Synthesis of [14C] acids from their unlabeled counterparts and application toward a novel [14C] labeled hepatitis C virus polymerase inhibitor

Article abstract of DOI:10.1002/jlcr.1762

A strategy has been developed for the rapid construction of a [ ¹⁴C] acid from either an aromatic or an alkyl acid via a decarboxylation-carboxylation sequence. A [¹⁴C] labeled heptatitis C virus polymerase inhibitor was prepared. Other examples and the limitations of this methodology are discussed. Copyright

Full text of DOI:10.1002/jlcr.1762

Abstracts
Published online 12 May 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1762
10th International Symposium on the
Synthesis and Applications of Isotopes and
Isotopically Labelled Compounds—Synthesis
of Compounds Labelled with Long-lived
Isotopes
Session 1: Monday, June 15, 2009
SESSION CHAIRS: DAVID HESKa AND VOLKER DERDAUb
aSchering-Plough, USA
bSanofi-Aventis, Germany
Abstract: This session outlined methods useful for the synthesis of intermediates labeled with carbon-13, carbon-14 or tritium.
Additional papers described the utilization of carbon-14 and tritium intermediates in the synthesis of labelled compounds for use in
the development of pharmaceutical and agricultural agents.
Keywords: 13C; 14C; Homogeneous metal-catalyzed exchange; tritium; vicarious nucleophilic substitution; electrophilic cyanating
reagent; labelling methods
HOMOGENEOUS METAL CATALYSED EXCHANGE LABELLING OF PHARMACEUTICALS WITH TRITIATED
WATER
D. HESK, P. MCNAMARA, D. KOHARSKI, K. VORONIN AND V. TRUONG
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Abstract: Low specific activity tritiated water in conjunction with homogeneous catalysts of rhodium, ruthenium and iridium has
3
been extensively used by the Radiochemistry group at Schering-Plough to rapidly prepare H labelled compounds for preliminary
drug metabolism work. Such studies are used to aid in the selection of drug candidates for further evaluation and development.
Through several examples, the paper will discuss the use of homogeneous metal catalysed exchange with tritiated water to label a
wide range of pharmaceutical structures.
Keywords: tritium; rhodium trichloride; tris-triphenylphosphine ruthenium (II) Chloride
Introduction: Tritium labelled compounds are extensively prepared by the Radiochemistry group at Schering-Plough for exploratory
metabolism work. A wide range of labelling strategies with both tritium gas and tritiated water are used by the group. Of these
methods, homogeneous metal catalysed exchange with tritiated water has proven to be one of the more widely applied techniques,
with two catalyst groups in particular used the most frequently. Rhodium trichloride trihydrate in conjunction with tritiated water is
well known to catalyze ortho hydrogen isotope exchange in aromatic acids, amides, aralkylamines and acetanilides (Figure 1).1
Figure 1. RhCl3 H2O catalyzed exchange with tritiated water.
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
Over more recent years, RuAcAc3, IrCOD(AcAc) and Ir COD(F6AcAc) have been developed as an improvement to the rhodium
trichloride system.2–4 Tris-triphenylphosphine ruthenium (II) chloride, while able to promote ortho exchange in aromatic acids, has
found an application as a promoter of hydrogen isotope exchange in alcohols and is particularly useful in amine systems (Figure 2).5,6
Figure 2. Ru(Ph3P)3Cl2 catalyzed exchange with tritiated water.
The late Professor J.R. Jones, to whom this paper is dedicated, spent many years researching this field. As a tribute to John, we
would like to illustrate though a series of examples how this chemistry has proved to be an invaluable tool in the preparation of 3H-
labelled pharmaceuticals.
Results and Discussion: The first two examples SCH A and SCH B (Figure 3) involved the use of rhodium trichloride hydrate to
label a-ethylbenzylamine and p-chlorobenzylamine as precursors.
Figure 3. [3H]SCH A and [3H]SCH B.
The resulting 3H labelled amines, which were purified by a simple extractive work-up and required no additional purification, were
3
then converted to the target compounds using the established medicinal chemistry routes. H NMR analysis showed the expected
sites of tritium incorporation.
SCH C (Figure 4) was labelled by rhodium trichloride hydrate with tritiated water on the free pyridine, followed by a rather
laborious oxidation process to generate the pyridine N-Oxide.
In the case of SCH D (Figure 4), after a pilot experiment with Ir(COD)dppePF76 and deuterium gas failed, presumably due to cyanide
poisoning of the catalyst, the acid intermediate was labelled using RuAcAc3 and tritiated water, which had been previously shown to
be a more effective promoter of exchange in aromatic acids than the rhodium trichloride system.2 The isolated acid was converted
to the desired product in two additional steps.
Figure 4. [3H]SCH C and [3H]SCH D.
Initial attempts to label SCH E involved Pt catalysed exchange with tritiated water, which gave a very poor yield. Treatment with
nBuLi followed by quenching with tritiated water while very effective in other structurally related analogs, resulted only in
decomposition products.8 Hence SCH E (Figure 5) was prepared by rhodium trichloride hydrate catalysed exchange of the
a-methylbenzylamine precursor. The resulting hplc purified amine was treated with triflic anhydride to generate the target
compound in respectable yield.
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Abstracts
Figure 5. [3H]SCH E.
SCH F (Figure 6) was prepared via reaction with IrCODAcAc and tritiated water with the intention of utilizing the urea and amide
functionalities to label the respective phenyl and pyridine rings in the ortho positions.
Figure 6. [3H]SCH F.
While a 3H labelled batch was isolated, 3H NMR analysis showed that virtually all of the tritium was incorporated in the urea N-methyl
group with a small amount ortho to the pyridine nitrogen. Such incorporation has been previously seen in Ir(I) catalysis with deuterium
gas.9 In the case of the structurally similar SCH G (Figure 7) a request was made for the label to be incorporated on both sides of the urea.
Figure 7. [3H]SCH G.
Hence the aminopyrazine was labelled via rhodium trichloride hydrate in the 3,5-difluorophenyl ring and subsequently converted
to the phenylcarbamate. The piperidine moiety was labelled by reacting benzoyl-N-methylpiperidine with Ru(Ph3P)3Cl2 and tritiated
3
water. After de-protection and acetylation it was coupled with the phenylcarbamate to generate the target compound. H NMR
showed tritium was largely incorporated in the expected sites with a small amount in the urea methyl group, which may be due to a
Ru complex formed between the carbamate carbonyl and the N-methyl group during the labelling step.
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
SCH H (Figure 8) was prepared via Ru(Ph3P)3Cl2 catalysed exchange of the piperidine precursor, followed by a standard coupling
3
of the pyrimidine acid. H NMR showed the expected labelling pattern. The same methodology was used for examples I to M.
Figure 8. [3H]SCH H.
In the case of SCH I (Figure 9), which was prepared via coupling of the 3H labelled piperazine with the chloro biaryl under
Finkelstein conditions, the 3H NMR showed that the presence of an axial t-butyl group, hindered the incorporation of the adjacent
equitorial position.
Figure 9. [3H]SCH I.
In the related SCH J (Figure 10), which was labelled via the piperazine containing an isopropyl group adjacent to the active NH,
the 3H NMR analysis showed that in addition to the expected hindrance of the opposite axial site, a substantial amount of label was
incorporated in the isopropyl methyl groups.
Figure 10. [3H]SCH J.
It is possible that a competing cyclometallation between the methyl group and the piperazine NH is responsible and is worthy of
further investigation. Interestingly for SCH K (Figure 11), and in contrast to SCH I, the axial methyl group had no effect on the axial/
equitorial distribution in the alpha positions.
Figure 11. [3H]SCH K.
Additional small amounts of tritium were also incorporated in the equitorial beta positions and the methyl group. SCH L (Figure
12) and SCH M (Figure 12) were labelled via the respective piperidine and piperazine intermediates and converted to the target
compounds in two steps and one step respectively.
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
Figure 12. [3H]SCH L and [3H]SCH M.
In the case of SCH L, the imidazole was protected with a trityl group during the labelling step and removed in the final step after
3
the coupling. H NMR analysis showed the expected distribution of tritium.
Acknowledgments: The authors would like to thank Dr. T.M. Chan and Dr. M. Senior from the Schering-Plough Research Institute
molecular spectroscopy group for the 3H NMR analysis. Thanks are also due to Dr. S. Saluja and Miss S. Hendershot from the
Radiochemistry Analytical group and also to SPRI medicinal chemistry for starting materials and procedures.
References
[1] D. Hesk, J. R. Jones, W. J. Lockley, J. Label. Compd. Radiopharm., 1990, 28, 1427–1436.
[2] W. J. Lockley. J, Label. Compd. Radiopharm., 1984, 21, 45–57.
[3] L. Kingston, W. J. Lockley, A. Mather, E. Spink, S. Thompson, D. Wilkinson, Tet. Lett. 2000, 41, 2705–2708.
[4] B. McAuley, M. Hickey, L. Kingston, J. R. Jones, W. Lockley, A. Mather, E. Spink, S. Thompson, D. Wilkinson, J. Label. Compd.
Radiopharm., 2003, 46, 1191–1204.
[5] E. Alexakis, M. Hickey, J. R. Jones, L. Kingston, W. Lockley, A. Mather, T. Smith, D. Wilkinson, Tet. Lett. 2005, 46, 4291–4293.
[6] D. Hesk, D. Cesarz, C. Magatti, K. Voronin, C. Lavey, P. McNamara, D. Koharski, S. Saluja, S. Hendershot, H. Pham, and V. Truong,
J. Label. Compd. Radiopharm., 2005, 48, 11-23.
[7] A. Shu, W. Chen, and J. R. Heys, J. Organometal. Chem., 1996, 524, 87-93.
[8] C. Lavey, D. Hesk, S. Hendershot, D. Koharski, S. Saluja, and P. McNamara, J. Label. Compd. Radiopharm., 2007, 50, 264–272.
[9] R. Salter, M. Chappelle, A. Morgan, T. Moenius, P. Ackermann, M. Studer, and F. Spindler, in ‘Synthesis and Applications of
Isotopically Labeled Compounds’, Vol. 7, Pleiss, U, and Voges R., Eds., Wiley, Chichester, 2001, 63–67.
SYNTHESIS OF LABELLED TERMINAL [14C]-ACETYLENES
¨
VICTOR MURRELL, CHARLES-HENRY FABRITIUS, DAVID SPEED, MICKAEL DENANCE, NATHALIE L’HELIAS,
´
´
HELENA MCALONAN, GUILLAUME PELE, LOIC LEMAN, AUSTIN MCMORDIE AND WILLIAM WATTERS
¨
Almac Sciences, Almac House, 20 Seagoe Industrial Estate, Craigavon, BT63 5QD, Northern Ireland
Abstract: There are several literature methods for conversion of 14C precursors into [14C]-acetylenes, and a number of these involve
elaboration of [14C]-aldehydes into a terminal acetylenes. One method that involves mild conditions, and in our experience is high
yielding and tolerant of a range of functional groups uses the Ohira-Bestmann reagent to effect this transformation. This document
briefly describes some of the different methods that have been used for the preparation of [14C]-acetylenes and discusses two
worked examples, the synthesis of [5-14C]-hex-5-ynoic acid and [benzene-(U)-14C]-7-methoxybenzofuran, both of which involve
Ohira-Bestmann chemistry being used for the preparation of terminal [14C]-acetylenes.
Keywords: terminal acetylene; Corey-Fuchs; Ohira-Bestmann; 7-methoxybenzofuran; hex-5-ynoic acid
Introduction: Acetylenes are versatile synthetic intermediates that allow access to a wide range of linear, cyclic and heterocyclic
molecules. It would be very useful, therefore, to be able to prepare radiolabelled acetylenes conveniently from readily available 14C
precursors, and there are various approaches to doing this that can be applied to carbon-14 radiosynthesis. One approach is to use
[(U)-14C]-metal acetylides, and while these are accessible, useful and permit higher specific activities in products than singly labelled
analogues, [(U)-14C]-metal acetylides are intolerant of many functional groups and can be difficult to handle. One alternative that has been
applied to preparation of labelled acetylenes is based upon Corey-Fuchs methodology1 (Scheme 1). [14C]-Aldehydes can be readily
prepared in a number of ways and are commonly made by reduction of [14C]-nitriles or by addition of an organometalic to [14C]-DMF.
