5-(1H-Indol-3-yl)-oxazoles, -oxadiazoles, and -furans as sperm motility enhancers

Abstract

The present invention relates to the use of compounds of formula (I), their stereoisomers and physiologically tolerable salts for enhancing the motility of sperm obtained from non-human mammals: #imgabs0# wherein in formula I: X is -NR' - (wherein R' is H or C 1‑3 alkyl, such as -CH3), -O -, -S - or -CH2 -; Y is -S -, -O -, -NR'' - (wherein R'' is H or C 1‑3 alkyl, such as -CH3), or -CH2 -; Z 1 and Z 2 are independently selected from N and CH; R 1 to R 3 are independently selected from hydrogen, halogen, C 1‑6 alkyl, C 1‑6 alkoxy, C 1‑6 haloalkyl, -OH, -CN, -NO2 and -NR 12 R 13 ; R 4 is hydrogen, halogen, C 1‑6 alkyl, C 1‑6 alkoxy, C 1‑6 haloalkyl or -OH; R 5 is hydrogen, halogen, C 1‑6 alkyl, C 1‑6 alkoxy or C 1‑6 haloalkyl; R 6 and R 7 are independently selected from hydrogen, halogen, C 1‑6 alkyl, C 1‑6 alkoxy, C 1‑6 haloalkyl, -OH, -CN, -NO2 and -NR 12 R 13 ; R 8 is hydrogen, halogen, C 1‑6 alkyl, C 1‑6 alkoxy, C 1‑6 haloalkyl, -CN, -NO2 or -NR 12 R 13 ; R 9 is hydrogen or C 1‑4 alkyl; R 10 and R 11 are independently selected from hydrogen and C 1‑3 alkyl; or R 10 and R 11 together with the intervening -O - and ring atoms are joined to form a dioxolane; and R 12 and R 13 are independently selected from H and C 1‑3Alkyl groups. These compounds are particularly useful in animal husbandry to increase the fertilization rate of artificial insemination in livestock, especially pigs. They can also be used for the preparation, pretreatment or storage of semen for artificial insemination procedures.

Description

Technical Field

[0001] The present invention relates to the use of a compound to enhance sperm motility. It is particularly applicable to the livestock industry to improve the fertilization rate of artificial insemination of livestock (especially pigs). The present invention can also be used for preparing, pre-treating or storing semen for artificial insemination procedures. Background Art

[0002] Pork is the most widely consumed meat worldwide due to its low price and high nutritional content. To meet the growing demand, the productivity of pork is also increasing.

[0003] Global production of meat for human consumption (especially pork and beef) depends on artificial insemination methods to produce sufficient livestock to meet consumer demand. Natural breeding requires more male animals, which is labor-intensive for farmers. However, artificial reproduction by artificial insemination of female animals requires fewer males, has a higher success rate, is more time-saving, and reduces the cost for farmers. For example, artificial insemination also allows for selective breeding, which can be used for breeding livestock or for breeding high-value animals (such as racehorses) or endangered species.

[0004] Artificial insemination is commonly used for breeding livestock, especially pigs and cattle, and usually requires storing the collected semen in liquid nitrogen before use, i.e., cryopreservation. Cryopreservation of semen is widely used in the beef industry but is less common in the pork industry because semen from pigs (i.e., boars) may be more sensitive to temperature than bull sperm. For example, compared with stallion, bull or ram semen, the temperature sensitivity of boar sperm may be due to a lower cholesterol-to-phospholipid ratio. A higher cholesterol content results in less sensitivity to temperature reduction. Cryopreservation of boar sperm can alter its protein profile. This may lead to the loss of sperm surface proteins and the inactivation of membrane-bound enzymes, which may result in a decrease in sperm cell activity and motility. Cryopreservation may also cause an early acrosome reaction, which occurs in the acrosome of sperm when the sperm approaches the egg and is crucial for fertilization to occur. In addition, cryopreserving boar sperm before fertilization may lead to sperm cell apoptosis, DNA fragmentation and the production of reactive oxygen species. After cryopreservation, the fertilization rate of boar sperm is only 40 - 70% of that of fresh sperm.

[0005] Despite these problems, cryopreservation must still be used when transporting boar sperm or when long-term storage is required. For example, storage may be needed to preserve genetic variation and valuable genes. In addition, the fertility of pigs depends on the month of the year. In warm months, pigs give birth to fewer offspring, and boar sperm collected during these months is more susceptible to the effects of freezing. These fluctuations within a year result in economic losses in the pork industry. To alleviate this economic burden and improve the quality of life of pigs, other methods for preserving boar sperm that do not affect sperm quality (sperm quantity, motility and activity) are needed.

[0006] In order to be able to store boar semen for several days, an extender is added to the semen sample. The extender contains buffers and nutrients that can prolong the half-life of the semen, but they do not increase the viability of sperm cells compared to freshly collected semen.

[0007] One strategy for increasing fertilization and litter size is to increase the viability of sperm cells. Holt et al. (J Androl 1997, 18:312 - 323) showed a significant correlation between the sperm viability of boars and the insemination results on the farm. If sperm cells have higher viability, they will have a higher chance of traveling up the female reproductive tract and reaching the egg. There are different patterns of sperm motility: hyperactivation, forward movement, and standard movement. All three parameters are important during the fertilization process.

[0008] The present invention attempts to solve at least some of these problems in the art, and in particular provides compounds that can enhance sperm viability, function, and / or activity. More specifically, it attempts to provide compounds that can be used to increase the conception rate and / or litter size in artificial insemination of non - human mammals (such as livestock). Summary of the Invention

[0009] The inventors have now surprisingly discovered certain compounds that act as "sperm activators". When added to fresh boar semen or boar semen samples that have been stored at low temperature for several days, these substances have been found to enhance sperm viability. It has also been found that adding such "activators" can increase the fertility rate and offspring rate in pig artificial insemination.

[0010] The "activator" compounds disclosed herein are particularly suitable for the reproduction and breeding of pigs in the pork industry. However, it is expected that the inventors' findings will extend to the treatment of sperm from other livestock and other non - human mammals. Therefore, these compounds can also be used in other meat - producing industries, such as raising cattle, as well as breeding high - value animals, such as horses. Although the sperm of stallions and bulls can be cryopreserved with a higher fertility rate than boar semen, increasing the success rate of artificial insemination by adding "activators" will also greatly benefit these industries, especially since the sperm doses of stallions and bulls are very expensive.

