Synthesis of novel 4'-acylamino modified 21E-benzylidene steroidal derivatives and their cytotoxic activities.

A series of 4'-acylamino modified Δ1,4-pregnadien-21E-benzylidene-3,20-dione derivatives (6a-v) was synthesized from the commercially available progesterone (1). These title compounds were evaluated for their toxicity against brine shrimp (Artemia salina) and cytotoxic activities against two human cancer cell lines (HeLa and MCF-7). The results revealed that compound 6f exhibited promising in vitro cytotoxic activity to the two cancer cell lines and the nature of acylamino functional group in the benzylidene moiety had a significant influence on cytotoxicity.

Synthesis and biological activity of two pregnane derivatives with a triazole or imidazole ring at C-21.

Pregnane derivatives are studied as agents for the treatment of different hormone-dependent diseases. The biological importance of these steroids is based on their potential use against cancer. In this study, we report the synthesis, characterization and biological activity of two pregnane derivatives with a triazole (3ß-hydroxy-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-20-one; T-OH) or imidazole (3ß-hydroxy-21-(1H-imidazol-1-yl)pregna-5,16-dien-20-one; I-OH) moieties at C-21. These derivatives were synthesized from 16-dehydropregnenolone acetate. The activity on cell proliferation of the compounds was measured on three human cancer cells lines: prostate cancer (PC-3), breast cancer (MCF7) and lung cancer (SK-LU-1). The cytotoxic and antiproliferative effects of T-OH and I-OH were assessed by using SBR and XTT methods, respectively. The gene expressions were evaluated by real time PCR. In addition, results were complemented by docking studies and transactivation assays using an expression vector to progesterone and androgen receptor. Results show that the two compounds inhibited the three cell lines proliferation in a dose-dependent manner. Compound I-OH downregulated the gene expression of the cyclins D1 and E1 in PC-3 and MFC7 cells; however, effect upon Ki-67, EAG1, BIM or survivin genes was not observed. Docking studies show poor interaction with the steroid receptors. Nevertheless, the transactivation assays show a weak antagonist effect of I-OH on progesterone receptor but not androgenic or antiandrogenic actions. In conclusion, the synthesized compounds inhibited cell proliferation as well as genes key to cell cycle of PC-3 and MCF7 cell lines. Therefore, these compounds could be considered a good starting point for the development of novel therapeutic alternatives to treat cancer.

Effect of new hybrids based on 5,16-pregnadiene scaffold linked to an anti-inflammatory drug on the growth of a human astrocytoma cell line (U373).

In spite of the fact that anaplastic astrocytoma is an uncommon disease, very often the pathology of this disease is associated with lethal effects due to the late diagnosis and unspecific treatments. This paper reports the synthesis and the biological effect on the growth of U373 cell line (human anaplastic astrocytoma) of new hybrid compounds based on 5,16-pregnadiene scaffold linked to an anti-inflammatory drug (6a-e). Moreover, we also determined the cell growth effect of five non-steroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, indomethacin and sulindac) as well as the free steroidal alcohol 5. The results from this study indicated that sulindac as well as compound 5 decreased the number of U373 cells at different concentrations. However, when an anti-inflammatory drug was bound to the steroidal structure (5), the resulting compounds (6a-e) showed an enhanced biological effect with exception of hybrid 6c. Furthermore, derivative 6e (sulindac hybrid) did not allow cell growth during six days of experiment at a concentration of 10 µM. The overall data indicated that these molecules showed an anti-proliferative activity on anaplastic astrocytoma cell line.

Pregna-5,17(20)-dien-21-oyl amides affecting sterol and triglyceride biosynthesis in Hep G2 cells.

Synthesis of series [17(20)Z]- and [17(20)E]-pregna-5,17(20)-dien-21-oyl amides, containing polar substituents in amide moiety, based on rearrangement of 17α-bromo-21-iodo-3ß-acetoxypregn-5-en-20-one caused by amines, is presented. The titled compounds were evaluated for their potency to regulate sterol and triglyceride biosynthesis in human hepatoma Hep G2 cells in comparison with 25-hydroxycholesterol. Three [17(20)E]-pregna-5,17(20)-dien-21-oyl amides at a concentrations of 5 µM inhibited sterol biosynthesis and stimulated triglyceride biosynthesis; their regulatory potency was dependent on the structure of amide moiety; the isomeric [17(20)Z]-pregna-5,17(20)-dien-21-oyl amides were inactive.

