Synthesis and Evaluation of 4-Halogeno-N-[4-[6-(isopropylamino

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Synthesis and Evaluation of 4-Halogeno-N-[4-[6(Isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide for Imaging of Metabotropic Glutamate 1 Receptor in Melanoma Masayuki Fujinaga, Lin Xie, Tomoteru Yamasaki, Joji Yui, Yoko Shimoda, Akiko Hatori, Katsushi Kumata, Yiding Zhang, Nobuki Nengaki, Kazunori Kawamura, and Ming-Rong Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501845n • Publication Date (Web): 20 Jan 2015 Downloaded from http://pubs.acs.org on January 25, 2015

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Journal of Medicinal Chemistry

Synthesis and Evaluation of 4-HalogenoN-[4-[6-(Isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2 -yl]-N-[11C]methylbenzamide for Imaging of Metabotropic Glutamate 1 Receptor in Melanoma Masayuki Fujinaga†,§, Lin Xie†,§, Tomoteru Yamasaki†, Joji Yui†, Yoko Shimoda†, Akiko Hatori†, Katsushi Kumata†, Yiding Zhang†, Nobuki Nengaki†,‡, Kazunori Kawamura†, and Ming-Rong Zhang*,†

†

Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku,

Chiba 263-8555, Japan; ‡SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

§

These authors contributed equally

Corresponding Author:

* Ming-Rong Zhang, Phone: +81-43-382-3709, Fax: +81-43-206-3261 E-mail: [email protected]

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ABSTRACT

Metabotropic glutamate 1 (mGlu1) receptor is found not only in the brain but also in melanomas and breast cancers. mGlu1 is a promising target for molecular imaging-based diagnosis and treatment of melanoma because its overexpression induces melanocyte carcinogenesis. Here, we developed three PET tracers: 4-halogeno-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol2-yl]-N-[11C]methylbenzamide ([11C]4–6), which exhibited high uptake in target tumor and decreased uptake in non-target brain tissues. In vitro binding assay indicated high to moderate binding affinities of 4–6 (Ki, 22–143 nM) for mGlu1 receptor. In vivo biodistribution studies in mice implanted with B16F10 melanoma cells confirmed high radioactive uptake in tumor and low uptake in blood, skin, and muscles. Inhibition of mGlu1 receptor using an mGlu1 selective ligand led to reduced radioactive uptake in the tumor. [11C]6 displayed the highest ratio of uptake between tumor and non-target tissue and may prove useful as a PET tracer for mGlu1 imaging in melanoma.


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INTRODUCTION Glutamate receptors are divided into iontropic and metabotropic types based on the mechanism by which their activation gives rise to a postsynaptic current.1,2 Metabotropic glutamate 1 (mGlu1) receptor, one of eight subtypes of metabotropic type receptors, was originally found to be highly expressed in the brain.1-3 Followed by brain, mGlu1 was also found ectopically in melanomas,4 breast cancer5 and other cancers6,7. The overexpressed mGlu1 receptor is reported to exhibit oncogenic characteristics that independently trigger melanocyte tumorigenesis. The melanocytes show 100% penetrance through continuous activation of mitogen-activated protein kinase and phoshatidylinositol-3-kinase / protein kinase B pathways in a B-Raf– and N-Ras– independent fashion.8-11 Contrary to normal skin and benign nevi, which do not express mGlu1, around 68 to 88% of human melanoma biopsy specimens and human melanoma cell lines show overexpression of mGlu1 receptor.12,13 Further, inhibition or inactivation of mGlu1 was demonstrated to prevent growth and progression of melanomas. Although a definite mechanistic role of mGlu1 in melanocyte tumorigenesis is not yet determined, emipirical data suggest that mGlu1 might prove to be a promising molecular imaging target and may be applied in the diagnosis and personalized treatment of melanomas.

Several positron emission tomography (PET) tracers specifically binding to mGlu1 in vivo and exhibiting high uptake of radioactivity have been developed to visualize mGlu1 receptors in living brain.14-20

Among

these

radiotracers,

4-[18F]fluoro-N-[4-(6-(isopropylamino)pyrimidin-4-yl)-

1,3-thiazol-2-yl]-N-methylbenzamide ([18F]FITM, [18F]1, Scheme 1) is a novel PET tracer displaying high affinity for mGlu1 (IC50: 5.1 nM),21 and is highly selective for human mGlu1 over ACS Paragon Plus Environment


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other subtypes (IC50: >7 µM).22,23 PET studies with [18F]1 and its derivatives, [11C]2 and [11C]3, have been used for qualitative and quantitative investigations on mGlu1 densities and change in animal models of neurodegenerative diseases.24-28 Moreover, [11C]2 has been used as the first PET tracer applied for clinical imaging studies of mGlu1 in human brain.29

Based on the success of various radiotracers in brain imaging, we recently reported the first experiment on visualization of mGlu1 receptor in melanoma using [18F]1 in PET.30 [18F]1 showed high uptake of radioactivity in a melanoma model implanted subcutaneously with a mGlu1-positive B16F10 melanoma cell line. However, owing to a high density of mGlu1 in brain, [18F]1 showed considerable uptake of radioactivity in the brain, which in turn decreased its selectivity for detection of tumors over non-targeted organs or tissues and hampered its clinical application.30 Furthermore, accumulation and slow clearance of radioactivity from brain aroused the possibility of radiation induced damage. Nevertheless, [18F]1 was considered useful as a lead compound for further development of radiotracers useful for specific detection of mGlu1-positive melanomas and noninvasive quantification of mGlu1 expression in tumors.