H
14C
O
14C
PPh3, CBr4
BuLi
14C
Br
R
R
H
R
Br
Scheme 1.
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
[14C]-Aldehydes readily undergo conversion to corresponding vinyl dibromide derivatives and subsequent reaction with butyl
lithium gives the terminal lithium acetylide which is then protonated to give the terminal acetylene.
One disadvantage associated with this approach is that yields for the elimination reaction can be low due to side reactions. One
side-product often encountered is the corresponding vinyl bromide, which is presumably formed when the vinyl dibromide
undergoes lithium halogen exchange as the first step as opposed to elimination (Scheme 2). If isolated, this vinyl bromide can be
converted to the desired terminal acetylene by treatment with potassium tert-butoxide. Another issue associated with this approach
is that it is not tolerant of functional groups that are reactive with butyl lithium.
While this approach is synthetically useful, both of these issues coupled with the fact that there are up to three radiochemical
steps required to convert aldehydes to acetylenes (depending upon whether conversion of the vinyl bromide to the terminal
acetylene is required to maximise radiochemical yields), have encouraged us to look for alternative literature methods that could be
used in radiosynthesis for performing this conversion.
H
14C
Li
14C
Br
Li
BuLi
H
'H+'
BuLi
Br
Br
14C
14C
14C
R
R
R
R
R
Br
Br
H
14C
H
14C
'H+'
KOtBu
H
Br
Br
14C
R
R
R
Li
H
Common Side Product
Scheme 2.
Several alternatives to Corey Fuchs methodology have been reported, including development of the Ohira-Bestmann reagent (1)2
(Scheme 3) which allows for a single step conversion of aldehydes to a terminal acetylenes under mild conditions and is tolerant of a
number of different functional groups.
O
P
O
MeO
MeO
1
N2
O
Ohira-Bestmann Reagent
K2CO3, MeOH
14C
14C
[14C]-Products
R
R
H
Scheme 3.
This communication discusses the synthesis of [5-14C]-hex-5-ynoic acid (2) and [benzene-(U)-14C]-7-methoxybenzofuran (3) (Figure 1)
which are two examples of carbon-14 labelled intermediates prepared in our laboratories that have involved elaboration of an
aldehyde into a terminal acetylene.
OH
14C
*
O
O
O
[14C]-3
[14C]-2
Figure 1. Structures of C-14 labelled 2 and 3.
Results and Discussion: [5-14C]-Hex-5-ynoic acid (2)
Initially it was envisaged that the synthesis would proceed as described in Scheme 4. Nitrile [14C]-5 would be prepared from
commercially available bromoorthoester (4) and [14C]-potassium cyanide. This would then undergo reduction to give aldehyde [14C]-
6 which could then be elaborate into the desired [14C]-hex-5-ynoic acid (2). As a back-up plan, it was envisaged that nitrile [14C]-5
could be converted to the methyl ketone [14C]-7 by reaction with methyl lithium which could be elaborated to the desired product.
Both of these routes were initially investigated using unlabelled material or low specific activity carbon-14 labelled materials
(Scheme 5).
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
OMe
OMe
OMe
K14CN
OMe
OMe
OMe
H
OMe
OMe
OMe
14C
14C
O
Br
N
[14C]-6
[14C]-5
4
OMe
OMe
OMe
14C
O
OH
14C
O
[14C]-7
[14C]-2
Scheme 4.
Initial formation of nitrile [14C]-5 worked well in 80–90 % yield. Subsequent reduction of the nitrile, however, failed to yield any
useful amounts of product, and it is suspected that the reason for this is that this particular aldehyde is unstable, therefore
synthesis via the methyl ketone was attempted. Preparation of methyl ketone [14C]-7 proceeded in good yield. Subsequent
de-hydration of the ketone, using a modified procedure3 involving conversion to the enol phosphate followed by elimination,
failed to yield any of the desired product at all, and all attempts to form the desired product from [14C]-7 afforded complex
mixtures.
OMe
OMe
OMe
K14CN
OMe
OMe
OMe
14C
Br
H
OMe
OMe
OMe
14C
O
N
80 - 90 %
DiBAL
[14C]-6
4
[14C]-5
or LiHAl(OtBu)3
MeLi
THF
96 %
OH
OMe
OMe
14C
14C
O
O
OMe
1. TMPLi, THF, -78 oC, 1h
[14C]-7
[14C]-2
2. ClPO(OEt)2, -78 oC 30 min
then RT, 1 h
3. TMPLi, -78 oC
then RT, overnight
Scheme 5.
A further approach was attempted (Scheme 6). Nitrile [14C]-5 was converted to acid [14C]-8 using a nitrilase enzyme in 70 % yield
and the acid was selectively reduced to the alcohol [14C]-9 in 87 % yield using borane-THF. Subsequent oxidation using PCC on
celite to give [14C]-10 appeared to work by TLC and NMR, however, reaction of the crude product solution failed to give useful yields
of the terminal acetylene when reacted with Ohira-Bestmann reagent 1 and on standing re-formed the acid [14C]-8, thereby
suggesting that this intermediate aldehyde [14C]-10 was also unstable.
OMe Nitrilase
OMe
BH3, THF
HO
OMe
14C
HO
14C
OMe
14C
O
N
OMe
87 %
O
H
H
70 %
O
[14C]-5
[14C]-8
[14C]-9
O
O
P
OMe
OMe
N+
OMe
H
OMe
14C
1
14C
O
N
PCC on Celite
O
K2CO3, MeOH
< 10 %
O
[14C]-10
HO
14C
OMe
Standing
O
O
[14C]-8
Scheme 6.
[14C]-Hex-5-enoic acid was finally prepared as illustrated in Scheme 7. THP protected bromobutanol 11 was converted to nitrile
[14C]-12 using [14C]-potassium cyanide in 71 % radiochemical yield. Reduction with DiBAL gave aldehyde [14C]-13 in 80 %
radiochemical yield and conversion to acetylenic ether [14C]-14 using Ohira-Bestmann reagent (1) was performed in 68 %
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
radiochemical yield. Finally, de-protection of the THP protecting group and oxidation of the alcohol to the acid in a single step gave
the desired product [14C]-2 in 74 % radiochemical yield.
O
O
14C
K14CN, EtOH
71 %
N
14C
Br
Br
DiBAL
80 %
OH
OTHP
OTHP
p-TsOH,
EtOH
H
OTHP
[14C]-12
11
[14C]-13
67 %
O
O
P
OMe
OMe
N+
N
O
CrO3, H2SO4,
14C
14C
OTHP
OH
H2O, acetone
74 %
K2CO3, MeOH
68 %
[14C]-2
[14C]-14
Scheme 7.
[Benzene-(U)-14C]-7-Methoxybenzofuran (3)
It was envisaged that the chemistry would proceed as illustrated in Scheme 8. After a number of exploratory experiments we
selected the MOM group as a protecting group and prepared the O-MOM, O-methyl catechol derivative from [(U)-14C]-phenol as
shown in Scheme 9.
O
Acetylene
Formation
Multiple Steps
Formylation
Deprotection
Cyclisation
*
*
*
*
*
O
O
OH
O
O
O
PG
O
PG
O
PG
O
[U- C]-Phenol
Protected [ C]-o-Vanillin
[
C]-7-Methoxybenzofuran 3
Scheme 8.
[(U)-14C]-Phenol was MOM protected in 95 % radiochemical yield to give [14C]-15. This was then ortho-lithiated with butyl lithium
and the anion reacted with iodine to give the iodophenol derivative [14C]-16. The iodine was then displaced with sodium
methoxide, mediated with copper (I) bromide to give catechol derivative [14C]-17 in 40 % combined yield for the iodination and
displacement. This yield was lower than would have been anticipated from the trial chemistry that we performed, and it was noted
that 31% radiochemical yield of an 86:13 mixture of [14C]-15 and [14C]-16 was isolated after the methanol displacement. This could
have been re-cycled to furnish more product if desired, but this was not done as it was felt that there was more than enough of the
catechol derivative [14C]-17 to complete the synthesis successfully.
1. n-BuLi, Et O
2. I
NaH, DMF
MOM-Cl
NaOMe
CuBr
*
*
I
*
*
O
O
O
40 %
over 2 steps
OH
95%
O
[U- C]-Phenol
O
O
O
[
C]-15
[
C]-16
[
C]-17
Scheme 9.
Conversion of catechol derivative [14C]-17 to terminal acetylene [14C]-20 was performed as illustrated in Scheme 10. [14C]-17
was ortho-lithiated with butyl lithium and the resulting anion quenched with DMF. This gave exclusively one regioisomer with
the aldehyde being formed ortho to the MOM group and this reaction proceeded in 56% yield, however, unfortunately,
the reaction or work-up procedures also caused the MOM de-protection to occur to give [14C]-18 in 56% radiochemical yield.
[
14C]-18 was re-protected in quantitative yield to give [14C]-19 using MOM chloride in acetone in the presence of potassium
carbonate. Formation of the terminal acetylene [14C]-20 was performed using Ohira-Bestmann reagent (1) to give [14C]-20 in
75% yield.
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
O
OH
K2CO3,
Acetone,
MOM-Cl
O
O
1. n-BuLi, Et2O
2. DMF
Ohira-Bestmann reag.
K2CO3, MeOH
*
*
*
*
O
56 %
quant.
O
75 %
O
O
O
O
O
O
O
14C]-18
[
14C]-17
[
14C]-20
[
14C]-19
[
Scheme 10.
Finally, conversion of the terminal acetylene [14C]-20 to the final product [14C]-7-methoxybenzofuran (3) was performed as shown
in Scheme 11.
O
*
[14C]-21
3M HCl, MeOH
OH
O
[Rh(COD)Cl2]2,
NaHSO4,
SiO2, DCM
P(4-FC6H4)3,
DMF
*
*
*
O
O
OH
93 %
55 %
O
O
O
O
[14C]-20
[14C]-22
[14C]-7-Methoxy benzofuran 3
Scheme 11.
Acetylene [14C]-20 was de-protected using a mixture of sodium hydrogen sulphate and silica gel in DCM to give the phenol [14C]-22.
Initially, this conversion was attempted using 3M HCl in methanol, but this caused the acetylene to hydrate to give acetophenone
derivative [14C]-21. Formation of benzofurans from acetylenes is often accomplished by Sonogashira reaction of an o-iodophenol
with an acetylene, and under Sonogashira coupling conditions, cyclisation often occurs spontaneously. Cyclisation of [14C]-22 was
initially attempted by replicating Sonogashira type conditions4 (heating with a mixture of PdCl2, CuCl2, DCE, and also heating with a
mixture of PdCl2(PPh3)2, CuI, Et3N, THF) but this resulted in complex mixtures of products. The final product was ultimately prepared
from [14C]-22 by reaction with a rhodium catalyst in the presence of a phosphine ligand using a literature procedure5, and this gave
the final [14C]-7-methoxybenzofuran (3) in 55 % yield from acetylene [14C]-22.
Conclusion: [5-14C]-Hex-5-ynoic acid (2) was prepared in 4 radiochemcial steps and 29 % radiochemical yield from [14C]-potassium
cyanide. [Benzene-(U)-14C]-7-Methoxybenzofuran (3) was prepared in 8 radiochemical steps and 8 % radiochemical yield from
[(U)-14C]-phenol.
Preparation of a labelled [14C]-aldehyde followed by conversion to the [14C]-terminal acetylene using the Ohira-Bestmann reagent
is a convenient and useful approach to a range of labelled compounds provided the corresponding aldehyde is accessible.
References
[1] E. J. Corey, P. L. Fuchs, Tetrahedron Lett. 1972, 36, 3769–3772.
[2] (a) S. Ohira, Synth. Commun. 1989, 19, 561-564. (b) Mu¨ller, S. G; Liepold, B.; Roth, G. J; Bestmann, H. J. Synlett 1996, 521–522.
[3] E. Negishi, O. Anthony, A. O. King, J. M. Tour, Organic Syntheses, Coll. Vol. 7 (1990), p.63; Vol. 64, (1986), p.44.
