Synthesis, 18F-Radiolabeling, and in Vivo Biodistribution Studies of N

Article pubs.acs.org/jmc

Synthesis, 18F‑Radiolabeling, and in Vivo Biodistribution Studies of N‑Fluorohydroxybutyl Isatin Sulfonamides using Positron Emission Tomography Panupun Limpachayaporn,†,‡ Stefan Wagner,§ Klaus Kopka,§ Sven Hermann,∥ Michael Schaf̈ ers,∥ and Günter Haufe*,†,∥ †

Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, Corrensstrasse 40, D-48149 Münster, Germany International NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 10, D-48149 Münster, Germany § Klinik für Nuklearmedizin, Universitätsklinikum Münster, Albert-Schweitzer-Campus 1, Gebäude A1, D-48149 Münster, Germany ∥ European Institute for Molecular Imaging (EIMI), Westfälische Wilhelms-Universität Münster, Mendelstrasse 11, D-48149 Münster, Germany ‡

S Supporting Information *

ABSTRACT: The effector caspases-3 and -7 play a central role in programmed type I cell death (apoptosis). Molecular imaging using positron emission tomography (PET) by tracking the activity of executing caspases might allow the detection of the early onset as well as therapy monitoring of various diseases induced by dysregulated apoptosis. Herein, four new fluorinated diastereoand enantiopure isatin sulfonamide-based potent and selective caspase-3 and -7 inhibitors were prepared by cyclic sulfate ringopening with fluoride. All fluorohydrins exhibited excellent in vitro affinities (up to IC50 = 11.8 and 0.951 nM for caspase-3 and -7, respectively), which makes them appropriate PET radiotracer candidates. Therefore, N-(4-[18F]fluoro-3(R)-hydroxybutyl)and N-(3(S)-[18F]fluoro-4-hydroxybutyl)-5-[1-(2(S)-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin were synthesized in 140 min with 24% and 10% overall radiochemical yields and specific activities of 10−127 GBq/μmol using [18F]fluoride in the presence of Kryptofix and subsequent acidic hydrolysis. In vivo biodistribution studies in wild-type mice using PET/computed tomography imaging proved fast clearance of the tracer after tail vein injection.

1. INTRODUCTION

degeneration (Alzheimer’s, Parkinson’s, and Huntington’s diseases), and developmental defects.1 Although apoptosis comprises of a complex network of biological pathways, cell death is mainly regulated by a set of intracellular enzymes called caspases (cysteine aspartate-specific proteinases). More than 12 different caspases in mammals and 7 caspases in Drosophila have been characterized. Only twothirds have been implicated to play roles in the activation of apoptosis.3 These enzymes are classified into two categories: initiator caspases (e.g., caspases-2, -8, -9, and -10) and executioner or effector caspases (e.g., caspases-3, -6, and -7). The former are not directly involved in cell death. They induce the activation of the effector caspases from the corresponding procaspases. The effector caspases catalyze the selective hydrolysis of proteins, leading to regular cell death, while the

Apoptosis, programmed type I cell death, is a complicated mechanism in organisms to remove overproduced, potentially dangerous, and damaged cells without inflammatory response.1 It strictly controls the number of cells, thereby maintaining homeostasis, and is responsible for development and regeneration of organs and structures.1,2 Apoptosis is triggered either by an extrinsic specific interaction of the CD95 ligand and its receptor, which induces an intracellular death signal (death receptor pathway), or by DNA damage, leading to a mitochondrial induction of cell death (mitochondrial pathway). Finally, intracellular enzymesexecuting caspasesare activated, resulting in the destruction of the cells targeted by apoptosis.1 This cell death program is a fundamental and essential process of the life cycle for all multicellular organisms. On the other hand, dysregulation of apoptosis is observed in pathological conditions and a wide variety of diseases such as cancer, autoimmune and cardiovascular (ischemia, stroke, myocardial infarction) diseases, persistent infections, neuro© XXXX American Chemical Society

Received: February 19, 2013

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dx.doi.org/10.1021/jm400257a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

benzyl,10−12 pyridine,10a triazole,10b,13 and aliphatic chains,6,14 including bromofluoroalkyl and fluorohydroxyalkyl groups,12 were substituted at the isatin nitrogen. The N-substitution of isatin sulfonamide was demonstrated to improve the activity significantly. In addition, substituted arenes,10b,13 heterocycles,10 including triazoles,13 and other fluoro functional groups15 were introduced into the 2-position of the pyrrolidine ring instead of phenoxy- and methoxymethyl functionalities. Even ligands with fluorinated ether side chains bind quite efficiently.16 2-Substituted azetidines and other heterocycles replaced the pyrrolidine ring, retaining the inhibitor activity.9,10 Most of the modified compounds, especially the N-benzyl and N-fluorohydroxyalkyl isatin sulfonamides and 2(phenoxymethyl)pyrrolidinyl analogues, showed high binding potency in vitro toward the target enzymes. However, substitutions at the 4- or 5-position of the 2-(methoxymethyl)pyrrolidine moiety resulted in a drop of binding affinity to micromolar or millimolar values, except for the 4-fluoro and 4,4-difluoro analogues, which showed activity against caspases-3 and -7 with IC50 values of less than 1 μM.7 In 2006, we demonstrated that the (S)-5-[1-(2(methoxymethyl)pyrrolidinyl)sulfonyl]isatin series exhibited superior inhibitory properties in an in vivo cell culture model, although the derivatives of (S)-5-[1-(2-(phenoxymethyl)pyrrolidinyl)sulfonyl]isatin were more potent in vitro.11 Therefore, (S)-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin (1) was selected to function as the lead compound in this study.12,14−16 Recently, several N-fluoroalkoxybenzyl isatinbased radiotracers were prepared and used for in vivo biodistribution studies.17 However, metabolic stability studies using electrochemical and microsomal oxidation revealed to some extent the cleavage of the N-benzyl group of (S)-N-[4-(2fluoroethoxy)benzyl]-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin.18 Consequently, the 18F-radiotracer might lose the label too early when it is used in vivo, leading to the undesired decrease of the signal to the background ratio in PET imaging and loss of specificity. To overcome this metabolic drawback, the benzyl group is replaced with fluorohydroxyalkyl substituents in the present study. Most recently, the isatin sulfonamide [18F]ICMT-11 was reported to have nanomolar affinity toward caspase-310b,13 and hence was evaluated in recombinant enzyme whole cells10b and many tumor-bearing mice models.19 The dynamic PET imaging demonstrated increased signal intensity related to increasing activation of apoptosis.19 Furthermore, a first automated good manufacturing practice (GMP) radiosynthesis of [18F]ICMT11 has been established for routine clinical use.20 Herein we report on the synthesis of four new diastereo- and enantiopure regioisomeric N-(4-fluoro-3-hydroxybutyl)- and N(3-fluoro-4-hydroxybutyl)-5-[1-(2(S)-(methoxymethyl)pyrrolidinyl)sulfonyl]isatins [(R)-8, (S)-8, (R)-9, and (S)-9], the elucidation of their binding affinities against caspases-3 and -7, and 18F-radiolabeling of two selected fluorohydrins using noncarrier-added [18F]fluoride. For synthesis, we used the strategy of cyclic sulfate ring-opening by fluoride. This method is milder than epoxide ring-opening. Forced conditions may lead to competing nucleophilic fluoride attack at the carbonyl carbon, resulting in a reversible isatin ring-opening and hence in a loss of specific activity.17d Furthermore, the in vivo biodistribution of the tracer candidate (R)-[18F]8 using PET/ computed tomography (CT) imaging was studied in healthy wild-type mice to evaluate its clearance characteristics.

