Chemistry 150, Fall 2008

The Pill: The Occurrence, Biological Activity, Biosynthesis, and Synthesis of Progesterone
Denise Mullery, Fall 2008
Introduction
Progesterone originates from the yam family, Dioscorea. Dioscorea produces large amounts of a steroid called diosgenin, which can be converted into progesterone. Progesterone plays a major role in pharmaceuticals by being a significant component of birth control pills to prevent women from becoming pregnant. There is evidence of biosynthesis of converting diosgenin into progesterone. As for synthesis of progesterone, there are many reactions that have been tested and produced variable yields, but only a couple of them will be discussed later on in this final paper. Along with the synthesis, the occurrence, biological activity, and biosynthesis of progesterone will also be talked about in this final paper.
O

(www.diplom-biologe.de/images/meiner.jpg)

Figure 2. Birth control pill. Source (http://www.canadiansforchoice.ca/images/contraception_img 03.gif) Another plant that has been discovered to indirectly contain progesterone is Dioscorea pseudojaponica that resides in Taiwan. Research has showed that the Taiwanese yam contained saponins, steroids, which are converted to diosgenin. From diosgenin, it can produce progesterone. 3 Another plant that is found to contain these steroids that convert to progesterone is a wild yam called Dioscorea villosa . One study showed that the Dioscorea villosa contains 3.5% of diosgenin.4 There are many other Dioscorea species of the yam family that contribute to the production of progesterone, but there is one new species found. Dioscorea polygonoides has been researched and found 2.64% of diosgenin that was recognized by testing it with gas chromatography-mass spectrometry. 5 Many of the Dioscorea species that originate from the yam family grow in countries that have tropical and subtropical climates. 6

H H O Progesterone H

Figure 1 . Dioscorea .

Progesterone,

the

steroidal

triterpenoid

of

I. Occurrence
Progesterone is mostly, but indirectly found in Dioscorea mexicana . Dioscorea mexicana is a plant that is part of the yam family and resides in Mexico.1 It contains a steroid called diosgenin that is taken from the plant and is converted into the valued product, progesterone. 2 On the other hand, diosgenin and progesterone are not only found in Dioscorea mexicana , but also in other Dioscorea species.

II. Biological Activity

Progesterone is a steroid hormone that plays a major part in female pregnancy. 7 Varying responses to transcription activity monitors it. Transcription activity is determined by two progesterone receptors, PR-A and PR-B. Both originate from the same gene and are somewhat the same, but for the most part vary on reactions to progesterone. 8 In one study, PR-A protein shows normal mammary gland response to progesterone but severe uterine hyperplasia and ovarian abnormalitiesPR-B protein does not affect biological responses of the ovary or uterus to progesterone but results in reduced pregnancy-associated mammary gland morphogenesis.7 Several studies have shown that antiprogestins prevent progesterone receptors from proceeding the pregnancy process.8 Both progesterone receptor proteins, PR-A and PR-B, have a discrete hormone-binding domain close to a blocked amino acid terminal region of the peptide chain.9 Since both proteins came from the same gene, they have similar codes for binding to hormones, but still vary in other parts of the gene.9 One of the differences between hPR-A and hPR-B is the carboxyl group at the end of hPR. Human progesterone

