Synthesis and Functional Characterization of Novel RU1968-Derived CatSper Inhibitors with Reduced Stereochemical Complexity

Abstract

The sperm-specific Ca²⁺ channel CatSper (cation channel of sperm) controls the intracellular Ca²⁺ concentration and, thereby, the swimming behavior of sperm from many species. The steroidal ethylenediamine RU1968 (1) represents a well-characterized, potent, and fairly selective cross-species inhibitor of CatSper. Due to its two additional centers of chirality in the amine-bearing side chain, RU1968 is a mixture of diastereomeric pairs of enantiomers and, thus, difficult to synthesize. This has hampered the use of this commercially not available inhibitor as a powerful tool for research. Here, simplifying both structure and synthesis, we introduced novel stereochemically less complex and enantiomerically pure aminomethyl RU1968 analogues lacking the C-21 CH₃ moiety. Starting from (+)-estrone, a five-step synthesis was developed comprising a Wittig reaction as the key step, leading to a diastereomerically pure 17β-configured aldehyde. Subsequent reductive amination yielded diastereomerically and enantiomerically pure amines. Compared to RU1968, the novel ethylenediamine 2d and homologous trimethylenediamine derivative 2e inhibited CatSper with similar and even twofold enhanced potency, respectively. Considering that these aminomethyl analogues are enantiomerically pure and much easier to synthesize than RU1968, we envisage their common use in future studies investigating the physiology of CatSper in sperm.

The function of sperm is controlled by changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i).¹⁻⁴ Although the make-up of Ca²⁺-signaling pathways in sperm is quite diverse,^5,6 the sperm-specific Ca²⁺ channel CatSper channel is a common component, controlling [Ca²⁺]_i in sperm from many species including mammals.^4,7,8 Across species, CatSper is activated by depolarization of the membrane potential and alkalization of the intracellular medium.⁹⁻¹² Moreover, in human sperm, CatSper is also promiscuously activated by steroids and prostaglandins contained in reproductive fluids.¹³⁻¹⁸ It is unequivocal that in mice and humans, CatSper is required for fertilization: loss of CatSper function affects the swimming behavior of sperm and, thereby, causes male infertility.^4,7,19−21 However, in humans, the precise role of CatSper and its control by steroids and prostaglandins during fertilization is still only ill-defined, and except for sea urchins,¹² its function in species other than mice and humans is largely unknown. To address these questions, pharmacological tools, e.g., inhibitors, are required that allow interfering with the function of the channel. Of note, inhibitors that selectively target CatSper in sperm might not only serve as a tool for basic research but also to scrutinize CatSper as a target for novel contraceptives.

In the past decade, several compounds that inhibit CatSper have been discovered and employed to gain insights into the function of the channel, including for example NNC-0396 (NNC),^16,18 Mibefradil,¹⁸ MDL12330A (MDL),²² HC-056456 (HC),²³ and RU1968.²⁴ Among these compounds, the steroidal sigma receptor ligand RU1968²⁵ represents the most thoroughly characterized and best-performing CatSper inhibitor and, thus, a promising lead structure.²⁴ Of note, the action of RU1968 on sperm does not involve activation of sigma receptors.^24,26 In brief, RU1968 rather directly inhibits CatSper across species with a half-maximal inhibitory concentration (IC₅₀) of a few μM, lacks toxic side effects, suppresses CatSper-mediated motility responses, and mimics loss of CatSper function.²⁴ Nevertheless, RU1968 also suffers from some shortcomings. In particular, the compound is not commercially available, and the synthesis reported by Rennhack et al.²⁴ started from racemic estrone leading to racemic products. During synthesis, four diastereomeric pairs of enantiomers were formed and RU1968 had to be isolated from a complex mixture of stereoisomers. This demanding procedure has hampered the common (re-)synthesis and use of the drug: thus far, RU1968 has been utilized only in a few studies^11,17,27,28 other than that by Rennhack et al.²⁴ to investigate the function of CatSper.

Here, we report the design, synthesis, and functional characterization of RU1968 analogues that are easy to synthesize and do not require separation of stereoisomers. The novel enantiomerically pure CatSper inhibitors were derived by removal of the methyl moiety in the side chain and, thus, differ from RU1968 by one center of chirality (Figure 1), reducing the number of possible stereoisomers. We show that these compounds inhibit CatSper in human sperm with a similar efficacy and, depending on the modification, twofold enhanced potency compared to their congener RU1968.

Figure 1.

Design of novel CatSper inhibitors 2 derived from prototypical CatSper inhibitor RU1968 (1). In 2, the C-21 CH₃ moiety (red circle) leading to a chiral center in the 20-position of RU1968 (1) is removed. * Compound 1 is a racemic mixture.

Results

Synthesis of RU1968 Analogues

The synthesis of aminomethyl derivatives 2a–c (Scheme 1) started with methylation of enantiomerically pure (+)-estrone (3) with CH₃I under phase-transfer catalysis to obtain the methyl ether 4 in 90% yield. A Wittig reaction of ketone 4 with methoxymethyltriphenylphosphonium chloride afforded the enol ether 5, which was hydrolyzed with HCl to give the aldehyde 6 in 38% yield over two steps. The aldehyde 6 lacking the additional methyl moiety of RU1968 was isolated as a mixture of 17β and 17α diastereomers in the ratio 92:8. Reductive amination of the aldehyde 6 with pyrrolidine, 1-methylpiperazine, or 1-phenylpiperazine and NaBH(OAc)₃ led to amines 7a–c in 41–74% yield. Removal of the methyl ether from 7a–c with concentrated HBr provided RU1968 analogues 2a–c in 7–56% yield (Scheme 1). The aminophenols 2a–c were isolated as mixtures of diastereomers in the ratio 91:9 to 98:2 (17β:17α). Unfortunately, it was not possible to separate the diastereomers at the stage of the aldehyde 6, the methyl ethers 7, or the phenols 2. Moreover, the amine 2d containing the RU1968 substituent was also prepared via this route, but the resulting diastereomeric amines could not be separated either.

Scheme 1. Synthesis of Enantiomerically Pure Estrol Derivatives 2a–c with an Aminomethyl Substituent in the 17-Position^a.

Reagents and reaction conditions: (a) CH₃I, TBAI, CH₂Cl₂, NaOH, 70 °C, 5 h, 90%. (b) H₃COCH₂PPh₃ Cl, KO^tBu, THF, −20 °C to rt, 24 h. (c) HCl, THF/H₂O, reflux, 40 min, 38% (over two steps). (d) R₂NH, NaBH(OAc)₃, HOAc, THF, Na₂SO₄, rt, 24 h, 74% (7a), 41% (7b) 42% (7c). (e) HBr, 100 °C, 6 h, 7% (2a), 52% (2b) 56% (2c).

To improve purification of the final products, the protective group of the phenol was modified. Instead of a methyl moiety, a benzyl moiety was introduced by alkylation of phenol 3 with benzyl bromide. Wittig reaction of benzylated ketone 8 and subsequent hydrolysis led to the aldehyde 10 (Scheme 2). Although a diastereomeric mixture of aldehyde 10 was formed, recrystallization provided pure 17β-configured aldehyde 10 in 48% yield. The configuration of aldehyde 10 was confirmed by X-ray crystal structure analysis (Figure 2). Amines 11d and 11e were prepared by reductive amination of aldehyde 10 with (N,N-dimethylamino)alkanamines and NaBH(OAc)₃ in 68 and 77% yield, respectively. The final removal of the O-benzyl group with H₂ and Pd/C catalysts produced single diastereomers of aminophenols 2d and 2e in 43 and 53% yield, respectively (Scheme 2).

Scheme 2. Synthesis of Enantiomerically Pure Aminomethyl Estranol Derivatives 2d and 2e^a.

Reagents and reaction conditions: (a) BnBr, CH₂Cl₂, NaOH, 70 °C, 5 h, 90%. (b) H₃COCH₂PPh₃ Cl, KO^tBu, THF, −20 °C to rt, 24 h, 99%. (c) HCl, THF, reflux, 40 min, 48%. (d) R₂NH, NaBH(OAc)₃, HOAc, THF, Na₂SO₄, rt, 24 h, 68% (11d), 77% (11e). (e) H₂, Pd/C, THF, rt, 3 h, 43% (2d), 53% (2e).

Figure 2.

X-ray crystal structure of aldehyde 10. Thermal ellipsoids are set at 30% probability. The structure clearly shows the β-orientation of the formyl moiety in the 17-position; i.e., 10 reveals (17S) configuration.

Action of RU1968 Analogues on CatSper in Human Sperm and on Human Slo3

We studied the action of compounds 2a–e in populations of human sperm loaded with a fluorescent Ca²⁺ indicator, using a fluorescence plate reader.^15,18,24,29 The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored before and after application of various concentrations of the respective compound and subsequent application of progesterone (2 μM) or PGE₁ (2 μM) to evoke Ca²⁺ influx via CatSper.

At concentrations ≥4 μM, compound 2a evoked a small transient Ca²⁺ increase (Figure 3A,B), whose dose dependence, amplitude, and time course were reminiscent of the Ca²⁺ transient evoked by its congener RU1968 (see the study of Rennhack et al.²⁴). The mechanism underlying these drug-evoked changes in [Ca²⁺]_i is unknown but does not involve a toxic action, considering that, like RU1968,²⁴ compound 2a as well as compounds 2b–e did not affect the viability of sperm (Supplementary Figure S1). Furthermore, compound 2a slowed down and completely suppressed in a dose-dependent fashion CatSper-mediated Ca²⁺ signals evoked by progesterone (Figure 3C) or prostaglandin E₁ (Supplementary Figure S2A).

Figure 3.