Scheme 1

To overcome the shortcomings in [18F]1 which undermined its usage in clinical applications, we designed three novel ([11C]6)-

11

C-labeled radiotracers: 4-chloro ([11C]4), 4-bromo ([11C]5), and 4-iodo

N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide

(Scheme 1), by introducing other halogen atoms (chlorine, bromine, or iodine) into [18F]1 instead of a fluorine atom. The synthesis of [11C]4–6 and the evaluation of their potentials in PET imaging of ACS Paragon Plus Environment

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mGlu1 receptors in melanoma formed the aim of this study. It was expected that the replacement of the smaller fluorine atom by other larger halogen atoms may limit entrance of [11C]4–6 into the brain while simultaneously maintaining high tumor uptakes. Owing to its short half-life, 20.2 min) is practically inconvenient compared to

18

11

C (half-life:

F (half-life: 109.8 min). We hypothesized that

the use of Br and I in these compounds would render compounds with longer half-lives. It would be possible to label them using the isotopes of non-standard PET (76Br, half-life: 16 h or

124

I, half-life:

4.18 days), SPECT (123I, half-life: 13.27 h) and radiotherapy (131I, half-life: 8 days) without altering their chemical structures or pharmacological profiles.

In this present study, we report the radiosynthesis of [11C]4–6 and their in vitro binding affinity for mGlu1, biodistribution, and PET imaging of mice bearing B16F10 melanoma.

RESULTS AND DISCUSSION

Chemical synthesis. Unlabeled target compounds 4–6 and their corresponding desmethyl precursors 7–9 for radiosynthesis were synthesized according to pathways represented in Scheme 2. Among these compounds, synthesis of the chlorine analog 4 was reported in a paper previously published from our laboratory.25 Ethoxyvinylpyrimidine 10 was prepared according to a procedure described previously.24 Bromination of 10 with 1-bromopyrrolidine-2,5-dione, followed by treatment with thiourea derivatives, gave 11 and 12 at 83% and 61% yields, respectively. Benzoylation of 11 and 12 with the appropriate 4-substituted benzoyl chlorides afforded 13–18 at yields ranging from 19 to 90%. Three precursor compounds 7–9 and targeted compounds 4–6 were obtained by the reaction of 13–15 and 16–18 with isopropylamine at yields of 26–65%, respectively. ACS Paragon Plus Environment


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Scheme 2

Radiosynthesis of [11C]4–6. Compounds 4–6 were labeled with

11

C by N-[11C]methylation of

their corresponding desmethyl precursors 7–9 (Scheme 3). Radiosynthesis was performed using an automated synthesis system which had been developed in our facility.31,32

Scheme 3

Radiosynthesis of the chlorine analog [11C]4 was performed by allowing 7 to react with conventionally

used

[11C]methyl

iodide

([11C]CH3I),

which

was

prepared

from

the

cyclotron-produced [11C]carbon dioxide ([11C]CO2). However, the radiochemical yield of [11C]4 using [11C]CH3I was low and less than 370 MBq of [11C]4 was obtained by starting with 17 GBq of [11C]CO2 at the end of synthesis (EOS). This amount of radioactivity was not enough for evaluation of animal experiments. Thus, to improve the [11C]methylation efficiency, compound 7 was allowed to react with the more reactive [11C]methyl trifluoromethanesulfonate ([11C]CH3OTf). [11C]CH3OTf was prepared by passing [11C]CH3I through a heated column coated with silver triflate on graphitized carbon33 (Scheme 3). The resulting [11C]CH3OTf was subsequently trapped in a solution of 7 and NaOH in anhydrous acetone at room temperature. Upon completion of reaction, purification of the reaction mixture by semi-preparative reversed phase HPLC resulted in [11C]4 at radiochemical yields of 18 ± 5% (n = 7). Similar to [11C]4, [11C]5 or [11C]6 was synthesized by the reaction of 8 or 9 with [11C]CH3OTf. Radiochemical yields were 17 ± 4% (n = 7) or 20 ± 5% (n = 7), respectively. Radiochemical yields for [11C]4–6 were decay-corrected based on [11C]CO2 at EOS. Starting from 15.5–22.2 GBq of [11C]CO2, 1.0–1.9 GBq of [11C]4–6 was produced within an ACS Paragon Plus Environment

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average synthesis time of 31 min from the end of bombardment (EOB). Specific activity and radiochemical purity of [11C]4–6 were determined in the final product solution and were 65–160 GBq/µmol and higher than 97% at EOS, respectively. These radioactive products did not undergo any obvious radiolysis at room temperature until 120 min after formulation, indicating their radiochemical stability within the duration of at least one PET scan. The analytical results of [11C]4–6 were in compliance with the quality control and assurance specifications for PET radiopharmaceuticals produced in our facility. In Vitro Binding Assays. The in vitro binding affinity (inhibition constant, Ki) of compounds 4–6 for mGlu1 receptor was measured by a binding assay using the mGlu1-seletive radiotracer [18F]1 in rat brain homogenates24. As shown in Table 1, compounds 4–6 exhibited either high or moderate binding affinities for mGlu1, Ki values were observed to be 22, 35, and 143 nM, respectively. Compared to compound 1 (Ki; 14 nM or 5.4 nM24), the in vitro binding affinity for mGlu1 decreased with an increase in atomic number in the halogen column. This suggested that the mGlu1 receptor binding affinity was affected by substitution at the 4-position of the benzene ring in these compounds. We assume that difference in size or electronic property among the substituted halogen atoms might have contributed to changed binding affinity of 4–6 for mGlu1. In the present PET study of melanoma, we used a well established B16F10 melanoma cell line34,35 and C57BL/6J mice implanted with B16F10 cells.30 Previously, immunohistochemistry assays and in vitro autoradiography could confirm abundance of mGlu1 in mice bearing B16F10.30 The Bmax of mGlu1 in the B16F10 tumor sections was estimated around 120 fmol/mg protein (120 nM). The binding affinity of 4–6 for mGlu1 enables their PET imaging of mGlu1 in melanoma, although the ACS Paragon Plus Environment


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affinity of 6 seemed insufficient for imaging brain mGlu1 mainly because of lower density of mGlu1 receptor in the brain (for example: 36 nM in rat thalamus26) than that in the melanoma. Table 1 Computation and Measurement of Lipophilicity. Theoretically computed lipophilicity values at pH 7.4 (cLogD) for 4–6 were 2.62–3.42 (Table 1). Following the labeling of these compounds with

11

C, lipophilicity (LogD) values of [11C]4–6 were measured using the Shake Flask method.16

The experimentally determined LogD values of [11C]4–6 were 3.04–3.33.

