ChinaXiv合作期刊 卷 期 结 构 化 学（JIEGOU HUAXUE） Vol., No. Chinese J. Struct. Chem. Structural Modification for Antitumor Nitrogenous Steroid XIE Tian-Peng(谢田鹏);YANG Kang(杨康);ZHANG Li-Zhi(张立志);CHEN De-Sheng(陈 德胜);ZOU Kun(邹坤);HUANG Nian-Yu (黄年玉) Hubei Key Laboratory of Natural Products Research and Development, College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang 443002, China ABSTRACT Natural steroids have been showing notable cytotoxic activities, which are quite interesting lead compounds for the development of anticancer drug including estramustine and prednimustine. Considering that these semi-synthetic molecules are nitrogen mustard functionalized steroidal derivatives, the present review is focused on the methodologies of introducing nitrogen atom or nitrogen-containing heterocycles on A～D rings or side chains of steroids, and analysis of the structure-activity relationship (SAR) for these man-made cytotoxic steroids. Keywords: steroids; structural modification; N-hetercycles;antitumor;structure-activity relationship; DOI: 10.14102/j.cnki.0254-5861.2011-1688 1 INTRODUCTION Cancer is one of the leading causes of morbidity and mortality worldwide according to the WHO, and chinaXiv:201711.00153v1 the incidence of most cancers including gastric cancer, 1iver cancer and esophageal cancer in our country has kept gradually increasing over the past 20 years from 1989 to 2008[1]. Despite the availability of a large number of chemotherapeutic drugs, the medical need remains still largely unmet because of drawbacks of the antineoplastic agents, such as drug resistance, the lack of selectivity and cancer metastasis[2]. Natural products are an important original source of many widely used anti-cancer drugs[3]. As one of the widespread class of natural organic compounds occurring in plants, animals and fungi, steroid family has exhibited potent antitumor effects through direct suppression of tumor growth, including reduced cell cycle progression and apoptosis, or inhibiting tumor metastasis[4, 5], and numerous steroidal derivatives have been discovered with potent antineoplastic activity and high tissue selectivity[6]. Cytotoxic steroids like estramustine phosphate, a hybrid structure of estradiol and nitrogen mustard combined through a carbamate ChinaXiv合作期刊 linkage with alkylating capability and estrogen-induced specificity, is marketed as a chemotherapy agent for the treatment of prostate cancer[7, 8], which could also be used as a single chemotherapeutic agent or along with other agents such as 5α-reductase inhibitors[9]. Due to the pivotal role of antineoplastic nitrogenous steroids, the present review will discuss the recent cases for the structural modification on the cyclic backbone (Fig. 1) and corresponding SAR for these steroidal derivatives as anticancer agents. Fig. 1. Cyclic backbone of steroids 2 MODIFICATION OF A～D RINGS OF STEROID SKELETON 2. 1 Modification on the A-ring Poirier’s group reported the preparation of five libraries of 2β-piperazino-5α-androstane-3α,17β-diol derivatives by parallel solid-phase synthesis in 2011[10], and biological evaluation against HL-60 leukemia cells found these aminosteroids revealed interesting SAR. For example, the amide coupling functionality showed stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines, and six of the most active amide derivatives (Fig. 2, 1 ～ 6) were obtained with low half-maximal inhibitory concentration (IC50) values of 1.7～3.1 μM and high selectivity indices of 5～55. In 2012, amino-pregnane derivatives (7～9) were further prepared by using a sequence of liquid and solid-phase reactions to extend the previous SAR data on anti-leukemic activities[11], and derivative 9 substituted by the 3-acetylbenzoyl group chinaXiv:201711.00153v1 exhibits strong anti-proliferation effect against HL-60 leukemia cells (IC50: 1.9 μM) and low toxicity on normal peripheral blood lymphocytes (IC50: 31 μM). Then, a 5α-androstane-3α,17β-diol derivative with a quinoline nucleus at the end of the piperazine-proline side-chain at position 2β and an ethinyl at position 17α [12, 13] (10) showed the most potent cytotoxic activity against five cancer cell lines studied (IC50: 0.1～1.1 μM for HL-60, MCF-7, T-47D, LNCaP and WEHI-3, respectively), and selectivity experiment towards liver enzymes CYP3A4 and CYP2D6 indicated the aminosteroid possessed a very low risk of drug-drug interactions. In order to increase the in vivo drug potency, an optimized 3-dimethylcarbamate aminosteroid derivative (11) was finally synthesized in 2016 according to the pro-drug strategy targeting on the 3α-hydroxyl group[14], and the lead compound are more selective for cancer cells over normal cells and much more stable in liver microsomes. - 2 - ChinaXiv合作期刊 2016 Vol. 