2a provides examples of substituted or unsubstituted alkyl radicals. Substituted or unsubstituted alkyl acyl and substituted or unsubstituted alkyl acyl aryl acyl radicals are illustrated in Figure 2b. Figure 2c is a schematic representation of substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted aryl, substituted or unsubstituted alkylsulfonyl or arylsulfonyl, and substituted or unsubstituted Examples of alkylphosphonyl or arylphosphonyl radicals are illustrated. Figure 2d provides examples of substituted or unsubstituted alkyloxycarbonyl or aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl or arylaminocarbonyl radicals. Substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, substituted or unsubstituted alkyloxytithiocarbonyl, substituted or unsubstituted aryloxytithiocarbo Nyl radicals. The provision of such exemplary substituents is intended to illustrate the variety of permissible substituents and should not therefore be construed as limiting the scope of the invention. It will be readily understood by those skilled in the art that oligonucleotide sequences are the most important factors for sequence-specific binding of oligonucleotides to target pre-mRNA sequences rather than substituents in the N-terminus or C-terminus.

The compound of formula I strongly binds to complementary DNA as exemplified in the prior art [PCT / KR2009 / 001256]. The duplex between the PNA derivative of formula (I) and its full-length complementary DNA or RNA of formula (I) has a T _m value too high to be reliably measured in an aqueous buffer. The PNA compounds of formula (I) produce high T _m values even with shorter lengths of complementary DNA.

3') UGUACAGAUUGUCU]의 14-머 서열은 인간 TYR 프리-mRNA 중의 "인트론 1" 및 "엑손 2"의 접합부에 위치하며, "인트론 1"로부터의 7-머 및 "엑손 2"로부터의 7-머로 이루어진다. 따라서 상기 14-머 프리-mRNA 서열은 [(5'

3') uguacag┃AUUGUCU]로서 전통적으로 나타낼 수 있으며, 여기에서 상기 인트론 및 엑손 서열을 각각 "소" 및 "대" 문자로 제공하고, 상기 인트론-엑손 접합부는 "┃"에 의해 표시한다. 프리-mRNA에 대한 전통적인 표시는 인간 TYR 프리-mRNA 중의 "인트론 1" 및 "엑손 2"의 접합부에 걸쳐 있는 [(5'

3') ggguguuuuguacag┃AUUGUCUGUAGCCGA]의 30-머 서열로 추가로 예시된다. 또한, 상기 30-머 프리-mRNA 서열은 상기 인간 TYR 프리-mRNA내의 상기 화학식 I의 화합물에 의해 타겟화된 3' 스플라이스 위치를 명확하게 한정하지만, TYR 엑손의 넘버링을 달리할 수도 있다.The compound of formula I is complementary to the 3 'splice site of the exon 2 of the human TYR pre-mRNA [NCBI Reference Sequence: NG_0008748]. [(5 '

3 ') UGUACAGAUUGUCU] is located at the junction of "intron 1" and "exon 2" in human TYR pre-mRNA, and the 7-mer from "intron 1" . Thus, the 14-mer-pre-mRNA sequence is [(5 '

3 ') uguacag┃AUUGUCU, wherein the intron and exon sequences are provided as "small" and "large" letters, respectively, and the intron-exon junction is indicated by "┃". A traditional indication for pre-mRNA is [(5 ') < / RTI > over the junction of " intron 1 "

3 ') ggguguuuuguacag┃AUUGUCUGUAGCCGA]. In addition, the 30-mer-pre-mRNA sequence clearly defines the 3 'splice site targeted by the compound of formula I in the human TYR pre-mRNA, but may also differ in the numbering of TYR exons.

The compound of formula I strongly binds to the target 3 'splice site of the human TYR pre-mRNA transcribed from the human TYR gene and prevents the formation of a " spliceome early complex " involving the target exon of the compound. Since the compounds of the present invention sterically inhibit the formation of the " spliceome early complex ", the TYR " exon 2 " is tethered to produce a TYR mRNA splice variant (s) without " exon 2 ". As a result, the compounds of the present invention induce skipping of TYR " exon 2 ".

Strong RNA affinity indicates that the compound of formula I is capable of inducing skipping of the TYR " exon 2 " even when the PNA derivative has one or two incompatibilities with the target 3 'splice site in the TYR pre-mRNA do. In addition, the PNA derivatives of formula (I) may still induce skipping of TYR " exon 2 " among TYR mutant pre-mRNAs having one or two SNPs (single nucleotide polymorphisms) in the target splice site.

The compounds of formula (I) have good cellular permeability and can be readily delivered to cells as " naked " oligonucleotides as exemplified in the prior art [PCT / KR2009 / 001256]. Thus, the compounds of the present invention induce skipping of " exon 2 " among the TYR pre-mRNAs and include TYR mRNA splice variants with no TYR " exon 2 " in cells treated with compounds of formula I as " naked " oligonucleotides (S). The compounds of formula I do not require any means or formulations for delivery into cells to efficently induce skipping of the targeted exon in the cells. The compounds of formula I readily induce skimming of TYR " exon 2 " in cells treated with compounds of the invention as " naked " oligonucleotides, even at femtomolar-

Thanks to good cell or membrane permeability, the PNA derivatives of formula I above can be administered locally as " naked " oligonucleotides to induce skipping of TYR " exon 2 " in the skin. The compounds of formula I do not require formulations to increase transdermal delivery into the target tissue for the intended therapeutic or biological activity. Usually, the compound of formula (I) is dissolved in water and a co-solvent, and administered topically or transdermally at a concentration of less than a picomole to elicit the desired therapeutic or biological activity in the target skin. The compounds of the present invention need not be formulated heavily or invasively to elicit such topical therapeutic activity. Nevertheless, the PNA derivatives of formula (I) above may be formulated with cosmetic ingredients or adjuvants as topical creams or lotions. Such topical cosmetic creams or lotions may be useful in the treatment of facial hypercholesterolemia.

The compounds of formula (I) may be used in conjunction with a pharmaceutically acceptable acid or base, such as, but not limited to, sodium hydroxide, potassium hydroxide, hydrochloric acid, methanesulfonic acid, citric acid, trifluoroacetic acid and the like.

The PNA derivatives of the above formula I or pharmaceutically acceptable salts thereof may be mixed with pharmaceutically acceptable adjuvants such as, but not limited to, citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethylene glycol, polypropylene glycol, ethanol, isopropanol, sodium Bicarbonate, distilled water, preservative (s), and the like.

The compounds of the present invention may be administered topically to a subject at therapeutically or biologically effective concentrations in the range of 1 aM to 1 nM or more, and the concentration range will vary depending on the schedule of administration, the condition or circumstance of the subject, and the like.

Preferably a PNA derivative of formula (I) or a pharmaceutically acceptable salt thereof:

n is an integer between 10 and 21;

3') UGUACAGAUUGUCU]의 14-머 프리-mRNA 서열과 적어도 10-머 상보성 오버랩을 갖고;The compounds of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-

Wherein the entire compound of Formula I is fully complementary to the human TYR pre-mRNA or is partially complementary to said human TYR pre-mRNA with one or two incompatibilities of the entire compound;

, S_{n -1}, S_n, T₁, T₂,

, T_{n -1}, 및 T_n이 독립적으로 듀테리도 또는 하이드리도 라디칼을 나타내고;S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T &_lt; _{n > -1} , and T &_lt; _n > independently represent a diterido or hydrido radical;

X and Y are independently selected from the group consisting of deuterido, hydrido, formyl, aminocarbonyl, aminothiocarbonyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl , Substituted or unsubstituted aryl acyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted aryl Substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, Substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, substituted or unsubstituted alkylphosphonyl radicals, or substituted Or or unsubstituted aryl phosphonic sulfonyl radical;

Z represents hydrido, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, or substituted or unsubstituted amino radicals;

, B_{n -1}, 및 B_n이 아데닌, 티민, 구아닌, 시토신 및 우라실을 포함한 천연 핵염기, 및 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;

, B_{n -1}, 및 B_n 중 적어도 4개가 하기 화학식 Ⅱ, 화학식 Ⅲ 또는 화학식 Ⅳ에 의해 나타내는 비천연 핵염기 중에서 독립적으로 선택되고:B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by the following formula (II), (III) or (IV)

[Formula II]

[Formula (III)

[Formula IV]

In the above equations,

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from hydrido, and substituted or unsubstituted alkyl radicals;

L &_lt; _{1 >} , L &_lt; _{2 >} , and L < ₃ > are covalent linkers represented by the following formula V that covalently attach a basic amino group to the nucleobase portion:

(V)

In this formula,

Q ₁ and Q _m are substituted or unsubstituted methylene (-CH ₂ -) radicals, and Q _m is directly bonded to said basic amino group;

, Q_{m -1}은 치환되거나 또는 비치환된 메틸렌, 산소(-O-), 황(-S-), 및 치환되거나 또는 비치환된 아미노 라디칼 [-N(H)-, 또는 -N(치환체)-] 중에서 독립적으로 선택되고;Q ₂ , Q ₃ ,

, Q _{m -1} is selected from the group consisting of substituted or unsubstituted methylene, oxygen (-O-), sulfur (-S-), and substituted or unsubstituted amino radicals [-N (H) -, ) -]; < / RTI >

m is an integer between 1 and 15;

Importantly, the PNA derivative of formula (I) or a pharmaceutically acceptable salt thereof is

n is an integer between 11 and 18;

3') UGUACAGAUUGUCU]의 14-머 프리-mRNA 서열과 적어도 10-머 상보성 오버랩을 갖고;The compounds of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-

Wherein the entire compound of formula I is fully complementary to human TYR pre-mRNA;

, S_{n -1}, S_n, T₁, T₂,

, T_{n -1}, 및 T_n이 하이드리도 라디칼이고;S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;

X and Y are independently selected from hydrido, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl acyl, substituted or unsubstituted aryl acyl, substituted or unsubstituted Alkyloxycarbonyl, or a substituted or unsubstituted aryloxycarbonyl radical;

Z represents a substituted or unsubstituted amino radical;

, B_{n -1}, 및 B_n이 아데닌, 티민, 구아닌, 시토신 및 우라실을 포함한 천연 핵염기, 및 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;

, B_{n -1}, 및 B_n 중 적어도 4개가 화학식 Ⅱ, 화학식 Ⅲ 또는 화학식 Ⅳ에 의해 나타내는 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from hydrido, and substituted or unsubstituted alkyl radicals;

Q ₁ and Q _m are substituted or unsubstituted methylene radicals, Q _m is directly bonded to a basic amino group;

