Novel progestin membrane receptor antagonist

Abstract

To provide novel antagonists for progestin membrane receptors. [Solution] A compound represented by the following formula (A) or a salt thereof. TIFF2026089212000015.tif41149

Description

[Technical Field]

[0001] This disclosure relates to progestin membrane receptor antagonists. [Background technology]

[0002] Progestin membrane receptors (mPRs) are seven-transmembrane cell membrane receptors whose existence was reported in 2003 by Peter Thomas et al.'s group at the University of Texas (Non-Patent Literature 1). mPRs form a novel G protein-coupled receptor family consisting of 11 genes homologous to AdipoQ receptors, and are named the progestin and adipoQ receptors (PAQR) family. There are five types of mPR molecules: α, β, γ, δ, and ε, corresponding to PAQR7, PAQR8, PAQR5, PAQR6, and PAQR9, respectively. Furthermore, mPRs are conserved in cells of a wide range of vertebrates, from humans to fish, and are known to be expressed in various tissues such as the brain and kidneys (Non-Patent Literature 2-4). The present inventors have succeeded in purifying steroid membrane receptors with steroid hormone binding activity, enabling the screening of substances using purified mPR (Patent Literature 1).

[0003] In animals, it is known that agonist binding to mPR promotes egg cell maturation and ovulation. Therefore, inhibiting mPR may suppress egg cell maturation and lower the probability of pregnancy, while promoting mPR may promote egg cell maturation and increase the probability of pregnancy. However, no naturally occurring substances that act on mPR have been reported to date (Non-Patent Literature 5). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2015-010064 [Non-patent literature]

[0005] Non-Patent Document 1 Y. Zhu et al., "Cloning, expression,and characterization of a membrane progestin receptor and evidence it is anintermediary in meiotic maturation of fish oocytes", Proc Natl Acad Sci U S A 100(5):2231-2236 (2003). Non-Patent Document 2 Peter Thomas et al.,"Steroid and G Protein Binding Characteristics of the Seatrout and HumanProgestin Membrane Receptor Subtypes and Their Evolutionary Origins",Endocrinology 148(2):705-718 (2007). Non-Patent Document 3 Jessica L. Smith et al.,"Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirmstheir ability to function as membrane progesterone receptors", Steroids 73(11):1160-1173(2008). Non-Patent Document 4 Yefei Pang et al., "Characterization, Neurosteroid Binding and Brain Distribution of Human MembraneProgesterone Receptors δ and ε(mPRδ and mPRε) and mPRδ Involvement in Neurosteroid Inhibition ofApoptosis", Endocrinology 154(1):283-295 (2013). [Non-Patent Document 5] Peter Thomas, "MembraneProgesterone Receptors (mPRs, PAQRs): Review of Structural and Signaling Characteristics", Cells 2022, 11(11):1785 (2022). [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure aims to provide novel antagonists for progestin membrane receptors. [Means for solving the problem]

[0007] The inventors of this invention discovered a novel mPR antagonist from an extract of the sea fan (Padina arborescens) and completed the present invention.

[0008] This disclosure relates, for example, to the following: [1] Formula (A): [ka] A compound represented by or a salt thereof. [2] A compound represented by formula (A); an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof, comprising a pharmaceutical product. [3] A contraceptive comprising a compound represented by formula (A); an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof. [4] Progestin membrane receptor antagonists comprising a compound represented by formula (A); an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof. [5] A compound represented by formula (A); an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof, comprising an inhibitor of the formation of mature egg cells. A contraceptive agent containing a compound represented by formula (A); an ester, prodrug or metabolite of the compound; or a pharmaceutically acceptable salt thereof.

Advantages of the Invention

[0009] According to the present disclosure, a novel antagonist of the progesterone membrane receptor and a medicament containing the same are provided.

[0010] Such a medicament can be, for example, an antagonist of the progesterone membrane receptor. According to such an antagonist of the progesterone membrane receptor, for example, activation of an intracellular signal caused by binding of an agonist of the progesterone membrane receptor to the progesterone membrane receptor can be suppressed.

[0011] Binding of an agonist to the progesterone membrane receptor is known to promote maturation of oocytes. Further, the inventors have clarified that the novel antagonist of the progesterone membrane receptor found suppresses maturation of oocytes. Therefore, a medicament containing the novel antagonist of the progesterone membrane receptor found can be, for example, an agent for suppressing formation of mature oocytes.

[0012] Binding of an agonist to the progesterone membrane receptor is known to promote ovulation. Further, the inventors have clarified that the novel antagonist of the progesterone membrane receptor found suppresses ovulation. Therefore, a medicament containing the novel antagonist of the progesterone membrane receptor found can be, for example, an agent for suppressing ovulation.

