Fluorine-Substituted Tanaproget As a Progesterone Receptor Imaging Agent for Positron Emission Tomography

中国科技论文在线 http://www.paper.edu.cn 1096 Bioconjugate Chem. 2010, 21, 1096–1104

Development of [F-18]Fluorine-Substituted Tanaproget as a Progesterone Receptor Imaging Agent for Positron Emission Tomography

Jae Hak Lee,‡ Hai-bing Zhou,†,‡ Carmen S. Dence,§ Kathryn E. Carlson,‡ Michael J. Welch,§ and John A. Katzenellenbogen*,‡ Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Matthews Avenue, Urbana, Illinois 61801, and Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South Kingshighway, St. Louis, Missouri 63110. Received February 24, 2010; Revised Manuscript Received March 29, 2010

The level of progesterone receptors (PRs) in breast tumors can be used to guide the selection of endocrine therapies for breast cancer patients. To this end, we have prepared a fluorine-18 labeled analogue of Tanaproget, a nonsteroidal progestin with very high PR binding affinity and low affinity for androgen and glucocorticoid receptors, and have studied its tissue distribution in estrogen-primed rats to evaluate its potential for imaging PR levels by positron emission tomography. 4-[18F]Fluoropropyl-Tanaproget ([18F]9, FPTP) was prepared in three steps, within 140 min at an overall decay-corrected yield of 5% and effective specific activity of >550 Ci/mmol. In biodistribution studies, [18F]9 uptake was high in target tissues at both 1 and 3 h (uterus, 4.55 and 5.26%ID/g; ovary, 2.32 and 2.20%ID/g, respectively) and was cleanly blocked by coinjection of excess unlabeled compound. Uterus to blood and muscle activity ratios were 9.2 and 5.2 at 1 h and 32 and 26 at 3 h, respectively. The biodistribution of [18F]9 compares favorably to that of previously prepared F-18 labeled steroidal progestins, FENP and FFNP. Its high target tissue uptake efficiency and selectivity, and prolonged retention, suggest that it has excellent promise as a PET imaging agent for PR-positive breast tumors.

INTRODUCTION limited progress has been made with PET imaging of PR (19-22). Receptor-targeted radiopharmaceuticals are designed to image specific molecular targets and thereby obtain quantitative Because PR levels are regulated through ER, breast tumor information about important metabolic, physiological, and PR levels have been used, together with ER levels, to predict pathological processes in living systems. Steroid receptors, patient response to ER-targeted therapies (either antiestrogens members of the nuclear hormone receptor superfamily, are key or aromatase inhibitors) (2). In fact, because PR levels are regulators of gene expression and cellular function, and have increased by estrogen treatment, measurement of an increase emerged as very attractive targets for imaging (1). The activity in PR levels in breast tumors by PET, after a brief exposure to of certain steroid receptors drives the growth of hormone- estrogen, might be useful in assessing whether the ER is responsive cancers, both the estrogen receptor (ER) and the functional and thus capable of mediating endocrine therapies progesterone receptor (PR) in breast cancer, and the androgen (19, 23). PR itself is also a target for endocrine therapy in breast receptor (AR) in prostate cancer. Consequently, these receptors cancer using antiprogestins (24). are both biomarkers for diagnosis and targets for cancer Currently, most PR ligands are steroidal compounds having endocrine therapies (1). similar core structures (Figure 1) (25, 26). In the search for Currently, measurements of tumor ER, PR, or AR levels are diagnostic PR tumor imaging agents, some of these have been 2-4 labeled with radionuclides such as F-18, I-123, and Br-76 done in vitro by immunohistochemical assays ( ), but there - ) + is great interest in developing methods to determine receptor (1, 21, 27 29). Because fluorine-18 (t1/2 110 min, levels by in vivo imaging using positron emission tomography emitting) has the most favorable nuclear and physical properties for PET imaging, we and others have investigated the in vivo (PET), which could potentially measure receptor levels at all 18 tumor sites, simultaneously and noninvasively (1, 5-8). This behavior of two F-18 labeled steroidal progestins, 21-[ F]fluoro- 16R-ethyl-norprogesterone ([18F]FENP, 2) and 21-[18F]fluoro- information could be used to select patients most likely to benefit 18 from endocrine therapies, thereby sparing some the morbidity furanyl-norprogesterone ([ F]FFNP, 5). FENP, based on the of radiation and chemotherapy. Clinical PET imaging of ER in well-known, potent Organon progestin, ORG2058 (1), has very breast cancer using 16R-[18F]fluoroestradiol (FES) (9-14) and high binding affinity and showed excellent target tissue uptake AR in prostate cancer using 16-[18F]fluoro-5R-dihydrotest- efficiency and selectivity in estrogen-primed rats, but surpris- osterone (FDHT) (15-18) is quite advanced, but thus far, only ingly failed to image PR in humans (20). This species difference was traced to 20-hydroxysteroid dehydrogenase (20-HSD) activity, present in the blood of humans but not rodents, that * To whom correspondence should be addressed: Prof. John A. rapidly converts FENP into the inactive 20-hydroxy analogue Katzenellenbogen, Department of Chemistry, University of Illinois at (27). FFNP (19), on the other hand, appears resistant to this Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801. + + dehydrogenase and is currently being evaluated as a PET PR Telephone: 1-217-333-6310, Fax: 1-217-333-7325, E-mail: jkatzene@ imaging agent in humans. uiuc.edu. † Current address: College of Pharmacy, Wuhan University, Wuhan, Despite the continuing promise of FFNP, there are potential 430072 China. liabilities in using 20-keto steroids as PR PET imaging agents, ‡ University of Illinois at Urbana-Champaign. because steroidal progestins often show cross reactivity with § Washington University School of Medicine. other steroid receptors, notably the glucocorticoid receptor (GR) 10.1021/bc1001054  2010 American Chemical Society Published on Web 05/24/2010 转载 中国科技论文在线 http://www.paper.edu.cn 18Fl-Substituted Tanaproget as a PR Imaging Agent Bioconjugate Chem., Vol. 21, No. 6, 2010 1097

