Shankar M. Singh

Androgen Receptor Antagonists (Antiandrogens) Structure-Activity Relationships

Prostate cancer, acne, seborrhea, hirsutism, and androgenic alopecia are well recognized to depend upon an excess or increased sensitivity to androgens or to be at least sensitive to androgens. It thus seems logical to use antiandrogens as therapeutic agents to prevent androgens from binding to the androgen receptor. The two predominant naturally occurring androgens are testosterone (T) and dihydrotestosterone (DHT). DHT is the more potent androgen in vivo and in vitro. All androgen-responsive genes are activated by androgen receptor (AR) bound to either T or DHT and it is believed that AR is more transcriptionally active when bound to DHT than T. The two classes of antiandrogens, presently available, are the steroidal derivatives, all of which possess mixed agonistic and antagonistic activities, and the pure non-steroidal antiandrogens of the class of flutamide and its derivatives. The intrinsic androgenic, estrogenic and glucocorticoid activities of steroidal derivatives have limited their use in the treatment of prostate cancer. The non-steroidal flutamide and its derivatives display pure antiandrogenic activity, without exerting agonistic or any other hormonal activity. Flutamide (89) and its derivatives, Casodex (108) and Anandron (114), are highly effective in the treatment of prostate cancer. The combination of flutamide and Anandron with castration has shown prolongation of life in prostate cancer. Furthermore, combined androgen blockade in association with radical prostatectomy or radiotherapy are very effective in the treatment of localized prostate cancer. Such an approach certainly raises the hope of a further improvement in prostate cancer therapy. However, all antiandrogens, developed so-far display moderate affinity for the androgen receptor, and thus moderate efficacy in vitro and in vivo. There is thus a need for next-generation antiandrogens, which could display an equal or even higher affinity for AR compared to the natural androgens, and at the same time maintain its pure antiandrogenic activity, and thus providing improved androgen blockade using possibly antiandrogens alone.

Characterization of the effects of the novel non-steroidal antiestrogen EM-800 on basal and estrogen-induced proliferation of T-47D, ZR-75-1 and MCF-7 human breast cancer cells in vitro

Since estrogens play a predominant role in the development and growth of human breast cancer, antiestrogens represent a logical approach to the treatment of this disease. The present study compares the effects of the novel non‐steroidal anti‐estrogen EM‐800 and related compounds with those of a series of anti‐estrogens on basal and 17β‐estradiol (E2)‐induced cell proliferation in human breast cancer cell lines. In the absence of added E2, EM‐800 and related compounds failed to change basal cell proliferation, thus showing the absence of intrinsic estrogenic activity in the ER‐positive T‐47D, ZR‐75‐1 and MCF‐7 cell lines. The stimulation of T‐47D cell proliferation induced by 0.1 nM E2 was competitively blocked by a simultaneous incubation with EM‐652, EM‐800, OH‐tamoxifen, OH‐toremifene, ICI 182780, ICI 164384, droloxifene, tamoxifen and toremifene at apparent Ki values of 0.015, 0.011–0.017, 0.040–0.054, 0.043, 0.044, 0.243 and 0.735 nM, approx. 10 nM and >10 nM, respectively. Similar data were obtained in ZR‐75‐1 and/or MCF‐7 cells. Moreover, EM‐652 was 6‐fold more potent than OH‐Tamoxifen in inhibiting the proportion of cycling MCF‐7 cells. Our data show that EM‐800 and EM‐652 are the most potent known antiestrogens in human breast cancer cells in vitro and that they are devoid of the estrogenic activity of OH‐tamoxifen and droloxifene suggested by stimulation of cell growth in the absence of estrogens in ZR‐75‐1 and MCF‐7 cells. Int. J. Cancer 73:104–112, 1997.