, Q_{m -1}이 치환되거나 또는 비치환된 메틸렌, 산소, 및 아미노 라디칼 중에서 독립적으로 선택되고;Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from substituted or unsubstituted methylene, oxygen, and amino radicals;

m is an integer between 1 and 11;

Especially of the PNA derivatives of formula (I), or a pharmaceutically acceptable salt thereof, under the following conditions:

n is an integer between 11 and 16;

3') UGUACAGAUUGUCU]의 14-머 프리-mRNA 서열과 적어도 10-머 상보성 오버랩을 갖고;The compounds of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-

Wherein the entire compound of formula I is fully complementary to human TYR pre-mRNA;

, S_{n -1}, S_n, T₁, T₂,

, T_{n -1}, 및 T_n이 하이드리도 라디칼이고;S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;

X and Y are independently selected from hydrido, substituted or unsubstituted alkyl acyl, and substituted or unsubstituted alkyloxycarbonyl radicals;

Z represents a substituted or unsubstituted amino radical;

, B_{n -1}, 및 B_n이 아데닌, 티민, 구아닌, 시토신 및 우라실을 포함한 천연 핵염기, 및 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;

, B_{n -1}, 및 B_n 중 적어도 4개가 화학식 Ⅱ, 화학식 Ⅲ 또는 화학식 Ⅳ에 의해 나타내는 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from hydrido, and substituted or unsubstituted alkyl radicals;

Q ₁ and Q _m are methylene radicals and Q _m is directly bonded to a basic amino group;

, Q_{m -1}이 메틸렌, 산소, 및 아미노 라디칼 중에서 독립적으로 선택되고;Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from methylene, oxygen, and amino radicals;

m is an integer between 1 and 9;

Very importantly, a PNA derivative of formula (I) or a pharmaceutically acceptable salt thereof:

n is an integer between 11 and 16;

3') UGUACAGAUUGUCU]의 14-머 프리-mRNA 서열과 적어도 10-머 상보성 오버랩을 갖고;The compounds of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-

Wherein the entire compound of formula I is fully complementary to human TYR pre-mRNA;

, S_{n -1}, S_n, T₁, T₂,

, T_{n -1}, 및 T_n이 하이드리도 라디칼이고;S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;

X and Y are independently selected from hydrido, substituted or unsubstituted alkyl acyl, and substituted or unsubstituted alkyloxycarbonyl radicals;

Z represents a substituted or unsubstituted amino radical;

, B_{n -1}, 및 B_n이 아데닌, 티민, 구아닌, 시토신 및 우라실을 포함한 천연 핵염기, 및 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;

, B_{n -1}, 및 B_n 중 적어도 4개가 화학식 Ⅱ, 화학식 Ⅲ 또는 화학식 Ⅳ에 의해 나타내는 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);

R ₁ , R ₃ , and R ₅ are hydrido radicals, R ₂ , R ₄ , and R ₆ are independently hydrido, or substituted or unsubstituted alkyl radicals;

Q ₁ and Q _m are methylene radicals and Q _m is directly bonded to a basic amino group;

, Q_{m -1}이 메틸렌, 산소 라디칼 중에서 독립적으로 선택되고;Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from methylene, oxygen radicals;

m is an integer between 1 and 9;

Very importantly, a PNA derivative of formula (I) or a pharmaceutically acceptable salt thereof:

n is an integer between 11 and 16;

3') UGUACAGAUUGUCU]의 14-머 프리-mRNA 서열과 적어도 10-머 상보성 오버랩을 갖고;The compounds of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-

Wherein the entire compound of formula I is fully complementary to human TYR pre-mRNA;

, S_{n -1}, S_n, T₁, T₂,

, T_{n -1}, 및 T_n이 하이드리도 라디칼이고;S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;

X and Y are independently selected from hydrido, substituted or unsubstituted alkyl acyl, and substituted or unsubstituted alkyloxycarbonyl radicals;

Z represents a substituted or unsubstituted amino radical;

, B_{n -1}, 및 B_n이 아데닌, 티민, 구아닌, 시토신, 및 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from adenine, thymine, guanine, cytosine, and non-natural nucleobases;

, B_{n -1}, 및 B_n 중 적어도 5개가 화학식 Ⅱ, 화학식 Ⅲ 또는 화학식 Ⅳ에 의해 나타내는 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n is independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrido radicals;

Q ₁ and Q _m are methylene radicals and Q _m is directly bonded to a basic amino group;

, Q_{m -1}이 메틸렌, 및 산소 라디칼 중에서 독립적으로 선택되고;Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from methylene, and an oxygen radical;

m is an integer between 1 and 9;

Most importantly a PNA derivative of formula (I) or a pharmaceutically acceptable salt thereof:

n is an integer between 11 and 16;

3') UGUACAGAUUGUCU]의 14-머 프리-mRNA 서열과 적어도 10-머 상보성 오버랩을 갖고;The compounds of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-

Wherein the entire compound of formula I is fully complementary to human TYR pre-mRNA;

, S_{n -1}, S_n, T₁, T₂,

, T_{n -1}, 및 T_n이 하이드리도 라디칼이고;S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;

X is a hydrido radical;

Y represents a substituted or unsubstituted alkyl acyl, and a substituted or unsubstituted alkyloxycarbonyl radical;

Z represents a substituted or unsubstituted amino radical;

, B_{n -1}, 및 B_n이 아데닌, 티민, 구아닌, 시토신, 및 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from adenine, thymine, guanine, cytosine, and non-natural nucleobases;

, B_{n -1}, 및 B_n 중 적어도 5개가 화학식 Ⅱ, 화학식 Ⅲ 또는 화학식 Ⅳ에 의해 나타내는 비천연 핵염기 중에서 독립적으로 선택되고;B ₁ , B ₂ ,

, B _{n -1} , and B _n is independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrido radicals;

L ₁ is _{- (CH 2) 2 -O- (} CH 2) 2 -, -CH 2 -O- (CH 2) 2 -, -CH 2 -O- (CH 2) 3 -, -CH 2 -O - (CH ₂ ) ₄ -, or -CH ₂ -O- (CH ₂ ) ₅ -, wherein the right end is directly bonded to a basic amino group;

L ₂ And L ₃ is _{- (CH 2) 2 -O- (} CH 2) 2 -, - (CH 2) 3 -O- (CH 2) 2 -, - (CH 2) 2 -O- (CH 2) 3 _{-, - (CH 2) 2} -, - (CH 2) 3 -, - (CH 2) 4 -, - (CH 2) 5 -, - (CH 2) 6 -, - (CH 2) 7 -, And - (CH ₂ ) ₈ -, wherein the right end is directly bonded to the basic amino group.

Of particular significance is a PNA derivative of formula (I), or a pharmaceutically acceptable salt thereof, selected from the group of the compounds provided below:

C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Fethoc-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;

C) Fethoc-AC(1/2)A(5)-GA(5)C-AA(5)T-CTG(6)-C(1/2)C-NH₂;(N

C) Fethoc-AC (1/2) A (5) -GA 5 C-AA 5 T-CTG 6 -C (1/2) C-NH ₂ ;

C) Ac-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Ac-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) Benzoyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Benzoyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;

C) Piv-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Piv-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) Methyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Methyl-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) n-Propyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) n-Propyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;

C) Fmoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Fmoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) p-Toluenesulfonyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) p-Toluenesulfonyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;

C) H-Lys-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) H-Lys-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;

C) Fethoc-Lys-Gly-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Fethoc-Lys-Gly-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;

C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-Lys-NH₂;(N

C) Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -Lys-NH ₂ ;

C) Fethoc-Val-Gly-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Fethoc-Val-Gly- CA (5) G-ACA (5) -ATC (1O2) -TG (6) TA (5) -NH 2;

C) Ac-Arg-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Ac-Arg-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) Benzyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Benzyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;

C) Phenyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Phenyl-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) [N-(2-Phenylethyl)amino]carbonyl-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) [N- (2-Phenylethyl ) amino] carbonyl-CA (5) G-ACA (5) -ATC (1O2) -TG (6) TA (5) -NH 2;

C) Benzoyl-Leu-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Benzoyl-Leu-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) Piv-Lys-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-Val-NH₂;(N

C) Piv-Lys-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -Val-NH ₂ ;

C) Fethoc-CA(7)G-AC(2O2)A-A(4)TC(1O2)-TG(6)T-A-NH₂;(N

C) Fethoc-CA (7) G-AC (2 O 2) AA (4) TC (10 2) -TG (6) TA-NH ₂ ;

C) Fethoc-CTG(6)-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Fethoc-CTG (6) -ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) Fethoc-CA(5)G-ATA(5)-ATC(1O2)-TG(5)T-A(5)-NH₂;(N

C) Fethoc-CA (5) G-ATA (5) -ATC (10 2) -TG (5) TA (5) -NH ₂ ;

C) Fethoc-TA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂;(N

C) Fethoc-TA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;

C) Fethoc-TCA(3)-GAC(1O5)-A(5)AT-C(1O2)TG(5)-TA(5)-NH₂;(N

C) Fethoc-TCA (3) -GAC (10) -A (5) AT-C (10 2) TG (5) -TA (5) -NH ₂ ;

C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)C-NH₂;(N

C) Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) C-NH ₂ ;

C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(6)TA(5)-C-NH₂;(N

C) Fethoc-AG (6) A-CA (5) A-TC (10 2) TG (6) TA (5) -C-NH ₂ ;

C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(6)TA(5)-CA(2O2)A-NH₂;(N

C) Fethoc-AG (6) A-CA (5) A-TC (10) TG (6) TA (5) -CA (20 2) A-NH ₂ ;

C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)C(1O2)-AA(6)-NH₂;(N

C) Fethoc-AC (1O2) A-GA (6) C-AA (6) T-CTG (6) -TA (6) C (1O2) -AA (6) -NH 2;

C) Fethoc-AC(1O3)T-GA(6)C-TA(6)T-CTG(6)-TAC(1O5)-A(4)A-NH₂;(N

C) Fethoc-AC (1O3) T-GA (6) C-TA (6) T-CTG (6) -TAC (1O5) -A (4) A-NH 2;

C) Fethoc-GA(6)C-AA(6)T-CTG(6)-TA(6)C(1O2)-AA(6)-NH₂;(N

C) Fethoc-GA (6) C-AA (6) T-CTG (6) -TA (6) C (10) -AA (6) -NH ₂ ;

C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)C-A(5)AA(2O2)-A-NH₂;(N

C) Fethoc-AC (10) A-GA (6) C-AA (6) T-CTG (6) -TA (6) CA (5) AA (20 2) -A-NH ₂ ;