Figure 1. Structure and PR binding affinity of some steroidal (1-5) and nonsteroidal (6-9) progestins in the series leading to the F-18 labeled analogues, FENP (2), FFNP (5), and FPTP (9). and AR (30), and the inactivating metabolism by the 20-HSD Our original route to unlabeled FTPT was not well suited is still possible (27). Additionally, steroids generally have high for F-18 labeling, because fluoride ion was introduced early in lipophilicity, which could increase nonspecific binding. the sequence, and three subsequent steps were required to A number of pharmaceutical companies have reported non- produce the final product. In our first approach to develop an steroidal progestins with high PR binding affinity and excellent alternative synthesis more amenable to F-18 labeling, we tried selectivity (26, 31-33). We were particularly attracted to to prepare a precursor in which F-18 could be introduced in Tanaproget (7), a nonsteroidal progestin developed by Wyeth, the last step by fluoride ion displacement of a methanesulfonate that is based on a dihydrobenzoxazinethione core structure. Its analogue of FTPT itself (or a BOC-protected version). How- PR binding affinity is very high, nearly twice that of the potent ever, the alcohol in the hydroxypropyl group was not stable steroidal progestin R5020, giving it an estimated KD of ∼0.2 under the harsh conditions required to convert the carbamate nM (34, 35); its affinity for GR and AR is also very low. Thus, to the thiocarbamate with Lawesson’s reagent. It was also very if the F-18 radionuclide could be introduced into Tanaproget unlikely that the electrophilic methanesulfonate group would or a high-affinity analogue, it might be an effective PET imaging have been compatible with the nucleophilic thiocarbamate agent for PR-positive tumors. function under the conditions required for fluoride ion substitu- We recently reported the syntheses and binding affinity of tion. We therefore turned to the use of the methanesulfonate of several Tanaproget derivatives containing fluoroethyl or fluo- the corresponding carbamate (12), thinking that the oxygen ropropyl substituents as potential PET PR imaging agents, would be less nucleophilic than the sulfur. As shown in Scheme including ones having affinities higher than Tanaproget itself 1, synthesis of this precursor began with allylcarbmate 10, which (36). In this report, we describe synthesis of [18F]fluoropropyl- was hydroborated and oxidized to give corresponding hydroxyl Tanaproget (FPTP, 9), and we evaluate its tissue distribution compound 11 in good yield (74%). Subsequent treatment with in estrogen-primed immature female rats. Its target tissue uptake methansulfonyl chloride and triethylamine furnished the desired efficiency, selectivity, and specificity are excellent, and compare labeling precursor 12 in good yield (79%). favorably with those of FENP and FFNP. Therefore, it appears In model reactions for F-18 labeling, and to prepare authentic to be an excellent prospect for further evaluation as an agent samples of the unlabeled compounds, mesylate 12 was treated for PET imaging of PR in breast tumors. with tetrabutylammonium fluoride (TBAF) in tert-butyl alcohol at 90 °C for 2 h, followed after solvent removal by heating at RESULTS 160 °C neat for 20 min to remove the BOC protecting group Chemistry. Synthesis of Fluoropropyl Tanaproget Deri- on the pyrrole. This one-pot/two-step sequence provided the VatiVes. Guided by the structure-affinity relationships in the fluorinated carbamate 13 in a high yield (81%). As has been Tanaproget series, available in the literature, we synthesized a observed in other systems (37-41), use of this protic solvent number of Tanaproget analogues containing 2-fluoroethyl and protocol for the fluoride substitution step was effective in 3-fluoropropyl groups. These are described in our recent reducing the formation of side products through elimination or publication (36). Substitution on the pyrrole nitrogen gave lower- hydrolysis, and gave high yields of the fluorinated product. affinity analogues, but replacement of one of the methyl groups FPTP (9) itself was then obtained in good yield (70%) by on the dihydrobenzoxazinethione core with a 2-fluoroethyl or treatment with Lawesson’s reagent, which converted the car- a 3-fluoropropyl group gave very high affinity Tanaproget bamate to the thiocarbamate. However, the reaction required analogues (Figure 1). We chose one of these, 3-fluoropropyl- heating in toluene at 100 °C for 2 h, conditions that were Tanaproget (FPTP, 9), to evaluate further. In competitive subsequently modified to be more suitable for labeling with the ) radiometric PR binding assays, where the standard progestin, short-lived F-18 radionuclide (t1/2 110 min) (see below). R5020, is given a relative binding affinity (RBA) value of 100, Synthesis of [18F]Fluoropropyl-Tanaproget. 4-[18F]Fluoro- Tanaproget has an RBA value of 150 and FTPT of 189 (36). propyl-Tanaproget 9 (FPTP) was prepared by the same 中国科技论文在线 http://www.paper.edu.cn 1098 Bioconjugate Chem., Vol. 21, No. 6, 2010 Lee et al.

