KR101970326B1 - Glycosyl-Attached Enclomifene, Method of Preparing the Same and Pharmaceutical Compositions Comprising the Same - Google Patents

Abstract

The compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention is a glucosaccharide derivative having an effect of enhancing bioavailability while maintaining antagonistic activity to the estrogen receptor alpha (ERa) Can be useful as prodrugs or alternative formulations that can treat estrogen receptor related diseases such as non-insulin dependent diabetes or lipid dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer, breast cancer,
[Chemical Formula 1]

In the above formula (1), R ₁ is CH ₂ CH ₃ or H and R ₂ is glucose, 2-deoxy-glucose or galactose.

Description

[0001] The present invention relates to novel clomiphene, a method for preparing the same, and a pharmaceutical composition comprising the same. [0002] Glycosyl-Attached Enclomifene, Method of Preparing the Same and Pharmaceutical Compositions [

The present invention relates to enum-clomiphene, a method for preparing the same, and a pharmaceutical composition containing the same. More specifically, the present invention relates to clomiphene, which is a selective estrogen receptor modulator (SERM) (Enclomifene, enclomiphene) of the structural isomer of the structural isomer, and a process for the enzymatic production of the compound and a pharmaceutical composition containing the same.

Clomiphene (clomiphene or clomiphene) is one of selective estrogen receptor modulators (SERMs) and is used in female infertility treatment to induce ovulation induction in anovulation or oligo-ovulation It is a frequently used prescription ( Fertil . Steril . 2013. 100 (2): p341-348).

Clomiphene was first synthesized by organic synthesis in 1956 and started to be used in the 1960s. In the early days, clomiphene was used as a treatment for oligomenorrhea syndrome and has been used as an infertility treatment according to ovulation induction effect. In vivo, clomiphene acts on the estrogen receptors in the hypothalamus to interfere with the feedback inhibition of estrogen on the gonadotropin gonadotropin, which is the hypothalamic-pituitary-gonadal axis, pituitary-gonadal axis) to induce ovulation (Goldstein, SR et al ., Human Reprod . , 2000. 6 (3): p212-224). Clomiphene is also known to be effective in the treatment of hypogonadism in men. This pharmacological function has the advantages of cost reduction and ease of administration compared to conventional testosterone replacement therapy (Hill, S. et al ., Drugs 2009. 12 (2): p109-119). On the other hand, adverse effects of clomiphene administration may be reversible ovarian enlargement, blurred vision, visual disturbances such as scotomata, headache and hot flash, and in rare cases, Hyperplasia, reversible alopecia, and ovarian hyperstimulation syndrome.

Clomiphene is a trans-isomer of a triphenylethylene structure and a geometric isomer of a cis type structurally similar to a typical SERM, tamoxifen, and is also known as an enclomifene (hereinafter referred to as "ENCLOM") and a main clomiphene ("zuclomifene" , Contributing to the mixed activity of estrogenic and anti-estrogenic properties of clomiphene (Adashi EY, Fertil Steril , 1984. 42 (3): p331-344). In particular, with respect to the therapeutic effect of clomiphene on secondary hypogonadism in men, recently, the simple trans isomer of clomiphene, orclopimene (or the brand name Androxal) has entered the clinical phase 3, and in addition, (Hill S., et al ., Drugs , 2009. 12 (2): p109-119).

Most of the previous literature on enchromiphene and its structural metabolites is mostly about metabolites that appear during metabolic processes after administration of clomiphene (Mazzarino M., et al ., Eur . J. Mass Spectrom . , 2008. 14 (3): p171-180; Ganchev B., et al, Anal Bioanal Chem, 2011. 400 (10):....... p3429-3441; Murdter, TE, et al, Hum Mol Genet,. 2012. 21 (5): p1145-1154). Enclomiphene is metabolized through the metabolic pathway of the cytochrome p450 enzyme system of liver microsomes after administration of the human body, similar to tamoxifen, as described above, namely 4-hydroxyenclomifene (hereinafter referred to as 4-OH N-desethyl-4-hydroxyenclomipene (hereinafter referred to as N-DE-4-OH-ENCLOM) metabolism by the continuous metabolism of CYP2D6 and CYP3A4 / do. The active metabolite are showing at least 100 times the estrogen receptor antagonist activity compared to the original compound ENCLOM and it can be seen that clomiphene is also a kind of prodrug through which (Murdter, TE, et al. , Hum. Mol. Genet. , 2012. 21 (5): p1145-1154). The development of novel derivatives or the development of novel prodrugs that can provide higher bioavailability than conventional clomiphene or ENCLOM bioavailability will be important in the future. That is, high bioavailability will allow some reduction in the clinical treatment dose, and it will reduce the risk of side effects that may occur in the treated patient, depending on the low dose.