[0013] In animals in which egg cell maturation and ovulation are suppressed, pregnancy can also be suppressed. Therefore, such a drug could be, for example, a contraceptive. Since the novel antagonist discovered by the inventors is a non-steroidal compound, such a contraceptive is expected to have a low risk of side effects. Furthermore, since the novel antagonist discovered by the inventors is highly water-soluble, such a contraceptive is expected to have an active ingredient that is easily absorbed into the body. Moreover, since the novel antagonist discovered by the inventors has a simple chemical structure and is thought to be inexpensive to manufacture, this disclosure is expected to contribute to the supply of a contraceptive that is cheaper than existing oral contraceptives. [Brief explanation of the drawing]

[0014] [Figure 1] In Example 1, the absorbance chromatograms at 215 nm were obtained when chromatography was performed using an ODS column for the first (C18 column (1)) and second (C18 column (2)) chromatography, a TSKgel Phenyl-5PW RP Glass column (Phenyl 5PW-RP column), and a C30 type silica-packed column (C30 column) for the isolation of components contained in the sea fan extract. [Figure 2] This is the 1H-NMR spectrum of the components contained in the isolated sea fan extract in Example 1. [Figure 3] This is the 13C-NMR spectrum of a component contained in the isolated sea fan extract in Example 1. [Figure 4] This is the DQF-COSY spectrum of the components contained in the isolated sea fan extract in Example 1. [Figure 5] This is the HMQC spectrum of a component contained in the sea fan extract isolated in Example 1. [Figure 6] This is the HMBC spectrum of a component contained in the isolated sea fan extract in Example 1. [Figure 7]This figure shows the percentage of oocytes that developed GVBD in the group to which ethanol, 100 nM Org OD 02-0, or 100 nM Org OD 02-0 and 1 μM Compound A were added in Example 2. [Figure 8] This figure shows the percentage of oocytes that produced GVBD and the number of ovulations in zebrafish administered ethanol, 100 nM Org OD 02-0, or 100 nM Org OD 02-0 and 1 μM Compound A in Example 3. [Figure 9] This figure shows the results of evaluating the binding affinity of compound A and progesterone (P4) to mPR according to membrane binding assays in Example 4. [Figure 10] This figure shows the number of ovulations per individual in control mice or mice orally administered compound A in Example 5. [Modes for carrying out the invention]

[0015] Embodiments of the present invention will be described below, but the present invention is not limited to these embodiments.

[0016] <Definition> An alkyl group is a group obtained by removing one hydrogen atom from an alkane. The alkyl group may be a group obtained by removing one hydrogen atom from a linear or cyclic alkane, preferably a group obtained by removing one hydrogen atom from a linear alkane, and the linear alkane may be linear or branched. In one embodiment, the alkyl group is C 1-5 Alkyl may also be used. 1-5 Alkyl is an alkyl group having 1 to 5 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, or cyclopentyl, and these are C 1-5 It is alkyl. 1-3The alkyl group may be, for example, methyl, ethyl, propyl, or isopropyl.

[0017] <Compound A> This disclosure is based on the following formula (A): [ka] This disclosure relates to a compound represented by formula (A) or a salt thereof. In this disclosure, the compound represented by formula (A) is also referred to as compound A. The chemical name of compound A is 1-carboxybutyl 2-hydroxypentanoate.

[0018] Compound A may exist in several ionization states, such as those shown below, but Compound A according to this disclosure encompasses all of these ionization states. [ka]

[0019] Compound A may take on several stereostructures, such as those shown below, but the compound A relating to this disclosure encompasses all of these stereostructures. [ka]

[0020] Compound A is not particularly limited, as long as it has the same substantial chemical structure and atomic number as the compound represented by formula (A) and the above-mentioned ionization state and / or stereostructure. For example, compound A may be in the form of a tautomer, isotope-labeled compound, solvate, cocrystal and / or polymorph of the compound represented by formula (A) and the above-mentioned ionization state and / or stereostructure.

[0021] The solvent in the solvate of compound A may be any solvent capable of forming a solvate, and is preferably a pharmaceutically acceptable solvent. Examples of pharmaceutically acceptable solvents include water, ethanol, propanol, isopropanol, diethyl ether, or tetrahydrofuran, and is preferably water.

[0022] The salt of compound A may be any salt of compound A, and may be a monovalent or divalent salt. Preferably, the salt of compound A is a pharmaceutically acceptable salt of compound A. A pharmaceutically acceptable salt may be a salt formed between at least one selected from the group consisting of carboxyl groups and hydroxyl groups of compound A and one or two pharmaceutically acceptable cations, for example, a sodium salt, magnesium salt, potassium salt, calcium salt, ammonium salt, meglumine salt, or ethanolamine salt of compound A.

