First synthesis of [1,3,5-13C3]gallic acid

Article abstract of DOI:10.1002/jlcr.1776

Gallic acid is a phenolic plant metabolite which is normally encountered in plant tissues in ester form. These esters include a group of catechins with antioxidant properties which are found in green tea. Epigallocatechin-3-gallate is the most abundant ca

Full text of DOI:10.1002/jlcr.1776

Abstracts
Published online 2 June 2010 in Wiley Interscience
(
www.interscience.wiley.com) DOI: 10.1002/jlcr.1776
10th International Symposium on the
Synthesis and Applications of Isotopes and
Isotopically Labelled Compounds – Poster
Presentations
Session 19, Sunday, June 14 to Thursday, June 18, 2009
SESSION CHAIR: WILLIAM WHEELER
IsotopicSolutions, LLC, USA
Abstract: The poster session is a series of papers dealing with a conglomeration of all of the previous sessions. Cristian Postolache,
the fourth of the Wiley Young Scientist Award winners gave a Poster in this session.
Keywords: carbon-14; tritium; fluorine-18; In-111m; panabinostat; sulphur-35; coLogica Isotope synthesis database; QAB149; corn
plant metabolism
MICROWAVE-ASSISTED SYNTHESIS OF A CARBON-14 LABELED PYRROLOTRIAZINE PRECURSOR FOR
CORE LABELED DRUG CANDIDATES
ALBAN J. ALLENTOFF, MICHAEL W. LAGO, SHARON GONG, SCOTT BACH TRAN, MARC D. OGAN, AND SAMUEL J. BONACORSI JR.
Department of Chemical Synthesis, Bristol-Myers Squibb Research and Development, One Squibb Drive, New Brunswick, NJ 08903, USA
Summary: The 5-methyl-pyrrolo[1,2-f][1,2,4]triazine system is a chemotype scaffold common to drug candidates within several
1
therapeutic areas. Core carbon-14 labeled ethyl 5-methyl-4-oxo-2-[ C]-3,4-dihydropyrrolo[1,2-f][1,2,4]triazine-6-carboxylate was
4
1
4
efficiently prepared via a microwave-assisted cyclization reaction of a known aminopyrrole with [ C]triethylorthoformate.
Keywords: microwave irradiation; pyrrolotriazine; core labeling; carbon-14; drug candidates; oncology; immunology
Introduction: The novel pyrrolotriazine 1 (Table 1, unlabeled) has been utilized by medicinal chemistry programs as a core chemotype for
1,2
elaboration into drug candidates within the oncology and immunology therapeutic areas. During the discovery and development of
drug candidates, radiolabeled analogues are required for profiling their disposition in humans and animals, and for in vitro reaction
phenotyping and transport studies. Therefore the preparation of 1 with carbon-14 ring labeling offered an opportunity to efficiently
prepare core labeled drug candidates for diverse discovery and development activities via a single carbon-14 labeled precursor. In order to
execute this approach, an efficient method for the preparation of substantial quantities of carbon-14 labeled 1 was required.
3
4
Previous work described by this group and others have shown the viability of utilizing carbon-14 labeled triethylorthoformate as
a labeled formate equivalent in acid catalyzed cyclization reactions affording ring radiolabeled heterocycles. We now report on the
1
cyclization of [ C]triethylorthoformate 3 with the known N-aminopyrrole 2 to efficiently prepare labeled 1.
4
5
Results and Discussion: Preparation of stable isotopically labeled 1 was previously described from the reaction of a stable-
labeled analogue of aminopyrrole 2 with 7.5 equivalents of unlabeled triethylorthoformate and catalytic amounts of p-toluene
5
sulfonic acid. These conditions afforded almost quantitative yield of the stable-labeled analogue of pyrrolotriazine 1 (Table 1, Entry
1
). In applying this reaction to carbon-14 incorporation, it was critical to lower the amounts of [ C]triethylorthoformate to
4
1
approximately one molar equivalent in comparison to 2 in order to obtain useful radiochemical yields. However, extensive heating
1
4
at 1201C of 2 with 1.1 equivalents of [ C]triethylorthoformate and p-toluenesulfonic acid (Table 1, Entry 2) gave crude product
containing large amounts of unreacted starting aminopyrrole 2. Purification via recrystallization and chromatography, afforded a
3
0% isolated radiochemical yield of 1.
1
Initially, we explored the 250 W microwave irradiation of aminopyrrole 2 with 0.66 molar equivalents of [ C]triethylorthoformate
4
1
Entry 3). These conditions allowed rapid reaction of 2 with [ C]triethylorthoformate affording crude product 1 which after
4
(
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.

Abstracts
Table 1. Effect of stoichiometry and reaction conditions on the yield of 1 from the cyclization of 2 and 3
O
O
O
NH2
NH2
1
4
NH
EtOOC
+
CH
EtOOC
N
14C
O
O
N
N
H
2
3
1
Entry
Molar equivalents
2
3
Reaction conditions (N,N-dimethylacetamide)
% yield 1 (isolated)
Ã
ÃÃ
ÃÃ
ÃÃ
1
2
3
4
1
1
1
1
7.5
1.1
0.66
1.3
731C, 40 min
701C, (18 h) then 1201C (18 h)
Microwave (200 W) 15 min, Temperature Max = 1501C
Microwave (200 W) 20 min, Temperature Max = 1501C
99%
30%
73%
53%
Ã
ÃÃ
Yield of 1 based on aminopyrrole 2. This reaction was conducted using stable-labeled reactants [5].
14
Radiochemical yield of 1 based on [ C]triethylorthoformate.
recrystallization followed by column chromatography yielded pure 1 in 73% radiochemical yield. In order to facilitate large scale
4
1
preparations, the microwave-assisted cyclization reaction was also performed using 1.3 equivalents of [ C]triethylorthoformate
Entry 4) affording crude 1 with no observed aminopyrrole 2 remaining. Simple precipitation of the product by addition of water
gave 1 in 53% radiochemical yield and radiochemical purity in excess of 97%.
(
Conclusions: We have developed a facile method for the preparation of a radiolabeled pyrrolotriazine chemotype scaffold using a
microwave-assisted cyclization reaction. This microwave-assisted process is readily scaled up and allowed the preparation of
sufficient quantities of the precursor to support multiple labeling projects.
References
[
1] J. Hynes Jr., A. J. Dykeman, S. Lin, S. T. Wrobleski, H. Wu, K. M. Gillooly, S. B. Kanner, H. Lonial, D. Loo, K. W. McIintyre, S. Pitt,
D. R. Shen, D. J. Shuster, X. Yang, R. Zhang, K. Behenia, H. Zhong, P. H. Marathe, A. M. Doweyko, J. S. Tokarski, J. S. Sack,
M. Pokross, S. E. Kiefer, J. A. Newitt, J. C. Barrish, J. Dodd, G. L. Schieven, K. Leftheris, J. Med Chem. 2008, 51, 4–16.
2] R. M. Borzilleri, X. Zheng, L. Qian, C. Ellis, Z.-W. Cai, B. S. Wautlet, S. Mortillo, R. Jeyaseelan Sr., D. W. Kukral, A. Fura, A. Kamath,
V. Vyas, J. S. Tokarski, J. C. Barrish, J. T. Hunt, L. J. Lombardo, J. Fargnoli, R. S. Bhide, J. Med. Chem. 2005, 48, 3991–4008.
3] M. D. Ogan, D. J. Kucera, Y. R. Pendri, J. K. Rinehart, J. Label. Compd. Radiopharm. 2005, 48, 645–655.
4] J. S. Valsborg, L. J. S. Knutsen, I. Lundt, C. Foged, J. Label. Compd. Radiopharm. 1995, 36, 457–464.
[
[
[
[
5] M. D. Ogan, S. B. Tran, J. K. Rinehart, J. Label. Compd. Radiopharm. 2006, 49, 139–145.
AN INNOVATIVE AND EFFICIENT SYNTHESIS OF STABLE ISOTOPE LABELLED N-METHYL PYRAZOLE
AUDREY ATHLAN, CALVIN MANNING, AND GEOFF BADMAN
Isotope Chemistry, GlaxoSmithKline Research and Development, Gunnels Wood Road, Stevenage SG1 2NY, UK
1
3
3
Abstract: Novel synthesis of stable isotopically labelled N-Methyl pyrazole in three steps from commercially available diethyl [ C ]
malonate. The pyrazole ring is obtained by condensation between bisacetal derivative and hydrazine.
Keywords: pyrazole synthesis; reductive acetylation; hydrazine; malonate ester
Introduction: The synthesis of pyrazoles is of great interest due to the wide application of such heterocylic motifs in the
s
s
s
1
2
3
pharmaceutical industry eg COX-2 inhibitor celecoxib (Celebrex ), CB1 antagonist acomplia (Rimonabant ) and sildenafil (Viagra ).
In support of metabolism studies, a stable isotope labelled (SIL) synthesis of an M16 N-methyl pyrazole buildling block was
required.
Synthesis of 1,2-pyrazole moiety is classically achieved by either a 1,3-dipolar cycloaddition of diazo compounds and alkynes or by
condensation of hydrazine with 1,3 difunctionalised molecules, generally diketones. Such syntheses of a SIL version of
N-methylpyrazole appeared quite challenging, since the number of isotopically labelled starting materials for either route is quite
limited and we had therefore to design an alternative strategy towards this molecule.
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
4
Results and discussion: A literature review showed a recent patent describing the synthesis of N-methylpyrazole from 1,3-
bis(ethyloxy)-1,3-bis(methyloxy)propane and methylhydrazine (Scheme 1).
Me
OMe OMe
OEt
2
MeNH -NH2
N
N
EtO
HCl, H O
2
9
3%
Scheme 1.
5
Furthermore, Kopecky and Rychnovsky reported the synthesis of bisacetals by reductive acetylation of acyclic ester such as
malonate esters. This reaction proceeds via an aluminium acetal intermediate, which subsequently undergoes an in situ acetylation
(
Scheme 2).
EtO
EtO
EtO
EtO
EtO
EtO
2
) Ac O, Pyridine, DMAP
1) DIBAL, DCM
2
O
O
OAl(iBu)2
OAl(iBu)2
OAc
OAc
75%
Scheme 2.
Encouraged by these results, we investigated the possibility of a Knorr condensation between hydrazine and bisacetal synthesised
1
3
from readily available diethyl [ C ] malonate (Scheme3).
3
1
2
) DIBAL, DCM
NH -NH
H
2
2
) Ac O, Pyridine,
OAc OAc
2
O
O
N13
MeOH
1
3
13
AcOH
DMAP
1
3
13
C
13
C
N
13
C
C13
C
O
C
O
13
O
C
O
58%
C C
7
7%
1
2
3
Scheme 3.
1
3
3
M1 6 labelled pyrazole moieties was then obtained by N-Methylation in presence of [ CD ] iodomethane. Purification and
isolation of this volatile material was problematic. We therefore designed a one-pot procedure, where the labelled pyrazole 3
undergoes an N-methylation followed by an in situ boronylation. The labelled pyrazole molecule 4 was successfully used in a Suzuki
reaction (Scheme 4).
X
X
Br
H
13
13
X
Pd(PPh ) K CO
3 4, 2 3
CD3
1
) nBuLi, 13CD I, THF
CD3
3
O
N1
3
DME
2) nBuLi, PINBOP
N13
N13
B
N
C
N
C
N
C
X
13
13
O
C
C
13 13
13 13
7
3%
69%
C
C
C
C
3
4
5
Scheme 4.
Conclusion: We have developed an economical and efficient route towards labelled pyrazoles using SIL malonaldehyde equivalent.
1
M17 pyrazole 4 was thus synthesised in three steps from commercially available diethyl [ C ] malonate with an overall yield of 33%.
3
3
1
5
2
This approach appears versatile and other alkyl pyrazoles could potentially be generated. [ N ] hydrazine could also be used to
prepare M15 pyrazole.
We have also demonstrated that the labelled pyrazole boronylated species could successfully be used as a building block in cross-
coupling reactions.
References
[
[
[
[
[
1] T. D. Penning, J. J. Talley, S. R. Bertenshaw, J. S. Carte, J. Med. Chem. 1997, 40, 1347–1365.
2] S. R. Donohue, C. Halldin, V. W. Pike, Tett. Lett. 2008, 49, 2789–2791.
3] N. K. Terrett, A. S. Bell, D. Brown, P. Ellis, Bioorg. Med. Chem. Lett. 1996, 6, 1819–1824.
4] Ger. Offen. 10057194, 29 May 2002.
5] D. J. Kopecky, S. D. Rychnovsky, J. Org. Chem. 2000, 65, 191–198.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
THE SYNTHESIS OF CARBON-14 LABELLED 3H-QUINAZOLIN-4-ONE BUILDING BLOCKS
RYAN A. BRAGG, NICK BUSHBY, JOHN R. HARDING, DAVID A. KILLICK AND ANGELA JORDAN
Isotope Chemistry, Clinical Pharmacology & DMPK, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK
Abstract: The synthesis of carbon-14 labelled, 5-, 6-, 7- and 6,7- substituted, 3H-quinazolin-4-one building blocks are presented.
Keywords: isotopic labelling; carbon-14; 3H-quinazolin-4-one
Introduction: Over the past decade, 4-anilinoquinazolines have emerged as an important class of anticancer agent, as exemplified
1
by gefitinib and erlotinib (Figure 1). To support drug metabolism and distribution studies of this class of compound, a number of
carbon-14 labelled materials were required. Early labelling work on these compounds focused upon labelling the side chains of
2
4-anilinoquinazolines, based on the assumption that labelling the quinazoline core required a large excess of the labelled reagent.
F
O
HN
N
Cl
N
O
N
MeO
gefitinib
HN
O
MeO
MeO
N
O
N
erlotinib
Figure 1. Structures of gefitinib and erlotinib.
However, labelling the quinazoline core of a molecule is often desirable as this provides material with the label in a metabolically
stable position. We detail here the synthesis of carbon-14 labelled, 5-, 6-, 7- and 6,7- substituted, 3H-quinazolin-4-one building
blocks, the precursors of substituted 4-anilinoquinazolines.
Results and Discussion: Our first approach to labelled quinazolinones is shown in Scheme 1. Reaction of 2-amino-4-fluoro-
1
4
14
[
this approach was successful, the cost of carbon-14 labelled, tri-substituted, aromatic carboxylic acids is often high.
1- C]-benzoic acid with 2.0 equiv. of formamidine acetate at 801C gave 7-fluoro-3H-[4- C]-quinazolin-4-one in 76% yield. Whilst
14
3
We therefore turned our attention to labelling the quinazolinone in the 2-position using commercially available [ C]-sodium formate.
As shown in Scheme 2, reaction of 2-amino-4,5-dimethoxybenzamide with 1.05 equiv. of [ C]-sodium formate gave 6,7-dimethoxy-
H-[2- C]-quinazolin-4-one in 84% yield. However, it was necessary to heat this reaction to 1401C in a Carius tube for 2 days.
1
4
14
3
O
O
14C
14C
formamidine acetate
,3-propane diol
0oC , 1 hr
OH
NH
1
F
NH2
F
N
8
5
6 mCi/mmol
Scheme 1.
O
O
MeO
MeO
MeO
MeO
[14C]-sodium formate
HCl, water
NH2
NH2
NH
14C
N
o
1
40 C (Carius tube), 2 days
57 mCi/mmol
Scheme 2.
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
F
O
F
O
[14
C]-formamidine acetate
EtOH
OMe
NH2
NH
14C
N
9
0oC, 20 hrs
5
5m Ci/mmol
Scheme 3.