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). In particular, since the enucleation of enchromiphen and its structurally similar compounds can improve the bioavailability by keeping the concentration of the drug in the blood above a certain level during the metabolism process through the first pass compared to the original substance, Bio-conversion technology that is useful for the improvement of the formulation of a drug, the development of new formulations, and the prodrug of the drug.

Most of the patents for the preparation and use of necromifen and its related derivatives have been filed by Repros Therapeutics, Texas, and representative patents thereof are disclosed in International Patent Publications WO 2013020017A1 and WO 2014197477A1. These patents disclose the structural novelty of the trans-type clomiphene, ENCLOM, and metabolites (including 4-OH-ENCLOM and N-DE-4-OH-ENCLOM) biotransformed into a variety of metabolic reactions including inter- Methods and pharmacological uses. However, in the case of these patents, since the patent scope for ENCLOM and its metabolites is limited through enzymatic bioconversion, the production of a compound having a sugar added to ENCLOM and its active metabolites has not been derived so far .

The present inventors have found that a Korean patent application No. 10-2015-0104325 (a method of producing an estrogen receptor modulating agent added with a sugar-conjugated enzyme) is a kind of SERM, which can be used as a preventive and therapeutic agent for breast cancer, Have succeeded in converting their lives. For this purpose, the discovery of a novel glycosyltransferase (GT) derived from a microorganism , Micromonospora rhodorangea actinomycetes, which enables the glycosylation reaction using a tamoxifen structural analogue having SERM activity as a substrate, SERM biosynthesis, and further improvement of formulation properties such as bioavailability as compared to the original compound of the sugar moiety.

Thus, the inventors of the present invention found that ENCLOM was used as a substrate and human recombinant cytochrome P450 and Micromonospora rhodorangea ) One-pot glucosyltransferase ( MRGT2 ) recombinant derived recombinant adenomatous enchromophene derivatives were synthesized by the combination of these derivatives with the original SERMs and their derivatives of estrogen receptor ER) and the pharmacokinetic bioavailability were improved, and the present invention was completed.

It is an object of the present invention to provide a method for producing a SERM derivative which can be used for treating diseases such as hypogonadal hypogonadism and non-insulin dependent diabetes mellitus or fat dystrophy, infertility, male prostate hyperplasia, prostate cancer, ovarian cancer, breast cancer, It is an object of the present invention to provide a one-pot biosynthesis method of a chromogenic enzyme and a microorganism-derived recombinant sugar transferase.

In order to achieve the above object, the present invention provides an enchromophene derivative represented by the formula (1), an isomer thereof or a pharmaceutically acceptable solvate, hydrate or prodrug thereof:

[Chemical Formula 1]

In the above formula (1), R ₁ is CH ₂ CH ₃ or H and R ₂ is glucose, 2-deoxy-glucose or galactose.

(A) synthesizing an enchromophen derivative represented by Formula 1 by reacting enchromophen and a sugar donor in the presence of a glycoconjugate (MrGT2) represented by the amino acid sequence of SEQ ID NO: 4 and cytochrome; ; And (b) recovering the synthesized enchromophene derivative. The present invention also provides a method for producing an enchromophene derivative represented by the following formula (1).

The present invention also provides a pharmaceutical composition for treating an estrogen receptor-related disease, comprising an enchromophene derivative represented by the formula (1), an isomer thereof, or a pharmaceutically acceptable solvate, hydrate or prodrug thereof as an active ingredient.

According to the present invention, there is provided a method for producing recombinant CYP enzymes comprising the steps of: (1) reacting a recombinant glycosyltransferase (MrGT2) expressed in Escherichia coli derived from a strain of Micromonospora rhodorangea with human recombinant CYP enzymes, Or a pharmaceutically acceptable salt thereof, exhibits some in vitro antagonistic activity against the estrogen receptor (ER), and has an improved bioavailability when compared with the sugar addition compounds.

The compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention is useful for the treatment of diseases such as hypogonadal hypogonadism and non-insulin dependent diabetes mellitus or fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer, It can be applied as a structurally or pharmacologically improved drug for the purpose, and it can be used as a new drug formulation which can reduce the side effects that can be caused by the administration of existing SERM or necromifen.