C) Fethoc-AA(6)T-CTG(6)-TA(6)C-A(5)AA(2O2)-A-NH₂;(N

C) Fethoc-AA (6) T-CTG (6) -TA (6) CA (5) AA (20 2) -A-NH ₂ ;

C) Fethoc-AC(1O2)A-GA(6)C-AA(6)T-CTG(6)-TA(6)C-A(5)AA(2O3)-A-NH₂;(N

C) Fethoc-AC (10) A-GA (6) C-AA (6) T-CTG (6) -TA (6) CA (5) AA (20 3) -A-NH ₂ ;

C) Fethoc-GC(1O4)T-AC(1O2)A-GA(4)C-AAT-CTG(6)-TA(6)C-NH₂;(N

C) Fethoc-GC (10) T-AC (10) A-GA (4) C-AAT-CTG (6) -TA (6) C-NH ₂ ;

C) Fethoc-GC(1O2)T-A(3)CA-GA(4)C-AAT-C(1O2)TG-NH₂;(N

C) Fethoc-GC (1O2) TA (3) CA-GA (4) C-AAT-C (1O2) TG-NH 2;

C) Fethoc-GC(1O2)T-A(3)CA-G(2O2)AC-A(5)AT-C(1O2)TG-NH₂; 및(N

C) Fethoc-GC (10) TA (3) CA-G (20 2) AC-A (5) AT-C (10 ₂ ) TG-NH ₂ ; And

C) Fethoc-GC(1O2)T-A(3)CA-GA(4)C-A(2O2)AT-C(1O2)TG-NH₂:(N

C) Fethoc-GC (1O2) TA (3) CA-GA (4) CA (2O2) AT-C (1O2) TG-NH 2:

In the above,

A, G, T, and C are PNA monomers with natural nucleobases of adenine, guanine, thymine and cytosine, respectively;

Wherein the non-natural nucleobases represented by the following formulas (VI), (VII), (VII), (VIII), (IX) and (X) ≪ / RTI > are:

(VI)

[Formula VII]

(VIII)

[Formula IX]

(X)

In the above equations,

p and q are integers;

The abbreviations for N- and C-terminal substituents are as specifically set forth as follows: "Fmoc-" is an abbreviation for "[(9-fluorenyl) methyloxy] carbonyl] -";"Fethoc-" means "[2- (9-fluorenyl) ethyl-1-oxy] carbonyl";&Quot; Ac- " is " acetyl- ";&Quot; Benzoyl- " refers to " benzenecarbonyl- ";&Quot; Piv- " is " pivial reel- ";&Quot; Methyl- " is " methyl- ";"p-Toluenesulfonyl" refers to "(4-methylbenzene) -1-sulfonyl" - "n-propyl- ; "-Lys-" is an amino acid residue "lysine";"-Val-" is an amino acid residue "valine";"Leu-" is an amino acid residue "leucine" -Gly- "is an amino acid residue" glycine "," [N- (2-Phenylethyl) amino] carbonyl- ""1 - (phenyl) -";"Phenyl-" is "phenyl -";"Me-" is "methyl -";"-NH_2" is an unsubstituted - an abbreviation for groups "amino".

Figure 3 shows the chemical structure of the PNA monomers abbreviated as A, G, T, C, C (pOq), A (p), A (pOq), G (p) . As discussed in the prior art [PCT / KR2009 / 001256], C (pOq) is recognized as a "modified cytosine" PNA monomer because it is complementary to "guanine". A (p) and A (p0q) are recognized as "modified adenine" PNA monomers because they are complementary to "thymine". Similarly, G (p) and G (p0q) are known as "modified guanine" PNA monomers because they are base-paired with "cytosine".

Figure 4 clearly illustrates the chemical structure of various abbreviations for the substituents used in the present invention for the N-terminal or C-terminal diversification of PNA derivatives of formula (I).

C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂"로서 약기된 13-머 PNA 유도체의 화학 구조를 도 5a에 제공한다. 또 다른 예시로서, "(N

C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(3O2)TA(5)-CA(2O2)A-NH₂"로서 약기된 15-머 PNA 유도체의 화학 구조를 도 5b에 제공한다.To illustrate the abbreviations used for such PNA derivatives, " (N

The chemical structure of the 13-mer PNA derivative abbreviated as C-Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ " As another example, " (N

C) Fethoc-AG (6) A-CA (5) A-TC (1O2) TG (3O2) TA (5) -CA ( the chemical structure of the 15-Merced PNA derivative abbreviated as 2O2) A-NH ₂ " 5B.

C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂"의 13-머 PNA 서열은 프리-mRNA에의 상보성 결합을 위한 "(5'

3') CAG-ACA-ATC-TGT-A"의 DNA 서열과 동등하다. 상기 13-머 PNA는 인간 TYR 프리-mRNA에서 "인트론 1" 및 "엑손 2"의 접합부에 걸쳐 있는 [(5'

3') ggguguuuug uacag ┃ AUUGUCUG UAGCCGA]의 30-머 RNA 서열내에 "진하게" "밑줄그어" 표시한 13-머 서열과 13-머 상보성 오버랩을 갖는다.The " (N

The 13-mer PNA sequence of Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ "'

3 ') CAG-ACA-ATC-TGT-A ". The 13-mer PNA is located at the junction of the " intron 1 "

3 ') ggguguuuug uacag ┃ AUUGUCUG UAGCCGA] has a 13-mer sequence and a 13-mer complementarity overlap marked "underlined" in the 30-mer RNA sequence.

C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(3O2)TA(5)-CA(2O2)A-NH₂"의 15-머 PNA 서열은 프리-mRNA에의 상보성 결합을 위한 "(5'

3') AGA-CAA-TCT-GTA-CAA"의 DNA 서열과 동등하다. 상기 15-머 PNA는 인간 TYR 프리-mRNA에서 "인트론 1" 및 "엑손 2"의 접합부에 걸쳐 있는 [(5'

3') ggguguu uuguacag ┃ AUUGUCU GUAGCCGA]의 30-머 RNA 서열내에 "진하게" "밑줄그어" 표시한 15-머 서열과 15-머 상보성 오버랩을 갖는다.The " (N

C) Fethoc-AG (6) A-CA (5) A-TC (1O2) TG (3O2) TA (5) -CA (2O2) A-NH 2 "15- Murray PNA is complementary to the sequence of the pre--mRNA &Quot; (5 '

3 ') AGA-CAA-TCT-GTA-CAA ". The 15-mer PNA is located at the junction of the intron 1 and exon 2 in the human TYR pre-

3 ') ggguguu uuguacag ┃ AUUGUCU GUAGCCGA] has a 15-mer sequence and a 15-mer complementarity overlap marked "underlined" in the 30-mer RNA sequence.

C) Fethoc-AC(1O3)T-GA(6)C-TA(6)T-CTG(6)-TAC(1O5)-A(4)A-NH₂"의 17-머 PNA 서열은 프리-mRNA에의 상보성 결합을 위한 "(5'

3') ACT-GAC-TAT-CTG-TAC-AA"의 DNA 서열과 동등하다. 상기 17-머 PNA는 인간 TYR 프리-mRNA에서 "인트론 1" 및 "엑손 2"의 접합부에 걸쳐 있는 [(5'

3') ggguguu uuguacag ┃ AU "U" GUC "U" GU AGCCGA]의 30-머 RNA 서열내에 "진하게" "밑줄그어" 표시한 15-머 서열과 15-머 상보성 오버랩을 갖는다. 두 개의 불일치에는 인용부호 ""로 표시하였습니다.
"(N

C) Fethoc-AC (1O3) T-GA (6) C-TA (6) T-CTG (6) 17- Murray PNA sequences -TAC (1O5) -A (4) A-NH 2 " are pre- " (5 ')< / RTI >

3 ') ACT-GAC-TAT-CTG-TAC-AA ". The 17-mer PNA is located at the junction of " 5 '

3 ') ggguguu uuguacag ┃ AU "U" GUC "U" GU AGCCGA] and a 15-mer complementary overlap with the 15-mer sequence indicated "underlined" in the 30-mer RNA sequence. Two discrepancies are indicated by the quotation marks "".

One aspect of the present invention provides a composition for the treatment of a disease or disorder accompanied by the expression of a human tyrosinase gene comprising a peptide nucleic acid derivative or a pharmaceutically acceptable salt thereof according to the present invention.

One aspect of the present invention provides compositions for treating hypercholesterolemia comprising a peptide nucleic acid derivative or a pharmaceutically acceptable salt thereof according to the present invention. Examples of hyperpigmentation due to melanin overgrowth include melasma, acne scarring, liver spots, freckle, age spots, and actinic damage. .

One aspect of the present invention provides a method of treating a disease or disorder accompanied by the expression of a human tyrosinase gene by administering a peptide nucleic acid derivative according to the present invention. Here, administration can be carried out, for example, using a local administration route.

One aspect of the present invention provides a method of treating hyperpigmentation by administering a peptide nucleic acid derivative according to the present invention. Here, administration can be carried out, for example, using a local administration route.

