Design and Synthesis of an Easily Obtainable Maleimide Reagent N

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Design and Synthesis of an Easily Obtainable Maleimide Reagent N-[2-(4-[ F]fluoro-N-methylbenzenesulfonamido)ethyl]maleimide ([ F]FBSEM) to Radiolabel Thiols in Proteins 18

18

Yuji Fujita, Yoshihiro Murakami, Akihiro Noda, and Sosuke Miyoshi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00707 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016

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Bioconjugate Chemistry

Design and Synthesis of an Easily Obtainable 18 N-[2-(4-[ F]fluoro-N-methylbenzenesulfonamido)ethyl]maleimide Radiolabel Thiols in Proteins

Yuji Fujita*, Yoshihiro Murakami, Akihiro Noda, Sosuke Miyoshi Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Japan

*Corresponding author: Yuji Fujita Astellas Pharma Inc. 21 Miyukigaoka, Tsukuba, Ibaraki, Japan, 305-8585 e-mail: [email protected] Phone: +81-29-829-6482 Fax: +81-29-854-1879

ACS Paragon Plus Environment

Maleimide Reagent 18 ([ F]FBSEM) to


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ABSTRACT: An

easily

obtainable

thiol-selective

labeling

[18F]FBSEM

reagent

(N-[2-(4-[18F]fluoro-N-metheylbenzenesulfonamido)ethyl]maleimide) was developed. The advantage of the design is that the precursor and [18F]FBSEM have the same backbone and backbone construction is not required; in contrast, known thiol-specific labeling reagents do require backbone construction, and this is thought to be the cause of their complicated synthesis. [18F]FBSEM was successfully obtained in higher yield (25%) and in a simpler way (2 fluorination and deprotection steps in 65 min) than the widely used [18F]FBEM (N-[2-(4-[18F]fluorobenzamide)ethyl]maleimide). The labeling efficacy of [18F]FBSEM was confirmed by conjugation with glutathione. [18F]FBSEM is a promising labeling agent for proteins.

INTRODUCTION: PET (positron emission tomography), a useful imaging technique with the ability to noninvasively detect radiolabeled molecules, is used as a tool in drug discovery and development in pharmacokinetic studies,1 pharmacodynamic studies,2 and evaluation of drug efficacy.3 The number of biopharmaceuticals, such as antibodies, is now increasing.4 The advantage of biopharmaceuticals is their high target specificity. Given that the outcome of therapy in some patients depends on target expression status, the ability to image the target is useful in patient stratification in both drug discovery and development and in clinical treatment.5 PET can play a major role in this trend, thanks to its high sensitivity, adequate resolution, and high quantitative capability. Among PET nuclides, 18F is often used for radiolabeling proteins due to its moderate half-life (109.77min). The most common way to incorporate 18F into proteins is the use of prosthetic groups labeled with 18F, because direct introduction of 18

F requires harsh conditions under which proteins are not stable.6

18

F is first introduced into reactive prosthetic

groups and then the prosthetic groups are conjugated with proteins. These prosthetic groups react with proteins in two major ways, by reaction with the amino group of a lysine residue or with the thiol group of a cysteine residue. The acylating reagent [18F]SFB is widely used as a prosthetic group in reagents used for labeling lysine residues.6 It reacts randomly with amino groups at lysines, and the radiochemical yield of labeling is not usually high.

In

contrast,

maleimide-type

prosthetic

groups

such

as

[18F]FBEM

(N-[2-(4-[18F]fluorobenzamide)ethyl]maleimide) can be introduced site-specifically at the thiol group of a cysteine residue with relatively high yield. The first reported maleimide reagents for labeling thiols by


18

F are

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[18F]FPPD and [18F]DDPFB, reported by Shiue et al.7 Many other labeling reagents have been reported since then ,8-17 among which the most widely used is [18F]FBEM, 5, 18-25 reported by Cai et al.12 However, the synthesis of [18F]FBEM is complicated and time-consuming. The original method involves the synthesis of [18F]SFB as an intermediate, and is both slow and has low radiochemical yield.12 Several improved methods with higher yield have since been reported, but they are still complicated.26-28 In our laboratory, [18F]FBEM is obtained by an original modified method and has an average radiochemical yield of 17% in 94 min (Scheme 1). Although [18F]FBEM is useful for labeling proteins, synthesis is a complicated operation which requires many reagents and a long synthesis time. Here, therefore, we developed a novel maleimide reagent that can be easily obtained, and confirmed its reactivity with thiols.

Scheme 1. Synthetic route for [18F]FBEM.

