WO2019031754A1 - Sugar-added ospemifene, preparation method therefor, and pharmaceutical composition containing same as active ingredient - Google Patents

Abstract

The present invention relates to a novel ospemifene derivative compound in which a sugar is added to ospemifene as one kind of selective estrogen receptor modulator (hereinafter, SERM), and to a preparation method for the compound. The ospemifene derivative compound has improved bioavailability compared with existing ospemifene, and also has partially improved bioavailability compared with the original compound ospemifene, which provides a disease treatment effect for dyspareunia, vaginal dryness, and the like appearing in menopausal women, and thus can be favorably used as a modified dosage form.

Description

A method for preparing the same, and a pharmaceutical composition containing the same as an active ingredient

The present invention relates to a novel ospemipene derivative compound to which a sugar is added to ospemifene, which is a selective estrogen receptor modulator (SERM), and a method for producing the compound.

Ospemiphen (or deaminohydroxy-toremifene) is one of the selective estrogen receptor modulators (SERMs) and has been known to provide mixed activities such as relatively low estrogenic and anti-estrogenic activities in the universal hormone test 1, 2, and 3), and anti-tumor activity in preclinical experiments on breast cancer animal models (Non-Patent Document 4). With the approval of the US FDA for the purpose of treating certain menopausal dysfunctions such as dyspareunia and vaginal dryness among healthy women's menopausal syndromes in 2013, it is now being marketed as Ospena by Japanese pharmaceutical company Shinogi Co., Prescription medicine that sells. Dyspareunia is a menopausal female disorder disease in which vaginal atrophy occurs due to a decrease in body estrogen levels after menopause and pain during sexual activity. The administration of ospemipene stimulates the endometrium by providing an estrogenic effect on the vaginal tissue, thereby thickening the tissue, thereby alleviating the pain experienced by menopausal women during sexual activity. Clinically reported side effects of ospemiphen were redness, increased vaginal discharge, muscle spasms and sweating, and boxed warnings suggest that they should be prescribed for the shortest period of time consistent with individual women's treatment goals and risks.

 In a global outlook and analysis of Postmenopausal Dysrhagia published by Global Data, a UK business information services and consulting firm in 2013, the postmenopausal quality of seven major countries (including the US, UK, France, Germany, Italy, Spain and Japan) The market for atypical antipsychotics is projected to grow at an average annual rate of 6% (Non-Patent Document 5). The US market is expected to surpass US $ 900 million in 2012, expanding to more than US $ 1.9 billion in 2022, and the Japanese market is expected to reach US $ 20 million in 2022. In particular, the FDA approval of osmempen was presented as a major growth driver for postmenopausal vaginal atrophy.

Ospemiphen is similar to the universal selective estrogen receptor (SERM), through metabolism of the cytochrome p450 enzyme system of hepatic microsomes after human administration, that is, by the action of CYP2D6 or CYP2C9, 4-hydroxysulfemiphen 4-hydroxyspemifene (hereinafter referred to as 4-OH-OSPEM) or 4'-hydroxyospemifene (hereinafter referred to as 4'-OH-OSPEM). Ospemiphen is a highly hydrophobic compound that provides excellent tolerability, but the resulting low solubility and hence markedly lower bioavailability means that the recommended dose of ospemiphene daily is 60 mg or less As a result, pharmaceutical improvement of the above medicament is necessary.

Therefore, development of new derivatives that can improve the bioavailability, which is a problem in administration of existing ospemiphen as described above, will be important in the future. That is, high bioavailability will allow some reduction in the dose of the clinical treatment and, depending on the low-dose dosage, may further reduce the risk of side effects that may occur in the treated patient, It will be possible.

The development of novel derivatives through structural modification of SERMs is recognized as a key technology for the development of new drug delivery systems. One of them is the transfer of sugar molecules to hydroxy functions in the chemical structure There is an enzymatic production method using this enzyme (GT). Particularly, since the pro-cisplatin of osfenamic acid and its structural analogues can improve the bioavailability by keeping the concentration of the drug in the blood above a certain level during the first pass metabolism process, It is a bio-conversion technology that is useful for improving pharmaceutical formulations and developing new formulations.

The patent for the production method and use of ospemiphen is described as a chemical synthesis method through an organic synthesis method and for the treatment and prevention of a female disease atrophy-related disease or disorder after menopause (Patent Documents 1 and 2). However, the production of compounds in which sugar is added to 4-OH-OSPEM and 4'-OH-OSPEM metabolites, the metabolites of ospemiphen (hereinafter referred to as OSPEM)

The inventors of the present invention have found that Korean Patent No. 10-1717212 (entitled "Method for preparing an estrogen receptor modulator added with a sugar-modified enzyme") and International Application No. PCT-KR2017-004022 (entitled " A method for producing the same, and a pharmaceutical composition containing the same) have succeeded in the conversion of tamoxifen and necromphene derivatives, which can be used as a preventive and therapeutic agent for breast cancer, as a kind of SERM. For this purpose, the discovery of a novel glycosyltransferase (GT) derived from a microorganism, Microomonospora rhodorangea actinomycetes, which permits a glycosylation reaction using a tamoxifen structural analogue having SERM activity as a substrate, Has confirmed the biosynthesis of SERM and further improvement of formulation properties such as bioavailability as compared with the original compound of the sugar moiety.