remaining caspases, i.e., caspases-1, -4, -5, and -13, are involved in inflammation.4 The activation of effector caspases is a key event in apoptosis (“point of no return”); the pharmaceutical inhibition of effector caspases has been shown to suppress apoptosis.5 Thus, targeting effector caspases might enable both new therapeutic approaches for prevention (prevention of cell loss following ischemic events such as stroke or myocardial infarction) or induction of apoptosis (e.g., to kill tumor cells) and diagnostic approaches. With respect to the latter, positron emission tomography (PET) with labeled effector caspase ligands would support noninvasive studies on the role of apoptosis in physiological conditions vs apoptosis-related diseases and on monitoring of anti- or proapoptotic treatments. However, employment of PET needs specific imaging agents (radiotracers), which possess high binding affinities toward the target enzyme and a suitable positron emitter. In this study, isatin sulfonamides serve as the recognition unit, which are potent and selective inhibitors of the target effector caspases-3 and -7.5,6 Thus, the tracer will be developed from isatin sulfonamides. For the positron emitter, fluorine-18 is selected due to its relatively long half-life (∼110 min), allowing relatively time-consuming radiosynthesis in particular cases.7 In addition, the nature of fluorine substituents can also enhance the efficiency of enzyme−ligand interactions, leading to improved binding affinity.8 (S)-5-[1-(2-(Methoxymethyl)pyrrolidinyl)sulfonyl]isatin (1) and (S)-N-methyl-5-[1-(2-(phenoxymethyl)pyrrolidinyl)sulfonyl]isatin (2) were proved to be potent and selective inhibitors for caspases-3 and -7.5,6 The interaction of the enzymes and isatin sulfonamides was confirmed by X-ray cocrystal studies of isatin sulfonamide 2 and the recombinant human caspase-3 (Figure 1).5 It was reported that the 2-keto

Figure 1. Chemical structures of (S)-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin (1) and (S)-N-methyl-5-[1-(2(phenoxymethyl)pyrrolidinyl)sulfonyl]isatin (2) highlighted with caspase-3 subpockets interacting with parts of the ligand.5

group of isatin serves as the cysteine-163 binding site. The nucleophilic attack of the cysteine moiety leads to the formation of the tetrahedral thiohemiketal intermediate. The side chain attached at the isatin nitrogen binds with the S1pocket, while the pyrrolidine ring interacts well in the hydrophobic S2 -subsite. Additional polar and/or bulky substituents in the 4- or 5-position of the pyrrolidine ring, however, lead to less active inhibitors.7 In contrast, the 2position at the pyrrolidine ring is quite tolerant of substituents other than methoxymethyl or phenoxymethyl groups to be accommodated in the S3-pocket of the enzyme. It was found that the pyrrolidine ring is important for the selectivity for caspases-3 and -7 over other caspases.5,6 Further structural modifications of isatin sulfonamides were conducted to study the structure−activity relationship and to improve the inhibitor activities toward the effector caspases-3 and -7. Various functional groups such as substituted B



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Scheme 1. Synthetic Routes to the Cyclic Sulfate Precursors (R)-7 and (S)-7 for the Preparation of Fluorohydrins

Scheme 2. Preparation of Fluorohydrins (R)-8 and (S)-9 Starting from the Cyclic Sulfate (R)-7

2. RESULTS AND DISCUSSION

The two diastereomeric cyclic sulfate precursors (R)-7 and (S)-7 were prepared separately starting from the lead compound 16 and tosylates (R)-322 and (S)-3,23 respectively, as illustrated in Scheme 1. The N-substitution of 1 with both components using Cs2CO3 in DMF led to the corresponding acetals (R)-4 and (S)-4 in excellent yields. Subsequently, compounds 4 were hydrolyzed to the diols (R)-5 and (S)-5 using several acidic conditions, such as AcOH/H2O (8:7), oxalic acid in the presence of CeCl3·7H2O in MeCN, camphorsulfonic acid (CSA) in MeOH, and 50% TFA in DCM. Among them, hydrolysis using 50% TFA in DCM at room temperature was the most efficient method and gave the diols within 1 h in quantitative yield for both diastereomers. Treatment of the diols (R)-5 and (S)-5 with SOCl2 in the presence of pyridine delivered the cyclic sulfites (R)-6 and (S)6 in 89% and 62% yields, respectively. Finally, oxidation of the sulfites with NaIO4 in the presence of RuCl3·H2O resulted in the formation of the desired cyclic sulfates (R)-7 and (S)-7 in 84% yield for both cases. The cyclic sulfates are the precursors for the fluorohydrins (R)-8, (S)-9, (S)-8, and (R)-9. The fluorohydrins 8 and 9 were obtained in two consecutive steps starting from cyclic sulfates 7: (i) ring-opening with fluoride and (ii) hydrolysis. However, the most appropriate conditions had to be elucidated. Thus, the cyclic sulfate (R)-7 was converted to the corresponding fluorohydrins (R)-8 and

2.1. Chemistry. The β-fluorohydrin structural feature can help to improve the interaction between the ligand and the target enzyme in most cases. The higher acidity of the hydroxyl group due to the inductive effect of the vicinal fluorine increases its hydrogen bond donor ability.9 This principle might work also for isatin-based caspase inhibitors. Previously, we prepared several N-fluorohydroxyalkyl isatin sulfonamides by ring-opening of epoxides using Olah’s reagent or Et3N·3HF under stirring at room temperature or at 80−90 °C for several hours.12 This strategy was also applied for radiolabeling. 18Flabeling by ring-opening of (S)-N-[4-(oxiran-2-yl)benzyl]-5-[1(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin occurred within 220 min using carrier-added Et3N·3H[18F]F, resulting in the formation of two diastereomeric (S)-N-[4-(1-[18F]fluoro-2hydroxyethyl)benzyl]-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatins in 7% radiochemical yield (d.c.) and expected poor specific radioactivity (95%. Compounds (R)-3 and (S)-3 were prepared according to the literature.22,23 Radiofluorinations were carried out on a modified PET tracer radiosynthesizer (TRACERLab FxFDG, GE Healthcare). The recorded data were processed by the TRACERLab Fx software (GE Healthcare). Separation and purification of the radiosynthesised compounds were performed on the following semipreparative radio-HPLC system A: K500 and K-501 pumps, K-2000 UV detector (Herbert Knauer GmbH), NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec), and a Nucleodur 100-10 column (250 mm × 16 mm). The recorded data were processed by the GINA Star software (Raytest Isotopenmessgeräte GmbH). Radiochemical purities and specific activities were determined using the analytical radio-HPLC system B: two Smartline 1000 pumps and a Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γ-detector (Raytest Isotopenmessgeräte GmbH), and a Nucleosil 100-5 C-18 column (250 mm × 4 mm). Non-carrieradded aqueous [18F]fluoride was produced on an RDS 111e cyclotron (CTI-Siemens) by irradiation of a 1.2 mL water target using 10 MeV proton beams on 97.0% enriched 18O-water by the 18O(p,n)18F nuclear reaction. 4.2. Syntheses. 4.2.1. (R)-Fluorohydrins (R)-8 and (S)-9. 4.2.1.1. (S)-N-[(2′R)-1,2-O-3-Pentylidene-1,2-butanediol]-5-[1-(2(methoxymethyl)pyrrolidinyl)sulfonyl]isatin [(R)-4]. Under argon, a

potentially result in abdominal background radioactivity resulting from metabolites excreted from the gallbladder.

