Synthesis and Evaluation of Some New Aza-B-homocholestane Derivatives as Anticancer Agents

Using analogues of some marine steroidal oximes as precursors, a series of aza-B-homocholestane derivatives possessing different substituted groups at the 3-position of the steroidal nucleus were synthesized. Their biological activity against cancer cell proliferation was determined with multiple cancer cell lines. Aza-B-homocholestane derivatives possessing 3-hydroxyl, 3-hydroximino and 3-thiosemicarbazone groups displayed remarkable cytotoxicity to cancer cells via apoptosis inducing mechanism. Compounds 5, 10, 12, 15 and 18 exhibited better potency to inhibit cancer cell proliferation. In addition, compound 15 was further evaluated with three dimensional (3D) multicellular spheroids assay to determine its potency against spheroid growth. The structure-activity relationship (SAR) generated in the studies is valuable for the design of novel chemotherapeutic agents.

In our previous work, a series of 3-aza-A-homo and 4-aza-A-homo steroidal lactams were prepared and evaluated against the proliferation of SGC 7901 (human ventriculi carcinoma cell line), HeLa (human cervical carcinoma cell line) and Bel 7404 (human liver carcinoma cell line) [17][18][19]. The results showed that some of these steroidal lactams significantly inhibited the proliferation of the tumor cell lines tested and induced cancer cell apoptosis as well. In order to find more active derivatives as potential antitumor agents, a series of the new aza-B-homecholestane derivatives possessing different aza-position and various substitute groups on the 3-position of steroidal nucleus were designed and synthesized taking analogues of compound 2 as precursors. The antiproliferative activities of these new compounds were evaluated with different cancer cell lines.

Chemistry
First, 3-hydroximino-7-aza-B-homocholest-4-en-6-one (7) was synthesized as listed in Scheme 1. The synthesis of steroidal oxime (3) had been reported by our group [7]. Beckman rearrangement of 3 in SOCl 2 /THF gave 3-acetoxy-7-aza-B-homocholest-4-en-6-one (4). The structure of 4 was confirmed by 1 H NMR chemical shifts of 7a-protons at 3.16-3.21 ppm (2H, m). Compound 5 was obtained by deacetylation of 4 in aqueous solution of 13% K 2 CO 3 . The oxidation of compound 5 with Jones reagent afforded 7-aza-B-homocholest-4-en-3,6-dione (6). Last, the oxime 7 was produced in a yield of 42% by the reaction of 6 with hydroxylamine hydrochloride in ethanol in the presence of NaOAc. The structure of 7 was confirmed by analysis of the proton and carbon NMR chemical shifts. The downfield chemical shift of 2β-H at 3.453 ppm (dd, J = 15.3 and 4.8 Hz, due to the deshielding influence of the OH in the hydroximino group) and C-3 at 158.6 ppm demonstrated the (E)-configuration and formation of 3-hydroximino in compound 7. Scheme 1. Synthesis of 3-hydroximino-7-aza-B-homocholest-4-en-6-one. Similarly, compounds 12-15 possessing the 3-substituted-6-aza-B-homocholest-7-one key feature in their structures were synthesized with 8 steps using cholesterol as starting material (Scheme 2). Compound 8 was synthesized based on the known methodology [8]. The structure of 9 was affirmed by proton and carbon NMR spectrums. In the 1 H NMR spectrum, the resonances showing of C 5 -αH at 3.47-3.40 ppm (1H, m), C 7a -H at 2.36-2.22 ppm (2H, m) and 7-C at 176.2 ppm demonstrated a formation of NH-CO-bond and a position of 6-NH. The oxidization of 10 by Jones reagent generated compound 11. The reaction of compound 11 with HONH 2 · HCl, CH 3 ONH 2 · HCl, PhCH 2 ONH 2 · HCl or thiosemicarbazide afforded the corresponding products 12-15. Their structures were confirmed by IR and NMR spectrum, and the mixture of Eand Z-stereoisomer was obtained in preparation of 13-15, respectively.
Interestingly, all compounds with 3-hydroxyl, hydroximino and thiosemicarbazone groups exhibited a higher cytotoxicity against HeLa cells. For example, compound 10 with 3-hydroxyl and compound 15 with 3-thiosemicarbazone exhibited IC 50 values of 3.2 and 5.5 µM to HeLa cells, respectively. Apparently, all compounds (5, 10, 18) bearing the same 3-hydroxyl and a different aza-position displayed a similar cytotoxicity against these cancer cells. However, compound 15 having same 3-thiosemicarbazone and 6-aza-B-homocholest-7-one key features in its structure showed a better antiproliferative activity to these cancer cells than compound 21 with the structure of 7a-aza-B-homocholest-5-ene-7-one except SGC 7901 cell.
Above results showed that the conversion of a carbonyl group at 3-position to a hydroxyl, hydroximino, or thiosemicarbazone groups would result in a significant increase of the antiproliferative activity, suggesting the importance of these functional groups in the biological function of the compounds. Obviously, the substitution of the 3-hydroxyl group remarkably increased its cytotoxic activity against these cancer cells in comparison with the 3-acetoxyl group.
Overall, compounds 5, 10, 12, 15 and 18 were found to be the most potent compounds as anticancer agents, and they displayed a similar antiproliferative activity, when compared to cisplatin (a positive control).