Scheme 1a

a Reagents and conditions: (a) (i) BH3 · THF, THF, -10 °C, N2, 90 min; (ii) 4 N NaOH, 10 min; (iii) H2O2, 2 h, 74%; (b) MsCl, CH2Cl2,0°C, 2 h, 79%; (c) (i) n-Bu4NF, tert-BuOH, 90 °C, 2 h; (ii) neat, 160 °C, 20 min, 81%; (d) Lawesson’s reagent, toluene, 100 °C, 2 h, 70%. Radiolabeling 18 conditions: (c) (i) n-Bu4N[ F]F, tert-amyl alcohol. 130 °C, 20 min, (ii) 160 °C, 10 min; (d) Lawesson’s reagent, toluene, 130 °C, 30 min.

Figure 2. HPLC Profile of reaction mixture from the production of [18F]9 (Panel A), and purified [18F]9 coinjected with unlabeled 9 (Panel B). HPLC conditions (Panel A): Altima, C18, 10 µm, 250 × 10 mm, MeOH/water ) 65:35, 3.5 mL/min, Rt ) 16.8 min; (Panel B): co-injection with 18 standard 9 and [ F]9: Econosil, C18, 10 µm, 250 × 4.6 mm, CH3CN/water ) 50:50, 0.8 mL/min, 254 nm. sequence from methanesulfonate precursor 12. Treatment with reaction volume to 300 µL. Under our original conditions for 18 n-Bu4N[ F]F in tert-amyl alcohol at 130 °C for 20 min gave synthesizing the unlabeled compound (1.2 equiv and 100 °C the F-18 labeled intermediate, which on further heating in the for 2 h), the carbamate to thiocarbamate conversion proceeded same vessel after solvent removal (160 °C for 10 min) in an overall yield of less than 10%, whereas the modified underwent deprotection to give the penultimate precursor, 13. conditions gave a 40% yield. Conversion of the carbamate group to the thiocarbamate was With these modifications, the overall radiochemical yield of achieved by heating with Lawesson’s reagent in toluene at 130 desired F-18 labeled compound [18F]8 was 5% (decay-cor- °C for 30 min. The final step, producing [18F]9, proceeded in rected), within a total synthesis time of 140 min from end of 5% yield (decay corrected), after purification by reversed-phase bombardment (EOB). The radiochemical purity of [18F]9 was HPLC (see Figure 2). determined to be >95% by reversed-phase HPLC. The effective To accelerate the last step and increase conversion yield, we specific activity of [18F]9 was >550 Ci/mmol, determined by a used 3.0 equiv of Lawesson’s reagent, rather than 0.6 equiv, competitive receptor binding assay (42). which was sufficient in preparative scale reactions. We also Tissue Distribution Studies with [18F]9 (FPTP). Purified F-18 increased the reaction temperature to 130 °C and reduced the fluoropropyl-Tanaproget ([18F]9) was reconstituted in 10% 中国科技论文在线 http://www.paper.edu.cn 18Fl-Substituted Tanaproget as a PR Imaging Agent Bioconjugate Chem., Vol. 21, No. 6, 2010 1099

Table 1. Tissue Biodistribution of 3-[18F]Fluoropropyl-Tanaparoget postinjection was comparable with that of the other progestins, Derivative [18F]9 in Immature Estrogen-Primed Female Rats [18F]FENP and [18F]FFNP; kidney and, in one case, liver uptake percentage injected dose/gram ( SDa (n ) 5) of [18F]FPTP, however, was somewhat higher. All three 1 h 1 h blocked 3 h 3 h blocked compounds showed very good retention in target tissues, so that at 3 h postinjection, activity in the ovaries and uterus is ( ( ( ( blood 0.49 0.08 0.48 0.05 0.16 0.02 0.17 0.02 essentially the same as at 1 h, but activity in nontarget tissues liver 3.52 ( 0.35 3.37 ( 0.41 1.40 ( 0.18 1.47 ( 0.15 spleen 0.88 ( 0.08 0.85 ( 0.15 0.28 ( 0.05 0.27 ( 0.04 (liver, kidney, muscle, and blood) has decreased. Of the three 18 kidney 2.56 ( 0.30 2.46 ( 0.41 0.76 ( 0.08 0.76 ( 0.07 compounds, bone uptake is the least with [ F]FPTP, indicating muscle 0.87 ( 0.09 0.89 ( 0.09 0.20 ( 0.03 0.20 ( 0.03 that metabolic defluorination of this compound is minimal. Bone bone 0.93 ( 0.11 0.90 ( 0.18 0.60 ( 0.04 0.56 ( 0.09 uptake with [18F]FFNP is considerable, but some F-18 labeled uterus 4.55 ( 1.08 0.98 ( 0.11 5.26 ( 0.50 0.37 ( 0.07 compounds that show high bone uptake in rats show little bone ( ( ( ( ovaries 2.32 0.23 1.27 0.11 2.20 0.42 0.37 0.06 uptake in primates and humans (43). uterus/blood 9.19 ( 1.52 2.06 ( 0.36 32.22 ( 1.57 2.20 ( 0.37 uterus/muscle 5.16 ( 0.77 1.12 ( 0.16 26.54 ( 2.84 1.87 ( 0.13 In previous work, we sometimes compared the biophysical ovaries/blood 4.84 ( 0.37 2.67 ( 0.48 13.39 ( 1.65 2.20 ( 0.41 properties of potential PET receptor imaging agents not just in ovaries/muscle 3.95 ( 1.26 1.45 ( 0.24 11.04 ( 1.90 1.90 ( 0.21 terms of their binding affinities (expressed as relative binding affinity or RBA values), but also in terms of their nonspecific a SD is standard deviation. binding (NSB), which we estimated from their lipophilicity, as expressed by their calculated or measured octanol-water EtOH/saline. This solution was injected (i.v., veil tail) into partition coefficients (log P )(1, 22). We also calculated the immature female rats that had been estrogen pretreated to induce o/w ratio of these two values, RBA/NSB, which we termed the the levels of PR in the uterus (19, 21). Doses employed were binding selectivity index (BSI) of the compound. Often, the BSI 26 µCi/rat. The tissue distribution was determined at 1 and 3 h of a compound proved to be a more accurate predictor of the postinjection, and tissue activity, as percent injected dose/gram, target tissue uptake