3. CONCLUSION The four diastereo- and enantiopure regioisomeric N-(4-fluoro3-hydroxybutyl)- and N-(3-fluoro-4-hydroxybutyl)-5-[1-(2(S)(methoxymethyl)pyrrolidinyl)sulfonyl]isatins (R)-8, (S)-8, (R)-9, and (S)-9 were fluorinated by ring-opening of the cyclic sulfates (R)-7 and (S)-7 followed by acidic hydrolysis of the intermediate sulfonic esters. The caspase inhibition assay of the fluorohydrins and the corresponding diols demonstrated their in vitro inhibition potencies were on the nanomolar scale against caspases-3 and -7. Replacement of one of the hydroxy groups with a fluorine atom, giving the corresponding fluorohydrins, improved the inhibition potencies of the inhibitors up to 16- and 135-fold for caspases-3 and -7, respectively, compared with those of the lead compound 1. The radiosynthesis of (R)-[18F]8 and (S)-[18F]9 was performed smoothly starting from cyclic sulfate (R)-7 using non-carrieradded [18F]fluoride in the presence of K222, resulting in satisfactory radiochemical yields and high radiochemical purities. The in vivo biodistribution study of the radiotracer (R)-[18F]8 using PET and CT showed fast clearance and elimination of the activity in a C57BL/6 mouse within 10 min. The activity mainly accumulates in the gallbladder and the urinary bladder. In future studies the suitability of (R)-[18F]8 and (S)-[18F]9 for apoptosis imaging will be evaluated in mouse models characterized by dysregulated apoptosis. 4. EXPERIMENTAL SECTION 4.1. Materials and Methods. All chemicals, reagents, and solvents for the reactions were analytical grade and were used as delivered without further purification. Concerning reactions under dry conditions, the glassware was heated under vacuum and flushed with argon gas prior to use. The reactions were performed under an argon atmosphere. To characterize the synthesized compounds, the melting F



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Figure 3. (a) Time−activity curves of radioactivity distribution in a wild-type mouse in vivo. Quantitative analysis of representative tissues/organs shows a fast elimination of (R)-[18F]8 via the liver and the kidneys and accumulation of radioactivity in the urinary bladder and gallbladder. (b) The blood activity decreased significantly within 10 min and reached a stable background level 20−30 min postinjection. No accumulation of (R)-[18F]8 was observed in the lungs, muscle tissue, or any other organ system. CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 182.1 (3-CO), 158.1 (2-CO), 153.9 (8-CN), 137.4 (6-CH), 133.7 (5-CSO2), 124.5 (4CH), 117.4 (9-CCO), 113.5 (OCO), 110.7 (7-CH), 74.9 (16-CH2), 73.6 (21-CH), 69.7 (22-CH2), 59.2 (12-CH), 59.1 (18-CH3), 49.3 (15-CH2), 38.1 (19-CH2), 31.2 (20-CH2), 29.8 (CH2), 29.4 (CH2), 28.8 (13-CH2), 24.1 (14-CH2), 8.2 (CH3), 7.9 (CH3) ppm. HRMS (ESI+, MeOH): m/z = 503.1820 [M + Na]+, 535.2081 [M + Na + MeOH]+; calcd 503.1822 for C23H32N2O7S + Na, 535.2085 for C23H32N2O7S + Na + MeOH. 4.2.1.2. (S)-N-[(3R)-3,4-Dihydroxybutyl]-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin [(R)-5]. A stirred solution of (R)-4 (2.87 g, 5.97 mmol, 1.00 equiv) in DCM (115 mL) was treated with 50% aq TFA (8 mL) slowly. The reaction mixture was stirred vigorously at room temperature for 60 min, monitored by TLC analysis, and then diluted with DCM (450 mL) and H2O (200 mL). The organic phase was washed with satd aq NaHCO3 (1 × 200 mL) and separated. The aq phase was extracted with DCM (3 × 200 mL). The combined organic phase was washed with brine (1 × 200 mL) and dried over MgSO4, and the solvent was removed under reduced pressure. The

solution of (S)-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin (1) (1.09 g, 3.35 mmol, 1.00 equiv) in dry DMF (40 mL) was treated with Cs2CO3 (1.20 g, 3.68 mmol, 1.10 equiv) at rt. After being stirred for 5 min, the mixture was then treated with (R)-4-(tosyloxy)1,2-O-3-pentylidene-1,2-butanediol [(R)-3]22 (3.30 g, 10.0 mmol, 3.00 equiv). The obtained mixture was stirred at this temperature for 24 h. Then EtOAc (150 mL) was added, and the mixture was filtered through Celite to remove Cs2CO3 and salts. The filtrate was concentrated under reduced pressure, and the crude was purified by flash column chromatography (silica gel, EtOAc/cyclohexane, 3:2) to yield a yellow solid (1.53 g, 95%). Mp: 130 °C. 1H NMR (400 MHz, CDCl3): δ = 8.09 (d, 3JH,H = 8.3 Hz, 1H, 6-CH), 8.04 (s, 1H, 4-CH), 7.18 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 4.20−4.01 (m, 3H, 19-CHa, 21CH, 22-CHa), 3.94−3.80 (m, 1H, 19-CHb), 3.81−3.69 (m, 1H, 12CH), 3.59 (dd, 2JH,H = 9.1 Hz, 3JH,H = 3.5 Hz, 1H, 16-CHa), 3.58−3.43 (m, 1H, 22-CHb), 3.49−3.37 (m, 1H, 15-CHa), 3.44−3.30 (m, 1H, 16CHb), 3.37 (s, 3H, 18-CH3), 3.21−3.06 (m, 1H, 15-CHb), 2.10−1.77 (m, 4H, 13-CHa, 14-CHa, 20-CH2), 1.74−1.60 (m, 2H, 13-CHb, 14CHb), 1.69−1.49 (m, 4H, 2 × CH2), 0.84 (t, 3JH,H = 7.2 Hz, 6H, 2 × G



