Synthesis and evaluation of 7α-(3-[18F]fluoropropyl) estradiol

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Abstract

Introduction

Several lines of evidence suggest that C-7α-substituted estradiol derivatives are well tolerated by estrogen receptor (ER). In line with this hypothesis, we are interested in the design and synthesis of C-7α-substituted estrogens as molecular probes to visualize ER function.

Methods

We have synthesized 7α-(3-[18F]fluoropropyl) estradiol (C3-7α-[18F]FES) as a potential radiopharmaceutical for ER imaging by positron emission tomography (PET). In vitro receptor binding and in vivo biodistribution and blocking studies in mature female mice, and in vivo metabolite analysis were carried out. Furthermore, in vivo ER-selective uptake was confirmed using ER-positive T-47D and ER-negative MDA-MB-231 tumor-bearing mice. We also compared the in vivo biodistribution of C3-7α-[18F]FES with 16α-[18F]FES.

Results

C3-7α-[18F]FES was produced in moderate yields (30.7% ± 15.1%, decay corrected) with specific activity of 32.0 ± 18.1 GBq/μmol (EOS). The in vitro binding affinity of C3-7α-FES to the ERα isoform was sufficient and equivalent to that of estradiol. C3-7α-[18F]FES showed selective uptake in ER-rich tissues, such as the uterus (4.7%ID/g ± 1.2%ID/g at 15 minutes) and ovary (4.0%ID/g ± 1.0%ID/g at 5 minutes). The tissue time activity curves of these organs showed reversible kinetics, indicating suitability for quantitative analysis. The highest contrast was obtained at 120 minutes after injection of C3-7α-[18F]FES in the uterus (uterus/blood = 18, uterus/muscle = 17.3) and ovary (ovary/blood = 6.3, ovary/muscle = 6.0). However, the level of selective uptake of C3-7α-[18F]FES was significantly lower than that of 16α-[18F]FES. Most radioactivity in the uterus was detected in unchanged form, although peripherally C3-7α-[18F]FES was rapidly degraded to hydrophilic metabolites. In accordance with this peripheral metabolism, gradual increases in bone radioactivity were observed, indicating defluorination. Coinjection with estradiol dose-dependently inhibited C3-7α-[18F]FES uptake in the uterus and ovary. The in vivo IC50 values of estradiol in the uterus and ovary were 34.4 and 38.5 nmol/kg, respectively. Furthermore, in vivo tumor uptake of C3-7α-[18F]FES was significantly higher (unpaired t test with Welch’s correction; p = 0.015) in ER-positive T-47D tumors (2.3%ID/g ± 0.4%ID/g) than ER-negative MDA-MB-231 tumors (0.9%ID/g ± 0.1%ID/g).

Conclusions

Although extensive metabolism was observed in rodents, C3-7α-[18F]FES showed promising results for quantitative analysis of ER density in vivo. However, the selective uptake of C3-7α-[18F]FES was lower than that of 16α-[18F]FES. Further optimizations and structure–activity relationship studies of the C-7α-substituted estradiol are needed.

Introduction

Determination of the estrogen receptor (ER) status (positive or negative) is of prime importance in therapeutic management of breast cancer patients [1], [2], [3]. While histopathological analysis is still the gold standard for evaluating ER status, in vivo imaging of ER status by positron emission tomography (PET) is an attractive alternative to determine in situ ER status [4], [5], [6]. First, noninvasive ER imaging allows the simultaneous visualization of primary and metastatic tumor sites. Second, ER imaging is not subject to intrinsic heterogeneity of ER expression within a tumor, or to the possible discordance between primary and metastatic tumors. Finally, serial ER imaging can evaluate tumor responsiveness to hormonal therapy.

