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UBC Theses and Dissertations

New methods for rapid radiohalogen labelling of organic molecules Balatoni, Julius Alexander 1985

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NEW METHODS FOR RAPID RADIOHALOGEN L A B E L L I N G OF ORGANIC MOLECULES by JULIUS ALEXANDER BALATONI S c . , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Depar tment o f C h e m i s t r y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA November 1985 © J u l i u s A l e x a n d e r B a l a t o n i , 1985 In p r e s e n t i n g this thesis in partial fu l f i lment of t h e requ i rements for an a d v a n c e d d e g r e e at the Univers i ty of Br it ish C o l u m b i a , I agree that the Library shall m a k e it f reely avai lable for re fe rence a n d s tudy . I further agree that pe rm iss ion for ex tens ive c o p y i n g o f this thesis for scho lar ly p u r p o s e s may be g ranted by the h e a d of m y d e p a r t m e n t o r by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f this thesis for f inancia l gain shall no t b e a l l o w e d w i t h o u t m y w r i t t e n pe rm iss ion . D e p a r t m e n t o f Chemistry T h e Un ivers i ty o f Brit ish C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a V 6 T 1Y3 D a t e Jan. 14, 1986 i i ABSTRACT The a im of t h i s s t u d y was t o d e v e l o p new and r a p i d p r o c e d u r e s f o r the i n c o r p o r a t i o n o f s h o r t - l i v e d r a d i o b r o m i n e and r a d i o i o d i n e i s o t o p e s i n t o o r g a n i c compounds. T h i s was a c c o m p l i s h e d by t h e e l e c t r o p h i l i c c l e a v a g e o f t i n - c a r b o n bonds w i t h B r C l and IC1 g e n e r a t e d i n - s i t u f r o m b r o m i d e and i o d i d e , r e s p e c t i v e l y , i n the p r e s e n c e o f an o x i d i z i n g a g e n t . The v i n y l - t i n d e r i v a t i v e s , [ 4 ] , [ 8 ] , [ 1 2 ] , [ 1 7 ] , [18] and [ 1 9 ] , were p r e p a r e d by t r e a t m e n t o f t h e c o r r e s p o n d i n g a c e t y l e n i c compounds w i t h t r i - n - b u t y l t i n h y d r i d e , i n t h e p r e s e n c e o f a r a d i c a l i n i t i a t o r c a t a l y s t , i n 51-98% y i e l d . E a c h o f t h e v i n y l - t i n d e r i v a t i v e s , e x c e p t [ 1 7 ] , was b r o m i n a t e d i n h i g h y i e l d (89-100%) w i t h s o d i u m b r o m i d e o x i d i z e d by C h l o r a m i n e - T u n d e r a c i d i c c o n d i t i o n s . Compound [17] was b r o m i n a t e d i n 13% y i e l d ; c o m p e t i n g b r o m i n a t i o n o f t h e A - r i n g o f [17] was r e s p o n s i b l e f o r t h i s low y i e l d . The v i n y l - t i n p r e c u r s o r s [ 4 ] , [ 8 ] , [ 1 7 ] , [18] and [19] were i o d i n a t e d w i t h s o d i u m i o d i d e i n t h e p r e s e n c e o f a c i d i f i e d C h l o r a m i n e - T i n 88-99% y i e l d , e x c e p t [ 1 7 ] , w h i c h was i o d i n a t e d i n 1.9% y i e l d b e c a u s e o f f a c i l e A - r i n g i o d i n a t i o n . Compound [18] was s u b s e q u e n t l y r a d i o - l a b e l l e d w i t h 8 2 B r , I23j a n ( j 131j f o r m i n g [ 4 1 ] , [42] and [ 4 3 ] , r e s p e c t i v e l y , u s i n g the C h l o r a m i n e - T m e t h o d . Compound [41] was i s o l a t e d i n 77-81% r a d i o c h e m i c a l y i e l d u s i n g low s p e c i f i c a c t i v i t y N H ^ B r . Compounds [42] and [43] were o b t a i n e d i n 82-90% r a d i o -c h e m i c a l y i e l d , i n r e l a t i v e l y h i g h s p e c i f i c a c t i v i t y . The m a j o r s i d e - p r o d u c t f o r m e d d u r i n g t h e n o - c a r r i e r - a d d e d r a d i o i o d i n a t i o n s was t h e c h l o r i n a t e d d e r i v a t i v e [ 3 8 ] . An a l t e r n a t e p r o c e d u r e , b a s e d on N - c h l o r o s u c c i n i m i d e as t h e o x i d a n t , was d e v e l o p e d w h i c h gave [41] and [42] i n 85% and 62% ( n o - c a r r i e r -added 1 2 3 I ) r a d i o c h e m i c a l y i e l d s , r e s p e c t i v e l y . i i i [17] R' =R 2=H [18] R'=Me, R 2=H [19] R' =R2= Me X [ 41 ] X =82Br U2]X = , 2 3 I U3]X= , 3 ,I [38] X=Cl i v TABLE OF CONTENTS Chapter Page I INTRODUCTION 1 1.1 Background 1 1.2 The Problem 6 1.3 Synthetic Strategy 15 II DEVELOPMENT OF METHODS FOR RAPID LABELLING WITH NON-RADIOACTIVE BROMINE AND IODINE 20 11.1 Introduction 20 11.2 Synthesis of V i n y l - T i n Precursors 23 11.3 Bromine L a b e l l i n g Studies 35 11.4 Iodine L a b e l l i n g Studies 44 II I LABELLING STUDIES WITH RADIOACTIVE BROMINE AND IODINE 52 111.1 Introduction 52 111.2 Radiobromination Studies 52 111.3 Radioiodination Studies 59 111.4 Summary and Conclusions 69 V Page IV EXPERIMENTAL 72 IV.1 General Methods 72 IV.2 Nmr Methods and Instrumentation 73 IV.3 Sources of Materials 74 IV.4 Synthesis of Acetylenic Precursors 75 IV.5 Synthesis of V i n y l - T i n Compounds 79 IV.6 L a b e l l i n g with Non-radioactive Bromine 85 IV.7 L a b e l l i n g with Non-radioactive Iodine 92 IV.8 Synthesis of Chromatographic Standards for Rad i o - l a b e l l i n g 99 IV.9 L a b e l l i n g with Radioactive Bromine 101 IV. 10 L a b e l l i n g with Radioactive Iodine 103 IV.11 Nmr Characterization Data 105 REFERENCES 113 APPENDIX 118 Selected S t r u c t u r a l Formulae 118 vi LIST OF TABLES Table Page CHAPTER I I-l Half-lives of Selected Radionuclides used in Medicine 8 1-2 Physical Properties of Some Radioisotopes 11 1-3 Medically Useful Radioisotopes of Bromine and Iodine 15 1-4 Summary of Major Radiohalogenation Methods 16 CHAPTER II II-l Chemical Shifts (ppm) and Coupling Constants (Hz) of the Vinyl Protons of the Tin Compounds 34 II-2 Yields of Vinyl-Tin Derivatives 34 II-3 Yields of Vinyl-Brominated Derivatives 37 II-4 Chemical Shifts (ppm) and Coupling Constants (Hz) of the Vinyl Protons of the Brominated Compounds 37 II-5 Yields of Vinyl-Iodinated Derivatives 45 II- 6 Chemical Shifts (ppm) and Coupling Constants (Hz) of the Vinyl Protons of the Iodinated Compounds 45 CHAPTER III III- l Summary of Radioiodination Results 60 CHAPTER IV IV- 1 Chemical Shifts (ppm) and Multiplet Splittings (Hz) for the Carbohydrate Derivatives 106 IV-2 Chemical Shifts (ppm) and Multiplet Splittings (Hz) for the Steroidal Derivatives 108 v i i LIST OF FIGURES Figure Page CHAPTER I 1-1 Decay of positron emitting isotope r e s u l t s i n an a n n i h i l a t i o n event 3 1-2 PET process has four basic steps: (a) detection, (b) data recorded as p r o f i l e s , (c) p r o f i l e s are f i l t e r e d (convolution) and (d) back-projection to form image 4 I- 3 General scheme for the production of a radiopharmaceutical 13 CHAPTER I I I I - l Synthetic schemes for the preparation of the v i n y l -halogenated model compounds [5], [6], [9], [10] and [13] 21 II-2 Synthesis scheme for the preparation of vinyl-halogenated e s t r a d i o l d e r i v a t i v e s 22 II-3 Synthetic schemes for the preparation of carbohydrate (scheme 1) and e s t r a d i o l (scheme 2) acetylenic precursors ... .24 II-4 Simulated mass s p e c t r a l isotope pattern f o r the M^-C^Hg fragment (base peak) of compound [4] 26 II-5 The numbering scheme of the carbon skeleton of the carbohydrate compounds 27 II-6 Hydrostannylation reaction of compound [14], i n r e f l u x i n g 1,4-dioxane, produces a major side-product [26] i n addition to the desired product [17] .'29 II-7 The numbering scheme of the carbon skeleton of the e s t r a d i o l compounds 30 II-8 270 MHz *H nmr spectrum of compound [17] i n deuter iochlorof orm ....31 II-9 270 MHz XH nmr spectra of the v i n y l - t i n precursor [18] (spectrum A) and the vinyl-brominated product [22] (spectrum B), obtained i n deuteriochloroform 39 11-10 Side-products obtained during the preparation of compound [20] 41 v i i i 11-11 270 MHz 1H nmr spectra of the vinyl-tin precursor [18] (spectrum A) and the vinyl-iodinated product [23] (spectrum B), obtained in deuteriochloroform 47 11-12 Side-products obtained during the preparation of compound [21] 49 II- 13 Partial lK nmr spectra of side-products [36] (spectrum A) and [37] (spectrum B), as compared to compound [21] (spectrum C) 50 CHAPTER III III- l Synthesis of compound [41] 54 III-2 HPLC chromatograms obtained during purification of compound [41] 55 III-3 Synthesis scheme for the preparation of the vinyl derivative [39] 58 III-4 Synthesis of the radioiodinated compounds [42] and [43] 60 III-5 HPLC chromatograms obtained during purification of compound [42]....' 61 III-6 HPLC chromatogram obtained during the purification of compound [43] 62 III-7 Synthesis of compound [38] 63 III-8 270 MHz 1H nmr spectrum of compound [38] in deuteriochlorof orm 65 III-9 HPLC chromatograms obtained during purification of compound [42] • 67 ACKNOWLEDGEMENT I w i s h t o t h a n k D r . L . D . H a l l f o r h i s c o n s t a n t s u p p l y o f e n c o u r a g e m e n t , p a t i e n c e , s t i m u l a t i n g d i s c u s s i o n s and m o r a l s u p p o r t . I am v e r y g r a t e f u l to D r . M . J . Adam f o r many h e l p f u l d i s c u s s i o n s , and h i s t e c h n i c a l a s s i s t a n c e and d i r e c t i o n i n t h e r a d i o - l a b e l l i n g p o r t i o n o f t h i s w o r k . I am p a r t i c u l a r l y t h a n k f u l f o r h i s p r o v i s i o n o f h o t l a b f a c i l i t i e s and i n s t r u m e n t a t i o n . I w o u l d l i k e to a c k n o w l e d g e D r . J . R . N e s s e r , who p r o v i d e d i n v a l u a b l e a s s i s t a n c e i n t h e e x p e r i m e n t a l and c o n c e p t u a l d e v e l o p m e n t o f t h i s p r o j e c t . A l t h o u g h t o o numerous t o name i n d i v i d u a l l y , I w o u l d l i k e t o t h a n k t h e g r a d u a t e s t u d e n t s who were p r e s e n t i n t h i s l a b d u r i n g my M . S c . work as t h e y c o n t r i b u t e d i n many ways t o my e d u c a t i o n . I w o u l d a l s o l i k e to t h a n k D r s . T . R u t h and P . L e g z d i n s f o r h e l p f u l d i s c u s s i o n s c o n c e r n i n g t h i s w o r k . F i n a l l y , my t h a n k s go t o L e s l i e I v a n y and K a r e n S h l a n k a f o r the s y n t h e s i s o f a number o f c h e m i c a l p r e c u r s o r s . CHAPTER I 1 INTRODUCTION I.1 Background During the l a s t f i f t e e n years, medical science has experienced a rev o l u t i o n i n the area of medical imaging. Although most of the imaging methods currently i n use have existed for a much longer period, i t was the widespread use of computers and d i g i t a l e l e c t r o n i c s i n the 1970's, combined with other technical improvements, that have f u e l l e d t h i s s i g n i f i c a n t growth. The many imaging methods, which now e x i s t , f a l l into two broad categories, "transmission imaging" and "emission imaging". Transmission imaging can be performed using beams of X-rays, gamma-rays, neutrons or charged p a r t i c l e s ; i n a l l cases the beam i s passed through an object and then observed by a detection system located on the side opposite to the transmitter. In contrast, i n emission imaging a radionuclide i s administered to the patient and the d i s t r i b u t i o n of the radionuclide i s then observed by an external detection system which monitors the emitted gamma-rays. Neither Ultrasound imaging nor the newest imaging technique, Nuclear Magnetic Resonance (NMR) imaging f i t e a s i l y into the above imaging c a t e g o r i e s . 1 - , t The general purpose of medical imaging i s to provide useful information leading to e i t h e r an understanding of human b i o l o g i c a l processes, forming or confirming a diagnosis, or for guiding and monitoring therapy. The information required for these purposes i s twofold; f i r s t , confirmation of the 2 p r e s e n c e , o r a b s e n c e , of p a t h o l o g y ; and s e c o n d , c h a r a c t e r i z a t i o n o f t h e p a t h o l o g y o b s e r v e d . D e p e n d i n g on t h e i m a g i n g method c h o s e n , an image o f a b r a i n t u m o r , f o r e x a m p l e , may be o b s e r v e d as a change i n m o r p h o l o g y ; t h i s w o u l d be s e e n i n a c o n v e n t i o n a l X - r a y i m a g e . A l t e r n a t i v e l y , t h a t same b r a i n tumor may be o b s e r v e d by a change i n p h y s i o l o g i c a l f u n c t i o n , s u c h as t h e g l u c o s e m e t a b o l i s m , o r b l o o d f l o w i n the b r a i n ; 2 e i t h e r o f t h e s e p h y s i o l o g i c a l f u n c t i o n s c o u l d be o b s e r v e d w i t h e m i s s i o n i m a g i n g u s i n g a p p r o p r i a t e r a d i o p h a r m a c e u t i c a l s . E m i s s i o n i m a g i n g , w h i c h has been d e v e l o p e d as p a r t o f n u c l e a r m e d i c i n e , has p r o d u c e d two d i f f e r e n t t o m o g r a p h i c i m a g i n g methods w h i c h d i f f e r i n t h e n a t u r e o f the r a d i o n u c l i d e l a b e l u s e d . The f i r s t , S i n g l e P h o t o n E m i s s i o n Computed Tomography ( S P E C T ) , i s b a s e d on t h e use o f g a m m a - e m i t t i n g r a d i o n u c l i d e s . 3 The o t h e r , w h i c h i s a more r e c e n t d e v e l o p m e n t , P o s i t r o n E m i s s i o n Tomography ( P E T ) , i s b a s e d on t h e u s e o f p o s i t r o n - e m i t t i n g r a d i o n u c l i d e s . 5 B o t h methods p r o d u c e images by s i m i l a r c o u n t i n g m e t h o d s . The k e y d i f f e r e n c e i s t h e p h y s i c a l p r o p e r t i e s o f t h e r a d i a t i o n e m i t t e d and hence i t s d e t e c t i o n . To i l l u s t r a t e how t h e s e t e c h n i q u e s w o r k , t h e p r o c e s s e s i n v o l v e d i n a PET measurement w i l l be b r i e f l y d e s c r i b e d . A r a d i o p h a r m a c e u t i c a l , l a b e l l e d w i t h a p o s i t r o n e m i t t i n g i s o t o p e , i s a d m i n i s t e r e d to t h e p a t i e n t . I n s i d e the b o d y , t h e r a d i o i s o t o p e d e c a y s by e m i t t i n g a p o s i t r o n ( p o s i t i v e e l e c t r o n ) . As t h e p o s i t r o n t r a v e l s a few m i l l i m e t e r s i t l o s e s s u f f i c i e n t e n e r g y t h a t i t c a n combine w i t h an e l e c t r o n , i t s a n t i m a t t e r t w i n , and u n d e r g o an a n n i h i l a t i o n e v e n t w h i c h p r o d u c e s two gamma-ray p h o t o n s o f 511 KeV e a c h . T h e s e t r a v e l o f f , i n o p p o s i t e d i r e c t i o n s a p p r o x i m a t e l y 1 8 0 ° a p a r t ( s e e f i g u r e 1-1) p e n e t r a t i n g 3 A POSITRON ANNIHILATION EVENT # Positron emitting atom V - ^ e + / \—1 r '7- ray 511 KeV 511 KeV nearly 180* apart F i g u r e I - l . Decay o f p o s i t r o n e m i t t i n g i s o t o p e r e s u l t s i n an a n n i h i l a t i o n e v e n t . t h e s u r r o u n d i n g t i s s u e , and a r e d e t e c t e d by a c i r c u l a r a r r a y o f c o i n c i d e n c e d e t e c t o r s ( s e e f i g u r e I - 2 a ) e n c i r c l i n g t h e b o d y ; a n n i h i l a t i o n e v e n t s r e g i s t e r e d by d e t e c t o r s l o c a t e d 1 8 0 ° a p a r t a r e the o n l y ones r e c o r d e d . As t h e r a d i a t i o n c o u n t s a r e a c c u m u l a t e d f r o m t h e s e l e c t e d r e g i o n o f the s u b j e c t , t h e r a d i a t i o n I n t e n s i t y a t d i f f e r e n t a n g l e s , o r p r o j e c t i o n s , a b o u t t h i s r e g i o n a r e i n i t i a l l y r e c o r d e d as p r o f i l e s ( see f i g u r e I - 2 b ) . T h e s e p r o f i l e s a r e t h e n s u b j e c t e d t o a m a t h e m a t i c a l p r o c e s s known as c o n v o l u t i o n ( s e e f i g u r e I - 2 c ) to remove a r t i f a c t s f r o m t h e f i n a l i m a g e . The s o - c a l l e d " f i l t e r e d p r o f i l e s " a r e t h e n b a c k - p r o j e c t e d ( s e e f i g u r e I - 2 d ) u s i n g a s e c o n d m a t h e m a t i c a l a l g o r i t h m a p p l i e d by c o m p u t e r , t o r e c o n s t r u c t an image c o r r e s p o n d i n g t o t h e o r i g i n a l 5 s p a t i a l d i s t r i b u t i o n of the radionuclide l a b e l ; t h i s i s f i n a l l y displayed on a cathode-ray screen as the image. 5> 6 In contrast to PET, i n SPECT the random gamma r a d i a t i o n d i r e c t l y emitted from the radioisotope, i s recorded by an area imager, usually a gamma s c i n t i l l a t i o n camera. A collimator i s used to determine the l i n e of d i r e c t i o n which the r a d i a t i o n has t r a v e l l e d ; t h i s i s i n contrast to the l i n e d i r e c t i o n obtained from the nearly a n t i p a r a l l e l tracks of the photons used i n PET. The energy of the emitted photon i s determined by a s c i n t i l l a t o r and photo-m u l t i p l i e r array which can then discriminate against scattered r a d i a t i o n . Thereafter, the image reconstruction i s exactly analogous to that used f o r PET; as the gamma camera systematically scans the subject, i t forms a se r i e s of p r o f i l e s at d i f f e r e n t projections which are mathematically manipulated as described above, to form the image. 2' 7 For both of these methods, the key aspect i s the r a d i o - l a b e l l e d compound i t s e l f . It i s the function of the radiopharmaceutical upon introduction into the body to become l o c a l i z e d i n a s p e c i f i c region or organ of i n t e r e s t . Hence, the observation and measurement of the r a d i a t i o n emitted from these areas, over time, provides the means f o r determination of ph y s i o l o g i c a l function. In the simplest s i t u a t i o n , a diagnostic test f o r pathology can be made by comparing the uptake of the radiopharmaceutical with that for the normal organ of i n t e r e s t . 8 One of the cornerstones of modern medicine i s the understanding that a l l c l i n i c a l symptoms r e s u l t from biochemical reactions and as a consequence every pathology has an underlying biochemical defect. Therefore, the i d e n t i f i c a t i o n of abnormal biochemical a c t i v i t y associated with a s p e c i f i c pathology i s a continuing goal of medicine today, along with the 6 e a r l y and d i r e c t observation of the abnormality i n the a f f l i c t e d organ.5 Thus, i n t h i s context, the radiopharmaceutical acts as a biochemical probe which enables a physician to d i r e c t l y study various parameters of ph y s i o l o g i c a l function such as blood volume, blood flow, oxygen and glucose metabolic rates, drug-receptor i n t e r a c t i o n s , protein synthesis, amino acid transport, t i s s u e pH and the l i k e . 9 C l e a r l y , the a b i l i t y to measure the various parameters i s dependent on having the appropriate radiopharmaceutical av a i l a b l e ; c e r t a i n l y t h i s i s the key to emission imaging. As a consequence, the a b i l i t y to produce the wide v a r i e t y of radionuclide l a b e l s and radiopharmaceuticals i s probably the most challenging aspect of emission imaging. 1.2 The Problem The goals of radiopharmaceutical synthesis have many elements i n common with t r a d i t i o n a l synthetic organic chemistry. Both the radiochemist and organic chemist are concerned with developing syntheses which w i l l y i e l d i n the most d i r e c t manner, the desired compound, i n the largest chemical y i e l d p ossible. In addition, the radiochemist i s concerned with obtaining high radiochemical y i e l d s . Radiochemical y i e l d i s defined as the amount of r a d i o a c t i v i t y incorporated into the product as a percentage of the i n i t i a l quantity of r a d i o a c t i v i t y used. Both the radiochemist and organic chemist require the f i n a l compound to be i s o l a t e d chemically pure. However, r a d i o - l a b e l l e d compounds must be, i n addition, radiochemically pure and free of other r a d i o n u c l i d i c i m p u r i t i e s . 1 0 Furthermore, organic compounds produced 7 as pharmaceuticals and formulated for intravenous i n j e c t i o n , whether r a d i o - l a b e l l e d or not, must also be s t e r i l e and pyrogen f r e e . 1 0 In order to ensure a successful radiopharmaceutical synthesis, several considerations hold which are not common to synthetic organic chemistry. The key aspects that need to be addressed by the radiochemist are as follows: ( i ) choice of radionuclide, ( i i ) p h y s i c a l properties of the radionuclide, ( i i i ) source and chemical form of the radionuclide, and ( i v ) s p e c i f i c a c t i v i t y , stoichiometry, and the "no c a r r i e r " problem. These topics w i l l now be discussed i n the above order. The choice of radioisotope that i s to be used, whether, f o r example, i t i s 1 1 C , 1 8 F or 1 2 3 I , w i l l determine the possible synthetic routes for incorporating the radioisotope, the l i k e l y p o s i t i o n of attachment, and the nature and strength of the chemical bond formed. With the radioisotope chosen, the p h y s i c a l properties of the radionuclide w i l l then have a profound Influence i n the synthesis of the desired radiopharmaceutical. This i s c h i e f l y due to the h a l f - l i f e of the radioisotope used. Table 1-1 i s a p a r t i a l l i s t of radionuclides used i n medicine; the h a l f - l i v e s of these radioisotopes range from a few minutes to a few days. The synthesis time, beginning with the incorporation of the radionuclide, any subsequent chemical modifications ( i . e . removal of protecting groups, etc.) through to the f i n a l p u r i f i c a t i o n of the radiopharmaceutical must be short with respect to the h a l f - l i f e of the radioisotope. C l e a r l y t h i s becomes c r i t i c a l i n the use of the very short-l i v e d radionuclides whose h a l f - l i v e s are i n the order of minutes. The synthesis time, as an approximation, must be no more than one or two h a l f -l i v e s of the radioisotope used. The actual imaging of the patient must i n 8 t u r n be c o m p l e t e d w i t h i n t h r e e o r f o u r h a l f - l i v e s o f t h e r a d i o i s o t o p e u s e d , 1 1 * 1 2 f o l l o w i n g commencement o f t h e e n t i r e p r o c e s s . A n o t h e r p h y s i c a l p r o p e r t y o f t h e r a d i o n u c l i d e s w h i c h i s o f i m p o r t a n c e i s t h e p o t e n t i a l r a d i a t i o n e x p o s u r e to t h e r a d i o c h e m i s t . The r a d i o i s o t o p e s u s e d f o r i n - v i v o m e d i c a l work p r o d u c e h i g h e n e r g y , b o d y - p e n e t r a t i n g r a d i a t i o n , t h a t i s n e c e s s a r y t o e n a b l e e x t e r n a l d e t e c t i o n f o r m e d i c a l i m a g i n g . H e n c e , TABLE I - l . H a l f - l i v e s o f S e l e c t e d R a d i o n u c l i d e s u s e d i n M e d i c i n e * N u c l i d e H a l f - l i f e 1 3 Decay m o d e 1 3 A p p l i c a t i o n 1 5 0 2.03 m i n 8 + ( 9 9 . 9 % ) ; EC (0.1%) PET 1 2 2 ! 3 .6 m i n g + (77%) ; EC (23%) PET 3 8 K 7.6 m i n 6 +(100%) PET 6 8 G a 68 .3 min g + (90%) , EC (10%) PET 7 5 B r 1.6 h e + ( 7 5 . 5 % ) ; EC (24.5%) PET 9 9 m T c 6 .0 h 1 1 * I T 1 1 * SPECT 123;r_ 13.02 h EC (100%) SPECT n i l n 67 .2 h EC (100%) SPECT 2 0 1 T 1 73 .5 h EC (100%) SPECT *No a t t e m p t i s made t o a s s i g n the r e l a t i v e i m p o r t a n c e o f t h e s e r a d i o n u c l i d e s to n u c l e a r m e d i c i n e . D e c a y modes : B + = p o s i t r o n e m i s s i o n , EC = e l e c t r o n c a p t u r e , IT = i n t e r n a l t r a n s i t i o n 9 i t i s p r u d e n t f o r s a f e t y r e a s o n s , f o r one t o work w i t h a p p r o p r i a t e l e v e l s o f s h i e l d i n g and u s e remote o p e r a t i o n s whenever p o s s i b l e . T h i s need f o r s a f e t y e x e r t s a d i r e c t i n f l u e n c e on how l a b o r a t o r y m a n i p u l a t i o n s a r e d o n e , s i n c e t h e d a n g e r o f r a d i a t i o n e x p o s u r e f r o m the r a d i o i s o t o p e s must a l w a y s be remembered and g u a r d e d a g a i n s t . 1 5 The s o u r c e and c h e m i c a l f o r m i n w h i c h a g i v e n r a d i o i s o t o p e i s a v a i l a b l e has t h e g r e a t e s t i m p a c t on t h e d e v e l o p m e n t o f a p r a c t i c a l s y n t h e t i c s t r a t e g y . The f i r s t p r o b l e m i s t h e a v a i l a b i l i t y o f the d e s i r e d r a d i o n u c l i d e . V e r y few r a d i o n u c l i d e s f o r n u c l e a r m e d i c i n e a r e a v a i l a b l e f r o m n u c l e a r r e a c t o r s ; a l t h o u g h 1 3 1 l i s a r e a c t o r - s o u r c e r a d i o n u c l i d e , 1 6 most r a d i o i s o t o p e s a r e p r o d u c e d on s i t e o r w i t h i n r e l a t i v e l y s h o r t t r a v e l l i n g d i s t a n c e ( t i m e ) o f t h e g e n e r a t o r , o r c y c l o t r o n . A number o f companies p r o d u c e c o m p a c t , " e a s y t o u s e " c y c l o t r o n s f o r m e d i c a l r e s e a r c h . O n - s i t e p r o d u c t i o n i s v i t a l l y i m p o r t a n t when any o f t h e v e r y s h o r t - l i v e d PET r a d i o n u c l i d e s a r e u s e d . 1 7 A s e r i o u s c o n c e r n , t h e r e f o r e , o f t h e r a d i o c h e m i s t i s t o be a b l e to p r o d u c e , o r o b t a i n , t h e d e s i r e d r a d i o i s o t o p e s i n a r e l i a b l e f a s h i o n . The c h e m i c a l f o r m o f a g i v e n r a d i o n u c l i d e i s t h e s e c o n d p r o b l e m t h a t has t o be c o n s i d e r e d . R a d i o i s o t o p e s as o b t a i n e d f r o m a c y c l o t r o n / g e n e r a t o r a r e u s u a l l y a v a i l a b l e i n a l i m i t e d r a n g e o f c h e m i c a l f o r m s . H e n c e , i f t h e c h e m i c a l f o r m o f a r a d i o i s o t o p e i s n o t a p p r o p r i a t e f o r t h e p r o p o s e d s y n t h e s i s , i t must be c o n v e r t e d t o t h e d e s i r e d r e a g e n t f o r m . A good example o f t h i s a p p r o a c h i s i n l a b e l l i n g w i t h X 1 C . The two r e a d i l y a v a i l a b l e c h e m i c a l forms o f 1 1 C , f r o m t h e c y c l o t r o n , a r e 1 1 C - l a b e l l e d c a r b o n d i o x i d e and c a r b o n m o n o x i d e . T h e s e c a n be c o n v e r t e d i n t u r n to o t h e r u s e f u l r e a g e n t s . 