Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Results and discussion
According to the literature method,7 the hydroxyl group of
compound 5 was converted into an amine group via a Ritter
reaction, followed by hydrolysis with HCl (aq) under microwave
irradiation. The resulting crude product was coupled with
3-indazole-carboxylic acid to give compound 6. This ketone was
alkylated and reduced to give two isomers, 8 and 9, in an overall
Adamantine 1 was oxidized using concentrated H2SO4 and
HNO3 at 10 °C to give 3-hydroxy-adamantine 2 in 74% yield,6
which was coupled with 3-indazole-carboxylic acid to selectively
form amide 3. Compound 4 was obtained after the alkylation of 3
with bromopentane in an overall yield of 54% (Scheme 1).
yield of ̴ 8% and 4%, respectively, over five steps (Scheme 2).
The yield of the final step was low due to the difficulty of
separating the two isomers. Their structures were elucidated
using 1D and 2D NMR (ESI).
The NPS are generally screened using LC-HRMS in
forensic toxicology laboratories. During routine LC methods
applied at the National Board of Forensic Medicine (RMV),
the three potential metabolites
4, 8 and 9 co-eluted. A
structured method development process was successfully
applied to establish an LC method, enabling the separation
of these compounds. In this method, an LC gradient from 26
to 67% MeCN in 10 mM ammonium acetate on a 100x2.1
mm Cortecs UPLC C18 column (Waters) over 7.5 min was
Scheme 1. i) conc. H2SO4, conc. HNO3, 2 h, 10 °C, 74%; ii) TBTU,
1H-indazole-3-carboxylic acid, Et3N, THF, rt, overnight, 81%; iii)
1-bromopentane, t-BuOK, THF/DMF (5:1), rt overnight, 90%.
applied. Using this method, compound
8 was found to be the
target metabolite (Fig. 2), which can be used as a biomarker
for detection of AKB-48 in urine sample.
Conclusion
In conclusion, we report the straightforward synthesis and
identification of an important metabolite of AKB-48 with a
single hydroxyl group on a secondary carbon of the
adamantyl moiety. The presence of this metabolite in urine
samples could therefore act as a biomarker to potentially
reveal the abuse of AKB-48. The study also indicates
another important, more general, metabolic picture of the
adamantyl moiety, namely exclusive metabolism of the
adamantyl moiety on a secondary carbon. Furthermore,
these results highlight the importance of having access to all
the potential metabolites. Without such a set of reference
substances, it could be difficult to obtain accurate
metabolism results of bioactive molecules containing the
adamantyl moiety.
Scheme 2. i) CH3CN, BF3·Et2O, TFA; ii) conc. HCl (aq), 150 °C MW
irradiation, 1 h; iii) TBTU, 1H-indazole-3-carboxylic acid, Et3N, THF, rt,
overnight, 30% over three steps; iv) 1-bromopentane, t-BuOK, DMF/THF
(1:5), rt, overnight, 72%; v) NaBH4, 8, 36% and 9, 20%.
Acknowledgments
8
9
Financial support from the National Board of Forensic
Medicine in Sweden is gratefully acknowledged. The authors
would like to thank Anna Åstrand for her assisting in analysis.
4
References
1. (a) Lamoureux G.; Artavia, G. Current Med Chem. 2010, 17, 2967; (b)
Zoidis, G.; Kolocouris, N.; Naesens L.; De Clercq, E. Bioorg. Med.
Chem. 2009, 17, 1534; (c) Wang, J. J.; Lee, J. Y.; Chen, Y. C.; Chen
Y.T.; Chi, C. W. Int. J. Oncol. 2006, 28, 1003; (d) Park, J. S.; Phee, S.
D.; Jung, W. H.; Kang, N. S.; Kim, H. Y.; Kang, S. K.; Ahn J. H.;
Kim, K. Y. Eur. J. Pharmacol. 2012, 691, 19; (e) Vennerstrom, J. L.;
Arbe-Barnes, S.; Brun, R.; Charman, S. A.; Chiu, F. C. K.; Chollet, J.;
Dong, Y.; Dorn, A.; Hunziker, D.; Matile, H.; McIntosch, K.;
Padmanilayam, M.; Tomas, J. S.; Scheurer, C.; Scorneaux, B.; Tange,
Y.; Urwyler, H.; Wittlin S.; Charman, W. N.; Nature 2004, 430, 900;
(f) Chakrabarti, J. K.; Hotten, T. M.; Sutton S.; Tupper, D. E. J. Med.
Chem. 1976, 19, 967.
2. (a) Gatch M. B.; Forster, M.; J. Behav. Pharmacol. 2015, 26, 460; (b)
Uchiyama, N.; Kawamura, M.; Kikura-Hanajiri R.; Goda, Y. Forensic
Toxicol. 2012, 30, 114; (c) Jankovics, P. T.; Váradi, A. S.; Tölgyesi, L.
S.; Lohner, S.; Németh-Palotás J. L.; Balla, J. Z. Forensic Sci. Int.
2012, 214, 27.
Retention time (min)
Figure 2. Analysis of reference materials from compounds 4, 8 and 9 (blue
trace), and a urine sample (red trace) by LC-MS.