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 thiadiazolidinedionyl GSK3 inhibitors for modulating sperm motility

Technical Field

The present invention relates to the use of thiadiazolidinedionyl GSK3 inhibitors for modulating sperm motility.

Background

In recent years, with the rapid development of society, the economy and culture are improved, and the sexual concept of people is also changed greatly. Meanwhile, the incidence of prenatal sexual behavior is increasing, and the incidence of involuntary pregnancy is increasing, which is an annoyance for many women. How to safely control contraception has been a global problem. The termination of pregnancy causes great harm to the psychological and physiological health of women; the normal life of the female is seriously influenced to a certain extent, which is one of the problems to be solved urgently in the medical field. Worldwide, 2.2 million couples do not receive effective contraceptive measures each year, resulting in 8000 million accidental pregnancies and 4500 million induced abortions, of which 500 and 7 million cases of illness and death, respectively, are due to unsafe abortions.

In the aspect of female, a plurality of intrauterine devices, condoms and the like are used as contraceptive tools, and the contraceptive device has the advantages of simple and convenient use, proper use, good effect, no toxic or side effect and low price, and is favored by a plurality of people. But not for some people with rubber allergy. Oral contraceptives are liable to cause side effects such as menstrual disorder, leukorrhagia, endocrine dyscrasia and the like in women, and are not suitable for some women suffering from diseases such as hepatitis, nephritis, diabetes, hypertension, heart disease and the like; in addition, oral contraceptives are generally not suitable for co-administration with other drugs, which causes a constriction in oral contraceptives.

For men, the use of condoms and ties has remained a limited alternative contraceptive modality for men over the past few decades. The condom has long use history and can also prevent sexually transmitted diseases. However, 12% of couples will develop involuntary pregnancy within the first year of condom use, indicating a higher rate of condom failure. While the failure rate of contraception by vasectomy is less than 1%, complete contraception can be achieved only after a few months. After vasovasostomy, pregnancy rates are low and anti-sperm antibodies are easily produced, resulting in infertility. Therefore, vasoligation is not an ideal contraceptive modality. It is seen that there is an urgent need in society to develop non-invasive, reversible, highly comfortable, safe contraceptive agents, whether for women or men.

The non-hormone external contraceptive medicament generally does not affect the endocrine function of women, has low price and simple and convenient use method, is popular with many women and has been widely applied. The most commonly used vaginal spermicide at home and abroad at present is nonoxynol-9 (N-9), which is a nonionic surfactant and can destroy sperm cell membranes non-specifically to play a spermicidal role. However, nonoxynol-9, N-9 has the disadvantage of greater epithelial cytotoxicity within its effective spermicidal concentration range, can destroy the integrity of vaginal mucosa, and cause inflammation, thereby promoting the transmission of sexually transmitted diseases. Foreign phase III clinical trials suggest that N-9 may promote the sexual transmission of HIV. Therefore, the WHO published a report in 2002 prohibiting N-9 from being used in high risk groups of sexually transmitted diseases. Currently, no safer and more effective medicine capable of replacing nonoxynol-9 (N-9) is available internationally.

GSK3 includes both α and β isomers, and also differs in amino acid sequence among different species. GSK-3 beta is only reported in connection with the meiotic process in rat sperm and this document also demonstrates the differenceGSK-3 β of species have different interacting protein and substrate specificities [ Guo TB, Chan KC, Hakovirta H, Xiao Y, Toppari J, Mitchell AP, Salame WA: evolution for a roll of glycogen synthases kinase-3beta condensation hybridization. J android 2003,24(3):332-342 ]. Other knowledge about GSK3 α, β mainly comes from reports of species such as mouse and cow. Mouse sperm GSK3 alpha and beta are derived partly from testis and partly from epididymis. GSK3 of mouse epididymal sperm is mostly GSK3 α and increases significantly in the process of passing sperm through the head, body and tail of epididymis, while the amount of GSK3 β is almost unchanged [ Reid AT, Anderson AL, Roman SD, McLaughlin EA, McCluskey A, Robinson PJ, Aitken RJ, Nixon B: Glycogen synthase 3 regulated endogenous metabolism in mouse sperm motility a vision dynamin phosphorylation. EB J2015,29(7):2872-2882 ]. Inhibition of GSK3 may reduce progesterone-induced acrosome responses, but highly selective GSK3 inhibitors, such as CHIR99021 (IC)₅₀: 6.7nM GSK3 β; 10nM GSK3 α); a1070722 (Ki: 0.6nM, for both isomers); TWS-119 (IC)₅₀: 30nM, GSK3 β) does not affect the motility and survival of mature spermatozoa in mice [ Reid AT, Anderson AL, Roman SD, McLaughlin EA, McCluskey A, Robinson PJ, Aitken RJ, Nixon B: Glycogen synthase 3 enzymes involved in dysosomal exocytosis in mosespermatozoa via dynamin phosphorylation. FASEB J2015,29(7):2872-2882 ].

