KR101934049B1 - Flavin-Dependent Halogenase and Method for Preparing Halogenated Indole Compounds Using the Same - Google Patents

Abstract

The present invention relates to the preparation of halogenated indole compounds using a recombinant flavin-dependent halo or azide (MrFDH) derived from a microorganism of the genus Micromonospora rhodorangea , and more particularly to the production of antagonists against melatonin receptors ) ≪ / RTI > activity of the novel halogenated indole compounds. Since the enzymatically biotransformed halogenated indole derivative compounds using the recombinant flavin-dependent halogenase (MrFDH) according to the present invention exhibit improved binding to the melatonin receptor and antitumor activity against the uveal melanoma, Insomnia-related diseases, or uveal melanoma.

Description

Flavin-Dependent Halogenase and Method for Preparing Halogenated Indole Compounds Using the Same}

The present invention relates to the production of a halogenated indole compound using a recombinant flavin-dependent halogenase (MrFDH) derived from Micromonospora rhodorangea actinomycetes, and in more detail, antagonist against melatonin receptors. ) It relates to the enzymatic preparation of a novel halogenated indole compound having activity.

Halogenation can add novel or improved biological activity and pharmaceutical efficiency to existing compounds, as well as provide clues for further organic chemical substitutions in the future. Halogenated organic compounds are currently widely used in the chemical and agricultural industries and pharmaceutical industries, and are particularly important building blocks in carbon-coupling chemical reactions through metal catalysts (Frese M et al., Angew) Chem Int Ed Engl . 54(1):298-301, 2015). Thus, although halogenated compounds are widely used in organic synthesis, synthesis of complex halogenated compounds is still not easy.

Traditional organic chemical halogenation is a hazardous process that is generally not environmentally friendly and requires vigorous reaction conditions while using highly toxic reagents and solvents. Furthermore, in the case of the product through this reaction, one or more isomers and unwanted by-products are simultaneously produced, requiring an additional purification process (Alonso F et al., Chem Rev. 102(11):4009-4091, 2002). Therefore, enzymatic catalysts, that is, biological halogenation, which can be an alternative to organic chemical synthesis methods, can be more environmentally friendly because their reaction conditions are similar to biological reactions, and the enzyme has high regio-specificity compared to organic synthesis methods. Hyperstereo-specificity can minimize the generation of by-products after the reaction.

Enzymes that catalyze halogenation can be broadly classified into two groups (Wagner C et al., J Nat Prod. 72(3):540-553, 2009). First of all, haloperoxidase is the first halogenating enzyme discovered in the 1960s and uses hydrogen peroxide as a coenzyme. On the other hand, a flavin-dependent halogenase (flavin-dependent halogenase, hereinafter, which requires a flavin _{adenine dinucleotide (FADH 2} ) in a reduced form with a coenzyme while catalyzing a site-specific halogenation reaction) FDH). In particular, tryptophan halogenase, which adds a halogen element using tryptophan, a kind of amino acid as a substrate, was the first reported in the FDH group and is known to exist in various microorganisms (Payne JT et al., Methods Enzymol . 575: 93-126, 2016). However, FDH requires a halogenated ion, an oxygen molecule, and FADH ₂ as a coenzyme for its activity, and generally exhibits site-specific activity, so it is limited to be used for biosynthesis of various halogenated compounds. Accordingly, there is a need for FDH with substrate flexibility that extends the specificity of the substrate by discovering a novel FDH capable of halogenating a wide range of substrates or by protein engineering an existing FDH.

Meanwhile, since tryptophan contains a hetero-aromatic indole scaffold, a compound having an indole skeleton can be halogenated as a substrate for the FDH enzyme. Melatonin (N-acetyl-5-methoxy tryptamine or melatonin) is a tryptophan-derived neurohormone that contains an indole skeleton and is a chronobiotic that is involved in the regulation of circadian rhythm in the human body. It is known to be associated with diseases such as sleep dosorder, neurodegenerative disease, cancer and stroke (Hardeland R et al., Int J Biochem Cell Biol . 38(3):313 -316, 2006). In addition, melatonin and its metabolites 6-hydroxymelatonin and 6-chloromelatonin, in addition to pharmacological activities related to sleep and insomnia, are metastatic uveal melanoma, a kind of ocular cancer. melanoma) cell lines have been reported to inhibit their growth in a dose-dependent manner (Hu DN et al ., Melanoma Res . 8:205-210, 1998). However, melatonin is limited in its use as a therapeutic agent due to its extremely short in vivo half-life, low oral bioavailability, and various unknown mechanisms, and is marketed as a food supplement only in some countries, such as the United States and Canada (Altun A et al. ., Int J Clin Pract. 61(5):835-845, 2007).

Therefore, the discovery of a novel FDH or a novel FDH having flexibility in reaction to various substrates not only allows the development of a novel library of halogenated compounds with a new structure by adding a specific halogen element to a compound containing an indole skeleton, but also prevents sleep disorders and The structural diversity of melatonin derivatives used as therapeutic agents such as insomnia and anti-tumor active compounds for metastatic eye cancer can be presented and new physiological activities can be additionally provided.

Accordingly, the present inventors produced a novel halogenated structure of melatonin derivatives and made diligent efforts to improve the antagonistic activity of the derivatives against melatonin receptors. As a result, the antagonistic activity against melatonin receptors was excellent using a recombinant FDH enzyme, and at the same time, uveal melanoma. It was confirmed that a novel halogenated indole compound, a melatonin derivative, can be prepared with antitumor activity targeting cell lines, and the present invention was completed.

An object of the present invention is to provide a flavin-dependent halogenase enzyme (MrFDH) represented by the amino acid sequence of SEQ ID NO: 6 and a method for preparing the recombinant flavin-dependent halogenase enzyme (MrFDH).

Another object of the present invention is to provide a method for preparing a halogenated indole derivative using the flavin-dependent halogenase enzyme (MrFDH) and a halogenated indole derivative compound prepared by the method.

Another object of the present invention is to provide a composition for treating sleep disorders or insomnia containing a halogenated indole derivative compound prepared by the flavin-dependent halogenase enzyme (MrFDH).

Another object of the present invention is to provide a composition for treating rare eye cancer, such as uveal melanoma, containing a halogenated indole derivative compound prepared by the flavin-dependent halogenase enzyme (MrFDH).

In order to achieve the above object, the present invention provides a flavin-dependent halogenase enzyme (MrFDH) represented by the amino acid sequence of SEQ ID NO: 6.

The present invention also provides a polynucleotide encoding the enzyme.

The present invention also provides an expression vector comprising the polynucleotide.

The present invention also provides a recombinant microorganism into which the polynucleotide or expression vector has been introduced.