[0011] Using the "activator" compounds as described herein to treat the sperm of endangered mammals, for example after cryopreservation, can also increase the chance of successful reproduction in artificial insemination. In cases where semen can be stored for a long time, artificial insemination procedures can even be carried out after the death of the male donor. Detailed Description

[0012] In one aspect, the present invention provides the use of a compound of formula (I), its stereoisomers or physiologically tolerable salts for enhancing the motility of sperm obtained from non-human mammals:

[0013]

[0014] Wherein:

[0015] X is -NR' - (where R' is H or C 1-3 alkyl, such as -CH3), -O-, -S- or -CH2-;

[0016] Y is -S-, -O-, -NR'' - (where R'' is H or C 1-3 alkyl, such as -CH3) or -CH2-;

[0017] Z 1 and Z 2 independently selected from N and CH;

[0018] R 1 to R 3 independently selected from hydrogen, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, -OH, -CN, -NO2 and -NR 12 R 13 ;

[0019] R 4 is hydrogen, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or -OH;

[0020] R 5 is hydrogen, halogen, C 1-6 alkyl, C 1-6 alkoxy and C 1-6 haloalkyl;

[0021] R 6 and R 7 independently selected from hydrogen, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, -OH, -CN, -NO2 and -NR 12 R 13 ;

[0022] R 8 is hydrogen, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, -CN, -NO2 or -NR12 R 13 ;

[0023] R 9 is hydrogen or C 1-4 alkyl

[0024] R 10 and R 11 are independently selected from hydrogen and C 1-3 alkyl;

[0025] or R 10 and R 11 together with the intervening -O- and ring atoms are joined to form a dioxolane; and

[0026] R 12 and R 13 are independently selected from H and C 1-3 alkyl;

[0027] As used herein, the term "alkyl" refers to a monovalent saturated straight-chain or branched-chain carbon chain. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl. The alkyl group preferably contains 1-6 carbon atoms, more preferably 1-4 carbon atoms, such as 1-3 carbon atoms.

[0028] The term "halogen" refers to -F, -Cl, -Br or -I. Preferred examples of such groups are -F, -Cl and -Br.

[0029] The term "haloalkyl" refers to an alkyl group as defined herein, wherein at least one of the hydrogen atoms of the alkyl group is replaced by a halogen atom (preferably -F, -Cl or -Br). Examples of such groups include, but are not limited to, -CH2F, -CHF2, -CF3, -CCl3, -CHCl2 and -CH2CF3.

[0030] In one embodiment of formula (I), X is -NR' - (where R' is H or C 1-3 alkyl, e.g., -CH3), -O- or -CH2-. Preferably, X is -NR' - (where R' is H or -CH3) or -O-. More preferably, X is -NH- or -O-. Even more preferably, X is -NH-.

[0031] In one embodiment of formula (I), Y is -S-, -O-, -NR'' - (where R'' is H or -CH3) or -CH2-. Preferably, Y is -S-.

[0032] In one embodiment of formula (I), X and Y may not both be -CH2-. In one embodiment, X is -NR'-(wherein R' is as defined herein) and Y is -S-.

[0033] In one embodiment, the compounds for use in the present invention are compounds of formula (II), their stereoisomers and their physiologically tolerable salts:

[0034]

[0035] (wherein Z 1 、Z 2 ,and R 1 to R 11 are as defined herein).

[0036] In one embodiment of formula (I), Z 1 and Z 2 are both N, or one of Z 1 and Z 2 is N, while the other of Z 1 and Z 2 is CH. In one embodiment, Z 1 and Z 2 are not both CH. Preferably, Z 1 and Z 2 are both N.

[0037] In one embodiment, the compounds for use in the present invention are compounds of formula (III), their stereoisomers and their physiologically tolerable salts:

[0038]

[0039] (wherein X, Y, and R 1 to R 11 are as defined herein).

[0040] In one embodiment of formula (I), (II) and (III), R 1 to R 3 are independently selected from hydrogen, halogen (such as -F, -Cl or -Br), C 1-4 alkyl and C 1-4 haloalkyl (such as -CF3).

[0041] In one embodiment of formula (I), (II) and (III), R 4 is hydrogen, halogen (such as -F, -Cl or -Br), C 1-4 alkyl or C 1-4 haloalkyl (such as -CF3).

[0042] In one embodiment of formula (I), (II) and (III), R 5 is hydrogen, halogen (e.g., -Cl or -Br), C 1-4 alkyl or C 1-4 haloalkyl (e.g., -CF3);

[0043] In one embodiment of formula (I), (II) and (III), R 6 and R 7 are independently selected from hydrogen, halogen (e.g., -F, -Cl or -Br), C 1-4 alkyl and C 1-4 haloalkyl (e.g., -CF3);

[0044] In one embodiment of formula (I), (II) and (III), R 8 is hydrogen, halogen (e.g., -F, -Cl or -Br), C 1-4 alkyl or C 1-4 haloalkyl (e.g., -CF3).

[0045] In one embodiment of formula (I), (II) and (III), R 9 is hydrogen or -CH3. Preferably, R 9 is hydrogen.

[0046] In one embodiment of the compounds of formula (I), (II) and (III), R 10 and R 11 are independently selected from hydrogen and -CH3. In another embodiment, R 10 and R 11 together with the intervening -O- and ring atoms form a dioxolane ring.

[0047] In one embodiment, the compound for use in the present invention is a compound of formula (IV), its stereoisomers and its physiologically tolerable salts:

[0048]

[0049] (wherein Z 1 , Z 2 , X, Y and R 1 to R 9 are as defined herein).

[0050] In one embodiment of the compounds of formula (I), (II), (III) and (IV), only one of R 1 to R 4 is not hydrogen. In another embodiment of the compounds of formula (I), (II), (III) and (IV), R 1 to R 5Only one of them is not hydrogen.

[0051] In one embodiment of the compounds of formula (I), (II), (III) and (IV), R 6 to R 8 Only one of them is not hydrogen.

[0052] In one embodiment of the compounds of formula (I), (II), (III) and (IV), R 1 to R 8 are independently selected from hydrogen, -Cl, -CF3 and -CH3.

[0053] In one embodiment of the compounds of formula (I), (II), (III) and (IV), R 1 to R 8 are each hydrogen.

[0054] Examples of the compounds for use in the present invention include, but are not limited to, the following compounds, their stereoisomers and any physiologically tolerable salts:

[0055]

[0056] Certain compounds described herein are new and these form another aspect of the present invention.

[0057] Accordingly, in another aspect, the present invention provides a compound of general formula (I) or a stereoisomer or a physiologically tolerable salt thereof, wherein the compound is not:

[0058]

[0059] Or

[0060]

[0061] Any compound described herein can be used in the form of a physiologically tolerable salt. "Physiologically tolerable" means any component suitable for administration to an animal body, in particular any component suitable for intravaginal administration. The term "physiologically tolerable salt" means any physiologically tolerable organic or inorganic salt of any compound described herein. Such salts may include one or more additional molecules, such as counterions. A counterion may be any organic or inorganic group that stabilizes the charge on the parent compound. If the compound used in the present invention is a base, a suitable pharmaceutically acceptable salt can be prepared by reacting the free base with an organic or inorganic acid. If the compound used in the present invention is an acid, a suitable pharmaceutically acceptable salt can be prepared by reacting the free acid with an organic or inorganic base.

[0062] Any compound described herein may be converted into its salts, especially its physiologically tolerable salts, using inorganic or organic acids or bases. Procedures for salt formation are conventional in the art.