[4] For a recent example of this type of transformation see: Bang, H. B; Han, S. Y; Choi, D. H; Yang, D. M; Hwang, J. W; Lee, H, S; Jun,
J-G, Synthetic Communications, 2009, 39, 506–515.
[5] B. M. Trost, A. McClory, Angewandte Chemie Int Edn, 2007, 46, 2074–2077.
DESIGN AND SYNTHESIS OF A CARBON-14 ELECTROPHILIC CYANATING REAGENT: 1-[14C]CYANO-
BENZOTRIAZOLE AND ITS APPLICATION IN ORGANIC SYNTHESIS
SUMEI REN, PAUL MCNAMARA, AND DAVID KOHARSKI
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-4545, Kenilworth, NJ 07033, USA
Abstract: 1-[14C]Cyanobenzotriazole (BtÃCN), a Carbon-14 electrophilic cyanating reagent, was synthesized from K14CN in
86% yield in one step. The crude 1-[14C]cyanobenzotriazole was used in the synthesis of two Carbon-14 labeled
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
compounds: 3-(2-[14C]cyanoacetyl)-1,1-dimethylethyl ester (1) and a substituted pyrazolo-triazolo-pyrimidine (2). The application of
1-[14C]Cyanobenzotriazole in these syntheses enabled us to prepare the targeted Carbon-14 compounds in two steps from K14CN
with high overall yield.
Keywords: Carbon-14; 1-[14C]Cyanobenzotriazole; Electrophilic cyanating reagent
Introduction: Nitriles are versatile intermediates and have broad use in organic synthesis. K14CN is a useful reagent to synthesize 14
C
nitriles. K14CN is widely used in labeled compound synthesis due to its ready availability, low price and high stability. It is used to
introduce a cyano group via carbon-carbon bond formation by CN- nucleophilic attack at an electrophilic carbon. Some typical
transformations are shown in Scheme 1.
R-14CN
RX
Ar-14CN
A
r
I
,
R'
P
0
d
RCOR'
K14CN
HO
R
14CN
RCOH/R'NH2/HOAc
Strecker reaction
CuSO4
Cu14CN
14CN
ArNH2/NaNO2/HCl
Sandmeyer coupling
R
NHR'
Ar-14CN
NH2
14
COOH
R
Scheme 1. Synthesis of [14C]Nitriles from K14CN.
An electrophilic cyanating reagent, CN1, not only broadens the chemistry of C-CN bond formation, but can also be used to form
S-CN and N-CN bonds. There are a limited number of reagents that can serve as a cyano cation (CN1) equivalent, such as cyanogen
bromide, cyanogen chloride, phenyl cyanate, tosyl cyanide, 1-cyanobenzotriazole, 1-cyanoimidazole, and 2-cyanopridazin-3(2H)-
ones.1,2 Carbon-14 cyanogen bromide was the only reagent reported as a 14CN1 reagent.3,4 However, 14CNBr is difficult to handle
due to its low boiling point (61.5oC) and toxicity. A more user friendly 14CN1 reagent was needed.
Results and Discussion: Synthesis of 1-[14C]Cyanobenzotriazole (BtÃCN)
1-[14C]Cyanobenzotriazole was chosen as the target electrophilic cyanating reagent based on its physical properties, reactivity and
easy preparation from K14CN. Katritzky et al. reported synthesis of 1-cyanobenzotriazole from CNBr and benzotriazole in high yield.1
Hughes et al. synthesized 1-cyanobenzotriazole from 1-chlorobenzotriazole and NaCN in 70% yield after purification by
sublimation.5 We adopted and modified the Hughes’ method to prepare 1-[14C]Cyanobenzotriazole from K14CN and
1-chlorobenzotriazole in high yield as shown in Scheme 2. The crude product was used directly without sublimation. Attempts
to purify the compound by silica gel column chromatography were unsuccessful; ꢀ10% impurity (imine 3) was generated.
HN
14C
N
N
N
i) K14CN, CH3CN, 0oC, 3h
N
N
N
N
N
N
+
ii) RT, overnight
86% yield
N
N
N
Cl
14CN
Bt*CN
3
Scheme 2. Synthesis of 1-[14C]Cyanobenzotriazole.
A typical synthetic procedure follows:
1-Chlorobenzotriazole (290 mg, 1.89 mmol, synthesized according the literature procedure5) was added to a suspension of K14CN
(100 mCi, 1.72 mmol) in CH3CN ( anhydrous, 8.0 ml) at 01C. The reaction was stirred at 01C under N2, for 3 h and then warmed to RT
and stirred for 24 h. The solvent was removed under reduced pressure. The residue was dissolved in EtOAc (50 ml), washed with H2O,
brine, dried over Na2SO4, filtered and concentrated to give 85.6 mCi of 1-[14C]Cyanobenzotriazole as a light yellow solid. The RCP
was 82% by radio-TLC (silica gel, 2:1 hexanes: EtOAc, Rf = 0.49). The crude product was used in the next step without further
purification. The reagent could be stored at À201C for two weeks. Some decomposition was observed during storage for a longer
time. Pretreating the EtOAc extraction solvent with base (NaHCO3) increased the stability of 1-[14C]cyanobenzotriazole.
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
Synthetic applications of 1-[14C]Cyanobenzotriazole
Synthesis of (R)-tert-butyl 3-(2-[14C]cyanoacetyl)piperidine-1-carboxylate (1) through electrophilic C-Cyanation
Compound 1 was required with a labeled cyano group. The retrosynthesis is as shown in Scheme 3:
O
Br
O
N
N
C
*
K
*
CN
Boc
4
B
t
*
O-
N
O
C
N
Boc
1
CH3
N
N
Boc
5
Boc
Scheme 3. Retrosynthesis of [14C]compound 1.
Synthesis of Compond 1 by nucleophilic substitution of 4 with K14CN failed. An alternative approach, eletrophilic cyanation of
enolized ketone 5 with 1-[14C]cyanobenzotriazole, was successful. The synthesis is shown in Scheme 4. Ketone 5, was synthesized
according to a literature procedure,6 enolized with LiHMDS and then reacted with BtÃCN to give compound 1 in 39.4% yield.
O
O-
O
*
CN
, THF, -78oC-RT
LiHMDS, THF, -78oC
Bt*CN
39.4% yield, 95% RCP
N
N
N
Boc
1
Boc
5
Boc
Scheme 4. Synthesis of [14C]Nitrile 1.
Synthesis of Pyrazolo-Triazolo-[14C]Pyrimidines by eletrophilic N-cyanation and cyclization
Gata and Baraldi have reported the synthesis of compounds containing the Pyrazolo-Triazolo-Pyrimidine core 7 by treating
compound 6 with large excess of cyanamide (12 eq) as shown in Scheme 5.7,8
NH2
N
N
NH2CN (12eq), NMP,
N
N
H2N
N
N
R2
N
p-TsOH, 140oC, overnight
R2
N
H
R1
R1
24-48% yield
N
N
N
6
7
Scheme 5. Synthesis of compound 7.
Kuo et al. reported the similar transformation, with much higher yields, by using eletrophilic cyanating reagents 2-methoxyphenyl
cyanate or CNBr as shown in Scheme 6.9
NH2
OCN
N
1.35 eq, CH3CN,
N
53-58oC, 1h, 95% yield
H2N
N
N
N
N
N
OCH3,
N
H
O
O
R3
R3
or: CNBr, 1.3 eq, CH3CN,
75-80oC, 3h, 68% yield
N
N
8
9
N
Scheme 6. Synthesis of compound 9.
Ã
Labeled compound 2 was synthesized using Bt CN as the eletrophilic cyanating reagent similar to Kuo’s approach. Compound 10
Ã
was treated with Bt CN (1.0 eq) in a mildly basic two phase mixture to give labeled compound 2 in 39.9% yield with 97.6% RCP after
purification. The synthesis is shown in Scheme 7.
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
N
*
NH2
N
CN
HN
N
N
R5
HN
Bt*CN, THF/CH3CN/K3PO4/H2O
R5
N
H
R4
50oC, 2h, RT 2h
39.9% yield
N
R4
N
N
N
10
NH2
N
N
N
*
R5
N
R4
N
N
2
Scheme 7. Synthesis of Carbon-14 labeled compound 2.
Ã
Synthesis of 1-Naphthyl thiocyanate through S-Cyanation with Bt CN
Preliminary S-Cyanation chemistry was explored. Naphthalene-1-thiol was reacted with BtCN. The desired 1-Naphthyl thiocyanate
11 and impurity 12 were detected by MS when the reaction was conducted in CH3CN solvent. Only impurity 12 was detected with
CH3CN/H2O as solvent as shown in Scheme 8. Further work is planned.
NH
S
S
h
2
,
T
0o
R
-
C
1
,
O
SH
H2
/
N
C
H3
C
N
N
12
N
+
C
H
3C
N
o
,
0
C
NH
,
3
h
SCN
CN
S
S
+
11
12
Scheme 8. Synthesis of 1-Naphthyl thiocyanate 11.
Conclusion: An efficient carbon-14 labeled cyanating reagent, 1-[14C]cyanobenzotriazole, was designed and synthesized in 86%
yield from K14CN in one step. It was used in the synthesis of two compounds by electrophilic C-cyanation and N-cyanation. The use
of 1-[14C]cyanobenzotriazole enabled us to introduce the carbon-14 at a late step in the synthesis.
Acknowledgements: Special thanks to Dr. Alexei Buevich and Dr. T. M. Chan from Structural Chemistry Group for
NMR analysis.
References
[1] A. R. Katritzky, R. Akue-Gedu, A. V. Vakulenko, ARKIVOC 2007, 3, 5–12.
[2] J. J. Kim, D. H. Kweon, S. D. Cho, H. K. Kim, E. Y. Jung, S. G. Lee, J. R. Falc, Y. J. Yoon, Tetrahedron 2005, 61,
5889–5894.
[3] R. C. Thomas, J. Label. Compd. Radiopharm. 1978, 14, 807–811.
[4] D. H. Thang, N. H. Nam, H. Hoellinger, L. Pichat, J. Label. Compd. Radiopharm. 1981, 18, 1009–1014.
[5] H. V. Hughes, S. D. Hammond, M. P. Cava, J. Org. Chem. 1998, 63, 401–402.
[6] M. Ledeboer, A. Pierce, G. Bemis, L. Farmer, T. Wang, D. Messersmith, J. Duffy, F. Salituro, J. Wang, U.S. Pat. Appl. Publ. US
2006183761 A1 20060817, 2006.
[7] F. Gata, M. R. D. Giudice, A. Borioni, P. A. Borea, S. Dionnisotti, E. Ongini, Eur. J. Med Chem. 1993, 28, 569–576.
[8] P. G. Baraldi, F. Fruttarolo, M. A. Tabrizi, D. Preti, R. Romagnoli, H. EI-Kashef, A. Moorman, K. Varani, S. Gessi, S. Merighi, P. A. Bore,
J. Med. Chem. 2003, 46, 1229–1241.
[9] S. C. Kuo, D. J. Tsai, L. T. Tran, P. Zhang, A. D. Jones, U. S. Patent 0090492 A1, 2005.
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
SYNTHESIS OF [14C] ACIDS FROM THEIR UNLABELED COUNTERPARTS AND APPLICATION TOWARD A
NOVEL [14C] LABELED HEPATITIS C VIRUS POLYMERASE INHIBITOR
JONATHAN Z. HO, MICHAEL A. WALLACE, YUI S. TANG, ANDY S. ZHANG AND MATTHEW P. BRAUN
Department of Process Research, Merck Research Laboratories, Merck & Co., Inc., RY800-B375, 126 East Lincoln Avenue, Rahway, New Jersey, USA
Abstract: A strategy has been developed for the rapid construction of a [14C] acid from either an aromatic or an alkyl acid via a
decarboxylation-carboxylation sequence. A [14C] labeled heptatitis C virus polymerase inhibitor was prepared. Other examples and
the limitations of this methodology are discussed.