Synthesis of 21-nitrogen substituted pregna-5,17(20)-dienes from pregnenolone.

The facile synthesis of six [17(20)Z]- and [17(20)E]-isomeric 3ß-hydroxy-pregna-5,17(20)-dien-21-oyl amides and three [17(20)E]-3ß-hydroxy-2-[prergna-5,17(20)-dien-20-yl]-oxazolines from pregnenolone is presented. The synthetic scheme consists of transformation of pregnenolone into the known 17α-bromo-21-iodo-3ß-acetoxypregn-5-en-20-one followed by reaction with ethanolamine, 2-methyl-2-aminopropanol, and (1-aminocyclohexyl)methanol resulted in mixture of [17(20)E]- and [17(20)Z]-pregna-5,17(20)-dien-21-(2-hydroxy)-oyl amides; separation of [17(20)E]- and [17(20)Z]-isomers; their cyclization into [17(20)E]-oxazolines under action of POCl(3) in pyridine, and removal of acetate protecting groups. Significantly different orientation of nitrogen containing substituents in [17(20)Z]- and [17(20)E]-isomers regarding to steroid backbone enables their configuration to be easily identified by NMR spectroscopy. All synthesized compounds did not exhibit marked toxic effects in three cell lines (MCF-7, Hep G2, and LNCaP). In androgen-sensitive LNCaP cells all testing compounds at concentrations of 50 nM potently stimulated proliferation.

Photo-conversion of two epimers (20R and 20S) of pregna-5,7-diene-3beta, 17alpha, 20-triol and their bioactivity in melanoma cells.

Pregna-5,7-dienes and their hydroxylated derivatives can be formed in vivo when there is a deficiency in 7-dehydrocholesterol (7-DHC) Delta-reductase function, e.g., Smith-Lemli-Opitz syndrome (SLOS). Ultraviolet B (UVB) radiation induces photoconversion of 7-DHC to vitamin D3, lumisterol3 and tachysterol3. Two epimers (20R and 20S) of pregna-5,7-diene-3beta,17alpha,20-triol (4R and 4S, respectively) were synthesized and their UVB photo-conversion products identified as corresponding 9,10-secosteroids with vitamin D-like and tachysterol-like structures, and 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol-like). The number and character of the products and the dynamics of the process were dependent on the UVB dose. At high UVB doses, the formation of multiple oxidized derivatives of the primary products, and the formation of 5,7,9(11)-triene, were observed. The production of vitamin D-like, tachysterol-like and lumisterol-like derivatives was also observed in human skin treated with 4R and 4S, and subjected to UV irradiation, as shown by RP-HPLC. Newly synthesized compounds inhibited melanoma growth in dose dependent manner, and some of them showed equal or higher potency than 1,25(OH)2D3. In summary, we have characterized for the first time the products of UV induced conversion of pregna-5,7-diene-3beta,17alpha,20-triols and documented that the newly synthesized compounds have antiproliferative properties against melanoma cells.

Synthesis and photo-conversion of androsta- and pregna-5,7-dienes to vitamin D3-like derivatives.

Calcitriol (3beta,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-1alpha,3beta,25-triol) is a powerful oncostatic form of vitamin D3 that is of limited clinical utility due to hypercalcemic (toxic) effects. Since the removal of the side chain reduces or eliminates the calcemic activity of vitamin D3, secosteroidal compounds lacking or with a shortened side chain are good candidates for anti-cancer drugs. In addition, 5,7-steroidal dienes without a side chain can be generated in vivo under pathological conditions. A series of androsta- and pregna-5,7-dienes was efficiently synthesized from their respective 3-acetylated 5-en precursors by bromination-dehydrobromination and deacetylation reactions. Ultraviolet B (UVB) irradiation was used to generate corresponding 9,10-secosteroids with vitamin D-like structures. Additional products with tachysterol-like (T-like) structures or 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol, L-like) were also detected. Different doses of UVB resulted in formation of various products. At low doses, previtamin D-, T- or L-like compounds were formed as the main products, while higher doses induced further isomerization, with formation of potentially oxidized derivatives. In summary, we describe dynamic UVB induced conversion of androsta- and pregna-5,7-dienes into vitamin D-like compounds and their rearranged analogues; additionally novel T-like and L-like structures were also produced and characterized. Further biological evaluation of newly synthesized compounds should help to select the best candidate(s) for potential treatment of hyperproliferative diseases including cancer.

