CN107151235B - Use of thiadiazolidinedionyl GSK3 inhibitors for modulating sperm motility - Google Patents

Abstract

The present invention relates to the use of thiadiazolidinedionyl GSK3 inhibitors for modulating sperm motility. In particular, the invention relates to the use of a compound of formula I, wherein X, Y, R is shown below, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting sperm motility, a medicament for spermicidal, a contraceptive or a contraceptive device^aAnd R^bAs defined herein. The invention also relates to contraceptive devices or topical contraceptive agents containing a compound of formula I or a pharmaceutically acceptable salt thereof. With the contraceptive agent or device of the present invention, a better spermicidal contraceptive effect and less epithelial cytotoxicity can be achieved than with the conventionally used N-9.

Description

Use of thiadiazolidinedione-based GSK3 inhibitors in regulating sperm motility

technical field

The present invention relates to the use of thiadiazolidinedione-based GSK3 inhibitors in regulating sperm motility.

Background technique

In recent years, with the rapid development of society, the economy and culture have been greatly improved, and people's sexual concepts have also undergone great changes. At the same time, the incidence of premarital sex continues to rise, and the incidence of unwanted pregnancy also increases, which has become a worry for many women. How to use safe contraception is already a global problem. Termination of pregnancy has caused great harm to women's mental and physical health; to a certain extent, it has seriously affected women's normal life, and this has become one of the problems that need to be solved urgently in the medical field. Every year, 220 million couples around the world do not have access to effective contraceptive measures, resulting in 80 million unintended pregnancies and 45 million induced abortions, of which 5 million and 70,000 are sicknesses and deaths caused by unsafe abortions, respectively.

In terms of women, some IUDs, condoms, etc. are now used as contraceptive tools, which are easy to use, properly used, good in effect, non-toxic and side effects, and cheap in price, and are favored by many people. But it cannot be used for some people with rubber allergies. Oral contraceptives are likely to cause side effects such as menstrual disorders, leucorrhea increase, and endocrine disorders in women, and are not suitable for some women who suffer from hepatitis, nephritis, diabetes, high blood pressure, heart disease and other diseases; in addition, in general, oral contraceptives The drug should not be taken together with other drugs, resulting in the narrowness of oral contraceptives.

For men, condom use and sterilization have remained the limited contraceptive options for men over the past few decades. Condoms have a long history of use and also protect against sexually transmitted diseases. However, 12% of couples had an unwanted pregnancy within the first year of condom use, indicating a high rate of condom failure. Although the contraceptive failure rate of vasectomy is less than 1%, it takes several months to achieve complete contraception after surgery. After vasectomy, the pregnancy rate is relatively low, and it is easy to produce anti-sperm antibodies, leading to infertility. Therefore, vasectomy is also not an ideal form of contraception. It can be seen that no matter for women or men, the development of non-invasive, reversible, comfortable and safe contraceptives (tools) is urgently needed by society.

Non-hormonal external contraceptives generally do not affect women's endocrine function, and are affordable and easy to use, so they are loved by many women and have been widely used. At present, the most commonly used topical vaginal spermicide at home and abroad is nonoxynol-9 (N-9), which is a non-ionic surfactant that can non-specifically destroy sperm cell membranes and play a spermicidal role. But the shortcoming of nonoxynol (nonoxynol-9, N-9) is that within its effective spermicidal concentration range, it has greater epithelial cytotoxicity, can destroy the integrity of vaginal mucosa, and cause inflammation, and then promote Transmission of sexually transmitted diseases. Phase III clinical trials abroad suggest that N-9 may promote the sexual transmission of HIV. Therefore, WHO issued a report in 2002 to prohibit the use of N-9 in high-risk groups of sexually transmitted diseases. Currently, there is no safer and more effective drug that can replace nonoxynol (nonoxynol-9, N-9) in the world.

GSK3 includes α and β isomers, and there are also differences in amino acid sequences in different species. The report of GSK-3β related to sperm is only found in the meiotic process of rat sperm, and the literature also proves that GSK-3β of different species has different interacting proteins and substrate specificities [Guo TB, Chan KC, Hakovirta H, Xiao Y, Toppari J, Mitchell AP, Salameh WA: Evidence for a role of glycogen synthase kinase-3betain rodent spermatogenesis. J Androl 2003,24(3):332-342〕. Other knowledge about GSK3α, β mainly comes from the reports of species such as mice and cattle. Part of the mouse sperm GSK3α, β came from the testis, and the part came from the epididymis. The majority of GSK3 in mouse epididymal sperm is GSK3α, and it increases significantly during the process of sperm passing through the head, body and tail of the epididymis, while the amount of GSK3β hardly changes [Reid AT, Anderson AL, Roman SD, McLaughlin EA, McCluskey A, Robinson PJ, Aitken RJ, Nixon B: Glycogen synthase kinase 3regulates acrosomal exocytosis in mouse spermatozoa via dynamin phosphorylation. FASEB J2015,29(7):2872-2882〕. Inhibition of GSK3 can reduce progesterone-induced acrosome reaction, but highly selective GSK3 inhibitors, such as CHIR99021 (IC ₅₀ : 6.7nM GSK3β; 10nM GSK3α); A1070722 (Ki: 0.6nM, for both isomers); TWS-119 (IC ₅₀ : 30nM, GSK3β) did not affect the motility and survival of mouse mature sperm [Reid AT, Anderson AL, Roman SD, McLaughlin EA, McCluskey A, Robinson PJ, Aitken RJ, Nixon B: Glycogen synthase kinase 3 regulates acrosomal exocytosis in mousespermatozoa via dynamin phosphorylation. FASEB J 2015,29(7):2872-2882〕.

In mouse sperm, Wnt/GSK3 signaling is involved in sperm maturation in the epididymis. Inhibition of GSK3 kinase activity by Wnt promotes sperm protein stabilization (Wnt/STOP), thereby promoting sperm motility [De Robertis EM, PloperD: Sperm Motility Requires Wnt/GSK3 Stabilization of Proteins.Dev Cell 2015,35(4) :401-402; Koch S, Acebron SP, Herbst J, Hatiboglu G, Niehrs C: Post-transcriptional Wnt Signaling Governs Epididymal Sperm Maturation. Cell 2015,163(5):1225-1236〕. Therefore, it is theoretically speculated that GSK3 inhibitors should promote the increase of sperm motility, rather than reduce or brake or kill sperm motility.