1 8 The 10 H " C N , H " C H O " C H 3 O H , N C H 3 l O B T A I N E D F R O M C Y C L O T R O N P R E P A R E D B Y A D D I T I O N A L S T E P S r a d i o i s o t o p e may be a l s o m o d i f i e d t o t h e d e s i r e d c h e m i c a l f o r m i n - s i t u d u r i n g t h e c o u r s e o f t h e s y n t h e s i s . An example o f t h i s s t r a t e g y i s i n t h e u s e o f e l e c t r o p h i l i c i o d i n a t i o n t o i n c o r p o r a t e r a d i o i o d i n e i n t o a r o m a t i c s t r u c t u r e s . A l l i s o t o p e s o f r a d i o i o d i n e a r e i n i t i a l l y p r o d u c e d i n t h e i o d i d e f o r m , b u t t h e i o d i d e c a n be c o n v e r t e d i n - s i t u t o " e l e c t r o p h i l i c " i o d i n e . T h i s i s r e a d i l y a c c o m p l i s h e d by a number o f o x i d i z i n g a g e n t s w h i c h p r o d u c e m o l e c u l a r i o d i n e , o r some f o r m o f h y p o i o d i t e . 1 9 N e v e r t h e l e s s , the b e s t a p p r o a c h , when p o s s i b l e , i s t o p r e p a r e the r a d i o -n u c l i d e i n t h e c h e m i c a l f o r m d e s i r e d d u r i n g the r a d i o n u c l i d e p r o d u c t i o n . A l t h o u g h the p r e p a r a t i o n o f r a d i o i s o t o p e s i s d e p e n d e n t on the n u c l e a r r e a c t i o n u s e d , t a r g e t d e s i g n and c y c l o t r o n c h a r a c t e r i s t i c s , t h e c h e m i c a l f o r m o f t h e r a d i o i s o t o p e w i l l be d e t e r m i n e d by a number of f a c t o r s , t h e most c r u c i a l b e i n g the c h e m i c a l c o m p o s i t i o n o f t h e t a r g e t and t h e e n e r g y d e p o s i t i o n i n t h e t a r g e t . 2 0 The l a s t t o p i c o f t h i s i n t r o d u c t i o n i s t h e i s s u e o f s p e c i f i c a c t i v i t y , s t o i c h i o m e t r y and the "no c a r r i e r " p r o b l e m ; t h e s e t h r e e s u b j e c t s a r e c l o s e l y r e l a t e d . S p e c i f i c a c t i v i t y i s d e f i n e d as t h e q u a n t i t y o f r a d i o a c t i v i t y p r e s e n t , g e n e r a l l y e x p r e s s e d i n c u r i e s , p e r mole o f compound. One c u r i e o f 11 a c t i v i t y p r o d u c e s 3 . 7 0 * 1 0 1 0 b e q u e r e l s ( d i s i n t e g r a t i o n s p e r s e c o n d ) ; 2 1 t h e maximum s p e c i f i c a c t i v i t y o f a r a d i o n u c l i d e depends on i t s h a l f - l i f e ( s e e T a b l e 1 - 2 ) , and i s o n l y a t t a i n a b l e when no o t h e r i s o t o p e s o f t h e same e l e m e n t ( i . e . c a r r i e r ) i s p r e s e n t ; t h i s i d e a l s t a t e i s r e f e r r e d to as t h e " c a r r i e r - f r e e " ( C F ) s t a t e . T h i s s t a t e c a n be a p p r o a c h e d o n l y t o w i t h i n an o r d e r o f m a g n i t u d e f o r some r a d i o i s o t o p e s so t h a t some c a r r i e r i s u n a v o i d a b l y p r e s e n t i n most c a s e s . F o r e x a m p l e , i n t h e p r o d u c t i o n o f H 1 1 C N , t h e r a t i o o f n C t o 1 2 C i s a p p r o x i m a t e l y 1 : 3 0 0 0 . In l i g h t o f t h i s p r o b l e m , a d d i t i o n a l t e r m i n o l o g y i s n e e d e d f o r s p e c i f y i n g t h e e x t e n t o f d i l u t i o n p r e s e n t i n a TABLE 1 - 2 . P h y s i c a l P r o p e r t i e s o f Some R a d i o i s o t o p e s N u c l i d e H a l f - l i f e Decay Mode Maximum E n e r g y (MeV) Range* (mm) Maximum S p e c i f i c A c t i v i t y ( C i / m o l ) 3H 12 .35 y 8 _(100%) 0.0186 0 .0072 2.90x10'* 1 4 C 5730 y g-(100%) 0 .155 0 .359 62 .4 i l C 20 .4 m i n 6+(99+%) 0 .96 4 .108 9 . 2 2 x l 0 9 13 N 9 .96 m i n B + (100%) 1 .19 5 .39 l . s g x i o 1 0 18 F 109.7 m i n g + (97%) 0 .635 2 .39 1 . 7 1 x l 0 9 EC (3%) * Maximum r a n g e i n w a t e r . Decay modes : 3~ = b e t a p a r t i c l e e m i s s i o n , g + = p o s i t r o n e m i s s i o n EC = e l e c t r o n c a p t u r e 12 r a d i o p h a r m a c e u t i c a l p r o d u c t . The "no c a r r i e r a d d e d " (NCA) s t a t e , as a p p l i e d to an e l e m e n t o r compound, means t h a t no c a r r i e r o f t h e same e l e m e n t o r compound has been added d u r i n g i t s p r e p a r a t i o n . The " c a r r i e r a d d e d " (CA) s t a t e means a known amount o f c a r r i e r has been added t o t h e e l e m e n t o r compound d u r i n g i t s p r e p a r a t i o n . 2 2 When d e a l i n g w i t h s h o r t - l i v e d r a d i o p h a r m a c e u t i c a l s n e a r t h e i r maximum s p e c i f i c a c t i v i t y , t h e mass o f t h e p r o d u c t i s n o t d e t e c t a b l e by o r d i n a r y c h e m i c a l o r s p e c t r o s c o p i c m e a n s . 2 2 To i l l u s t r a t e t h i s p o i n t , r e f e r r i n g to the d a t a p r e s e n t e d i n T a b l e 1 - 2 , t h e f o l l o w i n g r e s u l t s c a n be d e r i v e d . 1 m C i o f 1 4 C = 9 . 5 9 X 1 0 1 8 a t o m s = 0 . 2 2 m g 1 m C i o f n C = 6 . 5 3 X 1 0 1 0 a t o m s = 1 . 5 p g As a c o n s e q u e n c e , t h e p r o d u c t i o n o f h i g h s p e c i f i c a c t i v i t y r a d i o p h a r m a c e u -t i c a l s i n t h e CF o r NCA s t a t e s i s h i g h l y d e s i r a b l e b e c a u s e t h e mass o f t h e r a d i o p h a r m a c e u t i c a l s a r e t h e n so s m a l l t h a t i t i s u s u a l l y b e l o w t h e t h r e s h o l d where any p h y s i o l o g i c a l r e s p o n s e i s i n v o k e d , 2 3 y e t t h e r e i s a d e q u a t e r a d i o a c t i v i t y ( i n t h e o r d e r o f 0 . 1 t o 0 .5 u C i p e r gram o f t i s s u e i n t h e c a s e o f PET i n s t r u m e n t s ) p r e s e n t t o be d e t e c t e d w i t h s t a t i s t i c a l s i g n i f i c a n c e . E v e n h i g h l y t o x i c m o l e c u l e s c a n be u s e d f o r s t u d i e s i f a d e q u a t e s p e c i f i c a c t i v i t i e s c a n be a c h i e v e d . 2 2 H o w e v e r , w o r k i n g w i t h s m a l l amounts o f h i g h s p e c i f i c a c t i v i t y r a d i o -n u c l i d e s l e a d s t o two c l o s e l y r e l a t e d p r o b l e m s , t h e i s s u e o f s t o i c h i o m e t r y and t h e "no c a r r i e r " p r o b l e m . S i n c e NCA r a d i o s y n t h e s e s a r e p e r f o r m e d on a v e r y s m a l l s c a l e , t h e amount o f i m p u r i t i e s p r e s e n t i n t h e r e a g e n t s and s o l v e n t s 13 BASIC STEPS OF RADIOPHARMACEUTICAL PRODUCTION Preparation of organic substrate for labelling { Precursor) Production of radionuclide via Cyclotron / Generator Convert radionuclide into suitable reagent form Labelling reaction to incorporate radionuclide Perform additional reactions with labelled substrate ( Deprotection, etc. ) chemically purify final labelled product sterilize product and ensure apyrogenicity (For i.v. use only) formulate product for administration to patient F i g u r e 1 - 3 . G e n e r a l scheme f o r t h e p r o d u c t i o n o f a r a d i o p h a r m a c e u t i c a l ( t h i s p r o c e s s must be c o m p l e t e w i t h i n one to two h a l f - l i v e s o f t h e r a d i o i s o t o p e u s e d ) . u s e d may be c o m p a r a b l e t o t h e q u a n t i t y o f t h e r a d i o n u c l i d e u s e d i n t h e s y n t h e s i s . T h e s e i m p u r i t i e s may compete w i t h t h e r a d i o n u c l i d e i n a g i v e n r e a c t i o n l e a d i n g t o unwanted s i d e - p r o d u c t s , o r even s t o p p i n g t h e f o r m a t i o n o f the d e s i r e d compound a l t o g e t h e r . 2 4 One o f the i m p u r i t i e s p r e s e n t i n the s y n t h e s i s c a n be c a r r i e r , w h i c h w i l l l o w e r t h e s p e c i f i c a c t i v i t y o f t h e p r o d u c t ; t h i s i s known as t h e "no c a r r i e r " p r o b l e m . 1 9 ' 2 5 To i l l u s t r a t e t h i s p r o b l e m , a c a r e f u l s t u d y was r e p o r t e d 2 6 w h i c h c o n c e r n e d c a r r i e r b r o m i d e a d d i t i o n d u r i n g h i g h s p e c i f i c a c t i v i t y r a d i o b r o m i n a t i o n s ; s i g n i f i c a n t amounts o f c a r r i e r b r o m i d e were f o u n d i n s o l v e n t s , r e a g e n t s and s u b s t r a t e s . I t was c o n c l u d e d t h a t i f h i g h s p e c i f i c a c t i v i t y r a d i o b r o m i n a t i o n s a r e d e s i r e d , a l l components o f t h e r a d i o b r o m i n a t i o n r e a c t i o n s had t o be a n a l y z e d f o r c a r r i e r b r o m i d e c o n t e n t ( n e u t r o n a c t i v a t i o n a n a l y s i s was u s e d ) b e f o r e u s e . The "no c a r r i e r " p r o b l e m a l s o a p p l i e s t o t h e p r o d u c t i o n o f t h e r a d i o n u c l i d e i t s e l f . Not a l l c h e m i c a l f o r m s o f a g i v e n r a d i o i s o t o p e a r e a v a i l a b l e i n h i g h s p e c i f i c a c t i v i t i e s . 1 7 Once a s u c c e s s f u l r a d i o p h a r m a c e u t i c a l s y n t h e s i s has been d e v e l o p e d , i t s t i l l must be i m p l e m e n t e d i n t o a r o u t i n e p r o d u c t i o n s y s t e m . 1 7 F i g u r e 1-3 summarizes t h e g e n e r a l s t e p s i n v o l v e d i n a t y p i c a l r a d i o s y n t h e s i s . As c a n be s e e n f r o m t h i s d i s c u s s i o n , w o r k i n g w i t h s h o r t - l i v e d r a d i o - l a b e l l e d compounds i s a c o m p l e x r e s e a r c h a r e a . The r e a s o n f o r t h i s l e n g t h y i n t r o d u c t i o n on g e n e r a l b a c k g r o u n d has been t o f a m i l i a r i z e t h e r e a d e r who i s n o t aware o f t h e p u r p o s e and p r o b l e m s o f r a d i o p h a r m a c e u t i c a l d e v e l o p m e n t f o r n u c l e a r m e d i c i n e . 15 1.3 Synthetic Strategy The a i m of t h e p r e s e n t s t u d y was t h e p u r s u i t o f new methods f o r t h e i n c o r p o r a t i o n o f two r a d i o i s o t o p e s o f m e d i c a l i n t e r e s t , namely r a d i o b r o m i n e and r a d i o i o d i n e ( s e e T a b l e 1 - 3 ) , i n t o o r g a n i c compounds . As a c o n s e q u e n c e o f t h e s y n t h e t i c c o n s i d e r a t i o n s d i s c u s s e d a b o v e , t h e f o l l o w i n g f i v e c r i t e r i a were s e t f o r t h . 1) The r e a c t i o n u s e d t o i n c o r p o r a t e the r a d i o n u c l i d e must be f a s t and r e s u l t i n h i g h y i e l d . 2) The r e a c t i o n must be s i t e - and s t e r e o s p e c i f i c . TABLE 1 - 3 . M e d i c a l l y U s e f u l R a d i o i s o t o p e s o f Bromine and I o d i n e N u c l i d e H a l f - l i f e 1 * 1 * Decay mode**1* A p p l i c a t i o n 7 5 B r 95 m i n e + (75%),EC(25%) PET 7 7 B r 57 h EC SPECT 120J 1.4 h EC(54%) ;g + (46%) PET 122j 3 .6 m i n 1 3 g + ( 7 7 % ) ; E C ( 2 3 % ) 1 3 PET 123 x 13 h EC SPECT 1 3 ^ 8.04 d 2 1 8~ 21 SPECT D e c a y modes : 3 = b e t a p a r t i c l e e m i s s i o n , = p o s i t r o n e m i s s i o n EC = e l e c t r o n c a p t u r e 3) The r e a c t i o n must be c o m p a t i b l e w i t h h i g h s p e c i f i c a c t i v i t y r a d i o -h a l o g e n s . TABLE 1-4. Summary of Major Radiohalogenation Methods Method Advantages Disadvantages A l i p h a t i c n u cleophilic exchange Fast, convenient reaction, high y i e l d s , high s p e c i f i c a c t i v i t y possible with non-halogen leaving groups Medium s p e c i f i c a c t i v i t y with non-isotopic exchange, racemization possible Aromatic halogen exchange Fast reaction, high y i e l d , r e g i o s p e c i f i c High temperatures necessary,* harsh reaction conditions, low s p e c i f i c a c t i v i t i e s Dediazoniation Very high s p e c i f i c a c t i v i t i e s possible, mild reaction conditions with triazene, r e g i o s p e c i f i c Oxidative conditions and low pH needed for diazoniation step, v a r i e t y of side-products, optimization for d i f f e r e n t substrates complex Direct e l e c t r o p h i l i c s u b s t i t u t i o n Fast, convenient reaction, high y i e l d , high s p e c i f i c a c t i v i t i e s possible Oxidation and c h l o r i n a t i o n side reactions possible, isomeric mixtures formed,* useful only for activated aromatic substrates Demetallation Fast reaction, high y i e l d s , high s p e c i f i c a c t i v i t y (B, S i , Sn), mild reaction conditions, Precursor synthesis required, oxidation and c h l o r i n a t i o n side reactions depending on reagents r e g i o s p e c i f i c , a l i p h a t i c and aromatic substrates R e c o i l l a b e l l i n g 2 1 * 2 7 High s p e c i f i c a c t i v i t i e s Slow reaction times, isomeric possible, high y i e l d s mixtures formed possible for some substrates *Where non-isotopic halogen s t a r t i n g materials, or side-products, separation problems can occur requiring sophisticated chromatography. 17 4) P r o t e c t i n g g r o u p s , i f n e e d e d , must be r a p i d l y removed a f t e r the l a b e l l i n g s t e p . 5) The l a b e l l e d p r o d u c t must be e a s i l y and r a p i d l y p u r i f i e d . A number o f a p p r o a c h e s have been r e p o r t e d i n t h e l i t e r a t u r e 1 9 * 2 1 f o r r a d i o -h a l o g e n a t i o n and t h e s e a r e b r i e f l y compared i n T a b l e 1 - 4 . The b e s t a p p r o a c h i n v o l v e s t h e u s e o f h a l o g e n a t i o n - d e r a e t a l l a t i o n , p a r t i c u l a r l y when c o n t r a s t e d to d i r e c t e l e c t r o p h i l i c s u b s t i t u t i o n a t a c a r b o n - h y d r o g e n b o n d . Due t o t h e p o l a r i z e d n a t u r e o f a c a r b o n - m e t a l b o n d , an o r g a n o m e t a l l i c compound i s s e n s i t i v e to e l e c t r o p h i l i c a t t a c k . T h i s i s i m p o r t a n t b e c a u s e t h e e l e c t r o p h i l i c c l e a v a g e o f o r g a n o m e t a l l i c p r e c u r s o r s p o t e n t i a l l y c a n be p e r f o r m e d u n d e r m i l d c o n d i t i o n s w i t h s h o r t r e a c t i o n t i m e s . The d i r e c t i o n o f t h e bond p o l a r i z a t i o n , i n w h i c h t h e m e t a l c a r r i e s a p a r t i a l p o s i t i v e c h a r g e w h i l e t h e c a r b o n has a p a r t i a l n e g a t i v e c h a r g e , d e t e r m i n e s t h a t a p o s i t i v e l y c h a r g e d e l e c t r o p h i l i c s p e c i e s w i l l become a t t a c h e d a t t h e m e t a l - c a r r y i n g c a r b o n ; t h i s r e n d e r s t h e r e a c t i o n s i t e s p e c i f i c . I n o r d e r t o u s e h i g h s p e c i f i c a c t i v i t y r a d i o b r o m i n e and i o d i n e , w h i c h a r e most r e a d i l y a v a i l a b l e i n t h e h a l i d e f o r m , t h e r a d i o h a l o g e n s must be o x i d i z e d to a s u i t a b l e e l e c t r o p h i l i c s p e c i e s f o r u s e w i t h o r g a n o m e t a l l i c compounds . T h i s i s no p r o b l e m , as t h e r e a r e a number o f m i l d o x i d i z i n g a g e n t s a v a i l a b l e f o r t h i s p u r p o s e . H i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y (HPLC) has become t h e s t a n d a r d , i n r a d i o - l a b e l l i n g , f o r r a p i d p u r i f i c a t i o n o f t h e l a b e l l e d p r o d u c t . A k e y f e a t u r e h e r e i s t h e c h r o m a t o g r a p h i c b e h a v i o u r o f t h e s t a r t i n g o r g a n o m e t a l l i c w h i c h i n g e n e r a l w i l l be q u i t e d i f f e r e n t f r o m t h e p r o d u c t , e n a b l i n g e a s y s e p a r a t i o n of e x c e s s s t a r t i n g m a t e r i a l . 1 9 18 A number o f m e t a l s ( B 2 8 , S i 2 9 » 3 0 , G e 3 1 , S n 3 2 , H g 3 3 , T I 3 4 ) have been u s e d i n r a d i o h a l o g e n a t i o n - d e m e t a l l a t i o n r e a c t i o n s . G e n e r a l l y t h e i r p r i m a r y a p p l i c a t i o n has been i n t h e f o r m a t i o n o f r a d i o - l a b e l l e d a r o m a t i c s t r u c t u r e s , w i t h t h e e x c e p t i o n o f b o r o n 2 8 and s i l i c o n 3 0 w h i c h have been s u c c e s s f u l l y u t i l i z e d i n p r e p a r a t i o n o f l a b e l l e d a l k y l - h a l i d e compounds . C o m p a r a t i v e l y l i t t l e has been done to u s e t h e s e m e t a l s t o p r o d u c e r a d i o - l a b e l l e d v i n y l -h a l o g e n compounds . A l t h o u g h a l a r g e r number o f b i o l o g i c a l l y i n t e r e s t i n g m o l e c u l e s e x i s t w i t h a r o m a t i c s t r u c t u r e s , a number o f s i g n i f i c a n t compounds h a v i n g v i n y l s t r u c t u r e s e x i s t . 3 3 ' 3 5 - 3 7 I t was t h e s e p o t e n t i a l a p p l i c a t i o n s w h i c h prompted o u r i n t e r e s t i n d e v e l o p i n g g e n e r a l methods o f r a d i o b r o m i n a t i o n and i o d i n a t i o n f o r t h e s y n t h e s i s o f v i n y l l a b e l l e d compounds. B o r o n , m e r c u r y and t i n have been t h e o n l y m e t a l s u s e d f o r r a d i o - l a b e l l i n g v i n y l compounds. K a b a l k a e t a l l 8 u s i n g b o r o n , i n t r o d u c e d v i a h y d r o b o r a t i o n o f a c e t y l e n i c p r e c u r s o r s , have p r e p a r e d a r a n g e of CA 1 2 5 I - l a b e l l e d v i n y l i o d i d e s w i t h r a d i o c h e m i c a l y i e l d s r a n g i n g f r o m 41 t o 87%; more r e c e n t l y , an i o d o -a l k e n y l f a t t y a c i d was p r e p a r e d w i t h NCA N a 1 2 5 I and N a 1 2 3 I i n 38% r a d i o -c h e m i c a l y i e l d . 3 6 R e a c t i o n t i m e s were i n t h e o r d e r o f 30 m i n u t e s . The d i s a d v a n t a g e of u s i n g b o r o n i s t h a t i t i s s e n s i t i v e t o c a r r i e r l e v e l s , g i v i n g t h e p o o r e s t r a d i o c h e m i c a l y i e l d s a t t h e NCA l e v e l . B o - L i . e t a l 3 3 u s e d 6 - c h l o r o m e r c u r y c h o l e s t e r o l as a p r e c u r s o r f o r r e a c t i o n w i t h low s p e c i f i c a c t i v i t y [ 8 2 B r ] B r 2 i n c h l o r o f o r m t o p r o d u c e [ 8 2 B r ] 6 - b r o m o c h o l e s t e r o l i n 46% r a d i o c h e m i c a l y i e l d ( r e a c t i o n t i m e , 70 m i n ) . T h i s s t r a t e g y i s n o t a d v a n t a g e o u s owing t o t h e use o f l a b e l l e d m o l e c u l a r b r o m i n e w h i c h c a n g i v e o n l y a maximum r a d i o c h e m i c a l y i e l d o f 50%. Hanson e t a l l 9 u s e d v i n y l - t i n 19 p r e c u r s o r s f o r 1 2 5 I - l a b e l l i n g . The t i n p r e c u r s o r s were p r e p a r e d by e m p l o y i n g b i s ( t r i b u t y l s t a n n y l ) e t h y l e n e , ' t 0 w h i c h was t r a n s m e t a l l a t e d t o a m o n o l i t h i a t e d a n a l o g , and r e a c t e d w i t h a k e t o n e f u n c t i o n a l i t y , as i n t h e c a s e o f e s t r o n e . T h e t i n p r e c u r s o r , 1 7 a - E - t r i b u t y l s t a n n y l v i n y l e s t a d i o l p r e p a r e d f r o m e s t r o n e , was i n i t i a l l y l a b e l l e d w i t h low s p e c i f i c a c t i v i t y [ 1 2 5 I ] I 2 , 3 9 b u t more r e c e n t l y w i t h NCA N a 1 2 5 I , u s i n g h y d r o g e n p e r o x i d e i n a c e t i c a c i d as t h e o x i d a n t , w h i c h r e s u l t e d i n o n l y a 46% r a d i o c h e m i c a l y i e l d ( r e a c t i o n t i m e , 30 m i n ) . 1 * 1 O t h e r e s t r a d i o l d e r i v a t i v e s , h o w e v e r , were l a b e l l e d i n e x c e l l e n t r a d i o c h e m i c a l y i e l d s ( 8 9 - 9 5 % ) . 3 5 A l t h o u g h t h a t work seems s u i t a b l e f o r 1 2 3 i -l a b e l l i n g , the r e a c t i o n t i m e s a r e t o o l o n g f o r use w i t h e i t h e r 1 2 0 l o r 1 2 2 I . T h i s a n a l y s i s o f t h e l i t e r a t u r e s u g g e s t e d t o us t h a t v i n y l - t i n r e a g e n t s w o u l d be t h e p r e f e r r e d l a b e l l i n g p r e c u r s o r s f o r o u r own s t u d i e s . T i n compounds a r e s u f f i c i e n t l y s t a b l e t o be p r e p a r e d i n b u l k and s t o r e d , and t h e n u s e d i n a l i q u o t s as n e e d e d . 1 * 1 I n a d d i t i o n , v i n y l - t i n p r e c u r s o r s c a n be p r e p a r e d r e a d i l y by r e d u c t i o n o f t h e c o r r e s p o n d i n g a c e t y l e n i c compounds, u s i n g t i n h y d r i d e r e d u c i n g a g e n t s . 1 * 2 T h i s a f f o r d s a p o t e n t i a l l y more c o n v e n i e n t s y n t h e s i s o f v i n y l - t i n compounds t h a n t h a t o f Hanson and c o - w o r k e r s 1 * 3 d i s c u s s e d a b o v e . F u r t h e r m o r e , g i v e n t h e e x p e r i e n c e o f u s i n g a r y l - t i n p r e c u r s o r s f o r l a b e l l i n g , 3 2 * 1 * 5 i t was a n t i c i p a t e d t h a t a l l f i v e c r i t e r i a as d e s c r i b e d e a r l i e r c o u l d be met w i t h v i n y l - t i n compounds . 20 CHAPTER II DEVELOPMENT OF METHODS FOR RAPID LABELLING WITH NON-RADIOACTIVE BROMINE AND IODINE II.1 Introduction Although the intent of this work was to develop methods of labelling with bromine and iodine that would be generally applicable, i t was determined that a specific application was needed to evaluate the new methodology. To this end, the synthesis of vinyl-halogen labelled estradiols was chosen. Radio-labelled estradiol derivatives have been shown to be useful for the detection of estrogen-receptor-binding proteins which are present in estrogen-responsive mammary carcinoma (i.e. breast cancer). 4 6 The vinyl-iodinated estradiol derivatives, 17ct-E-iodovinyl-l,3,5(10)-estratriene-3,17B-diol [21] and 3-methoxy-17cx-E-iodovinyl-l,3,5(10)-estratriene-173-ol [23] (see figure II-2) have been labelled with 1 2 5 i and evaluated in animal models with good results. 3 5* 3 9 The corresponding bromo-analogues have not been synthesized previously. These estrogen-receptor-seeking radiopharmaceuticals provide an additional challenge in that these must be produced in high specific activities (minimum of 1000 Ci/mmol is deemed necessary) as the concentration of the estrogen receptors in the target tissue is very low (pmole per cc of tissue). 1* 6 Initial development, however, of these new labelling procedures was performed with model compounds. 21 scheme 1 [ 9 j X = B r [10]x = I scheme 2 [111 [12] [13] Figure 11-1. Synthetic schemes for the preparation of the v i n y l -halogenated model compounds [5], [6], [9], [10] and [13]. 22 [ U ] R ' = R 2 = H [15]R'=Me, R 2=H [ 1 6 ] R l = R 2 =Me (n-Bu) 3 SnH [20]R'=R 2=H [21]R' = R 2=H [22]R , = Me ,R 2 =H [ 23 ] R1 = Me,R 2=H [2U] R'=R 2=Me [25] R1 = R 2 = Me Figure II-2. Synthesis scheme f o r the preparation of vinyl-halogenated e s t r a d i o l d e r i v a t i v e s . 23 In t h i s c h a p t e r , t h e s y n t h e s i s o f v i n y l - t i n d e r i v a t i v e s w i l l be d e s c r i b e d . T h i s w i l l be f o l l o w e d by s t u d i e s i n b r o m i n a t i o n and i o d i n a t i o n o f t h e p r e p a r e d v i n y l - t i n compounds, u s i n g n o n - r a d i o a c t i v e b r o m i n e and I o d i n e . The r e a c t i o n s e q u e n c e s p e r f o r m e d a r e summarized i n f i g u r e s I I - l and I I - 2 . S i n c e the v i n y l - t i n p r e c u r s o r s , a l o n g w i t h t h e i r c o r r e s p o n d i n g b r o m i n a t e d and i o d i n a t e d p r o d u c t s , were i n most c a s e s new compounds, t h e s e were f u l l y c h a r a c t e r i z e d . I n t h i s r e g a r d , P r o t o n N u c l e a r M a g n e t i c Resonance ( *H nmr) was f o u n d t o be t h e most u s e f u l c h a r a c t e r i z a t i o n t o o l . The i n d i v i d u a l s t e p s shown i n f i g u r e s I I - l and I I - 2 w i l l now be d i s c u s s e d i n d e t a i l . II.2 Synthesis of Vinyl-Tin Precursors I n o r d e r to p r e p a r e t h e d e s i r e d v i n y l - t i n compounds v i a h y d r o s t a n n y l a t i o n , t h e a p p r o p r i a t e a c e t y l e n i c p r e c u r s o r s n e e d to be e i t h e r p u r c h a s e d c o m m e r c i a l l y o r s y n t h e s i z e d . F o r t h i s p r e s e n t s t u d y , t h e a c e t y l e n i c compounds, p h e n y l -a c e t y l e n e [11] and 1 7 a - e t h y n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 3 - d i o l [ 1 4 ] , were o b t a i n e d c o m m e r c i a l l y . However , t h e r e m a i n i n g a c e t y l e n i c p r e c u r s o r s u s e d were p r e p a r e d u s i n g l i t e r a t u r e m e t h o d s , as summarized i n f i g u r e I I - 3 . The a c e t y l e n i c compounds a r e known e x c e p t f o r 3 , 1 7 g - d i m e t h o x y - 1 7 o - e t h y n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e [ 1 6 ] . T h i s p r o d u c t was r e a d i l y c h a r a c t e r i z e d by X H n m r . The k e y f e a t u r e s o f t h e s p e c t r u m were t h e p r e s e n c e o f the a c e t y l e n i c p r o t o n a t 6 2 .62 and t h e two methoxy g r o u p s a t 6 3 .77 and 6 3 . 4 2 . The h y d r o s t a n n y l a t i o n r e a c t i o n h a s been known s i n c e t h e l a t e 1 9 5 0 ' s . 1 + 7 The a d d i t i o n o f t r i - n - p r o p y l t i n h y d r i d e w i t h a v a r i e t y o f s i m p l e a c e t y l e n i c compounds were r e p o r t e d by v a n d e r K e r k and c o - w o r k e r s i n 1 9 5 9 . 2 They f o u n d Figure II-3. Synthetic schemes for the preparation of carbohydrate (scheme 1) and e s t r a d i o l (scheme 2) acetylenic precursors. 25 t h a t h e a t i n g a m i x t u r e o f t h e t i n h y d r i d e and a c e t y l e n i c compound, i n t h e a b s e n c e o f s o l v e n t , f o r s e v e r a l h o u r s , gave the v i n y l - t i n p r o d u c t s i n modera te t o good y i e l d s (34 -82%) . 1 + 2 I t a l s o has been r e p o r t e d i n t h e l i t e r a t u r e t h a t t h e h y d r o s t a n n y l a t i o n r e a c t i o n c a n be m o d i f i e d o r a c c e l e r a t e d by t h e a d d i t i o n o f f r e e r a d i c a l i n i t i a t o r s , u l t r a - v i o l e t r a d i a t i o n , o r by t h e a d d i t i o n o f p o l a r s o l v e n t s . 