FIG. 1 is a schematic diagram showing the conversion of human recombinant CYPs to enchromiphenone single-stranded biosynthetic pathway through the MrGT2 enzyme reaction of recombinant peritonezyme derived from actinomycetes.
Fig. 2 is a graph showing the antagonistic activity of ingroups of the necromiopenic derivatives and necromiopenia before biotransformation on the estrogen receptor.
FIG. 3 is a graph comparing degrees of bioavailability improvement of the derivatives prepared by analyzing the concentrations of enchromiphen and its active metabolites in the serum of experimental animals.
FIG. 4 is a graph of pharmacokinetic curves of the concentrations of enchromiphen and its active metabolites in the serum of experimental animals for 72 hours after administration.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention separates the GT-encoding gene from the genome of the micro-monospore rhodoranidus strain, and produces a recombinant expression vector and a transformed E. coli containing the GT-encoding gene and then transforms the expressed glycosyltransferase into ENCLOM metabolism Recombinant CYP2D6 and CYP3A4 / 5 were added, respectively, and enuclei and nucleotidyl sugar Uridine diphosphate glucose (UDP-Glc) and thymidine phosphate 2'-deoxy-glucose (TDP-2'-deoxy-Glc) was reacted with thymidine diphosphate 2'-deoxy-Glc by a one-pot reaction. Next, the structure was confirmed by mass spectrometry and nuclear magnetic resonance spectrometry analysis.

Accordingly, one aspect of the present invention relates to an enchromophene derivative represented by the formula (I), an isomer thereof, or a pharmaceutically acceptable solvate, hydrate or prodrug thereof.

[Chemical Formula 1]

In the above formula (1), R ₁ is CH ₂ CH ₃ or H and R ₂ is glucose, 2-deoxy-glucose or galactose.

In another aspect of the present invention, there is also provided a method for producing an endopeptidase, comprising the steps of: (a) reacting enchromophen and a sugar donor in the presence of a saccharide transferase (MrGT2) represented by the amino acid sequence of SEQ ID NO: 4 with cytochrome, Synthesizing; And (b) recovering the synthesized enchromophene derivative. The present invention also relates to a method for producing an enchromophene derivative represented by the following formula (1).

Yen clomiphene derivatives according to the present invention yen clomiphene 4- O-glucoside, N- des ethyl-N clomiphene 4- O-glucoside, yen clomiphene 4- O -2'- deoxy glucoside, N- des ethyl-N clomiphene 4 -O < 2 > -dioxy glucoside.

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 may 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 enzyme reaction product obtained in the present invention may further be separated or purified by a Medium Pressure Liquid Chromatography (MPLC) method.

The present invention relates to the in vitro antagonistic activity of the original SERMs and their derivatives before saccharification to the ER, and to investigate the biochemical activity of SERM derivatives, Were tested for bioavailability and antagonistic activity and bioavailability were improved.

Accordingly, the present invention relates to a pharmaceutical composition for the treatment of estrogen receptor-related diseases, comprising an enchromophene derivative represented by the general formula (1), an isomer thereof or a pharmaceutically acceptable solvate, hydrate or prodrug thereof as an active ingredient .

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

In the present invention, the estrogen receptor-related diseases may be male secondary hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer or breast cancer.

As used herein, the term " treatment " refers to any action that improves or cures the symptoms of the above-mentioned diseases by the administration of a pharmaceutical composition comprising a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof.

The compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention exhibits antagonistic activity with some inhibitory effects on ER (Experimental Example 1), and the bioavailability of the enchromophen derivatives of the present invention is improved (Experimental Example 2), it can be used effectively as a formulation which can not only reduce the existing dose but also can partially improve side effects on long-term administration.

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, weight, sex, dosage form, health condition, and disease severity. In general, when referring to an adult patient weighing 70 kg, 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at a predetermined time interval according to the judgment of a doctor or pharmacist.

The present invention also provides a hereditary enzyme (MrGT2) gene comprising all of the nucleotide sequence of SEQ ID NO: 3.

In one embodiment of the present invention, the GT may be, but is not limited to, a herbal transcription enzyme MrGT2 derived from a micro-monospore rhododendron strain.

In addition, the present invention provides MrGT2 comprising all of the amino acid sequence of SEQ ID NO: 4.

