Synthesis and antibacterial activity of some heterocyclic chalcone analogues alone and in combination with antibiotics

Article abstract of DOI:10.3390/molecules17066697

A series of simple heterocyclic chalcone analogues have been synthesized by Claisen Schmidt condensation reactions between substituted benzaldehydes and heteroaryl methyl ketones and evaluated for their antibacterial activity. The structures of the synthe

Full text of DOI:10.3390/molecules17066697

Molecules 2012, 17, 6697-6704; doi:10.3390/molecules17066697
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Simple, Rapid and Reliable Preparation of [11C]-(+)--DTBZ of
High Quality for Routine Applications
Jinming Zhang *, Xiaojun Zhang, Yungang Li and Jiahe Tian *
Department of Nuclear Medicine, the PLA General Hospital, Beijing 100853, China
* Authors to whom correspondence should be addressed; E-Mails: zhangjm301@163.com (J.Z.);
tianjh@vip.sina.com (J.T.).
Received: 27 April 2012; in revised form: 28 April 2012 / Accepted: 30 April 2012 /
Published: 1 June 2012
Abstract: [11C]-(+)--DTBZ has been used as a marker of dopaminergic terminal densities
in human striatum and expressed in islet beta cells in the pancreas. We aimed to establish a
fully automated and simple procedure for the synthesis of [11C]-(+)--DTBZ for routine
applications. [11C]-(+)--DTBZ was synthesized from a 9-hydroxy precursor in acetone
and potassium hydroxide with [11C]-methyl triflate and was purified by solid phase
extraction using a Vac tC-18 cartridge. Radiochemical yields based on [11C]-methyl triflate
(corrected for decay) were 82.3% ± 3.6%, with a specific radioactivity of 60 GBq/mol.
Time elapsed was less than 20 min from end of bombardment to release of the product for
quality control.
Keywords: [11C]-(+)--DTBZ; VMAT2; position emission tomography; automated synthesis
1. Introduction
The vesicular monoamine transporter (VMAT) has two pharmacologically distinct isoforms,
VMAT1 and VMAT2 [1]. In contrast to VMAT1, VMAT2 is primarily found in the central nervous
system of rodents and humans. Over 95% of striatal VMAT2 binding sites are associated with
dopaminergic terminals, and the striatal VMAT2 binding site density is a linear function of the
mesencephalic nigrostriatal neuron number [2]. Type-2 vesicular monoamine transporter binding is
predicted to be unaffected by dopaminergic agents or synaptic dopamine levels because synaptic
vesicle function is apparently regulated by transfer of vesicles between the reserve and actively cycling
synaptic terminal pool with relatively stable vesicle numbers [2]. Experimental evidence is consistent