[0023] Compound A is a progestin membrane receptor antagonist. Progestin membrane receptors (mPRs) are seven-transmembrane cell membrane receptors whose existence was reported in 2003 by Peter Thomas et al.'s group at the University of Texas (Non-Patent Literature 1). mPRs form a novel G protein-coupled receptor family consisting of 11 genes that show homology to the AdipoQ receptor, and are named the progestin and adipoQ receptors (PAQR) family. There are five types of mPR molecules: α, β, γ, δ, and ε, corresponding to PAQR7, PAQR8, PAQR5, PAQR6, and PAQR9, respectively. Furthermore, mPRs are conserved in the cells of a wide range of vertebrates, from humans to fish, and are known to be expressed in various tissues such as the brain and kidneys (Non-Patent Literature 2-4). An mPR according to one embodiment of the present invention may be at least one selected from the group consisting of mPRα, mPRβ, mPRγ, mPRδ, and mPRε, in a preferred embodiment it may be at least one selected from the group consisting of mPRα, mPRβ, and mPRγ, and in a more preferred embodiment it may be mPRα.

[0024] Molecules that bind to the ligand-binding site of a receptor include agonists, which activate downstream intracellular signaling by binding to the receptor, and antagonists, which do not activate intracellular signaling even when bound to the receptor. Among these, antagonists can antagonistically inhibit the binding of endogenous agonists to the receptor, and are therefore widely used as active ingredients in drugs that suppress the activation of downstream intracellular signaling.

[0025] <Method for producing compound A> Compound A is a compound discovered by the present inventors from an extract of the sea fan (Padina arborescens). Therefore, Compound A can be produced by isolating and purifying an extract of the sea fan, for example, by isolating and purifying an extract obtained from the thallus of the sea fan using methanol as the extraction solvent. As a more detailed example, it can be produced by isolating and purifying an extract of the sea fan according to the method described in Example 1 of this application. The sea fan (Padina arborescens) is a seaweed belonging to the genus Padina in the family Dipterocarpaceae, order Dipterocarpales. The sea fan is widely distributed in the Pacific region, including Japan, and the Indian Ocean region.

[0026] Compound A can also be produced by organic chemical synthesis. 2-hydroxypentanoic acid (also known as 2-hydroxyvaleric acid), represented by the following structural formula, can be purchased from suppliers such as Fujifilm Wako Pure Chemical Industries, Ltd. and Sigma-Aldrich. Therefore, compound A can be produced, for example, by dehydration condensation of 2-hydroxypentanoic acid or its hydroxyl group protected derivative with 2-hydroxypentanoic acid or its carboxyl group protected derivative according to a conventional method, followed by deprotection as necessary. Alternatively, compound A can be produced, for example, by forming an ester using the active ester or acid halide of the hydroxyl group protected derivative of 2-hydroxypentanoic acid and 2-hydroxypentanoic acid or its carboxyl group protected derivative according to a conventional method, followed by deprotection as necessary. [ka]

[0027] <Pharmaceutical> The present disclosure also relates to a pharmaceutical composition containing Compound A; an ester, prodrug or metabolite of Compound A; or a pharmaceutically acceptable salt thereof. As used herein, Compound A; an ester, prodrug or metabolite of Compound A; or a pharmaceutically acceptable salt thereof is also referred to as "Compound A substances". As described above, Compound A substances include (i) Compound A, (ii) a pharmaceutically acceptable salt of Compound A, (iii) an ester, prodrug or metabolite of Compound A, or (iv) a pharmaceutically acceptable salt of an ester, prodrug or metabolite of Compound A. The pharmaceutical composition according to this embodiment may contain one selected from the group consisting of (i) to (iv), or may contain two, three, four or more selected from that group. Note that, similar to what was described above for Compound A, Compound A substances contain any ionization state and steric structure in a molecule having the chemical structure of Compound A substances, and are not particularly limited as long as they are compounds having substantially the same chemical structure and atomic element numbers for each atom. For example, they may be in the form of tautomers, isotope-labeled compounds, solvates, co-crystals and / or polymorphs. The pharmaceutical composition of this embodiment may contain Compound A substances as an active ingredient. Also, the pharmaceutical composition of this embodiment may contain a therapeutically effective amount of Compound A substances.

[0028] The ester and prodrug of Compound A may be any of the compounds represented by the following general formula, for example. [Chemical Formula]

[0029] In the case of the ester, in the above general formula, R 1 and R 2 each represent, for example, an alkyl group independently. The alkyl group may be linear or branched. In a preferred embodiment of the ester, R 1 and R 2 each represent an independent C 1-10 alkyl group, and more preferably, C1-8 Alkyl alkyl group, C 1-6 Alkyl alkyl group or C 1-3 It may represent an alkyl group. In a more preferred embodiment of the ester, R 1 and R 2 Each of these may be independently selected from the group consisting of a methyl group, an ethyl group, a propyl group, and a butyl group.

