Bioorganic & Medicinal Chemistry Letters 19 (2009) 5026–5028

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Synthesis and evaluation of 2-amino-dihydrotetrabenzine derivativesas probes for imaging vesicular monoamine transporter-2

Lin Zhu, Jingying Liu, Hank F. Kung *

Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, Beijing 100875, PR ChinaDepartments of Radiology and Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 May 2009Revised 7 July 2009Accepted 8 July 2009Available online 24 July 2009

Keywords:TetrabenazineVesicular monoamine transporterBrainParkinson’s disease

0960-894X/$ - see front matter � 2009 Elsevier Ltd.doi:10.1016/j.bmcl.2009.07.048

* Corresponding author. Tel.: +1 215 662 3096.E-mail address: kunghf@sunmac.spect.upenn.edu

A novel series of analogs of 2-amino-dihydrotetrabenazine derivatives, 4–6, targeting the vesicularmonoamine transporter have been prepared. In vitro binding was carried out in tissue homogenates pre-pared from rat striatal tissue homogenates with both [125I]-iodovinyl-TBZ and [3H]DTBZ. There was agood correlation (r2 = 0.925) between the affinities of the different compounds for [125I]-iodovinyl-TBZand [3H]-DTBZ binding. Compound 5 exhibited a better affinity for the vesicular monoamine transporter(Ki = 8.68 ± 1.26 nM and 7.01 ± 0.07 nM, respectively), which may be a good lead compound for furtherstructural modification to develop useful probes for VMAT2.

� 2009 Elsevier Ltd. All rights reserved.

N

11CH3O

CH3O H

OH

(+)-dihydrotetrabenzaine

N

O

CH3O

18F

9-fluoropropyl-dihydrotetrabenazine

H

OH

11

10

9 765

4321

8

11b

Parkinson’s disease (PD) is a movement disorder characterizedby tremor and dyskinesia. Degeneration of nigrostriatal dopamineneurons plays a central role in PD. On the basis of mechanisms oflocalization, current imaging agents for PD can be generally di-vided into three different target-categories: (a) Enzymatic activity(aromatic amino acid decarboxylase); (b) dopamine transporters;(c) vesicular monoamine transporters (VMAT2).1–7

Imaging of the VMAT2 has been proposed as an alternative forfollowing degeneration of monoaminergic neurons (primarily dopa-minergic) in PD. In animal models, the loss of monoamine neuronshas been shown to be directly correlated to the reduction of radioli-gand binding to VMAT2 in the terminal fields.8 Tetrabenazine (TBZ)and dihydrotetrabenzine (DTBZ), the major metabolites of TBZ, arespecific inhibitors of the vesicular storage of monoamines, and bindwith high affinity to the synaptic vesicular monoamine transporter.9

Several [11C] labeled TBZ derivatives have been successfully devel-oped targeting VMAT2 and tested in humans.6 Animal data stronglysuggested that (+)-[11C]-DTBZ (labeled at the 9-O-methyl group) (1)(Fig. 1) is less sensitive to drugs affecting dopamine levels in thebrain; therefore, it reflects more accurately the concentration of via-ble monoamine neurons.10–13 It is clear that the optically resolvedisomer (+)-[11C]-DTBZ is an excellent PET tracer for measuringVMAT2 in the brain.14,15 To further improve the availability of PETimaging agents for studying VMAT2 binding sites, we have beensearching for 18F (T1/2 = 110 min)-labeled TBZ analogs, which maysignificantly increase the application of these tracers to clinics that

All rights reserved.

(H.F. Kung).

are equipped with PET scanners, but do not have sufficient resourcesto make (+)-[11C]-DTBZ (T1/2 = 10 min).