Over the past 30 years, several derivatives of fluorine 18 (18F)-labeled 17β-estradiol have been synthesized and evaluated [7]. The most successful ligand advanced to date is 16α-[18F]fluoro-17β-estradiol (16α-[18F]FES), developed at Washington University [8]. Several reports of clinical studies of 16α-[18F]FES showed the feasibility and potential of PET for ER density examination [9], [10], [11]. However, 16α-[18F]FES is not an optimal radioligand for ER imaging due to its rapid conversion to circulating radiometabolites, which prevents optimal localization of ER-binding sites [12].

In another approach, several lines of evidence suggest that C-7α-substituted estradiol derivatives are well tolerated by the ER [13], [14], [15], [16], [17]. In line with this hypothesis, we are interested in the design and synthesis of C-7α-substituted estrogens as molecular probes to visualize ER function. Recently, we successfully synthesized a boron-dipyrromethene derivative of estradiol and visualized in situ hormone–receptor interactions in the nuclei of uterine epithelial cells [18]. Furthermore, 7α-(5-[18F]fluoropentyl) estradiol (C5-7α-[18F]FES) was synthesized and its biodistribution was studied in immature rats [13]. Although blocking studies showed selective uptake in target tissues, the levels of nontarget tissue uptake, especially in fatty tissue and the blood, were high. This low selectivity may be due to the increased lipophilicity of the additional five-carbon chain. These findings prompted us to develop shorter alkyl chain length derivatives of 18F-labeled C-7α-substituted estradiol.

In the present study, we synthesized three-carbon derivative of 18F-labeled C-7α-substituted estradiol (C3-7α-[18F]FES) and characterized its in vitro binding, in vivo distribution, and performed blocking studies in mature female mice. We also analyzed in vivo metabolites in the plasma and uterus. Furthermore, in vivo ER-selective uptake was confirmed using ER-positive T-47D and ER-negative MDA-MB-231 tumor-bearing mice. We also compared the in vivo biodistribution of C3-7α-[18F]FES with 16α-[18F]FES to discuss the contribution of shorter side chain length of C-7α-substituted estradiol.

Section snippets

General

3-O-(Methoxymethyl)-16,17-O-sulfuryl-16-epistriol (MMSE) and 16α-Fluoro-17β-estradiol (16α-FES) were purchased from ABX GmbH (Radeberg, Germany). [2,4,6,7-3H(N)]-Estradiol ([3H]estradiol; 3300 GBq/mmol, 37 MBq/mL) was purchased from PerkinElmer (Boston, MA). Human recombinant estrogen receptor α-subtype (ERα) was purchased from Sigma-Aldrich Co. (St. Louis, MO). Anti-estrogen receptor α antibody (mouse monoclonal 1D5), anti-estrogen receptor β antibody (rabbit monoclonal EPR3778), and anti-actin

Chemistry

The precursor 5 was synthesized in 3 steps from 7α-allyl-estradiol as shown in Fig. 1. The overall yield starting from 7α-allyl-estradiol was 43%. C3-7α-FES was prepared by fluorination of 5 with tetrabutylammonium fluoride, followed by acid deprotection of the methoxymethyl group.

Radiosynthesis

18F-Fluorination of the tosylate 5 was carried out using the 18F-Kryptofix222-potassium carbonate system in acetonitrile at 100 °C for 15 minutes and deprotected with 0.5 N HCl in 50% acetonitrile at 100 °C for 2 minutes

Discussion

French et al. synthesized C5-7α-[18F]FES and evaluated its biodistribution in immature rats [13]. They found selective uptake to a certain degree in target tissues. However, the levels of nontarget tissue uptake were high, possibly due to the increased lipophilicity o the additional five-carbon chain. In the present study, we further investigated the shorter three-carbon derivative of 18F-labeled C-7α-substituted estradiol (C3-7α-[18F]FES) and characterized its biological properties.

C3-7α-[18

Acknowledgments

We thank Mr. Kunpei Hayashi (SHI Accelerator Service Co. Ltd) 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 Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for the Global COE Program “Practical Chemical Wisdom”

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