[0063] For example, suitable physiologically tolerable salts of the compounds described herein are acid addition salts of compounds having sufficient basicity, e.g., acid addition salts with acids such as inorganic or organic acids. Acids that may be used for this purpose include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, sulfonic acid, methanesulfonic acid, phosphoric acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, malic acid, malonic acid, maleic acid, acetic acid, trifluoroacetic acid and ascorbic acid, p-toluenesulfonic acid, 2-mesitylenesulfonic acid, 1,2-ethanedisulfonic acid, fatty acids, aspartic acid, benzenesulfonic acid, benzoic acid, ethanesulfonic acid or nicotinic acid.

[0064] Furthermore, suitable physiologically tolerable salts of the compounds described herein are, for example, base addition salts of compounds having sufficient acidity, e.g., metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt or aluminum salt, ammonium salts or salts with organic bases that provide physiologically acceptable cations, which include quaternary ammonium hydroxides such as methylamine, ethylamine, diethylamine, trimethylamine, tert-butylamine, triethylamine, dibenzylamine, N,N-dibenzylethylamine, cyclohexylethylamine, tris-(2-hydroxyethyl)amine, hydroxyethyldiethylamine, (IR,2S)-2-hydroxyindan-1-amine, morpholine, N-methylpiperidine, N-ethylpiperidine, piperazine, methylpiperazine, amantadine, choline hydroxide, tetrabutylammonium hydroxide, tris-(hydroxymethyl)methylamine hydroxide, L-arginine, N-methyl-D-glucosamine, lysine or arginine, and organic amines such as diethylamine, triethylamine, ethanolamine, diethanolamine, cyclohexylamine and dicyclohexylamine.

[0065] It is understood that certain compounds described herein may contain one or more stereocenters and thus may exist in different stereoisomeric forms. The term "stereoisomers" refers to compounds having the same chemical composition but differing in the spatial arrangement of atoms or groups. Examples of stereoisomers are enantiomers and diastereoisomers. The term "enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other. The term "diastereoisomers" refers to stereoisomers having two or more stereocenters that are not mirror images of each other. The present invention is considered to extend to the use of diastereoisomers and enantiomers, as well as racemic mixtures and enantiomerically enriched mixtures in ratios other than 1:1.

[0066] The compounds described herein can be resolved into their enantiomers and / or diastereomers. For example, in cases where these compounds contain only one chiral center, these compounds can be provided in the form of a racemate or a racemic mixture (a 50:50 mixture of enantiomers), or can be provided in the form of pure enantiomers, i.e., the R form or the S form. Any of the compounds in a racemic form can be separated into its enantiomers by methods known in the art (such as column separation on a chiral phase) or by recrystallization from an optically active solvent. Those compounds having at least two asymmetric carbon atoms can be resolved into their diastereomers using methods known per se based on their physicochemical differences, e.g., by chromatography and / or fractional crystallization, and in cases where these compounds are obtained in a racemic form, they can subsequently be resolved into their enantiomers.

[0067] The compounds disclosed herein are either known in the art or can be prepared by methods known to those skilled in the art. Some compounds are commercially available from sources including Enamine. For example, Compound No. 1 described herein can be obtained from Enamine under the catalog number Z19304321.

[0068] Any of the compounds described herein that are unknown in the art can be prepared using synthetic methods known in the art (such as those described in known textbooks, e.g., Advanced Organic Chemistry (March, Wiley Interscience, 5 th Ed. 2001) or Advanced Organic Chemistry (Carey and Sundberg, KA / PP, 4 th Ed. 2001)) from readily available starting materials.

[0069] The following reaction scheme shows the general methods for preparing the compounds of formula (I) and key intermediates. The compounds used as starting materials are known in the literature or can be commercially available. Alternatively, these compounds can be readily obtained by methods known in the literature. It is understood that other synthetic routes can be used to prepare the compounds when using different starting materials, different reagents, and / or different reaction conditions.

[0070]

[0071] wherein

[0072] Ar 1 is Ar 2 is

[0073] LG = leaving group

[0074] R 1 -R 10 is Scheme 1 as defined herein

[0075] Scheme 1 shows a general method for preparing the compounds for the present invention, wherein: (a)(i) K2CO3, acetone, reflux; (b)(i) toluene, reflux; (c)(i) MeOH, aqueous NaI solution; (c)(ii) NaOH, H2O, heating; (d)(i) DCM or MeOH, room temperature; (d)(ii) NaOH, H2O, heating.

[0076] The starting materials used in Scheme 1 can be prepared from readily available starting materials and / or according to the method in Scheme 2:

[0077]

[0078] Scheme 2

[0079] Scheme 2 shows a general method for constructing fragment A, wherein: (a)(i) NEt3, DCM, room temperature (US2019 / 0031655); (b)(i) NaNO2, HBF4, H2O, 0 °C, then CuSO4, Cu2O, rt; (ii) pyridine, DCM; (c)(i) NaNO2, HBF4, H2O, 0 °C, then K((CH=CH2)(BF3)), Pd2(μ-OAc)2(P(o-Tol)3)2, MeOH, room temperature; (c)(ii) 9-BBN, THF, rt, then H2O2, NaOH, EtOH; (c)(iii) TEMPO, NaOCl, NaClO2, MeCN, room temperature; (c)(iv) iPrMgCl, THF, TBME, -10 °C to room temperature, then 2-chloro-N-methoxy-N-methylacetamide, 0 °C (J. Org. Chem. 2014, 79:8917-8925). 6-Amino-3,4-benzodioxane and 3,4-dimethoxyaniline are also commercially available; variants of these compounds and benzo[1,3]dioxol-5-amine in which one aromatic H is substituted may be commercially available or prepared according to the methods shown herein.

[0080]

[0081] Scheme 3

[0082] Scheme 3 shows the general methods for constructing Fragment B, where: (a)(i) SOCl2, rt, then NH2NH2·H2O, THF, 0 °C (WO 2008 / 118745); (a)(ii) CS2, KOH, EtOH, reflux (Chem. Pap. 2019, 73:17 - 25); (b)(i) SOCl2, rt, then NH2NH2·H2O, THF, 0 °C (WO 2008 / 118745); (b)(ii) triphosgene, DIPEA, DCM, room temperature; (c)(i) SOCl2, rt, then NH2NH2·H2O, THF, 0 °C (WO 2008 / 118745); (c)(ii) BrCN, MeOH, reflux; (d)(i) LiAlH4, THF, 0 °C to room temperature (J. Med. Chem. 2013, 56, 3725 - 3732); (d)(ii) MnO2, MeCN, room temperature (Tetrahedron 2002, 58, 2813 - 2819); (d)(iii) TMSCN, MeCN, reflux (Tetrahedron 2002, 58:2813 - 2819); (d)(iv) DDQ, 1,4 - dioxane (Tetrahedron 2018, 74:217 - 223); (v) Pd / C, H2, H3CCOOH, room temperature (Tetrahedron Lett. 2009, 50:4343 - 4345); (d)(vi) CS2, Na2CO3, EtOH, reflux; (e)(i) [Rh(OH)(cod)]2, DPPP, NaOH, H2O, PhMe, 100 °C (Org. Lett. 2010, 12:992 - 995); (e)(ii) ClH2CCOCl, 1,4 - dioxane, heated (.J. Heterocycl. Chem. 2007, 44:109 - 114); (e)(iii) H2O, N2NCHO, 110 °C (J. Org. Chem. 1988, 53:5446 - 5453); (e)(iv) H2NCN, EtOH, reflux; (f)(i) tBuONO, CuCl2, heated; (f)(ii) H2O, LiOH, THF. Variants of 1H - indole - 3 - carboxylic acid in which one aromatic H has been substituted are commercially available and can also be prepared according to the methods shown herein. Alkylation of the indole N can be achieved according to the method in Scheme 12.