characteristics of the compound than its RBA and target tissue to blood and muscle ratios are given in Table value (1, 22). This was true with both estrogens (44, 45) and 1. To confirm that the uptake was mediated by a high-affinity, progestins (19). Table 2 (left) summarizes these PR biophysical limited-capacity system, one set of rats in each time study was characteristics of the three F-18 labeled progestins and the coinjected with the [18F]9 and 33.3 µg/rat of unlabeled 9,to standard progestin R5020 and (right) gives their GR and AR saturate the PR (Table 1, 1 and 3 h “blocked”). binding affinities. Labeled FPTP ([18F]9) showed target tissue uptake that was The PR binding affinities of all three progestins are greater efficient, selective, and persistent. At 1 h, the %ID/g was 4.55% than that of the standard progestin, R5020, with FENP being in the uterus and 2.32% in the ovary; this level of target tissue the highest. The BSI values for all three progestins, however, uptake persisted at 3 h without diminution. While initial uptake are very similar, because both FFNP and FPTP have lower in certain nontarget tissues was high (liver, 3.52% ID/g; kidney, NSB values than FENP and R5020. Thus, in terms of these 2.56% ID/g), these levels cleared considerably at the later time. biophysical properties, all three F-18 labeled progestins appear Consequently, the ratio of activity in uterus to blood and muscle quite comparable. increased markedly with time, so that at 3 h, the ratios were 32 for uterus/blood and 26 for uterus/muscle; these ratios for the Although FPTP does not have biodistribution characteristics ovary were 13 and 11, respectively. in estrogen-primed immature rat model that are significantly The selectivity of target tissue uptake was evident from the better than those of FENP and FFNP, it has other very favorable marked displacement that occurred in uterus and ovary when attributes that make it a compound that should be strongly the unlabeled compound (9) was coinjected. At 3 h, uterine considered for further development: First, FPTP is a nonste- activity was blocked by 93% and ovary activity by 86%; no roidal progestin and thus would not be a substrate for the 20- block was observed in nontarget tissues. The uptake in the bone, hydroxysteroid dehydrogenase found in human blood (27); this which, if high and persistent, can be an indicator of in vivo dehydrogenase led to the failure of FENP in humans (27), defluorination, was relatively low, 0.93% ID/g at 1 h, and despite the excellent biodistribution of this compound in the declined at 3 h (0.6% ID/g). rat model (19 cf., Figure 3). FFNP shares a 20-keto group with FENP, but although the bulky acetal function fused to the D-ring DISCUSSION in FFNP is thought to retard or block this inactivating metabolism, this is not certain. Second, FPTP, again being a In a quest for effective agents for imaging progesterone nonsteroidal compound, has very low affinity for other receptors receptor (PR) in breast tumors by PET, we have synthesized a in the same receptor subfamily, namely, AR and GR; FFNP, fluoropropyl analogue of the high affinity, nonsteroidal proges- by contrast, binds to GR avidly (Table 2, right). Whether this tin, Tanaproget (7), that we term fluoropropyl-Tanaproget proves to be an important matter in breast tumor imaging needs (FPTP, 9)(36). We developed a method to label this compound to await further studies. Finally, FPTP is a chiral compound 18 with F-18 at high specific activity ([ F]FPTP, 9), and in an and thus far has been prepared and studied only as a racemate. estrogen-primed immature rat animal model, we demonstrated Because it is likely that one FPTP enantiomer will have higher 18 that [ F]FPTP (9) shows target tissue uptake that is efficient, binding affinity than the other, the high-affinity isomer might 18 selective, and persistent. The synthesis of [ F]9 required two have yet more favorable biodistribution properties. We are steps after F-18 incorporation, the most challenging of which currently developing methods to separate and/or prepare selec- involved conversion of a carbamate to a thiocarbamate function. tively the FPTP enantiomers, and if they have different The overall process of F-18 labeling and subsequent transforma- affinities, we will prepare them in F-18 labeled form for further tions and purifications took 140 min and gave the final material study. in 5% radiochemical yield (RCY) with an effective specific activity of >500 Ci/mmol. CONCLUSION It is instructive to compare the biodistribution of [18F]FPTP (9) with that of two other F-18 labeled progestins that we have We have prepared a fluoropropyl-Tanaproget derivative, studied, 21-[18F]fluoro-16R-ethyl-norprogesterone (FENP, 2) FPTP ([18F]9), as a potential agent for PET imaging of PR in and 21-[18F]fluoro-furanyl-norprogesterone (FFNP, 5) (Figure breast cancer. This nonsteroidal compound has very high PR 3). The ovarian and uterine uptake of [18F]FPTP (9)at1h binding affinity and low affinity for AR and GR and would be 中国科技论文在线 http://www.paper.edu.cn 1100 Bioconjugate Chem., Vol. 21, No. 6, 2010 Lee et al.