Keywords: 14C-labeled acid; aromatic acid; heptatitis C virus polymerase inhibitor
Introduction: The carboxylic acid group plays an important role in organic, medicinal, supramolecular, and material chemistry.1 For
example, recent reports have cited examples of acids as potent HIV-1 protease inhibitors,2 p38 MAP kinase inhibitors for the
treatment of inflammatory diseases,3a and peptidemimetics with increased metabolic stability.3b Although there are a vast number
of methodologies to make an acid, the standard preparation of [14C] acids generally involves use of [14C]CO2 with Grignard or
organic lithium reagents, or hydrolysis of [14C]cyanides.4 This can be attributed to the fact that halide precursors are readily available
commercially or synthetically. When those precursors are difficult to prepare, a method of synthesis of [14C] acids from their
unlabeled counterparts becomes very attractive. In this paper a method with mild reaction conditions to convert an unlabeled acid
to a [14C] labeled acid is reported.
Results and Discussion: It is known that decarboxylative halogenation of carboxylic acids (the Hunsdiecker-Borodin reaction)
leads to a bromide via radical mechanism,[5] This method works well with saturated aliphatic, especially primary, carboxylic acids.
The O-acyl derivatives of N-hydroxy-2-thiopyridone (Barton esters) are commonly used as precursors of radicals,6 and either primary,
secondary, or tertiary carboxylic acids can be converted to the corresponding bromides.7
In model studies, decarboxylative halogenations with Barton esters were conducted to obtain bromides, which were then treated
with commercially available [14C]KCN to form cyanides. Upon hydrolysis, the desired [14C] acids were realized. The results are listed in
Table 1. Thus, unlabeled acids have been transformed to [14C] labeled products. In each case the conversion was monitored by HPLC
and LCMS, whereas the yield was based on the isolated product.
Table 1. Synthesis of [14C] aliphatic acids
O
O
C
O
c
a
d
14C
Br
14CN
N
R
R
R
O
R
OH
R
OH
b
S
1a-e
2a-e
3a-e
4a-e
5a-e
entry
R
Product
% yield
1
2
3
4
n-C11H23 (1a)
n-C13H27 (1b)
n-C15H31 (1c)
n-C17H35 (1d)
[1-14C]Lauric acid (5a)
[1-14C]Myristic acid (5b)
[1-14C]Palmitic acid (5c)
[1-14C]Stearic acid (5d)
68
67
62
60
O
14C
OH
5
63
1e
5e
Conditions: (a) Oxalyl chloride (1 eq), DMF. (b) 2-Mercaptopyridine N-oxide sodium salt, DMAP (0.1 eq), CCl3Br, reflux, 4 h. (c)
[14C]KCN, DMF, reflux, 2 h. (d) 6M HCl, water, reflux, 12 h.
Compound 6 is a fermentation product identified with anti-fungal activities.8 In order to study its metabolic profile, a radio
isotopically labeled tracer was needed. Initially, a [3H] tracer 8 was made via an iodination-tritiation process (Scheme 1).
To make a [14C] labeled tracer, 6 was hydrolyzed to give 1e, which was then converted to [14C] labeled 5e. Standard EDC-coupling
conditions were used to give final [14C] labeled tracer (9) in a short period of time. This [14C] labeled 9 superseded the tritium labeled
8 to became the tracer of choice adopted in metabolic studies.
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
HO
OH
HO
OH
O
H
O
H
O
O
HO
HO
NH
NH
N
N
a
H
H
N
N
O
O
H
N
H
N
HO
HO
H
H
O
O
OH
OH
R1
HO
HO
R2
7: R1 = R2 = I
8: R1 = R2
6
b
=
3H
HO
OH
O
O
14C
O
HO
NH
N
H
H
HO
1e
N
O
H
N
HO
O
14C
H
O
OH
HO
c
HO
5e
9
Scheme 1. Synthesis of [3H] labeled 8 and [14C] labeled 9. (a) Chloroiodide (ICl), DCM; (b) 3H2, 5% PdCO3, DMF; (c) EDC, HOBt, NMM, MeCN, rt, 1 h, 59%.
The limitation of the preparation of [14C] labeled acids via the Hunsdiecker-Borodin reaction using Barton esters was that the
yields were good for aliphatic substrates but often poor for aromatic ones. As a complement to this labeling method, it was found
that [14C] labeled aromatic acids could be synthesized via Curtius rearrangement as a key step. Since a great deal of aromatic acids
could undergo Curtius rearrangement,[9] this route could expand the availability of [14C] aromatic acids. One example of this effort is
shown in Scheme 2. Compound 10 was a lead compound for treatment of hepatitis C virus (HCV) infection.[10] For an absorption,
distribution, metabolism, and excretion (ADME) study, a [14C] labeled tracer was needed. However, it was not acceptable to place a
label at the N-methyl position due to its metabolic instability. It was necessary to put a [14C] label either in the main frame work or at
the carboxylic acid. Putting a [14C] label in the skeleton of 10 would involve a 9-step linear sequence[10] based on the medicinal
chemistry route to 10, whereas labeling of the carboxylic acid appeared simpler.
N
N
N
N
O
O
14C
N
N
OH
OH
10
15
Curtius
rearrangement
c, from 13
f, from 14
c
a
N
N
N
1
2
2
SO4
N
N
N
NH2
R
b
N2
N
d
N
N
13: R = I
e
11
12
14: R = 14CN
Scheme 2. Synthesis of [14C] acid 15 from its unlabeled counterpart 10. (a) DPPA, TEA, DMF, 801C, 4 h, 65%; (b) NaNO2, H2SO4, DME, À151C, 1 h; (c) Pd(OAc)2, AcONa, 14CO,
1%; (d) NaI, water, 91% from 11; (e) Zn(14CN)2, Pd(PPh3)4, DMF, 801C, 12 h, 88%; (f) 6M HCl, 1101C, 57%.
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Abstracts
Curtius rearrangement of 10 with diphenylphosphoryl azide (DPPA) gave aniline 11 in 65% yield after flash chromatography.
Diazonium salt 12 was obtained by treatment of 11 with NaNO2. Without separation, iodide 13 was prepared, in a one-pot fashion,
by addition of NaI to the reaction mixture. Although Pd-catalyzed carbonylation reaction of both 12 and 13 led to the final acid 15,
the yields for these reactions were low (below 1%). Conversion of iodide 13 to cyanide 14 with Zn(14CN)2 was found to be a robust
reaction with much higher yield (88%). Hydrolysis of 14 gave the acid 15. Preparative HPLC purification of 15 was used to achieve an
excellent radiochemical purity of 99.4%.
In summary, either an unlabeled alkyl acid or an aromatic acid can efficiently be converted to its [14C] labeled counterpart with
only the last two steps involving handling of radio-chemicals.
Acknowledgements: The authors wish to thank Dr. David J. Schenk for analytical support. J.Z.H. gratefully acknowledges Dr. Lee
J. Silverberg for help in the preparation of this manuscript.
References
[1] T. P. Vishnyakova, I. A. Golubeve, E. V. Glebova, Russ. Chem. Rev. (Engl. Transl.) 1985; 54, 249–261.
[2] J. F. Guichou, J. Viaud, C. Metling, Y. L. Lin, A. Chavanieu, J. Med. Chem. 2006, 49, 900–910.
[3] (a) J. Regan, S. Breitfelder, P. Cirillo, L. Tong, C. Torcellini, J. Med. Chem. 2002, 45, 2994–3008; (b) Q. Han, C. H. Chang, R. Li, Y. Yu,
P. K. Jadhav, P. Y. S. Lam, J. Med. Chem. 1998, 41, 2019-2028.
[4] For examples of synthesis of [14C] labeled acids see (a) J. Z. Ho, C. S. Elmore, M. P. Braun, J. Label. Compd. Radiopharm. 2008, 51,
399-403; (b) C. S. Elmore, D. C. Dean, K. Liu, A. Jones, D. G. Melillo, Syn. Appl. Isotop. Label. Compd, Proc. Int. Sym. 7th, J. Wiley,
New York, 2001, pp. 224–227.
[5] (a) R. G. Johnson, R. K. Ingham, Chem. Rev. 1956, 56, 219-269; (b) C. V. Wilson, Org. React. 1957, 9, 332–387.
[6] D. H. R. Barton, S. Z. Zard, Janssen Chimica Acta 1987, 4, 3–10.
[7] D. H. R. Barton, W. Liu, Tetrahedron Lett. 1997, 38, 2431–2434.
[8] (a) J. M. Balkovec, F. A. Bouffard, J. F. Dropinski, J. M. Balkovec, F. A. Bouffard, J. F. Dropinski, PCT Int. Appl. WO 9613272 A1
19960509, (1996), pp 86; (b) K. M. Belyk, D. R. Bender, R. M. Black, D. L. Hughes, W. Leonard, U.S. US 5552521 A 19960903 (1996).
[9] For recent reviews on Curtius rearrangements see (a) S. Lang, J. A. Murphy, Chem. Soc. Rev. 2006, 35, 146-156; (b) W. Adam,
A. V. Trofimov, Acc. Chem. Res. 2003, 36, 571–577.
[10] (a) F. Narjes, I. Stansfield, PCT Int. Appl. WO 2007029029 A2 20070315 (2007); (b) I. Stansfield, C. Ercolani, A. Mackay, I. Conte, M.
Pompei, U. Koch, N. Gennari, C. Giuliano, M. Rowley, F. Narjes, Bioorg. Med. Chem. Lett. 2009, 19, 627–632.
SURVEY OF DIFFERENT TRITIUM-LABELLING METHODS USED AT RC TRITEC
¨
CHRISTIAN MEISTERHANS, STEFAN C. NUCKEL, AND ALBERT ZELLER
RC TRITEC AG, Speicherstrasse 60a, CH-9053 Teufen, Switzerland
Abstract: The radiosynthesis group of RC TRITEC is specialised in custom tritium synthesis and therefore has gained a lot of
experience in a large variety of different tritium-labelling methods. We present a survey of the projects carried out in the past years
focusing on the diverse characteristics and popularities of the different methods. All applied reaction types have been classified into
different groups of tritium-labelling methods. These groups are illustrated and discussed with regard to specific activities,
regioselective labelling, tritium efficiency, possible labelling candidates and precursors. The varying popularities of the different
methods used at RC TRITEC in the past years are statistically analyzed.
Keywords: tritium chemistry; tritiation; tritide; tritium-labelling, tritiated
Introduction: Radioisotopically labelled organic compounds are irreplaceable tools for research and development especially in the
pharmaceutical and agrochemical industry as well as in academic research. Among the most widely used radioisotopes C-14 and
H-3, the latter, tritium, is very much favoured for early applications in the R&D process. From a synthetic viewpoint it is relatively easy
to introduce tritium into organic compounds, this allows fast and cost efficient labelling which is of great importance in the fast-
paced early development. Beside tritium chemistry’s nature of a low-threshold labelling approach the high specific activity of tritium
at 29 Ci/milliatom allows the use of such labelled compounds for binding studies, imaging techniques, and many other applications
that require highest sensitivities. In contrast, C-14-labelling usually requires multi step syntheses, therefore this time and cost
consuming labelling approach is used predominantly for later phases in development.
In this article we present a survey over different methods that are used in tritium chemistry and in special at our facility at RC
TRITEC. We would like to offer the researcher the ability to pick out the most suitable tritiation method for his labelling candidate.
Classification of tritium chemistry
There are numerous ways to bring some systematic order to the world of tritium chemistry. Evans1 has categorised the different
preparations of tritium-labelled compounds into isotope exchange reactions, direct chemical synthesis, biochemical methods and
recoil labelling (hot-atom reactions). After three decades of advances in tritium chemistry with the introduction of numerous new
reagents and methods, Saljoughian and Williams2 ordered the methods in two groups: ‘Hydrogen Isotope Exchange Labeling’ and
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
‘Synthetic Labeling’. The recently published new standard work for tritium chemists by Voges, Heys and Moenius3 again
differentiates between isotope exchange methods and chemical synthesis.