As a result, the present inventors have made efforts to develop an OSPEM derivative capable of increasing the bioavailability of OSPEM and reducing the amount of OSPEM. As a result, CYP2C9, which is a type of recombinant human cytochrome P450, firstly added hydroxyl groups to OSPEM -OH-OSPEM and 4'-OH-OSPEM, and at the same time a novel glucosyltransferase (MrGT2) derived from a microorganism monospora rhodozyma actinomycetus The utilization rate is increased and the pharmacological activity is improved, and the present invention is completed.

Prior art literature

Patent literature

(Patent Document 1) WO 1996007402A

(Patent Document 2) WO 2003103649A1

Non-patent literature

(Non-Patent Document 1) Kangas L, Biochemical and pharmacological effects of toremifene metabolites. Cancer Chemother Pharmacol. 1990. 27: p. 8-12.

(Non-Patent Document 2) Unkila M., et al., Vaginal effects of ospemifene in the ovariectomized rat preclinical model of menopause. J. Steroid Biochem, Mol. Biol. 2013. 138: p. 107-115.

(Non-Patent Document 3) Kaur G., et al., Design, synthesis and evaluation of ospemifene analogs as anti-breast cancer agents. Eur. J. Med. Chem. 2014. 86: p. 211-218.

(Non-Patent Document 4) Nappi R. E., et al., Advances in pharmacotherapy for treating female sexual dysfunction. Exp. Opin. Pharmacother. 2015, 16 (6): p. 875-887.

(Non-Patent Document 5) GlobalData, PharmaPoint: Postmenopausal vaginal atrophy-Global drug forecast and market analysis to 2022. 2013.

(Non-Patent Document 6) Tolonen A., et al., Ospemifene metabolism in human in vitro and in vivo: metabolite identification, quantification, and CYP assignment of major hydroxylations. Drug Metabol. Drug Interact. 2013. 28 (3): p. 153-161.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel glucoside derivative having increased bioavailability of ospemiphen (OSPEM).

Another object of the present invention is to provide a method for converting ospemiphen to a glucosyl-omepemiphen derivative by a one-pot bioconversion method of a recombinant human cytochrome c enzyme and a microorganism-derived recombinant sugar transferase .

In order to attain the above object, the present invention provides a compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

Wherein R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, Or 2'-deoxy-glucose.

The present invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and having a pharmacological activity similar to that of osfemiphen (OSPEM), a dyspareunia and vaginal dryness treatment drug, Or a pharmaceutically acceptable salt thereof.

The present invention also provides a process for preparing an osmemphene derivative represented by formula (1), comprising the steps of:

(a) The one-pot reaction was carried out by mixing the ospemiphen solution with the glycosyltransferase, cytochrome P450, UDP-glucose or TDP-2-deoxy-glucose and glucose 6-phosphate, glucose 6-phosphate dehydrogenase and NADP + ; And

(b) obtaining an osmemimene derivative represented by formula (1).

[Chemical Formula 1]

In this formula,

R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, -Deoxy-glucose. ≪ / RTI >

The present invention also relates to a method of treating dyspareunia, vaginal dryness, osteoporosis, facial palsy, osteoarthritis, and osteoporosis of a postmenopausal woman, comprising administering a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. Such as hyperglycemia or hot flashes, vaginal atrophy, gynecomastia, hypogonadal hypogonadism, non-insulin dependent diabetes mellitus, lipid dystrophy, infertility, male prostate hypertrophy, prostate cancer, ovarian cancer and breast cancer The present invention provides a method for treating or preventing a disease selected from the group consisting of

The present invention also relates to the use of a compound of formula (I) for the treatment of menopausal women with dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia, A compound represented by Formula 1 for the treatment or prevention of a disease selected from the group consisting of hypogonadal hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer and breast cancer. Lt; RTI ID = 0.0 > acceptable < / RTI >

The present invention also relates to the use of a compound of formula (I) for the treatment of menopausal women with dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia, (1) for the manufacture of a medicament for the treatment or prevention of diseases selected from the group consisting of hypogonadal hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer and breast cancer, Or a pharmaceutically acceptable salt thereof.

FIG. 1 is a schematic diagram showing transformation of a recombinant CYP enzyme derived from human and a recombinant sugar transferase MrGT2 enzyme derived from actinomycetes into an osfempiren one pot.