1 * 8 The most f r e q u e n t l y u s e d c o n d i t i o n s f o r h y d r o s t a n n y l a t i o n o f a c e t y l e n e s i n t h e r e c e n t l i t e r a t u r e has b e e n t o h e a t t h e a c e t y l e n i c compound i n n e a t h y d r i d e t o a b o u t 8 0 - 9 0 ° , i n t h e p r e s e n c e o f AIBN ( 2 , 2 ' - a z o b i s ( 2 - m e t h y l p r o p i o n i -t r i l e ) ) . 1 * 9 However , i n e a c h o f t h e c a s e s c i t e d , t h e a c e t y l e n i c p r e c u r s o r s c o n t a i n e d o n l y p r o t e c t e d h y d r o x y l g r o u p s , whereas 7 , 8 - d i d e o x y - l , 2 : 3 , 4 - d i - 0 _ -i s o p r o p y l i d e n e - D - g l y c e r o - a - D - g a l a c t o - o c t - 7 - y n o p y r a n o s e [3] c o n t a i n s a n u n p r o t e c t e d h y d r o x y l g r o u p and was t h e f i r s t compound i n t h i s s t u d y t o be h y d r o s t a n n y l a t e d . I n c a s e s where u n p r o t e c t e d h y d r o x y l g r o u p s were p r e s e n t i n t h e a c e t y l e n i c s u b s t r a t e , s u c h as i n [ 3 ] , t h e h y d r o s t a n n y l a t i o n r e a c t i o n was p e r f o r m e d u n d e r t h e r m a l a d d i t i o n c o n d i t i o n s . 1 * 2 Compound [3] was h y d r o s t a n n y l a t e d i n r e f l u x i n g t e t r a h y d r o f u r a n (THF) w i t h 5 .5 e q u i v a l e n t s o f t r i - n - b u t y l t i n h y d r i d e . T h i n l a y e r c h r o m a t o g r a p h i c ( t i c ) a n a l y s i s showed t h e r e a c t i o n had gone t o p a r t i a l c o m p l e t i o n . T h e r e f o r e , t h e r e a c t i o n was r e p e a t e d i n r e f l u x i n g 1 , 4 - d i o x a n e ( 1 0 1 . 5 ° ) and was f o u n d t o go t o c o m p l e t i o n . A n a l y t i c a l t i c showed a p r o d u c t m i x t u r e o f 3 components had r e s u l t e d , i n w h i c h t h e compound o f h i g h e s t p o l a r i t y was s t r o n g l y d o m i n a n t . Upon c h r o m a t o g r a p h i c w o r k u p , t h e m a j o r p r o d u c t was i s o l a t e d and d e t e r m i n e d t o be ( E ) - 7 , 8 - d i d e o x y - l , 2 : 3 , 4 - d i - 0 - i s o p r o p y l i d e n e - 8 - C - t r i b u t y l s t a n n y l - D - g l y c e r o -a - D - g a l a c t o - o c t - 7 - e n o p y r a n o s e [ 4 ] . The i s o l a t e d y i e l d o f [4] was 52%. 26 1 0 0 - i 7 5 -g 5 0 - I ui > 2 5 H < mt Ul at 0 r t - r 0 515 5 2 0 5 2 5 M A S S F i g u r e I I - 4 . S i m u l a t e d m a s s s p e c t r a l i s o t o p e p a t t e r n f o r t h e M ^ - C ^ H g f r a g m e n t ( b a s e p e a k ) o f c o m p o u n d [ 4 ] . Compound [4] was r e a d i l y c h a r a c t e r i z e d u s i n g * H nmr and mass s p e c t r o m e t r y . T i n has t e n n a t u r a l abundance i s o t o p e s w h i c h c o n f e r a d i s t i n c t mass s p e c t r a l p a t t e r n f o r t i n - c o n t a i n i n g d e r i v a t i v e s . A c a l c u l a t e d mass s p e c t r a l i s o t o p e p a t t e r n f o r [4] i s shown I n f i g u r e I I - 4 , w h i c h was i n d e e d o b s e r v e d i n the mass s p e c t r u m o f [ 4 ] . T h i s c o n f i r m s the p r e s e n c e o f t i n i n [ 4 ] . The 1 H nmr e x h i b i t e d the p r e s e n c e o f two v i n y l p r o t o n s a t 6 6 .18 and 6 6 . 3 8 . E a c h r e s o n a n c e was s p l i t i n t o a d o u b l e t o f d o u b l e t s . The r e s o n a n c e a t 6 6 .18 was a s s i g n e d t o H-7 due t o c o u p l i n g to H-6 (J = 4 . 4 H z ) , whereas 6 6 .38 was a s s i g n e d to H-8 ( J g - g = H z ) . The v i n y l p r o t o n s were f o u n d t o be o f t r a n s s t e r e o c h e m i s t r y ( J 7 - 0 = 19 .2 H z ) . T h i s was f u r t h e r c o n f i r m e d by t h e v a l u e s o f Figure I I - 5 . The numbering scheme of the carbon skeleton of the carbohydrate compounds. 28 t h e t i n - p r o t o n c o u p l i n g c o n s t a n t s , * w h i c h were 64 Hz f o r H-7 and 70 Hz f o r H - 8 . I t i s known t h a t f o r v i n y l - t i n compounds J ( S n - H ) t r a n s > J ( S n - H ) g e m > J ( S n - H ) c i s 5 0 > 5 1 so t h a t H - 8 , w h i c h i s g e m i n a l t o t h e - S n ( n - B u ) 3 g r o u p , s h o u l d have a l a r g e r J s n - H v a l n e t h a n H - 7 , w h i c h i s c i s . T h i s was i n d e e d f o u n d t o be t h e c a s e f o r [ 4 ] . G i v e n t h e r e s u l t s o b t a i n e d i n p r e p a r i n g [ 4 ] , t h e same r e a c t i o n c o n d i t i o n s were u s e d t o h y d r o s t a n n y l a t e [ 1 4 ] . H o w e v e r , a n a l y t i c a l t i c showed t h e r e a c t i o n had s e e m i n g l y n o t gone t o c o m p l e t i o n a f t e r r e f l u x i n g o v e r n i g h t . T h e r e f o r e , t h e r e a c t i o n m i x t u r e was c h r o m a t o g r a p h e d and t h e " s t a r t i n g m a t e r i a l " was r e c o v e r e d a f t e r i s o l a t i o n o f t h e p r o d u c t m i x t u r e . 1 H nmr a n a l y s i s o f t h e " s t a r t i n g m a t e r i a l " c l e a r l y showed t h i s m a t e r i a l t o be a m i x t u r e o f [14] and 1 7 a - v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 g - d i o l [26] i n a r a t i o of 2 9 : 7 1 , r e s p e c t i v e l y ( s e e f i g u r e I I - 6 ) . Compound [26] was i s o l a t e d i n p u r e f o r m v i a H P L C , and a f t e r r e c r y s t a l l i z a t i o n gave w h i t e c r y s t a l s o f mp 1 6 9 . 5 - 1 7 0 ° , [ a ] D 5 = + 5 8 . 5 ° ( 1 , 4 - d i o x a n e ) { l i t . 5 2 mp 1 4 8 - 1 5 0 ° ; [a ] D = + 5 7 . 3 ° ( 1 , 4 - d i o x a n e ) } . The p r o d u c t m i x t u r e was f u r t h e r p u r i f i e d by s i l i c a g e l c h r o m a t o g r a p h y and t h e major p r o d u c t , 1 7 a - E - t r i b u t y l s t a n n y l v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 g -d i o l [ 1 7 ] , was i s o l a t e d i n 20% y i e l d . T h i s low y i e l d was c l e a r l y t h e r e s u l t o f p r o t o n o l y s i s o f t h e t i n - c a r b o n bond o f [17] w h i c h r e s u l t e d i n t h e f o r m a t i o n o f [ 2 6 ] . Two p o s s i b l e r e a s o n s may a c c o u n t f o r t h i s r e s u l t . The a c e t y l e n i c d e r i v a t i v e [14] may n o t be s u f f i c i e n t l y a n h y d r o u s , t h u s i n t r o d u c i n g w a t e r i n t o * T h e r e a r e t h r e e n a t u r a l l y o c c u r r i n g t i n i s o t o p e s i n w h i c h t h e n u c l e a r s p i n quantum number 1=1/2. S i n c e 1 1 5 S n i s so low i n abundance (0 .34%), t h e i m p o r t a n t n u c l e i a r e 1 1 7 S n (7.54% a b u n d a n c e ) and 1 1 9 S n (8.62% a b u n d a n c e ) . 5 1 The t i n - p r o t o n c o u p l i n g c o n s t a n t s a r e s l i g h t l y l a r g e r f o r 1 1 9 S n t h a n f o r 1 1 7 S n , h o w e v e r , t h e m a g n i t u d e o f t h e s e c o u p l i n g s i s a l w a y s g i v e n as t h e a v e r a g e o f t h e 1 1 7 S n and 1 1 9 S n v a l u e s t h r o u g h o u t t h i s t h e s i s . 29 Figure 11-6. Hydrostannylation reaction of compound [14], in refluxing 1,4-dioxane, produces a major side-product [26] in addition to the desired product [17]. 30 t h e r e a c t i o n m i x t u r e w h i c h c a n a c t as a p r o t o n s o u r c e . A l t e r n a t i v e l y , t h e a c i d i c p r o t o n f r o m t h e p h e n o l i c h y d r o x y l g r o u p c o u l d f u n c t i o n i n l i k e manner . W h i c h e v e r t h e c a s e , t h e f i n a l y i e l d o f [17] was d i s a p p o i n t i n g . Compound [17] was c h a r a c t e r i z e d by mass s p e c t r o m e t r y and *H nmr. The mass s p e c t r u m o f [17] c o n f i r m e d t h e p r e s e n c e o f t i n a s d e s c r i b e d e a r l i e r w i t h [ 4 ] . The * H nmr ( s e e f i g u r e I I - 8 ) e x h i b i t e d t h e c h a r a c t e r i s t i c v i n y l r e s o n a n c e s as a n AB q u a r t e t a t <5 6 .20 and 6 6 . 0 7 , w i t h a c o u p l i n g c o n s t a n t o f 19 .4 H z . The c o u p l i n g c o n s t a n t c o n f i r m s t h e t r a n s s t e r e o c h e m i s t r y o f t h e F i g u r e I I - 7 . The n u m b e r i n g scheme of the c a r b o n s k e l e t o n of t h e e s t r a d i o l compounds . F i g u r e I I - 8 . 270 MHz l H nmr s p e c t r u m of compound [17] i n d e u t e r i o c h l o r o f o r m . 32 v i n y l protons. Since these v i n y l protons were not coupled to other protons i n the molecule, assignment was based on the tin-proton coupling constants. The doublet at 6 6.07 had the largest Jsn-H value of 70 Hz and t h i s was assigned as H-21, whereas the other doublet at 6 6.20 had a Jsn-H value of 66 Hz and was assigned as H-20. Hanson and co-workers had reported the preparation of [17] i n 1982,39 and c h a r a c t e r i z a t i o n d e t a i l s were reported l a t e r i n 1984.1+3 The XH nmr d a t a 4 3 given d i f f e r s from that obtained i n t h i s study, but the f i e l d strength of the nmr instrument they used was not reported. Although the thermal a d d i t i o n of t r i - n - b u t y l t i n hydride provided the desired v i n y l - t i n d e r i v a t i v e s , [4] and [17], i t was not s a t i s f a c t o r y i n regard to chemical y i e l d and, more importantly, i t required laborious chromatography and rechromatography of the isomeric product mixtures to i s o l a t e the pure E-isomer. Furthermore, the quantity of t r i - n - b u t y l t i n hydride used was excessive, and while e a s i l y removed, i t caused poor chromatography performance during product i s o l a t i o n . Therefore, the thermal a d d i t i o n method was discarded i n favour of the AIBN catalyzed procedure recently reported by Jung and L i g h t , 5 3 and Ensley and co-workers. 5 1* Both groups reported successful hydrostannylations of a number of acetylenic alcohols and encountered no d i f f i c u l t i e s with the unprotected hydroxyl groups. The f i r s t a c e t y l e n i c d e r i v a t i v e to be hydrostannylated with the AIBN catalyzed method was 3,17f3-dimethoxy-17ci-ethynyl-l,3,5(10)-estratriene [16], which has both hydroxyl groups protected. Approximately two equivalents of t r i - n - b u t y l t i n hydride and a c a t a l y t i c amount of AIBN was added to [16], 33 and t h e m i x t u r e was h e a t e d t o 9 5 ° o v e r n i g h t . C h r o m a t o g r a p h i c workup y i e l d e d 3 , 1 7 6 - d i m e t h o x y - 1 7 a - E - t r i b u t y l s t a n n y l v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e [19] i n 98% y i e l d . Compound [19] was r e a d i l y c h a r a c t e r i z e d by mass s p e c t r o m e t r y and 1 H nmr. The s p e c t r a l p r o p e r t i e s e x h i b i t e d by t h i s v i n y l - t i n d e r i v a t i v e were a n a l o g o u s t o t h a t o f [ 1 7 ] , d i s c u s s e d e a r l i e r . The c h e m i c a l s h i f t s and c o u p l i n g c o n s t a n t s o f t h e v i n y l p r o t o n s a r e summarized i n T a b l e I I - l . B a s e d on t h e above r e s u l t , t h e same r e a c t i o n c o n d i t i o n s were employed f o r t h e h y d r o s t a n n y l a t i o n o f [14] and 3 - m e t h o x y - 1 7 a - e t h y n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e -1 7 g - o l [ 1 5 ] . The r e s u l t s a r e shown i n T a b l e I I - 2 . I n t h e c a s e o f [ 1 5 ] , v e r y good r e s u l t s were o b t a i n e d as 3 - m e t h o x y - 1 7 a - E - t r i b u t y l s t a n n y l v i n y l - l , 3 , 5 ( 1 0 ) -e s t r a t r i e n e - 1 7 g - o l [18] was i s o l a t e d i n 87% y i e l d . However , o n l y modera te y i e l d s o f [17] were o b t a i n e d . A n a l y t i c a l t i c c o n f i r m e d t h a t the b y p r o d u c t [ 2 6 ] , d i s c u s s e d p r e v i o u s l y ( s e e f i g u r e I I - 6 ) , was a g a i n p r e s e n t i n t h e r e a c t i o n m i x t u r e o f [ 1 7 ] . The p r o b l e m o f t i n - c a r b o n bond p r o t o n o l y s i s c o n t i n u e s t o e x i s t u n d e r t h e s e r e a c t i o n c o n d i t i o n s . I t i s i n t e r e s t i n g to n o t e t h a t t h i s p r o b l e m was not o b s e r v e d i n the p r e p a r a t i o n o f t h e o t h e r v i n y l - t i n d e r i v a t i v e s i n t h i s s t u d y . Compound [18] has been p r e v i o u s l y p r e p a r e d by F r a n k e and Hanson i n 1 9 8 4 , 3 5 but t h e y have y e t t o p u b l i s h any c h a r a c t e r i z a t i o n d a t a on t h i s compound. Compound [18] was c h a r a c t e r i z e d by mass s p e c t r o m e t r y and 1 H nmr ( s e e T a b l e I I - l ) . The s p e c t r a l p r o p e r t i e s e x h i b i t e d by [18] were a n a l o g o u s t o t h e p r e v i o u s v i n y l - t i n d e r i v a t i v e s d i s c u s s e d e a r l i e r . The o t h e r a c e t y l e n i c s u b s t r a t e s i n t h i s s t u d y , [ 3 ] , 7 , 8 - d i d e o x y - l , 2 : 3 , 4 -d i - 0 j - i s o p r o p y l i d e n e - I v - g l y c e r o - a - p _ - g a l a c t o - o c t - 7 - y n o p y r a n o s e [7] and [11] were a l l h y d r o s t a n n y l a t e d u s i n g t h e A I B N c a t a l y z e d m e t h o d . The r e s u l t s a r e TABLE I I - l . C h e m i c a l S h i f t s (ppm) and C o u p l i n g C o n s t a n t s (Hz) o f t h e V i n y l P r o t o n s o f t h e T i n Compounds Compound H-7 H - 8 H-20 H-21 J H _ H [ 4 ] a 6 .18 6 .38 - - 19.2 J S n - H 6 4 J S n - H 7 ° [ 8 ] b 6 .48 6 .86 - - 19.2 J S n - H 6 5 J S n - H 7 6 [17] ' 6 .20 J S n - H 6 6 6.07 J S n - H 7 ° 19 .4 [18 ] ' 6 .20 J S n - H 6 6 6 .06 J S n - H 7 1 19 .3 [19] a D e u t e r i o c h l o r o f o r m s o l u t i o n b D e u t e r i o b e n z e n e s o l u t i o n 6 .37 J S n - H 6 9 6 .23 J S n - H 7 5 19.7 TABLE I I - 2 . Y i e l d s o f V i n y l - T i n D e r i v a t i v e s 3 S t a r t i n g M a t e r i a l P r o d u c t % Y i e l d [3] [A] 61 ( 5 2 ) c [7] [8] 58 [11] [12] 8 2 b [14] [17] 51-59 ( 2 0 ) c [15] [18] 79-87 [16] [19] 90-98 a The p r o d u c t s i s o l a t e d were p u r e E - i s o m e r . b T h i s p r o d u c t was I s o l a t e d as an E / Z i s o m e r i c m i x t u r e (E t o Z r a t i o o f 88 :12 by * H n m r ) . c T h e s e a r e y i e l d s o b t a i n e d by t h e r e a c t i o n o f ( n - B u ) 3 S n H w i t h s t a r t i n g m a t e r i a l ( A I B N i s n o t p r e s e n t ) i n r e f l u x i n g 1 , 4 - d i o x a n e . 3 5 s u m m a r i z e d i n T a b l e I I - 2 . S u r p r i s i n g l y , t h e h y d r o s t a n n y l a t i o n o f [ 3 ] a n d [ 7 ] g a v e l o w e r y i e l d s t h a n e x p e c t e d . T h i s i s p r e s u m a b l y d u e t o a l a r g e r q u a n t i t y o f Z - i s o m e r b e i n g p r e s e n t . E a c h o f t h e t i n p r o d u c t s w a s c h a r a c t e r i z e d , a s b e f o r e , b y m a s s s p e c t r o m e t r y a n d 1 H n m r ( s e e T a b l e I I - l ) . I I .3 Bromine L a b e l l i n g Studies O u r a p p r o a c h t o l a b e l l i n g v i n y l - t i n p r e c u r s o r s w i t h b r o m i n e i n v o l v e s t h e d i r e c t a t t a c k o f t h e t i n - c a r b o n b o n d w i t h a s u i t a b l e e l e c t r o p h i l i c b r o m i n a t i n g s p e c i e s , t h u s g e n e r a t i n g t h e d e s i r e d p r o d u c t . H o w e v e r , a s d i s c u s s e d i n t h e i n t r o d u c t i o n , h i g h s p e c i f i c a c t i v i t y r a d i o b r o m i n e a n d r a d i o i o d i n e a r e o n l y r e a d i l y a v a i l a b l e i n t h e h a l i d e f o r m . T h e r e f o r e , t o g e n e r a t e t h e n e c e s s a r y e l e c t r o p h i l i c s p e c i e s o f b r o m i n e , a n o x i d i z i n g a g e n t m u s t b e e m p l o y e d t o c o n v e r t b r o m i d e t o s o m e s p e c i e s c o n t a i n i n g B r + . T h i s o x i d a t i o n i s m o s t c o n v e n i e n t l y c a r r i e d o u t i n - s i t u i n t h e r e a c t i o n m i x t u r e i t s e l f , a l l o w i n g t h e g e n e r a t e d e l e c t r o p h i l i c b r o m i n a t i n g s p e c i e s t o r e a c t w i t h t h e v i n y l - t i n p r e c u r s o r p r e s e n t . A n u m b e r o f o x i d i z i n g a g e n t s h a v e b e e n u s e d f o r s i m i l a r p u r p o s e s , 2 0 * 2 1 b u t t h e o x i d a n t c h o s e n f o r t h i s s t u d y i s C h l o r a m i n e - T ( t h e s o d i u m s a l t o f N - c h l o r o - p - t o l u e n e s u l f o n a m i d e ) w h i c h i s s h o w n b e l o w . 36 It i s thought under a c i d i c conditions that Chloramine-T generates BrCl from bromide, 1 9 which then reacts with the v i n y l - t i n substrate. This approach was then used f o r the bromination of each of the v i n y l - t i n d e r i v a t i v e s ; [4 ], (E)-7,8-dideoxy-l,2:3,4-di-0-isopropylidene-8-C-tributyl-stannyl-L-glycero-a-D-galacto-oct-7-enopyranose [8], B-E/Z-tributylstannyl-styrene [12], [17], [18] and [19]. Reaction of the v i n y l - t i n precursor (see figures I I - l and II-2) with a s l i g h t excess of NaBr with two equivalents of Chloramine-T and IM HC1 (saturated with NaCl) i n an aqueous-THF mixture at 0°, f o r one minute, gave the corresponding brominated product. The r e s u l t s are summarized i n Table II-3. Very good y i e l d s were obtained f o r each of the brominated products except f o r 17a-E-bromovinyl-l,3,5(lO)-estratriene-3,17 0-d i o l [20] which was i s o l a t e d i n poor y i e l d . A n a l y t i c a l t i c of the reaction mixture of [20] showed the formation of a number of side-products. Upon chromatographic separation, the major side-products were i s o l a t e d and i d e n t i f i e d by 1H nmr. These r e s u l t s w i l l be discussed l a t e r i n d e t a i l , but these data b a s i c a l l y showed that the aromatic A-ring of [17] underwent concomitant bromination and c h l o r i n a t i o n i n ad d i t i o n to tin-carbon bond cleavage. A number of general observations were made i n connection to the bromin-a t i o n r e s u l t s shown i n Table II-3. With the exception of [20], a l l the reactions resulted i n clean conversions from the v i n y l - t i n precursor to the brominated product. To achieve these r e s u l t s the correct amount of acid to be used i n the reaction needed to be determined. If i n s u f f i c i e n t acid i s used, the y i e l d i s low because the reaction does not go to completion. A l t e r n a t i v e l y , i f the optimum amount of acid i s surpassed, the y i e l d i s reduced due to the formation of u n i d e n t i f i e d by-products. Furthermore, to TABLE I I - 3 . Y i e l d s o f V i n y l - b r o r a i n a t e d D e r i v a t i v e s S t a r t i n g M a t e r i a l P r o d u c t % Y i e l d 3 [4] [5] 95% [8] [9] 89% [12] [13] 90%b [17] [20] 13% [18] [22] 100% [19] [24] 90% a T h e s e were y i e l d s o b t a i n e d a f t e r c h r o m a t o g r a p h i c i s o l a t i o n . b T h i s y i e l d was d e t e r m i n e d v i a HPLC a n a l y s i s . TABLE I I - 4 . C h e m i c a l S h i f t s (ppm) and C o u p l i n g C o n s t a n t s (Hz) o f t h e V i n y l P r o t o n s o f t h e B r o m i n a t e d Compounds Compound H-7 H - 8 H-20 H-21 J H - H [ 5 ] a 6 .38 6 .50 - - 13.7 [9 ]b 6 . 4 0 6 .63 - - 13 .5 [ 2 0 ] c - - 6.47 6 .26 13 .5 [ 2 2 ] a - - 6 .46 6 .29 13.5 [ 2 4 ] a - - 6 . 3 1 6 .13 13 .9 a D e u t e r i o c h l o r o f o r m s o l u t i o n b D e u t e r i o b e n z e n e s o l u t i o n c D e u t e r i o c h l o r o f o r m s o l u t i o n w i t h a few d r o p s o f D e u t e r i o m e t h a n o l added 38 a v o i d s i d e - p r o d u c t f o r m a t i o n due t o r a d i c a l r e a c t i o n s , t h e r e a c t i o n m i x t u r e must be s h i e l d e d f r o m l i g h t . I t was f o u n d t h a t the r e a c t i o n was c o m p l e t e w i t h i n one m i n u t e . L o n g e r r e a c t i o n t i m e s gave no improvement i n y i e l d . The b r o m i n a t e d p r o d u c t s l i s t e d i n T a b l e I I - 3 , e x c e p t f o r B - E / Z - b r o m o -s t y r e n e [ 1 3 ] , were p r e v i o u s l y unknown compounds . A l l of t h e s e p r o d u c t s gave s p e c t r a l ( *H nmr and mass s p e c t r o m e t r i c ) d a t a i n f u l l a c c o r d w i t h t h e a s s i g n e d s t r u c t u r e s . The p r e s e n c e o f b r o m i n e was c o n f i r m e d i n t h e mass s p e c t r a o f t h e p r o d u c t s , by o b s e r v a t i o n of t h e c h a r a c t e r i s t i c b r o m i n e i s o t o p e p a t t e r n ( 7 9 B r , 50.5% abundance and 8 1 B r , 49.5% a b u n d a n c e ) . The * H nmr d a t a e x h i b i t e d a marked change i n t h e v i n y l p r o t o n r e s o n a n c e s o f t h e v i n y l - b r o m i n a t e d p r o d u c t s as compared t o t h e s t a r t i n g t i n p r e c u r s o r s ( s e e T a b l e I I - 4 ) . A t y p i c a l * H nmr s p e c t r u m i s shown I n f i g u r e I I - 9 . The c o u p l i n g c o n s t a n t s between t h e v i n y l p r o t o n s r a n g e d f r o m 13 .5 t o 13 .9 Hz c o n f i r m i n g t h e t r a n s s t e r e o c h e m i s t r y . A l t h o u g h t h e v i n y l p r o t o n s c o u l d be u n a m b i g u o u s l y a s s i g n e d f o r the c a r b o h y d r a t e d e r i v a t i v e s ( [ 5 ] , [ 9 ] ) , t h e v i n y l p r o t o n r e s o n a n c e s o f t h e e s t r a d i o l d e r i v a t i v e s ( [ 2 0 ] , [ 2 2 ] , [ 2 4 ] ) c o u l d be a s s i g n e d o n l y t e n t a t i v e l y b a s e d on c a l c u l a t e d c h e m i c a l s h i f t s . S a t i s f a c t o r y e l e m e n t a l a n a l y s e s were o b t a i n e d f o r e a c h new p r o d u c t e x c e p t [ 2 0 ] . Compound [20] c o u l d o n l y be c r y s t a l l i z e d ( b e n z e n e - h e x a n e s ) as t h e s o l v e n t a d d u c t , t r a p p i n g o u t a h a l f mole o f b e n z e n e p e r mole o f compound. A t t e m p t s f r o m o t h e r s o l v e n t sys tems e i t h e r f a i l e d t o g i v e c r y s t a l s o r y i e l d e d s o l v e n t a d d u c t s as w e l l . Any a t t e m p t t o remove t h e t r a p p e d s o l v e n t by h e a t i n g ( 5 6 ° ) i n vacuo l e a d to d e c o m p o s i t i o n . H o w e v e r , h i g h r e s o l u t i o n mass s p e c t r o m e t r y gave a s a t i s f a c t o r y m o l e c u l a r w e i g h t d e t e r m i n a t i o n f o r [ 2 0 ] . I 8 7 F i g u r e I I - 9 . I 6 5 4 3 2 1 0 270 MHz * H nmr s p e c t r a o f the v i n y l - t i n p r e c u r s o r [18] ( s p e c t r u m A) and t h e v i n y l - b r o m i n a t e d p r o d u c t [22] ( s p e c t r u m B ) , o b t a i n e d i n d e u t e r i o c h l o r o f o r m . I 3 T T OJ 40 As was r e p o r t e d e a r l i e r , t h e b r o m i n a t i o n o f [17] gave [20] i n p o o r y i e l d (13%). T h i s d i r e c t l y r e s u l t e d f r o m s i d e - r e a c t i o n s w i t h the p h e n o l i c A - r i n g o f [ 1 7 ] . P h e n o l s a r e h i g h l y a c t i v a t e d toward e l e c t r o p h i l i c a r o m a t i c s u b s t i t u t i o n . 5 5 T h e r e f o r e , i t i s no s u r p r i s e t h a t t h e A - r i n g would be c a p a b l e o f r e a c t i n g w i t h t h e e l e c t r o p h i l i c b r o m i n a t i n g s p e c i e s p r e s e n t . 5 6 T h i s was e v i d e n c e d by t h e s i d e - p r o d u c t s o b t a i n e d i n t h e c o u r s e o f i s o l a t i o n o f [20] ( s e e e x p e r i m e n t a l s e c t i o n ) and t h e s e a r e shown i n f i g u r e 1 1 - 1 0 . The s i d e -p r o d u c t s were i d e n t i f i e d u s i n g * H nmr, a s s i s t e d i n some c a s e s by mass s p e c t r o m e t r y . A f u l l c h a r a c t e r i z a t i o n o f t h e s e compounds was n o t a t t e m p t e d . The * H nmr a s s i g n m e n t s were t e n t a t i v e l y made, i n c o m p a r i s o n to o t h e r compounds w i t h s i m i l a r s t r u c t u r a l f e a t u r e s , 5 7 and a r e c o n s i s t e n t w i t h the a s s i g n e d s t r u c t u r e s g i v e n i n f i g u r e 1 1 - 1 0 . O t h e r methods were b r i e f l y examined to f i n d a more s u i t a b l e method f o r b r o m i n a t i o n o f [ 1 7 ] . Compound [17] was t r e a t e d w i t h 1.1 e q u i v a l e n t s of NaBr and N - c h l o r o s u c c i n i m i d e (NCS) i n a T H F - m e t h a n o l m i x t u r e a t room t e m p e r a t u r e f o r 15 m i n u t e s . A n a l y t i c a l t i c i n d i c a t e d a m i x t u r e o f p r o d u c t s were o b t a i n e d t h a t was v e r y s i m i l a r t o t h a t o b s e r v e d i n t h e N a B r / C h l o r a m i n e - T r e a c t i o n . Compound [17] was a l s o t r e a t e d w i t h B r 2 i n c h l o r o f o r m a t 0 ° , b u t l i k e w i s e gave a m i x t u r e o f p r o d u c t s . L a s t l y , [17] was a l l o w e d to r e a c t w i t h an e q u i m o l a r amount o f NaBr i n t h e p r e s e n c e o f H 2 0 2 - a c e t i c a c i d ( b u f f e r e d w i t h a s o l u t i o n o f sodium a c e t a t e i n a c e t i c a c i d ) f o r 50 m i n u t e s a t room t e m p e r a t u r e . H o w e v e r , a n a l y t i c a l t i c showed p r e d o m i n a t e l y s t a r t i n g m a t e r i a l t o be p r e s e n t w i t h v e r y l i t t l e c o n v e r s i o n t o p r o d u c t . I t i s c l e a r t h a t t o p e r f o r m s u c c e s s f u l b r o m i n a t i o n s a t t h e t i n - c a r b o n s i t e r e q u i r e s p r o t e c t i o n o f t h e 41 Sn(Bu) Figure 11-10. Side-products obtained during the preparation of compound [20]. 4 2 phenolic hydroxyl group i f the Chloramine-T oxidation method i s to be employed. Concerning the mechanism for the oxidation of NaBr by Chloramine-T, the act u a l o x i d i z i n g species, i n aqueous s o l u t i o n , has been assumed to be H0C1 which e x i s t s i n a complex series of e q u i l i b r i a with Chloramine-T (see below). 5 8 More recent evidence has been claimed to show that at low pH TsNHCl T s N C I N a + v N T s N C I + H + v ^ 2 T s N H C l v s T s N C I 2 + H 2 0 v N T s N H C l + H 2 0 v N w h e r e T s = C H 3 C 6 T s N C I + N a + (1 ) T s N H C l ( 2 ) T s N C I 2 + T s N H 2 ( 3 ) T s N H C l + H O C I ( 4 ) T s N H 2 + H O C I ( 5 ) 4 s o 2 and TsNCl 2 are the ac t u a l oxidants, as these are predominate i n strongly a c i d i c s o l u t i o n . 5 8 The oxidant responsible then reacts with NaBr to form some type of e l e c t r o p h i l i c bromine ( B r + ) , but the actual complex containing B r + v a r i e s depending on the pH. 5 9 At low ( < 3 ) pH, the interhalogen species, B r C l , 5 9 i s thought to be formed, which i s s t a b i l i z e d at high a c i d c o n c e n t r a t i o n s . 