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resulting residue was purified by flash column chromatography (silica gel, acetone/EtOAc, 2:3) to obtain an orange-yellow oil (2.47 g, 100%). 1H NMR (300 MHz, CDCl3): δ = 8.07 (dd, 3JH,H = 8.3 Hz, 4 JH,H = 1.9 Hz, 1H, 6-CH), 8.00 (d, 4JH,H = 1.8 Hz, 1H, 4-CH), 7.22 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 4.15−3.95 (m, 1H, 19-CHa), 3.95−3.81 (m, 1H, 19-CHb), 3.80−3.64 (m, 2H, 12-CH, 21-CH), 3.68−3.58 (m, 1H, 22-CHa), 3.57 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.9 Hz, 1H, 16-CHa), 3.53−3.42 (m, 1H, 22-CHb), 3.49−3.36 (m, 1H, 15-CHa), 3.45−3.30 (m, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3), 3.18−3.05 (m, 1H, 15-CHb), 2.81 (br s, 2H, 21-COH, 22-COH), 2.00−1.80 (m, 2H, 13-CHa, 14CHa), 1.99−1.74 (m, 2H, 20-CH2), 1.76−1.60 (m, 2H, 13-CHb, 14CHb) ppm. 13C NMR (75 MHz, CDCl3): δ = 182.1 (3-CO), 158.5 (2CO), 153.6 (8-CN), 137.6 (6-CH), 133.6 (5-CSO2), 124.4 (4-CH), 117.4 (9-CCO), 111.0 (7-CH), 74.9 (16-CH2), 69.2 (21-CH), 66.5 (22-CH2), 59.2 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 37.5 (19CH2), 30.3 (20-CH2), 28.8 (13-CH2), 24.1 (14-CH2) ppm. HRMS (ESI+, MeOH): m/z = 435.1197 [M + Na]+, 467.1460 [M + Na + MeOH]+; calcd 435.1196 for C18H24N2O7S + Na, 467.1459 for C18H24N2O7S + Na + MeOH. 4.2.1.3. Cyclic Sulfite (R)-6. Under an argon atmosphere a stirred solution of (R)-5 (380 mg, 0.921 mmol, 1.00 equiv) in dry DCM (14 mL) was treated with pyridine (223 μL, 2.76 mmol, 3.00 equiv) at rt. After the mixture was stirred and cooled to 0 °C, SOCl2 (100 μL, 1.38 mmol, 1.50 equiv) was injected slowly. The reaction mixture was stirred at this temperature for 2 h and was filtered through a short column of silica gel which was eluted with EtOAc until no cyclic sulfite could be detected. The filtrate was concentrated under reduced pressure, and the crude product was purified by flash column chromatography (silica gel, EtOAc/DCM, 1:2) to obtain a yellow solid (339 mg, 89%). Mp: 164 °C. 1H NMR (400 MHz, CDCl3): δ = 8.03 (dd, 3JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 7.97 (d, 4JH,H = 1.8 Hz, 1H, 4-CH), 7.15 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 5.08−5.01 (m, 1H, 21CH), 4.80 (dd, 2JH,H = 8.6 Hz, 3JH,H = 6.3 Hz, 1H, 22-CHa), 4.09 (dd, 2 JH,H = 8.6 Hz, 3JH,H = 6.2 Hz, 1H, 22-CHb), 4.07−3.88 (m, 2H, 19CH2), 3.77−3.66 (m, 1H, 12-CH), 3.55 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.9 Hz, 1H, 16-CHa), 3.44−3.35 (m, 1H, 15-CHa), 3.38−3.32 (m, 1H, 16CHb), 3.35 (s, 3H, 18-CH3), 3.16−3.05 (m, 1H, 15-CHb), 2.20 (dtd, 2 JH,H = 14.7 Hz, 3JH,H = 7.4 Hz, 3JH,H = 3.3 Hz, 1H, 20-CHa), 2.12− 1.99 (m, 1H, 20-CHb), 1.96−1.82 (m, 2H, 13-CHa, 14-CHa), 1.73− 1.58 (m, 2H, 13-CHb, 14-CHb) ppm. 13C NMR (100 MHz, CDCl3): δ = 181.7 (3-CO), 158.1 (2-CO), 153.2 (8-CN), 137.5 (6-CH), 133.7 (5-CSO2), 124.5 (4-CH), 117.4 (9-CCO), 110.5 (7-CH), 77.5 (21CH), 74.8 (16-CH2), 71.5 (22-CH2), 59.2 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 37.2 (19-CH2), 30.3 (20-CH2), 28.8 (13-CH2), 24.1 (14-CH2) ppm. HRMS (ESI+, MeOH): m/z = 481.0710 [M + Na]+, 513.0970 [M + Na + MeOH]+; calcd 481.0710 for C18H22N2O8S2 + Na, 513.0972 for C18H22N2O8S2 + Na + MeOH. 4.2.1.4. Cyclic Sulfate (R)-7. A stirred solution of the cyclic sulfite (R)-6 (2.00 g, 4.36 mmol, 1.00 equiv) in MeCN (45 mL) was treated with NaIO4 (1.40 g, 6.54 mmol, 1.50 equiv) at room temperature. The mixture was stirred vigorously before RuCl3·H2O (30 mg, 0.131 mmol, 0.03 equiv) in H2O (18 mL) was added quickly. The reaction mixture was stirred at rt for 30 min. Diethyl ether (150 mL) and H2O (150 mL) were added, and the diethyl ether phase was collected. The aqueous phase was extracted with diethyl ether (3 × 150 mL). The combined organic phase was washed with brine (1 × 150 mL) and dried over MgSO4. The solvent was removed completely under reduced pressure, and the residue was purified by flash column chromatography (silica gel, EtOAc/cyclohexane, 4:1) to obtain a yellow gum (1.74 g, 84%). 1H NMR (400 MHz, CDCl3): δ = 8.08 (dd, 3 JH,H = 8.3 Hz, 4JH,H = 1.8 Hz, 1H, 6-CH), 8.01 (d, 4JH,H = 1.8 Hz, 1H, 4-CH), 7.12 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 5.16−5.02 (m, 1H, 21CH), 4.87 (dd, 2JH,H = 8.9 Hz, 3JH,H = 6.0 Hz, 1H, 22-CHa), 4.45 (dd, 2 JH,H = 9.0 Hz, 3JH,H = 6.4 Hz, 1H, 22-CHb), 4.07−3.90 (m, 2H, 19CH2), 3.77−3.67 (m, 1H, 12-CH), 3.55 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.8 Hz, 1H, 16-CHa), 3.46−3.37 (m, 1H, 15-CHa), 3.40−3.32 (m, 1H, 16CHb), 3.36 (s, 3H, 18-CH3), 3.17−3.07 (m, 1H, 15-CHb), 2.46−2.19 (m, 2H, 20-CH2), 1.97−1.59 (m, 4H, 13-CH2, 14-CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 181.4 (3-CO), 158.1 (2-CO), 152.8 (8-