Figure 3. Comparison of tissue biodistribution data of [18F]FPTP (9), [18F]FENP (2), and [18F]FFNP (5) in immature estrogen-primed female rats.

Table 2. Comparison of Receptor Binding Affinities and Biophysical purchased from Fisher Scientific (Pyrex no. 9825). Oasis HLB-6 Properties of Three F-18 Labeled Progestins cm3 cartridges (500 mg) as ion exchange cartridge were compd PR-RBAa cLogPb PR-NSBc PR-BSId GR-RBAf AR-RBAg purchased from Waters Corp. (part no. 186000115). [18F]Fluo- 18 18 FPTP (9) 189 3.03 0.400 472 0.9 0.04 ride ion was produced at Washington University by O(p,n) F FENP (2) 700 3.98 1.06 660e 1.3 0.11 reaction through irradiation of 95% enriched [18O]water, using FFNP (5) 190 2.94 0.364 521e 365 0.53 either the JSW BC16/8 cyclotron (The Japan Steel Works Ltd.) R5020 [100] 3.92 [1] [100] 2.6 0.26 or the CS15 cyclotron (The Cyclotron Corp.). Radiochemical 18 a RBA is relative binding affinity value, relative to the standard, purification of [ F]9 utilized a reversed-phase semipreparative b R5020 (KD [R5020] is 0.4 nM). Calculated log Po/w from ChemDraw. HPLC column (Alltima C18, 250 × 10 mm, 10 µm, 254 nm, c For calculation of NSB, see ref 46. d BSI ) RBA/NSB. e These BSI MeOH/water ) 65:35, 3.5 mL/min). For quality control, the values differ somewhat from those reported earlier (19), which were 18 f radiochemical purity of [ F]9 was determined by analytical based on experimentally measured log Po/w values. GR-RBA values are HPLC (Econosil C18, 250 × 4.6 mm, 10 µm, 254 nm, CH3CN/ relative to the standard, dexamethasone (KD [dexamethasone] is 20 nM). g ) AR-RBA values are relative to the standard, R1881 (KD [R1881] is 0.6 water 50:50, 0.8 mL/min). TLC plates were analyzed using nM). a Bioscan, Inc., System 200 imaging scanner. Radioactivity was determined with a dose calibrator. Radiochemical yields are resistant to 20-hydroxysteroid dehydrogenase inactivation. It also decay-corrected to the beginning of synthesis time. N-tert- shows very favorable target tissue distribution in an estrogen- Butoxycarbonylpyrrole-2-boronic acid is commercially available primed immature rat model. Further studies are planned on this (CAS no 135884-31-0). compound and its constituent enantiomers in other animal Synthesis of 5-(4-(3-Hydroxy)-4-methyl-2-oxo-2,4-dihydro- models. 1H-benzo[d][1,3]oxazin-6-yl)-N-tert-buthoxycarbonyl-pyrrole- 2-carbonitrile (11). To a solution of 2-(4-allyl-4-methyl-2-oxo- EXPERIMENTAL SECTION 1,4-dihydro-2H-benzo[d][1,3]oxazin-6-yl)-5-cyano-pyrrole-1- All chemical was commercially purchased from Sigma- carboxylic acid tert-butyl ester (10)(36) (200 mg, 681.849 µmol) · Aldrich Chemical Co. and TCI and used without further in dried THF (10 mL) was added BH3 THF (1.0 M, 3.41 mL, purification. Anhydrous THF solvent was obtained from an 3.409 mmol) at -10 °C and stirred for 90 min. The reaction anhydrous solvent dispensing system (47). Flash column chro- mixture was added aq. NaOH (4.0 M, 852 µL, 3.409 mmol) matography was performed with silica gel (Merck, 230-400 and stirred for 10 min, followed by addition of H2O2 (30%, mesh ASTM). Analytical thin layer chromatography (TLC) was 386 µL, 3.409 mmol) and further stirring for 2 h. The reaction performed with Merck silica gel F-254 glass-backed plates. mixture was added to water (200 mL) and extracted with EtOAc Visualization on TLC was monitored by UV light. 1H and 13C (20 mL × 3). The combined organic layer was dried over NMR spectra were obtained on Varian Unity 400 (400 MHz) Na2SO4 and concentrated under reduced pressure. The residue and 500 (500 MHz) and are reported in parts per million was purified by flash column chromatography on silica gel to downfield from internal tetramethylsilane. Mass spectra were give 3-hydroxypropyl benzo[d]oxazines 11 (170 mg, 81%) as 1 obtained on 70 eV using the micromass 70-VSE mass spec- a bright yellow oil. H NMR (500 MHz, CDCl3) δ 9.61 (s, 1H), trometer, and ESI mass spectra were achieved using the Quattro 7.20 (dd, J ) 8.5 Hz, 2.0 Hz, 1H), 7.07 (d, J ) 1.5 Hz, 1H), mass spectrometer. Melting points obtained using a Thomas- 6.96 (d, J ) 3.5 Hz, 1H), 6.93 (d, J ) 8.5 Hz, 1H), 6.23 (d, J Hoover Uni-Melt capillary melting point apparatus and are ) 3.5 Hz, 1H), 3.62 (td, J ) 6.2 Hz, 1.2 Hz, 2H), 2,23 (br s, uncorrected. High-performance liquid chromatography (HPLC) 1H), 2.17-2.01 (m, 2H), 1.71 (s, 3H), 1.69-1.62 (m, 2H), 1.52 13 was carried out on a Thermo Co. system with a semipreparative (s, 9H); C NMR (125 MHz, CDCl3) δ 152.7, 147.1, 139.6, 18 column. H2 O was purchased from Rotem Industries. The 134.4, 129.5, 127.3, 124.6, 124.4, 124.2, 114.3, 113.9, 113.3, screwcap test tubes used for fluoride incorporation were 105.4, 87.2, 85.5, 62.1, 37.2, 27.5, 27.1, 26.7; MS (FAB): m/z 中国科技论文在线 http://www.paper.edu.cn 18Fl-Substituted Tanaproget as a PR Imaging Agent Bioconjugate Chem., Vol. 21, No. 6, 2010 1101

