Patai S., Rappoport Z. 1997 The chemistry of functional groups. The chemistry of double-bonded functional groups / 0909 110

VI. SYNTHESIS AND USES OF COMPOUNDS CONTAINING CDC, CDO

OR CN GROUPS LABELLED WITH RADIOACTIVE SULPHUR . . . 1013 VII. ISOTOPE EFFECT STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1014 A. Isotope Effect Studies of Chemical Reactions . . . . . . . . . . . . . . . . 1014 B. Isotope Studies of Chemical Catalytic Reactions . . . . . . . . . . . . . . 1039 C. Isotope Studies of Enzymatic Biochemical Reactions . . . . . . . . . . . 1061 D. Isotope Effects in Photochemical and Physical Processes . . . . . . . . 1075

VIII. ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085 IX. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085

I. INTRODUCTION

As in the past, the majority of the sometimes very laborious syntheses of isotopically substituted or isotopically labelled compounds has been carried out to provide the biologically active substances useful as analytical, diagnostic or therapeutic agents. The first six parts of this chapter are devoted to a brief description of the methods used in the preparation of compounds isotopically labelled with stable and radioactive isotopes, including the shortlived positron emitters carbon-11 and fluorine-18 widely applied in nuclear medicine. This time, we give also in Section VII of the chapter a rather extensive presentation of isotopic studies aimed at a better understanding of the mechanisms of chemical processes proceeding in vitro and in vivo, including chemical catalytic and enzymatic biochemical reactions cited in Volumes 119, 120 and 121 of Chemical Abstracts. The growing number of isotope effect papers with each year reflects the existence of numerous questions which should be given immediate answers on the chemical, atomic and molecular levels, to develop a coordinated interdisciplinary strategy linking the life sciences and technology trends to meet both human and economic concerns of contemporary society.

II. SYNTHESES AND USES OF COMPOUNDS CONTAINING C=C, C=O OR

C=N GROUPS LABELLED WITH STABLE ISOTOPES

A. Compounds Labelled with Deuterium

1. Synthesis of deuterium-labelled benzyl cyanides

In the substitution of alkyl halides by cyanide ion in aprotic organic solvents in the presence of crown ethers1,2 some ˛, ˛-2H2 benzyl chlorides lose deuterium when the reaction is carried out in acetonitrile. This has been used3 for the synthesis of deuteriated benzyl cyanides by refluxing with cyanide ion and crown ether in deuteriosolvents (C2HCl3, C2HCl3/CH3O2H, C2H3CN, and less efficient C2H2Cl2 and C62H6) as shown in equation 1.

RC2 H2 CH2 NH2

Deuteriochloroform was the solvent of choice. The ˇ,ˇ-2H2 and ˛,˛,ˇ,ˇ-2H4 phenylethylamine, m- and p-tyramine and tryptamine obtained3,4 have been used as tracers in metabolic studies and as internal standards in quantitative mass spectrometry3 5.

2. Synthesis of triethoxy-[ 2H]methane 1, and ethoxy-[ 2H]methylene malononitrile, 3

1 has been prepared5 in 31% yield by reacting deuteriochloroform with monodeuteriated ethanol in the presence of sodium during 12 hours and subsequent work-up and separation of deuterioorthoester using a short Vigreux column. 1, reacted with deuteriomalononitrile, 2, in acetic anhydride provided 3 in 86% yield deuteriated in 96 97% (equation 2). Ring closure of 3 with acetamidine, CH3C(NH)NH2, in ethanol leads to 4-amino-5-cyano-2- methyl-62H-pyrimidine, 4, without loss of deuterium. 3 was synthesized6 in the course of studies on the mechanism of the cleavage of thiamine, 5, which, treated with sulphite, decomposed to sulphonic acid betaine, 6 (equation 3)7.

3. Synthesis of [6,7-2H2]trilostane

The title compound, (4˛, 5˛, 17ˇ)-4, 5-epoxy-[6,7-2H2]-3, 17-dihydroxyandrost-2-ene- 2-carbonitrile, 78,9, has been found to be of benefit in the treatment of some forms of breast cancer9. It has been synthesized10 in 6 steps as shown in equation 4. The label has been introduced into 7 in the first reaction step by catalytic deuteriation of androsta-4,6- dien-3,17-dione, 8. Similarly, [4-14C]trilostane (sp. act. 95 Ci/g, r.p. >98%, 57% overall yield) has been prepared10,11 in four steps from [4-14C]testosterone, 9.

