Imaging agent of a TRPA1 inhibitor

Article abstract of DOI:10.1002/jlcr.3030

A method for the preparation of [3'-³H]-4-(2'-chloro-6'- hydroxyphenyl)-2-thioxo-3,4-dihydro-1H-indeno[1,2-d]pyrimidin-5 (2H)-one (1), a TRPA1 inhibitor, was developed for the evaluation of imaging properties of a class of TRPA1 inhibitors. 1 was prepared via tritiation of a protected benzaldehyde followed by a tetrachlorosilane catalyzed multicomponent one-step fusion and was obtained at a specific activity of 0.9 TBq/mmol. A ³H-NMR spectrum on 13.5MBq at 75 μM was recorded. Copyright

Full text of DOI:10.1002/jlcr.3030

Note
Received 09 November 2012,
Revised 14 January 2013,
Accepted 15 January 2013
Published online in 16 February 2013 Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3030
Imaging agent of a TRPA1 inhibitor†
e
d,c
Jonas Malmquist,a,b Alexandra Bernlind, and Sandra Lindberg
*
A method for the preparation of [30-3H]-4-(20-chloro-60-hydroxyphenyl)-2-thioxo-3,4-dihydro-1H-indeno[1,2-d]pyrimidin-5
(2H)-one (1), a TRPA1 inhibitor, was developed for the evaluation of imaging properties of a class of TRPA1 inhibitors. 1
was prepared via tritiation of a protected benzaldehyde followed by a tetrachlorosilane catalyzed multicomponent one-step
3
fusion and was obtained at a specific activity of 0.9 TBq/mmol. A H-NMR spectrum on 13.5 MBq at 75 mM was recorded.
Copyright © 2013 John Wiley & Sons, Ltd.
Keywords: isotopically labeled synthesis; 3H; TRPA1; NMR
fact, the proton sensitivity of the probe head, because an 1H
Introduction
spectrum of the labeled substance is needed to confirm a
1
The transient receptor potential (TRP) family of ion channels is
structure by comparison with the H spectrum of the unlabeled
often found to be sensors of multiple chemical and physical
reference material.
stimuli (temperature, smell, taste, and noxious chemicals). In
Conclusions
particular, the non-selective cation channel transient receptor
potential ankyrin-repeat 1 (TRPA1), originally cloned as an
A method for the preparation of 1 has been developed provid-
ankyrin-like protein,1 is activated by multiple pungent and
ing material with specific activity sufficient for experimental
pro-algesic compounds.2 Several in vitro experiments on the
work on in vitro imaging of TRPA1. A 3H NMR with sufficient sig-
inhibition of TRPA1 in pain models have shown a link between
nal to noise was obtained on 13.5 MBq of 1.
TRPA1 and pain transmission3. It was suggested that in vitro
inhibition of the TRPA1 calcium and sodium influx will determine
a compound’s propensity to work as an analgesic in vivo.
Acknowledgements
The International Atomic Energy Agency (IAEA) and the IIS2012
organizing committee is acknowledged for a travel grant to
Jonas Malmquist, making it possible for him to attend the Inter-
national Symposium on the Synthesis and Application of
Results and discussions
Isotopes and Isotopically Labeled Compounds 2012 in Heidelberg,
Germany in times of travel budget restrictions. We would like to
thank Galina Bessidskaia for analysis of radiolabelled compounds.
To be able to more fully evaluate the properties of a class of
TRPA1 inhibiting substances, tritiated material was needed and
1 was the initial target as it had a high potency and appropriate
Log P (Scheme 1). Tritiodehalogenation of a brominated precur-
sor to 1 was attempted but did not succeed, most probably
because of sulfur poisoning of the catalyst. Hydrogen/tritium
exchange using Crabtree’s catalyst resulted in the recovery of
unlabeled material. Therefore, we opted to introduce the tritium
in an earlier stage of the synthetic sequence. Brominated alde-
hyde 2 was protected using acetic anhydride to obtain the triacy-
lated product 34. Treatment of 3 with tritium gas and palladium
on carbon afforded 45 and subsequent hydrolysis with sodium
hydroxide in methanol afforded the deprotected benzaldehyde
56. Finally, a tetrachlorosilane catalyzed multicomponent one-
step fusion7 of 5 with the indene 6 and thiourea gave 18 with
a specific activity of 0.90 TBq/mmol and a 99% radiochemical
purity after purification9. Four months later, a resynthesis was
needed as the substance had decomposed completely upon
storage at ꢀ20ꢁC.
Conflict of Interest
The authors did not report any conflict of interest.
aIsotope Chemistry, Screening and Profiling Global DMPK IM, AstraZeneca,
Research & Development | Innovative Medicines, SE- 151 85 Södertälje, Sweden
bCurrent address: Novandi Chemistry AB, Biovation Park Telge, SE-151 85 Södertälje,
Sweden
cMedicinal Chemistry, CNSP iMed Science Södertälje, AstraZeneca, Research &
Development | Innovative Medicines, SE- 151 85 Södertälje, Sweden
dCurrent address: FOI, Swedish Defence Research Agency, SE- 901 82 Umeå, Sweden
eCurrent address: SP Process Development, Box 36, SE- 151 21 Södertälje, Sweden
*Correspondence to: Malmquist, Jonas, Novandi Chemistry AB, Biovation Park
Telge, SE-151 85 Södertälje, Sweden.
E-mail: jonas.malmquist@novandi.se
We then sought to test the amount of radioactivity required to
3
obtain a satisfactory H NMR. Therefore, several low concentra-
† This article is published in Journal of Labelled Compounds and Radiopharma-
ceuticals as a special issue on IIS 2012 Heidelberg Conference,edited by Jens Atz-
rodt and Volker Derdau, Isotope Chemistry and Metabolite Synthesis, DSAR-DD,
Sanofi-Aventis Deutschland GmbH, Industriepark Höchst G876, 65926 Frankfurt
am Main, Germany.
tion samples were prepared and analyzed by 3H NMR. An 3H
NMR spectrum with sufficient signal-to-noise was obtained on
13.5 MBq dissolved in 200 mL deuterated methanol in a 3 mm
NMR-tube, that is, 75 mM (Figure 1).10,11 The limiting factor is, in
J. Label Compd. Radiopharm 2013, 56 536–537
Copyright © 2013 John Wiley & Sons, Ltd.

