Optimization of the alkyl side chain length of fluorine-18-labeled 7α-alkyl-fluoroestradiol

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Abstract

Introduction

Several lines of evidence suggest that 7α-substituted estradiol derivatives bind to the estrogen receptor (ER). In line with this hypothesis, we designed and synthesized 18F-labeled 7α-fluoroalkylestradiol (Cn-7α-[18F]FES) derivatives as molecular probes for visualizing ERs. Previously, we successfully synthesized 7α-(3-[18F]fluoropropyl)estradiol (C3-7α-[18F]FES) and showed promising results for quantification of ER density in vivo, although extensive metabolism was observed in rodents. Therefore, optimization of the alkyl side chain length is needed to obtain suitable radioligands based on Cn-7α-substituted estradiol pharmacophores.

Methods

We synthesized fluoromethyl (23; C1-7α-[18F]FES) to fluorohexyl (26; C6-7α-[18F]FES) derivatives, except fluoropropyl (C3-7α-[18F]FES) and fluoropentyl derivatives (C5-7α-[18F]FES), which have been previously synthesized. In vitro binding to the α-subtype (ERα) isoform of ERs and in vivo biodistribution studies in mature female mice were carried out.

Results

The in vitro IC50 value of Cn-7α-FES tended to gradually decrease depending on the alkyl side chain length. C1-7α-[18F]FES (23) showed the highest uptake in ER-rich tissues such as the uterus. Uterus uptake also gradually decreased depending on the alkyl side chain length. As a result, in vivo uterus uptake reflected the in vitro ERα affinity of each compound. Bone uptake, which indicates de-fluorination, was marked in 7α-(2-[18F]fluoroethyl)estradiol (C2-7α-[18F]FES) (24) and 7α-(4-[18F]fluorobutyl)estradiol (C4-7α-[18F]FES) (25) derivatives. However, C1-7α-[18F]FES (23) and C6-7α-[18F]FES (26) showed limited uptake in bone. As a result, in vivo bone uptake (de-fluorination) showed a bell-shaped pattern, depending on the alkyl side chain length. C1-7α-[18F]FES (23) showed the same levels of uptake in uterus and bone compared with those of 16α-[18F]fluoro-17β-estradiol.

Conclusions

The optimal alkyl side chain length of 18F-labeled 7α-fluoroalkylestradiol was the shortest: C1-7α-[18F]FES. Our results indicate that shorter chain lengths within the 4-Å ligand binding cavities of ERα are suitable for 7α-fluoroalkylestradiol derivatives.

Introduction

Fluorine-18-labeled steroid hormones are useful probes for positron emission tomography visualization of receptor-positive tumors such as breast and prostate cancer. Over the past 30 years, several derivatives of 18F-labeled 17β-estradiol have been synthesized and evaluated. Among them, 16α-[18F]fluoro-17β-estradiol (16α-[18F]FES) [1] is currently used as the standard radioligand for imaging both primary and metastatic estrogen receptor-positive tumors [2], [3].

On the other hand, several lines of evidence suggest that 7α-substituted estradiol derivatives bind to the estrogen receptor (ER), even though they have a long chain with complex functionality [4], [5], [6], [7], [8], [9], [10], [11]. In contrast, small polar groups at the 7α-position do not bind well [12], [13]. Furthermore, substitutions with 7α-alkyl chains bearing alcohol, carboxylic acid, and ester groups also have low affinity [6]. Thus, Anstead et al. recommended that groups at the 7α-position bind well to ERs, even if they are rather long; however, polar functions must be positioned away from the core of the steroid structure [14]. In line with this hypothesis, we are interested in the design and synthesis of 7α-fluoroalkylestradiol (Cn-7α-[18F]FES) as a molecular probe to visualize ER function. Previously, French et al. synthesized the five-carbon derivative 7α-(5-[18F]fluoropentyl)estradiol (C5-7α-[18F]FES) and evaluated its biodistribution in immature rats [5]. Although C5-7α-[18F]FES showed somewhat selective uptake in target tissues, the levels of uptake into non-target tissues were high, possibly due to the increased lipophilicity of the additional five-carbon chain. In a recent follow-up study, we investigated the shorter three-carbon derivative 7α-(3-[18F]fluoropropyl)estradiol (C3-7α-[18F]FES) and characterized its biological properties [15]. As expected, the shorter three-carbon chain resulted in lower uptake into non-target tissues, such as fat and blood, and the uterus-to-blood ratio at 60 min was double that of the five-carbon chain derivative. However, the three-carbon derivative underwent greater metabolic de-fluorination than the five-carbon derivative. These results indicate that opportunities still exist for further optimization of the alkyl side chain length of Cn-7α-[18F]FES derivatives.