[0030] In the case of prodrugs, R in the above general formula 1 and R 2 For example, each of these independently represents a medoxomyl group (5-methyl-2-oxo-1,3-dioxol-4-yl group).

[0031] The metabolites of compound A are substances obtained as a result of the metabolism of compound A within the administered animal, and which bind to mPR.

[0032] The content of compound A in the pharmaceutical according to this embodiment may be, for example, 1% by mass or more, 10% by mass or more, 25% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 92% by mass or more, 94% by mass or more, 96% by mass or more, 97% by mass or more, 98% by mass or more, 99% by mass or more, 99.5% by mass or more, or 100% by mass, and may also be 99.5% by mass or less, 99% by mass or less, 98% by mass or less, 97% by mass or less, 95% by mass or less, 93% by mass or less, 91% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 15% by mass or less, or 5% by mass or less, based on the total mass of the pharmaceutical.

[0033] The pharmaceutical product according to this embodiment may further contain, in addition to compound A, additives commonly used in the pharmaceutical technology field, such as at least one additive selected from the group consisting of excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers, and flow additive modifiers.

[0034] The target organism to which the pharmaceutical agent according to this embodiment is administered is not limited to eukaryotes that express mPR, but is preferably a vertebrate that expresses mPR, and more preferably a mammal or fish that expresses mPR. Examples of target organisms include mammals such as humans, dogs, cats, mice, rats, rabbits, guinea pigs, horses, pigs, and sheep, and fish such as zebrafish, goldfish, carp, rainbow trout, sea bream, and flounder. The target organism is preferably a human or a pet mammal (e.g., a dog or cat), and more preferably a human.

[0035] The administration method of the pharmaceutical agent according to this embodiment is not particularly limited and can include, for example, oral administration, vaginal administration, transfacial administration, intravenous injection, subcutaneous injection, intramuscular injection, and transdermal administration. When the target of administration is a mammal, the preferred administration method according to one embodiment may be oral administration or vaginal administration, and more preferably oral administration. When the target of administration is a fish, the preferred administration method according to one embodiment may be oral administration or transfacial administration. In the case of oral administration to a fish, the pharmaceutical agent may be administered mixed with feed.

[0036] The dosage form of the pharmaceutical according to this embodiment is not particularly limited, and examples thereof include capsules, solutions, syrups, granules, powders, tablets, pills, oral jelly, inhalants, sprays, gels, creams, lotions, ointments, aerosols, patches, tapes, plasters, injections, vaginal suppositories, and vaginal tablets. When the pharmaceutical according to this embodiment is orally administered to mammalian animals, the dosage form may be, for example, capsules, solutions, syrups, granules, powders, tablets, pills, oral jelly or inhalants. When the pharmaceutical according to this embodiment is vaginally administered to mammalian animals, the dosage form may be, for example, sprays, gels, creams, lotions, ointments, aerosols, patches, tapes, plasters, injections, vaginal suppositories, and vaginal tablets. Further, the pharmaceutical according to this embodiment may be an immediate-release preparation or a sustained-release preparation. As an example of the specific dosage, for example, when the administration target is an adult human female, the dosage of the pharmaceutical per day may be 0.0001 μg to 10000 mg / day / 60 kg body weight in terms of the amount of compound A. When the administration target is a fish animal, the dosage of the pharmaceutical per day may be 0.0001 μg to 10000 mg / day / g body weight in terms of the amount of compound A.

[0037] <Antagonist of mPR> The inventors have found that compound A antagonistically inhibits the binding of agonists to mPR. In one aspect, the pharmaceutical according to this embodiment may be an antagonist of mPR containing compound A. The antagonist of mPR may contain compound A as an active ingredient.

[0038] The antagonist of mPR means a preparation containing an antagonist of mPR and used to antagonistically inhibit mPR. The antagonistic effect on mPR can be confirmed, for example, by the in vitro maturity evaluation test using zebrafish oocytes shown in Example 2, the in vivo maturity evaluation test or ovulation number evaluation test of oocytes in animal solids shown in Examples 3 and 5, or the antagonistic inhibition test of agonist binding to purified mPR shown in Example 4.

[0039] The mPR antagonist effect of the mPR antagonist in this embodiment may mean that, when evaluated by the above method, maturation, ovulation, or agonist binding is suppressed by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the case where the drug is not present. Furthermore, the mPR antagonist effect of the mPR antagonist in this embodiment may mean that, when evaluated by the above method, maturation, ovulation, or agonist binding is statistically significantly suppressed compared to the case where the drug is not present, for example, that the p-value by Student's t-test is less than 0.05 or less than 0.01.