In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5alpha-reductase inhibitors.

In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives (11a-11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a-11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC50 values by measuring the steroid concentration that inhibits 50% of the activity of 5alpha-reductase present in human prostate. In order to study the mechanism of action of 11a-11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a-11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a-11d exhibited a high inhibitory activity for the human 5alpha-reductase enzyme with IC50 values of 1.4 x 10(-8), 1.8 x 10(-9), 1.0 x 10(-8) and 4 x 10(-5) respectively. However the IC50 value of 11a (1.8 x 10(-9)) was the only one lower than that of finasteride (8.5 x 10(-9)). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b-11d. The competition analysis for the androgen receptor indicated that the IC50 value of non-labeled mibolerone used in this experiment was 1nM, whereas steroids 10, 11a-11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives (11a-11d) cannot be considered as prodrugs of compound 10. In conclusion, the compounds containing chlorine 11a, bromine 11b, iodine 11c atoms, and 11d (without any substituent in the ester moiety) at C-3 produce a significant decrease of the prostate weight in castrated animals treated with T and inhibits the activity of the 5alpha-reductase. Apparently the presence of the halogen atoms in compounds 11a-11c enhances the inhibitory activity for the 5alpha-reductase enzyme.

Effect of chirality at C-20 of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate derivatives on antiinflammatory activity.

In an effort to determine the C-20 chirality effect on the antiinflammatory activity of 17beta-glycolate esters, methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate and its 9alpha-fluoro analog, their acetonide and their carbonate derivatives were synthesized and evaluated. The agents were tested for their binding potency to the macrophage glucocorticoid receptor, and their effect on LPS-induced nitric oxide generation in RAW 264.7 cells. The acetonide derivatives showed the highest binding affinity while the triols and carbonates bound rather poorly to the receptors. With the exception of the triols, the alpha-isomer in each pair of the agents exhibited higher binding affinity to the receptor than its corresponding beta-isomer, clearly indicating that C-20 chirality has a significant effect on antiinflammatory activity. In addition, the alpha-isomers of the acetonides showed substantially higher binding affinity than the parent compound, prednisolone. In contrast to the high binding activity exhibited by some of the acetonides, all of the agents showed weak inhibitory effect on NO generation. Metabolic inactivation during assessment of NO inhibition may play a role in the divergence noted between receptor affinity and the measured biologic activity resulting from the binding.

N-Butyl-N-methyl-11-(3'-hydroxy-21', 17'-carbolactone-19'-nor-17'alpha-pregna-1',3', 5'(10')-trien-7'alpha-yl)-undecanamide: an inhibitor of type 2 17beta-hydroxysteroid dehydrogenase that does not have oestrogenic or androgenic activity.