4. Synthesis of (š)-1-(1,3-dithiane-2-yl)-4-(1-hydroxy-1-[ 2H3]-methylethyl)-cyclohex- 2-en-1-ol, 10, and [ 2H10]- 1-THC-7-oic acid, 11

10 has been synthesized as shown in equation 5 and used for the preparation12 of 11, by condensation of 10 with [2H7]-olivetol, 12, 1-THC-7-oic acid, the major psychoactive

Cis-17 and trans-18 hexenols have been prepared in similar fashion from the acetylenic alcohol 21 (equations 7a and 7b),

(18)63% yield

6.Synthesis of 6,6,6-2H3-2E-hexanal (23)

23, ‘leaf-aldehyde’, product of the enzymatic and oxidative degradation of unsaturated fatty acids in processed foods, important aroma constituent in a number of fruit, vegetables, and leaves21,22, has been synthesized23 in 45% yield and 99.3% G.C. purity in a one-pot procedure (equation 8) of 3,3,3-2H3-n-propyl magnesium bromide with an

CD3

1. N HCl, work-up, separation

CD3 O

(23)

SiMe3 O OMgBr

(25)

[2-2H2]acetate generated by action of LDA with 1-trimethylsilyloxy-3-butanone, 31

(29) R = Me, (30) HMVA; R = Et

LDA = Lithium diisopropylamide

(11)

(b) Synthesis of 30 has been carried out29 in 70% yield by condensation of 1- trimethylsilyloxy-3-pentanone 34 with 32. The intermediate 33 was prepared in high yield as shown in equation 12.

No signals for protons at C 2 have been found in the 1H NMR spectrum of 30. This indicates complete deuteriation at this position and that no significant deuterium exchange took place under the conditions of the synthesis. Compounds labelled with stable isotopes are finding increasing applications in biosynthetic studies31. Labelled HMVA are required for metabolic studies in insects29.

9. Synthesis of 8- and 12-carbon deuterium-labelled aldehydic esters

Methyl 8-oxooctanoate-4,5-D2, 35, and methyl 12-oxododecarbate-4,5,8,9-D4, 36, have been synthesized32 as shown in equations 13 and 14 by monoozonization and sodium acetate cleavage of 1,5-cyclooctadiene and 1,5,9-cyclododecatriene, respectively. The resultant unsaturated aldehydic acids 37 and 38 have been converted to the corresponding acetal esters, which have been deuteriated with Wilkinson’s catalyst33 and hydrolysed to the deuterium-labelled aldehydic esters 35 and 36 in 47% and 49% overall yields and

isotopic purities of 97% and 89%, respectively.

O

(35) 95% step yield, b.p. 91−94 °C

The acetal ester does not trimerize and can be stored for years before hydrolysis and use34. The deuterium-labelled fats have been needed in multigram quantities for studies of the metabolism of configurational and positional fatty acid isomers in humans.

O

(MeO)2 CH(CH2 )2 CH CH(CH2 )2 CH CH(CH2 )2 COOMe

(14)

(MeO)2 CH(CH2 )2 CHDCHD(CH2 )2 CHDCHD(CH2 )2 (CH2 )2 COOMe

O

C(CH2 )2 CHDCHD(CH2 )2 CHDCHD(CH2 )2 (CH2 )2 COOMe

H

(36) 96% step yield

10. Synthesis of [ˇ, ˇ, , , υ, υ, υ-2H7]-n-butylbenzene (39)

39, required in substantial amount as a starting material for preparation of mass spectrometric standards, has been synthesized in a two-step procedure35. In the first step the ˇ, ˇ- and υ, υ, υ-hydrogens of 4-phenyl-2-butanone 40 have been replaced with deuterium atoms by base-catalyzed isotopic exchange of the five labile ˇ- and υ-hydrogens of 40 with excess of D2O MeOD containing a solution of NaOD in D2O under reflux. In the

second step the carbonyl group of the isolated intermediate product 41 has been reduced in THF with zinc dust, and the required amount of DCl was generated in situ by reaction of D2O with trimethylsilyl chloride36.

11. Synthesis of [3,3,-D2]-4-hydroxy-1-(3-pyridyl)-1-butanone, [3,3,-D2]HPB (42)

42 was needed as internal standard for quantification of HPB37 released upon hydrolysis of DNA and globin adducts with metabolically activated (˛-hydroxylated) ‘NNK’, 43, and ‘NNN’, 44, playing an important role in causing cancers of the lung, oral cavity, esophagus and pancreas in people who use tobacco38,39 products (equation 15). Recently, 42 has been produced40 in good yield as shown in equation 16, more efficiently than in the previous low-yield procedure used for production of [4,4-D2]HPB41.

N O

N

N

CH2 OH

HO

N

HBP (42) − unlabelled