Journal für Reproduktionsmedizin und Endokrinologie – Journal of Reproductive Medicine and Endocrinology –

Andrologie • Embryologie & Biologie • Endokrinologie • Ethik & Recht • Genetik Gynäkologie • Kontrazeption • Psychosomatik • Reproduktionsmedizin • Urologie

"The Rabbits are Prepared ..." - The Development of Ethinylestradiol and Ethinyltestosterone Frobenius W J. Reproduktionsmed. Endokrinol 2011; 8 (Sonderheft 1), 32-57

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Wir freuen uns auf Sie! Development of Ethinylestradiol and Ethinyltestosterone “The Rabbits are Prepared …” – The Development of Ethinylestradiol and Ethinyltestosterone

W. Frobenius

In an exciting scientific neck-and-neck race, European and American scientists in the late 1920s and early 1930s isolated the ovarian, placental, and testicular hormones. At the same time the constitution of the human sex steroids was elucidated. However, it soon emerged that with oral administration the therapeutic value of the natural substances was extremely limited. The first-pass effect in the estrogens, the sensitivity of progesterone to gastric acid, and the short plasma half-lives of natural ovarian hormones made treatment with them largely ineffective. The development by Hans Herloff Inhoffen and Walter Hohlweg of the orally effective sex steroids ethinylestradiol and ethinyltestosterone (ethisterone) in Berlin in 1937 can therefore be regarded as a milestone in the history of gynecological endocrinology. Ethinylestradiol is found even today as a highly effective estrogen component in almost all combined oral contraceptives. Ethinyltestosterone was the very first synthetic gestagen and can be regarded as the progenitor of the modern steroids in the 19-nortestosterone series. The present study describes details of the development of these two steroids and the history of their reception in the field of gynecology. In addition to the scientific literature, previously unexamined archival materials and German and American patent specifications were used in the study. The results show several surprising aspects, which are discussed in detail. J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1): 32–57. Key words: history, endocrinology, steroids, ethinylestradiol, ethisterone, contraception

 Introduction caments, over its history of more than This remarkably successful compound 50 years, if used at an appropriate dos- is nowadays linked with the names of Ethinylestradiol1 represents the most age, no serious undesired effects have Walter Hohlweg (1902–1992) and Hans effective and still most frequently thera- become known. This is true at least if Herloff lnhoffen (1906–1992)4b (Fig. 1, 2; peutically used derivative of the natural contraindications are observed and acted p. 40). estrogens. In contrast to the physiologi- upon. With ethinylestradiol, since the cal hormones, estrone, estradiol and es- end of the 30s, medicine has for the first From the literature we can see that the triol, it displays high endocrine activity time had available to it a follicular hor- work of these two chemists on the syn- even when taken orally. This property mone derivative, that by reason of its thesis of the substance and the nature of permits the use of ethinylestradiol in tab- biochemical properties makes possible its physiological action on laboratory let form at dosages in the microgram an inexpensive and uncomplicated estro- animals was carried out at the end of range and has made it the most widely gen treatment3. 1937 in Berlin, in the main laboratory of used estrogen component of oral contra- Schering AG5. ceptives2.

Although the substance as a component 3 Similarly promising was the non-steroidal diethylstilbestrol that was discovered in 1938 by a group of the contraceptive pill has been one of working under the English biochemist Charles Dodds (Dodds et al. 1938). See p. 47. Historically it is of the most thoroughly investigated medi- interest that Dodds obtained important hints for this development from the main laboratory of Schering AG in Berlin. See, e.g., Dodds and Lawson 1937, p. 1069 and Hohlweg 1953, p. 532. [Dodds EC, Goldberg L, Lawson W and Robinson R. Oestrogenic activity of certain synthetic compounds. Nature 1938; 141: 1 In the literature written in German, the English 247–8. Dodds EC, Lawson W. Oestrogenic activity of p-Hydroxy Propenyl Benzene (Anol). Nature 1937; way of spelling the names of substances is increas- 139: 1068–9. Hohlweg W. Die Hormone der Keimdrüsen. In: Seitz, Ludwig, Amreich (Hrsg.). Biologie ingly adopted, but this is much less often the case und Pathologie des Weibes, Bd. 1, 2. Aufl. Urban und Schwarzenberg, Berlin 1953; 525–639]. where the natural estrogens are concerned. In the 4 For biographical sketches see: Frobenius 1990, pp. 73–80. See in addition Rhode, Hinz 2010. [Frobenius present work – above all in the case of the presen- W. A triumph of scientific research. The development of ethinylestradiol and ethinyltestosterone: a tation of older investigations – the designation story of challenges overcome. The Parthenon Publishing Group, Carnforth, UK, and New Jersey, USA, “Äthinylöstradiol” is also used. The subject un- 1990. Rhode W, Hinz G. Endokrinologische Forschung an der Charité-Frauenklinik (II. Universitäts- der discussion is always the substance ethinylated Frauenklinik) 1908–1951, zugleich Keimzelle des 1951 gegründeten Instituts für Experimentelle in the 17α-position, formerly defined as 17α- Endokrinologie der Charité. In: David M, Ebert AD (Hrg.). Geschichte der Berliner Universitäts- ethinyl-1,3,5(10)-estratriene-3,17β-diol, but now Frauenkliniken. Berlin, New York, 2010; 131–62. α defined as 19-Nor-17 -pregna-1,3,5(10)-trien-20- 5 The company was founded by Ernst Schering (1824–1889) in North Berlin as a “Grüne Apotheke”. In yne-3,17-diol. 1855 he expanded the pharmacy laboratory into a works manufacturing chemical and pharmaceutical 2 See reviews on the composition of oral contra- products. In 1871 it was converted to the “Chemische Fabrik auf Actien (vorm. E. Schering)”. In 1927 ceptives (e.g. the “Rote Liste” as well as text- and it was amalgamated with the “Chemische Fabrik CAF Kahlbaum GmbH” and the name of the company hand-books of gynaecology). Mestranol, the 3- was changed to “Schering-Kahlbaum AG”. From 1937 the firm was called Schering AG. In 2006 the methylether of ethinylestradiol, that was likewise Schering AG was taken over by the Bayer AG, then named “Bayer Schering Pharma AG”. Since 2011 used as an estrogen component of oral contracep- the trade name Schering has been abandoned for conversion to “Bayer HealthCare Pharmaceuticals”. tives, first becomes active after demethylation. The Schering-Archive: www.wirtschaftsarchivportal.de/archiv/details/id/34. See in addition e.g, [Holländer behaviour of the 3-cyclopentyl ether, also known H. Geschichte der Schering Aktiengesellschaft. Herausgegeben von der Schering AG Berlin. Gedruckt as Quinestrol, is analogous. und verlegt bei Erich Blaschker, Berlin, 1955].

Reprint of: Frobenius W. A Triumph of Scientific Research. The development of ethinylestradiol and ethinyltestosterone: a story of challenges overcome. The Parthenon Publishing Group, Carnforth, UK, 1990. Reprint with permission from informa healthcare communications.

From the Clinic of Obstetrics and Gynecology, University Clinic Erlangen, Germany Correspondence: PD Dr. med. Wolfgang Frobenius, MME, Frauenklinik, Universitätsklinikum Erlangen, D-91054 Erlangen, Universitätsstraße 21–23; e-mail: [email protected]

32 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) For personal use only. Not to be reproduced without permission of Krause & Pachernegg GmbH. Development of Ethinylestradiol and Ethinyltestosterone

Appropriate publications in 1938 and presentations of the patents and publica- bances of the female hormone metabo- 1939 made ethinylestradiol known to the tions on the ethinylation of steroids, lism. The hormone preparations avail- professional world. However, years were which preceded the work, now consid- able for the treatment, however, left still to pass before this estrogen could be ered classic, of Hohlweg and Inhoffen. much to be desired, and for their isola- introduced into therapy. Thereafter reported on will be the publi- tion, enormous amounts of biological cations of Hohlweg and Inhoffen, as well raw material were necessary. This made Hitherto, historians have not pointed out as the significant clinical investigations production complicated and expensive. that examples of the preparation of the which led to the introduction into therapy Furthermore, it had become apparent estrogen derivative are to be found in of ethinylestradiol and ethinyltestoster- that for physiological reasons there were patents claimed by Schering AG in the one. Appropriate to its lasting practical strict limits to the oral use of estrogens10. years 1935 and 1936. In the correspond- importance, principal attention will then ing patent specifications indications of focus on the estrogen derivative. Progesterone could be administered the physiological action of the substance only by injection. were given6. The story of the discovery and reception of the two ethinyl steroids gives rise to With purification and elucidation of the Therefore the Schering chemists Arthur many questions. For example, how do constitution of the ovarian hormones, ef- Serini, Lothar Strassberger and Josef we account for the fact that processes for forts to achieve partial or total syntheses Kathol seemed to be credited with inven- the production of ethinylestradiol were of these substances had begun. At the tion of the process whereby ethinyl- to be found in patents dated as early as same time ways were sought to achieve estradiol was produced7. 1935 and 1936? Why did it take so long an increase in the hormonal activity by for the estrogen derivative to be intro- means of changes to the structure of the  The First Synthetic duced into therapy? A discussion of molecules of the estrogens. Through the these and other viewpoints occurs in the discovery that the physiologically sig- Gestagen closing chapter. nificant hormones of the supra-renal Closely coupled to their work on ethi- cortex belong to the steroids, research nylestradiol, Hohlweg and Inhoffen de- For the present study original papers were into the chemistry of this class of sub- veloped ethinyltestosterone8, the first viewed, so far as possible. This applies stances experienced enormous growth. synthetic preparation of a gestagen. Al- above all to chemical, physiological and Every effort to find simpler and cheaper though this hormone derivative was used clinical investigations directly linked to ways of synthesizing these substances in therapy for just a few years, consid- the ethinyl compounds. To discern the appeared justified. This – as will become ered historically its appearance has tre- major relationships, however, general re- apparent – would greatly benefit gynaeco- mendous importance. It is the precursor views had to be drawn upon, in which his- logical endocrinology. of a large group of artificial gestagens torical importance has not always been that can be delivered in tablet form, and taken into account to a desirable extent, Within the framework of the efforts to which are at present indispensable for Many of the events under consideration obtain more effective estrogenic sub- treatment with sexual hormones9. are, moreover, dealt with only in part in a stances, by the mid-1930s various estro- scientific fashion: even important ques- genic acids had been prepared, besides The present paper describes in detail the tions of priority still await detailed analy- the already mentioned benzoates of es- discovery and reception of these two sis. This circumstance means that many trone and of estradiol. The significance ethinyl compounds. Starting point are questions remain unanswered. of these compounds for the synthesis of ethinylestradiol and of ethinyltestoster- Access to archives also presented prob- one will be discussed later11. 6 See in addition pp. 34–7. lems. During the war years numerous 7 For biographical data see: p. 5, footnote (Serini) documents were irrecoverably lost. This The ethinylation of steroids, which was and p. 34, footnotes (Serini, Strassberger, Kathol). situation became painfully obvious dur- begun about 1935 and is the centre of 8 The substance was first called “pregneninolon” ing the attempt to obtain biographic in- interest in this discussion, initially by Hohlweg and Inhoffen because of its progest- erone-like action. lt was however – as became clear formation about the chemists working at served two objectives. On the one hand, later – not a compound of the pregnane series, be- Schering on research into the steroid the introduction of acetylene groups into cause the two carbon atoms of the ethinyl group hormones. certain steroids made possible the prepa- at C17 are in the α-position. In ignorance of this circumstance, one also spoke of anhydrohydroxy- ration of tertiary alcohols, from which it progesterone. Later the designation ethinyl- testosterone or ethisterone (17α-hydroxy-pregn-  Synthesis, Biological Test- 4-en-20-yn-3-one) was adopted. ing and Presentation of 10 9 The discussion here is of the so-called 19-nor- the Substance Here, only the estrogens occurring in women steroids, which are testosterone derivatives, and are considered. The so-called conjugated estro- which are demethylated at C10 (removal of C19) On the Ethinylation of Steroids gens, that are obtained from the urine of pregnant mares, behave differently. They contain, inter alia, and carry an ethinyl group in the C-17α-position, By the mid-1930s the female gonadal as in the compound already prepared by Hohlweg the equilins, that do not occur in women, and their and Inhoffen. The first of this group was devel- hormones had been purified and their metabolism is delayed. The first-pass effect thus oped by Djerassi and his colleagues at the begin- constitution elucidated. Joint efforts by does not play a great part. See in addition [Kuhl H, Taubert HD. Das Klimakterium. Pathophysio- ning of the 50s, with norethisterone. See in addi- biochemists, physiologists and medical tion [Fieser LF, Fieser M. Steroids. Reinhold Pub- logie, Klinik, Therapie. Thieme-Verlag, Stuttgart- lishing Corporation, New York, Chapmann and men had led to the establishment of a New York, 1987; p. 57 et 93]. Hall, London, 1959; 951]. rational therapy for numerous distur- 11 See in addition p. 37.

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 33 Development of Ethinylestradiol and Ethinyltestosterone was hoped increased physiological ac- The application was dated 10th Novem- greater than that of estradiol (1 RE = 3 γ); tivity would arise. On the other hand the ber, 1935 and the granting of the patent ethinylestradiol 1 RE= 0.50 γ (!)”18. ethinylated steroids would serve as start- was made known on the 3rd December, ing materials for syntheses that would go 1942. As examples of the process, be- The second patent, which is dated before further. sides the preparation of ethinylestradiol, the classic publications of Hohlweg and are cited four other syntheses with start- Inhoffen and in which ethinylestradiol is There are two sources of information as ing materials from the androstane se- described, was applied for on 22nd Sep- regards the early work on the ethinyla- ries16. tember, 1936. lt likewise was granted to tion of steroids: the scientific literature Schering AG and names as inventor and the patent specifications. The latter As the purpose of the process, the patent Josef Kathol19. are gaining in importance for historio- specification states very generally that graphy, because investigations that lead the reaction products achieved should Almost analogously to the patent speci- to patents are not in every case published “[…] find use as such or as intermediates fication by Serini and Strassberger, the anywhere else. Drawing upon patents to for the preparation of other physiologi- title is: “Process for the preparation of clarify questions of priority proved, cally valuable substances”. It goes on tertiary alcohols of the cyclopentano- however, problematic, at least for the pe- more specifically: “[…] e.g. substances polyhydrophenanthrene series”20. The riod before 1968. The reasons for this with the effectiveness of masculine sex granting of the patent was disclosed on will be discussed in more detail later12. hormones to be transformed into such 14th September, 1939. with the effectiveness of female sex hor- Here it must only be remembered that mones. In other cases […] the per oral Specially noteworthy in this patent the description of a substance – in this effectiveness compared to the starting specification is that it gives two ex- case, ethinylestradiol – in a patent in the material will be considerably in- amples for the conversion to ethinyl- 1930s does not inevitably mean that at creased”17. estradiol from estrone: one with a yield the date of the application it was actually of only 3% and the second with a yield already prepared. Worthy of protection The chemical details of the process de- of over 90%, In this case the synthesis at that time were processes only, but not scribed in the patent will be discussed with the high yield does not correspond the individual substances preparable here only insofar as they are of interest to to the in all eight preceding examples of thereby. Moreover then, as also today, the present work. Crucial is the fact that general process description. As will be during the years before the patent is the tertiary alcohol group striven for by apparent, it is much more a matter of a granted, examples of synthesis can be Serini and Strassberger was achieved, considerable modification. subsequently filed13. amongst other ways, by ethinylation of the keto group of steroids in position Only Low Yield The Early Patents C17. Thus the inventors went from es- In the first example, 1 g of “follicular The patent in which a preparation of trone to ethinylestradiol. This reaction hormone acetate” is dissolved in abso- ethinylestradiol from estrogen is de- appears in the patent specification also scribed for the first time, was filed in with the structural formulas. 1935. lt was granted to Schering AG in 18 DRP 730 050, p. 3. The abbreviation RE means Berlin. Named as inventors of the “Pro- The conversion of estrone requires parts “Ratteneinheit” (rat unit). This is the smallest cess or the preparation of tertiary car- of magnesium, phenyl bromide and amount of an estrogenically active substance that is sufficient to trigger in a castrated rat the cyto- binols of the cyclopentanopolyhydro- ether to be gently boiled for 30h whilst logical changes in the vaginal epithelium that are phenanthrene series” are Arthur Serini14 acetylene is continuously passed through. typical of “estrus”. In the case of the last weight and Lothar Strassberger15. Then estrogen dissolved in ether is datum, a printing error has obviously been made. lt ought to read: estradiol 1 RE 50γ. lf one as- added. After 3 days the conversion prod- sumes this to be correct, then the values deter- ucts formed are hydrolysed the ketone mined correspond exactly to those that lnhoffen 12 See in addition pp 53–4. that has not converted is removed and and Hohlweg have given in their classic paper of 13 See in addition commentaries on the Patent law, the end product is crystallised from 1938. See in addition p. 39. e g. Molter 1936. 19 Josef Kathol (b. 1899) after the “Abitur” at Easter 14 Arthur Serini (1897–1945). After his “Abitur” methanol. The yield from their process, 1918 enrolled at the Würzburg University to study at the “Königliches Gymnasium” in Bonn (1915) according to Serini and Strassberger, is mathematics. He was, however, immediately after- Serini served in the army until 1918. Then he be- 30%. ward called up for military service. After his dis- gan the study of chemistry in Freiburg that he com- charge in March 1919 he began to study chemis- pleted in Bonn in 1920. There he gained his doc- try in Freiburg/Breisgau, which he completed in torate with a paper “On the oxidation of pinacolin- In the patent, details are given of the Würzburg in 1923. There in 1924 he was awarded hydrazone with mercury oxide” (Serini 1922). In physiological importance of the ethinyl- his doctorate for a paper “On knowledge of the the 1930s, he was one of the most productive mem- tertiary butyl group” [Kathol J. Zur Kenntnis der bers of the staff of the Schering main laboratory. estradiol thus prepared: “When adminis- Tertiärbutylgruppe. Phil Diss Univ Würzburg He died in 1945, after the Russians entered Berlin. tered subcutaneously it displays an 1924]. After he joined Schering in the same year 15 Lothar Strassberger (b. 1902) gained his junior activity that is equal to that of estradiol Kathol first worked on terpene chemistry. Later he worked in the Schering main laboratory on high school diploma at the “Oberrealschule” in (1 RE = 0.1 γ) and in the case of oral ad- Würzburg in 1922. Then he studied chemistry at steroids. In 1936 he was transferred to manage the Würzburg University. In June 1927 he passed the ministration its activity is considerably hormone operation in Adlershof. After the final examination. In 1929 he was awarded his Adlershof works in East Germany had been ex- doctorate by the Philosophical Faculty of the Uni- propriated in 1951, he worked in the patent de- versity for a paper on the “Synthesis of syringin” partment where in 1959 be obtained a power of 16 [Strassberger L. Synthese des Syringins. Phil Diss DRP 730 050 attorney: Schering staff file. Univ Würzburg 1929]. 17 DRP 730 050, p. 2 20 DRP 681 869