The present invention also provides a method for producing a recombinant flavin-dependent halogenase enzyme (MrFDH) comprising the step of inducing the expression of a flavin-dependent halogenase enzyme by culturing the recombinant microorganism, and then recovering it. do.

The present invention also provides a halogenated indole derivative compound represented by the following Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof.

[Formula 1]

[Formula 2]

In the above formula, R ₁ is hydroxy, methyl or methoxy, and R ₂ and R ₃ are chloro or bromo.

The present invention also biosynthesizes a halogenated indole derivative represented by Formula 1 or Formula 2 by reacting an indole compound represented by Formula 3 or Formula 4 with a halogen ion donor in the presence of the flavin-dependent halogenase enzyme (MrFDH). Letting go; And recovering the synthesized halogenated indole derivative.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

In the above formula, R ₁ is hydroxy, methyl or methoxy, and R ₂ and R ₃ are chloro or bromo.

The present invention also provides a pharmaceutical composition for improving or treating sleep disorders or insomnia containing the compound represented by Formula 1 or Formula 2 as an active ingredient.

The present invention also provides a food for improving or preventing sleep disorders or insomnia containing the compound represented by Formula 1 or Formula 2 as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of ophthalmic cancer containing the compound represented by Formula 1 or Formula 2 as an active ingredient.

The present invention also provides a food for the improvement or prevention of and cancer containing the compound represented by the formula (1) or (2) as an active ingredient.

The halogenated indole derivative compound enzymatically bioconverted using a recombinant flavin-dependent halogenase (MrFDH) derived from Micromonospora rhodorangea actinomycetes according to the present invention has improved binding power to melatonin receptors and Since it exhibits anti-tumor activity against uveal melanoma, it is very useful as a pharmaceutical composition for the treatment of sleep disorders and insomnia-related diseases or rare eye cancers such as uveal melanoma.

1 shows the bioconversion of an indole compound to a halogenated melatonin derivative by a recombinant MrFDH flavin-dependent halogenase enzyme reaction.
Figure 2 shows the binding strength of the halogenated melatonin derivative, which is a bioconverted halogenated indole derivative, to the melatonin receptor hMT1.
3 is a view confirming the binding ability of the halogenated melatonin derivative, which is a bioconverted halogenated indole derivative, to the melatonin receptor hMT2.
Figure 4 shows the anti-tumor activity of a bioconverted halogenated indole derivative, a halogenated melatonin derivative, against a human-derived uveal melanoma cell line.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present invention, flavin-dependent halogenase (FDH) coding candidate open reading frames were isolated from the genome from the genome of the micromonospora rhodorangia strain, a kind of soil actinomycetes, and screened FDH A recombinant expression vector containing the gene and a transformed E. coli were prepared, and then, a recombinant flavin-dependent halogenase enzyme MrFDH was prepared. Thereafter, the recombinant flavin-dependent halogenase enzyme MrFDH and 5-demethoxy-5-methyl-melatonin (5MMEL), 5-demethoxy-5-hydroxy-melatonin ( 5-demethoxy-5-hydroxy-melatonin; 5-demethoxy-5, a novel halogenated indole derivative, by reacting three halogenated substrates of 5-demethoxy-5-hydroxy-melatonin; 5HMEL) and agomelatine (AGMEL) and sodium chloride or sodium bromide for halogen donating ions. -Methyl-7-chloro-melatonine (5-demethoxy-5-methyl-7-chloro-melatonine; 5M7CMEL), 5-demethoxy-5-methyl-7-bromo-melatonin (5-demethoxy-5-methyl- 7-bromo-melatonine; 5M7BMEL), 5-demethoxy-5-hydroxy-7-chloro-melatonine (5-demethoxy-5-hydroxy-7-chloro-melatonine; 5H7CMEL), 5-demethoxy-5-hydroxy Roxy-7-bromo-melatonine (5-demethoxy-5-hydroxy-7-bromo-melatonine; 5H7BMEL), 5-chloro-agomelatine (5CAGMEL) and 5-bromo-agomel Latin (5-bromo-agomelatine; 5BAGMEL) was biosynthesized. Afterwards, the structure was confirmed through mass spectrometry and nuclear magnetic resornance spectrometry analysis, while novel halogenated melatonin derivatives, which are indole derivatives added with halogen elements such as chlorine and bromine, in the structure of the indole compound It was confirmed that improved antagonistic activity against melatonin receptors.

The present invention confirms the halogenation catalytic function by performing an in vitro reaction using a recombinant FDH enzyme capable of adding a halogen element in the structural skeleton of an indole compound, and furthermore, an enzyme other than a chemical organic synthesis method using a novel FDH A novel halogenated melatonin derivative was prepared through a biotransformation method and the antagonistic activity of the derivatives to the melatonin receptor was compared.

Accordingly, the present invention relates to a flavin-dependent halogenase enzyme (MrFDH) represented by the amino acid sequence of SEQ ID NO: 6 in a consistent view.

In another aspect, the present invention relates to a polynucleotide encoding the enzyme.

In the present invention, the polynucleotide may be characterized in that it is represented by the nucleotide sequence of SEQ ID NO: 5, Micromonospora rhodorangia (Micromonospora rhodorangea ) may be characterized in that the strain is derived.

In another aspect, the present invention relates to an expression vector comprising the polynucleotide.

In another aspect, the present invention relates to a recombinant microorganism into which the polynucleotide or the expression vector has been introduced.

In the present invention, the microorganism is preferably E. coli, but is not limited thereto.

In another aspect, the present invention is to produce a recombinant flavin-dependent halogenase enzyme (MrFDH) comprising the step of inducing the expression of a flavin-dependent halogenase enzyme by culturing the recombinant microorganism, and then recovering it. It's about the method.

As used herein, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are currently the most commonly used form of vectors, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. For the purposes of the present invention, it is preferred to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a replication starting point that enables efficient replication to contain several to hundreds of plasmid vectors per host cell, and (b) host cells transformed with plasmid vectors are selected. It has a structure that includes an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method.

After ligation, the vector must be transformed into an appropriate host cell. Transformation can be easily achieved using the calcium chloride method or electroporation (Neumann, et al., EMBO J., 1:841, 1982) or the like. As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used. The skeleton vector that can be used in the method of the present invention is not particularly limited thereto, but various vectors capable of transforming E. coli selected from the group consisting of pT7, pET/Rb, pGEX, pET28a, pET-22b(+) and pGEX are used. Can be used.