Improved highly efficient synthesis of α,β-acetylenic ketones. Nature of the intermediate from the reaction of lithium acetylide with boron trifluoride etherate

Abstract A wide variety of α,β-acetylenic ketones were synthesized in very high yields via an exceptionally facile intermolecular reaction of lithium alkynyltrifluoroborates and carboxylic acid anhydrides.

Structural Characterization of the Human Androgen Receptor Ligand-binding Domain Complexed with EM5744, a Rationally Designed Steroidal Ligand Bearing a Bulky Chain Directed toward Helix 12

Antiandrogens are commonly used to treat androgen-dependent disorders. The currently used drugs unfortunately possess very weak affinity for the human AR (hAR), thus indicating the need to develop new high-affinity steroidal antiandrogens. Our compounds are specially designed to impede repositioning of the mobile carboxyl-terminal helix 12, which blocks the ligand-dependent transactivation function (AF-2) located in the AR ligand-binding domain (ARLBD). Using crystal structures of the hARLBD, we first found that H12 could be directly reached from the ligand-binding pocket (LBP) by a chain positioned on the C18 atom of an androgen steroid nucleus. A set of 5α-dihydrotestosterone-derived molecules bearing various C18 chains were thus synthesized and tested for their capacity to bind hAR and act as antagonists. Although most of those having very high affinity for hAR were agonists, several very potent antagonists were obtained, confirming the structural importance of the C18 chain. To understand the role of the C18 chain in their agonistic/antagonistic properties, the structure of the hARLBD complexed with one of these agonists, EM5744, was determined at a 1.65-Å resolution. We have identified new interactions involving Gln738, Met742, and His874 that explain both the high affinity of this compound and the inability of its bulky chain to prevent the repositioning of H12. This structural information will be helpful to refine the structure of the chains placed on the C18 atom to obtain efficient H12-directed steroidal antiandrogens.

Comparison of in vitro effects of the pure antiandrogens OH-flutamide, casodex, and nilutamide on androgen-sensitive parameters

OBJECTIVES A combination of flutamide (Eulexin) or nilutamide (Anandron) with a luteinizing hormone-releasing hormone (LHRH) agonist or orchiectomy is the only therapy demonstrated to prolong life in prostate cancer. Recently, the low 50-mg daily dose of Casodex, an analogue of the pure antiandrogen flutamide, was chosen for clinical studies on the basis that the compound was 5 to 10 times more potent than flutamide, as suggested by data obtained in the inappropriate intact rat model. The present study was designed to compare the in vitro antiandrogenic activity of OH-flutamide (OH-FLU), the active metabolite of flutamide, Casodex, and nilutamide. METHODS The effect of the antiandrogens was tested on two androgen-sensitive parameters, namely proliferation of the SEM-107 clone of Shionogi mouse mammary tumor cells and secretion of the GCDFP-15 (gross cystic disease fluid protein 15 kDa) in T-47D and ZR-75-1 human breast cancer cells. RESULTS The twofold stimulation of Shionogi cell proliferation caused by a 10-day exposure to 1 nM testosterone was competitively reversed by incubation with OH-FLU, Casodex, or nilutamide, at the respective IC50 values of 72, 243, and 412 nM. Moreover, the marked increase in GCDFP-15 release induced by 1 nM testosterone was blocked by OH-FLU. Casodex, or nilutamide at respective IC50 values of 29, 180, and 87 nM in T-47D cells and at 35, 142, and 75 nM in ZR-75-1 cells. Similar data were detected in 4-androstenedione-induced Shionogi cell proliferation and in dihydrotestosterone-induced GCDFP-15 secretion in T-47D cells. CONCLUSIONS OH-FLU is 3.1- to 7.8-fold more potent than Casodex, as measured on two in vitro androgen-sensitive parameters, in agreement with our recent in vivo data obtained in the model of castrated rats supplemented with 4-androstenedione implants, in which threefold greater potency of flutamide was observed. The present data, as well as other data from the literature, strongly indicate the need to choose a more appropriate dose of Casodex for the treatment of prostate cancer.