[0083]

[0084] Scheme 4

[0085] Scheme 4 shows a general method for constructing fragment C (if the corresponding phenol is not commercially available), where: (i) NaNO2, HBF4, H2O, 0 °C, then CuSO4, Cu2O, room temperature. 6-Amino-3,4-benzodioxane and 3,4-dimethoxyaniline are also commercially available. Variants of these compounds and benzo[1,3]dioxol-5-amine in which one aromatic H has been substituted may be commercially available or prepared according to the methods shown herein.

[0086]

[0087] Scheme 5

[0088] Scheme 5 shows a general method for constructing fragment D, where: (a)(i) SOCl2, room temperature, then NH2NH2.H2O, THF, 0 °C (WO 2008 / 118745); (a)(ii) ClCH2COOH, POCl3, 80 °C (Eur. J. Med. Chem. 2013, 63, 22 - 32); (a)(iii) H2C(CO2Me)2, NaH, THF, 0 °C, then 1H-indole-3-carboxylic acid hydrazide, NaI, room temperature; (a)(iv) LiOH, THF / MeOH / H2O, room temperature; (a)(v) DMF, heated; (a)(vi) SOCl2, toluene, reflux; (b)(i) SOCl2, room temperature; (b)(ii) methyl 5-aminolevulinate, NEt3, DCM, (b)(iii) Burgess reagent, NEt3, MeCN, reflux; (b)(iv) LiOH, THF / MeOH / H2O, room temperature; (b)(v) SOCl2, toluene, reflux; (c)(i-v) see Scheme 3d; (c)(vi) ethyl 4-chloro-4-oxobutyrate, pyridine, room temperature; (c)(vii) P2O5, CHCl3, heated; (c)(viii) LiOH, THF / MeOH / H2O, room temperature; (c)(ix) SOCl2, toluene, reflux. Variants of 1H-indole-3-carboxylic acid in which one aromatic H has been substituted are commercially available or may be prepared according to the methods shown herein. Alkylation of the indole N can be achieved according to the method in Scheme 12.

[0089]

[0090] Scheme 6

[0091] Scheme 6 shows a general method for constructing fragment E (all fragment A compounds can be used as starting materials), where: (i) NH3, EtOH, heated. Variants of these compounds in which one aromatic H is substituted may be commercially available or prepared according to the methods shown herein.

[0092]

[0093] Scheme 7

[0094] Scheme 7 shows a general method for constructing fragment F, where: (i) NH3, CDI, DMF, room temperature (Chim. Acta 1994, 77, 1886 - 1894); (ii) Cl3CCHO, PhMe, heating; (iii) PCl5, Et2O; (iv) PPh3, PhH, heating. Variants of these compounds in which one aromatic H is substituted can be commercially available or prepared according to the methods shown herein.

[0095]

[0096] Scheme 8

[0097] Scheme 8 shows a general method for constructing fragment G (all fragment A compounds can be used as starting materials), where: (a) (i) KI, acetone, reflux; (b) (i) Na2CO3, H2O, NMP, heating; (b) (ii) Tf2O, 2,6 - lutidine, DCM, -78 °C (Variants of these compounds in which one aromatic H has been substituted can be commercially available or prepared according to the methods shown herein).

[0098]

[0099] Scheme 9

[0100] Scheme 9 shows a general method for constructing fragment H, where: (a) (i) Na2S, MeOH, room temperature; (b) (i) PhCOCH2Br, DCM, room temperature; (b) (ii) NaOMe, MeOH, room temperature (Variants of these compounds in which one aromatic H is substituted can be commercially available or prepared according to the methods shown herein).

[0101]

[0102] Scheme 10

[0103] Scheme 10 shows methods for synthesizing various non-commercially available monosubstituted 1H-indole-3-carboxylic acids, where: (a)(i) CBr4, PPh3, DCM, 0 °C (Tetrahedron Lett. 2011, 52:1815 - 1818); (a)(ii) Cs2CO3, DMSO, 120 °C (Tetrahedron Lett. 2011, 52:1815 - 1818); (b)(i) POCl3, DMF, 0 °C to 40 °C (US2017 / 0066717); (b)(ii) NaOH, MeOH / THF, 80 °C (the isomers 4-(trifluoromethyl)-1H-indole, 6-(trifluoromethyl)-1H-indole, and 7-(trifluoromethyl)indole can also be used as starting materials in this order) (EP 1990335); (c)(i) NEt3, THF, 0 °C (J. Org. Chem. 2014, 79:2105 - 2110); (c)(ii) 200 °C (J. Org. Chem. 2014, 79:2105 - 2110); (c)(iii) PhNH2, Pd2(dba)3, NaHCO3, pivalic acid, DMA, 120 °C (CN104892485); (c)(iv) NaOH, MeOH / THF, 80 °C; (d)(i) H2SO4, MeOH, reflux; (d)(ii) Pd / C, H2, MeOH; (d)(iii) NaOH, MeOH / THF, 80 °C; (e)(i) H2SO4, MeOH, reflux (Chem. Pap. 2018, 72:1369 - 1378); (e)(ii) K((CH=CH2)(BF3)), PdCl2, PPh3, Cs2CO3, THF / H2O, heat; (e)(iii) Pd / C, H2, MeOH, room temperature; (e)(iv) NaOH, MeOH / THF, 80 °C. Other substitution patterns can be obtained using differently substituted bromoindoles; C3 and C4 alkyl isomers can be incorporated in a similar manner using suitable commercially available organotrifluoroborates or organoboric acids or esters; (f)(i) H2SO4, EtOH, reflux; (f)(ii) EtI, K2CO3, acetone, heat; (f)(iii) KOH, EtOH, heat (other substitution patterns can be obtained by using differently substituted hydroxyindoles; other C1 - C4 moieties can be incorporated using appropriate commercially available alkyl iodides or alkyl bromides).

[0104]

[0105] Scheme 11

[0106] Scheme 11 shows a general method for the preparation of N-alkyl-substituted indoles (5- and 6-nitro-1H-indole-3-carboxylic acids are also commercially available and can also be used as starting materials in this order; other alkyl groups can be incorporated using appropriate aldehydes or ketones), where: (i) H2SO4, MeOH, reflux; (ii) Pd / C, H2, MeOH, room temperature (Bioorg. Med. Chem. Lett. 2011, 21:1782-1785); (iii) H3CCH2CHO, Pd / C, H2, MeOH, room temperature; (iv) NaOH, MeOH / THF, 80 °C.