34 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone lute ether and brought to conversion with The processes presented in the two pat- tees the best yield, as already mentioned, an excess of sodium amide. Then, it says ents, in the outlined form, have appar- in the case of Kathol there is no hint of in the patent specification, for several ently not been described in any scientific this. He describes this method merely as hours acetylene is led into the ethereal publication before 1937. Certainly, no the last of his eight examples. Also, the solution, until no more absorption takes references were found by looking very generally expressed patent claims place. The reaction product is then de- through journals24 of those years, or con- by Kathol appear to be little matched to composed with water and the mixture sulting later publications on the ethi- this special process – quite in contrast to obtained is extracted with ether. The nylation of steroids – with the exception the claims of Inhoffen and Hohlweg in starting material that is not converted of a footnote which will be discussed the American patent, which is com- and still contains keto groups, can be fully later, because it must be considered pletely devoted to it27. brought to separation with the aid of keto in connection with a claim for priority. reagents. The yield of remaining “ethi- In the literature, papers on the ethinyla- nyldihydrofollicular hormone acetate” The US Patent of Inhoffen and tion of steroids first appear in the second was about 30 mg21. Hohlweg half of 1937. Whereas in the presented In contrast, that process for the ethinyla- patents the introduction of the tertiary In the second example22, by contrast, tion of estrone with a yield of more than alcohol at C17 is the primary interest, potassium is dissolved in liquid ammo- 90% which is the highest yielding pro- now the concentration is on the addi- nia (cooled with dry ice and acetone) cess given in the patent by Kathol, ap- tional carbon atoms coupled there with and acetylene is passed until the blue pears in a literal translation into English the ethinyl group. In this way a possible colour disappears. Further, the patent in a US patent granted to Inhoffen and way of adding side chains appeared to says that a solution, or suspension, of 3 g Hohlweg in December, 1941. The patent open, which was to provide a good prac- of estrone in benzene and ether is then specification carries the title “Tertiary ticable partial synthesis of difficultly ac- slowly added to it. The freezing mixture alcohols of the estrone series and their cessible steroid hormones. is removed, the batch is allowed to stand derivatives and a process for their manu- 25 for 2 hours and further stirred continu- facture” .  Papers of the Ruzicka ously overnight. Then the reaction solu- tion is treated with ice and water, acidi- The application was submitted in Octo- Group fied with sulphuric acid to a Congo acid ber, 1938. For the patent the priority of a The first of these publications28 was by reaction and the solution extracted 5 German application on 25th October, the Swiss chemists Leopold Ruzicka29 times with ether. The combined ether 1937 is claimed26. and K. Hofmann. lt carried the title “On extracts are washed twice with water, the deposition of acetylene at the keto once with 5% soda solution and again A comparison of the US patent with the group in the C17 position in the case of with water, until the wash water is neu- cited patent specification by Kathol trans-androsterone and Δ5-trans-dehy- tral. Then the ether is evaporated, the shows significant differences in the gen- droandrosterone”30 and was submitted to residue dissolved in a little methanol and eral process description which precedes diluted with water. The separated prod- the examples. Whereas in the patent by uct can be recrystallised from aqueous Hohlweg and Inhoffen it is expressly 27 US Patent 2 265 976, p. 1 methanol. The yield amounted to 2.77 g. emphasised that the conversion of the 28 Of the two publications cited below, by the Swiss The melting point of the ethinylestradiol starting material with potassium acetyl- and Berlin scientists, the exact date of appearance could be determined only for the paper that ap- thus obtained was 142–144°C. ide dissolved in liquid ammonia guaran- peared in Naturwissenschaften: it was the 15th October, 1937. The investigation published in Of the properties of all prepared ethinyl Helvetica Chimica Acta, however, should likewise have appeared in October, probably in the first compounds it is very broadly said that 24 Looked through were: Die Naturwissenschaften, half. See in regard to this Inhoffen and Köster they are distinguished “either by a high Berichte der Deutschen chemischen Gesellschaft 1939, p. 595. It can be said with certainty, that the physiological activity, that in many cases and Helvetica Chimica Acta (in each case the vol- paper by the Swiss was received at Helvetica Chimica Acta on 3rd September, 1937. considerably exceeds that of the starting umes for 1934 to 1937). In addition Decennial Index to chemical Abstracts, Vols. 21–30 (1927– 29 Leopold Ruzicka (1887–1976) was from 1926 product, or they can serve as intermedi- 1936) published by the American chemical Soci- to 1929 Professor of Organic Chemistry in Utrecht ates for the manufacture of other physi- ety, Easton (Pa). In the large handbook by in the Netherlands. Then he went in the same ca- ologically valuable compounds“23. Bomskov (1939) it is not possible to find refer- pacity to the Federal Technical University in ence to the ethinylation of estrone [Bomskov C. Zürich (ETH). Of his researches, the most out- Methodik der Hormonforschung. Bd. II. Thieme- standing were the investigations of the sex hor- Details, such as are given in the patent by Verlag, Leipzig 1939]. mones. In 1939 Ruzicka and Butenandt shared the Serini and Strassberger for ethinylestra- 25 US Patent 2 265 976 Nobel prize for chemistry. Ruzicka worked closely 26 with the Gesellschaft für Chemische Industrie in diol, are completely absent. Neither is A patent specification originating from this ap- plication could not be found during the investiga- Basel (CIBA) [Tausk M. Organon. The story of reference made to their patent specifica- tion. See in regard to this the research report. an unusual pharmaceutical enterprise. Published tion. Scheringianum, Archive no. B1/285. Also there is by Akzo Pharma bv, Oss, The Netherlands, 1984; no information available about the application it- 91]. See in addition his autobiography [Ruzicka self, because all the patent files from those years, L. In the Borderland between Bio-organic Chem- according to information from the Patent Office istry and Biochemistry. Ann Rev Biochem 1973; 42: 1–20]. 21 DRP 681 869, p. 2 in Munich, have already been destroyed. This cir- cumstance makes it impossible to report on de- 30 Ruzicka L, Hofmann K. Über die Anlagerung 22 DRP 681 869, p. 3. In Kathols patent the ex- tails of the patent processes that naturally, par- von Acetylen an die 17-ständige Ketogruppe bei ample of the synthesis carries the number 8. ticularly in regard to ethinylestradiol, would be trans-Androsteron und 5Δ-trans-Dehydro- 23 DRP 681 869, p. 2 of the greatest historical interest. androsteron. HeIv Chim Acta 1937; 20: 1280–2.

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 35 Development of Ethinylestradiol and Ethinyltestosterone

Helvetica Chimica Acta on 3rd Septem- ammonia. Ruzicka and Hofmann ob- the patent, as in the cited publication in ber, 1937. The paper initially points out tained by this means a yield of around Helvetica Chimica Acta, there is how- the new intentions for the ethinylation of 80%. They report nothing about the bio- ever no reference to the biological prop- steroids that have been indicated above, logical activity of their new compounds. erties of the substance. Ruzicka and Hofmann state that the re- action given in the title has been carried The results of this work are obviously At almost the same time as the work of out in order to obtain starting materials part of a patent that was applied for the Swiss chemists there appeared in that should later be convertible into by the „Gesellschaft für Chemische Die Naturwissenschaften a publication physiologically important steroids with Industrie“ in Basel (CIBA) on 25th May, from the main laboratory of Schering in a side chain at C17. The authors on this 193832. In it are taken into consideration Berlin. It also reports on ethinylations occasion mentioned progesterone and several priorities from applications in under the heading “A transition from “the compounds of the supra-renal cor- Switzerland that go back to 26th June, the androstane series to the pregnane tex”. It further states that the starting 1937. The patent specification is entitled series”34. The authors were Kathol, materials for the preparation of such “Process for the preparation of acetylene Logemann35 and Serini. steroids have been hitherto “extremely derivatives of the cyclopentanopoly- difficult of access”. The dehydroepi- hydrophenanthrene series”. In its con- The Berlin chemists emphasized the androsterone now used, in contrast, tents it goes considerably beyond what is special physiological importance of the allows easy steroid preparation by a new contained in the publication by Ruzicka compounds in the pregnane series: method of sterol degradation31. and Hormann. In the introduction, ear- Butenandt and his colleagues had shown lier processes for the ethinylation of ste- that progesterone belongs to this series, The authors then discuss special prob- roids are discussed33. while Reichstein36 with his group had lems in the synthesis of the desired side proved the close relationship of the su- chain. The keto groups in the C17 posi- With reference to this it is stated that the pra-renal gland cortex hormone corti- tion of the steroids are distinguished by a yield had been only very small because a costerone to progesterone. The easy certain inactiveness. Many of the usual large part of the material resinified. Ref- accessibility of the dehydroepiandros- ketone condensations therefore do not erences are not made to the extremely terone had then initiated the search for occur or the reaction products obtained high yielding method described in the transitions from the androstane to the display an “abnormal behaviour”. Thus patent by Kathol as well as that by pregnane series. they are, for example, easily split. The Inhoffen and Hohlweg. acetylene addition, in contrast, can be Kathol, Logemann and Serini also re- carried out extremely smoothly. Besides No Statements about the Bio- ported that the attachment of the two small amounts of unaltered starting ma- logical Activity carbon atoms to C17 took place very terial, only the ethinyl products will be It is noteworthy that in the Swiss scien- smoothly with the aid of acetylene and obtained. tists’ patent the preparation of ethinyl- its derivatives. The triply unsaturated estradiol from estrone is described in the pregnane compounds obtained are With the help of acetylene addition, examples of the use of the process “naturally particularly suitable for fur- Ruzicka and Hofmann prepared Δ5- Ruzicka and Hofmann obtained ethinyl- ther conversions: hydrogen, oxygen, 17-ethinyl-androsten-3-trans, 17-diol as estradiol with a yield of about 80%. In hydroxyl groups and the like may be well as the corresponding androstane derivative. Starting substances were – as Δ5 stated in the title of the paper – the - 32 DRP 702 063. An inventor is not named in the patent specification. There is much to suggest that trans-dehydroandrosterone and trans- Ruzicka and Hofmann were at least considerably involved in the patent specification. A corresponding androsterone. The latter is easily acces- US patent (no. 2 272 131) that was applied for in July, 1938 and which referred to an application in sible, by catalytic hydrogenation, from Switzerland in July, 1937, carried the name of Ruzicka. 33 the Δ5-compound, the paper indicates. References to sources are absent. 34 Kathol J, Logemann W, Serini A. Ein Übergang aus der Androstan-Reihe in die Pregnan-Reihe. Die The two new diols are distinguished by Naturwissenschaften 1937; 25: 682. great stability. 35 Willy Bernhard Logemann (b. 1909) passed his “Abitur” in the “Humanistisches Gymnasium” in Oldenburg in 1928. Then he studied chemistry in Marburg. After the examination in 1934, he worked as The details of the syntheses are set out in “Liebig assistant” in the Chemical Institute of the University and received his doctorate in 1935 with a paper on “Investigations of the autoxidation of mercaptans. At the same time, a contribution to the the experimental section of the publica- chemical nature of papain” [Logemann W. Untersuchungen über die Autoxydation von Merkaptanen. tion. Of particular interest is that the Zugleich ein Beitrag zur chemischen Natur des Papains. Phil Diss Univ Marburg 1935]. Logemann, Swiss authors use a method of ethinyla- who at Schering worked successfully on steroids for many years, was the actual discoverer of the so- called “Serini reaction”, that played a part in the chemistry of the adrenal cortex steroids [Fieser LF, tion that in its essential points corre- Fieser M. Steroids. Reinhold Publishing Corporation, New York, Chapmann and Hall, London, 1959; p. sponds to that given by Inhoffen and 628]. It made possible the synthesis of desoxycorticosterone, that in 1939 was put on the market by Hohlweg and is also to be found in the Schering for the therapy of insufficiency of the adrenal cortex [Laurent H. 50 Jahre Steroidchemie bei der Schering AG (1923–1973). Manuskript, datiert vom 24.5.1973 (beim Verfasser)]. patent by Kathol. The conversion of the 36 Tadeus Reichstein (1897–1996) in 1929 became a docent and later Professor of Organic chemistry in starting material likewise takes place in a the Federal Technische Hochschule in Zürich (ETH). From 1931 he worked as a personal assistant of solution of potassium acetylide in liquid Ruzicka, in 1938 he went to Basel. In 1932 Reichstein discovered the synthesis of Vitamin C and in 1936 the adrenal cortex hormone, corticosterone. In 1950 he received, together with the American biochemist Kendall and the rheumatologist Hench, the Nobel prize for medicine. See in addition: [Tausk M. Organon. The story of an unusual pharmaceutical enterprise. Published by Akzo Pharma bv, Oss, 31 Ibid. Ruzicka and Hofmann 1937; p. 1280. The Netherlands, 1984; pp. 6768].

36 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone added up to partial or complete satura- The absence of a full experimental sec- scription of this method was first given tion“37. tion in the publication by Kathol and his in a Schering patent, applied for on 22nd colleagues gives rise to the question as to November, 1936 and published on 3rd Examples of ethinylation also given which process was used by them. Hints October, 1939. are the synthesis of Δ5-17-ethinyl- are given in the footnote commented androsten-3,17-diol from dehydroepi- upon above, which relates to a paper by In the early Schering patents (1935 and androsterone and the preparation of Inhoffen and Köster, in which was de- 1936) on acetylene addition on steroids, 17-ethinylepiandrostan-3, 17-diol from scribed, in 1939, a new preparative pro- the preparation of ethinylestradiol had epiandrosterone. Report of further con- cess for ethinyltestosterone41. This says, been described. In them were also given versions is promised at a later date by in connection with the publications of details of the physiological effect of this the authors. the Swiss and Berlin groups: “The au- substance. From the present point of thors of our working group are named in view this appears astonishing, because In the paper the biological properties the first place, because their publication in the literature the discovery of the sub- of the substances prepared are reported. of a patent application, Dtsch. Reichs- stance and its hormonal activity is al- The authors write, and this may be Pat. Anmeld. Sch. 111 452 IV c/120 of ways attributed to a paper by Inhoffen of particular physiological interest, 21.11.1936 is the basis, whereas Ruzicka and Hohlweg in 1938. The possible “that the ethinyl compounds have the and colleagues published their first find- causes for this contradiction (Fig. 4), character of a female rather than a male ings on the same subject in October, briefly referred to in the introduction, hormone”. For example, the unit of 1937”. The cited application referred to will be discussed more fully later42. the monoacetate of Δ5-17-ethinyl- the above patent presented by Kathol. androsten-3,17-diol in the Allen-Doisy How did the Synthesis of Ethi- test38 on the female rat was 0.2mg, All Started with the Schering nylestradiol and Ethinyltestos- whereas 1mg on the cockscomb was Patent terone Come About? still ineffective39. To sum up, it can be established that The investigations, discussed above, into work had already been done in 1935 on the ethinylation of steroids by the groups In contrast to Ruzicka and Hofmann, the ethinylation of steroids. The early in- of workers in Switzerland and in Berlin Kathol and his colleagues give no more vestigations only found expression in ended only a few weeks after their publi- extensive detail about the synthesis of patent specifications. They referred to cation in the preparation of ethinyl- the substances. They simply say that the the preparation of tertiary alcohols from estradiol and ethinyltestosterone. Both attachment of the two carbon atoms compounds containing keto groups of syntheses and the discovery of the bio- takes place “by the use of the Nef reac- the cyclopentanoperhydrophenanthrene logical activity of the substances are now tion”40, whereupon they refer to a large series. lt was hoped, by these alterations – as mentioned – linked with the names survey, but give no indication of the rel- of steroids, to arrive at easily accessible of the chemists Walter Hohlweg and evant pages. Data about the yields that substances that act as hormones. Hans Herloff Inhoffen. We shall now were achieved in the ethinylations car- describe the circumstances that led to the ried out are likewise absent. No refer- Reports on ethinyl steroid compounds work of Hohlweg and Inhoffen, the re- ence is to be found in the paper to the appeared in the scientific literature for sults of which were first reported in the patents by Schering described in some the first time in October, 1937. Corre- scientific literature in 1938. detail above. sponding publications, by the Swiss chemists Ruzicka and Hofmann and by In 1937, both Hohlweg and Inhoffen the Schering chemists Kathol, Logemann were working in the Berlin main labora- 37 Kathol J, Logemann W, Serini A. Ein Übergang and Serini, appeared almost simulta- tory of Schering AG. One of the tasks of aus der Androstan-Reihe in die Pregnan-Reihe. Die neously. In these the centre of interest the then 35-year old Austrian, Hohlweg, Naturwissenschaften 1937; 25: 682. was the side chain added with the aide of was the biological testing of all the hor- 38 The “Allen-Doisy-Test” determines the number acetylene on C17: the endeavour was to mone derivatives developed by the com- of weight units of an estrogenic substance that is necessary to produce “estrus” in castrated rats. The achieve a partial synthesis of progester- pany. In the course of his almost 10 years “estrus” is manifested by the appearance of spe- one and the hormones of the supra-renal of work for Schering, Hohlweg’s main cific cornified epithelial cell forms in the vagina. cortex. interest had shifted from pure chemistry In contrast to other bioassays, such as the “Cor- ner-Allen test”, the “Allen-Doisy-test” retains its to the physiology of the endocrine sys- eponym even in its manifold variations. Various processes were used for the tem. Inhoffen was 31 years old and had 39 The cockscomb test was used as a semiquanti- ethinylation. Initially, the yield was worked in the main laboratory for just 1 tative bioassay for the testing of substances for meagre. Very good results were first year: in contrast, he worked exclusively androgenic activity. It goes back to a paper by the American McGee. The criterion is the growth of achieved when the condensation was in his original field. the comb of a castrated animal that can be brought completed in liquid ammonia by using about by a certain amount of a hormonally active the potassium salt of acetylene. The de- There exist various descriptions by both substance. Numerous modifications of the method were used. See in addition: [Bomskov C. Methodik scientists of the particular circumstances der Hormonforschung. Bd. II. Thieme-Verlag, that at the end of 1937 first led to the Leipzig, 1939; 445–76]. 41 [Inhoffen HH, Köster H. Untersuchungen in der preparation of ethinylestradiol. Essen- 40 John Ulric Nef (1862–1915) [Nef JU. Ueber das Sexualhormonreihe, IV. Mitteil.: Ein neues Dar- Phenylacetylen, seine Salze und seine Halogen- stellungsverfahren für Pregneninolon. Berichte der substitutionsprodukte. Liebig’s Annalen der Deutschen Chemischen Gesellschaft 1939; 72: Chemie 1899; 307–308: 264–328]. 595–6 (footnote 3)] 42 See in addition pages 52–5.