FIG. 2 is a graph showing the results of a comparison between two kinds of mammalian cell lines, MCF-1 and MCF-2, which are two kinds of ospemipene derivatives (4-OSPEM-G and 4-OSPEM-DG), osfemimene (OSPEM) and tamoxifene 7 and MDA-MB-231 in half-life inhibitory concentration (IC50).

FIG. 3 is a graph showing the effect of the osmotic pressure on osmolysis of osmotic pressure in a dose of 10 mg / kg / day for 2 weeks using osfemiphen (OSPEM), 2 kinds of oselpemiphen (4-OSPEM-G, 4-OSPEM-DG) This is a bar graph comparing vaginal epithelial changes in ovariectomized rats administered orally. After the oral administration to the ethinyl estradiol treatment group (low dose of 0.1 mg / kg / day) as a positive control for 2 weeks, the vaginal epithelium thickness and weight and uterine weight were 100% Relative epithelium thickness, relative quality of body mass, and relative uterine weight were calculated as percentages (%) of the above parameters in the derivative and the raloxifene treatment group.

Fig. 4 shows the results of ovariectomized rats administered orally at a dose of 10 mg / kg / day for 2 weeks with ospemiphen (OSPEM) and two different ospemipene derivatives (4-OSPEM-G and 4-OSPEM-DG) A graph comparing the bioavailability of the derivatives of the sugar moiety by analyzing the residual amount of ospemipene in the blood by analyzing samples collected 24 hours after the last administration.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In the present invention, a GT encoding gene is isolated from the genome of a micro-monospore rhodolagenic strain to develop an ospemiphen derivative capable of overcoming the disadvantages of osupimpen which has a low bioavailability, , And transformed E. coli were prepared. Next, recombinant CYP2C9 involved in the oxidative metabolism of OSPEM was added to the above expressed transgene, and uridine diphosphate glucose (UDP-Glc), which is a nucleotidyl sugar, One-pot reaction in which thymidine diphosphate 2'-deoxy-glucose (TDP-2'-deoxy-Glc) was reacted with a glycosyl donor resulted in the formation of a new sugar moiety as an OSPEM derivative O-ospemifene glucoside (hereinafter referred to as 4-OSPEM-G) and 4-O-ospemifene 2'-deoxyglucoside (hereinafter referred to as 4-OSPEM-DG) derivatives) And nuclear magnetic resonance spectrometry (MRI) spectroscopy. The results of this study are as follows. First, we compared the cytotoxicity of the original ospemiphen and its derivatives before inoculation with the in vitro cytotoxicity and the bioavailability Improving .

Accordingly, in one aspect, the present invention relates to a compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

In this formula,

R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, -Deoxy-glucose. ≪ / RTI >

In the present invention, the compound is selected from the group consisting of ospemifene glucoside, ospemiphenegalactoside, ospemiphenaloside, ospemiphentaloside, ospemipheniloside, ospemipene N-acetylglucosamine- 4-O-2'-N-acetylglucosaminide, ospemifene lactose, or ospemifene-4-O-2'-deoxyglucoside.

The compound of formula (I) according to the present invention may be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, lactic acid, maleic acid, pumaric acid, gluconic acid, Succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid and the like can be used.

The compounds of formula (I) according to the present invention include not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

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

The compound of formula (1) according to the present invention, or a pharmaceutically acceptable salt thereof, is an ospemipene derivative, and its sugar moiety exhibits relatively low in vitro breast cancer cell toxicity than ospemiphen, a former compound, It was confirmed that the bioavailability of the ovariectomized rats was improved, and it is possible to apply the present invention to a medicament for the treatment of diseases such as dyspareunia and vaginal dryness, It could be used as a new drug formulation that overcomes the disadvantages of low-bioavailability ospemiphen.

Accordingly, in another aspect, the present invention provides a method for treating menopausal female disorders, such as dyspareunia and vaginal dryness, comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, (Eg, dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, vaginal atrophy, gynecomastia, male secondary hypogonadism, non-insulin dependent Diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer or breast cancer.

In another aspect, the present invention provides a method of treating dyspareunia, vaginal dryness, osteoporosis, or osteoporosis in a postmenopausal woman, comprising administering a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. ), Facial flushing or hot flashes, vaginal atrophy, gynecomastia, male secondary hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostate hyperplasia, prostate cancer, ovarian Cancer and breast cancer. ≪ RTI ID = 0.0 > [0002] < / RTI >

In another aspect, the present invention provides a method of treating dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia, ), A compound of formula (1) for the treatment or prevention of diseases selected from the group consisting of male secondary hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male hyperplasia, prostate cancer, ovarian cancer and breast cancer, Or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method of treating dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia, ) For the manufacture of a medicament for the treatment or prophylaxis of diseases selected from the group consisting of male hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer and breast cancer. ≪ / RTI > or a pharmaceutically acceptable salt thereof.