Synthesis of these maleimide reagents involves construction of backbone; that is, the fluorinated moiety and maleimide moiety are linked after fluorination (Figure 1A). In the case of [18F]FBEM, fluorobenzoic acid is reacted with amine to construct the backbone. Considering this to be the cause of the complicated synthesis, we designed a novel maleimide reagent whose precursor has the same backbone as the labeling reagent (Figure 1B). It was desirable to obtain a labeling reagent with a single-step fluorination reaction, but this was not possible due to the instability of maleimide under typical nucleophilic radiofluorination conditions.11, 14, 17 Thus, the maleimide moiety of the precursor was protected and fluorination was followed by a deprotection step. The designed compound was [18F]FBSEM (N-[2-(4-[18F]fluoro-N-metheylbenzenesulfonamido)ethyl]maleimide) and the assumed synthetic route is shown in Scheme 2. The precursor has a fluorination moiety which is activated with sulfonamide and a maleimide moiety which is protected with an easily removable protective group.

18

F is

introduced in a benzene ring, so it is metabolically stable against delfuorination, as is the case with [18F]FBEM.



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Here we report the synthesis of [18F]FBSEM and confirmation of its reactivity with thiols using glutathione as a model substrate.

Figure 1. Synthetic strategy of maleimide reagent: (A) reported method; (B) our original method.

Scheme 2. Designed compound and planned synthetic route.

RESULTS AND DISCUSSION: Synthetic routes for standard FBSEM and the precursor of [18F]FBSEM are shown in Scheme 3. Maleic anhydride 1 was protected via the Diels-Alder reaction to afford 2,29 which was reacted with t-butyl(2-aminoethyl)carbamate to form 3. Removal of the Boc group with HCl, sulfonamidation with sulfonylchloride, and methylation with CH3I afforded 6a-b. FBSEM (7) was obtained from 6a via the retro-Diels-Alder reaction. Reduction of the nitro group of 6b, followed by dimethylation with CH3I and treatment with CH3OTf gave the precursor of [18F]FBSEM (10).


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Scheme 3a. Synthetic route for FBSEM and the precursor of [18F]FBSEM.

a

Reagents and conditions (a) benzene, rt, 63 h, 76%; (b) t-butyl(2-aminoethyl)carbamate, MeOH, reflux 10 h and

rt 2 days, 59%; (c) EtOAc, 4M HCl/EtOAc, rt, 5 h, 99%; (d) R-Ph-SO2Cl, Et3N, CH2Cl2, 0 °C-rt, 7-15 h, 99% (5a), 91% (5b); (e) K2CO3, CH3I, DMF, rt, 15 h, 76% (6a), 94% (6b); (f) anisole, 150 °C, 8 h, 84%; (g) Fe, NH4Cl, EtOH/H2O, 110 °C, 0.5 h, 90%; (h) K2CO3, CH3I, DMF, 70 °C, 4h, 37%; (i) CH3OTf, CH2Cl2, rt, 24 h, 82%

The primary synthetic route of [18F]FBSEM (Method A) is shown in Scheme 4A. The precursor was fluorinated and the reaction mixture was purified by HPLC. Solid phase extraction was performed and the fluorinated product [18F]6a was collected in a second reaction vessel and concentrated. After deprotection in xylene and subsequent concentration, [18F]FBSEM was obtained as an Et2O solution without further purification (decay-corrected radiochemical yield: 5.1%, radiochemical purity: 45%, synthesis time: 99 min). However, there were two problems. First, a byproduct was observed after deprotection and thus radiochemical purity was very low. Second, although the number of reaction steps was only two, the reaction was performed in two reaction vessels and the operation was complicated. Synthesis time was even longer than that of [18F]FBEM. As for the byproduct, collected [18F]FBSEM solution was analyzed by LC-MS and the structure of the byproduct was estimated. The obtained mass spectrum and the fact that the byproduct did not react with a thiol group indicated that the byproduct was an adduct of maleimide and that xylene used as solvent (Figure 2). As the byproduct was