CN), 137.7 (6-CH), 134.2 (5-CSO2), 124.7 (4-CH), 117.5 (9-CCO), 110.4 (7-CH), 80.0 (21-CH), 74.8 (16-CH2), 72.4 (22-CH2), 59.3 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 36.6 (19-CH2), 30.5 (20CH2), 28.8 (13-CH2), 24.1 (14-CH2) ppm. HRMS (ESI+, MeOH): m/z = 497.0654 [M + Na]+, 529.0916 [M + Na + MeOH]+; calcd 497.0659 for C18H22N2O9S2 + Na, 529.0921 for C18H22N2O9S2 + Na + MeOH. 4.2.1.5. (S)-N-[(3R)-4-Fluoro-3-hydroxybutyl]-5-[1-(2(methoxymethyl)pyrrolidinyl)sulfonyl]isatin and (S)-N-[(3S)-3-Fluoro-4-hydroxybutyl]-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin [(R)-8 and (S)-9]. Under an argon atmosphere, a stirred solution of the cyclic sulfate (R)-7 (400 mg, 0.843 mmol, 1.00 equiv) in dry DMF (4 mL) was treated with CsF (384 mg, 2.53 mmol, 3.00 equiv) at rt. The reaction mixture was stirred further for 1 h followed by complete removal of the solvent under reduced pressure. Subsequently, 20% aq H2SO4 (12 mL) was added to the resulting residue, and it was stirred at 90 °C for 3 h. After this time H2O (80 mL) was added, and the aq phase was extracted with EtOAc (3 × 80 mL). The combined organic phase was washed with satd aq NaHCO3 (1 × 80 mL) and brine (1 × 80 mL) and dried over MgSO4. The solvent was removed under reduced pressure, and the residue was purified by flash column chromatography (silica gel, EtOAc/cyclohexane, 4:1) to obtain primary fluorohydrin (R)-8 (171 mg, 49%) as a sticky yellow solid, secondary fluorohydrin (S)-9 (59 mg, 17%) as a yellow wax, and the corresponding diol 5 (28 mg, 8%) as a yellow oil. The following are data for (R)-8. 1H NMR (400 MHz, CDCl3): δ = 8.06 (dd, 3JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 8.00 (d, 4JH,H = 1.9 Hz, 1H, 4-CH), 7.21 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 4.42 (ddd, 2JH,F = 46.9 Hz, 2JH,H = 9.5 Hz, 3JH,H = 3.5 Hz, 1H, 22-CHa), 4.34 (ddd, 2JH,F = 47.7 Hz, 2JH,H = 9.6 Hz, 3JH,H = 6.1 Hz, 1H, 22-CHb), 4.11−3.87 (m, 2H, 19-CH2), 4.00−3.87 (m, 1H, 21-CH), 3.77−3.69 (m, 1H, 12-CH), 3.57 (dd, 2 JH,H = 9.4 Hz, 3JH,H = 3.9 Hz, 1H, 16-CHa), 3.46−3.38 (m, 1H, 15CHa), 3.39−3.32 (m, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3), 3.17−3.06 (m, 1H, 15-CHb), 2.78 (br s, 1H, 21-COH), 2.01−1.76 (m, 2H, 20CH2), 2.01−1.57 (m, 4H, 13-CH2, 14-CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 182.0 (3-CO), 158.4 (2-CO), 153.6 (8-CN), 137.6 (6-CH), 133.7 (5-CSO2), 124.4 (4-CH), 117.4 (9-CCO), 110.8 (7CH), 86.4 (d, 1JC,F = 170.4 Hz, 22-CH2), 74.8 (16-CH2), 67.5 (d, 2JC,F = 19.7 Hz, 21-CH), 59.2 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 37.3 (19-CH2), 29.5 (d, 3JC,F = 6.2 Hz, 20-CH2), 28.8 (13-CH2), 24.1 (14-CH2) ppm. 19F NMR (282 MHz, CDCl3): δ = −228.5 (s, 1F, 22CH2F) ppm. HRMS (ESI+, MeOH): m/z = 437.1155 [M + Na]+, 469.1419 [M + Na + MeOH]+; calcd 437.1153 for C18H23FN2O6S + Na, 469.1415 for C18H23FN2O6S + Na + MeOH. The following are data for (S)-9. 1H NMR (400 MHz, CDCl3): δ = 8.10 (dd, 3JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 8.05 (d, 4JH,H = 1.7 Hz, 1H, 4-CH), 7.12 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 4.78−4.56 (m, 1H, 21-CH), 4.00− 3.94 (m, 2H, 19-CH2), 3.89−3.67 (m, 2H, 22-CH2), 3.80−3.71 (m, 1H, 12-CH), 3.58 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.8 Hz, 1H, 16-CHa), 3.48−3.38 (m, 1H, 15-CHa), 3.38 (dd, 2JH,H = 9.4 Hz, 3JH,H = 7.6 Hz, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3), 3.18−3.08 (m, 1H, 15-CHb), 2.24−1.90 (m, 2H, 20-CH2), 1.99−1.81 (m, 2H, 13-CHa, 14-CHa), 1.78−1.61 (m, 2H, 13-CHb, 14-CHb) ppm. 13C NMR (100 MHz, CDCl3): δ = 181.8 (3-CO), 157.9 (2-CO), 153.4 (8-CN), 137.6 (6CH), 134.0 (5-CSO2), 124.6 (4-CH), 117.4 (9-CCO), 110.4 (7-CH), 91.7 (d, 1JC,F = 170.0 Hz, 21-CH), 74.8 (16-CH2), 64.4 (d, 2JC,F = 21.9 Hz, 22-CH2), 59.2 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 36.9 (d, 3 JC,F = 4.3 Hz, 19-CH2), 28.9 (d, 2JC,F = 20.7 Hz, 20-CH2), 28.8 (13CH2), 24.1 (14-CH2) ppm. 19F NMR (282 MHz, CDCl3): δ = −192.3 (s, 1F, 21-CHF) ppm. HRMS (ESI+, MeOH): m/z = 437.1160 [M + Na] + , 469.1423 [M + Na + MeOH] +; calcd 437.1153 for C18H23FN2O6S + Na, 469.1415 for C18H23FN2O6S + Na + MeOH. 4.2.2. Fluorohydrins (S)-8 and (R)-9. 4.2.2.1. (S)-N-[(2S)-1,2-O-3Pentylidene-1,2-butanediol]-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin [(S)-4]. (S)-4 was prepared from 1 (1.51 g, 4.66 mmol, 1.00 equiv) and (S)-4-(tosyloxy)-1,2-O-3-pentylidene-1,2-butanediol [(S)-3]23 (4.59 g, 14.0 mmol, 3.00 equiv) according to the procedure described in section 4.2.1.1. The compound was obtained as a yellow solid (1.91 g, 84%). Mp: 145 °C. 1H NMR (400 MHz, CDCl3): δ = 8.08 (dd, 3JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 8.03 (d, 4JH,H = H