Molecules 2012, 17
6698
with this prediction; VMAT2 binding is not affected by dopaminergic drug treatments that cause
changes in dopamine receptor or DAT expression [3,4]. VMAT2 has been used to monitor and
diagnosis neurodegenerative disorders such as Parkinson’s and Huntington’s disease [5]. More recently,
VMAT2 has been found expressed in human islet beta cells in the pancreas, and its use as a surrogate
marker for beta cell mass loss and progression of diabetes has been suggested [6]. Thus, imaging
VMAT2, particularly with high affinity PET, is an area of ongoing research interest.
(+)--Dihydrotetrabenazine [(+)--DTBZ] is the ideal ligand for VMAT due to its high affinity for
VMAT2 and lipophilicity, confirmed by pharmacological results of in vitro studies. In the early 1990s,
[11C]-(+)--DTBZ was investigated as a tracer for imaging VMAT2. Lang and Tong et al.
demonstrated the correlation between the DTBZ to VMAT2 binding levels in the stratum [7,8].
[11C]-(+)--DTBZ was superior to other tracers, such as [18F]-DOPA or [18F]-CFT in studies of
dopaminergic teriminals. The number of VMAT2 vesicles is not affected by pharmacological treatment
with dopaminergic analogues which affect other elements of the system, such as the dopamine, DAT
and D2 receptor. The simple synthesis of [11C]-(+)--DTBZ was developed by Jewett, who reacted a
9-hydroxy precursor with [11C]-methyl iodide on a column of alumina impregnated with KOH [9]. The
KOH-impregnated alumina column was dried of atmospheric moisture to avoid failure of the synthesis.
Quincoces later described the use of an alumina solid phase extraction cartridge to replace the
KOH-alumina column, which was eluted with ethyl ether and ethanol [10]. The residual solvents were
removed by evaporation. This method was not used due to low radiochemical purity. Instead, we
attempted to use a C-18 SPE cartridge rather than an alumina SPE cartridge to enable reliable, high
quality preparations of [11C]-(+)--DTBZ for routine applications.
2. Results and Discussion
2.1. Production
[11C]-(+)--DTBZ synthesis was automated using a cartridge purification method. Briefly
(+)-desmethyldihydrotetrabenazine and 3 M KOH in DMSO was mixed with [11C]-methyl triflate as a
methylation agent. After reaction and purification with Sep-Pak Vac tC-18 and an alumina cartridge,
the radiochemical yield of [11C]-(+)--DTBZ was 82.3 ± 3.6% based on [11C]-methyl triflate
(corrected for decay). It took about 20 min from the end of bombardment to release of the product for
quality control. About 5.5 ± 0.4 GBq of [11C]-(+)--DTBZ can be obtained after a bombardment time
of 10 min.
[11C]-methyl iodide was also used as the methylating agent for [11C]-(+)--DTBZ to obtain the
same synthesis yields as those observed for [11C]-methyl triflate. However, [11C]-methyl iodide
produced products of inferior radiochemical purity when compared with those prepared using
[11C]-methyl triflate. Because [11C]-methyl triflate is more reactive than [11C]-methyl iodide, it is
washed more easily from Sep-Pak, ultimately leading to a product of superior radiochemical purity.
2.2. Purification with Sep-Pak C-18
The reaction solution was passed through different Sep-Pak C-18 cartridges after mixing with 10 mL
of water. The Sep-Pak C-18 was washed with 10 mL water. The Sep-Pak cartridges used were Waters

Molecules 2012, 17
6699
C-18, tC-18 and Vac tC-18, respectively. The product was eluted from Sep-Pak C-18 with 1 mL
ethanol. The elution yields were calculated from radioactivity remaining on the Sep-Pak and the
radioactivity measured in the product fraction. The break-out efficiency was determined by analyzing
the wash fractions (waste) by HPLC for “break-out” from the SPE cartridges. The results are shown in
Table 1.
Table 1. Elution and break-out efficiencies with different Sep-Pak C-18.
Sep-Pak
C-18
Elution yields (%)
22.1 ± 7.8
Break out (%)
0.5 ± 0.4
tC-18
55.6 ± 10.2
1.2 ± 0.6
Vac tC-18
95.3 ± 2.6
2.5 ± 1.4
The Sep-Pak C-18 and tC-18 remained highly radioactive, which indicated low elution yields. We
selected the Sep-Pak Vac tC-18 as the best purification column, as it had a high elution yield and low
break-out. The final preparation was not clear but yellow. By placing an alumina Sep-Pak cartridge
immediately before the filter, we were able to eliminate the yellow colored impurities. The final
preparation was clear. Quincoces [10] used two Sep-Pak alumina N cartridges as purification columns,
and 5 mL of ethyl ether with 1% ethanol were eluted in two steps from the column. We were unable to
reproduce these results, as the observed radiochemical purity was less than 75% (Figure 1).
Figure 1. RCP of [11C]-(+)--DTBZ by two alumina N Sep-Pak purification.
Minutes
2.3. Quality Control
The radiochemical purity was determined by HPLC; the desired product eluted as a single peak
with a retention time of 4.2 min, which corresponded exactly with the observed UV retention of cold
DTBZ standard at 254 nm. (Figure 2).The radiochemical purity was >99%. The specific radioactivity
was 60 GBq/moL (n = 12).