In mouse sperm, Wnt/GSK3 signaling is involved in sperm maturation in the epididymis. Wnt inhibition of GSK3 kinase activity promotes Sperm protein Stabilization (Wnt/STOP) and thus Sperm Motility [ De Robertis EM, ploverD: farm Motility Wnt/GSK3 Stabilization of proteins. Dev Cell 2015,35(4): 401-402; koch S, Acebron SP, Herbst J, Hatibogllu G, Niehrs C Post-translational Wnt Signaling Governs epidemic term preservation. cell 2015,163(5):1225-1236 ]. Therefore, it is theorized that inhibitors of GSK3 should promote increased sperm motility, rather than reduce or arrest or kill sperm motility.

thus, there remains a need in the art for new contraceptive agents that are comparable or better than N-9 contraceptive efficacy, but with fewer side effects.

Disclosure of Invention

the present disclosure is directed to a novel class of compounds for use as contraceptives. Compared with the traditional N-9, the compound has better spermicidal contraceptive effect and smaller epithelial cytotoxicity, thereby being beneficial to preparing external spermicidal contraceptive medicaments/utensils or sanitary products which can be used by women independently.

In particular, the present disclosure provides the use of a compound of formula I below, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting sperm motility, a medicament for spermicidal, a contraceptive, or a contraceptive device:

In the formula (I), the compound is shown in the specification,

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

R^aSelected from: alkyl, aryl, aralkyl and-alkylene-CO₂-alkyl, wherein said alkyl, aryl and aralkyl are optionally substituted with 1 to 3 substituents selected from alkyl, alkoxy, haloalkyl and halogen; and

R^bSelected from: alkyl, cycloalkyl, aryl and aralkyl, wherein the alkyl, cycloalkyl, aryl and aralkyl are optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, haloalkyl and halogen.

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

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

In one or more specific embodiments, R^aIs aralkyl optionally substituted with 1 to 3 substituents selected from alkyl, alkoxy, haloalkyl and halogen, R^bIs aryl optionally substituted with 1-3 substituents selected from alkyl, alkoxy, haloalkyl and halogen.

In one or more specific embodiments, R^aIs optionally substituted by 1 to 3 substituents selected from alkyl, alkoxy, haloalkyl and halogenA substituted benzyl group; r^bIs naphthyl optionally substituted with 1-3 substituents selected from alkyl, alkoxy, haloalkyl, and halogen.

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

In the formula (I), the compound is shown in the specification,

R¹And R²Each independently selected from alkyl, alkoxy, haloalkyl, and halogen; and

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

In one or more embodiments, n and m are both 0.

The present disclosure also provides a contraceptive device comprising a compound of formula I or a pharmaceutically acceptable salt thereof.

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

The present disclosure also provides a contraceptive agent for external use comprising a compound of formula I as shown herein or a pharmaceutically acceptable salt thereof.

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

Drawings

FIG. 1: instant spermicidal Effect (EC) less than 20 seconds₁₀₀) After the desired concentrations of Tideglusib and N-9, respectively, were co-incubated with human sperm for different periods of time, the CASA system determined the ratio of motile (A + B + C%) and forward (A + B%) sperm ratios. FIGS. A and B are the ratio of motile sperm to forward motile sperm of Tideglusib, respectively; FIGS. C and D are the ratio of motile sperm to forward motile sperm of N-9, respectively. In the figure, "CN" refers to blank control.

FIG. 2: SYBR-14/PI staining of treated human sperm was performed and sperm membrane integrity was observed by fluorescence microscopy.

FIG. 3: comparison of inhibition rates of HeLa cell proliferation by Compound N9 (Panel A) and Tideglusib (Panel B).

Detailed Description

The present disclosure relates to the use of GSK3 inhibitors, in particular GSK3 β 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 particularly, the present disclosure relates to the use of compounds of formula I below, or pharmaceutically acceptable salts thereof, to inhibit sperm motility and achieve spermicidal contraceptive effects:

In the formula (I), the compound is shown in the specification,

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

R^aSelected from: alkyl, aryl, aralkyl and-alkylene-CO₂-alkyl, wherein said alkyl, aryl and aralkyl are optionally substituted with 1 to 3 substituents selected from alkyl, alkoxy, haloalkyl and halogen; and

R^bSelected from: alkyl, cycloalkyl, aryl and aralkyl, wherein the alkyl, aryl and aralkyl are optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, haloalkyl and halogen.

As used herein, "alkyl" includes straight or branched chain alkyl groups, typically C_1-6Alkyl, preferably C_1-4Alkyl or C_1-3alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, and the like.

As used herein, "alkylene" refers to a divalent group formed after an alkyl group has been further deprived of an H atom, and exemplary alkylene groups include, but are not limited to, methylene (-CH)₂-) ethylene (-CH₂CH₂-) and the like. The number of carbon atoms of the alkylene group is also usually in the range of 1 to 6, preferably 1 to 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.

As used herein, "cycloalkyl" refers to a saturated carbocyclic ring, typically having from 3 to 8 ring carbon atoms. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

as used herein, an "aryl" group is a monocyclic, bicyclic or tricyclic aromatic group containing 6 to 14 carbon atoms. Useful aryl radicals include C_6-14Aryl, preferably C_6-10And (4) an aryl group. Typical C_6-14Aryl includes phenyl, naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylene and fluorenyl. Preferred aryl groups herein are phenyl and naphthyl.