412.2 (M+H)+, 356.1, 311.1, 289.1, 269.1, 154.2 (100), 136.1, 160.6. MS m/z 329 ([M]+, 15%), HRMS (EI) calcd for 106.7. HRMS (FAB) calcd for C22H26N3O5 412.1872, found C17H16ON3SF 329.0998, found 329.0998. 412.1873. General Radiochemical Synthesis of 5-(4-(3-[18F]fluoropro- Synthesis of 5-(4-(3-Methylsulfonyloxy)-4-methyl-2-oxo-2,4- pyl)-4-methyl-2-thioxo-2,4-dihydro-1H-benzo[d][1,3]oxazin-6- dihydro-1H-benzo[d][1,3]oxazin-6-yl)-N-tert-buthoxycarbonyl- yl)-1H-pyrrole-2-carbonitrile ([18F]9). The fluorine-18 radionu- pyrrole-2-carbonitrile (12). To a solution of 3-hydroxypropyl clide was produced by the 18O(p,n)18F reaction on an enriched 18 benzo[d]oxazines 11 (170 mg, 413.173 µmol) in CH2Cl2 (50 water target. Oxygen-18 water containing the [ F]fluoride anion mL) was added triethylamine (173 µL, 1.240 mmol), and the was transferred to a reaction vessel containing tetrabutylam- ° mixture was cooled to 0 C. Methanesulfonyl chloride (48 µL, monium bicarbonate (TBAHCO3) (40% in water, 2 µL), and 619.759 µmol) was added, and stirring continued for 2 h. The CH3CN was added. The water in the reaction vessel was mixture was added to water (100 mL). The organic layer was removed by azeotropic distillation with CH3CN under an N2 separated, and aqueous layer was extracted with CH2Cl2 (20 stream at 110 °C. The reaction vessel was cooled to room × mL 2). The combined organic layer was dried over Na2SO4 temperature, and the remaining solvent was removed under a and concentrated by rotary evaporation. The residue was purified gentle N2 stream at room temperature. The 3-mesylated by flash column chromatography on silica gel (70% EtOAc/ Tanaproget substrate 12 was added to reaction vessel followed hexane) to obtain mesylated benzo[d]oxazines 12 (136 mg, 67%) by adding tert-amyl alcohol (500 µL). The capped reaction 1 as a bright yellow oil. H NMR (500 MHz, CDCl3) δ 9.54 (s, vessel was heated at 130 °C for 20 min and then cooled to room ) ) 1H), 7.23 (dd, J 8.5 Hz, 2.0 Hz, 1H), 7.06 (d, J 1.5 Hz, temperature. After radio-TLC analysis to confirm incorporation ) ) 1H), 6.97 (d, J 3.5 Hz, 1H), 6.92 (d, J 8.0 Hz, 1H), 6.25 of the radionuclide, the reaction solvent was removed completely (d, J ) 4.0 Hz, 1H), 4.23 (t, J ) 6.0 Hz, 2H), 2.98 (s, 3H), under a stream of N2 at 110 °C, and the residue in the vessel 2.21-2.06 (m, 2H), 1.94-1.83 (m, 2H), 1.71 (s, 3H), 1.51 (s, ° 13 was heated to 160 C for 10 min. The reaction mixture was 9H); C NMR (125 MHz, CDCl3) δ 152.4, 147.1, 139.4, 134.3, allowed to reach room temperature and dissolved with EtOAc; 129.8, 127.6, 124.45, 124.43, 123.6, 114.4, 114.0, 113.3, 105.5, water (2 mL) and brine (500 µL) were added. The resulting m z 87.2, 84.9, 69.4, 37.3, 36.6, 27.5, 27.3, 23.7; MS (FAB): / solution was extracted with EtOAc (1 mL × 2). The combined 490.1 (M+H)+, 460.1, 389.1, 307.1, 289.1, 273.1, 154.2 (100), organic layer was evaporated completely under a gentle N 136.1, 119.9, 106.7. HRMS (FAB) calcd for C H N O S 2 23 28 3 7 stream at 110 °C. The residue was treated with Lawesson’s 490.1648, found 490.1648. reagent followed by addition of toluene and then heated to 130 Synthesis of 5-(4-(3-Fluoropropyl)-4-methyl-2-oxo-2,4-dihy- °C for 30 min. The solvent removed under N2 stream at 110 dro-1H-benzo[d][1,3]oxazin-6-yl)-1H-pyrrole-2-carbonitrile (13). °C. After being cooled to room temperature, the residue was To a solution of mesylated benzo[d]oxazines 12 (680 mg, 1.390 dissolved with CH CN followed by passage through an activated · 3 mmol) in tert-BuOH (100 mL) was added n-Bu4NF 3H2O (658 silica Sep-Pak. The resulting solution was added to an equal mg, 2.085 mmol) at room temperature. The