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. 1998 Jan 1;329(Pt 1):151–156. doi: 10.1042/bj3290151

Enzymic aromatization of 6-alkyl-substituted androgens, potent competitive and mechanism-based inhibitors of aromatase.

M Numazawa 1, A Yoshimura 1, M Oshibe 1
PMCID: PMC1219026  PMID: 9405288

Abstract

To gain insight into the relationships between the aromatase inhibitory activity of 6-alkyl-substituted androgens, potent competitive inhibitors, and their ability to serve as a substrate of aromatase, we studied the aromatization of a series of 6alpha- and 6beta-alkyl (methyl, ethyl, n-propyl, n-pentyl and n-heptyl)-substituted androst-4-ene-3,17-diones (ADs) and their androsta-1,4-diene-3,17-dione (ADD) derivatives with human placental aromatase, by gas chromatography-mass spectrometry. Among the inhibitors examined, ADD and its 6alpha-alkyl derivatives with alkyl functions less than three carbons long, together with 6beta-methyl ADD, are suicide substrates of aromatase. All of the steroids, except for 6beta-n-pentyl ADD and its n-heptyl analogue as well as 6beta-n-heptyl AD, were found to be converted into the corresponding 6-alkyl oestrogens. The 6-methyl steroids were aromatized most efficiently in each series, and the aromatization rate essentially decreased in proportion to the length of the 6-alkyl chains in each series, where the 6alpha-alkyl androgens were more efficient substrates than the corresponding 6beta isomers. The Vmax of 6alpha-methyl ADD was approx. 2.5-fold that of the natural substrate AD and approx. 3-fold that of the parent ADD. On the basis of this, along with the facts that the rates of a mechanism-based inactivation of aromatase by ADD and its 6alpha-methyl derivative are similar, it is implied that alignment of 6alpha-methyl ADD in the active site could favour the pathway leading to oestrogen over the inactivation pathway, compared with that of ADD. The relative apparent Km values for the androgens obtained in this study are different from the relative Ki values obtained previously, indicating that there is a difference between the ability to serve as an inhibitor and the ability to serve as a substrate in the 6-alkyl androgen series.