Action of compounds 2a–e on the intracellular Ca²⁺ concentration and CatSper-mediated Ca²⁺ signals in human sperm. (A) Representative Ca²⁺ signals in human sperm loaded with a fluorescent Ca²⁺ indicator evoked by compound 2a and subsequent stimulation by progesterone (2 μM). ΔF/F (%) indicates the percentage change in fluorescence (ΔF) with respect to the mean basal fluorescence (F) before application of 2a. (B) Ca²⁺ signals evoked by 2a shown in (A) depicted on an extended time scale. (C) Progesterone-induced Ca²⁺ signals evoked in the presence of 2a shown in (A), depicted relative to the mean basal fluorescence before the application of progesterone. (D) Dose–response relation of the maximal Ca²⁺-signal amplitudes evoked by progesterone in the presence of 2a shown in (C), and dose–response relation of the PGE₁-evoked Ca²⁺ signal in the presence of compound 2a. To increase comparability between experiments, Ca²⁺ signal amplitude is normalized to the control signal amplitude evoked in the absence of the compound (set to 1). (E) Representative Ca²⁺ signals evoked by compound 2b and subsequent application of progesterone. (F) Ca²⁺ signal evoked by 2b shown on an extended time scale. (G) Progesterone-induced Ca²⁺ signals in the presence of 2b. (H) Dose–response relation of normalized maximal signal amplitudes evoked by progesterone or PGE₁ in the presence of 2b. (I) Representative Ca²⁺ signals evoked by compound 2c and subsequent application of progesterone. (J) Ca²⁺ signals evoked by 2c shown on an extended time scale. (K) Progesterone-induced Ca²⁺ signals in the presence of 2c. (L) Representative Ca²⁺ signals evoked by compound 2d and subsequent application of progesterone. (M) Ca²⁺ signal evoked by 2d shown on an extended time scale. (N) Progesterone-induced Ca²⁺ signals in the presence of 2d. (O) Dose–response relation of normalized maximal signal amplitudes evoked by progesterone or PGE₁ in the presence of 2d. (P) Representative Ca²⁺ signals evoked by compound 2e and subsequent application of progesterone. (Q) Ca²⁺ signals evoked by 2e shown on an extended time scale. (R) Progesterone-induced Ca²⁺ signals in the presence of 2e. (S) Dose–response relation of normalized maximum signal amplitudes evoked by progesterone or PGE₁ in the presence of 2e.

To quantify the inhibitory action of 2a on CatSper, we depicted and analyzed the progesterone- or PGE₁-evoked increases in fluorescence relative to the baseline right before application of the ligand, correcting for 2a-induced changes in fluorescence (compare Figure 3A,C). Analysis of the dose–response relations yielded IC₅₀ values of 7.6 ± 1.8 and 8.2 ± 1.8 μM (mean ± standard deviation, n = 3) for the inhibition of progesterone- and PGE₁-evoked Ca²⁺ signals, respectively (Figure 3D; Table 1), i.e., about twofold larger than those reported for RU1968²⁴ (Table 1). Thus, compared to RU1968, compound 2a inhibits CatSper with similar efficacy but slightly reduced potency.

Table 1. IC₅₀ Values of Compounds 2a–e for the Inhibition of Progesterone- and PGE1-Induced Ca²⁺ Signals^a.

^an.d., IC₅₀ values could not be determined; P4, progesterone; n = 3.

Like compound 2a, also compounds 2b (Figure 3E,F), 2c (Figure 3I,J), 2d (Figure 3L,M), and 2e (Figure 3P,Q) evoked small Ca²⁺ increases on their own. Yet, compared to 2a, the 2b- and 2c-evoked Ca²⁺ increase was longer lasting and rather brief, respectively, whereas for 2d and 2e, its amplitude was significantly smaller. Furthermore, compounds 2b (Figure 3E,G; Supplementary Figure S2B), 2d (Figure 3L,N; Supplementary Figure S2D), and 2e (Figure 3P,R; Supplementary Figure S2E) also suppressed in dose-dependent fashion Ca²⁺ signals evoked by progesterone or PGE₁. RU1968 and 2d, which bear the same substituent, exhibit similar IC₅₀ values, i.e., 3.5–4.0 μM (Figure 3O, Supplementary Figure S2D, and Table 1). Therefore, we conclude that the C-21 methyl group in RU1968 (see Figure 1, red circle) is neither essential for inhibition of CatSper nor does it affect the potency. For 2b, which structurally differs from 2d’s (dimethylamino)ethylamino moiety only by cyclization, the potency was reduced compared to 2d (and RU19681) and rather similar to that of 2a, i.e., ∼8 μM (Figure 3H,O,D; Supplementary Figure S2A, and Table 1). Thus, neither a methyl piperazine (2b) nor a pyrrolidine (2a) enhances the potency, indicating that cyclic motifs and loss of the second amine in the side chain are tolerated but not favorable. By contrast, the trimethylenediamine 2e (Figure 3R,S; Supplementary Figure S2E) featured IC₅₀ values of ∼1.5 μM, representing an at least twofold gain in potency compared to 2d and RU1968, rendering it the most potent inhibitor of our new set (Table 1). Finally, the phenylpiperazine 2c did not inhibit Ca²⁺ signals evoked by progesterone (Figure 3I,K) or PGE₁ (Supplementary Figure S2C), suggesting spatial limitations at CatSper’s inhibitory binding site.

Furthermore, the sperm-specific Slo3 channel represents the principal K⁺ channel in mouse^30,31 and human sperm.³² RU1968 does not affect mouse Slo3 but inhibits human Slo3, yet, with about tenfold lower potency than CatSper in electrophysiological recordings.²⁴ Therefore, we studied the action of 2d—as a representative of the series of RU1968 analogues—on human Slo3. We also studied the action of compound 2c, considering that it did not inhibit human CatSper.

To this end, we transiently expressed human Slo3 together with its auxiliary subunit LRRC52 in CHO cells and recorded K⁺ currents carried by Slo3 by whole-cell patch clamp recordings.³⁰ Compound 2d suppressed Slo3 currents in a dose-dependent fashion with an IC₅₀ of 10 ± 2 μM (n = 5, Figure 4A,B, Table 1), indicating that the RU1968 analogues also inhibit Slo3. Yet, the slight, statistically insignificant increase in the IC₅₀ value of 2d versus RU1968 (7 ± 6 μM)²⁴ might point toward a small gain in selectivity for CatSper over Slo3. Remarkably, compound 2c not only failed to inhibit CatSper but also Slo3 (Figure 4C,D), highlighting the overlapping pharmacology of the inhibitory binding sites of the channels in humans.

Figure 4.

Action of compounds 2d and 2c on heterologous human Slo3. (A) Representative current voltage relation recorded from a CHO cell transiently expressing human Slo3 in the absence and presence of different concentrations of 2d. (B) Dose–response relation of the suppression of mean Slo3 current amplitudes by 2d (n = 4); amplitudes are depicted relative to that recorded in the absence of 2d (set to 1). (C) Representative current voltage relation recorded from a CHO cell transiently expressing human Slo3 channels in the absence (control) and presence of 2d. (D) Mean current amplitude recorded in the presence of 2c (100 μM) relative to that in its absence (set to 1) (n = 4).

Discussion

Starting from enantiomerically pure (+)-estrone, we established a straightforward synthesis and isolation of diastereomerically and enantiomerically pure analogues of RU1968. Compared to RU1968 (1), the ethylenediamine 2d (IC₅₀ = 3.8 μM) and homologous trimethylenediamine 2e (IC₅₀ = 1.7 μM) inhibited CatSper with similar and twofold enhanced potency, respectively. Considering that 2d (TS116) and 2e (TS150) are much easier to synthesize and isolate, we envisage their common use instead of RU1986 in future studies investigating the role of CatSper.

Of note, future studies are also required to further improve the properties of this class of CatSper inhibitors. For example, by an unknown mechanism, RU1968 and its analogues 2 evoke small Ca²⁺ signals on their own. This reinforces that the pharmacology of CatSper is complex. The channel seems to harbor several activator- and inhibitor-binding sites, which might be allosterically coupled. Compounds of the RU1968 class might engage an inhibitory site and, at the same time, also represent weakly efficacious agonists, causing small elevations of [Ca²⁺]_i on their own. However, their precise mechanism and site of action on CatSper seems difficult to elucidate by structure–function analysis or site-directed mutagenesis, considering that CatSper resists functional expression.

Yet, the cryo-electron microscopy (cryo-EM) structure of the CatSper complex isolated from mouse sperm was recently achieved.³³ The cryo-EM analysis of CatSper in the presence of small-molecule modulators such as RU1968 might identify their binding sites and, thus, facilitate the synthesis of novel analogues based on structure-based drug design.³⁴

Moreover, like RU1968, also its analogues are not perfectly selective for CatSper. In human sperm, the compounds also inhibit Slo3. To further improve the inhibitor’s selectivity for CatSper, a structure–activity analysis is required to identify RU1968 analogues that do not act on human Slo3. The fact that human Slo3 can be functionally expressed in cultured cells facilitates this endeavor. Furthermore, RU1968²⁵ and most likely also its new analogues act on sigma receptors expressed in somatic cells. This action would need to be abolished prior to an in vivo use of the compounds.

Finally, only recently, a high-throughput screening (HTS) approach and ensuing comprehensive structure–activity and selectivity analysis identified seven new classes of CatSper inhibitors.³⁵ One of these classes of compounds inhibited CatSper at low micromolar concentrations, whereas at 10 μM, the drug did not affect human Slo3. If the drug might inhibit human Slo3 at >10 μM remains to be determined by studying its action on Slo3 in a dose-dependent fashion. Nevertheless, this HTS-based approach yielded another promising lead compound to develop potent and highly selective CatSper inhibitors for basic research and potential application as contraceptives.

Materials and Methods

Chemical Synthesis

Unless otherwise noted, moisture-sensitive reactions were conducted under dry nitrogen. Thin-layer chromatography (tlc): silica gel 60 F₂₅₄ plates (Merck). Flash chromatography (fc): silica gel 60, 40–64 μm (Merck); parentheses include the diameter of the column (d), fraction size (v), eluent, and R_f value. Melting point: melting point apparatus MP50 (Mettler Toledo), uncorrected. MS: MAT GCQ (Thermo-Finnigan): EI, MAT LCQ (Thermo Finnigan): ESI. ¹H NMR (400 MHz), ¹³C NMR (100 MHz): Agilent DD2 400 and 600 MHz spectrometers.; δ in ppm related to tetramethylsilane; coupling constants are given with 0.5 Hz resolution; the assignments of ¹³C and ¹H NMR signals were supported by 2D NMR techniques. IR: FT/IR IRAffinity-1 IR spectrometer (Shimadzu) using an ATR technique. Optical rotation: Polarimeter UniPoL 1000 (Schmidt Haensch); 1.0 dm tube; concentration c in g/100 mL; T = 20 °C; wavelength 589 nm (D-line of Na light); the unit of the specific rotation ([α]_D^T ° mL dm^–1 g^–1) is omitted for clarity.

HPLC Analysis of Compound Purities

HPLC: Merck Hitachi Equipment; UV detector: L-7400; autosampler: L-7200; pump: L-7100; degasser: L-7614; column: LiChrospher 60 RP-select B (5 μm); LiChroCART 250–4 mm cartridge; flow rate: 1.0 mL/min; injection volume: 5.0 or 10 μL; detection at λ = 210 nm; solvents: A: water with 0.05% (v/v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) trifluoroacetic acid: gradient elution: (A %): 0–4 min: 90%, 4–29 min: 90 → 0%, 29–31 min: 0%, 31–31.5 min: 0 → 90%, 31.5–40 min: 90%. Data acquisition: HSM software; manual integration. The purity of all test compounds was >95%.

