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

	17. Syntheses and uses of isotopically labelled compounds						919
S	S				S	S
				O
							O
	+ Br			n-BuLi
				THF
N				O			O
						THF (COOH)2
				1. CD3 OD, MeONa
S	S		O	2. D2 O	S	S
	CD2
		C					O
			H				C
							H
	THF LiA lH4						(16)
				t-BuMe2 SiCl,			Me
					S	S
				4-DMA P, Et3 N
						CD2	OSi-Bu-t
S	S			RT, overnight
	CD2		OH				Me
				MeCN, 0 °C, 1 h work-up
				O	A gNO3 , NCS,

CD2 OH

N

(42) [3,3-D2 ]HPB, 100% D2 by NMR 4-DMPA = 4-dimethylaminopyridine

The NlCl MS (negative ion chemical ionization mass spectrometry) of the pentafluoro-benzoate derivative of 42 has been identical to the corresponding derivative of [4,4-D2]HPB. The levels of HPB released from hemoglobin of NNK-treated rats, determined using [3,3-D2]- and [4,4-D2]-HPB standards, were the same40.

12. Synthesis of 3-[ 2H3methyl-3-buten-1-ol (45)

The use of stable isotopes in biosynthetic studies has increased greatly in recent years42. The title compound, the 3-isopentyl alcohol, IPA, 45, needed for the preparation of the corresponding 3-isopentyl pyrophosphate42, IPP, has been obtained according to equation 17, using perdeuterioacetic acid as the starting material43,44. The product 45

920	Mieczysław Ziełinski´ and Marianna Kanska´

consisted predominantly (95%) of [2H3] species.

C2 H3

1. H + / EtOH

CH2

C2 H3 COO2 H

CHCH2 CCH2 CH

CH2

2. Two equiv. of CH2

CHCH2 Br / Et2 O, THF, Mgo

OH

2. H2 O / A cOH

1. O3 / CH2Cl2 −A cOH

HO

C2 H3

HO

C2 H3

Cinchonine (Ref. 44) / MeOH-Et2 O

(17)

HOOC

COOMe

24 h stirred

O

O

O

overnight stirring

HC(O-neo-C5H11)2 NMe2

/ CHCl3

C2 H3

1a. 1N NaOH / EtOH

C2 H3

1b. acidification with HCl

CH2

CCH2 COOMe

1c. extractions

CH2

CCH2 CH2 OH

2. LiA lH4 / Et2 O, 2 h reflux work-up

(45) IPA; 72% isolated yield

13. Synthesis of 3-hydroxy[1,3-2H2]-2-propenal (46)

The title compound, 1,3-dideuteriated malondialdehyde, MDA-46, formed in a lipid peroxidation process involved in the pathogenesis of many human diseases45,46, has been needed for quantitative determination of MDA in human blood or urine by isotope dilution mass spectrometry. It has been synthesized47 by condensation of deuteriated butyl vinyl ether with deuteriated triethyl orthoformate in the presence of montmorillonite clay K-10 (equation 18).

BuO			BuO			BuO
	1. t-BuLi, −78 °C				1. n-BuLi, −78 °C	C CH2
HC CH2			C CH2
					2. D2 O
	2. Bu3 SnCl
			Bu3 Sn			D
			RT, 2.5 h
				DC(OEt)3 / Et2 O, clay K-10
BuO		OEt				(18)
D C CH2 C		D
EtO		OEt
(47) 75% yield			1. Dowex 50		D	O
2. Titration to pH 7 with aq. solution of NaOH					C CH	CD
				+	−
				Na O

(46) dihydrate sodium salt of malondialdehyde

17. Syntheses and uses of isotopically labelled compounds

921

A comparison of the mass spectra of the deuteriated diacetal 47 and the non-deuteriated analogue (given in parentheses) led to the complete assignment of the observed fragmentation peaks presented in equation 19.