[0040] The mPR antagonist according to this embodiment can be used, for example, as a research reagent to evaluate the physiological function of mPR. Specifically, the mPR antagonist according to this embodiment can be administered to a subject to suppress the activation of intracellular signals transmitted by mPR, and then the phenotypic changes, or changes in gene and protein expression, that occur in the administered subject can be evaluated. Through such evaluation, it is possible to elucidate the effects of intracellular signals transmitted by mPR on the subject, and the changes in the expression of molecules that contribute to the transmission of those intracellular signals.

[0041] The mPR antagonist according to this embodiment can be used, for example, to suppress physiological phenomena caused by the binding of an agonist to mPR. Examples of such physiological phenomena include egg cell maturation and ovulation.

[0042] The mPR antagonist according to this embodiment can be used for screening other mPR antagonists or mPR promoters.

[0043] <An inhibitor of mature egg cell formation> The inventors have found that compound A inhibits the maturation of egg cells. In one embodiment, the pharmaceutical agent according to this embodiment may be an inhibitor of the formation of mature egg cells containing compound A. The inhibitor of the formation of mature egg cells may contain compound A as an active ingredient.

[0044] A mature oocyte (ovum) refers to a germ cell, which is the female gamete in sexually reproducing vertebrates. Mature oocytes are formed when primary oocytes stored in the ovary of female vertebrates undergo meiosis (meiosis I) to produce secondary oocytes, and these secondary oocytes undergo further meiosis (meiosis II). The inhibitor of mature oocyte formation according to this embodiment can suppress the formation of such mature oocytes. The inhibitory effect on oocyte maturation can be confirmed, for example, in an in vitro maturation evaluation test using zebrafish oocytes as shown in Example 2, or in vivo oocyte maturation evaluation tests or ovulation count evaluation tests in animal organisms as shown in Examples 3 and 5.

[0045] The inhibitory effect of the mature egg cell formation inhibitor in this embodiment on egg cell maturation may mean that, when evaluated by the above method, maturation or ovulation is suppressed by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the case where the drug is not present. Furthermore, the inhibitory effect of the mature egg cell formation inhibitor in this embodiment on egg cell maturation may mean that, when evaluated by the above method, maturation or ovulation is suppressed statistically significantly compared to the case where the drug is not present, for example, that the p-value by Student's t-test is less than 0.05 or less than 0.01.

[0046] The inhibitor of mature egg cell formation according to this embodiment can be used, for example, in the same applications as ovulation inhibitors and contraceptives described later.

[0047] <Ovulation suppressant> The present inventors have found that compound A suppresses ovulation. In one embodiment, the pharmaceutical agent according to this embodiment may be an ovulation inhibitor containing compound A. The ovulation inhibitor may contain compound A as an active ingredient.

[0048] The ovulation-inhibiting effect can be confirmed, for example, by in vivo ovulation count evaluation tests in animal organisms as shown in Examples 3 and 5. The ovulation-inhibiting effect of the ovulation-inhibiting drug in this embodiment may mean that, when evaluated by the above method, ovulation is suppressed by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the case where the drug is not present. Furthermore, the ovulation-inhibiting effect of the ovulation-inhibiting drug in this embodiment may mean that, when evaluated by the above method, ovulation is suppressed statistically significantly compared to the case where the drug is not present, for example, that the p-value by Student's t-test is less than 0.05 or less than 0.01.

[0049] The ovulation-suppressing drug of this embodiment can be used, for example, for the same purposes as the contraceptives described later.

[0050] The ovulation-suppressing drug of this embodiment can be used, for example, as a treatment for diseases that can be treated by suppressing ovulation. Diseases that can be treated by suppressing ovulation include diseases that are treated with low-dose oral contraceptives (low-dose pills). Examples of such diseases include endometriosis, dysmenorrhea, and premenstrual syndrome (PMS). In other words, the ovulation-suppressing drug of this embodiment may, in one embodiment, be a treatment for endometriosis, a treatment for dysmenorrhea, and / or a treatment for premenstrual syndrome (PMS).

[0051] The ovulation-suppressing drug of this embodiment can be used, for example, as a preventive drug for diseases that can be prevented by suppressing ovulation. Diseases that can be prevented by suppressing ovulation include diseases that are prevented by low-dose oral contraceptives (low-dose pills). Such diseases include, for example, cervical cancer and ovarian cancer. That is, in one embodiment, the ovulation-suppressing drug of this embodiment can be a preventive drug for cervical cancer and / or ovarian cancer.