The term " treatment " as used herein means all the actions of improving or ameliorating the symptoms of the diseases by administration of the pharmaceutical composition comprising the compound represented by the above-mentioned formula 1 or a pharmaceutically acceptable salt thereof .

The compound of formula (I) or a pharmaceutically acceptable salt thereof of the present invention exhibits some in vitro cytotoxic activity against breast cancer cell lines and does not show cytotoxicity in normal cells (Experimental Example 1) In the in vivo animal test, the extracts of the mice showed activity against the vaginal epithelial tissue similar to that of the original compound ospemiphen (Experimental Example 2), and compared with administration of ospemipene And the bioavailability of the sugar portion is improved (Experimental Example 3). Therefore, it can be effectively used as an improved dosage form which can partially improve the low bioavailability of the existing ospemiphen.

The pharmaceutical compositions of the present invention may be formulated into oral or parenteral administration forms according to standard pharmaceutical practice. These formulations may contain, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, adjuvant or diluent.

When the composition of the present invention is used as a medicine, a pharmaceutical composition containing at least one compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient can be administered orally or parenterally But are not limited thereto.

The formulations for oral administration may be, for example, tablets, pills, hard capsules, soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and the like, (E.g., silica, talc, magnesium salt of stearic acid, calcium salt of stearic acid, and / or polyethylene glycol), such as sodium chloride, dextrose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine have. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt The same disintegrating or boiling mixture and / or absorbing agent, coloring agent, flavoring agent, and sweetening agent.

Formulations for parenteral administration may include subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection. In this case, in order to formulate the composition for parenteral administration, the pharmaceutical composition of the present invention contains at least one compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof, which is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, Which can be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and / or contain preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances and may be mixed, granulated Or a coating method.

The dose of the compound of the present invention to the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity. When the patient is 70 kg in body weight, And may be 0.01-100 mg / day, preferably 0.01-500 mg / day. Depending on the judgment of a doctor or pharmacist, it may be administered once to several times a day at intervals of a certain period of time.

In another aspect, the present invention provides a method for the production of a pharmaceutical composition for the treatment and / or prophylaxis of atherosclerosis, comprising the steps of: (a) contacting an osfemimene solution with a glycoconjugate, cytochrome P450, UDP- glucose or TDP-2-deoxy- glucose and glucose 6-phosphate, Mixing NADP + to perform a one-pot reaction; And (b) obtaining an osmemimene derivative represented by the formula (1).

The present invention relates to a process for obtaining a recombinant GT enzyme derived from actinomycetes in Escherichia coli and 2) obtaining a total of two saccharide-derived ospemiphene derivatives by a sequential enzymatic reaction of a recombinant CYP with human recombinant GT (i.e., MrGT2) .

In one aspect of the present invention, the production method may further include a step of separating, purifying, or separating and purifying the enzyme reaction product obtained in the above step by Medium Pressure Liquid Chromatography (MPLC) have.

In one embodiment of the present invention, the recombinant glycoprotein of step 1) can be isolated from micro-monospore rhododendron, but is not limited thereto.

In one aspect of the present invention, the recombinant glycoprotein is preferably MrGT2, more preferably MrGT2 expressed in E. coli.

In one aspect of the invention, the stationary phase used in the intermediate-pressure liquid chromatography method in this step may be, but is not limited to, reversed phase C18.

In one aspect of the present invention, the mobile phase used in the above intermediate-pressure liquid chromatography method may be, but not limited to, a mixed solution of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v).

In one aspect of the present invention, the medium pressure liquid chromatography retention time in this step may be 11 to 13 minutes, but is not limited thereto.

In the present invention, " vector " means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms " plasmid " and " vector " are sometimes used interchangeably in the context 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 cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, the preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5α, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, . However, E. coli strains such as FMB101, NM522, NM538 and NM539, as well as the speices and genera of other prokaryotes, and the like, can also be used. In addition to the E. coli mentioned above, Agrobacterium sp. Strain such as Agrobacterium A4, bacilli such as Bacillus subtilis, Salmonella typhimurium or Serratia marcensis marcescens) and various strains of the genus Pseudomonas can be used as host cells.

Transformation of prokaryotic cells can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used to transform these cells.

The expression " expression control sequence " in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked to a particular host organism. Such regulatory sequences include promoters for conducting transcription, any operator sequences for modulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. For example, regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells include promoters, polyadenylation signals and enhancers. The most influential factor on the expression level of the gene in the plasmid is the promoter. As the promoter for high expression, SRα promoter and cytomegalovirus-derived promoter are preferably used. In order to express the DNA sequences of the present invention, any of a wide variety of expression control sequences may be used in the vector. Useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, The promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of the phosphatase, such as Pho5, the promoter of the yeast alpha-mating system and the gene expression of prokaryotic or eukaryotic cells or viruses And other combinations known in the art. The T7 promoter may be useful for expressing the protein of the present invention in E. coli.

A nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way that the appropriate molecule (e. G., Transcriptional activator protein) is capable of gene expression when bound to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, " operably linked " means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

As used herein, the term " expression vector " is usually a recombinant carrier into which a fragment of different DNA is inserted, and generally means a fragment of double-stranded DNA. Herein, the heterologous DNA means a heterologous DNA that is not naturally found in the host cell. Once an expression vector is in a host cell, it can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterologous) DNA can be generated.

As is well known in the art, to increase the level of expression of a transfected gene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in the selected expression host. Preferably the expression control sequence and the gene are contained within an expression vector containing a bacterial selection marker and a replication origin.

Host cells transformed or transfected with the above expression vectors constitute another aspect of the present invention. As used herein, the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration. As used herein, the term " transfection " means that an expression vector, whether or not any coding sequence is actually expressed, is accepted by the host cell.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Preparation of Glucose-Derivative of Ospemiphen Derivatives

(1) Isolation of GT-encoding gene from micro-monospore laurogenase genome

In order to isolate the sterol glycosyltransferase from the microorganism Monospora rhodorans, first, the genome isolated from the microorganism Monospora rhodorans strain was used as a template, The polymerase chain reaction (PCR) was carried out using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, which were prepared based on the nucleotide sequence, as follows. The N-terminal primer is the sequence of SEQ ID NO: 1 below and the C-terminal primer is the sequence of SEQ ID NO: 2 below.

SEQ ID NO: 1: 5'-GGCATATGATCCACGCGCACGACTTCCGGATG-3 '(NdeI restriction site)

SEQ ID NO: 2: 5 '- CGCTCGAGATTCGGCCTGCGCCTCCCACGTCCA-3' (XhoI restriction site)

The genomic DNA of the strain and the primers were mixed together with Taq DNA polymerase to perform a total of 32 cycles (initial inactivation at 98 [deg.] C for 5 min, gradient from 52.8 [deg.] C to 69.3 [deg.] C, Terminated after 5 minutes of reaction). PCR product was purified and ligated to pGEMR-T easy vector (pGEMR-T easy vector, Promega, Madison, Wis., USA) and then ligated to E. coli XL1 blue (XL1 blue, Stratagene, La Jolla, Lt; 0 > C for 45 seconds, followed by transformation. After isolating and purifying the T-iso vector from the selected transformed E. coli, the nucleotide sequence thereof (SEQ ID NO: 3) and the amino acid sequence deduced therefrom (SEQ ID NO: 4) were determined.

(2) Expression of recombinant herpesvirus MrGT2 in E. coli

The MrGT2 DNA fragment treated with NdeI and XhoI restriction enzymes was inserted into pET-28 (a +) (Novagen, Madison, WI, USA), a protein expression vector treated with the same restriction enzymes, Were transformed into E. coli BL21 (DE3) (Stratagene, La Jolla, CA, USA). At this time, 50 ppm of kanamycin antibiotics was used for selection of transformants.

The recombinant E. coli was inoculated into the LB medium (Luria Bertani) supplemented with 1 M of sorbitol and 2.5 mM betaine at a final concentration of 1%, and cultured at 30 ° C. D-thiogalactopyranoside (IPTG; Sigma-Aldrich) was added at a final concentration of 0.5 mM to induce protein expression when growth was observed between 0.6 and 0.8 optical density. , St. Louis, Mo., USA). The recombinant E. coli strain was further cultured at 22 DEG C for 18 hours. After culturing, the culture was centrifuged at 2000 rpm for 10 minutes to collect the cells, and the cells were suspended in 50 mM sodium phosphate lysis buffer (300 nM NaCl, 10 mM imidazole, 10% glycerol, 1% Triton X-100) And sonication was carried out. After centrifugation at 12000 rpm for 20 minutes, the supernatant was collected separately and analyzed by 12% SDS-PAGE to confirm the expression level of the recombinant MrGT1 enzyme. In order to purify the expressed recombinant protein, the supernatant was diluted with Talon-metal affinity resin (Clontech, Mountain View, Calif., USA) equilibrated with 50 mM phosphate buffer (0.3 M NaCl, 20 mM imidazole) CA, USA) and incubated at 4 ° C for one hour. After refrigerated centrifugation at 2000 rpm for 5 minutes, the resin was introduced into a disposable column and washed with phosphate buffer containing 50 mM imidazole in a volume of 10 times the resin. Finally, 3 ml of a phosphate buffer solution containing 150 mM imidazole was used to purify the recombinant glycoconjugate MrGT2 bound to the resin.