[0107]

[0108] Scheme 12

[0109] Scheme 12 shows a general method for the preparation of N-alkylated indoles. (i) H2SO4, MeOH, reflux (Org. Biomol. Chem. 2012, 10:6885-6892); (ii) NaH, THF, then EtI, 70 °C (WO 2008 / 087529) (iii) NaOH, THF / MeOH (Bioorg. Med. Chem. Lett. 2005, 15:2734-2737). Other C1-C4 moieties can be incorporated using appropriate commercially available alkyl iodides or alkyl bromides.

[0110]

[0111] Scheme 13

[0112] Scheme 13 shows a method for the synthesis of non-commercially available trifluoromethyl-substituted anilines (some not shown here only differ in the substituents on O: these substitutions can be achieved by applying the general method shown in Scheme 20 at the appropriate stage of the synthesis), where: (a)(i) F2ClCCO2Me, KF, CuI, DMF, heating; (a)(ii) SnCl2.2H2O, EtOH, reflux; (b)(i) F3CCOOH, XeF2, DCM, room temperature (J. Org. Chem. 1988, 53, 4582-4585) (b)(ii) SnCl2.2H2O, EtOH, reflux; (c)(i) NaClO2, sulfonamide, MeOH, H2O, 0 °C (EP2557082); (ii) DPPA, NEt3, benzene, reflux, then LiOH, THF / H2O, room temperature.

[0113]

[0114] Scheme 14

[0115] Scheme 14 shows a synthetic method for non-commercially available cyano-substituted anilines (2,3-dihydro-benzo[1,4]dioxine-5-carbonitrile is also commercially available and can be used as a starting material in this sequence; the conversion to the corresponding dioxolane can be achieved by applying the general method shown in Scheme 20 at the appropriate stage of the synthesis), where: (i) trifluoromethanesulfonic anhydride, Me4N(NO3), DCM (J. Org. Chem. 2003, 68, 267-275); (ii) Fe, H3CCOOH, iPrOH, heating (J. Heterocycl. Chem. 1986, 23: 1821-1828); (iii) Fe, HCl, EtOH, reflux (WO 2018 / 232094).

[0116]

[0117] Scheme 15

[0118] Scheme 15 shows a synthetic method for non-commercially available halogen-substituted anilines (some not shown here only differ in the substituents on O: these substitutions can be achieved by applying the general method shown in Scheme 20 at the appropriate stage of the synthesis), where: (a)(i) Br2, H3CCOOH, room temperature (WO 2011 / 025798); (a)(ii) BBr3, DCM; )a)(iii) CH2I2, K2CO3, DMF, heating; (a)(iv) Pd / C, H2, EtOH; (b)(i) Br2, H3CCOOH (Tetrahedron Lett. 2008, 49, 5309-5311); (b)(ii) dimethyl sulfate, K2CO3 (Tetrahedron Lett. 2008, 49: 5309-5311); (b)(iii) Pd / C, H2, EtOH; (b)(iv) BBr3, DCM; (b)(v) CH2I2, K2CO3, DMF, heating; (b)(vi) Pd / C, H2, EtOH; (c)(i) DPPA, NEt3, PhMe, reflux, then 9-fluorenemethanol, reflux, then piperidine, room temperature; (d)(i) HNO3, 0-20 °C (WO 2013 / 052568); (d)(ii) Pd / C, H2, EtOH; (d)(iii) PtO2, H2, EtOH (WO2013 / 052568).

[0119]

[0120] Scheme 16

[0121] Scheme 16 shows a method for hydroxy-substituted and alkoxy-substituted anilines that are not commercially available (2H-benzo[d][1,3]dioxol-4-ol and 2,3-dihydro-1,4-benzodioxin-5-ol are also commercially available and can be used as starting materials for sequences a, b, and e; 4,5-dimethoxy-2-nitrophenol and 7-nitro-2,3-dihydro-benzo[1,4]dioxin-6-ol are also commercially available and can be used as starting materials in sequence d; 2,3-dimethoxyphenol and 2,3-dihydro-1,4-benzodioxin-5-ol can also be used as starting materials in sequence e; other C1-C4 moieties can be incorporated using appropriate commercially available alkyl iodides or alkyl bromides). In this scheme: (a)(i) BnBr, K2CO3, MeCN, reflux (Chem.: Eur. J. 2016, 22: 15058-15068); (a)(ii) HNO3, (CH3CO)2O, room temperature; (a)(iii) Pd / C, H2 (20 psi), EtOH, room temperature (US 6,048,866); (b)(i) HNO3, H2O, H3CCOOH, 5 °C (US 6,337,420); (b)(ii) Pd / C, H2, MeOH; (c)(i) EtI, K2CO3, DMF, room temperature; (c)(ii) Pd / C, H2, MeOH; (d)(i) MeI, K2CO3, acetone; (d)(ii) HNO3, (H3CCO)2O, room temperature (WO 2004 / 046120); (d)(iii) Pd / C, H2, MeOH; (e)(i) HNO3, H2O, H3CCOOH, 5 °C (US 6,337,420); (e)(ii) dimethyl sulfate, K2CO3, xylene (J. Chem. Soc. 1931, 2542-2549); (e)(iii) Sn, HCl (J. Chem. Soc. 1931, 2542-2549).

[0122]

[0123] Scheme 17

[0124] Scheme 17 shows a method for the synthesis of nitro-substituted anilines that are not commercially available (some not shown here differ only in the substituents on the O: these substitutions can be achieved by applying the general methods shown in Scheme 20 at the appropriate stages of the synthesis), where: (a)(i) DPPA, NEt3, PhMe, reflux, then 9-fluorenemethanol, reflux, then piperidine, room temperature; (b)(i) SOCl2, DMF, 1,2-DCE, heat, then NaN3, acetone / H2O, 5 °C, then H3CCOOH / H2O, heat (J. Med. Chem. 1999, 42: 2373-2382).

[0125]

[0126] Scheme 18

[0127] Scheme 18 shows a synthetic method for non-commercially available nitro-substituted anilines (some not shown here only differ in the substituents on O: these substitutions can be achieved by applying the general method shown in Scheme 20 at the appropriate stage of the synthesis; other C2-C4 alkyl isomers can be incorporated in a similar manner using suitable commercially available organotrifluoroborates or organic boronic acids or esters). In this scheme: (a)(i) HNO3, -10 °C (Tetrahedron 1985, 41:2355-2359); (a)(ii) Pd / C, H2, MeOH, <586 Torr, room temperature (Tetrahedron 1985, 41:2355-2359); (b)(i) Isopropenylboronic acid pinacol ester, Pd(dppf)Cl2, NaHCO3, 1,4-dioxane / H2O, 100 °C (WO 2017 / 160569); (b)(ii) Pd / C, H2 (50 psi), MeOH (WO 2017 / 160569); (c)(i) NaOH, DMSO / H2O, 90 °C, then CH2I2 (US2008 / 0234314); (c)(ii) HNO3, (H3CCO)2O, -10 °C (US 2008 / 0234314): (c)(iii) Fe, HCl, EtOH / H2O, reflux (US 2008 / 0234314); (d)(i) Isopropenylboronic acid pinacol ester, Pd(dppf)Cl2, NaHCO3, 1,4-dioxane / H2O, 100 °C; (d)(ii) Pd / C, H2 (50 psi), MeOH; (e)(i) NaH, then MeI (Czech.Chem.Commun. 2000, 65:924-940); (e)(ii) MeCN, nBu4NClO4 (Czech.Chem.Commun. 2000, 65:924-940); (e)(iii) NaOH, EtOH / H2O, heat.