In addition, the present invention provides a method for producing a recombinant GT-derived recombinant GT enzyme, which comprises the steps of: 1) obtaining a recombinant GT enzyme derived from actinomycetes in E. coli, and 2) sequential enzymatic reaction of recombinant CYP with human recombinant GT (i.e. MrGT2) And a step of obtaining an enklophen derivative in the sugar moiety.

The production method of the present invention may further comprise the step of 3) separating, purifying, or separating and purifying the enzyme reaction product obtained in the step 2) by Medium Pressure Liquid Chromatography (MPLC) method.

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

In the present invention, the recombinant glycoprotein of step 1) is preferably MrGT2, more preferably MrGT2 expressed in E. coli.

In the present invention, the stationary phase used in the intermediate-pressure liquid chromatography method in the step 3) may be, but is not limited to, the reversed-phase C18.

In the present invention, the mobile phase used in the intermediate pressure liquid chromatography method in the step 3) may be a mixed solution of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v), but is not limited thereto.

In the present invention, the medium pressure liquid chromatography retention time in step 3) may be 14-16 minutes, but is not limited thereto.

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 embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

[Example]

Example  1 to 2: Enclomidin  Preparation of derivatives

(One) Micro Monospore Rhodolajia  From the dielectric GT  Isolation of the coding gene

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' -GG CATATG ATCCACGCGCACGACTTCCGGATG-3 '( Nde I restriction site)

SEQ ID NO: 2: 5 '- CG CTCGAG ATTCGGCCTGCGCCTCCCACGTCCA-3' ( Xho I 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 98 for 5 minutes, 52.8 to 69.3 for slope, 72 for 5 minutes Termination). 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, For 45 seconds and then transformed. 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. The base sequence of the whole ORF is 1146 bp, and the translated protein is composed of 381 amino acids (total 41.1 kDa).

SEQ ID NO: 3:

SEQ ID NO: 4:

(2) recombination in E. coli The party  enzyme MrGT2's  Expression

The T- not the MrGT2 DNA fragments treated with the vector with Nde I and Xho I restriction enzymes, inserted into the same restriction as long as the pET-28 protein expression vector treated with the enzyme (a +) (Novagen, Madison, WI, USA) , And then 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 Escherichia coli was inoculated in an amount of 1% by volume in the LB medium (Luria Bertani) supplemented with 1 M of sorbitol and 2.5 mM betaine, and then cultured at 30, D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO) at a final concentration of 0.5 mM to induce protein expression when growth was observed at an optical density of between 0.6 and 0.8. Louis, MO, USA). The recombinant E. coli strain was further cultured at 22 for 18 hours. After the culture, the culture was centrifuged at 2000 rpm for 10 minutes to collect the cells, and then the cells were dissolved in 50 mM sodium phosphate lysis buffer (300 nM NaCl, 10 mM imidazole, 10% glycerol, 1% Triton X-100) Sonication was performed. After centrifugation at 12000 rpm for 20 minutes, the supernatant was collected separately, and some samples were analyzed by 12% SDS-PAGE to confirm the expression level of recombinant MrGT1. The purified supernatant was diluted with a Talon-metal affinity resin (Clontech, Mountain View, CA, USA) equilibrated with 50 mM phosphate buffer (0.3 M NaCl, 20 mM imidazole) to purify the expressed recombinant protein. USA) and incubated at 4 for one hour. After centrifugation at 2000 rpm for 5 minutes, the resin was introduced into a disposable column and then washed with a phosphate buffer solution 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 glycoprotein MrGT2 bound to the resin.

(3) Human-derived recombinant CYPs and  Recombinant derived from actinomycetes MrGT2  By a chain enzyme reaction Enclomene The party  As a derivative Live conversion , Separation and purification

(50 μM) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM, and then incubated in a reaction buffer (50 mM (CYP2D6, CYP3A4, and CYP3A5, 20 uM, respectively) purchased from BD Gentest (Woburn, Mass., USA) after dilution to a final concentration of 1 mM with phosphate buffered saline (10 mM magnesium chloride) (10 mM, Sigma) at 25 mM of the recombinant MrGT2 and at a level of 2 mM each of the nucleic acid UDP-Glc and TDP-2'-deoxy-Glc, and for the continuous supply of NADH coenzyme for the oxidation reaction of CYP (Sigma-Aldrich), NADP ⁺ (1 mM, Sigma-Aldrich) and glucose 6-phosphate dehydrogenase (8 U / ml level, Sigma-Aldrich) were added. 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. Acquity CSH C18 (Waters, 50 × 1.0 mm, 1.7 μm; Milford, MA, USA) was used as the mobile phase at a flow rate of 140 μl / min in methanol: water: formic acid 65: ).