O
O
[14
C]-formamidine acetate
Br
Br
OMe
NH2
NH
EtOH
14C
o
N
150 C (
), 20 mins
5
9 mCi/mmol
Scheme 4.
1
A more practical procedure was used for the synthesis of 5-fluoro-3H-[2- C]-quinazolin-4-one as shown in Scheme 3. In this
4
4
1
4
example, 2-amino-6-fluorobenzoic acid methyl ester was treated with 1.25 equiv. of [ C]-formamidine acetate to give the desired
product in 71% yield after heating in ethanol for 20 hours.
1
A further refinement of this approach was used for the synthesis of 6-bromo-3H-[2- C]-quinazolin-4-one (Scheme 4). Heating
4
1
4
2
-amino-5-bromobenzoic acid methyl ester and [ C]-formamidine acetate at 1501C in a microwave for 20 minutes gave the desired
compound in 83% yield. The equivalent thermal reaction required 20 hours at reflux.
Conclusions: In conclusion, we have demonstrated that carbon-14 labelled, 5-, 6-, 7- and 6,7- substituted 3H-quinazolin-4-ones
can be prepared by a number of different approaches. Furthermore, the use of labelled formamindine acetate in conjunction with
microwave heating has been shown to provide efficient access to these valuable building blocks.
1
4
Acknowledgements: The authors would like to acknowledge GE Healthcare for the synthesis of 6,7-dimethoxy-3H-[2- C]-
quinazolin-4-one.
References
[
[
1] G. Blackledge, S. Averbuch, Br. J. Cancer 2004, 90, 566–572.
2] J. A. Bergin, H. Booth, N. Bushby, J. R. Harding, D. Killick, C. D. King, D. J. Wilkinson, J. Label. Compd. Radiopharm. 2004, 47,
2
3] E. J. Merrill, J. Label. Compd. Radiopharm. 1969, 5, 346–350.
99–334.
[
[
4] Y. Zhang, Y. Huang, C. C. Huang, J. Label. Compd. Radiopharm. 2005, 48, 485–496.
1
THE SYNTHESIS, CHARACTERIZATION AND CHEMISTRY OF [METHYL- C] METHYL NOSYLATE
4
STEVEN A. CARR, LINDA P. LACY, SCOT POUNDS, AND CRIST N. FILER
PerkinElmer Health Sciences Inc., 940 Winter Street, Waltham, MA 02451, USA
1
Abstract: A method for the preparation of [methyl- C] methyl nosylate is described as well as representative examples of its use as
4
a carbon-14 alkylating reagent.
1
4
Keywords: [ C] methyl iodide; [ C] methyl nosylate; methylation
14
Introduction: The use of substances labelled with carbon-14 has been extraordinarily valuable in many aspects of the life sciences
for over half a century. One of the most versatile and simple ways to install carbon-14 in many compounds of interest is by
1
methylating available heteroatoms like nitrogen, oxygen and sulfur to afford corresponding methyl- C analogues. Over the years,
4
1
4
[
C] methyl iodide has been a convenient electrophilic reagent of choice to employ in this regard. However recently, safety
1
14
regulations have restricted the shipment of [ C] methyl iodide by air and its limited stability has made other forms of prolonged
transportation impractical and inadvisable. For that reason we decided to explore the synthesis, characterization and chemistry of
1
methyl- C] methyl nosylate (1) as a useful alternative carbon-14 radiomethylating reagent.
4
[
O
O14CH
3
O N
S
2
O
1
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
2
,3
Results and Discussion: The synthesis of 1 is analogous to that of the corresponding tritiated reagent first reported by Pounds
and is described in the experimental section. The methylation chemistry of 1 is similar to and equally straightforward as that of the
tritiated analogue and is illustrated in Scheme 1. Using 1, estrone (2) could easily be converted to methyl ether 3 in approximately
1
5% radiochemical yield, while [methyl- C] caffeine (5) and [methyl- C] yohimbine (7) were correspondingly prepared from their
4
14
9
respective desmethyl analogues in about 50% radiochemical yield. Employing 1 also permitted its safe addition to reaction vessels
1
4
without concern for volatile carbon-14 contamination as with [ C] methyl iodide. Also, subsequent portions of 1 could easily be
added to drive a radiomethylation reaction to completion.
Besides solving a transportation and regulatory challenge, the availability of reagent 1 has also been accompanied by another
1
4
valuable benefit; namely, significantly enhanced shelf life over that of [ C] methyl iodide. Preliminary studies have shown that the
stability of 1 when stored in a solution of hexane:ethyl acetate (4:1) at ꢀ201C is at least nine months before needing repurification. In
contrast, [ C] methyl iodide is well known to be stable for only a few weeks at best.
1
4
Experimental: All reagents and solvents were purchased from Sigma-Aldrich Chemical Company. Proton NMR spectra were
recorded on a Bruker 300 MHz spectrometer with internal TMS as reference standard.
1
4
Methyl- C] Methyl Nosylate (1)
[
Silver nosylate was freshly prepared in the following manner in the dark: Silver carbonate (2.08 g, 7.56 mmol) was placed in a round
bottom flask and suspended in 10 mL of acetonitrile under nitrogen. To the flask was then added 3.6 g (15.1 mmol) of
4-nitrobenzenesulfonic acid (85% pure) in 40 mL of acetonitrile with stirring over the course of 20 min. After the addition, the dark
green opaque solution was heated to 601C for 1 h. The reaction was then cooled and filtered free of fine black solids. The filtrate was
rotary evaporated and the resulting tan colored solid was crushed in a mortar and pestle in the dark. The silver nosylate was
transferred to a breakseal flask fitted with a septum and vacuum attachment followed by the addition of 65 mL of dry acetonitrile.
1
[
4
C] Methyl iodide (12.6 mmol at 51 mCi/mmol) was then added via syringe to the pre-evacuated reaction vessel and the syringe side
arm was flame sealed. The reaction was heated to 801C overnight and cooled to ambient temperature the next morning. The reaction
vessel was then attached to a vacuum line and the acetonitrile was distilled away leaving a residue which was dissolved in a minimum
amount of ethyl acetate. Silica gel flash chromatography purification using hexane:ethyl acetate (4:1) and pooling appropriate
fractions afforded 501 mCi (a 78% radiochemical yield) of 1 that was homogeneous on silica gel TLC (hexane:ethyl acetate (10:3)) and
provided a proton NMR (CDCl ) that was in concert with that of unlabelled methyl nosylate; d 8.35 (d, 2), 8.05 (d, 2), 3.75 ppm (s, 3)
3
1
4
General C Methylation Procedure. The compound to be methylated was dissolved in DMF at a concentration of 0.1–0.4 M. Excess
appropriate base was added, followed by stirring at ambient temperature for an hour. After this time, an equivalent of 1 was added
as a 0.02 M acetonitrile solution and stirring was continued at ambient temperature for several hours. The reaction could be
monitored by TLC and excess 1 was added if starting material was still seen to be present. When the reaction had progressed to
completeness, it was worked up by partitioning between an appropriate organic solvent and water followed by chromatographic
purification.
O
O
1
/ NaOH
14
HO
CH O
3
2
3
CH3
N
CH3
N
1
/ NaHCO
3
O
O
N
N
NH
N
N
N
14CH3
H C
O
H C
O
3
3
4
5
1
/ K CO
2
3
N
H
N
H
N
H
H
N
H
H
H
H
OH
OH
HO C
14
2
CH O C
3
2
6
7
Scheme 1. Methylation chemistry of 1.
References
[
[
[
1] US DOT Regulation 49CFR Part 172.
2] S. Pounds, Patent WO 2004/108636 A2, A3.
3] S. Pounds, Synthesis and Applications of Isotopically Labelled Compounds, Vol. 8 (Eds. D. C. Dean, C. N. Filer, K. E. McCarthy), 2004,
pp 469–472.
J. Label Compd. Radiopharm 2010, 53 406–489
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Abstracts
THE AUTOSYNTHETIC AND SOLID EXTRACTION METHOD DEVELOPED ON [F-18]FLUMAZENIL
RADIOSYNTHESIS
JENN-TZONG CHEN, KANG WEI CHANG, YEAN-HUNG TU, AND WUU-JYH LIN
Institute of Nuclear Energy Research, Taoyuan, Taiwan
Abstract: This study represents the newly developed autosynthetic and purification method for radiofluorinated flumazenil
1
8
18
F]FMZ as the brain GABA/BZR receptor antagonist imaging agent. It represents a fast and convenient way to produce [ F]FMZ
[
and is much easier to meet GMP compliance than preparative HPLC process. The process takes 45 minutes, decay corrected yield is
20–30%, and the radiochemical purity of the product is higher than 95%. The lipophilicity is a little higher partition coefficient with
1.4970.12 which represents the high capability of penetrating the blood-brain barrier (BBB). An ex vivo study of this product bound
to brain whole-hemisphere sections demonstrated complete inhibition of a BZR agonist which proved the capability of the newly
1
8
developed solid extraction purification method can be applied in non-carrier added F-flumazenil preparation for further animal
and clinical studies.
1
Keywords: flumazenil; [ F]FMZ; GABA receptor antagonist
8
Introduction: Gama-aminobutyric acid (GABA) is one of the most important inhibitory neurotransmitters in epilepsy, anxiety and other
psychiatric disorders. The GABA receptor is very sensitive to damaged and is abundant in the cortex of the brain. Part of the GABA
receptor with chloride ion channel is the central benzodiazepine receptor. It can be traced by radioactive flumazenil derivatives
1
8
1,2
and was found to be more sensitive and accurate than [ F]FDG image for focus localization in epilepsy foci
1
.
C-flunitrazepam, C-flumazenil, H-sarmazenil, F-fluoroalkylflumazenil and F-flumazenil ([ F]FMZ) have been studied for
1
11
3
18
18
18
3
–7
18
GABA/benzodiazepine receptor transmitting activity to determine their binding efficiency to the cortex . Non-carrier added [ F]FMZ
is the most potential radioligand for quantitative evaluation of the epileptogenic region, Alzheimer disease and for cortical damage
8
after stroke in the brain. PET brain metabolic studies and blocking experiments of [ F]FMZ in cynomolgus monkey have shown it to be
similar to C-flumazenil . However, a radio-HPLC unit is needed in the separation process and takes more than an hour to accomplish
the radiosynthesis and purification. Therefore a faster and a more convenient automatic synthetic method is needed for better
compliance with the GMP regulation. We investigated a solid phase extraction method based on a sequence controlled liquid target
1
1
1
3
1
8
system and an automated radiosynthesizer to produce [ F]FMZ for application to the study of whole-hemisphere sections.
Experimental: No carrier added Fluorine-18 in this experiment is produced from a recently designed target system with a
titanium or niobium target body and a programmable logical controlled target material delivering system with sixteen pneumatic
valves. Oxygen-18 enriched water (Trace, 98%) was irradiated with 17 MeV protons (EBCO, TR30). Nitro-flumazenil precursor, Ro
15-2344, was purchased from SYNCOM (The Netherlands). Other chemicals and solvents were purchased from Fluka and Merck and
all were of analytical grade. Thin layer chromatography silica gel plates (Si-60) were purchased from Merck. Radiofluorination
1
8
synthesis reaction and solid extraction purifications were completed on a Nuclear Interface [ F]FDG synthesizer.
Radiolabeling of nitro-flumazenil with F-18: For all radiolabeling experiments, the nitro precursor was dissolved in DMSO with
1
8
minimum of water content. [ F]fluoride from the pneumatic target water delivery system was delivered to the fluorine-18
separation unit to remove the cation impurities and oxygen-18 water by carbonated Bio-Rad AG1x8 resin. Tetra-butyl ammonia
1
bicarbonate was used to elute the [ F] as TBA F. Acetonitrile was used to remove water from TBA F before the radiofluorination
8
18
18
reaction. The nitro-flumazenil precursor was then delivered to the reactor to complete the fluorine-18 radiolabelling reaction at
8
1601C for 30 minutes (Figure 1).
N
N
CO C H
CO C H
2 2 5
2
2
5
N
N
TBAHCO3
DMSO, 160°C, 30 min
O N
2
N
18F
N
CH3
CH3
O
O
18
Figure 1. The non-carrier added [ F]FMZ radiofluorination reaction with tetra-butyl ammonia hydrogen carbonate phase catalyzed in DMSO nitro-flumazenil solution.
Solid phase extraction was completed by passing through a Waters Alumina-N cartridge. A Waters C-18 cartridge was used to
remove organics generated during the reaction. The product was collected and its radiochemical purity was measured and then
applied in brain tissue in ex vivo studies to check its inhibition with an agonist present.
Results and discussion: The radiochemical decay corrected yield was 15% to 20%. This yield is a little bit lower than the first
studies of non-carrier added radiofluorinated flumazenil completed using radio-HPLC which takes 90 minutes for a single run. This is
probably due to the pneumatic timer controlled valves in the system. This might cause some impurities during target water
1
8
transportation. However, this is the first water target system designed and fabricated in Taiwan and then applied to [ F]FDG
production for years. Radiochemical purity was determined by using Merck Si-60 silica gel plate and a radio-TLC imaging scanner
1
AR-2000, Bioscan). The mobile phase used for developing was a 90% acetonitrile solution. [ F]flumazenil had a Rf = 0.4–0.5, while
8
(
1
8
free [ F]-flouride remained at the origin (Rf = 0.0–0.1). The radiochemical purity was 4 95% (Figure 2).
www.jlcr.org
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J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
18
18
Figure 2. Radio-TLC analytical diagram of [ F]FMZ. [ F]FMZ is developed on Merck Si-60 plate with 90% acetonitrile solution.
1
8
Partition coefficients were measured by mixing [ F]FMZ (3.7–5.0 KBq) with 3 mL each of 1-octanol and 0.1 M phosphate buffer
saline, pH 7.4 in a test tube. The test tube was vortexed for 1 min at room temperature, followed by centrifugation (3000 g) for 3 min.
Two samples (1 mL each) from the 1-octanol and buffer layers were assayed for radioactivity in a gamma scintillation counter.
Samples from the 1-octanol layer were repartitioned until consistent partitions coefficient values were obtained.
1
8
Partition coefficient is an index of the lipophilicity of the probe in buffer. [ F]FMZ had a little higher partition coefficient
1.4970.12), suggesting it was 30 fold more lipophilic and was capable of penetrating the blood-brain barrier (BBB).
(
1
8
A 14–16 month old Sprague-Dawley(SD) rat, anesthetized with ether, was injected with 37 MBq/200 mL of [ F]FMZ through the
lateral tail vein. The rat was sacrificed by decapitation at 30 min postinjection. In a competition study, another rat was co-injected
1
8
2
00 mg of diazepam (BZR agonist) with [ F]FMZ. The brains were immediately removed and frozen in powdered dry ice. Sagittal
sections of 1 mm were cut and exposed to Kodak XAR film for 72 h. Ex vivo film autoradiograms were obtained. Results from the rat
1
brain study demonstrated the [ F]FMZ can be clearly inhibited by the BZR agonist (Figure 3).
8
18
Figure 3. [ F]FMZ rat autoradiography of whole-hemisphere sections in comparing with BZR agonist images.
The ex vivo autoradiography of rat whole-hemisphere sections demonstrated a high and similar uptake in areas known to contain
1
8
a high density of BZR. The binding of [ F]flumazenil demonstrated a high density of binding sites throughout the different cortical
regions (cerebellum ratio was 1.3070.13). Similar low binding ratios was observed between the cerebellum and hippocampus,
thalamus and pons. (ratios were 0.9970.07, 0.9870.10 and 0.9170.08, respectively) The binding was completely inhibited by the
addition of diazepam (BZR agonist) (Table 1).