As used herein, "aralkyl" refers to a residue substituted with any one of the above C_6-14Aryl substituted C_1-6Alkyl radical, C_1-4Alkyl or C_1-3An alkyl group. Exemplary aralkyl groups include, but are not limited to, benzyl, phenethyl, or naphthylmethyl, and the like.

as used herein, "halogen" includes fluorine, chlorine, bromine or iodine.

As used herein, "haloalkyl" refers to an alkyl group substituted with 1-5 halogens, e.g., trifluoromethyl, and the like.

Herein, when substituted, the number of substituents is typically 1,2, 3 or 4 unless otherwise specified, and the substituents may typically be selected from alkyl, alkoxy, haloalkyl, halogen, nitro, amino, aryl, acyl (e.g. C) as described herein_1-4Acyl), carboxyl, and the like.

compounds of formula I are GSK3 β inhibitors known to be useful in the treatment of diseases involving GSK3, including alzheimer's disease and non-insulin dependent diabetes, see CN 101045715a, the entire contents of which are 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^aPreferably an optionally substituted alkyl, aralkyl or aryl group. More preferably, R^aIs an optionally substituted aralkyl group. IntoIn one preferred step, R^aIs an optionally substituted benzyl group.

R^bPreferably an optionally substituted alkyl or aryl group. More preferably, R^bPreferably an optionally substituted aryl group. Further preferably, R^bPreferably an optionally substituted phenyl or naphthyl group.

In one or more embodiments, X is preferably O; y is preferably O or S; r^aPreferably an optionally substituted alkyl, aralkyl or aryl group; r^bPreferably an optionally substituted alkyl or aryl group.

in one or more specific embodiments, X and Y are both O; r^aIs optionally substituted aralkyl; r^bPreferably an optionally substituted aryl group.

In one or more specific embodiments, X and Y are both O; r^aIs an optionally substituted benzyl group; r^bPreferably an optionally substituted phenyl or naphthyl group.

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 group is preferably 1.

In particular, each of the compounds listed in Table 1 and IC in Table 3, including CN 101045715A, herein₅₀Compounds with values within 100 μ M. Specifically, these compounds include, but are not limited to:

TABLE 1

Ra	Rb	X	Y
				CH2Ph	Me	O	O
Et	Me	O	O
				Et	nPr	O	O
Et	Cyclohexane	O	O
				Ph	Me	O	O
CH2CO2Et	Me	O	O
				4-OMePh	Me	O	O
CH2Ph	Et	O	O
				Et	iPr	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

Other preferred compounds of formula I are also found in Emma M.Turner et al [ Small Molecule inhibitors of Regulars of G Protein signalling (RGS) Prateins, ACS Med.chem.Lett., 2012, 3, 146-150, the entire contents of which are herein incorporated by reference ], especially the 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 within the scope of the compounds of the present application.

In particular, these compounds include compounds of formula II below, wherein R^aAnd R^bAs 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 formula III below:

In the formula (I), the compound is shown in the specification,

R¹And R²Each independently selected from alkyl, alkoxy and halogen; and

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

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

Herein, examples of the 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 inorganic and organic base salts formed with bases such as sodium hydroxy, TRIS (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methylglucamine.

The compounds of formula I herein or pharmaceutically acceptable salts thereof may be formulated into various topical pharmaceutical compositions suitable for spermicidal contraception as are known in the art, including but not limited to dosage forms suitable for vaginal administration, preferably topical dosage forms such as gels, tablets, capsules, pessaries, creams, pastes, jellies, foams, sprays, lotions and the like, as described in CN105263472a (the entire contents of which are incorporated herein by reference).

alternatively, the compound or pharmaceutically acceptable salt thereof, or the topical pharmaceutical composition may be applied to a contraceptive device, such as a condom, film, ring, implant, tampon, or the like. For example, a condom is coated externally or internally with a compound described herein, or a pharmaceutically acceptable salt thereof, or a topical pharmaceutical composition thereof, such as a topical pharmaceutical composition in the form of a gel, cream, paste, foam, jelly, or the like.

in the topical pharmaceutical compositions, the amount of the compound of formula I of the present invention is generally in the range of 0.1 wt% to 10 wt%, such as in the range of 0.1 wt% to 5.0 wt%, 0.1 wt% to 3.0 wt%, 0.1 to 1.0 wt%, and the like.

The topical pharmaceutical composition may also contain other agents/drugs commonly used in contraceptives known in the art, for example antiretroviral agents such as protease inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleotide reverse transcriptase inhibitors and integrase inhibitors. These agents/drugs may 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; lopinavir; indinavir; atazanavir; lacinavir; (ii) palinavir; tipranavir; (ii) benazevir; darunavir; n-cycloalkylglycine, α -hydroxyarylbutanamide; α -hydroxy- γ - [ (carbocyclic or heterocyclic substituted) amino) carbonyl ] alkanamide derivatives; gamma-hydroxy-2- (fluoroalkylaminocarbonyl) -1-piperazine pentanamide; dihydropyrone derivatives and alpha-and beta-amino acid hydroxyethylamino sulfonamides; and N-amino acid substituted L-lysine derivatives.