A base sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. This may be a gene and regulatory sequence(s) linked in a manner that allows gene expression when an appropriate molecule (eg, a transcriptional activating protein) is bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide, and a promoter or enhancer is a sequence If it affects the transcription of the coding sequence, or if the ribosome binding site is operably linked to the coding sequence if it affects the transcription of the sequence, or if the ribosome binding site is arranged to facilitate translation coding Operably linked to the sequence. In general, "operably linked" means that the DNA sequence to which it is linked is in contact, and in the case of a secretory leader, is contacted and is in the reading frame. However, the enhancer does not need to be in contact. The ligation of these sequences is carried out by ligation (linkage) at a convenient restriction enzyme site. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.

As is well known in the art, in order to increase the level of expression of a transgene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that exert a function in the selected expression host. Preferably, the expression control sequence and the corresponding gene are included in a single recombinant vector including a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector must further contain an expression marker useful in the eukaryotic expression host.

Host cells transformed with the above-described recombinant vector constitute another aspect of the present invention. As used herein, the term "transformation" means that DNA is introduced into a host so that the DNA becomes replicable either as an extrachromosomal factor or by chromosomal integrity.

Of course, it should be understood that not all vectors function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, those skilled in the art can make an appropriate selection among various vectors, expression control sequences, and hosts without departing from the scope of the present invention without undue experimental burden. For example, when choosing a vector, you must consider the host, because the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered.

It will be apparent to those skilled in the art to which the invention pertains that even when the gene is inserted into the genomic chromosome of the host cell, it will have the same effect as when the recombinant vector is introduced into the host cell as described above.

In the present invention, as a method of inserting the gene onto the chromosome of a host cell, a conventionally known gene manipulation method can be used, for example, a retroviral vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector , A method of using a poxvirus vector, a lentiviral vector, or a non-viral vector.

In another aspect, the present invention relates to a halogenated indole derivative compound represented by the following Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof.

[Formula 1]

[Formula 2]

In the above formula, R ₁ is hydroxy, methyl or methoxy, and R ₂ and R ₃ are chloro or bromo.

In the present invention, the halogenated indole derivative is 5-demethoxy-5-methyl-7-chloro-melatonine (5-demethoxy-5-methyl-7-chloro-melatonine), 5-demethoxy-5-methyl-7 -Bromo-melatonine (5-demethoxy-5-methyl-7-bromo-melatonine), 5-demethoxy-5-hydroxy-7-chloro-melatonine (5-demethoxy-5-hydroxy-7-chloro-melatonine) ), 5-demethoxy-5-hydroxy-7-bromo-melatonine, 5-chloro-agomelatine or 5 -Bromo-agomelatine (5-bromo-agomelatine) is preferred, but is not limited thereto.

In the present invention, the compound of Formula 1 or 2 may be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Inorganic acids and organic acids can be used as free acids, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. can be used as inorganic acids, and citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfuric acid can be used as organic acids. Ponic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galucturonic acid, embonic acid, glutamic acid, aspartic acid, and the like can be used. Preferably, sulfuric acid can be used.

In addition, the compound of Formula 1 or 2 according to the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

In addition, the compound of Formula 1 or 2 according to the present invention may be prepared in a crystalline or amorphous form, and when the compound is prepared in a crystalline form, it may be optionally hydrated or solvated.

In another aspect of the present invention, (a) in the presence of the flavin-dependent halogenase enzyme (MrFDH) of claim 1, by reacting an indole compound represented by the following Formula 3 or Formula 4 with a halogen ion donor, Formula 1 or Formula 2 Biosynthesizing a halogenated indole derivative represented by; And (b) recovering the synthesized halogenated indole derivative.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

In the above formula, R ₁ is hydroxy, methyl or methoxy, and R ₂ and R ₃ are chloro or bromo.

In the present invention, the step (b) is a reverse phase C18 fixed phase, acetonitrile:water:formic acid 75:25:0.1 (v/v/v/v) mixed liquid mobile phase, and medium pressure liquid chromatography (MPLC). ) Is preferably detected under a 15-21 minute retention time condition, but is not limited thereto.

In the present invention, the indole compound is preferably 5MMEL (5-demethoxy-5-methyl-melatonin), 5HMEL (5-demethoxy-5-hydroxy-melatonin) or AGMEL (agomelatine), and the halogen ion donor is sodium chloride Or sodium bromide is preferred, but is not limited thereto.

In the present invention, the flavin-dependent halogenase enzyme (MrFDH) of step (a) is an isolated and purified flavin-dependent halogenase enzyme, that is, a flavin-dependent halo represented by the amino acid sequence of SEQ ID NO: 6 It may be any one selected from the group consisting of a genase enzyme, a recombinant strain expressing the enzyme, a culture of the recombinant strain, and a lysate of the recombinant strain, and the lysate is a lysate or a lysate of the recombinant strain. It refers to a supernatant obtained by centrifugation, and contains an enzyme protein produced from the recombinant strain. In the present specification, unless otherwise noted, the recombinant strain used for the production of flavin-dependent halogenase enzymes is at least one selected from the group consisting of the cells of the strain, the culture of the strain, and the lysate of the strain. It is used to mean.

Halogenation is one of the important manufacturing processes in the pharmaceutical and agrochemical industries, and about 25% of them contain halogen atoms added in the molecule (Herrera-Rodriguez LN et al., Chem . Today 29:31, 2011). In addition, halogenation of a specific compound may have a great influence on the biological activity of the compound, and this may include antibiotics, anticancer agents, and psychoactive drugs (Williams PG et al., J Org Chem . 70(16):6196-6203, 2005; Smith BM et al., J Med Chem . 51(2):305-313, 2008; Bunders CA et al., J Am Chem Soc. 133(50):20160-20163, 2011).

In addition, more than 5000 halogenated compounds have been continuously reported in nature until 2010 (Smith DR et al., Curr Opin Chem Biol . 17(2):276-283, 2013), and the continuous discovery of such halogenated natural products expands interest in the enzyme involved in halogenation inherent in biosynthesis, and most of the halogenated natural products are chlorine or bromine among halogen elements. And, in some cases, natural products containing iodine or fluorine have been identified.

On the other hand, since indole is widely distributed in nature, it is known to play an important role in mechanisms such as pathogenicity and antibiotic resistance of bacteria, as it is used as a signaling substance in several species of bacteria (Lee JH et al., Roles of indole as an interspecies and interkingdom signaling molecule.Trends Microbiol. 23(11):707-718, 2015). On the other hand, in plants and animals, it acts as oxidative stress, intestinal inflammation, and hormones. Some plant-derived indole derivatives such as Indole-3-carbinol and 3,3-diindolylmethane exhibit anticancer activity against various types of cancer cells (Biersack B et al., Indole compounds against breast cancer: recent developments. Curr Drug Targets. 13 (14):1705-1719, 2012), in particular, serotonin and melatonin, which can be called tryptophan analogs as human hormones, also contain indole structures as major skeletons.