J. Malmquist et al.
Scheme 1. Synthesis of [3’-3H]-4-(2’-chloro-6’-hydroxyphenyl)-2-thioxo-3,4-dihydro-1H-indeno[1,2-d]pyrimidin-5(2H)-one (1).
0.48 mmol) in a 72% yield as an oil which solidified upon standing.
1H NMR (500 MHz, DMSO-d6) d ppm 2.07 (s, 6 H), 2.33 (s, 3 H), 7.24 (d,
J = 8.83 Hz, 1 H), 7.96 (d, J = 8.83 Hz, 1 H), 8.00 (s, 1 H). 13C NMR
(126MHz, DMSO-d6) d ppm 20.3, 20.6, 86.1, 120.4, 125.2, 127.4, 133.3,
135.5, 149.4, 168.4, 169.0. GCMS (CI) m/z 319 (80%), 321 (100%), 323
(20%) ([M-OAc]ꢀ).
[5] [5-3H]-(2-acetoxy-6-chlorophenyl)methylene diacetate (4) (2-acetoxy-5-
bromo-6-chlorophenyl)methylene diacetate, 3, (12 mg, 32 mmol) was
mixed with palladium (10% on carbon) (1.4mg) in methanol (1mL).
The mixture was degassed and was connected to tritium (1084mbar)
at ambient temperature. After 1½ hour, the mixture was disconnected
from the tritium manifold, was filtered, and was evaporated with
methanol (1 mL) thrice. Compound 4 (7mg, 23 mmol) was obtained
in 72% yield and was used as is in the next step. GCMS (CI) m/z 241
(30%), 243 (100%), 245 (25%) ([M-OAc]ꢀ)
[6] [3-3H]-2-chloro-6-hydroxybenzaldehyde (5). A solution of [5-2H]-(2-
acetoxy-6-chlorophenyl)methylene diacetate, 4, (7 mg, 0.02 mmol)
and sodium hydroxide (10% aq., 0.074 mL, 0.19 mmol) in methanol
(1 mL) was heated at 65ꢁC under a nitrogen atmosphere. After 3 h,
the reaction was concentrated to dryness. Dichloromethane (2 mL)
was added and the mixture was acidified with hydrochloric acid
(2M, 0.116 mL, 0.23 mmol). Sodium sulfate was added. The dried
organic phase was filtered and concentrated to give 5 (2 mg,
0.01 mmol) in a 50% yield. The product was used as is in the next
step. GCMS (CI) m/z 157 (25%), 159 (100%), 161 (25%) (Mꢀ).
[7] C. Ramalingan, Y.-W. Kwak, Tetrahedron, 2008, 64, 5023–5031.
[8] [30-3H]-4-(20-chloro-60-hydroxyphenyl)-2-thioxo-3,4-dihydro-1H-indeno
[1,2-d]pyrimidin-5(2H)-one (1). To a stirred mixture of [3-3H]-2-chloro-6-
hydroxybenzaldehyde, 5, (2mg, 0.01 mmol), 1H-indene-1,3(2H)-dione,
6, (2 mg, 0.01 mmol) and thiourea (2 mg, 0.03 mmol) in N,N-dimethylfor-
mamide (0.5 mL) and acetonitrile (1 mL) under a nitrogen atmosphere
was added silicon(IV) chloride (0.1 mL, 1 mmol). After stirring overnight
at ambient temperature, the mixture was concentrated. The residue
was dissolved in N,N-dimethylformamide (0.4 mL) and was filtered. Pur-
ification by HPLC on a Kromasil™ C8 column (5 m, 10ꢂ 250 mm) using
53% acetonitrile in 50 mM ammonium acetate in MilliQ water at 2 mL/
min gave compound 1 (630 MBq, 0.90 TBq/mmol) in a 7% yield and a
radiochemical purity of 99%. The product was stored in ethanol (abs.,