reaction mixture volume of deionized water and purified by reversed-phase ° was stirred at 90 C for 2 h. The reaction mixture was heated semipreparative HPLC (MeOH/water ) 65:35, 3.5 mL/min) to ° to 160 C for 20 min under N2 gentle stream. The vial was give the desired product [18F]9, which eluted at 19-21 min (5% cooled to room temperature, dissolved with EtOAc (50 mL), decay-corrected radiochemical yield from end of bombardment; and absorbed onto a small amount of silica gel. Purification by a total synthesis time of approximately 140 min). The fractions flash column chromatography on silica gel (60% EtOAc/hexane) 1 were collected and diluted with deionized water (50 mL) and gave compound 13 (350 mg, 81%) as a bright yellow oil. H passed through an activated C18 Sep-Pak. The radioactivity on NMR (500 MHz, CDCl ) δ 10.42 (s, H), 9.14 (s, 1H), 7.44 3 C18 Sep-Pak was eluted with EtOH (1.5 mL). The solution was (dd, J ) 8.2 Hz, 1.8 Hz, 1H), 7.35 (d, J ) 2.0 Hz, 1H), removed under a N stream at room temperature, and the residue 6.92-6.90 (m, 2H), 6.46 (dd, J ) 4.0 Hz, 2.5 Hz, 1H), 4.47 2 was dissolved in 10% EtOH/saline (1.5 mL) for the tissue (dt, J ) 47.0 Hz, 5.8 Hz, 2H), 2.89-2.11 (m, 2H). 1.87-1.77 distribution studies. The radiochemical purity, determined by (m, 2H), 1.74 (s, 3H); 13C NMR (125 MHz, CDCl ) δ 152.5, 3 analytical reversed-phase HPLC, was in all cases greater than 136.9, 133.8, 126.7, 125.9, 125.0, 121.5, 120.4, 115.43, 115.36, ) ) 95%. The effective specific activity, determined by a competitive 107.0, 100.9, 85.5, 83.8 (d, J 164.0 Hz, 1C), 36.5 (d, J > 3.75 Hz, 1C), 27.5, 24.9 (d, J ) 20.1 Hz, 1C); MS (FAB): m/z radiometric ligand binding assay (42) was 550 mCi/mmol. The + + identity of the desired product was also confirmed by reversed- 314.1 (M H) , 307.1, 289.1, 273.1, 258.1, 235.2, 219.3, 195.2, ) 165.2, 154.1 (100), 136.1, 119.9, 106.7. HRMS (FAB) calcd phase analytical HPLC (CH3CN/water 50:50, 0.8 mL/min, for C H FN O 314.1305, found 314.1305. 254 nm), by coinjecting with an authentic sample of compound 17 17 3 2 9 with [18F]9. Synthesis of 5-(4-(3-Fluoropropyl)-4-methyl-2-thioxo-2,4-di- hydro-1H-benzo[d][1,3]oxazin-6-yl)-1H-pyrrole-2-carboni- Biological Procedures for Relative Binding Affinity (RBA) trile (9). The mixture of compound 13 (20.0 mg, 63.830 µmol) and Specific Activity Determinations. Relative binding affinities and Lawesson’s reagent (28.4 mg, 70.214 µmol) in toluene was of the progestins were determined in several receptor systems heated at 100 °C for 2 h. The reaction mixture was added to using competitive radiometric binding assays. Measurements water (10 mL) and extracted with EtOAc (5 mL × 3). The were carried out using procedures reported in previous publica- 28, 48 combined organic layer was dried over Na2SO4 and concentrated tions for progesterone receptor ( ), glucocorticoid in vacuo. The residue was purified by flash column chroma- receptor (49, 50), and androgen receptor (48, 51). The PR assay tography on silica gel (30% EtOAc/hexane) to give 3-fluoro- used purified recombinant full-length PR B (Invitrogen, Carls- propyl benzo[d]thiazines 9 (14.7 mg, 70%) as a yellow oil. 1H bad, CA) and R5020 as the standard and 3H tracer (Perkin- ) NMR (500 MHz, CDCl3) δ 9.23 (s, 1H), 9.12 (s, 1H), 7.46 Elmer, Boston, MA) (Kd 0.4 nM); GR used adrenalectomized (dd, J ) 8.5 Hz, 1.5 Hz, 1H), 7.29 (s, 1H), 6.93 (t, J ) 3.0 Hz, rat liver cytosol and dexamethasone as the standard and 3H tracer 1H), 6.89 (d, J ) 8.5 Hz, 1H), 6.51 (t, J ) 3.0 Hz, 1H), (Amersham Biosciences, Piscataway, NJ) (Kd ) 20 nM), and 3.62-3.54 (m, 2H), 2.36-2.30 (m, 1H), 2.22-2.16 (m, 1H), for AR, purified recombinant AR ligand binding domain 2.03-1.96 (m, 1H), 1.92-1.84 (m, 1H), 1.78 (s, 3H); 13C NMR (Invitrogen, Carlsbad, CA) and as the standard and 3H tracer ) (125 MHz, CDCl3) δ 25.1 (d, J ) 20.3 Hz, 1C), 26.0, 36.3 (d, R1881 (Perkin-Elmer, Boston, MA) (Kd 0.6 nM) for AR. By J ) 4.0 Hz, 1C), 83.6 (d, J ) 162.3 Hz, 1C), 85.8, 107.5, 114.7, definition, the standards have RBA values of 100%. Effective 115.1, 120.8, 121.0, 126.0, 126.2, 127.9, 132.2, 135.2, 136.9, specific activities of the 18F-labeled progestins were measured 中国科技论文在线 http://www.paper.edu.cn 1102 Bioconjugate Chem., Vol. 21, No. 6, 2010 Lee et al.