1
Table 1. Binding ratios of [ F]FMZ and agonist in cortex, hippocampus, thalamus, hypothalamus, pons and cerebellum
8
PSL/mm2
Cortex
Hippocampus
Thalamus
Hypothalamus
Pons
Cerebellum
F-18-FMZ
F-18-FMZ with
diazepam
F-18-FMZ
compared
with
1698.677158.68 1302.44782.98 1289.167122.85 1528.30795.79 1197.237107.30 1311.94773.86
654.35712.28
1.3070.13
677.1676.28
0.9970.07
692.2274.51
0.9870.10
702.8674.62
1.1770.09
701.51710.68
0.9170.08
662.24721.28
1
cerebullum
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
1
Conclusion: In conclusion, non-carrier added [ F]FMZ can be prepared in the simplified Nuclear Interface synthesizer such as
8
1
8
using the [ F]FDG module. A solid extraction purification unit can be easily adapted on the synthesizer to remove the impurities
generated in the reaction. This process is much easier to meet cGMP compliance in accordance with drug company standards. The
purified product behaves as an ideal brain GABA/BZR receptor imaging agent.
References
[
[
[
[
1] I. Savic, M. Ingvar, S. Stone-Elander, J. Neurol. Neurosurg. 1993, 56, 615–21.
2] B. Szelies, J. Sobesky, G. Pawlik, Eur. J. Neurol. 2002, 9, 137–42.
3] J. E. Litton, J. Neiman, S. Pauli, L. Farde, T. Hindmarsh, C. Halldin et al., Psychiatry Res. 1993, 50, 1–13.
4] P. Leveque, S. Sanabria-Bohorquez, A. Bol, A. D. Volder, D. Labar, K. V. Rijckevorsel et al., Eur. J. Nucl. Med. Mol. Imaging. 2003, 30,
1
630–36.
[5] Y. H. Yoon, J. M. Jeong, H. W. Kim, S. H. Hong, Y. S. Lee, H. S. Kil et al., Nucl. Med. Biol. 2003, 30, 521–27.
[6] A. D. Windhorse, R. P. Klok, C. L. Koolen, G. W. M. Visser, J. D. M. Herscheid, J. Label. Compd. Radiopharm. 2001, 44, S930.
[7] A. D. Windhorse, M. P. J. Mooijer, R. P. Klok, G. W. M. Visser, J. D. M. Herscheid, J. Label. Compd. Radiopharm. 2003, 46, S148.
[8] N. N. Ryzhikov, N. Seneca, R. N. Krasikova, N. A. Gomzina, E. Shchukin, O. S. Fedorova, D. A. Vassiliev, B. Gulyas, H. Hall, I. Savic,
C. Halldin, Nucl. Med. Biol. 2005, 32, 109–16.
AUTO-SYNTHESIS OF FLUORO-18-FDDNP ON ALZHEIMER’S RESEARCH
a,b
KANG-WEI CHANG, SHIH-YING LEE, WUU-JYH LIN, CHIA-CHIEH CHEN, JENN-TZONG CHEN
AND HSIN-ELL WANG
b
b
b
b
a
a
Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taiwan
b
Division of Isotope Application, Institute of Nuclear Energy Research, Atomic Energy Council, Taiwan
Abstract: Alzheimer’s disease (AD) is an epidemic neurodegenerative disorder-affecting millions of elderly adults. Senile plaques
1
SPs) and neurofibrillay tangles (NFTs) are hallmarks in AD. [ F]FDDNP (2-(1-{ 6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl}
8
(
ethylidene)malononitrile) was superior binding with SPs and NFTs. High quality [ F]FDDNP was produced by an auto synthesizer.
1
8
1
8
[
(
F]FDDNP has a partition coefficient of 1.9370.10 mean was it shows hydrophobic ability to penetrate the blood brain barrier
BBB). In vitro autoradiography saturated with Tg2576, showed high retention ratio for Ab rich regions (for example in Tg2576,
hippocampus and frontal cortex were 2.1070.34 and 1.9070.17, respectively) and human brain section, postcentral gyrus, and
occipital lobe showed significant different retention ratio (1.4870.04 and 2.3770.13, respectively). In ex vivo study, in hippocampus
and frontal cortex region, Tg2576 had better retention than control mice. (2.4070.33 and 2.1970.22, respectively). In this paper, we
1
8
show our modified protocol for the synthesis of [ F]FDDNP and report in vitro, in vivo and ex vivo results in transgenic mice as a
model for applications in Ab radiopharmaceutical research.
Keyword: alzheimer’s disease; FDDNP; auto-synthesizer
Introduction: Alzheimer’s disease (AD) is predicted to become the most common form of dementia among the neurodegenerative
4
1
–3
diseases in the new millennium. AD causes morbidity and mortality in about 40–60% of the population over 80 years of age. Two
hallmark lesions characterize this progressive neurodegenerative disease: (1) senile plaques (SPs) and (2) neurofibrillary tangles
1
NFTs). AD involves deterioration in a broad spectrum of cognitive processes, including memory, language, knowledge, attention,
,5
(
and executive functions. Conventional imaging techniques, such as computed tomography and magnetic resonance imaging, have
7
4
,6,8
limitations in diagnosis because they currently lack sensitivity and specificity.
A strong association of amyloid plaques with an ideal biomarker could be used to improve diagnosis in the early stages of AD and
3
,9,10,11
evaluate the efficacy of therapeutic agents.
When labeled with appropriate isotopes, these biomarkers could develop into
critical in vivo tools for monitoring the formation and aggregation of Ab plaques and assisting in the early diagnosis of patients with
2
,8,12
18
The solvent polarity- and viscosity-sensitive radioisotope probe, [ F]FDDNP (aminonaphthalene groups), can be used to
AD.
label SPs and NFTs and shows excellent in vitro fluorescence visualization of these fibrillar aggregates.
7
,13
18
When [ F]FDDNP
combined with PET is used to quantify the Ab peptides load in the brains of patients with AD, it can lead to early diagnosis and
could help elucidate the etiology of AD by correlating neuropathology with functional loss.
7
18
18
Reported herein, we modified the auto-synthesis of [ F]FDG for the synthesis of [ F]FDDNP. We obtained encouraging results with
this tracer in Tg2576 mice which overexpress a mutant form of the human amyloid precursor protein (APP) associated with an autosomal
dominant form of AD suggesting that the development of imaging agents for mapping Ab plaques in the living human brain may be
18
possible. We wish to present [ F]FDDNP in microPET imaging, to predict the pathogenic process and provide standard methods for AD.
1
8
Materials and methods: Synthesis of [ F]FDDNP
1
8
6
F]FDDNP was synthesized by a modification of the procedure published in 2007 and for the preparation of FDG. Nucleophilic
[
1
8
substitution of the tosyl group with the no-carrier-added precursor [ F]KF/Kryptofix 222 in acetonitrile yielded FDDNP. The product
was passed through an activated C-18 cartridge and then through the cartridge again to remove water and finally with 100%
ethanol. The product was identified by high-performance liquid chromatography (Figure 1).
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
Partition coefficient determination
1
8
[
F]FDDNP combined with 1-octanol and 0.1 M phosphate-buffered saline, pH 7.4 in a test tube and vortexes the tube for 1 min at
room temperature, followed by centrifugation (3000 ꢁ g) for 3 min. Two samples from the 1-octanol and buffer layers were assayed
for radioactivity in a gamma scintillation counter.
3
1
8
In vitro autoradiography: Frozen brain sections (20 mm thick) of Tg2576 and human brain tissue were incubated with [ F]FDDNP
(
water. After drying, sections were exposed to Cronex MRF-34 film for 72 h.
10.25 mM cold FDDNP) at a concentration of 5 mCi/mL. The sections were rinsed with saturated Li
2
CO
3
in 40% EtOH, 40% EtOH and
1
4
1
Ex vivo plaque binding with [ F]FDDNP: Tg2576 were anesthetized with ether, injected with 185 MBq of [ F]FDDNP through the
8
18
lateral tail vein. Mice were sacrificed by decapitation at 5, 30, and 60 min after injection. The brains were cut as sagittal sections of
5
1
2
0 mm and exposed to Kodak XAR film for 72 h. Ex vivo film autoradiograms were obtained (Figure 2).
18
Result and discussion: Synthesis of [ F]FDDNP
1
[
8
F]FDDNP was synthesized in high radiochemical yield (20–25%) and high radiochemical purity (490%).
Figure 1. The synthesis protocol of [18F]FDDNP. Take the precursor of DMTEAN (lysis in acetonitrile) and complete the SN2 reaction at 95. Add 0.1N HCl for hydrolysis for
0 mins. Pass the reaction solution through an activated C18 cartridge. Wash with water, and finally elute with ethanol.
1
1
8
Partition coefficient: The partition coefficient of [ F]FDDNP was 1.9370.10, indicates it was suitable for penetration of the blood-
brain barrier (BBB).
In vitro autoradiography
16
18
Tg2576 is a standard strain for AD research to develop AD pathology. In this assay, strong binding of [ F]FDDNP in brain sections b-
amyloid rich region was observed. And in competition assay added non-labeled FDDNP, the binding was significantly reduced Table 1.
18
Figure 2. Film autoradiograms of the in vitro assay showing the binding of [ F]FDDNP in sagittal sections of the brains of mice from A to C (A and B were Tg2576, and C
18
was control mice) and human brain section from D to E. B and E were the competition assay with non-labeled FDDNP(10.25 mM). It is evident that [ F]FDDNP labeled the
Ab plaques with high sensitivity and clarity, whereas binding was inhibited when non-labeled FDDNP was included.
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
1
Table 1. In vitro autoradiography results of [ F]FDDNP binding in the hippocampus and frontal cortex sections in Tg2576
8
mice and control normal mice. Competitive assay in Tg2576 mice brain sections involving co-incubation with cold FDDNP
(
10.25 mM). (N = 3, mean7SD)
Tg2576
w/w 10.25 mM FDDNP
Control mice
w/w 10.25 mM FDDNP
F-18-FDDNP
F-18-FDDNP
Cortex
109.41729.43
49.08717.24
73.57729.63
44.14728.48
39.53716.68
33.08710.53
38.19713.74
115.80750.36
33.39716.90
23.52710.53
17.75710.31
19.4678.37
20.96712.21
20.89710.34
23.7779.24
48.01276.28
7.1973.17
4.0270.75
9.4674.68
5.8272.97
1.1673.57
5.5073.63
9.8475.64
22.31714.23
2.1371.44
2.1371.66
10.6174.81
4.0670.19
2.3572.12
3.3471.74
2.1572.09
21.73710.21
Hippocampus
Thalamus
Hypothalamus
Midbrain
Pons
Medulla
Cerebellum
1
8
We noted two different in Tg2576 accumulated with [ F]FDDNP. One is in cerebellum, a different condition with AD patients. And
the other is the ratio comparison with Tg2576 and control mice. Although that, it maybe be explained by the control mice having
bred up to 15 months cause over-expression of APP protein and neurodegeneration. In addition, FDDNP not only binding with
8
tangles and plaques in Alzheimer’s disease also could associate with prion protein in neuro-disease (Table 2).
1
8
Table 2. In vitro autoradiography results of [ F]FDDNP binding in the AD patient brain section. Competitive assay in Tg2576
mice brain sections involving co-incubation cold FDDNP (10.25 mM). (N = 3, mean7SD)
Frontal
lobe
Temporal
lobe
Hippocampus
Amygdala
Precentral
gyrus
Postcentral
gyrus
Occipital
lobe
Comparison with
control human brain
Comparison with
nonlabeled FDDNP
1.4070.16
1.0870.12
0.9770.06
1.1570.18
0.7470.10
1.4870.06
2.3770.13
14.1670.93 125.9374.98
10.4570.74
14.4970.96 14.6771.09 14.6771.36 13.4570.69
1
8
18
Ex vivo plaque binding with [ F]FDDNP: The distribution of [ F]FDDNP in the brain is determined by ex vivo autoradiography.
F]FDDNP uptake in the whole brain of Tg2576 was higher than that in normal control mice (Table 3).
1
8
[
1
8
Table 3. Ex vivo autoradiography assay determined binding with [ F]FDDNP showing brain sections of Tg2576 and control
mice over a time course of 30 min. (N = 3, mean7SD)
Tg2576
Control mice
Cortex
48.24728.40
45.58725.13
51.55729.16
41.37720.47
54.79728.76
61.90734.96
73.54737.45
54.53726.50
10.9778.54
14.75710.88
12.5578.12
10.7175.69
16.1378.36
15.0878.20
13.2479.70
14.24711.86
Hippocampus
Thalamus
Hypothalamus
Midbrain
Pons
Medulla
Cerebellum
The ratio between the two animal models were about 3 to 4 fold at 30 min postinjection. The results were consistent with
biodistribution studies (data not shown).
1
8
Conclusion: In this article, we show the successful synthesis of [ F]FDDNP by an automatic synthesizer. It could be provide the
benefits to reduces the operator radiation dose for faster diagnosis. Encouraging results in Tg2576 and human brain section, suggest
that develop imaging Ab plaques in the living human brain may be possible. The present agent may prove a useful PET probe for
imaging Ab plaques in the living human brain.
References
[
1] R. P. Brendza, B. J. Bacskai, J. R. Cirrito, K. A. Simmons, J. M. Skoch, W. E. Klunk, C. A. Mathis, K. R. Bales, S. M. Paul, B. T. Hyman,
D. M. Holtzman, J. Clin. Invest. 2005, 115, 2.
[
[
2] M. P. Kung, C. Hou, Z. P. Zhuang, A. J. Cross, D. L. Maier, H. F. Kung, Eur. J. Nucl. Med. Mol. Imaging. 2004, 31, 8.
3] C. W. Lee, M. P. Kung, C. Hou, H. F. Kung, Nucl. Med. Biol. 2003, 30, 6.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
[
4] P. V. Kulkarni, V. Arora, A. C. Roney, C. White III, M. Bennett, P. P. Antich, F. J. Bonte, Nuclear Instruments and Methods in Physics
Research B. 2005, 241.
[5] L. Giovannelli, F. Casamenti, C. Scali, L. Bartolini, G. Pepeu, Neuroscience. 1995, 66, 4.
[6] J. Liu, V. Kepe, A. Zabjek, A. Petric, H. C. Padgett, N. Satyamurthy, J. R. Barrio, Mol Imaging Biol. 2007, 9, 1.
[7] E. D. Agdeppa, V. Kepe, A. Petri, N. Satyamurthy, J. Liu, S. C. Huang, G. W. Small, G. M. Cole, J. R. Barrio, Neuroscience. 2003, 117, 3.
[8] E. D. Agdeppa, V. Kepe, J. Liu, S. Flores-Torres, N. Satyamurthy, A. Petric, G. M. Cole, G. W. Small, S. C. Huang, J. R. Barrio, J.
Neurosci. 2001, 21, 24.
[
10] W. Zhang, M. P. Kung, S. Oya, C. Hou, H. F. Kung, Nucl. Med. Biol. 2007, 34, 1.
11] A. B. Newberg, N. A. Wintering, K. Pl o¨ ssl, J. Hochold, M. G. Stabin, M. Watson, D. Skovronsky, C. M. Clark, M. P. Kung, H. F. Kung,
J. Nucl. Med. 2006, 47, 5.
9] W. E. Klunk, Y. Wang, G. F. Huang, M. L. Debnath, D. P. Holt, C. A. Mathis, Life Sci. 2001, 69, 13.
