5-(1H-INDOL-3-YL)-OXAZOLE, -OXADIAZOLE AND -FURAN DERIVATIVES AS ENHANCERS OF SPERM MOTILITY

Abstract

The invention relates to the use of compounds of formula (I), stereoisomers, and physiologically tolerable salts thereof, to enhance the motility of sperm obtained from a non-human mammal: wherein in formula I: X is —NR′— (in which R′ is H or C.sub.1-3 alkyl, e,g, —CH.sub.3), —O—, —S— or —CH.sub.2—; Y is —S—, —O—, —NR″— (in which R″ is H or C.sub.1-3 alkyl, e.g. —CH.sub.3), or —CH.sub.2—; Z.sup.1 and Z.sup.2 are independently selected from N and CH; R1 to R3 are independently selected from hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, —OH, —CN, —NO.sub.2 and -NR.sup.12R.sup.13; R.sup.4 is hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, or —OH; R.sup.5 is hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, or C.sub.1-6 haloalkyl; R.sup.6 and R.sup.7 are independently selected from hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, —OH, —CN, —NO.sub.2 and -NR.sup.12R.sup.13; R.sup.8 is hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, —CN, —NO.sub.2 or -NR.sup.12R.sup.13; R.sup.9 is hydrogen or C.sub.1-4 alkyl; R.sup.10 and R.sup.1.sup.1 are independently selected from hydrogen and C.sub.1-3 alkyl; or R.sup.10 and R.sup.1.sup.1, together with the intervening —O—and ring atoms, are joined to form a dioxolane ring; and R.sup.12 and R.sup.13 are independently selected from H and C.sub.1-3 alkyl. The compounds find particular use in animal husbandry to increase the rate of fertilization in artificial insemination of livestock, especially pigs. They can also be used to prepare, pre-treat or store semen for use in artificial insemination procedures.

Claims

1. Use of a compound of formula (I), a stereoisomer, or a physiologically tolerable salt thereof, to enhance the motility of sperm obtained from a non-human mammal: ##STR00029## wherein: X is —NR′— (in which R′ is H or C.sub.1-3 alkyl, e.g. —CH.sub.3), —O—, —S— or —CH.sub.2—; Y is —S—, —O—, —NR″— (in which R″ is H or C.sub.1-3 alkyl, e.g. —CH.sub.3), or —CH.sub.2—; Z.sup.1 and Z.sup.2 are independently selected from N and CH; R.sup.1 to R.sup.3 are independently selected from hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, —OH, —CN, —NO.sub.2 and -NR.sup.12R.sup.13; R.sup.4 is hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, or —OH; R.sup.5 is hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, or C.sub.1-6 haloalkyl; R.sup.6 and R.sup.7 are independently selected from hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, —OH, —CN, —NO.sub.2 and -NR.sup.12R.sup.13; R.sup.8 is hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, —CN, —NO.sub.2 or -NR.sup.12R.sup.13; R.sup.9 is hydrogen or C.sub.1-4 alkyl; R.sup.10 and R.sup.11 are independently selected from hydrogen and C.sub.1-3 alkyl; or R.sup.10 and R.sup.11, together with the intervening —O— and ring atoms, are joined to form a dioxolane ring; and R.sup.12 and R.sup.13 are independently selected from H and C.sub.1-3 alkyl.

2. Use as claimed in claim 1, wherein X is —NR′— (in which R′ is H or —CH.sub.3) or —O—, preferably wherein X is —NH— or —O—, e.g. —NH—.

3. Use as claimed in claim 1 or claim 2, wherein Y is selected from —S—, —O—, —NR″— (in which R″ is H or —CH.sub.3), and —CH.sub.2—, preferably wherein Y is —S—.

4. Use as claimed in claim 1, wherein said compound is a compound of formula (II), or a stereoisomer or physiologically tolerable salt thereof: ##STR00030## (wherein Z.sup.1, Z.sup.2, and R.sup.1 to R.sup.11 are as defined in claim 1).

5. Use as claimed in any one of the preceding claims, wherein both Z.sup.1 and Z.sup.2 are N, or one of Z.sup.1 and Z.sup.2 is N and the other of Z.sup.1 and Z.sup.2 is CH.

