Drayer, N.M.

Hummelen, J.C.

Kraan, G.P.B.

Kruizinga, W.H.

Vaalburg, W.

English

13C2-17α-ethinylestradiol (13C2-EE2) was synthesized from estrone and 13C2-C2H2-gas to measure the metabolic clearance rate and the plasma concentration of 17α-ethinylestradiol (EE2) in tall girls, who are treated with high dosages of this estrogen. Interesting characteristics determined by (i) MS: the molecular ion m/z 298.183 is the only labelled ion in the spectrum, and by (ii)1H-NMR δ(ppm) 2.96 (dd, 1J(13C-H)=249 Hz, 2J(13C-H)=49.6 Hz; 1H; H20) and (iii) 13C-NMR δ 88.8(dd, 1J(13C-13C)=166 Hz, 2J(13C-H20)=49.8 Hz; C19); 80.2 (dd, 1J(13C17-13C19)= 73 Hz, 2J(13C17-13C20)=12.3 Hz; C17) and 74.6 (dd, 1J(13C-13C)=166 Hz, 1J(13C-H)= 249 Hz; C20) confirm the synthesis and structure of 13C2-EE2

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   University of GroningenSynthesis and Physicochemical Properties of [19,20-13C]-17α-EthinylestradiolKraan, G.P.B.; Drayer, N.M.; Kruizinga, W.H.; Vaalburg, W.; Hummelen, J.C.Published in:Synthesis and Applications of Isotopically Labelled CompoundsIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1989Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Kraan, G. P. B., Drayer, N. M., Kruizinga, W. H., Vaalburg, W., & Hummelen, J. C. (1989). Synthesis andPhysicochemical Properties of [19,20-13C]-17α-Ethinylestradiol. In Synthesis and Applications ofIsotopically Labelled Compounds: Proceedings of the Third International Symposium, Innsbruck, Austria,17 -21 July 1988 (pp. 705-708). Elsevier.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 07-06-2022Synthesis and Applications of Isotopically Labelled Compounds 1988 705Proceedings of the Third International Symposium, Innsbruck, Austria, 17 -21 July 1988,T.A. Baillie and J.R. Jones (Editors), 705-708© 1989 Elsevier Science Publishers B.V., Amsterdam - Printed in The NetherlandsSYNTHESIS AND PHYSICOCHEMICAL PROPERTIES OF [19,20-13C]-17α-ETHINYLESTRADIOLG.P.B. KRAAN1, N.M. DRAYER1, J. KOEK2, W.H. KRUIZINGA2, W. VAALBURG2 and J.C.HUMMELEN2.1. Department of Pediatrics, University of Groningen, Oostersingel 59, 9713EZ Groningen (The Netherlands)2. Department of Organic Chemistry, University of Groningen, Nijenborgh 16,9747 AG, Groningen (The Netherlands)SUMMARY13C2-17α-ethinylestradiol (13C2-EE2) was synthesized from estrone and 13C2-C2H2-gas to measure the metabolic clearance rate and the plasma concentrationof 17α-ethinylestradiol (EE2) in tall girls, who are treated with high dosagesof this estrogen. Interesting characteristics determined by (i) MS: themolecular ion m/z 298.183 is the only labelled ion in the spectrum, and by(ii)1H-NMR δ(ppm) 2.96 (dd, 1J(13C-H)=249 Hz, 2J(13C-H)=49.6 Hz; 1H; H20) and(iii) 13C-NMR δ 88.8(dd, 1J(13C-13C)=166 Hz, 2J(13C-H20)=49.8 Hz; C19); 80.2 (dd,1J(13C17-13C19)= 73 Hz, 2J(13C17-13C20)=12.3 Hz; C17) and 74.6 (dd, 1J(13C-13C)=166 Hz,1J(13C-H)= 249 Hz; C20) confirm the synthesis and structure of 13C2-EE2.INTRODUCTIONAdministration of estrogens to healthy, (pre)pubertal tall girls willcause a decreased growth rate, accelerated sexual maturation, epiphysialfusion and, as desired, a reduced final adult height (ref. 1).In order to keep the potential complications of the estrogen therapy,existing of 2x 50 µg 17α-ethinylestradiol (EE2) per os daily, as low aspossible it would be necessary to measure carefully the individualconcentration time relationship of EE2 in plasma in relation to growth rate,skeletal maturation and other parameters. For this purpose we decided tosynthesize 13C-labelled 17α-ethinylestradiol (13C2-EE2) acoording to thepreviously published method (ref.2).In this paper the synthesis and main physicochemical characteristics of13C2-EE2 are described.METHODSInstrumentationCentrifugal liquid chromatography