Synthesis of radioiodinated metyrapone—a potential adrenal imaging agent

Int. J. Appl. Radiat. Isot. Vol. 34. No. 6. pp. 907-9t4. 1983

0020-708X/83/060907-06503.00/0 Copyright © 1983 Pergamon Press Ltd

Printed in Great Britain.All rights reserved

Synthesis of Radioiodinated Metyrapone A Potential Adrenal Imaging Agent W. R O B I E N 1 a n d I. Z O L L E 2. tlnstitut fiir Organische Chemic, Universi~t Wien, W~hringerstr. 38, A.1090 Wien, and "Abteilung f'dr Nuklearmedizin, 2. Medizinische Universit~tsklinik, A-1090 Wien, Osterreich

(Received 27 July 1982) Metyrapone has been labelled with radioiodine selectively in the 4'-position of ring B. The synthesis of t23I-(131)-metyrapone involves four intermediate compounds. 4'-Bromo-metyrapone serves as a stable precursor that is labelled before use. Studies of the biodistribution of [tJWJmetyrapone in rats indicate the highest concentration in the adrenal gland 10 rain after the injection with a fast elimination of the radioactivity. However, the absolute uptake in the normal adrenal is low. When [t23I]rnetyrapone was used in a patient with bilateral hyperplasia, faint adrenal images were obtained.

Introduction Pharmacologically active compounds that possess aniline or pyridine moieties have been shown to inhibit adrenal cortex enzymes.(t'2) Metyrapone has been used clinically to evaluate pituitary ACTH-response to adrenal function. Its mechanism of action is based on the inhibition of steroid 1lfl-hydroxylation and thus of the biosynthesis of cortisol, corticosteron¢ and aldosterone. Because of its specific uptake in the adrenal cortex a high accumulation and fast elimination of labelled metyrapone was assumed. Beierwares et al. reported data on the biodistribution of a number of radiolabelled adrenocortical enzyme inhibitors of which C3H]metyrapoi showed the highest concentration in the adrenal cortex. (a) Since metyrapon¢ consists of two pyridin¢ rings on a propanone chain, the best position for a ",,-label appeared to be on one of the pyridine rings. The use of t2aI as a radioactive label would offer excellent imaging characteristics with the y-camera. Pyridines are highly resistant to direct halogenation. However, N-oxidation has been effectively used to introduce a number of functional groups. (4-6) Nitro-N-oxides are the key intermediates in the synthetic approach to halogen derivatives.(7) Halogenation may be accomplished via diazotation of the reduced nitrogroup or directly by halogen substitution. 5'-iodo-metyrapol was described by Wieland et al. in preference to the 4'-iodo-compound.(a) A comparison with tritiated metyrapol however, showed a considerable loss of selectivity indicated by a lower adrenal uptake. (9) Figure 1 illustrates our synthetic route to 4'-t23I-(131)-metyrapone. Metyrapone was selectively converted to its mono-N-oxide.T M t) 4"-Bromo* Author to whom reprint requests should be addressed.

metyrapone was obtained from the mono-N-oxide by a three step conversion. (s'~'t2) Exchange labelling of 4'-bromo-rnetyrapone with tZ3NaI-(131) in the melt gave radioiodinated metyrapone. (13-ts) Halogen exchange of 4-bromo substituted pyridines was found independent of the N-oxide group. (7'tr't~) The advantage of this procedure is the preparation of labelled iodo-metyrapone by halogen exchange as the final reaction step. This avoids further handling of the labelled compound, which is important in the case of short-lived t23i. Evaluation of [t3tI]metyrapone in rats indicated a rapid uptake in the adrenal gland with fast elimination of radioactivity. When [t23I]metyrapone was used in a patient with Cushing's syndrome the adrenal glands were visualized.(ta) Methods

Synthesis of 4'-bromo-metyrapone (a) Preparation of metyrapone-N-oxide (2)