6 0 The complex H 2OBr + has also been suggested. 5 9 However, at high ( > 5 ) pH, N-brominated species, such as TsNClBr, of Chloramine-T are 4 3 claimed to be the most l i k e l y s p e c i e s . 5 9 If BrCl i s the act u a l brominating species under a c i d i c conditions, the tin-carbon bond cleavage can be r a t i o n a l i z e d as a concerted reaction proceeding v i a a four-numbered t r a n s i t i o n state, l i k e that described f o r e l e c t r o p h i l i c substitutions of mercury. 6 1 Since BrCl i s po l a r i z e d with bromine car r y i n g a p a r t i a l p o s i t i v e charge, the bromine atom w i l l become attached to the tin-bearing carbon i n a s t e r e o s p e c i f i c fashion as shown above. Unfortunately, l i t t l e i s a c t u a l l y known about these types of reactions. 44 II.4 Iodine Labelling Studies The s t r a t e g y t o l a b e l v i n y l - t i n d e r i v a t i v e s w i t h i o d i n e i s e x a c t l y t h e same as t h a t d e s c r i b e d f o r b r o m i n e l a b e l l i n g , as d i s c u s s e d i n the p r e c e -e d i n g s e c t i o n . C h l o r a m i n e - T i s e q u a l l y s u i t a b l e f o r t h e o x i d a t i o n o f i o d i d e , s i n c e i o d i d e i s e a s i e r t o o x i d i z e t h a n b r o m i d e . The i o d i n a t i n g s p e c i e s e x p e c t e d t o be g e n e r a t e d w o u l d be I C 1 , w h i c h p o s s e s s e s a more p o l a r i z e d bond t h a n B r C l . 1 9 I t was f o u n d t h a t t h e c o n d i t i o n s f o r s u c c e s s f u l i o d i n a t i o n c o u l d be a c h i e v e d a f t e r m o d i f y i n g t h e r e a c t i o n c o n d i t i o n s u s e d f o r b r o m i n a t i o n . I t was o b s e r v e d t h a t t h e i o d i n a t i n g s p e c i e s was l e s s r e a c t i v e t h a n t h e c o r r e s p o n d i n g b r o m i n a t i n g s p e c i e s . As a r e s u l t , t h e r e a c t i o n n e e d e d t o be p e r f o r m e d a t room t e m p e r a t u r e and t h e q u a n t i t y o f a c i d u s e d had t o be i n c r e a s e d t o t h r e e e q u i v a l e n t s o f 1M H C 1 . O t h e r w i s e , t h e r e a c t i o n c o n d i t i o n s r e m a i n e d t h e same as t h o s e d e s c r i b e d f o r b r o m i n a t i o n i n s e c t i o n I I . 3 . The v i n y l - t i n d e r i v a t i v e s [M» [ 8 ] , [ 1 7 ] , [18] and [19] were i n t u r n i o d i n a t e d ( s e e f i g u r e s I I - l and I I - 2 ) as d e s c r i b e d above and t h e r e s u l t s a r e summarized i n T a b l e I I - 5 . V e r y good y i e l d s were o b t a i n e d f o r e a c h o f t h e i o d i n a t e d p r o d u c t s e x c e p t f o r 1 7 a - E - i o d o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 3 - d i o l [21] w h i c h was i s o l a t e d i n v e r y p o o r y i e l d . As was o b s e r v e d i n t h e b r o m i n a t i o n o f [ 1 7 ] , t h e a r o m a t i c A - r i n g was h i g h l y a c t i v a t e d t o w a r d e l e c t r o p h i l i c a t t a c k , 5 5 t h u s A - r i n g i o d i n a t i o n was r e s p o n s i b l e f o r t h e low y i e l d o f [ 2 1 ] . I n f a c t , an a l t e r n a t e method was u s e d t o p r e p a r e [21] i n q u a n t i t y ( s e e t h i r d e n t r y i n T a b l e I I - 5 ) , b u t t h e s e r e s u l t s w i l l be d e s c r i b e d l a t e r i n d e t a i l . The same g e n e r a l o b s e r v a t i o n s were made f o r t h e i o d i n a t i o n r e s u l t s , u s i n g t h e C h l o r a m i n e - T TABLE I I - 5 . Y i e l d s o f V i n y l - I o d i n a t e d D e r i v a t i v e s S t a r t i n g M a t e r i a l P r o d u c t % Y i e l d 3 [4] [6] 92% [8] [10] 97% [ 1 7 j c [21]c 9 3 % D » C [17] [21] 1.9% b [18] [23] 88% [19] [25] 99% a T h e s e were y i e l d s o b t a i n e d a f t e r c h r o m a t o g r a p h i c i s o l a t i o n . b T h e s e y i e l d s were d e t e r m i n e d v i a HPLC a n a l y s i s . c T h i s r e s u l t was o b t a i n e d u s i n g t h e method o f H 2 0 , -a c e t i c a c i d o x i d a t i o n o f N a l d e v e l o p e d by F r a n k e and H a n s o n . 3 5 TABLE I I - 6 . C h e m i c a l S h i f t s (ppm) and C o u p l i n g C o n s t a n t s (Hz) o f t h e V i n y l P r o t o n s o f t h e I o d i n a t e d Compounds Compound H-7 H-8 H-20 H-21 J „ „ H—n [ 6 ] a 6 .73 6 .53 - - 14 .5 [ 1 0 ] D 6 .76 6 .68 - - 14 .5 [ 2 1 ] c - - 6 .79 6 .25 14 .4 [ 2 3 ] a - . - 6 .79 6 .29 14 .3 [ 2 5 ] D - - 6 .67 5 .94 1 4 . 8 a D e u t e r i o c h l o r o f o r m s o l u t i o n D D e u t e r i o b e n z e n e s o l u t i o n c D e u t e r i o c h l o r o f o r m s o l u t i o n w i t h a few d r o p s o f D e u t e r i o m e t h a n o l added 46 o x i d a t i o n o f N a l , as was d e s c r i b e d f o r t h e b r o m i n a t i o n r e s u l t s i n s e c t i o n I I . 3 . The i o d i n a t e d p r o d u c t s l i s t e d i n T a b l e I I - 5 , e x c e p t f o r [21] and 3 - m e t h o x y - 1 7 a - E - i o d o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 1 7 g - o l [ 2 3 ] , were p r e v i o u s l y unknown compounds . Compound [21] was p r e v i o u s l y p r e p a r e d by Hanson and c o - w o r k e r s 3 9 i n 1982 u s i n g I 2 c l e a v a g e o f t h e v i n y l - t i n p r e c u r s o r [ 1 7 ] , and c h a r a c t e r i z a t i o n d e t a i l s were r e p o r t e d l a t e r i n 1984.** 3 The p r e p a r a t i o n o f [ 2 1 ] , by a d i f f e r e n t m e t h o d , was r e p o r t e d i n 1984 by F e r r e i r a and c o - w o r k e r s 6 2 u s i n g the c l e a v a g e o f v i n y l - b o r o n i c a c i d s by N a l o x i d i z e d by C h l o r a m i n e - T u n d e r n e u t r a l c o n d i t i o n s . Compound [23] was f i r s t s y n t h e s i z e d i n 1981 by K a b a l k a and c o - w o r k e r s 6 3 v i a IC1 c l e a v a g e o f t h e c o r r e s p o n d i n g v i n y l - b o r o n i c a c i d . F e r r e i r a and c o - w o r k e r s a l s o p r e p a r e d [23] f o r t h e i r s t u d i e s . 6 2 The i o d i n a t e d p r o d u c t s l i s t e d i n T a b l e I I - 5 gave s p e c t r a l ( 1 H nmr and mass s p e c t r o m e t r i c ) d a t a i n f u l l a c c o r d w i t h t h e a s s i g n e d s t r u c t u r e s . The * H nmr e x h i b i t e d v i n y l p r o t o n r e s o n a n c e s w i t h q u i t e d i f f e r e n t c h e m i c a l s h i f t s as compared t o e i t h e r t h e s t a r t i n g t i n p r e c u r s o r s o r t h e b r o m i n a t e d a n a l o g u e s ( s e e T a b l e I I - 6 ) . The X H nmr s p e c t r u m of [23] i s shown i n f i g u r e 1 1 - 1 1 . The c o u p l i n g c o n s t a n t s o f t h e v i n y l p r o t o n s c o n f i r m e d t h e t r a n s s t e r e o c h e m i s t r y o f the d o u b l e b o n d . The v i n y l p r o t o n r e s o n a n c e s of 3 , 1 7 g - d i m e t h o x y - 1 7 a - E - i o d o -v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e [25] were t e n t a t i v e l y a s s i g n e d b a s e d on c o m p a r i s o n w i t h o t h e r known v i n y l - i o d i n a t e d d e r i v a t i v e s i n c l u d i n g [21] and [ 2 3 ] . S a t i s f a c t o r y e l e m e n t a l a n a l y s e s were o b t a i n e d f o r e a c h p r o d u c t l i s t e d i n T a b l e I I - 5 , e x c e p t [ 2 1 ] . As was e x p e r i e n c e d w i t h t h e v i n y l - b r o m i n a t e d d e r i v a t i v e [ 2 0 ] , [21] c r y s t a l l i z e d w i t h t h e e n t r a p m e n t o f s o l v e n t m o l e c u l e s . T h i s was a l s o t h e e x p e r i e n c e o f F e r r e i r a and c o - w o r k e r s 6 2 as t h e y i n i t i a l l y i s o l a t e d I 8 7 F i g u r e I 4 11-11 . " i i i 1 1 r 6 5 4 3 2 1 0 270 MHz lE nmr s p e c t r a of the v i n y l - t i n p r e c u r s o r [18] ( s p e c t r u m A) and the v i n y l - i o d i n a t e d p r o d u c t [23] ( s p e c t r u m B ) , o b t a i n e d i n d e u t e r i o c h l o r o f o r m . 4 8 [21] as a c r y s t a l l i n e a d d u c t w i t h C H C l j . R e c r y s t a l l i z a t i o n o f t h e C H C l g a d d u c t f r o m e t h a n o l - w a t e r gave [21] as t h e h y d r a t e - 6 2 I n o u r c a s e , r e c r y s t a l l i z a t i o n f r o m b e n z e n e - h e x a n e s y i e l d e d t h e benzene a d d u c t . H e a t i n g ( 5 6 ° ) of t h e p r o d u c t i n vacuo l e d t o d e c o m p o s i t i o n . However , h i g h r e s o l u t i o n mass s p e c t r o m e t r y gave a s a t i s f a c t o r y m o l e c u l a r w e i g h t d e t e r m i n a t i o n f o r [ 2 1 ] . As was r e p o r t e d e a r l i e r , t h e i o d i n a t i o n o f [17] gave [21] i n v e r y p o o r y i e l d (1 .9%). As was d i s c u s s e d f o r t h e a n a l o g o u s b r o m i n a t i o n o f [17] e a r l i e r i n s e c t i o n I I . 3 , t h e r e a s o n f o r t h i s low y i e l d i s t h e c o m p e t i n g e l e c t r o p h i l i c r e a c t i v i t y o f t h e A - r i n g . As a r e s u l t , c o n c u r r e n t A - r i n g i o d i n a t i o n was o b s e r v e d t o t a k e p l a c e d u r i n g t i n - c a r b o n bond c l e a v a g e . T h i s was e v i d e n c e d by t h e s i d e - p r o d u c t s o b t a i n e d d u r i n g i s o l a t i o n o f [21] ( s e e e x p e r i m e n t a l s e c t i o n ) and t h e s e a r e shown i n f i g u r e 1 1 - 1 2 . The s i d e - p r o d u c t s were i d e n t i f i e d by *H nmr and mass s p e c t r o m e t r y . A f u l l c h a r a c t e r i z a t i o n o f t h e s e compounds was n o t a t t e m p t e d . The * H nmr a s s i g n m e n t s were r e a d i l y a c c o m p l i s h e d by c o m p a r i s o n t o s i m i l a r compounds and a r e c o n s i s t e n t w i t h t h e a s s i g n e d s t r u c t u r e s shown i n f i g u r e 1 1 - 1 2 . P a r t i a l * H nmr s p e c t r a o f some o f t h e s e s i d e - p r o d u c t s a r e shown i n f i g u r e 1 1 - 1 3 . I n o r d e r t o p r e p a r e [21] i n s u f f i c i e n t q u a n t i t y , t h e r a d i o - l a b e l l i n g p r o c e d u r e d e v e l o p e d by F r a n k e and H a n s o n 3 5 was a d a p t e d and u s e d f o r n o n - r a d i o a c t i v e i o d i n a t i o n . The v i n y l - t i n d e r i v a t i v e was s u c c e s s f u l l y i o d i n a t e d u s i n g t h i s p r o c e d u r e w h i c h c o n s i s t e d o f r e a c t i n g a s l i g h t e x c e s s o f N a l i n t h e p r e s e n c e o f H 2 0 2 ~ a c e t i c a c i d ( b u f f e r e d w i t h a s o l u t i o n o f s o d i u m a c e t a t e i n a c e t i c a c i d ) f o r 30 m i n u t e s a t room t e m p e r a t u r e . The p r o d u c t [21] was i s o l a t e d i n 77% y i e l d . I n t h i s r e a c t i o n , h o w e v e r , n o t a l l t h e p r o d u c t was 4 9 Figure 11-12. Side-products obtained during the preparation of compound [21]. 5 0 F i g u r e 1 1 - 1 3 . P a r t i a l 1 H nmr s p e c t r a o f s i d e - p r o d u c t s [36] ( s p e c t r u m A ) and [37] ( s p e c t r u m B ) , o b t a i n e d i n d e u t e r i o c h l o r o f o r m , as compared to compound [21] ( s p e c t r u m C) i n d e u t e r i o c h l o r o f o r m w i t h a few d r o p s o f d e u t e r i o m e t h a n o l a d d e d . i s o l a t e d as e f f i c i e n t l y as possible. The a n a l y t i c a l t i c c l e a r l y showed a quantitative conversion to have resulted. To properly a s c e r t a i n the chemical y i e l d s of t h i s reaction and the Nal/Chloramine-T method, small scale reactions were performed and the reaction mixtures were analyzed by HPLC. The r e s u l t s are presented i n Table II-5 (entries 3 and 4). C l e a r l y the NaI/H 20 2-acetic a c i d oxidation i s superior to Nal/Chloramine-T i n t h i s case. Indeed, Franke and Hanson found the Nal/H 20 2~acetic a c i d method to be very successful i n 1 2 5 I - l a b e l l i n g , however, they did not report the use of t h i s method f o r the synthesis of non-radioactive iodinated d e r i v a t i v e s . 3 5 Mechanistically, the i o d i n a t i o n of v i n y l - t i n d e r i v a t i v e s , using the oxidation of Nal v i a Chloramine-T, can be considered exactly analogous to the corresponding bromination process discussed i n section II.3. The only d i f f e r e n c e i s that instead of o x i d i z i n g NaBr to BrCl, Nal i s oxidized to IC1, under a c i d i c conditions. The same r a t i o n a l i z a t i o n can then be employed f o r the r e a c t i o n of IC1 with the v i n y l - t i n precursor. 52 CHAPTER I I I LABELLING STUDIES WITH RADIOACTIVE BROMINE AND IODINE 111.1 Introduction I n t h i s c h a p t e r , r a d i o a c t i v e l a b e l l i n g s t u d i e s w i t h 8 2 B r , 1 2 3 I and 1 3 1 I w i l l be d e s c r i b e d . R a t h e r t h a n l a b e l a number o f v i n y l - t i n compounds, t h e v i n y l - t i n d e r i v a t i v e [18] was u s e d i n a l l t h e l a b e l l i n g e x p e r i m e n t s . T h i s a p p r o a c h e n a b l e d a more t h o r o u g h e v a l u a t i o n o f t h e C h l o r a m i n e - T l a b e l l i n g p r o c e d u r e d e v e l o p e d i n c h a p t e r I I , g i v e n l i m i t e d a v a i l a b i l i t y o f t h e r a d i o -n u c l i d e s . S i n c e t h e q u a n t i t y o f t h e r a d i o - l a b e l l e d p r o d u c t i s so s m a l l * , t h e s t a n d a r d methods o f c h a r a c t e r i z a t i o n , s u c h as 1 H nmr, c a n n o t be u s e d f o r p r o d u c t i d e n t i f i c a t i o n . T h e r e f o r e , c h r o m a t o g r a p h i c a n a l y s i s , u s i n g n o n -r a d i o a c t i v e a n a l o g u e s as s t a n d a r d s , i s t h e b e s t means o f p r o d u c t i d e n t i f i c a -t i o n a v a i l a b l e . The c h r o m a t o g r a p h i c method b e s t s u i t e d f o r t h i s p u r p o s e i s H P L C 2 5 . The r a d i o b r o m i n a t i o n o f [18] w i l l now be d i s c u s s e d i n d e t a i l , f o l l o w e d by t h e c o r r e s p o n d i n g r a d i o i o d i n a t i o n s t u d i e s . 111.2 Radiobromination Studies The r a d i o b r o m i n e i s o t o p e s , 7 5 B r and 7 7 B r , a r e t h e most u s e f u l r a d i o n u c l i d e s o f b r o m i n e f o r d i a g n o s t i c n u c l e a r medicine.**** U n f o r t u n a t e l y , * A s was d i s c u s s e d i n s e c t i o n 1 . 2 , t h i s a p p l i e s o n l y t o s h o r t - l i v e d r a d i o n u c l i d e s p o s s e s s i n g h i g h s p e c i f i c a c t i v i t y . Compounds l a b e l l e d w i t h t r i t i u m , f o r e x a m p l e , c a n be c h a r a c t e r i z e d by s t a n d a r d m e t h o d s . 53 7 5 B r and 7 7 B r a r e n o t a v a i l a b l e c o m m e r c i a l l y o r f r o m TRIUMF a t t h e p r e s e n t t i m e . As a r e s u l t , 8 2 B r was c h o s e n f o r t h i s s t u d y as i t i s c o m m e r c i a l l y a v a i l a b l e f r o m a number o f s u p p l i e r s . 8 2 B r i s a r e a c t o r - s o u r c e r a d i o i s o t o p e t h a t d e c a y s by B~ e m i s s i o n ( h a l f - l i f e i s 35 .34 h o u r s ) . 6 5 The drawback o f u s i n g 8 2 B r I s t h a t i t i s p r o d u c e d i n low s p e c i f i c a c t i v i t y ( o u r b a t c h had a s p e c i f i c a c t i v i t y o f ~255 mCi/mmol) f r o m 8 1 B r , w h i c h means t h a t a s i g n i f i c a n t amount o f 8 1 B r i s p r e s e n t as c a r r i e r . 6 6 T h e r e f o r e , NCA r e a c t i o n s c a n n o t be p e r f o r m e d w i t h 8 2 B r . Compound [18] (4 mg, 6 umol) was t r e a t e d w i t h a b o u t 120-160 u C i o f N H l t 8 2 B r i n t h e p r e s e n c e o f a p p r o x i m a t e l y t h r e e e q u i v a l e n t s o f C h l o r a m i n e - T ( C A T ) , a c i d i f i e d w i t h 1M HC1 ( s a t u r a t e d w i t h N a C l ) , I n an a q u e o u s - e t h a n o l - T H F m i x t u r e a t room t e m p e r a t u r e , f o r one m i n u t e , as summarized I n f i g u r e I I I - l . A f t e r w o r k u p , HPLC i s o l a t i o n y i e l d e d t h e r a d i o b r o m i n a t e d p r o d u c t , 3 - m e t h o x y -1 7 o - E - ( 8 2 B r ) b r o m o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 1 7 B - o l [ 4 1 ] , i n 77-81% r a d i o -c h e m i c a l y i e l d . A t y p i c a l r a d i o - H P L C c h r o m a t o g r a m , o b t a i n e d d u r i n g t h e i s o l a t i o n o f [ 4 1 ] , i s shown i n f i g u r e I I I - 2 A . The chromatogram i n d i c a t e s a c l e a n c o n v e r s i o n f r o m t h e s t a r t i n g m a t e r i a l [18] (peak C) to t h e d e s i r e d p r o d u c t [41] (peak B ) . A s m a l l amount o f b y - p r o d u c t (peak A) i s formed and was l a t e r i d e n t i f i e d as 3 - m e t h o x y - 1 7 c t - v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 1 7 B - o l [ 3 9 ] , w h i c h w i l l be d i s c u s s e d l a t e r . The v e r y l a r g e p e a k , w h i c h e l u t e d f i r s t , i s due t o C A T . Two a d d i t i o n a l f e a t u r e s a r e p r e s e n t I n t h i s c h r o m a t o g r a m . F i r s t , t h e p r o d u c t [41] r e g i s t e r e d a s i g n i f i c a n t r e s p o n s e f r o m t h e u l t r a - v i o l e t ( u v ) d e t e c t o r . T h i s i n d i c a t e s a l a r g e q u a n t i t y o f c a r r i e r i s p r e s e n t , t h u s t h e p r o d u c t i s o f low s p e c i f i c a c t i v i t y . S e c o n d , t h e p r o d u c t , as i s o l a t e d , was o f 100% r a d i o c h e m i c a l p u r i t y s i n c e no o t h e r r a d i o - l a b e l l e d s i d e - p r o d u c t s a r e 54 F i g u r e I I I - l . S y n t h e s i s o f compound [ 4 1 ] . o b s e r v e d ( t h e t i n y peak a t ~ 2 m i n r e t e n t i o n t ime i n t h e r a d i o t r a c e i s r e s i d u a l N H 1 + 8 2 B r ) i n t h e r a d i o a c t i v e t r a c e . When the r a d i o b r o m i n a t i o n was p e r f o r m e d a t 0 ° , t h e r a d i o c h e m i c a l y i e l d d e c r e a s e d t o 40%. T h i s c a n be v i s i b l y o b s e r v e d i n t h e r a d i o - H P L C chromatogram f o r t h i s r e a c t i o n , shown i n f i g u r e I I I - 2 B . A number o f g e n e r a l o b s e r v a t i o n s were made w i t h r e g a r d to the r a d i o -b r o m i n a t i o n r e s u l t s . As was f o u n d d u r i n g t h e n o n - r a d i o a c t i v e b r o m i n a t i o n s t u d i e s ( s e e s e c t i o n I I . 3 ) , the c o r r e c t q u a n t i t y o f a c i d needed to be d e t e r -m i n e d q u i t e c a r e f u l l y , as an i n c r e a s e o r d e c r e a s e o f 2 u l o f IM HC1 s o l u t i o n c o u l d change t h e r a d i o c h e m i c a l y i e l d by 10-15%. T h i s b e g i n s t o show the t e c h -n i c a l p r o b l e m s i n v o l v e d w i t h p e r f o r m i n g m i c r o - s c a l e r a d i o - l a b e l l i n g r e a c t i o n s . A l l s o l u t i o n s o r m a t e r i a l s need t o be q u a n t i f i e d c a r e f u l l y t o o b t a i n r e p r o d u c -i b l e r e s u l t s . The most d i f f i c u l t m a n i p u l a t i v e p r o b l e m e n c o u n t e r e d was t h e t r a n s f e r , v i a s y r i n g e , o f t h e r e a c t i o n m i x t u r e to t h e HPLC i n s t r u m e n t . The compounds a r e : ( A , 5 .9 m i n ) , [ 3 9 ] ; ( B , 7 .4 m i n ) , [ 4 1 ] ; ( C , 16 .4 m i n ) , [ 1 8 ] . The HPLC c o n d i t i o n s a r e : f l o w r a t e = 6 m l / m i n ; s o l v e n t p r o g r a m : 0 t o 10 m i n i s o c r a t i c 85% MeOH/15% H 2 0 , 10 t o 12 m i n l i n e a r g r a d i e n t f r o m 85% MeOH/15% H 2 0 t o 100% MeOH, 12 t o 20 m i n i s o c r a t i c 100% MeOH; C-18 r e v e r s e phase c o l u m n ; uv d e t e c t o r s e t a t 254 nm. F i g u r e I I I - 2 HPLC chromatograms o b t a i n e d d u r i n g p u r i f i c a t i o n o f compound [ 4 1 ] . Chromatograms A and B r e s u l t e d f rom t h e r a d i o b r o m i n -a t i o n o f [18] w i t h CAT a t room t e m p e r a t u r e and 0 ° , r e s p e c t i v e l y . I n v a r i a b l y , a s m a l l amount o f s o l u t i o n w o u l d r e m a i n i n t h e g l a s s s y r i n g e (1 m l volume) w h i c h s t i l l c o n t a i n e d 10-20 u C i o f r a d i o a c t i v i t y . T h i s amount o f r a d i o a c t i v i t y c o u l d r e p r e s e n t a l o s s o f 10% r a d i o c h e m i c a l y i e l d . When d i s p o s a b l e p l a s t i c s y r i n g e s were u s e d , t h e r u b b e r p l u n g e r was f o u n d t o a b s o r b some o f t h e r a d i o a c t i v i t y , t h u s making t h e s e u n s u i t a b l e . A c o m p l e t e l y s a t i s f a c t o r y s o l u t i o n was not f o u n d . F o r c o m p a r a t i v e p u r p o s e s , an a l t e r n a t i v e p r o c e d u r e u s i n g NCS (shown above) as t h e o x i d a n t , was u s e d f o r t h e r a d i o b r o m i n a t i o n o f [ 1 8 ] . Compound t h r e e e q u i v a l e n t s of NCS i n a T H F - m e t h a n o l m i x t u r e , f o r 30 m i n u t e s . HPLC i s o l a t i o n a f f o r d e d [41] i n 85% r a d i o c h e m i c a l y i e l d . The r a d i o - H P L C chromatogram showed t h a t a d d i t i o n a l u n i d e n t i f i e d s i d e - p r o d u c t s had f o r m e d ( o b s e r v e d f r o m uv t r a c e ) , o v e r l a p p i n g p a r t l y w i t h t h e p r o d u c t p e a k . The r a d i o a c t i v i t y t r a c e showed the p r o d u c t t o be r a d i o c h e m i c a l l y p u r e . The t o t a l s y n t h e s i s t i m e , as measured f r o m t h e a d d i t i o n o f N H l + 8 2 B r to t h e HPLC i s o l a t i o n o f [ 4 1 ] , was 47 m i n u t e s . The C h l o r a m i n e - T p r o c e d u r e r e q u i r e d a s y n t h e s i s t ime o f 17-18 m i n u t e s . [18] was t r e a t e d w i t h 125 y C i o f N H 1 + 8 2 B r i n t h e p r e s e n c e o f a p p r o x i m a t e l y 57 NCS i s a p o p u l a r o x i d i z i n g agent f o r b r o m i d e and i o d i d e . A l t h o u g h t h e NCS p r o c e d u r e was n o t o p t i m i z e d i n t h i s c a s e , i t does show p r o m i s e . However , t h e CAT p r o c e d u r e , b a s e d on t h e s h o r t r e a c t i o n t i m e r e q u i r e d and t h e m i n i m a l s i d e - p r o d u c t f o r m a t i o n , w o u l d seem t o be t h e p r e f e r r e d m e t h o d . The t r u e t e s t o f w h i c h method w o u l d be b e s t i s t o compare e a c h method u s i n g NCA 7 5 B r o r 7 7 B r . T h i s i s i m p o r t a n t as n o t a l l l a b e l l i n g p r o c e d u r e s , s u c c e s s f u l w i t h low s p e c i f i c a c t i v i t y r a d i o n u c l i d e s , w i l l p e r f o r m w e l l w i t h h i g h s p e c i f i c a c t i v i t y r a d i o n u c l i d e s . 1 9 As was d i s c u s s e d e a r l i e r , p r o d u c t i d e n t i f i c a t i o n was p e r f o r m e d u s i n g r a d i o - H P L C a n a l y s i s . The v i n y l - b r o m i n a t e d d e r i v a t i v e [22] e x h i b i t e d t h e same r e t e n t i o n t i m e as t h e r a d i o - p e a k s l a b e l l e d B i n f i g u r e I I I - 2 . T h i s p r o v i d e s s t r o n g e v i d e n c e f o r t h e i d e n t i t y o f t h e r a d i o - l a b e l l e d p r o d u c t a s [ 4 1 ] . The s i d e - p r o d u c t (peak A ) was t h o u g h t t o be due t o e i t h e r c h l o r i n a t i o n o r p r o t o n o l y s i s o f t h e s t a r t i n g v i n y l - t i n p r e c u r s o r [ 1 8 ] . S i n c e t h e p r o t o n a t e d s i d e - p r o d u c t seemed more l i k e l y , a sample o f [39] was p r e p a r e d as summarized i n f i g u r e I I I - 3 , as a s t a n d a r d . Compound [39] was i s o l a t e d i n p u r e f o r m v i a s i l i c a g e l c h r o m a t o g r a p h y , and upon r e c r y s t a l l i z a t i o n a f f o r d e d w h i t e c r y s t a l s o f mp 1 1 0 . 5 ° , [ a ] g 5 = + 5 6 . 9 ° ( 1 , 4 - d i o x a n e ) { l i t . 6 7 mp 9 8 - 1 0 0 ° , [<x]g5 = + 6 0 ° ( c h l o r o f o r m ) } . S p e c t r a l ( 1 H nmr and mass s p e c t r o m e t r i c ) d a t a were c o n s i s t e n t w i t h t h e a s s i g n e d s t r u c t u r e . The r e t e n t i o n t i m e o f [39] was f o u n d t o be t h e same as t h e s i d e - p r o d u c t (peak A ) , t h u s p r o v i d i n g s t r o n g e v i d e n c e f o r t h e i d e n t i f i c a t i o n o f t h e s i d e - p r o d u c t as [ 3 9 ] . Figure I I I - 3 . Synthesis scheme for the preparation of the v i n y l d e r i v a t i v e [39]. 59 III.3 Radioiodination Studies The most i m p o r t a n t r a d i o i o d i n e i s o t o p e s , c u r r e n t l y i n u s e i n d i a g n o s t i c n u c l e a r m e d i c i n e , a r e 1 3 1 I and 1 2 3 I . However , 1 3 1 I i s b e i n g r e p l a c e d more and more by 1 2 3 I as i t becomes more w i d e l y a v a i l a b l e . 2 1 F o r t u n a t e l y , 1 2 3 I i s r e a d i l y a v a i l a b l e a t TRIUMF t h r o u g h A t o m i c E n e r g y o f Canada L t d . (AECL) w h i c h i s a c o m m e r c i a l s u p p l i e r o f 1 2 3 I and o t h e r c y c l o t r o n p r o d u c e d r a d i o n u c l i d e s . T h e r e f o r e , 1 2 3 I was c h o s e n f o r t h i s s t u d y and i t s p h y s i c a l p r o p e r t i e s a r e summarized i n T a b l e 1 - 3 . * 2 3 i i s p r o d u c e d i n h i g h s p e c i f i c a c t i v i t y o f g r e a t e r t h a n 1000 C i / m m o l . Some r a d i o i o d i n a t i o n s were a l s o p e r f o r m e d w i t h 1 3 1 I # The 1 2 5 I - l a b e l l e d a n a l o g u e o f [23] has b e e n p r e p a r e d p r e v i o u s l y by s e v e r a l r e s e a r c h g r o u p s . 3 5 > 3 8 > 6 2 However , n e i t h e r t h e 1 2 3 l - n o r 1 3 1 l -l a b e l l e d a n a l o g u e s has b e e n s y n t h e s i z e d b e f o r e . I t i s i m p o r t a n t t o n o t e t h a t methods s u c c e s s f u l i n i n c o r p o r a t i n g 1 2 5 I have n o t a l w a y s worked as w e l l when r e p e a t e d w i t h h i g h s p e c i f i c a c t i v i t y l 2 3 i . 