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 in which a mixed liquid of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v) was flowed to a mobile phase at a rate of 15 ml per minute in a reverse-phased C18 cartridge, After injecting a crude extract, the peak detected in 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, enclromphene was collected on a MPLC system at a retention time of about 17 to 18 minutes, and the desired sugar moiety was detected as 14 to 16 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 40%, but the yield of biotransformation was less than 25% , Indicating differences in specificity for the sugar receptor and sugar donor substrates of this enzyme.

NMR samples were prepared by dissolving the respective 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 standard. All NMR data were calculated using Mnova Suite 5.3.2 software and compared to SERMs using the ¹³ C-NMR spectra of SERM derivatives as substrates.

Yen clomiphene 4- O - glucoside (E1; 22.3mg; lifetime rates ^{42%; 13 C NMR [125} MHz, DMSO-d6] δ 13.2, 49.7, 54.0, 62.1, 66.8, 71.4, 73.2, 76.8, 81.3, 109.0, 114.1, 120.3, 127.6, 128.4, 129.1, 130.1, 131.1, 131.8, 137.3, 156.2, 158.3)

Death N- ethyl-N clomiphene 4- O-glucoside (E2, 20.7mg; lifetime rates ^{38%; 13 C NMR [125} MHz, DMSO-d6] δ 15.5, 44.2, 49.2, 62.1, 68.9, 71.4, 73.4, 76.8 , 81.5, 109.1, 114.1, 120.3, 127.6, 128.4, 129.1, 130.1, 131.1, 131.8, 137.3, 156.2, 158.3)

Yen clomiphene 4- O -2'- deoxy glucoside (E3, 13.0mg; lifetime rates ^{24%; 13 C NMR [125} MHz, DMSO-d6] δ 13.2, 37.6, 49.8, 54.1, 62.1, 66.8, 68.9, 71.3 , 81.4, 104.4, 114.1, 120.3, 127.6, 128.4, 129.1, 130.1, 131.1, 131.8, 137.5, 156.4, 158.6)

Death N- ethyl-N clomiphene 4- O -2'- deoxy glucoside (E4, 11.3mg; lifetime rates ^{20%; 13 C NMR [125} MHz, DMSO-d6] δ 15.5, 37.5, 44.2, 49.3, 62.1, 68.8, 71.4, 81.4, 104.6, 114.1, 120.3, 127.6, 128.4, 129.1, 130.1, 131.1, 131.7, 137.5, 156.6, 158.6)

Experimental Example  1: Enclomidin  Estrogens of derivatives Receptor alpha (ER alpha)  Antagonistic Activity Assay

An NR peptide ERα ELISA kit (ActiveMotif, Carlsbad, Calif., USA) was used to test the binding affinity of the SERM derivatives to the estrogen receptor α (ERα) Diol (17 [beta] -estradiol) was used. The detailed experimental method was performed according to the protocol of the manufacturer. Each control dissolved in DMSO was diluted with the buffer solution contained in the kit to a level of 25 uM and the nuclear extracts were prepared according to the protocol of the kit manufacturer per well in 96-well microplates. ) Was added at a level of 15 ug. On the other hand, the samples were prepared at 25 uM level even for the sugar derivatives. Activity against estrogen receptor α (ERα) was quantitated with a microplate reader at a final absorbance of 450 nm, and all assays were expressed as means and standard deviations of at least three iterations. As a result, the antagonistic activity of antiprotozoal antioxidant, anticholinergic antagonist, was observed at 25 uM level. On the other hand, the total amount of the four nucleoprotein derivatives showed statistically significant antagonistic activity at the level of 25 uM. The above results indicate that the glucosidase is converted into the former enchynopenic active metabolite through the reaction of the metabolic enzyme system (i.e., glucosidase, etc.) present in the nuclear extract, And the other is that it is not.

Therefore, it can be confirmed that the antagonist of the present invention has antagonistic activity similar to that of the antagonist of the endocrine receptor α (ERα).