Melatonin is known to function as a biological rhythm regulator by acting on two types of melatonin receptors MT1 and MT2, a type of G-protein coupled receptor existing in the human body. Melatonin receptor ligands and agonists have been developed (Spadoni G et al., Melatonin receptor agonists: new options for insomnia and depression treatment. CNS Neurosci Ther. 17(6):733-741, 2011). They bind to the melatonin receptor and activate the melatonin receptor, some of which are currently being used in clinical practice. For example, ramelteon (brand name Rozerem), chemically synthesized as a melatonin structure derivative, was developed by Takeda Pharmaceutical company (European Patent EP 0885210 A1). Compared to melatonin, this melatonin derivative provided stronger binding affinity to the two melatonin receptors, and through clinical trials, it lowered sleep latency and increased total sleep time, thereby treating insomnia in 2005. (Wurtman R. Ramelteon: a novel treatment for the treatment of insomnia. Expert Rev Neurother . 6(7):957-964, 2006). In addition, agomelatine (brand name Valdoxan or Melitor) and tasimelteon (brand name Hetlioz) are also being used in clinical trials (US 5194614 A; WO 1998025606 A1). It is being used as a 24-hour non-sleep disorder treatment. Recently, melatonin receptor antagonists TIK-301 and piromelatine are also currently undergoing clinical trials as therapeutic drugs and candidate new drugs for rare diseases (Rivara S et al., Therapeutic uses of melatonin and melatonin derivatives: a patent review (2012-2014). Expert Opin Ther Pat. 25(4):425-441, 2015; Zisapel N. Current Phase II investigational therapies for insomnia.Expert Opin Investig Drugs . 24(3):401-411, 2015).

In another aspect, the present invention relates to a pharmaceutical composition for improving or treating sleep disorders or insomnia containing a halogenated indole derivative compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient. .

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating ophthalmic cancer containing a halogenated indole derivative compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the oram is preferably an uveal melanoma, but is not limited thereto.

The term "improvement" as used in the present invention means preventing, ameliorating, treating or delaying the onset of symptoms, and "prevention" is a compound represented by Formula 1 or 2, or a pharmaceutically acceptable thereof It refers to any action that suppresses or delays cancer disease by administering a pharmaceutical composition containing a possible salt. In addition, the term "treatment" used in the present invention refers to diseases such as sleep disorders and insomnia and uveal melanoma by administration of a pharmaceutical composition containing a compound represented by Formula 1 or 2, or a pharmaceutically acceptable salt thereof. This refers to any action in which the symptoms of rare and cancer are improved or cured.

The compound of Formula 1 or 2 of the present invention, or a pharmaceutically acceptable salt thereof, exhibits some improved avidity to human-derived melatonin receptors, and thus can be usefully used in the prevention or treatment of diseases such as sleep disorders and insomnia.

The compound of Formula 1 or 2 of the present invention, or a pharmaceutically acceptable salt thereof, exhibits in vitro anti-tumor activity against human uveal melanoma cell lines, and is useful for the prevention or treatment of rare eye cancers such as uveal melanoma. Can be used.

In addition, the present invention provides a pharmaceutical composition comprising the compound represented by Formula 1 or 2, or a pharmaceutically acceptable salt and carrier thereof.

The pharmaceutical composition of the present invention can be formulated in an oral or parenteral dosage form according to standard pharmaceutical practice. These formulations may contain additives such as a pharmaceutically acceptable carrier, adjuvant, or diluent in addition to the active ingredient.

When the composition of the present invention is used as a pharmaceutical, a pharmaceutical composition containing at least one or more active ingredients of a compound represented by Formula 1 or 2, or a pharmaceutically acceptable salt thereof, may be administered orally or It may be formulated and administered in a parenteral dosage form, but is not limited thereto.

Formulations for oral administration may be, for example, tablets, pills, hard capsules, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations can be formulated as a diluent (e.g., lacquer) in addition to the active ingredient. Tods, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, magnesium salt of stearic acid, calcium salt of stearic acid and/or polyethylene glycol). have. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, optionally with starch, agar, alginic acid or sodium salt thereof. It may contain the same disintegrant or boiling mixture and/or absorbent, colorant, flavoring, and sweetening agent.

Formulations for parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. At this time, in order to formulate a formulation for parenteral administration, a compound represented by Formula 1 or 2, or a pharmaceutically acceptable salt thereof, is included, and is mixed in water with a stabilizer or buffer to prepare a solution or suspension. And, it can be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and/or contain preservatives, stabilizers, hydrating agents or emulsifying accelerators, adjuvants such as salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances, and mixing, granulating, which are conventional methods Or it can be formulated according to the coating method.

In addition, the dosage of the compound of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and when based on an adult patient weighing 70 kg, generally It is 0.001 to 1,000 mg/day, preferably 0.01 to 500 mg/day, and may be dividedly administered once a day or several times a day at regular time intervals according to the judgment of a doctor or pharmacist.

In another aspect, the present invention relates to a food for improving or preventing sleep disorders or insomnia, containing a halogenated indole derivative compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention relates to a food for the improvement or prevention of and cancer containing a halogenated indole derivative compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the oram is preferably an uveal melanoma, but is not limited thereto.

The food of the present invention may be added as it is to the halogenated indole derivative compound or used with other foods or food ingredients, and may be appropriately used according to a conventional method.

There is no particular limitation on the type of the food. Examples of such foods include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamins. There is a combination drug, etc., and includes all foods in the usual sense.

The halogenated indole derivative compound according to the present invention may be added to food as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, the amount of the halogenated indole derivative compound in the health food may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g based on 100ml. I can. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be below the above range, and there is no problem in terms of safety, so the active ingredient may be used in an amount above the above range.

The health functional beverage composition of the present invention is not particularly limited to other ingredients other than containing the halogenated indole derivative compound as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates, etc. as additional ingredients, as in ordinary beverages. I can. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained.

In addition, the halogenated indole derivative compound of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. Although the proportion of these additives is not so important, it is generally selected from 0 to about 20 parts by weight based on 100 parts by weight of the halogenated indole derivative compound of the present invention.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1: Isolation of FDH-encoding gene from micromonospora rhodorangia genome

A set of degenerate primers that considers the codon usage of the micromonospora genome after selecting an amino acid sequence conservation site with high homology through comparison and analysis of the previously reported amino acid sequences of FDHs (SEQ ID NO: 1/2) was prepared.