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Selected References

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  1. Akhtar M., Calder M. R., Corina D. L., Wright J. N. Mechanistic studies on C-19 demethylation in oestrogen biosynthesis. Biochem J. 1982 Mar 1;201(3):569–580. doi: 10.1042/bj2010569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banting L., Nicholls P. J., Shaw M. A., Smith H. J. Recent developments in aromatase inhibition as a potential treatment for oestrogen-dependent breast cancer. Prog Med Chem. 1989;26:253–298. doi: 10.1016/s0079-6468(08)70242-x. [DOI] [PubMed] [Google Scholar]
  3. Brodie A. M., Coombes R. C., Dowsett M. Aromatase inhibitors: basic and clinical studies. J Steroid Biochem. 1987;27(4-6):899–903. doi: 10.1016/0022-4731(87)90166-x. [DOI] [PubMed] [Google Scholar]
  4. Brodie A. M., Santen R. J. Aromatase and its inhibitors in breast cancer treatment--overview and perspective. Breast Cancer Res Treat. 1994;30(1):1–6. doi: 10.1007/BF00682736. [DOI] [PubMed] [Google Scholar]
  5. Brodie H. J., Kripalani K. J., Possanza G. Studies on the mechanism of estrogen biosynthesis. VI. The stereochemistry of hydrogen elimination at C-2 during aromatization. J Am Chem Soc. 1969 Feb 26;91(5):1241–1242. doi: 10.1021/ja01033a050. [DOI] [PubMed] [Google Scholar]
  6. Cole P. A., Robinson C. H. Mechanism and inhibition of cytochrome P-450 aromatase. J Med Chem. 1990 Nov;33(11):2933–2942. doi: 10.1021/jm00173a001. [DOI] [PubMed] [Google Scholar]
  7. Covey D. F., Hood W. F. A new hypothesis based on suicide substrate inhibitor studies for the mechanism of action of aromatase. Cancer Res. 1982 Aug;42(8 Suppl):3327s–3333s. [PubMed] [Google Scholar]
  8. Fishman J., Guzik H., Dixon D. Sterochemistry of estrogen biosynthesis. Biochemistry. 1969 Nov;8(11):4304–4309. doi: 10.1021/bi00839a013. [DOI] [PubMed] [Google Scholar]
  9. GUAL C., MORATO T., HAYANO M., GUT M., DORFMAN R. I. Biosynthesis of estrogens. Endocrinology. 1962 Dec;71:920–925. doi: 10.1210/endo-71-6-920. [DOI] [PubMed] [Google Scholar]
  10. Kellis J. T., Jr, Vickery L. E. Purification and characterization of human placental aromatase cytochrome P-450. J Biol Chem. 1987 Mar 25;262(9):4413–4420. [PubMed] [Google Scholar]
  11. Kitawaki J., Yoshida N., Osawa Y. An enzyme-linked immunosorbent assay for quantitation of aromatase cytochrome P-450. Endocrinology. 1989 Mar;124(3):1417–1423. doi: 10.1210/endo-124-3-1417. [DOI] [PubMed] [Google Scholar]
  12. MEYER A. S. Conversion of 19-hydroxy-delta 4-androstene-3,17-dione to estrone by endocrine tissue. Biochim Biophys Acta. 1955 Jul;17(3):441–442. doi: 10.1016/0006-3002(55)90395-4. [DOI] [PubMed] [Google Scholar]
  13. Meigs R. A., Ryan K. J. Enzymatic aromatization of steroids. I. Effects of oxygen and carbon monoxide on the intermediate steps of estrogen biosynthesis. J Biol Chem. 1971 Jan 10;246(1):83–87. [PubMed] [Google Scholar]
  14. Milewich L., Bradfield D. J., Coe L. D., Masters B. S., MacDonald P. C. Metabolism of 1,4-androstadiene-3,17-dione by human placental microsomes. Enzyme properties and kinetic parameters in the formation of estrogens and 17 beta-hydroxy-1,4-androstadien-3-one. J Steroid Biochem. 1981 Nov;14(11):1115–1125. doi: 10.1016/0022-4731(81)90041-8. [DOI] [PubMed] [Google Scholar]
  15. Numazawa M., Oshibe M. 6-Alkyl- and 6-arylandrost-4-ene-3,17-diones as aromatase inhibitors. Synthesis and structure-activity relationships. J Med Chem. 1994 Apr 29;37(9):1312–1319. doi: 10.1021/jm00035a011. [DOI] [PubMed] [Google Scholar]
  16. Numazawa M., Oshibe M. Further studies on 6-alkylandrost-4-ene-3,17-diones as aromatase inhibitors: elongation of the 6-alkyl chain. Steroids. 1995 Aug;60(8):506–511. doi: 10.1016/0039-128x(95)00058-x. [DOI] [PubMed] [Google Scholar]
  17. Numazawa M., Oshibe M., Yamaguchi S., Tachibana M. Time-dependent inactivation of aromatase by 6-alkylandrosta-1,4-diene-3,17-diones. Effects of length and configuration of 6-alkyl group. J Med Chem. 1996 Mar 1;39(5):1033–1038. doi: 10.1021/jm950720u. [DOI] [PubMed] [Google Scholar]
  18. Numazawa M., Satoh S., Ogura Y., Nagaoka M. Determination of estradiol 2- and 16 alpha-hydroxylase activities in rat liver microsomes using high-performance liquid chromatography. Anal Biochem. 1985 Sep;149(2):409–414. doi: 10.1016/0003-2697(85)90591-3. [DOI] [PubMed] [Google Scholar]
  19. Oh S. S., Robinson C. H. Mechanism of human placental aromatase: a new active site model. J Steroid Biochem Mol Biol. 1993 Mar;44(4-6):389–397. doi: 10.1016/0960-0760(93)90242-o. [DOI] [PubMed] [Google Scholar]
  20. Osawa Y., Yoshida N., Fronckowiak M., Kitawaki J. Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes. Steroids. 1987 Jul-Sep;50(1-3):11–28. doi: 10.1016/0039-128x(83)90058-2. [DOI] [PubMed] [Google Scholar]
  21. RYAN K. J. Biological aromatization of steroids. J Biol Chem. 1959 Feb;234(2):268–272. [PubMed] [Google Scholar]
  22. Siiteri P. K., Thompson E. A. Studies of human placental aromatase. J Steroid Biochem. 1975 Mar-Apr;6(3-4):317–322. doi: 10.1016/0022-4731(75)90149-1. [DOI] [PubMed] [Google Scholar]
  23. Spaeth D. G., Osawa Y. Estrogen biosynthesis. 3. Stereospecificity of aromatization by normal and diseased human ovaries. J Clin Endocrinol Metab. 1974 May;38(5):783–786. doi: 10.1210/jcem-38-5-783. [DOI] [PubMed] [Google Scholar]
  24. Thompson E. A., Jr, Siiteri P. K. The involvement of human placental microsomal cytochrome P-450 in aromatization. J Biol Chem. 1974 Sep 10;249(17):5373–5378. [PubMed] [Google Scholar]
  25. Thompson E. A., Jr, Siiteri P. K. Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J Biol Chem. 1974 Sep 10;249(17):5364–5372. [PubMed] [Google Scholar]
  26. Townsley J. D., Brodie H. J. Studies on the mechanism of estrogen biosynthesis. 3. The stereochemistry of aromatization of C19 and C18 steroids. Biochemistry. 1968 Jan;7(1):33–40. doi: 10.1021/bi00841a005. [DOI] [PubMed] [Google Scholar]
  27. Yoshida N., Osawa Y. Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization. Biochemistry. 1991 Mar 26;30(12):3003–3010. doi: 10.1021/bi00226a004. [DOI] [PubMed] [Google Scholar]

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