[
[
[12] M. P. Kung, C. Hou, Z. P. Zhuang, B. Zhang, D. Skovronsky, J. Q. Trojanowski, V. M. Lee, H. F. Kung, Brain Res. 2002, 956, 2.
[13] L. Ye, J. L. Morgenstern, J. R. Lamb, A. Lockhart, Biochem. Biophys. Res. Commun. 2006, 347, 3.
[
14] Z. P. Zhuang, M. P. Kung, C. Hou, K. Pl o¨ ssl, D. Skovronsky, T. L. Gur, J. Q. Trojanowski, V. M. Lee, H. F. Kung, Nucl. Med. Biol. 2001, 28, 8.
[15] J. R. Barrios, V. Kepea, N. Satyamurthy, S. C. Huang, W. Gary, Small International Congress Series. 2006, 1290.
[16] S. Oddo, A. Caccamo, J. D. Shepherd, M. P. Murphy, T. E. Golde, R. Kayed, R. Metherate, M. P. Mattson, Y. Akbari, F. M. LaFerla,
Neuron. 2003, 39, 3.
LABELLING OF PEPTIDE DERIVATIVE WITH IN-111 FOR RECEPTOR IMAGING
HUNG-MAN YU, MEI-HUI WANG, JENN-TZONG CHEN, AND WUU-JYH LIN
Institute of Nuclear Energy Research, Taoyuan, Taiwan
1
11
177
Abstract: DTPA-conjugated peptides can be labelled with radionuclides such as In and Lu. These radiolabelled peptides used
for peptide receptor imaging (PRI) and radionuclide therapy (PRRT) require high specific activities. In this study, parameters
influencing the kinetics of labelling of DTPA-peptides, including ligand concentration, type and pH of buffer, reaction temperature
and time, were investigated and conditions were optimised to obtain the highest achievable specific activity. The labelling yield of
DTPA-peptides was decreased with ligand concentration and was optimal at pH 2; pH ^4 strongly decreased the labelling yield.
1
11
Labelling with In in sodium citrate buffer was completed after 30 min at 601C. The results in this study were helpful in optimising
the specific activity of In labelled DTPA-peptide derivatives, and in increasing their effectiveness in PRI and PRRT.
1
11
Keyword: peptide; DTPA; indium-111; radiolabelling; specific activity
Introduction: Cells express on their cellular membranes a variety of receptor proteins with a high affinity for regulatory peptides.
Alterations of receptor expression during disease, such as overexpression in many neoplasias, can be exploited by imaging
techniques. Therefore, peptide receptor scintigraphy with the small radioactive peptide analogs appeared to be a sensitive and
1
,2
specific technique to show in vivo the presence of receptors on various tumors.
Peptides can be easily chemically synthesized, modified and stabilized to obtain optimized pharmacokinetic parameters. Peptides
are rapidly taken up and retained in target tissues, have fast plasma clearance due to the renal excretion, rapid tissue penetration
3
and low antigenicity . Since the late 1980s, peptide radiopharmaceuticals have become an important class of tracers for tumor
detection and localization by peptide receptor imaging (PRI) and for therapy by peptide receptor radiotherapy (PRRT). The most
99m
1
11
frequently used isotopes for peptide labeling are radiometals, such as In,
68
64
90
Tc, Ga, or Cu for imaging purposes and Y and
1
77
4,5
Lu for therapeutic approaches.
DTPA-conjugated peptides can be readily labeled with radionuclides such as
peptides to be successfully used in PRI or PRRT, high specific activities are required . The aim of this study is to determine the
optimal conditions for radiolabelling DTPA-peptides with the In. We investigated parameters that influence radiochemical yields
and reaction kinetics in order to obtain maximal achievable specific activities.
Experimental: Materials and methods
1
11
177
In and
Lu. In order for these radiolabelled
6
1
11
All chemicals used in the present work were of analytical grade. Sodium citrate, sodium acetate, citric acid and acetic acid were
purchased from Sigma-Aldrich. Instant thin layer chromatography silica gel (ITLC-SG) plates were purchased from Pall Life Science.
1
11
InCl
Labeling of peptide with In-111: For all radiolabeling experiments, the individual components were added in the following
order: specific buffer solution (0.1 M, 20 mL), specific concentration of the ligand solution (0.26 mM - 13 mM, 5 mL), 1.1 MBq no-
3
was produced at our institute.
1
11
carrier-added
InCl solution (0.55 GBq/1 mL, 2 mL) were added in the Eppendorf microcentrifuge tubes (0.5 mL). The reaction
3
111
parameters that affect the radiochemical yield of In-DTPA-peptide like peptide concentration, reaction temperature (251C, 601C,
901C), reaction time, type of buffer solution (sodium citrate, sodium acetate) and pH of buffer solution (2–6) were studied.
Radiochemical yield: The radiochemical yield was determined using ITLC-SG and radio-TLC imaging scanner (AR-2000, BIOSCAN).
The mobile phase used for developing was 10 mM sodium citrate solution (pH 5). The labeled compound of peptide remained near
the origin (Rf = 0–0.1), while free In-111 moved to the solvent front (Rf = 0.9–1).
Results and discussion: Peptide concentration studies
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
111
The effect of peptide concentration on the radiochemical yield of In-DTPA-peptide was shown in Figure 1. The results indicate
1
11
that the radiochemical yield of In-DTPA-peptide increased from 27% to 499% and 67% to 499% by increasing the amount of
peptide for sodium citrate buffer and for sodium acetate buffer respectively. Under the same reaction conditions, 44 ng and 110 ng
of ligand were needed to achieve 499% radiochemical yield for sodium citrate buffer and for sodium acetate buffer respectively.
1
00
8
6
4
2
0
0
0
0
0
sodium citrate
sodium acetate
0
50
100
150
200
250
Ligand amounts (ng)
Figure 1. Influence of ligand amount on radiochemical yield in complexation of DTPA-peptide with In-111 at 25 (5 mL ligand 1 2 mL 1.1 MBq In-111 1 20 mL buffer of pH 3).
Reaction temperature studies: The results in Figure 2 show that for both buffers and all ligand concentrations, by increasing the
reaction temperature from 251C to 601C, the labeling yield was increased and the extent was greater for lower ligand concentration. By
raising the reaction temperature to 901C, the labeling yield was decreased (except labeling condition with 4.4 ng in sodium citrate
1
11
buffer). The decreased yield of In-DTPA-peptide at 901C may be attributed to the thermal decomposition of the labeled compound.
1
00
2
5 °C 60 °C 90 °C
8
6
4
2
0
0
0
0
0
citrate
22)
citrate
(11)
citrate
(4.4)
acetate
(44)
acetate
(22)
(
Ligand amounts (ng)
Figure 2. Influence of reaction temperature on radiochemical yield in complexation of DTPA-peptide with In-111 (5 mL ligand 1 2 mL 1.1 MBq In-111120 mL buffer of pH 3).
Reaction time studies: Figure 3 shows that the labeling yield increased with reaction time. At 251C, the labeling yield for sodium citrate
buffer and sodium acetate bufferincreased gradually with reaction time. At 601C, the labeling yield for sodium citrate buffer increased with
time, but for sodium acetate buffer, the labeling yield reached maximum to 47% at 30 min and then decreased to 38% at 120 min. These
111
results confirm that reaction time is more important at low reaction temperature for radiolabeling of In-DTPA-peptide in our study.
1
00
8
6
4
2
0
0
0
0
0
2
2
5°C citrate
5°C acetate
5
10
15
Reaction time (minute)
Figure 3. Influence of reaction time on radiochemical yield in complexation of DTPA-peptide with In-111 at 25 or 60 (5 mL 11ng ligand 1 2mL 1.1MBq In-111 1 20 mL buffer of pH 3).
30
60
120
240
360
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
1
11
Buffer pH studies: Figure 4 shows the variation of the radiochemical yield of
citrate buffer solution. The maximum yield was obtained at pH 2. The yield had a steep decrease from pH 3 to pH 4. The results
In-DTPA-peptide as a function of pH in sodium
111
clarify that the pH is a significant factor that affects the labeling yield with In.
1
00
8
6
4
2
0
0
0
0
0
0
2
4
6
8
pH of buffer
Figure 4. Influence of buffer pH on radiochemical yield in complexation of DTPA-peptide with In-111 at 25 (5 mL 11ng ligand 1 2mL 1.1MBq In-111 1 20 mL soudium citrate buffer).
Conclusion: DTPA-peptides can be radiolabelled at high specific activity. Conditions were optimal at pH 2 and the reactions were
complete after 30 min at 601C. This study has shown that several parameters (ligand concentration, buffer solution, reaction temperature,
reaction time and buffer pH) are important for radiolabelling of peptides with In-111 to obtain maximal achievable specific activities.
Acknowledgement: This research was supported by grant (98-2001-01-D-10) from National Science and Technology Program for
Biotechnology and Pharmaceuticals (NSTPBP), National Science Council, Taiwan.
References
[
[
1] D. J. Kwekkeboom, E. P. Krenning, M. de Jong, J. Nucl. Med. 2000, 41, 1704–1713.
2] W. A. Breeman, M. de Jong, D. J. Kwekkeboom, R. Valkema1, W. H. Bakker, P. P. Kooij, T. J. Visser, E. P. Krenning, Eur. J. Nucl. Med.
2001, 28, 1421–1429.
[
[
[
[
3] M. de Jong, D. Kwekkeboom, R. Valkema, E. P. Krenning, Eur. J. Nucl. Med. 2003, 30, 463–469.
4] A. Signore, A. Annovazzi, M. Chianelli, F. Corsetti, C. Van de Wiele, R. N. Watherhouse, Eur. J. Nucl. Med. 2001, 28, 1555–1565.
5] H. J. Wester, M. Schottelius, T. Poethko, K. Bruus-Jensen, M. Schwaiger, Cancer Biother. Radiopharm. 2004, 19, 231–244.
6] W. A. Breeman, M. de Jong, T. J. Visser, J. L. Erion, E. P. Krenning, Eur. J. Nucl. Med. Mol. Imaging 2003, 30, 917–920.
PHARMACOKINETIC EVALUATION OF RADIOLABELED HYALURONIC ACIDS: A POTENTIAL AGENT FOR
THE EARLY OSTEOARTHRITIC THERAPY
a
HAO-WEN KAO, CHI-JEN CHAN, SUNG-CHING CHEN, SHAIN-JY WANG, MING-HSIEN LIN, WUU-JYH LIN,
a
b
c
a,d
e
e
JENN-TZONG CHEN, AND HSIN-ELL WANG
a,
Ã
a
Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan
b
Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan
Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan
c
d
e
Department of Nuclear Medicine, Zhongxiao Branch of Taipei City Hospital, Taipei, Taiwan
Institute of Nuclear Energy Research, Department of isotope application, Taoyuan, Taiwan
Ã
Correspondence to: Hsin-Ell Wang, No. 155, Sec. 2, Linong st., Beitou District, Taipei 112, Taiwan
Abstract: Viscosupplementation treatment is a accepted therapeutic modality for osteoarthritis (OA). Intraarticular injection of
hyaluronic acid (HA) provides pain relief by lessening the frictional resistance of the cartilage and improving joint function. This study
1
evaluated the pharmacokinetics of I-labeled HAs in an osteoarthritis rabbit model. HA22/HA142 were conjugated with tyramine and
31
1
labeled with I-131 to afford I-Tyr-HAs. Osteoarthritis in the left knee joint was surgically induced in New Zealand white rabbits. After
31
1
31
intraarticular administration of
I-Tyr-HAs into the OA and normal knee joints, the whole-body distribution of radioactivity was
longitudinally monitored using scintigraphic imaging. Most of the radioactivity was found retained in the synovial joints. A small
portion accumulated slowly in the liver and bladder, indicating that HA, after exiting synovial joints, was metabolized in the liver and
excreted in urine. The pharmacokinetics was evaluated based on the counts of ROI derived from the scintigraphic images. The results
1
31
revealed that HA of higher molecular weight ( I-Tyr-HA142) enjoyed longer distribution half-life in normal and OA knee joints than
1
31
131
that of lower molecular weight ( I-Tyr-HA22). This study demonstrated that I-labeled tyramine-conjugated HA can be used as a
scintigraphic probe that allows non-invasive long-term pharmacokinetic evaluation of HA in an OA rabbit model.
Keywords: viscosupplementation treatment; hyaluronic acid; scintigraphic imaging; pharmacokinetics
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
Introduction: Osteoarthritis (OA) is a common form of arthritis, characterized by the slow degeneration of cartilage, pain, and
1
restriction of mobilit. Although increasing age and excessive articular surface contact stress increase the risk of degeneration in all
2
joints, the pathophysiology of the joint degeneration that leads to clinical syndrome of OA remains poorly understood. The
treatment toward OA, including nonpharmacologic modalities, pharmacologic therapy and surgery, can help patients reduce pain,
maintain or improve joint mobility, and limit functional impairment. The nonpharmacologic modalities contain weight loss, bracing,
muscle-strengthening exercises and so on, while the prescription used in pharmacologic therapy includes oral analgesic agents,
3
,4
intraarticular (i.a.) relief supplements and topical treatment modalities.
Hyaluronic acid (HA) is a high molecular weight unbranched polymer chain of repeating N-acetyl- D-glucosamine-D-glucuronic acid
5
disaccharides. HA, one of the components of trinary macromolecular complex in articular cartilage matrix, endowed articular cartilage
with many specific mechanical properties, such as viscoelasticity. HA is also a major component of synovial fluid, which supplies
nutrients to the articular cartilage and lubricates the joints, thereby minimizing friction on the surface of the articular cartilage. I.a.
exogenous HA therapy, which has been approved by the US Food and Drug Administration since 1997, has recently become widely
accepted for OA of the knee based on its anti-inflammatory effect and for reducing pain. The decrease of endogenous HA in the synovial
5
fluid is believed to accelerate cartilage destruction in OA. The effect of i.a. therapy with HA is to help replace synovial fluid that has lost
its viscoelastic properties and provide improvement in pain, physical function, and quality of daily living. Studies on animals have shown
5
–9
that HA effectively reduces articular pain and slow down the degenerative process of OA. Clinical trials and current studies have
demonstrated that i.a. HA injection effectively relieves pain and improves motility function. However, the mechanism by which this
10
occurs have not yet been elucidated. The viscoelasticity of HA in situ depends on its molecular weight and local concentration.
Although the effectiveness of the different molecular weight HAs are still debated, some previous studies revealed that the HA with
5
higher molecular weight is more effective than that with lower molecular weight in inhibiting cartilage degeneration in early OA.
Iodine-131, a radionuclide with a gamma emission of 364 keV and a physical half-life of 8.02 d, is suitable for longitudinal
scintigraphic monitoring within one month post injection. Since HA does not contain any convenient group for radiolabeling, it is
1
first conjugated with tyramine (Tyr) as a prosthetic group and then labeled with I to afford I-Tyr-HA. This study demonstrated
31
131
the in vivo whole- body distribution and evaluated the pharmacokinetics in OA knees and normal knees of a rabbit model using non-
1
invasive planar gamma imaging after i.a. administration with I-Tyr-HAs of different molecular weights.