Biodistribution Study in B16F10 Melanoma Mice. The distribution of radioactivity in mGlu1-positive B16F10 melanoma mice was measured at six experimental time points (1, 5, 15, 30, 60, and 90 min) after the injection of each radiotracer ([11C]4, Table 2; [11C]5, Table 3; and [11C]6, Table 4). Table 2 Table 3 Table 4 Radiotracers [11C]4–6 showed similar pharmacokinetic profiles of radioactivity in the major peripheral organs. High radioactive uptakes (> 5% ID/g) were observed in the blood, lung, heart, and kidney 1 min after the injection and then decreased rapidly to low levels ( [11C]5 > [11C]6. Radioactivity uptake in the tumor itself increased gradually after the early phases in case of [11C]4 and [11C]5 and reached the maximum values at 60 min (4.91% ID/g for [11C]4 at 30 min and 4.24% ID/g for [11C]5). Uptake reduced to 3.91% ID/g and 3.61% ID/g at 90 min, respectively. In case of [11C]6, radioactivity level was higher than 4% ID/g 15 min after the injection and reached a maximum value of 4.61% ID/g at 90 min. The order of uptakes in the tumor was [11C]6 > [11C]5 > [11C]4 at 90 min. mGlu1 is abundantly expressed in the brain leading to a high uptake of the mGlu1 PET radiotracers. High radioactive uptakes may hinder usefulness of radiotracers in tumor imaging. Thus, a simultaneous low brain uptake and a high tumor uptake is indispensable for development and application of a PET tracer for imaging mGlu1 levels in the tumor. In our previous experiments, [18F]1 was reported to show a high uptake at 90 min not only in the tumor (5% ID/g) but also in the brain (10% ID/g), limiting its application.30 Compared to [18F]1, [11C]4–6 showed significantly reduced uptakes and highest radioactivity levels of 5.19% ID/g for [11C]4 at 15 min, 2.97% ID/g for [11C]5 at 5 min, and 1.65% ID/g for [11C]6 at 5 min after the injection, respectively. Thus, [11C]6 ACS Paragon Plus Environment


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showed the lowest uptake (0.57% ID/g) in the brain while maintaining the highest uptake (4.67% ID/g) in the tumor at 90 min after injection. Figure 1 The ratio of radioactivity in tumor and in surrounding regions, i.e. in target regions compared to healthy non-targeted organs or tissues, is an important index reflective of the specificity of a radiotracer for the tumor in imaging studies.36 Figure 1 shows the ratios of radioactivity in the tumor compared to brain, blood, skin, and muscle between 1 and 90 min after the injection of [11C]4–6. All the four ratios increased with time and [11C]6 showed the highest ratio of tumor to brain, while the corresponding ratio of tumor to brain for [18F]1 was only 0.5 at 90 min. No significant difference was observed for [11C]4–6 in the ratios of tumor to blood, tumor to skin, and tumor to muscle, respectively. This suggested specific accumulation of radioactivity in the tumors. Thus, [11C]4–6 could be further tested for in vivo PET imaging of melanoma in mice owing to their high specificity for target tissue along with a high degree of portioning of radioactivity in tumors compared to the background. PET Study of Mice Bearing B16F10 Melanoma. We performed PET imaging study with [11C]4–6 for the subcutaneous B16F10-bearing mice. Since the biodistribution study showed relatively definite radioactivity levels in the tumor between 30 and 90 min after the injection of each radiotracer (Table 1–3), a time frame between 45 and 75 min was chosen for performing dynamic PET scans. Images were acquired by summing the uptake of radioactivity within this duration (Figs. 2–4). Figure 2 ACS Paragon Plus Environment

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Fig. 2 shows the time-activity curves (TACs) in the tumor and brain of mice between 45 and 75 min after the injection of [11C]4–6. Radioactivity uptake (% ID/g) was similar to biodistribution data reported above (Table 2–4). These radiotracers displayed similar tumor uptakes (~4.0–4.2% ID/g at 60 min), which were close to previous PET based [18F]1 radioactivity level measurements (4.5% ID/g) for identical B16F10-bearing mice. Definite radioactive levels of [11C]4–6 were also observed in the brain. However, values were different from each other. Uptake in brain decreased with an increase in atomic number of the halogen atom. In particular, the iodo [11C]6 showed much lower brain uptake than the chloro [11C]4 and bromo [11C]5. Figure 3 Fig. 3 summarizes the uptakes of radioactivity, which were quantified using AUC45-75 min (% ID/g × min), in the mouse tumors and brains. Both [18F]1 and [11C]4–6 showed similar AUC45-75 min values of ~ 120–130% ID/g in the tumor (p > 0.05). Interestingly, compared to the AUC45-75 min value in the brain for [18F]1 (380% ID/g ), [11C]4–6 showed significantly reduced AUC45-75 min values which were 110, 90, and 30% ID/g, respectively. Thus, substitution of fluorine to iodine atom at position 4 in the benzene ring of [11C]4–6 resulted in limited effects on their uptake, and [11C]6 exhibited the lowest uptake in the brain (p < 0.001). Figure 4 Representative summed PET images (45–75 min) were acquired under the same scanning conditions and are shown in Fig. 4. Radioactive signals in the sagittal brain images of control, particularly in cerebellum and thalamus, decreased in the order: [11C]4 > [11C]5 > [11C]6. High



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amounts of radioactivity was observed to accumulate in the tumor as observed in the horizontal tumor images of control and low uptake was noted in muscle. To determine whether uptake for these radiotracers in the tumor was specific to mGlu1 receptor, we performed inhibitory experiments using the mGlu1-specific and selective ligand 1. Injection of excess 1 led to significantly reduced [11C]4–6 radioactivity levels in both the brain and the tumor. Comparative differences in the TACs (Fig. 2), AUC45-75 min values (Fig. 3), and PET images (Fig. 4) between the control and treatment with 1 implied specificity of radiotracers for mGlu1 in the tumor. As shown in the AUC45-75

min

values (Fig. 3), the specific binding of [11C]4–6 for mGlu1 accounted

for 51–58% of the total binding. In the brain, about 43% of total binding of [11C]4 and [11C]5 was specific to mGlu1, whereas no specific binding of [11C]6 was found in the brain. In fact, the brain uptake of [11C]6 was low.