SEQ ID NO: 1: 5-GGSGTSGGSGARGCSAC-3 (FDH_F)

SEQ ID NO: 2: 5-GGGATCTYCCASGTCCASCC-3 (FDH_R)

In the fosmid library (Hoang NH et al., J. Microbiol . Biotechnol . 26(3):477-482, 2016) of the genome of micromonospora rhodorangia strain, a type of soil actinomycetes that has been previously reported. The isolated genome is used as a template, and a polymerase chain reaction (PCR) using a primer set of SEQ ID NO: 1 (forward primer, FDH_F) and SEQ ID NO: 2 (reverse primer, FDH_R) specific to the prepared conserved site. ) Was carried out. The fosmid library DNA and primers were mixed with Taq DNA polymerase to perform a polymerase chain reaction, and positive fosmid libraries showing the amplified PCR products were first selected, and then the insertion sequence in these fosmids Was subjected to full-length sequence analysis.

As a result, it was possible to select an open reading frame predicted to encode only one type of FDH in the fosmid genome. Polymerase chain reaction again using a primer set of SEQ ID NO: 3 (forward primer, MRFDH_F) and SEQ ID NO: 4 (reverse primer, MRFDH_R) specific to the open reading frame using a fosmid library containing an open reading frame as a template Was carried out as follows.

SEQ ID NO: 3: 5' -GG CATATG GACCTTCAAGAACGTCGCCA-3'(MRFDH_F, Nde I restriction site)

SEQ ID NO: 4: 5' -CG CTCGAG CGGGGTGGTGATCAGGTAGA-3'(MRFDH_R, Xho I restriction site)

After the PCR product was purified, it was conjugated to pGEMR-T easy vector (pGEMR-T easy vector, Promega, Madison, WI, USA), and then 42 in E. coli XL1 blue (XL1 blue, Stratagene, La Jolla, CA, USA). After heat treatment at °C for 45 seconds, transformation was performed. After separating and purifying the T-easy vector from the selected transformed E. coli, the base sequence (SEQ ID NO: 5) and the amino acid sequence inferred therefrom (SEQ ID NO: 6) were determined.

As a result, the nucleotide sequence (SEQ ID NO: 5) of the entire open reading frame was 1467 bp, and the protein after translation was composed of a total of 488 amino acids (SEQ ID NO: 6) (total 53.2 kDa). As a result of blast (Basic Local Aligment Search Tool, NCBI) analysis of the amino acid sequence of the protein, NADB-Rossman superfamily and geranylgeranyl reductase, which are conserved domains of tryptophan halogenase, FDH. ) It contained all of the superfamily. On the other hand, it showed the highest homology of about 86% with the tryptophan halogenase coding estimated amino acid sequence (GenBank accession number WP_007074591) derived from micromonospora species ATCC 39149 in the NCBI database, and at least 6 kinds of Streptomyces and micromono Spora-derived halogenase or dehydrogenase (dehydrogenase) encoding putative amino acid sequences (GenBank accession numbers SCF43367, WP_040892142, WP_062150269, WP_037886771, ELS56116 and EME97269) showed more than 60% homology.

Example 2: Expression of the recombinant flavin-dependent halogenase enzyme MrFDH in E. coli

The T-easy vector of Example 1 was treated with Nde I and Xho I restriction enzymes, and the MrFDH DNA fragment was treated with the same restriction enzymes, the protein expression vector pET-28(a+) (Novagen, Madison, WI, USA) After insertion into, E. coli BL21 (DE3) (Stratagene, La Jolla, CA, USA) was transformed. At this time, 50 ppm of kanamycin antibiotic was used for selection of transformants.

The recombinant E. coli was inoculated at a 1% volume ratio in LB medium (Luria Bertani) to which kanamycin antibiotics and a final concentration of 1 M sorbitol and 2.5 mM betaine were added, and then incubated at 30°C, and then optically When growth is confirmed at an optical density between 0.6 and 0.8, in order to induce protein expression, a final concentration of 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO, USA) was added. After culturing the recombinant E. coli strain at 22℃ for 20 hours, the culture solution was centrifuged at 2000 rpm for 10 minutes to recover the cells, and then 50 mM sodium phosphate lysis buffer solution (300 nM NaCl, 10 mM imidazole, 10% glycerol). , 1% Triton X-100) was dissolved in the cells, and sonication was performed. Thereafter, refrigerated centrifugation at 12000 rpm for 15 minutes was performed, and the supernatant was collected separately, and some samples were analyzed by 12% SDS-PAGE to confirm the expression level of the recombinant prenyltransferase enzyme MrPT.

To purify the recombinant protein whose expression has been confirmed, the supernatant was equilibrated with 50 mM phosphate buffer (0.3 M NaCl, 20 mM imidazole), and Talon-metal affinity resin (Clontech, Mountain View, CA) , USA) and incubated for two hours at 4°C. After refrigerating centrifugation at 2000 rmp for 5 minutes, the resin was introduced into a disposable column, and washed with a phosphate buffer solution containing 10 times the volume of 50 mM imidazole. Finally, the recombinant flavin-dependent halogenase enzyme MrFDH bound to the resin was purified with 3 ml of a phosphate buffer containing 180 mM imidazole.

Example 3: Recombinant MrFDH flavin-dependent halogenase enzyme bioconversion to halogenated derivatives of indole compounds

3-1: Halogenase activity of the recombinant halogenase enzyme MrFDH

Before bioconversion of the halogenated indole derivative, the halogenase activity of the recombinant halogenase enzyme MrFDH, which was long-limited in Example 2, was assayed. L-tryptophan (Sigma, St. Louis, MO, USA) and 1-aminoanthracene (Sigma), previously reported to have been used as substrates for tryptophan halogenase, were used as substrates with a final concentration of 500 μM, for the production of coenzyme FADH ₂ commercially available recombinant flavin reductase kinase (flavin reductase; EC1.5.1.29; Novocib, Lyon, France; # E-Nov8; 10 units) , and catalase (catalase; Sigma; # C9322; 30 units) at a concentration of 1 mM flavin adenine dinucleotide (FAD; Sigma) was added. After adding the above mixtures and purified recombinant MrPT in a reaction buffer solution (25 mM phosphate buffer; pH 7.4; 20 mM sodium chloride), an enzymatic reaction was performed at 30° C. for 10 hours to investigate whether a halogenation reaction was performed. Thereafter, the reaction product was heat-treated in boiling water for 5 minutes and centrifuged for 5 minutes to recover the supernatant. The production of halogenated L-tryptophan and 1-aminoanthracene to which chlorine was added as a reaction product was confirmed by HPLC-ESI-MS analysis.