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not observed in cold reaction, it was estimated to be formed via radiolysis. Considering the relatively high stability of benzyl radicals, we supposed that the benzyl radical was generated from xylene and reacted with maleimide. Therefore, the solvent used in the deprotection reaction was changed to anisole, which does not have a benzyl position. Radiochemical purity was then extensively raised to 92%. Furthermore, by changing the solvent of fluorination to CH3CN, which can be concentrated as the boiling point is relatively low, synthesis was performed with a one-pot reaction followed by HPLC purification and solid phase extraction. As maleimide is not stable under heating in radiofluorination conditions, MsOH was used for acidification before deprotection by the retro-Diels-Alder reaction. By these modifications, radiochemical purity was raised to >99% and synthesis time was slightly shortened. However, the operation was still complicated and the synthesis time was not sufficiently decreased. This was due to the two concentration steps, so the synthetic route was further changed to remove the concentration steps. The modified final version of the synthetic route (Method B) is shown in Scheme 4B. The solvent of the fluorination reaction was changed to DMSO to allow deprotection at a high temperature without changing the solvent, and fluorination temperature was raised. The deprotection temperature was also raised to 170 °C (measured with a thermocouple set outside the reaction vessel) to shorten the reaction time. In our test runs, setting of the reaction temperature in the deprotection reaction was important. When the temperature was not high enough, the proportion of protected compound was high, whereas a temperature which was too high resulted in the generation of a byproduct. In the modified process, fluorination, deprotection with the addition of MsOH, HPLC purification, and solid phase extraction afforded [18F]FBSEM as a CH2Cl2 solution using automated synthesis apparatus (irradiation time: 30 min, radioactivity: 136.9 mCi, decay-corrected radiochemical yield: 25%, radiochemical purity: >99%, specific activity: 2.1 Ci/µmol, synthesis time: 65 min). In the process of solid phase extraction with a reversed-phase cartridge, CH2Cl2, CH3CN, and Et2O were tested as an eluting solvent. CH2Cl2 and CH3CN showed a higher collection rate of [18F]FBSEM (>90%) than Et2O. In view of the time required for concentration of the organic solvent before conjugation with thiols, CH2Cl2 was chosen as an eluent.


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Scheme 4. Synthetic route of [18F]FBSEM: (A) primary route; (B) modified route.

Figure 2. Estimated structure of a byproduct.

A comparison of the synthesis of [18F]FBEM and [18F]FBSEM is shown in Table 1. With the same irradiation time, we could obtain [18F]FBSEM with more radioactivity, higher radiochemical yield, shorter synthesis time, and fewer reagents and solid phase cartridges (cartridges used for F- collection, solid phase extraction, and anhydration) in the automated synthesis apparatus than [18F]FBEM. Synthesis time of 65 min includes 22 min for collection of F- from the cyclotron and azeotropic drying, so the time needed from fluorination through collection of [18F]FBSEM is about only 40 min. Thus, by designing [18F]FBSEM and its precursor with the same backbone, we successfully obtained [18F]FBSEM while overcoming the problems with the synthesis of [18F]FBEM. Table 1. Comparison of the synthesis of [18F]FBEM and [18F]FBSEMa [18F]FBEMb [18F]FBSEM

a

Radioactivity

70.5 mCic

136.9 mCid

Radiochemical yielde

17%

25%

Specific activity

-f

2.1 Ci/µmolg

Radiochemical purity

-f

>99%

Synthesis timeh

94 min

65 min

Reagents and cartridges

19

12

Data for 30 min irradiation. b Average (n=3). c At 15-17 min after SPE elution (radioactivity was measured after



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concentration). d At the end of synthesis (just after SPE elution). e Decay-corrected (calculated from [18F]F-). f Not measured. g At the end of synthesis. h Including 22 min for collection of F- and azeotropic drying.

Obtained [18F]FBSEM was conjugated with glutathione (GSH) as a model substrate to confirm its reactivity with thiols (Scheme 5). [18F]FBSEM was reacted with glutathione (1 mg/mL in PBS) at room temperature for 30 min. [18F]FBSEM was completely consumed and radiochemical purity of [18F]FBSEM-GSH was almost 90%, which showed its high reactivity with thiols like [18F]FBEM, as expected. Scheme 5. Conjugation of [18F]FBSEM with glutathione.

CONCLUSION: We have designed a novel maleimide reagent [18F]FBSEM, which can be prepared easily by an original synthetic strategy. [18F]FBSEM was obtained with fewer reagents, simpler operation, and a shorter synthesis time than [18F]FBEM, which is a widely used maleimide reagent for labeling thiols. The obtained radioactivity and radiochemical yield of [18F]FBSEM was high and its reactivity with thiols was confirmed by conjugation with glutathione. [18F]FBSEM is promising as a labeling agent for proteins such as antibodies. An in vivo preclinical PET imaging study is underway.