C) Fethoc-CA(5)G-ACA(5)-ATC(1O2)-TG(6)T-A(5)-NH₂"의 화학 구조.
도 5b. 15-머 PNA 유도체 "(N

C) Fethoc-AG(6)A-CA(5)A-TC(1O2)T-G(3O2)TA(5)-CA(2O2)A-NH₂"의 화학 구조.
도 6. 본 발명의 PNA 유도체의 합성에 사용된 Fmoc-PNA 단량체의 화학 구조.
도 7a-7b. 각각 HPLC 정제 전 및 후의 "ASO 1"의 C₁₈-역상 HPLC 크로마토그램.
도 8. C₁₈-RP 프렙 HPLC에 의해 정제된 "ASO 1"의 ESI-TOF 질량 스펙트럼.
도 9a. 0(음성 대조용), 1, 10 또는 1,000 aM "ASO 1M"로 처리된 B16F10 마우스 흑색종 세포에서 네스티드(nested) PCR 산물의 전기영동 분석.
도 9b. 엑손(2-3)의 스키핑에 지정될 수 있는 PCR 산물에 대한 상거(Sanger) 서열분석 데이터.
도 10a. 0(음성 대조용), 1, 10, 100 또는 1,000 aM의 "ASO 1M"로 처리된 B16F10 마우스 흑색종 세포에서 완전길이 TYR mRNA 수준의 변화 (표준 오차에 의한 오차 막대).
도 10b. 0 zM(음성 대조용), 10 zM, 100 zM, 1 aM 또는 10 aM(즉 10,000 zM)의 "ASO 1M"로 24시간 동안 처리된 B16F10 마우스 흑색종 세포에서 TYR 웨스턴 블럿 데이터.
도 11. 1, 10, 100 또는 1,000 aM의 "ASO 1M", 또는 10 ㎍/㎖ 또는 100 ㎍/㎖의 알부틴으로 처리된 B16F10 마우스 흑색종 세포에서 멜라닌 함량의 변화 (표준 오차에 의한 오차 막대).
도 12. 0 zM(음성 대조용), 1 zM, 100 zM 또는 10 aM의 "ASO 1"로 처리된 인간 1차 상피 멜라닌 세포에서 qPCR에 의한 완전길이 TYR mRNA 수준의 변화 (표준 오차에 의한 오차 막대).Figures 1a-1c. Examples of natural or non-natural (modified) nucleobases that can be selected for the peptide nucleic acid derivatives of formula (I).
2a-2e. Examples of substituents that are selectable for peptide nucleic acid derivatives of formula (I).
Figure 3. Chemical structures of PNA monomers with natural or modified nucleobases.
Figure 4. Chemical structure for abbreviations of N- or C-terminal substituents.
5a. 13-mer PNA derivative " (N

Chemical structure of Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ".
5b. 15-mer PNA derivative " (N

C) Fethoc-AG (6) A-CA (5) A-TC (1O2) TG (3O2) TA (5) -CA (2O2) A-NH 2 The chemical structure of ".
6. Chemical structure of the Fmoc-PNA monomers used in the synthesis of the PNA derivatives of the present invention.
Figures 7a-7b. C ₁₈ - reversed-phase HPLC chromatograms of "ASO 1" before and after HPLC purification, respectively.
8. ESI-TOF mass spectrum of "ASO 1" purified by C ₁₈ -RP prep HPLC.
9a. Electrophoretic analysis of nested PCR products in B16F10 mouse melanoma cells treated with 0 (negative control), 1, 10 or 1,000 aM "ASO 1M".
9b. Sanger sequencing data for PCR products that can be assigned to the skipping of exons (2-3).
10a. Changes in the level of full length TYR mRNA in B16F10 mouse melanoma cells treated with "ASO 1M" of 0 (negative control), 1, 10, 100 or 1,000 aM (error bars by standard error).
10b. TYR Western blot data from B16F10 mouse melanoma cells treated with " ASO 1M " of 0 zM (negative control), 10 zM, 100 zM, 1 aM or 10 aM (i.e. 10,000 zM) for 24 hours.
Figure 11. Change in melanin content (error bars by standard error) in B16F10 mouse melanoma cells treated with 1, 10, 100 or 1,000 aM of "ASO 1M", or with 10 or 100 μg / .
12. Changes in the level of full-length TYR mRNA by qPCR in human primary epithelial melanocytes treated with "ASO 1" at 0 zM (negative control), 1 zM, 100 zM or 10 aM rod).

PNA Oligomeric  General process for manufacturing

PNA oligomers with some, but reasonable variations, of the prior art [US 6,133,444; (Solid phase peptide synthesis) based on Fmoc-chemistry according to the method disclosed in WO 96/40685. The solid support used in this study was H-Rink Amide-ChemMatrix purchased from PCAS BioMatrix Inc. (Quebec, Canada). Fmoc-PNA monomers with modified nucleobases were synthesized as described in the prior art [PCT / KR 2009/001256] or with slight modifications. PNA derivatives of the present invention were synthesized using Fmoc-PNA monomers having the modified nucleobases as described above and Fmoc-PNA monomers having natural nucleobases. The PNA oligomer was purified by C ₁₈ -reversed-phase HPLC (water / acetonitrile with 0.1% TFA / water / methanol) and characterized by mass spectrometry including ESI / TOF / MS.

Scheme 2 illustrates a typical monomer elongation cycle employed in the SPPS of the above study and provides synthesis details as follows. However, it is apparent to those skilled in the art that there are a number of apparently small variations in effectively practicing such SPPS reactions on an automatic or passive peptide synthesizer. Each of the reaction steps of Scheme 2 is briefly provided as follows.

[Reaction Scheme 2]

[Activation of H-Link-Chem Matrix Resin]

0.01 mmol (20 mg resin) of the chemmatrix resin in 1.5 mL of 20% piperidine / DMF was vortexed in a libra tube for 20 minutes and the DeFmoc solution was filtered. The resin was washed successively with 1.5 mL of methylene chloride (MC), 1.5 mL of dimethylformamide (DMF), 1.5 mL of MC, 1.5 mL of DMF, and 1.5 mL of MC for 30 seconds each. Coupling with the Fmoc-PNA monomer or Fmoc-protected amino acid derivative was performed on the free amine produced on the solid support.

[DeFmoc]

The resin was vortexed in 1.5 mL of 20% piperidine / DMF for 7 min and the DeFmoc solution was filtered. The resin was washed successively with 1.5 mL MC, 1.5 mL DMF, 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC for 30 seconds each. Coupling with the Fmoc-PNA monomer was immediately performed on the free amine generated on the solid support.

[Coupling with Fmoc-PNA Monomer]

The free amines on the solid support were coupled with Fmoc-PNA monomers as follows. 0.04 mmol of PNA monomer, 0.05 mmol of HBTU, and 10 mmol of DIEA were incubated in 1 ml of anhydrous DMF for 2 min and added to the resin with free amine. The resin solution was vortexed for 1 hour and the reaction medium was filtered. The resin was then washed successively with 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC for 30 seconds each. The chemical structure of the Fmoc-PNA monomer with modified nucleobases used in the present invention is provided in FIG. The Fmoc-PNA monomers with modified nucleobases provided in Figure 6 above should be considered as examples and should not be considered as limiting the scope of the invention. One of ordinary skill in the art can readily understand many of the changes in the Fmoc-PNA monomers for synthesizing the PNA derivatives of formula (I).

[Capping]

Following the coupling reaction, the unreacted free amine was capped by shaking in 1.5 ml of capping solution (5% acetic anhydride in DMF and 6% 2,6-lutidine) for 5 minutes. The capping solution was then filtered and washed successively with 1.5 mL MC, 1.5 mL DMF, and 1.5 mL MC for 30 seconds each.

[Introduction of the " Fethoc- " radical in the N-terminus]

The "Fethoc-" radical was introduced at the N-terminus by reacting the free amine on the dendrites with "Fethoc-OSu" under basic coupling conditions. &Quot; Fethoc-OSu " 179337-69-0, the chemical structure of _{C 20 H 17 NO 5, MW} 351.36] is provided as follows.

[Separation from resin]

The PNA oligomer bound to the resin was separated from the resin by shaking for 3 hours in 1.5 mL of a separate solution (2.5% triisopropylsilane in trifluoroacetic acid and 2.5% water). The resin was filtered and the filtrate was concentrated under reduced pressure. The resulting residue was treated with diethyl ether and the resulting precipitate was collected by filtration and then purified by reverse phase HPLC.

[HPLC analysis and purification]

Following separation from the resin, the crude product of the PNA derivative was purified by C ₁₈ -reversed phase HPLC using water / acetonitrile or water / methanol (gradient method) containing 0.1% TFA as eluent. Figures 7a and 7b are exemplary HPLC chromatograms for " ASO " before and after HPLC purification, respectively. The oligomer sequences of " ASO 1 " are as provided in Table 1.

Synthesis of PNA Derivatives of Formula I Example

In order to complementarily target the 3 'splice site of "exon 2" in human TYR pre-mRNA, the PNA derivatives of the invention were prepared according to the synthetic procedure provided above or with slight modifications thereof. The provision of such PNA derivatives to target the human TYR pre-mRNA is intended to illustrate the PNA derivatives of formula I and should not be construed as limiting the scope of the invention.

PNA derivatives that complementarily target the 3 'splice site of " exon 2 " in human TYR pre-mRNA and structural characterization data thereof by mass spectrometry PNA example PNA sequence (N? C) Exact mass, m / z theor. ^a obs. ^b ASO 1 Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ 4258.96 4260.99 ASO 2 Fethoc-AC (12) A- GA (5) C-AA (5) T-CTG (6) -TA (5) C (1O2) -AA (5) -NH 2 5532.55 5532.54 ASO 3 Fethoc-AC (102) A (5) -GA 5 C-AA 5 T-CTG 6 -C (10 ₂ ) C-NH ₂ 4592.11 4592.11 ^{a) the} theoretical exact mass, ^b) the exact mass observed

Table 1 shows PNA derivatives complementarily targeting the 3 'splice site of " exon 2 " in human TYR pre-mRNA read from human TYR gene [NCBI reference sequence: NG_0008748] and their mass spectrometric analysis Provides structural characterization data. The provision of the TYR ASOs of Table 1 is intended to illustrate the PNA derivatives of Formula I and should not be construed as limiting the scope of the invention.

3') ggguguuuug uacag ┃ AUUGU - CUG UAGCCGA]의 30-머 RNA 서열내에 "진하게" "밑줄그어" 표시한 13-머 서열과 13-머 상보성 오버랩을 갖는다. 따라서 "ASO 1"은 상기 인간 TYR 프리-mRNA 내에 "인트론 1"과의 5-머 오버랩 및 "엑손 2"와의 8-머 오버랩을 갖는다.
&Quot; ASO 1 " is the [(5 ')< / RTI > sequence extending across the junction of " intron 1 "

3 ') ggguguuuug uacag ┃ AUUGU - CUG UAGCCGA] with a 13 - mer sequence and a 13 - mer complementary overlap indicated "underlined" in the 30 - mer RNA sequence. Thus, "ASO 1" has a 5-mer overlap with "intron 1" and an 8-mer overlap with "exon 2" in the human TYR pre-mRNA.

Table 2 summarizes "ASO 1M" and its mass spectroscopy "complementary" to the 3 'splice site of "exon 2" in mouse TYR pre-mRNA read from mouse genomic DNA [NCBI reference sequence: NC_000073] Provides structural characterization data by analytical method. The provision of " ASO 1M " is for assessing antisense activity in cells of mouse origin.

PNA derivatives that complementally target the 3 'splice site of " exon 2 " in mouse TYR pre-mRNA and structural characterization data by their mass spectrometry PNA example PNA sequence (N? C) Exact mass, m / z theor. ^a obs. ^b ASO 1M Fethoc-CA (5) AA (5) TG-A (5) TC (10 2) -TG (6) TG-NH ₂ 4289.95 4289.96 ^{a) the} theoretical exact mass, ^b) the exact mass observed

3') aauuguuuuucacag│AUCAUUUGUAGCAGA]의 30-머 RNA 서열을 마우스 TYR 유전자 [NCBI 수납 번호: NC_000073]로부터 판독하였다.[(5 ') < / RTI > over the junction of intron 1 and exon 2 in mouse TYR pre-

3 ') aauuguuuuucacagAUCAUUUGUAGCAGA] was read from the mouse TYR gene [NCBI Accession Number: NC_000073].