When we started to systematically classify all the different chemical methods that have been used at our facility, we
found that virtually all methods could be categorised according to the following questions: Does the method involve a direct isotope
exchange or a chemical synthesis? Does it use gaseous tritium, a triton containing solvent, tritiated methyl iodide or
tritiated intermediates as the tritium source? In cases where a catalyst is used, is it a heterogeneous or a homogeneous one? Are
special reagents like tritides utilized? According to this approach, the methods could be divided into five classes distinguished as follows:
1. Direct isotope exchange on the target compound.
2. Catalytic tritium gas reductions of precursors.
3. Tritide reagents are used for selective reductions of suitable precursors.
4. Methylation reactions using tritiated methyl iodide or methyl sulfonates.
5. Coupling of tritiated intermediates.
Tritium-labelling methods
Class 1: Hydrogen isotope exchange
In the past 20 years direct tritium-labelling has gone through ground-breaking developments. The thorough investigation
of heterogeneous (Pt, Pd, Rh) and homogeneous catalysts (Rh, Ru, Ir)4–13 for hydrogen isotope exchange paved the way
for new methods affording products with high specific activities. The most obvious advantage of direct isotope exchange labelling is
its speed as no precursor is required. Nowadays, a whole bunch of different catalysts, especially Ir(I) based homogeneous
ones11, 13 offer a versatile toolbox for hydrogen exchange on various substrates. Nevertheless, the major drawback is the poor
predictability of both extent and position of tritium incorporation resulting in a need for preliminary studies. The problematic
generation of high activity tritium waste by Crabtree’s catalyst has been relieved by the use of an immobilised version
of this versatile Ir(I) catalyst.14 Alternatively, carrier free tritium oxide or other triton sources like alcohols and carboxylic acids
can be used for acid- or base-catalysed exchange reactions or to quench deprotonated C-H acids, e.g. lithiated aromates,
see Figure 1.
HN
N
NH2
N
R
N
R
T
F
T
N
N
H
T
NH2
R
T
O
70 mol% Crabtree's catalyst
DCM/THF 1:3
49 Ci/mmol
PtO2 (Adam's catalyst)
DMF
30 Ci/mmol
3 eq. LDA, THF
20 Ci T2O, -80˚C to rt
13 Ci/mmol
Figure 1. Hydrogen isotope exchange products.
Class 2: Tritium gas reductions
The catalytic reduction of unsaturated or halogenated precursor compounds with carrier free tritium gas is probably the most
popular method to introduce tritium into organic molecules. As soon as a precursor is available, heterogeneous catalysis allows
rapid tritiation yielding products with high to very high specific activities. A single tritiodebromination typically affords 15–20 Ci/
mmol, whereas saturation of an olefinic precursor can give products well above 100 Ci/mmol, especially due to isotope exchange of
allylic protons, as can be seen from the second example in Figure 2.
Class 3: Tritide reductions
The development and application of tritide reagents in the last 20 years2, 15, 16 have dramatically diversified the tritium chemist’s
lab work, as reagents are now available for almost any synthetic task. LiT derived tritide reagents offer both maximum specific
activities (29 Ci/mmol per incorporated tritide) and 100% predictability of the label’s position, which is of particular importance for
metabolic studies. As stoichiometric quantities of tritiated butane result as a side product from the LiT production, special worker
and environmental safety precautions have to be taken. The handling of these reagents in multi Ci quantities asks for dedicated
equipment which is available in the form of stainless steel vacuum manifold systems manufactured by RC TRITEC.17–19 Figure 3
illustrates the use of tritide reducing agents.
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
T
T
Br
Br
Double debromination
700 mbar T2, 10% Pd/C
DIPEA, MeOH
35 Ci/mmol
O
O
S
S
R2N
R2N
O
O
(
)
T
T
T
R
C,C-double bond saturation
R
700 mbar 60% T2, 10% Pd/C
MeOH
94 Ci/mmol
T
T
R
R
T
Oxime reduction
600 mbar T2, Ra-Ni
EtOH
25 Ci/mmol
NH2
N
OH
Figure 2. Catalytic gas reductions.
T
NaBT4 reduction
CeCl3 . 7 H2O, NaBT4
MeOH/EtOH
R
R
R
R
R
R
OH
T
14 Ci/mmol
low tritium efficiency !
+
O
OH
T
T
N
LiBEt3T reduction
R
R
H2N
2.5 eq tritide, THF
58 Ci/mmol !!
high tritium efficiency !
LiAlT4 reduction
3 eq. tritide, THF
56 Ci/mmol !!
O
T
T
H
H
N
R
N
R
O
OH
Figure 3. Various applications of tritide reagents.
Class 4: Methylations
The introduction of highly tritiated methyl groups (60–87 Ci/mmol) has become an important option as various target compounds
can be prepared by methylation at hydroxy-, amino-, amido-, and thiol-groups. Furthermore, methyl-acetylenes and methyl-
aromates are accessible through C-methylation, either by direct methylation of a carbanion as shown in the second example in
Figure 4 or by Pd-catalysed coupling reactions. The already intrinsic sensitivity of these reactions is even worsened by the very small
scales of tritium synthesis, as 10 mmol of methylating reagent correspond to 600–870 mCi of activity. The disadvantages of methyl
iodide, namely its volatility, low stability, and poor UV absorbance, have been overcome by the introduction of non-volatile, stable,
and UV-active aromatic methyl sulfonates.20
CT3
OH
O
O
O
Methylation of a phenolate
phenol, DMF, 4 eq. Cs2CO3
0.13 eq. CT3ONos, 40˚C
60 Ci/mmol
O
O
N
N
H
H
N
N
3H,19F-Fallypride
(PET prestudies)
F
F
Methylation of a carbanion
acetylene, THF, 1.1 eq. n-BuLi
1 eq. CT3I
-16˚C to rt
70 Ci/mmol
R2N
R2N
CT3
Figure 4. Methylations at phenolic oxygen and acetylenic carbon.
J. Label Compd. Radiopharm 2010, 53 239–268
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
Class 5: Coupling of intermediates
In case the target compound bears a terminal group that can be coupled easily to an available precursor, one may employ
tritiated intermediates, such as methyl amine, ethanol amine, carboxylic acids, amino acids etc. The site specific and high degree of
labelling has to be paid with multiple active steps and difficult coupling reactions on small scales (ofteno10 mmol), see Figure 5.
HOBt, EDC.HCl
Coupling of 3H-ethanolamine
O
O
carboxylic acid, EDC.HCl, HOBt, DMF
OH
3H-ethanolamine
OH
Ar
N
H
Ar
OH
36 Ci/mmol
H2N
T
T
T
T
BOP, TEA
CT3 NH2
Coupling of 3H-methyl amine
carboxylic acid, 1.1 eq. BOP
DMF, 0.2 eq. 3H-methyl amine HCl, 4 eq. TEA
70 Ci/mmol
O
O
Ar
CT3
Ar
HO
N
H
C4H9
C4H9
Coupling of 3H-stearic acid
i) 3H-stearic acid, 1.2 eq. CDI, THF
ii) 1.6 eq. CoA . 3 Li, THF/water, 40˚C
45-60 Ci/mmol
T
T
T
T
i) CDI, THF
ii) CoA.3 Li
O
O
CoA
C3H6
C3H6
S
OH
T
T
T
T
Coupling of 3H-Boc-L-Ala-OH
3H-amino acid, 1.4 eq. EDC.HCl
1.4 eq. HOBt, 0.9 eq. peptide, DMF, 30˚C
25 Ci/mmol
O
i) HOBt, EDC.HCl
H
N
OH
H2N
Pentapeptide
O
N
H
ii) 4 N HCl in dioxane
O
O
Figure 5. Coupling reactions of selected tritiated intermediates.
Popularity statistics of methods used at RC TRITEC
The statistical analysis of all the tritiations projects carried out at RC TRITEC between 2005 and 2008 revealed the distribution
depicted in Figure 6.
Coupling of
intermediates
H/T exchange
18%
4%
Methylation
14%
Tritide reduction
19%
T2 reduction
45%
Figure 6. Statistical distribution of the projects over the five method classes.
A surprisingly low 18% of the projects involved an exchange methodology. Among these projects, most were carried out using
tritium gas and a homogeneous or heterogeneous catalyst, for the rest a triton source was used. In almost every second project
(45%) a gas reduction method was applied, either tritiodehalogenation, C-C-double/triple bond saturation, rarely C-heteroatom-
double/triple bond saturation (e.g. nitrile reduction). Tritide chemistry plays a most important role in our facility, as almost every fifth
tritiation involved the preparation and use of a tritide reagent, mainly made from lithium tritide (aluminotritides, lithium borotritides,
tin tritide), to a lesser extent made from sodium borotritide prepared by thermal exchange of sodium borohydride in tritium gas.
Every seventh tritium-labelled product resulted from a methylation, mostly at heteroatoms like oxygen, nitrogen and sulphur,
occasionally C-methylation was applied successfully. The remaining 4% of all projects accounted for the production and coupling of
tritium-labelled intermediates, a strategy that is rarely used due to its time-consumption and thus costly nature.
Conclusion: We have systematically classified the tritium-labelling methods used at our facility in five different classes and
illustrated them with typical examples. The discussion of the main features of the different methods like suitable target compounds,
observed specific activities, and site specific labelling should be helpful for researchers to choose a tailor-made tritium-labelling
strategy for their labelling candidates. The statistical analysis of all the applications from the past four years done at RC TRITEC gives
an idea of the diversity of today’s tritium chemistry and its methods.
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
References
[1] E. A. Evans, Tritium and its compounds, Butterworths, London 1974.
[2] M. Saljoughian, P. G. Williams, Current Pharmaceutical Design, 2000, 6, 1029–1056.
[3] R. Voges, R. Heys, T. Moenius, Preparation of Compounds Labelled with Tritium and Carbon-14, Wiley, Chichester, 2009.
[4] P. G. Williams, C. A. Lukey, M. A. Long, J. L. Garnet, J. Labelled Compd. Radiopharm. 1990, 29, 175–192.
[5] P. G. Williams, C. Than, S. Rabbani, M. A. Long, J. L. Garnett, J. Labelled Compd. Radiopharm. 1995, 36, 1–14.
[6] V. Derdau, J. Atzrodt, W. Holla, J. Labelled Compd. Radiopharm. 2007, 50, 295–299.
[7] W. J. S. Lockley, J. Labelled Compd. Radiopharm. 1985, 22, 623–630.
[8] D. Hesk, J. R. Jones, W. J. S. Lockley, J. Labelled Compd. Radiopharm. 1990, 28, 1427–1436.
[9] D. Hesk, J. R. Jones, W. J. S. Lockley, J. Labelled Compd. Radiopharm. 1991, 30, 887–890.
[10] W. Chen, K. T. Garnes, S. H. Levinson, D. Saunders, S. G. Senderoff, A. Y. L. Shu, A. J. Villani, J. R. Heys, J. Labelled Compd.
Radiopharm. 1997, 39, 291–298.
[11] G. J. Ellames, J. S. Gibson, J. M. Herbert, A. H. McNeill, Tetrahedron 2001, 57, 9487–9497.
[12] J. R. Heys, J. Labelled Compd. Radiopharm. 2007, 50, 770–778.
[13] W. J. S. Lockley, J. Labelled Compd. Radiopharm. 2007, 50, 779–788.
[14] M. J. Hickey, L. P. Kingston, W. J. S. Lockley, P. Allen, A. Mather, D. J. Wilkinson, J. Labelled Compd. Radiopharm. 2007, 50, 286–289.
[15] H. Andres, H. Morimoto, P. G. Williams, E. M. Zippi, in Synthesis and applications of isotopically labelled compounds, Vol. 5, (Eds:
J. Allen, R. Voges), Wiley, Chichester, 1994, pp. 83–90.
[16] E. M. Zippi, H. Andres, H. Morimoto, P. G. Williams, Synth. Commun. 1995, 25, 2685–2693.
¨
[17] E. Bannwart, A. Zeller, P. Strom, M. Skrinjar, in Synthesis and applications of isotopically labelled compounds, Vol. 7, (Eds: U.
Pleiss, R. Voges), Wiley, Chichester, 2001, pp. 664–666.
[18] K. Pfanner, A. Zeller, J. Labelled Compd. Radiopharm. 1998, 41, 1033–1034.
¨
[19] P. Strom, J. Labelled Compd. Radiopharm. 1998, 41, 1032.