EXPERIMENTAL PROCEDURES General Procedures. All reagents and solvents were purchased and used as received, unless otherwise noted. 1

H-NMR spectra were recorded with a Varian UNITY spectrometer (Varian Inc., Palo Alto, CA, USA) or an

AV-III HD 500 spectrometer (Bruker Biospin K.K., Yokohama, Japan). 13C-NMR spectra were recorded with a


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AV-III HD 500 spectrometer. Chemical shifts are expressed in δ units using tetramethylsilane as the internal standard. Mass spectral data were obtained using a liquid chromatography-mass spectrometer (LC-MS) method with a UPLC/SQD LC-MS system (Nihon Waters K.K., Tokyo, Japan). HRMS data were obtained using a LC-MS method with a LCT Premier (Nihon Waters K.K., Tokyo, Japan) or an Exactive Plus (Thermo Fisher Scientific K.K., Tokyo, Japan). Lyophilization was performed with a DC401 (Yamato Scientific Co., Ltd., Tokyo, Japan). Chemistry.

N-[2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl]-4-fluorobenzenesulfonamide (5a). To a suspension of 4 (798 mg, 3.26 mmol) in CH2Cl2 (16 mL) was added Et3N (1.00 mL, 7.17 mmol). The solution was cooled on ice and 4-fluorobenzenesulfonylchloride (702 mg, 3.61 mmol) was added. The mixture was stirred at room temperature for 15 h and NaHCO3aq was added. The solution was extracted with CH2Cl2. The organic layer was dried over MgSO4 and concentrated. The residue was triturated with Et2O. The precipitate was collected to afford 5a (1.18 g, 3.22 mmol, 99% yield) as a colorless solid. 1H NMR (400 MHz, (CD3)2SO): δ 7.87 (1H, t, J = 6.3 Hz), 7.82 (2H, dd, J = 8.9, 5.2 Hz), 7.44 (2H, dd, J = 8.9, 8.9 Hz), 6.54 (2H, dd, J = 0.9, 0.9 Hz), 5.10 (2H, dd, J = 0.9, 0.9 Hz), 3.37-3.40 (2H, m), 2.89 (2H, s), 2.83-2.88 (2H, m).

13

C NMR (125 MHz,

dioxane-d8): δ 176.8, 165.5 (d, J = 251.5 Hz), 138.3 (d, J = 3.0 Hz), 137.2, 130.4 (d, J = 9.3 Hz), 116.7 (d, J = 22.6 Hz), 81.4, 48.1, 40.6, 38.7. MS (ESI+): 367.1 [M+H]+, 389.1 [M+Na]+. HRMS (ESI): Calcd for C16H16O5N2FS, 367.0758 [M+H]+, found 367.0755.

N-[2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl]-4-nitrobenzenesulfonamide (5b). To a suspension of 4 (2.05 g, 8.36 mmol) in CH2Cl2 (41 mL) was added Et3N (2.55 mL, 18.3 mmol). The solution was cooled on ice and 4-nitrobenzenesulfonylchloride (2.06 g, 9.31 mmol) was added. The mixture was stirred at 0 °C for 0.5 h and at room temperature for 6.5 h. The precipitate was collected by filtration and washed with CHCl3 to afford 5b (1.92 g, 4.87 mmol, 58% yield) as a colorless solid. The filtrate was concentrated and the residue was triturated with CHCl3. The precipitate was collected to afford additional 5b (1.08 g, 2.75 mmol, 33% yield). 1H NMR (400 MHz, (CD3)2SO): δ 8.42 (2H, d, J = 8.9 Hz), 8.23 (1H, t, J = 6.2 Hz), 8.02 (2H, d, J = 8.9 Hz), 6.54 (2H, br s), 5.10 (2H, br s), 3.38-3.42 (2H, m), 2.91-2.96 (2H, m), 2.90 (2H, s). 13C NMR (125 MHz, dioxane-d8): δ 176.8, 151.1, 147.3, 137.2, 128.6, 125.0, 81.4, 48.1, 40.7, 38.6. MS (ESI+): 416.1 [M+Na]+. HRMS (ESI): Calcd for C16H16O7N3S, 394.0709 [M+H]+, found 394.0700.



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N-[2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl]-4-fluoro-N-methylbenzenesulfona mide (6a). To a solution of 5a (798 mg, 2.18 mmol) and K2CO3 (910 mg, 6.58 mmol) in DMF (16 mL) was added CH3I (273 µL, 4.38 mmol) and the mixture was stirred at room temperature for 15 h. H2O was added and the precipitate was collected by filtration. The collected solid was dried at 60 °C under vacuum to afford 6a (630 mg, 1.66 mmol, 76% yield). 1H NMR (500 MHz, (CD3)2SO): δ 7.80 (2H, dd, J = 8.9, 5.2 Hz), 7.46 (2H, dd, J = 8.9, 8.9 Hz), 6.55 (2H, br), 5.14 (2H, br), 3.53 (2H, t, J = 6.2 Hz), 3.11 (2H, t, J = 6.2 Hz), 2.94 (2H, s), 2.71 (3H, s). 13C NMR (125 MHz, (CD3)2SO): δ 176.7, 164.9 (d, J = 251.7 Hz), 136.9, 134.2 (d, J = 3.0 Hz), 130.5 (d, J = 9.7 Hz), 117.1 (d, J = 22.6 Hz), 80.8, 47.7, 47.1, 36.4, 35.3. MS (ESI+): 381.2 [M+H]+, 403.2 [M+Na]+. HRMS (ESI): Calcd for C17H18O5N2FS, 381.0915 [M+H]+, found 381.0915.