31
Results and discussion: Preparation and analysis of radiolabeled HA
6
Tyr-HA22/142 (molecular weight 0.22/1.42 ꢁ 10 dalton) conjugates were synthesized by a carbodiimide/active ester-mediated
coupling reaction in distilled water (Scheme 1). Based on the H NMR measurement, about one tyramine molecule was present per
1
1
1
3
.0 repeat units of HA22, and 3.6 repeat units of HA142. After radioiodinated with chloramine-T and centrifugally purified with
1
31
Microcon YM-30, I-Tyr-HAs with different molecular weights were prepared with moderate radiochemical yield (about 30%) and
high radiochemical purity (495%). The radiochemical purity was determined using thin layer chromatography (TLC) performed with
a reverse phase silica gel on aluminum sheets and methanol as developing agent.
OH
O
CH3
OH
HOOC
O
OH
O
NH
O
OH
HO
HO
O
O
O
HOOC
O
O
O
*
HO
O
O
HO
HO
EDC/HOBt
pH=6.8
O
OH
NH
O
m
n
HO
OH
NH
OH
NH
n
O
CH3
O
CH3
NH
2
HO
HA22/142
Scheme 1. The synthesis of Tyr-HA22/HA142 conjugates.
Tyramine
Tyr-HA22/142
1
31
131
In vitro stability studies: The in vitro stability of I-Tyr-HA22 and I-Tyr-HA142 in PBS at 41C was good; the radiochemical purities
were 496% after 72 h incubation. The stability of the two tracers in rabbit plasma at 371C was fair; the radiochemical purities
1
31
131
declined to 84.271.9% for I-Tyr-HA22 and 84.170.5% for I-Tyr-HA142 (Figure 1) after 72 h incubation.
1
00
8
0
0
2
HA22 in PBS
HA142 in PBS
HA22 in plasma
HA142 in plasma
0
0
20
40
Time of incubation (h)
Figure 1. The stability of I-Tyr-HA22 and I-Tyr-HA142 in PBS at 41C and in rabbit plasma at 371C after incubation for 0.083, 0.5, 1, 2, 4, 8, 24, 48 and 72 h.
60
80
131
131
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
The planar gamma imaging studies New Zealand white rabbits (female, age 1471 weeks, body weight 2.570.2 kg) were
examined in this study. Experimental osteoarthritis in the left knee joint of the rabbit was surgically induced at six weeks after
7
,9,12,13
anterior cruciate ligaments transaction (ACLT) and posterior cruciate ligaments transaction (PCLT).
1
was conducted to assess the whole-body distribution up to 3 weeks post i.a. injection of
The planar gamma imaging
131
I-Tyr-HA22 and I-Tyr-HA142 in the
31
normal and OA knees of the rabbits. The scintigrams showed that most of the radioactivity was retained in the knees for 504 h post
injection (Figure 2). A slight accumulation of radioactivity in the liver and bladder was noticed, indicating that HA, after leaking from
the synovial joint, might be degraded or metabolized in liver and excreted in the urine. Longitudinal monitoring the distribution of
1
31
intravenously injected I-Tyr-HA142 in the same rabbit model using scintigraphic imaging was also performed to investigate the
1
fate of I-labeled HA after leaving the synovial joints. The image showed significant accumulation in liver from 3 h post injection till
31
363 h (data not shown), indicating that the HA in systemic circulation was first trapped and metabolized in the liver and then
excreted to urine.
1
4,15
In comparison with hepatic endothelial cells, normal synovial lining cells have limited capability for the
1
6
metabolic degradation of HA.
131
Figure 2. Scintigraphic images of New Zealand white rabbits with normal (right) and OA (left) knees at designated time points after intraartucular injection of (a) I-Tyr-
131
HA22 (right, 158 mCi/0.5 mL; left, 165 mCi/0.5 mL) (b)
I-Tyr-HA142 (right, 62 mCi/0.5 mL; left, 49 mCi/0.5 mL) in the knee joints. Imaging was performed under initial
anesthesia with intravenous injection of propofol (10 mg/kg) and constant anesthesia with isoflurane using a vaporizer system during 20 min.
1
31
131
131
I.a. injection of
joints. The results of scintigraphic imaging (data not shown) revealed that the
rapidly from the synovial joint. Most of the radioactivity was excreted in the urine within 6 h. Hence the low but persistent
I-tyramine was conducted to clarify whether the
I-tyramine were degraded from
I-Tyr-HAs in synovial
1
31
I-tyramine injected in the knee joint eliminated
1
31
131
radioactivity accumulation in liver after i.a. injection of I-Tyr-HAs was most probably attributed to the escaped I-Tyr-HAs, or the
radioactive metabolites with smaller molecular weight, from the joints.
Pharmacokinetics of
normal knee derived from planar gamma images are shown in Figure 3a. The difference in the retention of radiolabeled HAs in
131
normal knee and OA knee was not obvious (P = 0.82 and 0.64 for I-Tyr-HA22 and I-Tyr-HA142).
1
31
131
131
I-Tyr-HA22 and I-Tyr-HA142 in OA knee and
I-Tyr-HA22/142: The radioactivity-time curves of
1
31
131
131
Figure 3. Radioactivity in ROI of (a) knee joints and (b) livers after intraarticular injection of I-Tyr-HA22 and I-Tyr-HA142 were derived from the scintigraphic images of
rabbits at designated time points (n = 3). The radioactivity concentration were expressed as the percentage of injection dose (%ID) according to the following formula:
%
knee joint, A
ID = (A
i
/A
0
) ꢁ 100%, where A
i
= decay-corrected radioactivity (counts) of ROI (knee joint and liver) is derived from the gamma images at designated time points. For the
is the total counts in the whole imaging field at 4 h post injection.
0
is the counts in the specific knee at 4 h post injection; for the liver, A
0
However, the differences in the retention of HA22 and HA142 in the joints of both normal and OA knees are significant (Po0.05).
131
The pharmacokinetic parameters derived from the radioactivity-time curves of I-Tyr-HAs were summarized in Table 1. The half-life
(
longer and higher than that of
T1/2) and AUC(o-N) of radiolabeled HA142 in synovial joints of normal and OA knees after 1.a. administration were significantly
1
31
I-Tyr-HA22. The maximum radioactivity accumulation in liver after i.a. administration of
I-Tyr-HA22 and I-Tyr-HA142 (Figure 3b) were 46.81 and 22.06 %ID at 24 h post injection, respectively. The higher the molecular
1
31
131
1
31
weight of I-Tyr-HA administered, the lower the accumulation of radioactivity in liver was observed. These results indicate that HA
with higher molecular weights escape the knee joints (both normal and OA) less readily.
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
131
Table 1. Pharmacokinetics parameters derived from the scintigraphic images of rabbits after intraarticular injection of I-Tyr-
1
31
HA22 and
analysis model
I-Tyr-HA142 in normal and OA knees using Matlab 7.0 (The MathWorks, Natick, MA) with one-compartmental
Normal knee
Osteoarthritis knee
Ã
Ã
Half-life (T1/2) (h)
AUC(0-N) (h %ID)
Half-life (T1/2) (h)
AUC(0-N) (h %ID)
HA22
HA142
19.2972.7
45.8471.1
3107.27
6778.87
22.0279.1
48.13711.3
3383.67
6834.37
Although the pathogenesis of OA is still unclear, many experimental methods have been used to induce OA in animals, including
6
,17
7,9,12,13
joint immobilization
previous studies. In our study, both ACLT and PCLT were carried out in the left knee to ensure OA would developed within six
weeks.
3
and resection of cruciate ligaments.
ACLT has been used to induce knee OA in rabbit models in some
1
2
H-labeled HAs with different molecular weights have been applied as radiotracers to study the pharmacokinetics of HA in normal
1
rabbits. The pharmacokinetic parameters were evaluated based on the metabolic H O formed in the plasma. The mean
8
3
2
3
intrasynovial half-life of H-hyaluronic acid with high molecular weight (M
6
r
4 6.0 ꢁ 10 , 13.2 h) was longer than that of low
6
molecular weight (M 40.09 ꢁ 10 , 10.2 h). In this study, HAs were conjugated with tyramine and labeled with iodine-131 to afford
1
I-Tyr-HAs with high in vitro stability and medium in vivo stability.
r
31
131
I-Tyr-HAs was employed and scintigraphic imaging was
performed to monitor the distribution and to evaluate the pharmacokinetics of HA in normal and OA knees joints longitudinally
after i.a. administration. Only a trace amount of radioactivity was noticed in rabbit thyroid revealed that free Iodine-131 was not an
1
31
3
131
immediate metabolite of I-Tyr-HAs even after the radiotracer has escaped the knee joints. Compared with H-hyaluronic acid, I-
Tyr-HA might be a more pertinent radiotracer that would allow direct monitoring of the distribution and pharmacokinetics of HA in
a rabbit OA models for up to 3 weeks.
1
31
Conclusion: This study demonstrated that the pharmacokinetics of
420 kDa) in the OA knee joints was not significantly different from those in normal knee joints after intraarticular injection.
I-Tyr-HAs (molecular weight ranged from 220 to
1
HA with higher molecular weights was retained longer than that with lower molecular weights was in both normal and
OA knee and may provide better therapeutic efficacy, at least in the lubrication, after single injection for treatment of
OA knees.
Acknowledgements: This work was supported by grant (96002-62-074) from the Department of Health, Taipei City Government,
Taiwan. HA22 and HA142 were kind gifts from the Industrial Technology Research Institute, Taiwan.
References
[
[
[
[
[
[
[
1] N. Gerwin, C. Hops and A. Lucke, Adv. Drug. Deliv. Rev. 2006, 58, 226–242.
2] J. A. Buckwalter and J. A. Martin, Adv. Drug. Deliv. Rev. 2006, 58, 150–167.
3] Arthritis Rheum 2000, 43, 1905–1915.
4] M. C. Barron and B. R. Rubin, J. Am. Osteopath. Assoc. 2007, 107, ES21–27.
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6] L. L. Fu, N. Maffulli and K. M. Chan, Clin. Rheumatol. 2001, 20, 98–103.
7] T. Kawano, H. Miura, T. Mawatari, T. Moro-Oka, Y. Nakanishi, H. Higaki and Y. Iwamoto, Arthritis Rheum. 2003, 48,
1
8] L. W. Moreland, Arthritis. Res. Ther. 2003, 5, 54–67.
9] K. D. Brandt, G. N. Smith, Jr. and L. S. Simon, Arthritis. Rheum. 2000, 43, 1192–1203.
923–1929.
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10] P. Juni, S. Reichenbach, S. Trelle, B. Tschannen, S. Wandel, B. Jordi, M. Zullig, R. Guetg, H. J. Hauselmann, H. Schwarz, R. Theiler,
H. R. Ziswiler, P. A. Dieppe, P. M. Villiger and M. Egger, Arthritis. Rheum. 2007, 56, 3610–3619.
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13] Y. S. Park, S. W. Lim, I. H. Lee, T. J. Lee, J. S. Kim and J. S. Han, Arthritis Res. Ther. 2007, 9, R8.
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17] A. Wigren, J. Falk and O. Wik, Acta. Orthop. Scand. 1978, 49, 121–133.
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www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
COMBINING DOCKING, MOLECULAR DYNAMICS AND MM/PBSA METHODS TO IDENTIFY THE BINDING
MODES OF CONGO RED TOWARDS AMYLOID PROTOFIBRILS
a
JIAN-HUA ZHAO, HSUAN-LIANG LIU, KUNG-TIEN LIU, and JENN-TZONG CHEN
a,b
c
d
a
Department of Chemical Engineering and Biotechnology National Taipei University of Technology 1 Sec. 3 ZhongXiao E. Rd. Taipei, 10608, Taiwan
b
Graduate Institute of Biotechnology National Taipei University of Technology 1 Sec. 3 ZhongXiao E. Rd. Taipei, 10608, Taiwan
Chemical Analysis Division Institute of Nuclear Energy Research 1000, Wunhua Rd., Longtan Township Taoyuan County, 32546, Taiwan
c
d
Isotope Application Division Institute of Nuclear Energy Research 1000, Wunhua Rd., Longtan Township Taoyuan County, 32546, Taiwan
Abstract: In this study, molecular docking, molecular dynamics (MD) simulations and binding free energy calculation were
conducted to investigate the binding modes of Congo red toward the protofibril formed by an amyloidogenic fragment
(
GNNQQNY) from yeast prion protein Sup35. Four specific binding sites were indentified in our simulations. In the primary binding
site, it shows that Congo red bound to a regular groove formed by the first three residues (G1-N2-N3) of the b-strands along the b-
sheet extension direction with a strong binding free energy as estimated by the MM-PBSA method (ꢀ24.4 kcal/mol), which is
consistent with recent theoretical measurements using the MM-GBSA method (ꢀ22.871.6 kcal/mol). Moreover, our simulations
further indentified other binding sites: the long groove between two b-sheet layers, the aromatic pitch of Y7 and the top or bottom
of the protofibril, implying that these binding sites could be bound by Congo red and other long flat molecules. These results not
only yield valuable insight into the nature of the molecular form in which the amyloid dye binds the fibril and the binding locations
on the fibril, but also provide a clue to design new compounds for clinical purposes.
Keywords: docking; molecular dynamics; binding modes; Congo red; GNNQQNY
Introduction: Congo red has been commonly used to identify amyloid fibrils in tissues for more than 80 years (Figure 1A).
1
1
11
Derivatives, such as [ C]PIB, [ C]SB-13, [ F]FDDNP, have been developed as positron emission tomography (PET) tracers
18
1
,2
and helped to detect and visualize amyloid plaques and neurofibrillary tangles in living subjects. However, the specificity
and the stabilities of these binding modes and their roles in amyloid fibril detection remain elusive. It is clear that obtaining a
molecular level of understanding of binding is essential in order to interpret binding experiments as well as for the design of better
dyes for clinical purposes. In this study, molecular docking, molecular dynamics (MD) simulations and binding free energy
calculation were used to explore the nature of the molecular form in which the amyloid dye binds the fibril and the binding
locations on the fibril.
Figure 1. (a) Structures of Congo red molecule. (b) The top and (c) side view of protofibril structure formed by amyloidogenic fragment (GNNQQNY) from the yeast prion
protein Sup35.
Results and discussion: We identified and characterized four specific binding modes of Congo red molecules (Figure 1A) to a
3
protofibril formed by an amyloidogenic fragment (GNNQQNY) from the yeast prion protein Sup35 (Figure 1B and C) as follows:
groove between two b-sheet layers (Figure 2A), groove formed by the first three residues G1-N2-N3 (Figure 2B), the aromatic pitch
of Y7 (Figure 2C) and the top or bottom of protofibril (Figure 2D). Our analysis, consistent with recent MD simulation results, suggest
that Congo red primarily binds to a regular groove of the first three residues (G1-N2-N3) of the b-strands along the b-sheet extension
4
direction (Figure 2B). This primary binding mode exhibits a strong binding free energy of ꢀ24.4 kcal/mol, is in qualitative
4
agreement with the previous theoretical measurements (ꢀ22.871.6 kcal/mol). Moreover, our simulations also indicate that Congo
red could stably bind to the other three binding sites during the 500-ps MD simulations (Figure 2A, C and D).These results led us to
propose that these four binding interaction could be general recognition modes of amyloid fibrils by Congo red, Thioflavin T, and
other long flat molecules. To examine the generality of the conclusions, we further explored the binding of Thioflavin T to a
5
protofibrillar oligomer of Ab(9–40) peptide, and our preliminary results reveal multiple binding sites on Ab(9–40) protofibril surface
6
–8
(
data not shown), which are also consistent with the previous experimental observations.