[20] S. Pounds, J. Labelled Compd. Radiopharm. 2004, 47, 243–288.
THE SYNTHESIS OF 5, 7-DIMETHYL-1, 2, 4-TRIAZOLO[1, 5A(14C)]PYRIMIDINE-2-CARBALDEHYDE FROM
[14C]AMINOGUANIDINE: A KEY INTERMEDIATE FOR THE RADIOCHEMICAL GMP SYNTHESIS OF A DRUG
CANDIDATE
ANIL M. RANE AND KLAAS SCHILDKNEGT
Pfizer Global Research & Development, Eastern Point Road, Groton, CT 06340, USA
Abstract: Hydrazination of barium [14C]cyanamide and hydrazinolysis of S-methylisothio[14C]urea were compared for their ability to
efficiently prepare [14C]aminoguanidine bicarbonate. [14C]Aminoguanidine bicarbonate was converted in three steps to 5, 7-
dimethyl-1,2,4-triazolo[1,5a(14C)]pyrimidine-2-carbaldehyde, a key radiochemical GMP starting material.
Keywords: Barium [14C]cyanamide, S-methylisothio[14C]urea, [14C]aminoguanidine bicarbonate, 5,7-Dimethyl-1,2,4-triazo-
lo[1,5a(14C)]pyrimidine-2-carbaldehyde
Introduction: We required a large quantity of 5,7-dimethyl-1,2,4-triazolo[1,5a(14C)]pyrimidine-2-carbaldehyde (4) for use as a
radiochemical GMP starting material. The established synthesis of non-labeled 4 started from aminoguanidine bicarbonate1–a
readily available reagent. However, [14C]aminoguanidine bicarbonate (1) was not commercially available and required an efficient
synthesis to enable the production of 4 (Scheme 1).
Scheme 1.
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
The journal and patent literature describing both non-labeled and labeled syntheses of aminoguanidine2 can be categorized into
three general strategies: reduction of nitroguanidine, hydrazination (hydrazine nucleophilic addition) of cyanamide and
hydrazinolysis (hydrazine nucleophilic addition-elimination) of urea derivatives (Scheme 2). Of these strategies, we focused our
attention on both hydrazination and hydrazinolysis approaches to the synthesis of 1 because they utilize starting materials (BaCN2
and thiourea, respectively) that are readily available in carbon-14 form.
Scheme 2.
Results and Discussion: Several generalizations can be made from published hydrazination syntheses of non-labeled/labeled
aminoguanidine. Firstly, CaCN2 or NaHCN2 are used as cyanamide sources in unlabeled aminoguanidine syntheses and Ba14CN2 is
the only reported reagent used in labeled aminoguanidine syntheses. Hydrazine is typically introduced as a hydrate or sulfate salt.
Reactions are typically run with excess cyanamide and in water at low concentration to avoid cyanamide dimerization. Reaction pH
is often kept at 5-6 to avoid formation of a variety of impurities and maximize product yield. Precipitation of aminoguanidine as its
bicarbonate salt (the most commonly sited salt form and the one we desired) is typically accomplished by saturating the product
solution with KHCO3 and/or CO2 gas and adjusting reaction pH with H2SO4. Within this body of work, reported yields of
aminoguanidine range widely from 30–95%.
After exploring all established hydrazination procedures to prepare aminoguanidine bicarbonate, we were able to confirm several
process observations. Maintaining tight control of reaction pH was indeed critical to product formation, but reaction heterogeneity
throughout the course of the reaction made pH measurement difficult. Precipitation of the product bicarbonate salt proved difficult
if aminoguanidine was not present in high concentration. Additionally, since unlabeled BaCN2 is not commercially available
(and Ba14CN2 is the only carbon-14 cyanamide commercially available) we necessarily performed our exploratory work with CaCN2.
The dramatic solubility differences between these reagents made adequate development of labeled chemistry impossible. Our
optimized hydrazination synthesis of 1, performed on 110 mCi of Ba14CN2, proved only modestly successful, Scheme 3.
Scheme 3.
In light of our inability to prepare [14C]aminoguanidine in high yield via hydrazination of Ba14CN2, we turned our attention to
hydrazinolysis methodology. Specifically, S-methylisolthiourea 5 was an appealing aminoguanidine precursor because it can be
directly prepared from commercially available carbon-14 labeled thiourea, and can also be derived from Ba14CN2. Numerous
literature reports showcase the conversion of S-methylisolthiurea salts to aminoguanidine in water by reaction with
hydrazine sulfates or hydrates in the presence of base.2a,3 This approach has been used to prepare both 13C/15N2 and 14C
aminoguanidine.3
Using unlabeled thiourea, we quickly confirmed the utility of this approach, Scheme 4. Methylation of this reagent with either
methyliodide or dimethylsulfate in ethanol solvent afforded unlabeled S-methylisothiourea 5a and 5b in good yields as H2SO4
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
(insoluble) or HI (soluble) salts, respectively. These salts could be converted in high yield to aminoguanidine bicarbonate by either
stepwise or one pot reaction with hydrazine hydrate followed by CO2/KHCO3 treatment.
Scheme 4.
The efficient conversion of barium [14C]cyanamide to S-methyliso[14C]thiourea 5 was central to our choice for pursuing a
hydrazinolysis approach to 1. In practice, a suspension of 124 mCi of Ba14CN2 in water in a pressure tube was treated with 3.4
equivalents of Ba(OH)2 (to maintain basic reaction pH and a common counterion) and saturated with H2S gas. The reaction tube was
sealed and heated to affect cyanamide sulfurization.4 The reaction was then saturated with carbon dioxide to purge excess H2S gas
and to precipitate barium as BaCO3. The reaction was filtered to remove the suspended BaCO3, the aqueous filtrate was
concentrated and the residue was diluted with ethanol to suspend remaining traces of the salt. Additional filtration and
concentration delivered 122 mCi of chemical and radiochemical pure [14C]thiourea.
Two methylation procedures were then investigated for their ability to efficiently S-alkylate [14C]thiourea. Specifically, reaction of
[14C]thiourea with dimethyl sulfate3 in water proved slightly more effective than the same reaction performed with iodomethane5 in
ethanol for formation of S-methyliso[14C]thiourea salts, 5a/5b, respectively. Of note, the methylation to form 5a directly provided
high purity product, while the analogous formation of 5b required purification (Scheme 5).
Scheme 5.
Carbon-14 salts 5a and 5b were processed to [14C]aminoguanidine bicarbonate (1) separately to determine whether the
intermediate HSO-4 and I- counterions in any way effected reaction efficiency. To that end, 5a and 5b were reacted with hydrazine in
water at room temperature–both reactions were deemed complete within 3 hours, and the reaction of 5b was allowed to continue
for an extended time period to demonstrate product stability. Each reaction solution was then saturated with carbon dioxide gas to
purge liberated methyl mercaptan and introduce carbonate. Dissolution of solid KHCO3 into each solution initiated precipitation of 1
as white solids that were isolated by filtration. Overall, syntheses of 1 via dimethyl sulfate or iodomethane methylations and
subsequent hydrazinolyses of the S-methyliso[14C]thiourea intermediates proceeded with equal efficiency. The overall yield from
hydrazinolysis approach starting from Ba14CN2 was 78% over three steps compared to hydrazination approach which afforded in
only 37%.
J. Label Compd. Radiopharm 2010, 53 239–268
Copyright r 2010 John Wiley & Sons, Ltd.
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Abstracts
Having developed high yielding synthesis of 1, we turned our attention on efficiently constructing aldehyde 4. First, the
reaction of unlabeled 1 with glycolic acid (2 equiv) in water with catalytic HNO3 at 100–1101C afforded intermediate 5 in
moderate yield. Product degradation resulted from unavoidable evaporation during this reaction. Because of this we explored
alternate reaction heating methods. Microwave heating and sealed vessel heating proved equally superior to the original
open reflux reaction conditions, with the sealed vessel method being more appropriate for processing radioactivity. With this
information in hand a solution of 99 mCi of 1, 2 equivalents of glycolic acid, water and catalytic HNO3 was heated at 1101C in a
sealed vessel for 40 hours to afford high radiochemical purity 2, Scheme 1. Azeotropic removal of water by ethanol afforded
solid 2 that was used without purification in the next step. A suspension of 2 in ethanol was reacted with 1.8 equivalents
of acetylacetone and catalytic AcOH at 701C for 1.5 h to afford 78 mCi of triazolopyrimidine 3 after flash column chromato-
graphy. Finally, 3 was oxidized with PhI(OAc)2 in the presence of TEMPO radical to afford 62 mCi of 4 after flash column
chromatography.
Summary: [14C]Aminoguanidine bicarbonate (1) was initially prepared in low yield by hydrazination of Ba14CN2. A more efficient
process for synthesizing 1 was developed that involved converting Ba14CN2 to S-methyliso[14C]thiourea and reacting that
intermediate with hydrazine. Reaction of 1 with glycolic acid in a sealed vessel afforded triazole 2 which was directly reacted with
acetylacetone to afford triazolopyrimidine 3. PhI(OAc)2/TEMPO mediated oxidation of
triazolo[1,5a(14C)]pyrimidine-2-carbaldehyde (4) in high overall yield.
3 delivered 5,7-dimethyl-1,2,4-
References
[1] D. Camp, C. F. Mathews, S. T. Neville, M. Rouns, R. W. Scott, Y. Truong, Org. Process Res. Dev., 2006, 10, 814–821.
[2] (a) F. Kurzer, L. Godfrey, Chemistry & Industry 1962, 1584-1594; (b) E. Lieber, G. B. L. Smith, Chem. Rev. 1939, 25, 213–271.
[3] C. O. Manning, A. H. Wadsworth, I. Fellows, J. Label. Compd. Radiopharm. 2002, 45, 611–618.
[4] (a) M. M. Cashyap, M. B. Mitchell, S. D. Osborne, D. Saunders, J. Label. Compd. Radiopharm. 1985, 22, 1239–1247; (b) M. A.
Armitage, M. M. Cashyap, D. Saunders, J. Label. Compd. Radiopharm. 1987, 24, 431–445.
[5] D. Plano, C. Sanmartin, E. Moreno, C. Prior, A. Calvo, J. A. Palop, Bioorg. Med. Chem. Lett. 2007, 17, 6853–6859.
CHALLENGES AND DIFFICULTIES IN SYNTHESIS OF TRITIUM LABELED FLUOCINOLONE ACETONIDE
DESONG ZHONG AND ANITA H. LEWIN
Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC 27709, USA
Abstract: Tritium labeled fluocinolone acetonide was synthesized through five-step procedure. Many challenges and difficulties
were encountered in the synthesis, especially in the regeneration of 1,2 double bond from reaction of oxidative de-hydrogenation
of tritium labeled 4-ene analog. Selenium oxide oxidative de-hydrogenation of 11,21-dihydroxy un-protected 4-ene fluocinolone
acetonide analog gave a complex product mixture. The reaction gave a clean unlabeled intermediate after protection of
21-hydroxy group by acetylation, but the same procedure failed to give desired tritium labeled intermediate due to fast
tritium-hydrogen exchange reaction. The de-hydrogenation proceeded well with DDQ in refluxing benzene for unlabeled
intermediate, but no reaction at all for the tritium labeled one. Benzeneseleninic anhydride/toluene refluxed with tritium
labeled 4-ene analog was found to be the best de-hydrogenation reaction conditions after exploring suitable conditions. Detailed
examination of the de-hydrogenation product, 1,4-diene analog by radio-HPLC, HPLC-MS, proton and tritium NMR found that not
only had the de-hydrogenation taken place, but also 11-hydroxy group was oxidized to ketone at the same time. This problem was
resolved by acetylation protection of both 11,21-dihydoxy groups. De-protection of tritium labeled 11,21-diacetyl 1,4-diene
intermediate in K2CO3/CH3OH/H2O gave expected final product, [1,2-3H]fluocinolone acetonide with a specific activity of 36.8 Ci/
mmol (radiochemical purity: 97%) after purification.