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 37 Development of Ethinylestradiol and Ethinyltestosterone tially, their accounts complement each To the great surprise of all participants, main laboratory at the beginning of the other. In a letter43 Hohlweg says, in con- physiological testing of the ethinyl- 1940s titled “Progress in the chemistry nection with the search for estrogen de- estradiol showed the enormous estro- of the cyclopentanophenanthrene de- rivatives that would be very effective genic activity of this substance. lnhoffen rivatives”, no particular credit was given. when administered orally, that he had reported that about 2 weeks after the first The comment merely was that “[…] it proposed to the manager of the main preparation of the estrone derivative, [the ethinyltestosterone] did not show laboratory at that time, Walter Hohlweg “burst in” with this news46. as one could have expected the action of Schoeller44, that a 17-carboxylic estra- Special importance was attributed to the the masculine hormone, but instead, as diol be manufactured. Schoeller was im- circumstance that ethinylestradiol dis- Hohlweg and Inhoffen found, surpris- mediately enthusiastic about this idea. played its power even when orally ad- ingly gave that of the corpus luteum hor- He complied with Hohlweg’s request ministered. mone […]”49. that a steroid chemist be made available to him for this work: Hohlweg’s immedi- After this success, both scientists imme- Inhoffen remembered, however, that ate choice was Inhoffen. The two men diately agreed to modify the masculine Schoeller had recognized “the character had previously collaborated successfully gonad hormone, testosterone, in analo- of the molecule as a pregnane deriva- in the synthesis of equilenin acid. gous fashion. They hoped in this way to tive” and had recommended the test for obtain an orally effective androgen. The progesterone activity. The request of “You Can Have the Acid” preparation of ethinyltestosterone pre- the patentees that he also sign the corre- Inhoffen commented in a lecture45 on sented no difficulties. However, in ani- sponding patent, “he modestly declined”. how Hohlweg approached him in this mal trials, the expected effects of the This was characteristic of Schoeller’s case: “One morning my colleague preparation were largely absent. “We “generosity in giving suggestions”, that Walter Hohlweg […] came to me and were […] very disappointed that this he, Inhoffen, had witnessed and had said: ‘Make a derivative of the follicular substance had scarcely any androgenic himself experienced50. Schoeller himself hormone for me, the 17-carboxylic acid; effect and thus we had developed no has referred to his participation in the I believe it would be effective, adminis- orally effective testosterone prepara- discovery only as an example of „what a tered orally.’ I gazed into the air for a few tion”, wrote Hohlweg at the time47. laboratory manager can achieve by a seconds and then said: ‘You can have the well-timed suggestion”51. acid in 2 weeks. I add acetylene to the The Great Surprise estrone and then ozonise’.” Further biological experiments with “The Rabbits are Prepared” ethinyltestosterone led, a little later, to Hohlweg confirmed that Schoeller had Inhoffen thus wanted to use the addition another very surprising result, namely spoken with him about the possible pro- of acetylene to the 17-keto group in or- that the new substance displayed consid- gestagenic activity of the ethinyltestos- der, in 2 steps, to get the estrogen car- erable progesterone activity. In contrast terone and had suggested appropriate boxylic acid desired by Hohlweg. He to the highly purified progesterone then tests. The suggestion arose when the further said that the planned ethinylation used in therapy, the new substance dis- close chemical relationship of the sub- of estrone had been carried out in 2 days. played its effect not only when injected stance to progesterone was in mind. At Of the ethinylestradiol thus prepared, he but also when administered orally. that time Hohlweg had himself started handed over to Hohlweg 50 mg for bio- Hohlweg and Inhoffen had thus in their the appropriate trials: “I [Hohlweg] logical testing. The intended ozonising search for an active androgenic substance could tell him [Schoeller] that the rabbits of the substance had, according to discovered the first semi-synthetic pro- were already being prepared”52. Inhoffen, to be put off because of exter- gestagen! Because of its physiological nal circumstances. action, the substance was initially named The preparation and biological testing of pregneninolone. the two ethinyl compounds were re- ported in several publications in 1938 lt is not quite clear, from whom the deci- and 1939. The details will be discussed 43 Hohlweg 1967 to Dr. Raspé, Schering AG. sive impulse came to test the ethinyl- later. The precise moment at which Scheringianum. Archive no. B1/266. testosterone for progestagenic action. Hohlweg and Inhoffen for the first time 44 Walter Schoeller (1880–1965), after his “Abitur” Schoeller wrote in a letter in 1955 that it held ethinylestradiol in their hands, can in 1899, studied chemistry in Berlin. He prepared had been him, “[…] that gave Hohlweg be established: it was 6th September, his dissertation under the famous chemist and pro- fessor Emil Fischer, who habilitated him in 1915. and Inhoffen the good advice, to test 1937. In 1919 Schoeller was given the title of Professor. their ethinyltestosterone for corpus lu- In 1923 he joined Schering, where later as man- teum activity, after they had told me, ager of the main laboratory he decisively influ- with great sadness, that the hoped-for enced for decades the development of hormone 49 48 From the manuscript neither the occasion nor the research. On the importance of this scientist, see masculine effect had failed to appear” . exact date of the lecture is apparent. The content [Inhoffen HH. Walter Schoeller zum 80. Geburts- In a lecture given by the manager of the however shows that it was given at the end of the tag. Chemiker-Ztg–Chem Apparatur 1960; 84: 1930s or the beginning of the 40s. Scheringianum, 709–11]. Archive no. B1/285. 45 “On the problems of the unpredictability of the 50 [Inhoffen HH. Walter Schoeller zum 80. paths from fundamental research to applied chem- 46 Inhoffen 1983 (lecture). Geburtstag. Chemiker-Ztg–Chem Apparatur 1960; istry.” This is an account of a lecture that Inhoffen 47 84: 711]. gave in 1983 at several universities. The manuscript Hohlweg 1967 (letter). 51 was kindly entrusted to the author: Scheringianum, 48 Schoeller 1955 in a letter to Dr. Schmidt, Schoeller 1055 (letter). Archive no. B1/267. Schering AG. Scheringianum, Archive no. B1/267. 52 Hohlweg 1967 (letter).

38 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone

Starting point for the discovery of the work was dedicated for his 60th birthday, Inhoffen and Hohlweg in the paper also two orally effective ethinyl steroids was spoke in this connection of “horrible ef- report the results of trials on rats: the thus the proposal by Hohlweg that 17- forts”57. Both substances proved also to new compound had proved effective at a carboxylic estradiol should be made. For be “nearly inactive” as estrogens58. dosage of 3 mg administered orally to the historian this gives rise to the ques- castrated animals. An accompanying tion, why the Austrian scientist hoped to In closing, it remains to be said that pre- table showed that this performance, in get from this substance, what he, to- paration of ethinyltestosterone, shortly part, considerably surpasses the physi- gether with Inhoffen, discovered in ethi- after Hohlweg and lnhoffen had achieved ologically occurring follicular hor- nylestradiol. it, was also reported by Ruzicka’s mones. In addition, the test on a cas- group59. The Swiss scientists had, how- trated female baboon had shown very The answer to this is to be found in a ever, to recognise the priority of the good activity of the new substance when publication by Schering scientists (one Schering researchers. In contrast to the orally administered. of whom was Hohlweg) that appeared in first publication by Hohlweg and 197153. Inhoffen, the paper by Ruzicka and his In the publication it is pointed out that colleagues contains a detailed account of clinical trials of the new estrogen deriva- In this it states that the preparation of the the method of preparation. tive have already begun. A decision 17-carboxylic estradiol was undertaken about its therapeutic use was to be ex- at that time because of the already The Papers by Hohlweg and pected from the results. The two scien- known, good estrogenic activities of the Inhoffen tists give no further details. estrogen acids. As examples, the authors Die Naturwissenschaften, 1938 cite doisynol acid and equilin acid54. The first publication on the synthesis of Secondly, the authors report on the Ethinylestradiol, however, pharmaco- ethinylestradiol was in the journal Die analogously carried out synthesis of a logically surpassed all the synthetic es- Naturwissenschaften60. This was a brief testosterone derivative that they initially trogens known at the time, “[…] so that initial communication, one page in called pregnen-in-on-3-ol-17 (Fig. 4). the estrogen acids and attempts to syn- length, under the heading “New orally This molecule, that had been prepared in thesize similar compounds were uninter- effective female sex hormone deriva- view of the “interesting biological prop- esting”55. tives: 17-ethinylestradiol and pregnen- erties of ethinylestradiol”62 had yielded in-on-3-ol-17”. The authors were Hans very surprising results in the biological Horrible Efforts Herloff lnhoffen and Walter Hohlweg testing: “Tested for corpus luteum activ- In the cited work, it was a matter of and the manuscript was dated 31st Janu- ity, the substance proved active when an investigation in which the project that ary, 1938. 2 mg were tested on a baby rabbit, pre- Hohlweg and Inhoffen had tackled in treated with follicular hormone. Thus, 1937, namely the preparation of the 17- Without going into details of the synthe- when administered subcutaneously the 1 carboxylic estradiol in a slightly modi- sis, the two Berlin scientists reported substance had about /3 of the activity of fied form, had been brought to a success- briefly on the preparation of ethinyl- progesterone. The test, using 4 mg ad- ful completion. Decisive for this may well estradiol from estrone. The synthesis of ministered orally, gave a positive result! have been a suggestion by Hohlweg56. the substance was the result of efforts to […] Progesterone itself, tested in the The 17α- and the 17β-estradiol acid make, from estrone, follicular hormone same way, i.e. using 60 mg administered were each made as its 3-methylether. In derivatives with good activity when ad- orally, is still completely inactive”63. the process it appeared that, contrary ministered orally. The observation had to expectation, this synthesis was ex- certainly been made in the past that, Inhoffen and Hohlweg remark at this tremely difficult. lnhoffen, to whom the “there are connections between the point, in a single sentence, that the new chemical constitution and the strength of compound has shown no activity of the the activity when orally administered”61. sort associated with a male sex hormone. 53 [Prezewowsky K, Wiechert R, Hohlweg W. Synthese von racem-3-Methoxy-17β-hydroxy- 1.3.5 (10)-östratrien-17α-carbonsäure und 3- In closing, the publication says that if the Methoxy-17α-hydroxy-1.3.5. (10)-östratrien-17β- new progestin proves successful in clini- carbonsäure. Liebigs Ann Chem 1971; 752: 68– 77]. cal trials, it will be of great therapeutic 57 Inhoffen 1983 (lecture). 54 See in addition also [Hohlweg W, Inhoffen HH. importance, because a hormone hitherto 58 Equileninsäure, ein oral hochwirksames Östrogen. [Prezewowsky K, Wiechert R, Hohlweg W. only effective when injected could, in β Dtsch med Wchschr 1947; 72: 86–7]. Synthese von racem-3-Methoxy-17 -hydroxy- 1.3.5 (10)-östratrien-17α-carbonsäure und 3- the future, be administered orally. The 55 [Prezewowsky K, Wiechert R, Hohlweg W. Methoxy-17α-hydroxy-1.3.5. (10)-östratrien-17β- authors announce that more comprehen- Synthese von racem-3-Methoxy-17β-hydroxy- α carbonsäure. Liebigs Ann Chem 1971; 752: 68– 1.3.5 (10)-östratrien-17 -carbonsäure und 3- 77]. Methoxy-17α-hydroxy-1.3.5. (10)-östratrien-17β- 59 carbonsäure. Liebigs Ann Chem 1971; 752: 68– [Ruzicka L, Hofmann K, Meldahl F. Bereitung Δ 77]. des 17-Äthinyltestosterons und des 5-17-Vinyl- 62 [Inhoffen HH, Hohlweg W. Neue per os wirk- 3-trans, 17-dioxyandrostens. Helv Chim Acta 56 same weibliche Keimdrüsenhormon-Derivate: 17- Hohlweg 1967 (letter). He wrote: “Was an at- 1938; 21: 371–4]. tempt ever made to make the estradiol acid that I Aethinyloestradiol und Pregnen-in-on-3-ol-17. 60 wanted? As my new laboratories in Graz will soon [Inhoffen HH, Hohlweg W. Neue per os wirksa- Die Naturwissenschaften 1938; 26: 96]. be available and I have a capable chemist I would me weibliche Keimdrüsenhormon-Derivate: 17- 63 [Inhoffen HH, Logemann W, Hohlweg W, Serini attempt the synthesis, if it has not already been Aethinyloestradiol und Pregnen-in-on-3-ol-17. A. Untersuchungen in der Sexualhormon-Reihe. done elsewhere”. Scheringianum. Archive no. B Die Naturwissenschaften 1938; 26: 96]. Berichte der Deutschen Chemischen Gesellschaft 1/266. 61 Ibid, p. 96. 1938; 71: 1024–32].

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 39 Development of Ethinylestradiol and Ethinyltestosterone

Figure 1. Hans Herloff Inhoffen. Source: Schering Archiv, Bayer AG.

Figure 2. Walter Hohlweg. Source: Schering Archiv, Figure 4. Inhoffen and Hohlweg’s classic publication in “Naturwissenschaften” 1938. The ethisterone was then Bayer AG. still called pregnen-in-on-3-ol-17.

Figure 5. Advertisement for oral ethinltestosterone from just after World War II. Source: Schering Archiv, Bayer AG.

Figure 6. Commercial packag- ing of ethinylestradiol (Progynon C). It arrived first on the Ger- Figure 3. Walter Hohlweg (right side with chalk) in front of the formula for ethinyl- man market in 1949. Source: testosterone (ethisterone). Source: Schering Archiv, Bayer AG. Schering Archiv, Bayer AG.