1-Propanone- l-( 3-p yridin yl)- 2-( 3-p yridin yl- 1-oxide)-2-methyl. A cooled solution (0°C) of m-chloroperbenzoic acid (6.7 g, 85%) in CHCIa (80 mL) was added to a solution of metyrapone (5 g) in CHCI 3 (40 mL) at 0°C during 2 h. The mixture was stirred for a further 4 h at 0°C. The organic,phase was then washed twice with saturated aquaeous sodium hydrogencarbonate (200 mL) and once with water (150 mL). Evaporation of the dried (Na2SO4) chloroform phase yielded a crystalline residue, which on recrystallization from ethyl acetate gave 3.8 g of (2) (71%). m.p. 165--167°C (Lit.(1°) m.p. 144-150°C). tH-NMR: 6(ppm): 1.60(H-9), 7.12(H-4'), 7.27(H-5'), 7.30(H-5), 7.86(H-4), 8.15(H-6'), 8.30(H-2'), 8.66(H-6), 8.77(H-2). Anal. calcd. for C1,Ht4N202: (2, 69.40; H, 5.82; N, 11.56. Found: C, 69.24; H, 5.77; N, 11.54.

907

908

~: Robien and I. Zolle

(b) Preparation of 4"-nitro-met37,apone-N-oxide (3)

l-Propanone- l-( 3-p ~ridinyl)-2-14-nirro-3-p ~Tidinyl- loxide)-2-methyl. The N-oxide (2) (2.75 g) was dissolved in concentrated sulphuric acid (7 mL) at 0~C and added dropwise to a stirred mixture of fumic nitric acid (12 mL) and concentrated sulphuric acid (7 mL). The mixture was stirred at room temperature for 1 h and then heated to 95°C for 18 h. After cooling, water (80 mL) was added, keeping the vessel in an ice bath. The pH of the solution was adjusted to 5.3 by addition of 6 N NaOH. The product was extracted with chloroform (6 x 50 mL). The organic layer was dried (Na,SO4) and evaporated. The residue was recrystallized from ethyl acetate giving 1.65 g of (3) as yellow needles (51%). m.p. 169-172°C. tH-NMR: 6(ppm): 1.82(H-9), 7.36(H-5), 8.00(H-5'). 8.04(H-4), 8.19(H-6'), 8.53(H-2'), 8.68(H-6), 8.79(H-2); Anal. calcd, for Cl,~HtaNaO,~: C, 58.53; H, 4.56; N. 14.63. Found: C, 58.37; H, 4.55; N, 14.35. (c) Preparation of 4'-bromo-met)Tapone-N-oxide (4)

l- Propanone- l-( 3- pyridin y l)- 2-I 4-bromo- 3-pyridin- yll-oxide)-2-methyl. The nitro-N-oxide (3) (1.6g) was added in small portions to ice-cooled acetyl bromide (12 mL). The suspension was stirred for 30 min at 0~C and for additional 30 rain at room temperature, followed by heating at 75°C for 45 rain. The cooled mixture was poured onto crushed ice (100g), the aquaeous phase made alkaline (KzCOa) and the product extracted with CHCIa (3 x 50 mL). The dried (K2CO3) solvent was evaporated and the product purified as above giving 1.2 g of (4) (67~o). m.p. 185-187°C, IH-NMR: 6(ppm): 1.78(H-9), 7.29(H-5), 7.33(H-5'), 7.97(H-4), 7.99(H-6'), 8.52(H-2'), 8.69(H-6), 8.88(H-2). Anal. calcd, for Ct,LHtaBrN202: C, 52.35; H, 4.08; Br. 24.88; N, 8.72. Found: C, 52.03; H, 4.06; Br, 24.49; N, 8.67. (d) Preparation of 4'-bromo-metyrapone (5)