Therefore, there is still a need in the art for a new contraceptive drug with a contraceptive effect equivalent to or better than that of N-9 and with lower side effects.

Contents of the invention

The present disclosure aims to provide a new class of compounds for use as contraceptives. Compared with the traditionally used N-9, the compound has better spermicidal and contraceptive effect and less epithelial cytotoxicity, thereby facilitating the preparation of external spermicidal and contraceptive drugs/tools or sanitary products that women can use independently.

Specifically, the present disclosure provides the use of a compound represented by the following formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting sperm motility, a medicament for killing sperm, a contraceptive or a contraceptive device:

In the formula,

X and Y are each independently selected from O and S; and

R ^a is selected from the group consisting of: alkyl, aryl, aralkyl and -alkylene-CO ₂ -alkyl, wherein the alkyl, aryl and aralkyl are optionally replaced by 1 to 3 members selected from alkane substituents of radical, alkoxy, haloalkyl and halogen; and

R ^b is selected from: alkyl, cycloalkyl, aryl and aralkyl, wherein the alkyl, cycloalkyl, aryl and aralkyl are optionally replaced by 1-3 members selected from alkyl, alkane Substituents of oxy, haloalkyl and halogen are substituted.

In one or more specific embodiments, both X and Y are O.

In one or more specific embodiments, said X is O and Y is S.

In one or more specific embodiments, R ^a is aralkyl optionally substituted with 1-3 substituents selected from alkyl, alkoxy, haloalkyl, and halogen, and R ^b is aralkyl optionally substituted by An aryl group substituted with 1-3 substituents selected from alkyl, alkoxy, haloalkyl and halogen.

In one or more specific embodiments, R ^a is benzyl optionally substituted with 1-3 substituents selected from alkyl, alkoxy, haloalkyl, and halogen; R ^b is benzyl optionally substituted with 1 - naphthyl substituted with 3 substituents selected from alkyl, alkoxy, haloalkyl and halogen.

In one or more specific embodiments, the compound of formula I has the structure shown in the following formula III:

In the formula,

R and R ^are each independently selected from alkyl, alkoxy, haloalkyl, and halogen ^; and

n and m are 0, 1, 2 and 3 each independently.

In one or more embodiments, both n and m are zero.

The present disclosure also provides a contraceptive device, which contains the compound represented by formula I herein or a pharmaceutically acceptable salt thereof.

In one or more specific embodiments, the contraceptive device is a condom, membrane, ring, implant or tampon.

The present disclosure also provides an external contraceptive agent, which contains the compound represented by formula I herein or a pharmaceutically acceptable salt thereof.

In one or more specific embodiments, the external contraceptive agent is a topical gel, tablet, capsule, pessary, cream, paste, jelly, foam, spray, lotion or lubricant.

Description of drawings

Figure 1: After the concentration of Tideglusib and N-9 required for the instant spermicidal effect (EC ₁₀₀ ) of less than 20 seconds, after co-incubating with human sperm for different times, the proportion of motile sperm (A+B+C%) and Proportion of forward motile (A+B%) sperm. Figures A and B are the motile sperm ratio and forward motile sperm ratio of Tideglusib, respectively; Figures C and D are the motile sperm ratio and forward motile sperm ratio of N-9, respectively. In the figure, "CN" refers to blank control.

Figure 2: Human sperm after SYBR-14/PI staining, the integrity of the sperm membrane was observed by fluorescence microscopy.

Figure 3: Comparison of compound N9 (panel A) and Tideglusib (panel B) on the inhibition rate of HeLa cell proliferation.

Detailed ways

The present disclosure relates to the use of GSK3 inhibitors, particularly GSK3β inhibitors, to inhibit sperm motility. GSK3 inhibitors suitable for use in the present disclosure are preferably thiadiazolidinedione derivatives, especially [1,2,4]thiadiazolidine-3,5-dione derivatives.

More specifically, the present disclosure relates to the use of compounds represented by the following formula I or pharmaceutically acceptable salts thereof to inhibit sperm motility and achieve spermicidal and contraceptive effects:

In the formula,

X and Y are each independently selected from O and S; and

R ^a is selected from the group consisting of: alkyl, aryl, aralkyl and -alkylene-CO ₂ -alkyl, wherein the alkyl, aryl and aralkyl are optionally replaced by 1 to 3 members selected from alkane substituents of radical, alkoxy, haloalkyl and halogen; and

R ^b is selected from: alkyl, cycloalkyl, aryl and aralkyl, wherein the alkyl, aryl and aralkyl are optionally replaced by 1-3 members selected from alkyl, alkoxy, haloalk Substituents of radicals and halogens.

Herein, "alkyl" includes straight chain or branched chain alkyl, usually C _1-6 alkyl, preferably C _1-4 alkyl or C _1-3 alkyl, such as methyl, ethyl, n-propyl Base, isopropyl, n-butyl, tert-butyl, isobutyl, etc.

Herein, "alkylene" refers to a divalent group formed after an alkyl group further loses one H atom. Exemplary alkylene groups include but are not limited to methylene (-CH ₂ -), ethylene (-CH ₂ CH ₂ -), etc. The number of carbon atoms in the alkylene group is also usually in the range of 1-6, preferably 1-3.

As used herein, "alkoxy" refers to a group of formula -O-alkyl, wherein alkyl is as previously described. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and the like.

Herein, "cycloalkyl" refers to a saturated carbocyclic ring, usually having 3-8 ring carbon atoms. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

Herein, "aryl" is a monocyclic, bicyclic or tricyclic aromatic group containing 6 to 14 carbon atoms. Useful aryl groups include C _6-14 aryl groups, preferably C _6-10 aryl groups. Typical C _6-14 aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylene and terfenyl. Preferred aryl groups herein are phenyl and naphthyl.

