Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

Abstract

Na,K-ATPase α4 is a testis-specific plasma membrane Na⁺ and K⁺ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca²⁺, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.

Figure 1

(A) Structures of ouabain, strophanthidin, cymarin, and digoxin and their IC₅₀ values for Na,K-ATPase isoform inhibition. IC₅₀ values for ouabain are taken from ref (35). (B) Dose–response curves for the inhibition of Na,K-ATPase α4 activity by strophanthidin, cymarin, and digoxin. Values are the mean of two experiments performed in quadruplicate.

Figure 2

Design of ouabain analogues.

Scheme 1. Synthesis of C17 Hydroxymethylene Triazole Analogues

Reagents and conditions: (a) acetone, concentrated HCl, rt, 84%; (b) MOM-Cl, DIPEA, CH₂Cl₂, rt, 68%; (c) (i) O₃, −78 °C then Zn/AcOH, CH₂Cl₂, (ii) KHCO₃, MeOH, rt, (iii) NaBH₄, MeOH, 42% (for 3 steps); (d) NaIO₄, THF/H₂O (8:2), rt, 63%; (e) ethynylmagnesium bromide, THF, −78 °C, 74%; (f) benzyl azide or 4-fluorobenzyl azide, Cu₂SO₄·5H₂O (20 mol %), sodium ascorbate (40 mol %), DMF/H₂O, 6 (40%), 7 (64%); (g) 4 N HCl in MeOH, rt, 8 (42%), 9 (40%).

Scheme 2. Synthesis of C17 Hydroxymethyl and Nitrile Analogues

Reagents and conditions: (a) NaBH₄, MeOH, 78%; (b) (i) NH₂OH·HCl, NaOAc, (ii) CDI, CH₂Cl₂, 70% (for 2 steps).

Scheme 3. Synthesis of C3 and C17 Modified Triazolylmethyl Analogues

Reagents and conditions: (a) acetone, concentrated HCl, rt, 55%; (b) MOM-Cl, DIPEA, rt, 75%; (c) (i) O₃, −78 °C then Zn/AcOH, CH₂Cl₂, (ii) KHCO₃, MeOH, rt, 63% (for 2 steps); (d) NaBH₄, MeOH, 76%; (e) NaIO₄, THF/H₂O (8:2), rt, 62%; (f) NaBH₄, MeOH, 72%; (g) TsCl, pyridine, 73%; (h) NaN₃, DMSO, 60 °C, 70%, (i) 4-methoxyphenyl acetylene, Cu₂SO₄·5H₂O (20 mol %), sodium ascorbate (40 mol %), DMF/H₂O, 68%; (j) 4 N HCl in MeOH, rt, 51%.

Scheme 4. Synthesis of C17 Triazole Analogues

Reagents and conditions: (a) K₂CO₃, MeOH, 71%; (b) benzyl azide (22, 58%), 4-chlorobenzyl azide (23, 56%), or 4-fluorobenzyl azide (24, 66%), Cu₂SO₄·5H₂O (20 mol %), sodium ascorbate (40 mol %), DMF/H₂O; (h) 4 N HCl in MeOH, rt, 25 (47%), 26 (51%), 27 (53%).

Scheme 5. Synthesis of C17 Substituted Analogues

Reagents and conditions: (a) NH₂OH·HCl, NaOAc, EtOH, rt, 81%; (b) CDI, CH₂Cl₂ rt, 78%; (c) TMSCF₃, TBAF, THF, rt, 46%; (d) ethynylmagnesium bromide, THF, −78 °C, 79%; (e) NaIO₄, H₂O/AcOH (2:1), EtOH, rt, 52%.

Figure 3

Selectivity of ouabain analogues on Na,K-ATPase α4 over other isoforms. Dose–response curves for the inhibition of Na,K-ATPase activity by compounds 10 (A), 17 (B), and 25 (C) were determined on rat α1β1, α2β1, and α3β1 produced in Sf9 insect cells and were compared to that of α4β1. Hydrolysis of ATP in the presence of saturating concentrations of Na⁺, K⁺, and Mg²⁺ was measured using γ[³²P]-ATP. The curves represent the best fit of the experimental data, assuming a single population of binding sites. Each value is the mean ± SEM of three independent experiments. The corresponding IC₅₀ values are shown in Table 2 and exhibit a much lower affinity, in the micromolar and millimolar range for Na,K-ATPases α1, α2, and α3, compared to the nanomolar to picomolar range observed for Na,K-ATPase α4.