Synthetic Procedures

3-Methoxyestra-1,3,5(10)-trien-17-one (4)

A suspension of estrone (3, 1.0 g, 3.70 mmol, 1.0 equiv), TBAI (0.07 g, 0.19 mmol, 0.05 equiv), an aqueous solution of NaOH (10%_w/w, 20 mL), and CH₃I (0.87 mL, 14.1 mmol, 3.8 equiv) were added in CH₂Cl₂. After heating to 70 °C for 5 h, the mixture was cooled down to room temperature. Layers were separated, and the aqueous phase was extracted with CH₂Cl₂ (3 × 70 mL). The organic layers were combined, dried (Na₂SO₄), and filtered, and the solvent was evaporated under reduced pressure. The product was not further purified. (TLC: cyclohexane/ethyl acetate 9:1, R_f = 0.28). C₁₉H₂₄O₂ (284.4 g/mol). Colorless solid, mp 170–171 °C, yield 1.04 g (90%). ¹H NMR (400 MHz, CDCl₃): δ [ppm] = 0.91 (s, 3H, 18-CH₃), 1.37–1.71 (m, 6H, 7-CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H)), 1.88–2.31 (m, 5H, 7-CH₂ (1H), 9-CH, 12-CH₂ (1H), 15-CH₂ (1H), 16-CH₂ (1H)), 2.35–2.56 (m, 2H, 11-CH₂ (1H), 16- CH₂ (1H)), 2.86–2.95 (m, 2H, 6-CH₂), 3.78 (s, 3H, OCH₃), 6.62–6.68 (d, J = 2.7 Hz, 1H, 4-CH), 6.72 (dd, J = 8.6/2.8 Hz, 1H, 2-CH), 7.21 (dd, J = 8.7/1.1 Hz, 1H, 1-CH). ¹³C NMR (101 MHz, CDCl₃): δ [ppm] = 13.8 (1C, C-18), 21.6 (1C, C-15), 25.8 (1C, C-11), 26.4 (1C, C-7), 29.5 (1C, C-6), 31.4 (1C, C-12), 35.9 (1C, C-16), 38.2 (1C, C-8), 43.8 (1C, C- 9), 47.9 (1C, C-13), 50.3 (1C, C-14), 55.0 (1C, OCH₃), 111.4 (1C, C-2), 113.7 (1C, C-4), 126.2 (1C, C-1), 131.9 (1C, C-10), 137.6 (1C, C-5), 157.4 (1C, C-3), 220.7 (1C, 17-C=O). IR (neat): ν̃ [cm^–1] = 2955 (C–H_aliph), 2916 (C–H_aliph), 2874(C–H_aliph), 1730 (C=O), 1504 (C=C_arom), 1454 (C=C_arom). Exact mass (APCI): (m/z) = 285.1863 (calcd. 285.1849 for C₁₉H₂₅O₂ [M + H]⁺). Purity (HPLC): t_R = 22.4 min, purity 97.8%.

3-Methoxyestra-1,3,5(10)-triene-17-carbaldehyde (6)

Under N₂, a suspension of Wittig reagent CH₃OCH₂PPh₃Cl (1.80 g, 5.25 mmol, 1.50 equiv) in tetrahydrofuran (THF) (4.20 mL) was cooled to −15 °C. Then, KOtBu (0.59 g, 5.25 mmol, 1.50 equiv) was added to the suspension. The mixture was stirred for 15 min. Ketone 4 (1.00 g, 3.35 mmol, 1.0 equiv) was added, and the cooling was removed after 1 h. After stirring overnight, water was added followed by the extraction with cyclohexane (3 × 30 mL). The organic layers were combined, dried (Na₂SO₄), and filtered, and the solvent was removed in vacuo. A yellow solid was obtained. The residue was purified by flash chromatography (Ø = 5 cm, h = 20 cm, V = 20 mL, cyclohexane/ethyl acetate = 99:1 → 98.5:1.5 → 97.5:2.5 → 97:3 → 95:5). A transparent waxy solid (enol ether 5) was obtained. The crude product was dissolved in THF (60 mL). Then, 20 mL of a 5 %_V/V HCl (20 mL) was added. The mixture was heated to 73 °C for 40 min. The mixture was extracted with cyclohexane (3 × 30 mL). The combined organic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo, and the residue was purified using a flash column (Ø = 5 cm, h = 18 cm, V = 20 mL, cyclohexane/ethyl acetate = Ø = 5 cm, h = 18 cm, V = 20 mL, cyclohexane/ethyl acetate = 99:1 → 98.5:1.5 → 97.5:2.5 → 97:3 → 95:5, cyclohexane/ethyl acetate 9:1 R_f = 0.50). Colorless solid, mp 122–123 °C, yield 400 mg (38%). C₂₀H₂₆O₂ (298.4 g/mol). ¹H NMR (600 MHz, CDCl₃): δ [ppm] = 0.80 (s, 3 × 0.92 H, 18–CH₃), 0.96 (s, 3 × 0.08 H, 18–CH₃), 1.32–1.56 (m, 5H, 7-CH₂ (1H), 8-CH, 11-CH₂ (1H), 14-CH, 16-CH₂ (1H)), 1.65 (td, J = 12.9/4.0 Hz, 1H, 12-CH₂ (1H)), 1.73–1.96 (m, 3H, 7-CH₂ (1H), 11-CH₂ (1H), 15-CH₂ (1H)), 2.10–2.22 (m, 2H, 12-CH₂ (1H), 15-CH₂ (1H)), 2.24–2.30 (m, 2H, 9-CH), 2.34 (dtd, J = 13.4/4.1/2.8 Hz, 1H, 16–CH₂), 2.39 (td, J = 9.2/2.1 Hz, 1H, 17-CH), 2.81–2.93 (m, 2H, 6-CH₂), 3.78 (s, 3H, OCH₃), 6.59–6.65 (d, J = 2.8 Hz, 1H, 4-CH), 6.7 (dd, J = 8.6/2.9 Hz, 1H, 2-CH), 7.17–7.22 (m, 1H, 1-CH), 9.82 (s, 1 H, HC=O). ¹³C NMR (151 MHz, CDCl₃): δ [ppm] = 14.3 (1C, C-18), 21.5 (1C, C-15), 24.9 (1C, C-11), 26.5 (1C, C-16), 28.4 (1C, C-7), 29.9 (1C, C-6), 38.4 (1C, C-12), 38.8 (1C, C-8), 44.2 (1C, C- 9), 45.4 (1C, C-13), 55.6 (2C, C-14, OCH₃), 63.3 (1C, C-17), 111.9 (1C, C-2), 114.2 (1C, C-4), 126.6 (1C, C-1), 132.7 (1C, C-10), 138.2 (1C, C-5), 157.9 (1C, C-3), 205.2 (1C, C=O). IR (neat): ν̃ [cm^–1] = 2934 (C–H_aliph), 2913 (C–H_aliph), 2870 (C–H_aliph), 1734 (C=O), 1472 (C=C_arom), 1447 (C=C_arom). Exact mass (APCI): (m/z) = 299.1987 (calcd. 299.2006 for C₂₀H₂₇O₂ ([M + H]⁺). Purity (HPLC): t_R = 23.5 min, purity 95.5%.

1-[(3-Methoxyestra-1,3,5(10)-trien-17-yl)methyl]pyrrolidine (7a)

Aldehyde 6 (194 mg, 0.65 mmol, 1.0 equiv), pyrrolidine (0.267 mL, 3.25 mmol, 5.0 equiv), CH₃COOH (0.112 mL, 1.95 mmol, 3.0 equiv), and NaBH(OAc)₃ (0.83 g, 3.90 mmol, 6.0 equiv) were suspended in THF (3 mL). The mixture was stirred at room temperature for 24 h. H₂O (20 mL) was added, and the mixture was extracted with CH₂Cl₂ at a basic pH value of 12 (3 × 20 mL). The organic layers were combined, dried (Na₂SO₄), and filtered. The solvent was removed in vacuo, and the residue was purified by flash chromatography (Ø = 2 cm, h = 18 cm, V = 10 mL, CH₂Cl₂/CH₃OH = 9/1, R_f = 0.75).

Colorless solid, 143–144 °C, yield 170 mg (74%). C₂₄H₃₅NO (353.3 g/mol). ¹H NMR (600 MHz, CDCl₃): δ [ppm] = 0.63 (s, 3 × 0.81 H, 18–CH₃), 0.82 (s, 3 × 0.19 H, 18-CH₃), 1.19–1.58 (m, 7H, 7- CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H), 16-CH₂ (1H)), 1.74–2.08 (m, 8H, 7-CH₂ (1H), 12-CH₂ (1H), 15-CH₂ (1H), 16-CH₂ (1H), N(CH₂CH₂)₂ (4H)), 2.16–2.31 (m, 2H, 9-CH, 11-CH₂ (1H)), 2.39 (m, 1H, R₂CH₂N), 2.55–2.67 (m, 5H, N(CH₂CH₂)₂ (4H), 17-CHCH₂N (1H)), 2.79–2.91 (m, 2H, 6-CH₂), 3.77 (s, 3H, OCH₃), 6.62 (d, J = 2.7 Hz, 1H, 4-CH), 6.70 (dd, J = 8.6/2.8 Hz, 1H, 2-CH), 7.20 (dd, J = 8.6/1.1 Hz, 1H, 1-CH). ¹³C NMR (151 MHz, CDCl₃): δ [ppm] = 12.5 (1C, 18-CH₃), 23.6 (2C, N(CH₂CH₂)₂, 24.7 (1C, C-15), 26.5 (1C, C-11), 28.0 (1C, C-7), 28.7 (1C, C- 16), 30.1 (1C, C-6), 38.0 (1C, C-12), 38.8 (1C, C-8), 44.2 (1C, C-9), 45.7 (1C, C-13), 50.3 (1C, C-17), 54.9 (2C, N(CH₂CH₂)₂), 55.3 (1C, C-14), 58.3 (1C, 17-CHCH₂N), 111.6 (1C, C-2), 113.9 (1C, C-4), 126.4 (1C, C-1), 133.1 (1C, C-10), 138.2 (1C, C- 5), 157.5 (1C, C-3). IR (neat): ν̃ [cm^–1] = 2932 (C–H_aliph), 2866 (C–H_aliph), 2778 (C–H_aliph), 1501 (C=C_arom), 1447 (C=C_arom). Exact mass (APCI): (m/z) = 354.2791 (calcd. 354.2791 for C₂₄H₃₆ NO [M + H]⁺). Purity (HPLC): t_R = 20.8 min, purity 95.1%.