3-2: Bioconversion to novel halogenated indole derivatives by recombinant MrFDH halogenase enzyme

For bioconversion of novel halogenated indole derivatives, 5-demethoxy-5-methyl-melatonin (hereinafter 5MMEL) as halogenated substrates; Aurora Fine Chemicals LLC, San Diego, CA, USA ; #K00.821.433), 5-demethoxy-5-hydroxy-melatonin (5-demethoxy-5-hydroxy-melatonin; hereinafter 5HMEL; Sigma; #A1824) and agomelatine (Sigma; hereinafter AGMEL; #A1362) Three types were used, and an enzymatic reaction was performed at 30° C. for 10 hours with a sodium chloride (Sigma) or sodium bromide (Sigma) solution for a halogen donating ion to a final level of 20 mM (FIG. 1). HPLC-ESI-MS analysis was performed to confirm the biosynthesis of the target halogenated indole derivatives in the reaction product. Acetonitrile:water:formic acid 75:25:0.1 (v/v/v/v) as a mobile phase at a rate of 120 μl per minute, and as a stationary bed column, Acquity CSH C18 (Waters, 50x1.0 mm, 1.7 μm; Milford, MA, USA).

Next, the CombiFlash Rf MPLC system was applied to separate and purify the desired halogenated indole derivatives from the crude extract. Using an MPLC system flowing a mixture of acetonitrile:water:formic acid 75:25:0.1 (v/v/v/v) into a reverse-phased C18 cartridge at a rate of 15 ml per minute as a mobile phase, the same mobile phase 3 After the crude extract dissolved in ml was injected, the detected peak on the chromatogram was automatically fractionated. After concentrating the individual fractions under reduced pressure, the desired halogenated indole derivatives were separated by analyzing a mass spectrum with an ion trap mass spectrometer (LCQ ion-trap mass spectrometer, ThermoFinnigan, San Jose, CA, USA).

As a result, 5MMEL (5-demethoxy-5-methyl-melatonin), 5HMEL (5-demethoxy-5-hydroxy-melatonin), and AGMEL (agomelatine) used as substrates were maintained with a retention time of about 26 to 33 minutes on the MPLC system. The fractions were collected, and the desired halogenated indole derivatives were detected with a retention time of 15 to 21 minutes depending on their higher hydrophilicity than the substrate. These purified halogenated indole derivatives are shown in FIG. 1. The yield of the bioconversion reaction using sodium chloride as a halogen ion donor was approximately 40% or more, but in the case of sodium bromide, the bioconversion yield was very low at a level of 25% or less. In addition, in the case of halogen ion donors such as sodium fluoride (NaF) or sodium iodide (NaI), the bioconversion reaction product was not detected by instrumental analysis. This suggested the relative specificity of the recombinant MrFDH enzyme for the halogen ion donor.

3-3: NMR of a novel halogenated indole derivative

NMR samples were prepared by dissolving each of the indole derivatives in 200 μl of DMSO-d6, and then leaving the solvent in a 5 mm Shigemi advanced NMR microtube (Sigma, St. Louis, MO). ¹³ C NMR spectra were acquired at 298 K using a Varian INOVA 500 spectrophotometer, and chemical shifts were recorded in ppm using TMS as an internal reference. All NMR data calculations were performed using Mnova Suite 5.3.2 software, and ¹³ C-NMR spectra of halogenated indole derivatives were compared with aromatic compounds used as substrates. The addition position of the halogen in the separated and purified halogenated indole derivative was shown at the 7th or 5th position, respectively, and as a result of HPLC-ESI-MS analysis, in the case of the indole derivatives estimated to be double halogenated, separation and purification Did not carry out.

5-demethoxy-5-methyl-7-chloro-melatonine; 5M7CMEL; bioconversion rate 49%; ¹³ C NMR [125 MHz, DMSO-d6] δ 21.1, 23.2, 25.5, 41.3, 112.8, 116.2, 119.2, 120.8, 123.1, 128.7, 130.1, 134.9, 170.3)

5-demethoxy-5-methyl-7-bromo-melatonine; 5M7BMEL; bioconversion rate 25%; ¹³ C NMR [125 MHz, DMSO-d6] δ 21.0 , 23.2, 25.5, 41.4, 99.7, 112.9, 117.0, 123.0, 123.8, 129.6, 131.3, 132.5, 170.4)

5-demethoxy-5-hydroxy-7-chloro-melatonine; 5H7CMEL; bioconversion 46%; ¹³ C NMR [125 MHz, DMSO-d6] δ 23.2 , 25.4, 41.4, 101.5, 107.2, 113.3, 120.9, 123.1, 130.1, 130.6, 153.6, 170.3)

5-demethoxy-5-hydroxy-7-bromo-melatonine; 5H7BMEL; bioconversion 23%; ¹³ C NMR [125 MHz, DMSO-d6] δ 23.2, 25.5, 41.4, 101.4, 102.8, 106,6, 113.4, 123.1, 128.2, 130.9, 154.4, 170.3)

5-chloro-agomelatine (5CAGMEL; bioconversion rate 41%; ¹³ C NMR [125 MHz, DMSO-d6] δ 23.2, 33.3, 40.6, 55.6, 109.6, 118.2, 122.5, 124.2, 125.3 , 126.2, 133.6, 134.2, 134.9, 157.3, 170.5)

5-bromo-agomelatine; 5BAGMEL; Bioconversion rate 20%; ¹³ C NMR [125 MHz, DMSO-d6] δ 23.2, 33.3, 40.6, 55.8, 110.1, 121.5, 124.1, 124.9, 125.2, 125.9, 127.2, 134.0, 134.5, 157.3, 170.4)

Example 4: Adhesion of halogenated indole derivatives to melatonin receptors

In order to compare and assay the antagonistic activity of 6 types of halogenated indole derivatives against 2 types of melatonin receptors among the compounds of Example 3-2, human-derived melatonin receptors hMT1 and hMT2 overexpressing CHO cells (Chinese hamster ovarian cells) Two commercial kits containing membranes (Chemiscreen Melatonin Receptor Membrane Preparation; Eurofins Scientific, Petaluma, CA, USA; #HTS065M and #HTS106M) were used. As a control group, three kinds of indole compounds (5MMEL, 5HMEL, AGMEL) before the bioconversion of halogenated were used.

First, the 6 kinds of halogenated indole derivatives dissolved in DMSO, the 3 kinds of indole compounds before halogenation, and the positive control melatonin were mixed in a cell membrane homogenous suspension in the kit and 0.5 nM (in the case of hMT1) or 0.25 nM (in the case of hMT2). ) Of 2-[ ¹²⁵ I]iodomelatonin and incubated at 25° C. for 120 minutes. Thereafter, the radioactivity intensity was measured through a TopCount (Perkin-Elmer corp., Boston, MA, USA) microplate reader, and the IC ₅₀ (50% inhibitory concentration) was calculated by nonlinear regression analysis. Meanwhile, the dissociation constant (hereinafter Ki) of the samples for the melatonin receptors was calculated according to Equation 1 below. Here, L is the use concentration of 2- ^[125 I] iodomelatonin and Kd refers to 2- ^[125 I] affinity constant (affinity constant) of iodomelatonin. The number of repetitions of all experiments was carried out in at least 3 repetitions, and the Ki value was measured.