1-Propanane- l-( 3°pyridinyl)- 2-( 4-bromo- 3-p yridinyl)-2-methyl. A solution of the bromo-N-oxide (4) (2 g) in ethanol (200 mL) was stirred with Raney-Nickel W-2H9"2°~ (400rag) under hydrogen for 6 h. After filtration and evaporation the residual oil was distilled (150~C/0.001Torr) giving a crystalline product. Further purification was carried out by recrystallization (ethyl acetate) yielding 1.46 g of (5) (77%). m.p. 89-90°C, ~H-NMR: 6(ppm): 1.83(H-9), 7.22(H-5), 7.40(H-5'), 7.95(H-4), 8.36(H-6'). 8.61(H-6), 8.80(H-2), 8.91(H-2'), mass spectrum: m/e: 304/306 (M+), 198/200, 170/172, 106, 78 Anal. calcd, for C1,~HtaBrN20: C, 55.10; H, 4.29; Br, 26.19; N. 9.18. Found: C, 54.97; H, 4.20; Br. 26.00; N, 9.09.

(e) Analytical procedures NMR-spectra were determined for approximately 0.05 M (IH), 0.4-0.6 M (lsC) solutions respectively in CDCl a with tetramethylsilane as the internal standard on a Bruker WM-250 spectrometer. Typical par-

ameters: *H: SF = 250.132MHz, PW = 1 #s (15"~), SW = 2500 Hr. digital resolution: 0.3 Hz. tsC: SF = 62.89 MHz, PW = 6/as (35'), SW = 15000 Hz, digital resolution: 0.9 Hz. The mass spectra were determined on a Varian MAT CH-7 spectrometer. The melting points are uncorrected. Analytical TLC: Merck Silica Gel F2s,L plates in two solvent systems.

Exchange Labelling with Nai-123(131) (a) Preparation of 4'-~ "3l-(131)-met)rapone To the tzaI-(131) activity (IRE. Belgium respectively IBS-500, Amersham, En~and), up to 10 mCi in less than 0.1 mL of 0.01 N NaOH, 20#g of NazS205 in 0.005 mL aquaeous solution are added. Then 1-2 mg of 4'-bromo-metyrapone dissolved in 0.05-0.1 mL ethanol are added and the solvent evaporated under vacuum at 65-'C. The dried reaction mixture is placed into an oil bath and heated at 165:C for 2 h. After cooling, the reaction product is dissolved in 0.5 mL chloroform respectively ethanol for subsequent purification. (b) Separation of free iodide. (1) Extraction with chloroform and an equal volume of an aquaeous alkaline solution containing 0.5mg KL 0.Stag Na2S2Oa and ling K, CO3. Extraction is repeated twice, finally the chloroform phase, containing [~2aI]metyrapone, is washed once with water. (2) Anion-exchange chromatography on a 9 x 20 mm Cellex-D column (ethanol-suspension, weakly basic DEAE-groups). To assure that unbound iodine is reduced, 0.02 mL of a Na2SzOs-solution (4 mg/mL) is added to the reaction product in 0.5 mL ethanol. Elution is performed with 2 mL chloroform. (c) Radiochemical purity The radiochemical purity of the product is determined by radio-TLC using Silica Gel F_~5,~plates in two solvent systems: (1) Chloroform/methanol (9:1) for identification of the product, R r = 0.55-0.65, single spot by u.v.quenching. (2) 1 N HCI for determination of free iodide, R j- = 0.8-0.9, Rf for t2al-(131)-metyrapone 0.25-0.35. For pharmaceutical use, tzaI-(131)-metyrapone is dissolved in ethanol, diluted with physiological saline, membrane filtered and used for injection as a 5%-ethanol solution in saline.