was performed on a Hitachi CLC-5instrument. A rotating disk of 3 mm silicagel 60-G (Merck, Darmstadt, G.) and10 cm path length was eluted with chloroform (Merck, p.a.) at a rate of 8.3ml. min-1 at 480 rpm approximately. Mass spectra were recorded on a706AEI-MS-9 spectrometer. The infrared spectrum was recorded on a Unicam SP-200spectrophotometer in a KBr pellet. Nuclear magnetic resonance spectra wererecorded on a Varian-VXR-300 at 300 MHz for 1H-NMR and at 75.48 MHz for 13C-NMRin CD3OD. 1H-chemical shifts are reported in δ units (ppm) relative to TMS and13C-chemical shifts relative to CD3OD.SynthesisApproximately 3 mmoles 13C2-C2H2 gas(MSD Isotopes) was condensed in a 250 mlthree necked flask containing a mixture of 1.5 ml 1.5 N n-Buli/n-hexane and 15ml distilled THF at -115°C. During warming up to 0°C the mixture was stirred.Then a solution of 1.1 mmole estrone (Aldrich) in 20 ml dry THF was addeddropwise and the mixture was stirred during 30 minutes at 0°C and 15 minutesat 40°C. After cooling to room temperature a solution of 12 g NH4Cl in 60 mlH2O was added dropwise and the resulting mixture was stirred 30 minutes. Theproduct was extracted with CHCl3 (2x 100 ml) and the organic layer was driedon MgSO4, filtered and evaporated to dryness to yield 365 mg of crude steroidas a white powder. Spectroscopically pure 13C2-EE2 was obtained afterchromatography on a Hitachi CLC-5 chromatograph using CHCL3 as the eluent andmonitoring at 254 nm. After two runs 0.67 mmole of pure 13C2-EE2 was obtained asa white mass.RESULTSFig.1 shows the structure and the numbering of the carbon atoms of 13C2-EE2(13C indicated by an asterix).The mass spectra of 13C2-EE2 (a) and EE2 (b) are shown in fig. 2. Apart fromvery small fragments at m/z > 250 the only labelled fragment is the molecularion of m/z 298. The exact mass of the molecular ion was measured andcalculated to be 298.183 and 298.184, respectively.Absorbance maxima of 13C2-EE2 in the infrared were recorded at 3350 (s),2950 (s), 1620 (m), 1510 (m), 1460 (m), 1290 (m), 1240 (m) and 1025 (s) cm-1.1H-NMR of 13C2-EE2 was recorded in CD3OD at 300 MHz. The chemical shiftsare given in δ units(ppm) relative to TMS: 7.21(d,J=8.4 Hz;1H;H2);6.72(d, J=8.4 Hz; 1H;H1);6.65(s,1H;H4);2.96(dd,1J(13C-H)=249 Hz,2J(13C-H)=49.6 Hz;1H;H20);2.9(m, 2H);2.45(m;2H);2.33-1.8(m;7H);1.65-1.3(m;4H);1.00(s,3H;H18).13C-NMR of 13C2-EE2 was recorded in CD3OD at 75.48 MHz. The chemical shiftsare given in δ units (ppm) relative to CD3OD. The assignmentsof these shiftsto the carbon atoms were derived from the APT spectrum and the coupled anduncoupled spectra. The uncoupled spectrum (fig. 3) shows strong resonance at88.8(dd,1J(13C-13C)=166Hz,2J(13C-H20)=49.8 Hz;C19);74.6(dd,1J(13C-13C)=166 Hz,1J(13C-H)=249Hz;C20),707708and weak resonance at 155.5(s;C3),138.7(s;C5);132.6(s;C10);127.1(d;C1);116.1(d;C4); 113.7(d;C2);80.2(dd,1J(13C17-13C19)=73 Hz,2J(13C17-13C20)= 12.3Hz;C17);50.7(d;C14);48.1(s;C13);44.8(d;C9);40.9(d;C8); 39.9(t;C16);133.9(t;C12);30.5(t;C6);28.5(t;C7);27.6(t;C11);23.6(t;C15);13.3(q; C18).CONCLUSIONFrom the characteristics of the steroid determined by IR, MS and NMR itcan be concluded that indeed [19,20-13C]-EE2 is synthesized.REFERENCES1 N.J. Charest and J.J. Van Wyk, Tall stature, in: S.S. Gellis and B.M. Kagan(Eds), Current Pediatric Therapy, W.B. Saunders Cy, Philadelphia, 1986, pp.299-301.2 W. Vaalburg, S. Reiffers, E. Beerling et al., The preparation of carbon-11labeled 17α-ethinylestradiol, J. Label Cmpds Radiopharm, 12 (1977), 200-201.

Synthesis and Physicochemical Properties of [19,20-13C]-17α-Ethinylestradiol

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Synthesis and Physicochemical Properties of [19,20-13C]-17α-Ethinylestradiol

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