3D Multicellular Spheroids Screening
Screening and initial characterization of anticancer drugs typically use monolayer cultures of tumor cells. However, such monolayer cultures do not represent the characteristics of threedimensional (3D) solid tumors. The multicellular tumor spheroid model has an intermediate complexity between in vivo tumors and in vitro monolayer cell cultures. Considering the complexity of the in vivo situation, it is not surprising that many drugs that are effective in a twodimensional cell culture will lose efficacy with the in vivo assays. Limited penetration of the drug into tumor cell masses is one main factor that will lead to poor drug efficacy in animal models. Testing the efficacy of the drug with tumor spheroids may help to predict their in vivo potency [20]. Herein, compound 15 was examined with the 3D spheroid growth assay.
Spheroids were photographed in an inverted phase contrast microscope. A micrometer scale was photographed at the same magnification, and spheroid size was determined and compared.
In Figure 2, the spheroids treated with 15 µM of 15 had a smaller size after 6 days than the untreated control spheroids. The result indicated that compound 15 showed good tumor penetration ability.

Compound 5 Induced Apoptosis of Cancer Cells
To determine the molecular mechanism by which compound 5 inhibited cancer cell proliferation, we further analyzed the cytotoxicity of compound 5 in SGC-7901 cells. As shown in Figure 3, compound 5-induced SGC-7901 cell death could be clearly observed. To determine whether the decreased viability of SGC-7901 cells was due to compound 5 induced apoptosis, the cells were treated with compound 5, and Annexin V assay was performed. FITC-conjugated Annexin V is commonly used to determine apoptotic cells at an early stage. As shown in Figure 4, treatment with 5 μg/mL of compound 5 resulted in 8.12% PI/Annexin V double-labeled apoptotic cells (control: 0.57%; cisplatinum: 8.94%) after 24 h incubation (the lower right quadrant and the upper right quadrant which contains early and late apoptotic cells, respectively), suggesting compound 5 is a potent apoptotic inducer in gastric carcinoma cells.