6 8 Compound [18] (2 mg, 3 umol) was a l l o w e d t o r e a c t w i t h between 50 and 270 y C i of NCA N H ^ 1 2 3 ! , o r N a 1 2 3 I , i n t h e p r e s e n c e o f a p p r o x i m a t e l y two e q u i v a l e n t s o f C A T , a c i d i f i e d w i t h 1M HC1 ( s a t u r a t e d w i t h N a C l ) , i n an a ' q u e o u s - e t h a n o l - T H F m i x t u r e a t room t e m p e r a t u r e , f o r one m i n u t e ( see f i g u r e I I I - 4 ) . A f t e r w o r k u p , HPLC i s o l a t i o n a f f o r d e d the d e s i r e d p r o d u c t , 3 - m e t h o x y -1 7 a - E - ( 1 2 3 I ) i o d o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 1 7 6 - o l [ 4 2 ] . The r e s u l t s a r e summarized i n T a b l e I I I - l . The r a d i o i o d i n a t i o n o f [18] was a l s o p e r f o r m e d , u n d e r t h e same c o n d i t i o n s , w i t h NCA N a 1 3 1 I and t h e c o r r e s p o n d i n g p r o d u c t , F i g u r e I I I - 4 . S y n t h e s i s o f t h e r a d i o i o d i n a t e d compounds [42] and [ 4 3 ] . TABLE I I I - l . Summary o f R a d i o i o d i n a t i o n R e s u l t s X~ O x i d a n t R e a c t i o n R a d i o c h e m i c a l Time ( m i n ) Y i e l d (%) NCA N H ^ 1 2 31 CAT 1 .0 82-90 NCA N a 1 3 1 I CAT 1 .0 82-85 NCA N a 1 2 3 I CAT 1 .0 82-88 CA N a 1 2 3 I CAT 1 .0 91 NCA N a 1 2 3 I NCS 30 62 CA N a 1 2 3 I NCS 30 85 NCA N a 1 2 3 I H 2 0 2 - H O A c 30 85 CA N a 1 2 3 I H 2 0 2 - H O A c 30 81 61 i — > U J 00 z o Q_ 00 Ixt rr rr o i-o U J I -U J Q V IB u o o "O o > U J 00 U J z 00 o ~z. Q_ o 00 Q_ U J 00 rr U J rr rr o cr 1- o O r— U J O r - U J U J I -Q U J Q 1 1 1 1 i r 0 4 8 12 16 20 T I M E ( m i n ) u B VivJ u o o o Ixl 00 z o 0. oo Ixl rr or o h-O U J r-n i i i r 0 4 8 12 16 T I M E ( m i n ) 20 The compounds a r e : ( A , 6 .9 m i n ) , [ 3 8 ] ; ( B , 8 . 1 m i n ) , [ 4 2 ] ; ( C , 16.4 m i n ) , [ 1 8 ] . T h e HPLC c o n d i t i o n s a r e : f l o w r a t e = 6 m l / m i n ; s o l v e n t p r o g r a m : 0 t o 10 m i n i s o c r a t i c 85% Me0H/15% H , 0 , 10 t o 12 rain l i n e a r g r a d i e n t f r o m 85% Me0H/15% H 2 0 t o 100% MeOH, 12 t o 20 m i n i s o c r a t i c 100% MeOH; C-18 r e v e r s e phase c o l u m n ; uv d e t e c t o r s e t a t 254 nm. F i g u r e I I I - 5 . HPLC chromatograms o b t a i n e d d u r i n g p u r i f i c a t i o n o f compound [ 4 2 ] . Chromatograms A and B r e s u l t e d f r o m t h e r a d i o -i o d i n a t i o n o f [18] w i t h CAT u n d e r NCA and CA c o n d i t i o n s , r e s p e c t i v e l y . 62 TIME (min) The compounds a r e : ( A , 5 .9 m i n ) , [ 3 9 ] ; ( B , 6 .9 m i n ) , [ 3 8 ] ; ( C , 8 . 1 m i n ) , [ 4 3 ] ; ( D , 16 .4 m i n ) , [ 1 8 ] . The HPLC c o n d i t i o n s a r e : f l o w r a t e = 6 m l / m i n ; s o l v e n t p r o g r a m : 0 t o 10 m i n i s o c r a t i c 85% MeOH/15% H , 0 , 10 to 12 rain l i n e a r g r a d i e n t f r o m 85% MeOH/15% H 2 0 t o 100% MeOH, 12 t o 20 min i s o c r a t i c 100% MeOH; C-18 r e v e r s e phase c o l u m n ; uv d e t e c t o r s e t a t 254 nm. F i g u r e I I I - 6 . HPLC chromatogram o b t a i n e d d u r i n g the p u r i f i c a t i o n o f compound [43] w h i c h r e s u l t e d f r o m the r a d i o i o d i n a t i o n o f [18] w i t h CAT u n d e r NCA c o n d i t i o n s . 63 F i g u r e I I I - 7 . S y n t h e s i s o f compound [ 3 8 ] . 3 - m e t h o x y - 1 7 a - E - ( 1 3 1 I ) i o d o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 1 7 e - o l [ 4 3 ] , was s u c c e s s f u l l y o b t a i n e d . The NCA s y n t h e s e s o f [42] and [43] were f o u n d to be v e r y s u c c e s s f u l . R e p r e s e n t a t i v e r a d i o - H P L C chromatograms a r e shown i n f i g u r e I I I - 5 A ( 1 2 3 I -l a b e l l i n g e x p e r i m e n t ) and f i g u r e I I I - 6 ( 1 3 1 I - l a b e l l i n g e x p e r i m e n t ) . The key f e a t u r e s o f t h e s e chromatograms a r e e s s e n t i a l l y t h e same. I n f i g u r e I I I - 5 A t h e s t a r t i n g m a t e r i a l ( p e a k C) f o r m e d a s i n g l e new r a d i o i o d i n a t e d p r o d u c t [42] (peak B) w h i c h e x h i b i t e d t h e same c h r o m a t o g r a p h i c p r o p e r t i e s as t h e v i n y l -i o d i n a t e d d e r i v a t i v e [ 2 3 ] . T h i s p r o v i d e s s t r o n g e v i d e n c e f o r t h e i d e n t i t y o f 64 t h e r a d i o - p e a k . H o w e v e r , n o t e t h e a b s e n c e o f a uv r e s p o n s e a t t h e same r e t e n t i o n t i m e as [ 4 2 ] . T h i s shows t h a t [42] p o s s e s s e s r e l a t i v e l y h i g h s p e c i f i c a c t i v i t y ( c a r r i e r i o d i n e n o t d e t e c t a b l e ) , i n c o m p a r i s o n t o t h e r a d i o b r o m i n a t e d a n a l o g u e [41] d i s c u s s e d e a r l i e r ( s e c t i o n I I I . 2 ) . A m a j o r s i d e - p r o d u c t (peak A ) was f o r m e d i n t h e r e a c t i o n , and was c o l l e c t e d d u r i n g a number o f e x p e r i m e n t s . T h i s m a t e r i a l was a n a l y z e d by mass s p e c t r o m e t r y . The s p e c t r a l d a t a i n d i c a t e d t h e s i d e - p r o d u c t t o be 3 - m e t h o x y -1 7 a - E - c h l o r o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 1 7 6 - o l [38] ( s e e f i g u r e I I I - 7 ) . T h e r e f o r e , [38] was p r e p a r e d by t r e a t i n g [18] w i t h two e q u i v a l e n t s of CAT and IM HC1 ( s a t u r a t e d w i t h N a C l ) , i n an a q u e o u s - e t h a n o l - T H F m i x t u r e a t room tem-p e r a t u r e , f o r two h o u r s . Compound [38] was i s o l a t e d v i a s i l i c a g e l c h r o m a t o -g r a p h y i n 69% y i e l d . S i n c e [38] has n o t b e e n p r e v i o u s l y r e p o r t e d , a c o m p l e t e c h a r a c t e r i z a t i o n was p e r f o r m e d . The 1 H nmr s p e c t r u m , shown i n f i g u r e I I I - 8 , c o n f i r m e d t h e s t r u c t u r e o f [ 3 8 ] . The v i n y l p r o t o n s (H-20 and H-21 ) a r e o b s e r v e d as a two p r o t o n s i n g l e t a t 6 6 .20 i n d e u t e r i o c h l o r o f o r m . H o w e v e r , i n d e u t e r i o b e n z e n e , an AB q u a r t e t i s o b s e r v e d w i t h a c o u p l i n g c o n s t a n t o f 1 3 . 1 H z . The low f i e l d d o u b l e t a t 6 6 . 11 was t e n t a t i v e l y a s s i g n e d t o H - 2 1 , whereas t h e h i g h f i e l d d o u b l e t a t 6 6 .04 was a c c o r d i n g l y a s s i g n e d t o H - 2 0 . The c h l o r i n a t i o n s i d e - r e a c t i o n i s q u i t e f a c i l e , as i s shown by t h e p r e p -a r a t i o n o f [ 3 8 ] . The amount o f c h l o r i n a t i o n o b s e r v e d d u r i n g r a d i o i o d i n a t i o n v a r i e s f r o m e x p e r i m e n t t o e x p e r i m e n t . The amount o f c h l o r i n a t i o n o b s e r v e d i n t h e r a d i o - H P L C chromatogram shown i n f i g u r e I I I - 6 was o f t e n o b s e r v e d d u r i n g 1 2 3 x - l a b e l l i n g s t u d i e s . The p r e s e n c e o f t h e c h l o r i n a t e d s i d e - p r o d u c t [38] makes f o r a d i f f i c u l t s e p a r a t i o n p r o b l e m . O v e r l a p o f [42] w i t h [ 3 8 ] , CHCI H 2 0 , H 2 1 \ in Benzene 39 H i H 2 i H 20 1 I ii J V 3 - O C H , 1 7 - O H - C H 7 a n d - C H -f rom steroid r ing 1 8 - C H , T M S J r 8 7 F i g u r e I I I I 6 I 5 I 4 I 2 I 0 - 8 . 270 MHz * H nmr spec t rum of compound [38] i n d e u t e r i o c h l o r o f o r m . The i n s e r t shows the H-20 and H-21 r e s o n a n c e s of [38] i n d e u t e r i o b e n z e n e . O* 66 r e s u l t i n g f r o m band t a i l i n g , d u r i n g p r o d u c t i s o l a t i o n w i l l c a u s e a d e c r e a s e i n t h e f i n a l s p e c i f i c a c t i v i t y of [42] ( [38] i s f u n c t i o n i n g as pseudo c a r r i e r ) . T h i s i s a v e r y u n d e s i r a b l e s i t u a t i o n and must be a v o i d e d . As was o b s e r v e d d u r i n g r a d i o b r o m i n a t i o n s , p r o t o n o l y s i s a l s o o c c u r s as a s i d e - r e a c t i o n ( s e e f i g u r e I I I - 6 ) . The i d e n t i t y o f peak A was r e a d i l y c o n f i r m e d u s i n g [39] as a c h r o m a t o g r a p h i c s t a n d a r d . To s t u d y t h e a f f e c t of c a r r i e r i o d i d e on the r a d i o i o d i n a t i o n r e a c t i o n u s i n g CAT as t h e o x i d a n t , t h e same p r o c e d u r e was r e p e a t e d w i t h t h e a d d i t i o n o f 1.6 umol o f N a l as c a r r i e r and t h e r e s u l t s a r e shown i n T a b l e I I I - l . When p e r f o r m e d b a c k t o b a c k t o NCA r a d i o i o d i n a t i o n s , a s m a l l i n c r e a s e i n r a d i o -c h e m i c a l y i e l d was o b t a i n e d . The r a d i o - H P L C chromatogram o f t h e CA e x p e r i m e n t i s shown i n f i g u r e I I I - 5 B . A l t h o u g h a s m a l l c a r r i e r a f f e c t was o b s e r v e d , t h e CAT p r o c e d u r e n o n e t h e l e s s p r o d u c e d h i g h r a d i o c h e m i c a l y i e l d s w i t h NCA 1 2 3 I . I n a d d i t i o n , t h e same g e n e r a l o b s e r v a t i o n s t h a t were d e s c r i b e d f o r t h e r a d i o -b r o m i n a t i o n s t u d i e s , u s i n g CAT as t h e o x i d a n t , were o b s e r v e d f o r t h e above r a d i o i o d i n a t i o n s t u d i e s . The use o f o t h e r o x i d i z i n g a g e n t s f o r r a d i o i o d i n a t i o n was a l s o i n v e s t i -g a t e d . Compound [18] was a l l o w e d t o r e a c t w i t h NCA N a 1 2 3 I i n t h e p r e s e n c e o f 1 .5 e q u i v a l e n t s o f NCS i n an a q u e o u s - m e t h a n o l - T H F m i x t u r e a t room t e m p e r a t u r e , f o r 30 m i n u t e s . HPLC i s o l a t i o n a f f o r d e d t h e d e s i r e d p r o d u c t [42] i n 62% r a d i o c h e m i c a l y i e l d . The r a d i o - H P L C chromatogram of t h i s r e a c t i o n i s shown i n f i g u r e I I I - 9 A . Compound [18] was a l s o s u c c e s s f u l l y r a d i o i o d i n a t e d w i t h NCA N a 1 2 3 I u s i n g the method o f F r a n k e and H a n s o n , 3 5 b a s e d on H 2 0 2 ~ a c e t i c a c i d as t h e o x i d a n t , i n w h i c h t h e 1 2 5 I - l a b e l l e d a n a l o g u e o f [42] was o b t a i n e d i n 95% r a d i o c h e m i c a l y i e l d . Compound [42] was i s o l a t e d i n 85% r a d i o c h e m i c a l 67 S [1 0 4 8 12 16 20 0 4 8 12 16 20 T I M E ( m i n ) T I M E ( m i n ) The compounds a r e : ( A , 6 .9 m i n ) , [ 3 8 ] ; ( A ' , 5 .9 m i n ) , [ 3 9 ] ; ( B , 8 . 1 m i n ) , [ 4 2 ] ; ( C , 16.4 m i n ) , [ 1 8 ] . T h e HPLC c o n d i t i o n s a r e : f l o w r a t e = 6 m l / m i n ; s o l v e n t p r o g r a m : 0 t o 10 m i n i s o c r a t i c 85% MeOH/15% H 2 0 , 10 t o 12 m i n l i n e a r g r a d i e n t f r o m 85% MeOH/15% H 2 0 t o 100% MeOH, 12 t o 20 m i n i s o c r a t i c 100% MeOH; C-18 r e v e r s e phase c o l u m n ; uv d e t e c t o r s e t a t 254 nm. F i g u r e I I I - 9 . HPLC chromatograms o b t a i n e d d u r i n g p u r i f i c a t i o n o f compound [ 4 2 ] . Chromatograms A and B r e s u l t e d f r o m t h e r a d i o -i o d i n a t i o n o f [18] u s i n g t h e o x i d a n t s NCS and H 2 0 2 ~ a c e t i c a c i d , r e s p e c t i v e l y . 68 y i e l d , b u t t h i s r e s u l t was o b t a i n e d u s i n g a f r e s h l y made H 2 0 2 - a c e t i c a c i d s o l u t i o n ( s e e e x p e r i m e n t a l s e c t i o n ) . When the o x i d a n t was p r e p a r e d 24 h o u r s i n a d v a n c e , as u s e d by Hanson and c o - w o r k e r s , 1 + 3 t h e r a d i o c h e m i c a l y i e l d was s i g n i f i c a n t l y r e d u c e d . The r a d i o - H P L C chromatogram o f t h i s r e a c t i o n i s shown i n f i g u r e I I I - 9 B . B o t h t h e NCS and H 2 0 2 ~ a c e t i c a c i d p r o c e d u r e s were r e p e a t e d i n t h e p r e s e n c e o f 1 .6 umol of c a r r i e r N a l and the r e s u l t s a r e summarized i n T a b l e I I I - l . Compound [42] was s u c c e s s f u l l y s y n t h e s i z e d by b o t h t h e NCS and H 2 0 2 ~ a c e t i c a c i d p r o c e d u r e s , a l t h o u g h t h e NCS method gave a l o w e r y i e l d . The r a d i o - H P L C chromatograms i n d i c a t e t h a t [42] was i s o l a t e d i n 100% r a d i o c h e m i c a l p u r i t y and s i d e - p r o d u c t f o r m a t i o n was much r e d u c e d o v e r t h a t o b s e r v e d d u r i n g t h e CAT p r o c e d u r e . The s i d e - p r o d u c t (peak A ' , f i g u r e I I I - 9 A ) g e n e r a t e d d u r i n g t h e NCS p r o c e d u r e was d e t e r m i n e d t o be v i n y l - c h l o r i n a t e d d e r i v a t i v e [ 3 8 ] . A l t e r n a t i v e l y , t h e s i d e - p r o d u c t (peak A , f i g u r e I I I - 9 B ) o b t a i n e d i n t h e H 2 0 2 - a c e t i c a c i d p r o c e d u r e was i d e n t i f i e d as t h e v i n y l compound [ 3 9 ] . B o t h methods a r e c o m p a t i b l e w i t h NCA 1 2 3 I , p r o d u c i n g [42] i n p o t e n t i a l l y h i g h s p e c i f i c a c t i v i t y . H o w e v e r , t h e c a r r i e r - a d d e d s t u d i e s seem t o i n d i c a t e t h a t t h e NCS method i s somewhat c a r r i e r s e n s i t i v e as t h e r a d i o c h e m i c a l y i e l d was n o t i c e a b l y i m p r o v e d w i t h t h e a d d i t i o n o f N a l . The H 2 0 2 ~ a c e t i c a c i d p r o c e d u r e , i n c o n t r a s t , i s s e e m i n g l y u n a f f e c t e d by c a r r i e r . The r e s u l t s s u g g e s t t h a t t h e H 2 0 2 _ a c e t i c a c i d method i s s u p e r i o r t o t h e NCS method f o r h i g h s p e c i f i c a c t i v i t y r a d i o i o d i n a t i o n s . However , f o r u s e w i t h s h o r t - l i v e d r a d i o n u c l i d e s o f i o d i n e ( s e e T a b l e 1 - 3 ) , t h e CAT method w o u l d seem t o be t h e b e s t c h o i c e due t o i t s s h o r t r e a c t i o n t i m e and h i g h r a d i o c h e m i c a l y i e l d s . 69 The mechanism f o r the r a d i o i o d i n a t i o n s , and radiobrominations, using CAT as the oxidant would be expected to be the same as discussed e a r l i e r i n chapter I I . Nonetheless, a change i n k i n e t i c behaviour would be anticipated between the non-radioactive and NCA radioactive iodine l a b e l l i n g experiments. 1 9 Since the tin-carbon bond cleavage by IC1 i s thought to be a concerted bimolecular process, a decrease of several orders of magnitude of one of the reacting species should cause a reduction of the re a c t i o n rate. This can be observed from the so c a l l e d c a r r i e r e f f e c t , i n which added Nal produces a greater radiochemical y i e l d due to the increased amount of IC1 a v a i l a b l e f o r reaction. This problem i s usually overcome by using a large excess of s t a r t i n g material and oxidant. Of course, side-reactions can predominate under NCA conditions which may not be normally observed. In the case of NCS, i t i s thought that IC1 i s the i o d i n a t i n g species formed. 2 9 It i s i n t e r e s t i n g to see that a noticeable c a r r i e r e f f e c t i s observed i n t h i s procedure. It has been suggested that peracetic acid i s the actual o x i d i z i n g agent formed from the H 2 0 2 - a c e t i c acid m i x t u r e . 6 9 From iodide, the peracetic acid probably generates HOI as the i o d i n a t i n g species; t h i s i s suggested from the analogous oxidation of bromide by H 2 0 2 ~ a c e t i c a c i d . 6 9 III.4 Summary and Conclusions The purpose of t h i s study was to develop a general methodology to incorporate s h o r t - l i v e d radioisotopes of bromine, and iodine, onto the v i n y l moiety of an organic compound. This was s u c c e s s f u l l y accomplished by the e l e c t r o p h i l i c c l e a v a g e o f t i n - c a r b o n bonds w i t h B r C l and I C 1 g e n e r a t e d i n - s i t u f r o m b r o m i d e and i o d i d e , r e s p e c t i v e l y , i n the p r e s e n c e o f a n o x i d i z i n g a g e n t . T h i s method was f o u n d t o y i e l d t h e d e s i r e d p r o d u c t s i n h i g h r a d i o c h e m i c a l y i e l d and p u r i t y i n s h o r t r e a c t i o n t i m e s . The v i n y l - t i n p r e c u r s o r s were r e a d i l y p r e p a r e d v i a t h e AIBN c a t a l y z e d h y d r o s t a n n y l a t i o n o f t h e c o r r e s p o n d i n g a c e t y l e n i c d e r i v a t i v e s , w i t h t r i - n -b u t y l t i n h y d r i d e , i n 51-98% y i e l d . The v i n y l - t i n d e r i v a t i v e s [ 4 ] , [ 8 ] , [ 1 2 ] , [18] and [ 1 9 ] , were b r o m i n a t e d i n h i g h y i e l d (89-100%), w i t h s o d i u m b r o m i d e o x i d i z e d by C h l o r a m i n e - T u n d e r a c i d i c c o n d i t i o n s . Under t h e same c o n d i t i o n s , [17] was b r o m i n a t e d i n p o o r y i e l d (13%). T h i s r e s u l t e d f r o m c o m p e t i n g b r o m i n a t i o n a t t h e A - r i n g o f [ 1 7 ] . The v i n y l - t i n d e r i v a t i v e s [ 4 ] , [ 8 ] , [18] and [ 1 9 ] , were i o d i n a t e d w i t h s o d i u m i o d i d e i n t h e p r e s e n c e o f a c i d i f i e d C h l o r a m i n e - T i n 88-99% y i e l d , whereas [17] was i o d i n a t e d i n 1.9% y i e l d . The f a c i l e e l e c t r o p h i l i c r e a c t i v i t y o f t h e p h e n o l i c A - r i n g r e s u l t e d i n p r e d o m i n a t e i o d i n a t i o n o c c u r r i n g a t t h e A - r i n g o f [17] r a t h e r t h a n a t t h e t i n - c a r b o n s i t e . To b r o m i n a t e o r i o d i n a t e s u c c e s s f u l l y [17] u s i n g t h e C h l o r a m i n e - T o x i d a t i o n p r o c e d u r e w o u l d r e q u i r e p r i o r p r o t e c t i o n o f t h e p h e n o l i c h y d r o x y l g r o u p , f o l l o w e d by d e p r o t e c t i o n a f t e r t h e l a b e l l i n g s t e p . Compound [18] was u s e d f o r r a d i o - l a b e l l i n g s t u d i e s w i t h 8 2 B r , ! 2 3 j a n ( j 1 3 1 I u s i n g t h e C h l o r a m i n e - T m e t h o d . The r a d i o b r o m i n a t e d p r o d u c t [41] was i s o l a t e d i n 77-81% r a d i o c h e m i c a l y i e l d u s i n g low s p e c i f i c a c t i v i t y N H 1 + 8 2 B r . The r a d i o i o d i n a t e d p r o d u c t s [42] and [43] were o b t a i n e d i n 82-90% r a d i o c h e m i c a l y i e l d , i n r e l a t i v e l y h i g h s p e c i f i c a c t i v i t y . The m a j o r s i d e - p r o d u c t f o r m e d d u r i n g t h e NCA r a d i o i o d i n a t i o n s was t h e c h l o r i n a t e d d e r i v a t i v e [ 3 8 ] . The r e a c t i o n t i m e s n e e d e d were s h o r t (1 m i n ) , t h u s r e q u i r i n g a s y n t h e s i s t ime o f a p p r o x i m a t e l y 17-18 m i n u t e s t o o b t a i n t h e f i n a l r a d i o - l a b e l l e d p r o d u c t a f t e r HPLC p u r i f i c a t i o n . An a l t e r n a t i v e r a d i o - l a b e l l i n g p r o c e d u r e , b a s e d on NCS as t h e o x i d a n t , was d e v e l o p e d w h i c h gave [41] and [42] i n 85% and 62% (NCA 1 2 3 I ) r a d i o c h e m i c a l y i e l d s , r e s p e c t i v e l y . The d e g r e e o f c o m p e t i n g c h l o r i n a t i o n i n t h i s p r o c e d u r e was much r e d u c e d o v e r t h a t o b s e r v e d w i t h t h e C h l o r a m i n e - T m e t h o d . However , t h e NCS method seems more s e n s i t i v e t o c a r r i e r l e v e l s when p e r f o r m i n g NCA r a d i o i o d i n a t i o n s , and r e q u i r e s l o n g e r r e a c t i o n t i m e s i n t h e o r d e r o f 30 m i n u t e s . T h e r e f o r e , t h e s e r e s u l t s s u g g e s t t h a t t h e C h l o r a m i n e - T b a s e d p r o c e d u r e w o u l d be b e s t f o r NCA r a d i o h a l o g e n l a b e l l i n g . I t i s a p p r o p r i a t e a t t h i s s t a g e t o s u g g e s t some l i k e l y e x t e n s i o n s o f t h i s w o r k . The r a d i o b r o m i n a t i o n s t u d i e s s h o u l d be r e p e a t e d w i t h h i g h s p e c i f i c a c t i v i t y 7 5 B r o r 7 7 B r t o a s c e r t a i n t h e p e r f o r m a n c e o f t h e C h l o r a m i n e - T and NCS methods u n d e r NCA c o n d i t i o n s . I t w o u l d a l s o be p r u d e n t t o s u r v e y t h e u s e o f o t h e r o x i d i z i n g a g e n t s w h i c h w o u l d g e n e r a t e d i f f e r e n t e l e c t r o p h i l i c forms o f b r o m i n e and i o d i n e and compare t h e i r b e h a v i o u r w i t h i n - s i t u g e n e r a t e d B r C l and I C 1 . L a s t l y , i t w o u l d be v e r y i n t e r e s t i n g t o d e t e r m i n e w h e t h e r v i n y l - t i n d e r i v a t i v e s c o u l d be s u c c e s s f u l l y f l u o r i n a t e d w i t h some f o r m of e l e c t r o p h i l i c f l u o r i n e . T h i s w o u l d be v e r y u s e f u l f o r r a d i o f l u o r i n a t i o n s w i t h 1 8 F . CHAPTER IV 72 EXPERIMENTAL IV.1 General Methods A l l s o l u t i o n s were c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e w i t h a B u c h i r o t a r y e v a p o r a t o r . A l l m e l t i n g p o i n t s were d e t e r m i n e d on a F i s h e r - J o h n s m e l t i n g p o i n t a p p a r a t u s and a r e u n c o r r e c t e d . O p t i c a l r o t a t i o n s were d e t e r m i n e d u s i n g a P e r k i n - E l m e r m o d e l 241 M C , o r model 141, p o l a r i m e t e r . T h i n l a y e r c h r o m a t o g r a p h y ( t i c ) was p e r f o r m e d on p r e - c o a t e d s i l i c a g e l p l a t e s ( B a k e r - f l e x S i l i c a g e l 1B2-F o r E . M e r c k S i l i c a g e l 60 , N o . 5 5 3 4 ) . V i s u a l i z a t i o n was e f f e c t e d i n one o f t h e f o l l o w i n g m a n n e r s : ( i ) by s p r a y i n g w i t h 30% s u l f u r i c a c i d i n e t h a n o l , and h e a t i n g , ( i i ) by s p r a y i n g w i t h 5% ammonium m o l y b d a t e d i s s o l v e d i n 10% s u l f u r i c a c i d i n e t h a n o l , and h e a t i n g , o r ( i i i ) w i t h s h o r t - w a v e l e n g t h u l t r a - v i o l e t l i g h t . R a d i o - t i c p l a t e s were a s s a y e d by e x p o s i n g i n s t a n t f i l m ( P o l a r o i d 545 L a n d f i l m ; 3000 ASA) i n c o n t a c t w i t h t h e e l u t e d p l a t e s . The t i c s o l v e n t s y s t e m s u s e d were v e r y s i m i l a r t o t h o s e l i s t e d f o r t h e c o l u m n c h r o m a t o g r a p h y o f t h e i n d i v i d u a l compounds . Column c h r o m a t o g r a p h y was p e r f o r m e d u s i n g S i l i c a g e l 60 ( E . M e r c k , 70-230 m e s h ) . F l a s h c h r o m a t o g r a p h y was p e r f o r m e d u s i n g S i l i c a g e l 60 ( E . M e r c k , 230-400 mesh) by t h e method o f S t i l l e t a l . 7 0 H i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y (HPLC) was done w i t h a s y s t e m c o n s i s t i n g o f a S p e c t r o - P h y s i c s SP 8700 s o l v e n t d e l i v e r y s y s t e m , a Rheodyne 7126 i n j e c t o r f i t t e d w i t h a 0 .5 o r 1 .0 m l sample l o o p ( 1 . 0 2 mm i d ) , a C-18 73 r e v e r s e phase Whatman P a r t i s i l 10 ODS-3 c o l u m n (25 cm x 9 mm) w i t h a Waters C-18 g u a r d c o l u m n , a n ISCO (model V 4 ) v a r i a b l e w a v e l e n g t h U . V . d e t e c t o r o p e r a t e d a t 254 nm o r 280 nm, and a S p e c t r o - P h y s i c s SP 4270 i n t e g r a t o r r e c o r d e r . R a d i o - H P L C a n a l y s i s and p u r i f i c a t i o n was p e r f o r m e d w i t h t h e same HPLC s y s t e m f i t t e d w i t h a N a l ( T l ) s c i n t i l l a t i o n d e t e c t o r s y s t e m and a d u a l c h a n n e l s t r i p c h a r t r e c o r d e r . R a d i o a c t i v e samples were a s s a y e d by e i t h e r a Beckman 8000 s c i n t i l l a t i o n c o u n t e r o r i n a C a p i n t e c w e l l c o u n t e r ( m o d e l C R C - 5 4 3 X ) . T e t r a h y d r o f u r a n (THF) was d i s t i l l e d f r o m c a l c i u m h y d r i d e o r sodium b e n z o p h e n o n e k e t y l . 7 1 P y r i d i n e was d i s t i l l e d f r o m p o t a s s i u m h y d r o x i d e and was o s t o r e d o v e r 4A m o l e c u l a r s i e v e s . F o r HPLC w o r k , s i n g l y d i s t i l l e d w a t e r and HPLC g r a d e m e t h a n o l were u s e d , w h i c h were f i l t e r e d (0 .45 pm M i l l i p o r e b r a n d D u r a p o r e membrane) b e f o r e u s e . A l l o t h e r s o l v e n t s u s e d were o f s p e c t r o o r r e a g e n t g r a d e , and were u s e d w i t h o u t f u r t h e r t r e a t m e n t . Low r e s o l u t i o n mass s p e c t r a were r e c o r d e d w i t h a V a r i a n MAT A t l a s CH4-B o r a K r a t o s / A E I MS-50 mass s p e c t r o m e t e r . S p e c t r a a r e q u o t e d as m/z v a l u e s , w h i l e s e l e c t e d i o n f r a g m e n t a t i o n s a r e r e p o r t e d as p e r c e n t a g e s o f t h e b a s e p e a k . H i g h r e s o l u t i o n mass measurements were d e t e r m i n e d u s i n g a K r a t o s / A E I MS-50 mass s p e c t r o m e t e r . A l l m i c r o a n a l y s e s were p e r f o r m e d by M r . P . B o r d a , M i c r o a n a l y t i c a l L a b o r a t o r y , U n i v e r s i t y o f B r i t i s h C o l u m b i a . IV.2 Nmr Methods and Instrumentation P r o t o n nmr s p e c t r a were measured a t room t e m p e r a t u r e a t 270 MHz w i t h a h o m e - b u i l t s p e c t r o m e t e r b a s e d on a B r u k e r WP-60 c o n s o l e , a N i c o l e t 1180 74 computer ( 3 2 K ) , a N i c o l e t 293B p u l s e programmer , a one Megabyte D i a b l o d i s k d r i v e ( m o d e l 3 1 ) , and an O x f o r d I n s t r u m e n t s S u p e r c o n d u c t i n g s o l e n o i d m a g n e t . D a t a were a c c u m u l a t e d i n t h e q u a d r a t u r e d e t e c t i o n mode. P r i o r t o F o u r i e r t r a n s f o r m a t i o n , a l l t h e d a t a were m u l t i p l i e d by an e x p o n e n t i a l l i n e b r o a d e n i n g f a c t o r ( 0 . 