Currently, the most common route for preparing 16α-[18F]FES uses 3-methoxymethyl-16β,17β-epistriol-O-cyclic sulfate [16]. However, some difficulties remain regarding the time required for (> 30 min) acid hydrolysis of the bisulphate intermediate [17], and further optimization is required for routine clinical use in individual facilities [18]. In contrast, di-methoxymethyl-protected groups of labeling precursors of Cn-7α-[18F]FES derivatives may be removed more quickly and have acceptable times required for radiosynthesis.

In this study, we further synthesized fluoromethyl (22; C1-7α-[18F]FES) to fluorohexyl (25; C6-7α-[18F]FES) derivatives of Cn-7α-[18F]FES, except fluoropropyl (C3-7α-[18F]FES) and fluoropentyl derivatives (C5-7α-[18F]FES), which were synthesized previously [5], [15]. We characterized the in vitro binding and in vivo distribution of these derivatives in mature female mice compared to the previously published data for C3-7α-[18F]FES and 16α-[18F]FES, and we discuss the optimization of the alkyl side chain length.

Section snippets

Chemical synthesis

The methods of synthesis of non-radioactive compounds are outlined in Scheme 1, Scheme 2, Scheme 3, and the details are summarized in the supporting information.

Radiochemical synthesis

[18F]Fluoride was produced by proton irradiation of 18O-enriched water (Taiyo Nippon Sanso, Tokyo, Japan) at 50 μA for 5 min using the HM-20 cyclotron (Sumitomo Heavy Industries, Tokyo, Japan). Isolation of [18F]fluoride from enriched water and subsequent 18F-fluorination, de-protection, purification, and formulation were carried out

ER binding assay

[2,4,6,7-3H(N)]-Estradiol ([3H]estradiol; 3300 GBq/mmol, 37 MBq/mL) was purchased from PerkinElmer (Boston, MA). 16α-fluoro-17β-estradiol (16α-FES) was purchased from ABX GmbH (Radeberg, Germany). C3-7α-FES was prepared from estradiol as reported previously [15]. The purity of C3-7α-FES was confirmed by HPLC (condition 1): tR = 18.2 min, purity = 94.5%. Human recombinant α-subtype (ERα) of the ER was purchased from Life Technologies (Carlsbad, CA).

Relative binding affinity (RBA) was determined by a

Design consideration

The first reported C5-7α-[18F]FES was synthesized via a key reaction involving addition of a 1,6 conjugate to a dienone body using the acetyl protecting group of dihydrotestosterone as the starting material [5]. This method of synthesis cannot be universally used with other labels, and synthesis of derivatives with short side chains is particularly challenging. Therefore, we aimed to establish a novel synthesis method for Cn-7α-FES that can be used to synthesize derivatives with a variety of

Conclusion

In conclusion, we optimized the alkyl side chain length of Cn-7α-[18F]FES derivatives. The current study revealed the impact of the alkyl side chain length of Cn-7α-[18F]FES on the in vitro binding affinity, biodistribution, and in vivo metabolism. C1-7α-[18F]FES (23) showed uterus-to-blood and ovary-to-blood ratios that were comparable to those of 16α-[18F]FES. Combined with in vivo stability as indicated by low bone uptake and reasonably good affinity for ERα and its high target-to-non-target

Acknowledgments

We thank Mr. Kunpei Hayashi (SHI Accelerator Service) for his technical support with the cyclotron operation and radiosynthesis and Dr. Seijiro Hosokawa (Waseda University) and Dr. Kazuo Nagasawa (Tokyo University of Agriculture and Technology) for their valuable advice. This work was supported in part by a Grant-in Aid for Scientific Research (B) 25293271 from the Japan Society for the Promotion of Science (JSPS) and an Early Bird support project for young researchers at Waseda Research

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