Chemistry
The sterols were purchased from the Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. All chemicals and solvents were analytical grade and solvents were purified by general methods before being used. Melting points were determined on an X4 apparatus and were uncorrected. The solution of thionyl chloride (1.5 mL) in 5 mL dry THF was added to a solution of the oxime 3 (300 mg) in dry THF (30 mL). The solution was stirred under anhydrous condition for 1 h at 0 °C. The reaction was terminated and water was added to the solution. The solution was neutralized with ammonia and the product was extracted with CH 2 Cl 2 (20 × 3 mL). The combined extract was washed with water, 5% NaHCO 3 , and saturated brine, dried over anhydrous Na 2 SO 4 Figure S1). (5) 20 mL of K 2 CO 3 solution (13%) was added to a solution of compound 4 (1520 mg, 3.32 mmol) in CH 3 OH (200 mL). The reaction mixture was heated under reflux condition for 1 h. After completion of the reaction as indicated by TLC, the solvent was removed under reduced pressure. 200 mL of CH 2 Cl 2 was added to dissolve solid and the resulting solution was washed with cold water and saturated brine. After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the resulting crude product was purified by chromatography on silica gel using methanol/dichloromethane  Figure S3). (7) CH 3 COONa.3H 2 O (120 mg, 0.33 mmol) and NH 2 OH.HCl (23 mg, 0.33 mmol) were added to the solution of 120 mg (0.27 mmol) 6 in 20 mL 95% ethanol. After the solution was heated to 60 °C, the mixture was stirred at the temperature for 1 h. Then the reaction was terminated and the majority of solvent was evaporated under reduced pressure. Water was added into the reaction mixture, and the product was extracted with ethyl acetate (20 × 3 mL). The combined extracts were washed with saturated brine, dried, and evaporated under reduced pressure. The residue was subjected to chromatography using petroleum ether/ethyl acetate (5:1) as the eluent to give 50 mg of 7 as white solid. Yield: 42%, mp 285-286 °C. IR (KBr) ν: 3317, 2941, 2859, 2348, 1645, 1600, 1449, 1367, Compounds 13 and 14 were prepared similarly according to the procedure of 11, but CH 3 ONH 2 · HCl and PhCH 2 ONH 2 · HCl were used as reagents instead of NH 2 OH· HCl.  CO2). For drug treatment, 100 μL fresh medium with various concentration of drugs were added at day 3 (final matrigel concentration became 2.5% as well). Spheroid morphological images in 96-well microplate were carried out manually on an inverted microscope equipped with camera. Spheroid diameters and volumes were determined from their images. The treatment was quart replicated, and the spheroid images were taken every other day. The suppression of the spheroid growth was normalized with control treatment (0.1% DMSO) [21,22].

Annexin V Staining Assay
Apoptosis was detected with an annexin V-FITC kit purchased from BD Pharmingen (San Diego, CA, USA) according to the manufacturer's instructions. SGC-7901 cells were seeded in 35 mm culture dishes and allowed to attach overnight. The cells were treated with different concentration of compound 5 for 24 h respectively, collected, and washed twice with PBS. To detect early and late apoptosis, both adherent and floating cells were harvested together and resuspended in annexin V binding buffer at a concentration of 10 6 cells/mL. Subsequently, 5 μL of FITC-conjugated annexin V and 5 μL of propidium iodide were added to 100 μL of the cell suspension (10 5 cells). The cells were incubated for 15 min at room temperature in the dark. Finally, 400 μL of annexin V binding buffer was added to each tube, and cells were analyzed by a two color cytometry using FACS Calibur (Becton Dickinson, Biosciences, Franklin Lakes, NJ, USA).

Conclusions
We have prepared a series of aza-B-homocholestane derivatives having different substituted groups at 3-postion of the steroidal nucleus, taking analogues of marine steroidal oximes as precursors. The antiproliferative activity of the synthesized compounds against SGC-7901, HeLa, Bel-7404, GNE 2, SPC-A and Tu 686 cell lines was investigated. The results showed that aza-B-homocholestane derivatives possessing 3-hydroxyl, 3-hydroximino and 3-thiosemicarbazone groups showed remarkable cytotoxic activity. In the synthesized compounds, compounds 5, 10, 12, 15 and 18 were found to be the most potent compounds as anticancer agents, and they displayed a similar antiproliferative activity as cisplatin did. The result of 3D multicellular spheroids screening of 15 showed also distinct antiproliferative activity, and Annexin V staining assay indicated that compound 5 was able to effectively induce tumor cells apoptosis. Compounds 5 and 15 are now submitted to further acute toxicity and antitumor activity studies in animal models, and the relative possible results will be reported in due course. Our findings provide new evidence showing the relationship between the chemical structure and biological activities, and may be useful for the design of novel chemotherapeutic drugs.