Keywords: fluocinolone acetonide; oxidative de-hydrogenation; tritium labeled, benzeneseleninic anhydride
Introduction: Fluocinolone acetonide is a compound with anti-inflammatory, anti-pruritic properties and it has been used for
treatment of various kinds of skin disorders since 1965 for its glucocorticoid activity. In addition, it has also been used for ear, eye,
and nose inflammatory disorders.1 Recent uses of fluocinolone acetonide include treatment of melasma when combined with other
components in a cream,2 treatment of retinopathy3 and uveitis.4 Tritium labeled fluocinolone acetonide with high specific activity
was needed in receptor binding studies. To our best knowledge, tritium labeling of fluocinolone acetonide has not been reported
although carbon-14 labeled version was used in mice5 and human.6
Results and Discussion: Based on literature data7 and our knowledge of the reactions of this compound class, Scheme 1, shown
below, seemed a straightforward approach to tritium labeled fluocinolone acetonide [3H]-1.
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Abstracts
OH
O
OAc
OAc
O
O
O
O
1. HCO2H
2. KOH
(Ac)2O
HO
O
HO
OH
OAc
HO
O
F
F
F
3
O
2
O
1
O
F
F
F
/
T2 [(Ph)3P]3RhCl
OH
OAc
OAc
O
O
O
HO
HO
OH
OH
OH
OAc
KOH
HO
OH
OAc
SeO2
T
T
T
F
F
F
T
T
T
[3H]-4
O
O
[3H]-6
O
[3H]-5
F
F
F
acetone
HClO4
OH
O
O
HO
O
T
F
T
[3H]-1
O
F
Scheme 1.
The key steps would be the selective catalytic tritiation of 1,2-double bond of unlabeled fluocinolone acetonide and regeneration
of the 1,2-double bond by oxidative de-hydrogenation after tritiation reaction.
In order to save time and to improve radiochemical yield, we briefly investigated the shortest synthesis route (Scheme 2, shown
below). Hydrogenation of fluocinolone acetonide under the modified literature condition[vii] using Wilkinson’s catalyst gave the
expected unlabeled intermediate 7. Oxidative de-hydrogenation of 7 using selenium oxide as oxidizing agent gave a multi-
component mixture (HPLC). This result was not totally surprising since the reported[ix] yield for this kind of reaction was only about
10% even when 21-hydroxy group was acetyl protected.
OH
O
OH
O
OH
O
O
O
O
SeO2
[(Ph)3P]3RhCl
toluene/THF/T2
HO
HO
O
HO
O
O
T
T
F
F
F
T
T
[3H]-7
[3H]-1
O
O
1
O
F
F
F
Scheme 2.
It was therefore decided to protect the 21-hydroxy group and obtain tritium labeled fluocinolone acetonide as shown in Scheme 3.
Selective protection of the 21-hydroxy group (secondary alcohol) was achieved with acetic anhydride using trimethylsilyltri-
fluoromethanesulfonate in methylene chloride. Selective hydrogenation of 21-acetate gave the expected 4-ene ([3H]-8) analogue.
J. Label Compd. Radiopharm 2010, 53 239–268
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Abstracts
Oxidative de-hydrogenation of the 4-ene using selenium dioxide in pyridine and t-butanol under reflux (at 951C) re-generated the
1,2-double bond. Selective de-protection of the 21-acetate without hydrolyzing the acetonide under separately optimized condition
(NaHCO3/CH3OH/H2O) gave fluocinolone acetonide.
Problems were encountered when the same approach was applied to the radiosynthesis. Selective tritiation of 21-acetate 2
with tritium gas using Wilkinson’s catalyst in toluene/THF gave tritiated 4-ene in good yield (317 mCi, 26% radiochemical
yield). Refluxing of a mixture of tritiated 4-ene with selenium dioxide in pyridine/t-butanol did not give the expected 1,4-diene
[3H]-2 although the radio-HPLC peak of tritiated 4-ene ([3H]-8) disappeared. Instead, most of the radioactivity appeared in
the solvent front. This implied that tritium-hydrogen exchange preceded oxidative de-hydrogenation under the reaction
condition
OH
O
OAc
O
OAc
O
O
O
O
(Ac)2O/CH2Cl2
CF3SO3Si(CH3)3
[(Ph)3P]3RhCl
HO
O
HO
HO
O
O
T
F
F
F
T
toluene/THF/T2
[3H]-8
O
2
O
1
O
F
F
F
SeO2
pyridine
t-butanol
95 oC
OH
O
OAc
O
O
O
NaHCO3
HO
O
HO
O
T
T
F
F
T
T
CH3OH/H2O
[3H]-1
[3H]-2
O
O
F
F
Scheme 3.
When oxidative de-hydrogenation of unlabeled 4-ene 8 was tried using DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) in
refluxing benzene10 for 14 hours, clean unlabeled 1,4-diene 2 was obtained. Surprisingly, no oxidative de-hydrogenation took place
when tritium labeled 4-ene [3H]-8 was treated under the same condition. Instead, most of [3H]-8 was recovered.
It was concluded that oxidative de-hydrogenation conditions should be explored with tritium labeled 4-ene [3H]-8.
Different oxidative de-hydrogenation conditions, including refluxing selenium dioxide/dioxane,11,hn/DIB/I2/CH2Cl2,hn/DIB/I2/
toluene,12, refluxing iodoxybenzene/benzeneseleninic anhydride/toluene,13 and refluxing benzeneseleninic anhydride/toluene,8
were tried to regenerate the 1,2-double bond from tritium labeled 4-ene [3H]-8. Only the last condition (refluxing
benzeneseleninic anhydride/toluene), followed by de-acetylation gave a mixture displaying two vinyl resonances and two
aliphatic resonances in the tritium NMR spectrum (Figure 1). The chemical shifts of the latter were not consistent with those of
the tritiated starting material. Moreover, HPLC co-injection of this material, which was expected to contain the tritium labeled
final product [3H]-1, with authentic fluocinolone acetonide (1) generated two very closely eluting UV peaks (Figure 2).
Treatment of the product with metal scavengers showed that the differences in the NMR and HPLC profiles of the unlabeled
reference and the tritiated product were not caused by complexation of transition metal (from Wilkinson’s catalyst) with the
product. The different retention time could also be caused by an isotope effect, but this possibility was removed by observing
that the proton NMR spectra of purified product (thought-to-be [3H]-1) and the authentic unlabeled fluocinolone acetonide
were different. Mass spectrometric analysis of the de-hydrogenation product obtained from unlabeled 4-ene 8 under the same
condition followed by de-protection showed that the 11-hydroxy group was oxidized to ketone. In other words, the de-
hydrogenation product from tritium labeled 4-ene [3H]-8 using refluxing benzeneseleninic anhydride/toluene was [3H]-9, not
expected [3H]-2 (Scheme 4). This is noteworthy since it has been reported that the 11-hydroxy was intact when DDQ was used
for the same class of compound.14,15
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Abstracts
Figure 1. Tritium NMR: top: [3H]-8; bottom: de-protected intermediate from refluxing benzeneseleninic anhydride/toluene.
Figure 2. HPLC co-injection of de-protected intermediate from refluxing benzeneseleninic anhydride/toluene with authentic unlabeled fluocinolone acetonide: top: UV
channel (235 nm); bottom: b-RAM channel.
OAc
OH
OAc
O
O
O
HO
O
benzeneseleninic anhydride
toluene/reflux
O
O
O
O
K2CO3
T
T
T
F
O
F
O
F
O
T
T
T
CH3OH/H2O
[3H]-8
[3H]-10
O
[3H]-9
O
O
F
F
F
Scheme 4.
J. Label Compd. Radiopharm 2010, 53 239–268
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
Based on these findings, the synthetic approach shown in Scheme 5 was used to complete the synthesis of [3H]-11. Reaction
conditions were probed using 11,21-diacetate, which was generated by reacting unlabeled 4-ene 8 with acetic anhydride in pyridine
and DMAP. Longer reaction time was needed in the oxidative de-hydrogenation of 4-ene-11,21-diacetate 11. The de-protection
condition (NaHCO3/CH3OH/H2O) used for 21-acetate 2 was found inappropriate for the 11,21-diacetate as the de-acetylation of
11-acetyl was sluggish and the de-protected product decomposed during longer reaction time. The best de-protection condition
found for 11,21-diacetate was K2CO3/CH3OH/H2O.
The radiosynthesis was continued by recovering tritium labeled 11-hydroxy-4-ene- [3H]-8 (261 mCi) from an unsuccessful DDQ
oxidation attempt using an alumina solid phase extraction cartridge. Acetylation of the recovered material with acetic anhydride in
pyridine and DMAP gave 11,21-diacetate (238 mCi) and this material was oxidatively de-hydrogenated using refluxing
benzeneseleninic anhydride/toluene to afford 1,4-diene [3H]-12. This required a much longer period of time (37 hours) than the
analogous reaction with unlabeled material (7 hours), but the reaction was clean. After C-18 solid phase extraction cartridge
purification, two fractions, 71.3 mCi (98% radiochemical purity), and 21 mCi (76% radiochemical purity), were obtained. Separate
de-protection of the two fractions gave crude final product [3H]-1 (60.4 mCi with 49% radiochemical purity and 15.9 mCi with 49%
radiochemical purity, respectively). The pure final product [3H]-1 (30.8 mCi with 97% radiochemical purity and 95% chemical purity)
was obtained from prep-HPLC purification of the combined fractions. The specific activity (36.8 Ci/mmol) was determined using an
HPLC calibration curve generated from the authentic unlabeled fluocinolone acetonide (1). The product identity was confirmed by
HPLC co-injection with the authentic unlabeled fluocinolone acetonide.
OH
OAc
OAc
O
O
O
(Ac)2O
[(Ph)3P]3 RhCl
toluene/THF/T2
HO
O
HO
HO
O
O
T
F
O
F
O
F
O
CF3SO3Si(CH3)3
T
[3H]-8
O
2
1
O
O
F
F
F
(Ac)2 O
pyridine
DMAP
OH
O
OAc
O
OAc
O
O
O
O
benzeneseleninic anhydride
HO
O
K2CO3
AcO
O
AcO
O
T
T
T
F
F
F
T
T
T
CH3OH/H2O
toluene/reflux
[3H]-1
[3H]-12
[3 H]-11
O
O
O
F
F
F
Scheme 5.
Conclusion: Tritium labeled fluocinolone acetonide (30.8 mCi) with high specific activity (36.8 Ci/mmol) was synthesized from
unlabeled fluocinolone acetonide by selective tritiation of the 1,2-double bond, followed by re-generation of the 1,2-double bond
by oxidative de-hydrogenation with benzeneseleninic anhydride in refluxing toluene. Protection of both the 21- and 11-hydroxy
functionalities was found to be necessary in the syntheses.
Acknowledgement: The authors are grateful to the NCI (Contract No. N01-CM-44208) for financial support and for the authentic
fluocinolone acetonide.
References
[1] W. Martindale in Martindale the Extra Pharmacopoeia, 30th ed. (Ed.: J. E. F. Reynolds), London, American Pharmaceutical
Association, 1993, p. 732.
[2] T. Cestaro, L. Adjadj, M. Hux, M. R. Shimizu, V. P. Rives, J. Drugs Mermato. 2007, 6(2), 153–160.
[3] S. G. Schwartz, H. W. Flynn Jr., Exp. Diabetes Res. 2007, 2007, 52487.
[4] M. V. Brumm, Q. D. Nguyen, Int. J. Nanomed. 2007, 2(1), 55–64. T. Takahashi, T. Kimura, T. Meshi, Y. Sato, Chem. Pharm. Bull.
(Tokyo) 1971, 19(2), 309–316.
[5] T. Takahashi, T. Kimura, T. Meshi, Y. Sato, Chem. Pharm. Bull. (Tokyo) 1971, 19(2), 309–316.
[6] M. D. Chaplin, W. C. Cooper, E. J. Segre, J. Oren, R. E. Jones, C. Nerenberg, J. Allergy Clin. Immunol. 1980, 65(6), 445–453.
[7] B. A. Thalen, B. I. Axelsson, P. H. Anderson, R. L. Brattsand, B. Nylander, Steroids 1998, 63(1), 37–43.