1-[(3-Methoxyestra-1,3,5(10)-trien-17-yl)methyl]-4-methylpiperazine (7b)

Aldehyde 6 (170 mg, 0.61 mmol, 1.0 equiv), 1-methylpiperazine (0.34 mL, 3.00 mmol, 5.0 equiv), CH₃COOH (0.105 mL, 1.80 mmol, 3.0 equiv), and NaBH(OAc)₃ (0.776 g, 3.66 mmol, 6.0 equiv) were suspended in THF (3 mL). The mixture was stirred at room temperature for 24 h. Water was added, and the mixture was extracted with CH₂Cl₂ at a basic pH value of 12 (3 × 25 mL). The organic layers were combined, dried (Na₂SO₄), and filtered. The solvent was removed in vacuo, and the residue was purified by flash chromatography (Ø = 2 cm, h = 18 cm, V = 10 mL, CH₂Cl₂/CH₃OH = 9/1, R_f = 0.43).

Colorless solid, mp 118–119 °C, yield 90 mg (41%). C₂₅H₃₈N₂O (382.3 g/mol). ¹H NMR (400 MHz, CDCl₃): δ [ppm] = 0.64 (s, 3 × 0.88 H, 18–CH₃), 0.82 (s, 3 × 0.12 H, 18–CH₃), 1.17–1.56 (m, 7H, 7- CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H), 16-CH₂ (1H)), 1.60–1.81 (m, 2H, 15-CH₂ (1H), 17-CH), 1.81–1.97 (m, 2H, 7-CH₂ (1H), 16-CH₂ (1H)), 1.99–2.10 (m, 1H, 12-CH₂), 2.14–2.28 (m, 3H, 9-CH, 11-CH₂ (1H), R₂CH₂N(1H)), 2.31 (s, 3H, NCH₃), 2.40–2.58 (m, 9H, R₂CH₂N(1H), N(CH₂CH₂)₂N (8H)), 2.77–2.94 (m, 2H, 6-CH₂), 3.77 (s, 3H, OCH₃), 6.60–6.64 (d, J = 2.8, 1H, 4-CH), 6.70 (dd, J = 8.6/2.8 Hz, 1H, 2-CH) 7.20 (dd, J = 8.7/1.1 Hz, 1H, 1-CH). ¹³C NMR (101 MHz, CDCl₃): δ [ppm] = 12.1 (1C, 18-CH₃), 24.4 (1C, C-15), 26.3 (1C, C-11), 27.7-28.2 (2C, C-7, C-16), 29.7 (1C, C-6), 37.9 (1C, C-12), 38.5 (1C, C-8), 42.4 (1C, C-13), 44.0 (1C, C-9), 45.7 (1C, NCH₃), 47.0 (1C, C-17), 53.254.7 (4C, N(CH₂CH₂)₂N), 54.9 (1C, C-14), 55.0 (1C, OCH₃), 60.1 (1C, R₂CH₂N), 111.2 (1C, C-2), 113.6 (1C, C-4), 126.1 (1C, C-1), 132.8 (1C, C-10), 137.9 (1C, C-5), 157.2 (1C, C-3). IR (neat): ν̃ [cm^–1] = 2932 (C–H_aliph), 2866 (C–H_aliph), 2843 (C–H_aliph), 1504 (C=C_arom), 1447 (C=C_arom). Exact mass (APCI): (m/z) = 383.3050 (calcd. 383.3057 for C₂₂H₃₂ N₂O [M + H]⁺). Purity (HPLC): t_R = 17.0 min, purity 97.1%.

1-[(3-Methoxyestra-1,3,5(10)-trien-17-yl)methyl]-4-phenylpiperazine (7c)

Aldehyde 6 (150 mg, 0.50 mmol, 1.0 equiv), 1-phenylpiperazine (0.28 mL, 2.50 mmol, 5.0 equiv), CH₃COOH (0.0859 mL, 1.5 mmol, 3.0 equiv), and NaBH(OAc)₃ (0.636 g, 3.00 mmol, 6.0 equiv) were suspended in THF (3 mL). The mixture was stirred at room temperature for 24 h. Water was added, and the mixture was extracted with CH₂Cl₂ at a basic pH value of 12 (3 × 25 mL). The organic layers were combined, dried (Na₂SO₄), and filtered. The solvent was removed in vacuo, and the residue was purified by flash chromatography (Ø = 2 cm, h = 18 cm, V = 10 mL, CH₂Cl₂/CH₃OH = 9/1, R_f = 0.78). Colorless solid, yield 94 mg (42%). C₃₀H₄₀N₂O (444.7 g/mol). ¹H NMR (400 MHz, CDCl₃): δ [ppm] = 0.67 (s, 3 × 0.90 H, 18-CH₃), 0.84 (s, 3× 0.10 H, 18-CH₃), 1.18–1.57 (m, 7H, 7- CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H), 16-CH₂ (1H)), 1.68–1.83 (m, 2H, 15-CH₂ (1H), 17-CH), 1.85–2.00 (m, 2H, 7-CH₂ (1H), 16-CH₂ (1H)), 2.08 (dt, J = 12.6/3.1 Hz, 1H, 12-CH₂), 2.15–2.32 (m, 3H, 9-CH, 11-CH₂ (1H), 17-CHCH₂NR₂ (1H)), 2.43–2.69 (m, 5H, 17-CHCH₂NR₂ (1H), N(CH₂CH₂)₂N (4H)), 2.78–2.95 (m, 2H, 6-CH₂), 3.20 (t, J = 5.0 Hz, 4H, N(CH₂CH₂)₂N (4H)), 3.78 (s, 3H, OCH₃), 6.63 (d, J = 2.7 Hz, 1H, 4-CH), 6.71 (dd, J = 8.6/2.8 Hz, 1H, 2-CH), 6.85 (tt, J = 7.3, 1.1 Hz, 1H, C-4_ph), 6.90–6.98 (m, 2H, C_arom), 7.16–7.23 (dd, J = 9.2/1.2, 1H, 1-CH), 7.24–7.30 (m, 2H, C_arom). ¹³C NMR (101 MHz, CDCl₃): δ [ppm] = 12.1 (1C, 18-CH₃), 24.4 (1C, C-15), 26.3 (1C, C-11), 27.7 (1C, C-16), 28.1 (1C, C-7), 29.7 (1C, C-6), 38.1 (1C, C-12), 38.5 (1C, C-8), 42.4 (1C, C-13), 44.0 (1C, C-9), 47.1 (1C, C-17), 49.0 (2C, N(CH₂CH₂)₂N), 53.6 (2C, N(CH₂CH₂)₂N), 54.79 (1C, OCH₃), 55.0 (1C, C-14), 60.2 (1C, 17-CHCH₂NR₂), 111.2 (1C, C-2), 113.6 (1C, C-4), 115.8 (2C, C_arom), 119.3 (1C, C_arom), 126.1 (1C, C-1), 128.9 (2C, C_arom), 132.9 (1C, C- 10), 137.9 (1C, C-5), 151.3 (1C, C_arom), 157.2 (1C, C-3). IR (neat): ν̃ [cm^–1] = 2936 (C–H_aliph), 2816 (C–H_aliph), 1497 (C=C_arom). Exact mass (APCI): (m/z) = 445.3198 (calcd. 445.3213 for C₂₂H₃₂ N₂O [M + H]⁺). Purity (HPLC): t_R = 22.7 min, purity 97.2%.

17-[(Pyrrolidin-1-yl)methyl]estra-1,3,5(10)-trien-3-ol (2a)

Amine 7a (150 mg, 0.34 mmol) was suspended in an excess of HBr 48%. The mixture was heated to 100 °C in a sealed tube for 5 h. After cooling to room temperature, the mixture was neutralized to pH 7–8 with a saturated solution of Na₂CO₃ and extracted with ethyl acetate (3 × 25 mL). The organic layers were combined, dried (Na₂SO₄), and filtered. The solvent was removed in vacuo, and the residue was recrystallized from CH₃OH (TLC: CH₂Cl₂/CH₃OH = 9/1 + 2 drops of ammonia, R_f = 0.54). Colorless solid, mp 141–142 °C (decomposition), yield 9.0 mg (7%). C₂₃H₃₃NO (339.5 g/mol). ¹H NMR (400 MHz, DMSO-D₆): δ [ppm] = 0.60 (s, 3 × 0.91 H, 18–CH₃), 0.78 (s, 3 × 0.09 H, 18-CH₃), 1.12–1.40 (m, 7H, 7-CH₂, 8-CH, 11-CH₂, 12-CH₂, 14-CH, 15-CH₂, 16-CH₂), 1.46–1.69 (m, 6H, 15-CH₂, 17-CH, N(CH₂CH₂)₂), 1.82 (ddd, J = 18.6, 12.5, 8.0 Hz, 2H, 7-CH₂, 16-CH₂), 1.92–2.01 (m, 1H, 12-CH₂), 2.09 (d, J = 7.1 Hz, 1H, 9-CH), 2.16–2.31 (m, 2H, 11-CH₂, 17-CHCH₂N), 2.34–2.42 (m, 4H, N(CH₂CH₂)₂), 2.42–2.47 (m, 1H, 17-CHCH₂N), 2.67–2.80 (m, 2H, 6-CH₂), 6.42 (d, J = 2.6 Hz, 1H, 4-CH), 6.49 (dd, J = 8.4/2.7 Hz, 1H, 2-CH), 6.99–7.06 (dd, J = 8.4/1.1 1H, 1-CH), 8.96 (s, 1H, OH). ¹³C NMR (151 MHz, DMSO-D₆): δ [ppm] = 12.1 (1C, C-18), 23.1 (1C, N(CH₂CH₂)₂) 24.0 (1C, C-15), 26.2 (1C, C-11), 27.5 (1C, C-16), (1C, C-7), 29.2 (1C, C-6), 37.7 (1C, C-12), 38.4 (1C, C-8), 42.0 (1C, C-13), 43.7 (1C, C-9), 49.3 (1C, C-17), 53.8 (2C, N(CH₂CH₂)₂), 54.4 (1C, C-14), 57.5 (1C, 17-CHCH₂N), 112.7 (1C, C-2), 114.9 (1C, C-4), 126.0 (1C, C-1), 130.5 (1C, C-10), 137.1 (1C, C-5), 154.9 (1C, C-3). IR (neat): ν̃ [cm^–1] = 2928 (C–H_aliph), 1582 (C=C_arom), 1470 (C=C_arom). Exact mass (APCI): (m/z) = [M + H]⁺ calculated for C₂₃H₃₄NO, 340.2635; found 340.2668. Purity (HPLC): t_R1 = 17.5 min, diastereomer 1, intensity 91.8%, t_R2 = 17.1 min, diastereomer 2, intensity 2.3%.

