Supplement A3: The Chemistry of Double-Bonded Functional Groups. Edited by Saul Patai Copyright 1997 John Wiley & Sons, Ltd.
ISBN: 0-471-95956-1
CHAPTER 17
Syntheses and uses of isotopically labelled compounds containing C=C, C=O or C=N groups
´
MIECZYSŁAW ZIEŁINSKI
Isotope Laboratory, Faculty of Chemistry, Jagiellonian University, Cracow, Poland Fax: 48-12-34-05-15
and
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MARIANNA KANSKA |
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Department of Chemistry, Warsaw University, Warsaw, Poland |
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Fax: (48)-(22)-225996; e-mail: mkanska@chem.uw.edu.pl |
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I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . |
910 |
II. SYNTHESES AND USES OF COMPOUNDS CONTAINING CDC, CDO |
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OR CDN GROUPS LABELLED WITH STABLE ISOTOPES . . . . . . . |
910 |
A. Compounds Labelled with Deuterium . . . . . . . . . . . . . . . . . . . . . |
910 |
B. Compounds Labelled with Carbon-13 . . . . . . . . . . . . . . . . . . . . . |
925 |
C. Compounds Labelled with Nitrogen-15 . . . . . . . . . . . . . . . . . . . . |
939 |
III. SYNTHESIS AND USES OF COMPOUNDS CONTAINING CDC, CDO |
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OR CN GROUPS LABELLED WITH TRITIUM . . . . . . . . . . . . . . . |
943 |
A. Compounds Labelled with Tritium . . . . . . . . . . . . . . . . . . . . . . . |
943 |
B. Double-labelled Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . |
959 |
IV. SYNTHESES AND USES OF COMPOUNDS CONTAINING CDC, CDO |
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OR CN GROUPS LABELLED WITH RADIOACTIVE CARBON . . . . |
964 |
A. Compounds Labelled with Carbon-11 . . . . . . . . . . . . . . . . . . . . . |
964 |
B. Compounds Labelled with Carbon-14 . . . . . . . . . . . . . . . . . . . . . |
980 |
V. SYNTHESES AND USES OF COMPOUNDS CONTAINING CDC, CDO |
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OR CN GROUPS LABELLED WITH RADIOACTIVE HALOGEN . . |
997 |
A. Compounds Labelled with Fluorine-18 . . . . . . . . . . . . . . . . . . . . |
997 |
B. Compounds Labelled with Heavier Radiohalogens . . . . . . . . . . . . |
1004 |
909
910 |
Mieczysław Ziełinski´ and Marianna Kanska´ |
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
RCH2 Cl |
KCN |
LiA lH4 |
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RC2 H2 CN |
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C2 HCl3 , 18-crown-6, 24−72 h reflux |
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or LiA l2 H4 |
RC2 H2 C2 H2 NH2 |
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R = Ph-, m- and p-benzyloxyphenyl-, or 3-n-indolyl |
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(1) |
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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.
17. Syntheses and uses of isotopically labelled compounds |
911 |
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.
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Na |
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CD2 (CN)2 , (2) |
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EtO |
CN |
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CDCl3 |
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C2 H6 OD |
DC(OEt)3 |
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C |
C |
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(2) |
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12 h |
A c2O, reflux |
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D |
CN |
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(1) |
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(3) |
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NH2 |
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CN |
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N |
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Me |
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(4) |
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NH2 |
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NH2 |
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Me |
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N |
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N |
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SO3 |
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CH2 OH |
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N |
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Me |
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(5) |
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(6) |
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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
912 |
Mieczysław Ziełinski´ and Marianna Kanska´ |
constituent of Cannabis, is the major urinary metabolite of 1-tetrahydrocannabinol ( 1- THC) in man13,14. The carbon-14 labelled terpene synthon has been obtained12 by using carbon-14 labelled methyl iodide in the conversion of the keto function in 13 to the tertiary alcohol in compound 14. Compound 11 is suitable as internal standard for MS assays.