[0128]

[0129] Scheme 19

[0130] Scheme 19 shows a synthetic method for non-commercially available N-substituted anilines (some not shown here differ only in the substituents on O: these substitutions can be achieved by applying the general method shown in Scheme 20 at the appropriate stage of the synthesis; other alkyl groups can be incorporated using suitable aldehydes, ketones or alkyl halides). In this scheme: (a) and (b) (i) H3CCH2CHO, Pd / C, H2, MeOH, room temperature; (ii) NaOH, MeOH / THF, 80 °C; (iii) DPPA, NEt3, PhMe, reflux, then 9-fluorenemethanol, reflux, then piperidine, room temperature; (iv) EtBr, NaOH, PhH, heat; (v) NaOH, MeOH / THF, 80 °C; (vi) DPPA, NEt3, PhMe, reflux, then 9-fluorenemethanol, reflux, then piperidine, room temperature; (c) (i) HCl, MeOH / H2O (US 4,287,341); (c) (ii) H3CCH2CHO, Pd / C, H2, MeOH, room temperature; (c) (iii) NaOH, MeOH / THF, 80 °C; (c) (iv) DPPA, NEt3, PhMe, reflux, then 9-fluorenemethanol, reflux, then piperidine, room temperature; (c) (v) HCl, MeOH / H2O (US 4,287,341); (c) (vi) EtBr, NaOH, PhH, heat; (c) (vii) NaOH, MeOH / THF, 80 °C; (c) (viii) DPPA, NEt3, PhMe, reflux, then 9-fluorenemethanol, reflux, then piperidine, room temperature. Compounds having NH2 instead of being alkylated can be obtained by reducing the corresponding NO2 using the exemplary procedures described herein at the appropriate stage of the synthesis.

[0131]

[0132] Scheme 20

[0133] Scheme 20 shows a general method for the interconversion of substituents on the 1,2-dihydroxybenzene moiety, where: (i) BBr3, DCM; (ii) dimethyl sulfate, K2CO3, acetone, heat; (iii) BBr3, DCM; (iv) CH2Br2, K2CO3, DMF, heat.

[0134] The compounds described herein have useful properties in enhancing sperm motility. On the one hand, the present invention provides the use of a compound of formula (I), its stereoisomers or physiologically tolerable salts for enhancing the motility of sperm obtained from non-human mammals.

[0135] Sperm cells exhibit different motility patterns to adapt to their functional requirements in space and time. For example, these motilities can be monitored by a computer-aided sperm analyzer (CASA), which records and analyzes the movement of the sperm head. It has been shown that factors such as average path velocity (VAP), curvilinear velocity (VCL), straightness (STR), linearity (LIN), and amplitude of lateral head displacement (ALH) are directly related to semen quality. "Sperm motility" is defined herein as the percentage of sperm that are in motion and can be further divided into progressive motility, non-progressive motility, and immotility (i.e., no movement). In semen, sperm cells mostly exhibit rapid forward movement (termed "progressive motility"), which is necessary for the cell to traverse the cervix. "Non-progressive motility" refers to all other motility patterns that are not progressive. The complex process of sperm capacitation (the penultimate step in mammalian sperm maturation that is necessary to enable them to fertilize an egg) begins once the cell enters the uterus. Hyperactivation or "overmovement" is a hallmark of capacitation and this motility behavior is observed as a vigorous and asymmetric swimming pattern that aids in the release of sperm from the oviductal epithelium and subsequent penetration of the egg. Since sperm motility affects the ability of sperm to reach the egg, either in the female reproductive tract (in vivo) or through water (in vitro), it is a factor in successful fertilization. In mammals, sperm motility also aids in the passage of sperm through the cumulus oophorus and the zona pellucida surrounding the mammalian oocyte.

[0136] The compounds described herein have an effect of enhancing general sperm motility when contacted with mature mammalian sperm cells, i.e., they act as "sperm activators". In particular, they are capable of increasing the forward motility of sperm. Given these properties, these compounds are suitable for enhancing the fertilization of eggs, either in vivo or in vitro, and can therefore be used in fertility programs.

[0137] Any reference herein to "enhancing sperm motility" means an increase in sperm motility as compared to the sperm motility of a control mammal (or from the same mammal) that has not been contacted with the compounds described herein. Any reference herein to the use of a compound or composition "for enhancing sperm motility" shall be construed accordingly.

[0138] In some embodiments, the compounds described herein are capable of increasing sperm motility in all forms. In some embodiments, the compounds may be capable of increasing progressive and hyperactivated motility. In some embodiments, they may be capable of increasing forward motility. For example, in boars, progressive sperm may be defined as those cells that exhibit an average path velocity (VAP) greater than 45 μm / s and a straightness (STR) greater than 45%. For example, hyperactivated motility of boar sperm may be defined according to the parameters discussed in Schmidt and Kamp (Induced Hyperactivity in Boar Spermatozoa and its Evaluation by Computer-Assisted Sperm Analysis, Reproduction, 128:171-179, 2004). In particular, hyperactive sperm in boars may be defined as those cells that exhibit a curvilinear velocity (VCL) greater than 97 μm / s and a linearity (LIN) less than 32% and an average lateral head displacement (LHD 平均值 ) greater than 3.5 μm and a wobble (WOB) less than 71%.

[0139] In some embodiments, the compounds described herein are capable of increasing the proportion of progressive and / or hyperactivated mature sperm cells in a sperm sample from a non-human mammal by up to 20%, such as up to 15%, such as 10-15%.

[0140] In another aspect, the present invention provides a method for enhancing sperm motility obtained from a non-human mammal, the method comprising the step of contacting the sperm in vitro with an effective amount of a compound of formula (I) or a stereoisomer or a physiologically tolerable salt thereof as described herein. Optionally, the method may additionally comprise the step of obtaining or collecting semen from the mammal. Such methods are well known in the reproduction of livestock and other animals.

[0141] As used herein, the term "effective amount" is intended to denote a sperm motility enhancing concentration. The concentration required to achieve the desired activity will depend on various factors such as the particular compound and its intended use and may vary or be adjusted as needed. Generally, the required concentration may be in the range of 50 to 100 μM, such as 55 to 95 μM, 60 to 90 μM, 70 to 85 μM or 75 to 80 μM.

[0142] In view of their ability to increase sperm motility, such as increasing progressive sperm motility, the compounds described herein are particularly suitable for use in artificial insemination methods.