40 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone sive publications about the compounds after saponification with alkali it yielded a derivative of pregnane, that in addition will appear shortly. the starting material with unaltered has the action of progesterone, and not properties. This finding underlined the that of testosterone69. Berichte der Deutschen Chemischen uniformity of the ethinyl compound. Gesellschaft, 1938 On the preparation of pregneninolone, it The first of these more detailed publica- Inhoffen and his co-authors then once is stated one can start from ethinyl- tions is found in the Berichte der again discuss the physiological activity androstendiol, which is obtained by ad- Deutschen Chemischen Gesellschaft. of ethinylestradiol, that had previously dition of acetylene on dehydroandroster- The manuscript was received by the edi- been shown in the first publication by one. The oxidation of the secondary al- torial staff of the journal on 14th April, means of experiments with rats. Whereas cohol group on C3 to a keto group fi- 193864. The publication was based on a then only comparisons of the subcutane- nally leads to pregneninolone. The melt- lecture that had been given by Inhoffen ous and oral effectiveness of ethinyl- ing point of the compound is given as on 26th February, 1938 at the convention estradiol with those of the physiological 264–266°C. of the North-west German Chemistry gonadal hormones had been used, now Docents in Göttingen65. additional substances are employed: it is With regard to the progestin action of a matter of the likewise ethinylated de- pregneninolone, the authors speak as in The title of the paper was very general: rivatives of equilin and equilenin67. their first publication of the “very sur- “Investigations of the sex hormone se- prising results” of the biological test. ries”. Besides Inhoffen, the co-authors Supporting the fact of high oral effec- Why the testosterone derivative for cor- named were, in order, Willy Logemann, tiveness of ethinylestradiol (emphasized pus luteum action had been investigated Walter Hohlweg and Arthur Serini. Ini- in the first paper), the authors go on to at all is not stated. On the topic of activ- tially, reference was made to the two pre- question the basis for this effect. ity, lnhoffen and his coauthors again cite viously published papers on steroid ethi- the published results of trials on baby nylation. The first of these, which had On this they say one has to assume, “[…] rabbits pretreated with follicular hor- originated at Schering, had a list of au- that the marked gastric and intestinal re- mone. In addition they say, “the ethinyl thors beginning with the steroid chemist sorption that is apparent in the case of group in this compound, with a constitu- Josef Kathol, followed by Logemann and ethinyl compounds must be attributable tion very close to that of progesterone, Serini. The other paper was by the Swiss to the presence of the ethinyl group”. To thus leaves the subcutaneous activity es- researchers, Ruzicka and Hofmann. examine this assumption, the triple bond sentially unchanged and, what is more, has been changed, by hydrogenation, to causes the appearance of an oral activity The technique of adding acetylene on a double bond. Biological tests on rats that the natural hormone lacks“70. the 17th carbon atom of steroids from the by oral administration has then shown a androstane series, as discussed in the decrease in activity of the substance (17- Remarkably Effective two cited papers, is pointed out as having ethenylestradiol) to values that again In the case of pregneninolone also, the been applied to estrone by Inhoffen and correspond to those of estradiol. “The special relation between the ethinyl Hohlweg. By this means, the expected marked oral effectiveness of ethinyl- group and the physiological effect has ethinylestradiol had been obtained. The estradiol is thus in causal connection been demonstrated. As in the case of melting point of the compound was 145– with the ethinyl group”68. Administered ethinylestradiol, the appropriate ethinyl 146°C. subcutaneously, ethenylestradiol has group had been obtained by partial hy- proved somewhat more active than even drogenation of ethinyl androstendiol and The authors point out that the addition of the ethinylated compound. subsequent oxidation of the oxy group acetylene can lead to two isomers, the on C3. The effective dose of pregnadien- cis- and the trans-forms. However, the Inhoffen and colleagues then deal with ol-ons is 7.5 mg if administered subcuta- formation of only one of these isomers is ethinyltestosterone, made in analogous neously, and 15mg if orally. The activity very obviously strongly favoured, “since fashion to the estrone derivative, that in has thus decreased by about 1/4 when the product obtained at a 90% yield is the paper in Die Naturwissenschaften applied in both ways. The effectivity of undoubtedly uniform”66. To characterise they had called pregnen-in-on-3-ol-17. the ethinyl derivative when administered the substance the monobenzoate, which They now call this compound pregnen- orally, compares with progesterone strik- crystallizes well, had been prepared, but in-ol-on. This name is justified in a foot- ingly; it is notably greater. note by the statement that it is a matter of In the paper it is additionally pointed out

64 that the progestin effect of pregneni- [Inhoffen HH, Logemann W, Hohlweg W, Serini 67 These substances, whose preparation is com- A. Untersuchungen in der Sexualhormon-Reihe. prehensively described in the cited paper, will not nolone disappears completely if one re- Berichte der Deutschen Chemischen Gesellschaft be discussed in detail here. They remain unim- 1938; 71: 1024–32]. portant. 65 69 [Inhoffen HH, Hohlweg W. Neue per os wirk- 68 [Inhoffen HH, Logemann W, Hohlweg W, Serini It was – however – as became clear later – not a same weibliche Keimdrüsenhormon-Derivate: 17- A. Untersuchungen in der Sexualhormon-Reihe. compound of the pregnane series, because the two Aethinyloestradiol und Pregnen-in-on-3-ol-17. Berichte der Deutschen Chemischen Gesellschaft carbon atoms of the ethinyl group at C 17 are in α Die Naturwissenschaften 1938; 26: 96]. 1938; 71: 1025]. The assumption that the ethinyl the -position. 66 [Inhoffen HH, Logemann W, Hohlweg W, Serini group improves the resorption of the hormone in 70 [Inhoffen HH, Logemann W, Hohlweg W, Serini A. Untersuchungen in der Sexualhormon-Reihe. the gastric and intestinal tract, later proved to be A. Untersuchungen in der Sexualhormon-Reihe. Berichte der Deutschen Chemischen Gesellschaft false. The high oral activity is much more attrib- Berichte der Deutschen Chemischen Gesellschaft 1938; 71: 1024–32]. utable to a delay of metabolisation in the liver. 1938; 71: 1027.

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 41 Development of Ethinylestradiol and Ethinyltestosterone moves the tertiary hydroxyl group on estrogens that differ only in one oxygen solved in alcohol orally administered to C17. In a footnote71 the authors state and two hydrogen atoms73. baby rabbits pretreated with estrogens. that, according to a recently published paper, Ruzicka and colleagues had like- Moreover, Hohlweg and Inhoffen recall The test was carried out for 5 days. This wise prepared pregneninolone and con- the preparation of estradiol from es- entailed the preparation of two groups of firmed the already publicised results. trone, that was successfully carried out three rabbits for each preparation, and by Schwenk and Hildebrandt in 1933. In two different doses of progesterone or Inhoffen and his colleagues then de- this case the addition of two hydrogen pregneninolone were administered with scribe in detail the experiments that led atoms led to a substance whose activity, a pharyngeal probe. to the synthesis of all the substances even when used on people, was far supe- mentioned in the publication. The clini- rior to that of the starting material74. On the 6th day, histological examinations cal testing of ethinylestradiol, which was were carried out of the endometrium of mentioned in the previous publication is The variations mentioned in the molecu- the animals, to assess progestin activity. no longer mentioned. Whether or not lar structure of estrogens had, however, The results showed that progesterone something of the same kind is intended led only to a strengthening or weakening administered orally in total doses of 30 for pregneninolone remains obscure. of the specific action. Schoeller and his and 60 mg remained completely inac- colleagues were the first to succeed in tive, whereas in the uteri of the animals Klinische Wochenschrift, 1939 transforming completely the biological treated with 5 and 10 mg of pregneninol- The next publication on the ethinyl activity of the active component of a hor- one, a progestin influence could be de- compounds of the female gonadal hor- mone by chemical means: by complete tected. Further: “The precise testing of mones from the main laboratory of hydrogenation of estrone they had ob- pregneninolone by oral administration Schering is found in January, 1939 in tained substances without estrogenic but showed that 4mg represents the effective the Klinische Wochenschrift. The au- with androgenic activity (hexahydro- limiting dose […]”76. thors on this occasion are Hohlweg and estradiol). In trans-dehydroandrosterone Inhoffen only. The paper is entitled a substance with estrogenic and andro- Finally, the authors report on trials of „Pregneninolone, a new corpus luteum genic activity had at last been prepared75. progesterone and pregneninolone ad- hormone preparation that is effective ministered to rabbits by injection. Here, when administered orally“72. Hohlweg and Inhoffen then report anew with either substance, a 10 mg dose was how it came about that pregneninolone effective. Hohlweg and Inhoffen first deal very was prepared, that according to its for- comprehensively in the introductory re- mula could also be described as ethinyl- It is noticeable in the presented paper marks with the circumstance that there testosterone. They then point out again that ethinylestradiol is practically no are only small differences between the that corpus luteum hormone prepara- longer mentioned, although in the chemical structures of the various go- tions hitherto could be administered meantime a paper by Clauberg has ap- nadal hormones. All these compounds only by injection, because when admin- peared, in which clinical experiences have the same polycyclic ring structure; istered orally they are inactive. Preg- with both ethinyl compounds are re- the differences are only in degree of neninolone was thus the first orally ef- ported77. The Berlin scientists mention saturation, in the kind and location of the fective corpus luteum hormone prepara- the publication by Clauberg in only one oxygen atoms and in the structure of the tion. sentence, saying that he had „[…] been side chain. Modifications can therefore able to show the corpus luteum hor- bring about great changes not only in the Comparison with Progesterone mone action of pregneninolone in hu- quantity but also the quality of the physi- The paper goes on to give further details mans when orally administered […]“78. ological activity. of the way in which the pregneninolone Clauberg’s investigations will be re- was tested for its progestin action. In or- ported comprehensively in connection The authors refer in this connection to der to have a direct and reliable compari- with the history of the reception of the history of the discovery of estrone son with physiological progesterone, the ethinylestradiol. and estriol, where the small dissimilari- same process was employed, which ties in the constitution and the great dif- tested crystallized progesterone dis- Ethinylestradiol had Hardly a Role ferences in the physiological activity, af- Ethinylestradiol has scarcely played ter the isolation of the active estrogens any part in later publications by from the urine of pregnant women, led to 73 See in addition [Simmer HH. Zur Entdeckung considerable confusion on the part of the des Östriols. Endokrinologie-Informationen 1980; 76 [Hohlweg W, Inhoffen HH. Pregneninolon, ein investigators. It took some time before it 6: 252–62]. neues per os wirksames Corpus luteum-Hormon- was clear that the urine of pregnant 74 [Schwenk E, Hildebrandt F. Reduktion des präparat. Klin Wochschr 1939; 18: 77–9.] women contained two different active Follikelhormons. Die Naturwissenschaften 1933; 77 [Clauberg C, Ustün Z. Menstruation – per os 21: 177]. See in addition [Neumann F. History of erzeugt. Beweise der Wirksamkeit von Progynon the Endocrinological Research of Schering AG. C, einem neuen Follikelhormonderivat, und Pro- In: Kracht J, von zur Mühlen A, Scriba PC (eds). luton C, einem neuen Luteohormonpräparat, bei Endocrinology Guide. Verlag der Brühlschen peroraler Verabreichung. Zbl Gynäkol 1938; 62: 71 Ibid. Universitätsdruckerei Gießen 1976]. 1745–61.] 72 [Hohlweg W, Inhoffen HH. Pregneninolon, ein 75 [Schoeller W, Schwenk E, Hildebrandt F. Neue 78 [Hohlweg W, Inhoffen HH. Pregneninolon, ein neues per os wirksames Corpus luteum-Hormon- Hydrierungsprodukte des Follikelhormons. Die neues per os wirksames Corpus luteum-Hormon- präparat. Klin Wochschr 1939; 18: 77–9]. Naturwissenschaften 1933; 21: 286]. präparat. Klin Wochschr 1939; 18: 78.]

42 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone

Hohlweg and Inhoffen. In a survey by of the new substances had already be- Buschbeck’s Investigations Hohlweg of the oral activity of natural gun82. In the article published in Die In addition, Herbert Buschbeck, docent and synthetic estrogens, which ap- Naturwissenschaften it is true that fur- at the Universitäts-Frauenklinik Würz- peared in 1950 in the Wiener Klinische ther details about the place and scope of burg, reported on the first clinical expe- Wochenschrift79, the substance is men- the investigations are absent; in the later riences with ethinylestradiol and ethi- tioned only in passing. Of the synthetic papers there are no references at all to nyltestosterone85. He did so during a lec- estrogens, the author favours the use of clinical trials, either in progress or com- ture given to the Physical-Medical Asso- dienestrol in the form of the diacetate, pleted. ciation in Würzburg on 24th November, which is a stilbene derivative. Hohlweg 1938. This lecture was reported in referred to the results of clinical tests On looking through the literature83, only the Klinische Wochenschrift in 1939. in the Universitäts-Frauenklinik der one comprehensive publication (in Buschbeck’s own paper on this has not Charité, Berlin, where he was at the 1938) is available on the action on the been found. time head of the laboratory. sex organs of women of orally used ethinylestradiol and pregneninolone. In the report, which is principally con- In detail the 1950 survey about ethinyl- This paper deals with investigations by cerned with the orally effective estrogen estradiol says: “Unfortunately, in the Clauberg84, who at the time was senior stilbestrol86, it is noted that 90 mg of course of comprehensive clinical tests, consultant at the Universitäts-Frauen- ethinylestradiol, administered orally, “is this substance was not well tolerated. klinik at Königsberg in Prussia. Together sufficient to build up completely the In the case of 20–30% of the women with Ziya Üstün, a Turkish doctor at the mucous membrane”. Prevention of lac- treated, nausea attacks occurred, similar hospital, Clauberg published the results tation was achieved with 24 mg, distrib- to those which occur after treatment with relating to eight patients who had been uted over 4 days. When used for the stilbestrol. The preparation was there- treated with the new substance for sec- treatment of dysmenorrhoea, amenor- fore not initially introduced into therapy. ondary amenorrhoea, uterus hypoplasia rhoea was observed for 2–3 months. In During and after the war, however, it has and glandular-cystic hyperplasia of the summing up it was stated: “The great been clinically tested abroad and be- endometrium. Clauberg’s paper will be activity when orally administered is very cause of its high activity it is used thera- comprehensively presented below. gratifying. The risks if inexpertly used peutically, although its therapeutic range are great. Schering AG are therefore for is relatively small”80. the present not putting the preparation on the market“87. What these risks are is Only in his large contribution on not stated, although in connection with 82 [Inhoffen HH, Hohlweg W. Neue per os wirk- “The hormones of the gonads” for the same weibliche Keimdrüsenhormon-Derivate: 17- stilbestrol, the poor tolerance of it is ex- second edition of the handbook by Aethinyloestradiol und Pregnen-in-on-3-ol-17. pressly referred to. Seitz-Amreich titled Biology and Die Naturwissenschaften 1938; 26: 96.] Pathology of the Woman that appeared 83 In the search for papers on ethinylestradiol and Of pregneninolone, the reporter writes ethinyltestosterone the 1938 to 1956 volumes of in 1953, does Hohlweg state that the Index Medicus and the “Berichte über Fort- that this is the first “corpus luteum hor- ethinylestradiol had acquired “great schritte in der Geburtshilfe und Gynäkologie” mone” that is effective when orally ad- therapeutic importance”81. He does not were looked through. In addition the author has ministered. With 250–400 mg of the considered all the literature references available refer here to the previously mentioned in Scheringianum. For newer papers on the sub- substance a secretory phase of the endo- secondary effects, but emphasizes only ject it was reverted to literature references in rel- metrium had been brought about. The that when administered orally to hu- evant handbooks. preparation was harmless, and its use in mans, the substance is 10 times as 84 Clauberg and Üstün, 1938. Carl Clauberg cases of spastic dysmenorrhoea, emesis (1898–1957): The scientist ranked at that time as active as intramuscularly injected estra- one of the authorities in gynaecological endo- and hyperemesis gravidarum, as well as diol benzoate. crinology. After graduation in medicine (1925) he habitual abortions, was conceivable. In- obtained his training in gynaecology and obstet- vestigations of it must be continued. rics from Robert Schröder in Kiel. In 1933  On the Reception of the Clauberg became “Privatdozent” at the university in Königsberg, where he worked until 1940 as Treatment Trials at the Charité Ethinylsteroids supplementary professor and consultant physician. Obviously at that time treatment trials After 1940, Clauberg was a chief medical officer First Clinical Tests in Germany outside the university sector. Because of criminal with ethinylestradiol were also run at the As already mentioned, Inhoffen and experiments on people such as the compulsory Universitäts Frauenklinik at the Charité Hohlweg reported in 1938 in their first sterilization of women in the Auschwitz concen- in Berlin. A corresponding reference is tration camp, Clauberg who belonged to several publication on the preparation of the Nazi organizations, was in 1956 excluded from ethinyl compounds, that the clinical tests the German Society for Gynaecology and obstet- rics (DGGG). In 1957 there was a provisional ban against the practice of medicine by him, in the 85 [Buschbeck H. Über neue Stoffe mit hormonaler same year he died whilst in detention awaiting Wirkung. Referat vor der Physikalisch-Medizini- 79 [Hohlweg W. Über die orale Wirksamkeit natür- trial. See e. g. Grosch 1985. 77 [Clauberg C, Ustün schen Gesellschaft Würzburg am 15. Dezember licher und synthetischer Östrogene. Wien KIin Z. Menstruation – per os erzeugt. Beweise der 1938. Klin Woschr 1939; 25: 899.] Wirksamkeit von Progynon C, einem neuen Wochschr 1950; 62: 313–6.] 86 Follikelhormonderivat, und Proluton C, einem Details of the discovery and importance of stil- 80 Literature is not cited here. neuen Luteohormonpräparat, bei peroraler bestrol, see pp. 46–7. 81 [Hohlweg W. Die Hormone der Keimdrüsen. In: Verabreichung. Zbl Gynäkol 1938; 62: 1745–61, 87 [Buschbeck H. Über neue Stoffe mit hormonaler Seitz L, Amreich A (Hrsg). Biologie und Patho- Grosch H. Carl Clauberg, ein biographischer Wirkung. Referat vor der Physikalisch-Medizini- logie des Weibes, Bd. 1, 2. Aufl., Urban und Hinweis. Endokrinologie-Informationen 1985; 9: schen Gesellschaft Würzburg am 15. Dezember Schwarzenberg, Berlin, 1953, 531.] 103–8.] 1938. Klin Woschr 1939; 25: 899.]

