New progesterone esters as 5alpha-reductase inhibitors

The pharmacological activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione (7), 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione (8), 4-bromo-17alpha-(p-chlorobenzoyloxy)-pregnene-3,20-dione (9) and 4-bromo-17alpha-(p-toluoyloxy)-4-pregnene-3,20-dione (10) was determined. These compounds were evaluated as 5alpha-reductase inhibitors on gonadectomized hamster seminal vesicles and flank organs. The pharmacological data of this study indicate that compounds 7 and 9 having at C-17 p-fluorobenzoyloxy and p-chlorobenzoyloxy ester functions respectively showed the highest antiandrogenic effect as measured by the reduction of the weight of the seminal vesicles. In the flank organ model, the same compounds 7 and 9 exhibited a smaller diameter, 1.8 and 1.0 mm, respectively, than the commercially available finasteride 3 (2.3 mm), thus indicating a higher inhibitory effect on 5alpha-reductase enzyme. Steroid 7 showed a higher inhibitory activity on the conversion of T to DHT (Fig. 3) than the presently used finasteride, thus indicating a higher antiandrogenic effect. The nonsubstituted benzoyloxy ester (compound 15) showed a lower antiandrogenic activity as measured in the seminal vesicles model than the p-substituted benzoyloxy compounds.     

Studies on topical antiinflammatory agents. V. 17-(Alkylthio)- and methoxyalkanoates of corticosteroids

As part of our search for new topical antiinflammatory agents, a series of corticosteroid 17-(alkylthio)- and methoxyalkanoate derivatives was prepared and tested for vasoconstrictive activities. Several compounds were proved to have activity superior or comparable to that of 9 alpha-fluoro-11 beta,21-dihydroxy-16 beta-methyl-17 alpha-valeryloxy-1,4-pregnadiene-3,20-dione (betamethasone 17-valerate, BV). Among these compounds, 21-chloro-11 beta-hydroxy-17 alpha-(methylthio)acetoxy-4-pregnene-3,20-dione (5Aa) was found to have the most potent activity, being more active than BV. The structure-activity relationships of the series revealed that introduction of a (methylthio)acetate function into the 17-position as well as the 21-position of corticosteroids was effective for enhancing the topical antiinflammatory activity.     

Synthesis and pharmacological evaluation of new 16-methyl pregnane derivatives

The pharmacological activity of several new pregnane derivatives 15-19 were determined on gonadectomized male hamster flank organs, seminal vesicles and in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors. Steroids 15-19 decreased the diameter of the pigmented spot in the flank organs as compared to the T treated animals; in this model, steroids 16 and 19 showed a higher activity than the commercially available finasteride 3. Injection of T increased the weight of the seminal vesicles. Compounds 15-19 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. The trienone 19 exhibited a considerably higher activity than finasteride. Steroids 15-19 inhibited the in vitro metabolism of [3H]T to [3H]DHT in seminal vesicles homogenates of gonadectomized male hamsters. Compounds 18 and 19 showed a much higher antiandrogenic effect than finasteride. This enhancement of the biological activity could probably be attributed to the coplanarity of the steroidal skeleton as previously observed by our group. The high antiandrogenic activity of the epoxy compound 16 is probably the result of the ring opening of the oxiran ring with the nucleophilic part of the enzyme 5alpha-reductase thus leading to a stable adduct with concomitant deactivation of this enzyme.     

5 Alpha-reductase inhibitory and antiandrogenic activities of novel steroids in hamster seminal vesicles

The pharmacological activity of several 16-bromosubstituted trienediones 4 and 5, 16-methyl substituted dienediones 6 and 7 and the 16-methyl substituted trienedione 8 was determined on gonadectomized hamster seminal vesicles by measuring the in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors and also the ability of these steroids to bind to the androgen receptor. Steroids 6 and 7 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. Compounds 5 and 6 reduced substantially the conversion of T to DHT and therefore can be considered good inhibitors for the enzyme 5alpha-reductase; however both steroids failed to form a complex with the androgen receptor. On the other hand compound 7 which showed a very small inhibitory activity for the enzyme 5alpha-reductase, exhibited a very high affinity for the androgen receptor and thus can be considered an effective antiandrogen. This compound also reduced substantially the weight of the seminal vesicles. Steroids 4 and 8 did not reduce the weight of the seminal vesicles and exhibited a low affinity for the androgen receptor; 8 showed a weak 5alpha-reductase inhibitory activity, whereas 4 exhibited a weak androgenic effect.     