6. Use as claimed in any one of claims 1 to 4, wherein both Z.sup.1 and Z.sup.2 are N.

7. Use as claimed in claim 1, wherein said compound is a compound of formula (III), or a stereoisomer or physiologically tolerable salt thereof: ##STR00031## (wherein X, Y and R.sup.1 to R.sup.11 are as defined in any one of claims 1 to 3).

8. Use as claimed in any one of the preceding claims, wherein R.sup.1 to R.sup.3 are independently selected from hydrogen, halogen (e.g. —F, —Cl or —Br), C.sub.1-4 alkyl, and C.sub.1-4 haloalkyl (e.g. —CF.sub.3).

9. Use as claimed in any one of the preceding claims, wherein R.sup.4 is hydrogen, halogen (e.g. —F, —Cl or —Br), C.sub.1-4 alkyl, or C.sub.1-4 haloalkyl (e.g. —CF.sub.3).

10. Use as claimed in any one of the preceding claims, wherein R.sup.5 is hydrogen, halogen (e.g. —Cl or —Br), C.sub.1-4 alkyl, or C.sub.1-4 haloalkyl (e.g. —CF.sub.3).

11. Use as claimed in any one of the preceding claims, wherein R.sup.6 and R.sup.7 are independently selected from hydrogen, halogen (e.g. —F, —Cl or —Br), C.sub.1-4 alkyl, and C.sub.1-4 haloalkyl (e.g. —CF.sub.3).

12. Use as claimed in any one of the preceding claims, wherein R.sup.8 is hydrogen, halogen (e.g. —F, —Cl or —Br), C.sub.1-4 alkyl, or C.sub.1-4 haloalkyl (e.g. —CF.sub.3).

13. Use as claimed in any one of the preceding claims, wherein R.sup.9 is hydrogen or —CH.sub.3, preferably hydrogen.

14. Use as claimed in any one of the preceding claims, wherein R.sup.10 and R.sup.11 are independently selected from hydrogen and C.sub.1-3 alkyl (e.g. —CH.sub.3).

15. Use as claimed in any one of claims 1 to 13, wherein R.sup.10 and R.sup.11, together with the intervening —O— and ring atoms, are joined to form a dioxolane ring.

16. Use as claimed in claim 1, wherein said said compound is a compound of formula (IV), or a stereoisomer or physiologically tolerable salt thereof: ##STR00032## (wherein Z.sup.1, Z.sup.2, X, Y and R.sup.1 to R.sup.9 are as defined in any one of claims 1 to 3, 5, 6 and 8 to 13).

17. Use as claimed in claim 1, wherein the compound of formula (I) is: ##STR00033## or a stereoisomer, or physiologically tolerable salt thereof.

18. Use as claimed in any one of the preceding claims in enhancing progressive sperm motility.

19. A composition comprising a compound as defined in any one of claims 1 to 17, in combination with one or more physiologically tolerable carriers, excipients or diluents.

20. A composition as claimed in claim 19, wherein said carriers, excipients or diluents are selected from sugars, antibiotics, proteins, membrane protecting agents, antioxidants, phospholipids, glycerol, citric acid, solvents (e.g. DMSO or water), and buffers.

21. Use of a composition as claimed in claim 19 or claim 20 to enhance the motility of sperm obtained from a non-human mammal, for example to enhance the progressive motility of said sperm.

22. A method of treating or preparing sperm for use in an artificial insemination procedure, said method comprising: (a) providing a sperm sample from a non-human mammalian male subject; and (b) contacting said sperm sample with a compound as defined in any one of claims 1 to 17 or a composition as defined in claim 19 or claim 20.

23. A method of artificial insemination, said method comprising: (a) providing a sperm sample from a non-human mammalian male subject; (b) contacting said sperm sample with a compound as defined in any one of claims 1 to 17 or a composition as defined in claim 19 or claim 20; and (c) introducing the sperm sample contacted with said compound or composition into the vaginal tract of a non-human mammalian female subject.

24. A method as claimed in claim 23, wherein the sperm sample is from a ram, bull or boar, preferably from a boar.

25. A compound of general formula (I), or a stereoisomer or physiologically tolerable salt thereof, wherein said compound is other than: ##STR00034## ##STR00035## ##STR00036## .