[Equation 1]

Ki = IC ₅₀ /(1+L/Kd)

As a result, as shown in Figs. 2 and 3, in the case of melatonin, which is a positive control, average dissociation constants of 0.28 and 0.25 nM were shown for hMT1 and hMT2, respectively, while two indole compounds 5MMEL and 5HMEL among the comparative controls Was in the range of 0.24 to 0.64 nM, whereas AGMEL in clinical use showed mean dissociation constants of 0.06 and 0.29 nM for hMT1 and hMT2, respectively. Halogenated indole derivatives generally exhibit higher average dissociation constants compared to the comparative groups, indicating that the antagonistic activity of these derivatives to the melatonin receptor decreased. In addition, compared to the chlorinated indole derivatives, the average dissociation constant of the brominated derivatives was remarkably increased, indicating a significantly lower antagonistic activity of the indole derivatives halogenated with a polymer.

However, among the halogenated indole compounds, 5M7CMEL targets the hMT1 receptor, shows no significant antagonistic activity compared to the control group 5MMEL, and exhibits improved antagonistic activity within 90% significance level when compared to the positive control melatonin. In addition, the halogenated AGMEL derivatives (5CAGMEL and 5BAGMEL) also show a higher dissociation constant value compared to the control group AGMEL, but show a lower average dissociation constant compared to the control melatonin. That is, as a result, most of the new melatonin structural analogs halogenated to hydrophilicity showed lower antagonistic activity against melatonin receptors than the compounds before biotransformation, but in the case of a limited number of three derivatives (i.e., 5M7CMEL, 5CAGMEL, and 5BAGMEL). Compared to N melatonin, human-derived melatonin receptors (especially hMT1) showed somewhat improved antagonistic activity.

Example 5: Antitumor activity of halogenated indole derivatives against uveal melanoma

In order to compare and assay the antitumor activity of the six halogenated indole derivatives of the compound of Example 3-2 against the metastatic uveal melanoma cell line, the tumor cell line MP41 (ATCC, CRL-3297) derived from uveal melanoma patients was targeted. Changes in the number of cells before and after treatment were measured. As a positive control, melatonin, which has been reported to have antitumor activity against the cells, was used.

The metastatic uveal melanoma cell lines were pre-cultured in RPMI-1640 medium (0.2% sodium bicarbonate, glutamine 4 mM, streptomycin 100 ppm, penicillin 10 ppm) containing 20% FBS, followed by centrifugation and a 24-well plate. (24-well plate, Corning, New York, USA) were individually dispensed into 3,000 per well and incubated at 37°C in 5% carbon dioxide atmosphere. One day after incubation, the six halogenated indole derivatives of the example compounds, the indole compounds before bioconversion, and the positive control were treated to be in three concentration ranges from 0.1 nM to 10 nM, and then additional culture was performed for 5 days, and required In case, the medium was changed every 3 days. After the cells were treated with Tiripsin-EDTA from the final culture solution, the number of dissociated cells was measured. The degree of anti-tumor activity was measured as a percentage percentage of the number of cells before treatment with each compound and the number of cells after treatment, and the results are shown in FIG. 4.

As shown in FIG. 4, in the case of the indole compound comparative groups before halogenation, the antitumor activity was up to about 36% compared to before treatment. On the other hand, the previously reported positive control melatonin showed a maximum of about 44% of antitumor activity in the assay concentration range, and in the case of a total of six halogenated indole derivatives of the example compounds, a maximum of about 62%. Presented. In contrast to the excellent antagonistic activity of the AGMEL compound and its halogenated derivatives, 5CAGMEL and 5BAGMEL, to the melatonin receptors shown in FIG. 2, antitumor activity against uveal melanoma cell lines did not appear at all. In contrast, the halogenated derivatives of 5MMEL, 5M7CMEL and 5M7BMEL, exhibit antitumor activities of up to 62% and 58%, respectively, at a concentration of 1 nM among the example compounds. In other words, the hydrophilic halogenated melatonin derivatives show improved antitumor activity related to uveal melanoma, a type of metastatic eye cancer, compared to the original material, melatonin structural analogs before biotransformation, and also markedly improved anti-tumor activity compared to the positive control melatonin. It provides tumor activity and shows the potential for its pharmacological application. On the other hand, in the case of AGMEL and its related halogenated derivatives, antitumor activity against uveal melanoma cell lines did not appear at all. do.