Recently, a series of fluoroalkyl DTBZ derivatives targetingVMAT2 binding sites in the brain have been developed (Fig. 1).16,17

Optically resolved (+)-9-fluoropropyl-DTBZ (2) displayed very highbinding affinity for VMAT2 (Ki = 0.10 ± 0.01 nM). Biodistributionstudies on mice after an intravenous injection of the (+)-[18F]-FP-DTBZ (2) exhibited a ratio of striatum (ST, target) to cerebellum(CB, background) of 4.51 at 30 min postinjection. Following thesesuccessful results, we decided to further extend our research onpreparation and testing of previously reported 2-amino-DTBZ deriv-atives18 (Fig. 2) as potential VMAT2 imaging agents. By substitutingthe amino group with an N-alkyl group and forming the secondaryamino-DTBZ derivatives, we hoped to develop ligands that wouldbe sufficiently lipophilic to reach the intracellular target (VMAT2)and bind with high affinity and selectivity, while exhibiting a degreeof hydrophilicity which might minimize nonspecific binding during

(+)-[11C]-DTBZ, 1 (+)-[18F]-FP-DTBZ, 2

Figure 1. Previously reported ligands, 1-2, for vesicular monoamine transporter-2(VMAT2).

N

MeO

MeO

NH2

N

MeO

MeO

NH

N

MeO

MeO

NHFn

4: n=25: n=3

3 6

(a) (b)

Figure 2. (a) Amino-DTBZ; (b) N-Alkyl or N-fluoroalkyl substituted amino-DTBZ.

N

MeO

MeO

O

N

MeO

MeO

NHX

a b

4: X=F, n=26: X=H, n=1

7n

Scheme 2. Reagents and conditions: (a) CH3NH2, MeOH, reflux 4 h, rt 10 h; orFCH2CH2NH2�HCl, EtOH, Et3N, reflux 4 h, rt 12 h; (b) NaBH4, 0 �C to rt, 10 h.

Table 1Inhibition constants of compounds on the binding of [125I]-iodovinyl-TBZ and [3H]-DTBZ to rat stiatal homogenates

Compounds Ki (nM ± S.E.M.)a

[125I]-iodovinyl-TBZ binding [3H]-DTBZ binding

DTBZ 2.45 ± 0.18 3.75 ± 0.29(+)-FP-DTBZ (2) 0.24 ± 0.02 0.36 ± 0.10Amino-DTBZ (3) 45 ± 1.48 150 ± 374 28 ± 6 230 ± 384-I 9.21 ± 0.99 8.63 ± 0.584-II 226 ± 20 686 ± 1175 8.68 ± 1.26 7.01 ± 0.076 199 ± 27 269 ± 33

a Each Ki value represents data from at least three independent experiments, eachperformed in duplicate.

L. Zhu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5026–5028 5027

in vitro binding assays. (Fig. 2b) We report herein the synthesis offluoro substituted amino-DTBZ derivatives and the ability of thesecompounds to inhibit the specific binding of [125I]-iodovinyl-TBZand [3H]-DTBZ as improved probes for VMAT2 imaging.

The synthesis procedures for these target molecules are illus-trated in Scheme 1 and 2. The known compound, amine 3, was syn-thesized using a previously published procedure.19 A methanolicsolution of TBZ and ammonium acetate was treated with NaCNBH3,and the mixture was stirred at room temperature to afford amine 3in moderate yield (87%). When the amine 3 directly reacted withfluoroalkyl p-toluenesulfonate, the secondary N-fluoroalkyl substi-tuted amines (4–5) were formed. The secondary N-fluoroalkylsubstituted amine (4, 6) can also be synthesized from TBZ andalkylamine, with imine as the intermediate, and then treated withNaBH4. Using the latter synthetic procedure to make the N-fluoro-ethyl substituted amine (4), we found two components in the finalproduct. The two components were tested by LC–MS, which wereboth demonstrated to be N-fluoroethyl substituted amine (4-I, 4-II). We collected both the components (4-I, 4-II) and the complexof the two (4) for further study.

