Synthesis of Pregnane Derivatives, Their Cytotoxicity on LNCap and PC-3 Cells, and Screening on 5α-Reductase Inhibitory Activity

A series of epoxy- and/or 20-oxime pregnanes were synthesized from commercially available pregnenolone. Compounds 1, 3, 7, 8 and 11-13 were evaluated for cytotoxicity activity towards LNCaP (androgen-dependent) and PC-3 (androgen-independent) prostate cancer cells. Compound 13 showed the highest activity on both LNCaP (IC50 15.17 μM) and PC-3 (IC50 11.83 μM) cell lines. Compound 11 showed weak activity on LNCaP cells (IC 50 71.85 μM) and 8 showed the weak activity on PC-3 cells (IC50 68.95 μM), respectively. The 5α-reductase II (5AR2) inhibitory effects of compounds 1-3, 5 and 7-13 were investigated in a convenient screening model, in which compounds 5, 8, 11 and 12 were observed to be potential inhibitors of 5α-reductase, in particular, the 4-azasteroid 11, that also inhibited cell proliferation of androgen-dependent cells and 8, that in addition inhibited PC-3 cells more potently than LNCaP cells.


Introduction
Testosterone (T) and dihydrotestosterone (DHT) play a key role in the maintenance of cell proliferation in the prostate gland. The enzyme steroid 5α-reductase (5AR) catalyzes the NADPHdependent reductive conversion of T to DHT. The deficiency of 5AR in males results in an incomplete differentiation of external genitalia at birth [1]. On the other hand, abnormally high 5AR activity in

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humans results in excessively high DHT levels in peripheral tissues, which is implicated in the pathogenesis of prostate cancer, benign prostatic hyperplasia (BPH), acne and male pattern baldness [2]. It is presently recognized that there are two genes encoding two distinct isozymes of 5AR that are differentially expressed in human tissues and referred to as type 1 5AR (5AR1) and type 2 5AR (5AR2) [3][4][5]. Although 5AR1 is predominantly expressed in the skin and liver, 5AR2 is mainly expressed in prostate, seminal vesicles, liver and epididymis [6].
Various steroidal [7][8][9] and non-steroidal [10,11] inhibitors have been synthesized and tested against 5AR. Of these, finasteride (PROSCAR ® ), a type II-selective 5α-reductase inhibitor, was the first 5AR inhibitor approved in the USA for the treatment of BPH and prostate cancer. Finasteride was also reported to reduce the proliferation rate in vitro of DU145 and PC-3 prostate cancer cell lines, although several reports in the literature classified them as being "hormone-independent" [12]. Bologna reported that the growth rate of the LNCap cell line can be dose-dependently inhibited by a 5α-reductase inhibitor (finasteride) and antiandrogens (cuproterone and hydroxyflutamide) under culture conditions with 5% FCS DMEM; on the other hand, growth of these cells is only modestly stimulate by T and by DHT when those hormones are added under the same culture conditions [13]. However, finasteride is slow acting and produces side effects affecting sexual function [14,15]. It has also been demonstrated that dutasteride acts as a 5AR1 and 5AR2 inhibitor and blocks the activation of the androgen receptor with the consequences of decreased proliferation and increased death of LNCaP cells [16], but is unresponsive on androgen-independent PC-3 cells [17].
On the basis of the facts described in these references and in order to obtain the skeleton structure required for 5AR inhibitory activity, we describe here the synthesis of some 20-oximes and/or epoxy pregnane, pregnene and pregnadiene derivatives, their in-vitro cytotoxicity on LNCaP (androgendependent) and PC-3 (androgen-independent) prostate cancer cells, and the screening test results as potential inhibitors of 5AR2.