Description

[0105] FIG. 1: A: % Activation of boar sperm in the presence of Compound No. 1; and B: Motility and progressive motility of boar sperm in the presence of Compound No. 1.

[0106] FIG. 2: Progressive motility and motility of boar sperm following the addition of Compound No. 1 to a sperm sample stored at 18° C. for 1 to 8 days.

[0107] FIG. 3: In vitro fertility rates (% fertilization) in mice oocytes contacted with mice sperm cells incubated in the presence of Compound No. 1.

[0108] FIG. 4: Genotoxicity of different concentrations of Compound No. 1 against human T-cells.

[0109] FIG. 5: Average number of embryos per sow following artificial insemination with untreated “control” boar sperm and boar sperm incubated with Compound No. 1 (“activator”).

Example 1 - Determination of Sperm Motility and Progressive Motility

Method:

[0110] Duroc boar sperm samples were received from Norsvin AS and contained pooled semen from three individual boars. Semen samples were stored in Androstar® extender (mini-tube) at 18° C. prior to use. Compound No. 1 was tested in duplicates, using 2-fold serial dilutions including 10 individual concentrations starting from 100 .Math.M. 995 .Math.L semen were aliquoted in Eppendorf tubes and 5 .Math.L of Compound No. 1 diluted in DMSO were added leading to the described final concentrations. The resulting semen samples were mixed gently and incubated for 12 min at 37° C. in a water bath. Subsequently, 10 .Math.L of each semen sample was added to a counting chamber slide (Leja) and incubated on a plate incubator at 37° C. Sperm motility, progressive motility and curvilinear velocity were determined using a CASA (Hamilton Thorne), starting from 14 min. For each chamber A and B 15 individual frames were analysed and motility as well as progressive motility data was read out. The data were converted into activation-% using DMSO-only controls (set to 100% activity) by determining the percental increase of either motility or progressive motility relative to the control sample without added Compound No. 1. For the determination of EC.sub.50 values, data were fitted using Prism8 (non-linear fit, absolute IC.sub.50, x is concentration, graphpad.com).

Results:

[0111] Activation (“activation-%”) of boar sperm in the presence of Compound No. 1 is shown in FIG. 1A for the different concentrations tested (n=6). The respective curve fits for the determination of EC.sub.50 values are shown for motility and progressive motility. Absolute ECso motility: 3.31 .Math.M; absolute EC.sub.50 progressive motility: 11.17 .Math.M. As shown in FIG. 1B, Compound No. 1 increases both motility and progressive motility in a dose-dependent manner (n=4).

Example 2 - Effect of Long-Term Storage - Boar Sperm

[0112] Method:

[0113] The effect of Compound No. 1 (“activator”) on the shelf-life of boar sperm was evaluated. Compound No. 1 was freshly added to stored boar sperm aliquots.

[0114] The sperm samples were stored as aliquots in Eppendorf tubes (995 .Math.l) at 18° C.

[0115] During the course of one week 5 .Math.l 10 mM activator solution in DMSO (50 .Math.M final concentration) was added to one aliquot and the resulting solution was incubated in a water bath at for 12 min at 37° C. Subsequently, 10 .Math.l semen samples were taken out and added to a counting chamber slide, incubated for 2 min at 37° C. and analysed as described in Example 1.

Results:

[0116] Motility and progressive motility for the stored sperm samples after the addition of Compound No. 1 are shown in FIG. 2. The results show that the addition of the activator solution to the stored boar sperm sample increases motility and progressive motility, even after 7 days of storage and therefore has a positive impact on boar sperm shelf life.

Example 3 - In Vitro Fertilization - Mouse

Method:

[0117] In vitro fertilization (IVF) studies were conducted to determine whether the activator compound No. 1 has any adverse effects on the fertilization process. Male mice were sacrificed, and the epididymis was opened to release the sperm into the appropriate buffer in a petri dish. The sperm cells were incubated with 50 .Math.M of the activator compound for one hour before an aliquot was transferred to a petri dish containing oocytes. The cells were further incubated at 37° C. and 1-cell/ 2-cell eggs were counted after 24 hours. The experiment was performed in duplicates using sperm from the same epididymis and oocytes from two different mice.

Results:

[0118] Sperm treated with Compound No. 1 showed fertility rates comparable to the control samples. The results are within the normal fertilization rate of mouse IVF ranging from 50 to 80% (see FIG. 3). Compound No. 1 therefore does not interfere with the fertilization process.