Biological Sttdies (a) Biodistribution of [1~i l]metyrapone in rats Female rats (215 g), received 0.5 mL of a solution containing 1.4/aCi/0.2 mg of [ ~a t ] metyrapone in travenously. Another 0.5 mL was diluted to 2.0 mL and 0.2 mL used as a standard. Rats were sacrificed under ether anesthesia at specified times. Blood samples and organs were removed and the radioactivity measured

it ,.Sl]Metyrapone fo r adrenal imaging CH3 I

909 0 CH-~

4' Pef~GI(I

CH3 ~'E'/ A

0

I

B

~tympot~

0

CH3

0

CH3

N~,z

.NO-d~,SC~. "~"tq"/

95oC

CH3 "~'~//

/

I

0

/

,

0

7'5o

0 CH-~ Br .

C~

~'~/

H2

',,,"if'.,,'

CH3

~'-a'/

0

N,r~ lilac Fig. 1. Synthetic scheme for the preparation of 4"-iodo-123(131)-metyrapone.

in a well-type scintillation counter. The obtained values were corrected and expressed as % kg dose/g tissue based on a minimum of 4 animals. The values obtained in the adrenal, liver, thyroid and blood at various dines after injection are plotted semilogarithmically and the half-life determined graphically.

Table I. ~3C chemical shifts of 4'.substituted metyrapones (ppm from internal TMS, 0.4--0.6M in CDCI 3) 9

R2

5

5'

~a) Adrenal imagin0 A patient with Cushing's syndrome, scheduled for adrenalectomy, received 1.25mCi of l'xz3I]metyrapone intravenously. Imaging with a y-camera was performed with the patient in prone position. Data accumulation was started immediately after injection of the tracer and continued for 90 rain.

Results (a) Synthesis of 4'-bromo-met~Tapone N-Oxidation of metyrapone with m-chloroperbenzoic acidT M zj leads under specified conditions to the mono-N-oxide (2)-structural evidence was established by t3C-NMR spectroscopy. N-Oxidation of ring B causes a downfield shift of C-3' (see Table I), which is easily identified in the off-resonance decoupled spectrum: 2t'22) The 4'-nitro-derivative (3) was obtained from (2) by treatment with a mixture of fumic nitric acid and sulphuric acid(s) at 95"C for 18 h.

I Ri

Compound

(I)

(2)

(3)

(4)

(5)

Rz

--

O

O

O

--

R2 Carbon

H

H

NOz

Br

Br

2 3 4 5 6 2' 3' 4' 5' 6' 7 8 9

150.7 131.6 136.6 123.0 152.2 147.8 139.9 133.5 123.8 148.6 201.3 50.5 27.3

150.4 '131.I 136.5 123.2 152.6 137.4 144.3 123.3 126.2 137.9 200.0 50.3 26.9

149.2 132.2 136.3 123.5 152.5 139.6 139.9 142.0 123.3 138.3 198.5 50.9 27.2

150. I 131.4 136.1 123.2 152.8 138.5 144.1 119.1 131.5 138.6 199.1 52.0 26.4

150.1 131.2 136.3 123.0 152.4 148.3 140.2 133.6 129.3 149.4 200.3 51.8 26.6

I4/. Robien and I. Zolle

910

~

I ~ o r . C.4~4kmaO

R~o3

C~I~x D

~wCt,~.mUs

TIC: SIkica gel

TLC: $aica S~!

I n HCI

~o.3

|"

efo.o

J J

$

Fig. 2. TLC in 1 N HCI. 1"~23I'lmetyraponebefore and after separation of free iodide by Cellex-D chromatography.

For evaluation of the subsequent reaction steps, 4-nitro-3-methyl-pyridine-N-oxide~:3) was adopted as a model compbund, because of its similiarity with ring B of (3). It was converted to 4-bromo-3-methylpyridine-N-oxide respectively 4-bromo-3-methyl-pyridine by known reactions. "2"z*'-'5) Both compounds were refluxed for 24 h with 48~o HI giving 4-iodo-3-methylpyridine in good yield~v't6"t7) contaminated with a trace amount of 4-bromo-3-methylpyridine. These results showed, that halogen exchange of 4-bromo substituted pyridines is independent of the N-oxide group and thus would offer a simple and effective route to carrier-free 4'-iodo-metyrapone. Consequently 4'-nitro-metyrapone-N-oxide (3) was treated with acetyl bromide at 75°C for I h cleanly affording 4'-bromo-metyrapone-N-oxide (4). Catalytic hydrogenation of (4) with Raney-Nickel W-2 in ethanol gave