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

<110> Korea University Research and Business Foundation <120> Flavin-Dependent Halogenase and Method for Preparing Halogenated Indole Compounds Using the Same <130> P16-B291 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> FDH_F <400> 1 ggsgtsggsg argcsac 17 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FDH_R <400> 2 gggatctycc asgtccascc 20 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> MRFDH_F <400> 3 ggcatatgga ccttcaagaa cgtcgcca 28 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> MRFDH_R <400> 4 cgctcgagcg gggtggtgat caggtaga 28 <210> 5 <211> 1467 <212> DNA <213> Micromonospora rhodorangea <400> 5 atggtctcca ccgtcctggt catcggcggc ggcccggccg gcgccaccgc cgccgccctg 60 ctggcccgct ccggcctgtc cgtcaccctg ctggagaagg agaccttccc gcgctaccac 120 atcggcgagt ccgtcgcctc ctcctgccgc accatcgtcg acttcgtcgg cgccctggac 180 gaggtcgact cccgcggcta cccgcaggag aacggcgtcc tgctgcgctg gggcaacaag 240 gactggtcca tcgactgggc caagatcttc ggcccgggca tccgctcctg gcaggtcgac 300 cgcgacgact tcgaccacat cctgctgaac aactccggca agcagggcgc caagatcatc 360 cagggcgccg ccgtcgagcg cgtcctgttc gacggcgagc gcgccaccga ggccgagtgg 420 ttcgacccgg agtccggcga ggtccgcacc atcgacttcg actacatcat cgactcctcc 480 ggccgcgccg gcctgatccc gtcccagcac ttcaagcacc gccgcccgac caagaccttc 540 aagaacgtcg ccatctgggg ctactggcag ggcggctccc tgctgccgaa ctccccgtcc 600 ggcggcatca acgtcatcgc cgccccggac ggctggtact ggatcgtccc gctgcgcggc 660 gaccgctacg ccatcggctt cgtctgccac cagtcccgct tcctggagcg ccgcgaggag 720 cacgcctccc tggaggacat gctggcctcc ctggtccagg agtccccgac cgtccgcggc 780 ctgacctcca acggcaccta ccagccgggc gtccgcgtca agcaggactt cgcctacgtc 840 tccgactcct tctgcggccc gggctacttc gccgccggcg actccgcctg cttcctggac 900 ccgctgctgt ccaccggcgt ccacctggcc ctgtactccg gcatgctggc ctccgcctcc 960 atcctggcca ccatccacgg cgacgtcacc gaggaggagg cccgcgcctt ctacgagtcc 1020 ctgtaccgca acgcctacca gcgcctgttc tacctggtcg ccggcgtcta ccagcagcag 1080 gccggccgcc gcgcctactt cggcctgccg gaggccctgg tcggcgactc cggcgacacc 1140 gagtacaagg aggtcgacgg cgcccgcccg ttcgcccagc tggtctccca cctggccgac 1200 ctggacgagg ccgccgaggg ccgccacgac tccccggccg ccgccgccac cgcccgccag 1260 gagaactcca tccgccagct gttcctggcc ccgcgcgagg cccgcaagat ggccgacacc 1320 cgcccgggct ccgccggcat ctccgaggcc ccgggcgagc tggactccgg cgacctgttc 1380 gagtccgccc cgcacgtcta cctgatcacc accccgcgca tcggcgtccg ccgcccggag 1440 accgccgaca cccagtccgc ctcctga 1467 <210> 6 <211> 488 <212> PRT <213> Micromonospora rhodorangea <400> 6 Met Val Ser Thr Val Leu Val Ile Gly Gly Gly Pro Ala Gly Ala Thr 1 5 10 15 Ala Ala Ala Leu Leu Ala Arg Ser Gly Leu Ser Val Thr Leu Leu Glu 20 25 30 Lys Glu Thr Phe Pro Arg Tyr His Ile Gly Glu Ser Val Ala Ser Ser 35 40 45 Cys Arg Thr Ile Val Asp Phe Val Gly Ala Leu Asp Glu Val Asp Ser 50 55 60 Arg Gly Tyr Pro Gln Glu Asn Gly Val Leu Leu Arg Trp Gly Asn Lys 65 70 75 80 Asp Trp Ser Ile Asp Trp Ala Lys Ile Phe Gly Pro Gly Ile Arg Ser 85 90 95 Trp Gln Val Asp Arg Asp Asp Phe Asp His Ile Leu Leu Asn Asn Ser 100 105 110 Gly Lys Gln Gly Ala Lys Ile Ile Gln Gly Ala Ala Val Glu Arg Val 115 120 125 Leu Phe Asp Gly Glu Arg Ala Thr Glu Ala Glu Trp Phe Asp Pro Glu 130 135 140 Ser Gly Glu Val Arg Thr Ile Asp Phe Asp Tyr Ile Ile Asp Ser Ser 145 150 155 160 Gly Arg Ala Gly Leu Ile Pro Ser Gln His Phe Lys His Arg Arg Pro 165 170 175 Thr Lys Thr Phe Lys Asn Val Ala Ile Trp Gly Tyr Trp Gln Gly Gly 180 185 190 Ser Leu Leu Pro Asn Ser Pro Ser Gly Gly Ile Asn Val Ile Ala Ala 195 200 205 Pro Asp Gly Trp Tyr Trp Ile Val Pro Leu Arg Gly Asp Arg Tyr Ala 210 215 220 Ile Gly Phe Val Cys His Gln Ser Arg Phe Leu Glu Arg Arg Glu Glu 225 230 235 240 His Ala Ser Leu Glu Asp Met Leu Ala Ser Leu Val Gln Glu Ser Pro 245 250 255 Thr Val Arg Gly Leu Thr Ser Asn Gly Thr Tyr Gln Pro Gly Val Arg 260 265 270 Val Lys Gln Asp Phe Ala Tyr Val Ser Asp Ser Phe Cys Gly Pro Gly 275 280 285 Tyr Phe Ala Ala Gly Asp Ser Ala Cys Phe Leu Asp Pro Leu Leu Ser 290 295 300 Thr Gly Val His Leu Ala Leu Tyr Ser Gly Met Leu Ala Ser Ala Ser 305 310 315 320 Ile Leu Ala Thr Ile His Gly Asp Val Thr Glu Glu Glu Ala Arg Ala 325 330 335 Phe Tyr Glu Ser Leu Tyr Arg Asn Ala Tyr Gln Arg Leu Phe Tyr Leu 340 345 350 Val Ala Gly Val Tyr Gln Gln Gln Ala Gly Arg Arg Ala Tyr Phe Gly 355 360 365 Leu Pro Glu Ala Leu Val Gly Asp Ser Gly Asp Thr Glu Tyr Lys Glu 370 375 380 Val Asp Gly Ala Arg Pro Phe Ala Gln Leu Val Ser His Leu Ala Asp 385 390 395 400 Leu Asp Glu Ala Ala Glu Gly Arg His Asp Ser Pro Ala Ala Ala Ala 405 410 415 Thr Ala Arg Gln Glu Asn Ser Ile Arg Gln Leu Phe Leu Ala Pro Arg 420 425 430 Glu Ala Arg Lys Met Ala Asp Thr Arg Pro Gly Ser Ala Gly Ile Ser 435 440 445 Glu Ala Pro Gly Glu Leu Asp Ser Gly Asp Leu Phe Glu Ser Ala Pro 450 455 460 His Val Tyr Leu Ile Thr Thr Pro Arg Ile Gly Val Arg Arg Pro Glu 465 470 475 480 Thr Ala Asp Thr Gln Ser Ala Ser 485

Claims (7)

5-chloro-agomelatine, 5-bromo-agomelatine, or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for improving or treating sleep disorder or insomnia containing the compound of claim 1 as an active ingredient.
A food for improving or preventing sleep disorder or insomnia containing the compound of claim 1 as an active ingredient.
A process for the preparation of a compound of claim 1 comprising the steps of:
(a) reacting an indole compound represented by the following formula (4) with a halogen ion donor in the presence of a flavin-dependent halogenase enzyme (MrFDH) represented by the amino acid sequence of SEQ ID NO: 6 to produce a halogenated indole derivative represented by the formula ; And
(b) recovering the synthesized halogenated indole derivative.
(2)

[Chemical Formula 4]

Wherein R &lt; ₃ &gt; is chloro or bromo.
5. The method of claim 4, wherein step (b) is performed using a reversed phase C18 stationary phase, a mobile phase of acetonitrile: water: formic acid 75: 25: 0.1 (v / v / v / v) mixed liquid and a medium pressure liquid chromatography , MPLC) under conditions of 15-21 min retention time.
5. The method according to claim 4, wherein the indole compound is AGMEL (agomelatine).
5. The process according to claim 4, wherein the halogen ion donor is sodium chloride or sodium bromide.

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