(3) Recombinant CYPs derived from human and recombinant MrGT2 chain enzymes derived from actinomycetes were used to convert, isolate and purify

Ospemiphen (OSPEM, Sigma-Aldrich) used as a substrate was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM, and the resultant was dissolved in a reaction buffer (50 mM phosphate buffer, 10 mM magnesium chloride) (CYP2C9 20 μM level) purchased from BD Gentest (Woburn, MA, USA), 25 μM recombinant herpesvirus MrGT2, and UDP-Glc (10 mM, Sigma-Aldrich), NADP + (1 mM, Sigma-Aldrich) at a level of 2 mM each of TDP-2'-deoxy-Glc and for the continuous feeding of NADH coenzyme for CYP oxidation reaction. Aldrich) and glucose 6-phosphate dehydrogenase (8 U / ml level, Sigma-Aldrich) were added and the one-pot reaction was performed at 37 ° C for 2 hours (FIG. After the reaction, an equal amount of ethyl acetate was added to stop the reaction. The reaction mixture was centrifuged at 6000 rpm for 10 minutes, and only the upper organic solvent layer was collected and dried under reduced pressure. HPLC-ESI-MS analysis was performed to confirm the biosynthesis of the target compound in the extract. The mobile phase was Acquity CSH C18 (Waters, 50x1.0 mm, 1.7 μm; Milford, MA) as a stationary phase column at a rate of 140 μl / min in methanol: water: formic acid 65: , USA).

On the other hand, the following Combi Flash Rf MPLC system was applied to isolate and purify desired sugar-derived SERM derivatives from the crude extract. To a MPLC system running a mixed solution of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v) in a reverse-phased C18 cartridge at a flow rate of 15 ml / minute into the mobile phase, dissolved in 6 ml of the same mobile phase After the crude extract was injected, the peak detected on the chromatogram was automatically collected. The individual fractions were again concentrated under reduced pressure and the mass spectra were analyzed with an ion trap mass spectrometer (LCQ ion-trap mass spectrometer, ThermoFinnigan, San Jose, Calif., USA) to isolate the desired sugar moieties. As a substrate, ospemiphen was aliquoted at a retention time of about 15 to 16 minutes on the MPLC system, and the desired sugar moiety was detected in 11 to 13 minutes, which is a faster retention time than the substrate. These purified sugar moieties are shown in FIG. In the case of TDP-2'-deoxy-Glc, the yield of biotransformation using nucleic acid per UDP-Glc donor was about 48%, but the yield of biotransformation was lower than 30% , Which suggests that the enzyme has some substrate flexibility for the sugar donor substrate and a difference in preference for the sugar donor substrates. In addition, the fact that the sugar portion of the produced osfemiphen derivative is limited to the hydroxyl group at the 4-position rather than the 4'-position during the one-pot reaction of CYP and MrGT2 indicates that the specificity of the glycosyltransferase Respectively.

  NMR samples were prepared by dissolving each derivative in 200 μl of DMSO-d6 followed by leaving the solvent in 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 internal reference. All NMR data were calculated using Mnova Suite 5.3.2 software and compared to osmemphen using the 13C-NMR spectra of SERM derivatives as substrate.

DMSO-d6) [delta] 35.8, 42.7, 60.7, 62.2, 69.4, 71.5, 73.3, 76.7, 81.4, 109.2, 114.1, 126.3, 127.5, 127.9, 128.7, 129.1, 131.6, 139.2, 156.4, 161.5)

DMSO-d6) [delta] 35.8, 37.5, 42.7, 60.7, 62.2, 68.9, 69.4, 71.4, 81.2, 104.1, 114.1, 126.3, 127.5, 127.9, 128.6, 129.1, 131.6, 139.3, 156.4, 161.5)

Experimental Example 1: Comparison of cytotoxicity against breast cancer and normal cell lines

Two types of breast cancer cell lines, MCF-7 (ER positive, ATCC, Manassas, VA) and MDA-MB-231 cell lines (ER negative and ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum Serum), penicillin (100 units / ml level), and streptomycin (100 μg / ml level) were added at a concentration of 5% CO 2 at 37 ° C. The cytotoxicity of OSPEM and two derivatives (4-OSPEM-G and 4-OSPEM-DG) of the control group and tamoxifen, a comparative group, in the two types of breast cancer cell lines were confirmed by MTT assay. Each activated cell line (2 x 10 3) was dispensed into individual wells in a 96-well plate and OSPEM (Sigma-Aldrich) at various concentrations (0.1, 1, 3, 10, 30, The cells were incubated for 4 days (96 hours), MTT reagent was added to each well, and the cells were further cultured for an additional 3 hours. After addition of DMSO, the absorbance was measured with a Tecan microwell plate reader (Tecan AG, Mannedorf, Switzerland) set at a wavelength of 570 nm, and nonlinear regression analysis with Prism software revealed that the half-inhibition concentration ) Were measured.

On the other hand, the OSPEM and the OSPEM derivatives of the present invention were compared with the cytotoxicity of normal cells to confirm the cytotoxicity of breast cancer cell lines. Mouse embryonic fibroblast (ATCC) was used as a normal cell and the cytotoxicity of the treated group was compared with the above MTT assay.