BuO	OEt
D C CH2	C D
EtO	OEt

+	+	BuO	+	EtO	+
BuO	EtO		OH		OH
C OEt	C OEt	C CHC		C	CHC
D	D	D	D	D	D	(19)
m/z 132 (131);	m/z 104 (103);	m/z 131 (129);		m/z 103 (101);

+	+	HO	+
BuO	EtO		OH
C OH	C OH	C	CHC
D	D	D	D
m/z 104 (103)	m/z 76 (75)	m/z 75 (73)

14. Synthesis of multiply deuterium-labelled prednisone and prednisolone

[1,19,19,19-2H4]Prednisone, 48, and [1,19,19,19-2H4]prednisolone, 49, containing four deuterium atoms at chemically stable sites, have been synthesized48 starting from [1,1,19,19,19-2H5]cortisone, 50 (equations 20a, 20b and 20c). No loss of deuterium from the C 19 and C 1 positions has been observed in the course of synthetic sequence, which involved the oxidation of the intermediates 51 and 52 with selenium dioxide in t-butanol.

				HO	O
		O		O	O
		D			O
	D	CD3	H	OH	HCl / HCHO / CHCl3
					RT,15 h

O

H H

(51)

O

(50) cortisone −D5

(20a)

922		Mieczysław Ziełinski´ and Marianna Kanska´
		D
		CD3
(51)	SeO2 /t-BuOH	EtOH / THF / 46% HF
	24 h reflux	0 °C, 90 min
	under argon	2−5 °C, 12 h		HO
	O	polyethylene
		test tube
		(53)		O
		O
		D
		CD3	H	OH
		H	H
		O	(48)

[1,19,19-2 H4 ]-17α, 21-Dihydroxypregna-1,4-diene-3,11,20-trione (prednisone-D4 )

D

CD3

NH2 CONHNH2 • HCl

53

in CH2 Cl2 / MeOH / Py

RT, 40 h

NH2 CONHN

KBH4 / THF / H2 O		(20b)
RT, 16 h, work-up, extractions		O
		O
HO		O
D
CD3	H	O

CH3 COCOOH / H2 O /A cOH

H H

RT, 15 h

NH2 CONHN

(54)

Prednisolone-D4 -BMD

EtOH / THF

HO

46% HF

HO		O
D
CD3	H	OH
H	H
O
	(49) 55% yield

17. Syntheses and uses of isotopically labelled compounds

923

The route 20c has been less satisfactory because of the formation of by-products, especially in the oxidation of 52.

D

D CD3

NH2 CONHNH2 • HCl

51

NH2 CONHN

HO

D

KBH4

D CD3 H

SeO2	deprotection
H	54
	at C(3 ) and C(17)

NH2 CONHN

(52)

(20c)

49 + by-products

48 and 49 with 2H-label in chemically and biologically stable C 19 and C 1 positions are suitable for use in stable isotope methodology (coupled with GC-MS49,50) of investigations on steroid hormones in humans51.

15. Synthesis of deuteriumand tritium-labelled (3-xenyl)cyclohexane

The use of PCBs (polychlorinated biphenyls), organic heating medium causing skin affliction in humans52 and producing non-metastasizing neoplastic liver nodules in rats and mice52, is prohibited53 in Japan. Hydrogenated terphenyls (HTPS) are used as a substitute. (3-Xenyl)cyclohexane, known to be a major component of the HTPs, has been tritium and deuterium labelled54 to study its metabolic fate in living organisms by Clemmensen reduction of 2-(3-xenyl)cyclohexanone, 55, and by Wolf Kishner reduction of 55.

(a) 2-(3-xenyl)cyclohexanone-2,6,6-[3H3], 55, has been prepared by deuterium exchange catalysed by 40% NaOD in dry benzene and MeOD (equation 21).

		D
Ph	Ph		CD2
	D2 O, 40% NaOD, benzene/MeOD		O
O	2 days reflux
(56)		(55)	(21)

(b) The deuteriated product 57 has been obtained by Clemmensen reduction of 55 (equation 22).

The deuteriated product 58 has been prepared as shown in equation 23 using concentrated DCl in D2O.

924		Mieczysław Ziełinski´ and Marianna Kanska´
				D
			Ph	C(D)x	(22)
	A malgamated Zn, toluene, conc. HCl
55
		15 h reflux, separations
				(57)
				D
		Ph		CD2
	A malgamated Zn, DCl, D2 O			C	(23)

55

heat

(D)x

(58)

The deuterium labelling has been achieved also by Wolf Kishner reduction of 2-(3- xenyl)cyclohexanone. The hydrazone of 56 dissolved in diethylene glycol containing D2O was heated first at 120 °C for 15 min and, after addition of KOH, heated at 230 °C for an additional two hours. The product 59 has been identified by the mass spectrum as the predominant constituent.