[0052] <Contraceptives> The inventors have found that compound A suppresses egg cell maturation and ovulation. In sexually reproducing vertebrates, pregnancy eventually occurs when a mature egg cell released by ovulation is fertilized by sperm, which are male gametes, to form a fertilized egg. Therefore, in animals in which egg cell maturation and ovulation are suppressed, pregnancy may also be suppressed. Accordingly, the pharmaceutical product according to this embodiment may, in one embodiment, be a contraceptive containing compound A. The contraceptive may contain compound A as an active ingredient. The contraceptive according to this embodiment may preferably be an oral contraceptive or a vaginal contraceptive, and more preferably an oral contraceptive.

[0053] The contraceptive effect of the contraceptive according to this embodiment means that the pregnancy rate of a woman of the same age (or, in the case of an animal, a female of the same age or month of birth) is reduced by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more compared to a woman of the same age (or, in the case of an animal, a female of the same age or month of birth) without the use of a contraceptive.

[0054] The contraceptive according to this embodiment can be used, for example, for contraception in humans. Since compound A is a non-steroidal compound, the contraceptive according to this embodiment is expected to be a contraceptive with a low risk of side effects. Furthermore, since compound A is highly water-soluble, the contraceptive according to this embodiment is expected to be a contraceptive in which the active ingredient is easily absorbed into the body. In addition, since compound A has a simple chemical structure and is thought to be inexpensive to manufacture, the contraceptive according to this embodiment is expected to contribute to the supply of a contraceptive that is cheaper than existing oral contraceptives.

[0055] The contraceptive according to this embodiment can be used, for example, to prevent reproduction in pets such as dogs, cats, and rabbits, where keeping multiple animals together is a problem.

[0056] The contraceptive of the present invention can be used, for example, to reduce production and promote growth in aquaculture, livestock farming, and poultry farming. In aquaculture, livestock farming, and poultry farming, if animals become pregnant unintentionally, it can lead to an uncontrolled increase in production, and in addition, the growth of the female parent is inhibited because nutrients are used for the development of the individual from the fertilized egg. Therefore, by preventing pregnancy with the contraceptive of this embodiment, it is possible to suppress such uncontrolled increases in production and growth inhibition. [Examples]

[0057] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0058] <Example 1: Isolation of components contained in sea fan extract> 150 g of sun-dried thallus of the sea fan (Padina arborescens) was crushed into a powder using a ball mill (Retsch Ball Mill PM 100, Verder Scientific Co., Ltd., Germany) that crushes the sample by impact with small stainless steel balls, and the powder was suspended in 1.5 L of 100% methanol. The suspension was incubated for 7 days with occasional stirring to extract the thallus components. After filtering the extract through filter paper, the filtrate was subjected to a rotary evaporator (AS ONE, ARE-V1200) at 40°C to remove methanol. The solid sample remaining after methanol removal was dissolved in an equal mixture of 80% methanol and n-hexane. The resulting solution was placed in a separatory funnel and liquid-liquid separation was performed. The 80% methanol layer (polar molecular layer) was collected and subjected to another rotary evaporator at 40°C to remove methanol. The sample remaining after methanol removal was dissolved in ethanol. The obtained solution was diluted 10-fold with ultrapure water to obtain a sample, which was then supplied to an ODS column (50 μm, 1.8 × 11.4 cm, 7 g, manufactured by Yamazen Corporation) using a roller pump to load the ODS column with sea fan extract. Approximately 3 g of dried sea fan powder was loaded onto each ODS column.

[0059] The secretion loaded onto an ODS column was separated by reverse-phase liquid chromatography by supplying a water-acetonitrile mixture in a gradient (100:0-0:100) under acidic conditions containing 0.05% trifluoroacetic acid. The resulting fraction was then loaded onto an ODS column (4.6 × 30 cm, manufactured by Tosoh Corporation), and the secretion was separated again by reverse-phase liquid chromatography by supplying a water-acetonitrile mixture in a gradient (100:0-0:100). This purification using an ODS column was performed in two steps. The resulting fraction was then loaded onto a TSKgel Phenyl-5PW RP Glass (8 mm × 7.5 cm) column (manufactured by Tosoh Corporation), and the secretion was separated again by reverse-phase liquid chromatography by supplying a water-acetonitrile mixture in a gradient (100:0-0:100). Finally, the secretions were separated by reverse-phase liquid chromatography by loading a C30 (triacontyl group) type silica-packed column (Wako Pack Navi C30-5 2.0 × 150 mm) and supplying a water-acetonitrile mixture in a gradient (100:0-0:100). The elution of substances in these chromatography sessions was monitored by absorbance at 215 nm, and the temperature was maintained at 40°C.

[0060] Figure 1 shows chromatograms of absorbance at 215 nm during chromatography using an ODS column (1st and 2nd C18 column (2)), a TSKgel Phenyl-5PW RP Glass column (Phenyl 5PW-RP column), and a C30 silica-packed column (C30 column) for the isolation of components contained in the sea fan extract. In chromatography, the peaks indicated by thick black lines in each chromatogram in Figure 1 were recovered. Through four column chromatographys, we successfully recovered fractions containing single components from the sea fan extract.