The cytotoxicity results of the cancer cell lines are shown in Fig. In the control group, the half-inhibition concentration was about 52.7 μM and 100 μM for the breast cancer cell lines MCF-7 and MDA-MB-231, respectively, whereas the half-inhibition concentration in the control group, tamoxifen- 62.6 [mu] M and 77.7 [mu] M. On the other hand, in the case of the two new omephemiphen derivatives (4-OSPEM-G, 4-OSPEM-DG) of the present invention, the MCF-7 cell line, which is an ER positive breast cancer cell line, lt; / RTI > On the other hand, MDA-MB-231, an ER-negative breast cancer cell line, showed that the half-inhibitory concentrations of two sugar derivatives were more than 100 μM as in the case of osmempen treatment. Thus, the two derivatives of the glucosidase exhibit relatively low cytotoxicity against two types of breast cancer cell lines rather than the ospemipene control group, but they provide selective cytotoxicity to ER-positive breast cancer cell lines similar to ospemifene Showed the same tendency. In the cytotoxicity test for mouse fetal fibroblasts, which are normal cells, the toxicity was not observed in all treatment groups up to 100 μM level, and osfemiphen and tamoxifen and glucoside were found to be the novel osmemiphen derivatives 2 All species have cytotoxicity specific to cancer cells.

Experimental Example 2: Comparison of vaginal epithelial changes in ovariectomized rats

Female Sprague Dawley rats (DBL, Chungbuk, Korea) were housed in an individual cage for 12 hours in an amnion cycle. Female rats from 9 to 11 weeks of age were used for oral administration and ovaries of experimental rats were harvested 10 to 14 days prior to administration. The following experiment was performed on ovariectomized rats. In each experiment, treatment groups (between 5 and 6) were randomly selected and the mean body weight of the treatment groups was the same. Compared with two new derivatives of osfemiphen (4-OSPEM-G and 4-OSPEM-DG) and positive control group 17α-ethinyl estradiol (Sigma-Aldrich) for oral administration, The raloxifene (Sigma-Aldrich) was first dissolved in ethanol and diluted to the appropriate concentration with the PEG-treated untreated group. The dose was 3 mL per kg body weight. In order to test the relationship between dose and effect, oral administration was performed once a day for a total of 2 weeks.

(10 mg / kg / day dose) and 4-OSPEM-G (10 mg / kg / day dose) were used as the control group, PEG no treatment group, positive control group ethinyl estradiol (10 mg / kg / day), 4-OSPEM-DG (10 mg / kg / day) and the comparative group treated with raloxifene Was fixed at a dose of 10 mg / kg / day with reference to the capacity of the existing literature (Unkila M. et al., J Steroid Biochem Mol. Biol. 138: 107, 2013). Twenty-four hours after the last dose, rats in each treatment group were weighed and drawn. After blood collection, rats in each group were euthanized by cervical dislocation in a carbon dioxide chamber. The vaginal specimens except the cervix were weighed and the uterus was preserved in 10% buffered formalin. Each vaginal site was cut in half, immersed in 10% buffered formalin in a paraffin block, and cut into 4 μm sections for hematoxylin-eosin staining. Relative mass weight was converted to Equation 1 below.

[Equation 1]

The vaginal epithelium thickness was measured by image analysis using Image Pro Plus software after capturing images using a microscope equipped with a CCD camera. In order to measure the epithelial thickness of the vagina, the prepared cross section was magnified 100 times and measured from 3 to 4 times. Extraction and histological analysis of all organs were performed after the blindfolding of the individual treatment groups. On the other hand, for the comparison of the epithelial thickness, the relative quality weight, and the relative uterine weight measured for each treatment group, the measurement average of the ethinyl estradiol treatment group as the positive control group was taken as 100, and the relative value, that is, the percentage of ethinylestradiol reaction In terms of percentage (%), the vaginal epithelium thickness, relative quality and relative uterine weight of the other treatment groups were expressed. One-way ANOVA ANOVA with Dunnett's post-validation method and Kruskal-Wallis method as a nonparametric test method were used for the statistical analysis of the three parameters and SPSS version 14.0 was used as the statistical analysis program .

The results of the comparison of vaginal epithelium thickness, vaginal weight and uterine weight performed on oviposited rats exposed to 2 weeks of ospemiphen and the new glucosin derivatives of the present invention and raxoxifen, a comparative group, Respectively. Particularly, all results were obtained by fixing the parameters shown in the positive control group ethinylestradiol treatment group to 100% and comparing the values of osfemiphen (OSPEM) and the novel glucosides according to the present invention to the two osmemphene derivatives (4-G-OSPEM , 4-DG-OSPEM), and the relative changes in vaginal epithelium thickness, vaginal weight, and uterine weight in the individual treatment groups of raloxifene.