Evaluation of new pregnane derivatives as 5alpha-reductase inhibitor

The objective of this study was to synthesize several new pregnane derivatives and evaluate them as antiandrogens. From the commercially available 16-dehydropregnenolone acetate (7), two new steroidal compounds were synthesized: 17alpha-hydroxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dione (18) and 17alpha-acetoxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dione (19). The 5alpha-reductase inhibitory effect of the new compounds 18 and 19 together with the previously synthesized intermediates 7, 8, 13, 16, and 17 was determined in three different models: gonadectomized hamster flank organs diameter size, incorporation of [1,2-(14)C]sodium acetate into lipids in flank organs and conversion of [3H]testosterone (T) to [3H]dihydrotestosterone (DHT) by Penicillium crustosum. The evaluation of these steroids was carried out with three different controls: one group was treated with vehicle, the second with T and the third group with T plus finasteride. The pharmacological results from this work demonstrated that T significantly increases the diameter of the pigmented spot on the flank organs (p<0.05) as well as the incorporation of labeled sodium acetate into lipids in gonadectomized hamster flank organs (from 0.125 to 0.255 nmol per gland). In this study we also observed that broth of Penicillium crustosum converted [3H]T to [3H]DHT in a manner comparable to that of the flank organs. All experiments indicated that finasteride as well as steroids 7, 8, 13, 16-19 reduced significantly the conversion of T to DHT in P. crustosum. These compounds also decrease the size of the pigmented spot in the flank organs as well as reducing the incorporation of radiolabeled sodium acetate into lipids; T and the control sample (treated with vehicle only) were used for comparison. Apparently the presence of the 4,6-diene-3,20-dione moiety and also the C-17 ester group produce a higher inhibitory effect on the parameters used. PPThe data from this study indicated also that the three models used for the pharmacological evaluation exhibited comparable results.     

Antiandrogenic effect of 16-substituted, non-substituted and D-homopregnane derivatives

The pharmacological activities of 12 pregnane derivatives (4-15) were determined on gonadectomized male hamster flank organs and seminal vesicles as antiandrogens and as 5alpha-reductase inhibitors. The results from this study indicate that subcutaneous injection of testosterone for 3 d increased the diameter of the pigmented spot in the flank organs, whereas finasteride when injected with testosterone decreased the size of the spot significantly when steroids 4-15 were injected together with testosterone, the diameter of the flank organs of gonadectomized male hamsters, decreased significantly (p<0.005) compared to testosterone. Compound 11 was the most active steroid and reduced the diameter of the pigmented spot more than the other synthesized steroids or finasteride. Subcutaneous injections of testosterone to gonadectomized animals restore the seminal vesicle size lost upon castration. Injection of testosterone plus finasteride decreased significantly the weight of these glands (p<0.005). Steroids 5-15 when injected with testosterone decreased the weight of the seminal vesicles compared to testosterone. Finasteride is a good inhibitor of the conversion of testosterone to dihydrotestosterone (DHT) (low formation of DHT) measured as pmole of DHT/g of protein/h. Steroids 6-15 inhibited the conversion of testosterone to DHT as compared to testosterone however finasteride and 10 appeared to be the most effective compounds. Castration increases the protein content of the seminal vesicles (control) expressed as microg/mg of tissues. Testosterone tends to decrease it significantly, as did compounds 4, 5, 7, 9, and 15. We demonstrated that DHT as well as cyproterone acetate and steroids 5, 6, 8, 9, 11, and 14 at increasing non radioactive steroid concentration, inhibited the binding of [3H]DHT to cytosolic androgen receptor (AR), as indicated by its Ki values. However, 4, 7, 10, 12, and 13 did not have any inhibitory effect.     