O
OH(β)
H(α)
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5% Pd/C, D2 |
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DIBA L/toluene |
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O |
MeOH |
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0 °C, 1 h |
RT, 1 h |
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O |
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(8) |
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0 °C |
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RT, 16 h under N2, work-up |
Py, NaH/HCOOMe |
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OH |
N |
NH2 OH.HCl / NaA cO / H2 O / A cOH |
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RT, 16 h |
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HO |
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RT, 16 h |
m-CPBA /CHCl3 |
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D |
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OH |
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1. KOH / THF / H2 O, RT, 16 h |
NC |
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2. conc. HCl / H2 O, 50 min |
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3. Work-up |
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D |
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m-CPBA = 3-chloroperbenzoic acid |
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(7) 42% yield, m.p. 255.7−256.7 °C |
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OSiMe3 |
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OSiMe3 |
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90 °C, 4 h |
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OMe |
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(13a) (13b)
1.C[2 H3 ]MgI/Et2 O, 40 °C, 20 min
2.Work-up
(4)
(5)
Me |
C[2 H3 ] |
+ Me |
C[2 H3 ] |
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17. Syntheses and uses of isotopically labelled compounds |
913 |
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O |
O |
(14a) + |
(14b) |
1. 1% CCl3 COOH/H2 O |
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2. Work-up, separation |
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S |
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Me |
C[2 H3 ] Me |
C[2 H3 ] |
OH |
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S
1. Li |
/ THF (Ref. 17) |
S
−45 °C 4 °C, overnight
+
2.H2 O, work-up, separations purification, silica gel, chrom.
Me |
C[2 H3 ] |
Me |
C[2 H3 ] |
(5 continued) |
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OH |
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OH |
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(10b) |
(10a) |
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40% yield, m.p. 116.6−117.2 °C |
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HO |
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1. |
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COOH |
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H)4 C[ H3 |
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(12) |
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benzene, MeSO3 H catal., 40 °C, work-up
2.A c2 O / Py
3.HgO /Et2 O.BF3 /aq. THF (Ref. 16)
4.NaCN / A cOH / MnO2 / neutral pH
5.KOH / H2 O / EtOH hydrolysis of acetylated ∆1-THC-7-oic acid
Me |
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(CH2 H)4 C[2 H3 ] |
[2 H3 ]C |
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−+ -(11), |
5−10% overall yield |
5. Synthesis of deuterium-labelled hexenols
Hexenols are important flavour components of fruit and vegetables. Syntheses of cis- 3-hexen-1-ol-6,6,6-2H3, 15, hexan-1-ol-6,6,6-2H3, 16, cis-2-hexen-1-ol-6,6,6-2H3 17, and trans-2-hexen-1-ol-6,6,6-2H3, 18, have been accomplished18 in order to incorporate these precursors into the biochemical system of living fruit tissue (to study the complex multienzyme systems of their production and metabolism). Deuteriated cis-hexenol 15 and hexanol 16 have been obtained18 20 by alkylation of (THF)ether of 3-butyn-1-ol, 19, with 1-bromoethane-2,2,2-2H3 followed by partial hydrogenation of alkynol 20 leading to 15, or by hydrogenation of 20 using 5% palladium on charcoal giving product 16
914 |
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Mieczysław Ziełinski´ and Marianna Kanska´ |
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(equations 6a and 6b). |
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HC |
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CCH2 CH2 OTHP |
1. LiNH2 , liquid NH3 |
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CD3 CH2 C |
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CCH2 CH2 OH |
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2. CD3 CH2 Br, −35 °C, overnight |
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(19) |
3. MeOH/Amberlite (THP removal) |
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(20) |
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(6a) |
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H2 /Lindlar |
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CD3 CH2 CH |
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(15) 71% |
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Pd/charcoal/pentene/Et2O/H2,RT(overnight) |
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20 ! CD3(CH2)4CH2OH |
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(16) 40% |
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Cis-17 and trans-18 hexenols have been prepared in similar fashion from the acetylenic alcohol 21 (equations 7a and 7b),
HC |
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CCH2 OTHP |
1. LiNH2 , liq. NH3 |
CD3 (CH2 )2 C |
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CCH2 OH |
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2. CD3 CH2 CH2 Br |
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3. MeOH / Amberlite |
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H2 / Lindlar |
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OH |
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CD3 |
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(17) 70% yield |
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22 in Et2 O |
Na / liq. NH3 |
CD3 |
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− 35 |
°C, 14 h |
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(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
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CD3 CH2 CH2 MgBr |
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SiMe3 O |
O |
Et2 O, −78 °C, RT, 30 min |
SiMe3 O |
OMgBr |
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CD3
1. N HCl, work-up, separation
CD3 O
(23)
SiMe3 O OMgBr
(25)
17. Syntheses and uses of isotopically labelled compounds |
915 |
ethereal solution of 3-trimethylsiloxy-2-propenal, 24, prepared in situ (equation 9). The side products, RCHDCHCH(OH)R and RCHDCHCHR(OSiMe3), R D n-Pr, Ph, isolated in test experiments, are consistent with the 1,4-addition of Grignard reagent to 24 and formation of the intermediate 25 prior to hydrolysis. 24 has been obtained by silylation of potassium malondialdehyde24 with Me3SiCl in the presence of catalytic amounts of N, N-dimethylaminopyridine (equation 9).