Ph

C

D (D)

(59)

The tritium-labelled 1-(3-xenyl)cyclohexane-2-[3H], 60, has been obtained similarly (in 84.3% chemical yield and 0.89% radiochemical yield based on the total radioactivity of 3H2O used); see equation 24.

	80% hydrazine hydrate		1. Diethylene glycol,		Ph
			3 H2 O (80 mCi), 120	°C, 15 min
56	abs. EtOH, dry benzene	oily product				1H
	1 hour reflux	hydrazone	2. KOH pellets, 230 °C, 2 h
						3 H
			3. Work-up

(60)

(24)

The Clemmensen reduction was accompanied by D/H exchange via acid-catalysed enolization of the carbonyl group, resulting in the production of deuteriated compounds 57 and 58 with various numbers of deuterium atoms. The mixture of the compound 59 obtained by the Wolf Kishner reduction was predominantly labelled at position 2. This has been proved by the 13C-NMR spectrum. Isotope shift and loss intensivity on substituted C 2 carbon signals have been observed54,55.

17. Syntheses and uses of isotopically labelled compounds

925

B. Compounds Labelled with Carbon-13

1. Synthesis of 13C-labelled verapamil compounds

Verapamil, a slow calcium channel antagonist used in treatment of angina, hypertension and superventricular tachycardia56,57, labelled with 13C in the 1-position of the phenethyl side chain, 61, and with 13C-labels in all four O-methyl groups, 62, and also containing 50% of 13C in the N-methyl group, 62, has been prepared58 in multistep syntheses which involved, in the case of the synthesis of 61, the displacement of mesylate 63 with N- methyl-2-(3,4-dimethoxyphenyl)-1-[13C]-ethyl amine, 64 (equation 25).

MeO			OCH3
	CHMe2
MeO	C(CH2 )3 N 13 CH2 CH2		OCH3
	CN	Me
		(61)
13 CH3 O			O13 CH3
	CHMe2

13 CH3 O	C(CH2 )3 NCH2 CH2
	CN	R

(62) R = 12 CH3 and 13 CH3

CHMe2

64

Ar-C(CH2 )3 OMs 61

CN

(63)

Synthesis of 62 commenced from verapamil hydrochloride, demethylated by heating in aqueous 48% HBr (equation 26).

HO

−

CN MeBr

O13 CH3

(25)

which has been O-

OH

Verapamil	48% HBr	HO		C(CH2 )3 NHCH+ 2 CH2		OH
				CHMe2
			acetone			(26)
			13 CH3 I, K2 CO3
13 CH3 O		HCMe2	−	O13 CH3
			CH3 Br
13 CH3 O		+		O13 CH3	EtSH	62
		C(CH2 )3	N CH2 CH2		HMPA

CN 13 CH3

926 Mieczysław Ziełinski´ and Marianna Kanska´

2. Synthesis of L-[4-13C] and L-[3,4-13C2]aspartic acid

Biosynthetically prepared, isotopically labelled serine has been N-protected by conversion to the L-N-(t-Boc)-[3-13C]serine, 65, which has been cyclized by treatment with Ph3P and dimethyl azodicarboxylate (DMAD) at 78 °C to yield the ˇ-lacton, 66, The solution of 66 and dialkyl azodicarboxylate in DMSO added to a solution of Na13CN in DMSO gave L-ˇ-[cyano, 3-13C2]cyanoalanine, 67. Acid hydrolysis of 67, provided L- [3,4-13C2]aspartic acid, 68 (equation 27), in 96% enantiomeric excess and 13.3% overall yield based on 65. Similarly, L-[4-13C]aspartic acid has been prepared from L-serine and K13CN. L-Configuration of the labelled amino acid has been required for studies of amino acid metabolism and for studies of peptide and protein structure and dynamics58,59.

H

O

H

C

H

N Boc

O

H

N

H

N

Boc

C

KCN

C

C

HO

C

C

COO−

NHBoc

C COO−

C

H

H

H

H

H

H

(65)

(66)

(67)

(27)

+

COO−

6M HCl

H3 N

C H

CH2

COO−

(68)

3. Synthesis of ˛-13C-glycine (69)

Gram quantities of 69 have been prepared60 from K13CN as shown in equation 28 and its incorporation into thioredoxin studied by heteronuclear NMR60.