Molecules 2012, 17
Figure 2. RCP of [11C]-(+)--DTBZ analysised by HPLC (UV was standard).
6700
2.4. Biodistribution of [11C]-(+)--DTBZ in Normal Mice
NH mice were sacrificed at 0, 10, 20, 30, 60 min post-injection of [11C]-(+)--DTBZ. Radioactivity
passed through the blood-brain barrier [BBB] as indicated by cortex uptake of 12.1% ± 1.86% ID/g,
and the striatum of 19.18% ± 1.25% ID/g immediately post-injection. As expected, the ratio of
striatum to cortex increased to about 3.9 at 20 min and remained 3.4 at 30 min. The radioactivity
distribution (Table 2) was greatest at the liver with excretion occurring through the kidney.
Table 2. Biodistribution of [11C]-(+)--DTBZ in mice (mean ± SD, n = 5, %ID/g).
0 min
10 min
20 min
30 min
60 min
Blood
Heart
Liver
Spleen
Lung
Kidney
Striatum
Cortex
12.10 ± 1.85 1.83 ± 0.19 1.93 ± 0.30 1.13 ± 0.07 1.33 ± 0.06
20.87 ± 2.40 3.81 ± 0.69 2.56 ± 0.66 1.82 ± 0.12 1.43 ± 0.08
2.85 ± 0.20 10.27 ± 1.47 13.16 ± 2.26 9.50 ± 0.99 10.61 ± 1.69
1.79 ± 0.51 5.36 ± 0.59 5.35 ± 0.69 3.19 ± 0.15 2.57 ± 0.28
27.24 ± 9.56 5.03 ± 0.63 5.70 ± 0.30 3.34 ± 0.33 1.85 ± 0.44
3.32 ± 1.30 7.57 ± 0.87 7.62 ± 1.12 4.70 ± 0.04 3.42 ± 0.19
19.17 ± 1.25 13.00 ± 0.29 10.85 ± 1.56 5.75 ± 0.39 2.32 ± 0.16
16.29 ± 1.97 3.42 ± 0.08 2.76 ± 0.44 1.71 ± 0.10 0.93 ± 0.11
Striatum/cortex
1.2
3.8
3.9
3.4
2.5

Molecules 2012, 17
6701
3. Experimental
3.1. Materials
1,3,4,6,7,11-Hexahydro-10-methoxy-3-(2-methylpropyl)-(2R,3R,11bR)-2H-benzo[a]quinolizine-2,
9-diol), the precursor for 2H-benzo[a]-quinolizine-2-ol,1,3,4,6,7,11b-hexahydro-9,10-dimethoxy-3-(2-
methylpropyl)-(2R,3R,11bR)-[11C]-(+)--dihydroterabenazine, the reference standard for [11C]-di-
hydroterabenazine; and 1 M lithium aluminum hydride in THF were purchased from ABX Compounds
(Radeberg, Germany). 57% HI, DMSO and acetone were obtained from Aldrich (city, country) and
used without further purification. Sep-Pak C-18 (400 mg), tC-18 (400 mg), Vac tC-18 (500 mg) and
Alumina cartridges (1 g) were purchased from Waters (Milford, MA, USA), 0.22 m sterile filters
were purchased from Millipore (Billerica, MA, USA). The analytical HPLC column (C-18, 250 × 4.6 mm)
was obtained from Phenomenex (Torrance, CA, USA).
3.2. Synthesis of 11C-(+)--DTBZ
This was accomplished as shown in Scheme 1. (+)Desmethyldihydrotetrabenazine (0.8 mg) in
DMSO (0.2 mL) with 3 M KOH (4 L) was mixed with [11C]-methyl triflate as a methylation agent.
Purification was accomplioshed using a Sep-Pak Vac tC-18 and an alumina cartridge.
Scheme 1. Synthesis route of 11C-(+)--DTBZ.
HO
H311CO
N
11C-CH3-Triflate
N
H3CO
CH3
H3CO
CH3
H
H
Base
CH3
CH3
OH
OH
3.3. Fully Automated Radiosynthesis
A scheme of the synthesis module is presented in Figure 4. All details given in the following section
refer to this Figure.
Three kinds of C-18 cartridge were preconditioned using ethanol and water; one of them was
connected between the V6 and V10 position. The alumina cartridge was treated with water and
connected between V10 and the collection vial.
3.4. Instruments
For the fully automated preparation of [11C]-DTBZ, a Methiodine Module and Methylation Module
(PET Beijing Science and Technology Co., Ltd., Beijing, China) were remotely controlled by a
standard PC. Analytical HPLC was performed on the following systems: Waters 515 pump, 2487
UV-detector, and BioScan Flow-Count (Washington, DC, USA).