Figure 1. Dioscorea mexicana. Source

Chemistry 150, Fall 2008

receptor-B (hPR-B) starts the transcription of progesterone genes, while hPR-A blocks the transcription of progesterone receptors. An example of this was tested in a study where hPR-A mediated repression of human estrogen receptor (hER) transcriptional activity is not dependent on hER expression level but depends largely on expression level of hPR-A.10 hPR-A is able to stop transcription of hER because there is no competition to prevent hPR-A from performing its function. hPR has agonists and antagonists that are attached to each other by ligand-receptors. A study proved that a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists.11 Genes that react to the progesterone receptors are partial agonists. Overall, this shows that different ligands networking in different areas of different binding locations of PRs will vary in multiple biological outcomes. 11 One example of this was a study on rats being tested with gonadotropin (hCG). In the study, hCG stimulated the production of progesterone and this effect was blocked by tyrosine The essential functional groups necessary for progesterone production antagonism were they tyrosine amino and phenolhydroxyl groups, whereas the carboxyl group was seemingly essential for the antagonistic effect. 12 Another example of having varied processes, but a common result is the study of the level of affinity for progesterone receptors. In this study, 19-Nor-progesterone (19NP) is a potent progestagen, which possesses a high affinity for the progesterone receptor. In contrast, 17 -hydroxylated-progesterone (170HP) had no hormonal activity and does not compete for the progesterone receptor.13 One way to achieve a high affinity for 170HP is the attachment of a 17 -hydroxyl group on 19NPaddition of a Me group combined with the formation of a double-bond at C6 results in nomegestrol (NOM)and the protection of the free 17 -hydroxyl group of NOM by an acetate led to a potent progestin with high affinity. 13 One way to measure the affinity of progesterone receptors attaching to steroids is by using the Minimal Topological Difference (MTD) method. This method measures the hydrophobic level of progesterone to see if it has high chances of bonding to the steroid. 14 These attachments between PR and steroid are related to the human body by the uterine endometrium and myometrium uniting with the progesterone receptors. This was proven in a test where the gonane progestogen dl-norgestrel bound strongly to the uterine receptors, but only the d-isomer was active. All three pregnane progestogens, i.e. chlormadinone acetate, medroxyprogesterone acetate, and megestrol acetate also showed significant binding to receptors, but for estrane progestogens, only norethisterone had a high binding capacity. 15 Overall, in order to see if progesterone was effective in birth control pills, a study was commenced. In one study, 50 [women] took combined low-dose estrogen and progesterone pills for a year or more and the other 50 used different methods of birth control. 16 In the end, there were no negative effects of taking the progesterone pills for birth control. As a word of caution, if the progesterone receptors

are disrupted during the first 28 days of pregnancy, there will be negative consequences towards the ability to reproduce. 17

III. Biosynthesis
Progesterone can be biosynthetically produced from steroidal diosgenin, which originates from triterpenoids. Diosgenin undergoes acetylation to make 2 . It then oxides to produce 3 and performs ester hydrolysis to form dehydropregnenolone acetate 4 . Afterwards, hydrogenation turns 4 into pregnenolone acetate 5 . Then a hydroxyl group is added to replace the -OAc group to form pregnenolone 6 . Lastly, Oppenauer oxidation and tautomerism occurs to produce progesterone.18 This reaction is very similar to converting cholesterol converting into progesterone based on the fact that both reactions rely on oxidation to form progesterone. 19 A study has proven that pregenolone and progesterone can also be biosynthesized by yeast. 20
H H H HO H 1, diosgenin O O H O H H H H 2 O O AcO AcO O H CrO3

Ac2O heat AcO

H H AcO H

HOAc heat

H H H 3 O

AcO 4, dehydropregnenolone H2/Pd acetate O

H H AcO H

OHH HO

H H

Al(OPr)3 Me2CO O

5, pregnenolone acetate

6, pregnenolone

H H O H

Figure 3. Biosynthesis of progesterone18

progesterone

IV. Synthesis
The synthesis of progesterone begins with 7 reacting with PhLi in THF to produce 8. Then it mixes with the reagent of step 1 while the temperature increases from -70C to -30C and follows it up with reacting again with PhLi along with a surplus amount of MeOH at -30C to make 9 . Afterwards, 0.1 N HCl at an increased temperature of 40C is added into the reaction that leads to 10 . EtOH and 2% of NaOH then close the ring to form 11 yielding 40%. The next step is the reaction undergoes methylation and hydroxylation to develop 12 at a high yield of 99%. The reactant next transforms into chair conformation 13 after reacting in TFA at 0C. In the following step, it undergoes cation- cyclization to produce 14 . The next step produces 15 after yielding 72% by breaking off the cation. After that, the ring is opened and oxidized at -