Highly efficient nucleophilic addition of alkyl Grignard reagents to 17-ketosteroids in the presence of cerium(III) chloride: Synthesis of 17α-propyl-17β-hydroxy-4-androsten-3-one, an androgen receptor antagonist

Abstract The addition of alkyl Grignard reagents to sterically hindered 17-ketosteroids was significantly enhanced by anhydrous cerium(III) chloride with notable suppression of abnormal reactions, while the addition products were obtained in good to excellent yields and high stereoselectivity.

Laboratory scale preparation of 4,4,5,5,5-pentafluoropentan-1-thiol: An important chain of anti-breast cancer agents

Abstract Quantitative free radical addition of perfluoroethyl iodide to propargyl alcohol in the presence of sodium hydrosulfite gave E/Z-2-iodo-4,4,5,5,5-pentafluoro-2-penten-1-ols, which were converted to 4,4,5,5,5-pentafluoropentan-1-ol in one step in excellent yield by catalytic hydrogenation over platinum oxide in the presence of triethylamine. 4,4,5,5,5-Pentafluoropentan-1-thiol was obtained in good yield via modified Mitsunobu reaction of the alcohol.

Synthesis and in vitro study of 17β-[N-ureylene-N,N′-disubstituted]-4-methyl-4-aza-5α-androstan-3-ones as selective inhibitors of type I 5α-reductase

A series of 17β-(N-ureylene-N,N′-disubstituted)-4-azasteroids as inhibitors of human type I 5α-reductase (5α-Rc) were prepared from 17β-N-alkyl-4-methyl-4-aza-5α-androstan-3-ones and various isocyanates. For the measurement of 5α-Re activity, 293 cells transfected with human type I 5α-Re, cDNA were used. Azasteroids with an N-cyclopropyl ring exhibited potent inhibitory activity against type I 5α-Re. As the chain length increased, from the N′-ethyl to the N′-butyl chain, activity of compounds also increased and azasteroids with the N′-butyl chain showed strong inhibitory activity (IC50 = 5.3 nM). Branching of alkyl chains decreased the potency of compounds. Introduction of the 1,2-double bond significantly reduced the activity of azasteroids. Replacement of the N′-alkyl chain with the phenyl moiety gave the most active compound of this series (IC50 = 1.3 nM). Other variations such as the replacement of a N-cyclopropyl ring with the N-methyl or the N-butyl chain decreased the activity of compounds (compounds were less active compared with above). The IC50 values of N-methyl-N′-cyclohexyl- and N-butyl-N′-phenyl-ureylenes were 31.5 and 11.5 nM, respectively. In general, all azasteroids were poor inhibitors of Type II 5α-Re.

Synthesis and in vitro antiandrogenic activity of 17β-hydroxy-17α-(ω-hydroxy/haloalkyn-1′-yl)-4-methyl-4-aza-3-oxo-5α-androstan-(1-ene)-3-ones

Abstract Synthesis of 17β-hydroxy-17α-(ω-hydroxy/haloalkyn-1′-yl)-4-methyl-4-aza-(1-ene)-5α-steroids ( 7–22 ) was achieved by the addition of THP protected hydroxy alkynyllithium to 4-methyl-4-aza-(1-ene)-5α-androstan-3,17-diones ( 1 and 2 ), followed by deprotection and halogenation of 17α-(ω-hydroxy) compounds ( 7–10 ). Chloro- compounds 13 and 14 , and iodo- compound 21 are potent antiandrogens. Introduction of a 1,2-double bond increased the potency by 2-fold compared to the parent compounds.

Synthesis of Atamestane (SH 489): An Aromatase Inhibitor

Abstract Atamestane (SH 489) was synthesized from 17β-Acetoxy-1α-methyl-5α-androstan-3-one. Thus dibromination followed by dehydrobromination of the A-ring gave dienone (5) in good yield, which was hydrolyzed and oxidized to give the title compound in 60% overall yield.