[0143] For use in accordance with the present invention, the compounds described herein are contacted with mature sperm cells, thereby increasing their motility. For example, they can be mixed with semen samples taken from non-human mammals. In any of the uses or methods described herein, the compounds are typically formulated into a composition that can be added to sperm (e.g., a semen sample) to enhance sperm viability. Such compositions can comprise an active compound of formula (I), its stereoisomers or a physiologically acceptable salt, in combination with one or more physiologically tolerable carriers, excipients or diluents. Such compositions form another aspect of the present invention. Carriers, excipients and diluents acceptable for this purpose are well known in the art and can be readily selected. Examples include solvents, solubilizers, preservatives (e.g., antioxidants), semen extenders, buffers (e.g., TRIS - (tris(hydroxymethyl)aminomethane)), pH regulators (e.g., citric acid), viscosity improvers, stabilizers and tonicity regulators. For example, suitable carriers can include DMSO, water, phosphate buffer and saline.

[0144] In one embodiment, the compounds for use in the present invention can be formulated into a liquid composition that is suitable for vaginal administration when mixed with a semen sample. For example, the compound can be dissolved or dispersed in a suitable solvent. Those skilled in the art can readily select suitable such solvents taking into account that the solvent is physiologically tolerable.

[0145] As used herein, the term "physiologically tolerable" with respect to any compound or composition means that the compound or composition is chemically and / or toxicologically compatible with the other components of the formulation or with the subject to which it will be administered.

[0146] As used herein, the term "semen extender" (or "extender") refers to any component or combination of components capable of maintaining the viability of a semen sample, i.e., preserving its fertilizing ability. Its role is to protect sperm cells from their own toxic by-products, while also protecting sperm cells from cold shock and osmotic shock during refrigeration and / or transportation. Cooling of sperm reduces its metabolism and thus aids in its storage. For example, ingredients commonly known to be used as semen extenders include egg yolk, milk proteins, membrane protectants, proteins, sugars, antioxidants, antibiotics (e.g., ticarcillin, timentin, amikacin sulfate, penicillin, gentamicin, tylosin, spectinomycin, lincomycin, streptomycin or combinations thereof, or other antibiotics in accordance with EU Directive 88 / 407), phospholipids, glycerol, buffers (e.g., TRIS), citric acid and combinations thereof. Commercially available semen extenders for pigs include (which contains phospholipids, TRIS, citric acid, sugars, antioxidants, buffers, glycerol, water and antibiotics), CCS and (All contain TRIS, citric acid, sugar, buffer, glycerol, water, and antibiotics (such as tylosin, gentamicin, spectinomycin, and lincomycin) that comply with EU Directive 88 / 407).

[0147] The compositions according to the present invention can be prepared using conventional techniques, such as dissolution and / or mixing procedures.

[0148] In one embodiment, an activator compound or a composition containing the compounds described herein can be added to a semen sample before cryopreservation. Cryopreservation of sperm allows for longer storage of sperm, for example, when it is necessary to transport for use, or when it will be used at a later time. When used in this way, the cryo-composition can additionally include a cryoprotectant (commonly also referred to as a "cryoprotective agent" or "cryopreservation adjuvant"), which helps to protect sperm during freezing and subsequent thawing. Typical cryoprotectants include, for example, glycerol, mannitol, sucrose, trehalose, DMSO, and dimethylformamide (DMF).

[0149] In one embodiment, an activator compound or a composition containing the compounds described herein can be added to a fresh semen sample. Alternatively, this can be added to a cryopreserved semen sample after thawing.

[0150] For use in artificial insemination, the compounds and compositions can be used to pretreat or prepare sperm for such procedures. For example, sperm for an artificial insemination procedure can be contacted (i.e., "pretreated") in vitro with the compound or the composition before vaginal administration to a female mammal. Contact can include incubation with the compound or the composition. The incubation time can vary, but can be in the range of about 15 to 45 minutes before use, for example, about 15 to 20 minutes before use. After incubation, the treated sperm sample is "ready-to-use". By pretreating sperm intended to be used in such procedures, there is a possibility of increasing the fertilization rate and / or the number of offspring.

[0151] In another aspect, the present invention thus provides a method for treating or preparing sperm for an artificial insemination procedure, the method comprising the steps of:

[0152] (a) providing a sperm sample from a mammalian male subject; and

[0153] (b) contacting the sperm sample with the compound or composition described herein.

[0154] The sperm sample can be a fresh sperm sample or a cryopreserved and thawed sperm sample. The treated sperm obtained or obtainable by this method also constitutes an aspect of the present invention.

[0155] In another aspect, the present invention provides a method for artificial insemination of female non-human mammals, the method comprising the step of inseminating the female with the treated sperm as described herein. It is understood that the female will be fertile.

[0156] For use in artificial insemination, a fixed volume of semen can be combined with a single dose of a composition containing the activator compound as described herein and gently agitated (e.g., carefully inverted) to ensure thorough mixing to form an "activated" dose of semen. This can then be used to inseminate the female using conventional methods in the art. A typical fixed volume of semen can range from about 70 to 100 mL, such as 80 to 95 mL, preferably about 90 mL. For example, a single dose of the composition can contain a 100 mM solution of the activator compound in an amount of 20 to 100 μL, preferably 30 to 80 μL, such as about 50 μL.

[0157] Non-human mammalian subjects that can be treated according to the present invention include, in particular, farm animals (such as sheep, cattle, pigs, etc.), but can also include domestic animals (such as dogs, cats, etc.) and laboratory animals (such as mice, rats, monkeys, etc.). Particularly preferred mammals are pigs. Other mammals that can benefit from the present invention include horses (such as racehorses) and endangered animal species.

[0158] Examples

[0159] The present invention will now be described in more detail by the following non-limiting examples and with reference to the following accompanying drawings:

[0160] Figure 1: A: % activation of boar sperm in the presence of Compound 1; and B: motility and forward movement of boar sperm in the presence of Compound 1.

[0161] Figure 2 : Forward movement and motility of boar sperm after addition of Compound 1 to sperm samples stored at 18 °C for 1 to 8 days.

[0162] Figure 3 : In vitro fertilization rate (% fertilization) of mouse oocytes contacted with mouse sperm cells incubated in the presence of Compound 1.

[0163] Figure 4 : Genotoxicity of different concentrations of Compound 1 on human T cells.

[0164] Figure 5 : Average number of embryos per sow after artificial insemination with untreated "control" boar sperm and boar sperm incubated with Compound 1 ("activator").

[0165] Example 1- Determination of sperm motility and forward movement

[0166] Method:

[0167] The Duroc boar sperm samples were from Norsvin AS and contained pooled semen from three boars. Before use, the semen samples were stored in diluent (micropipettes) at 18 °C. The testing of Compound 1 was repeated using 2-fold serial dilutions (including 10 individual concentrations starting from 100 μM). 995 μL of semen was aliquoted into Eppendorf tubes and 5 μL of Compound 1 diluted in DMSO was added to reach the stated final concentration. The resulting semen samples were gently mixed and incubated in a 37 °C water bath for 12 minutes. Subsequently, 10 μL of each semen sample was added to a counting chamber slide (Leja) and incubated in an incubator at 37 °C. Starting from 14 minutes, sperm motility, progressive motility, and curvilinear velocity were determined using CASA (Hamilton Thorne). For each chamber A and B, 15 individual frames were analyzed and motility and progressive motility data were read out. Using only DMSO control (set as 100% active), the data was converted to activation-% by determining the percentage increase in motility or progressive motility relative to the control sample without the addition of Compound 1. To determine the EC 50 value, the data was fitted using Prism8 (nonlinear fit, absolute IC 50 , where x is the concentration, graphpad.com).