Antiandrogen. I. 2-azapregnane and 2-oxapregnane steroids.

2-Azachlormadinone acetate (5a, 17-acetoxy-6-chloro-2-azapregna-4,6-diene- 3,20-dione), 2-oxachlormadinone acetate (6, 17-acetoxy-6-chloro-2-oxapregna-4,6-diene-3,20-dione) and the derivatives were prepared as potential antiandrogenic agents. Biological evaluation showed that 5a and 6 had a potent antiandrogenic activity when tested in the castrated male rat.     

Synthesis and pharmacological evaluation of new progesterone esters as 5alpha-reductase inhibitors

In this study we report the synthesis and pharmacological evaluation of four new progesterone derivatives; 17alpha-hydroxy-16beta-methylpregna-4,6-diene-3,20-dione 12, 17alpha-cyclopropylcarbonyloxy-16beta-methylpregna-4,6-diene-3,20-dione 13, 17alpha-cyclobutylcarbonyloxy-16beta-methylpregna-4,6-diene-3,20-dione 14, 17alpha-acetoxy-16beta-methylpregna-4,6-diene-3,20-dione 15 and the pregnatriene compound 17alpha-cyclobutylcarbonyloxy-16beta-methylpregna-1,4,6-triene-3,20-dione 16. The pharmacological effect of these compounds was determined in vivo as well as in vitro. The evaluation in vivo was carried out on gonadectomized male hamsters that were injected subcutaneously daily with testosterone (T) and/or finasteride, or with the novel compounds. At the end of the treatments the animals were sacrificed and the prostates were weighed. It was observed that when testosterone (T) and finasteride or compounds 12-16 were injected together, the weight of the prostate decreased significantly as compared to that of the testosterone-treated animals. The 5alpha-reductase inhibitory activity was evaluated in vitro using human prostate homogenates. These experiments showed the following IC50 values: compound 12 (alcohol at C-17) 1.2 x 10(-6) M, 13 (cyclopropyl substituent at C-17) 7.9 x 10(-10) M, 14 (cyclobutyl substituent) 3.2 x 10(-8) M, 15 (acetoxy substituent) 6.3 x 10(-11) M and 16 (cyclobutyl substituent) 3.9 x 10(-6) M. It is evident from these data that when the size of the substituent at C-17 is decreased, the 5alpha-reductase inhibitory activity increases. Apparently, in this biological model, the 5alpha-reductase inhibitory activity depends upon the steric effect of the substituent at C-17. However, the free alcohol 12 showed much lower 5alpha-reductase inhibitory activity.     

Steroids

Chemistry of Natural Compounds - The opening of the epoxide ring of the 3,20-diketal of 16α, 17α-epoxy-Δ5-pregnene-3, 20-dione and of the acetate of the 20-ketal of 16α,...     

Synthesis of deuterium-labeled 16 alpha,19-dihydroxy C19 steroids as internal standards for gas chromatography-mass spectrometry.

[2 beta,7,7,16 beta-2H4]16 alpha,19-Dihydroxyandrost-4-ene-3,17-dione (14) and [7,7,16 beta-2H3]3 beta,16 alpha,19-trihydroxyandrost-5-en-17-one (16), with high isotopic purity, respectively, were synthesized from unlabeled 3 beta-(tert-butyldimethylsiloxy)-androst-5-ene-17 beta-yl acetate (1). The deuterium introduction at C-7 was carried out by reductive deoxygenation of the 7-keto compound 3 with dichloroaluminum deuteride and that at C-2 beta and/or C-16 beta by controlled alkaline hydrolysis of 16-bromo-17-ketone 11 or 12 with NaOD in D2O and pyridine. [7,7-2H2]3 beta-Hydroxyandrost-5-en-17-one (6), obtained from compound 1 by a five-step sequence, was converted to compound 14 or 16 by an eight-step or seven-step sequence, respectively. The labeled steroids 14 and 16 are useful as internal standards for gas chromatography-mass spectrometry analysis of the endogenous levels.     