[SiMe3OCH(R)CHDCHOMgBr]
25
(RO)2CHCH2CH(OR)2
R D Me (68% yield) R D Et (75% yield)
1. 2 N HCl, 50 |
°C |
C |
TMSCl, Et |
N (0.1 eq) |
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! KOCHDCHCHDO ! 24 (9)
2. 5 N KOH |
DMAP (cat.), Et2O, RT |
7. Synthesis of D35-1-dodecylhexahydro-2H-azepin-2-one (26)
The title compound, perdeuterioazone, 26, a dermal penetration enhancer25 increasing the passage of the wide range of molecules through the skin, has been synthesized26 in 45% yield by the base-catalyzed coupling of D11-hexahydro-2H-azepin-2-one, 27, with D25-1-chlorododecane, 28 (equation 10), in one step. Comparison of the mass spectra of 27 and 26 and use of the mass calculation program showed that 26 contains more than 98% atom% D. 26 will be applied to further investigate the mechanism of action of azone by use of Fourier transform IR spectroscopy27 and neutron reflectometry28.
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K2 CrO7 , D2 SO4 , D2 O |
D2 C |
CD2 |
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CD2 |
ether, 30 min, 0−5 °C |
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CD2 |
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1. 125-130 °C |
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2. 36 ° C, neutralization with |
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28% NH3 aq., separation |
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D2 C |
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(10) |
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D2 C |
CD2 |
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D2 C |
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1. NaH, o-xylene, 15-crown-5, 10 h reflux |
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D2 C |
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2. C1 2 D2 5Cl, 28 in o-xylene, 6 h reflux |
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3. Work-up |
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(27) |
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O
CD2 (CD2 )1 0 CD3
N
D2 C
CD2
D2 C
CD2 CD2
(26) b.p. 170 °C/0.06 mm Hg
916 |
Mieczysław Ziełinski´ and Marianna Kanska´ |
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8. Synthesis of (š)[2-2H2]mevalonolactone (29) and (š)[2-2H2]homomevalonol- |
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acetone (30) |
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(a) 29 has been |
prepared29,30 in 77% yield by condensation |
of lithiotrimethylsilyl |
[2-2H2]acetate generated by action of LDA with 1-trimethylsilyloxy-3-butanone, 31
(equation 11). The regiospecificity of the |
labelling retention of 2H in |
29 was |
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than 95%. |
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R |
OH |
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CD3 COOD N-TMS imidazole CD3 COOTMS |
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1. LDA , −78 ° C |
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2. 1-trimethylsilyloxy-3-butanone (31) |
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(32) |
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Me3 Si N |
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(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.
Et |
CH2 COOEt |
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Et |
CH2 CH2 OH |
O |
O |
LiAlH4 |
O |
O |
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(12) |
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supported on silica gel |
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p-toluenesulphonic acid |
O |
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O |
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N-TMS - imidazole |
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EtCCH2 CH2 OTMS |
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EtCCH2 CH2 OH |
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(34) |
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(33) 93% yield |
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
17. Syntheses and uses of isotopically labelled compounds |
917 |
isotopic purities of 97% and 89%, respectively.
O
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1. O3 |
/ A cOH |
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C(CH2 )2 CH |
CH(CH2 )2 COOH |
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2. A cONa, A c2 O |
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H |
(37) |
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MeOH / HC(OMe)3 / HCl |
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(MeO)2 CH(CH2 )2 CH |
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CH(CH2 )2 COOMe |
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D2 , benzene |
(13) |
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(Ph3 P)3 RhCl |
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(MeO)2 CH(CH2 )2 CHDCHD(CH2 )2 COOMe |
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H2 O / MeCN, HCl |
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O |
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C(CH2 )2 CHDCHD(CH2 )2 COOMe |
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H |
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(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
as in equation 13 |
C(CH2 )2 CH |
CH(CH2 )2 CH CH(CH2 )2 COOH |
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H |
(38) |
(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
918 |
Mieczysław Ziełinski´ and Marianna Kanska´ |
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.
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O |
O |
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PhCH2 CD2 CD2 CD3 |
PhCH2 CH2 |
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PhCH2 CD2 |
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CCH3 |
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CCD3 |
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(39) |
(40) |
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(41) |
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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.
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O |
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N |
O |
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(CH2 )3 N |
N |
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Me |
N |
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N O |
(43) NNK |
N |
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(44) NNN |
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N O
N
N
CH2 OH
HO
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N O |
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(15) |
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O |
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(CH2 )3 N NOH |
N |
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O |
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(CH2 )3 OH |
[DNA and globin adducts] |
H + or −OH |
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N
HBP (42) − unlabelled