[0061] Next, the isolated fractions were dried using a freeze-dryer (FDU-810 freeze-dryer, EYELA) to isolate the components contained in the sea fan extract. The isolated samples were analyzed by nuclear magnetic resonance (CRS) and mass spectrometry. For CRS, the isolated samples were dissolved in 500 μL of CDCl3 and measured using a JNM-ECZ500R spectrometer (JEOL, Tokyo, Japan). Mass spectrometry was performed in negative mode using a liquid chromatography-mass spectrometer (LC-MS) Agilent 1100 (Agilent Technologies, USA).

[0062] Figure 2 shows the components contained in the isolated sea fan extract. 1 This is the 1H-NMR spectrum. Figure 3 shows the components contained in the isolated sea fan extract. 13 Figure 4 shows the 1C-NMR spectrum. Figure 4 shows the DQF-COSY spectrum of the isolated component contained in the sea fan extract. Figure 5 shows the HMQC spectrum of the isolated component contained in the sea fan extract. Figure 6 shows the HMBC spectrum of the isolated component contained in the sea fan extract. Furthermore, a peak was observed at the position 217.107 as a result of negative-mode mass spectrometry. From these results, the isolated component contained in the sea fan extract was identified as compound A, whose structural formula is shown below. The calculated mass number of the anion produced by the elimination of one proton from compound A is 217.108. In the following examples, the isolated product obtained in Example 1 was used as compound A. [ka]

[0063] <Example 2: Inhibition of oocyte maturation and ovulation by compound A in zebrafish> Org OD-02 (10-Ethenyl-19-norprogesterone), an agonist of mPR, is known to induce oocyte maturation and ovulation by binding to mPR present on the cell surface of oocytes. Therefore, in Examples 2, 3, and 5, the inhibitory effect of compound A on oocyte maturation and ovulation was evaluated by whether compound A inhibited oocyte maturation and ovulation induced by Org OD-02.

[0064] Fertilized female zebrafish with well-developed immature oocytes were pre-treated with the mPR agonist Org OD 02-0 (Axon Medchem, catalog number Axon2085, CAS No.: 13258-85-0) to induce oviposition. The oviposition female zebrafish were then reared for another 7-10 days to produce newly developed immature oocytes. On the day of the experiment, females that had not ovulated were selected and used in the experiment.

[0065] Selected female zebrafish were transferred to tanks containing 100 mL of water per fish. Ethanol, 100 nM Org OD 02-0, or 100 nM Org OD 02-0 and 1 μM compound A were added, and the tanks were incubated at 28.5°C under room light for 4 hours. After sacrificing the female zebrafish, the ovaries were isolated. The isolated ovaries were manually separated into ovarian segments containing 1 to 10 oocytes each. The obtained ovarian segments were imaged with a light microscope to evaluate oocyte maturation and ovulation. Oocyte maturation was evaluated as the percentage of cleaved oocytes among more than 20 oocytes in each group, indicating the occurrence of germinal vesicle breakdown (GVBD). Ovulation was evaluated as the percentage of cells with a clear membrane.

[0066] Figure 7 shows the percentage of oocytes that developed GVBD in groups treated with ethanol, 100 nM Org OD 02-0, or 100 nM Org OD 02-0 and 1 μM compound A. In the figure, ** indicates that the p-value in Student's t-test is less than 0.01. According to Figure 7, GVBD induced in the presence of Org OD 02-0 was suppressed in the presence of compound A. This indicates that compound A can inhibit oocyte maturation.

[0067] <Example 3: Inhibition of oocyte maturation and ovulation by compound A in zebrafish> Fertilized female zebrafish with well-developed immature oocytes were pre-treated with the mPR agonist Org OD 02-0 to induce oviposition. These oviposition female zebrafish were then reared for another 7-10 days to produce newly developed immature oocytes. On the day of the experiment, females that had not ovulated were selected and used in the experiment.

[0068] Selected female zebrafish were transferred to tanks containing 100 mL of water per fish. A final concentration of 100 nM of Org OD 02-0, or 100 nM of Org OD 02-0 and a final concentration of 1 μM of compound A, was added, and the tanks were incubated at 28.5°C under room light for 4 hours. After sacrificing the female zebrafish, the ovaries were isolated. The isolated ovaries were manually separated into ovarian segments containing 1 to 10 oocytes each. The obtained ovarian segments were imaged under a light microscope to evaluate oocyte maturation and ovulation. Oocyte maturation was evaluated as the percentage of cleaved oocytes among more than 20 oocytes in each group, representing the proportion of GVBD (Gross Vascular Blight). Ovulation was evaluated as the proportion of cells with a clear membrane.