4'-bromo-metyrapone (5) suitable as a precursor (26) for exchange labelling. (b) Factors affecting labelling The labelling yield is mainly affected by the radioiodine solution used for labelling, and the heating temperature. Highest non-isotopic exchange with 4'-bromo-metyrapone (1-2 nag) occurred at temperatures between 160° and 170°C with a radiocbemical yield between 65 and 75%. At 120°C labelling was below 20*//,. 4'-bromo-metyrapone is resistant to heating showing no degradation products on TLC: zv) Optimal labelling conditions require t23I-(131) solutions with minimum salt content. Concentrations of NaOH corresponding to 0.05 mL of a 0.1 N solution have totally prevented labelling. Separation of free iodide from t23I-(131)-metyrapone is quantitative

Table 2. Relative tissue distribution of l'tstI]metyrapone in rats at different times after injection (~ kg dose/g tissue; mean ± SEM) Organ

I0 + ± ± ±

30

Blood Heart Lung Liver

0.12 0.11 0.17 0.33

0.02 0.01 0.01 0.03

Intestine

0.13 ± 0.07

Spl~n Kidney Ovary Adrenal Thyroid

0.12 ± 0.17 ± 0.18 + 0.91 + 0.36 ±

0.01 0.01 0.03 0.30 0.02

X=8

60

-0.II ± 0.01 0.17 ± 0.01 0.31 ± 0.02

-0.12 ± 0.21 ± 0.17 ± 0.79 ± 1.60 +

0.08 0.09 0.19 0.17

1 2 0 m in

± 0.01 ± 0.01 ± 0.02 _+ 0.03

0.27 ± 0.07 0.01 0.02 0.02 0.22 0.26

X=4

0.I0 + 0.16 ± 0.14 ± 0.50 ± 2.29 +

0.01 0.02 0.03 0.15 0.15

X--4

0.04 ± 0.01 0.14 ± 0.02 0.I0 ± 0.01

-0.05 ± 0.I0 ± 0.07 ± 0.21 ± 4.02 +

0.01 0.02 0.01 0.03 1.45

X--5

Fig. 4. Adrenal scan with [ “‘l]metvrapone _

(1.25 mCi) in a patient with bilateral hyperpiasia. 20 min after injection.

911

[ t "Jl] Met vrapone for adrenal intaging S-

J ~ t.

Li~er ox Adrenol i, Thyroid T

• Blood

,It i o'.s

to

is

i.o

Time.h

Fig. 3. Rate of disappearance of [t3tl]metyrapone in various organs in rats expressed as % kg dose/g as a function of time after i.v. injection. both by extraction and ion-exchange chromatography. Radio-TLC revealed t23I-(131)-metyrap0ne as single spot, identical with the 4'-bromo-metyrapone standard. No free iodide was measured after separation of the product (Fig. 2). (c) Biodistribution in rats Table 2 shows the relative concentration of radioactivity in the excised organs at 10, 30, 60 and 120 rain after the injection. The adrenal gland shows the highest value 10 rain after the injection, with a fast elimination of radioactivity. From a semilogarithmic plot, the biological half-life in the adrenal was calculated as 47rain. (Fig. 3). The elimination of the labelled compound from the liver occurred with a similar half-time. Deiodination of [t3trJmetyrapone is indicated by uptake of radioactivity in the thyroid gland.