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
Figure 2. Snapshots of four binding modes of Congo red to GNNQQNY protofibril structure at 0, 250 and 500 ps. Four binding sites are as follows: (A) groove between two
b-sheet layers, (B) groove formed by the first three residues G1-N2-N3, (C) the aromatic pitch of Y7 and (D) the top or bottom of protofibril.
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[
SYNTHESIS OF RADIO- AND STABLE-LABELED LBH589 (PANABINOSTAT) TO SUPPORT ANIMAL AND
HUMAN ADME STUDIES
GRAZYNA CISZEWSKA, BOHDAN MARKUS, LAWRENCE JONES, AMY WU, AND TAPAN RAY
Isotope Laboratories, Translational Sciences, Drug Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research, East Hanover, NJ, USA
Abstract: Radiolabeled LBH589 was required to support animal and human ADME studies and also in vitro protein binding study.
Stable labeled LBH589 (with an appropriately high number of labeled mass units) was also needed as an LC/MS internal standard for
use in bioanalytical assays.
Keywords: radiolabeled; stable labeled; LBH589 (Panabinostat)
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
Introduction: Histone deacetylases (HDACs) act on chromatin and on transcription factors themselves, and modulate gene
regulation including the function of tumor suppressor genes, p53 and Rb. Thus, HDACs are a compelling therapeutic target for
cancer therapy. Histone deacetylase inhibitors (HDACi) have been shown to inhibit the growth of tumor cells in vitro and activate
genes regulating cell cycle arrest, apoptosis and differentiation in a tumor cell specific manner. LBH589 (Panobinostat) was design as
a synthetic deacetylase inhibitor (DACi) belonging to a structurally novel cinnamic hydroxamic acid class of compounds.
1
4
14
14
[
benzene ring of the indole moiety (Figure 1). Stable labeled LBH589 was prepared with six- C situated in the phenyl ring of the
C]LBH589 was labeled in two different positions: C-methylene-phenyl position and with C uniformly incorporated into the
1
3
indole part of molecule (Figure 2).
O
*
H
N
O
N
H
H
N
14
C
N
H
OH
N
H
N
H
OH
[14
C]LBH589
[14C]LBH589
labeled at 14C-methylene-phenyl position
uniformly labeled in the benzene ring of the indole moiety
14
Figure 1. Structures of [ C]LBH589 isotopomers.
1
3
1
1
3
3
C 13
C
C
13
C 1
3
C
O
C
H
N
14
N
H
C
N
H
OH
[13
C ]LBH589
6
labeled with six-13C at benzene ring
of the methyltryptamine moiety
13
6
Figure 2. Structure of [ C] LBH589.
1
4
13
Results and Discussion: The procedures used for the preparation of [ C] and [ C]LBH589 were a modification of the synthetic route
1
] 13
2
developed by Novartis Chemical and Analytical Development.
Syntheses of (2E)-N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl-benzene- C)ethyl]amino]methyl-]phenyl]-2-propenamide
6
C -Phenyl hydrazine was synthesized based on a literature procedure.
1
4
1
and (2E)-N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl-benzene- C
1
The syntheses were done in the same way using two different starting materials: C-uniformly labeled aniline or [ C
3
6
)ethyl]amino]methyl]phenyl]-2-propenamide (Scheme 1).
4
13
6
]aniline.
In both cases the following synthesis route was applied: Starting from labeled aniline 1, the aryldiazonium-salt was formed, which
was reduced to the appropriate phenylhydrazine 2. The Fischer indole synthesis was applied to produce compound 4 from labeled
phenylhydrazine 2 and ketone 3 under acidic conditions. Reductive amination of 4 with compound 5 afforded ester intermediate 6,
which reacted with hydroxylamine to provide labeled LBH589 (7).
NH2
O
Cl
1. NaNO2
HCl/H O
3
2
*
*
*
N
H
NH2 2. NaHCO
HCl/H O
ZnCl
2
3
N
H
NH2
2
2
EtOH
4
1
* = [14
C(U)] or [13C6]
O
NaBH4
MeOH
OCH3
O
HCl
5
H
*
O
*
O
H NOH
x HCl
2
H
H
N
OH
N
N
H
N
N
O
H
NaOH
H
6
7
Scheme 1.
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
1
4
Synthesis of (2E)-N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl- C]phenyl]-2-propenamide lactate (Scheme 2).
The synthesis consisted of seven steps. (2E)-3-(4-bromophenyl)-2-propenoic acid (8) was converted to methyl ester 9. Starting
1
4
from compound 9, by utilizing the Rosenmund-von Braun reaction 10 was formed. Compound [ C]5 was obtained by hydrolysis of
cyano-group to an aldehyde, which was achieved by treatment of 10 with the mixture of formic acid/water (3:1) in the presence of
1
powdered Raney nickel alloy. Reductive amination of [ C]5 with 4 in the presence of sodium borohydride in methanol provided 11,
4
which reacted with hydroxylamine to form the crude drug substance. HPLC purification of crude reaction mixture provided
1
4
compound 12. Treatment of 12 with lactic acid in ethanol/water afforded [ C]LBH589 as a monohydrate lactate salt (13).
O
OH
K14CN + CuSO .5H O + Na S O /NaHSO
8
4
2
2
2
5
3
Br
H SO4
MeOH
2
Cu14CN
O
O
DMF/ reflux overnight
OCH3
OCH3
1
4
N
C
Br
9
10
Formic acid:water
3:1)
Al-Ni catalyst
(
O
NaBH4
MeOH
OCH3
x HCl
O
H
O 14
HCl
N
C
H
O
NH2
N
H
14
C
[14C]5
11
N
H
4
NaOH
H NOH
2
O
lactic acid
EtOH / H O
H
N
OH
14C]LBH589 monohydrate lactate
[
N
N
H
H
14
C
2
13
12
Scheme 2.
Analytical Data: The radiochemical purity of the drug substance was determined by high performance liquid chromatography
HPLC) and thin layer chromatography (TLC). The chemical identity was confirmed by retention time comparison using HPLC and Rf
(
values for TLC. The chemical identity was further confirmed by Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR) and
Differential Scanning Calorimetry (DSC).
1
Conclusion: [ C]LBH589 is not radiochemically stable. Esters 6 and 11 were much more stable than the drug substance, so it was
4
possible to store these intermediates and synthesized the drug substance when needed for the studies.
3
1
[
6
C ]LBH589 was labeled at benzene ring of the methyltryptamine moiety. For
[
M16]LBH589, no parent peak was detected by mass spectrometry (MS).
References
[
[
1] J. Slade and coworkers: CHAD-US Laboratory Process Description.
2] M. Hunsberger, E. Shaw, J. Fugger, R. Ketcham and D. Lednicer, J. Org. Chem. 1956, 21, 394–396.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
EXPLORING A METHYLSULFINATION APPROACH TO HIGH SPECIFIC ACTIVITY ARYL METHYL
3
5
[
S]SULFONES
RICHARD V. COELHO, JR., AND KLAAS SCHILDKNEGT
Pfizer Global Research & Development, Groton/New London Laboratories, Pfizer Inc, Groton, CT 06340, USA
3
Abstract: An experimentally straightforward procedure has been developed to prepare functionally diverse aryl methyl [ S]sulfones
5
3
5
35
35
from sodium methane[ S]sulfonate. High specific activity methane[ S]sulfonyl chloride was prepared from sodium methane[ S]-
3
sulfonate and reduced with zinc metal to afford zinc methane[ S]sulfinate. This intermediate was immediately subjected to copper
3
I) catalyzed coupling with a variety of aryl iodides to afford aryl methyl [ S]sulfones.
5
5
(
Keywords: sulfur-35; methanesulfonyl chloride; sulfinate, sulfone
Introduction: Sulfur-35 enjoys wide use in the radiosynthesis of small molecules primarily because it’s specific activity is
3
5
comparable to that of iodine-125, and it is a pure b- emitter. However, within this ever-growing body of work the primary
functional group form is a methyl sulfonamide – undoubtedly a result of the ready commercial availability of methane[ S]sulfonate.
S
3
5
3
We sought to expand the use of this reagent toward the synthesis of aryl methyl [ S]sulfones, another commonly encountered high
5
3
5
oxidation state sulfur functionality. Two endgame strategies appeared to be amendable to the use of methane[ S]sulfonate to
access aryl methyl sulfones: Friedel-Crafts sulfonylation of arenes with methanesulfonyl chloride, and metal catalyzed
1
methanesulfinic acid couplings to functionalized aromatics. Because the former strategy had been explored in sulfur-35 form
(
and was shown to be non-regioselective), we chose to explore the latter approach.
3
Results and Discussion: Central to realizing a successful methane[ S]sulfinic acid approach to aryl methyl[ S]sulfones was
demonstrating a straightforward partial reduction of methane[ S]sulfonate. The literature did not provide useful procedures for this
exact transformation, but did showcase transition metal and hydride mediated methods for accessing methanesulfinic acid via
reduction of methanesulfonyl chloride. After reviewing these methods for their probable ease of processing radioactivity, we
5
35
3
5
2
focused our attention on a zinc metal reduction, Scheme 1.
Scheme 1. Zinc Methanesulfinate Formation.
Initial experimentation was performed on gram scale using nonlabeled reagents. Methanesulfonyl chloride was added drop-wise
to a refluxing suspension of zinc powder (1.1 eq) in ethanol. At the end of the addition the reaction was cooled and filtered to
remove trace zinc solids. Water was added to the filtrate to precipitate zinc methanesulfinate as a white solid. We envisioned
3
adapting a recently published copper catalyzed sulfinate based sulfone synthesis to prepare sulfur-35 sulfones. Although this
process utilized sodium methanesulfinate as the sulfur source, we demonstrated that our synthesized zinc reagent participated in
0
the reaction as well. Experimentally, a suspension of zinc sulfinate, aryl iodide and N,N -dimethylethylenediamine in DMSO was
treated with catalytic copper (I) triflate and heated at 1101C for 20 hours. Reaction workup involved an ethyl acetate/brine partition,
4
to the reaction (for the purpose of in situ sodium methanesulfinate generation ) did
Scheme 2. Of note, adding excess Na
increase reaction yield.
2
CO
3
Scheme 2. Model Aryl Methyl Sulfone Synthesis.
3
Several adaptations were necessary to efficiently process methane[ S]sulfonate through the demonstrated zinc metal reduction,
5
3
5
Scheme 3. A 10 mCi portion of purchased methane[ S]sulfonate (1400 Ci/mmol) in water was treated with 10 mL of 1N NaOH (to
5
3
5
ensure reagent existed in non-volitile sodium methane[ S]sulfonate form) and the solution was evaporated to dryness with a
stream of N gas. The residue was treated with CH Cl solvent, excess (COCl) and catalytic DMF. After 18 hours the reaction was
washed with 1% aq NaHCO and dried over Na SO to afford 6 mCi of methane[ S]sulfonyl chloride in CH
purity of methane[ S]sulfonyl chloride prepared in this way was separately confirmed by its efficient use in the formation of a
known sulfonamide. The CH Cl solution was distilled to a minimum volume and this solution was added directly to refluxing EtOH
2
2
2
2
3
5
3
2
4
2 2
Cl solvent. The high
3
5
2
2
containing excess zinc powder. After 30 minutes the EtOH solution was cooled and decanted from residual zinc – no water was
added to the solution as previously described. The EtOH solution was assayed to contain 3.5 mCi of radioactivity, presumably in the
3
form of zinc methane[ S]sulfinate.
5
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
35
Scheme 3. Zinc Methane[ S]sulfinate Formation.
35
Portions of the zinc methane[ S]sulfinate solution were transferred to reaction tubes, evaporated to residues with N gas and reacted
2
2 3
with a series of functionally diverse aryl iodides under the previously tested reaction conditions - with added Na CO , Table 1.
3
Table 1. Aryl Methyl [ S]Sulfone Examples
5
1
2
0.50
0.50
0.15 (30)
0.11 (22)
3
4
0.50
0.50
0.24 (48)
0.22 (44)
5
0.25
0.08 (32)
6
0.50
0.50
0.11 (22)
0.06 (12)
7
a. Product activity corrected for radio-HPLC purity
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
As reported in the non-radioactive account of this reaction, both electron-rich and electron-poor aryl iodides delivered sulfone products.
However, we did identify a trend favoring electron-rich aryl iodides (entries 1, 3, 4 and 5 vs. 6 and 7) which was not clearly evident within
the unlabeled work. It is plausible that this reactivity difference is rooted in our introduction of a zinc counterion. Although clearly
35
evident from reaction design, it is worth reinforcing that this process delivers single isomer products. Finally, the [ S]sulfone products
were not isolated from these reactions, but rather identified and quantified by radio-HPLC and LSC.
References
[
[
[
[
[
1] M. A. Wallace, C. Raab, D. Dean, D. Melillo, J. Label. Compd. Radiopharm. 2007, 50, 347–349.
2] N. Chumachenko, P. Sampson, Tetrahedron, 2006, 62, 4540–4548.
3] J. M. Baskin, Z. Wang, Org. Lett. 2002, 4, 4423–4425.
4] F. C. Whitmore, F. H. Hamilton, Org. Synth. 1941, Collect. Vol. I, 492–494.
5] PerkinElmer Life and Analytical Sciences.
EXPEDIENT DEUTEROLABELLING OF LIGNANS USING IONIC LIQUID–DCL/D O UNDER MICROWAVE
2
HEATING
¨
MONIKA POHJOISPAA, ULLASTIINA HAKALA, GUDRUN SILVENNOINEN, AND KRISTIINA WAHALA
¨
¨
¨ ¨
Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, 00014 University of Chemistry, Finland
Abstract: We have developed a fast and high-yielding deuteration method for lignans using 35% DCl/D O and ionic liquids, namely
2
1
-butyl-3-methylimidazolium chloride [bmim]Cl or 1-butyl-3-methylimidazolium bromide [bmim]Br, as co-solvents, under MW
irradiation. This methodology is generally applicable and simple to carry out with reaction times shortened from several days or
5 hours to 20–40 minutes. The ionic solvent is easily recycled.
1
Keywords: deuterium labelling; lignan; ionic liquid; microwave; anhydrosecoisolariciresinol; matairesinol; enterolactone
Introduction: The lignans are a major group of compounds found in the plant kingdom from where they end up as
mammalian metabolic products. They vary widely in structure, but most of the lignans appear as polyphenols. The growing
1
interest in these compounds is due to their beneficial health effects. The analytical techniques used for quantitation of
2
lignans are based on HPLC-MS and ID-GC-MS. In these methods stable isotope polylabelled compounds are needed as internal
standards.
Dibenzylbutyrolactone type lignans are readily accessed by a tandem Michael addition-alkylation reaction. Thus we have studied
3
H/D exchange reactions within the lignan molecule framework. Synthetic labelling schemes often rely on laborious and expensive
total synthesis using prelabelled starting materials.
All aromatic region lignan labelling methods have been based on H/D exchange reactions under acidic conditions. In our earlier
0
0
0
0
4
work, enterolactone was refluxed with a mixture of PBr /D O to give [2,4,6,2 ,4 ,6 -D ]-enterolactone. Later, we carried out
6
3
2
5
deuterium labelling under strongly acidic conditions by deuterated phosphoric acid-boron trifluoride complex, prepared in situ. The
6
method was found to be expedient for various butyrolactone type lignans but required rather long reaction times. We have now
developed faster method to deuterate lignans and other polyphenolic compounds, using microwave irradiation and ionic liquids.