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 43 Development of Ethinylestradiol and Ethinyltestosterone to be found in a paper by Hahn88, pub- With the aid of hysterosalpingography Of particular interest for the purposes of lished in 1951, to which we will return and histology of the endometrium, the present paper are the data relating to later. According to the data given by this Clauberg gave proof of the effectiveness three women who suffered from second- author, who at the time of the publication of the orally administered preparations. ary amenorrhoea. The first patient re- was an assistant physician at the hospi- Using X-ray presentation of the size of ceived up to 4 mg Progynon C daily for tal, treatment with the preparation was the uterine cavity both before and after 19 days, then for 5 days she was given a not introduced into therapy because of completion of the hormone treatment, the combination of the estrogen with preg- considerable sideeffects. The investiga- growth of the womb could be established. neninolone (daily 2 mg Progynon C and tions mentioned have apparently not led On the endometrium the known histo- 20 mg Proluton C92. The total dose of to a publication. Hohlweg later indicated logical criteria concerning the effect of ethinylestradiol was 65 mg. An endo- that it was Kaufmann who tested ethinyl- the ovarian hormones were used. metrium biopsy after 19 days showed estradiol at the Charité89. proliferated mucous membrane, the hys- As mentioned previously, the eight terosalpingography uterine growth. lt Until long after World War II, therefore, patients were women who came for was 5 days after completion of the treat- the paper by Clauberg remained in therapy because of secondary amenor- ment that bleeding started. Biopsy in the German-speaking countries the sole rhoea, primary sterility, or menstrual dis- luteal phase was omitted. comprehensive publication concerned turbances because of glandular-cystic hy- with the action of ethinylestradiol on perplasia of the endometrium. For the The second patient was treated with women’s organs. For subsequent investi- purposes of his investigation Clauberg 3 mg EE per day for 10 days, then again gators – primarily scientists in the USA – formed three groups. In the first therapy for 10 days with 5 mg EE per day and it presented an important source on the was aimed at uterine growth, in the thereafter for 11 days with, in all, a fur- clinical use of the substance and conse- second at proliferation as well as transfor- ther 30 mg EE and 300 mg Proluton C93. quently was mentioned in almost all lists mation of a dormant endometrium and in The total amount of estrogen given for of references. In the case of pregneni- the third group at the conversion of a hy- the 31 days of the treatment is noted in nolone, the situation was – as will be perplastic mucous membrane into secre- the paper as 110 mg. An endometrium shown – somewhat different. tory transformation. With this division, biopsy at the conclusion of therapy three patients were found in both the first showed secretorial conversion of the The Paper by Clauberg and the second group90. Thus in the mucous membrane, and the hysterosalp- Through the investigations by Clauberg framework of the same investigation were ingography showed considerable growth and Üstün the information should be assessed the growth of the womb and the of the uterus. Once more, bleeding given, that an effective oral hormone change of the uterine mucous membrane. started 5 days after medication ceased. therapy for women had become possible with the arrival of ethinylestradiol and Of Special Interest: Secondary The third patient initially received 1mg pregneninolone. In the introduction to Amenorrhoea Progynon C per day over 10 days, and the paper it is noted that, of even the The women were treated for very differ- then 2 mg of estrogen daily for the next strongest of the hitherto usual follicular ent periods (from 25 days to barely 10 days. The investigators then gave her hormone preparations, when orally ap- 3 months) with markedly differing doses 2 mg EE daily for a further 12 days, plied, at most a fifth of the amount of of ethinylestradiol (EE) and pregneni- combined with, in all, 100 mg Proluton hormone introduced reaches the target nolone. The considerations, by which C (daily dose not stated). After 20 days organs to act upon them. In the case of Clauberg and Üstün allowed themselves of such therapy with estrogens, growth the corpus luteum hormone, oral appli- to be guided, cannot be inferred from the of the uterus was again ascertained, and cation had not been considered hitherto, paper. Also, the principle remains un- after the 12th day of the combined treat- because, as it was stated, the substance is mentioned according to which the histo- ment menstruation occurred, after a de- immediately inactivated in the digestive logical examination of the endometrium lay of 5 days. In this case a histological tract. in the various phases of the treatment investigation of the endometrium was was carried out. The authors merely omitted. The results of the investigations were briefly state that “the proof (of the effec- published in August, 1938 in the tiveness of both preparations) after oral Zentralblatt für Gynäkologie. The case administration was therefore here […] 92 Clauberg deviates here and elsewhere with the combined dose of estrogen/progestin in the sec- history discloses that the treatments took carried out in the same way as it had ear- ond half of the cycle from the then widely used place between February and May, 1938. lier been carried out by Clauberg for the “Kaufmann scheme”. The ethinylestradiol and pregneninolone injected preparations […]”91. 93 [Clauberg C, Ustün Z. Menstruation – per os were made available by Schering AG, erzeugt. Beweise der Wirksamkeit von Progynon C, einem neuen Follikelhormonderivat, und Pro- 90 and the substances carried the designa- This circumstance repeatedly led later investi- luton C, einem neuen Luteohormonpräparat, bei tions „Progynon C“ and „Proluton C“. gators astray in regard to the total number of pa- peroraler Verabreichung. Zbl Gynäkol 1938; 62: tients: they then spoke of 11 instead of 8 treated 1749]. At this place in the paper there is obvi- women. ously an error in the dosage data. It says here, “[…] 91 [Clauberg C, Ustün Z. Menstruation – per os then from the 27th April to the 7th May, 1938 in all erzeugt. Beweise der Wirksamkeit von Progynon 30 mg Progynon C orally (twice daily, each of 88 [Hahn H. Testierung, Wirkung und Bewertung C, einem neuen Follikelhormonderivat, und Pro- 5 mg) […]”. If one starts from the 10 mg per day, des Äthinylöstradiols (Progynon C). Zbl Gynäkol luton C, einem neuen Luteohormonpräparat, bei then the improbable total dose of 110 mg would 1951; 73: 783.] peroraler Verabreichung. Zbl Gynäkol 1938; 62: be given for the last days of the therapy. There- 89 Personal communication. 1747.] fore 30 mg is assumed as a more probable value.

44 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone

Optimal Dosage was of Little The omission of endometrium biopsies the brief entry there is no information Interest after combined estrogen/progestin treat- about the chemical nature of the medica- Looked at as a whole, Clauberg and his ment was limited to the cases in which ment. It is merely characterised as a co-author appear to have regarded that considerably less than the mentioned “corpus luteum preparation for oral ad- proof of the oral effectiveness of the 300 mg of Proluton C had been adminis- ministration”. Indications were habitual ethinyl compounds had been provided, tered. In these cases the investigators ex- abortion and spastic dysmenorrhoea. in that the substances succeeded in pro- pected no transformation. This conclu- The preparation was in the form of ducing growth of the uterus, menstrua- sion is supported by the statement of dragées each containing 5 or 10 mg of the tion and secretorial transformation of a Clauberg, namely that the bleeding oc- active substance. The recommended dose hyperproliferated mucous membrane. curring in these cases is a consequence was 1 or 2 dragees, 2 or 3 times per day. The question of the optimal dosage was of the breakdown of mucous membrane obviously of less interest. There were no in the proliferation because of a shortage Comprehensive information about Pro- indications as to why women with the of hormone. luton C was contained in a scientific data same illness were given such different sheet issued by Schering AG in 1955101. doses. The authors do not clearly explain Despite the enormously high doses (by In this it is noted, without reference to the criteria according to which they de- today’s standards) that Clauberg and the introduction of the preparation, so termined the dosages. Üstün used in their estrogen treatment, no long ago, that hitherto corpus luteum word is said of sideeffects. Furthermore, hormone could be administered only by As to the important question of the par- nothing is said of patients who had to be injection, because enteral administration ticular illnesses for which the ethinyl- eliminated from the study at some stage led to inactivation. By experiments estradiol should be given, the paper only because of intolerance of the medica- which attempted to increase the oral ef- says that one must in the case of oral ad- ment. Finally, there is no evaluation with fectivity of gonadal hormones by intro- ministration reckon to use about 2–3 regard to the rank that the new substances ducing specific substituents, Inhoffen times the amount that is necessary in the could severally occupy in therapy. and Hohlweg had found in pregneni- case of intramuscular injection of folli- nolone an “active substance very close to cular hormone. In reckoning the basis of The Introduction of Pregneni- progesterone”, that even when adminis- the dose, Clauberg was bearing in mind nolone into Therapy tered orally was hardly less effective the amount of generally used estradiol In contrast to ethinylestradiol, pregneni- (Fig. 5; p. 40). benzoate94, that would achieve a good nolone97 was introduced into therapy proliferation of the mucosa uteri. Fur- by Schering AG a very short time after Secretory Transformation by thermore, it was explained that there was its preparation. According to company Proluton C no wish to establish a general standard documents, this first orally effective pro- The company then referred briefly to the for the orally administered dose95. gestin was put on the market in 193998. results of experimental tests of the new Primarily on account of wartime dam- substance. In the case of baby rabbits Side-Effects not Mentioned age, archives are not now available pretreated with follicular hormone, a In the case of Proluton C the approach which might indicate what preparations secretory transformation of the mucous was somewhat different. Here the au- were involved and what the symptoms membrane of the uterus had been thors obviously started from the findings were for which the preparation was rec- achievable with 4 mg of orally adminis- of Hohlweg and Inhoffen, which indi- ommended99. tered Proluton C. Pure progesterone had cated that pregneninolone administered in contrast been ineffective even at a subcutaneously to a rabbit had about a In the “Rote Liste”, which provided in- dosage of 60 mg. In the case of rabbits third of the effectiveness of progester- formation about medicaments that were castrated after impregnation, Courrier one; if administered orally twice the on the market, the preparation first ap- and Fost had maintained the pregnancy dose was necessary. So Clauberg and peared under the name Proluton C in by feeding with Proluton C102. Üstün reckoned with a factor of at least 1940100. At that time the reference book five to six when determining the trans- was issued in an emergency format. In In connection with the clinical testing of formation dose of pregneninolone for a the preparation, reference is exclusively woman. In the event at least 300 mg 97 Schering at that time preferred the name preg- to Clauberg. He had succeeded, in were finally given96. neninolone for ethinyltestosterone (ethisterone). women who had acquired an artificial 98 Scheringianum, Archive no. XC 1.29/03. mucous membrane by means of estro- 99 The Berlin factories of Schering AG were al- gens, in transforming by oral adminis- 94 Estradiol benzoate is distinguished by prolonged most completely destroyed by bombs and shell- tration of pregneninolone the membrane effectiveness. The endometrium build-up dose was fire in the last years of the war. In April 1945, the 25–30 mg, distributed over five injections at in- factories in Wedding, location of the main labora- into the secretory phase. After treatment tervals of 4 days. See in addition e.g. [Ufer J. tory, were from time to time in the main fighting with the progestin, a genuine menstrua- Hormontherapie in der Frauenheilkunde. Grund- line. In May 1945, it was completely dismantled. tion could be observed. Also a uterine lagen und Praxis. 5. Aufl., De Gruyter, Berlin-New See in addition [Holländer H. Geschichte der York, 1978; 100]. Schering Aktiengesellschaft. Herausgegeben von mucous membrane, pathologically al- 95 [Clauberg C, Ustün Z. Menstruation – per os der Schering AG Berlin. Gedruckt und verlegt bei erzeugt. Beweise der Wirksamkeit von Progynon C, Erich Blaschker, Berlin 1955; 70–3]. 101 einem neuen Follikelhormonderivat, und Proluton 100 “Rote Liste” (Red List), Supplement 1940, Proluton C. For oral corpus luteum hormone C, einem neuen Luteohormonpräparat, bei peroraler p. 101. The next issue appeared in 1949. Here therapy. Schering AG (West) Berlin, 1955. Verabreichung. Zbl Gynäkol 1938; 62: 1761]. Proluton C is chemically characterized as Preg- Scheringianum, Archive no. S1/106. 96 Ibid., p. 1758. neninolone: Rote Liste, 1949, p. 513. 102 Literature references are missing.

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 45 Development of Ethinylestradiol and Ethinyltestosterone tered in the sense of a glandular cystic range of pregneninolone, did not in the In 1944 the former assistant of hyperplasia, had been brought to physi- least play a role in the fundamental accep- Windaus106, Maximilian Ehrenstein107, ological secretion. In this way a histo- tance of the preparation. This compares succeeded at the Pennsylvania Univer- logically ensured corpus luteum hor- not at all with the case of the investiga- sity in preparing a mixture of the isomers mone effect had been achieved for the tions into ethinylestradiol, which will be of 19-norprogesterone from strophanthi- first time by oral administration. discussed in detail later. din108. These steroids proved, in an in- vestigation by Willard Allen, to be pro- With regard to the necessary doses, it The Father of Modern Progestins gestins at least as effective as progester- says in the information sheet that Rapid progress in steroid chemistry had one109. Finally, in 1951 the young Mexi- according to the investigations by already begun in the 40s, which served can chemist Luis Miramontes under the Clauberg the sixfold amount of Proluton only to reduce the importance of the first tutelage of Carl Djerassi prepared nor- C orally administered is, in some cases, synthetic progestin to historical interest. ethisterone from 19-nortestosterone, and sufficient to achieve the same effect as In 1940 the American chemist Russell E. this new substance is about five times as injected progesterone. In general, how- Marker104 discovered a method by which effective as ethinyltestosterone110. Nor- ever, as a basis for the dosage a ratio of the vegetable compound diosgenin ethisterone led to the many highly effec- 10:1 should be the starting point. Ad- could be converted to progesterone (and tive progestins now known to us, which ministered orally, 200–300 mg preg- pregneninolone). This process was are ethinylated testosterone derivatives neninolone thus correspond to 25 mg of easily employed and could be based on and are primarily used in oral contracep- progesterone administered by injection. an almost inexhaustible raw material tives111. source. Such raw material was acces- The new preparation could be used in all sible, however, only to the Allies during  Early Papers by American complaints attributable to a deficiency of World War II. The availability of syn- corpus luteum hormone. As important thetic substances via the new process Authors on Ethinylestra- indications, the information sheet men- quickly led to a slump in the expensively diol tions habitual abortion, juvenile and cli- priced natural hormone105. Competition from the Stilbenes macteric bleeding as a consequence of After the publication of the much cited glandular-cystic hyperplasia of the en- investigations by Clauberg and Üstün, dometrium, as well as functional dys- 104 Russell E. Marker (1902–1995) is one of the nothing was at first said about ethinyl- most colourful personalities amongst the Ameri- menorrhoea. Finally it says, Proluton C can chemists of this century. Against the wishes estradiol. In the years 1939–1942 – as is excellently tolerated: sideeffects of of his father, a farmer of German descent in Mary- any kind had never been observed. land, he decided to study and enrolled at Mary- land university in the Department of chemistry. After the examination he refused to fulfil a for- 106 Adolf Otto Reinhold Windaus (1876–1959); Great Therapeutic Range mal requirement for the awarding of his doctorate studied chemistry under Emil Fischer in Berlin Although this Schering information sheet (he felt this to be “time-wasting”) and left the Uni- and Heinrich Kiliani in Freiburg/Breisgau. From versity in 1925 without a title, although his dis- 1915 to 1945 Windaus was the Director of the In- of 1955 for the clinical testing sertation was completed. His doctor-father proph- stitute for Chemistry at Göttingen University. of ethinyltestosterone referred only to esied at the time, that without his doctorate he Windaus isolated the vitamins, now classed as D1, Clauberg, at the time, however, the results would end up as a “urine analyst”. From 1928 D2 and D3. In 1928 he received a Nobel prize for Marker, after several appointments in industry, his work on the constitution of the sterols and their of other investigations were also avail- worked at the Rockefeller Institute in New York. relation to other natural substances. He discov- able. These investigations had been car- During this period he published about 25 trend- ered histamine and contributed to rhe synthesis of setting papers. Then the chemist decided to de- vitamin B1: [CDSB 1981: Concise Dictionary of ried out in the early 1940s in Germany Scientific Biography. Published under the auspices 103 vote himself to the steroids. As the Rockefeller and abroad , and they will not be consid- Institute would not give him the possibility to do of the American Council of Learned Societies. ered here because, by and large, they con- so, he resigned in 1935. In the following years he Charles Scribner’s Sons, New York, 1981; 737.] firm the previously available results. Ad- devoted himself almost single-handed to the 107 Maximilian Ehrenstein (1899–1968) studied trailblazing work on the conversion of diosgenin chemistry at Göttingen and from 1921 to 1923 was ditional points were merely concerned to progesterone mentioned above. It took place to assistant to Windaus. In 1934 he emigrated to the with dosages considered necessary for a large extent under apparently adventurous con- USA. Previously he had acquired a doctorate in the transformation of the endometrium. ditions in Mexico. In 1949, Marker abruptly with- pharmaceutical chemistry at Berlin University. In drew from chemistry. Below his last paper the 1949 Ehrenstein became Professor of Biochemis- The question of ascertaining the dose, author’s address does not appear as that of a labo- try at the University of Pennsylvania: [Strauß H, however, because of the great therapeutic ratory, instead simply “Hotel Geneve, Mexico Röder W (eds). International Biographical Dic- City”, Marker got from his work neither the wealth tionary of Central European Emigrés 1933–1944. to be expected nor particular honour. After his Vol l und II. K. G. Saur, München-New York-Lon- 103 See in addition, e.g.: [Zondek B, Rozin S. Uter- break with chemistry, for 30 years he dealt in don-Paris, 1983; 239.] ine haemorrhage induced by oral administration handicraft articles from Mexico. In an outline of 108 [Ehrenstein M. Investigations on steroids. VIII: of pregneninolon. Lancet 1939; 1: 504. Engelhart his life, he wrote that he had given up chemistry, Lower homologs of the pregnane series: 19-nor- E. Über die Auswertung eines peroral wirksamen feeling that he had achieved what he had set out 11-desoxycorticosterone acetate and 19-norpro- Gelbkörperhormonpräparates bei der kastrierten to do. About Marker, see [Marker RE. [Lebenslauf gesterone. J Org Chem 1944; 435.] ohne Titel.] Scheringianum, Archiv Nr. B 1/267. Frau. Zbl Gynäk 1940; 64: 98–103. Lauterwein 109 [Allen W, Ehrenstein M. 19-norprogesterone, C. Perorale Progesterontherapie. Zbl Gynäk 1940; Fieser LF, Fieser M. Steroids. Reinhold Publish- ing Corporation, New York, Chapmann and Hall, a physiologically active lower homolog of proges- 64: 108–20. Cohen M, Stein I. Laboratory and terone. Science 1944; 100: 251.] clinical experience with oral pregneninolone. Am London, 1959; 547–9. Djerassi C. The chemical 110 J Obst Gynec 1940; 40: 713–24. Preissecker E. history of the pill. In: Parnham MJ, Bruinvels J. [Fieser LF, Fieser M. Steroids. Reinhold Pub- Zur peroralen Gelbkörperhormon-Anwendung. Discoveries in Pharmacology, Vol. II. Elsevier, lishing Corporation, New York, Chapmann and Wien Med Wochschr 1941; 91: 94–5. Salmon UJ, Amsterdam-New York-Oxford, 1984; 345–7.] Hall, London, 1959; 591–2.] Geist SH, Walter RI, Mintz N. Therapeutic effec- 105 [Fieser LF, Fieser M. Steroids. Reinhold Pub- 111 See in addition [Djerassi C. The pill at 50 (in tiveness of orally administered ethinylestradiol. lishing Corporation, New York, Chapmann and Germany): thriving or surviving. J Repr Med Endo J Clin Endocrin 1941; 1: 556–8.] Hall, London, 1959; 547–54.] 2011; 8 (Special Issue 1): 14–31.]