Experimental Example  2: The party Enclomidin  Comparison of bioavailability of derivatives

In order to compare the bioavailability of the nucleoside derivative produced by the above example with the clomiphene used in the clinical trial using the experimental rat model of the hysterectomy, Detailed experimental examples were carried out as follows. A week after uterine circulation was performed on female rats of about 150 g, this experiment was used. Four groups were designed as one treatment group, and a total of six groups (i.e., one group was placebo, another group was treated as a clomiphene control group, and the other four groups were treated with each sugar as a derivative treatment group) The body weight was measured before administration. After one week of oral administration at 3 μmol / day, 24 hours after the last administration, the body weight of rats in each group was measured and blood was collected. After collecting blood, whole blood was treated with the same volume of ethyl acetate, mixed vigorously for 30 seconds, and centrifuged at 4000 rpm for 5 minutes. Thereafter, the organic solvent upper layer was separated, dried under reduced pressure, and stored at -70 ° C. until analysis. The quantification of 4-OH-ECLOM and N-DE-4-OH-ECLOM and enclomin (ECLOM), which are active metabolites in the blood, was carried out by HPLC-ESI-MS instrumental analysis proposed in the present invention. 1, 2, 4, 6, 12, 18, 24, 36, and 40 days after the last administration for the comparison of the bioavailability in the total of the five groups of the clomiphene and glucose- 48, 60 and 72 hours. The blood samples taken at different time intervals were prepared in the same manner as described above. The amounts of active metabolites 4-OH-ECLOM, N-DE-4-OH-ECLOM and ECLOM were analyzed by HPLC-ESI-MS Respectively.

As a result, the body weight of rats before administration was constant in all groups, but the average body weight of each group was measured 24 hours after the last administration, and the increase in body weight (about 28 ~ 33g) Respectively. On the other hand, the plasma concentrations of 4-OH-ECLOM and N-DE-4-OH-ECLOM, which are active metabolites of clomiphene, were found to be slightly higher than those of clomiphene-treated control group (Fig. 3). At this time, the umbilical cord blood was injected orally to the mice at a dose of 3 umbilicals once a week for 24 hours after the uterine circulation. As shown in FIG. 3, as shown in Experimental Example 1, it has been shown that the glucoside derivatives can function as prodrugs metabolized to 4-OH-ECLOM and N-DE-4-OH-ECLOM as active metabolites Suggesting that the glucosides may remain in the blood circulation for a relatively long period of time than the direct oral administration of the active metabolite.

On the other hand, pharmacokinetic analysis was performed to more accurately determine the degree of improvement of bioavailability of clomiphene to clomiphene. That is, after analyzing the total concentration of necromifen and its active metabolites from plasma samples of 12 compartments up to 72 hours prepared in each treatment group, the concentration of enchromiphen and its active metabolites in the plasma over time The change is shown graphically. Pharmacokinetic parameters were measured by non-compartmental analysis of WinNonlin Professional software (version 5.1; Pharsight Co., Mountain View, Calif., USA). The parameters of C _max , peak concentration, T _max , paek time, and area under curve (AUC 72 _hr ) were measured as the pharmacokinetic index of each treatment group. Respectively.

[Table 1]

As shown in Fig. 4, the curves of the four groups treated with clomiphene were higher than those of the control group treated with clomiphene. However, no significant difference was observed between the four groups of glucoside derivatives treated with clomiphene derivatives. However, the addition of deoxyglucose-added clomiphene deoxyribose (E1 and E3) to the glucose-added clomiphene glucoside derivatives The results of treatment with glucoside derivatives (E2 and E4) showed somewhat higher curvilinear values. On the other hand, the distribution of the highest blood concentrations of all the treatment groups including the control group did not show any significant difference within the significance level, but in the case of the maximum blood concentration reaching time, Somewhat higher. This shows that there is no difference in the rate of hepatic metabolism of the administered compound in each treatment group, but it shows that there is a difference in the retention time in the blood of the administered compounds, and the bioavailability of the cholimephen derivatives Which is 1.2 times to 1.4 times higher than the bioavailability of the total added clomiphene. Thus, the relatively improved bioavailability of dioxy glucoside derivatives (E2 and E4), even within the sugar derivatives, suggests its potential for prodrugs. Therefore, it is possible to solve the problems caused by the long - term medication, which is one of the problems in administering the estrogen receptor modulating agent, by using the nucleoprotein derivatives produced by the examples.

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

Formulation example  1: Tablet (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 pressed to prepare tablets.

Formulation example  2: Tablet (wet assembly)

After 5.0 mg of the compound was sieved, 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 pulverized. 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, followed by mixing 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.