913

halogen exchange in the melt. N-oxidation of metyrapone at a low temperature leads to the mono-Noxide. Formation of the dioxide is minimal. Consequently, only the activated ring B is undergoing substitution reactions. Structural evidence by t3C-NMR spectroscopy verified N-oxidation of ring B exlusively. The synthesis includes nitration in the 4'-position and subsequent substitution with bromine. It was shown with model compounds that halogen exchange in the 4-position is independent of the N-oxide group. Thus 4'-bromo-metyrapone serves as a precursor that can be labelled with radioiodine before use. Labelling may be performed with tZ3NaI-(131) from commercial sources. However, the nucleophilic I- for Br-exchange in the melt requires radioiodine as no-carrier-added iodide with minimum salt content in dry form. To fulfill this requirement, a reducing agent is added to convert oxydized forms quantitatively into iodide. 2s Low salt concentration is maintained by using radioiodine with a sodium hydroxide content corresponding to less than 10 -6 mol. We generally used t23I-(131) in aquaeous or 0.01 N alkaline solution. The heating temperature had the most influence on the exchange rate. While practically no labelling was obtained at 120°C, heating at 165°C for 2 h produced 65-75% incorporation of the label. Labelled metyrapone showed a high in-vitro stability indicated by less than 1%/ofree iodide after 10 days suspension in saline. When kept in chloroform, only 0.3% free label was measured on TLC. Evaluation of [t3tI]metyrapone as a radiopharmaceutical in rats showed the highest concentration of radioactivity in the adrenals with a rapid uptake and fast elimination of the labelled compound (tl/2 = 47 rain). Although liver, bile and intestinal tract showed a lower concentration, the total organ uptake expressed as a percentage of the injected dose is highest, Since metyrapone accumulates in the bile, the main route of excretion of radioactivity is the intestinal tract. ['123FJmetyrapone was used for adrenal imaging in a patient with bilateral hyperplasia of the gland. Both adrenals were visualized 20 rain after the injection, blood activity measured over the lumbal region remained constant throughout the measurement.

(d) Patient stud)' [-t ,313.metyrapone was used for adrenal imaging in a patient with Cushing's syndrome. Figure 4 shows the scintigram obtained 20 rain after injection of the tracer. Both adrenals were visualized, the left adrenal gland is well outlined. At peak uptake in the adrenals, the contribution of background radioactivity is minimal and not interfering with the measurement. Discussion The preparation of ~23I-(131)-metyrapone can be seen as two separate processes: (1) synthesis of 4'-bromo-metyrapone as a precursor and (2) labelling by

Conclusion Using 4'-bromo-metyrapone as a precursor, l~3I-(131)-metyrapone may be Obtained with approximately 60% overall radiochen~cal yield within 3 h. The radiopharmaceutical is radiochemicaUy pure with a high in-vitro and in-vivo stability. Imaging of the adrenals was successfully performed in a patient with Cushing's syndrome. A!though radiolabelled cholesterol (13 t I-6AS-iodomethyl-19-norcholest-5(10)en-3fl-ol) has high diagnostic value, the prolonged imaging time and high radiation dose to the patient present a considerable limitation. The biokinetics of Ct23IJmetyrapone with its short effective half-life

W. Robien and I. Zolle

914

would offer a considerable reduction of the radiation dose and would permit imaging right after the injection of the tracer. So far adrenal visualization has only been successful in a patient with hyperfunctioning adrenals.

Acknowledgements--We wish to express our gratitude to Dr E. HASLINGER for his continued interest in this work. Support was given by the Fonds zur FOrderung der wissenschaftlichen Forschung in Osterreich for the development. Project 3500 and NMR-spectroscopy, Project 4009.