This study indicated that substitution of fluorine atom in [18F]1 to iodine atom in [11C]6 could decrease uptake of radioactivity in brain tissue. This may partially be explained by the lipophilicity of [11C]6 (LogD: 3.33) along with an decreased binding affinity for mGlu1 receptor compared to other radiotracers. A high lipophilicity may allow binding of [11C]6 to blood albumin and limit its entrance into the brain. Although this might also limit the uptake of [11C]6 in the tumor, the effects of lipophilicity on the brain uptake than on the tumor uptake seem limited. Because the density of mGlu1 in the brain is lower than that in the melanomas, [11C]6 with its moderate mGlu1 binding affinity may be able to bind mGlu1 only in the tumor but not in the brain and may selectively bind melanoma tumors.


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Considering the presence of iodine atom in compound 6 and definite radioactivity level of [11C]6 in melanoma during extended periods, we plan to label 6 with radioactive iodine isotopes 131

124

I and

I . This would afford longer half-lives to the radiotracers than those based on 11C. Prolonging the

starting time of PET scan after radiotracer injection may be useful for acquiring PET images with higher signal ratio of tumor to background. More importantly, utilization of the radiotherapy isotope in the form of 131I-labeled 6 may achieve therapeutic effect for melanoma. SUMMARY In summary, three novel radiotracers [11C]4–6 were developed and evaluated for application in PET-based visualization of mGlu1 receptor in melanoma. [11C]4–6 could be synthesized in a short synthesis time and reliable radiochemical yields by the N-[11C]methylation of the corresponding desmethyl precursors 7–9 with [11C]CH3OTf. These PET radiotracers showed high to moderate in vitro binding affinities for mGlu1 receptor and high uptakes in the melanoma. The ratio of radioactivity between tumor compared to blood, skin, and muscle was very high. The iodine analog [11C]6 showed the highest ratio of radioactivity of tumor to brain and is thus regarded as a useful PET tracer for imaging mGlu1 in melanoma in the future. [11C]6 can be further developed by and

131

124

I

I radiolabeling for long-duration PET scan and radiotherapeutic usage. These labeling and

evaluation studies are in progress in our laboratory.

EXPERIMENTAL SECTION



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Materials and Methods. Melting points were measured using a micro melting point apparatus (MP-500P, Yanaco, Tokyo, Japan) and are uncorrected. 1H–NMR (300 MHz) spectra were recorded on a JEOL-AL-300 spectrometer (JEOL, Tokyo) using tetramethylsilane (TMS) as an internal standard. All chemical shifts (δ) are reported as parts per million (ppm) downfield relative to the TMS signal. Signals are annotated as s (singlet), d (doublet), t (triplet), br (broad), or m (multiplet). Fast atom bombardment mass spectra (FAB–MS) and high-resolution mass spectra (HRMS) were recorded on a JEOL-AL-300 spectrometer (JEOL) and recorded on the spectrometer. Silica gel column chromatography was performed using Wako gel C-200 (70–230 mesh, Wako Pure Chemical Industries, Osaka, Japan). HPLC analysis was performed using a JASCO HPLC system (JASCO, Tokyo) and Capcell Pack C18 column (4.6 mm i.d. × 250 mm, Shiseido, Tokyo). Chemical purity of compounds 4–6 and 7–9 determined by analytical high performance liquid chromatography (HPLC) was more than 95%. Conditions used for HPLC were as follows: flow rate 1.0 mL/min; solvents, acetonitrile (MeCN)/H2O/triethylamine (Et3N), (7/3/0.01, v/v/v) for compounds 4–6 and MeCN/H2O/Et3N, (6/4/0.01, v/v/v) for 7–9.

Carbon-11 (11C) was produced by a 14N (p, α) 11C nuclear reaction using CYPRIS HM-18 cyclotron (Sumitomo Heavy Industry, Tokyo). Unless stated otherwise, radioactivity was measured with an IGC-3R Curiemeter (Aloka, Tokyo). [18F]1 used for the in vitro binding assays was prepared according to the previously described procedures.22 Chemical synthesis

4-Bromo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide


(5).

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Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 17 (150 mg, 0.37 mmol) and K2CO3 (102 mg, 0.74 mmol) in 1,4-dioxane (5 mL) at room temperature, and the reaction mixture was heated at 80 °C for 8 h. The reaction mixture was quenched with water and extracted with CH2Cl2. The organic layer was washed with brine, dried over Na2SO4, and evaporated under reduced pressure. Residue was purified by column chromatography using n-hexane/ethyl acetate/Et3N (1/1/0.001, v/v/v) to give 5 (89 mg, 55.6%) as a colorless solid; mp 180–182 °C. 1H NMR (CDCl3): δ 1.29 (6H, d, J = 6.6 Hz), 3.73 (3H, s), 4.10 (1H, br), 4.84 (1H, br), 7.04 (1H, s), 7.46 (2H, d, J = 8.4 Hz), 7.66 (2H, d, J = 8.4 Hz), 7.93 (1H, s), 8.57 (1H, s). HRMS m/z: 432.0465 (calculated for C18H19ON5BrS: 432.0494). 4-Iodo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide

(6).

Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 18 (150 mg, 0.33 mmol) and K2CO3 (91 mg, 0.66 mmol) in 1,4-dioxane (12 mL) was added at room temperature. The reaction mixture was heated at 80 °C for 8 h. The mixture was treated as described for the synthesis of 5. Purification by column chromatography using n-hexane/ethyl acetate/Et3N (1/1/0.001, v/v/v) gave 6 (103 mg, 65.1%) as a colorless solid; mp 210–211 °C. 1H NMR (CDCl3): δ 1.29 (6H, d, J = 6.2 Hz), 3.73 (3H, s), 4.12 (1H, br), 4.85 (1H, br), 7.04 (1H, s), 7.32 (2H, d, J = 8.4 Hz), 7.87 (2H, d, J = 8.1 Hz), 7.93 (1H, s), 8.57 (1H, s). HRMS m/z: 480.0386 (calculated for C18H19ON5SI: 480.0355). 4-Chloro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]benzamide

(7).

Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 13 (100 mg, 0.29 mmol) and K2CO3 (79 mg, 0.57 mmol) in 1,4-dioxane (5 mL) was added at room temperature. The reaction mixture was stirred at 80 °C for 10 h. The mixture was treated as described for the synthesis of 5. Column ACS Paragon Plus Environment


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chromatography of the residue on silica gel under CH2Cl2/CH3OH (15/1, v/v) gave 7 (53 mg, 49.7%) as a white powder; mp 198–200 °C. 1H NMR (DMSO-d6): δ 1.19 (6H, d, J = 6.2 Hz,), 4.13 (1H, br), 6.99 (1H, s), 7.41 (1H, d, J = 7.3 Hz), 7.65 (2H, d, J = 8.4 Hz), 7.92 (1H, s), 8.14 (2H, d, J = 8.4 Hz), 8.42 (1H, s), 12.87 (1H, s). HRMS m/z: 374.0873 (calculated for C17H17ON5ClS: 374.0842). 4-Bromo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]benzamide

(8).

Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 14 (150 mg, 0.38 mmol) and K2CO3 (105 mg, 0.76 mmol) in 1,4-dioxane (7 mL) at room temperature. The reaction mixture was stirred at 80 °C for 11 h. The mixture was treated as described for the synthesis of 5. Column chromatography of the residue on silica gel under CH2Cl2/CH3OH (15/1, v/v) gave 8 (93 mg, 58.5%) as a white powder; mp 202–204 °C. 1H NMR (DMSO-d6): δ 1.19 (6H, d, J = 6.4 Hz), 4.14 (1H, br), 6.99 (1H, s), 7.41 (1H, d, J = 7.5 Hz), 7.79 (2H, d, J = 8.4 Hz), 7.92 (1H, s), 8.06 (2H, d, J = 8.6 Hz), 8.42 (1H, s), 12.86 (1H, s). HRMS m/z: 418.0371 (calculated for C17H17ON5BrS: 418.0337). 4-Iodo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]benzamide (9). Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 15 (182 mg, 0.41 mmol) and K2CO3 (86 mg, 0.62 mmol) in 1,4-dioxane (7 mL) at room temperature. The reaction mixture was stirred at 80 °C for 11 h. The mixture was treated as described for the synthesis of 5. Column chromatography of the residue on silica gel under CH2Cl2/CH3OH (15/1, v/v) gave 9 (50 mg, 26.3%) as a white powder; mp 201–203 °C. 1H NMR (DMSO-d6): δ 1.19 (6H, d, J = 6.6 Hz), 4.14 (1H, br), 6.99 (1H, s), 7.40 (1H, d, J = 7.7 Hz), 7.87–7.97 (5H, m), 8.42 (1H, s), 12.84 (1H, s). HRMS m/z: 466.0188 (calculated for C17H17ON5SI: 466.0199). 4-(6-Chloropyrimidin-4-yl)-thiazol-2-amine (11). 1-Bromopyrrolidine-2,5-dione (2.55 g, 14.3 ACS Paragon Plus Environment

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mmol) was added to a solution of 4-chloro-6-(1-ethoxyvinyl)pyrimidine24 (10; 2.40 g, 13.0 mmol) in THF/H2O (40 mL, 1/1) and stirred at room temperature for 2 h. Thiourea (0.99 g, 13.0 mmol) was added to the reaction mixture and the reaction mixture was stirred for 3 h. The resulting precipitate was collected by filtration. Moreover, the filtrate was extracted with CH2Cl2. The organic layer was washed with brine, dried over Na2SO4, and evaporated under reduced pressure. The residue was purified by column chromatography using n-hexane/ethyl acetate (1/1, v/v) to give 11 (2.3 g, 83.3%) as a yellow solid; mp. 203–206 °C. 1H NMR (DMSO-d6): δ 7.32 (2H, s), 7.67 (1H, s), 7.76 (1H, s), 8.95 (1H, s). HRMS m/z: 212.9979 (calculated for C7H6N4ClS: 213.0002). 4-Chloro-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]benzamide

(13).

4-Chlorobenzoyl

chloride (294 µL, 2.3 mmol) was added to a solution of 11 (319 mg, 1.5 mmol) and Et3N (627 µL, 4.5 mmol) in THF (5 mL) under N2 atmosphere. The reaction mixture was stirred at 60 °C for 14 h. This mixture was quenched with water and extracted with CH2Cl2. The organic layer was dried over Na2SO4 and evaporated under reduced pressure. Column chromatography of the residue on silica gel under CH2Cl2 gave 13 (100 mg, 19%) as a white powder; mp 245–247 °C. 1H NMR (DMSO-d6): δ 7.66 (2H, d, J = 8.4 Hz), 8.01 (1H, s), 8.15 (2H, d, J = 8.4 Hz), 8.32 (1H, s), 9.07 (1H, s), 13.00 (1H, s). HRMS m/z: 350.9884 (calculated for C14H9ON4Cl2S: 350.9874) 4-Bromo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]benzamide

(14).

