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

949

7. Synthesis of methyl [5,6,8,9,14,15-3H6]-hepoxilin B3 (138a and 138b) and methyl [5,6,8,9,14,15-3H6]-hepoxilin A3 (139a and 139b)

The biologically active hepoxilins HxA3, 139a, 139b and HxB3, 138a, 138b, stimulating insulin secretion, enhancing Ca2C transport across membranes, potentiating hormone-induced vascular permeability and contraction, etc.125 127, have been tritium labelled128 to investigate their metabolism both in vitro and in vivo, by selective tritiation of the triacetylenic analog of the HxB3 i.e. 10(R/S)-hydroxy-11(R),12(S)-epoxyeicosa- 5,8,14-triynoate, 137, using larger amounts of Lindlar catalyst (equation 67) to form 138a and 138b. The ratio of two [3H6]-HxB3 epimers 138a : 138b was 65 : 35.

OH

Me(CH2 )4 C CCH2 CH CHCHC CCH2 C C(CH2 )3 COOMe

(137)O

1.100% 3 H2 , Lindlar, quinoline

2.TLC (A gNO3 )

3.HPLC

OH

R1

OH

R1

R2

CH

CHCHC

C

R2

CH

CHCHC

C

O

(R) 3 H

3 H

O (S)

3 H

3 H

(138a)

(138b)

20°C, 10 min

1. PPh3 , DEA D

20°C, 10 min

1. PPh3 , DEA D

PhCOOH / THF

PhCOOH / THF

2. MeONa / MeOH

2. MeONa / MeOH

OPPh3

OPPh3

R2

CH

CHCH

+

1R

R2

CH

CHCH

+

1R

O

(R)

3 H

O

(S)

3 H

(67)

3 H

3 H

−

−

PhCOO

PhCOO

OH

1R

OH

1R

R2

CH

CHCHC

C

R2

CH

CHCHC

C

O

(S)

3 H

O

(R)

3 H

3 H

3 H

OH

3 H

3 H

OH

R2

CH

CHCH

CC

R2

CH

CHCH

CC

O

3 H (R) 1R

O

3 H (S) 1R

1R

1R

3 H

R2

CH

CHCH

CC

OH

R2

CH

CHCH

CC

O

3 H (R) 3 H

O

3 H (S) OH

(139a)

(139b)

Preferred conformations

R1 = −C3 H(CH2 )3 COOMe

R2 = Me(CH2 )4 C3 H=C3 H−

DEAD = diethylazodicarboxylate

950 Mieczysław Ziełinski´ and Marianna Kanska´

138a and 138b have been subsequently transformed into [3H6]-HxA3 methyl esters (139ab) in almost equal ratio by allylic rearrangement129 via the corresponding [3H6]- HxA3 benzoates (equation 67) followed by mild treatment with MeONa in MeOH to hydrolyse the benzoates.

8. Synthesis of (˛S ,Z ,1R,3R)-[40,400 -3H]cyhalothrin, 140a, and -[400,60 -3H]-40- fluorcyhalothrin, 140b

These highly potent insectides, 141 and 140, have been synthesized130 (equations 68 and 69) for identifying and for the studies of the mechanism of pyrethroid-sodium channel interaction131,132. The 3H incorporation was detected by 1H NMR of [3H]-140 and by proton coupled and decoupled 3H-NMR determinations of 140, 140a and 140b. These are suitable radioligands for binding studies with neuronal membranes on the molecular level.

		Br		F
Cl	O
				O
F3 C	O	CN	Br2	- (141)
	K2 CO3 , EtOA c	PtO2 , 2 H2
2 H	F		2 H
	O
	[2 H] - (141)
		R1		R2
H	H
Cl	O
				O
F3 C	O	CN
			EtOH abs.
			T2 , 10% Pd / C
R1	R2		R3
	O

Br

(68)

R3

(69)

(140a)	R1	= H, R2 = R3 = l	[3 H]- (140a)	R1	= H, R2 = R3 = 3 H
(140b)	R1	= R3 = l, R2 = F	[3 H]- (140b)	R1	= R3 = 3 H, R2 = F

17. Syntheses and uses of isotopically labelled compounds				951
9. Synthesis of (C)-N	-(6-ethoxy[2,4-3		H]phenyl)-N -(1,2,2-trimethylpropyl)-2-nitro-
	3
ethene-1,1-diamine ([		H]Ba Yx9228), 142

The novel potassium channel opener, 142, hyperpolarizing the cellular membrane and increasing the efflux of KC (or RbC ) from tissues133 has been tritium labelled134 by dehalogenization of the dibrominated racemic precursor, 143, with Pearlman’s catalyst135 which dehalogenated 143 more rapidly (within two minutes) than it reduced the nitro group (equation 70). BAYx9227 has no potassium channel activity. The tritiated 142 was needed for the search for specific binding sites in different cell types, since the potassium channel openers are considered as the new drugs for hypertension, angina pectoris, asthma and other treatments.