Chemistry 150, Fall 2008

70C and then increases from -15C to 23C to produce 16 . Finally, it reacts with water and 5% of KOH with a ratio of 5:2, at 23C, to produce progesterone at a decent yield of 51%.21
Me Me O O Me PPh3+ITHF, PhLi (1 equiv. in Et2O) O O Me PPh3 1. O -70C to -30C

lower production amount of 78%.22

O Me Me H O 1, desoxycorticosterone H H O 2, progesterone 90% (CHCl3) 78% (CH3CN) OH Me2SiI, solvent Me H H H O Me Me

Figure 5. Semi-Synthesis of Progesterone22

O 7

O 8 Me Me

2. PhLi (1 equiv.); then excess MeOH, -30C Me Me O O Me

Conclusion
Progesterone had and still does play a major role in the pharmaceutical world of birth control. Millions of women use it to prevent pregnancy. Preventing pregnancy is not the only benefit of using birth control pills. It also reduces acne, protects against certain types of cancer related to the reproductive system, adjusts and keeps the menstrual cycle in order, etc. On the other hand, there are negative side effects such as weight gain, headaches, etc. In some countries, women can now buy the morning-after pill, or Plan B, over-the-counter. This has only been passed in India, Canada, Sweden, Norway, and the Netherlands. There has been some recent news of making a birth control pill for men, but it is currently undergoing clinical trials. The one factor is it cannot be taken as a pill, but by skin cream or injection. Overall, progesterone as an important component of the birth control pill is commonly used around the world and will continue to grow as it becomes more available to the public.

Me O EtOH/2% NaOH in H2O, reflux (40% from 1.) 0.1 N HCl in H2O/ MeOH (3:10), 40C O Me Me Me MeLi, Et2O (99%) Me O Me O H 14 O Me Me H Me 15 H H Me H O3, MeOH/CH2Cl2, 70C; then Zn, AcOH H2O, -15C to 23C (88%) O Me 10

O Me

O 9

Me O

O O

Cl(CH2)2Cl, Me OH 12 TFA, 0C Me O Me Me cation-! cyclization Me 13

11

Me H Me

K2CO3, H2O/MeOH (72% from 12)

O Me Me H O Me 16 H H H 2O 5% KOH (5:2), 23C (51%) O Me Me H O progesterone H H Me H Me H

References

________________
1 2 Applezweig, N. Steroids. Chem. Week 1969, 104, 57-72. Noguchi, E.; Fujiwara, Y.; Matsushita, S.; Ikeda, T.; Ono, M.; Nohara, T. Metabolism of Tomato Steroidal Glycosides in Humans. Chem. Pharm. Bull. 2006, 54, 1312-1314. Yang, D. J.; Lu, T. J.; Hwang, L. S. Isolation and Identification of Steroidal Saponins in Taiwanese Yam Cultivar (Dioscorea Pseudojaponica Yamamoto). J. Agric. Food. Chem. 2003, 51, 6438-6444. Hooker, E. Final Report of the Amended Safety Assessment of Dioscorea Villosa (Wild Yam) Root Extract. Int. J. Toxicol. 2004, 23, 49-54. Nino, J.; Jimenez, D. A.; Mosquera, O. M.; Correa, Y. M. Diosgenin Quantification by Hplc in a Dioscorea Polygonoides Tuber Collection from Columbian Flora. J. Braz. Chem. Soc. 2007, 18, 1073-1076. Myoda, T.; Nagai, T.; Nagashima, T. Properties of Starches in Yam (Dioscorea Spp.) Tuber. Curr. Top. Food. Sci. Technol. 2005, 105-114.

Figure 4. Synthesis of progesterone21 The next semi-synthesis of progesterone is much simpler than the previous one because it will only focus on the regioselectivity. In this semi-synthesis, there are two ways to produce progesterone, but each way ends with a different yield. For the first semi-synthesis, desoxycorticosterone reacts with Me3 SiI in CHCl3 to lose a hydroxyl group and produce progesterone. Using CHCl3 as a solvent, it gives the product at lower yield, but it produces progesterone at a greater amount of 90%. As for the second semi-synthesis, descoxycorticosterone reacts with Me3 SiI in MeCN to make progesterone as a result of losing a hydroxyl group. MeCN as a solvent produces progesterone at a higher yield, but at a