2 9 H z ) . D e u t e r a t e d s o l v e n t s were o b t a i n e d f r o m M e r c k , S h a r p and Dohme Canada L t d . o r S t o h l e r I s o t o p e C h e m i c a l s , and t e t r a m e t h y l s i l a n e was u s e d as t h e i n t e r n a l s t a n d a r d . IV.3 Sources of Materials S o u r c e s f o r t h e k e y c h e m i c a l s u s e d i n t h e s y n t h e s i s o f compounds i n t h i s s t u d y a r e as f o l l o w s : 1 , 2 : 3 , 4 - d i - £ - i s o p r o p y l i d e n e - a - D - g a l a c t o p y r a n o s e ( K o c h - L i g h t L a b o r a t o r i e s L t d . ) ; 1 7 o t - e t h y n y l e s t r a d i o l ( S i g m a ) ; t r i - n - b u t y l t i n h y d r i d e and N - c h l o r o s u c c i n i m i d e ( A l f a ) ; P h e n y l a c e t y l e n e (vacuum d i s t i l l e d p r i o r to u s e ) and 2 , 2 ' - a z o b i s ( 2 - m e t h y l p r o p i o n i t r i l e ) r e f e r r e d t o as AIBN ( A l d r i c h ) ; C h l o r a m i n e - T (Eas tman and MCB) ; chromium t r i o x i d e ( F i s h e r S c i e n t i f i c L t d . ) was k e p t d r y i n v a c u o o v e r p h o s p h o r u s p e n t a o x i d e ; a c e t y l e n e , 99.6% p u r e ( M a t h e s o n Gas P r o d u c t s C a n a d a ) . HPLC g r a d e m e t h a n o l was o b t a i n e d f r o m F i s h e r S c i e n t i f i c L t d . N H ^ 8 2 B r (~255 mCi /mmol) was p u r c h a s e d f rom New E n g l a n d N u c l e a r C a n a d a . N H ^ 1 2 3 ! ( h i g h s p e c i f i c a c t i v i t y ) was d o n a t e d by A t o m i c E n e r g y Canada L t d . ( A E C L ) , a n d N a 1 2 3 I ( h i g h s p e c i f i c a c t i v i t y ) was d o n a t e d by C r o c k e r N u c l e a r L a b o r a t o r y ( U n i v e r s i t y o f C a l i f o r n i a , D a v i s ) . N a 1 3 l I ( h i g h s p e c i f i c a c t i v i t y ) was o b t a i n e d f r o m C h a r l e s E . F r o s t and C o . ( M o n t r e a l , Q u e . ) . 75 IV.4 Synthesis of Acetylenic Precursors Preparation of 1,2:3,4-di-0_-isopropylidene-a-D-galacto-hexodialdo-l,5-pyranose [ 2 ] . 7 2 Method A : 7 3 Under an i n e r t a t m o s p h e r e ( N 2 ) chromium t r i o x i d e (23 .05 g , 0 .231 mol) was added t o d r y p y r i d i n e ( 3 7 . 1 m l , 0 .461 mol ) d i s s o l v e d i n C H ^ C l ^ (600 m l ) . The m i x t u r e was s t i r r e d f o r 20 m i n u t e s a t room t e m p e r a t u r e t o o b t a i n a d e e p - r e d s o l u t i o n . W i t h s t i r r i n g , a s o l u t i o n of 1 , 2 : 3 , 4 - d i - 0 _ -i s o p r o p y l i d e n e - a - D - g a l a c t o p y r a n o s e [1] ( 5 . 0 g , 19 .2 mmol) i n C H 2 C 1 2 (50 m l ) was a d d e d i n one p o r t i o n t o t h e C r O g / p y r i d i n e s o l u t i o n , g i v i n g a d a r k brown p r e c i p i t a t e . The m i x t u r e was s t i r r e d f o r an a d d i t i o n a l 20 m i n u t e s a t room t e m p e r a t u r e . The s u p e r n a t a n t s o l u t i o n was d e c a n t e d i n t o i c e - c o l d , s a t u r a t e d , aqueous s o d i u m b i c a r b o n a t e (650 m l ) and t h e r e s i d u e was e x t r a c t e d w i t h a s m a l l amount o f e t h e r w h i c h was a l s o added t o t h e sodium b i c a r b o n a t e s o l u t i o n . The m i x t u r e was s t i r r e d f o r a few m i n u t e s and t h e o r g a n i c l a y e r was s e p a r a t e d , washed w i t h w a t e r (3x350 m l ) , and d r i e d o v e r a n h y d r o u s magnesium s u l f a t e . The s o l v e n t was removed and t h e r e s u l t i n g d a r k brown s y r u p was d r i e d u n d e r h i g h vacuum ( 0 . 0 1 t o r r ) . S h o r t - p a t h ( K u g l e r o h r ) d i s t i l l a t i o n a f f o r d e d [2] ( 2 . 6 0 g) as a c l e a r , p a l e y e l l o w s y r u p i n 52% y i e l d . S p e c t r a l ( 1 H nmr) d a t a a r e c o n s i s t e n t w i t h t h e s t r u c t u r e and a r e i n agreement w i t h t h e l i t e r a t u r e . 7 2 The p r o d u c t was s t o r e d i n v a c u o , o v e r p h o s p h o r u s p e n t a o x i d e , f o r s u b s e q u e n t u s e . M e t h o d B : 7 1 * Under an i n e r t a t m o s p h e r e ( N 2 ) chromium t r i o x i d e ( 7 . 6 8 g , 7 6 . 8 mmol) was a d d e d t o d r y p y r i d i n e (12 .4 m l , 0 .154 m o l ) d i s s o l v e d i n C H 2 C 1 2 (160 m l ) . The m i x t u r e was s t i r r e d f o r 20 m i n u t e s a t room t e m p e r a t u r e to 76 o b t a i n a d e e p - r e d s o l u t i o n . W i t h s t i r r i n g , [1] ( 5 . 0 g , 19.2 mmol) i n C H 2 C 1 2 (40 ml) was added i n one p o r t i o n t o the C r 0 3 / p y r i d i n e s o l u t i o n . A c e t i c a n h y d r i d e (7 .25 m l , 76 .8 mmol) was added a t once and t h e r e a c t i o n m i x t u r e was s t i r r e d f o r 9 m i n u t e s a t room t e m p e r a t u r e . The r e a c t i o n was quenched w i t h t h e a d d i t i o n o f e x c e s s e t h a n o l ( 4 . 5 m l , 77 mmol) d i s s o l v e d i n e t h y l a c e t a t e (25 m l ) . The m i x t u r e was t h e n added t o e t h y l a c e t a t e (250 ml) i n w h i c h the chromium compounds p r e s e n t were p r e c i p i t a t e d . The r e s u l t i n g m i x t u r e was t r a n s f e r r e d to t h e t o p o f a s h o r t pad of s i l i c a g e l (12 cm x 3 . 5 cm, M a l l l n c k r o d t S i l i c A r C C - 4 , 60-200 mesh) i n e t h y l a c e t a t e and t h e p r o d u c t was e l u t e d w i t h e t h y l a c e t a t e i n one f r a c t i o n . A f t e r r e m o v a l o f t h e s o l v e n t , t o l u e n e was added t o t h e c r u d e s y r u p and was e v a p o r a t e d . T h i s was r e p e a t e d a few t i m e s t o remove any a c e t i c a c i d and p y r i d i n e p r e s e n t , a f t e r w h i c h the c r u d e p r o d u c t was d r i e d u n d e r h i g h vacuum. K u g l e r o h r d i s t i l l a t i o n gave [2] ( 3 . 3 5 g) i n 68% y i e l d . T h i s p r o d u c t e x h i b i t e d t h e same * H nmr s p e c t r u m as t h e m a t e r i a l o b t a i n e d i n method A . Preparation of 7,8-dideoxy-l,2:3,4-di-fJ-isopropylidene-D-glycero-a-D-galacto-oct-7-ynopyranose [3] and i t s L-glycero-ot-D-galacto epimer [ 7 ] . 7 5 A s o l u t i o n o f e t h y l m a g n e s i u m b r o m i d e was p r e p a r e d i n t h e f o l l o w i n g manner . U n d e r an N 2 a t m o s p h e r e , a s o l u t i o n o f e t h y l bromide ( 4 3 . 8 g , 0 .40 m o l ) i n THF (115 m l ) was added d r o p w i s e t o a s t i r r e d m i x t u r e o f magnesium t u r n i n g s ( 3 . 0 g , 0 .12 mol ) i n THF (35 ml) a t a r a t e s u f f i c i e n t t o m a i n t a i n a g e n t l e r e f l u x o f t h e m i x t u r e . A f t e r t h e a d d i t i o n o f e t h y l b r o m i d e s o l u t i o n was c o m p l e t e d , s t i r r i n g was c o n t i n u e d u n t i l t h e m i x t u r e had c o o l e d t o room t e m p e r a t u r e . The r e s u l t i n g m i x t u r e had a g r e e n i s h - b r o w n c o l o u r a t i o n . W i t h s t i r r i n g , a c e t y l e n e was p a s s e d t h r o u g h THF (150 ml ) f o r one h o u r a t room t e m p e r a t u r e . The p r e p a r e d s o l u t i o n o f e t h y l m a g n e s i u m b r o m i d e was i n t u r n added d r o p w i s e t o t h e a c e t y l e n e s a t u r a t e d THF s o l u t i o n , w h i l e a c o n t i n u e d f l o w o f a c e t y l e n e was p a s s e d t h r o u g h . The r e s u l t i n g s o l u t i o n g r a d u a l l y t u r n e d b r i g h t r e d , t h e n f i n a l l y became d a r k r e d d i s h - b r o w n i n c o l o u r , and was s t i r r e d an a d d i t i o n a l h o u r w i t h c o n t i n u e d a c e t y l e n e f l o w . To t h e r e s u l t i n g e t h y n y l m a g n e s i u m b r o m i d e m i x t u r e , a s o l u t i o n o f [2] ( 9 .45 g , 36 .6 mmol) i n THF (100 ml) was a d d e d d r o p w i s e , w i t h s t i r r i n g , a t room t e m p e r a t u r e . A f l o w o f a c e t y l e n e was c o n t i n u e d t h r o u g h o u t t h e a d d i t i o n o f [2] and f o r a f u r t h e r t h r e e h o u r s . The r e a c t i o n m i x t u r e was d i l u t e d w i t h e t h e r (500 m l ) , washed w i t h i c e - c o l d 10% aqueous ammonium c h l o r i d e (3x300 ml ) and w a t e r (3x300 m l ) , and d r i e d o v e r a n h y d r o u s magnesium s u l f a t e . Removal of t h e s o l v e n t a f f o r d e d t h e c r u d e p r o d u c t s [3] and [7] as a d a r k y e l l o w , c r y s t a l l i n e s o l i d (8 .80 g) i n 85% y i e l d . 1 H nmr a n a l y s i s o f t h e c r u d e p r o d u c t m i x t u r e i n d i c a t e d , by t h e r a t i o o f t h e a n o m e r i c p r o t o n s , t h a t [3] and [7] were p r e s e n t i n a r a t i o of 6 2 : 3 8 , r e s p e c t i v e l y . F u r t h e r m o r e , t h e a n o m e r i c s i g n a l o f [2] was c o m p l e t e l y a b s e n t . Column c h r o m a t o g r a p h y o f a 1 .70 g p o r t i o n o f t h e c r u d e p r o d u c t m i x t u r e was p e r f o r m e d o n s i l i c a g e l (150 g) w i t h C ^ C l j / h e x a n e s / e t h e r 6 : 2 : 1 . The f i r s t f r a c t i o n ( 0 . 8 5 g) c o n t a i n e d p u r e [3] as w h i t e c r y s t a l s , mp 1 3 0 - 1 3 1 ° { l i t . 7 5 mp 1 3 1 . 0 - 1 3 1 . 5 ° } . S p e c t r a l ( *H nmr) d a t a a r e c o n s i s t e n t w i t h t h e s t r u c t u r e and a r e i n agreement w i t h t h e l i t e r a t u r e . 7 5 The s e c o n d f r a c t i o n ( 0 . 1 3 g) o b t a i n e d c o n s i s t e d o f a m i x t u r e o f [3] and [ 7 ] . The f i n a l f r a c t i o n (0 .44 g) c o n t a i n e d o n l y [7] as w h i t e c r y s t a l s , mp 1 2 5 - 1 2 8 ° { l i t . 7 5 mp 1 3 6 - 1 3 7 ° } . R e c r y s t a l l i z a t i o n o f a sample f r o m e t h e r -h e x a n e s , f o l l o w e d by a f u r t h e r r e c r y s t a l l i z a t i o n f r o m b e n z e n e - h e x a n e s gave [7] 78 as w h i t e n e e d l e s , mp 1 3 8 - 1 3 9 ° . S p e c t r a l ( 1 H nmr) d a t a a r e c o n s i s t e n t w i t h t h e s t r u c t u r e and a r e i n agreement w i t h t h e l i t e r a t u r e . 7 5 The r e m a i n i n g c r u d e p r o d u c t m i x t u r e was c h r o m a t o g r a p h e d i n s e v e r a l p o r t i o n s as d e s c r i b e d above i n o r d e r t o i s o l a t e [3] and [7] I n p u r e f o r m . Preparation of 3-methoxy-17a-ethynyl-l,3,5(10)-estratriene-17f$-ol [15] 7 6 and 3,178-dimethoxy-17a-ethynyl-l,3,5(10)-e8tratriene [16]. T h i s p r o c e d u r e was a d a p t e d f r o m t h e l i t e r a t u r e 7 7 i n w h i c h o t h e r u n r e l a t e d compounds were p r e p a r e d . Powdered p o t a s s i u m h y d r o x i d e ( 1 . 5 1 g , 2 6 . 9 mmol) was a d d e d t o DMSO (40 m l ) . A f t e r s t i r r i n g f o r f i v e m i n u t e s a t room t e m p e r a t u r e , 1 7 a - e t h y n y l e s t r a d i o l [14] ( 2 . 0 0 g , 6 .75 mmol) was a d d e d , f o l l o w e d i m m e d i a t e l y by m e t h y l i o d i d e (1 .44 g , 1 0 . 1 m m o l ) . S t i r r i n g was c o n t i n u e d f o r 25-30 m i n u t e s a f t e r w h i c h t h e m i x t u r e was p o u r e d i n t o w a t e r (100 ml ) and was e x t r a c t e d w i t h d i c h l o r o m e t h a n e (3x100 m l ) . The c o m b i n e d o r g a n i c e x t r a c t s were washed w i t h w a t e r (2x100 m l ) , d r i e d o v e r a n h y d r o u s magnesium s u l f a t e and t h e s o l v e n t was r e m o v e d . F l a s h c h r o m a t o g r a p h y , o f t h e c r u d e m i x t u r e , on s i l i c a g e l ( 3 . 5 cm x 15 cm) w i t h h e x a n e s / e t h e r 2 : 1 , was p e r f o r m e d . Compound [16] e l u t e d f i r s t i n one p o r t i o n (0 .67 g ) , i n 31% i s o l a t e d y i e l d as w h i t e n e e d l e s , mp 1 4 2 . 5 - 1 4 4 ° , [ a ] £ 5 = + 0 . 8 ° ( c = l , CHCI3). A n a l , c a l c d . f o r C 2 2 H 2 8 0 2 : C 8 1 . 4 4 , H 8 . 7 0 , 0 9 . 8 6 ; f o u n d : C 8 1 . 4 7 , H 8 . 8 6 , 0 1 0 . 0 . Mass s p e c t r u m , m / z : 324 ( M + , 1 0 0 ) , 309 ( M + - C H 3 , 7 0 ) . C o n t i n u e d e l u t i o n a f f o r d e d [15] ( 1 . 4 0 g) i n 67% i s o l a t e d y i e l d , as w h i t e c r y s t a l s , mp 1 5 0 . 5 - 1 5 1 ° { l i t . 7 6 mp 1 5 0 - 1 5 1 . 5 ° } . S p e c t r a l ( X H nmr and mass s p e c t r a ) p r o p e r t i e s were c o n s i s t e n t w i t h t h e s t r u c t u r e . 79 I n o r d e r t o p r e p a r e [16] i n h i g h e r y i e l d , t h e f o l l o w i n g m o d i f i e d p r o c e d u r e was e m p l o y e d . Powdered p o t a s s i u m h y d r o x i d e ( 1 . 5 1 g , 2 6 . 9 mmol) was added t o DMSO (15 m l ) and was s t i r r e d f o r f i v e m i n u t e s , a t room t e m p e r a t u r e . Compound [14] ( 1 . 0 0 g , 3 .37 mmol) was t h e n a d d e d , f o l l o w e d by m e t h y l i o d i d e ( 1 . 9 1 g , 13 .4 m m o l ) . The m i x t u r e was s t i r r e d f o r one h o u r , and was p o u r e d i n t o w a t e r (150 m l ) , t h e n e x t r a c t e d w i t h d i c h l o r o m e t h a n e (3x150 m l ) . The o r g a n i c e x t r a c t s were washed w i t h w a t e r (2x75 m l ) , d r i e d o v e r a n h y d r o u s magnesium s u l f a t e and the s o l v e n t was r e m o v e d . F l a s h c h r o m a t o g r a p h y , o f t h e c r u d e m i x t u r e , on s i l i c a g e l ( 3 . 5 cm x 15 cm) w i t h h e x a n e s / e t h e r 4 : 1 y i e l d e d 0 .90 g (82%) o f [ 1 6 ] . A n a l y t i c a l t i c and s p e c t r a l ( *H nmr) p r o p e r t i e s o f t h i s m a t e r i a l were i d e n t i c a l w i t h t h o s e r e p o r t e d e a r l i e r . F u r t h e r e l u t i o n w i t h h e x a n e s / e t h e r 1 :1 a f f o r d e d 0 . 1 1 g (18%) of [ 1 5 ] . A n a l y t i c a l t i c and s p e c t r a l (*H nmr) p r o p e r t i e s o f t h i s compound were t h e same as t h o s e o b t a i n e d p r e v i o u s l y . IV.5 Synthesis of Vinyl-Tin Compounds Hydrostannylation of acetylenic precursors. P r o c e d u r e A : Under an N 2 a t m o s p h e r e , a s o l u t i o n o f a c e t y l e n i c s u b s t r a t e i n 1 , 4 - d i o x a n e was p r e p a r e d . F i v e e q u i v a l e n t s o f t r i - n - b u t y l t i n h y d r i d e were t h e n added and t h e s t i r r e d m i x t u r e was r e f l u x e d ( 1 0 1 . 5 ° ) o v e r n i g h t u n d e r N 2 . A f t e r t h e s o l v e n t was removed , t h e c r u d e m i x t u r e was c h r o m a t o g r a p h e d on a s i l i c a g e l c o l u m n t o o b t a i n t h e d e s i r e d p r o d u c t . P r o c e d u r e B : 5 3 > 5 1 + Under an N 2 a t m o s p h e r e , a m i x t u r e o f a c e t y l e n i c s u b s t r a t e , a c a t a l y t i c amount o f A I B N and two e q u i v a l e n t s o f t r i - n - b u t y l t i n 80 h y d r i d e were h e a t e d o v e r n i g h t ( o i l b o t h t e m p e r a t u r e 9 5 ° ) , w i t h s t i r r i n g . The p r o d u c t was i s o l a t e d by c h r o m a t o g r a p h y o f t h e c r u d e m i x t u r e on s i l i c a g e l . G e n e r a l : I n a l l c a s e s , t h e v i n y l - t i n d e r i v a t i v e s , a f t e r c h r o m a t o g r a p h y , were t h o r o u g h l y d r i e d u n d e r h i g h vacuum and were s t o r e d i n v a c u o o v e r s o d i u m h y d r o x i d e p e l l e t s . A l t h o u g h v i n y l - t i n d e r i v a t i v e s a r e s u f f i c i e n t l y a i r - s t a b l e t o be e a s i l y h a n d l e d i n t h e open u n d e r n o r m a l c o n d i t i o n s , t h e y w i l l decompose upon e x p o s u r e t o t h e a t m o s p h e r e o v e r l o n g e r p e r i o d s o f t i m e . P r o p e r s t o r a g e i s t h e r e f o r e v i t a l . Preparation of (E)-7,8-dideoxy-l,2:3,4-di-0-isopropylidene-8-C-tributyl-stannyl-D-glycero-o-D-galacto-oct-7-enopyranose [ 4 ] , 7 8 Method A : Compound [3] (390 mg, 1.37 mmol) i n 1 , 4 - d i o x a n e (3 m l ) was h y d r o s t a n n y l a t e d as o u t l i n e d i n p r o c e d u r e A . Column c h r o m a t o g r a p h y o f t h e c r u d e m i x t u r e was p e r f o r m e d on s i l i c a g e l (80 g) w i t h C H 2 C l 2 / h e x a n e s / e t h e r 6 : 6 : 1 . The f i r s t f r a c t i o n (390 mg) c o n t a i n e d t h r e e p r o d u c t s i n w h i c h [4] was d o m i n a n t , whereas t h e s e c o n d f r a c t i o n (330 mg) was p u r e [ 4 ] . R e c h r o m a t o g r a p h y ( s i l i c a g e l ; 60 g) o f t h e f i r s t f r a c t i o n gave an a d d i t i o n a l 80 mg o f [ 4 ] . Compound [4] was i s o l a t e d as a c o l o u r l e s s s y r u p i n 52% o v e r a l l y i e l d , [ a ] 2 , 3 = - 3 6 . 1 ° ( c = l , C H C 1 3 ) . A n a l , c a l c d . f o r C j g R ^ g O g S n : C 5 4 . 2 8 , H 8 . 4 1 ; f o u n d : C 5 4 . 2 1 , H 8 . 5 2 . Mass s p e c t r u m , m / z : 5 6 1 ( 1 2 0 S n : M + - C H 3 , 3 ) , 519 ( 1 2 0 S n : M + ^ H g , 1 0 0 ) . Method B : Compound [3] (884 mg, 3 . 1 1 mmol) was h y d r o s t a n n y l a t e d as d e s c r i b e d i n p r o c e d u r e B . Column c h r o m a t o g r a p h y on s i l i c a g e l (200 g) w i t h C H 2 C l 2 / h e x a n e s / e t h e r 6 : 6 : 1 y i e l d e d i n one f r a c t i o n 1093 mg (61%) o f [ 4 ] . 81 A n a l y t i c a l t i c and s p e c t r a l ( 1H nmr) properties of t h i s material were i d e n t i c a l with those reported e a r l i e r . Preparation of (E)-7,8-dideoxy-l,2:3,4-di-0_-isopropylidene-8-C-tributyl-stannyl-L-glycero-a-D-galacto-oct-7-enopyranose [8]. Compound [7] (1016 mg, 3.57 mmol) was hydrostannylated as outlined i n procedure B. Column chromatography on s i l i c a g e l (200 g) with CH 2Cl 2/hexanes/ ether 6:6:1 yielded 1199 mg (58%) of [8] as a colourless syrup, [a]2)1* = -37.0° (c=1.3, CHC1 3). Anal, c a l c d . for C^H^OgSn: C 54.28, H 8.41, 0 16.68; found: C 54.57, H 8.47, 0 16.55. Mass spectrum, m/z: 561( 1 2 0 S n : M+-CH3, 2), 5 1 9 ( 1 2 0 S n : M+^Hg, 100). Preparation of 0-E/Z-tributylstannylstyrene [12]. 7 9 Phenylacetylene [11] (2.05 g, 20.0 mmol) was hydrostannylated using a modified version of procedure B. 8 0 The changes were, using t r i - n - b u t y l t i n hydride (5.52 g, 18.9 mmol) as the l i m i t i n g reagent, and heating the r e s u l t i n g mixture at 83° ( o i l bath temperature) for twelve hours. Short-path d i s t i l -l a t i o n ( a i r bath temperature; 116-156°, at 0.70 t o r r ) afforded 6.58 g of crude [12]. The crude product was r e d i s t i l l e d twice using short-path d i s t i l l a t i o n . Compound [12], i n the f i n a l d i s t i l l a t i o n ( a i r bath temperature; 117-143°, at 0.60 t o r r ) , was i s o l a t e d as a pale yellow o i l (6.09 g) i n 82% y i e l d . HPLC analysis of the product on a C-18 column using methanol as the eluant (flow rate 6.0 ml/min; detector set at 280 nm) showed the product to be 98% pure (re t e n t i o n time, 3.96 min). *H nmr analysis shows the r a t i o of E-isomer to Z-isomer to be 88:12. The s p e c t r a l (*H nmr and mass spectra) data are 82 c o n s i s t e n t w i t h t h e s t r u c t u r e and a r e i n agreement w i t h t h e l i t e r a t u r e . 7 9 The p r o d u c t was s t o r e d u n d e r an N 2 a t m o s p h e r e . Preparation of 17a-E-tributylstannylvinyl-l,3,5(10)-estratriene - 3,17B-diol [17]1*3 and l7o-vinyl-l,3,5 (10)-estratriene-3,178-diol [26]. 5 2 Method A : Compound [14] (1986 mg, 6 .70 mmol) i n 1 , 4 - d i o x a n e (10 m l ) was h y d r o s t a n n y l a t e d as d e s c r i b e d i n p r o c e d u r e A . F l a s h c h r o m a t o g r a p h y o f t h e c r u d e m i x t u r e was p e r f o r m e d on s i l i c a g e l ( 3 . 5 cm x 15 cm) w i t h C H 2 C l 2 / h e x a n e s / e t h e r 6 : 2 : 1 . The f i r s t f r a c t i o n c o n t a i n e d [17] as t h e d o m i n a n t p r o d u c t c o n t a m i n a t e d w i t h two u n i d e n t i f i e d b y p r o d u c t s , w h i l e f u r t h e r e l u t i o n i s o l a t e d a m i x t u r e o f [14] and [26] (1090 mg) i n a r a t i o o f 2 9 : 7 1 as measured f r o m t h e 1 H nmr s p e c t r u m . T h e r e f o r e , 774 mg o f [26] i s e s t i m a t e d t o be p r e s e n t i n t h e m i x t u r e f o r a y i e l d o f 38%. The f r a c t i o n c o n t a i n i n g [17] was p u r i f i e d by c o l u m n c h r o m a t o g r a p h y on s i l i c a g e l (150 g) w i t h C H 2 C l 2 / h e x a n e s / e t h e r 6 : 4 : 1 w h i c h a f f o r d e d 780 mg o f [17] (20% o v e r a l l i s o l a t e d y i e l d ) as a v e r y v i s c o u s s y r u p , w h i c h u p o n d r y i n g o v e r n i g h t i n v a c u o , c r y s t a l l i z e d as a n o f f - w h i t e s o l i d . An a n a l y t i c a l sample was o b t a i n e d by f l a s h c h r o m a t o g r a p h y on s i l i c a g e l w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 4 : 1 , mp 8 6 - 8 7 . 5 ° , [a]fck = + 1 9 . 0 ° ( c = 1 . 7 , 1 , 4 - d i o x a n e ) . A n a l , c a l c d . f o r C 3 2 H 5 2 0 2 S n : C 6 5 . 4 3 , H 8 . 9 2 ; f o u n d : C 6 5 . 6 0 , H 8 . 7 8 . Mass s p e c t r u m , m / z : 531 ( 1 2 0 S n : M + - C , t H g , 1 0 0 ) . I n an a t t e m p t t o s e p a r a t e [26] f r o m [14] f o r a n a l y s i s , c o l u m n c h r o m a t o -g r a p h y o f a p o r t i o n o f t h e m i x t u r e o f [26] and [14] was p e r f o r m e d on s i l i c a g e l w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 4 : 1 . U n f o r t u n a t e l y , c o m p l e t e s e p a r a t i o n was n o t e f f e c t e d as [14] c o - e l u t e d w i t h a l l f r a c t i o n s c o n t a i n i n g [ 2 6 ] , w i t h t h e 83 largest percentage of [14] being present in the i n i t i a l fractions. Therefore, a sample was obtained by combining several of the late fractions which contained the lowest percentage of [14]. This sample was recrystallized from benzene-hexanes and analyzed by HPLC using a C-18 column with methanol/water 75:25 as the eluant (flow rate 6.0 ml/min; detector set at 280 nm). The HPLC analysis showed that a ratio of [14] (retention time, 3.60 min) to [26] (retention time, 5.00 min) of 19:81 was present. As a result, to properly isolate [26], an HPLC separation was carried out by injecting the sample (dissolved in THF) in several portions onto the C-18 column and eluting the product with the same solvent system as described before (flow rate 3.0 ml/min). The collected product was repurified by HPLC again, as described above. The product was recrystallized from benzene-hexanes, which afforded [26] as white crystals, mp 169.5-170°, [a] 2, 5 =+58.5° (c=l, 1,4-dioxane). Anal, calcd. for C 2 ( )H 2 60 2: C 80.50, H 8.78; found: C 80.54, H 8.72. Mass spectrum, m/z: 298 (M+, 51), 280 (M+^O, 15). Exact mass calcd. for C 2 0 H 2 6 ° 2 : 2 9 8 - 1 9 3 4 > found: 298.1934. Method B: Compound [14] (1.00 g, 3.37 mmol) was hydrostannylated as outlined in procedure B. Column chromatography on s i l i c a gel (100 g) with CH2C12/hexanes/ether 6:4:1 yielded in one portion 1.17 g (59%) of [17]. Physical and spectral (*H nmr) properties of this material were identical with those reported earlier. Analytical t i c confirmed the presence of [26] in the reaction mixture, but the compound was not eluted off the column. 8 4 Preparation of 3-methoxy-17o-E-tributylstannylvinyl-l,3,5(10)-estratriene-170-ol [18]. 3 5 Compound [15] ( 1 . 0 0 g , 3 .22 mmol) was h y d r o s t a n n y l a t e d as d e s c r i b e d i n p r o c e d u r e B . Column c h r o m a t o g r a p h y on s i l i c a g e l (200 g) w i t h h e x a n e s / e t h e r 5 : 1 y i e l d e d 1 .68 g (87%) o f [18] as a c o l o u r l e s s o i l , [ a ] ^ = + 1 7 . 1 ° ( c=1 .2 , 1 , 4 - d i o x a n e ) . A n a l , c a l c d . f o r C 3 3 H 5 1 + 0 2 S n : C 6 5 . 9 0 , H 9 . 0 5 , 0 5 . 3 2 ; f o u n d : C 6 5 . 8 3 , H 9 . 0 0 , 0 5 . 2 8 . Mass s p e c t r u m , m / z : 6 O 2 ( 1 2 0 S n : M+, 0 . 2 ) , 5 4 5 ( 1 2 0 S n : M + ' C ^ H g , 1 0 0 ) . Preparation of 3,178-dimethoxy-17o-E-tributylstannylvinyl-l,3,5(10)-estratriene [19]. Compound [16] ( 1 . 0 0 g , 3 .08 mmol) was h y d r o s t a n n y l a t e d a s d e s c r i b e d i n p r o c e d u r e B . Column c h r o m a t o g r a p h y on s i l i c a g e l (200 g) w i t h h e x a n e s / e t h e r 2 0 : 1 a f f o r d e d , i n t h e f i r s t f r a c t i o n , 0 .23 g o f [19] c o n t a m i n a t e d w i t h a m i n u t e amount o f u n i d e n t i f i e d b y p r o d u c t , and i n t h e s e c o n d f r a c t i o n , 1 .56 g o f p u r e [ 1 9 ] . Compound [19] was i s o l a t e d i n an o v e r a l l y i e l d o f 94% as an o i l , w h i c h a f t e r d r y i n g i n h i g h vacuum f o r 15-30 m i n u t e s , c r y s t a l l i z e d as a w h i t e s o l i d . The m a t e r i a l f r o m t h e s e c o n d f r a c t i o n e x h i b i t e d mp 5 0 - 5 2 . 5 ° , [a]^ = + 4 4 . 1 ° ( c = 1 . 2 , C H C 1 3 ) . A n a l , c a l c d . f o r C 3 I t H 5 6 0 2 S n : C 6 6 . 3 5 , H 9 . 1 7 , 0 5 . 2 0 ; f o u n d : C 6 6 . 3 7 , H 9 . 1 8 , 0 5 . 3 5 . Mass s p e c t r u m , m / z : 6 1 6 ( 1 2 0 S n : M+, 1 ) , 6 0 1 ( 1 2 0 S n : M + - C H 3 , 5 ) , 5 5 9 ( 1 2 0 S n : M ^ - C ^ H g , 6 6 ) . 85 IV.6 Labelling with Non-Radioactive Bromine Bromination of vinyl - t i n precursors; General Procedure. To a s o l u t i o n o f v i n y l - t i n s u b s t r a t e ( 0 . 6 - 0 . 9 mmol) d i s s o l v e d i n THF (100 mg o f s u b s t r a t e p e r m l ) , 1 .00 t o 1 .05 e q u i v a l e n t s o f s o d i u m b r o m i d e i n H 2 0 ( 0 . 5 M) was a d d e d . The m i x t u r e was c o o l e d t o 0 ° and s h i e l d e d f r o m l i g h t . Two e q u i v a l e n t s o f C h l o r a m i n e - T i n T H F / H 2 0 1 :1 ( 0 . 6 7 M) were a d d e d , and t h e n two e q u i v a l e n t s o f aqueous HC1 s o l u t i o n ( 1 M ) , w h i c h had b e e n s a t u r a t e d w i t h s o d i u m c h l o r i d e , were added i n one p o r t i o n t o t h e m i x t u r e . A f t e r one m i n u t e o f s t i r r i n g a t 0 ° , t h e m i x t u r e was p o u r e d i n t o an a q u e o u s s o d i u m a c e t a t e s o l u t i o n (1M, 20 ml ) and was e x t r a c t e d w i t h e t h e r (3x25 m l ) . The o r g a n i c e x t r a c t s were t h e n washed w i t h w a t e r (2x25 m l ) and d r i e d o v e r a n h y d r o u s magnesium s u l f a t e . The s o l v e n t was removed and the p r o d u c t was i s o l a t e d by s i l i c a g e l c h r o m a t o -g r a p h y . Preparation of (E)-8-C-bromo-7,8-dideoxy-l,2:3,4-di-O-isopropylidene-D-glycero-a-D-galacto-oct-7-enopyranose [5] . 7 8 Compound [4] (483 mg, 0 .84 mmol) was b r o m i n a t e d as d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e u s i n g 1 .05 e q u i v a l e n t s o f s o d i u m b r o m i d e . Column c h r o m a t o g r a p h y on s i l i c a g e l (100 g) w i t h C H 2 C l 2 / h e x a n e s / e t h e r 6 : 2 : 1 y i e l d e d 291 mg (95%) o f [ 5 ] . An a n a l y t i c a l sample was p r e p a r e d by r e c r y s t a l l i z a t i o n f r o m e t h e r - h e x a n e s w h i c h gave powdery w h i t e c r y s t a l s , mp 8 1 - 8 2 ° , [ a ] 2 , 5 = - 6 6 . 5 ° ( c = l . l , C H C 1 3 ) . A n a l , c a l c d . f o r C ^ H ^ B r O g : C 4 6 . 