		NO2		Br	Br
		Br2 / CCl4 , FeCl3 / EtOH / H2 O
	NH	NH	5 h, work-up		NH
OEt					OEt
		NEt3 / THF, work-up	T2 , Pd(OH)2 on charcoal		(143)
		T	T
			NH
		OEt			(70)
		(144)
		(enantiomeric separations)	Chiralcel OD column
T	T	NO2	T	T	NO2
		+
	NH	NH		NH	NH
	OEt		OEt
(142)	[3 H]BAYx9228 (+)		[3 H]BaYx9227

sp. activity 47.9 Ci / mmol, enantiomeric purity = 98.3%

10. Synthesis of tritium-labelled thapsigargin (145)

The tumor-promoting 145, activating a broad number of cells by inhibition of endoplasmic calcium ATPase136,137, has been tritium labelled138 (equation 71) by butanoylation of debutanoylthapsigargin, 146, prepared by stereoselective reduction of ketene 147 with sodium boro[3H]hydride. The fluorescent derivative 148 of 145 has also been synthesized

952	Mieczysław Ziełinski´ and Marianna Kanska´

by treating 146 with N-dansyl-ˇ-alanine followed by acetylation, for studies on the distribution of the molecule in the cell138.

		O
Me(CH2 )6		O		O
	H
O		(10)	R
O				O
	(2)	(1)
O		(6)	OH
				OH
O		O
					MeOH
(145) 90% r. purity			O	(n-PrCO)2	O
						O
				Me(CH2 )6	O
					O H
						R
				O		OH
					O	OH
						OH
					O	O
*H = H,2 H or 3 H					(146)	O
				O	R = H2, H or 3 H
						(71)
		O		H2 CrO4
Me(CH2 )6					NaB*H4
		O H
				O
O
	O			OH
				OH

O	O

(147)O

			O
O		N	S
OR	O	R	O
	OR		N

O

O (148)

R = Ac

17. Syntheses and uses of isotopically labelled compounds

953

11. Synthesis of 3H-labelled levamisole (149)

149, of high specific activity, a potent anthelmintic also showing immunotropic properties, has been synthesized139,140 (equation 72).

O

o-BrC6 H4

CCH3

o-BrC6 H4

CCH2 Br

o-BrC6 H4 CCH2 N

O

O

O

O

O

o-BrC6 H4

CHCH2 NH2

.2HCl

o-BrC6 H4 CHCH2 N

NH2

rac.

NH2

rac.

O

NH2

T2 (30 Ci)

Pd 10% on charcoal

NH2 .tartrate

CaO, (i-Pr)2O, RT, 18h

Br

NH2

(S) - (++)

(72)

1. RT 2h

NH2 .2HCl

2. 105 °C, 13 h

T (S) - (+)

S

NH

NaOH / EtOH

CS2 under N2

NH

H

T

aqueous KOH (16%), BrCH2CH2Br, NaHCO3,

2-propanol, 80 °C under N2, 16h

work-up

S

S

N

N

+

N . HCl

N .

HCl

H

H

T

T

(S) - (-) (149) 51% of radioactivity

(S) 47% of radioactivity

954	Mieczysław Ziełinski´ and Marianna Kanska´

12. Synthesis of [ 3H]U-86170, 150, and [ 3H]U-91356, 151

The title compound 150 and a significant by-product 151 a potent dopamine D2 agonist and radioligand, were used in receptor binding studies141 and have been tritium labelled142 (equation 73). 150 has shown good stability. Its radiochemical purity, when stored in MeOH (1 and 10 mCi/ml) at 70 °C for 18 months, declined from 99% to 83%. Partially degraded samples of 150 have been purified by HPLC142.

	N
NH2	R
12 steps
	N
N	HN
	O
(153)	(152)
R = H or	CH2 CH CH2 (9% yield from 153)

				EtOA c		(73)
					(N.C.A .) T2 , 5% Pt / C
		N		T	NH	T
			T	+		T
	N	T			N
HN		O	T		HN
					O
	(150) 73% yield				(151) 22% yield

13. Synthesis of tritium-labelled guanidineacetic acids (156) and tritiated photoaffinity labelling reagent

156 sweeteners, binding to the receptor molecules on the surface of the tongue143 with potencies in excess of 105 times that of sucrose144, have been synthesized144 from isothiourea 155 (equation 74). Ditritioglycine has been used for the synthesis of radioglycine 156a.