[0168] Results:

[0169] For different test concentrations (n = 6), the activation of boar sperm in the presence of Compound 1 (“activation-%”) is as Figure 1A shown. The corresponding curve fits used to determine the EC 50 value are shown for motility and progressive motility. Absolute EC 50 Motility: 3.31 μM; absolute EC 50 Progressive motility: 11.17 μM. As Figure 1B shown, Compound 1 increased motility and progressive motility in a dose-dependent manner (n = 4).

[0170] Example 2 –Effect of long-term storage – boar sperm

[0171] Method:

[0172] The effect of Compound 1 (“activator”) on the shelf life of boar sperm was evaluated. Compound 1 was freshly added to aliquots of the stored boar sperm.

[0173] Sperm samples were stored as aliquots at 18 °C in Eppendorf tubes (995 μl). During the course of one week, an activator solution (final concentration of 50 μM) in 5 μl of 10 mM DMSO was added to one aliquot, and the resulting solution was incubated in a water bath at 37 °C for 12 minutes. Subsequently, 10 μl of the semen sample was taken out and added to a counting chamber slide, incubated at 37 °C for 2 minutes and analyzed as described in Example 1.

[0174] Results:

[0175] The viability and forward motility of the stored sperm samples after the addition of Compound 1 were as Figure 2 shown. The results indicate that even after 7 days of storage, the addition of the activator solution to the stored boar sperm samples increases viability and forward motility, thus having a positive effect on the shelf life of boar sperm.

[0176] Example 3 -In vitro fertilization - mouse

[0177] Method:

[0178] In vitro fertilization (IVF) studies were conducted to determine whether Activator Compound 1 has any side effects on the fertilization process. Male mice were sacrificed, the epididymis was opened, and sperm were released into an appropriate buffer in a Petri dish. Sperm cells were incubated with the activator compound at 50 μM for one hour before transferring the aliquot to a Petri dish containing oocytes. The cells were incubated at 37 °C for another 24 hours, and 1-cell / 2-cell eggs were counted. Repeated experiments were performed using sperm from the same epididymis and oocytes from two different mice.

[0179] Results:

[0180] Sperm treated with Compound 1 showed a fertility rate comparable to that of the control samples. The results were within the range of 50% to 80% of the normal fertilization rate in mouse IVF (see Figure 3 ). Therefore, Compound 1 does not interfere with the fertilization process.

[0181] Example 4 -Genotoxicity

[0182] Method:

[0183] Human T cells (1 compound at four concentrations including 0 μM; two exposure times) were embedded in 1% agarose and lysed in pH 10, cold 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris buffer and 1% Triton X-100. After incubation with dithiothreitol and lithium diiodosalicylate, they were then incubated in cold 0.3 M NaOH and 1 M EDTA for 20 minutes and then electrophoresed at 0.8 V / cm for 20 minutes in a cold room. After neutralization with PBS and staining with SYBRGold (Invitrogen), the slides were scored using the Comet Assay IV image analysis program (Perceptive Instruments).

[0184] Results:

[0185] The results are summarized in Table 1 and presented graphically in Figure 4 . No genotoxic effects of the activator were observed.

[0186] Table 1

[0187]

[0188] Example 5 - In-field pilot study – Sow insemination for livestock reproduction

[0189] Methods:

[0190] The pilot in - vivo study included 27 sows, of which 13 received untreated control sperm and 14 received sperm treated with Compound 1 (“activator”). During the experiment, insemination doses were collected from the same animals and contained pooled semen from three boars. Immediately before insemination, 45 μl of a 100 mM DMSO activator solution was added directly to 89 ml of boar sperm (final concentration 50 μM), and insemination was carried out within 15 to 45 minutes. The sperm was transferred to an insemination bottle, and then the activator was added. After the insemination catheter was placed in the sow's cervix, the bottle was connected and the sperm sample was released into the sow. The sows received the insemination dose on the following two days. The animals were sacrificed 30 to 35 days later, the uterus was removed and the embryos were counted for all 30 sows. Optical examinations were performed on organ material from 5 sows receiving the activator and 5 sows from the control group, and samples from the uterus, cervix and vagina were fixed in formalin for histological examination. The organ material of the control group and the activator group mostly showed normal mucosa, and there were no signs of lesions in the cervix, uterus and vagina. The formalin specimens were sectioned and stained with hematoxylin and eosin for histological examination. Histopathological evaluation included a comprehensive assessment of the mucosal epithelium and the underlying lamina propria. In the epithelium and lamina propria, particular emphasis was placed on acute changes. In the epithelium, acute changes included single - cell necrosis / apoptosis and polymorphonuclear leukocyte infiltration. In the lamina propria, infiltration of polymorphonuclear leukocytes was considered the most important acute change. Three of the five sows in the activator group and one sow in the control group showed extensive polymorphonuclear leukocyte infiltration mainly in the cervix and uterus. Cell necrosis / apoptosis was detected in one sow in the control group and one sow in the activator - receiving group.

[0191] Infiltration of lymphocytes in the epithelium and lamina propria was considered to be within the normal range for all individuals. It was difficult to evaluate flattening of the surface epithelium and possible loss of parts. Lamina propria edema and hemorrhage were detected in several individuals. Estrus and age, which are indicators of leukocyte infiltration in sows, were considered normal.

[0192] The average number of embryos was determined for sows in the control group (n = 11, 11 sows after insemination) and sows receiving the activator (n = 13, 13 sows after insemination). The activator not only increased the pregnancy rate in sows but also resulted in an average increase of 2 embryos per sow (see Figure 5 ).

[0193] Example 6 - In - vivo pilot study

[0194] Following the success of the pilot study reported in Example 5, a larger scale field trial study involving 441 sows is underway. 224 sows received untreated control sperm, and 217 sows received sperm treated with Compound 1 (“activator”). The results of this larger study showed that the pregnancy rate of sows treated with the activator compound increased by 5%, and on average, each sow produced approximately 1 more live piglet. By increasing the conception rate and the number of piglets produced per sow, the use of a sperm activator can shorten the weaning to successful mating interval (“WSI”), i.e., the interval between consecutive pregnancies.

Claims

1. Use of a compound of formula (I) or a physiologically tolerable salt thereof in the preparation of a medicament for enhancing sperm motility obtained from non-human mammals:

2. Use of a compound of formula (I) or a physiologically tolerable salt thereof in the preparation of a medicament for enhancing sperm motility in rams; 3. Use of a compound of formula (I) or a physiologically tolerable salt thereof in the preparation of a medicament for enhancing sperm motility in bulls; 4. Use of a compound of formula (I) or a physiologically tolerable salt thereof in the preparation of a medicament for enhancing sperm motility in boars; 5. Use of a compound of formula (I) or a physiologically tolerable salt thereof in the preparation of a medicament for enhancing sperm motility in stallions;

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