Herein, "aralkyl" refers to a C _1-6 alkyl, C _1-4 alkyl or C _1-3 alkyl substituted by any of the above C _6-14 aryl groups. Exemplary aralkyl groups include, but are not limited to, benzyl, phenethyl, or naphthylmethyl, and the like.

Herein, "halogen" includes fluorine, chlorine, bromine or iodine.

Herein, "haloalkyl" refers to an alkyl group substituted by 1 to 5 halogens, such as trifluoromethyl and the like.

Herein, when substituted, unless otherwise stated, the number of substituents is usually 1, 2, 3 or 4, and the substituents can usually be selected from alkyl, alkoxy, haloalkyl, halogen as described herein , nitro, amino, aryl, acyl (such as C _1-4 acyl) and carboxyl, etc.

Compounds of formula I are GSK3β inhibitors known to be useful in the treatment of diseases involving GSK3, including Alzheimer's disease and non-insulin-dependent diabetes mellitus, see CN 101045715A, the entire content of which is incorporated herein by reference.

More specifically, in the compound of formula I, X is preferably O, and Y is preferably O or S. More preferably, both X and Y are O.

R ^a is preferably optionally substituted alkyl, aralkyl or aryl. More preferably, R ^a is optionally substituted aralkyl. Further preferably, R ^a is optionally substituted benzyl.

^Rb is preferably optionally substituted alkyl or aryl. More preferably, R ^b is preferably optionally substituted aryl. Further preferably, R ^b is preferably optionally substituted phenyl or naphthyl.

In one or more specific embodiments, X is preferably O; Y is preferably O or S; ^Ra is preferably optionally substituted alkyl, ^aralkyl or aryl; R is preferably optionally substituted alkyl or aryl.

In one or more specific embodiments, both X and Y are O; R ^a is optionally substituted aralkyl; R ^b is preferably optionally substituted aryl.

In one or more specific embodiments, both X and Y are O; ^Ra is optionally substituted benzyl; R ^is preferably optionally substituted phenyl or naphthyl.

In one or more of the above specific embodiments of the present disclosure, when substituted, the number of substituents on the alkyl, aryl or aralkyl is preferably one.

In particular, each compound listed in Table 1 of CN 101045715A and each compound with an _IC50 value within 100 μM in Table 3 is included herein. Specifically, these compounds include, but are not limited to:

Table 1

R^a R^b x Y CH₂Ph Me o o Et Me o o Et wxya o o Et Cyclohexane o o Ph Me o o CH₂CO₂Et Me o o 4-OMePh Me o o CH₂Ph Et o o Et iP o o CH₂Ph Et o S CH₂Ph CH₂Ph o S Ph Ph o S Et Et o S 4-MePh Me o o 4-BrPh Me o o 4-FPh Me o o 4-ClPh Me o o

[Small Molecule Inhibitars of Regulatars of G Protein Signaling (RGS) Prateins, ACS Med.Chem.Lett., 2012, 3, 146-150, its entire content herein with incorporated herein by reference], especially compounds 1a-1i, 2a-2i, 3-8, 9a-9b, 10a-10d, 11a-11d, 12a-12d and 13-15 disclosed in Table 1 thereof. These compounds are also included in the scope of the compounds of the present application.

Specifically, these compounds include compounds of the following formula II, wherein R ^a and R ^b are shown in Table 2 below:

Table 2

In Table 2, Bn is benzyl.

In one or more preferred embodiments, the compound of formula I has the structure shown in the following formula III:

In the formula,

R and R ^are each independently selected from alkyl, alkoxy and halogen ^; and

n and m are 0, 1, 2 and 3 each independently.

In a preferred embodiment, both m and n are 0, at this time, the compound of formula I is shown in the following formula, which is Tideglusib (CAS: 865854-05-3):

Herein, examples of pharmaceutically acceptable salts include, but are not limited to, inorganic acid salts and organic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, Maleate, fumarate, mandelate, and oxalate; and with bases such as sodium hydroxy, tris(hydroxymethyl)aminomethane (TRIS, tromethamine), and N-methylglucamine Inorganic and organic base salts formed.

As described in CN 105263472 A (the entire content of which is incorporated herein by reference), the compound of formula I herein or a pharmaceutically acceptable salt thereof can be formulated into various topical drug combinations known in the art and suitable for spermicidal contraception substances, including but not limited to dosage forms suitable for vaginal administration, preferably external dosage forms, such as gels, tablets, capsules, pessaries, creams, pastes, jellies, foams, sprays, lotions and the like.

Alternatively, the compound, or a pharmaceutically acceptable salt thereof, or the topical pharmaceutical composition may be applied to a contraceptive device, such as a condom, membrane, ring, implant, or tampon, or the like. For example, a condom coated with a compound described herein or a pharmaceutically acceptable salt thereof, or a topical pharmaceutical composition thereof, such as a gel, cream, paste, foam, jelly, etc. Topical pharmaceutical composition.

In the topical pharmaceutical composition, the content of the compound of formula I of the present invention is usually in the range of 0.1wt% to 10wt%, such as 0.1wt% to 5.0wt%, 0.1wt% to 3.0wt%, 0.1 to 1.0wt%, etc. within the range.

The topical pharmaceutical composition may also contain other agents/drugs commonly used in contraceptives known in the art such as antiretroviral agents such as protease inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and integrase inhibitors. These agents/drugs can be added to the topical pharmaceutical compositions of the present invention in amounts well known in the art.

For example, suitable protease inhibitors useful in the topical pharmaceutical compositions of the present invention may include saquinavir; ritonavir; nelfinavir; amprenavir; Zanavir; Lacinavir; Palinavir; Tipranavir; Fosamprenavir; Darunavir; N-cycloalkylglycine, α-hydroxyarylbutanamide; α-hydroxy-γ- [(carbocyclic or heterocyclic substituted)amino)carbonyl]alkanamide derivatives; γ-hydroxy-2-(fluoroalkylaminocarbonyl)-1-piperazine valeramides; dihydropyrone derivatives and α - and β-amino acid hydroxyethylsulfamate; and N-amino acid substituted L-lysine derivatives.