									S
(R1C6 H4 )2 CHNH2 + SCNC6 H4 R2 - p						p-R2 C6 H4 NH				NHCH(C6 H4 R1 )2
	SCH3						MeI				NHCH2 COOH
p-R2 C6 H4 N	NHCH(C6 H4 R1 )2			Glycine / NaOH							(74)
									p-R2 C6 H4 N		NHCH(C6 H4 R1 )2
				EtOH / H2 O
	(155)
155 (R1 = Br, R2 = CN)		T2 / Pd / C			154		(R1 = H, R2 = CN)
				156			(R1	= T, R2 = CN)
				157			(R1	= H, R2 = N )
								3
				158			(R1	= T, R2 = N )
								3

17. Syntheses and uses of isotopically labelled compounds

955

NHCT2 COOH

N NH

(156a)

14. Synthesis of [ 3H]t-butyl 8-chloro-5,6-dihydro-5-methyl-6-oxo-4H -imidazo[1,5- a][1,4]benzodiazepine 3-carboxylate (159)

159, [3H]TCIB, has been obtained145 starting with 5-chloroisatoic anhydride 160 and catalytic tritiolysis of 161 (equation 75). 159 is considered a valuable tool to study the structure, function and pharmacological role of DI receptors (‘diazepine insensitive’ subtype of benzoadepine receptor146, BZR).

			O
		Br	N
			O Bu-t
	NH	O
			N
		3 steps
	Cl	O
		Cl	N
	O		O (161)
	(160)
		(NCA ) T2 , (25 Ci), EtOH, 1 h, RT	(75)
		10% Pd / C

O

N

T

O Bu-t

N

Cl

N

O

(159)

15. Biosynthesis of tritium-labelled 3˛-hydroxy-5˛-pregnan-20-one (162)

The title compound, 3˛-hydrohy-5˛-[1,2,6,7-3H]-pregnan-20-one (3H-HPO), 162, has been prepared147 from [1,2,6,7-3H]-progesterone, 163, via 5˛-pregnanedione, 164, using a microsomal preparation from rat liver and NADPH, containing two metabolizing enzymes, 5˛-reductase and 3˛-hydroxysteroidoxidoreductase147 (equation 76). 162 was used to

956	Mieczysław Ziełinski´ and Marianna Kanska´
study the allosteric binding site for HPO in vitro147.
	O	O
	37 °C, 60 min
	TGT-buffer
O	O
	H
(163)	(164)

(76)

O

HO

H

(162)60% yield, purity > 98%

16.Microscale synthesis of norgestrel-[9,11-3H] (165)

165, has been prepared148 from 166 (from norgestrel itself (equation 77)) used for the preparation of 18-methyl-4-estren-[9,11-3H]-3,17-dione (167), which was transformed subsequently into 165 3H2 following equation 78. 165 is an orally active progestational agent149 and 165-3H2 will be applied in highly sensitive radioimmunoassay (RIA) methodologies connected with the development of enhanced formulations148.

	OH	OAc
	4 steps	(77)
O	MeO
		H
(165)		(166)

17. Syntheses and uses of isotopically labelled compounds

957

(166)

1.3 H2 - 10 % Pd / C, EtOA c

2.K2 CO3 / MeOH, reflux

OH

3 H

3 H

Na / NH3

THF / EtOH

MeO	MeO
	H

aq. HCl, MeOH

O

3 H

CrO3 / H2 SO4

3 H

O

O

(167)

(77 continued)

O

3 H

3 H

Pyrrolidine / MeOH

(167)

N

Li-acetylide-EDA comp lex

THF

(78)

OH

3 H

3 H

NaOA c, HOA c / MeOH

4 h reflux

O

(165 - 3 H2 )

958	Mieczysław Ziełinski´ and Marianna Kanska´
17. Synthesis of	tritium-labelled [3-O-(3-hydroxypropyl)]-17˛-estradiol chromium
tricarbonyl (168)

168 has been deuterium or tritium labelled150 by reduction of the corresponding estrone by NaBD4 or NaB[3H]4 (equation 79). The specific activity of 168 (4.1 Ci/mmol) is sufficiently high for hormone receptor binding studies.

O O

NaOH, Br(CH2 )3 OH

HO	HO(CH2 )3 O

Cr(CO)6

O

O

β-Cr(CO)3

(79)

HO(CH2 )3 O

α-Cr(CO)3	HO(CH2 )3 O
12% yield	12% yield
	OH

H (or D, or 3 H)

NaBH4 (or D,3 H), i-propanol

HO(CH2 )3 O

α-Cr(CO)3

(168)94% step yield

18.Synthesis and tissue distribution of N -[2-(hydroxyethoxy)methyl]-5-[3H]methyl- uracil, 3H-169

3H-169 has been synthesized151 for evaluation as tumor diagnostic agent, by converting 5-[3H]-methyluracll, 3H-170, to the 2,4-bis-trimethylsilyl intermediate152, and further to

3H-171 and then to 3H-169 (equation 80). The tissue distribution of 3H-169 has been examined in mice bearing Lewis Lung (LL) carcinomas. The title compound was found to be an unsatisfactory diagnostic agent for LL carcinoma because of low tumor uptake and rapid urinary elimination of injected radioactivity from the body.