Molecules 2012, 17
6702
Figure 4. A scheme of the synthesis module.
Reactor and Vessels were filled as follows: Reactor 1: 150 L 1.0 M lithium aluminium hydride
solution in THF at RT; Injector 2: 200 L of 57% HI; Reactor 3: 0.8 mg precursor in DMSO
or acetonitrile or acetone with KOH or base; Vial 4: 10 mL water; Vial 5: 10 mL water; Vial 6:
1 mL ethanol.
3.5. Production of [11C]-Methyl Iodide and [11C]-Methyl-Triflate [11,12]
Irradiation of a 99.5/0.5 mixture of N2/O2 (v/v) with a 20 Mev proton beam at 40 A in a Sumitomo
HM-20S (Osaka, Japan) cyclotron for 10 min yielded about 30 GBq of [11C]-carbon dioxide. At the
end of bombardment, the [11C]-CO2 was passed through stainless steel tubes to the synthesis unit. The
[11C]-CO2 was frozen in a stainless steel loop at −190 C using liquid nitrogen. The frozen [11C]-CO2
was warmed up to −20 C and transferred by a stream of nitrogen gas at 15 mL/min to the reactor
containing 150 L 1.0 M lithium aluminium hydride solution in THF at RT. The reactor was heated to
170 C to remove the THF by a stream of nitrogen gas at 40 mL/min. After cooling the reactor, 200 L
of 57% HI was added and the [11C]-methyl iodide was carried by a stream of nitrogen gas. The
[11C]-methyl iodide was passed over a silver triflate/C on-line with the gas/solid exchange reaction at
210 C yielding the [11C]-methyl triflate.
3.6. Automated On-Line Synthesis of [11C]-(+)--DTBZ
Gaseous [11C]-methyl triflate produced by the Methiodine Module was delivered through V6 into
the reactor of the previously prepared synthesizer system and trapped on-line in the reaction mixture
containing (+)-desmethyldihydrotetrabenazine (0.2 mg) in DMSO or acetone (0.2 mL) with KOH or
NaOH. The methylation reaction was completed in three minutes according to the reaction illustrated
in Figure 4. After the reaction, the solution was passed through Sep-Pak C-18 after mixing with 10 mL
water in Vial 4. The Sep-Pak C-18 was then washed with 10 mL water in Vial 5. The preparation was
eluted from Sep-Pak C-18 with 1 mL ethanol, passed through an alumina cartridges, reconstituted with
9 mL saline and filtered through 0.22 m filter.