4-Bromobenzoyl

chloride (241 mg, 1.1 mmol) was added to a solution of 11 (160 mg, 0.75 mmol) and Et3N (314 µL, 2.25 mmol) in THF (5 mL) at room temperature under N2 atmosphere. The reaction mixture was stirred at 60 °C for 16 h. The mixture was treated as described for the synthesis of 13. Column chromatography of the residue on silica gel under n-hexane/ethyl acetate (3/1, v/v) gave 14 (188 mg, ACS Paragon Plus Environment


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63.5%) as a pale yellow powder; mp 239–241 °C. 1H NMR (DMSO-d6): δ 7.80 (2H, d, J = 8.4 Hz), 8.01 (1H, s), 8.07 (2H, d, J = 8.4 Hz), 8.31 (1H, s), 9.06 (1H, s), 13.00 (1H, s). HRMS m/z: 394.9410 (calculated for C14H9ON4ClBrS: 394.9369). 4-Iodo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]benzamide (15). 4-Iodobenzoyl chloride (400 mg, 1.5 mmol) was added to a solution of 11 (213 mg, 1.0 mmol) and Et3N (419 µL, 3.0 mmol) in THF (5 mL) at room temperature under N2 atmosphere. The reaction mixture was stirred at 60 °C for 17 h. The mixture was treated as described for the synthesis of 13. Column chromatography of the residue on silica gel under n-hexane/ethyl acetate (3/1, v/v) gave 15 (340 mg, 76.8%) as a white powder. mp 227–229 °C. 1H NMR (DMSO-d6): δ 7.90 (2H, d, J = 8.4 Hz), 7.97 (2H, d, J = 8.4 Hz), 8.01 (1H, s), 8.31 (1H, s), 9.07 (1H, s), 12.98 (1H, s). HRMS m/z: 442.9267 (calculated for C14H9ON4ClSI: 442.9230) 4-Bromo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide

(17).

4-Bromobenzoyl chloride (1.65 g, 7.5 mmol) was added to a solution of 12 (1.14 g, 5.0 mmol) and Et3N (2.1 mL, 15.0 mmol) in toluene (30 mL) at room temperature under N2 atmosphere. The reaction mixture was stirred at 100 °C for 6 h. This mixture was quenched with water and extracted with CH2Cl2. The organic layer was dried over Na2SO4 and evaporated under reduced pressure. Column chromatography of the residue on silica gel under CH2Cl2/Et3N (1/0.001, v/v) and then CH2Cl2/ethyl acetate (10/1, v/v) gave 17 (1.1 g, 54%) as a white powder; mp 225–226 °C. 1H–NMR (CDCl3): δ 3.76 (3H, s), 7.48 (2H, d, J = 8.4 Hz), 7.68 (2H, d, J = 8.4 Hz), 8.06 (1H, s), 8.13 (1H, s), 8.95 (1H, s). HRMS m/z: 408.9545 (calculated for C15H11ON4ClBrS: 408.9525). 4-Iodo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (18). 4-Iodobenzoyl ACS Paragon Plus Environment

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chloride (264 mg, 0.99 mmol) was added to a solution of 12 (150 mg, 0.66 mmol) and Et3N (275 µL, 2.0 mmol) in toluene (5 mL) at room temperature under N2 atmosphere. The reaction mixture was stirred at 100 °C for 6 h. The mixture was treated as described for the synthesis of 17. Column chromatography of the residue on silica gel under CH2Cl2/Et3N (1/0.001, v/v) and then CH2Cl2/ethyl acetate (10/1, v/v) gave 18 (210 mg, 70.0%) as a white powder; mp 281–282 °C. 1H–NMR (CDCl3): δ 3.75 (3H, s), 7.34 (2H, d, J = 8.6 Hz), 7.89 (2H, d, J = 8.6 Hz), 8.05 (1H, s), 8.13 (1H, s), 8.95 (1H, s). HRMS m/z: 456.9354 (calculated for C15H11ON4ClSI: 456.9387). Radiochemistry 4-Chloro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide ([11C]4). [11C]CO2 produced in the cyclotron after irradiation was bubbled into 0.4 M lithium aluminum hydride in anhydrous tetrahydrofuran (THF, 0.3 mL). After evaporation of THF, the remaining complex was treated with 57% hydroiodic acid (0.3 mL) to give [11C]CH3I, which was distilled and transferred through a glass tube containing sliver trifluoromethanesufonate and graphitized carbon under N2 gas flow to yield [11C]methyl trifluoromethanesulfonate ([11C]CH3OTf). [11C]CH3OTf was immediately transferred into a solution containing 7 (1.0 mg) and 0.5 M aqueous NaOH solution (5 µL) in anhydrous acetone (300 µL) at room temperature. The reaction mixture was kept at room temperature for 5 min after the radioactivity reached a plateau. After removal of acetone, 1.0 mL of the preparative HPLC mobile phase was added. HPLC separation was completed on a Capcell Pack C18 column (10 mm i.d. × 250 mm; Shiseido) using MeCN/H2O/Et3N (7/3/0.01, v/v/v) at 4.5 mL/min. The radioactive fraction corresponding to [11C]4 (tR: 7.7 min) was collected in a sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and ACS Paragon Plus Environment