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1.8 Hz, 1H, 4-CH), 7.19 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 4.18−4.05 (m, 1H, 21-CH), 4.14−4.04 (m, 1H, 22-CHa), 4.11−3.99 (m, 1H, 19CHa), 3.87 (dt, 2JH,H = 14.1 Hz, 3JH,H = 6.7 Hz, 1H, 19-CHb), 3.78− 3.68 (m, 1H, 12-CH), 3.59 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.8 Hz, 1H, 16CHa), 3.55−3.45 (m, 1H, 22-CHb), 3.48−3.37 (m, 1H, 15-CHa), 3.42−3.31 (m, 1H, 16-CHb), 3.37 (s, 3H, 18-CH3), 3.18−3.04 (m, 1H, 15-CHb), 2.04−1.84 (m, 2H, 20-CH2), 1.96−1.83 (m, 2H, 13-CHa, 14-CHa), 1.72−1.59 (m, 2H, 13-CHb, 14-CHb), 1.59 (q, 3JH,H = 7.5 Hz, 2H, CH2), 1.55 (q, 3JH,H = 7.5 Hz, 2H, CH2), 0.84 (t, 3JH,H = 7.4 Hz, 3H, CH3), 0.83 (t, 3JH,H = 7.4 Hz, 3H, CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 182.1 (3-CO), 158.0 (2-CO), 153.9 (8-CN), 137.3 (6-CH), 133.5 (5-CSO2), 124.4 (4-CH), 117.3 (9-CCO), 113.4 (OCO), 110.7 (7-CH), 74.8 (16-CH2), 73.5 (21-CH), 69.7 (22-CH2), 59.2 (12-CH), 59.1 (18-CH3), 49.3 (15-CH2), 38.0 (19-CH2), 31.2 (20-CH2), 29.8 (CH2), 29.4 (CH2), 28.8 (13-CH2), 24.1 (14-CH2), 8.2 (CH3), 7.9 (CH3) ppm. HRMS (ESI+, MeOH): m/z = 503.1818 [M + Na]+, 535.2081 [M + Na + MeOH]+, 983.3734 [2M + Na]+; calcd 503.1822 for C23H32N2O7S + Na, 535.2090 for C23H32N2O7S + Na + MeOH, 983.3753 for 2(C23H32N2O7S) + Na. 4.2.2.2. (S)-N-[(3S)-3,4-Dihydroxybutyl]-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin [(S)-5]. (S)-5 was prepared from (S)-4 (3.00 g, 6.24 mmol, 1.00 equiv) according to the procedure described in section 4.2.1.2. The crude product was purified by flash column chromatography (silica gel, MeOH/DCM, 1:20) to obtain a yellow wax (2.56 g, 99%). 1H NMR (600 MHz, CDCl3, 328 K): δ = 8.05 (dd, 3 JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 7.98 (d, 4JH,H = 1.8 Hz, 1H, 4-CH), 7.19 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 3.98 (ddd, 2JH,H = 14.7 Hz, 3 JH,H = 8.2 Hz, 3JH,H = 6.7 Hz, 1H, 19-CHa), 3.89 (ddd, 2JH,H = 14.3 Hz, 3JH,H = 7.1 Hz, 3JH,H = 5.2 Hz, 1H, 19-CHb), 3.80−3.74 (m, 1H, 12-CH), 3.77−3.71 (m, 1H, 21-CH), 3.63 (dd, 2JH,H = 11.1 Hz, 3JH,H = 3.5 Hz, 1H, 22-CHa), 3.54 (dd, 2JH,H = 9.6 Hz, 3JH,H = 3.9 Hz, 1H, 16CHa), 3.47 (dd, 2JH,H = 11.1 Hz, 3JH,H = 6.9 Hz, 1H, 22-CHb), 3.41− 3.36 (m, 1H, 15-CHa), 3.37 (dd, 2JH,H = 9.6 Hz, 3JH,H = 7.3 Hz, 1H, 16-CHb), 3.34 (s, 3H, 18-CH3), 3.18 (dt, 2JH,H = 9.7 Hz, 3JH,H = 6.9 Hz, 1H, 15-CHb), 2.64 (br s, 2H, 21-COH, 22-COH), 1.94−1.84 (m, 3H, 13-CHa, 14-CHa, 20-CHa), 1.83−1.75 (m, 1H, 20-CHb), 1.75− 1.65 (m, 2H, 13-CHb, 14-CHb) ppm. 13C NMR (150 MHz, CDCl3, 328 K): δ = 182.1 (3-CO), 158.5 (2-CO), 153.8 (8-CN), 137.6 (6CH), 134.4 (5-CSO2), 124.4 (4-CH), 117.6 (9-CCO), 111.0 (7-CH), 75.0 (16-CH2), 69.4 (21-CH), 66.6 (22-CH2), 59.5 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 37.7 (19-CH2), 30.6 (20-CH2), 29.0 (13CH2), 24.2 (14-CH2) ppm. HRMS (ESI+, MeOH): m/z = 435.1198 [M + Na]+, 467.1457 [M + Na + MeOH]+; calcd 435.1196 for C18H24N2O7S + Na, 467.1459 for C18H24N2O7S + Na + MeOH. Note: The corresponding diol [(S)-5] was obtained as a yellow wax. As DCM or CDCl3 was added, it became a yellow gel-like solution, and it melted at ca. 50−60 °C. NMR spectra at room temperature showed broad peaks without fine structure. Thus, NMR spectra were recorded at 328 K. 4.2.2.3. Cyclic Sulfite (S)-6. (S)-6 was prepared from (S)-5 (2.57 g, 6.23 mmol, 1.00 equiv) and SOCl2 according to the procedure described in section 4.2.1.3. For purification, the solvent was removed completely under reduced pressure and the residue was taken up in EtOAc (150 mL). It was washed with water (3 × 100 mL) and brine (1 × 100 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc/DCM, 1:2) to obtain a yellow foam (1.76 g, 62%). Mp: 138 °C. 1H NMR (300 MHz, CDCl3): δ = 8.04 (dd, 3JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 7.98 (d, 4JH,H = 1.8 Hz, 1H, 4-CH), 7.15 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 5.10−4.98 (m, 1H, 21-CH), 4.80 (dd, 2JH,H = 8.5 Hz, 3JH,H = 6.3 Hz, 1H, 22-CHa), 4.10 (dd, 2JH,H = 8.6 Hz, 3JH,H = 6.2 Hz, 1H, 22-CHb), 4.05−3.86 (m, 2H, 19-CH2), 3.77− 3.65 (m, 1H, 12-CH), 3.55 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.8 Hz, 1H, 16CHa), 3.47−3.33 (m, 1H, 15-CHa), 3.40−3.30 (m, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3), 3.17−3.04 (m, 1H, 15-CHb), 2.20 (dtd, 2JH,H = 14.8 Hz, 3JH,H = 7.5 Hz, 3JH,H = 3.3 Hz, 1H, 20-CHa), 2.13−1.97 (m, 1H, 20-CHb), 1.97−1.78 (m, 2H, 13-CHa, 14-CHa), 1.75−1.58 (m, 2H, 13-CHb, 14-CHb) ppm. 13C NMR (75 MHz, CDCl3): δ = 181.7 (3CO), 158.0 (2-CO), 153.2 (8-CN), 137.6 (6-CH), 133.7 (5-CSO2), 124.5 (4-CH), 117.4 (9-CCO), 110.5 (7-CH), 77.5 (21-CH), 74.8