3') CAA-ATG-ATC-TGT-G"의 DNA 서열과 동등하다. "ASO 1M"은 마우스 TYR 프리-mRNA 중의 "인트론 1" 및 "엑손 2"의 접합부에 걸쳐 있는 [(5'

3') aauuguuuuu cacag │ A UCAUUUG UAGCAGA]의 30-머 RNA 서열내에 "진하게" "밑줄그어" 표시한 13-머 서열과 13-머 상보성 오버랩을 갖는다. &Quot; ASO < RTI ID = 0.0 > 1M "

3 ') CAA-ATG-ATC-TGT-G "." ASO 1M "

3 ') aauuguuuuu cacag A UCAUUUG UAGCAGA] has a 13-mer sequence and a 13-mer complementarity overlap marked "underlined" in the 30-mer RNA sequence.

Like "ASO 1" for human TYR pre-mRNA, "ASO 1M" has a 5-mer overlap with "intron 1" and an 8-mer overlap with "exon 2" in mouse TYR pre-mRNA. "ASO 1M" can act as a good substitute for the antisense activity of "ASO 1" in cells of human origin.

Figure 7a is the HPLC chromatogram obtained for the crude product of " ASO 1 ". The crude product was purified by C ₁₈ -RP preparative HPLC. 7B is an HPLC chromatogram of the purified product of " ASO 1 ". The purity of " ASO 1 " was significantly improved with the preparative HPLC purification. Figure 8 provides the ESI-TOF mass spectrum obtained for the purified product of " ASO 1 ". The provision of analytical data for " ASO 1 " is intended to illustrate how the PNA derivatives of formula I are purified and identified in the present invention and should not be construed as limiting the scope of the invention.

The binding affinity of the model PNA derivative to complementary DNA

In Tables 1 and 2, PNA derivatives were evaluated for their binding affinity for 10-mer DNAs that are targeting N-terminal or C-terminal complementarily. The binding affinity was assessed by the T _m value for the duplex of PNA and 10-mer complementary DNA. The duplex between the PNA derivative and the fully complementary DNA exhibits a T _m value that is too high to be reliably measured in an aqueous buffer because the buffer tends to boil during the T _m measurement.

The _Tm values were measured on a UV / Vis spectrometer as follows. A mixed solution of 4 μM PNA oligomer and 4 μM complementary 10-mer DNA in 4 ml aqueous buffer (pH 7.16, 10 mM sodium phosphate, 100 mM NaCl) in a 15 ml polypropylene falcon tube was maintained at 90 ° C. for 1 minute, Lt; / RTI > The solution was then transferred to a 3 ml quartz UV cuvette with airtight cap and a T _m measurement at 260 nm on a UV / visible spectrophotometer was performed as described in the prior art [PCT / KR2009 / 001256] or with slight modifications . 10-mer complementary DNA for T _m measurement was purchased from Bioneer ( www.bioneer.com , Daejeon, Korea) and used without further purification.

C) Fethoc- CA(5)G-ACA(5)-ATC(1O2)-T G(6)T-A(5)-NH₂]에 "진하게" "밑줄그어" 표시한 바와 같은 PNA 중의 N-말단 10-머를 타겟화하는 10-머 상보성 DNA와의 듀플렉스에 대해서 78.0 ℃의 T_m 값을 나타내었다. 반면에, "ASO 1"은 [(N

C) Fethoc-CA(5)G- ACA(5)-ATC(1O2)-TG(6)T-A(5) -NH₂]에 "진하게" "밑줄그어" 표시한 바와 같은 PNA 중의 C-말단 10-머를 타겟화하는 10-머 상보성 DNA와의 듀플렉스에 대해서 73.0 ℃의 T_m 값을 나타내었다.The observed T _m values of the PNA derivatives of formula I are very high for complementarity binding to 10-mer DNA and are provided in Table 3. For example, "ASO 1" is [(N

C) Fethoc- CA (5) G -ACA (5) -ATC (1O2) -T G (6) TA (5) -NH 2] "dark" to "underlined" N- terminus of the PNA as shown The T _m value at 78.0 ° C was shown for the duplex with 10-mer complementary DNA targeting 10-mer. On the other hand, " ASO 1 " is [(N

C) Fethoc-CA (5) G - ACA (5) -ATC (1O2) -TG (6) TA (5) -NH 2] "dark" to "underlined" C- terminus of PNA as a display 10 And a T _m value of 73.0 [deg.] C for a duplex with 10-mer complementary DNA targeting the target.

T _m values between PNA and 10-mer complementary DNA targeting the N-terminal or C-terminal of PNA PNA T _m value, ° C The 10-mer DNA for the N-terminus The 10-mer DNA to C-terminus ASO 1 78.0 73.0 ASO 1M 72.0 72.0

For biological activities of PNA derivatives of formula (I) Example

In the present invention, PNA derivatives were evaluated for in vitro TYR antisense activity in B16F10 mouse melanoma cells and human melanocytes. Biological embodiments are provided as an example to illustrate the biological profile of PNA derivatives of formula I and should not be construed as limiting the scope of the invention.

Example 1. Exon skipping induced by " ASO 1M "

3') aauuguuuuu cacag │ AUCAUUUG UAGCAGA]의 30-머 RNA 서열내에 "진하게" "밑줄그어" 표시한 13-머 서열과 13-머 상보성 오버랩을 갖는다. 반면에, "ASO 1M"은 인간 TYR 프리-mRNA 중의 "인트론 1" 및 "엑손 2"의 접합부에 걸쳐 있는 [(5'

3') ggguguuuug"u" acag ┃ AU "UG" U "C" UG UAGCCGA]의 30-머 RNA 서열내에 "진하게" "밑줄그어" 표시한 바와 같은 4개의 불일치와 함께 9-머 상보성을 갖는다. 상기 4개의 불일치는 따옴표(" ")로 표시되었다.&Quot; ASO 1M " as shown in Table 2 indicates that [(5 ') < / RTI > spanning the junction of " intron 1 "

3 ') aauuguuuuu cacag AUCAUUUG UAGCAGA has a 13-mer sequence and a 13-mer complementarity overlap, which are marked "underlined" in the 30-mer RNA sequence. On the other hand, " ASO 1M " spans the junction of " intron 1 " and " exon 2 " in human TYR pre-

3 ') ggguguuuug "u" acag ┃ AU "UG" U "C" UG UAGCCGA] with the four mismatches indicated as "underlined" in the 30-mer RNA sequence. The four discrepancies are indicated by quotation marks ("").

Although "ASO 1M" has four mismatches with the 3 'splice position of "exon 2" in the human TYR pre-mRNA, the "ASO 1M" of the mouse B16F10 melanoma cells induces skipping of the TYR "exon 2" Ability was evaluated. "ASO 1M", which is completely complementary to mouse TYR pre-mRNA, can serve as a good substitute for "ASO 1", which is completely complementary to human TYR pre-mRNA.

[Cell culture & ASO treatment]

B16F10 mouse melanoma cells (Cat. No. CRL-6475, ATCC) were cultured in DMEM (Dulbecco's Modified Eagles essential minimum medium) supplemented with 10% FBS, 1% streptomycin / penicillin, and 0.01 mg / . B16F10 cells grown in a 60 mm culture dish containing 5 ml DMEM were incubated with 0 (negative control), 1, 10, 100 or 1000 aM of "ASO 1M" for 5 hours.

[RNA Extraction & cDNA Synthesis by 1-Step PCR]

3') GTAAGTTTGGATTTGGGG; 및 엑손 4_역방향: (5'

3') AGAGC-GGTATGAAAGGAA]의 엑손-특이성 프라이머 세트에 대해 플래티늄(Platinum)(등록상표) Taq 폴리머라제(Cat. No. 10928-042, 인비트로젠(Invitrogen))를 갖는 슈퍼스크립트(Super Script)(등록상표) 원 스텝(One-Step) RT-PCR 키트를 사용하여, 25 ㎕ 역전사 반응에 사용하였다: 50 ℃ 30분 및 94 ℃ 2분에 이어서, 15 주기의 94 ℃ 30초, 52 ℃ 30초 및 72 ℃ 40초.Following 5 hours of incubation, total RNA was extracted according to the manufacturer's instructions using " Universal RNA Extraction Kit " (Cat. No. 9767, Takara). 200 ng of the RNA template was subjected to the following cycle conditions [exon 1 - forward direction: (5 '

3 ') GTAAGTTTGGATTTGGGG; And exon 4_ reverse direction: (5 '

(Super Script) with Platinum TM Taq Polymerase (Cat. No. 10928-042, Invitrogen) for a set of exon-specific primers of the 3 'AGAGC-GGTATGAAAGGAA. PCR was carried out using 25 占 퐇 of a reverse transcription reaction using one-step RT-PCR Kit: 50 占 폚 for 30 minutes and 94 占 for 2 minutes followed by 15 cycles of 94 占 폚 for 30 seconds, 52 占 폚 30 Sec and 72 ° C for 40 seconds.

[Nested PCR amplification]

3') GAGAACTAACTGGGGATGA; 및 엑손 4n_역방향: (5'

3') CGATAGGTGCATTGGCTT]의 프라이머 세트에 대해 20 ㎕ 네스티드 PCR 반응(Cat. No. K2612, 바이오니어)에서 추가로 증폭시켰다: 95 ℃ 5분에 이어서 30 주기의 95 ℃ 30초, 52 ℃ 30초 및 72 ℃ 40초.1 < / RTI > of the cDNA was amplified in accordance with the specified cycle conditions as follows:

3 ') GAGAACTAACTGGGGATGA; And exon 4n_ reverse direction: (5 '

3 ') CGATAGGTGCATTGGCTT] was further amplified in 20 μl nested PCR reaction (Cat. No. K2612, Bioni): 95 ° C for 5 minutes followed by 30 cycles of 95 ° C for 30 seconds, 52 ° C for 30 seconds And 72 캜 for 40 seconds.

[Identification of exon skipping product]

The PCR product was subjected to electrophoresis separation on 2% agarose gel. Bands of target size were collected and analyzed by Sanger sequencing.