Simultaneous determination of 15 steroids in rat blood via gas chromatography-mass spectrometry to evaluate the impact of emasculation on adrenal

Adrenal was believed to affect the prostate tumor tissue growth by its secretion of adrenal androgens. However, the mechanisms regulating these effects were not fully understood. In this work, a sensitive and specific method for the determination of 15 steroids in blood via gas chromatography-mass spectrometry in selective ion storage detection mode was established to evaluate the impact of emasculation on adrenal steroids metabolism. Steroids were isolated by solid-phase extraction using Oasis HLB cartridge, and then derivated with heptafluorobutyric anhydride before analysis. The limits of detection were between 0.15 and 1.0 ng/mL and limits of quantification were between 0.62 and 2.6 ng/mL. The recoveries of steroids were above 83%, and both the intra-day and inter-day precisions (RSD%) were lower than 8%. Pregnenolone, progesterone, 17α-hydroxyprogesterone (17αP), 17α-hydroxypregnenolone (17αH), dehydroepiandrosterone (DHEA), estrone, 17β-estradiol, dihydrotestosterone (DHT), testosterone (T), 4-androstenedione (4-A), 1,4-androstadiene-3,17-dione, 11-deoxycortisol, 11-deoxycorticosterone, cortisol and aldosterone were quantified in 156 major male SD rats at 0, 1, 2, 4, 7 days, 2, 4, 6, 8, 10, 12, 14, and 16 weeks following emasculation. T and DHT decreased by 86.2% and 73.4%, respectively in the first 7 days following emasculation, but adrenal androgens (DHEA, 4-A) stabled at the normal level accordingly. Adrenal androgens and their precursors (17αH, 17αP) increased from the 2nd week along with the increase of androgens and the decrease of mineralocorticoids. These facts revealed that adrenal possibly enhanced its function of producing adrenal androgens from the 2nd week responding to the low androgens level induced by emasculation.     

3-Aryl- and 2,3-Diaryl-4-oxo-4,5,6,7-tetrahydroindazoles. 1. Reactions of Phenyl- and Tosylhydrazones of Dimedone and Cyclohexane-1,3-dione with Substituted Benzaldehydes

Sixteen 3-aryl-4-oxo-2-phenyl-4,5,6,7-tetrahydroindazoles were obtained from the reaction of the phenylhydrazones of dimedone and cyclohexane-1,3-dione with 3-bromo-, 4-bromo-, 4-chloro-, 4-fluoro-, 2-hydroxy-, 4-hydroxy-, 4-methoxy-, 2-nitro-, 3-nitro-, 4-nitro-, and 4-dimethylamino-benzaldehydes. The interaction of the tosylhydrazones of dimedone and cyclohexane-1,3-dione with the substituted benzaldehydes gave thirteen 3-aryl-4-oxo-4,5,6,7-tetrahydroindazoles. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 11, pp. 1662–1668, November 2005.     

Mass spectrometry in structural and stereochemical problems—CCXLIV : The influence of substituents and stereochemistry on the mass spectral fragmentation of progesterone

The complete high resolution mass spectra of progesterone (Δ⁴-pregnene-3,20-dione) and twenty-nine stereoisomers and alkyl substituted analogs have been analyzed with the aid of the recently developed computer program INTSUM. Progesterone analogs with “normal” configuration at the six chiral skeletal carbon atoms give rise to abundant ions corresponding to cleavage of the 1–2 and 3–4 bonds (ketene elimination), to cleavage of the 6–7 and 9–10 bonds (ring B cleavage), and to cleavage of the 13–17 and 15–16 bonds (partial ring D cleavage); these reactions are frequently followed by elimination of alkyl radicals. Alkyl groups at C-6 and C-10 exert a pronounced influence on the formation and fragmentation of the [M-ketene] ions. Reversal of configuration at C-10 increases the importance of ring B cleavage, whereas reversal at C-17 favors the partial cleavage of ring D. The fragmentation of 17-alkylprogesterones differs significantly from the general pattern, with acetyl loss (cleavage of the 17–20 bond) and partial ring D cleavage as the predominating reactions. Loss of ring D by cleavage of the 13–17 and 14–15 bonds is not an important reaction of progesterones. Direct interaction of the two ketonic functions was not observed.     