17-[(4-Methylpiperazin-1-yl)methyl]estra-1,3,5(10)-trien-3-ol (2b)

Amine 7b (130 mg, 0.43 mmol) was suspended in an excess of HBr 48%. The mixture was heated to 100 °C in a sealed tube for 5 h. After cooling down to room temperature, the mixture was neutralized to pH 7–8 with a saturated solution of Na₂CO₃ and extracted with ethyl acetate (3 × 30 mL). The organic layers were combined, dried (Na₂SO₄), and filtered. The solvent was removed in vacuo. (TLC: CH₂Cl₂/CH₃OH = 9/1, R_f = 0.58). Colorless solid, mp 200–201 °C (decomposition), yield 65 mg (52%).

C₂₄H₃₆N₂O (368.6 g/mol). ¹H NMR (600 MHz, DMSO-D₆): δ [ppm] = 0.59 (s, 3 × 0.93 H, 18–CH₃), 0.78 (s, 3 × 0.08 H, 18–CH₃), 1.10–1.36 (m, 7H, 7-CH₂, 8-CH, 11-CH₂, 12-CH₂, 14-CH, 15-CH₂, 16-CH₂), 1.60–1.71 (m, 2H, 15–CH₂, 17-CH), 1.75 (m, 2H, 7-CH, 11-CH₂), 1.94–2.12 (m, 3H, 17-CHCH₂N, 9-CH, 12-CH₂), 2.14 (s, 3H, NCH₃), 2.15–2.46 (m, H, 16-CH₂, N(CH₂CH₂)₂N (8H), 17-CHCH₂N), 2.66–2.75 (m, 2H, 6-CH₂), 6.40 (d, J = 2.6 Hz, 1H, 4-CH), 6.47 (dd, J = 8.4/2.6 Hz, 1H, 2-CH), 7.01 (d, J = 8.5 Hz, 1H, 1-CH), 8.95 (s, 1H, OH). ¹³C NMR (151 MHz, DMSO-D₆): δ [ppm] = 12.0 (1C, 18-CH₃), 24.0 (1C, C-15), 26.2 (1C, C-11), 27.5 (2C, C-7, C-16), 29.2 (1C, C-6), 37.8 (1C, C-12), 38.4 (1C, C-13), 40.0 (1C, NCH₃), 42.0 (1C, C-8), 43.7 (1C, C-9), 45.63, 46.4 (1C, C-17), 53.1 (2C, N(CH₂CH₂)₂N), 54.5 (1C, C-14), 54.8 (2C, N(CH₂CH₂)₂N), 59.8 (1C, 17-CHCH₂N), 112.7 (1C, C-2), 114.9 (1C, C-4), 126.0 (1C, C-1), 130.5 (1C, C-10), 137.1 (1C, C-5), 154.9 (1C, C-3). IR (neat): ν̃ [cm^–1] = 2928 (O–H), 2851 (C–H_aliph), 2820 (C–H_aliph), 1462 (C=C_arom), 1442 (C=C_arom). Exact mass (APCI): (m/z) = 369.2933 (calcd. 369.2900 for C₂₂H₃₂ N₂O [M + H]⁺). Purity (HPLC): t_R1 = 14.1 min, diastereomer 1, intensity 93.1%; t_R2 = 17.1 min, diastereomer 2, intensity 5.8%.

17-[(4-Phenylpiperazin-1-yl)methyl]estra-1,3,5(10)-trien-3-ol (2c)

Amine 7c (94 mg, 0.33 mmol) was suspended in an excess of HBr 48%. The mixture was heated to 100 °C in a sealed tube for 5 h. After cooling to room temperature, the mixture was neutralized to pH 7–8 with a saturated solution of Na₂CO₃ and extracted with ethyl acetate (3 × 30 mL). The organic layers were combined, dried (Na₂SO₄), and filtered. The solvent was removed in vacuo. (TLC: CH₂Cl₂/CH₃OH = 9/1, R_f = 0.65). Colorless solid, mp 210–211 °C (decomposition), yield 51 mg (56%). C₂₉H₃₈N₂O (430.6 g/mol). ¹H NMR (600 MHz, DMSO-D₆): δ [ppm] = 0.64 (s, 3 × 0.98 H, 18-CH₃), 0.80 (s, 3 × 0.02 H, 18-CH₃), 1.19–1.37 (m, 7H, 7-CH₂, 8-CH, 11-CH₂, 12-CH₂, 14-CH, 15-CH₂, 16-CH₂), 1.65–1.75 (m, 2H, 15-CH₂, 17-CH), 1.76–1.85 (m, 2H, 7-CH₂, 16-CH₂), 2.07–2.14 (m, 2H, 9-CH, 12-CH₂), 2.15–2.23 (m, 2H, 11-CH₂, 17-CHCH₂N), 2.40–2.56 (m, 5H, 17-CHCH₂N, N(CH₂CH₂)₂N), 2.65–2.77 (m, 2H, 6-CH₂), 3.11 (t, J = 5.1 Hz, 4H, N(CH₂CH₂)₂N), 6.43 (d, J = 2.6 Hz, 1H, 4-CH), 6.49 (dd, J = 8.4/2.6 Hz, 1H, 2-CH), 6.76 (t, J = 7.2 Hz, 1H, 4-CH_ph), 6.92 (d, J = 8.2 Hz, 2H, 2-CH_ph, 6-CH_ph), 7.03 (d, J = 8.4 Hz, 1H, 1-CH), 7.13–7.23 (m, 2H, 3-CH_ph, 5-CH_ph), 9.87 (s, 1H, OH). ¹³C NMR (151 MHz, DMSO-D₆): δ [ppm] = 11.79 (1C, C-18), 23.78 (1C, C-15), 25.87 (1C, C-11), 27.24 (2C, C-7, C-16), 28.92 (1C, C-6), 37.50 (1C, C-12) 38.16 (1C, C-8), 41.86 (1C, C-13), 43.39 (1C, C-9), 46.16 (1C, C-17) 48.01 (2C, N(CH₂CH₂)₂N), 53.01 (2C, N(CH₂CH₂)₂N), 54.16 (1C, C-14), 59.57 (1C, 17-CHCH₂N) 112.41 (1C, C-2), 114.63 (1C, C-4), 115.02 (1C, C-1), 118.42 (1C, C_ph), 125.71 (2C, C_ph), 128.62 (2C, C_ph), 130.20 (1C, C-10) 136.85 (1C, C-5), 145.9 (1C, C_ph), 154.60 (1C, C-3). IR (neat): ν̃ [cm^–1] = 3209 (O–H), 2924 (C–H_aliph), 2831 (C–H_aliph), 1497 (C=C_arom), 1450 (C=C_arom). Exact mass (APCI): (m/z) = 431.3049 (calcd. 431.3057 for C₂₂H₃₂ N₂O [M + H]⁺). Purity (HPLC): t_R1 = 20.0, min, diastereomer 1, intensity 84.9%, t_R2 = 22.6 min, diastereomer 2, intensity 11.9%.

3-Benzyloxyestra-1,3,5(10)-trien-17-one (8)

Tetrabutylammonium bromide (0.596 g, 1.85 mmol, 0.1 equiv), benzyl bromide (4.40 mL, 6.33 g, 37 mmol, 2 equiv), and 10% aqueous NaOH solution (90 mL) were added to a suspension of estrone (3, 5.00 g, 18.5 mmol, 1.0 equiv) in CH₂Cl₂ (90 mL). The mixture was heated to reflux for 6 h. After cooling to room temperature (rt), layers were separated, and the aqueous phase was extracted with CH₂Cl₂ (3 × 50 mL). The organic phases were combined, washed with brine (50 mL), dried (Na₂SO₄), and filtered, and the solvent was removed in vacuo. The residue was recrystallized from 96% ethanol. Colorless crystalline solid, mp 128–130 °C, yield 6.00 g (90%). R_f (cHex/EtOAc 7:3) = 0.34. C₂₅H₂₈O₂ (360.5). ¹H NMR (600 MHz, CDCl₃): δ (ppm) = 0.91 (s, 3H, CH₃), 1.39–1.69 (m, 6H, 7-CH₂ (1H), 8-CH (1H), 11-CH₂ (1H), 12-CH₂ (1H), 14-CH (1H), 15-CH₂ (1H)), 1.92–1.98 (m, 1H, 12-CH₂), 2.01 (ddt, J = 12.8/5.7/2.9 Hz, 1H, 7-CH₂), 2.06 (dddd, J = 11.9/8.8/5.5/1.0 Hz, 1H, 15-CH₂), 2.15 (dt, J = 19.0/9.0 Hz, 1H, 16-CH₂), 2.26 (td, J = 10.8/4.4 Hz, 1H, 9-CH), 2.36–2.43 (m, 1H, 11-CH₂), 2.51 (ddd, J = 19.1/8.8/1.0 Hz, 1H, 16-CH₂), 2.85–2.96 (m, 2H, 6-CH₂), 5.04 (s, 2H, OCH₂), 6.74 (d, J = 2.8 Hz, 1H, 4-CH), 6.80 (dd, J = 8.6/2.8 Hz, 1H, 2-CH), 7.21 (d, J = 8.7 Hz, 1H, 1-CH), 7.30–7.35 (m, 1H, 4-H_benzyl), 7.35–7.41 (m, 2H, 3-H_benzyl, 5-H_benzyl), 7.41–7.46 (m, 2H, 2-H_benzyl, 6-H_benzyl). ¹³C NMR (151 MHz, CDCl₃): δ (ppm) = 14.0 (1C, C-18), 21.7 (1C, C-15), 26.1 (1C, C-11), 26.7 (1C, C-7), 29.8 (1C, C-6), 31.7 (1C, C-12), 36.0 (1C, C-16), 38.5 (1C, C-8), 44.1 (1C, C-9), 48.2 (1C, C-13), 50.6 (1C, C-14), 70.1 (1C, PhCH₂O), 112.5 (1C, C-2), 115.0 (1C, C-4), 126.0 (1C, C-1), 127.6 (2C, C-2_benzyl, C-6_benzyl), 128.0 (1C, C-4_benzyl), 128.7 (2C, C-3_benzyl, C-5_benzyl), 132.5 (1C, C-10), 137.4 (1C, C-1_benzyl), 137.9 (1C, C-5), 157.0 (1C, C-3), 221.1 (1C, C-17). Exact mass (ESI): (m/z) = 361.2191 (calcd. 361.2162 for C₂₅H₂₉O₂ [M + H⁺]). IR (neat): ν̃ [cm^–1] = 2920 (C–H_aliph), 2870 (C–H_aliph), 1732 (C=O), 1601 (C=C_arom). Purity (HPLC): 99.6% (t_R = 24.4 min). Specific rotation: [α]²⁰_D = 130.1 (c = 0.229 g/100 mL, CH₂Cl₂).