N-[2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl]-N-methyl-4-nitrobenzenesulfona mide (6b). To a solution of 5b (2.73 g, 6.94 mmol) and K2CO3 (1.93 g, 14.0 mmol) in DMF (55 mL) was added CH3I (650 µL, 10.4 mmol) and the mixture was stirred at room temperature for 15 h. H2O was added and the precipitate was collected by filtration. The collected solid was dried at 60 °C under vacuum to afford 6b (2.65 g, 6.51 mmol, 94% yield) as a colorless solid. 1H NMR (500 MHz, (CD3)2SO): δ 8.41 (2H, d, J = 8.9 Hz), 8.00 (2H, d, J = 8.9 Hz), 6.55 (2H, br), 5.14 (2H, br), 3.55 (2H, t, J = 6.1 Hz), 3.18 (2H, t, J = 6.1 Hz), 2.95 (2H, s), 2.77 (3H, s). 13C NMR (125 MHz, (CD3)2SO): δ 176.1, 149.8, 142.8, 136.3, 128.4, 124.6, 80.2, 47.0, 46.6, 35.8, 34.7. MS (ESI+): 408.2 [M+H]+, 430.1 [M+Na]+. HRMS (ESI): Calcd for C17H18O7N3S, 408.0860 [M+H]+, found 408.0855.

N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-4-fluoro-N-methylbenzenesulfonamide (N-[2-(4-fluoro-N-methylbenzenesulfonamido)ethyl]maleimide, 7, FBSEM). A solution of 6a (28.8 mg, 75.7 µmol) in anisole (20 mL) was stirred at 150 °C for 8h. The solution was concentrated and purified by silica gel column chromatography (hexane/EtOAc = 100/0-50/50) to afford 7 (19.9 mg, 63.7 µmol, 84% yield) as a colorless solid. 1H NMR (400 MHz, (CD3)2SO): δ 7.77 (2H, dd, J = 8.9, 5.1 Hz), 7.44 (2H, dd, J = 8.9, 8.9 Hz), 7.04 (2H, s), 3.58 (2H, t, J = 5.7 Hz), 3.14 (2H, t, J = 5.7 Hz), 2.72 (3H, s). 13C NMR (125 MHz, (CD3)2SO): δ 170.7, 164.3 (d, J = 251.4 Hz), 134.5, 133.7 (d, J = 3.1 Hz), 129.8 (d, J = 9.6 Hz), 116.5 (d, J = 22.6 Hz), 47.4, 34.6, 34.3. MS (ESI+): 313.0 [M+H]+, 335.0 [M+Na]+. HRMS (ESI): Calcd for C13H14O4N2FS, 313.0653 [M+H]+, found 313.0650. 4-amino-N-[2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl]-N-methylbenzenesulfon