Molecules 2012, 17
6703
3.7. Quality Control
Chemical and radiochemical impurities were detected using radio-HPLC. Quality was assessed with
analytical RP-HPLC; acetonitrile and 0.1 M ammonium formate (25/75; v/v, pH = 4.0) were used as
the mobile phase with a flow rate of 1 mL/min. The entire quality control was completed within 8 min;
the retention time of the precursor (Nor-DTBZ) was 3.5 min and the product [11C]-(+)--DTBZ was
eluted with a retention time of 4.2 min. The chemical identity of [11C]-(+)--DTBZ was determined by
co-injection of the unlabelled reference compound, (+)--DTBZ.
3.8. Biodistribution of [11C]-(+)--DTBZ in Normal Mice
[11C]-(+)--DTBZ (0.2 mL, 3.7 MBq) was injected through the tail vein into five groups of NH
mice (20~22 g, n = 5/group). The mice were sacrificed at 0, 10, 20, 30, and 60 min post-injection. The
organs (blood, heart, liver, spleen, lung, kidneys, striatum and cortex) were dissected and weighed,
prepared for counting with a well-type Na(I) detector, and the uptake of each organ was expressed as
the percentage of injection dose per gram (%ID/g). All biodistributions were carried out in compliance
with the national laws related to the conduct of animal experimentation.
4. Conclusions
An automated [11C]-(+)--DTBZ synthesis based on a Sep-Pak Vac tC-18 cartridge purification
procedure is reported. In contrast to HPLC purification, the solid phase extraction (SPE purification)
procedure was easier to include in radiosynthesis under good manufacturing practice (GMP)
conditions. No modification of a commercial module was needed. The [11C]-(+)--DTBZ cartridge
purification is easily transferable to other commercial synthesizers such as GE TracerLab FXc.
Acknowledgment
The authors are very grateful to the National Natural Science Foundation of China (81071170).
References
1. Zhang, G.; Dwoskin, L.P.; Crooks, P.A. Vesicular Monoamine Transporter 2: Role as a Novel
Target for Drug Development. AAPS J. 2006, 8, E682–E692.
2. Bohnen, N.I.; Albin, R.L.; Koeppe, R.A.; Wernette, K.A.; Kilbourn, M.R.; Minoshima, S.; Frey, K.A.
Positron emission tomography of monoaminergic vesicular binding in aging and Parkinson
disease. J. Cerebr. Blood Flow Metab. 2006, 26, 1198–1212.
3. Kilbourn, M.R.; Frey, K.A.; Borght, T.V.; Sherman, P.S. Effects of dopaminergic drug treatments
on in vivo radioligand binding to brain monoamine transporters. Nucl. Med. Biol. 1996, 23,
467–471.
4. Kemmerer, E.S.; Jdesmond, T.; Albin, R.L.; Kilbourn, M.R.; Frey, K.A. Treatment effects on
nigrostriatal projection integrity in partial 6-OHDA lesions: Comparison of L-DOPA and
pramipexole. Exp. Neurol. 2003, 183, 81–86.

Molecules 2012, 17
6704
5. Anlauf, M.; Eissele, R.; Schäfer, M.K.; Eiden, L.E.; Arnold, R.; Pauser, U.; Klöppel, G.; Weihe E.
Expression of the Two Isoforms of the Vesicular Monoamine Transporter (VMAT1 and VMAT2)
in the Endocrine Pancreas and Pancreatic Endocrine Tumors. J. Histochem. Cytochem. 2003, 51,
1027–1040.
6. Harris, P.E.; Ferrara, C.; Barba, P.; Polito, T.; Freeby, M.; Maffei, A. VMAT2 gene expression
and function as it applies to imaging β-cell mass. J. Mol. Med. 2008, 86, 5–16.
7. Lang, A.E. Clinical rating scales and videotape analysis. In Therapy of Movement Disorders,
2nd ed.; Koller, W.C., Paulson, G., Eds.; Marcel Decker Inc.: New York, NY, USA, 1995;
pp. 21–46.
8. Tong, J.; Wilson, A.A.; Boileau, I.; Houle, S.; Kish, S.J. Dopamine Modulating Drugs Influence
Striatal (+)-[11C]DTBZ Binding in Rats:VMAT2 Binding is Sensitive to Changes in Vesicular
Dopamine Concentration. Synapse 2008, 62,873–876.
11
9. Jewett, D.M.; Kilbourn, M.R.; Clee, L. A simple synthesis of C-dihydrotetrabenazine (DTBZ).
Nucl. Med. Biol. 1997, 24, 197–199.
10. Quincoces, G.; Collantes, M.; Catalan, R.; Ecay, M.; Prieto, E.; Martino, E.; Blesa, F.J.;
Álvarez-erviti, L.; Areses, P.; Arbizu, J.; et al. Quick and simple synthesis of
11C-(+)--dihydrotetrabenazine to be used as a PET radioligand of vesicular monoamine
transporters. Rev. Esp. Med. Nucl. 2008, 27, 13–21.
11. Zhang, J.M.; Tian, J.H.; Wang, W.S.; Liu, B.L. An automated synthesis of 11C-methyl iodide with
single vessel. Chin. J. Nucl. Med. 2004, 24, 243–244.
11
12. Zhang, J.M.; Tian, J.H.; Wang, W.S.; Liu, B.L. On-line Production of C-CH3-Triflae. Isotopes
2006, 19, 124–128.
Sample Availability: Not available.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).