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 such, as a substitute prodrugation and a new dosage form of the existing drug The possibility of application as a raw material for pharmaceuticals and medicines including the applicability of the present invention can be confirmed.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the invention will be defined by the claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Method for Preparing Glycosylated Estrogen Receptor Modulators          Using Glycosyltransferase <130> P15-B199 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> P1 <400> 1 ggcatatgat ccacgcgcac gacttccgga tg 32 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> P2 <400> 2 cgctcgagat tcggcctgcg cctcccacgt cca 33 <210> 3 <211> 1146 <212> DNA <213> Micromonospora rhodorangea <400> 3 atgcgcaccc tcagcgccac cgccggcccg cggggcgacg gggccccgta caccggccgc 60 ggcgcggccc ggcggcgcgc ggggcacgac gtggccgtcg ccacgaccga cacgtcggcc 120 cgagtggtcc gggagcccgg gccagggttc cgggccctcc cggccgacca ccgcgcgcac 180 gcgggctggc ggccccaccg cgaggtcgtg cgcgcggccg gcgggcaccg cggaactccg 240 ccagccgttc gccgacgcgc cgtctcggag ggcacggacc tccggccgcg gccgacgacc 300 accgcgccgc tgcggtggct cagcggcgag gcgacggccc cccgggacct ccggcgcgac 360 caccagccca ccgccaccac cgccgacggc ccgccggtcg tcaccggcgc gcgctccctg 420 gggcggcccg gcaaccgcac ggccggccgc ctggccctgc gcatggccga ccggatctac 480 gccgaggccg tcgcgcgcct gcgggagcgg ctgtgcctgc cgccgctgtc cgcggccgcg 540 atgccggcgc ggcaggagcg ggccggctgg cccgtcctgc acggcttcgg cacggccctc 600 gtcccgcggc cggccgactg gcgccccggc ctcgaggtcg tcgggccgtg gtggccgcac 660 ccgaggccg gcgagcgcct gccctcggaa ctcgaggact tcctggacgc gggaccgcgg 720 ccggtcctgg tgcgcttcgg cagcatggcc gcccggcacg gcgagcggct cagcgaactg 780 gcggtgcgcg cgctgcgccg cgccggcctc cgcggcatcc tccagtccgg caacgcctgc 840 ctcgcggcgg agggcgacga cgtcctgacg gtcggcgacg tcccccacgc cctcctgttc 900 ccccggctcg cggcggtggt gcaccacgcg cgccggcacc agcgccgcga ccctgcgggc 960 ggccgtaccc tcggtggcgg tcccggtgac cgccgaccag agccgttctg ggccggccgg 1020 ctggcgcgga tccgggccgc cccagccccg gtccccttca cgtcgtcggc gacgaggcgt 1080 cgccgcgcgc gtgtga 1146 <210> 4 <211> 381 <212> PRT <213> Micromonospora rhodorangea <400> 4 Met Arg Thr Leu Ser Ala Thr Ala Gly Pro Arg Gly Asp Gly Ala Pro   1 5 10 15 Tyr Thr Gly Arg Gly Ala Ala Arg Arg Ala Gly His Asp Val Ala              20 25 30 Val Ala Thr Thr Asp Thr Ser Ala Arg Val Val Arg Glu Pro Gly Pro          35 40 45 Gly Phe Arg Ala Leu Pro Ala Asp His Arg Ala His Ala Gly Trp Arg      50 55 60 Pro His Arg Glu Val Val Arg Ala Gly Gly His Arg Gly Thr Pro  65 70 75 80 Pro Ala Val Arg Arg Ala Val Ser Glu Gly Thr Asp Leu Arg Pro                  85 90 95 Arg Pro Thr Thr Thr Ala Pro Leu Arg Trp Leu Ser Gly Glu Ala Thr             100 105 110 Ala Pro Arg Asp Leu Arg Arg Asp His Gln Pro Thr Ala Thr Thr Ala         115 120 125 Asp Gly Pro Pro Val Val Thr Gly Ala Arg Ser Leu Gly Arg Pro Gly     130 135 140 Asn Arg Thr Ala Gly Arg Leu Ala Leu Arg Met Ala Asp Arg Ile Tyr 145 150 155 160 Ala Glu Ala Val Ala Arg Leu Arg Glu Arg Leu Cys Leu Pro Pro Leu                 165 170 175 Ser Ala Ala Met Pro Ala Arg Gln Glu Arg Ala Gly Trp Pro Val             180 185 190 Leu His Gly Phe Gly Thr Ala Leu Val Pro Arg Pro Ala Asp Trp Arg         195 200 205 Pro Gly Leu Glu Val Val Gly Pro Trp Trp Pro His His Hisp Ala Gly     210 215 220 Glu Arg Leu Pro Ser Glu Leu Glu Asp Phe Leu Asp Ala Gly Pro Arg 225 230 235 240 Pro Val Leu Val Arg Phe Gly Ser Met Ala Ala Arg His Gly Glu Arg                 245 250 255 Leu Ser Glu Leu Ala Val Arg Ala Leu Arg Arg Ala Gly Leu Arg Gly             260 265 270 Ile Leu Gln Ser Gly Asn Ala Cys Leu Ala Ala Glu Gly Asp Asp Val         275 280 285 Leu Thr Val Gly Asp Val Pro His Ala Leu Leu Phe Pro Arg Leu Ala     290 295 300 Ala Val Val His His Ala Arg Arg His Gln Arg Arg Asp Pro Ala Gly 305 310 315 320 Gly Arg Thr Leu Gly Gly Gly Pro Gly Asp Arg Arg Pro Glu Pro Phe                 325 330 335 Trp Ala Gly Arg Leu Ala Arg Ile Arg Ala Ala Pro Ala Pro Val Pro             340 345 350 Phe Thr Ser Ser Ala Thr Arg Arg Thr Arg Gly Pro Leu Gly Arg Arg         355 360 365 Arg Pro Asp Leu Ala Ala Glu Asp Gly Ala Ala Arg Val     370 375 380