by in vitro competitive binding assays performed on fully Morgan, R. J., Jr., Siegel, B. A., Singhal, S., Tyler, D. S., and decayed samples (42). Wong, R. J. (2009) NCCN task force: clinical utility of PET in Tissue Distribution Studies. The progesterone receptor levels a variety of tumor types. J. Natl. Compr. Canc. Netw. 7, S1–26, in the uteri of immature Sprague-Dawley rats (21 days old, Suppl 2. 40-50 g, five rats per time point) were induced by daily (9) McGuire, A. H., Dehdashti, F., Siegel, B. A., Lyss, A. P., subcutaneous injections for 2 days of 5 µg of estradiol in 0.2 Brodack, J. W., Mathias, C. J., Mintun, M. A., Katzenellenbogen, mL of 20% ethanol/sunflower seed oil (19). The experiments J. A., and Welch, M. J. (1991) Positron tomographic assessment were begun 24 h after the last injection. The 18F-labeled of 16 alpha-[18F] fluoro-17 beta-estradiol uptake in metastatic Tanaproget derivative [18F]9, purified by reversed-phase HPLC, breast carcinoma. J. Nucl. Med. 32, 1526–1531. concentrated in vacuo, and redissolved in 10% EtOH/isotonic (10) Mintun, M. A., Welch, M. J., Siegel, B. A., Mathias, C. J., Brodack, J. W., McGuire, A. H., and Katzenellenbogen, J. A. saline, was passed through a filter. Anesthetized rats were (1988) Breast cancer: PET imaging of estrogen receptors. injected in the tail vein with 26 µCi/100 µLofthe18F-labeled 18 Radiology 169, 45–48. compound [ F]9. At specified time points postinjection, the rats (11) Mortimer, J. E., Dehdashti, F., Siegel, B. A., Katzenellenbo- were sacrificed by decapitation, and blood and organs were gen, J. A., Fracasso, P., and Welch, M. J. (1996) Positron removed, weighed, and counted in a Beckman Gamma 6000 emission tomography with 2-[18F]Fluoro-2-deoxy-D-glucose and counter. To determine whether the uptake was mediated by a 16alpha-[18F]fluoro-17beta-estradiol in breast cancer: correlation high-affinity, limited-capacity system, one set of animals was with estrogen receptor status and response to systemic therapy. coinjected with the 18F-labeled fluoropropyl-Tanaproget deriva- Clin. Cancer Res. 2, 933–939. tive [18F]9 together with 33.3 µg of unlabeled fluoropropyl- (12) Mortimer, J. E., Dehdashti, F., Siegel, B. A., Trinkaus, K., Tanaproget derivative 9 in 14% ethanol/∼30% 2-hydroxypropyl- Katzenellenbogen, J. A., and Welch, M. J. (2001) Metabolic flare: -cyclodextrin in saline as standard compound to fully occupy indicator of hormone responsiveness in advanced breast cancer. the progesterone receptors. J. Clin. Oncol. 19, 2797–2803. (13) Dehdashti, F., Flanagan, F. L., Mortimer, J. E., Katzenellen- ACKNOWLEDGMENT bogen, J. A., Welch, M. J., and Siegel, B. A. (1999) Positron emission tomographic assessment of “metabolic flare” to predict This work has been presented at the 18th International response of metastatic breast cancer to antiestrogen therapy. Eur. Symposium on Radiopharmaceutical Sciences, S218, Edmonton, J. Nucl. Med. 26, 51–56. Canada, 12-17 July 2009. Supported by grants from the NIH (14) Dehdashti, F., Mortimer, J. E., Trinkaus, K., Naughton, M. J., (PHS R01 CA025836 to JAK; R24 CA086307 to MJW). Ellis, M., Katzenellenbogen, J. A., Welch, M. J., and Siegel, Funding for NMR and MS instrumentation at the University of B. A. (2009) PET-based estradiol challenge as a predictive Illinois is from the Keck Foundation, NIH, and NSF. We are biomarker of response to endocrine therapy in women with grateful to Terry Sharp, Nicole Fettig, Margaret Morris, Paul estrogen-receptor-positive breast cancer. Breast Cancer Res. Eisenbeis, and Amanda Roth for help with the animal Treat. 113, 509–517. experiments. (15) Beattie, B. 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