Figure 9A provides electrophoresis data of the PCR products. Cells without the "AS0 1M" treatment produced another one for the two strong PCR bands, one for full-length TYR mRNA, and the splice variant TYR mRNA without exons 2 and 3. Therefore, it is considered that skipping of the exon 2-3 occurs spontaneously. However, cells treated with " ASO 1M " of 1 to 1,000 aM produced only splice variant TYR mRNA without exons 2 and 3. Thus, "ASO 1M" increases the tendency of exon 2-3 skipping in B16F10 melanoma cells. The exon-skipping PCR product was sequenced as being the skipping of exon 2-3 as shown in Figure 9b.

Example 2. qPCR for TYR mRNA in B16F10 mouse melanoma cells treated with " ASO 1M ".

&Quot; ASO 1M " was assessed by TYR nested qPCR for its ability to induce a change in mouse TYR mRNA levels in B16F10 cells as described below.

[Cell culture & ASO treatment]

B16F10 cells grown in 60 mm culture dishes containing 5 ml DMEM were treated with 0 (negative control), 1, 10, 100 or 1000 aM of "ASO 1M" (2 culture dishes per dose).

[RNA Extraction & cDNA Synthesis by 1-Step PCR]

3') GTAAGTTTGGATTTGGGG; 및 엑손 4_역방향: (5'

3') AGAGCGGTATGAAAGGAA]의 엑손-특이성 프라이머 세트에 대해 원 스텝 RT-PCR 키트(인비트로젠, 미국 소재)를 사용하여 25 ㎕ 역전사 반응에 사용하였다: 50 ℃ 30분 및 94 ℃ 2분에 이어서, 15 주기의 94 ℃ 30초, 52 ℃ 30초 및 72 ℃ 40초.Following incubation with "ASO 1M" for 5 hours, total RNA was extracted from the cells using the "Universal RNA Extraction Kit" (Cat. No. 9767, Takara) according to the manufacturer's instructions. 200 ng of the RNA template was subjected to the following cycle conditions [exon 1 - forward direction: (5 '

3 ') GTAAGTTTGGATTTGGGG; And exon 4_ reverse direction: (5 '

3 ') AGAGCGGTATGAAAGGAA] was used in a 25 μl reverse transcription reaction using a one step RT-PCR kit (Invitrogen, USA): 50 ° C. for 30 minutes and 94 ° C. for 2 minutes, 15 cycles of 94 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 40 seconds.

[Nested qPCR amplification]

The cDNA solution was diluted up to 100-fold, and 1 μl of each diluted PCR product was subjected to a Taqman probe set (Cat. No. Mm00495818_m1, Thermofix Scientific Thermo Fisher Scientific) performed real time PCR reactions: 95 ° C for 3 minutes followed by 30 cycles of 95 ° C for 10 seconds, and 60 ° C for 30 seconds.

[Statistical Analysis]

The nested qPCR experiment was repeated four times independently and the individual mRNA levels from each experiment were normalized to mRNA levels without " ASO 1M " treatment. The mRNA levels obtained from all four separate experiments were collected for statistical analysis by Student's t-test. Thus, the number of the RNA samples is 8 per concentration.

Figure 10a provides qPCR data collected here, wherein the full-length mRNA level was significantly reduced by about 40% in cells treated with " ASO 1M " of 1 to 1,000 aM.

Example 3. Inhibition of TYR protein expression in B16F10 melanoma cells by " ASO 1M ".

&Quot; ASO 1M " was evaluated for its ability to down-regulate TYR protein expression in B16F10 mouse melanoma cells as described below.

B16F10 cells grown in a 60 mm culture dish containing 5 ml DMEM were treated with " ASO 1M " of 0 zM (negative control), 10 zM, 100 zM, 1 aM or 10 aM. After 24 hours, cells were washed twice with cold PBS (phosphate buffered saline) and then incubated with 200 [mu] l 1X cell lysis buffer (Cat. No. No) supplemented with 1X protease inhibitor cocktail (Cat. No. P8340, Sigma) 9803, Cell Signaling Tech). 200 [mu] l of each lysate in a 1.5 ml e-tube was mixed with 100 [mu] l 5X sample buffer and boiled at 100 [deg.] C for 5 minutes. 20 μl of the lysate was subjected to electrophoretic separation on a 4 to 15% gradient TGX gel (Cat No. 456-1086, Bio-Rad) and protein transfer was performed on a 0.45 μm PVDF membrane. The membrane was probed with anti-TYR antibody (Cat. No. 9319, Cell Signaling Tech) and anti-beta-actin antibody (Cat. No. a3845, Sigma).

Figure 10b provides Western blot data for TYR protein expression in B16F10 cells. The band intensity of TYR expression was significantly weaker in the "ASO 1M" treated lysates than in the negative ones, ie, negative control. The TYR protein and mRNA levels were comparatively reduced by the ASO treatment (see " Example 2 ").

Example 4. Inhibition of melanin formation in B16F10 melanoma cells by " ASO 1M "

&Quot; ASO 1M " was evaluated for its ability to inhibit melanin formation in B16F10 mouse melanoma cells as described below.

Transfected B16F10 cells in a 60 mm culture dish containing 5 ml DMEM were treated with untreated (negative control), 1 to 1,000 aM of "ASO 1M", or 10 or 100 μg / ml of arbutin for positive control (2 culture dishes per dose). After 24 hours, cells were washed twice with cold PBS and lysed with 200 [mu] l 1N NaOH. Each lysate was collected in a 1.5 ml e-tube and kept at room temperature overnight. The melanin content in each lysate was measured by absorbance at 475 nm on an ELISA reader. The same experiment was repeated 4 times using cells in different passages. Four melanin content data sets were collected for statistical analysis by Student's t-test for untreated melanin content (negative control).

Figure 11 summarizes the change in melanin content in B16F10 cells following incubation with " ASO 1M " or arbutin for 24 hours. The melanin content significantly decreased by about 15% and 25% in cells treated with 10 μg / ml and 100 μg / ml arbutin, respectively. In the case of cells treated with " ASO < RTI ID = 0.0 > 1M, < / RTI > the melanin content significantly decreased by about 15% The inhibitory activity of " ASO 1M " was comparable to that of 10 μg / ml arbutin.

Example 5. Preparation of topical cream containing the compound of formula (I)

A compound of formula I, for example " ASO2 " is formulated as a cream for topical application to the subject. The topical cream was a formulation as described below. Considering that a number of different topical creams may be present, the formulations should be considered as an example and should not be construed as limiting the scope of the invention.

[Preparation of Solution A]

(196 g), disodium EDTA (0.06 g), glycerin (15 g), dipropylene glycol (30 g), phenoxyethanol (0.9 g), ethylhexyl glycerin (0.9 g), hydroxy Ethyl acrylate / sodium acryloyldimethyltaurate copolymer (2.4 g), and 1 nM of "ASO 2" aqueous solution (0.3 ml) were mixed. The mixture was homogenized at 70-75 [deg.] C using a homomixer.

[Preparation of solution B]

In another beaker, cetearyl ethylhexanoate (12 g), dimethicone (6 g), stearic acid / hydrogenated vegetable oil / behenyl alcohol / cetearyl alcohol (15 g) at 70-75 ° C, (9 g) of Butyrose Pseudomonas Fischer (shea) butter and polyglyceryl-3-methylglucose distearate / glyceryl stearate SE / methyl glucose sesquistearate (6 g) were mixed.

[Production of topical cream]

&Quot; Solution A " and " Solution B " were mixed and homogenized using a homomixer at 7,200 rpm and 70-75 ° C for 3 minutes. The mixture was slowly cooled to room temperature to produce a topical cream containing 1 pM " ASO 2 ".

Example 6 qPCR for TYR mRNA in human melanocytes treated with " ASO 1 "

3') ggguguuuug uacag ┃ AUUGUCUG UAGCCGA]의 30-머 인간 TYR 프리-mRNA 서열에 "진하게" "밑줄 그어" 표시한 바와 같은 인간 TYR 중의 인트론 1 및 엑손 2의 접합부에 걸쳐 있는 3' 스플라이스 위치에 완전히 상보성인 13-머 TYR ASO이다."ASO 1" specified in Table 1 is [(5 '

The 3 ') ggguguuuug uacag ┃ AUUGUCUG UAGCCGA ] 30- Murray human TYR free -mRNA "dark" in sequence "underlined" show a person 3 that spans the junction of intron 1 and exon 2 of TYR as described in the' splice location It is a fully complementary 13-mer TYR ASO.

"ASO 1" was assessed by TYR nested qPCR for its ability to induce changes in TYR mRNA levels in human primary epithelial melanocytes as follows.

[Cell culture & ASO treatment]

Primary epithelial melanocytes (Cat. No. PCS-200-013, ATCC) were cultured in a skin cell basal medium supplemented with Adult Melanocyte Growth Kit Component (Cat. No. PCS-200-042, ATCC) (Dermal Cell Basal Medium) (Cat Number PCS-200-030, ATCC). Melanocytes proliferated in a 60 mm culture dish containing 5 ml culture medium were treated with " ASO 1 " of 0 zM (negative control), 1 zM, 100 zM, or 10 aM (3 culture dishes per concentration) .

[RNA Extraction & cDNA Synthesis by 1-Step PCR]

3') CTCTTTGTCTGGATGCATT; 및 엑손 5_역방향: (5'

3') CTGTGGTAATCCTCTTTCT]의 엑손-특이성 프라이머 세트에 대해 플래티늄(등록상표) Taq 폴리머라제(Cat. No. 10928-042, 인비트로젠)를 갖는 슈퍼 스크립트(등록상표) 원 스텝 RT-PCR 키트를 사용하여 25 ㎕ 역전사 반응을 수행하였다: 50 ℃ 30분 및 94 ℃ 2분에 이어서, 15 주기의 94 ℃ 30초, 50 ℃ 30초 및 72 ℃ 1분.Following incubation with "ASO 1" for 5 hours, total RNA was extracted according to the manufacturer's instructions using "RNeasy Mini Kit" (Cat. No. 74106, Qiagen). 200 ng of the RNA template was subjected to the following conditions [cycle 1 (forward) (2): (5 '

3 ') CTCTTTGTCTGGATGCATT; And exon 5_ reverse direction: (5 '

Step RT-PCR kit with Platinum (TM) Taq Polymerase (Cat. No. 10928-042, Invitrogen) for the exon-specific primer set of 3 'CTGTGGTAATCCTCTTTCT 25 [mu] l of a reverse transcription reaction were carried out: 50 [deg.] C for 30 min and 94 [deg.] C for 2 min followed by 15 cycles of 94 [deg.] C for 30 sec, 50 [deg.] C for 30 sec and 72 [

[Nested qPCR amplification]

3') 5,6-FAM-CACTGG-ZEN- AAGGATTTGCTAGTCCAC-3IABkFQ]에 대한 [엑손 2n_순방향: (5'

3') GATAAAGCTGCCAATTTC; 및 엑손 3n_역방향: (5'

3') TTGTGCATGCTGCTTTGA]의 엑손-특이성 프라이머 세트에 대해 20 ㎕ 네스티드 PCR 반응(Cat. No. K2612, 바이오니어)에서 추가로 증폭시켰다. 주기 조건: 95 ℃ 3분에 이어서 40 주기의 95 ℃ 10초, 및 60 ℃ 30초.1 < / RTI > of the cDNA was amplified using a Taqman probe [(5 '

3 ') 5,6-FAM-CACTGG-ZEN- AAGGATTTGCTAGTCCAC-3IABkFQ]

3 ') GATAAAGCTGCCAATTTC; And exon 3n_ reverse direction: (5 '

3 ') TTGTGCATGCTGCTTTGA] was further amplified in a 20 [mu] l nested PCR reaction (Cat. No. K2612, Bioniard) for the exon-specific primer set. Cycle conditions: 95 ° C for 3 minutes followed by 40 cycles of 95 ° C for 10 seconds, and 60 ° C for 30 seconds.