6.64 mL) at 95MBq/mL. 1H NMR (600MHz, 13.5 MBq, i.e., 75 mM in
200 mL methanol-d4 in a 3 mm NMR-tube) d ppm 6.19 (s, 1 H) 6.73 (d,
J= 7 Hz, 1 H) 7.07 (t, J= 8 Hz, 1 H) 7.29–7.36 (m, 2 H) 7.38 (t, J=7 Hz, 1 H)
Figure 1. NMR spectra showing (A) 1H spectrum of unlabeled reference to 1; (B) 1
H
spectrum of 13.5 MBq 1; (C) 3H spectrum of 13.5 MBq 1; and (D) 3H spectrum with 1H
decoupling of 13.5 MBq 1. A total of 13.5 MBq 1 corresponds to 75 mM in 200 mL deut-
erated methanol in a 3 mm NMR tube, and this low amount of material is quite suffi-
cient to obtain spectra with sufficient signal-to-noise ratios. The 3 mm NMR tube was
flame sealed and placed in a standard 5 mm NMR tube to enable the use of a standard
spinner and add extra protection from breaking the 3 mm NMR tube.
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3
7.53 (d, J= 7 Hz, 1 H). H NMR (640 MHz, 13.5 MBq, i.e., 75 mM in 200mL
in a 3 mm NMR-tube) d ppm 6.91 (d, J=8 Hz, 1 T). LCMS (ES+) m/z 343.1
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MPO and TRPA1 with a pinch of sulfur presented at the International
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pically Labelled Compounds 2012 in Heidelberg, Germany, which
also included: a) J. Malmquist, P. Ström, J. Lab. Comp. Radiopharm.,
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[4] 2-acetoxy-5-bromo-6-chlorophenyl)methylene diacetate (3). 5-bromo- 6-
chloro-2-hydroxybenzaldehyde, 2, (157mg, 0.67 mmol) was mixed
with acetic anhydride (1.5mL, 16mmol) and sulfuric acid (5mL, [10] A lock signal could not be obtained with 29 mM (5 MBq) in 25 mL
0.09mmol) and the mixture was stirred at ambient temperature for
2 min. The mixture was heated to reflux for 2 min, then cooled in an
ice/water bath and was diluted with water (5mL). A precipitate was
formed which was dissolved in chloroform (5mL). The organic phase
was dried over sodium sulfate and was filtered and the resulting solu-
tion was concentrated. The residue was chromatographed on silica gel
(25 g) using ethyl acetate as eluent to obtain compound 3 (181 mg,
deuterated methanol in a 1 mm NMR tube in the NMR at the time
the experiment was performed. Subsequently, a hardware malfunc-
tion was discovered in the equipment; therefore, the 5 MBq might
have succeeded otherwise.
[11] For experimental details see: J. Malmquist, A. Bernlind, J. Sandell, P.
Ström, M. Waldman, J. Lab. Comp. Radiopharm. 2012, 55, 80–83.
Tritium-gas was handled in a tritium manifold from RC Tritec AG, Teufen.
J. Label Compd. Radiopharm 2013, 56 536–537
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