结 构 化 学（JIEGOU HUAXUE）Chinese J. Struct. Chem. Fig. 2. Chemical structures of compounds 1～11 To develop more effective steroidal drugs, we synthesized a library of (25R)-2-[(1H-1,2,3-triazol-4-yl)methoxy]-spirostan-1,4,6-triene-3-ones from diosgenin for anti-tumor screening, and three steroidal triazoles (Fig. 3, 12～14) possessed potent anti-proliferative effects against [15] Caski cells with IC50 of 9.4～11.8 μM . The structure/cytotoxicity investigations implied that the benzyl, 2-oxopropyl or 3-hydroxyphenylethyl substituted steroidal triazoles exhibited excellent anti-tumor activities. Fig. 3. Synthesis of compounds 12～14 chinaXiv:201711.00153v1 Krstić[16] reported the synthesis and anticancer activity of mono-/bis-thiosemicarbazones and mono-/bis-thiadiazolines substituted steroid at the C-3 position, and the best cytotoxic products were 3-thiosemicarbazones (Fig. 4, 15～17) and 3,17-bis(thiadiazolines) (18, 19) against six cancer cell lines (HeLa, K562, MDA-MB-361, MDA-MB-453, LS174 and A549), which were comparable to cisplatin. The spiro heterocyclic substituent at the C-17 position, as well as the presence of an α,β-unsaturated thiosemicarbazone moiety at C-3, enhanced the activity of the tested compounds. Fig. 4. Chemical structures of compounds 15～19 ChinaXiv合作期刊 Bufadienolides are a type of natural cardiac steroids with potent antitumor activities from the Traditional Chinese medicine Chan’Su. In order to improve their biological activity and water solubility, Hu’s group[17] designed and synthesized the bufalin 3-nitrogen-containing-ester derivatives in 2013, and found the C3 moiety of A-ring had important influence on the cytotoxic activity in SAR study. The bufalin-3-piperidinyl-4-carboxylate (Fig. 5, 21) displayed a significant cytotoxic potency compared to the parent compound bufalin with IC50 values of 0.76 and 0.34 nM against HeLa and A549 cell lines, respectively. Several years later, they found bufalin-3-yl nitrogen-containing-carbamate hydrochloride (22, IC50: 0.30 to 1.09 nM against ten tested tumor cell lines) and C4΄-substituted oleandrin analogues (23 and 24, IC50: 10.9 to 21.7 nM against human cervical carcinoma cell line) exhibited significant in vitro anti-proliferative activities[18, 19]. Fig. 5. Chemical structures of compounds 20～24 To clarify the SAR of anti-tumor activity of diosgenin derivatives in vitro, Fan’s group[20, 21] designed and synthesized 3-azole bromides substituted diosgenin derivatives (Fig. 6, 25 ～ 30) based on the three-dimensional pharmacophore docking simulation for Bcl-2 inhibitors, and SAR results indicated that chinaXiv:201711.00153v1 those with larger hydrophobic heterocyclic group have better activity through the mechanism of hydrogen bonding interaction and dipole-dipole interaction. Due to the restriction of molecular length, the side chain of compound 30 could not occupy the P2 and D3 sites compared with other derivatives, which therefore resulted in the poor antitumor activity. Fig. 6. Chemical structures of compounds 25～30 - 4 - ChinaXiv合作期刊 2016 Vol. 结 构 化 学（JIEGOU HUAXUE）Chinese J. Struct. Chem. Duan’s group firstly prepared E-salignone (Fig. 7, 31)[22] by eight steps of reactions in 16.3% total yields, and found it could inhibit the invasion of human breast cancer MDA-MB-231 cells and non-small cell lung cancer cells (A549) induced by the chemokine epidermal growth factor (EGF) with IC50 values of 0.36 and 5.77 μM, respectively. After modification, a series of antimetastatic E-salignone amide derivatives (32～34) were synthesized[23], and compound 32 was found to possess anti-migration effects in wound-healing assay. Recently, they have further prepared pregn-17(20)-en-3-amine derivatives (35～42) as anti-breast cancer [24] agents . Compared with the positive control LY294002 (IC50: 0.38 μM), the most potent anti-metastatic compound 40 exhibited 30 nM of IC50 value through the mechanism of inhibiting the phosphorylations of integrin β1, PI3K, Akt and PKCζ. Preliminary SAR indicated 3β-substituted steroid derivatives exhibited better anti-invasion activities than the 3α-substituted ones, and the α,β-unsaturated fragment in ring D might be critical for their anti-metastatic activities. Fig. 7. Chemical structures of compounds 31～42 Apart from the above acyclic chain nitrogen-containing derivatives on the A-ring of steroid skeleton, some heterocycle fused steroids were also reported as anticancer agents. Cephalostatin 1 (Fig. 8, 43)[25], a remarkably cytotoxic bis-steroidal pyrazine isolated from the marine tube worm Cephalodiscus gilchristi, has chinaXiv:201711.00153v1 become an important target for total syntheses and structural modification model for the drug discovery. Tian’s lab illustrated an efficient and practical synthesis