Ionic liquids have achieved wide recognition as green alternatives to conventional volatile organic compounds (VOCs) used as
solvents in many chemical processes. Ionic liquids are defined as salts that are in a liquid form at or below 1001C. Their chemical and
physical properties can be adjusted by varying the choice of cations, anions and substituents. Ionic liquids have essentially no vapor
7
pressure under normal conditions and thus serve as potential replacements for VOCs in the chemical industry.
8
Use of dielectric microwave (MW) heating has been increasingly exploited in organic synthesis, including deuteration reactions.
9
Using dipolar or ionic solvents, MW energy can be transferred to the reaction media using two primary mechanisms — dipole
rotation and ionic conduction. Utilizing ionic liquids as a primary or co-solvent, both mechanisms for energy transfer can operate,
1
0
thus making ionic liquids a highly suitable medium for MW-assisted organic synthesis.
Experimental: Microwave heating was provided in a CEM Discover microwave reactor with single mode irradiation at 2.45 GHz.
Reaction temperature and pressure were monitored using the built-in, on-line IR temperature sensor and pressure sensor.
s
1
The reactions were carried out in sealed pressure-proof vials under argon with magnetic stirring. H NMR spectra were recorded
using a 300 MHz instrument. Mass spectra (ESI1 and ESIꢀ) were measured on a ESI-TOF mass spectrum.
3
5% DCl/D O was purchased from Aldrich and was used directly as received. Matairesinol, enterolactone
11
3
and
2
1
2
anhydrosecoisolariciresinol were prepared in our laboratory as previously reported by us.
The predeuterated (evaporated from D O/acetone) lignan compound (15–70 mg, 0.04–0.23 mmol) and [bmim]Cl or [bmim]Br (8
eqvs) were dried under high vacuum (1–0.5 mbar) after which they were placed in a dried 5 ml or 10 ml pressure-proof reaction vial.
5% DCl/D O (0.2–1.0 ml) was added and the vial was sealed. The vial was irradiated for 20–40 min with 20 W microwave power (with
2
3
2
continuous compressed air cooling, 2 bar) at 701C. After completion of the reaction, 0.5–1 ml of D
2
O was added and the mixture was
extracted with EtOAc. The organic layer was washed with water and brine, dried over MgSO , concentrated in a rotary evaporator
4
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
and the product was dried under high vacuum. The deuterated lignan (87–95%) was obtained as white solid compound. If
necessary, the deuteration was repeated until the isotopic purity was more than 90%.
1
The H NMR spectra were found to be the same as for unlabeled compounds,
3,11,12
with the exception that deuterated aromatic
protons were lacking and in D -labelled enterolactone the unlabelled 5- ja 5 -protons had undergone a change to singlets.
0
6
0
0
0
0
0
0
0
0
0
1
[
2,5,6,2 ,5 ,6 -D
4H, m, 7’ 8, 8’), 2.89–2.95 (2H, m, 7), 3.81 (6H, s, 2 ꢁ OCH
C H D O m/z (ESI–) 363.
6
]-Matairesinol [(2,5,6,2 ,5 ,6 -D
6
)-4,4 -dihydroxy-3,3 -dimethoxylignano-9,9 -lactone]. H NMR (CDCl
3
): d = 2.41–2.63
), 3.85 (1H, m, 9 a), 4.14 (1H, m, 9 b), 5.52 (1H, s, OH), 5.53 (1H, s, OH).
0
0
(
3
2
0 16 6 6
0
0
0
0
0
0
0
0
1
[
, 8 ), 2.64–2.73 (2H, m, 7 ), 2.87–3.01 (2H, m, 7), 3.85–3.91 (1H, m, 9 a), 4.01–4.07 (1H, m, 9 b), 7.09, 7.13 (2H, s, 5, 5 ), 8.21, 8,25 (2H, s,
6 6 6
2,4,6,2 ,4 ,6 -D ]-Enterolactone [(2,4,6,2 ,4 ,6 -D )-3,3 -dihydroxylignano-9,9 -lactone]. H NMR (d -acetone): d = 2.47–2.58 (2H, m,
0
0
0
0
0
8
2
x OH). C H D O m/z (ESI1) 304.
1
8
12 6 4
0
0
0
0
0
0
0
0
0
1
2,5,6,2 ,5 ,6 -D ]-Anhydrosecoisolariciresinol [(2,5,6,2 ,5 ,6 -D )-4,4 -dihydroxy-3,3 -dimethoxylignano-9,9 -furan]. H NMR (CDCl ):
[
6
6
3
0
0
0
0
d = 2.15–2.21 (2H, m, 8, 8 ), 2.48–2.62 (4H, m, 7, 7 ), 3.50–3.55 (2H, m, 9a, 9 a), 3.82 (6H, s, 2 ꢁ OCH
2H, s, 2 ꢁ OH). C20 m/z (ESI1) 350.
Results and discussion: We have developed aryl-ring-selective acid catalyzed deuteration method for representative
polyphenols in ionic liquid, namely 1-butyl-e-methylimidazolium chloride [bmim]Cl or 1-butyl-3-methylimidazolium bromide
3
), 3.89–3.94 (2H, m, 9b, 9 b), 5.47
(
18 6 4
H D O
1
3
[
bmim]Br, using 35% DCl/D2O as an inexpensive deuterium source under microwave irridiation. Scheme 1. shows the
reaction scheme for deuteration of matairesinol, enterolactone and anhydrosecoisolariciresinol. Our method is fast and simple to
carry out as reaction times are shortened from several days or hours to 20–40 minutes. Other green chemistry considerations are also
satisfied in our method: the deuterated products are easy to isolate, purification steps are minimized and the reaction solvent is
recyclable.
2
Scheme 1. Deuteration of matairesinol (MAT), enterolactone (ENL) and anhydrosecoisolariciresinol under microwave conditions using DCl/D O – IL reaction media.
Acknowledgements: KW is grateful for the Finnish Academy (the grant number 126431) support.
References
[
[
[
[
[
[
[
[
1] B. Raffaelli, A. Hoikkala, E. Lepp a¨ l a¨ , K. W a¨ h a¨ l a¨ , J. Chromatogr. B. 2002, 777, 29–43.
2] A. Hoikkala, E. Schiavoni, K. W a¨ h a¨ l a¨ , Br. J. Nutr. 2003, 89(suppl.1), 5–18.
3] B. Raffaelli, E. Lepp a¨ l a¨ , C. Chappuis, K. W a¨ h a¨ l a¨ , Environ. Chem. Lett. 2006, 4, 1–9.
4] K. W a¨ h a¨ l a¨ , T. M a¨ kel a¨ , R. B a¨ ckstr o¨ m, G. Brunow, T. Hase, J. Chem. Soc. Perkin Trans. I. 1986, 95–98.
5] E. Lepp a¨ l a¨ , K. W a¨ h a¨ l a¨ . J. Label. Compd. Radiopharm. 2004, 47, 25–30.
6] E. Lepp a¨ l a¨ , M. Pohjoisp a¨ a¨ , J. Koskimies, K. W a¨ h a¨ l a¨ . J. Label. Compd. Radiopharm. 2008; 51, 407–412.
7] P. Wasserscheid, T. Welton (eds.) Ionic Liquids in Synthesis. WILEY-VCH, Germany, 2003.
8] (a) O. Kappe. Angew. Chem., Int Ed. 2004; 43, 6250–6284; b) A. Loupy. Chim. 2004, 7, 103–112; c) M. Larhed, A. Hallberg. Drug
Discovery Today. 2004, 6, 406–416.
[
10] J. Hoffmann, M. Nuchter, B. Ondruschka, P. Wasserscheid. Green Chem. 2003, 5, 296–299.
11] T. H. M a¨ kel a¨ , K. T. W a¨ h a¨ l a¨ , T. A. Hase. Steroids. 2000, 65, 437–441.
12] (a) W. Mazur, T. Fotsis, K. W a¨ h a¨ l a¨ , S. Ojala, A. Makkonen, H. Adlercreutz. Anal. Biochem. 1996, 233, 169–180; b) S. Rasku,
W. Mazur, H. Adlercreutz, K. W a¨ h a¨ l a¨ . J. Med. Food. 1999, 2, 103–105.
9] J. R. Jones, S-Y. Lu in Microwaves in Organic Synthesis (Loupy ed.) Wiley-VCH, New York, USA, 2006.
[
[
[
[
13] U. Hakala, K. W a¨ h a¨ l a¨ . J. Org. Chem. 2007, 72, 5817–5819.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
14
14
PREPARATION OF C-LABELED 1,2,4-TRIAZOLES FROM [ C]-LABELED TETRAZOLES VIA DIELS-ALDER
REACTION: SYNTHESIS OF 11b-HYDROXYSTEROID DEHYDROGENENASE TYPE 1 (HSD1) INHIBITORS
JONATHAN Z. HO AND MATTHEW P. BRAUN
Department of Process Research, Merck Research Laboratories, Merck & Co., Inc., RY800-B375, 126 East Lincoln Avenue, Rahway, New Jersey, USA
1
Abstract: [ C]--labeled HSD1 inhibitors were synthesized for metabolic profiling and selection of drug candidates. A three-step
4
1
reaction sequence was applied. 1-Iodo-2-trifluoromethylbenzene was labeled via a Pd-catalyzed cyanation with Zn( CN)
4
2
to give
cyano- C]2-trifluorobenzonitrile (3). The nitrile was then converted to C-labeled tetrazole (4). Diels-Alder reaction of 4 with the
1
4
14
[
appropriate imidate yielded the desired C-labeled triazole tracers exemplified by 7c.
1
4
F C
3
N
N
CF3
N
N
CF3
*
*
*
N
N
NC
*
N
N
H
F
O
N
7
c
3
4
*
denotes 14Clabel
1
Keywords: C-labeled triazole; C-labeled tetrazole; C-labeled synthesis
4
14
14
Introduction: The 11b-hydroxysteroid dehydrogenase type 1 (HSD1) enzyme regulates intracellular concentrations of
glucocorticoids by generating cortisol in humans whereas corticosterone in rodents from 11-hydrocoticosterone. Cortisol is a
1
2
metabolically active hormone and cortisone is an inactive analogue. Increased intracellular cortisol levels may induce metabolic
3
4
syndrome which is a constellation of unhealthy factors such as low HDL levels, elevated triglycerides, hypertension, elevated
5
6
fasting glucose, hypercholesterolemia, diabetes and increased waist circumference with visceral adiposity. When those
7
8
9
10
1
multiple risk factors occur together, the likelihood of incidence of cardiovascular disease increases exponentially. Since the cortisol
1
1
hormone actions on tissues depend on both intracellular metabolism and circulating glucocorticoid levels, the inhibition of HSD1
2
1
3
would lower intracellular cortisol concentration, and, therefore, it would serve as a treatment of metabolic syndrome. Compound 1
1
was discovered by Merck medicinal chemists as the lead compound for HSD1 (Figure 1). In order to conduct metabolic profiling,
4
1
4
a
C-labled tracer was needed.
F3C
N
N
N
N
F
O
N
1
Figure 1. New HSD1 inhibitor 1.
1
4
Results and Discussion: Previous studies from our laboratory demonstrated that cyanation of an aryl bromide with Zn( CN)
2
is
an important method for the introduction of a [ C]-labeled CN group. In this study, we examined aryl bromides with both
electron-withdrawing and electron-donating groups. The desired cyanides were generated in the presence of Pd(PPh) (10%
loading). It is crucial that the reaction mixture be purged with nitrogen gas for several minutes before it was heated to the reaction
1
4
15
4
1
6
14
temperature. After cyanation, NaN was added, affording the corresponding C-labeled tetrazoles in good yields (Table 1).
3
1
4
14
After obtaining C-labeled tetrazoles, compound 4a was converted to several C-labeled triazoles and the results are listed in
Table 2.
We envisioned that a tetrazole compound would react like a diene with a dienophile under Diels-Alder conditions to give a
1
4
14
[
2.2.1]bycyclo adduct. When [ C]-labeled tetrazole 4a reacted with chloroimidate 6, freshly prepared from amide 5, [ C]-labeled
triazoles 7 and 8 were formed, with 7 as the favored isomer in each case. Each of 7a-c was isolated but the ratios of 7:8 were
determined by analyzing the reaction mixtures. The results are located in Table 2. When R was an admantyl group as in 6c, the
product ratio was 499:1 (entry 3 in Table 2). However, when R was a phenyl group as in 6a, the product ratio of 7a to 8a was 89:11
(
entry 1 in Table 2), due to less steric interaction.
The stereoselectivity can be explained as follows (Scheme 1). When chloroimidate 6 reacts with tetrazole 4a, intermediate 9 is
favored over 10 due to less steric interactions in the transition state (TS). Upon release of nitrogen from 9 and 10, tetrazoles 7 and 8
are obtained. When R was a bulky tert-butyl group as in 6b, the product ratio of 7b to 8b increased to 96:4 (entry 2 in Table 2).
J. Label Compd. Radiopharm 2010, 53 406–489
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www.jlcr.org

Abstracts
1
Table 1. Preparation of C-labeled tetrazoles
4
*
Zn(CN) (1 eq)
2
R
R
N
N
R
NaN (3 eq)
Pd(PPh ) (0.1 eq)
3
3
4
N
Br
NC
*
*
*
DMF
DMF
water (0.01%)
N
H
o
8
0 C, 16 h
o
8
0 C, 16 h
2
4
3
entry
SM
R
Product
yield of 4 (%)
1
2
3
4
5
2a
2b
2c
2d
2e
CF3
4a
4b
4c
4d
4e
84
87
85
81
72
3
CH O
CH
H
3
NO2
Ã
14
denotes a C label.
1
Table 2. Synthesis of C-labeled triazoles via Diels-Alder Reaction
4
CF3
CF3
N
N
N N
Cl
*
N
*
O
b)
a)
R
H3C
N
+
4a
+
R
NCH3
R
R
NHCH3
CH3
5
6
7
Major
8
Minor
entry
Amide
Substrate R =
imidate
% yield
ratio of 7:8
1
2
3
5a
5b
5c
Ph
t-Bu
6a
6b
6c
7a (62%)
7b (64%)
7c (65%)
89:11
96:4
>99:1
F
N
O
N
a) Oxalyl chloride, catalytical amount of DMF, CH Cl , 01C, 1h; b) DMF, 1001C, 16 h.
2
2
Cl
R
H
N
H
N
N
Cl
N
N
(N2)
N
R
N
*
Ar
N
N
*
N
Ar
11
9
-
HCl
H
CF3
N
N
N
*
R
*
Cl
R
HN
Ar
N
Major
N
CH3
N
N
N
N
CF3
Cl
7
N
*
favored TS
+
N
R
NCH3
H
H
CF3
N
N
N
N
6
4a
*
H3C
N
R
HN
Ar
N
Minor
Cl
*
R
N
8
less favored TS
-
HCl
CF3
H
Ar =
H
N
Cl
N
N
N
N
N
N
N
(N2)
Cl
*
denotes 14C label
*
*
Ar
N
N
R
R
Ar
1
0
12
Scheme 1. Proposed mechanism for the synthesis of C-labeled trizoles 7 and 8.
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 406–489

Abstracts
1
4
[
inhibitor with an excellent HSD1/HSD2 selectivity, outstanding PK profile, low clearance, long half life and very high oral
C]-labeled 7c was a radio-tracer of the HSD1 inhibitor 1, which later was identified as a very potent human HSD1 (h-HSD1)
1
bioavailability. Details of those studies will be published in due course.
This methodology for preparation of C-labeled triazoles is a general one. It is known that a wide variety of pharmaceutical
products have the triazole functionality and a great deal of molecules with a triazole display biological activity such as
4
1
4
17
1
7a
17b
17c
14
A good method for the preparation of C-labeled triazoles could help to
antibacterial,
antitumor
and antiviral agents.
conduct much needed drug metabolism studies in a timely manner.