Suitable nucleoside reverse transcriptase inhibitors useful in the pharmaceutical compositions of the present invention may include zidovudine; didanosine; stavudine; lamivudine; abacavir; adefovir dipivoxil; (ii) lobucavir; entecavir; (ii) an alecitabine; emtricitabine; zalcitabine; (ii) dexelvucitabine; alovudine; amdoxovir; elvucitabine; a phosphine azide; β -L-2',3' -dideoxy-5-fluorocytidine; a purine nucleoside; and lodenosine.

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

Suitable integrase inhibitors that may be used in the pharmaceutical compositions of the present invention may include raltegravir, eltamivir.

The topical pharmaceutical compositions herein may be in the form of controlled release formulations, delayed release formulations, extended release formulations, pulsed release formulations, and mixed immediate release and controlled release formulations, among others. The topical pharmaceutical composition is formulated to release the active ingredient at a rate that produces an effective concentration at the site of administration.

It will be appreciated that the pH of the topical pharmaceutical composition may be varied as desired by the skilled person, preferably having a pH of 3.0 to 5.5, more preferably between 3.5 and 5.0.

The topical pharmaceutical composition preferably has lubricating properties to facilitate the sexual intercourse process 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/lubricating agents, surfactants, pH regulators, solvents/co-solvents, and tonicity adjusting or osmotic agents. Excipients of the above mentioned classes may be as described in CN105263472A, the entire content of which is herein incorporated by reference.

For example, gelling agents useful 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, cellulose derivatives such as carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, ethylcellulose and hydroxyethylmethylcellulose, methacrylate polymers, polyvinylpyrrolidone, bentonite, alginic acid, carbomer, gelatin, guar gum, glyceryl behenate, algae extract, gums, polyethylene oxide, poloxamers, pectin, hydrolyzed proteins, polymers containing pendant carboxylic acid groups or esters thereof, polymers containing anhydrides of pendant dicarboxylic acid groups and block copolymers based on ethylene oxide and/or propylene oxide and the like or mixtures thereof. The gelling agent may comprise about 0.05% to 10% of the total weight of the topical pharmaceutical composition.

Chelating agents useful in topical pharmaceutical compositions include, but are not limited to, disodium ethylenediaminetetraacetate, sodium condensed phosphate, diethylenetriaminepentaacetic acid, and the like, or combinations thereof. The chelating agent may comprise about 0.01% to 1% of the total weight of the topical pharmaceutical composition.

Preservatives that may be used in the topical pharmaceutical compositions include, but are not limited to: hydroxybenzoate esters (e.g., parabens, such as methyl paraben, propyl paraben), benzyl alcohol, benzoic acid, chlorphenesin, sorbic acid, phenoxyethanol, and the like, or combinations thereof. The preservative may comprise 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, hydroxypropylcellulose, ethylcellulose, polyethylene glycol, polysaccharide hyaluronic acid, polyvinylpyrrolidone, sodium alginate, sodium carboxymethylcellulose, methylcellulose, starch, and the like, or combinations thereof. The bioadhesive or polymer may comprise from about 0.05% to 2% by weight of the total weight of the topical pharmaceutical composition.

The wetting agent and/or lubricant provides lubricity and lubricity, thereby facilitating filling and dispensing of the topical pharmaceutical composition. Humectants and/or lubricants that may be used in the topical pharmaceutical composition include, but are not limited to, polyhydric alcohols 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, polyglycerol, cholesterol, squalene, fatty acids, octyldodecanol, myristyl alcohol, urea, lanolin, lactic acid, esters such as isopropyl stearate, isopropyl myristate, isopropyl palmitate, and isopropyl laurate, and the like, or combinations thereof. The wetting agent and/or lubricant may comprise about 2% to 20% of the total weight of the topical pharmaceutical composition.

Viscosity modifiers or regulators improve gel formation. Suitable viscosity modifiers or modulators 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, polymethacrylates, maleic anhydride-styrene copolymers, hydrogenated copolymers of styrene-butadiene, hydrogenated styrene-isoprene polymers, hydrogenated isoprene polymers, polyacrylates, polyalkylstyrenes, alkenyl aryl conjugated diene copolymers, esters of maleic anhydride-styrene copolymers, ethylene-propylene copolymers functionalized with the reaction product of maleic anhydride and an amine, polymethacrylates functionalized with an amine, styrene-maleic anhydride copolymers reacted with an amine, polymethacrylate polymers, esterified polymers of vinyl aromatic monomers and unsaturated carboxylic acids or derivatives thereof, or combinations thereof. The viscosity modifier or regulator may be present in an amount of about 0.05% to 2% by weight of the total composition.

Suitable tonicity adjusting agents or osmotic agents to match the osmotic properties of physiological fluids include, but are not limited to, glycerin, sodium chloride, potassium chloride, mannitol, sucrose, lactose, fructose, maltose, glucose, anhydrous glucose, propylene glycol, glycerol, and the like or combinations thereof. The tonicity adjusting agent or osmotic agent may be present in an amount of about 0.001% to 1% by weight of the total composition.