0 4 , H 5 . 8 0 , Br 86 2 1 . 8 8 ; f o u n d : C 4 6 . 2 1 , H 5 . 8 5 , Br 2 1 . 7 3 . Mass s p e c t r u m , m / z : 3 5 1 ( 8 1 B r : M + - C H 3 , 3 ) , 3 4 9 ( 7 9 B r : M + - C H 3 , 3 ) . Preparation of (E)-8-C-bromo-7,8-dideoxy-l,2:3,4-di-0-isopropylidene-L-glycero-a-D-galacto-oct-7-enopyranose [9]. Compound [8] (375 mg, 0 .65 mmol) was b r o m i n a t e d a s d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e u s i n g 1 .05 e q u i v a l e n t s o f s o d i u m b r o m i d e . Column c h r o m a t o g r a p h y on s i l i c a g e l (100 g) w i t h C H 2 C l 2 / h e x a n e s / e t h e r 6 : 2 : 1 y i e l d e d 211 mg (89%) o f [ 9 ] . A f t e r d e c o l o u r i z a t i o n w i t h c h a r c o a l , f o l l o w e d by r e c r y s t a l l i z a t i o n f r o m e t h a n o l - w a t e r , [9] was o b t a i n e d as w h i t e c r y s t a l s , mp 1 0 7 - 1 0 8 ° , [ a ] 2 , 5 = - 7 0 . 6 ° ( c = l , C H C 1 3 ) . A n a l , c a l c d . f o r C 1 1 + H 2 1 B r 0 6 : C 4 6 . 0 4 , H 5 . 8 0 , Br 2 1 . 8 8 ; f o u n d : C 4 5 . 9 8 , H 5 . 6 9 , Br 2 1 . 7 9 . Mass s p e c t r u m , m / z : 3 5 1 ( 9 l B r : M + - C H 3 , 4 ) , 3 4 9 ( 7 9 B r : M + - C H 3 , 4 ) . Preparation of B-E/Z-bromostyrene [13]. Compound [12] (102 mg, 260 umol) was b r o m i n a t e d , on s m a l l s c a l e , as d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e u s i n g 0 .90 e q u i v a l e n t s o f s o d i u m b r o m i d e . Changes i n t h e workup p r o c e d u r e were t h e u s e o f 5 m l o f aqueous s o d i u m a c e t a t e ( I M ) , 3 ml o f e t h e r f o r e x t r a c t i o n ( 3 x ) , and 3 m l o f w a t e r f o r w a s h i n g ( 2 x ) . A f t e r s o l v e n t r e m o v a l , t h e r e s i d u e was d i s s o l v e d i n a known v o l ume w i t h T H F . A s t a n d a r d s o l u t i o n o f a u t h e n t i c [13] ( E a s t m a n ; r a t i o o f E t o Z i s o m e r s o f 87 :13 by X H nmr) was p r e p a r e d . The p r o d u c t m i x t u r e was a n a l y z e d by HPLC u s i n g t h e s t a n d a r d s o l u t i o n o f [13] as a n e x t e r n a l s t a n d a r d . The a n a l y s i s was p e r f o r m e d on a C-18 c o l u m n u s i n g m e t h a n o l / w a t e r 75 :25 a s t h e e l u a n t ( f l o w 87 rate 6.0 ml/min; detector set at 280 nm). The analysis indicated that 38.4 mg of [13] (retention time, 5.02 min; both isomers co-elute) was present, for a yield of 90% based on the sodium bromide added. Preparation of 17a-E-bromovinyl-l,3,5(10)-estratriene-3,17B-dlol [20] Compound [17] (448 mg, 0.76 mmol) was brominated as described in the general procedure using 1.05 equivalents of sodium bromide. Column chromato-graphy on silica gel (200 g) with CH2Cl2/hexanes/ether 6:4:1 yielded 32 mg of [20] in one portion, followed by 8 mg of a mixture of [20] and [26], in a ratio of 67:33, respectively. Compound [20] was obtained in an overall yield of 13%. An analytical sample was obtained by flash chromatography on silica gel with CH2Cl2/hexanes/ether 6:4:1, followed by decolourization with charcoal and recrystallization from benzene-hexanes. Compound [20] was obtained as white needles, mp 87-98°C (dec), [a]g5 = +11.3° (c=0.9, 1,4-dioxane). Anal, calcd. for C20H25BrO2'O.5C6Hg: C 66.35, H 6.78; found: C 66.26, H 6.76. Mass spectrum, m/z: 378(81Br: M+, 0.1), 376(79Br: M+, 0.1); 360(81Br: tfM^O, 0.4), 358(79Br: M+-H20, 0.5), 297(M+-Br, 0.7). Exact mass calcd. for C 2 QH 2 5 8 1Br0 2: 378.1018; found: 378.1017. Exact mass calcd. for C 2 0H 2 5 7 9BrO 2: 376.1038; found 376.1042. In the course of isolating [20] during column chromatography, a number of other products were isolated and partially characterized (!H nmr and mass spectrometry). The first fraction (83 mg) collected contained a mixture of 4-bromo-17a-E-tributylstannylvinyl-l,3,5(10)-estratriene-3,176-diol [27] with some unidentified impurities, where [27] was dominant. Compound [27] 88 e x h i b i t e d t h e f o l l o w i n g X H nmr ( C D C 1 3 ) 6 : 0 . 7 1 - 3 . 0 4 ( s e v e r a l m, 46H, 1 7 - O H , I 8 - C H 3 , - S n ( n - B u ) 3 and s t e r o i d n u c l e u s ) , 5 . 6 1 ( s , 1H, 3 - O H ) , 6 . 0 6 ( d , 1 H , H - 2 1 , J=19.4 H z ) , 6 . 2 0 ( d , 1H, H - 2 0 , J=19.4 H z ) , 6 . 8 4 ( d , 1H, H - 2 , J=8 .5 H z ) , 7 . 1 7 ( d , 1H, H - l , J=8.5 H z ) . I n o r d e r t o p u r i f y [ 2 7 ] , f l a s h c h r o m a t o g r a p h y o n s i l i c a g e l ( 2 . 0 cm * 15 cm) w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 6 : 1 was p e r f o r m e d . However , the sample o f [27] had undergone s i g n i f i c a n t d e c o m p o s i t i o n and no improvement i n p u r i t y was g a i n e d . N o n e t h e l e s s , an i n t e r e s t i n g b y - p r o d u c t o f t h e d e c o m p o s i t i o n , a m i x t u r e o f 4 - b r o m o - 1 7 a - v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 B - d i o l [28] and 4 - c h l o r o - 1 7 a - v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 B - d i o l [ 2 9 ] , was i s o l a t e d . The r a t i o o f [28] t o [29] was a p p r o x i m a t e d to be 6 :4 by * H n m r . Mass s p e c t r o s c o p i c a n a l y s i s o f t h e m i x t u r e gave t h e f o l l o w i n g r e s u l t s ; mass s p e c t r u m of [ 2 8 ] , m / z : 3 7 8 ( 8 1 B r : M+, 2 2 ) , 3 7 6 ( 7 9 B r : M+, 2 2 ) , 3 6 0 ( 8 1 B r : M + - H 2 0 , 1 4 ) , 3 5 8 ( 7 9 B r : M + - H 2 0 , 16 ) ; mass s p e c t r u m o f [ 2 9 ] , m / z : 3 3 4 ( 3 7 C 1 : M+, 1 8 ) , 3 3 2 ( 3 5 C 1 : M+, 4 5 ) . Compound [28] e x h i b i t e d the f o l l o w i n g X H nmr ( C D C 1 3 ) 6 : 0 . 9 4 ( s , 3 H , 1 8 - C H 3 ) , 1 . 2 1 - 2 . 3 3 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 6 3 - 3 . 0 4 ( m , 2H, m e t h y l e n e p r o t o n s ) , 5 . 1 5 ( d , 1H, H - 2 1 a , J 2 0 - 2 1 a = 1 0 ' 8 H z ) » 5 . 2 0 ( d , 1H, H - 2 1 b , J 2 0 - 2 1 b = 1 7 , 3 H z > » 5 « 6 1 ( b r s , 1H, 3 - 0 H ) , 6 . 1 0 ( d d , 1H, H - 2 0 , J " 2 0 _ 2 1 a = 1 0 . 8 H z , J 2 Q _ 2 1 b = 1 7 . 3 H z ) , 6 . 8 4 ( d , 1H, H - 2 , J=8 .5 H z ) , 7 . 1 6 ( d , 1H, H - l , J=8.5 H z ) . Compound [29] e x h i b i t e d the f o l l o w i n g * H nmr ( C D C 1 3 ) 6 : 0 . 9 4 ( s , 3 H , 1 8 - C H 3 ) , 1 . 2 1 - 2 . 3 3 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 6 3 - 3 . 0 4 ( m , 2H, m e t h y l e n e p r o t o n s ) , 5 . 1 5 ( d , 1H, H - 2 1 a , J 2 0 - 2 1 a = 1 0 ' 8 H z ) » 5 . 2 0 ( d , 1 H , H - 2 1 b , J 2 0 - 2 1 b = 1 7 ' 3 H z ) » 5 . 6 1 ( b r s , 1H, 3 - 0 H ) , 6 . 1 0 ( d d , 1H, H - 2 0 , J " 2 0 - 2 i a = 1 0 * 8 H z > J 2 0 - 2 l b = 1 7 * 3 H z ) , 6 . 8 4 ( d , 1 H , H - 2 , J=8 .5 H z ) , 7 . 1 2 ( d , 1H, H - l , J=8 .5 H z ) . 89 The second fraction (18 mg) eluted contained a mixture of 2,4-dibromo-17a-E-bromovinyl-l,3,5(10)-estratriene-3,17B-diol [30] with small amounts of unidentified impurities. Compound [30] exhibited the following *H nmr (CDC13)6: 0.91 (s, 3H, I 8 - C H 3 ) , 1.07-2.37 (several ra, 14H, 17-OH and steroid nucleus), 2.55-2.98 (m, 2H, methylene protons), 5.84(br s, 1H, 3-0H), 6.31 (d, 1H, H-21, J=13.5 Hz), 6.44(d, 1H, H-20, J=13.5 Hz), 7.38(s, 1H, H-l). Unfortunately, [30] decomposed before further purification could be effected. After continued elution, a third fraction (43 mg) was collected which contained a mixture of three compounds, 4-bromo-17a-E-bromovinyl-l,3,5(10)-estratriene-3,176-diol [31], 4-chloro-17a-E-bromovinyl-l,3,5(10)-estratriene-3,176-diol [32] and 4-bromo-17a-E-chlorovinyl-l,3,5(10)-estratriene-3,173-diol [33]. Small amounts of unidentified impurities were also present. The ratio of [31] to [32] to [33] was approximated to be 51:34:15, respectively, by *H nmr. To separate the mixture, column chromatography was performed on silica gel (10 g) using hexanes/ether 1:1. However, l i t t l e separation was achieved as the mixture of compounds co-eluted. The ratio of [31] to [32] to [33] was found to be approximately 62:26:12, respectively, by 1H nmr. Mass spectro-scopic analysis of the mixture gave the following results; mass spectrum of [31], m/z: 458(81Br, 8 1Br: M+, 2),456(81Br, 7 9Br: M+, 4), 454(79Br, 7 9Br: M+, 2), 440(81Br, 8 1Br: M+-H20, 52), 438(81Br, 7 9Br: M+-H20, 100), 436(79Br, 7 9Br: M+-H-0, 55); mass spectrum of isomeric mixture of [32] and [33], m/z: 90 4 1 4 ( 8 1 B r , 3 7 C 1 : M+, 0 . 9 8 ) , 4 1 2 ( 7 9 B r , 3 7 c l : M + > 3 . 7 ) > 4 1 0 ( 7 9 B r , 3 5 d : M+, 3 . 9 ) , 3 9 8 ( 8 1 B r , 3 7 C 1 : M + ^ O , 2 5 ) , 3 9 6 ( 7 9 B r , 3 7 C 1 : M + - H 2 0 , 8 8 ) , 3 9 4 ( 7 9 B r , 3 5 C 1 : M + ^ O , 7 3 ) . Compound [31] e x h i b i t e d t h e f o l l o w i n g * H nmr ( C D C 1 3 ) 6 : 0 . 9 2 ( s , 3H, I8-CH3), 1 . 1 9 - 2 . 4 2 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 5 9 - 3 . 0 2 ( m , 2 H , m e t h y l e n e p r o t o n s ) , 5 . 5 9 ( b r s, 1H, 3 - 0 H ) , 6 . 30(d , 1H, H - 2 1 , J=13.5 H z ) , 6 . 4 6 ( d , 1H, H - 2 0 , J - 1 3 . 5 H z ) , 6 . 8 5 ( d , 1H, H - 2 , J=8.6 H z ) , 7 . 1 6 ( d , 1H, H - l , J=8.6 H z ) . Compound [32] e x h i b i t e d t h e f o l l o w i n g X H nmr ( C D C 1 3 ) 5 : 0 . 9 2 ( s , 3H, 1 8 - C H 3 ) , 1 . 1 9 - 2 . 4 2 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 5 9 - 3 . 0 2 ( m , 2H, m e t h y l e n e p r o t o n s ) , 5 . 5 9 ( b r s , 1H, 3 - 0 H ) , 6 . 3 0 ( d , 1H, H - 2 1 , J=13.5 H z ) , 6 . 46(d , 1H, H - 2 0 , J=13.5 H z ) , 6 . 8 5 ( d , 1H, H - 2 , H=8.6 H z ) , 7 . 1 1 ( d , 1H, H - l , J=8 .6 H z ) . Compound [33] e x h i b i t e d t h e f o l l o w i n g * H nmr (CDC1 3)<5: 0 . 9 2 ( s , 3 H , 1 8 - C H 3 ) , 1 . 1 9 - 2 . 4 2 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 5 9 - 3 . 0 2 ( m , 2 H , m e t h y l e n e p r o t o n s ) , 5 . 5 9 ( b r s , 1H, 3 - 0 H ) , 6 . 1 8 ( d , 1H, H - 2 0 , J=12.8 H z ) , 6 . 2 1 ( d , 1H, H - 2 1 , J=12.8 H z ) , 6 . 85(d , 1H, H - 2 , J=8.6 H z ) , 7 . 1 6 ( d , 1H, H - l , J=8 .6 H z ) . The f o u r t h f r a c t i o n (25 mg) c o l l e c t e d c o n t a i n e d a m i x t u r e o f [28] and [ 2 9 ] , b u t was h i g h l y c o n t a m i n a t e d w i t h u n i d e n t i f i e d i m p u r i t i e s . The X H nmr s p e c t r a l p r o p e r t i e s o f [28] and [29] were the same as t h o s e o b s e r v e d p r e v i o u s l y . L a s t l y , t h e d e s i r e d p r o d u c t [20] was i s o l a t e d as d e s c r i b e d e a r l i e r . Preparation of 3-methoxy-17a-E-bromovinyl-l,3,5(10)-estratriene-17fJ-ol [22]. Compound [18] (539 mg, 0 .90 mmol) was b r o m i n a t e d a s o u t l i n e d i n t h e g e n e r a l p r o c e d u r e u s i n g 1 .00 e q u i v a l e n t o f s o d i u m b r o m i d e . F l a s h c h r o m a t o g r a p h y o n s i l i c a g e l ( 3 . 5 cm x 15 cm) w i t h h e x a n e s / e t h e r 2 : 1 y i e l d e d 351 mg (100%) o f [ 2 2 ] . A f t e r c r y s t a l l i z a t i o n f r o m e t h a n o l - w a t e r , [22] was o b t a i n e d as w h i t e c r y s t a l s , mp 1 0 7 . 5 - 1 0 9 ° , [ a ] 2 , 5 = + 1 4 . 5 ° ( c = 0 . 9 , 1 , 4 -d i o x a n e ) . A n a l , c a l c d . f o r C 2 1 H 2 ? B r 0 2 : C 6 4 . 4 5 , H 6 . 9 5 , B r 2 0 . 4 2 ; f o u n d : C 6 4 . 1 1 , H 7 . 1 7 , B r . 2 0 . 2 5 . Mass s p e c t r u m , m / z : 3 9 2 ( 8 1 B r : M + , 1 0 ) , 3 9 0 ( 7 9 B r : M+, 1 0 ) , 3 7 4 ( 8 1 B r ; M + - H 2 0 , 9 9 ) , 3 7 2 ( 7 9 B r : M + - H 2 0 , 9 9 ) , 310(M+-HBr, 1 0 0 ) . Preparation of 3,17p-dimethoxy-17o-E-bromovinyl-l,3,5(10)-estratriene [24]. Compound [19] (507 mg, 0 .82 mmol) was b r o m i n a t e d as d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e u s i n g 1.02 e q u i v a l e n t s o f s o d i u m b r o m i d e . Column c h r o m a t o -g r a p h y on s i l i c a g e l (200 g) w i t h h e x a n e s / e t h e r 2 4 : 1 y i e l d e d 300 mg (90%) o f [ 2 4 ] . A f t e r d e c o l o u r i z a t i o n w i t h c h a r c o a l , f o l l o w e d by r e c r y s t a l l i z a t i o n f r o m e t h a n o l , [24] was o b t a i n e d as w h i t e n e e d l e s , mp 1 0 4 . 5 - 1 0 5 ° , [a] 2 , 1* = + 5 8 . 2 ° ( c = l , C H C 1 3 ) . A n a l , c a l c d . f o r C 2 2 H 2 g B r 0 2 : C 6 5 . 1 8 , H 7 . 2 1 , Br 1 9 . 7 1 ; f o u n d : C 6 5 . 2 4 , H 7 . 1 0 , Br 1 9 . 5 5 . Mass s p e c t r u m , m / z : 4 0 6 ( 8 1 B r : M+, 3 ) , 4 0 4 ( 7 9 B r : M + , 3 ) , 325(M+-Br , 1 0 0 ) . 92 IV.7 Labelling with Non-radioactive Iodine Iodination of vinyl-tin precursors; General Procedure To a s o l u t i o n o f v i n y l - t i n s u b s t r a t e ( 0 . 7 - 0 . 9 mmol) i n THF (100 mg o f s u b s t r a t e p e r m l ) , s h i e l d e d f r o m l i g h t , was a d d e d 1.05 e q u i v a l e n t s o f s o d i u m i o d i d e i n H 2 0 ( I M ) . Two e q u i v a l e n t s o f C h l o r a m i n e - T i n T H F / H 2 0 1 : 1 (0 .67 M) were a d d e d , and t h e n t h r e e e q u i v a l e n t s o f aqueous HC1 s o l u t i o n ( I M ) , w h i c h had b e e n s a t u r a t e d w i t h s o d i u m c h l o r i d e , were added i n one p o r t i o n t o t h e m i x t u r e . The m i x t u r e was s t i r r e d f o r one m i n u t e a t room t e m p e r a t u r e , t h e n q u e n c h e d w i t h e x c e s s aqueous s o d i u m t h i o s u l f a t e ( I M ) . The m i x t u r e was p o u r e d i n t o an aqueous s o d i u m a c e t a t e s o l u t i o n ( I M , 20 ml) and was e x t r a c t e d w i t h e t h e r (3x25 m l ) . The o r g a n i c e x t r a c t s were t h e n washed w i t h w a t e r (2x25 m l ) and d r i e d o v e r a n h y d r o u s magnesium s u l f a t e . The s o l v e n t was removed and t h e p r o d u c t was i s o l a t e d by s i l i c a g e l c h r o m a t o g r a p h y . Preparation of (E)-8-C-iodo-7,8-dideoxy-l,2:3,4-di-0-isopropylidene-D-glycero-a-D-galacto-oct-7-enopyranose [6]. Compound [4] (416 mg, 0 .72 mmol) was i o d i n a t e d as d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e . Column c h r o m a t o g r a p h y on s i l i c a g e l (100 g) w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 2 : 1 y i e l d e d 273 mg (92%) o f [ 6 ] . A f t e r d e c o l o u r i z a t i o n w i t h c h a r c o a l , f o l l o w e d by r e c r y s t a l l i z a t i o n f r o m e t h a n o l - w a t e r , [6] was o b t a i n e d as w h i t e c r y s t a l s , mp 8 0 - 8 1 . 5 ° , [O]D"* = - 6 5 . 6 ° ( c = l , C H C l g ) . A n a l , c a l c d . f o r C 1 1 + H 2 1 I 0 6 : C 4 0 . 7 9 , H 5 . 1 3 , I 3 0 . 7 9 ; f o u n d : C 4 0 . 8 0 , H 5 . 1 7 , I 3 0 . 6 6 . Mass s p e c t r u m , m / z : 4 1 2 ( M + , 0 . 1 ) , 3 9 7 ( M + - C H 3 > 1 1 ) , 3 3 9 ( 3 ) . 93 Preparation of (E)-8-C-iodo-7,8-dideoxy-l,2:3,4-di-0-isopropylldene-L-glycero-a-D-galacto-oct-7-enopyranose [10]. Compound [8] (402 mg, 0 .70 mmol) was i o d i n a t e d as d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e . Column c h r o m a t o g r a p h y on s i l i c a g e l (100 g) w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 2 : 1 y i e l d e d 278 mg (97%) o f [ 1 0 ] . A f t e r d e c o l o u r i z a t i o n w i t h c h a r c o a l , f o l l o w e d by r e c r y s t a l l i z a t i o n f r o m e t h a n o l - w a t e r , [10] was o b t a i n e d as w h i t e c r y s t a l s , mp 1 1 9 - 1 1 9 . 5 ° , [ a ] 2 , 5 = - 6 4 . 4 ° ( c = l , C H C l g ) . A n a l , c a l c d . f o r C 1 1 + H 2 1 I 0 6 : C 4 0 . 7 9 , H 5 . 1 3 , I 3 0 . 7 9 ; f o u n d : C 4 0 . 7 0 , H 5 . 2 0 , I 3 0 . 6 9 . Mass s p e c t r u m , m / z : 3 9 7 ( M + - C H 3 , 5 ) , 3 3 9 ( 4 ) . Preparation of 17a-E-iodovinyl-l,3,5(10)-estratriene-3,l7&-diol [21]**3>62 using hydrogen peroxide/acetic acid as the oxidizing agent. 3 5 To a s o l u t i o n o f [17] (219 mg, 0 .37 mmol) i n THF ( 2 . 0 m l ) , s h i e l d e d f r o m l i g h t , was a d d e d s o d i u m i o d i d e (59 mg, 0 .39 mmol) i n w a t e r (0 .37 m l ) , a 5% ( w e i g h t / v o l u m e ) s o l u t i o n o f s o d i u m a c e t a t e i n g l a c i a l a c e t i c a c i d ( 0 . 6 0 m l ) and a s o l u t i o n o f 30% h y d r o g e n p e r o x i d e i n g l a c i a l a c e t i c a c i d ( 2 : 1 , v o l u m e / v o l u m e ; 0 .60 m l ) . The m i x t u r e was s t i r r e d f o r 30 m i n u t e s a t room t e m p e r a t u r e . The r e a c t i o n was t e r m i n a t e d w i t h t h e a d d i t i o n o f aqueous s o d i u m t h i o s u l f a t e ( 3 . 6 m l , 0 .95 M) and was p o u r e d i n t o w a t e r (20 m l ) , w h i c h was e x t r a c t e d w i t h e t h e r (3x10 m l ) . The o r g a n i c e x t r a c t s were washed w i t h aqueous s a t u r a t e d s o d i u m b i c a r b o n a t e (2x15 m l ) , f o l l o w e d by w a t e r (2x15 m l ) and f i n a l l y d r i e d o v e r a n h y d r o u s magnesium s u l f a t e . A f t e r r e m o v a l o f t h e s o l v e n t , c o l u m n c h r o m a t o g r a p h y on s i l i c a g e l (50 g) w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 4 : 1 y i e l d e d 121 mg o f [21] i n one f r a c t i o n , f o l l o w e d by 51 mg o f a m i x t u r e o f [21] 94 and some u n i d e n t i f i e d b y p r o d u c t s . The y i e l d o f p u r e [21] was 77%. A f t e r d e c o l o u r i z a t i o n w i t h c h a r c o a l , f o l l o w e d by r e c r y s t a l l i z a t i o n ( t w i c e ) f r o m b e n z e n e - h e x a n e s , [21] was o b t a i n e d as w h i t e n e e d l e s , mp 8 8 - 9 7 ° ( d e c . ) { l i t . 1 * 3 mp 9 3 - 1 0 0 ° ( d e c . ) } , [a ] f t 5 = - 3 . 8 ° ( c = l , 1 , 4 - d i o x a n e ) . A n a l , c a l c d . f o r C 2 0 H 2 5 I 0 2 * C 6 H 6 : C 6 2 * 1 5 > H 6 . 2 2 ; f o u n d : C 6 1 . 8 1 , H 6 . 3 5 . Mass s p e c t r u m , m / z : 424(M+, 2 ) , 4 0 6 ( M + - H 2 O , 2 ) , 297(M+-I , 2 ) . E x a c t mass c a l c d . f o r C 2 Q H 2 5 I 0 2 : 4 2 4 . 0 9 0 1 ; f o u n d : 4 2 4 . 0 9 0 2 . I n o r d e r t o compare d i r e c t l y t h e c h e m i c a l y i e l d o f t h e above r e a c t i o n to t h a t o f u s i n g C h l o r a m i n e - T as t h e o x i d a n t ( s e e f o l l o w i n g p r e p a r a t i o n o f [ 2 1 ] ) , t h e above r e a c t i o n was r e p e a t e d on a s m a l l e r s c a l e u s i n g HPLC a n a l y s i s t o q u a n t i f y t h e y i e l d o f p r o d u c t . To a s o l u t i o n o f [17] ( 6 1 . 5 mg, 105 umol) i n THF ( 0 . 6 m l ) , s h i e l d e d f r o m l i g h t , was added s o d i u m i o d i d e ( 1 5 . 6 mg, 104 ymol) i n w a t e r ( 0 . 1 1 m l ) , a 5% ( w e i g h t / v o l u m e ) s o l u t i o n o f s o d i u m a c e t a t e i n g l a c i a l a c e t i c a c i d ( 0 . 1 5 ml ) and a s o l u t i o n o f 30% h y d r o g e n p e r o x i d e i n g l a c i a l a c e t i c a c i d ( 2 : 1 v o l u m e / v o l u m e ; 0 .15 m l ) . The m i x t u r e was s t i r r e d f o r 30 m i n u t e s , a t room t e m p e r a t u r e , and was s t o p p e d w i t h t h e a d d i t i o n o f aqueous sodium t h i o -s u l f a t e ( 0 . 9 0 m l , 0 .95 M ) . The r e a c t i o n m i x t u r e was p o u r e d i n t o w a t e r (5 m l ) and e x t r a c t e d w i t h e t h e r (3x3 m l ) . The o r g a n i c e x t r a c t s were washed w i t h a q u e o u s s a t u r a t e d s o d i u m b i c a r b o n a t e (2x3 m l ) , t h e n w i t h w a t e r (2x3 m l ) and were d r i e d o v e r a n h y d r o u s magnesium s u l f a t e . A f t e r s o l v e n t r e m o v a l , t h e r e s i d u e was d i s s o l v e d i n a known volume w i t h T H F . A s t a n d a r d s o l u t i o n o f a u t h e n t i c [21] ( s y n t h e s i z e d p r e v i o u s l y ) was p r e p a r e d . The p r o d u c t m i x t u r e was a n a l y z e d by HPLC u s i n g t h e s t a n d a r d s o l u t i o n o f [21] as an e x t e r n a l s t a n d a r d . The a n a l y s i s was p e r f o r m e d on a C-18 co lumn u s i n g t h e f o l l o w i n g g r a d i e n t s o l v e n t p r o g r a m : f r o m 0 t o 10 m i n , an i s o c r a t i c m i x t u r e o f M e 0 H / H ? 0 75 :25 was u s e d , f o l l o w e d by a s y s -95 t e m a t i c i n c r e a s e t o 100% MeOH d u r i n g 10 t o 13 m i n , and a c o n s t a n t c o m p o s i t i o n o f 100% MeOH was m a i n t a i n e d f r o m 13 t o 20 m i n . A c o n s t a n t f l o w r a t e o f 6 .0 m l p e r m i n u t e was u s e d and t h e d e t e c t o r was s e t a t 280 nm. The a n a l y s i s i n d i c a t e d t h a t 4 1 . 2 mg o f [21] ( r e t e n t i o n t i m e , 6 .78 min) was p r e s e n t , f o r a y i e l d of 93%. Preparation of 17a-E-iodovinyl-l,3,5(10)-estratrlene-3,17e-diol [21]"»3>62 using Chloramine-T as the oxidizing agent. Compound [17] (104 mg, 176 y m o l ) was i o d i n a t e d , on s m a l l s c a l e , as d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e u s i n g 1 .00 e q u i v a l e n t o f s o d i u m i o d i d e and 2 e q u i v a l e n t s o f aqueous HC1 s o l u t i o n . Changes i n t h e workup p r o c e d u r e were t h e u s e o f 10 m l o f aqueous s o d i u m a c e t a t e ( L M ) , 6 ml o f e t h e r f o r e x t r a c t i o n ( 3 x ) , and 6 m l o f w a t e r f o r w a s h i n g ( 2 x ) . A f t e r s o l v e n t r e m o v a l , t h e r e s i d u e was s t o r e d f o r two m o n t h s . The r e a c t i o n m i x t u r e was t h e n c h r o m a t o g r a p h e d on s i l i c a g e l (35 g) w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 6 : 1 . The r e a c t i o n m i x t u r e c o n t a i n e d s e v e r a l p r o d u c t s a c c o r d i n g t o t i c a n a l y s i s , b u t o n l y t h e compounds p r e s e n t i n s i g n i f i c a n t amounts were c o l l e c t e d . The f i r s t f r a c t i o n c o n t a i n e d a m i x t u r e i n w h i c h 2 - i o d o - 1 7 a - E - i o d o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 B - d i o l [34] was d o m i n a n t . T h i s m i x t u r e was f u r t h e r p u r i f i e d by f l a s h c h r o m a t o g r a p h y on s i l i c a g e l (2 cm x 15 cm) w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 4 : 1 . A m i x t u r e ( 4 . 1 mg) o f [34] w i t h 3 m i n o r , u n i d e n t i f i e d s i d e - p r o d u c t s was o b t a i n e d and * H nmr a n a l y s i s i n d i c a t e d t h e r a t i o o f [34] t o the t h r e e s i d e - p r o d u c t s was 6 7 : 3 3 . T h i s m a t e r i a l e x h i b i t e d t h e f o l l o w i n g p r o p e r t i e s ; mass s p e c t r u m , m / z : 5 5 0 ( M + , 100 ) , 5 3 2 ( M + - H 2 0 , 1 2 ) , 423(M+-I , 4 3 ) ; 1 H nmr ( C D C 1 3 ) 5 : 96 0 . 9 2 ( s , 3 H , 1 8 - C H 3 ) , 1 . 2 5 - 2 . 2 7 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 7 9 ( m , 2H, m e t h y l e n e p r o t o n s ) , 5 . 1 2 ( s , 1H, 3 - O H ) , 6 . 3 0 ( d , 1H, H - 2 1 , J=14.4 H z ) , 6 . 7 2 ( s , 1H, H - 4 ) , 6 . 7 7 ( d , 1H, H - 2 0 , J=14.4 H z ) , 7 . 4 9 ( s , 1H, H - l ) . The s e c o n d f r a c t i o n c o n t a i n e d a m i x t u r e o f 4 - i o d o - 1 7 a - E - i o d o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 B - d i o l [35] and 2 - i o d o - 1 7 a - v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e -3 , 1 7 6 - d i o l [ 3 6 ] . T h i s m i x t u r e was f u r t h e r p u r i f i e d by f l a s h c h r o m a t o g r a p h y on s i l i c a g e l (2 cm x 15 cm) w i t h C H 2 C l 2 / h e x a n e s / e t h e r 6 : 4 : 1 . A m i x t u r e (12 .5 mg) o f [35] and [36] was o b t a i n e d i n a r a t i o o f 1 2 : 8 8 , r e s p e c t i v e l y , by *H nmr a n a l y s i s . Mass s p e c t r o s c o p i c a n a l y s i s o f t h e m i x t u r e gave t h e f o l l o w i n g r e s u l t s ; mass s p e c t r u m o f [ 3 5 ] , m / z : 550 (M"1", 3 ) ; mass s p e c t r u m of [ 3 6 ] , m / z : 424(M+, 100) , 4 0 6 ( M + - H 2 O , 5 4 ) . Compound [35] e x h i b i t e d t h e f o l l o w i n g X H nmr ( C D C 1 3 ) 6 : 0 . 9 1 ( s , 3H, 1 8 - C H 3 ) , 1 . 2 5 - 2 . 2 3 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 79 (m, 2H, m e t h y l e n e p r o t o n s ) , 3 -OH n o t o b s e r v e d , 6 . 3 0 ( d , 1H, H - 2 1 , J=14.4 H z ) , 6 . 7 8 ( d , 1H, H - 2 0 , J - 1 4 . 4 H z ) , 6 . 8 3 ( d , 1H, H - 2 , J=8.6 H z ) , 7 . 1 8 ( d , 1H, H - l , J=8 .6 H z ) . Compound [36] e x h i b i t e d t h e f o l l o w i n g X H nmr ( C D C 1 3 ) 5 : 0 . 9 4 ( s , 3H, 1 8 - C H 3 ) , 1 . 2 5 - 2 . 2 3 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 7 9 ( m , 2 H , m e t h y l e n e p r o t o n s ) , 3 - 0 H n o t o b s e r v e d , 5 . 1 5 ( d , 1H, H - 2 1 a , J 2 Q _ 2 l a = 1 0 . 8 H z ) , 5 . 2 0 ( d , 1H, H - 2 1 b , J 2 0 _ 2 l b = 1 7 . 3 H z ) , 6 . 0 9 ( d d , 1H, H - 2 0 , J 2 0 _ 2 i a = 1 0 . 8 H z , J 2 Q _ 2 l b = 1 7 . 3 H z ) , 6 . 7 1 ( s , 1H, H - 4 ) , 7 . 5 0 ( s , 1H, H - l ) . The t h i r d f r a c t i o n c o n t a i n e d an i s o m e r i c m i x t u r e o f [36] and 4 - i o d o - 1 7 a -v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 3 , 1 7 6 - d i o l [ 3 7 ] . T h i s m i x t u r e was f u r t h e r p u r i f i e d by f l a s h c h r o m a t o g r a p h y on s i l i c a g e l (2 cm x 15 cm) w i t h C H 2 C 1 2 / h e x a n e s / e t h e r 6 : 4 : 1 . A m i x t u r e ( 6 . 6 mg) o f [36] and [37] was o b t a i n e d i n a r a t i o o f 8 : 9 2 , r e s p e c t i v e l y , by * H nmr a n a l y s i s . Compound [37] e x h i b i t e d t h e f o l l o w i n g p r o p e r t i e s ; mass s p e c t r u m , m / z : 424(M+, 5 2 ) , 4 0 6 ( ^ - ^ 0 , 3 1 ) ; *H 97 nmr ( C D C 1 3 ) 6 : 0 . 9 3 ( s , 3 H , 1 8 - C H 3 ) , 1 . 2 4 - 2 . 3 3 ( s e v e r a l m, 14H, 17-OH and s t e r o i d n u c l e u s ) , 2 . 6 1 - 2 . 8 8 ( m , 2 H , m e t h y l e n e p r o t o n s ) , 5 . 1 5 ( d d , 1H, H - 2 1 a , J 2 0 - 2 1 a = 1 0 . 8 H z , J 2 0 - 2 l b = < l H z ) , 5 . 2 0 ( d d , 1H, H - 2 1 b , J 2 0 - 2 l b = 1 7 . 3 H z , J 2 0 - 2 1 b = < l H z ) , 5 . 4 2 ( b r s , 1H, 3 - 0 H ) , 6 . 1 0 ( d d , 1H, H - 2 0 , J 2 0 _ 2 i a = 1 0 . 8 H z , J 2 0 - 2 1 b = 1 7 . 3 H z ) , 6 . 8 3 ( d , 1H, H - 2 , J=8 .5 H z ) , 7 . 1 9 ( d , 1H, H - l , J=8.5 H z ) . Compound [36] e x h i b i t e d t h e same * H nmr s p e c t r u m as o b t a i n e d e a r l i e r . The f i n a l f r a c t i o n o b t a i n e d c o n t a i n e d t h e d e s i r e d p r o d u c t [21] w h i c h e x h i b i t e d t h e same s p e c t r a l ( 1 H nmr) p r o p e r t i e s as p r e v i o u s l y r e p o r t e d . I n o r d e r t o d e t e r m i n e the c h e m i c a l y i e l d of [21] p r o d u c e d i n t h e above r e a c t i o n , and t o compare t h i s v a l u e w i t h t h a t o b t a i n e d u s i n g h y d r o g e n p e r o x i d e as t h e o x i d a n t , t h e above r e a c t i o n was r e p e a t e d on a s m a l l e r s c a l e u s i n g HPLC a n a l y s i s t o q u a n t i f y t h e y i e l d . Compound [17] ( 5 4 . 