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amide (8). A suspension of 6b (1.30 g, 3.19 mmol), Fe (574 mg, 10.3 mmol), and NH4Cl (414 mg, 7.74 mmol) in EtOH (13 mL) and H2O (4.0 mL) was stirred at 110 °C for 0.5 h. The mixture was cooled to room temperature and filtered through Celite, which was washed with CHCl3. NaHCO3aq was added to the filtrate and the solution was extracted with CHCl3. The organic layer was dried over MgSO4 and concentrated. The residue was triturated with Et2O and the precipitate was collected to afford 8 (1.09 g, 2.88 mmol, 90% yield) as a colorless solid. 1H NMR (400 MHz, (CD3)2SO): δ 7.33 (2H, d, J = 8.7 Hz), 6.62 (2H, d, J = 8.7 Hz), 6.54 (2H, dd, J = 0.9, 0.9 Hz), 6.02 (2H, s), 5.13 (2H, dd, J = 0.9, 0.9 Hz), 3.49 (2H, t, J = 6.3 Hz), 2.97 (2H, t, J = 6.3 Hz), 2.92 (2H, s), 2.61 (3H, s). 13C NMR (125 MHz, (CD3)2SO): δ 176.1, 152.9, 136.3, 128.9, 121.5, 112.7, 80.2, 47.1, 46.5, 35.9, 34.8. MS (ESI+): 378.3 [M+H]+. HRMS (ESI): Calcd for C17H20O5N3S, 378.1118 [M+H]+, found 378.1115. 4-(dimethylamino)-N-[2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl]-N-methylbenz enesulfonamide (9). To a solution of 8 (400 mg, 1.06 mmol) and K2CO3 (738 mg, 5.34 mmol) in DMF (20 mL) was added MeI (1.30 mL, 20.8 mmol) and the mixture was stirred at 70 °C for 4 h. Water was added and the solution was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel column chromatography (CHCl3/MeOH = 100/0-100/1) and the eluate was concentrated. The residue was triturated with Et2O and the precipitate was collected by filtration to afford 9 (159 mg, 0.393 mmol, 37% yield) as a colorless solid. 1H NMR (500 MHz, CDCl3): δ 7.57 (2H, d, J = 9.1 Hz), 6.66 (2H, d, J = 9.1 Hz), 6.51 (2H, dd, J = 0.9, 0.9 Hz), 5.27 (2H, dd, J = 0.9, 0.9 Hz), 3,64 (2H, t, J = 5.8 Hz), 3.14 (2H, t, J = 5.8 Hz), 3.03 (6H, s), 2.95 (2H, s), 2.78 (3H, s).

13

C NMR (125 MHz, dioxane-d8):

δ 176.7, 153.5, 137.1, 129.5, 125.1, 111.6, 81.3, 48.1, 47.6, 39.9, 36.3, 35.0. MS (ESI+): 406.3 [M+H]+. HRMS (ESI): Calcd for C19H24O5N3S, 406.1431 [M+H]+, found 406.1429. 4-{[2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl](methyl)sulfamoyl}-N,N,N-trimet hylanilinium trifluoromethanesulfonate (10). To a solution of 9 (122 mg, 0.300 mmol) in CH2Cl2 (5.0 mL) was added MeOTf (36.2 µL, 0.330 mmol) and the mixture was stirred at room temperature for 24 h. Et2O was added and the precipitate was collected by filtration and washed with Et2O to afford 10 (140 mg, 0.246 mmol, 82%) as a colorless solid. 1H NMR (400 MHz, (CD3)2SO): δ 8.20 (2H, d, J = 9.2 Hz), 7.99 (2H, d, J = 9.2 Hz), 6.55 (2H, br), 5.14 (2H, br), 3.64 (9H, s), 3.56 (2H, t, J = 6.1 Hz), 3.17 (2H, t, J = 6.1 Hz), 2.94 (2H, s), 2.77 (3H, s). 13C NMR (125 MHz, (CD3)2SO): δ 176.1, 150.0, 139.0, 136.4, 128.6, 122.2, 120.6 (q, J = 322.4 Hz), 80.2, 56.3, 47.1, 46.6, 35.8, 34.8. MS (ESI+): 420.3 [M]+. HRMS (ESI): Calcd for C20H26O5N3S, 420.1588 [M]+, found 420.1586.



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FBSEM-GSH (Conjugation of FBSEM (7) with glutathione). To a solution of glutathione (40.1 mg, 130 µmol) in PBS (500 µL) was added a solution of 7 (44.1 mg, 141 µmol) in CH3CN (500 µL) and the mixture was stirred at room temperature for 1 h. The solution was concentrated and purified by reversed-phase liquid chromatography (H2O/CH3CN = 80/20-70/30). The fraction containing FBSEM-GSH was collected, concentrated, and lyophilized to afford FBSEM-GSH (34.8mg, 56.2 µmol, 43% yield, a mixture of diastereomers) as a colorless solid. 1H NMR (500 MHz, CD3OD): δ 7.82 (2H, d, J = 8.9 Hz), 7.81 (2H, d, J = 8.6 Hz), 7.33 (2H, d, J = 8.6 Hz), 7.32 (2H, d, J = 8.9 Hz), 4.69-4.75 (2H, m), 4.03-4.08 (2H, m), 3.92 (2H, s), 3.91 (2H, s), 3.65-3.74 (6H, m), 3.44 (1H, dd, J = 14.1 Hz, 5.2 Hz), 3.16-3.29 (8H, m), 2.97 (1H, dd, J = 14.1 Hz, 8.9 Hz), 2.81 (6H, s), 2.53-2.61 (6H, m), 2.09-2.19 (4H, m). MS (ESI+): 620.3 [M+H]+, 642.3 [M+Na]+, 618.4 [M-H]-. HRMS (ESI): Calcd for C23H31O10N5FS2, 620.1491 [M+H]+, found 620.1491. Radiochemistry. No-carrier-added aqueous [18F]fluoride ion was produced by the