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passed through a 0.22 µm Millipore filter to give 1.9 GBq of [11C]4. The identity of [11C]4 (tR: 5.8 min) was confirmed by analytical HPLC with 4. Specific activity was calculated by comparing the assayed radioactivity to the mass measured at UV 254 nm. The synthesis time was 30 min from EOB; radiochemical yield (decay-corrected), 20% based on [11C]CO2; radiochemical purity, > 99%; specific activity at EOS, 130 GBq/µmol. 4-Bromo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide ([11C]5). [11C]CH3OTf was transferred immediately upon synthesis into a solution containing 8 (1.0 mg) and 0.5 M aqueous NaOH solution (5 µL) in anhydrous acetone (300 µL) at room temperature. The reaction mixture was kept further for 5 min at room temperature after radioactivity reached a plateau. After removal of acetone, 1.0 mL of the preparative HPLC mobile phase was added. HPLC separation was completed on a Capcell Pack C18 column (10 mm i.d. × 250 mm; Shiseido) using MeCN/H2O/Et3N (7/3/0.01, v/v/v) at 4.5 mL/min. The radioactive fraction corresponding to [11C]5 (tR: 8.1 min) was collected in a sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and passed through a 0.22 µm Millipore filter to give 1.4 GBq of [11C]5. The identity of [11C]5 (tR: 6.3 min) was confirmed by analytical HPLC using 5. Specific activity was calculated by comparing the observed radioactivity with the mass measured at UV 254 nm. The synthesis time was 31 min from EOB; radiochemical yield (decay-corrected), 23% based on [11C]CO2; radiochemical purity, > 99%; specific activity at EOS, 160 GBq/µmol. 4-Iodo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide ([11C]6). The resulted [11C]CH3OTf and transferred under N2 gas flow into a solution containing 9 (1.0 mg) and 0.5 M aqueous NaOH solution (5 µL) in anhydrous acetone (300 µL) at room ACS Paragon Plus Environment

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temperature. The reaction mixture was kept at room temperature for 5 min after the radioactivity reached a plateau. After removal of acetone, 1.0 mL of the preparative HPLC mobile phase was added, the final mixture was applied to the HPLC system. HPLC separation was completed on a Capcell Pack C18 column (10 mm i.d. × 250 mm; Shiseido) using MeCN/H2O/Et3N (7/3/0.01, v/v/v) at 4.5 mL/min. The radioactive fraction corresponding to [11C]6 (tR: 8.5 min) was collected in a sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and passed through a 0.22 µm Millipore filter to give 1.8 GBq of [11C]6. The identity of [11C]6 (tR: 7.1 min) was confirmed by analytical HPLC with 6. The synthesis time was averaged to be 31 min from EOB; radiochemical yield (decay-corrected), 25% based on [11C]CO2; radiochemical purity, > 99%; specific activity at EOS, 100 GBq/µmol. Computation and Measurement of Lipophilicity. The cLogD values of compounds 4–6 were determined computationally using Pallas 3.4 software (CompuDrug, Sedona, AZ). The LogD value was measured by mixing [11C]4–6 (radiochemical purity: 100%; about 200,000 cpm) with n-octanol (3.0 g) and phosphate buffered saline (PBS; 3.0 g, 0.1 M, pH 7.4) in a test tube. After vortexing for 3 min at room temperature, contents were centrifuged at 2,330 g for 5 min. An aliquot of 1 mL PBS and 1 mL n-octanol was removed, weighed, and its radioactivity was counted using a 1480 Wizard autogamma counter (Perkin-Elmer, Waltham, MA), respectively. Each sample from the remaining organic layer was removed and repartitioned until a consistent LogD value was obtained. The LogD value was calculated by comparing the ratio of cpm/g of n-octanol to that of PBS and expressed as LogD = Log[cpm/g (n-octanol)/cpm/g (PBS)]. All measurements were performed in triplicate.



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Animal Experiments. Animal studies were approved by the Animal Ethics Committee of National Institute of Radiological Sciences (Chiba. Japan). The animals were maintained and handled in accordance with the recommendations of National Institute of Health and the institutional guidelines of National Institute of Radiological Sciences. In Vitro Binding Affinity for mGlu1 Receptor. Sprague-Dawley rats (8 week, 250–290 g, n = 5) were sacrificed by decapitation under anesthesia with isoflurane (5 % in air). Entire rat brains were rapidly removed after sacrifice and homogenized in 10 volumes of 50 mM Tris-HCl (pH, 7.4) containing 120 mM NaCl with a Silent Crusher S homogenizer (Heidolph Instruments). The homogenate was centrifuged in polypropylene tube at 40,000 g for 15 min at 4°C using an Optima-TLX (Beckman Coulter). Supernatant and the pellet was resuspended and centrifuged in the same buffer. This procedure was repeated twice and the final brain homogenate pellet was stored at −80°C until further experimental use. In vitro binding assay to measure affinity for mGlu1 receptor using brain homogenate was performed according to a method described elsewhere.24 Briefly, the brain homogenate was incubated with [18F]1 (3 nM in PBS) and multi-dose tested compounds (10−6 to 10−10 M in 0.1% dimethylsulfoxide). The results of inhibitory experiments were subjected to nonlinear regression analysis using Prism 5 (GraphPad Software, La Jolla, CA) by which IC50 and the inhibition constant (Ki) values were calculated.

Preparation of Tumor Model Mice. Melanoma B16F10 cell line (American Type Culture Collection, Manassas, VA) was maintained and passaged in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 unit/mL) and streptomycin ACS Paragon Plus Environment

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(0.1 mg/mL). Animal experiments were performed in 6–8-week-old male C57BL/6J mice (Japan SLC, Shizuoka, Japan). Subcutaneous tumor-bearing models were created via injection into the left flank with a single-cell suspension of 1 × 106 B16F10 cells in 100 µL DMEM medium without serum. Mice with a maximum tumor diameter of 9–12 mm were selected for evaluation studies 10 days after subcutaneous inoculation.

Biodistribution Studies. The B16F10-bearing mice were sacrificed by cervical dislocation at designated time points after injection of [11C]4–6 (1.70 to 1.85 MBq/0.1 mL). Tumors and major organs and tissues (blood, lung, heart, kidney, liver, pancreas, spleen, testis, intestine, muscle, and skin, brain), were promptly excised, harvested, and weighed. Radioactivity was counted using a g-counter and expressed as a percentage of the injected dose per gram of wet tissue (% ID/g). All radioactivity measurements were corrected for decay. The ratios of tumor-to-blood, tumor-to-muscle, and tumor-to-skin were calculated.