9 mg, 93 umol) was i o d i n a t e d , on s m a l l s c a l e , as d e s c r i b e d i n the g e n e r a l p r o c e d u r e u s i n g 1 .00 e q u i v a l e n t o f s o d i u m i o d i d e . Changes i n t h e workup p r o c e d u r e were t h e u s e of 5 m l o f aqueous s o d i u m a c e t a t e ( 1 M ) , 3 m l o f e t h e r f o r e x t r a c t i o n ( 3 x ) , and 3 m l o f w a t e r f o r w a s h i n g ( 2 x ) . A f t e r s o l v e n t r e m o v a l , t h e r e s i d u e was d i s s o l v e d i n a known volume w i t h T H F . A s t a n d a r d s o l u t i o n o f a u t h e n t i c [21] ( s y n t h e s i z e d p r e v i o u s l y ) was p r e p a r e d . The p r o d u c t m i x t u r e was a n a l y z e d by HPLC u s i n g t h e s t a n d a r d s o l u t i o n o f [21] as an e x t e r n a l s t a n d a r d . The a n a l y s i s was p e r f o r m e d u s i n g t h e same HPLC c o n d i t i o n s as d e s c r i b e d i n t h e p r e v i o u s a n a l y s i s o f [ 2 1 ] . The a n a l y s i s i n d i c a t e d t h a t 0 .76 mg o f [21] ( r e t e n t i o n t i m e , 6 . 8 1 min) was p r e s e n t , f o r a y i e l d o f 1.9%. 98 Preparation of 3-methoxy-17a-E-iodvinyl-l,3,5(10)-estratriene-17B-ol [23]. 6 2 Compound [18] (472 mg, 0 .79 mmol) was i o d i n a t e d a s d e s c r i b e d i n t h e g e n e r a l p r o c e d u r e . F l a s h c h r o m a t o g r a p h y on s i l i c a g e l ( 3 . 5 cm x 15 cm) w i t h h e x a n e s / e t h e r 2 : 1 y i e l d e d 304 mg (88%) o f [ 2 3 ] . A f t e r d e c o l o u r i z a t i o n w i t h c h a r c o a l , f o l l o w e d by r e c r y s t a l l i z a t i o n f r o m e t h a n o l - w a t e r , [23] was o b t a i n e d as w h i t e c r y s t a l s , mp 8 5 - 9 1 ° ( d e c ) , [ a ] 2 ) 5 = - 2 . 7 ° ( c = l , 1 , 4 - d i o x a n e ) . A n a l , c a l c d . f o r C 2 1 H 2 ? I 0 2 : C 5 7 . 5 4 , H 6 . 2 1 , I 2 8 . 9 5 ; f o u n d : C 5 7 . 6 8 , H 6 . 1 5 , I 2 8 . 9 0 . Mass s p e c t r u m , m / z : 438(M+, 3 4 ) , 4 2 0 ( M + - H 2 0 , 8 2 ) , 3 1 1 ( M + - I , 2 2 ) . Preparation of 3,17B-dimethoxy-17ct-E-iodovinyl-l,3,5(10)-estratriene [25]. Compound [19] (519 mg, 0 .84 mmol) was i o d i n a t e d as o u t l i n e d i n t h e g e n e r a l p r o c e d u r e e x c e p t 1 .10 e q u i v a l e n t s o f sodium i o d i d e and 2 . 2 0 e q u i v a l e n t s o f C h l o r a m i n e - T were u s e d i n s t e a d . Column c h r o m a t o g r a p h y on s i l i c a g e l (100 g) w i t h h e x a n e s / e t h e r 2 0 : 1 y i e l d e d 379 mg (99%) o f [ 2 5 ] . A f t e r d e c o l o u r i z a t i o n w i t h c h a r c o a l , f o l l o w e d by r e c r y s t a l l i z a t i o n f r o m e t h a n o l , [25] was o b t a i n e d as w h i t e n e e d l e s , mp 6 5 - 7 5 ° ( d e c ) , [ a ] f j 5 = + 4 7 . 7 ° ( c = l , C H C 1 3 ) . A n a l , c a l c d . f o r C 2 2 H 2 9 I 0 2 : C 5 8 . 4 1 , H 6 . 4 6 , I 2 8 . 0 5 ; f o u n d : C 5 8 . 5 4 , H 6 . 6 0 , I 2 7 . 9 1 . Mass s p e c t r u m , m / z : 4 5 2 ( M + , 3 ) , 3 2 5 ( M + - I , 7 6 ) . I V . 8 S y n t h e s i s o f C h r o m a t o g r a p h i c S t a n d a r d s f o r R a d i o - l a b e l l i n g 99 P r e p a r a t i o n o f 3 - m e t h o x y - 1 7 a - E - c h l o r o v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 1 7 ( $ -[ 3 8 ] . To a s o l u t i o n o f [18] (206 mg, 0 .34 mmol) d i s s o l v e d i n THF ( 2 . 0 m m o l ) , s h i e l d e d f r o m l i g h t , was added e t h a n o l ( 5 . 0 m l ) , C h l o r a m i n e - T (193 mg, 0 .69 mmol) i n T H F / H 2 0 1 : 1 ( 1 . 0 m l ) and aqueous HC1 s o l u t i o n ( 0 . 6 9 m l , 1M) , w h i c h had been s a t u r a t e d w i t h s o d i u m c h l o r i d e . The m i x t u r e was s t i r r e d f o r 2 h o u r s a t room t e m p e r a t u r e . An e x c e s s o f aqueous s o d i u m t h i o s u l f a t e ( 0 . 8 0 m l , 0 .95 M) was added t o t e r m i n a t e the r e a c t i o n . The m i x t u r e was p o u r e d i n t o an aqueous s o d i u m a c e t a t e s o l u t i o n (10 m l , 1M) and was e x t r a c t e d w i t h e t h e r (3x10 m l ) . The o r g a n i c e x t r a c t s were t h e n washed w i t h w a t e r (2x10 ml ) and d r i e d o v e r a n h y d r o u s magnesium s u l f a t e . The s o l v e n t was removed and [38] was i s o l a t e d u s i n g f l a s h c h r o m a t o g r a p h y on s i l i c a g e l ( 2 . 0 cm x 15 cm) w i t h h e x a n e s / e t h e r 4 : 1 w h i c h a f f o r d e d 81 mg (69%) o f [ 3 8 ] . An a n a l y t i c a l sample was p r e p a r e d by c o l u m n c h r o m a t o g r a p h y on s i l i c a g e l w i t h h e x a n e s / e t h e r 4 : 1 , f o l l o w e d by c r y s t a l l i z a t i o n f r o m e t h a n o l - w a t e r w h i c h gave [38] as w h i t e n e e d l e s , mp 4 7 . 5 - 4 9 . 5 ° , [ a ] § 5 = + 2 6 . 0 ° ( c = 0 . 9 , 1 , 4 - d i o x a n e ) . A n a l , c a l c d . f o r C 2 1 H 2 7 C 1 0 2 : C 7 2 . 7 1 , H 7 . 8 4 , f o u n d : C 7 2 . 5 2 , H 7 . 7 3 . Mass s p e c t r u m , m / z : 3 4 8 ( 3 7 C 1 : M+, 9 ) , 3 4 6 ( 3 5 C 1 : M+, 2 4 ) , 3 3 0 ( 3 7 C 1 : M + ^ O , 2 0 ) , 3 2 8 ( 3 5 C 1 : M + - H 2 0 , 5 8 ) , 3 1 0 ( M + - H C 1 , 4 2 ) . E x a c t mass c a l c d . f o r C 2 1 H 2 ? 3 7 C 1 0 2 : 3 4 8 . 1 6 7 1 ; f o u n d : 3 4 8 . 1 6 7 4 . E x a c t mass c a l c d . f o r C 2 1 H 2 7 3 5 C 1 0 2 : 3 4 6 . 1 7 0 1 ; f o u n d : 3 4 6 . 1 7 0 1 . 100 Preparation of 3-methoxy-17a-vInyl-l,3,5(10)-estratriene-17B-ol [39] . 6 7 T h i s p r o c e d u r e i s a m o d i f i c a t i o n o f t h e one u s e d i n t h e p r e p a r a t i o n o f [15] and [ 1 6 ] . Powdered p o t a s s i u m h y d r o x i d e ( 0 . 3 0 g , 5 .4 mmol) was added t o DMSO (3 m l ) and was s t i r r e d f o r 5 m i n u t e s , a t room t e m p e r a t u r e . A m i x t u r e o f [14] and [26] (200 mg, 0 .67 mmol) , w h i c h was i s o l a t e d p r e v i o u s l y ( r a t i o o f [14] t o [26] was 18 :82 by HPLC a n a l y s i s ) , was a d d e d , f o l l o w e d i m m e d i a t e l y by m e t h y l i o d i d e (380 mg, 2 .68 m m o l ) . S t i r r i n g was c o n t i n u e d f o r 60 m i n u t e s , -t h e n t h e m i x t u r e was p o u r e d i n t o w a t e r (30 m l ) and was e x t r a c t e d w i t h d i c h l o r o m e t h a n e (3x30 m l ) . The o r g a n i c e x t r a c t s were washed w i t h w a t e r (3x30 m l ) , d r i e d o v e r a n h y d r o u s magnesium s u l f a t e and t h e s o l v e n t was r e m o v e d . F l a s h c h r o m a t o g r a p h y o f t h e c r u d e m i x t u r e , on s i l i c a g e l (2 cm x 15 cm) w i t h h e x a n e s / e t h e r 4 : 1 , a f f o r d e d 90 mg o f a m i x t u r e o f [16] and 3 , 1 7 ( 3 - d i m e t h o x y -1 7 c t - v i n y l - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n e [ 4 0 ] . The r a t i o o f [16] ( r e t e n t i o n t i m e , 2 .54 min) t o [40] ( r e t e n t i o n t i m e , 3 .10 min) was d e t e r m i n e d t o be 35 :65 by HPLC a n a l y s i s on a C-18 c o l u m n u s i n g m e t h a n o l as t h e e l u a n t ( f l o w r a t e 6 .0 m l / m i n ; d e t e c t o r s e t a t 280 nm). T h i s m i x t u r e was c h a r a c t e r i z e d by 1 H nmr and mass s p e c t r o m e t r y . No a t t e m p t was made to i s o l a t e [40] i n a p u r e f o r m . Mass s p e c t r o s c o p i c a n a l y s i s o f t h e m i x t u r e gave t h e f o l l o w i n g r e s u l t s ; [16] e x h i b i t e d the same s p e c t r a l p r o p e r t i e s as r e p o r t e d e a r l i e r ; mass s p e c t r u m f o r [ 4 0 ] , m / z : 326(M+, 4 4 ) . F u r t h e r e l u t i o n w i t h h e x a n e s / e t h e r 1 :1 y i e l d e d 90 mg o f [ 3 9 ] , i n one p o r t i o n , f o r a y i e l d of 52%, b a s e d on t h e i n i t i a l amount o f [26] u s e d . HPLC a n a l y s i s showed t h a t [39] ( r e t e n t i o n t i m e , 5 .52 min) was t h e o n l y compound p r e s e n t , w i t h no t r a c e o f [15] o b s e r v e d . The HPLC a n a l y s i s was p e r f o r m e d on a 101 C-18 c o l u m n w i t h m e t h a n o l / w a t e r 85 :15 ( f l o w r a t e 6 .0 m l / m i n ; d e t e c t o r s e t a t 280 n m ) . A f t e r r e c r y s t a l l i z a t i o n f r o m e t h a n o l - w a t e r , [39] was o b t a i n e d as w h i t e c r y s t a l s , mp 1 1 0 . 5 ° , [ a ] u 5 = + 5 6 . 9 ° ( c = l , 1 , 4 - d i o x a n e ) . A n a l , c a l c d . f o r C 2 1 H 2 8 0 2 : C 8 0 . 7 3 , H 9 . 0 3 ; f o u n d : C 8 0 . 7 7 , H 9 . 0 9 . Mass s p e c t r u m , m / z : 3 1 2 ( M + , 1 0 0 ) , 2 9 4 ( M + - H 2 0 , 8 ) . E x a c t mass c a l c d . f o r C 2 1 H 2 8 0 2 : 312 .2090 ; f o u n d : 3 1 2 . 2 0 9 0 . IV.9 Labelling with R a d i o a c t i v e Bromine General procedures for r a d i o b r o m i n a t i o n P r o c e d u r e A : To a s t i r r e d s o l u t i o n o f [18] ( 3 . 7 mg, 6 .2 y m o l ) i n THF (40 y l ) , s h i e l d e d f r o m l i g h t , was added e t h a n o l (100 y l ) , N H ( + 8 2 B r (20 y l aqueous s t o c k s o l u t i o n ) and 14-18 y l o f aqueous HC1 s o l u t i o n (1 M) w h i c h had been s a t u r a t e d w i t h s o d i u m c h l o r i d e . C h l o r a m i n e - T ( 3 . 8 mg i n 20 y l , T H F / H 2 0 1 :1 ) was added and t h e m i x t u r e was s t i r r e d a t e i t h e r 0 ° o r room t e m p e r a t u r e f o r one m i n u t e . The r e a c t i o n was q u e n c h e d w i t h aqueous s o d i u m t h i o s u l f a t e (100 y l , 0 . 1 M ) . P r o c e d u r e B : To a s t i r r e d s o l u t i o n o f [18] ( 3 . 7 mg, 6 .2 ymol) i n THF (40 y l ) , s h i e l d e d f r o m l i g h t , was a d d e d N H 4 8 2 B r (20 y l aqueous s t o c k s o l u t i o n ) . N - C h l o r o s u c c i n i m i d e ( 2 . 3 mg i n 60 y l , THF/MeOH 1 :1 ) was added and t h e m i x t u r e was s t i r r e d f o r 30 m i n u t e s a t room t e m p e r a t u r e . The r e a c t i o n was q u e n c h e d w i t h aqueous s o d i u m t h i o s u l f a t e (120 y l , 0 . 1 M ) . G e n e r a l Workup: F o r b o t h p r o c e d u r e s , A and B , t h e workup and i s o l a t i o n o f t h e r a d i o - l a b e l l e d p r o d u c t was p e r f o r m e d i n t h e same manner . The r e a c t i o n m i x t u r e was w i t h d r a w n w i t h a s y r i n g e f r o m t h e r e a c t i o n v e s s e l . T h e n t h e 102 v e s s e l was r i n s e d w i t h a s o l v e n t m i x t u r e of T H F / H 2 0 1 : 1 (100 u l ) t h r e e t i m e s ; e a c h t i m e t h e s o l v e n t r i n s e was w i t h d r a w n i n t o t h e s y r i n g e c o n t a i n i n g t h e r e a c t i o n m i x t u r e . The c o n t e n t s o f t h e s y r i n g e were i n j e c t e d , i n one p o r t i o n , o n t o a r e v e r s e - p h a s e HPLC c o l u m n . E l u t i o n w i t h t h e f o l l o w i n g g r a d i e n t s o l v e n t p r o g r a m a f f o r d e d t h e d e s i r e d r a d i o - l a b e l l e d p r o d u c t : f r o m 0 t o 10 m i n , an i s o c r a t i c m i x t u r e o f M e O H / H 2 0 85 :15 was u s e d , f o l l o w e d by a s y s t e m a t i c i n c r e a s e t o 100% MeOH d u r i n g 10 t o 12 m i n , and a c o n s t a n t c o m p o s i t i o n o f 100% MeOH was m a i n t a i n e d f r o m 12 t o 20 m i n . A f l o w r a t e o f 6 .0 ml p e r m i n u t e was u s e d and t h e d e t e c t o r was s e t a t 254 nm. The c o l l e c t e d r a d i o - l a b e l l e d p r o d u c t was i m m e d i a t e l y a s s a y e d to d e t e r m i n e t h e r a d i o c h e m i c a l y i e l d of t h e r e a c t i o n . Preparation of 3-methoxy-17o-E-(82Br)bromovinyl-l,3,5(10)-estratriene-17B-ol [41]. Method A : Compound [18] was r a d i o b r o m i n a t e d as d e s c r i b e d i n p r o c e d u r e A u s i n g 131 u C i o f N H , t 8 2 B r and 14 y l o f aqueous HC1 s o l u t i o n . The r e a c t i o n was p e r f o r m e d a t room t e m p e r a t u r e . HPLC i s o l a t i o n o b t a i n e d 105 u C i o f [41] ( r e t e n t i o n t i m e , 7 .4 min) f o r a r a d i o c h e m i c a l y i e l d o f 81%. T h i s m a t e r i a l has t h e same r e t e n t i o n t i m e as an a u t h e n t i c c o l d sample o f [ 2 2 ] . Method B : Compound [18] was r a d i o b r o m i n a t e d as o u t l i n e d i n p r o c e d u r e B u s i n g 125 u C i o f N H 1 + 8 2 B r . HPLC i s o l a t i o n o b t a i n e d 106 y C i o f [41] f o r a r a d i o c h e m i c a l y i e l d o f 85%. T h i s m a t e r i a l has t h e same r e t e n t i o n t i m e as an a u t h e n t i c c o l d sample o f [ 2 2 ] . 103 IV.10 Labelling with Radioactive Iodine General procedures for radioiodination. P r o c e d u r e A : To a s t i r r e d s o l u t i o n o f [18] (2 mg, 3 .3 y m o l ) i n THF (20 y l ) , s h i e l d e d f r o m l i g h t , was added e t h a n o l (50 y l ) , and i n t h e c a s e o f c a r r i e r a d d e d (CA) s y n t h e s e s , aqueous n o n - r a d i o a c t i v e s o d i u m i o d i d e (5 y l , 0 .33 M ) . T h i s was f o l l o w e d by t h e a d d i t i o n o f 10 y l o f aqueous s t o c k s o l u t i o n o f e i t h e r N a 1 3 1 I , N a 1 2 3 I o r N H ^ 1 2 3 1 , t h e n o f 5-8 y l o f aqueous HC1 s o l u t i o n (1 M ) , w h i c h had been s a t u r a t e d w i t h sodium c h l o r i d e . C h l o r a m i n e - T (10 y l , 0 .67 M i n T H F / H 2 0 1 :1 ) was a d d e d and t h e m i x t u r e was s t i r r e d f o r one m i n u t e a t room t e m p e r a t u r e . The r e a c t i o n was quenched w i t h aqueous s o d i u m t h i o s u l f a t e (63 y l , 0 . 1 M ) . P r o c e d u r e B : To a s t i r r e d s o l u t i o n o f [18] (2 mg, 3 .3 ymol) i n THF (20 y l ) , s h i e l d e d f r o m l i g h t , was added aqueous n o n - r a d i o a c t i v e s o d i u m i o d i d e (5 y l , 0 .33 M ) , i n t h e c a s e o f CA s y n t h e s e s , and N a 1 2 3 I (10 y l aqueous s t o c k s o l u t i o n ) . N - C h l o r o s u c c i n i m i d e ( 0 . 7 mg i n 20 y l , THF/MeOH 1 :1 ) was added and t h e m i x t u r e was s t i r r e d f o r 30 m i n u t e s a t room t e m p e r a t u r e . The r e a c t i o n was q u e n c h e d w i t h a q u e o u s s o d i u m t h i o s u l f a t e (63 y l , 0 . 1 M ) . P r o c e d u r e C : 3 5 To a s t i r r e d s o l u t i o n of [18] (2 mg, 3 .3 ymol ) i n THF (20 y l ) , s h i e l d e d f r o m l i g h t , was added aqueous n o n - r a d i o a c t i v e s o d i u m i o d i d e (5 y l , 0 .33 M ) , i n t h e c a s e o f CA s y n t h e s e s , and N a 1 2 3 I (10 y l aqueous s t o c k s o l u t i o n ) . To t h i s was a d d e d 50 y l o f a 5% ( w e i g h t / v o l u m e ) s o l u t i o n o f sodium a c e t a t e i n g l a c i a l a c e t i c a c i d and 50 y l of a s o l u t i o n o f 30% h y d r o g e n p e r o x i d e i n g l a c i a l a c e t i c a c i d ( 2 : 1 , v o l u m e / v o l u m e ) . The m i x t u r e was s t i r r e d 104 f o r 30 m i n u t e s a t room t e m p e r a t u r e and t h e r e a c t i o n was s t o p p e d w i t h t h e a d d i t i o n o f aqueous s o d i u m t h i o s u l f a t e (63 u l , 0 . 1 M ) . G e n e r a l Workup: F o r e a c h p r o c e d u r e , t h e workup and i s o l a t i o n o f t h e r a d i o - l a b e l l e d p r o d u c t was c o n d u c t e d i n t h e same f a s h i o n . The c o n t e n t s o f t h e r e a c t i o n v e s s e l were w i t h d r a w n w i t h a s y r i n g e , a f t e r w h i c h , t h e v e s s e l was r i n s e d w i t h a s o l v e n t m i x t u r e o f T H F / H 2 0 1 :1 (150 y l ) [ f o r p r o c e d u r e C , E t O H / H 2 0 3 : 1 (100 y l ) ] t h r e e t i m e s ; e a c h t i m e t h e s o l v e n t r i n s e was w i t h d r a w n i n t o t h e s y r i n g e c o n t a i n i n g the r e a c t i o n m i x t u r e . The c o n t e n t s o f t h e s y r i n g e were i n j e c t e d , i n one p o r t i o n , o n t o a r e v e r s e - p h a s e HPLC c o l u m n . E l u t i o n o f t h e r a d i o - l a b e l l e d p r o d u c t was e f f e c t e d u s i n g t h e same g r a d i e n t s o l v e n t p r o g r a m as p r e v i o u s l y d e s c r i b e d i n t h e r a d i o b r o m i n a t i o n p r o c e d u r e s . The c o l l e c t e d r a d i o - l a b e l l e d p r o d u c t was t h e n i m m e d i a t e l y a s s a y e d to d e t e r m i n e t h e amount o f r a d i o a c t i v i t y p r e s e n t . Preparation of 3-methoxy-17a-E-( 1 2 31)iodovinyl-1,3,5(10)-estratriene-17B-ol [42] Method A : Compound [18] (2 .2 mg, 3 .7 y m o l ) was r a d i o i o d i n a t e d as d e s c r i b e d i n p r o c e d u r e A u s i n g 256 y C i o f N H ^ 1 2 3 ! , 6 y l o f aqueous HC1 s o l u t i o n and 1 .9 mg (6 .7 y m o l ) o f C h l o r a m i n e - T . HPLC i s o l a t i o n a f f o r d e d 221 y C i o f [42] ( r e t e n t i o n t i m e , 8 . 1 min) f o r a r a d i o c h e m i c a l y i e l d o f 86%. T h i s m a t e r i a l has t h e same r e t e n t i o n t i m e a s an a u t h e n t i c c o l d s a m p l e o f [ 2 3 ] . Method B : Compound [18] ( 2 . 1 mg, 3 .5 y m o l ) was r a d i o i o d i n a t e d as o u t l i n e d i n p r o c e d u r e B us i n g 74 .5 y C i o f N a 1 2 3 I . HPLC i s o l a t i o n y i e l d e d 4 6 . 2 y C i o f [42] f o r a r a d i o c h e m i c a l y i e l d o f 62%. T h i s m a t e r i a l has t h e same r e t e n t i o n t i m e as an a u t h e n t i c c o l d sample o f [ 2 3 ] . 105 Method C : Compound [18] ( 2 . 1 mg, 3 .5 y m o l ) was r a d i o b r o m i n a t e d as d e s c r i b e d i n p r o c e d u r e C u s i n g 62 .7 y C i o f N a 1 2 3 I . HPLC i s o l a t i o n o b t a i n e d 53 .5 y C i o f [42] f o r a r a d i o c h e m i c a l y i e l d of 85%. T h i s m a t e r i a l has t h e same r e t e n t i o n t ime as a n a u t h e n t i c c o l d sample o f [ 2 3 ] . P r e p a r a t i o n o f 3 - m e t h o x y - 1 7 o - E - ( 1 3 1 I ) i o d o v i n y l - 1 , 3 , 5 ( 1 0 ) - e s t r a t r i e n e -17B - o l [43] Compound [18] ( 2 . 2 mg, 3 .7 y m o l ) was r a d i o i o d i n a t e d as d e s c r i b e d i n p r o c e d u r e A u s i n g 124 y C i o f N a 1 3 1 I , 7 y l o f aqueous HC1 s o l u t i o n and 1.9 mg ( 6 . 7 y m o l ) o f C h l o r a m i n e - T . HPLC i s o l a t i o n a f f o r d e d 105 y C i o f [43] ( r e t e n t i o n t i m e , 8 .1 m i n ) f o r a r a d i o c h e m i c a l y i e l d o f 85%. T h i s m a t e r i a l has t h e same r e t e n t i o n t i m e as an a u t h e n t i c c o l d sample o f [ 2 3 ] . IV.11 Nmr C h a r a c t e r i z a t i o n D a t a TABLE IV-1. Chemical Shifts (ppm) and Multiplet Splittings (Hz) for the Carbohydrate Derivatives Compound H-l H-2 H-3 H-4 H-5 H-6 H-7 H-8 6-OH Others [ 4 ] a , g 5.59 4.32 4.61 4.45 3.68 4.34 6.18 6.38 2.81 1.21-1.55 (d) (dd) (dd) (dd) (dd) (m) (dd) (dd) (d) (2m, 24H)C J x _ 2 5.0 J 2 _ 3 2.3 Jg-^ 8.0 J u _ 5 1.7 J 5 _ 6 6.5 J 6 _ 7 4.4 J ? _ 8 19.2 J 6 _ 8 1.4 J 6_ Q H7.0 0.81-0.97 JSn-H 6 4 JSn-H 7° <«• ^ [5] 3 5.57 4.33 4.64 4.44 3.67 -4.35 6.38 6.50 2.82 1.52 (d) (dd) (dd) (dd) (dd) (m) (dd) (dd) (d) 1.48 J x _ 2 5.1 J 2 _ 3 2.4 J3^ 8.0 J„_ 5 1.8 J 5 _ 6 6.9 J 6 _ ? 5.1 J ? _ 8 13.7 J 6 _ 8 1.0 J 6 _ Q H 6.6 1.36 1.34 (4s, ea. 3H) e [6] a 5.56 4.33 4.63 4.43 3.66 -4.31 6.73 6.53 2.92 1.52 (d) (dd) (dd) (dd) (dd) (m) (dd) (dd) (d) 1.48 h-2 5 ' ° J 2-3 2 ' 4 V - 7 ' 9 V s X ' 9 J 5"6 6 ' 9 J 6 - 7 5'X J7S 1 4 ' 5 V e X - 4 J 6 - 0 H 6 * 6 1 , 3 6 1.33 (4s, ea. 3H) e TABLE IV-1. Continued Compound H-l H-2 H-3 H-4 H-5 H-6 H-7 H-8 6-OH Others 18] b,g 5.50 (d) 4.18 (dd) 2 - 3 4.50 (dd) 4.30 (dd) 3.82 (dd) 4.73 (m) 6.48 (dd) 6.86 (dd) J l . 2 4.9 J,_, 2.3 J ' 3 - 4 7.9 J,._c 1.5 J 5 _ 6 7.6 J c _ 7 5.1 J , _ 0 19.2 J c _ 0 1.0 ' 4 " 5 6-7 J7"8 '6-8 3.07 1.24-1.71 (s) (2m, 18H)f 1.17 J n „65 Sn-H JSn-H 7 6 1.04 (2s, ea. 3H) e 0.86-1.01 (m, 15 H) d [9] 5.39 4.09 4.29 3.68 3.56 4.40 6.40 6.63 2.77 1.36 (d) (dd) (dd) (dd) (dd) (m) (dd) (dd) (br s) 1.33 J l . 2 5.0 J 2 _ 3 2.3 J j . ^ 7.9 J^.g 1.7 J 5 _ 6 8.0 J g _ 7 5.2 J ? _ 8 13.5 J g _ 8 1.4 1.03 1.01 (4s, e a . 3H)« [10] b 5.39 4.10 4.30 3.71 3.56 4.38 6.76 6.68 2.83 1.36 (d) (dd) (dd) (dd) (dd) (m) (dd) (d) (br s) 1.33 J t _ 2 4.9 J 2 _ 3 2.3 J 3_,, 7.9 J ^ _ 5 1.7 J 5 _ 6 7.9 J 6 _ 7 4.4 J 7 _ 8 14.5 1.03 1.01 (4s, ea. 3H) e a Deuteriochloroform solution, b Deuteriobenzene solution d c 2 x C(CH 3) 2 and -Sn(CH2CH^CH2CH3 ) 3 , d -S^CH^C^CH^Hj ) 3 , e -CH3 from C(CH ?) 2  f 1 x C(CH 3) 2 and -S^CHjCHjCH^CHg)3, 8 Jsn-H values are an average of the i^ 7Sn and 1 1 9 S n values. TABLE IV-2. Chemical Shifts (ppm) and Multiplet Splittings (Hz) for the Steroidal Derivatives Compound H-l H-2 H-4 H-20 H-21 3-OH 3-0CH. 17-OCH, 18-CH, Others [17] a,d 7.13 (d) 6.62 (dd) Jj—2 8.8 ^2~h 6.57 (d) 6.20 (d) J 2 0 - 2 1 1 9 - 4  JSn-H 6 6 6.07 (d) JSn-H 7 0 5.30 (s) over- 2.80 lapped (m, 2H) e 1.20-2.37 (several m, 26H)' 0.81-0.97 (m, 18H)J [20]' 7.10 (d) ' 1 - 2 6.62 (dd) 8.4 J 2~k 2.5 6.56 (d) 6.47 (d) J 2 0 - 2 1 1 3 , 5 6.26 (d) not observed 0.91 2.82 (s) (m, 2H)e 1.24-2.34 (several m, 14H)^ [21]' 7.10 (d) J x _ 2 8.4 6.62 (dd) J2-„ 2.5 6.56 (d) 6.79 (d) J 2 0 ~ 2 1 1 4 , 4 6.25 (d) not observed 0.91 2.82 (s) (m, 2H) e 1.24-2.37 (several m, 14H)* TABLE IV-2. Continued Compound H-l H-2 H-4 H-20 H-21 3-OH 3-0CH 3 17-0CH 3 I8-CH3 Others [26]' 7.14 (d) 1-2 8.2 6.62 (dd) J2-4 2' 8 6.56 (d) 6.11 (dd) J20-21a 1 0 * 8 1.2 H-2 la 5.15 (dd) J21a-21b H-2 lb 5.20 (dd) J 20-21b 1 7 , 3 4.68 (br s) 0.95 2.80 (s) (m, 2H) e 1.24-2.37 (several m, 14H)^ [18] a,d 1 - 2 7.19 (d) 8.5 6.69 (d) 6.62 (s) 6.20 (d) J20-21 1 9' Sn-H 66 6.06 (d) JSn-H 7 1 3.76 (s) over-lapped 2.84 (m, 2H) e 1.20-2.31 (several m, 26H) 0.82-0.97 (m, 18H)J TABLE IV-2. Continued Compound H-l H-2 H-4 H-20 H-21 3-0H 3-0CH3 17-OCH3 18-CH3 Others [22]* 7.18 (d) J^_2 8.6 J 2 " 4 6.71 (dd) 2.5 6.63 (d) 6.46 (d) J 2 0 ~ 2 1 13.5 6.29 (d) 3.77 ( s ) 0.92 2.86 (a) (m, 2H) e 1.23-2.35 (several m, 14H)f [23]' ' 1 ~ 2 7.18 (d) 8.6 6.70 (dd) 2.6 ' 2 - 4 6.63 (d) 6.79 (d) J 2 0 ~ 2 1 14.3 6.29 (d) 3.77 (s) 0.92 2.85 (s) (m, 2H) e 1.23-2.38 (several m, 14H)^ [38]' 7.19 (d) 8.5 6.71 (dd) J 2 - 4 2 ' 6 6.63 (d) 6.20 (s) 6.04 (d) 6.20 (s) 6.11 (d) 3.77 (s) 0.92 (s) 2.86 (m, 2H) e 1.20-2.37 (several m, 14H/ J 2 0 - 2 1 1 3 , 1 TABLE IV-2. Continued Compound H-l H-2 H-4 H-20 H-21 3-OH 3-OCH, 17-OCH, I8-CH3 Others [39]' 7.19 (d) 6.70 (dd) J , _ o 8.6 Jo-1. 2.6 '1-2 2~k 6.63 (d) 6.11 (dd) J20-21a 1 0 - 8 H-2 la 5.15 (d) H-2 lb 5.20 (d) J20-21b 1 7 ' 3 3.77 (s) 0.94 (s) 2.86 (m, 2H) e 1.24-2.34 (several m, 14H)^ [16]' 7.21 (d) 6.71 (dd) Jj—2 8.6 <• 2.7 6.63 (d) 2.62 (s) 3.77 (s) 3.42 (s) 0.88 (s) 2.86 (m, 2H)« 1.47-2.39 (several m, 13H)8 [19] b,d -7.16 (d) 6.75 (dd) J j_2 8.6 ^2~it 6 6.68 (d) 6.37 (d) 6.23 (d) J20-21 1 9 ' 7 J Sn-H 7 5  JSn-H 6 9 3.39 (s) 3.23 1.12 2.63-2.86 (s) (s) (m, 2H) e 1.21-2.29 (several m, 25H) 1.01 (m, 6H) k 0.93 (t, 9H)1 TABLE IV-2. Continued Compound H-l H-2 H-4 H-20 H-21 3-OH 3-0CH, 17-0CH, 18-CH- Others [24] 7.17 (d) Jj_2 8.6 6.69 (dd) J 2 - 4 2 ' 6 6.62 (d) 6.31 (d) J 2 0_ 2 113.9 6.13 (d) 3.77 (3) 3.19 0.90 2.83 (s) (s) (m, 2H) e 1.23-2.35 (several m, 13H)8 [25]' 7.10 (d) Jj-2 8.7 6.78 (dd) 2.6 ' 2 - 4 6.68 (d) 6.67 (d) J 2 0 . 2 1 14.8 5.94 (d) 3.42 (s) 2.95 0.90 2.70 (s) (s) (m, 2H) e 1.02-2.08 (several m, 13H)8 [40]' 7.16 (d) J j _ 2 8.4 -6.68 (d) 6.61 (s) 5.92 H-2 la (dd) 5.35 J20-21a 10.9 (d) H-2 lb 5.12 (d) J20-21b 3.75 (s) 3.16 0.91 2.83 (s) (s) (m, 2H) e 1.22-2.37 (several m, 13H)8 17.6 a Deuteriochloroform solution, b Deuteriobenzene solution, d e h j c Deuteriochloroform solution with a few drops of Deuteriomethanol added. J S n _ j j values are an average of the l l 7 S n and 1 1 9 S n values. Methylene protons of steroid nucleus, * 17-0H and steroid nucleus, 8 Steroid nucleus 17-0H, -Sn(CH2CH2CH2CH3)3 and steroid nucleus, 1 -Sn(CH2CH2CH2CH3)3 and steroid nucleus k -Sn<'r;H-rH.rH-CH3)3, 1 -Sn(CH2CH2CH3 18-CH3 and -Sn(CHjCH2CH2013)3, Sn(CH2CH2CH2CH3)3  113 REFERENCES 1 . K . K o u r i s , N . M . S p y r o u and D . F . J a c k s o n , I m a g i n g w i t h I o n i z i n g R a d i a t i o n , S u r r e y U n i v e r s i t y P r e s s , L o n d o n , 1982. p p . 2 - 3 . 2 . R . W . R e d i n g t o n and W . H . B e r n i n g e r , P h y s i c s T o d a y , 34 , 36 ( 1 9 8 1 ) . 3 . J . L l a c e r , I E E E S p e c t r u m , 1_8, 33 ( 1 9 8 1 ) . 4 . M . M . T e r - P o g o s s i a n , S e m i n a r s i n N u c l e a r M e d i c i n e , 11 , 13 ( 1 9 8 1 ) . 5 . M . M . T e r - P o g o s s i a n , M . E . R a i c h l e and B . E . S o b e l , S c i e n t i f i c A m e r i c a n , 243 , 170 ( 1 9 8 0 ) . 6 . R e f e r e n c e 1 . p p . 142-143 . 7 . D . A . Weber and R . E . 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N a k a d a t e , C a r b o h y d r . R e s . , 2_5, 205 ( 1 9 7 2 ) . 76 . F . B . C o l t o n , L . N . N y s t e d , B . R i e g e l and A . L . Raymond, J . Am. Chem. S o c , 79_, 1123 ( 1 9 5 7 ) . 77 . R . A . W . J o h n s t o n e and M . E . R o s e , T e t r a h e d r o n , 3_5, 2169 ( 1 9 7 9 ) . 7 8 . J . R . N e e s e r , L . D . H a l l and J . A . B a l a t o n i , H e l v . C h i m . A c t a . , 66_, 1018 ( 1 9 8 3 ) . 7 9 . J . C . C o c h r a n , S . C . B a y e r , J . T . B i l b o , M . S . Brown, L . B . C o l e n , F . J . G a s p i r i n i , D . W . G o l d s m i t h , M . D . J a m i n , K . A . N e a l y , C T . R e s n i c k , G . J . S c h w a r t z , W . M . S h o r t , K . R . S k a r d a , J . P . S p r i n g and W . L . S t r a u s s , O r g a n o m e t a l l i c s , _1, 586 ( 1 9 8 2 ) . 8 0 . M . L . S a ' i h i and M. P e r e y r e , B u l l . S o c . C h i m . F r a n c e , 1252 ( 1 9 7 7 ) . APPENDIX Selected Structural Formulae 118 Sn(Bu), Sn(Bu) f-OH _ h o t HO—I HO — -H-l ^ J o C) 0 " ^ - J o i 3 i i - V K i n n (TTO^- i^ i -v o-v-^ J * " 1 M O l x - i a-[11] [12] Sn(Bu), Br cr [13] R'O RO & [ U ] R ' = R 2 = H [15]R '=Me, R 2 = H [ 16] R 1 = R 2 = Me R'O [20]R'=R2=H [22]R'=Me,R2=H [24] R'=R2=Me HQ — MeO MeO Sn(Bu), [17]R'=R2=H [18]R'=Me,R2=H [19]R'=R2=Me [ 21 ]R' = R2=H [23]R'=Me,R2=H [25] R' = R 2 = Me U l ] X = 8 2 B r [42]X = , 2 3 I [43] X= I 3 I I [38] X=Cl 

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