46 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone will be shown – few clinicians worked 1960s. It was given in large doses of up amination of the endometrium before with the new substance, although inter- to 300 mg per day for the treatment of and after the therapy did not take place. est in highly effective oral estrogen pre- impending abortion. In Germany the ap- parations steadily increased. This phe- proach to the substance was consider- Salaber and del Castillo thus used a con- nomenon, whose causes will be compre- ably more reserved, primarily due to the siderably smaller dose of ethinylestra- hensively discussed later, may be attrib- influence of the gynaecologist Carl diol than had Clauberg and Ustün. Con- utable to, inter alia, the discovery of di- Kaufmann115. This caution was to prove sidered as a whole, they reduced the ethylstilbestrol in 1938. This substance a blessing for at the beginning of the daily dose by at least a factor of five. In was the first and may well be the most 1970s it became apparent that young their paper, in which they refer to important representative of a group of girls whose mothers had been treated Clauberg and Ustün as well as to non-steroidal compounds with high es- with stilbestrol during their pregnancies, Inhoffen and Hohlweg, this reduced dos- trogenic activity, an activity undimin- consequently developed neoplasias of age is specially emphasized119. Justifica- ished when administration is oral. What the vagina116. The preparation is there- tion for this is not given; all that is noted is more, stilbestrol could be made easily fore no longer used in gynaecological is that the therapeutic doses can be deter- and cheaply. Because of their impor- therapy. mined, only when a larger number of tance for the reception of ethinylestra- patients has been treated. diol, the stilbenes will be discussed here In the case of the few investigations of in considerable detail. ethinylestradiol which at the end of the The two South Americans, in contrast to 1930s and beginning of the 1940s had Clauberg, additionally reported on the The synthesis of diethylstilbestrol was been somewhat overshadowed by the toleration of the medication. The sub- achieved by the English biochemist stilbene derivatives, they were a matter stance had been well tolerated. The Edward Charles Dodds112 and his col- of clinical studies by American authors. effect was comparable with that of intra- leagues after having worked on the prob- In Europe, then preoccupied by war, no muscularly injected estradiol mono- lem for many years. lt is not without in- one was intensively occupied with the benzoate. The authors were convinced terest to learn that Dodds obtained im- steroids, although the paper by Clauberg that the use of ethinylestradiol would portant, if not decisive, hints for his had certainly created interest117. represent a real step forward in the field work from Schering’s Berlin main labo- of hormone therapy for menstruation ratory113. The new substance, because of The question of the causes of this devel- anomalies. The ethinylestradiol for their its described properties, excited great in- opment will be considered later. study was made available by the Schering terest amongst clinicians soon after its subsidiary, Quimica Schering S.A. in preparation, and it was shortly intro- Two Argentinians Made the Start Buenos Aires. It carried the designation duced into therapy. Numerous investiga- Probably the first foreign workers to be- “Neo-Estrona”. tions of its usability became public in the come involved were the Argentinian 1940s via a wealth of papers in the spe- gynaecologists J. A. Salaber and E. B. Probably the first experiences of North cialist literature114. del Castillo who tested ethinylestradiol American clinicians with ethinylestra- on three patients118. The results of their diol were published by Robert Frank and German Gynecologists were investigations were published in Janu- his colleagues in 1940 in the journal En- Reserved ary, 1939 in the journal published in docrinology120. They considered the sub- Although intolerance phenomena simi- Buenos Aires La Semana Medica. The stance within the framework of a survey lar to those experienced with overdoses indication for the therapy was, in all of newer preparations of sex hormones. of ethinylestradiol had been reported three cases, secondary amenorrhoea. The authors were members of a group of earlier, stilbestrol was widely used, workers from the Endocrinology Clinic above all in the USA, until the late The patients were treated for 24, 29, or and the laboratories of the Mount Sinai 32 days with a total dose between 7, 4 Hospital in New York City. and 15 mg of the synthetic hormone. 112 Edward Charles Dodds (1899–1973) was one of the most important English biochemists. He was The authors reported that uterine bleed- The publication was concerned prima- Courtauld Professor at London University and ing had been started in all three patients, rily – as one would expect at that time – Director of the Courtauld Institute of Biochemis- and in each case it occurred 4–6 days af- with diethylstilbestrol. All that was said try at the Middlesex Hospital. He received a life peerage in 1964. He was a Fellow of the Royal ter medication ceased. Histological ex- Society and President of the Royal College of Phy- sicians as well as an honorary member of the 119 Ibid, p. 9. American Association for cancer Research [Can- 115 120 cer Research 1983; 43: no. 9 (cover).] See in addition [Zander J. Carl Caufmann. 21. [Frank RT, Goldberger MA, Felshin G. Clini- August 1900–18. August 1980 [Nachruf]. cal and laboratory investigations of some of the 113 See in addition, e.g. [Dodds EC, Lawson W. Geburtsh Frauenheilk 1981; 41: 84–5.] newer sex hormone preparations. Endocrinology Oestrogenic activity of p-hydroxy propenyl ben- 116 See in addition [Ufer J. Hormontherapie in der 1940; 27: 381–4.] A preparation made by CIBA zene (Anol). Nature 1937; 139: 1069. Tausk M. Pharmaceutical Products was tested. This com- Organon. The story of an unusual pharmaceutical Frauenheilkunde. Grundlagen und Praxis. 5. Aufl., De Gruyter, Berlin-New York, 1978; 108–8.] pany, like Roche-Organon and Schering in the enterprise. Published by Akzo Pharma bv, Oss, The USA made ethinylestradiol available for clinical 117 Netherlands 1984; 103–4. Hohlweg W. Die Hor- No references can be found to publications that investigations. The Schering corporation had, mone der Keimdrüsen. In: Seitz L, Amreich A go beyond the already mentioned clinical tests at however, the major share of the clinical studies. (Hrsg.). Biologie und Pathologie des Weibes. Bd. the Charité and the University of Würzburg. The question as to why other firms had ethinyl- 1, 2. Aufl., Urban und Schwarzenberg, Berlin, 118 [Salaber JA, del Castillo EB. Acción del estradiol at their disposal so soon after the dis- 1953; 532.] etinilestradiol, derivado de la hormona follicular, covery of this new substance, will be briefly dis- 114 See in addition Index Medicus 1938–1956. por via oral. La semana medica 1939; 1: 6–10.] cussed later.

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 47 Development of Ethinylestradiol and Ethinyltestosterone about ethinylestradiol was that clinical The effect of the preparation was as- gations in the United States that finally, tests of the substance had been broken sessed by the degree to which these at the end of the 1940s, led to the intro- off at a very early stage, because the ma- symptoms disappeared, but also by the duction of ethinylestradiol into general jority of the patients after the first dose estrogen effect on vaginal smears or by therapy. Within the framework of this of the medicament had shown the most vaginal biopsy. Patients who cytologi- monograph it would be excessive to de- severe side-effects in the form of sick- cally or histologically showed no sign of scribe, in full detail and in chronological ness and vomiting. Information about hormone deficiency were not included order, the studies that increased in num- the indications and the doses is not in the study. ber with the years. Representative of the given. The literature cited in the paper whole collection therefore, will be ex- gives no indication as to which of the Salmon’s group of workers also used emplary investigations, which will be ar- then available publications about ethi- considerably smaller doses of the medi- ranged according to the indications pre- nylestradiol had been consulted by the cament than had Clauberg. Although the sented. New York working group. New York investigators expressly refer in their paper to the dosages used by the The Treatment of Ovarian Despite these unencouraging results in Königsberg doctor, it remains unex- Insufficiency therapy, the new substance was persisted plained why they did not follow his sug- Therapy for the symptoms characteristic with at the Mount Sinai Hospital. In the gestions. They also refrained from any of the menopause, whether a conse- following year another team from the comment on the explicitly ascertained quence of natural or iatrogenic ovarian hospital (Udall J. Salmon and col- fact that in Clauberg’s paper nothing was insufficiency, was clearly in the 1940s leagues) published again about ethinyl- said about the side effects of ethinyl- the principal occasion for further trials estradiol121. On this occasion the result estradiol. with ethinylestradiol. Besides women of a clinical trial on 22 patients was de- who suffered cessation phenomena due scribed, all the patients having suffered Clear Estrogenic Effects to age, patients were also treated whose from the menopause syndrome. Not a In the study by Salmon, at the start of ovaries, because of certain illnesses, had word was said about the treatments bro- the therapy the patients were first treated had to be removed surgically or whose ken off because of the side-effects dur- for 4–7 days with 1.8–3.15 mg of the ovaries had been made functionally in- ing the previous year. preparation. The average daily dose was active by irradiation. 0.45 mg. Within this period, with two The Ideal Drug: Low Dose, Cheap, exceptions, an improvement in the clini- The limitations that Salmon and his col- Orally Applicable cal symptoms was observable in all the leagues had placed in 1941 on the use of The authors concentrated on explanation women. In the vaginal mucous mem- ethinylestradiol because of the observed in detail of the importance of an orally brane, clear estrogenic effects were ap- side effects are not to be found in a paper effective estrogen for the therapy of cli- parent. In the case of four women, the published in 1942 by B. A. Watson123. macteric complaints in particular. The treatment had to be broken off because Although the author, a worker in the En- treatment of the menopause syndrome of sickness, vomiting and stomach- docrinological Department of the sana- requires a hormone substitute over a aches, three further patients reacted to a torium in Battle Creek, Michigan, did long period, it was said. The ideal prepa- dose decrease with improvement in the not use doses significantly smaller than ration for the purpose must fulfil several side-effects. After the initial phase, the those used by Salmon’s group, the unde- conditions: high activity in small doses, treatment could be continued with daily sirable reactions that occurred did not simple administration by the patient her- doses between 0.15 and 0.3 mg. The au- appear serious to him. “From our experi- self, high tolerance and cheap manufac- thors report only one instance of uterine ence it is possible to conclude that ethi- ture. Salmon and his colleagues did not bleeding, which occurred 2 weeks after nylestradiol is an effective and safe drug regard the preparations then available as therapy ended, and this in the case of a capable of fulfilling these requirements patient who had received a total dose of in ideal fashion. Therefore ethinylestra- 23.1 mg of ethinylestradiol over a period diol was tested. of 50 days. 123 [Watson BA. Clinical experiences with oral ethinylestradiol. J Clin Endocr 1942; 2: 447–9.] Strange to say, at the start of the paper “ethinyl- Among the 22 patients who were treated In the assessment of their results the au- estradiol benzoate” is spoken of as the tested sub- with ethinylestradiol tablets were some thors came to the conclusion that the stance. Later the author speaks only of “ethinyl- women castrated by surgery and others tested substance orally administered in estradiol”. In the literature references he cites Clauberg 1938 and Salmon 1941, without point- by radiation therapy. They all showed small doses shows high estrogenic activ- ing out a likely chemical difference of the tested “[…] characteristic menopausal symp- ity. lt therefore appeared to be extremely substances. These two papers were the first on the toms: hot flushes, nervousness, sleep- promising with regard to the above clinical use of the new medicament, now also in- vestigated by him, he explains (p. 447). There- lessness, gastrointestinal disturbances, stated requirements for a substitution fore it must be assumed that the designation vertigo, headaches and arthralgia”122. therapy in the climacterium. Before a “ethinylestradiol benzoate” is an error. [Clauberg recommendation for general use, how- C, Ustün Z. Menstruation – per os erzeugt. Be- weise der Wirksamkeit von Progynon C, einem ever, the cause of the side-effects was to neuen Follikelhormonderivat, und Proluton C, 121 [Salmon UJ, Geist SH, Walter RI, Mintz N. be elucidated in further investigations. einem neuen Luteohormonpräparat, bei peroraler Therapeutic effectiveness of orally administered Verabreichung. Zbl Gynäkol 1938; 62: 1745–61, ethinylestradiol. J Clin Endocrin 1941; 1: 556– Salmon UJ, Geist SH, Walter RI, Mintz N. Thera- 8.] The study outlined above represented peutic effectiveness of orally administered ethinyl- 122 Ibid, p. 556. the prelude to numerous further investi- estradiol. J Clin Endocrin 1941; 1: 556–8.]

48 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone to use in the treatment of menopausal daily dose and this was gradually de- for a certain period, occasional endo- symptoms […],” wrote Watson124. creased after 7 to 14 days”127. metrium biopsies showed proliferation phases, occasionally also hyperplasia The investigation, published in the The principal concern of the investiga- with expanded and cystic glands. Ac- Journal of Clinical Endocrinology, was tion was the question of the effectiveness cording to the paper, various patients re- based on observations of 18 patients. of the preparation in the control of the ported a white discharge, that obviously The women were treated for several clinical symptoms associated with hor- was attributable to an increase in the months, and they all suffered from typi- mone deficiency. The estimation of the maturing and keratinisation of the vagi- cal menopausal phenomena, including objectively detectable estrogen activity nal epithelium. hot flushes. “No patients were included of the substance had been of secondary in this study who did not experience interest, wrote Groper and his co-author. Similar experiences to those of Groper flashes because it was felt that the disap- In 27 out of 33 cases, first-class results in and Biskind were those of S. D. Soule, pearance of these would more clearly in- treatment had been achieved and a mod- who in 1943 published in the American dicate subjective improvement”, it is erate improvement of the symptoms had Journal of Obstetrics and Gynecology said in the paper125. occurred in two women. Only in four the results of treatment with ethinyl- cases did failure of the therapy have to estradiol in cases of menopausal syn- Successful Treatment with be admitted. drome in 30 women. He too used a daily “Minimal Doses” hormone dose of 0.05 mg. He also re- Watson said concerning the doses that Side-Effects Scarcely Occurred ferred to the side-effects that Salmon after some time a very specific scheme In the therapy described by Groper and and his colleagues had observed when of application had proved especially Biskind, side-effects scarcely occurred. giving larger doses of the medicament. favourable, because with it side-effects None of the treated women suffered The 0.05 mg ethinylestradiol per day had been avoidable: three times 0.15 mg from nausea or vomiting, the paper re- that he had administered were well tol- per day for the first 3 days, then re- ports. Headache was observed in only erated by 28 of the 30 patients. In one duction of the daily dose to 0.15 mg three cases. Apart from one case, in case there had been complaints about twice and finally, as long-term therapy, which a termination of therapy was nec- acute sickness, and in another the ter- 0.15 mg ethinylestradiol daily. The essary, in other cases the symptom, de- mination of the therapy had been neces- treatment extended over 2 to 13 succes- spite continuation of the treatment, dis- sary129. sive months. In the process only one fail- appeared of its own accord. Conse- ure of the therapy and one “toxic reac- quently the comment is made: “The “Excellent” Success tion” (urticaria) had been observed. toxic manifestations that have been de- The success of the treatment in 19 cases scribed with the nonsteroid oral estro- was rated by Soule as “excellent”; nine Morris J. Groper and Gerson A. Biskind gens did not occur.” And in another patients had responded well. One patient reported on a successful treatment of the place: “The development of an orally showed little or no improvement, so one menopause syndrome with still smaller effective steroid estrogen […], which in case of complete therapeutic failure had doses of ethinylestradiol (“minimal our hands has shown practically no to be recorded. Amongst the patients doses”) in a paper that likewise appeared toxic symptoms, is a welcome ad- were 16 women whose hormone defi- in 1942 in the Journal of Clinical Endo- vance”128. ciency was the consequence of an opera- crinology126. According to the experi- tion. The problem of uterine bleeding ences of these two authors, which were The therapy with ever smaller doses of after prolonged treatment with ethinyl- based on the treatment of 33 women, ethinylestradiol was soon taken into ac- estradiol is not discussed in detail. Soule some over 2 years, the dose of the medi- count by the manufacturer. In the cited merely states that after the administra- cament could be reduced to 0.05 mg paper it is pointed out that the Schering tion of in all 0.85 mg over 17 days, on daily, without relapses of the patients. Corporation in Bloomfield (New Jersey) cessation of treatment bleeding had been initially supplied the medicament in tab- observable. A recommendation for treat- It is apparent from the study that the let form with 0.15 mg of effective mate- ment interruptions is not given. treatment initially employed substan- rial per tablet. Groper and Biskind write tially larger doses of the semi-synthetic that the dose per tablet was later de- Since most of the patients in the study estrogen. “When ethinylestradiol was creased to 0.05 mg. had been treated with other preparations first employed, the majority of patients before the ethinylestradiol therapy was was given 0.15 mg daily for a period of Although the authors were primarily started, the author had an opportunity to 14 to 21 days, and the dosage was then concerned with the clinical symptoms of compare the subjective efficacies. Soule decreased to 0.15 mg every other day. In the menopause, the objective signs of the wrote in regard to this that “[…] twelve a few instances 0.30 mg was the initial estrogen effect that occurred during the of the patients who previously had taken treatment were recorded: in seven cases from 0.25 to 1.0 mg stilbestrol daily ap- – as expected – uterine bleeding oc- proximately were equally benefited by curred. After the therapy had continued 0.05 mg ethinylestradiol.” He referred 124 Ibid, p. 449. 125 Ibid, p. 447. 127 126 [Groper MJ, Biskind GR. Ethinylestradiol: a Ibid, p. 703. 129 [Soule SD. A clinical trial of ethinylestradiol. clinical evaluation. J Clin Endocr 1942; 2: 703–6.] 128 Ibid, p. 706. Am J Obstet Gynec 1943; 45: 315–7.]