[8] D. Hesk, P. Delduca, D. Koharski, P. McNamara, C. Magatti, S. Saluja, L. Thomas. J. Labelled Comp. Radiopharm. 1993, 33(5),
439–442.
[9] J. S. Mills, A. Bowers, C. Djerassi, H. J. Ringold, J. Am. Chem. Soc. 1960, 82(13), 3399–3404.
[10] G. Woods, J. Cairns, G. McGarry, Alkylierte 3,20-Diketo-Delta4-steroide der Pregnanreihe. German Patent No. 2301317, 1973.
[11] C. Meystre, H. Frey, W. Voser, A. Wettstein, Helv. Chim. Acta. 1956, 39(3), 734–742.
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
[12] A. S. Veleiro, P. J. Taich, L. D. Alvarez, P. H. Di Chenna, G. Burton, Tetrahedron Lett. 2005, 46, 4235–4238.
[13] D. H. R. Barton, C. R. A. Godfrey, J. W. Morzycki, W. B. Motherwell, and A. Stobie, Tetrahedron Lett. 1982, 23(9), 957–960.
[14] D. H. Barton, N. K. Basu, M. J. Day, R. H. Hesse, M. M. Pechet, A. N. Starratt, J. Chem. Soc. [Perkin 1] 1975, 22, 2243–2251.
[15] F. M. Kaspersen, C. W. Funke, J. Vader, G. N. Wagenaars J. Labelled Compd. Radiopharm. 1993, 33(5), 409-418.
SYNTHETIC STUDIES OF [M15] HYDROXYMIDAZOLAM
ZHIGANG JIAN, AMY WU, MELISSA LIN, LAWRENCE JONES AND TAPAN RAY
Isotope Lab, Novartis Institutes for BioMedical Research Translational Sciences, One Health Plaza, East Hanover, NJ 07936, USA
Abstract: Midazolam is often used for drug interaction research and stable isotope labeled hydroxymidazolam is needed for those
studies. Although the synthesis of an M13 hydroxymidazolam has been published, it is much more desirable to have a stable isotope
labeled hydroxymidazolam which has a molecular weight of 4 or more mass units higher than the parent compound because of the
presence of a chlorine atom in the molecule. Herein we report our efforts on the synthesis of M15 hydroxymidazolam, in which the key
step is the use of 3-(4-tosyl)imidazo[1,5-a][1,4]-benzodiazepines. Details of the synthesis will be discussed.
Keywords: stable isotope labeling; midazolam; hydroxymidazolam; synthesis
Introduction: Hydroxymidazolam is a metabolite of midazolam which is an ultra short-acting benzodiazepine derivative that has
found many therapeutical applications including anxiolytics, hypnotic, anticonvulsant, and as a skeletal muscle relaxant1. Stable
isotope labeled hydroxymidazolam is often used as an internal standard in bio-analytical studies.
Two chemical syntheses have been reported for hydroxymidazolam, one of which used midazolam as the key intermediate. The
oxidation of midazolam and subsequent rearrangement furnished hydroxymidazolam in very low yield.2 The other reported synthesis
of a stable isotope labeled hydroxymidazolam with 3 mass unites higher than the non-labeled parent compound.3 It avoided the low
yield oxidation step and is a better synthetic route from chemical synthesis point of view. But it is much more desirable to have a
stable isotope labeled hydroxymidazolam with a molecular weight of 4 or more mass units higher than the parent compound
because of the presence of a chlorine atom in the molecule. A recent publication reported the formation of imidazo[1,5-
a][1,4]benzodiazepines (2) by the reaction of 1,4-benzodiazepinic N-nitrosoamidines (1) with tosylmethyl isocyanide (Scheme 1). The
treatment of 2 with butyllithium and methyl iodide, followed by the removal of tosyl group gave rise to midazolam (4).4
This looked attractive to us since the anion of 2 could react with an oxygen carrying precursor to form a compound that could
later on be converted to hydroxymidazolam.
N
N
Tos
Tos
N NO
N
N
N
CH3C6H4SO2CH2NC
Cl
BuLi/CH3I
N
N
N
Cl
Cl
F
BuLi
F
F
3
2
1
N
N
2. Na-Hg
N
Cl
F
4
Scheme 1. Synthesis of Midazolam (4)
Results and Discussion: The synthesis of stable isotope labeled key intermediate 5 is outlined in Scheme 2. Thus, [13C, 15N]
methylamine was vacuum transferred to a methanol solution of methylformate with a catalytic amount of NaOMe to afford [13C, 15N]
methyl formamide, which was then treated with tosyl chloride in quinoline to afford [13C, 15N] methyl isocyanide. [13C, 15N] Methyl
isocyanide was converted to [13C, 15N] tosyl methylisocyanide by first treating with n-butyllithium, followed by reacting with tosyl
fluoride. The coupling of [13C, 15N] tosyl methylisocyanide with N-nitrosoamidine (1) furnished [13C, 15N] imidazo[1,5-
a][1,4]benzodiazepines (5) in a reasonable overall yield (25%).
J. Label Compd. Radiopharm 2010, 53 239–268
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
O
TosCl
LDA
TosF
Methyl formate
Quinoline
CH NH
CH NH
Tos CH
N
CH
N
H
NaOMe/MeOH
93%
75%
43%
#
N
*
Tos
N
NO
N
N
Tos CH
N
N
N
Cl
Cl
F
F
BuLi/82%
1
5
# Denotes N-15 and * denotes C-13
Scheme 2. Synthesis of 5.
Our initial plan was to introduce a [13C] carboxylic group in the imidazole ring to form the carboxylic acid (6); the removal of the
tosyl group and the reduction of the carboxylic group with LiAlD4 should provide [M15] hydroxymidazolam (7) (Scheme 3).
#
#
#
N
D
*
O
Tos
N
*
N
Tos
*
*
HO
HO
*
N
N
N
D
1. Na-Hg amalgam
2. LiAlD
BuLi/
O
C
N
N
Cl
N
Cl
Cl
F
F
F
H
5
7
6
Scheme 3. Planned Synthetic Route for M15 Hydroxymidazolam (7).
Unfortunately, the carboxylation reaction failed to provide carboxylic acid 6. Compound 6 was so unstable, due to the presence of
a strong electron withdrawing tosyl group in the imidazole ring, that decarboxylation occurred after the reaction mixture was
neutralized. LC-MS analysis of the reaction mixture showed the presence of a small amount of desired product (9%) immediately
after the reaction mixture was acidified. But no product was detected by LC-MS the next day.
We then decided to synthesize the tosyl hydroxymidazolam directly from [13C, 15N] imidazo[1,5-a][1,4]benzodiazepines (5) and
[13CD2] paraformaldehyde. Thus, 5 was first treated with one equivalent of n-butyllithium in THF, and then [13CD2] formaldehyde,
obtained by heating [13CD2] paraformaldehyde at 1801C, was passed through the reaction mixture with the help of a slow stream of
nitrogen gas. The desired product 8 was obtained in moderate yield (about 40%), along with unreacted starting material, and was
used directly in the next step. Unfortunately, treatment of 8 with sodium-mercury amalgam did not afford any desired product 7.
Instead, it produced the hydroxydihydromidazolam (10) as the major product. LC-MS analysis of incomplete reaction mixture
indicated that the reaction occurred in a stepwise manner: the tosyl hydroxydihydromidazolam (9) formed first, and subsequent
de-tosylation of 9 afforded 10 as the final product (Scheme 4).
The result was a surprise to us. We did not expect the hydroxymethyl group in 8 to have such an effect over the methyl group in
compound 3, both in the imidazole ring, that it induced hydrogenation at a distance carbon-nitrogen double bond prior to the
removal of the tosyl group, while compound 3 afforded midazolam (4) with the carbon-nitrogen double bond intact, under the
exact same reaction conditions.4
This prompted us to prepare stable isotope labeled midazolam according to the published procedure.4 Our intention was to verify
that the removal of the tosyl group would not cause saturation of the carbon-nitrogen double bond, as the authors had claimed,
and if this was true, to synthesize [M15] hydroxymidazolam from midazolam according to the published procedure.2
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J. Label Compd. Radiopharm 2010, 53 239–268

Abstracts
#
#
#
N
*
Tos
BuLi/13CD2O
N
N
Tos
*
*
*
HOD2C
*
HOD2C
N
N
N
Na-Hg Amalgam
N
Cl
N
N
Cl
Cl
F
F
F
5
8
7
#
#
N
N
Tos
*
*
*
*
HOD2C
Na-Hg Amalgum
HOD2C
N
N
NH
NH
Cl
Cl
F
F
9
10
Scheme 4. Attempted Synthesis of M15 Hydroxymidazolam (7).
Stable isotope labeled tosyl midazolam (11) was synthesized by treating 5 with n-butyllithium and [13CD3] methyl iodide. Contrary
to what the authors had claimed4, no stable isotope labeled midazolam (12) was detected when 11 was treated with Na-Hg
amalgam. LC-MS analysis of the reaction mixture indicated that tosyl dihydromidazolam (13) and the corresponding de-chlorinated
product 14 was produced initially, and de-tosylation products 15 and 16 formed afterwards (Scheme 5).
#
D
D
#
#
N
N
N
Tos
*
Tos
*
*
*
*
D
D
N
N
N
D
D
Na-Hg
BuLi/CD3I
N
N
N
Cl
Cl
Cl
F
F
F
5
11
12
D
#
#
N
N
D
D
#
Tos
*
*
D
N
Tos
*
Tos
*
*
D
*
N
D
D
Na-Hg
N
N
D
D
N
Incomplete
Reaction
Cl
NH
+
NH
Cl
F
F
F
11
14
13
D
#
D
#
N
N
*
*
D
*
*
D
D
N
N
D
Na-Hg
NH
+
NH
Cl
Complete
Reaction
F
F
16
15
Scheme 5. Attempted Synthesis of M16 Midazolam (12).
This result was partially consistent with our work with tosyl hydroxymidazolam and clearly indicated that de-tosylation reaction
will not lead to the formation of desired product directly.
J. Label Compd. Radiopharm 2010, 53 239–268
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
We are currently pursuing the synthesis of stable isotope labeled hydroxymidazolam through a modified synthetic plan (Scheme 6).
The key of the new synthesis is to re-introduce the carbon-nitrogen double bond to form 19 after the removal of the tosyl group in
acetyl hydroxydihydromidazolam (18). Hydrolysis of 19 should provide [M15] hydroxymidazolam (7).
#
#
#
N
*
N
*
N
Tos
*
HOD2C
*
*
Tos
*
AcOD2C
AcOD2C
N
N
N
Na/Hg
N
NH
Cl
N
Cl
Cl
F
F
F
8
18
17
#
#
N
*
N
HOD2C
*
*
*
AcOD2C
N
N
MnO2
N
N
Cl
Cl
F
F
7
19
Scheme 6. Revised Synthetic Plan for M15 Hydroxymidazolam (7).
Conclusion: The synthesis of [M15] hydroxymidazolam through a key intermediate of [13C, 15N] imidazo[1,5-a][1,4]benzodia-
zepines (5) was investigated. The de-tosylation reaction of both tosyl hydroxymidazolam (8) and tosyl midazolam (11),
unfortunately, failed to afford [M15] hydroxymidazolam (7) and [M16] midazolam (12) respectively. A modified synthesis of [M15]
hydroxymidazolam through the re-introduction of double bond after the removal of the tosyl group is currently under investigation.
References
[1] C. C. Eduardo, E. M. Isidro, O. G. Mellado. J. Heterocyclic Chem., 2002, 39, 1189-1193 and the references cited therein.
[2] Armin Walser, Louis E. Benjamin, Sr., Thomas Flynn, Carl Mason, Robert Schwartz, and R. Ian Fryer, J. Org. Chem., 1978, 43(5),
936–944.
[3] Yinsheng Zhang, Peter W.K. Woo, John Hartman, Norman Colbry, Yun Huang, and Che C. Huang, Tetrahedron Lett., 2005, 46,
2087–2091.
[4] Carlos del Pozo, Alberto Macias, Eduardo Alonso, and Javier Gonzalez, Synthesis, 2004, 16, 2697–2703.
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J. Label Compd. Radiopharm 2010, 53 239–268