Chemistry 150, Fall 2008

Mulac-Jericevic, B.; Conneely Orla, M. Reproductive Tissue Selective Actions of Progesterone Receptors. Reproduction 2004, 128, 139-146. Gadkar-Sable, S.; Shah, C.; Rosario, G.; Sachdeva, G.; Puri, C. Progesterone Receptors: Various Forms and Functions in Reproductive Tissues. Front Biosci. 2005, 10, 2118-2130. Birnbaumer, M.; Weigel, N. L.; Grody, W. W.; Minghetti, P. P.; Schrader, W. T.; O'Malley, B. W. Progesterone Receptor: Structure and Function. Progesterone Progestins, [Int. Symp.] 1983, 19-32. Wen, D. X.; Xu, Y.-F.; Mais, D. E.; Goldman, M. E.; McDonnell, D. P. The a and B Isoforms of the Human Progesterone Receptor Operate through Distinct Signaling Pathways within Target Cells. Mol. Cell. Biol. 1994, 14, 8356-8364. Wagner, B. L.; Pollio, G.; Leonhardt, S.; Wani, M. C.; Lee, D. Y. W.; Imhof, M. O.; Edwards, D. P.; Cook, C. E.; McDonnell, D. P. 16-Substituted Analogs of the Antiprogestin Ru486 Induce a Unique Conformation in the Human Progesterone Receptor Resulting in Mixed Agonist Activity. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 87398744. Wang, X.; Zhang, Q.; Gao, E.; Cheng, Z. Effects of Tyrosine and Its Analogs on Hcg-Induced Progesterone Production by Rat Corpus Luteum in Vitro. Chin. Med. J. (Beijing, Engl. Ed.) 1987, 100, 859-864. Botella, J.; Duc, I.; Delansorne, R.; Paris, J.; Lahlou, B. Structure-Activity and Structure-Affinity Relationships of 19-nor-Progesterone Derivatives in Rat Uterus. J. Endocrinol. Invest. 1990, 13, 905-910. Bohl, M.; Simon, Z.; Vlad, A.; Kaufmann, G.; Ponsold, K. Mtd Calculations on Quantitative Structure-Activity Relationships of Steroids Binding to the Progesterone Receptor. Z. Naturforsch., C: Biosci. 1987, 42, 935-940. Briggs, M. H. Contraceptive Steroid Binding to the Human Uterine Progesterone-Receptor. Curr. Med. Res. Opin. 1975, 3, 95-98. Ishak, R.; Mohamed, A. B.; Hassan, K. Platelet Function Studies in Women on Oral Contraceptive Pills. Malays J Reprod Health 1990, 8, 1-4. Ordian, N. E.; Akulova, V. K.; Mironova, V. I.; Pivina, S. G.; Rakitskaia, V. V. Influence of Progesterone Receptors Deficit in Early Stage of Development on Formation of the Reproduction Functions of Female Rats. Ross Fiziol Zh Im I M Sechenova 2008, 94, 465-473. Dewick, P. Medicinal Natural Products: A Biosynthetic Approach. 2nd ed.; Wiley&Sons: West Sussex, England, 2001; p 266. Tialowska, B.; Klimek, J.; Zelewski, L. Progesterone Biosynthesis Supported by Fatty Acid Oxidation in the Mitochondrial Fraction of Human Term Placenta. Acta Biochim. Pol. 1983, 30, 11-21.

20

Duport, C.; Spagnoli, R.; Degryse, E.; Pompon, D. SelfSufficient Biosynthesis of Pregnenolone and Progesterone in Engineered Yeast. Nat. Biotechnol. 1998, 16, 186-189. Johnson, W. S. Synthesis of Progesterone J. Am. Chem. Soc. 1971, 4332. Numazawa, M.; Nagaoka, M.; Kunitama, Y. Regiospecific Deoxygenation of the Dihydroxyacetone Moiety at C-17 of Corticoid Steroids with Iodotrimethylsilane. Chem. Pharm. Bull. 1986, 34, 3722-3726.

21 22

10

11

12

13

14

15

16

17

18

19