Figure 12 provides the qPCR data, wherein the full length TYR mRMA level was reduced by about 30% in human melanocytes treated with " ASO 1 " from 1 zM to 10 aM. The observed decrease was significant at cells treated with 1 zM and 10 aM of "ASO 1"(Student's t-test).

                         SEQUENCE LISTING <110> Jung, Daram        Park, HyeMi        Han, Seon-Young        Kim, Soyoung   <120> Tyrosinase antisense oligonucleotides <130> 2018op101pct & 2018dp103 <150> KR10-2017-0093605 <151> 2017-07-24 <150> KR10-2017-0167558 <151> 2017-12-07 <160> 38 <170> PatentIn version 3.2 <210> 1 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 1 cngacnatnt ntn 13 <210> 2 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (3) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 2 AnngNCantc tnnc 14 <210> 3 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 3 cngacnatnt ntn 13 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 4 cngacnatnt ntn 13 <210> 5 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 5 cngacnatnt ntn 13 <210> 6 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Mono-alkylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 6 cngacnatnt ntn 13 <210> 7 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Mono-alkylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 7 cngacnatnt ntn 13 <210> 8 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 8 cngacnatnt ntn 13 <210> 9 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Sulfonylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 9 cngacnatnt ntn 13 <210> 10 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 10 cngacnatnt ntn 13 <210> 11 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 11 cngacnatnt ntn 13 <210> 12 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 12 cngacnatnt ntn 13 <210> 13 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 13 cngacnatnt ntn 13 <210> 14 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 14 cngacnatnt ntn 13 <210> 15 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Mono-alkylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 15 cngacnatnt ntn 13 <210> 16 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Mono-arylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 16 cngacnatnt ntn 13 <210> 17 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Ureated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 17 cngacnatnt ntn 13 <210> 18 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 18 cngacnatnt ntn 13 <210> 19 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Acetylated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 19 cngacnatnt ntn 13 <210> 20 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature <222> (7) (7) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <400> 20 cnganantnt nta 13 <210> 21 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature &Lt; 222 > (3) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 21 ctnacnatnt ntn 13 <210> 22 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 22 cngatnatnt ntn 13 <210> 23 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 23 tngacnatnt ntn 13 <210> 24 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature &Lt; 222 > (3) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature <222> (7) (7) <223> modified base <220> <221> misc_feature &Lt; 222 > (10) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (14) <223> modified base <400> 24 tcngannatn tntn 14 <210> 25 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 25 cngacnatnt ntnc 14 <210> 26 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature &Lt; 222 > (10) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <400> 26 anacnatntn tnc 13 <210> 27 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature &Lt; 222 > (10) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (14) <223> modified base <400> 27 anacnatntn tncna 15 <210> 28 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) (17) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (14) <223> modified base <220> <221> misc_feature &Lt; 222 > (15) <223> modified base <220> <221> misc_feature &Lt; 222 > (17) <223> modified base <400> 28 anagncantc tntnnan 17 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) (17) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (15) <223> modified base <220> <221> misc_feature &Lt; 222 > (16) <223> modified base <400> 29 antgnctntc tntanna 17 <210> 30 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (14) <223> modified base <400> 30 gncantctnt nnan 14 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) (19) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (14) <223> modified base <220> <221> misc_feature &Lt; 222 > (16) <223> modified base <220> <221> misc_feature <222> (18). (18) <223> modified base <400> 31 anagncantc tntncnana 19 <210> 32 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (6) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature &Lt; 222 > (10) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <400> 32 antctntncn master 13 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) (19) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature (12). (12) <223> modified base <220> <221> misc_feature &Lt; 222 > (14) <223> modified base <220> <221> misc_feature &Lt; 222 > (16) <223> modified base <220> <221> misc_feature <222> (18). (18) <223> modified base <400> 33 anagncantc tntncnana 19 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) (18) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature &Lt; 222 > (5) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature &Lt; 222 > (15) <223> modified base <220> <221> misc_feature &Lt; 222 > (17) <223> modified base <400> 34 gntanagnca atctntnc 18 <210> 35 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (4) (4) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 35 gntncagnca atntg 15 <210> 36 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (4) (4) <223> modified base <220> <221> misc_feature <222> (7) (7) <223> modified base <220> <221> misc_feature &Lt; 222 > (10) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 36 gntncanacn atntg 15 <210> 37 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> human tyrosinase targetted artificial sequence <220> <221> misc_feature &Lt; 222 > (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (4) (4) <223> modified base <220> <221> misc_feature &Lt; 222 > (8) <223> modified base <220> <221> misc_feature &Lt; 222 > (10) <223> modified base <220> <221> misc_feature &Lt; 222 > (13) <223> modified base <400> 37 gntncagncn atntg 15 <210> 38 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> mouse tyrosinase targetted artificial sequence <220> <221> misc_feature <222> (1) <223> PNA modified oligonucleotide <220> <221> misc_feature <222> (1) <223> Carbamated N-terminal <220> <221> misc_feature <222> (2) (2) <223> modified base <220> <221> misc_feature <222> (4) (4) <223> modified base <220> <221> misc_feature <222> (7) (7) <223> modified base <220> <221> misc_feature &Lt; 222 > (9) <223> modified base <220> <221> misc_feature &Lt; 222 > (11) <223> modified base <400> 38 cnantgntnt ntg 13

Claims (12)

A peptide nucleic acid derivative represented by formula (I): &lt; EMI ID =
Formula I

In this formula,
n is an integer between 10 and 21;
The compound of formula (I) can be used in combination with [(5 '

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-
Wherein the entire compound of Formula I is fully complementary to the human tyrosinase pre-mRNA or is partially complementary to the human tyrosinase pre-mRNA with one or two incompatibilities of the entire compound;
S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n Are independently selected from the group consisting of deuterido, hydrido, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl radicals;
X and Y are independently dew Terry also, hydrido [H], formyl [HC (= O) -] , aminocarbonyl _{[NH 2 -C (= O)} -], amino thiocarbonyl [NH ₂ -C (= S) -], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl acyl, substituted or unsubstituted aryl acyl, substituted or unsubstituted alkyl Substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, Substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, substituted or unsubstituted alkyloxytithiocarbonyl, substituted or unsubstituted aryloxytithiocarbonyl, substituted or unsubstituted alkylsulfonyl, Substituted arylsulfonyl, substituted or unsubstituted aryl Or a substituted or unsubstituted arylphosphonyl radical;
Z is selected from the group consisting of hydrido, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amino, substituted or unsubstituted alkyl, or substituted or unsubstituted An aryl radical;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among non-natural nucleobases having a substituted or unsubstituted amino radical covalently bonded to the nucleobase moiety. 2. The peptide nucleic acid derivative of claim 1, wherein the peptide nucleic acid derivative or pharmaceutically acceptable salt thereof is selected from the group consisting of:
n is an integer between 10 and 21;
Compounds of formula (I) can be used in combination with [(5 ', &lt; RTI ID = 0.0 &gt;

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-
Wherein the entire compound of Formula I is fully complementary to the human tyrosinase pre-mRNA or is partially complementary to said human tyrosinase pre-mRNA with one or two incompatibilities of the entire compound;
S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T &_lt; _{n &gt; -1} , and T &_lt; _n &gt; independently represent a diterido or hydrido radical;
X and Y are independently selected from the group consisting of deuterido, hydrido, formyl, aminocarbonyl, aminothiocarbonyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl , Substituted or unsubstituted aryl acyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted aryl Substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, Substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, substituted or unsubstituted alkylphosphonyl radicals, or substituted Or or unsubstituted aryl phosphonic sulfonyl radical;
Z represents hydrido, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, or substituted or unsubstituted amino radicals;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by the following formula (II), (III) or (IV)
[Formula II]

[Formula (III)

[Formula IV]

In the above equations,
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from hydrido, and substituted or unsubstituted alkyl radicals;
L &_lt; _{1 &gt;} , L &_lt; _{2 &gt;} , and L &lt; ₃ &gt; are covalent linkers represented by the following formula V that covalently attach a basic amino group to the nucleobase portion:
Formula V

In this formula,
Q ₁ and Q _m are substituted or unsubstituted methylene (-CH ₂ -) radicals, and Q _m is directly bonded to said basic amino group;
Q ₂ , Q ₃ ,

, Q _{m -1} is selected from the group consisting of substituted or unsubstituted methylene, oxygen (-O-), sulfur (-S-), and substituted or unsubstituted amino radicals [-N (H) -, ) -]; &lt; / RTI &gt;
m is an integer between 1 and 15; 3. The peptide nucleic acid derivative according to claim 2, or a pharmaceutically acceptable salt thereof, having the following conditions:
n is an integer between 11 and 18;
Compounds of formula (I) can be used in combination with [(5 ', &lt; RTI ID = 0.0 &gt;