18

O(p, n)18F nuclear reaction

(30 µA) using a Cyclone 18/9 cyclotron (IBA, Louvain-La-Neuve, Belgium). Radiosynthesis was performed in an automated synthesis apparatus (Dainippon Seiki Co., Ltd., Kyoto, Japan). Purification of the reaction mixture by reversed-phase HPLC in radiosynthesis was performed using a YMC-Pack Pro C18 10×250 mm column (YMC Co., Ltd., Kyoto, Japan). Analytical chromatography was performed with an Agilent 6460 LC-MS system (Agilent Technologies Japan, Tokyo) and an Aloka positron detector RLC-700 (Aloka, Tokyo, Japan) using a YMC-Pack Pro C18 4.6×150 mm column (YMC Co., Ltd., Kyoto, Japan), eluted with 0.05 M NH4OAc-0.1% AcOH/CH3CN (60/40, 60/40-5/95, or 95/5-5/95) at 1.0 mL/min.

N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-4-[18F]fluorobenzamide (N-[2-(4-[18F]fluorobenzamide)ethyl]maleimide, [18F]FBEM). Aqueous [18F]fluoride ion was passed through a Sep-Pak Light Waters Accell Plus QMA Cartridge (Nihon Waters K.K., Tokyo, Japan) and eluted with a solution of K2CO3 (2.7 mg/mL in H2O, 1.0 mL) and Kryptofix 222 (20 mg/mL in CH3CN, 1.0 mL) into a reaction vessel. The solvent was evaporated under a stream of N2 at 140 °C. CH3CN (1.0 mL) was added and the solvent was evaporated at 140 °C. Again CH3CN (1.0 mL) was added and the solvent was evaporated at 120 °C. To the residue was added a solution of 4-(ethoxycarbonyl)-N,N,N-trimethylanilinium trifluoromethanesulfonate (5.0 mg, 14 µmol) in DMSO (800 µL) and the mixture was heated at 120 °C for 10 min. After cooling, 1 M NaOHaq (800 µL) was added and the mixture was heated at 100 °C for 5 min. After cooling, 1 M HClaq (1.0 mL) was added and the solution was transferred to a bottle containing H2O (10 mL). The solution was passed through an Oasis


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HLB Plus cartridge (Nihon Waters K.K., Tokyo, Japan). The cartridge was washed with H2O (5.0 mL). 4-[18F]fluorobenzoic acid was eluted with CH3CN (3 mL) into a second reaction vessel containing nPr4NOH (25% in H2O, 5 µL) in CH3CN (200 µL). The solvent was evaporated under a stream of N2 at 120 °C. CH3CN (1.0 mL) was added and the solvent was evaporated at 120 °C. To the residue was added 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate (8.9 mg, 35.0 µmol) in DMF (300 µL), HATU (13.3 mg, 35.0 µmol) in DMF (300 µL), and DIPEA (50 µL, 0.29 mmol) in DMF (100 µL) and the reaction was performed at 25 °C for 7 min. The mixture was transferred to a semi-preparative HPLC system, which included a wash of the reaction vessel with 0.05 M NH4OAc-0.1%AcOH (1.7 mL). After purification by reversed-phase HPLC (eluted with H2O/CH3CN = 70/30, 4.0 mL/min, 40 °C), the fraction containing [18F]FBEM was collected and diluted with H2O (15 mL). [18F]FBEM was trapped on the Oasis HLB Plus cartridge, eluted with CH2Cl2 (4.0 mL), and passed through two Sep-Pak Dry cartridges (Nihon Waters K.K., Tokyo, Japan) in series. The solution was evaporated under a stream of N2 with gentle heating (15-17min) and radioactivity was measured.

N-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-4-fluoro-N-methylbenzenesulfonamide (N-[2-(4-[18F]fluoro-N-methylbenzenesulfonamido)ethyl]maleimide, [18F]7, [18F]FBSEM). Method A: To the residue containing [18F]fluoride, K2CO3, and Kryptofix 222 prepared in the same way as [18F]FBEM was added a solution of 10 (5.0 mg, 8.8 µmol) in DMSO (800 µL) and the mixture was heated at 90 °C for 10 min. After cooling, the mixture was combined with 0.05 M NH4OAc-0.1%AcOH (500 µL) and transferred to a semi-preparative HPLC