S

NaOCl, NaOH

Ph

N

C

N

Ph

Ph

NHCNH

Ph

(71)

+

−

K13 CN, N (CH2 CH3 )3 H Br

(28)

13 CN

13 CH2 NH2

LiA lH4

Ba(OH)2

NH

13 CH COOH

Ph

N C NH Ph

Ph

N

C

NH

Ph

2

2

(70)

(69)

69 had been synthesized previously61 from the intermediate 70 (equation 29).

PbO, K13CN

71 ! 70

29

17. Syntheses and uses of isotopically labelled compounds

927

4.Synthesis of υ-aminolevulinic acid (ALA) labelled with 13C

(a)Synthesis of [5-13C]ALA, 72, has been carried out62 as shown in equation 30.

O

K CN CuSO4 , 0.5 N NaOH

Cu CN

EtOOCCH2 CH2 COCl

EtOOC(CH2 )2 C CN

sodium sulphite

MeCN, reflux 30 min

+

Zn/A cOH, A c2 O

ultrasonic waves

O

40 ° C, 90 min

O (30)

6N HCl or DCl

EtOOCCH2 CH2 C CH2 NH2

.HCl

EtOOC(CH2 )2 C CNHAc

2 h reflux

(72)

98.8% yield

13

1

H or

2

H

145 °C

C = C;

H =

m.p.

(b) [1-13C]ALA, 73, has been synthesized62 as indicated in equation 31. [2-13C]ALA, 74, has been synthesized similarly starting with [2-13C]sodium acetate. [3-13C]ALA, 75 and [4-13C]ALA, 76, have been synthesized also according to equation 31 but using [2- 13C]ethyl phthalimidoacetoacetate, 77, and [3-13C]ethyl phthalimidoacetatoacetate, 78, as reactants.

CH3 13 COONa		1. Br2 , benzyl bromide			BrCH2	13 COOEt
		2. Dry EtOH
					59% yield
		3. Work-up
NaH/MeOCH2 CH2 OMe, RT, 2 days					O
					O
					NCH2 CCH2 COOEt					(31)
O					O
O		CH2 13 COOEt					O
NCH2 CCH				A cOH / conc. HCl. 1 : 1						13 COOH
							H2 NCH2		CCH2 CH2
		COOEt			overnight
O							(73) 71% yield
74% yield

O

O

NCH2 C(3 ) C(2 )H2 C(1)OOEt

O

(77)*C(3 ) = 13 C(3 )

(78)*C(2 ) = 13 C(2 )

928	Mieczysław Ziełinski´ and Marianna Kanska´
(c) The	enzymatic transformation of the obtained 13C-labelled ALA to 13C-

porphobilinogen (PBG), 79, has been investigated62 by direct observation of 13C-NMR of the 13C-labelled PBG (the intermediate in biosynthesis of heme) without chemical degradation. Formation of PBG in vivo from two molecules of ALA is catalysed by ALA dehydratase, obtained from human erythrocytes or rat liver63 (equations 32a and 32b).

	COOH					COOH
					COOH
			A LA - D
O						(32a)
H2 N						NH
				NH2
						* = 13 C
[5-13					(79)
	C]ALA					PBG
	COOH					COOH
					COOH
			A LA - D
O						(32b)
H2 N						NH
					NH2
= 13 C
[3-13	C]ALA				(79)	PBG

5. Synthesis of n-butyl [3-13C]acrylate (80)

80 has been obtained64,65 by methylation of butyl acetoacetate, subsequent bromination, alkaline cleavage and dehydrobromination with cyclohexyldimethylamine (CDA), (equation 33). 80 has been required for studies of its metabolism in small animals.

			*							*
					CH3						CH3
*
	CH3 I					1. NaH / THF
MeCOCH2 COOBu	NaH / THF	MeCOCHCOOBu				2. Br	/ CH Cl			MeCOCBrCOOBu
						2	2	2
								0 °C, 1 h; RT, 1 h			Ba(OH2 ) / t-BuOH
*							CDA			*
		CH2		CHCOOBu						CH3 CHBrCOOBu
						190−200 °C, 3 h

C* =13 C

(80) 98% purity,

75.6%13 C enriched by MS

(33)