3-Benzyloxy-17-(methoxymethylen)estra-1,3,5(10)-triene (9)

Under N₂, KO^tBu (0.467 g, 4.16 mmol, 1.5 equiv) was added to a −20 °C stirred suspension of (methoxymethyl)triphenylphosphonium chloride (1.43 g, 4.16 mmol, 1.5 equiv) in dry THF (3.3 mL). After 30 min, ketone 8 (1.00 g, 2.77 mmol, 1.0 equiv) was added, and after stirring for 1 h at −20 °C, the cooling bath was removed. After 3 h, the mixture was cooled to −20 °C again and a suspension of (methoxymethyl)triphenylphosphonium chloride (0.475 g, 1.39 mmol, 0.5 equiv) and KOtBu (0.156 g, 1.39 mmol, 0.5 equiv) in THF (1.1 mL), which had been prepared separately and stirred at −20 °C for 30 min, was added. After 30 min, the cooling bath was removed and the transformation was terminated after another 2.5 h by addition of H₂O (2 mL). The mixture was transferred to a separation funnel and diluted with H₂O (25 mL), and the mixture was extracted with cHex (3 × 25 mL). The organic phases were combined and dried (Na₂SO₄), and the solvent was evaporated in vacuo. The residue was purified by flash chromatography (Ø = 4.5 cm, l = 16 cm, V = 30 mL, eluent: cHex/EtOAc 99:1). Colorless resin, yield 1.08 g (99%). R_f (cHex/EtOAc 9:1) = 0.34. C₂₇H₃₂O₂ (374.5 g/mol). ¹H NMR (600 MHz, CDCl₃): δ (ppm) = 0.84 (s, 1.2H, CH₃), 0.91 (s, 1.8H, CH₃), 1.20–1.69 (m, 6H), 1.73–1.98 (m, 2.4H), 2.14–2.40 (m, 4H), 2.45–2.52 (m, 0.6H), 2.79–2.94 (m, 2H), 3.49 (s, 1.8H, OCH₃), 3.58 (s, 1.2H, OCH₃), 5.04 (s, 2H, PhCH₂O), 5.71–5.76 (m, 1H, C=CHOCH₃), 6.70–6.74 (m, 1H, 4-H), 6.78 (dd, J = 8.5/2.8 Hz, 1H, 2-H), 7.22 (dt, J = 8.9/1.9 Hz, 1H, 1-H), 7.28–7.35 (m, 1H, 4-H_benzyl), 7.35–7.41 (m, 2H, 3-H_benzyl, 5-H_benzyl), 7.41–7.45 (m, 2H, 2-H_benzyl, 6-H_benzyl). The ratio of (E)/(Z)-configured diastereomers is 60:40. Exact mass (APCI): (m/z) = 389.2478 (calcd. 389.2475 for C₂₇H₃₃O₂ [M + H⁺]). IR (neat): ν̃ [cm^–1] = 2924 (C–H_aliph), 2874 (C–H_aliph), 1605 (C=C_arom). Purity: >95%, estimated by ¹H NMR spectroscopy.

3-Benzyloxyestra-1,3,5(10)-triene-17β-carbaldehyde (10)

Enol ether 9 (1.07 g, 2.75 mmol) was dissolved in THF (60 mL) and 5% aqueous HCl (20 mL). After heating to reflux for 1 h followed by cooling to rt, the solution was extracted with CHCl₃ (3 × 25 mL). The organic layers were combined, dried (Na₂SO₄), and concentrated in vacuo. The residue was purified by flash chromatography (Ø = 4.5 cm, l = 19 cm, V = 30 mL, eluent: cHex/EtOAc 9:1), and the product was recrystallized from EtOH (abs.). Colorless solid, mp 124–125 °C, yield 0.497 g (48%). C₂₆H₃₀O₂ (374.5 g/mol). R_f (cHex/EtOAc 7:3) = 0.49. ¹H NMR (600 MHz, CDCl₃): δ (ppm) = 0.80 (s, 3H, CH₃), 1.32–1.48 (m, 4H, 7-CH₂ (1H), 8-CH (1H), 12-CH₂ (1H), 14-CH (1H)), 1.52 (tdd, J = 13.3/11.9/3.8 Hz, 1H, 11-CH₂), 1.65 (td, J = 12.9/4.0 Hz, 1H, 15-CH₂), 1.75–1.94 (m, 3H, 7-CH₂ (1H), 12-CH₂ (1H), 16-CH₂ (1H)), 2.12–2.21 (m, 2H, 15-CH₂ (1H), 16-CH₂ (1H)), 2.27 (ddd, J = 14.2/10.1/4.3 Hz, 1H, 9-CH), 2.34 (dtd, J = 13.5/4.2/2.8 Hz, 1H, 11-CH₂), 2.39 (td, J = 9.2/2.2 Hz, 1H, 17-CH), 2.81–2.93 (m, 2H, 6-CH₂), 5.04 (s, 2H, PhCH₂O), 6.73 (d, J = 2.8 Hz, 1H, 4-CH), 6.79 (dd, J = 8.6/2.8 Hz, 1H, 2-CH), 7.20 (d, J = 8.7 Hz, 1H, 1-CH), 7.29–7.35 (m, 1H, 4-H_benzyl), 7.35–7.41 (m, 2H, 3-H_benzyl, 5-H_benzyl), 7.41–7.45 (m, 2H, 2-H_benzyl, 6-H_benzyl), 9.82 (d, J = 2.1 Hz, 1H, CH=O). ¹³C NMR (151 MHz, CDCl₃): δ (ppm) = 14.1 (1C, C-18), 21.3 (1C, C-16), 24.7 (1C, C-12), 26.3 (1C, C-11), 27.9 (1C, C-7), 29.9 (1C, C-6), 38.4 (1C, C-8), 38.6 (1C, C-15), 44.0 (1C, C-9), 45.2 (1C, C-13), 55.4 (1C, C-14), 63.1 (1C, C-17), 70.1 (1C, PhCH₂O), 112.5 (1C, C-2), 115.0 (1C, C-4), 126.4 (1C, C-1), 127.6 (2C, C-2_benzyl, C-6_benzyl), 128.0 (1C, C-4_benzyl), 128.7 (2C, C-3_benzyl, C-5_benzyl), 132.8 (1C, C-10), 137.4 (1C, C-1_benzyl), 138.1 (1C, C-5), 157.0 (1C, C-3), 205.0 (1C, CH=O). Exact mass (APCI): (m/z) = 375.2353, calcd. 375.2319 for C₂₆H₃₁O₂ [M + H⁺]). IR (neat): ν̃ [cm^–1] = 2924 (C–H_aliph), 2874 (C–H_aliph), 2712 (C–H_aldehyde), 1717 (C=O_aldehyde), 1605 (C=C_arom). Purity (HPLC): 98.7% (t_R = 25.8 min). Specific rotation: [α]²⁰_D = 90.0 (c = 0.183 g/100 mL, CH₂Cl₂).

N¹-[(3-Benzyloxyestra-1,3,5(10)-trien-17-yl)methyl]-N²,N²-dimethylethane-1,2-diamine (11d)

N,N-Dimethylethane-1,2-diamine (58.3 μL, 47.1 mg, 534 μmol, 2.0 equiv), HOAc (36.3 μL, 38.5 mg, 641 μmol), NaBH(OAc)₃ (80%, 212 mg, 801 μmol, 3 equiv), and molecular sieves 4 Å (∼0.5 g) were added to a solution of aldehyde 10 (100 mg, 267 μmol, 1.0 equiv) in dry THF (5.0 mL). The mixture was stirred at rt for 2 d, filtered through a Celite plug, and rinsed thoroughly with EtOAc. The collected filtrate was washed once with 1 M NaOH (15 mL), dried (Na₂SO₄), and concentrated in vacuo. The residue was purified by flash chromatography (l = 20 cm, Ø = 2 cm, V = 12 mL, eluent CH₂Cl₂/MeOH/7 M NH₃ in MeOH = 90:8:2). Colorless solid, mp 85–88 °C, yield 94 mg (77%). R_f (CH₂Cl₂/MeOH 9:1 + 1 drop conc. NH₃) = 0.51. C₃₀H₄₂N₂O (446.7 g/mol). ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 0.65 (s, 3H, CH₃), 1.17–1.56 (m, 7H, 7-CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H), 16-CH₂ (1H)), 1.61–1.73 (m, 1H, 17-CH), 1.73–1.81 (m, 1H, 15-CH₂), 1.82–2.04 (m, 3H, 7-CH₂ (1H), 12-CH₂ (1H), 16-CH₂ (1H)), 2.10–2.35 (m, 2H, 9-CH, 12-CH₂ (1H), 2.24 (s, 6H, N(CH₃)₂), 2.41–2.53 (m, 3H, CHCH₂NH (1H), CH₂N(CH₃)₂), 2.68–2.93 (m, 5H, 6-CH₂, CHCH₂NH (1H), NCH₂CH₂,), 5.03 (s, 2H, PhCH₂O), 6.71 (d, J = 2.5 Hz, 1H, 4-CH), 6.77 (dd, J = 8.6/2.8 Hz, 1H, 2-CH), 7.20 (dd, J = 8.3/0.8 Hz, 1H, 1-CH), 7.28–7.34 (m, 1H, 4-CH_benzyl), 7.34–7.40 (m, 2H, 3-CH_benzyl, 5-CH_benzyl), 7.40–7.45 (m, 2H, 2-CH_benzyl, 6-CH_benzyl). A signal for the NH-proton is not seen in the spectrum. ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 12.8 (1C, CH₃), 24.6 (1C, C-15), 26.6 (1C, C-11), 27.7 (1C, C-16), 28.0 (1C, C-7), 30.0 (1C, C-6), 38.4 (1C, C-12), 38.7 (1C, C-8), 42.4 (1C, C-13), 44.2 (1C, C-9), 45.7 (2C, N(CH₃)₂), 48.0 (1C, NHCH₂CH₂NMe₂), 50.7 (1C, C-17), 51.8 (1C, NHCH₂CH), 55.0 (1C, C-14), 59.0 (1C, CH₂N(CH₃)₂), 70.1 (1C, PhCH₂O), 112.4 (1C, C-2), 115.0 (1C, C-4), 126.4 (1C, C-1), 127.6 (1C, C-4_benzyl), 128.0 (2C, C-2_benzyl, C-6_benzyl), 128.7 (1C, C-3_benzyl, C-5_benzyl), 133.4 (1C, C-10), 137.5 (1C, C-1_benzyl), 138.3 (1C, C-5), 156.8 (1C, C-3). Exact mass (ESI): (m/z) = 447.3395, calcd. 447.3370 for C₃₀H₄₃N₂O [M + H⁺]. IR (neat): ν̃ [cm^–1] = 2978 (C–H_aliph), 2931 (C–H_aliph), 1613 (C=C_arom), 1497 (C=C_arom). Purity (HPLC): 99.0% (t_R = 19.3 min). Specific rotation: [α]²⁰_D = +52.6 (c = 0.270 g/100 mL, CH₂Cl₂).