Small-animal PET Scans. PET scans were performed for B16F10-bearing mice using a small-animal Inveon PET scanner (Siemens, Knoxville, TN) after intravenous injection of [11C]4–6 (12–17 MBq/0.1 mL). The scans provided 159 transaxial slices with 0.796 mm (center-to-center) spacing, a 10 cm transaxial field of view (FOV), and a 12.7 cm axial FOV. Emission scans were acquired in a 3-dimensional list mode with an energy window of 350–650 keV under isoflurane anesthesia starting from 45 to 75 min after injection in the subcutaneous tumor models. Blocking experiments for [11C]4–6 uptake were performed by injecting excess unlabeled 1 (10 mg/kg) at 1 min before radiotracer injection, to inhibit the binding of radiotracers to mGlu1 receptor. All



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list-mode acquisition data were sorted into 3-dimensional sinograms, which were then Fourier-rebinned into 2-dimensional sinograms and corrected for scanner dead time, randoms, and decay of the injected radiotracer. Dynamic images were reconstructed with filtered back-projection using a Hanning’s filter and a Nyquist cutoff of 0.5 cycles/pixel. Regions of interest in the tumors and right flank muscles were drawn using the Siemens Inveon Research Workplace (IRW) 4.0 software. The average radioactivity concentration in the tumors and tissues was obtained from mean pixel values in the region of interest volume, which was manually positioned based on the tumor and tissue contour in the different orthogonal planes with the largest diameter. Regional uptake of radioactivity was decay-corrected to injection time, normalized to weights, and expressed as % ID/g. Time-activity curves (TACs) of [11C]4–6 were determined. Values of area under time-activity curves (AUC45–75 min, % ID/g × min) in the subcutaneous tumors were calculated.

Statistical analysis. All data are presented as the mean of the values ± the standard error of mean (SEM). Comparisons of brain or tumor uptake among [11C]4–6 were performed using one-way ANOVA test. The threshold for statistical significance was set at p < 0.05. All in vivo experimental results included data from 4–6 mice in the respective groups, and all in vitro experimental results are representative of 3 independent experiments and indicated as the mean ratio of triplicate results in each experiment.

ASSOCIATED CONTENT Supporting Information Available. Purities of compounds 4–6 and 7–9 determined by HPLC,


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HPLC analytic charts of 4–9, HPLC purification charts for [11C]4–6; HPLC analysis charts for [11C]4–6. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

*

Phone: +81-43-382-3709; Fax: +81-43-206-3261; E-mail: [email protected]. Address: Molecular

Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

We would like to thank the staff at the National Institute of Radiological Sciences for their support with the cyclotron operation, radioisotope production, radiosynthesis, and animal experiments. This study was supported in part by Grants-in-Aid for Scientific Research (Basic Research B: 22591379) from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

ABBREVIATIONS USED AUC, area under the time-curve; [11C]CH3I, [11C]methyl iodide; [11C]CH3OTf, [11C]methyl trifluoromethanesulfonate, [11C]CO2, [11C]carbon dioxide; EOB, end of bombardment; EOS, end of synthesis; Et3N, triethylamine; FAB-MS, fast atom bombardment mass spectra; HPLC, high



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performance liquid chromatography; HRMS, high resolution mass spectra; % ID/g, percentage of the injected dose per gram of wet tissue; Ki, inhibition constant; MeCN, acetonitrile; mGlu1, metabotropic glutamate 1 receptor; PBS, phosphate buffered saline; PET, positron emission tomography; SUV, standardized uptake value; TAC, time-activity curve; tR, retention time.

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Table 1. In vitro binding affinity (Ki) for mGlu1 receptor and lipophilicity



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Table 2. Biodistribution of [11C]4 in tissues of C57BL/6J mice bearing B16F10 melanoma


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Scheme 1. Chemical structures of PET radiotracers for imaging of mGlu1 receptor in brain or tumor



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Scheme 2. Synthesis of 4–6 and desmethyl precursors 7–9a

a

Reagents and conditions: (a) THF, H2O, 1-bromopyrrolidine-2,5-dione, room temperature, 2 h; (b)

thiourea derivatives, room temperature, 3 h; (c) 4-substituted benzoyl chlorides, Et3N, THF, 60 °C, 14–17 h; (d) isopropylamine, K2CO3, 1,4-dioxane, 80 °C, 10–11 h; (e) 4-substituted benzoyl chlorides, Et3N, toluene, 100 °C, 6 h; (f) isopropylamine, K2CO3, 1,4-dioxane, 80 °C, 8 h.


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Scheme 3. Radiosynthesis of [11C]4–6 a

a

Reagents and conditions: (a) LiAlH4, THF, –15 °C, 2 min; (b) HI, 180 °C, 2 min; (c) NaOH, DMF,

70 °C, 5 min; (d) AgOTf, 150 °C; (e) NaOH, acetone, room temperature, 5 min.



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Figure 1. Tumor to brain ratios, tumor to blood ratios, tumor to skin ratios and tumor to muscle ratios for [11C]4–6 in B16F10-bearing mice (n = 6 for each group).


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Figure 2. Time-activity curves for [11C]4–6 in tumor and brain of B16F10-bearing mice (n = 6 for each group) between 45 and 75 min after the radiotracer injection.

Figure 3. Area under time-activity curves (AUC45–75

min)

for [11C]4–6 in tumor and brain of

B16F10-bearing mice (n = 6 for each group) between 45 and 75 min after the radiotracer injection. Asterisks indicate significant difference in tumor or brain uptake among [11C]4, [11C]5, and [11C]6 (**p < 0.001). NS, not significant.



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Figure 4. Representative PET images of the isoflurane-anesthetized mice bearing B16F10 (n = 6 for each group). These PET images were acquired by summing the radioactivity between 45 and 75 min after injection of [11C]4–6, respectively. Upper: sagittal images of brain. Lower: horizontal images of tumor and muscle.


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Table of Contents graphic


Synthesis and Evaluation of 4-Halogeno-N-[4-[6-(isopropylamino

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