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 49 Development of Ethinylestradiol and Ethinyltestosterone besides to another phenomenon that re- the climacteric syndrome. Bleedings belonged to Kurzrok’s group of workers peatedly appears in the literature. “lt was when the treatment was interrupted were in New York, published figures in 1947 noted quite frequently that the patient not the rule, and in certain cases they for the successes in weaning with who obtains a similar result with natural could be avoided by further reductions ethinylestradiol136. In this publication, in estrogens experiences a peculiar ‘lift’ or of the dose, without a corresponding de- which in addition other indications for sense of well-being that is difficult to crease in therapeutic effect. The undesir- the steroid hormone are discussed, it is explain but which is nevertheless fre- able side-effects that occurred were de- said that effective suppression of lacta- quently definite”130. scribed by the author as “minimal and tion had been achieved in 75% of the 145 transitory”133. patients. In the case of these puerperae The described small doses of ethinyl- also, no side effects were observed. The estradiol were also successfully used, in Hans Wiesbader and William Filler ob- doses used were, in all, 1.5 mg, distrib- the years after 1943, in other studies of tained similar results from an investiga- uted over 9 days. the treatment of ovarian insufficiency. tion of 53 patients with symptoms of Floyd E. Harding reported in 1944 in ovarian insufficiency134. In their study Other Indications the American Journal of Obstetrics and published in 1946, and also in the Ameri- In the USA after 1942 increasing num- Gynecology that even daily doses of can Journal of Obstetrics and Gynecol- bers of other indications for the treat- 0.02 mg of the substance had in certain ogy, they also reported on biopsies of the ment with ethinylestradiol were tested. cases produced a clear therapeutic suc- endometria of 10 patients that were car- Thus Robert A. Lyon in 1943 reported in cess131. ried out before and during the therapy. the journal Surgery, Gynecology and These showed that when treated with Obstetrics on the therapy of essential As optimal daily doses for most patients 0.05mg ethinylestradiol per day, after dysmenorrhoea in the case of 12 pa- with complaints in the natural or artifi- 14–20 days good proliferation of the tients137. The plan of the treatment was cial postmenopause he indicated 0.05– mucosa was observable in each case. based on observations, according to 0.1 mg of the estrogen derivative. He ob- which this illness occurred only in ovu- served bleeding in women previously Suppression of Lactation latory cycles. The administration of suffering from amenorrhoea in 5 of 17 As well as for the treatment of the post- ethinylestradiol was seen to prevent ovu- cases. menopausal syndrome, ethinylestradiol lation and the formation of a corpus lu- was used in early clinical trials in the teum. In another investigation in 1944 pub- USA to achieve the suppression of lacta- lished by Robert A. Lyon at the Medical tion during the puerperium. In the Jour- For the study the author monitored, by School of the University of California in nal of Clinical Endocrinology a group means of basal temperature curves, a to- San Francisco ethinylestradiol was used led by Lawrence Kurzrok reported in tal of 138 cycles of his patients. In 44 of cyclically132. The author reported on 1942 on the treatment of 59 women in these cycles he treated for 21–24 days about 45 patients with the climacteric childbed135. In these cases daily doses up with 0.05 mg ethinyl steroid. In all syndrome who had received daily doses to 2.4 mg were used. The therapy in each cases, ovulation was suppressed and the of 0.05 mg for 21–24 days. This was fol- case extended over 3–4 days. occurrence of dysmenorrhoic com- lowed by a treatment-free interval of 4 or plaints was prevented. Lyon, however, 7 days. This scheme, so says the paper, Commenting on the results it is noted pointed out in the paper that according to should give the endometrium the possi- that at daily doses below 1.5 mg ethinyl- his and the experiences of others, this bility of regression or bleeding. The estradiol there were, relatively, many treatment could guarantee no permanent duration of the treatment-free interval failures (9 out of 26 cases). If 1.5– cure. The preparation had been tolerated was made dependent on how quickly the 2.4 mg were given, however, lactation well by all the patients. patients observed a recurrence of their would almost always be effectively sup- complaints. pressed (one failure). Side effects did not In 1946, William Bickers reported (in appear. The treatment was in most cases the American Journal of Obstetrics and With Lower Dosage no Loss in begun 1 or 2 days post partum; in one Gynecology) on the treatment of metror- Therapeutic Effect case, however, it was begun 8 weeks af- rhagia with ethinylestradiol and pro- Lyon summarized the results of his study, ter birth. gesterone138. His 12 patients were in which 348 cycles were investigated, to younger women with strong and pro- the result that the ethinyl compound In later publications, with a larger num- longed bleeding from proliferated endo- made possible an effective therapy for ber of patients, the results were similar. metrium with no occurrence of ovula- Charles H. Birnberg for example, who tion. The patients first received 0.3 mg

130 Ibid, p. 316. The author here classes ethinyl- estradiol, in contrast to the stilbene derivates, as a 136 [Birnberg CH, Livingstone SH, Kurzrok L, natural estrogen. 133 Ibid, p. 535. Sherber DA. Ethinylestradiol. Am J Obstet Gynec 131 [Harding FE. Oral substitution therapy with 134 [Wiesbader H, Filler W. Oral therapy with 1947; 54: 855–60.] ethinylestradiol and alpha-estradiol. Am J Obstet ethinylestradiol in the menopause. Am J Obstet 137 [Lyon RA. Relief of essential dysmenorrhea Gynec 1944; 48: 181.] Gynec 1946; 51: 75–81.] with ethinylestradiol. Surgery, Gynecology and 132 [Lyon BA. Management of the climacteric with 135 [Kurzrok L, Birnberg CH, Livingstone S. Use Obstetrics 1943; 77: 657–60.] ethinylestradiol. Am J Obstet Gynec 1944; 47: of ethinylestradiol to prevent lactation in puerperal 138 [Bickers W. Ethinylestradiol in the treatment 532–6. Correction: Am J Obstet Gynec 1944; 48: patients. J Clin Endoc 1942; 2: 471–2 (Letters to of metrorrhagia. Am J Obstet Gynec 1946; 51: 146.] the Editor).] 100–3.]

50 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone of estrogen per day for 15 days, then on been accomplished, likewise it had been stance in the USA. Recommended appli- 5 further days the ethinylestradiol was possible to achieve, in all cases of hypo- cations were initially in the area of cli- combined in each case with 5 mg of pro- plasia, a growth of the uterus and the cer- macteric complaints. The tablets put on gesterone administered by subcutaneous vix. In the case of the patients with pri- the market each contained 0.02mg of injection. mary amenorrhoea and slight develop- hormone (Fig. 6). ment of the breasts (9 women), in the The paper records that, under the course of the therapy over several months In a letter written by the company to doc- therapy the bleeding had stopped within a noteworthy growth of the breasts and tors introducing the new preparation142, 6 days (with just one exception). Men- nipples had been observable. Only two it is stated that Progynon C has “[…] the strual bleeding began 5 days (at the patients in the whole group had com- great advantage of being orally fully ef- most) after medication was interrupted. plained about slight sickness. fective, well tolerated and particularly Bickers then continued in each case for economical.” With it the difficulties, that two further cycles, whereby the adminis- Towards the end of the 1940s ethinyl- in the past arose because of the cost of a tration of estrogen was begun always on estradiol was also used for induction consistent hormone therapy, could be the fifth day after the beginning of depri- of labour in pregnant women at term, avoided. vation bleeding. In this way, in 90% of in the case of missed abortion, acne, the women under treatment, a cyclic chronic haemospermia and in the With reference to the clinical investiga- bleeding pattern had been induced. After therapy of carcinoma. In connection tions available by 1949, a first scientific completion of the treatment, 70% of the with the last-named indication, particu- information sheet from Schering143 treated women retained the cycle, and in lar mention is made of prostate carci- about ethinylestradiol emphasized that the case of half the patients ovulation noma and mammary carcinoma. These the hormone possesses the same physi- had occurred (observation period 3–12 studies will however not be discussed in ological effects as natural estradiol, months). detail here. namely, formation of the proliferation phase of the endometrium, growth of the The author pointed out that similar treat- In a survey of the literature on ethinyl- uterus as well as influencing the second- ment trials had already been carried out estradiol Kenneth Wade Thompson in ary sex characteristics and the vaginal in the past with stilbestrol. In these, how- 1948 wrote in the Journal of Clinical epithelium. Furthermore, for those rea- ever, incompatibility reactions had fre- Endocrinology, that the substance had in sons indications for the preparation, “are quently forced the therapy to be stopped. the course of its testing clearly been practically all branches of follicular hor- In the treatment with ethinylestradiol in proved to be the most effective oral es- mone therapy.” Since initially the com- only one case had a small degree of nau- trogen140. Its therapeutic effects were pany had to hand only comprehensive sea occurred. To sum up, it could be said comparable to those of estradiol. The results of clinical tests on symptoms of that in the therapy of anovulatory bleed- preparation was – after initial uncer- deficiency in the climacterium, the use ing, daily doses of 0.03 mg ethinylestra- tainty about the dosage had been over- of Progynon C was at first restricted diol had been comparatively as effective come – used successfully and without to that area. As examples of complaints as 5 mg diethylstilbestrol. serious side-effects in all cases in which that could be treated with the prepara- natural estrogens had proved useful. tion, the information sheet listed, be- Therapy of Hypoplasias sides the class hot flushes, vertigo, In the paper by Birnberg and his col- The author, however, pointed out at the sleeplessness, depression, itchy skin leagues cited above the treatment with same time that further investigations eruptions, inflamed joints, circulatory ethinylestradiol of primary and second- of the metabolism of the substance disturbances and migraine. ary amenorrhoea was also discussed139. in the human body were desirable. At The therapy was undertaken in accor- that time scarcely anything was known Warning against Overdosage dance with a uniform scheme (20 days about it. In the advice concerning use, Schering each 0.05 mg ethinylestradiol, then a 10 expressly gave warning of the conse- day interval). The objective in the  The Introduction of Ethi- quences of overdosing. Besides gener- administration of estrogen was to start ally undesirable effects (such as head- cyclic bleeding as well as to treat hypo- nylestradiol into Therapy aches, dizziness and nausea) delays of plasias of uteri and mammae. The by Schering (1949) menstrual periods, hypermenorrhoea, authors refrained from the administra- Ethinylestradiol was introduced into hyperplasia bleeding up to severe tion of progestins, without giving a rea- therapy by Schering in the Federal Re- haemorrhages as well as painful swell- son for this. The participants in the study public of Germany in 1949, under the ing of the breasts could occur. lt was rec- were 24 patients aged from 16 to 35 trade name Progynon C141. In so doing, years. the company was relying primarily on the clinical experience with the sub- 142 Progynon C (ethinylestradiol). Schering AG. Berlin 1949. Scheringianum Archive no. S1/115. Concerning the results, it is recorded that 143 See in addition to this specialist literature on in all these women periodic bleeding had hormone treatment in gynaecology in the 1950s 140 [Thompson KW. Ethinylestradiol – an endo- and 1960s, e.g. [Hoffmann F. Die Sexualhormon- crine review. J Clin Endoc 1948; 8: 1088–97.] theraple in der Gynäkologie. 3., neu bearb. Aufl. 139 [Birnberg CH, Livingstone SH, Kurzrok L, 141 Circular issued in March, 1949. Scheringianum, Johann Ambrosius Barth Verlag, Leipzig, 1959.]. Sherber DA. Ethinylestradiol. Am J Obstet Gynec Archive no S1/115. The price for an original pack The term “Pille danach” refers to the use of hor- 1947; 54: 855–60.] with 30 tablets was then DM 1.95. mones for post-coital antinidation.

52 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone ommended to begin the treatment of cli- ity after metabolism to ethinylestradiol, steroids possible. What follows will be macteric complaints with one 0.02 mg scarcely still play a part. The future must an attempt to honour the performances tablet 3 times a day, and once it began to show whether other preparations can of the scientists who directly or indi- have an effect, slowly to reduce the dose displace ethinylestradiol in hormonal rectly participated in the development of to one tablet a day or every other day in contraceptives. ethinylestradiol and ethinyltestosterone severe cases or after castration an in- (ethisterone). It is intended that the crease of the daily amount of up to two  Discussion analysis will relate to the synthesis as tablets three times a day was possible. well as the biological and clinical testing If one regards the history of ethinylestra- of both substances. Particular impor- The initial limitation of the indication diol and ethinyltestosterone in retro- tance in this case must be allotted to the for ethinylestradiol was soon abandoned spect, the question that first arises is cited German patent specifications, in following years, with increasing clini- where are the origins of the chemical which appear to make questionable cal use of the substance in the Federal preparation of these two steroids. Al- Inhoffen’s claim for priority in the first Republic of Germany also. In combina- though details of the development of or- preparation of ethinylestradiol. tion with progestins the preparation later ganic chemistry cannot be discussed at served to bring on cyclic bleeding in this point, it should be established here Problematic Location of cases of primary and secondary amenor- that papers by the American J. Nef Sources rhoea. Ovulatory bleeding and premen- (1862–1915) and the Frenchman Victor The first point to be mentioned is that strual bleeding could be treated with Grignard (1871–1935) provided deci- where there have been problems locating ethinylestradiol only. In addition, the sive pre-requisites. Nef contributed to sources, questions must remain unan- preparation found use at greater doses science the fundamental knowledge of swered. On many points, only probabili- for weaning, as a “Pille danach”, as well the ethinylation of aldehydes or ketones ties can be put forward. The problems as in the therapy of certain carcinomas. with sodium acetylide to yield substi- that arise in the discussion of the facts tuted alkinols. Grignard later modified presented in this monograph have in part The most Important Indication: this principle. Both procedures, respec- already been addressed. Essentially, it is Contraception tively known as the Nef and the Grignard a matter of many documents having The most important area of indication, reactions, belong now to especially ef- been lost during the war. Also, in con- even today, for ethinylestradiol was fective methods of synthesis in prepara- nection with patents, access cannot be opened – as mentioned in the introduc- tive chemistry145. had to official files. Only the patent tion to this monograph – at the end of the specifications still exist. The documents 1950s with the introduction of hormonal The discovery that liquid ammonia is an that led to their drawing up and the infor- contraception by Gregory Pincus (1903– excellent solvent and reagent goes back mation about supplements that were sent 1967) and his colleagues. Only 3 years to H. P. Cady (b. 1897) and E. C. in during the course of the patenting pro- after the US registration of Enovid as the Franklin (1862–1937). They recognized cess over many years have, in accor- first hormonal contraceptive in the that this compound possesses properties dance with the usual procedure, been world, Schering in 1961 was the first that are extremely similar to those of destroyed decades ago147. At Schering a pharmaceutical company in Europe to water. Analogously to the aquo-system relevant exchange of letters with the put its own contraceptive pill Anovlar on of acids, bases and salts, Franklin after “Reichspatentamt” is likewise no longer the market. Enovid contained as its es- 1912 proposed a corresponding ammo- to be found. trogen component 150µg of mestranol, no-system. Subsequently liquid ammo- the 3-methyl ether of 17α-ethinylestra- nia (NH) was increasingly used in syn- It can be stated with certainty, that ste- diol, and 9.58 mg of norethynodrel. In theses as the solvent146. Decisive is the roids had been ethinylated already by Anovlar there were used, in contrast, ability of liquid ammonia to dissolve 1935. The cited patent by the Schering 50µg of the unetherified substance144 and alkali metals: they are then in a very chemists, Serini and Strassberger, that 4mg of norethisterone – an estrogen favourable form for organic reactions. had been applied for on the 10th Novem- dose that already came very close to the ber, 1935, leaves no doubt about this, presently widely used 30 µg. In the mid-30s, the steroid chemists in since the corresponding process is the their efforts to modify as well as to syn- subject of the patent claim. But what is Thus ethinylestradiol had found a range thesize these newly discovered sex hor- now the answer to the question, as to of application that is granted to very few mones could draw on these insights. whether ethinylestradiol had already pharmaceutical products. Nothing has They thus already had available the tools been prepared in 1935? Synthesis of the altered that, despite the fact that newer, that made the preparation of the ethinyl- substance at a yield of about 30% is de- orally easily used estrogen preparations scribed in fourth place amongst the five have displaced ethinylestradiol for cer- examples of the application in this tain indications. The later developed de- 145 See in addition [Nef JU. Ueber das Phenylacety- patent. len, seine Salze und seine Halogensubstitutions- rivatives of the compound, such as produkte. Liebig’s Analen der Chemie 1899; 307– mestranol, that display hormonal activ- 8: 264–328. Walden P. Geschichte der Organischen Chemie seit 1880. Zweiter Band zu: Graebe C. Geschichte der Organischen Chemie. Verlag von 144 See in addition [Schering AG (Hrsg). Die Pille Julius Springer, Berlin 1941. Reprint: Springer wird 20. So begann es … und da stehen wir heute. Verlag, Berlin-Heidelberg-New York, 1972; 101–3.] 147 Information from the German Patent Office in Symposium, Berlin, 1981; 63.] 146 Walden 1941, p. 111 et seq. Munich.