1
In conclusion, a novel and convenient synthesis of C-labeled tetrazoles and triazoles has been developed. We utilized this
4
1
method in the preparation of a C-labeled HSD1 inhibitor tracer 7c.
4
Acknowledgements: The authors wish to thank Mr. Steven J. Staskiewicz for analytical support. J.Z.H. gratefully acknowledges Dr.
Lee J. Silverberg for help in the preparation of this manuscript.
References
[
1] Recent reviews on HSD1 see: (a) M. Causevic, M. Mohaupt, Molecular Aspects of Medicine 2007, 28(2), 220–6; (b) M. Wang,
Current Opinion in Investigational Drugs 2006, 7(4), 319–323; (c) M. C. Holmes, J. R. Seckl, Molecular and Cellular endocrinology
2006, 248(1–2), 9–14.
[
2] (a) J. P. Monson, Clinical Endocrinology (Oxford) 1998, 49(3), 281–282. (b) J. Tourniaire, M. G. Daumont, Annales de
l’Anesthesiologie Francaise 1976, 17(4), 406–10.
[
3] (a) P. H. J. van der Voort, R. T. Gerritsen, A. J. Bakker, E. C. Boerma, M. A. Kuiper, l. Heide, Inten. Care Med. 2003, 29(12),
2
4] K. K. Mishra, H. P. Pandey, R. H. Singh, Ind. J. Clin. Biochem. 2007, 22(2), 41–43.
5] J. P. F. Chin-Dusting, B. A. Ahlers, D. M. Kaye, J. J. Kelly, A. Whitworth, Hypertension 2003, 41(6), 1336–1340.
6] P. J. Hornnes, C. Kuhl, Diabete Metabol. 1984, 10(1), 1–6.
7] K. Yamaguchi, N. Nakamura, H. Uzawa J. Clin. Endocrin. Metabol. 1984, 58(5), 786–9.
8] G. B. Phillips, C. H. Tuck, T. Y. Jing, B. Boden-Albala, I. F. Lin, N. Dahodwala, R. L. Sacco Diabetes Care 2000, 23(1), 74–9.
9] R. Rosmond, G. Holm, P. Bjorntorp, Int. J. Obes. Metabol. Disorders: J. Int. Ass. Study Obes. 2000, 24(4), 416–22.
199–203; (b) A. Hautanen, H. Adlercreutz, J. Int. Med. 1993, 234(5), 461–9.
[
[
[
[
[
[
[
[
[
10] G. Gueder, J. Bauersachs, S. Frantz, D. Weismann, B. Allolio, G. Ertl, C. E. Angermann, S Stoerk, Circulation 2007, 115(13),1754–1761.
11] A. Steptoe, S. R. Kunz-Ebrecht, L. Brydon, J. Wardle, Int. J. Obes. 2004, 28(9), 1168–1173.
12] D. A. Ehrmann, D. Breda, M. C. Corcoran, M. K. Cavaghan, J. Imperial, G. Toffolo, C. Cobelli, K. S. Polonsky, Am. J. Physio.
Endocrino. Metabol. 2004, 287(2), E241–6.
[
[
13] D. Sousa, R. A. Peixoto, S. Turban, J. H. Battle, K. E. Chapman, J. R. Seckl, N. M. Morton. Endocrinology 2008, 149(4), 1861–1868.
14] (a) X. Gu, J. Dragovic, K. G. C. Jasminka, S. L. Koprak, C. LeGrand, S. S. Mundt, K. Shah, S. Kashmira, M. S. Springer, E. Y. Tan,
R. Thieringer, A. Hermanowski-Vosatka, H. J. Zokian, J. M. Balkovec, S. T. Waddell, Bioorg. Med. Chem. Lett. 2005, 15(23),
5
266–5269; (b) J. M. Balkovec, R. Thieringer, S. S. Mundt, A. Hermanowski-Vosatka, G. F. Patel, S. D. Aster, S. T. Waddell,
S. H. Olson, M. Maletic, PCT Int. Appl. WO 2003065983 A2 20030814 2003; (c) N. J. Kevin, X. Gu, S. T. Waddell, PCT Int. Appl. WO
007087150 A2 20070802 2007; (d) S. T. Waddell, J. M. Balkovec, N. J. Kevin, X. Gu, PCT Int. Appl. WO 2007047625 A2 20070426
007; (e) L. J. Mitnaul, J. Tian, C. Burton, M. H. Lam, Y. Zhu, S. H. Olson, J. E. Schneeweis, P. Zuck, S. Pandit, M. Anderson,
2
2
M. Maletic, S. T. Waddell, S. D. Wright, C. P. Sparrow, E. G. Lund. J. Lipid Res. 2007, 48(2), 472–482.
[
[
[
15] C. S. Elmore, D. C. Dean, T. M. Marks, M. P. Braun, N. X. Yu, Y. Zhang, C. E. Raab, R. Singh, L. Jin, D. G. Melillo, C. J. Dinsmore,
T. M. Williams, Abs. Papers 224th ACS National Meeting, Boston, MA, USA, August 18–22, 2002, MEDI-132.
16] T. Yoshioka, M. Kitagawa, M. Oki, S. Kubo, H. Tagawa, K. Ueno, W. Tsukada, M. Tsubokawa, A. Kasahara, J. Med. Chem. 1978,
2
1(7), 633–9.
17] (a) P. D. Cook, G. Wang, T. W. Bruice, V. Rajappan, K. Sakthivel, K. D. Tucker, J. L. Brooks, J. M. Leeds, M. E. Ariza, P. C. Fagan,
PCT Int. Appl. WO 2003073989 A2 20030912 (2003); (b) R. S. Varma, J. Ind. Chem. Soc. 2004, 81(8), 627–638; (c) S. M. Rida,
S. A. M. El-Hawash, H. T. Y. Fahmy, A. A. Hazzaa, M. M. M. El-Meligy. Arch. Pharm. Res. 2006, 29(10), 826–833.
EFFORTS ON THE SYNTHESIS OF TRITIUM LABELED PEG-PEPTIDE TRACERS
JONATHAN Z. HO, ANDY S. ZHANG, YUI S. TANG, STEVEN J. STASKIEWICZ, 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: PEG-peptides refer to polyethylene glycol (PEG) peptides. PEGylation is a commonly used strategy to improve the
pharmacokinetics and metabolic stability of bioactive peptides intended for therapeutic use. The synthesis and purification of
tritium labeled PEG-peptide tracers are discussed.
3
3
Key words: H-labeled synthesis; H-labeled PEG-peptide; Oxyntomodulin.
1
Introduction: It is of a great deal of interest in developing biomimetic polymers that control interactions of a biological system.
This issue is particularly important in designing polymers, such as polyethylene glycol peptides (PEG-peptides), for drug targeting or
J. Label Compd. Radiopharm 2010, 53 406–489
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
2
tissue engineering. PEG, dextran, poly(vinyl alcohol) and polyacrylamide are hydrophilic and nonionic polymers which are non
3
conductive to protein and peptide adsorption. Although these polymers are water-soluble, they are not unambiguously suited to
4
tissue engineering because they can not form a stable three-dimensional constructs for cell attachment and proliferation. However,
when these materials are appropriately copolymerized with another polymer (e.g. peptide B), new peptides with a general structure
illustrated in Figure 1 are formed.
To support metabolism and pharmacokinetic (PK) studies of PEG-peptide conjugates, both radioisotope and stable isotope
labeled tracers are typically needed. Radiolabeling of select residues (e.g. Tyr) within the primary sequence affords tracers that are
commonly used to determine the proteolytic fate of the peptide moiety. Incorporation of radiolabels into the PEG linker, however,
while synthetically more challenging allows the disposition of PEGylated metabolites to be monitored independent of proteolytic
events that could otherwise result in loss of the radiolabel. In this paper, the synthesis and purification of PEG-peptide tracers
incorporating radiolabel within the PEG linker are discussed.
O
H
Pg
N
N
S
Peptide B
O
O
GL2- A
O
1
Figure 1. PEGylated co-polymers.
Results and discussion: The retro-synthetic analysis for the preparation of the tracer is outlined in Scheme 1. There are three
building blocks, 2, 3, and 4, on which tritium label could be placed. Succinimidyl maleimido propionate (SMP) 3 serves as a linker
between the fragments 2 and 4, therefore, putting a label on 3 would maximize the possibility of making a series of the tracers to
support SAR effort for this program
O
NH2
O
O
H
Pg
Pg
N
N
O
N
N
S
Peptide B
O
HS
Peptide B
O
GL2- A
O
O
O
O
GL2- A
O
2
3
4
1
3
Scheme 1. Oxyntomodulin analog 1 and the retro-synthetic strategy for making [ H] labeled tracers.
3
Raney-Ni catalyzed tritiation of 5 in IPA provided [ H
2
] labeled b-alanine tert-butyl ester 6, with a specific activity of 35 mCi/mmol.
It was crucial to conduct preparative HPLC purification and lyophilization of 6, because using crude 6 resulted in low yield in the
subsequent reaction. The Boc group was removed under acidic conditions to afford acid 7. Many coupling conditions were
investigated and dicyclohexylcarbodiimide (DCC) was the only reagent found to give the key intermediate 8. It was noted that 8 was
stable under mild acid conditions but rapidly decomposed when a base was present, and 8 can be stored in EtOH solution at ꢀ801C
for 2 months. With different building blocks 2 and 3, many targets 10 were obtained by using common peptide coupling techniques
(
Scheme 2). The major issue, however, is to purify the final compound with general structure 10. The best purification condition
found was follows. A TSKgel SP-5PW HPLC column eluted with 0.2% HCO H and 1M NaCl aqueous solution, followed by a Jupiter-
00 C18 column eluted with water and 0.2% HCO H in MeCN provided the final tracers with radiochemical purity 497.6%. Final
2
3
2
lyophilization of 10 resulted in very little changes in radiochemical purity. In this way, seven tracers of this kind have been prepared.
This methodology is a general one in preparation of tritium labeled PEG-peptide tracers.
O
O
O
a
O
NH2
b
c
O
N
HO
NH2
t-Bu
CN
t-Bu
N
T
T
T
T
O
T
T
O
O
O
O
O
5
6
7
8
d
O
O
H
Pg
Peptide B
H
N
N
e
N
N
S
T
T
O
T T
O
O
O
GL2- A
O
O
GL2- A
1
0
9
3
Scheme 2. (a)
6
MeCN.
H
h, TSKgel SP-5PW HPLC column eluted with water with 0.2% HCO
2
, Raney-Ni, IPA, rt, 0.5 h, 62%; (b) TFA/DCM, 01C, 2 h, 90%; (c) N-Hydroxysuccinimide, DCC, DMF, 55%; (d) 2, TEA/DCM, reflux, 18 h, 32–66%; (e) 4, buffer,
H & 1 M NaCl, followed by HPLC column Jupiter-300 C18 column eluted with water and 0.2% HCO H in
2
2
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Abstracts
Acknowledgements: The authors would like to thank Dr. Elisabetta Bianchi for providing reference samples and procedures for
the PEGylation purification; Dr. George Doss, Dr. Vijay Reddy and Mr. Tom Bateman from Merck-DMPK Rahway for their help in
analysis and characterization; Dr. Joseph Lynch, Dr. Ping Zhuang and Ms. Lisa Dimichele from Process Research for their help in
providing 2 and sharing previous analytical experience on this program. J.Z.H. gratefully acknowledges Dr. Lee J. Silverberg for help
in the preparation of this manuscript.
References
[
[
[
[
1] B. Ahren, Nature Reviews Drug Discovery 2009, 8(5), 369–385.
2] S. P. Baldwin, W. M. Saltzman, Trends Polym. Sci. 1996, 4, 177–182.
3] M. Zhang, T. Desai, M. Ferrari, Biomaterals 1998, 19, 953–960.
4] S. Zito, J. Shinde, I. C. S. Chen, T. Taldone, M. Barletta, Cur. Bioact. Compd. 2008, 4(2), 68–85.
REGIO-SELECTIVE IRIDIUM-CATALYZED TRITIUM EXCHANGE LABELING OF NON-STEROIDAL ANTI-
INFLAMMATORY DRUGS (NSAIDS) ZOMEPIRAC AND TOLMETIN USING BROMINE AS A PROTECTIVE
GROUP
YUI S. TANG, WENSHENG LIU, MATTHEW P. BRAUN, AND JONATHAN Z. HO
Department of Process Research, Merck Research Laboratories, 126 E. Lincoln Avenue, PO Box 2000, RY800-B375, Rahway, New Jersey 07065, USA
Abstract: Regio-selective tritium labeled Zomepirac and Tolmetine were obtained with Ir-catalyzed tritium exchange in which the
undesired labeling position was temporary protected by bromine.
3
Keywords: H-label; Zomepirac and Tolmetine
Introduction: Zomepirac (1a) and Tolmetin (1b) are non-steroidal anti-inflammatory drugs (NSAIDs) used to treat a wide variety of
1
inflammatory conditions such as osteo- and rheumatoid arthritis. These two structurally similar drugs present an interesting case in
2
the role of bioactivation in idiosyncratic adverse drug events (ADEs). While Zomepirac was removed from the market due to a
3
relatively high rate of anaphylactic liver toxicity, Tolmetin use has continued in Europe due to its relatively clean profile.
The cause of the different ADE profiles remains obscure and has drawn research interest in understanding the metabolic
4
bioactivation mechanisms. Radiolabeled Zomepirac (1a) and Tolmetin (1b) are important tools in this endeavor provided the label
5
is placed in a position that is not involved in the metabolic activation process. We describe herein a selective catalytic tritium
3
3
isotope exchange approach to its tritium labeled tracers, [ H]-1a and [ H]-1b, through temporary blocking of the pyrrole
functionality with bromine.
T
O
H
O
N
N
OH
OH
R1
R2
R1
T
O
R2
H
O
undesired label
positions
Zomepirac (1a): R1 = Cl, R2 = CH3
Tolmetin ( 1b): R1 = CH , R = H
3
3
3
2
[
[
H ]Zomepirac (9a): R = Cl, R2 = CH3
2 1
H ]Tolmetin ( 9b): R = CH , R = H
2
1
3
2
Results and Discussion: The study of drug metabolism and the identification of metabolic pathways is greatly aided by using
radiolabeled tracers, which allow researchers to track what the drug does through the complex biological matrix and to detect a low
6
abundance of bio-adducts. For this purpose, it is ideal that the label is located at those positions which will not be cleaved through
metabolism. The mechanism of catalytic tritiation to reduce a carbon-halide bond to a carbon-tritium bond is different than that of
7
the catalytic exchange of a hydrogen atom with a tritium. Application of these two techniques in a controlled way could achieve
radiolabeled tracers in a highly regio-selective manner.
As shown in Scheme 1, Zomepirac (1a) and Tolmetin (1b) were converted to their bromide intermediates (2a and 2b), and
6
subsequent tritiation gave both tracers, 3a and 3b. However, this strategy only put tritium label in the pyrrole ring. A preliminary
metabolism study showed that the glutathione-bearing pyrrole adducts (6a and 6b) were obtained from biooxidation (4a and 4b),
1
glutathione addition (5a and 5b) and dehydration. Based on this study, the tracers with tritium labels on the pyrrole were deemed
to be unacceptable, and new tracers with tritium labels at the phenyl ring were desired.
J. Label Compd. Radiopharm 2010, 53 406–489
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