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-
The entire compound of formula I is completely complementary to human tyrosinase pre-mRNA;
S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;
X and Y are independently selected from hydrido, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl acyl, substituted or unsubstituted aryl acyl, substituted or unsubstituted Alkyloxycarbonyl, or a substituted or unsubstituted aryloxycarbonyl radical;
Z represents a substituted or unsubstituted amino radical;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from hydrido, and substituted or unsubstituted alkyl radicals;
Q ₁ and Q _m are substituted or unsubstituted methylene radicals, Q _m is directly bonded to a basic amino group;
Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from substituted or unsubstituted methylene, oxygen, and amino radicals;
m is an integer between 1 and 11; 4. The peptide nucleic acid derivative or a pharmaceutically acceptable salt thereof according to claim 3,
n is an integer between 11 and 16;
Compounds of formula (I) can be used in combination with [(5 ', &lt; RTI ID = 0.0 &gt;

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-
The entire compound of formula I is completely complementary to human tyrosinase pre-mRNA;
S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;
X and Y are independently selected from hydrido, substituted or unsubstituted alkyl acyl, and substituted or unsubstituted alkyloxycarbonyl radicals;
Z represents a substituted or unsubstituted amino radical;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently selected from hydrido, and substituted or unsubstituted alkyl radicals;
Q ₁ and Q _m are methylene radicals and Q _m is directly bonded to a basic amino group;
Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from methylene, oxygen, and amino radicals;
m is an integer between 1 and 9; 5. The peptide nucleic acid derivative or a pharmaceutically acceptable salt thereof according to claim 4,
n is an integer between 11 and 16;
Compounds of formula (I) can be used in combination with [(5 ', &lt; RTI ID = 0.0 &gt;

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-
The entire compound of formula I is completely complementary to human tyrosinase pre-mRNA;
S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;
X and Y are independently selected from hydrido, substituted or unsubstituted alkyl acyl, and substituted or unsubstituted alkyloxycarbonyl radicals;
Z represents a substituted or unsubstituted amino radical;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);
R ₁ , R ₃ , and R ₅ are hydrido radicals, R ₂ , R ₄ , and R ₆ are independently hydrido, or substituted or unsubstituted alkyl radicals;
Q ₁ and Q _m are methylene radicals and Q _m is directly bonded to a basic amino group;
Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from methylene, oxygen radicals;
m is an integer between 1 and 9; 6. The peptide nucleic acid derivative or the pharmaceutically acceptable salt thereof according to claim 5,
n is an integer between 11 and 16;
Compounds of formula (I) can be used in combination with [(5 ', &lt; RTI ID = 0.0 &gt;

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-
The entire compound of formula I is completely complementary to human tyrosinase pre-mRNA;
S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;
X and Y are independently selected from hydrido, substituted or unsubstituted alkyl acyl, and substituted or unsubstituted alkyloxycarbonyl radicals;
Z represents a substituted or unsubstituted amino radical;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from adenine, thymine, guanine, cytosine, and non-natural nucleobases;
B ₁ , B ₂ ,

, B _{n -1} , and B _n is independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrido radicals;
Q ₁ and Q _m are methylene radicals and Q _m is directly bonded to a basic amino group;
Q ₂ , Q ₃ ,

, Q _{m -1} is independently selected from methylene, and an oxygen radical;
m is an integer between 1 and 9; 7. The peptide nucleic acid derivative of claim 6, or a pharmaceutically acceptable salt thereof,
n is an integer between 11 and 16;
Compounds of formula (I) can be used in combination with [(5 ', &lt; RTI ID = 0.0 &gt;

3 ') UGUACAGAUUGUCU] having at least a 10-mer complementarity overlap with the 14-mer-
The entire compound of formula I is completely complementary to human tyrosinase pre-mRNA;
S ₁ , S ₂ ,

, S _{n -1} , S _n , T ₁ , T ₂ ,

, T _{n -1} , and T _n are hydrido radicals;
X is a hydrido radical;
Y represents a substituted or unsubstituted alkyl acyl, and a substituted or unsubstituted alkyloxycarbonyl radical;
Z represents a substituted or unsubstituted amino radical;
B ₁ , B ₂ ,

, B _{n -1} , and B _n are independently selected from adenine, thymine, guanine, cytosine, and non-natural nucleobases;
B ₁ , B ₂ ,

, B _{n -1} , and B _n is independently selected from among the non-natural nucleobases represented by formula (II), (III) or (IV);
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrido radicals;
L ₁ is _{- (CH 2) 2 -O- (} CH 2) 2 -, -CH 2 -O- (CH 2) 2 -, -CH 2 -O- (CH 2) 3 -, -CH 2 -O - (CH ₂ ) ₄ -, or -CH ₂ -O- (CH ₂ ) ₅ -, wherein the right end is directly bonded to a basic amino group;
L ₂ And L ₃ is _{- (CH 2) 2 -O- (} CH 2) 2 -, - (CH 2) 3 -O- (CH 2) 2 -, - (CH 2) 2 -O- (CH 2) 3 _{-, - (CH 2) 2} -, - (CH 2) 3 -, - (CH 2) 4 -, - (CH 2) 5 -, - (CH 2) 6 -, - (CH 2) 7 -, And - (CH ₂ ) ₈ -, wherein the right end is directly bonded to the basic amino group. The method according to claim 1,
A peptide nucleic acid derivative selected from the group of peptide nucleic acid derivatives provided below or a pharmaceutically acceptable salt thereof:
(N

C) Fethoc-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;
(N

C) Fethoc-AC (1/2) A (5) -GA 5 C-AA 5 T-CTG 6 -C (1/2) C-NH ₂ ;
(N

C) Ac-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) Benzoyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;
(N

C) Piv-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) Methyl-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) n-Propyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;
(N

C) Fmoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) p-Toluenesulfonyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;
(N

C) H-Lys-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;
(N

C) Fethoc-Lys-Gly-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;
(N

C) Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -Lys-NH ₂ ;
(N

C) Fethoc-Val-Gly- CA (5) G-ACA (5) -ATC (1O2) -TG (6) TA (5) -NH 2;
(N

C) Ac-Arg-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) Benzyl-CA (5) G-ACA (5) -ATC (10) -TG (6) TA (5) -NH ₂ ;
(N

C) Phenyl-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) [N- (2-Phenylethyl ) amino] carbonyl-CA (5) G-ACA (5) -ATC (1O2) -TG (6) TA (5) -NH 2;
(N

C) Benzoyl-Leu-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) Piv-Lys-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -Val-NH ₂ ;
(N

C) Fethoc-CA (7) G-AC (2 O 2) AA (4) TC (10 2) -TG (6) TA-NH ₂ ;
(N

C) Fethoc-CTG (6) -ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) Fethoc-CA (5) G-ATA (5) -ATC (10 2) -TG (5) TA (5) -NH ₂ ;
(N

C) Fethoc-TA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) -NH ₂ ;
(N

C) Fethoc-TCA (3) -GAC (10) -A (5) AT-C (10 2) TG (5) -TA (5) -NH ₂ ;
(N

C) Fethoc-CA (5) G-ACA (5) -ATC (10 2) -TG (6) TA (5) C-NH ₂ ;
(N

C) Fethoc-AG (6) A-CA (5) A-TC (10 2) TG (6) TA (5) -C-NH ₂ ;
(N

C) Fethoc-AG (6) A-CA (5) A-TC (10) TG (6) TA (5) -CA (20 2) A-NH ₂ ;
(N

C) Fethoc-AC (1O2) A-GA (6) C-AA (6) T-CTG (6) -TA (6) C (1O2) -AA (6) -NH 2;
(N

C) Fethoc-AC (1O3) T-GA (6) C-TA (6) T-CTG (6) -TAC (1O5) -A (4) A-NH 2;
(N

C) Fethoc-GA (6) C-AA (6) T-CTG (6) -TA (6) C (10) -AA (6) -NH ₂ ;
(N

C) Fethoc-AC (10) A-GA (6) C-AA (6) T-CTG (6) -TA (6) CA (5) AA (20 2) -A-NH ₂ ;
(N

C) Fethoc-AA (6) T-CTG (6) -TA (6) CA (5) AA (20 2) -A-NH ₂ ;
(N

C) Fethoc-AC (10) A-GA (6) C-AA (6) T-CTG (6) -TA (6) CA (5) AA (20 3) -A-NH ₂ ;
(N

C) Fethoc-GC (10) T-AC (10) A-GA (4) C-AAT-CTG (6) -TA (6) C-NH ₂ ;
(N

C) Fethoc-GC (1O2) TA (3) CA-GA (4) C-AAT-C (1O2) TG-NH 2;
(N

C) Fethoc-GC (10) TA (3) CA-G (20 2) AC-A (5) AT-C (10 ₂ ) TG-NH ₂ ; And
(N

C) Fethoc-GC (1O2) TA (3) CA-GA (4) CA (2O2) AT-C (1O2) TG-NH 2:
In the above,
A, G, T, and C are PNA monomers with natural nucleobases of adenine, guanine, thymine and cytosine, respectively;
Wherein the non-natural nucleobases represented by the following formulas (VI), (VII), (VII), (VIII), (IX) and (X) &Lt; / RTI &gt; are:
(VI)

[Formula VII]

(VIII)

[Formula IX]

(X)

In the above equations,
p and q are integers;
The abbreviations for N- and C-terminal substituents are as specifically set forth as follows: "Fmoc-" is an abbreviation for "[(9-fluorenyl) methyloxy] carbonyl] -";"Fethoc-" means "[2- (9-fluorenyl) ethyl-1-oxy] carbonyl";&Quot; Ac- " is " acetyl- &quot;;&Quot; Benzoyl- " refers to " benzenecarbonyl- &quot;;&Quot; Piv- " is " pivial reel- &quot;;&Quot; Methyl- " is " methyl- &quot;;"p-Toluenesulfonyl" refers to "(4-methylbenzene) -1-sulfonyl" - "n-propyl- ; "-Lys-" is an amino acid residue "lysine";"-Val-" is an amino acid residue "valine";"Leu-" is an amino acid residue "leucine" -Gly- "is an amino acid residue" glycine "," [N- (2-Phenylethyl) amino] carbonyl- ""1 - (phenyl) -";"Phenyl-" is "phenyl -";"Me-" is "methyl -";"-NH_2" is an unsubstituted - an abbreviation for groups "amino". A composition for the treatment of a disease or disorder accompanied by the expression of a human tyrosinase gene, comprising a peptide nucleic acid derivative according to claim 1 or a pharmaceutically acceptable salt thereof. A composition for treatment of hyperpigmentation comprising a peptide nucleic acid derivative according to claim 1 or a pharmaceutically acceptable salt thereof. A method for treating a disease or disorder accompanied by the expression of a human tyrosinase gene by administering the peptide nucleic acid derivative according to claim 1. A method for treating hyperpigmentation by administering a peptide nucleic acid derivative according to claim 1.

Images