Surfactants may include, but are not limited to, one or more of the following: coconut diethanolamide, polysorbate, sodium lauryl sulfate, lauryl dimethyl amine oxide, docusate sodium, Cetyl Trimethyl Ammonium Bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, N-dimethyldodecylamine-N-oxide, cetyl trimethyl ammonium bromide, polyoxyethylene 10 lauryl ether, Brij, bile salts (sodium deoxycholate, sodium cholate), polyoxyethylene castor oil, nonylphenol ethoxylate, cyclodextrin, lecithin, benzethonium chloride, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfated natural oils and fats, sulfated esters, sulfated alkanolamides, ethoxylated and sulfated alkylphenols, ethoxylated aliphatic alcohols, polyoxyethylene surfactants, Carboxylic acid esters, polyethylene glycol esters, sorbitan esters and ethoxylated derivatives thereof, ethylene glycol esters of fatty acids, carboxamides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines having amide linkages, polyoxyethylene alkylamines and polyoxyethylene alkyl cycloaliphatic amines, N-tetrasubstituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline, N-cocoyl-3-aminopropionic acid/sodium salt, N-tallow-3-iminodipropionic acid disodium salt, N-carboxymethyl-N-dimethyl-N-9-octadecenylammonium hydroxide, N-cocamidoethyl-N-hydroxyethylglycine sodium salt, and the like, or combinations thereof. The surfactant may comprise about 0.05% to 20% of the total weight of the topical pharmaceutical composition.

Suitable pH adjusting agents or buffers that can 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, triethanolamine, sodium borate, sodium carbonate, potassium hydroxide, and the like, or combinations thereof. The pH adjusting agent or buffer may comprise about 0.1% to 2% of the total weight of the composition.

Solvents/co-solvents, solubilizing agents or vehicles that may be used in the topical pharmaceutical compositions described herein include, but are not limited to, water, glycerol, coconut diethanol amide, medium and/or long chain fatty acids or glycerides, monoglycerides, diglycerides, triglycerides, structured triglycerides, soybean oil, peanut oil, corn oil monoglycerides, corn oil diglycerides, corn oil triglycerides, polyethylene glycols, octyl hexanoyl macrogolglycerides, Caproyl90, propylene glycol, polyoxyethylene sorbitan fatty acid esters, 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. The solvent/co-solvent, solubilizer, or vehicle may comprise about 0.05% to 20% of the total weight of the topical pharmaceutical composition.

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

The topical pharmaceutical compositions and contraceptive devices herein may be prepared using methods conventional in the art. For example, topical pharmaceutical compositions may be prepared by selecting different excipients for different dosage forms and then mixing the different excipients together or separately with a compound of formula I, depending on the physicochemical properties of the respective excipients, to obtain the corresponding dosage form. The pharmaceutical compositions of the present invention may be applied or smeared onto conventional contraceptive devices, which may be obtained as described herein.

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

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

Also provided herein are uses of a compound of formula I, or a pharmaceutically acceptable salt thereof, including use as a contraceptive, use for inhibiting sperm motility, spermicidal or contraceptive, and use for the manufacture of a medicament for inhibiting sperm motility, a medicament for spermicidal, a contraceptive or a contraceptive device.

it is to be understood that the various agents, drugs, compounds, devices, methods, uses, and the like herein are applicable to a variety of animals, particularly mammals, such as humans, in need of sperm motility inhibition, spermicidal or contraception.

The present invention will be illustrated below by way of specific examples. It is to be understood that these examples are illustrative only and are not limiting upon the scope of protection herein. The methods, instruments and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Examples

Materials and reagents

Sperm culture solution: 25ml of 10 XBWW, 875mg of BSA, 7.5mg of sodium pyruvate, 525mg of sodium bicarbonate, 250 mgD-glucose, 0.925ml of sodium lactate, and 250ml of ultrapure water were diluted to 250ml, mixed well, filtered through a 0.2. mu.M filter membrane, and stored at 4 ℃.

DMEM incomplete medium: pouring one bag of the bagged DMEM medium powder into a bottle, adding 3.5g of sodium bicarbonate and tertiary water to 1000ml, fully shaking to dissolve, and filtering with a 0.2 mu m filter membrane for later use.

DMEM complete medium: taking 450ml of DMEM incomplete culture medium, adding 45ml of fetal bovine serum and 5ml of antibiotic, and mixing uniformly.

Freezing and storing liquid: fetal bovine serum + 10% DMSO.

Cell proliferation/toxicity detection kit CCK-8: shanghai Biyuntian Biotech Co., Ltd, China.

SYBR-14/PI cytotoxicity assay kit: molecular probes, USA.

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

olympus IX3 inverted and olympus BX3 upright microscope system: olympus corporation.

Tideglusib: selleck China.

other GSK3 inhibitors were prepared according to the methods disclosed in CN 101045715 a.