N¹-[(3-Benzyloxyestra-1,3,5(10)-trien-17-yl)methyl]-N²,N²-dimethylpropane-1,3-diamine (11e)

N,N-Dimethylpropane-1,3-diamine (36.7 μL, 30 mg, 293 μmol, 1.1 equiv), HOAc (10 μL, 10.6 mg, 176 μmol), and NaBH(OAc)₃ (80%, 113 mg, 427 μmol, 1.6 equiv). were added to a solution of aldehyde 10 (100 mg, 267 μmol, 1.0 equiv) in dry THF (1.0 mL). The mixture was stirred at rt for 24 h. H₂O (15 mL) was added, and the mixture was extracted with EtOAc (3 × 10 mL). The organic layers were combined, dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash chromatography (l = 13 cm, Ø = 1.5 cm, V = 12 mL, eluent CH₂Cl₂/MeOH/7 M NH₃ in MeOH = 90:9:1). Yellow waxy solid, mp 115–126 °C, yield 68 mg (68%). R_f (CH₂Cl₂/MeOH 9:1 + 1 drop conc. NH₃) = 0.21. C₃₁H₄₄N₂O (460.7 g/mol). ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 0.65 (s, 3H, C-18), 1.20–1.53 (m, 7H, 7-CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H), 16-CH₂ (1H)), 1.66–1.82 (m, 4H, 15-CH₂ (1H), 17-CH, NCH₂CH₂CH₂N), 1.83–2.01 (m, 3H, 7-CH₂ (1H), 12-CH₂ (1H), 16-CH₂ (1H)), 2.14–2.34 (m, 2H, 9-CH, 11-CH₂ (1H)), 2.25 (s, 6H, N(CH₃)₂ ), 2.37 (t, J = 6.9 Hz, 2H, CH₂NMe₂), 2.51 (dd, J = 11.3/9.1 Hz, 1H, CHCH₂NH), 2.64–2.96 (m, 5H, 6-CH₂, CHCH₂NH(1H), NHCH₂CH₂), 5.03 (s, 2H, PhCH₂O), 6.71 (d, J = 2.7 Hz, 1H, 4-CH), 6.77 (dd, J = 8.6/2.8 Hz, 1H, 2-CH), 7.19 (d, J = 8.6 Hz, 1H, 1-CH), 7.29–7.33 (m, 1H, 4-CH_benzyl), 7.34–7.40 (m, 2H, 3-CH_benzyl, 5-CH_benzyl), 7.40–7.45 (m, 2H, 2-CH_benzyl, 6-CH_benzyl). A signal for the NH proton is not seen in the spectrum. ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 12.8 (1C, C-18), 24.5 (1C, C-15), 26.5 (1C, C-11), 27.0 (1C, CH₂CH₂N(CH₃)₂, 27.5 (1C, C-16), 27.9 (1C, C-7), 30.0 (1C, C-6), 38.2 (1C, C-12), 38.7 (1C, C-8), 42.4 (1C, C-13), 44.2 (1C, C-9), 45.7 (2C, N(CH₃)₂, 49.5 (1C, NHCH₂CH₂), 50.2 (1C, C-17), 51.4 (1C, CHCH₂NH), 54.9 (1C, C-14), 58.7 (1C, CH₂N(CH₃)₂), 70.1 (1C, PhCH₂O), 112.4 (1C, C-2), 114.9 (1C, C-4), 126.4 (1C, C-1), 127.6 (2C, C-2_benzyl, C-6_benzyl), 127.9 (1C, C-4_benzyl), 128.7 (2C, C-3_benzyl, C-5_benzyl), 133.3 (1C, C-10), 137.5 (1C, 1-C_benzyl), 138.2 (1C, C-5), 156.8 (1C, C-3). Exact mass (ESI): (m/z) = 461.3525 (calcd. 461.3526 for C₃₁H₄₅N₂O [M + H⁺]). IR (neat): ν̃ [cm^–1] = 2978 (C–H_aliph), 2881 (C–H_aliph), 1612 (C=C_arom), 1574 (C=C_arom), 1501 (C=C_arom). Purity (HPLC): 98.1% (t_R = 19.4 min). Specific rotation: [α]²⁰_D = +42.1 (c = 0.240 mg/100 mL, CH₂Cl₂).

17-{[2-(Dimethylamino)ethylamino]methyl}estra-1,3,5(10)-trien-3-ol (2d)

Pd/C 10% (6 mg, 5.8 μmol, 0.05 eq.) was added to a solution of amine 11d (60 mg, 134 μmol, 1.0 equiv) in THF (10.0 mL) and EtOH (5.0 mL). The mixture was stirred at rt under a H₂ atmosphere (balloon) for 20 h. Then, it was filtered through a Celite plug and rinsed thoroughly with EtOAc. After concentrating the filtrate in vacuo, the residue was purified by flash chromatography twice (fc1: l = 16.5 cm, Ø = 1.5 cm, V = 10 mL, eluent CH₂Cl₂/MeOH/7 M NH₃ in MeOH = 99:9:1; fc2: l = 19.5 cm, Ø = 1 cm, V = 3 mL, CH₂Cl₂/MeOH/7 M NH₃ in MeOH = 99:9:1). Yellow waxy solid, mp 41–51 °C, yield 20 mg (43%). R_f (CH₂Cl₂/MeOH 9:1 + 1 drop conc. NH₃) = 0.29. C₂₃H₃₆N₂O (356.6 g/mol). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 0.60 (s, 3H, CH₃), 1.12–1.36 (m, 7H, 7-CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H), 16-CH₂ (1H)), 1.50–1.59 (m, 1H, 17-CH), 1.63–1.71 (m, 1H, 15-CH₂), 1.74–1.81 (m, 1H, 7-CH₂), 1.81–1.88 (m, 1H, 16-CH₂), 1.88–1.93 (m, 1H, 12-CH₂), 2.07–2.15 (m, 1H, 9-CH), 2.13 (s, 6H, N(CH₃)₂), 2.18–2.26 (m, 1H, 11-CH₂), 2.31 (t, J = 6.3 Hz, 2H, CH₂CH₂N(CH₃)₂), 2.36–2.43 (m, 1H, CHCH₂NH), 2.59 (t, J = 6.4 Hz, 2H, NHCH₂CH₂), 2.66 (dd, J = 11.3/5.4 Hz, 1H, CHCH₂NH), 2.69–2.77 (m, 2H, 6-CH₂), 6.42 (d, J = 2.6 Hz, 1H, 4-CH), 6.50 (dd, J = 8.4/2.7 Hz, 1H, 2-CH), 7.03 (d, J = 8.5 Hz, 1H, 1-CH), 8.97 (s, 1H, OH). A signal for the NH-proton is not seen in the spectrum. ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 12.3 (1C, CH₃), 24.0 (1C, C-15), 26.1 (1C, C-11), 27.2 (1C, C-16), 27.5 (1C, C-7), 29.2 (1C, C-6), 37.9 (1C, C-12), 38.4 (1C, C-8), 41.8 (1C, C-13), 43.6 (1C, C-9), 45.3 (2C, N(CH₃)₂), 47.3 (1C, NHCH₂CH₂), 50.0 (1C, C-17), 51.0 (1C, CHCH₂NH, 54.4 (1C, C-14), 58.4 (1C, CH₂N(CH₃)₂), 112.7 (1C, C-2), 114.9 (1C, C-4), 126.0 (1C, C-1), 130.5 (1C, C-10), 137.1 (1C, C-5), 154.9 (1C, C-3). Exact mass (ESI): (m/z) = 357.2929, calcd. 357.2900 for C₂₃H₃₇N₂O [M + H⁺]. IR (neat): ν̃ [cm^–1] = 2978 (C–H_aliph), 1608 (C=C_arom), 1581 (C=C_arom), 1458 (C=C_arom). Purity (HPLC): 97.4% (t_R = 14.3 min). Specific rotation: [α]²⁰_D = +56.7 (c = 0.177 mg/100 mL, MeOH).

17-{[3-(Dimethylamino)propylamino]methyl}estra-1,3,5(10)-trien-3-ol (2e)