system,

which

included a wash of the reaction vessel with 0.05 M

NH4OAc-0.1%AcOH/CH3CN (80/20, 700 µL). After purification by reversed-phase HPLC (eluted with 0.05 M NH4OAc-0.1%AcOH/CH3CN = 60/40, 4.0 mL/min, 40 °C), the fraction containing [18F]6a was collected and diluted with H2O (20 mL). [18F]6a was trapped on an Oasis HLB Plus cartridge. The cartridge was washed with 1 M HCl (10 mL) and H2O (5.0 mL). [18F]6a was eluted with CH3CN (3.0 mL) into second reaction vessel. The solvent was evaporated under a stream of N2 at 90 °C and xylene (2.0 mL) was added. The mixture was heated at 160 °C for 10 min. After evaporation of solvent under a stream of N2 at 100 °C, [18F]7 was dissolved in Et2O (3.0 mL) and collected. Method B: To the residue containing [18F]fluoride, K2CO3, and Kryptofix 222 was added a solution of 10 (5.0 mg, 8.8 µmol) in DMSO (800 µL) and the mixture was heated at 100 °C for 10 min. After cooling, MsOH (20 µL, 0.308 mmol) in DMSO (150 µL) was added and the solution was heated at 170 °C for 5 min. The mixture was



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transferred to a semi-preparative HPLC system with the addition of H2O (600 µL), which included a wash of the reaction vessel with 10%DMSO in H2O (1.2 mL). After purification by reversed-phase HPLC (eluted with 0.05 M NH4OAc-0.1%AcOH/MeOH = 50/50, 4.0 mL/min, 50 °C), the fraction containing [18F]7 was collected and diluted with H2O (20 mL). [18F]7 was trapped on the Oasis HLB Plus cartridge and the cartridge was washed with H2O (5.0 mL). [18F]7 was eluted with CH2Cl2 (3.0 mL) and passed through two Sep-Pak Dry cartridges in series. LC-MS (ESI+): 313.0 [M+H]+ (detected as [19F]FBSEM). [18F]FBSEM-GSH (Conjugation of [18F]7 ([18F]FBSEM) with glutathione). An aliquot (200 µL) of [18F]7 in CH2Cl2 was concentrated under a stream of N2. To the residue was added a solution of glutathione (1.0 mg/mL) in PBS (500 µL) and the mixture was incubated at room temperature for 30 min. The solution was analyzed by LC-MS. [18F]7 was completely consumed and radiochemical purity of [18F]FBSEM-GSH was almost 90%. LC-MS (ESI+): 620.1 [M+H]+ (detected as [19F]FBSEM-GSH).

ACKNOWLEDGEMENT

The authors would like to thank Dr. Shintaro Nishimura for his fruitful discussion.

SUPPORTING INFORMATION Experimental procedures of some compounds; NMR and MS spectra of new compounds; analytical and semi-preparative HPLC charts of radiosynthesis; and LC-MS data used to elucidate the structure of the by-product.

ABBREVIATIONS [18F]DDPFB,

N-[3-(2,5-

dioxo-2,5-dihydropyrrol-1-yl)phenyl]-4-[18F]fluorobenzamide;

N-[2-(4-[18F]fluorobenzamide)ethyl]maleimide; N-[2-(4-[18F]fluoro-N-methylbenzenesulfonamido)ethyl]maleimide;

[18F]FBEM, [18F]FBSEM, [18F]FPPD,

1-(4-[18F]fluorophenyl)pyrrole-2,5-dione; GSH, glutathione; K.222, Kryptofix 222; PBS, phosphate buffered saline; PET, positron emission tomography; [18F]SFB, N-succinimidyl-4-[18F]fluorobenzoate; SPE, solid phase extraction


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FUNDING The authors are employees of Astellas Pharma Inc. There is no other funding to declare.

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24x7mm (600 x 600 DPI)



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Figure 1. Synthetic strategy of maleimide reagent: (A) reported method; (B) our original method. 103x48mm (600 x 600 DPI)


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Figure 2. Estimated structure of a byproduct. 30x15mm (600 x 600 DPI)



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Scheme 1. Synthetic route for [18F]FBEM. 70x22mm (600 x 600 DPI)


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Scheme 2. Designed compound and planned synthetic route. 32x5mm (600 x 600 DPI)



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Scheme 3a. Synthetic route for FBSEM and the precursor of [18F]FBSEM. 102x48mm (600 x 600 DPI)


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Scheme 4. Synthetic route of [18F]FBSEM: (A) primary route; (B) modified route. 72x16mm (600 x 600 DPI)



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Scheme 5. Conjugation of [18F]FBSEM with glutathione. 60x18mm (600 x 600 DPI)


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Design and Synthesis of an Easily Obtainable Maleimide Reagent N

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