Biologically stable [18F]-labeled benzylfluoride derivatives1

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Abstract

Use of the [18F]-fluoromethyl phenyl group is an attractive alternative to direct fluorination of phenyl groups because the fluorination of the methyl group takes place under milder reaction conditions. However, we have found that 4-FMeBWAY showed femur uptake equal to that of fluoride up to 30 min in rat whereas 4-FMeQNB had a significantly lower percent injected dose per gram in femur up to 120 min. For these and other benzylfluoride derivatives, there was no clear in vivo structure-defluorination relationship. Because benzylchlorides (BzCls) are known alkylating agents, benzylfluorides may be alkylating agents as well, which may be the mechanism of defluorination. On this basis, the effects of substitution on chemical stability were evaluated by the 4-(4-nitro-benzyl)-pyridine (NBP) test, which is used to estimate alkylating activity with NBP. The effect of substitution on the alkylating activity was evaluated for nine BzCl derivatives: BzCl; 3- or 4-methoxy (electron donation) substituted BzCl; 2-, 3-, or 4-nitro (electron withdrawing) substituted BzCl; and 2-, 3-, or 4-chloro (electron withdrawing) substituted BzCl. Taken together, the alkylating reactivity of 3-chloro-BzCl was the weakest. This result was then applied to [18F]-benzylfluoride derivatives and in vivo and in vitro stability were evaluated. Consequently, 3-chloro-[18F]-benzylfluoride showed a 70–80% decrease of defluorination in both experiments in comparison with [18F]-benzylfluoride, as expected. Moreover, a good linear relationship between in vivo femur uptake and in vitro hepatocyte metabolism was observed with seven 18F-labeled radiopharmaceuticals, which were benzylfluorides, alkylfluorides, and arylfluorides. Apparently, the [18F]-fluoride ion is released by metabolism in the liver in vivo. In conclusion, 3-chloro substituted BzCls are the most stable, which suggests that 3-chloro benzylfluorides will be the most chemically stable compound. This result should be important in future design of radioligands labeled with a benzylfluoride moiety.

Introduction

Low molecular weight radiopharmaceuticals labeled with positron emitting radionuclides such as 11C, 13N, and 18F, with half-lives of 20.3, 9.97, and 109.7 min, respectively, have been used together with positron emission tomography (PET) for imaging of brain, heart, and tumors 2, 6, 19. 18F-labeled tracers are often desired for receptor imaging because the longer half-life allows 18F-labeled tracers to be detected for a few hours after injection. [18F]-Fluorine can be introduced into an aromatic ring or aliphatic group of the target molecule at high specific activity using nucleophilic substitution.

Most of the radiopharmaceuticals injected are metabolized in the body and the metabolites are excreted in the bile or urine. If free radioactive halogen ion is released in the blood by dehalogenation reactions, they will accumulate in the thyroid or stomach for iodine and bone for fluorine 10, 20. Consequently, dehalogenation of radiohalogen labeled radiopharmaceuticals in vivo becomes a hindrance for imaging or leads to needless radiation exposure of patients. We have evaluated and reported on the usefulness of several compounds containing the [18F]-fluoro-benzyl group 8, 11, 12, but the structure and bone accumulation relationship has not been established.

Some defluorination reactions in vivo have been reported to take place by enzymatic reactions 1, 7. On the other hand, defluorination is easily induced with chemically unstable compounds. The 4-(4-nitro-benzyl)-pyridine (NBP) test 4, 5, a test of alkylating reactivity of an alkyl halogen, may be useful as an index of chemical stability. In this study, benzylchloride (BzCl) derivatives were selected as model compounds and the effect of substitution on alkylating reactivity were evaluated by the NBP test as guide for developing a stable 18F-labeled radiopharmaceutical containing benzylfluoride. Moreover, although it is generally said that released [18F]-fluoride ion after defluorination in vivo is accumulated in the bone as mentioned above, it remains unclear how 18F-labeled radiopharmaceuticals are defluorinated in vivo. On this basis, comparison of in vivo uptake in femur of rats and in vitro metabolism by rat hepatocytes was performed using currently available 18F-labeled compounds at our facility.

Section snippets

Materials and methods

All chemicals were purchased from Aldrich Chemical Company (Milwaukee, WI USA). The ultraviolet absorbance was measured with a Hewlett Packard 8452A Diode Array Spectrophotometer (Palo Alto, CA USA). Nuclear magnetic resonance (NMR) spectra were obtained on a Varian Gemini 2000 (200 MHz) (Palo Alto, CA USA) with tetramethylsilane as the internal standard. Mass spectra (MS) data were obtained on a Hewlett Packard GC-MS system with a 5890A series Gas Chromatograph and a 5989B series Mass

Synthesis of 18F-labeled compounds

(VII), the 18F labeling precursor of 4-[18F]-fluoromethylbenzoic acid methyl ester, was easily synthesized by esterification of 4-bromomethylbenzoic acid in methanol with anhydrous acid. (V), the precursor of 2-chloro-4-[18F]-fluoromethylbenzoic acid methyl ester, was synthesized from 3-chlorotoluene by the Friedel-Crafts reaction, oxidization of the acetyl group to carboxylic acid, bromination of the methyl group, and esterification of the carboxylic group.

Labeling of [18F]-FMeB(Me) and 4-[18

Discussion

We found that 3-chloro substitution on BzCl (3-Cl-BzCl) produced the weakest alkylating agent (Fig. 2). For electron withdrawing groups, the chloro substitution was less reactive at each position compared with the nitro substitution. The observed order of reactivity of both substituted derivatives was 2- > 4- > 3. For electron donating groups, the 4-methoxy BzCl showed the highest reactivity. It is likely that this high reactivity of 4-methoxy BzCl is due to the resonance effect that is

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Current address: Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

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