Suitable nucleoside reverse transcriptase inhibitors that may be used in the pharmaceutical composition of the present invention may include zidovudine; didanosine; stavudine; lamivudine; abacavir; adefovir; Lobucavir; Entecavir; Aritabine; Emtricitabine; Zalcitabine; Dexevutabine; Alovudine; Amdosovir; Eftabine; Phosphine azide; β-L -2',3'-dideoxy-5-fluorocytidine; purine nucleoside; and Lordadenosine.

Suitable non-nucleotide reverse transcriptase inhibitors that can be used in the pharmaceutical composition of the present invention may include nevirapine, rilpivirine, delavirdine, efavirenz, etravirine, furopyridine-thiopyrimidine; Capvirine; 5-(3,5-dichlorophenyl)-thio-4-isopropyl-1-(4-pyridyl)methyl-1H-imidazol-2-ylmethyl carbonate; 1-(ethoxymethyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione; coumarin derivatives; Dapivirine.

Suitable integrase inhibitors that can be used in the pharmaceutical composition of the present invention may include raltegravir, eltgravir.

The topical pharmaceutical compositions herein may be in the form of controlled release formulations, delayed release formulations, extended release formulations, pulsed release formulations, mixed immediate and controlled release formulations, and the like. The topical pharmaceutical compositions are formulated so as to release the active ingredient at a rate that produces an effective concentration at the site of application.

It should be understood that the pH of the topical pharmaceutical composition can be changed by those skilled in the art as required, preferably having a pH of 3.0-5.5, more preferably between 3.5 and 5.0.

The topical pharmaceutical composition is preferably lubricious to facilitate sexual intercourse to ensure user compliance.

The topical pharmaceutical compositions herein may also comprise one or more pharmaceutically acceptable excipients. Suitable excipients that may be used in the topical pharmaceutical composition include, but are not limited to, gelling agents, chelating agents, preservatives, bioadhesives or polymers, viscosity modifiers or regulators, wetting agents/lubricants, Surfactants, pH adjusters, solvents/co-solvents and tonicity adjusters or osmotic agents. The various excipients mentioned above can be as described in CN105263472A, the entire content of which is incorporated herein by reference.

For example, gelling agents that can be used in the topical pharmaceutical compositions described herein include, but are not limited to: xanthan gum, sodium alginate, ETD2020, polycarbophil, polysaccharides, natural gums, acacia, tragacanth, starch, fibers Vein derivatives such as carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, ethylcellulose and hydroxyethylmethylcellulose , Methacrylate Polymer, Polyvinylpyrrolidone, Bentonite, Alginic Acid, Carbomer, Gelatin, Guar Gum, Glyceryl Behenate, Algae Extract, Gum, Polyethylene Oxide, Poloxamer , pectin, hydrolyzed protein, polymers containing pendant carboxylic acid groups or their esters, polymers of anhydrides containing pendant dicarboxylic acid groups and blocks based on ethylene oxide and/or propylene oxide Copolymers etc. or mixtures thereof. The gelling agent may comprise from about 0.05% to 10% of the total weight of the topical pharmaceutical composition.

Chelating agents that can be used in topical pharmaceutical compositions include, but are not limited to, disodium edetate, sodium condensed phosphate, diethylenetriaminepentaacetic acid, and the like, or combinations thereof. Chelating agents may comprise from about 0.01% to 1% of the total weight of the topical pharmaceutical composition.

Preservatives that may be used in the topical pharmaceutical composition include, but are not limited to: hydroxybenzoates (such as parabens, e.g. methylparaben, propylparaben), benzyl alcohol, benzene Formic acid, chlorphenesin, sorbic acid, phenoxyethanol, etc. or combinations thereof. Preservatives may comprise from about 0.05% to 2% of the total weight of the topical pharmaceutical composition.

Bioadhesives or polymers that may be used in the topical pharmaceutical composition include, but are not limited to, hydroxyethylcellulose, gelatin, carbopol, polycarbophil, cross-linked polymethacrylic acid, hydroxypropylmethylcellulose Hydroxypropyl cellulose, ethyl cellulose, polyethylene glycol, polysaccharide hyaluronic acid, polyvinylpyrrolidone, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, starch, etc. or combinations thereof. Bioadhesives or polymers may comprise from about 0.05% to 2% of the total weight of the topical pharmaceutical composition.

Wetting agents and/or lubricants provide smoothness and lubricity, thereby facilitating filling and dispensing of the topical pharmaceutical composition. Wetting agents and/or lubricants that may be used in the topical pharmaceutical composition include, but are not limited to, polyols such as glycols, and polysaccharides such as glycerin, ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, dipropylene glycol , Diglycerin, Sorbitol, Maltitol, Trehalose, Raffinose, Xylitol, Mannitol, Polyethylene Glycol, Propylene Glycol, Polyglycerin, Cholesterol, Squalene, Fatty Acid, Octyldodecanol, Myristyl alcohol, urea, lanolin, lactic acid, esters such as isopropyl stearate, isopropyl myristate, isopropyl palmitate and isopropyl laurate, etc. or combinations thereof. Wetting agents and/or lubricants may comprise from about 2% to 20% of the total weight of the topical pharmaceutical composition.

Viscosity modifiers or regulators improve gel formation. Suitable viscosity modifiers or modifiers that may be used in the topical pharmaceutical composition include, but are not limited to, polyethylene, polypropylene, polyalphaolefins, ethylene-propylene copolymers, polyisobutylene, maleic anhydride and its diene derivatives , polymethacrylate, maleic anhydride-styrene copolymer, hydrogenated copolymer of styrene-butadiene, hydrogenated styrene-isoprene polymer, hydrogenated isoprene polymer, polyacrylate, Polyalkylstyrenes, alkenyl aryl conjugated diene copolymers, esters of maleic anhydride-styrene copolymers, ethylene-propylene copolymers functionalized with reaction products of maleic anhydride and amines, functionalized with amines Polymethacrylates, styrene-maleic anhydride copolymers reacted with amines, polymethacrylate polymers, vinyl aromatic monomers and esterified polymers of unsaturated carboxylic acids or their derivatives or combination. Viscosity modifiers or regulators may be present in an amount from about 0.05% to 2% by weight of the total composition.