(16-CH2), 71.5 (22-CH2), 59.2 (12-CH), 59.1 (18-CH3), 49.4 (15CH2), 37.2 (19-CH2), 30.4 (20-CH2), 28.8 (13-CH2), 24.0 (14-CH2) ppm. HRMS (ESI+, MeOH): m/z = 481.0714 [M + Na]+, 513.0966 [M + Na + MeOH]+; calcd 481.0710 for C18H22N2O8S2 + Na, 513.0972 for C18H22N2O8S2 + Na + MeOH. 4.2.2.4. Cyclic Sulfate (S)-7. A stirred solution of the cyclic sulfite (S)-6 (800 mg, 1.74 mmol, 1.00 equiv) in MeCN (19 mL) was treated with NaIO4 (560 mg, 2.62 mmol, 1.50 equiv) followed by quick addition of a solution of RuCl3·H2O (20 mg, 87.2 μmol, 0.05 equiv) in H2O (6.8 mL) at ambient temperature. The reaction mixture was stirred for 30 min, and then H2O (90 mL) was added. The mixture was extracted with EtOAc (3 × 90 mL), and the combined organic phase was dried over MgSO4. After removal of the solvent, the crude product was purified by flash column chromatography (silica gel, 3% MeOH in DCM) to obtain a yellow foam (691 mg, 84%). 1H NMR (400 MHz, DMSO-d6): δ = 8.08 (dd, 3JH,H = 8.4 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 7.79 (d, 4JH,H = 1.9 Hz, 1H, 4-CH), 7.40 (d, 3JH,H = 8.4 Hz, 1H, 7-CH), 4.58 (dd, 2JH,H = 12.1 Hz, 3JH,H = 2.7 Hz, 1H, 22CHa), 4.47−4.40 (m, 1H, 21-CH), 4.36 (dd, 2JH,H = 12.2 Hz, 3JH,H = 5.0 Hz, 1H, 22-CHb), 3.99−3.83 (m, 1H, 12-CH), 3.78−3.60 (m, 2H, 19-CH2), 3.45 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.8 Hz, 1H, 16-CHa), 3.42− 3.21 (m, 2H, 15-CHa, 16-CHb), 3.27 (s, 3H, 18-CH3), 3.15−3.04 (m, 1H, 15-CHb), 2.00−1.64 (m, 4H, 13-CHa, 14-CHa, 20-CH2), 1.61− 1.43 (m, 2H, 13-CHb, 14-CHb) ppm. 13C NMR (100 MHz, DMSOd6): δ = 182.0 (3-CO), 158.3 (2-CO), 153.4 (8-CN), 136.6 (6-CH), 131.3 (5-CSO2), 122.6 (4-CH), 118.1 (9-CCO), 111.2 (7-CH), 77.5 (21-CH), 74.6 (16-CH2), 72.5 (22-CH2), 58.7 (12-CH), 58.5 (18CH3), 49.0 (15-CH2), 36.6 (19-CH2), 28.3 (13-CH2), 28.1 (20-CH2), 23.6 (14-CH2) ppm. HRMS (ESI+, MeOH): m/z = 497.0675 [M + Na] + , 529.0934 [M + Na + MeOH] +; calcd 497.0659 for C18H22N2O9S2 + Na, 529.0921 for C18H22N2O9S2 + Na + MeOH. 4.2.2.5. (S)-N-[(3S)-4-Fluoro-3-hydroxybutyl]-5-[1-(2(methoxymethyl)pyrrolidinyl)sulfonyl]isatin and (S)-N-[(3R)-3-Fluoro-4-hydroxybutyl]-5-[1-(2-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin [(S)-8 and (R)-9]. (S)-8 and (R)-9 were prepared from (S)-7 (401 mg, 0.845 mmol, 1.00 equiv) according to the procedure described in section 4.2.1.5. After the reaction EtOAc (25 mL) was added, and the mixture was neutralized by slow addition of satd aq NaHCO3 until CO2 evolution stopped. After phase separation the aq layer was extracted with EtOAc (3 × 30 mL). The combined organic phase was dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 80% EtOAc in cyclohexane and then 10% MeOH in DCM) to obtain primary fluorohydrin (S)-8 (169 mg, 48%) as a yellow wax, secondary fluorohydrin (R)-9 (68 mg, 19%) as a yellow oil, and when eluted with MeOH/DCM (1:10) the corresponding diol 5 (17 mg, 5%) as a yellow oil. The following are data for (S)-8. 1H NMR (600 MHz, CDCl3, 328 K): δ = 8.06 (dd, 3 JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 8.01 (d, 4JH,H = 1.9 Hz, 1H, 4-CH), 7.17 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 4.40 (ddd, 2JH,F = 47.0 Hz, 2 JH,H = 9.6 Hz, 3JH,H = 3.7 Hz, 1H, 22-CHa), 4.33 (ddd, 2JH,F = 47.0 Hz, 2JH,H = 9.6 Hz, 3JH,H = 6.2 Hz, 1H, 22-CHb), 4.02 (ddd, 2JH,H = 14.8 Hz, 3JH,H = 8.3 Hz, 3JH,H = 6.7 Hz, 1H, 19-CHa), 3.97−3.89 (m, 1H, 21-CH), 3.95−3.89 (m, 1H, 19-CHb), 3.78 (tt, 3JH,H = 7.5 Hz, 3 JH,H = 3.2 Hz, 1H, 12-CH), 3.55 (dd, 2JH,H = 9.6 Hz, 3JH,H = 3.9 Hz, 1H, 16-CHa), 3.41−3.37 (m, 1H, 15-CHa), 3.38 (dd, 2JH,H = 9.6 Hz, 3 JH,H = 7.2 Hz, 1H, 16-CHb), 3.34 (s, 3H, 18-CH3), 3.22−3.15 (m, 1H, 15-CHb), 2.45 (br s, 1H, 21-COH), 1.97−1.87 (m, 2H, 13-CHa, 14CHa), 1.97−1.79 (m, 2H, 20-CH2), 1.76−1.65 (m, 2H, 13-CHb, 14CHb) ppm. 13C NMR (150 MHz, CDCl3, 328 K): δ = 181.9 (3-CO), 158.4 (2-CO), 153.6 (8-CN), 137.5 (6-CH), 134.5 (5-CSO2), 124.5 (4-CH), 117.6 (9-CCO), 110.7 (7-CH), 86.4 (d, 1JC,F = 170.7 Hz, 22CH2), 75.0 (16-CH2), 67.7 (d, 2JC,F = 20.0 Hz, 21-CH), 59.4 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 37.4 (19-CH2), 29.7 (d, 3JC,F = 6.1 Hz, 20-CH2), 29.0 (13-CH2), 24.2 (14-CH2) ppm. 19F NMR (564 MHz, CDCl3, 328 K): δ = −228.9 (s, 1F, 22-CH2F) ppm. HRMS (ESI+, MeOH): m/z = 437.1153 [M + Na]+; 469.1414 [M + Na + MeOH]+; calcd 437.1153 for C18H23FN2O6S + Na, 469.1415 for C18H23FN2O6S + Na + MeOH. The following are data for (R)-9. 1H NMR (400 MHz, CDCl3): δ = 8.08 (dd, 3JH,H = 8.3 Hz, 4JH,H = 1.9 Hz, 1H, 6-CH), 8.03 I