Figure 4

Simulation interaction diagram obtained from the MD simulations of the ligand–protein complexes (from 5 to 18 ns). (A) Ouabain with the rat α4 isoform, (B) ouabain with the rat α1 isoform, (C) compound 25 with the rat α4 isoform, and (D) compound 25 with the rat α1 isoform. The amino acid residue numbers are based on those for the rat α1 in the uniprot database (ID: P06685). The solid magenta arrows represent hydrogen bonding interactions with the backbone of the protein, the dashed magenta arrows hydrogen bonding interactions with the side chains of the protein, green dotted lines hydrophobic surface, and the blue hashed lines π–π interactions.

Figure 5

Effect of compounds 10, 17, and 25 on rat sperm motility. (A) Dose–response curve for the effect of compounds 10, 17, and 25 on total sperm motility after 1 h of incubation. Sperm were collected from the cauda epididymis of rats, and after 1 h incubation, sperm movement was determined by CASA. Values are the mean ± SEM of three determinations. (B) Time dependence for the effect of 10, 17, and 25 on total sperm motility. Rat sperm were treated in the absence (control) or presence of 10^–8 M of the indicated compound. Sperm total motility was assessed as mentioned in part A. Values are the mean ± SEM of three determinations. The data points of compound 25, after treatment for 30 min and longer were statistically different from those of compounds 10 and 17 and the control, with P < 0.001.

Figure 6

Effect of 10, 17, and 25 on different parameters of rat sperm motility. Rat sperm was collected from the cauda epididymis and treated in the absence or presence of the indicated concentrations of each compound. After 1 h of incubation, different patterns of sperm movement were determined by CASA. (A) Progressive motility, (B) straight line velocity, (C) curvilinear velocity, (D) average path velocity, (E) linearity, and (F) beat cross frequency. Values are the mean ± SEM of three determinations. Asterisks indicate statistical differences between compound 25 vs compounds 10 and 17, with P < 0.05.

Figure 7

Persistent reduction in sperm motility by compounds 10, 17, and 25. Rat sperm, obtained from the cauda epididymis, was treated in the absence (black circles) and presence of 10^–8 M of each of the compounds (empty circles, compound 10; gray squares compound 17; and black triangles, compound 25). Sperm motility was measured by CASA, before and after washing the cells three times in Tyrode’s modified medium, at the indicated times. Values are the mean ± SEM of three determinations. Comparison of the data points for each compound and the untreated controls as well as among the different compounds showed statistical differences with P < 0.05. No statistical differences were found for the values of any of the particular compounds before and after the wash.

Figure 8

Effect of 25 on different biomarkers of Na,K-ATPase α4 activity. Sperm from the cauda epididymis was isolated in modified Tyrode’s medium and treated in the absence and presence of 10^–8 M 25 for 1 h. Then, (A) sperm V_m was measured using the fluorescent marker DiSC3(5); (B) sperm pH was determined with SNARF-1, and (C) sperm [Ca²⁺]_i was assessed by calcium green. Bars are the mean ± SEM of three determinations, and asterisks show statistically significant differences, with P < 0.001.

Figure 9

Effect of compound 25 on sperm hyperactivated motility. Sperm from the cauda epididymis were isolated and capacitated in Tyrode’s modified medium supplemented with albumin, bicarbonate, and calcium and in the absence (black bar) or presence (gray bar) of 10^–8 M compound 25 for 1 h. Sperm motility was determined using CASA. Bars represent the mean ± SEM of three experiments. Statistical significance between samples treated with or without compound 25 are indicated with an asterisk, with P values ranging between 0.05 and 0.001.

Figure 10

Effect of compound 25 on rat total and progressive sperm motility in vivo. Compound 25 was administered by oral gavage at different doses (5, 10, and 20 mg/kg of body weight) for 3 days (A, B) or at 5 mg/kg of body weight for the indicated times (C, D). Total and progressive sperm motility was determined on sperm from the cauda epididymis using CASA. Bars represent the mean ± SEM of three experiments. Values significantly different from the untreated controls are indicated with an asterisk, with P ≥ 0.05.