Suitable tonicity or osmotic agents to match the osmolarity of physiological fluids include, but are not limited to, glycerin, sodium chloride, potassium chloride, mannitol, sucrose, lactose, fructose, maltose, glucose, anhydrous dextrose, propylene glycol , glycerin, etc. or combinations thereof. Tonicity modifiers or penetrants may be present in an amount of about 0.001% to 1% by weight of the composition.

Surfactants may include, but are not limited to, one or more of the following: cocoamide, polysorbate, sodium lauryl sulfate, lauryl dimethylamine oxide, docusate sodium, cetyl trimethylammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynyl alcohol, N,N-dimethyldodecylamine-N-oxide, Hexaalkyltrimethylammonium Bromide, Polyoxyethylene 10 Lauryl Ether, Brij, Bile Salts (Sodium Deoxycholate, Sodium Cholate), Polyoxyethylene Castor Oil, Nonylphenol Ethoxylate, Cyclodextrin , lecithin, methylbenzethonium chloride, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated Natural oils and fats, sulfated esters, sulfated alkanolamides, ethoxylated and sulfated alkylphenols, ethoxylated aliphatic alcohols, polyoxyethylene surfactants, carboxylates, polyethylene glycols Alcohol esters, sorbitan esters and their ethoxylated derivatives, glycol esters of fatty acids, carboxamides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines with amide bonds, poly Oxyethylene alkylamines and polyoxyethylene alkylcycloaliphatic amines, N,N,N,N-tetrasubstituted ethylenediamines, 2-alkyl-1-hydroxyethyl-2-imidazolines, N-coco Oleyl-3-alanine/sodium salt, N-tallow-3-iminodipropionic acid disodium salt, N-carboxymethyl-n-dimethyl-n-9-octadecenyl hydroxide Ammonium, n-cocamidoethyl-n-hydroxyethylglycine sodium salt, etc. or a combination thereof. Surfactants may comprise from about 0.05% to 20% of the total weight of the topical pharmaceutical composition.

Suitable pH adjusters or buffers that may be used in the topical pharmaceutical compositions described herein include, but are not limited to, acidifying agents (e.g., hydrochloric acid, acetic acid, citric acid, tartaric acid, propionic acid), sodium hydroxide, sodium phosphate, ammonia solution, tris Ethanolamine, sodium borate, sodium carbonate, potassium hydroxide, etc. or combinations thereof. pH adjusters or buffers may comprise from about 0.1% to 2% of the total weight of the composition.

Solvents/cosolvents, solubilizers or vehicles useful in the topical pharmaceutical compositions described herein include, but are not limited to, water, glycerin, cocoyldiethanolamine, medium and/or long chain fatty acids or glycerides, monoglycerides , Diglycerides, Triglycerides, Structured Triglycerides, Soybean Oil, Peanut Oil, Corn Oil, Corn Oil Monoglycerides, Corn Oil Diglycerides, Corn Oil Triglycerides, Polyethylene Glycol, Capryl Caproyl Macroglycerides, Caproyl90, Propylene Glycol, Polyoxyethylene Sorbitan Fatty Acid Ester, Polyoxyethylene Castor Oil Derivatives, Castor Oil, Cottonseed Oil, Olive Oil, Safflower Oil, Peppermint Oil, Coconut Oil, Palm Seed oil, beeswax, oleic acid, methanol, ethanol, isopropanol, butanol, acetone, methyl isobutyl ketone, methyl ethyl ketone or mixtures thereof. Solvents/co-solvents, solubilizers or vehicles may comprise from about 0.05% to 20% by weight of the total topical pharmaceutical composition.

Another example of a gel composition can be found in CN 101559036, the entire content of which is incorporated herein by reference. For example, the gel composition of the present invention may be a gel composition containing a buffer, a bioadhesive, water, a phase change temperature regulator, and one or more of the following poloxamers Species: poloxamer 237, poloxamer 338 and poloxamer 407; the content of the poloxamer is 15wt%～30wt%; the content of the phase change temperature regulator is 1.5wt%～ 6.75 wt%; the mass ratio of the poloxamer to the phase change temperature regulator is 20:1-20:9; the pH of the vaginal gel composition is 3.0-5.5. The content of the compound of formula I of the present invention in the gel composition can be in the range of 0.1wt% to 10.0wt%, such as 0.1wt% to 3.0wt%, 0.1wt% to 1.0wt%, etc.

The topical pharmaceutical compositions and contraceptive devices herein can be prepared using methods conventional in the art. For example, different excipients can be selected according to different dosage forms, and then according to the physicochemical properties of each excipient, different excipients and the compound of formula I can be mixed together or separately, so that a topical pharmaceutical composition with a corresponding dosage form can be prepared. The contraceptive device described herein can be obtained by applying or smearing the pharmaceutical composition of the invention to a conventional contraceptive device.

It will be understood that the term "topical" as used herein includes application to body cavities as well as the skin.

Also provided herein are methods of contraception by administering the topical pharmaceutical compositions described herein.

Also provided herein is the use of the compound described in formula I or a pharmaceutically acceptable salt thereof, including use as a contraceptive, use for inhibiting sperm motility, spermicidal or contraceptive use, and for preparing a drug for inhibiting sperm motility Potential agents, agents used to kill sperm, use of contraceptive pills or contraceptive devices.

It should be understood that the various medicaments, drugs, compounds, devices, methods and uses herein are applicable to various animals, especially mammals, such as humans, that need to inhibit sperm motility, spermicide or contraception.

The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are only illustrative, not limiting the scope of protection herein. The methods, instruments and reagents used in the examples are conventional methods, instruments and reagents in the art unless otherwise stated.