The binding affinities of the compounds for VMAT2 were deter-mined by using [125I]-iodovinyl-TBZ as the radioligand in rat stria-tal tissue homogenates (Table 1). It shows that when the hydroxylgroup at the 2-position of DTBZ was changed to amino group, thebinding affinity to VMAT2 was decreased. Since the monoaminetransporter binds only the neutral form of substrates and inhibi-tors,20,21 the decrease in affinity of the amino analogs might bedue to a positive charge being carried by the amino compound un-der physiological pH. After N-methylation of the amine group, thebasicity of the amine group also increase and the binding affinitydecreased. Comparing compound 3 and 6 (Ki = 45 and 199 nM,respectively) to DTBZ, the increasing basicity may be an importantfactor, which reduced the binding affinity. However, when thesubstituted carbon chain-length increased to n = 3, the bindingaffinity also improved. The length of the N-alkyl chain to the 2-amino group of DTBZ may impart considerable flexibility and allowa better fit to the VMAT2 binding site. Besides the individual dia-stereomers of compound 4 (Ki = 9.21 and 226 nM for 4-I and 4-II,respectively), we also give the data of the mixtures of diastereo-mers and the binding affinity is in between the two individual dia-stereomers. Truly, based on the data from the mixtures of thediastereomers we can’t give the precious conclusion, but at leastit can reflect the general trend between different derivatives. In

N

MeO

MeO

O

a, bMeO

MeO

7

Scheme 1. Reagents and conditions: Reagents and conditions: (a) CH3COONH4, MeO

the future it may be possible to obtain crystallography data toidentify the specific diastereomer.

To confirm the specific binding for VMAT2, we also performedin vitro binding using [3H]-DTBZ as the radioligand (Table 1). Bothligands exhibited consistent binding for the vesicular monoaminetransporter. There was a good correlation (r2 = 0.925) betweenthe affinities of the different compounds for [125I]-iodovinyl-TBZand [3H]-DTBZ binding. Therefore, it is reasonable to conclude thatthe new 2-N-substituted DTBZ derivatives are likely competing forthe same binding site on VMAT2 (Fig. 3).

In summary, we have completed the synthesis of the novel ana-logs of VMAT2 antagonist, 2-amino-DTBZ. The compounds wereevaluated for their ability to inhibit the binding of specificallybound [125I]-iodovinyl-TBZ and [3H]-DTBZ to rat striatal membranehomogenates. Of the compounds evaluated here, secondary amine4-I and 5 exhibited a better affinity for the vesicular monoamine

N

NH2

c

4: n=25: n=3

3

N

MeO

MeO

NHFn

H, rt, 24 h; (b) NaCNBH3, rt, 4 h; (c) F(CH2)nOTs, n = 2–3, K2CO3, DMF, 65 �C, 6 h.

N

MeO

MeO

NHF

N

MeO

MeO

NHF

N

MeO

MeO

NHF

H H

4 4-I or 4-II 4-I or 4-II

Figure 3. Possible structures of N-fluoroethyl substituted amine 4, 4-I, and 4-II. When using the synthetic procedure (illustrated in Scheme 2) to make the N-fluoroethylsubstituted amine 4, we could separate the two components (4-I and 4-II), both of which are identified by NMR and LC/MS. The data suggest that they presumably are a pairof diastereomers. It is important to note that two isomers, 4-I and 4-II, were isolated. We do not know the exact nature of the geometry at the 2-position; however, it is likelythat these isomers may be related to the chiral center at the 2-position. In addition, all three N-substituted compounds (4, 5, and 6) have a chiral center at the 2-position.However, for compound 5 and 6, we were not successful in separating the two isomers using the synthetic procedure illustrated in Scheme 1 and 2. So compounds 5 and 6reported herein should be mixtures of diastereomers.

5028 L. Zhu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5026–5028

transporter-2. More detailed structure–activity relationship stud-ies are currently being pursued to further enhance the bindingaffinity of this series of 2-amino-dihydrotetrabenazine derivatives.

Acknowledgments

This work was supported by grants from the National 973 Pro-gram (2006CB500705) and the National 863 Program(2006AA02A408) from Ministry of Science and Technology, China.Authors thank Catherine Hou for her editorial assistance.