[0069] The results are shown in Figure 8. In the figure, * and ** indicate that the p-values ​​in Student's t-test were less than 0.05 and less than 0.01, respectively. According to Figure 8, oocyte maturation and ovulation induced in zebrafish reared in the presence of Org OD 02-0 were suppressed in the presence of compound A, and the proportion of cells that formed an egg membrane was significantly reduced. This indicates that compound A can inhibit oocyte maturation and ovulation in animal organisms.

[0070] <Example 4: Evaluation of the specific binding affinity of compound A to mPR> The binding affinity of compound A and progesterone (hereinafter also referred to as "P4") to mPR was evaluated according to the method (membrane binding assays) described in the paper (Toshinobu Tokumoto, Mika Tokumoto, and Peter Thomas. "Interactions of Diethylstilbestrol (DES) and DES Analogs with Membrane Progestin Receptor-α and the Correlation with Their Nongenomic Progestin Activities.", Endocrinology, Volume 148, Issue 7, Pages 3459-3467 (2007)). Membrane binding assays are assays that evaluate the binding affinity of a substance to mPR using a cell membrane expressing the mPR protein by introducing the mPR gene into breast cancer cells, and tritium (3-H) labeled P4. When a cell membrane expressing the mPR protein, tritium (3-H)-labeled P4, and the substance under evaluation are present together, if the substance under evaluation has binding affinity to mPR, the binding of tritium (3-H)-labeled P4 to the cell membrane expressing the mPR protein is antagonistically inhibited, and the amount of tritium (3-H) bound to the cell membrane decreases. The amount of tritium (3-H) bound can be expressed as DPM (disintegration per minute, the number of radioactive nuclides per minute). Therefore, the amount of this decrease in DPM was used as an indicator to evaluate the binding affinity to mPR. That is, when a cell membrane expressing the mPR protein, tritium (3-H)-labeled P4, and the substance under evaluation are present together, if the radioactivity of the substance under evaluation decreases in a concentration-dependent manner, the substance can be evaluated as having mPR binding affinity. Compound A was tested at a concentration of 10 μM, and P4 at a concentration of 1 μM.

[0071] Figure 9 shows the results of evaluating the binding affinity of compound A and progesterone (P4) to mPR according to membrane binding assays. The vertical axis in Figure 9 shows the amount of reduction in the amount of tritium (3-H) bound to the cell membrane (DPM) resulting from the antagonistic inhibition of the binding of tritium (3-H)-labeled P4 in the presence of the substance being evaluated. According to Figure 9, compound A, like the positive control progesterone, reduced the amount of tritium (3-H) bound to the cell membrane. This confirms that compound A does indeed have binding affinity for mPR.

[0072] <Example 5: Ovulation Inhibition Test with Compound A in Mice> This study investigated whether the administration of compound A made a difference in the amount of ovulation in mice that had undergone superovulation. Female mice (ICR mice, 6 weeks old) were intraperitoneally injected with serotropin for animals (Asuka Animal Health Co., Ltd.), a serum gonadotropin, at a dose of 7.5 IU per individual. Forty-eight hours later, an aqueous solution of compound A (150 μL) was orally administered using a mouse oral feeding tube (FCR & Bio Co., Ltd.) (dose: 10 mg / kg). Immediately afterward, human chorionic gonadotropin for injection (HCG Mochida for intramuscular injection: Mochida Pharmaceutical Co., Ltd.) was intraperitoneally injected at a dose of 7.5 IU to induce superovulation. Seventy hours later, the mice were dissected, and the ovulated eggs were extracted from the oviducts and counted under a microscope.

[0073] Figure 10 shows the number of ovulations per individual in control mice and mice orally administered compound A. In the figure, * indicates that the p-value in Student's t-test is less than 0.05. According to Figure 10, the number of ovulations was reduced in mice orally administered compound A, indicating that ovulation was suppressed. This demonstrates that compound A can inhibit ovulation in individual mammals.

Claims

1. Formula (A) below: 【Chemistry 1】 A compound represented by or a salt thereof.

2. Formula (A) below: 【Chemistry 2】 A pharmaceutical product containing a compound represented by ; an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof.

3. Formula (A) below: 【Transformation 3】 A contraceptive comprising a compound represented by ; an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof.

4. Formula (A) below: 【Chemistry 4】 A progestin membrane receptor antagonist comprising a compound represented by ; an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof.

5. Formula (A) below: 【Transformation 5】 An inhibitor of mature egg cell formation comprising a compound represented by ; an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof.

6. Formula (A) below: 【Transformation 6】 An ovulation inhibitor containing a compound represented by ; an ester, prodrug or metabolite of said compound; or a pharmaceutically acceptable salt thereof.

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