Example

Materials and Reagents

Sperm culture medium: 25ml 10×BWW, 875mg BSA, 7.5mg sodium pyruvate, 525mg sodium bicarbonate, 250mg D-glucose, 0.925ml sodium lactate, dilute to 250ml with third-grade ultrapure water, mix thoroughly, filter with 0.2μM Membrane filtration, stored at 4°C.

Incomplete DMEM medium: Pour a bag of DMEM medium powder into a bottle, add 3.5g sodium bicarbonate, add third-grade water to 1000ml, fully shake and dissolve, and filter with a 0.2μm filter membrane for later use.

DMEM complete medium: Take 450ml of DMEM incomplete medium, add 45ml of fetal bovine serum and 5ml of antibiotics and mix well.

Freezing medium: fetal bovine serum + 10% DMSO.

Cell Proliferation/Toxicity Assay Kit CCK-8: China Shanghai Biyuntian Biotechnology Co., Ltd.

SYBR-14/PI Cytotoxicity Detection Kit: American molecular probe company.

CASA system (computer-aided semen analysis system): Cyto-S, VideoTesT Co., Saint-Petersburg, Russia.

Olympus IX3 inverted and Olympus BX3 upright microscope systems: Olympus Corporation.

Tideglusib: Selleck China Company.

Other GSK3 inhibitors were prepared according to the methods disclosed in CN 101045715A.

Example 1, the compound Tideglusib regulates the motility of human sperm

The standard of semen used in this study is: the total number of sperm is 60×10 ⁶ -100×10 ⁶ , the concentration of semen is >20×10 ⁶ /ml, and the motility rate of sperm is >60%.

Sperm motility assays were performed under a microscope with a heated 37°C stage, and samples were prepared using preheated cytometers. Sperm motility is divided into grades a, b, c, and d according to the following criteria: a, fast forward movement: sperm actively move in a straight line or along a large circle, and the speed is extremely fast; b, slow or sluggish forward movement Movement: Sperm actively move in a straight line or along a large circle, and the speed is relatively slow; C, non-forward movement: all other forms of non-forward movement, such as swimming in a small circle, the tail power can hardly drive the head to move, Or only tail wagging can be observed; d, immobile: no movement. See World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical ^Mucus Interaction, 4th Ed., Cambridge University Press, Cambridge.

1.1 Acquisition of high motility sperm

High motility spermatozoa were obtained using the sperm upstream method. Add 500 μl of liquefied semen sample under 1000 μl of sperm culture solution and incubate for 1 h at 37°C in a 5% CO ₂ incubator. Take 600μl of culture fluid from the upper layer to obtain high-motility sperm, and adjust the sperm concentration to 5-10×10 ⁶ /ml with sperm culture fluid.

1.2 Screening and effects of rapid braking/spermicidal compounds

Use the method shown in 1.1 to obtain high-motility sperm from the collected semen, and adjust the sperm concentration to 10-20×10 ⁶ /ml. The compounds/drugs were diluted with sperm culture fluid on a 96-well plate at room temperature by doubling ratio, 50 μl per well. After the dilution was completed, the 96-well plate was placed in a 5% CO ₂ incubator at 37°C for 30 min. Another 50 μl of semen was drawn and dropped into this hole, and the stopwatch was immediately counted, and the pipette was blown 5 times at a uniform speed to mix well. Observe the motility of sperm under a microscope (×200), and calculate whether all sperm lose motility at this drug concentration within 20 seconds. Add well by well, and finally measure the minimum concentration of drug required to inactivate all sperm in 20s. Each test compound/drug concentration was tested three times consecutively for each semen sample, and the final results were averaged.

Through the screening of more than 5,000 compounds and the analysis of the sperm immobilization effect, we found that Tideglusib has the smallest effective concentration of sperm immobilization. The minimum effective concentration of Tideglusib's 20-second instant spermicidal effect (EC ₁₀₀ , that is, the lowest drug concentration that can immobilize sperm 100% in 20 seconds) is about 6.5±1.3μM (1.74μg/ml～2.61μg/ml), while the reference The EC100 of N-9 is _about 259 μM (160 μg/ml). Experimental results show that Tideglusib has a lower minimum effective concentration for instant spermicide than N-9.

In order to investigate the immobilization effect of low concentrations of compounds on sperm, low concentrations of Tideglusib and N-9 were co-incubated with human sperm. Use the method shown in 1.1 to obtain high-motility sperm from the collected semen, and adjust the sperm concentration to 10-20×10 ⁶ /ml. The compounds/drugs were diluted with sperm culture fluid on a 96-well plate at room temperature by doubling ratio, 50 μl per well. After the dilution was completed, the 96-well plate was placed in a 5% CO ₂ incubator at 37°C for 30 min. Another 50 μl of semen was drawn and dropped into the well, and incubated for 30 minutes, 60 minutes, 120 minutes, and 180 minutes, respectively, and the CASA system quantitatively measured the sperm motility parameters.

The measurement results of motile sperm ratio and forward motile sperm ratio after sperm were treated with different concentrations of Tideglusib and N-9 are shown in Figure 1, respectively. Tideglusib inhibited sperm motility in a dose-dependent manner, mainly affecting the proportion of forward motile sperm. SYBR-14/PI staining fluorescence microscope observation found that increasing the concentration of Tideglusib can lead to sperm death (PI staining positive, SYBR-14 negative), membrane permeabilization, that is, Tideglusib has a significant spermicidal effect. See Figure 2.

Example 2, Tideglusib has lower epithelial cell proliferation inhibition/cytotoxicity compared with N-9

2.1 Recovery of Hela cells

Quickly take out the cryopreservation tube containing the cells frozen in the liquid nitrogen tank, and put it into warm water close to 40°C to thaw quickly, and the best time is controlled within 1-2min. Transfer all the Hela cell cryopreservation solution contained in the cryopreservation tube into a 15ml centrifuge tube. After blowing evenly, put it in a centrifuge at 1000rpm and centrifuge for 4min. After centrifugation, discard the supernatant, then add 3ml of complete medium, and gently pipette and mix until ready to use. Aspirate the evenly mixed cell fluid into a 10cm petri dish, then add 8ml of DMEM complete medium to the petri dish, gently pipette and mix well, put it in a 37°C, 5% _CO2 incubator for cultivation, and replace it with a fresh one the next day Culture medium continued to cultivate. Subculture the cells in the original dish at a ratio of 1:3, mix well, and culture in a 37°C, 5% CO ₂ incubator.