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 53 Development of Ethinylestradiol and Ethinyltestosterone

Subsequent Filing of Examples Data from the Year 1937 Kathol’s patent has likewise been subse- was Usual A further indication of the subsequent fil- quently filed. What, however, is the posi- The patent law valid in those years, un- ing of ethinylestradiol in the patent in tion of the synthesis example that is cited til the conclusion of the notification, question is presented by the data cited in in the patent specification in the first allowed the subsequent filing of ex- the patent specification concerning the place? With it could Kathol have suc- amples that could then be included in physiological effectiveness of the sub- ceeded in preparing ethinylestradiol be- the patent specification148. A comple- stance. These data correspond apart from fore Inhoffen? Purely theoretically, this ment of the patent by Serini and one obvious misprint exactly to those that cannot be excluded. On the contrary, it Strassberger with regard to the prepara- are cited in the classic paper by Hohlweg even appears to give some clear hints of tion specifically of ethinylestradiol was and Inhoffen from the year 1938. The this. thus fundamentally possible up to 3rd data would in 1935, had they then been December, 1942. The substance could available, certainly have been considered Different Nomenclature therewith even after a first preparation as sensational as they were later. One In the patent specification it is striking at the end of 1937 by Inhoffen still be would therefore scarcely have refrained that Kathol uses a different nomen- included in the already applied for from a publication immediately after the clature in the two examples of the prepa- patent as an additional example of the patent application. Ethinylestradiol ration of ethinylestradiol. The first ex- ethinylation of steroids containing keto would thus have gone into clinical testing ample, actually already revised in 1937, groups. This likewise applies to the data many years earlier. speaks of formulations of “follicle hor- ascertained by Hohlweg on the physi- mone” and “ethinyldihydrofollicular ological effectiveness of the substance. In addition, Hohlweg has repeatedly em- hormone”. The second, in contrast, re- In view of the protractedness of the then phasized that at the time in question, all fers to “estrone” and “17-ethinylestra- procedure for the granting of a patent, a newly prepared steroids were immediately diol-3,17”. This gives the impression sequence of events of this kind was en- subjected to a physiological test. The as- that the first example must have been tirely usual. sumption that ethinylestradiol had possi- older and written before the papers by bly already been prepared in 1935, but Inhoffen. Also the position of the two Several indications can be found in the not tested, is therefore scarcely probable. examples within the patent specification available sources that in the patent by appears to support this view. Serini and Strassberger, actual use was More complicated is the situation of the made of subsequent filing of process ex- cited patent filed by Kathol, that was Rated still more significant could be the amples, and that ethinylestradiol was not applied for on 22nd November, 1936 and circumstance that Kathol, together with contained in the original application. granted on 14th September, 1939. In con- lnhoffen and Hohlweg, had received from Thus for the development of the sub- nection with this, the author of the present Schering the largest inventor payment for stance Schering later made payments to monograph had to learn that limits are to the development of ethinylestradiol. In several chemists for participation in the be set to the theoretical consideration of comparison to this, the already mentioned invention149. In contrast to Hohlweg, scientific historical sources, disregard of lower payments to several other company Inhoffen and some other chemists, which in some circumstances leads to chemists are of secondary importance. Serini was not considered. Strassberger false conclusions. The danger of false in- Kathol’s contribution must have been of did indeed receive a share, though a rela- terpretations, as will be shown, is particu- considerable importance. tively small one. It may, however, be as- larly great in the case of the theoretical sumed that these payments can be ex- analysis of old patent specifications. That being so, explanation of the cir- plained in relation to another contribu- cumstance whereby ethinylestradiol was tion to steroid chemistry: identical pay- In the patent by Kathol it happens as was first subjected to a physiological test in ments went also to Hildebrandt and pointed out that there are two processes 1937 caused difficulties, and Kathol Logemann whose names appear in none for the preparation of ethinylestradiol, plainly never claimed priority for the of the patent specifications directly that differ enormously with regard to their preparation of the substance. The former linked with ethinylestradiol. efficiency. Whereas the first example of only appeared plausible, if it was as- the synthesis (with a stated yield of just sumed that Kathol had refrained from 3%) in principle corresponds to the other the physiological test because the small specified analogous ethinylation of sub- yield of his process given in the patent 148 See in addition for example, Möller 1936, pp. 51–3. Material patents were inadmissable for food- stances from the androstane series, the did not allow an economical utilization. stuffs, semi-luxuries, medicaments and substances last stated second possibility of preparing The refraining from a claim of priority made by chemical means. Physical chemistry was ethinylestradiol falls completely outside may be understood – admittedly unsatis- an exception: mixtures and metal alloys could mostly be protected by material patents. On alter- this framework. This second process factorily – only as the consequence of a ation of the application, see Möller 1936, p. 231 makes use already of the elegant and par- possible in-company agreement. et seq. ticularly productive conversion in liquid 149 See in addition the file about inventor shares ammonia, that was first described in the “Paper Example” not Repro- by Dr. Josef Kathol (Bestand PA, Schering AG). The chemists participating in the development of literature in 1937! ducible the process received all together, 6% of the net The foundation of all the considerations profit from the sale of Progynon C. Of this, In this state of the sources it can be as- set out above was the assumption that Hohlweg and Inhoffen each received 1.56% and Kathol 1.04%. The rest went to Hildebrandt, sumed that the conversion of estrone to the process, given in Kathol’s patent Logemann and Strassberger (each 0.28%). ethinylestradiol in liquid ammonia in as the first example of the synthesis

54 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone of ethinylestradiol, is actually practi- inventor’s payment received by Kathol estrogen. The estrogen acid aimed at, in cable. This assumption has in retrospect in connection with the development of contrast, could as was shown decades proved to be wrong: in experiments that ethinylestradiol. For what contribution later in no way fulfil the expectations were carried out in the main laboratory of the researcher was it made, if not for imposed on it. Ethinyltestosterone, pre- of Schering AG in February, 1989, es- that patent example? Here too, only pared analogously to ethinylestradiol, trone acetate could be converted to speculation is possible. It appears not and conceived as a possible androgen, ethinylestradiol only in vanishingly improbable, however, that with the pay- proved, likewise quite unexpectedly, to small amounts. Even with modern pre- ment the company made remuneration be an active progestin. The historian parative and analytical procedures it for the development of the process for must here ask himself whether chance was not very easy to isolate the small ethinylation of ketosteroids in liquid am- directed events rather than purposeful amount of ethinylestradiol from the monia, a process which Inhoffen could scientific deliberation. (obtained) mixture of substances in make use of in his preparation of ethi- pure crystalline form. At that time one nylestradiol. Retrospective consideration of the out- had not been able to detect it analyti- lined events shows clearly that as a result cally, to say nothing of isolating it, it Patents do not Affect Inhoffen’s of the elucidation of the structure of says in the discussion of the corre- Priority estrogens, derivatives of these hormones sponding experiments150. To sum up, it is thus established that with better therapeutic applicability ethinylestradiol syntheses in patents ap- were very purposefully sought. Further- Thus it can now be considered certain, plied for before 1937 do not, as at first more, steroid chemists endeavoured to that in the first process given by Kathol appeared, affect Inhoffen’s priority for find ways of partially or completely syn- for the synthesis of ethinylestradiol it the preparation of this steroid. Two of thesizing the only laboriously accessible was a matter of a socalled “paper ex- these examples are of processes that, in natural substances. Thus were first ob- ample”. Patent examples were labelled accordance with the legal requirements tained the estrogen esters whose effect with this term when they were theoreti- in the course of the granting of a patent, was protracted, and the estrogen acids cally conceivable, though their practica- had been subsequently filed. The third, in the course of well considered investi- bility had never been closely investi- in retrospect, turns out to be a “paper gations. The cited efforts are those most gated. They served to word a patent example” that is not practicable. This closely linked with the names of Hohl- claim as widely as possible and thus to whole situation shows that the use of weg, Inhoffen, Doisy and Butenandt. ensure advantages over the economic patent specifications for the clarification competition. of questions of priority can be extremely Several Factors were Decisive problematic. It appears as a rule to be Immediately decisive in the discovery of With regard to the Kathol patent, there permissible only if the practicability of ethinylestradiol were several factors: are only two questions still unanswered. the processes described therein is proved Hohlweg’s urging to prepare a 17-estra- The first concerns the time at which the to be beyond doubt. diol acid, the work of Kathol, Logemann paper example was introduced. Here the and Serini on ethinylation and Inhoffen’s use of the revised nomenclature justifies Consistent Continuation of transfer of this reaction to estrone, the assumption that it had already been a Research as well as the physiological testing of component of the original application. In the case of the synthesis of ethinyl- the intermediate obtained, again by Final certainty could, however, be given testosterone (ethisterone), there are from Hohlweg. only by the files, no longer existing, the very beginning no difficulties with about the course of the patentgranting regard to the question of priority. The The importance of the preparatory work procedure. For the judging of the ques- preparation of the first artificial orally in Schering’s main laboratory on the tion of priority, this now appears point- effective progestin by Inhoffen must be ethinylation of steroids that finally less. The final clarification of this ques- regarded as the result of consistent con- ended in the productive process with the tion would only be of weight for a dis- tinuation of his and Hohlweg’s work on conversion in liquid ammonia, cannot be cussion of the patent law situation in ethinylestradiol. This work was in turn too highly esteemed. Without this deci- which Schering found itself at that time. based on the investigations of the sive progress, Inhoffen would perhaps In another connection, this will be Schering chemists Kathol, Logemann have sought for another way of synthe- briefly discussed later. and Serini, who at about the same time sizing the desired estradiol acid, and the as the Swiss chemists Ruzicka and experimental test of ethinylestradiol on The second, still open, question is Hofmann had developed an elegant animals would not have been done for thrown up by the relatively large method of ethinylating steroids with a some time. yield of over 90%. However, here it must also be pointed 150 The author thanks Dr.-Ing. Henry Laurent, the lt has been fully outlined that ethinyl- out that the elegant and productive then manager of the Scientific Secretariat in the estradiol was actually only intended to ethinylation of steroids in liquid ammo- Institute for Medicament Chemistry of Schering AG, for the idea of testing Example 1 in Kathol’s be an intermediate in the synthesis of a nia was to some extent “in the air” in patent for its practicability, and carrying out the 17-estradiol acid. There were no great 1937. Ruzicka and Hofmann discovered corresponding experiment. Only in this way may hopes linked with this intermediate sub- the process at least at the same time as a false interpretation of available sources be avoided. See in addition the experiment protocol stance and it was a matter of surprise the Schering chemists, if not before. lt in Frobenius 1990 (Appendix, p. 97). when it proved to be a highly effective appears quite conceivable that Example

J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) 55 Development of Ethinylestradiol and Ethinyltestosterone

8 was subsequently inserted in Kathol’s use in 1939. Closer consideration of the clinicians could continue their work patent, because the method had previ- course of the clinical trials of ethi- comparatively unhindered, for their col- ously been patented for Ruzicka or nylestradiol is a good example of what leagues active in Germany, conditions CIBA, and therefore could not be pat- difficulties have to be overcome in the in- became steadily worse: many of them ented by Schering. Since the example troduction of a highly effective new medi- were called up for military service, and also occurred in the claim of Kathol’s cament, and how decisive in this sort of the exchange of scientific information patent, it could be “saved” in this patent case is the effect of systematic procedure. was limited as investigations important specification. Another application in to the war effort and the basic care of which the example was originally con- An essential reason for the delayed in- patients had absolute priority. Further- tained must possibly have been with- troduction of ethinylestradiol is cer- more, since 1933 the National Socialists drawn or was rejected; only the parallel tainly to be found in the fact that the sub- had discriminated against numerous out- application in the USA for Hohlweg and stance, when orally administered to hu- standing researchers because of their po- Inhoffen succeeded. This view of the man beings, displays such extreme es- litical attitude or their race, and had matter is supported by the fact that a trogenic activity. The results of the tests forced them to emigrate or had even German patent corresponding to the US on animals could give only an inad- killed them. Under these circumstances patent cannot be found, although in the equate indication of this. Hohlweg’s in- clinical studies of an estrogenic hor- US patent reference is made to a German vestigations had indeed shown that in the mone could scarcely thrive152. application on 25th October, 1937. Allen-Doisy test on rats, 3 µg of the sub- stance, administered orally, were effec- When looking through the publications The physiological test of ethinylestradiol tive and thus ethinylestradiol exceeded of Hohlweg and Inhoffen on the ethinyl itself can, quite simply, not be regarded as the natural estrogens by a factor of 3 to compounds, however, the impression a product of chance. Hohlweg has repeat- 20. The actual potency of the medica- arises that the observed incompatibility edly pointed out that in his laboratory all ment in the treatment of women could, phenomena in the first clinical tests of the substances produced by syntheses had however, not be estimated from this. ethinylestradiol led the people in the to be tested for their hormonal activity. To Schering main laboratory too quickly to this systematic procedure, to which For the reasons stated, the first clinical a pessimistic assessment of the future Dohrn had already in the 1920s ascribed investigators used doses that in retro- chances of the preparation. As an indica- the greatest importance at Schering151, spect certainly were in the toxic region. tion of this an instance may be taken many other successes of the company are The administered amounts of the estrone which has already been referred to: in due. The discovery resulting from the derivative as in the case of the natural the publication about pregneninolone in physiological test is accounted for in what estrogens, their esters and also stilbestrol the Klinische Wochenschrift of 1939 Inhoffen (in his lecture) has called the were in the milligram region and must there is practically no longer any men- “problem of the unpredictability of the have triggered massive incompatibility tion of ethinylestradiol, although its ex- paths from fundamental research to ap- phenomena. Relevant observations were cellent effectiveness in women after plied chemistry”. then soon made and communicated by Clauberg’s publication in 1938 must Buschbeck. It is, however, extremely re- have been unquestionable. In 1950, From this aspect also is to be viewed the markable that there is no mention of this Hohlweg himself still favoured a stil- preparation of ethinyltestosterone and problem in Clauberg’s paper of 1938. bene derivative amongst the estrogens the discovery of its progestin effect. Other investigators were caused by the that could be administered orally. Here, obviously, considerations of the observed sideeffects to discontinue their assumed structure of the molecule had studies at an early stage. The reasons the Schering Corporation in already led to the correct path, after the the USA had for nevertheless making originally expected androgen effect had Thus the incompatibility phenomena ethinylestradiol available for clinical not been detectable in experiments on consequent upon overdosing formed the trials, can only be speculated upon. Pos- animals. most substantial hurdle that ethinylestra- sibly the responsible staff in the Ameri- diol had to take on its triumphant ad- can subsidiary viewed the preparation First Tests in the Toxic Region vance towards the goal of being the more optimistically. Perhaps they saw Finally, the circumstances should now be therapeutically most used estrogen. The themselves induced to do so for com- discussed which accompanied the question now arises as to why so few petitive reasons, since ethinylestradiol introduction of ethinylestradiol into efforts were made in Germany to help was soon offered there by other compa- therapy. As was comprehensively shown the preparation to overcome this ob- nies (CIBA Pharmaceutical Products after the first clinical test by Clauberg, it stacle. The present monograph shows took more than 10 years before the sub- that the foundations for its clinical use stance was put on the market in the Fed- had been laid almost exclusively through 152 Carl Kaufmann for example, who never made eral Republic of Germany. In marked investigations by American authors dur- a secret of his rejection of National Socialism and contrast to this, ethinyltestosterone had ing the 1940s. therefore got into great difficulties, did not pub- lish from about 1941 until the immediate post- already been released for general medical war period. Zander wrote in his obituary on the Chances Wrongly Estimated? scientist: “Heavy personal burdens, the air attacks Here an important part was certainly on Berlin, besides clinical responsibilities in the 151 Letter by Dohrn to the Board of Directors of Gynaecological Hospital of the Charité, no longer Schering on 15th December 1918. Scheringianum, played by World War II. Whereas at this allowed scientific publications” [Zander 1981, p. Archive no B1/84. time researchers among the American 83].

56 J Reproduktionsmed Endokrinol 2011; 8 (Special Issue 1) Development of Ethinylestradiol and Ethinyltestosterone and RocheOrganon) for clinical trials, as range) were soon abandoned. Already proliferation of the endometrium is at- the papers of American clinicians show. from 1942 onward (Groper et al.) the tained. The dose of 5 µg is adequate to Questions of patent law that arise from hormone doses used ranged from 150 to restore an atrophied vaginal epithelium this fact cannot be considered here. lt 50 µg per day, and such doses were later to its premenopausal condition. In a may be appropriate here to say no more also used for the first generation of con- group of climacteric women, 80% than that the European steroid producers traceptives. Unfortunately, nowhere is it could be freed of hot flushes with a in those years had entered into various possible to find any indication as to what dose of l5 µg per day, but an increase of agreements not to block each others’ pat- considerations or investigations were this dose brought no further improve- ents. Tausk speaks in this connection of determinants for this. Thus it can only be ment in the result. Ovulation can cer- the “Hormone cartel”153. guessed that the maximal tolerable doses tainly be stopped only with 100 µg, a were approached, so to speak, from circumstance that because of the clearly Undoubtedly of great importance for the above. increased morbidity risk at this dose, relatively slow reception of ethinylestra- leads to a contraceptive based only on diol was the discovery of the estrogenic From the present standpoint this appears ethinylestradiol appearing contraindi- effect of the stilbenes. These prepara- surprising, since with the Kaufmann ex- cated154. tions could – as mentioned – be made periment there already existed a model easily and cheaply. They were likewise that made possible the determination of The position that ethinylestradiol at the orally highly active and in the initial a threshold dose of a new estrogen by present time still occupies, as the most phase of the clinical testing (because of means of morphological findings. The used estrogen component of hormonal their wide therapeutic range) they were investigators in the USA, however, ini- contraceptives, more than 70 years after easier to use than the estrone derivative. tially restricted themselves, as Clauberg its discovery, has already been indicated They were therefore able to attract the had earlier done, to fundamental detec- in the introduction. lt has also already attention of a considerably wider range tion of an estrogenic effect and testing of been mentioned, that ethinyltestosterone of clinicians. The innumerable investi- the compatibility of the substance. In must be considered as forerunner of a gations of these compounds, carried out addition to clinical observations and large group of artificial progestins, that at the end of the 1930s and in the 1940s, endometrium findings, in the many stud- at the present time are indispensable for provide evidence of this. But they also ies of the treatment of the postmeno- treatments with sex hormones. Both are show, that even at that time there was a pausal syndrome, use was also made of here in conclusion once again called to lively interest in a cheap estrogen, with vaginal cytology. First indications of the mind, in order to underline the impor- good oral activity, although hormonal endometrium synthesis dose came after tance of the work that chemists, physi- contraception was still far away. The first 1944 in papers by W. Allen, Bickers and ologists and clinicians have done in con- indication for use was the climacteric Birnberg. Its more accurate determina- nection with the discovery of both sub- syndrome. tion remained reserved – so far as can be stances. seen to German authors at the beginning The Endometrium Synthesis of the 1950s. At that time, ethinylestra- Dose was Determined much diol had already been introduced into  Conflict of Interest Later therapy. In the early American papers on clinical The research for this article was sup- trials of ethinylestradiol, the originally lt is now known, that with ethinylestra- ported by a grant from the former used high daily doses (in the milligram diol at 50 µg per day a plateau in the Schering AG Berlin.

153 See in addition [Tausk M. Organon. The story of an unusual pharmaceutical enterprise. Published by Akzo Pharma bv, Oss, The Netherlands 1984; 88–93.] 154 See in addition [Kuhl H, Taubert HD. Das Klimakterium. Pathophysiologie, Klinik, Therapie. Georg Thieme Verlag Stuttgart-New York, 1987; 118.]

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