References and notes

1. Mozley, P. D.; Schneider, J. S.; Acton, P. D.; Plössl, K.; Stern, M. B.; Siderowf, A.;Leopold, N. A.; Li, P. Y.; Alavi, A.; Kung, H. F. J. Nucl. Med. 2000, 41, 584.

2. Brooks, D. J. Ann. Neurol. 1998, 44, S10.3. Frey, K. A. Eur. J. Nucl. Med. Mol. Imaging 2002, 29, 715.4. Lee, C. S.; Samii, A.; Sossi, V.; Ruth, T. J.; Schulzer, M.; Holden, J. E.; Wudel, J.;

Pal, P. K.; de la Fuente-Fernandez, R.; Calne, D. B.; Stoessl, A. J. Ann. Neurol. 2000,47, 493.

5. Kung, H. F.; Kim, H.-J.; Kung, M.-P.; Meegalla, S. K.; Plössl, K.; Lee, H.-K. Eur. J.Nucl. Med. 1996, 23, 1527.

6. Albin, R. L.; Koeppe, R. A.; Bohnen, N. I.; Nichols, T. E.; Meyer, P.; Wernette, K.;Minoshima, S.; Kilbourn, M. R.; Frey, K. A. Neurology 2003, 61, 310.

7. Kung, M. P.; Hou, C.; Goswami, R.; Ponde, D. E.; Kilbourn, M. R.; Kung, H. F. Nucl.Med. Biol. 2007, 34, 239.

8. Vander Borght, T. M.; Sima, A. A.; Kilbourn, M. R.; Desmond, T. J.; Kuhl, D. E.;Frey, K. A. Neuroscience 1995, 68, 955.

9. Mehvar, R.; Jamali, F.; Watson, M. W.; Skelton, D. Drug Metab. Dispos. 1987, 15,250.

10. Kilbourn, M. R.; Frey, K. A.; Vander Borght, T.; Sherman, P. S. Nucl. Med. Biol.1996, 23, 467.

11. Frey, K. A.; Koeppe, R. A.; Kilbourn, M. R. Adv. Neurol. 2001, 86, 237.12. Bohnen, N. I.; Albin, R. L.; Koeppe, R. A.; Wernette, K. A.; Kilbourn,

M. R.; Minoshima, S.; Frey, K. A. J. Cereb. Blood Flow Metab. 2006, 26,1198.

13. Lee, C. S.; Schulzer, M.; de la Fuente-Fernandez, R.; Mak, E.; Kuramoto,L.; Sossi, V.; Ruth, T. J.; Calne, D. B.; Stoessl, A. J. Arch. Neurol. 2004, 61,1920.

14. Kilbourn, M. R.; Lee, L. C.; Heeg, M. J.; Jewett, D. M. Chirality 1997, 9, 59.15. Frey, K. A.; Koeppe, R. A.; Kilbourn, M. R.; Vander Borght, T. M.; Albin, R. L.;

Gilman, S.; Kuhl, D. E. Ann. Neurol. 1996, 40, 873.16. Goswami, R.; Ponde, D. E.; Kung, M. P.; Hou, C.; Kilbourn, M. R.; Kung, H. F. Nucl.

Med. Biol. 2006, 33, 685.17. Kilbourn, M. R.; Hockley, B.; Lee, L.; Hou, C.; Goswami, R.; Ponde, D. E.; Kung, M.

p.; Kung, H. F. Nucl. Med. Biol. 2007, 34, 233.18. Canney, D. J.; Kung, M.-P.; Kung, H. F. Nucl. Med. Biol. 1995, 22, 527.19. Isambert, M. F.; Gasnier, B.; Laduron, P.; Henry, J. P. Biochemistry (Mosc.) 1989,

28, 2265.20. Scherman, D.; Henry, J. P. Eur. J. Biochem. 1981, 116, 535.21. Scherman, D.; Henry, J. P. Mol. Pharmacol. 1983, 23, 431.