2.2 Cell Proliferation Inhibition/Toxicity Experiment

Take the Hela cell culture dish in the logarithmic growth phase, wash it twice with PBS, add 1ml 0.25% trypsin to digest, observe under a high-power microscope that the digestion has been completed, quickly add DMEM complete medium to stop the digestion, and absorb a little cell suspension in the blood cells Count the cells on a counting plate and adjust the single cell suspension to 1×10 ⁵ /ml. The prepared Hela cell suspension was inoculated in a 96-well culture plate, 100 μl per well, and 5 parallel wells were set up in each group. The outermost circle of wells was replaced with PBS instead of cell suspension to prevent edge effects. Affects cell state and experimental results. Place the 96-well plate with the cells in an incubator with 5% CO ₂ at 37°C for 24 hours to make the cells adhere to the wall. The next day, 100 μl of fresh complete culture medium containing compound/drug (N-9 or other compound/drug) was replaced, and the cultivation was continued. After adding the compound/drug-containing complete culture solution, continue to cultivate for 24 hours, discard the medium, add 100 μl of serum-free medium, and then add 10 μl of CCK-8 reagent to each well. It is best to add it quickly to avoid CCK-8 remained on the tip of the pipette. After adding, lightly hit the 96-well plate to distribute CCK-8 evenly in the wells. After standing still for 30 minutes, put the 96-well plate added with CCK-8 into an incubator at 37° C. and 5% CO ₂ to continue culturing for 1-4 hours. Hela cells generally choose 2 hours as the test point. The OD value was determined by the enzyme label method. Before the test, the microplate reader was preheated for 30 minutes, and then the test was performed. The measurement wavelength is 450 nm. By measuring the OD value, the cell survival rate and IC ₅₀ value were obtained. Calculate the inhibition rate of cells according to the following formula:

Cell inhibition rate (%)=[(AC-Ab)-(AS-Ab)]/(AC-Ab)×100%

AS: average value of experimental wells (medium containing cells, CCK-8, toxic substances);

AC: average value of control wells (medium containing cells, CCK-8, no toxic substances);

Ab: average value of blank wells (medium without cells and toxic substances, CCK-8).

_IC50 is the compound/drug concentration required for 50% inhibition. The results are shown in Figure 3, N-9 and Tideglusib on the proliferation inhibition rate of epithelial cell HeLa cell line is about IC _50N-9 : 6 μ M, IC _{50 Tideglusib} : 30 μ M. Therapeutic index (TI) is the safety index of a drug, usually half toxic dose (TD50)/half effective dose (ED50) or half lethal dose (LD50)/half effective dose (ED50) is called therapeutic index. Here, TI was estimated by IC ₅₀ /EC ₁₀₀ , TI _N-9 =0.023; TI _Tideglusib =3.85-5.76. It can be seen that Tideglusib has a larger TI value than N-9, indicating that Tideglusib has better safety than N-9.

It is known that N-9 inhibits cell proliferation by disintegrating mucosal epithelial cells through detergent-like activity, while Tideglusib exhibits completely different kinetic characteristics, suggesting that Tideglusib inhibits the proliferation of HeLa cell lines through a pathway different from that of N-9. SYBR14/PI fluorescence staining analysis showed that the contact of N-9 with HeLa cells directly led to cell membrane permeabilization, while the contact of Tideglusib with HeLa cells did not lead to cell membrane permeabilization or cell disintegration of HeLa cells. Therefore, the cytotoxicity of N-9 manifests as direct cell membrane destruction, while Tideglusib only manifests as inhibition of epithelial cell proliferation.

2.3 Discussion

Known as an irreversible, non-ATP competitive GSK-3β inhibitor (IC50: 60nM), Tideglusib has entered the market as a drug for the treatment of Alzheimer disease and progressive supranuclear palsy (PSP) Phase II clinical trials [Dominguez JM, Fuertes A, Orozco L, delMonte-Millan M, Delgado E, Medina M: Evidence for irreversible inhibition of glycogen synthase kinase-3beta by tideglusib. J Biol Chem2012, 287(2): 893-904].

The clinical safety of Tideglusib has been verified by clinical trials. In small-scale clinical trials, the oral dose of patients was increased to 1000 mg per day, which still showed good safety and tolerance, and adverse reactions were not different from those in the control group [del Ser T, Steinwachs KC, Gertz HJ, Andres MV, Gomez-Carrillo B, Medina M, Vericat JA, Redondo P, Fleet D, Leon T: Treatment of Alzheimer's disease with the GSK-3 inhibitor tideglusib: a pilot study. J Alzheimers Dis 2013,33(1):205-215〕. A larger multinational, double-blind, controlled phase II clinical trial found that for patients with progressive supranuclear palsy, Tideglusib reduced the progression of brain atrophy, especially in the parietal and occipital lobes [Hoglinger GU, Huppertz HJ, Wagenpfeil S, Andres MV, Belloch V, Leon T, Del Ser T: Tideglusib reduces progression of brain atrophy in progressive supranuclear palsy in arandomized trial. Mov Disord 2014,29(4):479-487〕. Another randomized double-blind controlled trial of 146 patients with mild and moderate progressive supranuclear palsy (PSP) with a period of 52 weeks showed that Tideglusib was safe and well tolerated [Tolosa E, Litvan I, Hoglinger GU, Burn D, Lees A, Andres MV, Gomez-Carrillo B, Leon T, Del Ser T: A phase 2trial of the GSK-3 inhibitor tideglusibin progressive supranuclear palsy.Mov Disord 2014,29(4):470-478〕. Clinical trials for the treatment of Alzheimer's disease in Europe also proved the safety of Tideglusib, although the benefit of Tideglusib in clinical treatment of Alzheimer's disease was not observed [Lovestone S, Boada M, Dubois B, Hull M, Rinne JO, HuppertzHJ, Calero M, Andres MV, Gomez-Carrillo B, Leon T et al: A phase II trial oftideglusib in Alzheimer's disease. J Alzheimers Dis 2015,45(1):75-88〕.