Example 1 Compound Tideglusib regulates human sperm motility

The semen standards selected for this study were: total number of sperm cells 60X 10⁶-100×10⁶concentration of seminal fluid>20×10⁶Perml, sperm motility>60％。

Sperm motility was measured under a microscope with a heated 37 ℃ stage and samples were prepared using a pre-heated cytometric plate. Sperm motility was classified into grades a, b, c, d according to the following criteria: a, fast forward motion: the sperm actively moves linearly or along a large circle with extremely high speed; b, slow or sluggish forward motion: the sperm actively moves linearly or along a large circle with a slow speed; c, non-forward motion: all other forms of non-forward motion, e.g. swimming in small circles, tail power hardly drives head movement, orOnly tail swing can be observed; d, immobility: there is no movement. Reference is made to World health organization (1999) WHO Laboratory Manual for the administration of Human Senmenan and Sperm-scientific music Interaction,4^th Ed.,Cambridge University Press,Cambridge。

1.1 acquisition of high-motility sperm

High-activity sperms are obtained by adopting a sperm upstream method. 500 μ l of a liquefied semen sample was placed under 1000 μ l of sperm medium at 37 ℃ with a volume fraction of 5% CO₂Incubate in incubator for 1 h. Collecting upper 600 μ l culture solution to obtain high-activity sperm, and regulating sperm concentration to 5-10 × 10⁶/ml。

1.2 screening of Rapid arresting/spermicidal Compounds and their Effect

Collecting semen by the method shown in 1.1 to obtain high-activity semen, and adjusting semen concentration to 10-20 × 10⁶And/ml. The compound/drug was diluted in duplicate in a 96-well plate at room temperature using sperm cell culture medium at 50. mu.l per well. After dilution, the 96-well plate was placed at 37 ℃ with a volume fraction of 5% CO₂Incubate in incubator for 30 min. Another 50 mul of semen is sucked and dropped into the hole, and the stopwatch is used for timing immediately, and the pipette is used for blowing and beating for 5 times at a constant speed, and the mixture is mixed evenly. Sperm motility was observed under a microscope (x 200), and it was determined whether all sperm had lost motility within 20s at this drug concentration. Add well by well and finally measure the minimum concentration of drug required to inactivate all sperm for 20 s. Each test compound/drug concentration was continuously tested 3 times for each semen sample, and the final results were averaged.

Through screening of over 5000 compounds and analysis of sperm immobilization effect, we found that Tideglusib has the smallest effective concentration for sperm immobilization. Minimum effective concentration of 20 second instant spermicidal Effect (EC) of Tideglusib₁₀₀I.e., the lowest drug concentration capable of 100% arresting of sperm at 20 s) was about 6.5. + -. 1.3. mu.M (1.74. mu.g/ml to 2.61. mu.g/ml), and the EC for N-9 was used as a reference₁₀₀About 259. mu.M (160. mu.g/ml). The experimental results show that Tideglusib has a lower instantaneous spermicidal minimum effective concentration than N-9.

To investigate chemical combination at low concentrationsthe braking effect of the substance on sperms is that the low-concentration Tideglusib and N-9 are co-cultivated with human sperms respectively. Collecting semen by the method shown in 1.1 to obtain high-activity semen, and adjusting semen concentration to 10-20 × 10⁶And/ml. The compound/drug was diluted in duplicate in a 96-well plate at room temperature using sperm cell culture medium at 50. mu.l per well. After dilution, the 96-well plate was placed at 37 ℃ with a volume fraction of 5% CO₂incubate in incubator for 30 min. Another 50 μ l of semen was pipetted into the wells and incubated for 30min, 60 min, 120 min and 180 min, respectively, and the CASA system quantified the sperm motility parameters.

The results of the measurement of the ratio of motile sperm to forward motile sperm after Tideglusib and N-9 treatment of sperm with different concentrations are shown in FIG. 1. Tideglusib has a dose-dependent relationship to the inhibition of sperm motility, mainly affecting the proportion of forward motile sperm. Through observation of a SYBR-14/PI staining fluorescence microscope, the increase of the concentration of the Tideglusib can cause sperm death (PI staining positive and SYBR-14 negative) and membrane permeability, namely the Tideglusib has a remarkable spermicidal effect. See fig. 2.

example 2 Tideglusib has lower epithelial cell proliferation inhibition/cytotoxicity than N-9

2.1Hela cell Resuscitation

And (3) quickly taking out the freezing tube filled with the cells frozen in the liquid nitrogen tank, and quickly thawing the freezing tube in warm water at the temperature of approximately 40 ℃ within the optimal time of 1-2 min. And (3) transferring all the Hela cell frozen stock solution contained in the frozen stock tube into a 15ml centrifuge tube, uniformly blowing, putting into a centrifuge, and centrifuging for 4min at the rotating speed of 1000 rpm. After centrifugation, the supernatant was discarded, 3ml of complete medium was added, and the mixture was repeatedly and gently blown and beaten for use. Sucking the uniformly mixed cell liquid into a10 cm culture dish, adding 8ml of DMEM complete culture medium into the culture dish, slightly blowing, uniformly mixing, placing at 37 ℃ and 5% CO₂Culturing in an incubator, and replacing fresh culture solution for continuous culture the next day. Passing the cells in the original dish at a ratio of 1:3, mixing, adding 5% CO at 37 deg.C₂Culturing in an incubator.