As a result, the relative vaginal epithelium thickness, the weight and the difference in uterine weight differed significantly from the raloxifene treatment group in the two treatment groups of the osfemiphen derivative, and compared with the original compound ospemiphene treatment group Statistically very similar trends were shown.

Experimental Example 3: Bioavailability comparison of ospemiphen and glucosuccinimide derivatives

The in vivo animal test results (FIG. 3) as described above show that in vitro breast cancer cytotoxicity, which is relatively low compared to the osmemphene derivative of the ospemipene sugar derivative shown in Experimental Example 1 2). As shown in FIG. 2, in the case of two new oregus-free glucoside derivatives administered orally for 2 weeks in the ovariectomized rats, ≪ / RTI > activity. That is, in the case of two kinds of saccharide-added ospemiphen derivatives, the possibility of metabolism at a rate different from that of the original compound ospemiphen during the metabolic process in experimental animals after oral administration is raised, The residual capacity of ospemipene in the blood was measured from whole blood samples collected 24 hours after the administration, and the bioavailability of the sugar derivatives was confirmed.

Detailed experiments were carried out as follows. That is, after collecting blood, whole blood was treated with the same volume of ethyl acetate and mixed vigorously for 30 seconds, followed by centrifugation at 4000 rpm for 5 minutes. Then, the organic solvent upper layer was separated, dried under reduced pressure, and stored at 70 ° C until analysis. The residual amount of 4-OH-OSPEM, which is the main metabolite of osfemipene in blood, was analyzed by HPLC-ESI-MS instrumental analysis.

As a result, the plasma concentrations of 4-OH-OSPEM in the two groups administered with 4-OSPEM-G and 4-OSPEM-DG, which are sugar derivatives, compared with the osmemphene control group (2.1 ng / 8.4 and 9.3, respectively (Fig. 4). This suggests that the sugar moiety may remain in the blood circulation for a relatively long period of time through basic ADME processes such as absorption, distribution, metabolism and excretion after oral administration It is anticipated that it will be possible to improve the low bioavailability of the existing ospemiphen and to prolong the sustained efficacy thereof.

As shown in the above results, it is possible to improve the existing compound through transformation of the structure-modified derivative by sugar transfer of the existing pharmacologically active compound, and as an improvement thereof, the possibility of application to a new dosage form of existing drug The potential for use as pharmaceuticals and medicinal materials can be confirmed.

The compounds produced in the above examples were formulated as follows.

Formulation Example 1: Tablets (Direct Pressurization)

After 5.0 mg of the compound was sieved, 14.1 mg of lactose, 0.8 mg of crospovidone (USNF) and 0.1 mg of magnesium stearate were mixed and pressurized to prepare tablets.

Formulation Example 2: Tablet (wet assembly)

5.0 mg of the compound was sieved, and 16.0 mg of lactose and 4.0 mg of starch were mixed. After 800.3 mg of polysorbate was dissolved in pure water, an appropriate amount of this solution was added to make it finely-ground. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The mixture was pressurized to prepare tablets.

Formulation Example 3: Powder and Capsule

5.0 mg of the compound was sieved and then mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was filtered through a hard No. 5 gelatin capsules.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The compound of formula (I) according to the present invention, or a pharmaceutically acceptable salt thereof, is a glucosoxifene derivative prepared by the conversion of one-pot, which shows lower cytotoxicity than ospemiphen, has improved bioavailability, It is possible to use it as a new drug formulation for existing diseases of ospemipen.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

I attached an electronic file.

Claims (5)

1. Claims 1. A compound represented by the following formula (1): < EMI ID =

   [Chemical Formula 1]

   

   In this formula,

   R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, -Deoxy-glucose. ≪ / RTI >
2. 3. The composition of claim 1, wherein the compound is selected from the group consisting of ospemipene glucoside, osmemphenol galactoside, ospemiphenaloid, osmempentalloside, ospemiphenylazoide, ospemipen N-acetylglucosamine 4-O-2'-N-acetylglucosaminide, ospemiphenlactoside and ospemifene-4-O-2'-deoxyglucoside. Or a pharmaceutically acceptable salt thereof.
3. Vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy of the menopausal women, including the compounds of claims 1 or 2, , Gynecomastia, male hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostate hyperplasia, prostate cancer, ovarian cancer and breast cancer.
4. A process for producing an osmemphene derivative represented by formula (1), comprising the steps of:

   (a) The one-pot reaction was carried out by mixing the ospemiphen solution with the glycosyltransferase, cytochrome P450, UDP-glucose or TDP-2-deoxy-glucose and glucose 6-phosphate, glucose 6-phosphate dehydrogenase and NADP + ; And

   (b) obtaining an osmemimene derivative represented by formula (1).

   [Chemical Formula 1]

   

   In this formula,

   R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, -Deoxy-glucose. ≪ / RTI >
5. 5. The method according to claim 4, wherein the glycoprotein has an amino acid sequence represented by SEQ ID NO: 1.

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