It is well known that 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the formation and inactivation, from circulating precursors, of several active androgens and oestrogens. These enzymes can thus regulate tumoural cell proliferation in androgen- and oestrogen-dependent cancers. Recently, we discovered that adding a spiro-gamma-lactone to the oestradiol nucleus results in a novel inhibitor of type 2 17beta-HSD, an enzyme that catalyses the interconversions between 4-androstene-3,17-dione and testosterone, and between oestrone and oestradiol. This finding motivated our introducing the spiro-gamma-lactone moiety onto an anti-oestrogenic nucleus. The N-butyl-N-methyl-11-(3'-hydroxy-21', 17'-carbolactone-19'-nor-17'alpha-pregna-1',3', 5'(10')-trien-7'alpha-yl)-undecanamide (4) was then efficiently synthesized and its biological activity was assessed in vitro. Despite the presence of a bulky alkylamide side chain, the spiro-gamma-lactone function conserved its ability to inhibit type 2 17beta-HSD (IC(50) = 0.35 and 0.25 microM, with and without side chain, respectively). Furthermore, the selective inhibition by lactone 4 toward type 2 17beta-HSD (microsomal fraction of human placenta) was demonstrated by the absence of inhibitory activity toward type 1 17beta-HSD (cytosolic fraction of human placenta). Cell proliferation assays indicated that compound 4 had no oestrogenic activity but did show anti-oestrogenic activity on ER(+) cell line ZR-75-1. No androgenic activity could be detected when assayed on the AR(+) cell line Shionogi either. Based on these facts, we report the synthesis of a new steroidal derivative, one that inhibits type 2 17beta-HSD while possessing anti-oestrogenic activity.

Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase.

Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole).

Regioselective reactions of 1,2-dehydroprogesterone: syntheses of pregnane derivatives as possible contragestational agents.

A few pregnane derivatives were synthesized from 1,2-dehydroprogesterone (1). Ring A of 1,2-dehydroprogesterone was aromatized without affecting C-20, and the resulting acetoxy compound (2) after hydrolysis yielded 1-hydroxy-4-methyl-19-norpregna-1,3,5(10)-trien-20-one (3). Reactions of the phenol (3) with alkyl halides yielded the ethers 6a-6b and 7. Opening of the oxirane ring in 7 with secondary amines furnished the aminoalcohols 8a-8b. Friedelcraft's reaction of 3 with maleic anhydride and chloracetyl chloride led to the formation of 9 and 10, respectively. Base-catalyzed ring closure of 10 yielded 1-acetyl-12a-methyl-8-oxo-5[H]-1,2,3,3a,3b,4,8,9,10b,11,12, 12a-dodecahydrocyclopenta (7,8)-phenanthro (3,4-b) furan (11), which reacted with aromatic aldehydes regioselectively to furnish 12a-12b. Reaction of 1 with triethylorthoformate in the presence of boron trifluoride etherate involved the participation of C-21, and the carbonyl at C-3 remained unaffected. The product 13 was identified as 21-[2-hydroxyvinyl]-21-norpregna-1,4-diene-3,20-dione. Reductive amination with sodium cyanoborohydride in the presence of ammonium acetate did not attack ring A and smoothly furnished the amine 14 which, on reaction with succinic anhydride, gave 20-succinamylpregna-1,4-dien-3-one (15).

Aldosterone antagonists. 3. Synthesis and activities of steroidal 7 alpha-(alkoxycarbonyl)-15,16-methylene spirolactones.

Several A- and D-ring substituted steroidal 7 alpha-alkoxycarbonyl spirolactones were synthesized with the purpose of increasing the aldosterone antagonistic potency and reducing the endocrinological side effects relative to the standard drug spironolactone. It was found that the 15 beta,16 beta-methylene derivative 17 exhibited a 2-fold higher aldosterone antagonistic activity compared to either spironolactone or the 15,16-unsubstituted derivative 29 while showing remarkably reduced antiandrogenicity.

An efficient synthesis of 3 alpha-hydroxy-5 alpha-pregna-9(11),16-diene-20-one.

3 alpha-Hydroxy-5 alpha-pregna-9(11),16-diene-20-one (6) was synthesized for the first time from the commercially available 16,17 alpha-epoxy-3 beta-hydroxy-5-pregnen-20-one(1) in several steps. The key step involves a remote chlorination reaction utilizing m-iodobenzoate group as a free-radical guide.

Synthesis of 5alpha-pregna-1,20-dien-3-one.

5ALPHA-Pregna-1,20-dien-3-one has been synthesized from 3beta-hydroxy-5alpha-pregnan-20-one by reaction of the tosylhydrazone with butyllithium to introduce the vinyl moiety. Oxidation of the alcohol and treatment of the anion of the resulting ketone with benzeneselenenyl chloride gave the 2-phenylseleno derivative. This afforded the conjugated enone on oxidative elimination.