2.2 cell proliferation inhibition/toxicity test

Washing Hela cell culture dish in logarithmic growth phase with PBS for 2 times, adding 1ml of 0.25% trypsin for digestion, observing under high power microscope, adding DMEM complete culture medium to stop digestion, sucking a little cell suspension on blood cell counting plate, counting cells, adjusting single cell suspension to 1 × 10⁵And/ml. The prepared Hela cell suspension is inoculated into a 96-well culture plate, each well is 100 mu l, each group is provided with 5 parallel wells, and the outermost circle of wells are not added with the cell suspension and are replaced by PBS (phosphate buffer solution) so as to prevent edge effect and influence the cell state and the experimental result. The 96-well plate with the cells laid thereon was placed at 37 ℃ in 5% CO₂The culture chamber of (2) for 24h to allow the cells to adhere. The next day, 100. mu.l of fresh complete culture medium (N-9 or other compound/drug) containing compound/drug was replaced, and the culture was continued. After addition of the complete medium containing the compound/drug, incubation was continued for 24h, the medium was discarded, 100. mu.l of serum-free medium was added, 10. mu.l of CCK-8 reagent was added to each well, preferably quickly, to avoid CCK-8 remaining on the tip of the pipette, and after addition, the 96 well plate was gently tapped to evenly distribute CCK-8 in the wells. Standing for 30min, placing 96-well plate containing CCK-8 at 37 deg.C and 5% CO₂the incubator is continuously cultured for 1-4h, and Hela cells are generally selected for 2h as test points. And (3) determining the OD value by adopting an enzyme labeling method, preheating an enzyme labeling instrument for 30min before testing, and then testing. The measurement wavelength was 450 nm. By measuring OD value, cell survival rate and IC were determined₅₀The value is obtained. The inhibition rate of the cells was calculated according to the following formula:

The cell inhibition ratio (%) ═ [ (AC-Ab) - (AS-Ab) ]/(AC-Ab) × 100%

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

AC: control well mean (medium with cells, CCK-8, no toxic substance);

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

IC₅₀The compound/drug concentration required for 50% inhibition. The results are shown in FIG. 3, where N-9 and Tideglusib are on the HeLa cell line of epithelial cellsRespectively, about IC_50N-9：6μΜ，IC_50Tideglusib: 30 μm. Therapeutic Index (TI) is an indicator of the safety of a drug, and usually half toxic (TD 50)/half effective (ED50) or half lethal (LD 50)/half effective (ED50) is referred to as therapeutic index. Here with IC₅₀/EC₁₀₀Estimating TI, TI_N-9＝0.023；TI_Tideglusib3.85 to 5.76. Tideglusib is seen to have a larger TI value than N-9, indicating that Tideglusib has better safety than N-9.

n-9 is known to cause inhibition of cell proliferation by detergent-like activity disintegration of mucosal epithelial cells, and Tideglusib exhibits completely different kinetic profiles, suggesting that Tideglusib inhibits proliferation of HeLa cell lines through a different pathway than N-9. SYBR14/PI fluorescence staining analysis showed that contact of N-9 with HeLa cells directly resulted in cell membrane permeabilization, whereas contact of Tideglusib with HeLa cells did not result in cell membrane permeabilization or cell disintegration of HeLa cells. Thus, N-9 cytotoxicity appears as a direct cell membrane disruption, whereas Tideglusib appears to inhibit epithelial cell proliferation only.

2.3 discussion

Tideglusib is an irreversible, non-ATP-competitive GSK-3 β inhibitor (IC 50: 60nM) known to have entered the second phase of clinics as a drug for the treatment of Alzheimer's disease and Progressive Supranuclear Palsy (PSP) [ dominguz JM, Fuerts A, Orozco L, delMonte-Millan M, Delgado E, Medina M: evolution for irreversible inhibition of collagen synthesis kinase-3beta by tideglusib.J Biol Chem2012,287(2):893-904 ].

The clinical safety of Tideglusib has been validated in clinical trials. In small-scale clinical trials, patients ' oral doses increased to 1000 mg per day still showed good safety and tolerability with no adverse effects distinguished from controls [ del Ser T, Steinwaters KC, Gertz HJ, Andres MV, Gomez-Carrillo B, MedinaM, Vericat JA, Redondo P, Fleet D, Leon T: Treatment of Alzheimer's disease with the GSK-3inhibitor deglusib: a pilot study. J Alzheimer's diseases 2013,33(1):205-215 ]. On a larger scale, a two-phase clinical trial with multi-national, double-blind controls found that Tideglusib reduced the progression of brain atrophy, especially in patients with progressive supranuclear palsy [ Hoglinger GU, Huppertz HJ, Wagenpfeil S, Andres MV, Belloch V, Leon T, Del Ser T: Tideglusib reduction progression of brain atrophies in crude supra-lung palsy in ariandomized al III mole Disord 2014,29(4):479-487 ]. Another randomized double-blind control trial of patients with mild and moderate Progressive Supranuclear Palsy (PSP) at a period of 52 weeks, up to 146, showed that Tideglusib is 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 2 tertiary of the GSK-3inhibitor depletion in developmental subangler palsy. Mov disease 2014,29(4):470-478 ]. The safety of Tideglusib was also demonstrated in European clinical trials for treating Alzheimer 'S disease, although no clinical benefits of Tideglusib for treating Alzheimer' S disease were 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 Soft in Alzheimer 'S disease. J Alzheimer' S Dis 2015,45(1):75-88 ].

Thus, for both females and males, Tideglusib is expected to be developed as a non-invasive, reversible, highly comfortable, safe contraceptive.