Claims (9)

The enchromophene derivative represented by the formula (1), an isomer thereof or a pharmaceutically acceptable solvate or hydrate thereof:
[Chemical Formula 1]

In the above formula (1), R ₁ is CH ₂ CH ₃ or H and R ₂ is glucose, 2-deoxy-glucose or galactose.
The method of claim 1, wherein the derivative is ¥ ¥ clomiphene clomiphene 4- O-glucoside, N- des ethyl-N clomiphene 4- O-glucoside, yen clomiphene 4- O -2'- deoxy glucoside or N- des-ethyl- Encmrompene 4- O- 2'-dioxy glucoside, an isomer thereof or a pharmaceutically acceptable solvate or hydrate thereof.
A process for preparing an enchromophen derivative represented by the following formula (1), comprising the steps of:
(a) synthesizing an enchromophene derivative represented by the following formula (I) by reacting enchromophen and a sugar donor in the presence of a saccharide transferase (MrGT2) represented by the amino acid sequence of SEQ ID NO: 4 and cytochrome; And
(b) recovering the synthesized enchromophen derivative:
[Chemical Formula 1]

In the above formula (1), R ₁ is CH ₂ CH ₃ or H and R ₂ is glucose, 2-deoxy-glucose or galactose.
The method of claim 3, wherein said derivative is ¥ ¥ clomiphene clomiphene 4- O-glucoside, N- des ethyl-N clomiphene 4- O-glucoside, yen clomiphene 4- O -2'- deoxy glucoside, N- des-ethyl- &Lt; RTI ID = 0.0 &gt; 4- O -2 &apos; -dioxyglucoside. &Lt; / RTI &gt;
4. The method according to claim 3, further comprising separating or purifying the obtained enzyme reaction product by a Medium Pressure Liquid Chromatography (MPLC) method.
A method for the treatment of hypogonadism, infertility, male prostate hyperplasia, prostate cancer, ovarian cancer or breast cancer, which comprises the enchromophene derivative represented by the general formula (1) of claim 1, an isomer thereof or a pharmaceutically acceptable solvate or hydrate thereof as an active ingredient Pharmaceutical composition:
[Chemical Formula 1]

In the above formula (1), R ₁ is CH ₂ CH ₃ or H and R ₂ is glucose, 2-deoxy-glucose or galactose.
7. The method of claim 6 wherein the derivative is ¥ ¥ clomiphene clomiphene 4- O-glucoside, N- des ethyl-N clomiphene 4- O-glucoside, yen clomiphene 4- O -2'- deoxy glucoside, N- des-ethyl- &Lt; RTI ID = 0.0 &gt; 4- O -2 &apos; -dioxyglucoside. &Lt; / RTI &gt;
7. The pharmaceutical composition according to claim 6, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
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