Synthesis and photochemical transformation of 3β,21-dihydroxypregna-5,7-dien-20-one to novel secosteroids that show anti-melanoma activity

Abstract

We have synthesized 3β,21-dihydroxypregna-5,7-dien-20-one (21(OH) 7DHP) and used UVB radiation to induce its photoconversion to analogues of vitamin D (pD), lumisterol (pL) and tachysterol (pT). The number and character of the products and the dynamics of the process were dependent on the UVB dose. The main products: pD and pT compounds were characterized by UV absorption, MS and NMR spectroscopy after RP-HPLC chromatography. In addition, formation of multiple oxidized derivatives of the primary products was detected and one of these derivatives was characterized as oxidized 21-hydroxyisotachysterol compound (21(OH)oxy-piT). These newly synthesized compounds inhibited growth of human melanoma cells in a dose dependent manner, with greater or equal potency to calcitriol. 3β,21-Dihydroxy-9β,10α-pregna-5,7-dien-20-one (21(OH)pL) and 21(OH)oxy-piT had higher potency against pigmented melanoma cells, while the EC(50) for compounds 21(OH)7DHP and (5Z,7E)-3β,21-dihydroxy-9,10-secopregna-5,7,10(19)-trien-20-one (21(OH)pD) were similar in both pigmented and non-pigmented cells. Moreover, 21(OH)7DHP and its derivatives inhibited proliferation of human epidermal HaCaT keratinocytes, albeit at a lower activity compared to melanoma cells. Importantly, 21(OH)7DHP derivatives strongly inhibited the colony formation of human melanoma cells with 21(OH)pD being the most potent. The potential mechanism of action of newly synthesized compounds was similar to that mediated by 1,25(OH)(2)D(3) and involved ligand-induced translocation of vitamin D receptor into the nucleus. In summary, we have characterized for the first time products of UVB-induced conversion of 21(OH)7DHP and documented that these compounds have selective, inhibitory effects on melanoma cells.

Figure 1

UVB- driven photolysis of 21(OH)7DHP. Representative UV spectra of irradiated sample of 21(OH)7DHP. Peaks were assigned as follows: dotted line – 21(OH)7DHP (substrate), solid line – 21(OH)pre-pD, dashed line – 21(OH)pD, dash double dot line – iso-pT or oxidized iso-pT. The UV spectra of fractions collected from RP-HPLC separation of UVB-irradiation products were acquired by Nanodrop.

Figure 2

Identification of oxidized derivatives of 21(OH)7DHP by mass spectrometry. Sample were purified after UV treatment using RP-HPLC and analyzed with LC-MS. Representative spectrum of potentially oxidized derivative of 21(OH)7DHP is shown on panel B in comparison to starting material (Panel A). The compound 21(OH)piT shows m/z = 401.3. The parental compound (C₂₁H₃₀O_3, mass = 330.25) gave m/z = 353.25 and compound 21(OH)oxy-piT could be calculated as C₂₁H₃₀O₅, 362.3 [M+K]⁺ or as C₂₁H₃₀O₆, 378.3 [M+Na]⁺.

Figure 3

Proton NMR spectra of 21(OH)7DHP (A), 21(OH)pL (B), 21(OH)pD (C), and oxidized derivative – 21(OH)oxy-piT (D) analogs as well as their predicted structures (as insert for each panel).

Figure 4

Inhibition of growth of SKMEL-188 melanoma cells by 21(OH)7DHP and its derivatives. SKMEL-188 were seeded into 96-well plates and incubated in Ham’s F10 medium containing low tyrosine levels, 10 μM (amelanotic phenotype) or in DMEM:Ham’s F10 (50:50, v:v) media containing high tyrosine levels, 200 μM (melanotic phenotype). The media were supplemented with serial dilutions of compound 21(OH)7DHP and its derivative 21(OH)pD, 21(OH)pL and 21(OH)oxy-piT. After 48 hours the cells were submitted to an MTT test. The experiment was repeated with similar results. Examples of amelanotic and melanotic cells are shown as inserts for each graph. 1,25(OH)₂D₃ was used as positive control. Data is presented as means ±SEM for 6 independent measurements. *p < 0.05, **p < 0.005, ***p < 0.0005 versus control.

Figure 5

21(OH)7DHP (A), 21(OH)pD (B) and 21(OH)pL (C) inhibit DNA synthesis in SKMEL-188. (D) 1,25(OH)₂D₃ was used as a positive control. The cells were incubated with compounds for 72 h in Ham’s F10 media containing 5% charcoal treated FBS. [³H]-thymidine was added for last 4 hours of incubation. DNA synthesis was measured by counting the radioactivity incorporated into TCA precipitable material. Data are presented as means ±SD (n = 4). The dose dependent inhibition was analyzed by one-way ANOVA with #, P < 0.05 and ##, P < 0.01. The differences between control and the chemicals were analyzed with student’s t-test; *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 6

Inhibition of colony formation of melanoma SKMEL-188 treated with photo derivatives of 21(OH)7DHP. Ethanol -solvent control (A); 21(OH)pD, (B), 21(OH)pL, (C) and 21(OH)oxy-piT (D). The inhibition of melanoma SKMEL-188 colony formation was used to assay biological activity of newly synthesized compounds at 0.1, 10 and 1000 nM concentrations. Ethanol was used as a solvent control. The average value (n=6) for vehicle-treated cells is presented as control for drug treated cells. The total number of colonies was expressed in colony forming units (CFU). Data are presented as means ±SEM (n=3 or 4); *P<0.05, **P<0.005, and ***P<0.001.

Figure 7

21(OH)7DHP, 21(OH)pD and 21(OH)pL (10 nM) induced translocation of VDR from the cytoplasm to the nucleus. (A) Data are presented as mean ±SD (n ≥ 10). The differences were analyzed with student’s t-test. ***P < 0.001: differences between control and chemicals. ^#P < 0.05: differences between chemicals. (B) Representative epifluorescence microphotographs of cells expressing VDR-EGFP fusion protein.

Scheme 1

Synthesis of 3β,21-dihydroxypregna-5,7-dien-20-one (4) and its UVB-driven photolysis to vitamin D, tachysterol and lumisterol analogues. Reagents and conditions: (a) Ac₂O, microwave, p-toluenesulfonic acid monohydrate; (b) Dibromantin, 2,2′-azobisisobutyronitrile, benzene/hexane (1:1), 100°C, reflux; (c) Bu₄NBr, Bu₄NF, THF, room temperature; (d) K₂CO₃, MeOH-THF, H₂O, argon, room temperature.