Synthesis of 4-Amino-substituted-6-hydroxy and 11-hydroxynaphtho[2,3-g]quinoline-5,12-diones,and the unexpected formation of disubstituted imidazo[4,5,l-i,j]naphtho[2,3-g]quinolin-7-ones

4-Chloroquinoline-5,8-dione ( 8a ) and 6-bromo-4-chloroquinoline-5,8-dione ( 8b ) were reacted with homophthalic anhydride to give tetracyclic compounds 10 and 11 respectively. The 6,11-dihydroxy derivative 12 was prepared in low yield by photochemical addition of benzocyclobutenedione to 4-chloroquinoline-5,8-dione ( 8a ) and in better yield through a Friedel-Crafts reaction of phthalic anhydride with 4-chloro-5,8-dimethoxyquinoline ( 7a ). Whereas 4-chloro-6-hydroxynaphtho[2,3-g]quinoline-5,12-dione ( 11 ) was substituted by amines in the usual way to the corresponding 4-amino-substituted derivatives, 4-chloro-11-hydroxynaph-tho[2,3-g]quinoline-5,12-dione ( 10 ) led to a mixture of 4-amino derivatives and the unexpected 2,6-disubstituted-imidazo[4,5,l-I-j]naphtho[2,3-g]quinolin-7-ones, 13a-b .     

Synthesis of 5-fluoro-2-methyl-3-(2-trifluoromethyl-1,3,4-thiadiazol-5-yl)-4(3H)-quinazolinone and related compounds with potential antiviral and anticancer activity

The synthesis of ten new substituted 1,3,4-thiadiazolyl-4(3H)-quinazolinones 8–11, 13, 17 , and 20–23 is reported. Compounds 8–11 were prepared by condensation of 5-fluoro-2-methyl-3,1-benzoxazin-4-one (3) and 5-substituted 2-amino-1,3,4-thiadiazoles 4–7. Compound 13 was obtained by condensation of 5-fluoro-2-methyl-3,1-benzoxazin-4-one (3) with DL-α-amino-?-caprolactam (12) . Compound 17 was synthesized by condensation of 6-bromo-2-methyl-3,1-benzoxazin-4-one (16) and 2-amino-5-t-butyl-1,3,4-thiadiazole (5) . Compounds 20–23 were obtained by condensation of 5-chloro-6,8-dibromo-2-methyl-3,1-benzoxazin-4-one (19) and 5-substituted 2-amino-1,3,4-thiadiazoles 4–7, respectively. The substituted 3,1-benzoxazin-4-ones 3, 16, and 19 were obtained in good yield by refluxing the appropriate anthranilic acid, 1,15 , and 18 with acetic anhydride (2) .     

Studies on topical antiinflammatory agents. III. Synthesis of 17 alpha-acyloxy-9 alpha-fluoro-11 beta-hydroxy-16 beta-methyl-1,4-pregnadiene-3,20-dione 21-thio derivatives and related compounds

A series of 21-thio derivatives of 9 alpha-fluoro-11 beta,17 alpha-dihydroxy-16 beta-methyl-1,4-pregnadiene-3, 20-dione 17-esters and related compounds were synthesized and evaluated as topical antiinflammatory agents. These compounds were prepared by the reaction of 9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-16 beta-methyl-1,4-pregnadiene-3, 20-dione (betamethasone, I) 17-ester derivatives and various mercapto compounds. A structure-activity relationship study revealed that the structural combination of a thio group at the 21-position and an ester group at the 17-position contributed to vasoconstrictive activity. Among these compounds, the 21-methylthio 17-propanoate compound (6) was found to have the most potent activity, being more potent than betamethasone 17-valerate (BV).     

Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes

A method for the analysis of neutral oxosteroids by electrospray mass spectrometry is described. The oxosteroids are converted into their oximes by treatment with hydroxyammonium chloride in aqueous methanol. Intense peaks corresponding to protonated oxime molecules are observed in nano-electrospray mass spectra. The detection limits for the oximes of progesterone, pregnenolone and dehydroepiandrosterone were 2.5, 5 and 25 pg/microL, respectively, approximately 20 times lower than for the underivatised steroids. The signal intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/microL. Fragmentation by collision-induced dissociation (CID) was studied using oximes of 28 model steroids carrying an oxo group at C-3, C-17 or C-20. Some of the steroid oximes were labelled with deuterium or (15)N. Fragment ions were observed which yielded useful structural information. Upon CID, protonated oximes of 3-oxo-Delta(4)-steroids produced abundant ions by cleavage through the B-ring and by loss of the side chain, while protonated oximes of saturated 3-oxosteroids did not give abundant ions by cleavage through the B-ring. Protonated oximes of 20-oxosteroids unsubstituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 containing the side chain, C-16 and C-17. Protonated oximes of steroids containing only a 17-oxo group gave fewer ions of diagnostic value. Coupled with the selective isolation of steroid oximes from a biological matrix this method of derivatisation and CID may be used for the analysis of neutral oxosteroids in biological samples.     

Molecular interactions of new pregnenedione derivatives

The in vitro inhibitory activity of five new progesterone derivatives: 17alpha-hydroxy-16beta-methylpregna-1,4,6-triene-3,20-dione 1; 16beta-methyl-17alpha-toluoyloxypregna-1,4,6-triene-3,20-dione 2; 17alpha-hydroxy-6-methylenepregn-4-ene-3,20-dione 3; 6-methylene-17alpha-toluoyloxypregn-4ene-3,20-dione 4 and 17alpha-(p-bromobenzoyloxy)-6-methylenepregn-4-ene-3,20-dione 5 was determined. These compounds were evaluated as 5alpha-reductase inhibitors as well as antagonists for the androgen receptor. Compounds 1, 2, 3, 4 and 5 showed the following inhibitory activity for the 5alpha-reductase enzyme with IC(50) values of: 1 (1.65 microM), 2 (10 microM), 3 (19 nM), 4 (100 nM) and 5 (100 nM). The results of this study also showed the effect of increasing concentrations of the novel steroids upon [(3)H]dihydrotestosterone binding to androgen receptors from male hamster prostate. The K(i) values for compounds 1, 2, 3, 4, 5 and dihydrotestosterone showed the following order of affinity for the androgen receptor: 4>5>dihydrotestosterone>2>3>1. The overall data indicated that all synthesized compounds 1, 2, 3, 4 and 5 are inhibitors of the 5alpha-reductase enzyme present in the hamster prostate. In addition compounds 1, 2, 3, 4 and 5 also presented an affinity for the androgen receptor.     

Photochemistry of desonide, a non-fluorinated steroidal anti-inflammatory drug

The photochemistry of anti-inflammatory drug desonide (De, 1) was studied in aerobic as well as in anaerobic condition with different irradiation wavelengths (254, 310 nm) in acetonitrile and 2-propanol. All photoproducts obtained were isolated and characterized on the basis of IR, (1)H-, (13)C-NMR spectroscopy and elemental analysis study. The products were: 11beta,21-dihydroxy-16alpha,17alpha-(1-methylethylidenedioxy)-1,5-cyclopregn-3-ene-2,20-dione 2 (254 nm), 11beta-hydroxy-16alpha,17alpha-(1-methylethylidenedioxy)androsta-1,4-diene-3-one 3 (310 nm/2-propanol), 17beta-hydroperoxy-11beta-hydroxy-16alpha,17alpha-(1-methylethylidenedioxy)androsta-1,4-diene-3-one 4 (310 nm/O(2)/2-propanol). Cyclohexadienone moiety in ring A and keto group at C(17) were found to be deeply modified by UV light therefore, loss of biological activity both during storage and in vivo can not be ruled out.