References 1. SATRE M. and VIGNAIS P. V. Biochemsistry 13, 2201 (1974). 2. BEtERWALTESW. H., WIELAND D. M., YU T., SWANSON D. P. and MOSLEY S. T. Semin. NuLl. Med. 8, 5 (1978). 3. BEIERWALTESW. H.. WIELAND D. M., ICE R. D., SEABOLD J. E., SARKAR S. D.. GILL S. P. and MOSLEY S. T. J. NuLl. Med. 17, 998 (1976). 4. OCHIAI E. Aromatic Amine Oxides (Elsevier, Amsterdam, 1967). 5. DEN HERTOG J. H. and O',ZRHOFF J. Rec. Trar. Chim. 69, 468 (1950). 6. ABRAMOVITCHR, A. and SMITH E. M. In The Chemistry of Heterocyclic Compounds. Vol. 14. 2 (Supplement). IEds WEISSBERGERA. and TAYLOR E. C.) (Wiley, New York. 1974). 7. MER'I-ELH. E. In The Chemistry of Heterocyclic Compounds Vol. 14. 2. (Interscience. New York. 1961}. 8. WIELAND D. M., KENNEDY W. P., ICE R. D. and BEIERWALIES W. H. J, Labelled Compd. Radiopharm. 13, 229 (1977). 9. BEIERWALTESW. H., WIELANO D. M., Yu T.. SWANSON D. P. and MOSLEY S. T. Semin. NuLl. Med. 8, 14 (1978). 10. CROOKS P. A.. DAMANI L. A. and COWAN D. A. Chemistry and Industry 10, 335 (1981).

II. DELIA T. J.. OLSEN M. J. and BROWN G. B. J. Or9. Chem. 30, 2766 (1965). 12. KaJUI~ARAS. J. Chem. Soc. Jap. 86. 1060 (19651. 13. THAKUR M. L. and WATERS S. L. Int. J. Appl. Radiat. lsot. 27, 585 (1976). 14. ELIAS H.. ARNOLD C. and KLOSS G. Int. J. Appl. Radiat. lsot. 24, 463 (1973). 15. ELIAS H. and LOT~RHOS H. F. Chem. Bet. 109, 1580 (1976). 16. BAKERW.. CURTIS R. F. and EOWARDS M. G. J. Chem. Soc. 83, (1951). 17. KLINGSBERGE. J. Am. Chem. Soc. 72, 1031 (1950). 18. ZOLLE 1., ROBIEN W.. BERGMANN H. and HOVER R. In Radioaktive Isotope in Klinik und Forschung (Eds H6EER R. and BERGMAYN H.) Voi. 15, 2 Tell, p. 589 (Verlag H. Egermann. Vienna. 1982). 19. MOZlNGO R. In Organic Synthesis, Collect. Vol. lII. p. 181, (Wiley. New York, 1955). 20. Organikum. p. 804 (VEB. Deutscher Verlag der Wissenschaften. Berlin, 1976). 21. YAVARI I. and RO~ERIS J. D. Org. Magn. Res. 12, 87 (1979). 22. STOTHERS J. B. Carbon-13 N M R Spectroscopy. (Academic Press. New York, 1972). 23. TAYLORE. C. and CROVE'rrl H. J. In Organic Synthesis, Collect. Vol. IV. (Wiley, New York, 1963). 24. LYLE R. E.. BRISTOL J. A.. KANE M. J. and PORTLOCK D. E. J. Org. Chem. 38, 3268 (1973). 25. ABRAMOVlTCH R. A. and SAHA M. Can. J. Chem. 44, 1765 (1966}. 26. ZOLLE I., ROmEN W. and HOFER R. J. Labelled Compd. Radiopharm. (Abstr.) 19, 1526 (1982). 27. ZOLLE I., ANGELBERGERP. and ROBIEN W. In Progress in Radiopharmacology. Vol. 3 (Ed. Cox P. H.) (Martinus Nijhoff, The Hague, 19821. 28. ANGELBERGER P.. WAONER-LOFFLER M.. DUDCZAK R. and HRUBY R. In Radioaktive Isotope in Klinik und Forschung (Eds H6EER R. and BERGMANNH.) Vol. 15, ! Tell, p. 249 [Verlag H. Egermann, Vienna. 1982).