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(d, 4JH,H = 1.8 Hz, 1H, 4-CH), 7.14 (d, 3JH,H = 8.3 Hz, 1H, 7-CH), 4.80−4.55 (m, 1H, 21-CH), 4.01−3.92 (m, 2H, 19-CH2), 3.88−3.67 (m, 2H, 22-CH2), 3.80−3.70 (m, 1H, 12-CH), 3.58 (dd, 2JH,H = 9.4 Hz, 3JH,H = 3.8 Hz, 1H, 16-CHa), 3.47−3.38 (m, 1H, 15-CHa), 3.42− 3.33 (m, 1H, 16-CHb), 3.36 (s, 3H, 18-CH3), 3.17−3.08 (m, 1H, 15CHb), 2.23−1.90 (m, 2H, 20-CH2), 2.01−1.81 (m, 2H, 13-CHa, 14CHa), 1.76−1.60 (m, 2H, 13-CHb, 14-CHb) ppm. 13C NMR (100 MHz, CDCl3): δ = 181.8 (3-CO), 158.0 (2-CO), 153.4 (8-CN), 137.6 (6-CH), 133.9 (5-CSO2), 124.6 (4-CH), 117.4 (9-CCO), 110.5 (7CH), 91.7 (d, 1JC,F = 170.1 Hz, 21-CH), 74.8 (16-CH2), 64.3 (d, 2JC,F = 21.9 Hz, 22-CH2), 59.2 (12-CH), 59.1 (18-CH3), 49.4 (15-CH2), 37.0 (d, 3JC,F = 4.4 Hz, 19-CH2), 28.9 (d, 2JC,F = 20.8 Hz, 20-CH2), 28.8 (13-CH2), 24.1 (14-CH2) ppm. 19F NMR (282 MHz, CDCl3): δ = −192.2 (s, 1F, 21-CHF) ppm. HRMS (ESI+, MeOH): m/z = 437.1148 [M + Na]+, 469.1406 [M + Na + MeOH]+; calcd 437.1153 for C18H23FN2O6S + Na, 469.1415 for C18H23FN2O6S + Na + MeOH. Note: The fluorohydrin (S)-8 was obtained as a yellow wax. As DCM or CDCl3 was added, it became a yellow gel-like solution, and it melted at ca. 50−60 °C. The NMR spectra at rt showed broad peaks without fine structure. Therefore, the NMR spectra were recorded at 328 K. 4.3. In Vitro Enzyme Inhibition. The inhibition potencies against caspases of the target isatin sulfonamides 5, 8, and 9 are indicated as IC50 values. The recombinant human caspases-1, -3, -6, and -7, including their peptide-specific substrates Ac-YVAD-AMC (Ac-TyrVal-Ala-Asp-AMC) for caspase-1, Ac-DEVD-AMC (Ac-Asp-Glu-ValAsp-AMC) for caspases-3 and -7, and Ac-VEID-AMC (Ac-Val-Glu-IleAsp-AMC) for caspase-6, were purchased from Alexis Biochemicals (Switzerland). As already described,10,11 reaction rates showing inhibitory potencies of the inhibitors were assessed by measuring the accumulation of the cleaved fluorogenic product AMC (7-amino-4methylcoumarin) with a Fusion universal microplate analyzer (PerkinElmer) at excitation and emission wavelengths of 360 and 460 nm, respectively. All assays were performed at a volume of 200 μL at 37 °C in reaction buffer.10,11 Buffers contained the target compounds 5, 8, and 9 in DMSO in single doses (end concentration 500 μM, 50 μM, 5 μM, 500 nM, 50 nM, 5 nM, 500 pM, 50 pM, or 5 pM). Recombinant caspases were diluted into the appropriate buffer to a concentration of 0.5 unit per assay (= 500 pmol of substrate conversion after 60 min). After a 10 min incubation time, the peptide substrates (end concentration 10 μM) were added and reacted for a further 10 min. The IC50 values were determined by nonlinear regression analysis using the XMGRACE program (Linux software). 4.4. 18F-Radiolabeling. In a computer-controlled TRACERLab FxFDG synthesizer, the batch of aqueous [18F]fluoride ions (0.24−3.8 GBq) from the cyclotron target was passed through an anion exchange resin (Sep-Pak Light Waters Accell Plus QMA cartridge, preconditioned with 5 mL of 1 M K2CO3 and 10 mL of water for injection). [18F]Fluoride ions were eluted from the resin with a mixture of 46 μL of 1 M K2CO3, 200 μL of water for injection, and 800 μL of DNAgrade MeCN containing 27 mg (72 μmol) of K222 in the reactor. Subsequently, the aqueous K(K222)[18F]F solution was carefully evaporated to dryness under reduced pressure. A 12.7 mg (26.8 μmol) portion of precursor compound (R)-7 in 1000 μL of DNAgrade MeCN was added, and the mixture was heated at 110 °C for 20 min. The reaction mixture was cooled to 55 °C and evaporated to dryness under reduced pressure. Then 1000 μL of 20% aqueous sulfuric acid was added, and the mixture was heated at 110 °C for 20 min. After the mixture was cooled to 40 °C, 9 mL of water was added, and the mixture was passed through a Waters Sep-Pak Light C18 cartridge (preconditioned with 10 mL of EtOH and 10 mL of water). The cartridge was washed with an additional 10 mL of water and eluted with 3 mL of hot DMF. DMF was distilled off under reduced pressure, and 1 mL of DCM was added. The crude product was purified by gradient radio-HPLC system A (λ = 254 nm, flow 8.0 mL/ min, eluent DCM/2-propanol (97:3, v/v)). The product fraction of compound (R)-[18F]8 with tR = 16 min and (S)-[18F]9 with tR = 26 min was evaporated to dryness under reduced pressure and redissolved in 1 mL of water for injection/EtOH (4:1, v/v). Product compound (R)-[18F]8 was obtained in an overall radiochemical yield of 24 ± 3%

(d.c., based on cyclotron-derived [18F]fluoride ions, n = 4) within 142 ± 7 min from the end of radionuclide production with a radiochemical purity of 97 ± 4% with specific activities in the range of 10−127 GBq/ μmol at the end of the synthesis. (S)-[18F]9 was isolated in an overall radiochemical yield of 10 ± 2% (d.c., based on cyclotron-derived [18F]fluoride ions, n = 2) within 160 ± 17 min from the end of radionuclide production with a radiochemical purity of 92% (n = 1). The specific activity of (S)-[18F]9 was not determined. Radiochemical purities of (R)-[18F]8 (tR = 10.0 min) and (S)-[18F]9 (tR = 9.8 min) and specific radioactivities of (R)-[18F]8 were determined by analytical radio-HPLC B (λ = 254 nm, flow 1.0 mL/ min; eluents: (A) MeCN/TFA, 1000:1 (v/v), (B) H2O/TFA, 1000:1 (v/v); time program: eluent A from 10% to 90% in 15 min, from 90% to 10% in 3 min). 4.5. In Vivo Biodistribution Studies. Anesthesia was induced by placing a mouse in an anesthesia induction chamber filled with 3% isoflurane until the animal was completely anesthetized. Then the mouse was transferred to a heated PET scanner bed, isoflurane anesthesia was maintained (1.5−2% in oxygen), and biosignals (respiration, ECG, body temperature) were monitored. A vein catheter (27G diameter) was placed in one of the tail veins, flushed with saline (50 μL), and connected to the injection line. To prevent the eyes from running dry, they were moistened with eye salve. The setup for radiotracer injection consists of (a) the aforementioned tail vein catheter, (b) a 100 μL reservoir filled with the radiotracer (R)[18F]8 (9.8 MBq), and (c) the injection pump holding a syringe filled with saline. For PET imaging (32 module quadHIDAC, Oxford Positron Systems Ltd., Oxford, U.K.), the scanner bed was automatically positioned in the center of the camera field-of-view and PET list mode acquisition was started. Simultaneously the radiotracer reservoir was injected into the mouse by an injection pump controlled saline flush (300 μL/min). After 120 min of PET scanning, the scanning bed was transferred to the CT scanner (Inveon, Siemens Medical Solutions, Malvern, PA), and a CT acquisition with a spatial resolution of 80 μm was performed for each mouse. Reconstructed image data sets were coregistered on the basis of extrinsic markers attached to the multimodal scanning bed and the image analysis software (Inveon Research Workplace 3.0, Siemens Medical Solutions). Three-dimensional volumes of interest (VOI) were defined over the respective organs in CT data sets, transferred to the coregistered PET data, and analyzed quantitatively. Regional uptake was calculated as a percentage of the injected dose by dividing the counts per milliliter in the VOI by the total counts in the mouse multiplied by 100% ID/mL.

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S Supporting Information *

1

H, 13C, and 19F NMR spectra and numeration of the assigned signals of compounds 4−9 as well as radio-HPLC of the 18Flabeled compounds (R)-[18F]8 and (S)-[18F]9. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 251 83 33281. Fax: +49 251 83 39772. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the Deutsche Forschungsgemeinschaft (Collaborative Research Center, SFB 656, Projects B1, A3, C6, and Z5), the International NRW Graduate School of Chemistry, Münster (GSC-MS), the Interdisciplinary Centre for Clinical Research (core unit PIX), Münster, and the Development and Promotion of Science and Technology (DPST) talent project, Thailand, is gratefully acknowledged. J



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We thank Sandra Schröer and Wiebke Gottschlich for enzyme inhibition assays, Daniel Burkert for setting up the radiosynthesizer, and Roman Priebe for executing the in vivo biodistribution studies.

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DEDICATION Dedicated to Professor Dr. Otmar Schober with best wishes on the occasion of his 65th birthday.

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Synthesis, 18F-Radiolabeling, and in Vivo Biodistribution Studies of N

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