MTT test
Human prostate carcinoma cell lines have been available since 1977 and represent a good experimental model to assess new hormonal therapies [12]. Charcoal stripped-dextran treated heat inactivated 10% FBS was used for these assays to remove the endogeneous steroids.
We assessed the cytotoxicity of synthesized compounds 1, 3, 7, 8 and 11-13 on LNCap (androgendependent) and PC-3 (androgen-independent) cancer cells. (Figures 1 and 2) As shown in Figure 1, the 4-azasteroid 11 showed a dose dependently inhibitory effect on LNCaP cell and also exerted a higher cytotoxicity effect than finasteride, but 8 inversely inhibited the cell proliferation of PC-3 cell. 1,2-Epoxy compound 13 showed the highest cytotoxicity on both LNCaP (IC 50 15.17 μM) and PC-3 (IC 50 11.83 μM) cells. (Table 1) We thought that the cytotoxicity ability of 13 was due to epoxy ring especially on 1,2-position of steroid structure independent of cell type.
The inhibitory mechanism of enone compounds 8 and 12 towards the enzyme 5α-reductase can be generally explained by the fact that 5AR forms an enzyme-steroid activated complex in the first step and the nucleophilic portion of the enzyme attacks their double bond instead of testosterone in a Michael type addition reaction and NADPH subsequently donates a hydrogen to the C-5 position of the compounds [1]. It is assumed that the carbonyl group of the 3-acetoxy substituent of 5 and the 3-carbonyl group of 11 bind to the enzyme and then next step is same as that of the enones. However all tested compounds showed lower activity than finasteride, therefore a substituent at the C-20 position more bulky than an oxime group seems to be required. Consequently, the carbonyl group at the C-3 position, a substituted oxime at the C-20 position and an unsaturated bond in the A and/or B rings of the pregnane skeleton seem to be important for 5α-reductase inhibitory activity. Compound 8 is more active than the 4-azasteride analog 11, consequently we don't think that 4-azasteride is a necessary feature for activity. Compound 11 also showed cytotoxicity on androgen-dependent LNCaP cells, therefore the cytotoxicity of this compound was seems to relate with the androgens.

MTT assay on LNCaP and PC-3 cell
LNCaP and PC-3 prostate cancer cells were grown in RPMI medium 1640 containing charcoal stripped-dextran treated heat inactivated 10% fetal bovine serum (FBS) (Gibco BRL, Rockville, MD, USA) supplemented with 1% penicillin-streptomycin. Cells (2 × 10 3 ) were planted in 96 well cell plates and incubated in 5% CO 2 incubator (NAPCO Water-Jacketed CO 2 incubator) for 24 hours at 37 °C. Then cells were treated with 0, 1, 5, 10, 20, 50, 100 μM concentrations of sample (5 μL/well) and incubated in 5% CO 2 incubator for 48 hours at 37 °C. Triplicate wells were used for each concentration. After incubation for 48 hours, 50 μL/well of MTT (2 mg/mL in PBS) was added and further incubated for four hours in the dark. After removal of MTT solution, 150 μL/well DMSO was added to dissolve purple crystal for 15 minutes, after which the absorbance of the solution was measured at 540 nm with an ELISA reader (ELx 808, BIO-TEK). The inhibition effect of LNCaP and PC-3 cell growth was expressed relative cell survival percentage.

Statistical analysis
Triplicate analyses were performed for MTT test and all the data were subjected to analysis of variance using ANOVA and Duncan's multiple range test for significant difference comparison.

Conclusions
Thirteen epoxy-and/or 20-oxime pregnane, pregnene and pregnadiene derivatives were synthesized and evaluated for cytotoxicity activity and 5AR2 inhibition effects. The 5AR2 inhibition screening test results suggested that compounds 5, 8, 11 and 12 were potential inhibitors of 5α-reductase type II. Especially, 11 was active in the 5AR2 inhibitory test, and inhibited cell proliferation of androgendependent cell and 8 was the most active in the 5AR2 inhibitory test, but interestingly, it inhibited PC-3 cells more potently than LNCaP cells. Compound 13 showed the highest cytotoxic effects on both LNCap and PC-3 prostate cancer cells.