Therefore, whether it is for women or men, Tideglusib is expected to be developed into a non-invasive, reversible, comfortable and safe contraceptive drug (tool).

The preparation and test of embodiment 3, Tideglusib gel

Prepare Tideglusib gel with reference to CN 101559036A. The acid of the strong buffer is dissolved in water, and the alkali of the metal cation contained in the human body is added under stirring conditions such as (one or more in sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate, preferably hydrogen sodium oxide) to adjust the pH value to 3.0-5.5, more preferably to 3.3-3.6; add one or more of the phase change temperature regulator and poloxamer, stir evenly, and store at 0-8°C for 12-24 hours, add the bioadhesive and stir for 30-180 minutes until a homogeneous gel is obtained.

Specifically, the drug-loaded gel of this example can be prepared with reference to Example 2 of CN 101559036A. The proportions of the components of the drug-loaded gel are as follows: 20% poloxamer 407; 3% poloxamer 188; 4% lactic acid; 1% citric acid; 0.4% potassium hydrogen tartrate; 0.2% benzoic acid; 8% glycerol; Sodium Lactate, Sodium Citrate, Sodium Tartrate, Sodium Potassium Tartrate, Sodium Benzoate (the content of the above salts is the amount required to form pH3.5 with the corresponding acid), 0.8% Carbopol 934; the water content is to make up 100%.

The dissolved Tideglusib and nonoxynol were respectively uniformly dispersed in the drug-loaded gel prepared above to obtain 4% nonoxynol gel and 0.4% Tideglusib gel.

Rabbits were divided into random groups, 3 in each group, and administered after one week of adaptation. The above-mentioned 4% nonoxynol gel was used as a positive control, 0.9% sodium chloride was used as a blank control, and 0.4% tideglusib gel was used as a drug group. After 10 minutes of vaginal administration, the female rabbits in each group were mated with male rabbits known to be fertile at a ratio of 1:1, and the semen was taken out from the vagina of the female rabbits at 5, 10, 15, and 20 minutes after mating, and examined under a microscope. Observe sperm viability. Observe more than 10 fields of view, one or more motile sperm is recorded as "+", and no motile sperm is recorded as "-". The results are shown in Table 3 below.

Continue to raise, 15-20 days after mating, kill by air embolism, check by laparotomy whether there is conception. The results are shown in Table 4 below.

Table 3: Observation record of spermicidal effect in rabbits

Table 4: Observation record of contraceptive effect in rabbits

This example proves that Tideglusib has a good contraceptive effect.

Embodiment 4, other compounds regulate the method test of human sperm motility

Prepare and purify the compound shown in Table 1 of this application according to the method disclosed in CN 101045715A, and prepare the compound 1a-1i in Table 1 according to EmmaM.Turner et al [ACS Med.Chem.Lett., 2012, 3, 146-150] , 2a-2i, 3-8, 9a-9b, 10a-10d, 11a-11d, 12a-12d, and 13-15.

According to the description in Example 1 of the present application, the compound prepared above was tested to regulate the motility of human sperm. Specifically, the collected semen was obtained by the method shown in 1.1 of Example 1 to obtain high-motility sperm, and the sperm concentration was adjusted to 10-20×10 ⁶ /ml. At room temperature, the aforementioned compounds were diluted with sperm culture fluid on a 96-well plate in a ratio of 50 μl per well. After the dilution was completed, the 96-well plate was placed in a 5% CO ₂ incubator at 37°C for 30 min. Another 50 μl of semen was drawn and dropped into this hole, and the stopwatch was immediately counted, and the pipette was blown 5 times at a uniform speed to mix well. Observe the motility of the sperm under a microscope (×200), and measure whether all the sperm lose their motility under the concentration of the compound within 20s. Add wells one by one, and finally measure the minimum concentration of the compound required to inactivate all spermatozoa within 20 seconds. The concentration of each test compound was detected three times consecutively for each semen sample, and the final results were taken as the average value.

Thiadiazolidinediones are known to be used as GSK3β inhibitors (CN 101045715A) or RGS inhibitors (Emma M. Turner et al., ACS Med. Chem. Lett., 2012, 3, 146-150). However, RGS proteins are known to be expressed in a tissue-specific manner, and in particular RGS4, the most well-studied, is ubiquitously expressed in the central nervous system and rarely expressed in peripheral tissues (Emma M. Turner et al., supra). In addition, RGS22 has only been found to be expressed in testicular germ cells and Leydig cells in humans and mice so far, and may be involved in spermatogenesis and meiosis. Besides RGS22, no other RGS proteins have been found in mature sperm, and RGS22 Known functions were not found to be related to sperm motility. Therefore, it is reasonable to expect that Tideglusib acts as a GSK3β inhibitor through the GSK3 pathway to act as a spermicidal contraceptive. Therefore, it is believed that other thiadiazolidinedione compounds as GSK3β inhibitors, especially the compounds prepared and tested in this example can also play a spermicidal and contraceptive role.

Claims (6)

1. The compound represented by the following formula III or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting sperm motility, a medicament for killing sperm, a contraceptive or a contraceptive device: In the formula, R and R are each independently selected from C ^1-4 alkyl, C _{1-4 alkoxy, haloC 1-4} ^alkyl _and halogen; and n and m are 0, 1, 2 or 3 each independently. 2. purposes as claimed in claim 1, is characterized in that, described formula III compound has following formula structure: 3. A contraceptive device containing the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof. 4. The contraceptive device of claim 3, wherein the contraceptive device is a condom, membrane, ring, implant or tampon. 5. An external contraceptive agent comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof. 6. external contraceptive medicament as claimed in claim 5, is characterized in that, described external contraceptive medicament is external gel, tablet, capsule, vaginal suppository, cream, paste, jelly, foam, spray, lotion or lubricant.