Pd/C 10% (13 mg, 5.8 μmol, 0.1 equiv) was added to a solution of amine 11e (51 mg, 111 μmol, 1.0 equiv) in THF (5.0 mL) and EtOH (1.0 mL). The mixture was stirred at rt under a H₂ atmosphere (balloon) for 11 h. Then, it was filtered through a Celite plug twice and rinsed thoroughly with EtOAc. After concentrating the filtrate in vacuo, the residue was purified by flash chromatography twice (fc1: l = 15 cm, Ø = 1.5 cm, V = 10 mL, eluent CH₂Cl₂/MeOH/7 M NH₃ in the MeOH gradient from 95:4:1 to 80:15:1). Colorless solid, mp 122–127 °C, yield 22 mg (53%). R_f (CH₂Cl₂/MeOH 9:1 + 1 drop conc. NH₃) = 0.13. C₂₄H₃₈N₂O (370.6 g/mol). ¹H NMR (600 MHz, CDCl₃): δ (ppm) = 0.62 (s, 3H, C-18H₃), 1.14–1.51 (m, 7H, 7-CH₂ (1H), 8-CH, 11-CH₂ (1H), 12-CH₂ (1H), 14-CH, 15-CH₂ (1H), 16-CH₂ (1H)), 1.59–1.78 (m, 4H, 15-CH₂ (1H), 17-CH, NHCH₂CH₂), 1.79–2.00 (m, 3H, 7-CH₂ (1H), 12-CH₂ (1H), 16-CH₂ (1H)), 2.09–2.31 (m, 2H, 9-CH, 12-CH₂ (1H)), 2.25 (s, 6H, N(CH₃)₂), 2.38 (t, J = 7.2 Hz, 2H, CH₂N(CH₃)₂), 2.46 (dd, J = 11.4/8.9 Hz, 1H, CHCH₂NH), 2.61–2.89 (m, 5H, 6-CH₂, CHCH₂NH (1H), NHCH₂CH₂), 6.52 (d, J = 2.6 Hz, 1H, 4-CH), 6.58 (dd, J = 8.4/2.8 Hz, 1H, 2-CH), 7.10 (d, J = 8.3 Hz, 1H, 1-CH). Signals for the NH proton and the phenol proton are not seen in the spectrum. ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 12.8 (1C, C-18), 24.5 (1C, C-15), 26.6 (1C, C-11), 27.0 (1C, CH₂CH₂N(CH₃)₂), 27.5 (1C, C-16), 28.0 (1C, C-7), 29.9 (1C, C-6), 38.3 (1C, C-12), 38.8 (1C, C-8), 42.4 (1C, C-13), 44.1 (1C, C-9), 45.5 (2C, N(CH₃)₂, 49.1 (1C, NHCH₂CH₂), 50.3 (1C, C-17), 51.3 (1C, CHCH₂NH), 54.9 (1C, C-14), 58.3 (1C, CH₂N(CH₃)₂, 113.1 (1C, C-2), 115.8 (1C, C-4), 126.4 (1C, C-1), 131.7 (1C, C-10), 138.1 (1C, C-5), 154.9 (1C, C-3). Exact mass (ESI): (m/z) = 371.3083, calcd. 371.3057 for C₂₄H₃₉N₂O [M + H⁺]. IR (neat): ν̃ [cm^–1] = 2978 (C–H_aliph), 1612 (C=C_arom), 1582 (C=C_arom), 1462 (C=C_arom). Purity (HPLC): 95.3% (t_R = 14.2 min). Specific rotation: [α]²⁰_D = +54.2 (c = 0.205 g/100 mL, MeOH).

X-ray Diffraction Measurement

Data sets for compound 10 were collected with a Bruker D8 Venture Photon III diffractometer. Programs used: data collection: APEX3 V2019.1–0,³⁶ cell refinement: SAINT V8.40A;³⁷ data reduction: SAINT V8.40A;³⁷ absorption correction, SADABS V2016/2;³⁸ structure solution SHELXT-2015;³⁹ structure refinement SHELXL-2015(40) and graphics XP.(41)R-values are given for observed reflections, and wR² values are given for all reflections.

X-ray crystal structure analysis of 10: A colorless needle-like specimen of C₂₆H₃₀O₂, approximate dimensions 0.040 mm × 0.082 mm × 0.272 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured using a Bruker D8 Venture Bruker D8 Venture Photon III diffractometer system equipped with a micro focus tube CuKα (CuKα, λ = 1.54178 Å) and a MX mirror monochromator. A total of 1810 frames were collected. The total exposure time was 23.88 h. The frames were integrated with the Bruker SAINT software package using a wide-frame algorithm. The integration of the data using a triclinic unit cell yielded a total of 13,467 reflections to a maximum θ angle of 66.66° (0.84 Å resolution), of which 3461 were independent (average redundancy 3.891, completeness = 99.7%, R_int = 7.48%, and R_sig = 5.95%) and 2882 (83.27%) were greater than 2σ(F²). The final cell constants of a = 6.4778(2) Å, b = 8.1459(2) Å, c = 10.1467(3) Å, α = 71.796(2)°, β = 86.666(2)°, γ = 82.824(2)°, and volume = 504.53(3) ų are based upon the refinement of the XYZ-centroids of 5436 reflections above 20 σ(I) with 9.176° < 2θ < 132.7°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.877. The calculated minimum and maximum transmission coefficients (based on the crystal size) are 0.8570 and 0.9770, respectively. The structure was solved and refined using the Bruker SHELXTL software package, using the space group P1, with Z = 1 for the formula unit, C₂₆H₃₀O₂. The final anisotropic full-matrix least-squares refinement on F² with 254 variables converged at R1 = 4.87% for the observed data and wR² = 12.44% for all data. The goodness-of-fit was 1.071. The largest peak in the final difference electron density synthesis was 0.145 e^–/ų, and the largest hole was −0.176 e^–/ų with an RMS deviation of 0.039 e^–/ų. On the basis of the final model, the calculated density was 1.233 g/cm³ and F(000), 202 e^–.

CCDC-2181677 (compound 10) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Functional Analysis of Drug Actions

Isolation of Motile Sperm

Experiments on human sperm were conducted in agreement with the standards set by the Declaration of Helsinki. Healthy volunteers provided human ejaculate samples with prior written consent and under approval of the institutional ethics committees of the medical association Westfalen-Lippe and the Medical Faculty of the University of Münster (4INie). Motile sperm used for Ca²⁺ fluorimetric experiments were purified by the swim-up procedure as described previously.^18,24 In brief, in 50 mL falcon tubes angled at 45°, 4 mL of human tubular fluid (HTF) medium, containing (in mM) 93.8 NaCl, 4.69 KCl, 0.2 MgSO₄, 0.37 KH₂PO₄, 2.04 CaCl₂, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, 21 HEPES, and 4 NaHCO₃ (pH adjusted to 7.35 with NaOH) was underlaid with 0.7 to 1.2 mL of ejaculate. After incubation for 1 h at 37 °C, supernatants were collected, and sperm were washed twice with HTF by centrifugation at 700 × g for 20 min. After the second centrifugation, sperm were resuspended to 10⁷ cells·mL^–1 in HTF containing 3 mg mL^–1 human serum albumin (HSA, Irvine Scientific). Before experiments, sperm were incubated at 37 °C.

Measurement of Changes of the Intracellular Ca²⁺ Concentration

Changes of [Ca²⁺]_i in motile human sperm were measured in population using a plate reader (Fluostar Omega, BMG Labtech) with 384-well plates and fluorescence readout as described previously.^18,24 Sperm were loaded with the fluorescent Ca²⁺ indicator Fluo4 by incubation with the prodrug ester Fluo4-AM (5 μM, ThermoFisher) in the presence of Pluronic F-127 (0.05% w/v) for 20 min. Centrifugation at 700 × g for 5 min and resuspension to 5 × 10⁶ cells·mL^–1 in HTF removed excess dye. For the experiments, wells were filled with 50 μL of the dye-loaded sperm suspension. Fluorescence was excited at 480 nm, and emission was recorded at 520 nm. After recording a baseline, 25 μL of inhibitor solutions was added and fluorescence response recorded. After 4 min, 7.5 μL of stimulus (progesterone or PGE₁; 2 μM final) was added and ensuing changes in fluorescence were recorded. Solutions were added using an electric multichannel Eppendorf pipette. Changes in Fluo-4 fluorescence, i.e., [Ca²⁺]_i, are depicted as ΔF/F (%), i.e., the change in fluorescence (ΔF) relative to the mean basal fluorescence (F) before application of buffer or compounds/ligands to correct for intra- and inter-experimental variations in basal fluorescence among individual wells or to correct for compound-evoked changes in fluorescence (see below). For examination of compound-evoked Ca²⁺ signal, basal fluorescence (F) was determined before addition of compounds. For determination of inhibitory effects of compounds on ligand-evoked Ca²⁺ signals, basal fluorescence (F) was determined before addition of the respective ligand. IC₅₀ values were calculated by determining the maximal signal amplitudes evoked by the ligand in the absence and presence of various compound concentrations. For some analyses, ΔF/F was normalized to the maximal signal amplitude evoked in the absence of the compound to ease comparisons between experiments. Data from fluorescence recordings were analyzed in GraphPad Prism version 9.4.0.

Electrophysiological Recordings of Slo3 Currents

CHO cells were co-transfected with a pcDNA3.1(+) vector containing the full length coding sequence of human Slo3 modified with a carboxy-terminal hemagglutinin tag (HA-tag) and in which the sequence coding for the neomycin resistance gene was replaced by the coding sequence for citrine and a pcDNA3.1(+) vector containing a sequence encoding hLRRC52-mCherry.³² Cells were co-transfected with 2–5 ng/μL of the respective vector for 6–8 h using Lipofectamine 2000 in Opti-MEM (Gibco). Afterward, cells were cultivated in the F-12 medium; after another 24 h, cells were transferred onto poly-l-lysine-coated cover slides and incubated in 5 mM Na-butyrate at least 12 h before the experiment.

Electrophysiological recordings were performed in the whole-cell configuration using an Axopatch 200B patch clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) controlled by Clampex 10.7 (Molecular Devices). Signals were low-pass filtered at 10 kHz with a four-pole Bessel filter and digitized with a Digidata 1440A data acquisition system (Molecular Devices). Series resistance and cell capacitance were compensated. A step protocol with steps from −100 to +150 mV followed by a step to 50 mV from a holding potential of −80 mV was used. The intracellular solution contained 130 mM K-aspartate, 10 mM NaCl, 1 mM EGTA, 5 mM HEPES, and 15 mM glucose, pH 7.3 with KOH, and the pipette resistance was 4–6 MΩ. Cells were perfused with extracellular solution (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂,1.8 mM CaCl₂, 5 mM HEPES, and 10 mM glucose, pH 7.4 with NaOH). All substances were diluted in extracellular solution and applied to the cells using a gravity-driven perfusion system. Experiments were performed at rt.

Flow-Cytometry Analysis of Cell Viability

Sperm were incubated in parallel for 20 min at 37 ° C in HTF in the absence (control) and presence of the respective compound at a concentration of 60 μM. For the last 10 min, propidium iodide (PI) was added at a concentration of 3 μM. The fluorescence of PI in individual sperm was determined by an excitation wavelength of 488 nm and a detection filter of 690/50 BP at a gain of 250 in a CytoFlexS flow cytometer (BD Biosciences). At least 10,000 events were analyzed per condition, and the fraction of viable sperm was calculated based on the intensity of the PI fluorescence.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00188.

• (Figures S1 and S2) Action of compounds 2a–e on the viability of human sperm and on PGE₁-evoked Ca²⁺ signals in human sperm and HPLC chromatograms and ¹H and ¹³C NMR spectra (PDF)

Author Contributions

T.S. and B.W. contributed equally to this work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. B.W. and T.S. conceived and designed the study and coordinated the experiments. T.S., B.W., and T.S. wrote the manuscript. T.S., B.T., C.E., S.R., C.G.D., and C.B. acquired, analyzed, and/or interpreted data and revised the manuscript critically for important intellectual content. All authors approved the manuscript.

The authors declare no competing financial interest.