Example 3 preparation and testing of Tideglusib gel

Tideglusib gel was prepared with reference to CN 101559036 a. Dissolving acid of strong buffer in water, adding alkali (one or more of sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate, preferably sodium hydroxide) of metal cation contained in human body under stirring to adjust pH to 3.0-5.5, preferably 3.3-3.6; adding one or more of phase transition temperature regulator and poloxamer, stirring, storing at 0-8 deg.C for 12-24 hr, adding bioadhesive, and stirring for 30-180 min until uniform gel is obtained.

Specifically, the drug-loaded gel of the present example can be prepared with reference to example 2 of CN 101559036 a. The medicine carrying gel comprises the following components in proportion: 20% poloxamer 407; 3% poloxamer 188; 4% lactic acid; 1% citric acid; 0.4% potassium bitartrate; 0.2% benzoic acid; 8% of glycerol; sodium lactate, sodium citrate, sodium tartrate, sodium potassium tartrate, sodium benzoate (the amount of salt required to form a ph of 3.5 with its corresponding acid), 0.8% carbopol 934; the water content is the complement of 100%.

And respectively and uniformly dispersing the dissolved Tideglusib and the nonoxynol into the prepared medicine-carrying gel to obtain 4% nonoxynol gel and 0.4% Tideglusib gel.

Rabbits were randomly grouped into 3 animals per group, and were dosed one week later. The 4% nonoxynol gel was used as a positive control, 0.9% sodium chloride was used as a blank control, and 0.4% tedaglusib gel was used as a drug group. After administration for 10 minutes in vagina, each group of female rabbits was mated with male rabbits with confirmed fertility at a ratio of 1:1, and sperm was taken out from vagina of the female rabbits at 5, 10, 15, and 20 minutes after mating, respectively, and the sperm survival condition was observed under a microscope. More than 10 fields of view were observed, with one or more motile sperm recorded as "+" and non-motile sperm recorded as "-". The results are shown in table 3 below.

Continuously feeding, killing by air embolism method 15-20 days after mating, and performing laparotomy to check whether the pregnant woman exists. The results are shown in table 4 below.

Table 3: observation and record table for spermicidal effect in rabbit body

Table 4: instrument for observing and recording contraceptive effect in rabbit body

This example demonstrates that Tideglusib has good contraceptive efficacy.

Example 4 testing of the ability of other Compounds to modulate human sperm motility

Compounds shown in table 1 of the present application were prepared, purified according to the method disclosed in CN 101045715a, and compounds 1a-1i, 2a-2i, 3-8, 9a-9b, 10a-10d, 11a-11d, 12a-12d and 13-15 of table 1 thereof were prepared according to Emma m.turner et al (ACS med.chem.lett., 2012, 3, 146-150).

The compounds prepared as described above were tested for their ability to modulate human sperm motility as described in example 1 of the present application. Specifically, the collected semen was used to obtain high-motility sperm by the method shown in 1.1 in example 1, and the concentration of the sperm was adjusted to 10-20X 10⁶And/ml. The aforementioned compound was diluted in a 96-well plate at room temperature by a method of dilution in a sperm culture solution at a rate of 50. mu.l per well. After dilution, the 96-well plate was placed at 37 ℃ with a volume fraction of 5% CO₂Incubate in incubator for 30 min. Another 50 mul of semen is sucked and dropped into the hole, and the stopwatch is used for timing immediately, and the pipette is used for blowing and beating for 5 times at a constant speed, and the mixture is mixed evenly. Sperm motility was observed under a microscope (x 200), and it was determined whether all sperm had lost motility at the compound concentration within 20 s. Add well by well and finally measure the minimum concentration of compound required to inactivate all sperm for 20 s. The concentration of each test compound is continuously detected for 3 times aiming at each semen sample, and the final result is taken as the average value.

Thiadiazolidinediones are known to be useful as GSK3 β 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, especially the most extensively studied RGS4 is widely expressed in the central nervous system and rarely expressed in peripheral tissues (Emma M. Turner et al, supra). Furthermore, RGS22 was only found to date in humans and mice expressed in testicular spermatogenic cells and Leydig cells, possibly involved in spermatogenesis and meiosis, and other RGS proteins were not found to be present in mature sperm in addition to RGS22, and the known function of RGS22 was not found to be related to sperm motility. Therefore, it is reasonable to expect that Tideglusib plays a spermicidal contraceptive role as a GSK 3beta inhibitor through a GSK3 pathway. Thus, it is believed that other thiadiazolidinediones, especially those prepared and tested in this example, which are GSK3 β inhibitors, also function as spermicidal contraceptives.

Claims (6)

1. Use of a compound of formula III below, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting sperm motility, a medicament for spermicidal, a contraceptive, or a contraceptive device:

In the formula (I), the compound is shown in the specification,

R¹And R²Each independently selected from C_1-4Alkyl radical, C_1-4Alkoxy, halo C_1-4Alkyl and halogen; and

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

2. The use according to claim 1, wherein the compound of formula III has the formula:

3. A contraceptive device comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

4. The contraceptive device of claim 3, wherein the contraceptive device is a condom, a membrane, a ring, an implant, or a tampon.

5. A contraceptive for external use comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

6. The contraceptive agent for external use of claim 5, wherein the contraceptive agent for external use is a gel, tablet, capsule, pessary, cream, paste, jelly, foam, spray, lotion, or lubricant.