Example 4 - Genotoxicity

Method:

[0119] Human T-cells (four concentrations of Compound No. 1 including 0 .Math.M; two exposure times) were embedded in 1% agarose, lysed in cold 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris buffer, and 1% Triton X-100, pH 10. Incubation with dithiothreitol and lithium diiodosalicylate was followed by incubation in cold 0.3 M NaOH, and 1 mM EDTA for 20 min and then electrophoresis at 0.8 V/cm for 20 min in a cold room. After neutralization with PBS, and staining with SYBRGold (Invitrogen), slides were scored using the Comet Assay IV image analysis programme (Perceptive Instruments).

Results:

[0120] The results are summarised in Table 1 and graphically represented in FIG. 4. No genotoxic effects of the activator were observed.

TABLE-US-00001 Treatment % Tail DNA Control - Lysis 0.1 Control + H.sub.2O.sub.2 75.4 0.5 hr exposure 500 .Math.M 0.4 50 .Math.M 0.6 5 .Math.M 0.2 0 .Math.M 0.1 3 hr exposure 5 .Math.M 0.5 50 .Math.M 0.1 500 .Math.M 0.2 0 .Math.M 0.0

Example 5 - Field Trial Pilot Study - Insemination of Sows for Life-Stock Breeding

Method:

[0121] A pilot field study included 27 sows of which 13 received untreated control sperm and 14 received sperm treated with Compound No. 1 (“activator”). insemination doses were collected from the same animals during the course of the experiment and contained pooled semen from three individual boars. 45 .Math.l 100 mM activator solution in DMSO was added to 89 ml boar sperm (final concentration of 50 .Math.M) directly before insemination which was carried out within 15 to 45 min. The sperm was transferred to an insemination bottle and then the activator was added. After placing the insemination catheter into the cervix of the sow, the bottle was connected, and the sperm sample was released into the sow. The sows received insemination doses on two subsequent days. After 30 to 35 days the animals were sacrificed, the uteri were removed and embryos were counted for all 30 sows. Organ material from 5 activator-receiving sows and 5 sows from the control group were optically inspected and samples from the uterus, cervix and vagina were formalin-fixated for histology examination. The organ material from both the control group and the activator group showed mostly normal mucosa and no signs of lesions in the cervix, uteri and vaginae. The formalin preparations were cut and stained with hematoxylin eosin for histological examination. The histopathological assessment included a thorough evaluation of the epithelium of the mucosa and the underlying lamina propria. In both the epithelium and lamina propria, particular emphasis was placed on acute changes. In the epithelium, the acute changes included single cell necrosis/apoptosis and infiltration of polymorphonuclear leukocytes. In the lamina propria, infiltration of polymorphonuclear leukocytes was considered as the most important acute change. Three of the five sows in the activator group and one sow in the control group showed abundant infiltration of polymorphonuclear leukocytes mostly in the cervix and uterus. Necrosis/apoptosis was detected in one of the sows of the control group and one of the sows in the activator-receiving group.

[0122] Infiltration of lymphocytes in the epithelium and lamina propria was considered to be within the normal range of all individuals. Flattening and possibly loss of surface epithelium were partly difficult to assess. Edema and bleeding in the lamina propria were detected in several individuals. Heat and age are the denominators of leukocyte infiltration in sows and are considered normal.

[0123] The average number of embryos was determined for the sows of the control group (n=11, 11 sows were in pig after insemination) and the activator-receiving group (n=13, 13 sows in pig after insemination). Not only did the activator increase pregnancy rates in the sows, but it also led to an average increase of 2 embryos per sows (see FIG. 5).

Example 6 - Field Trial Study

[0124] Following the success of the pilot study reported in Example 5, a larger field trial study was conducting which included 441 sows. 224 sows received untreated control sperm and 217 received sperm treated with Compound No. 1 (“activator”). Results from this larger study revealed a 5% increase in pregnancy rate and an average increase of ~1 more live piglet born per sow for the sows which received sperm treated with the activator compound. By improving the pregnancy rate and number of piglets born per sow, use of the sperm activator improved the weaning to service interval (“WSI”), i.e. the interval between consecutive pregnancies.