Article pubs.acs.org/jmc
Synthesis and Evaluation of Fluorinated Fingolimod (FTY720) Analogues for Sphingosine-1-Phosphate Receptor Molecular Imaging by Positron Emission Tomography Rizwan S. Shaikh,†,‡,◆ Stefanie S. Schilson,†,◆ Stefan Wagner,§ Sven Hermann,∥ Petra Keul,# Bodo Levkau,# Michael Schaf̈ ers,§,∥,⊥ and Günter Haufe*,†,∥,⊥ †
Organisch-Chemisches Institut and International NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany ‡ 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, ⊥Cells-in-Motion Cluster of Excellence, Westfälische Wilhelms-Universität Münster, Waldeyerstraße 15, D-48149 Münster, Germany # Institute of Pathophysiology, Westdeutsches Herz- und Gefäßzentrum, University Hospital Essen, University of Duisburg-Essen, Hufelandstraße 55, D-45122 Essen, Germany S Supporting Information *
ABSTRACT: Sphingosine-1-phosphate (S1P) is a lysophospholipid that evokes a variety of biological responses via stimulation of a set of cognate G-protein coupled receptors (GPCRs): S1P1−S1P5. S1P and its receptors (S1PRs) play important roles in the immune, cardiovascular, and central nervous systems and have also been implicated in carcinogenesis. Recently, the S1P analogue Fingolimod (FTY720) has been approved for the treatment of patients with relapsing multiple sclerosis. This work presents the synthesis of various fluorinated structural analogues of FTY720, their in vitro and in vivo biological testing, and their development and application as [18F]radiotracers for the study of S1PR biodistribution and imaging in mice using small-animal positron emission tomography (PET).
1. INTRODUCTION
linase-catalyzed degradation and converts into ceramide. Deacylation of ceramide results in sphingosine, which is phosphorylated by sphingosine kinases (SphK1 and SphK2) to S1P.2 Both of these enzymes possess broad tissue distribution, with SphK1 being widely expressed in lungs and spleen, and SphK2, in heart, brain, and liver.3 Extracellular S1P acts in autocrine and paracrine manners by engaging a set of five high-affinity S1P receptors (S1P1−S1P5) that belong to the family of G-protein coupled receptors (GPCRs).4 The S1P1, S1P2, and S1P3 receptors are broadly expressed throughout the body, whereas S1P4 is mostly expressed in immune cells, and S1P 5, in natural killer cells (NKs), lymphocytes, and oligodendrocytes.5 S1P receptors are therapeutic targets for the treatment of certain autoimmune diseases such as multiple sclerosis (MS), the most common inflammatory disorder of the central nervous system.6,7 Fingolimod (2) is the representative of its class of compounds approved for the treatment of relapsing multiple
Sphingosine-1-phosphate (S1P) 1 (Figure 1) is a zwitterionic lysophospholipid synthesized as a part of the sphingomyelin cycle in yeasts, plants, insects, and animals. S1P plays a vital role in cell growth, programmed cell death, angiogenesis, immunity, and the cardiovascular (CVS) and central nervous systems (CNS).1 In living cells, sphingomyelin undergoes sphingomye-
Figure 1. Sphingosine-1-phosphate (S1P, 1) and phosphate of Fingolimod (FTY720, 2). © 2015 American Chemical Society
Received: December 31, 2014 Published: March 31, 2015 3471
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
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Figure 2. Structures of BZM055 (3), W146 (4), and the radiofluorinated analogue of W146 [18F]5.
Scheme 1. Synthesis of 2, Its ω-Fluorinated Analogue 17, and Its Deoxyfluoro Analogue 18a
a Reagents: (i) bromoacetyl bromide, AlCl3, DCM, rt, overnight; (ii) diethylacetamidomalonate, Cs2CO3, MeCN, reflux, 4 h; (iii) PBSF, Et3N·3HF, EtiPr2N, DCM, rt, 2 days; (iv) Et3SiH, TiCl4, DCM, rt, overnight for 10, or Et3SiH, cat. PdCl2, EtOH, rt, overnight for 12; (v) LiCl, NaBH4, THF/ EtOH, rt, 3 days for 13, or LiBH4 solution in THF, rt, overnight for 14; (vi) 1 M NaOH, MeOH, reflux, 5 h; (vii) DAST, DCM, −78 °C → rt, overnight.
there.16,17 The presence of iodine resulted in a significantly increased lipophilicity and decreased rate of phosphorylation as compared to that of 2. In blood and brain, compound 3 showed similar properties as those of 2. These favorable properties encouraged moving forward with 3 as a SPECT tracer for S1PR imaging in brain. Unfortunately, the much lower penetration into brain made 3 a less suitable tracer for in vivo imaging studies.18 In 2014, we reported [18F]5 as a radiolabeled structural analogue of (R)-{3-amino-4-[(3-hexylphenyl)amino]-4-oxobutyl}phosphonic acid (W146, 4) for PET imaging studies of S1PRs (Figure 2).19 W146 was originally synthesized by Sanna20 as a chiral S1P1 receptor antagonist that led to vascular leakage and pulmonary edema after application in vivo.21 The nonradioactive isotopomer of 5 had equal in vitro efficacy as that of parent compound 4. Although it was stable for hours in serum ex vivo, PET images showed fast defluorination and accumulation of free [18F]fluoride in bone.19 The in vivo defluorination of [18F]5 prevented its application as a tracer for PET imaging of S1PRs. Here, we report the synthesis of different structural analogues of 2 as [18F]-labeled potential radiotracers for molecular imaging of S1PRs.
sclerosis by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in 2010 and 2011, respectively. Compound 2 was discovered as a structural analogue of myriocin and has led to great interest in S1P signaling and physiology.8,9 After phosphorylation of 2 by SphK2, the formed Fingolimod-phosphate engages four out of the five S1P receptors (all except S1P2)13 and, most importantly, acts as a functional antagonist to S1P1,10 thereby sequestering lymphocytes in secondary lymphoid organs and leading to a profound reduction of T- and B-cells in peripheral blood.11,12 The efficacy of 2 in animal models and humans results from its prevention of lymphocyte infiltration into sites of autoimmunity, transplanted organs, and the central nervous system.8,14 Studying organ distribution of 2 and pharmacokinetics using an imaging tracer based on 2 in vivo would thus be an extremely valuable tool. During the past 2 decades, noninvasive molecular imaging techniques have been developed for application in biomedical studies and particularly for applications in the early detection of various diseases such as cancer, cardiovascular diseases, and CNS related disorders.15 These techniques include positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). The high sensitivity of SPECT and PET have attracted researchers to develop molecular imaging agents.15 There have been no reports on S1PR imaging until the 123Iradiolabeled SPECT tracer BZM055 (3), an agonist of S1PRs, was described in 2011. This structural analogue of 2 was used to image the myelin sheath, as 2 has been shown to accumulate
2. RESULTS AND DISCUSSION Compound 2 possesses a simple structural scaffold consisting of a 2-amino-1,3-diol polar headgroup, which is phosphorylated in vivo by SphK, with the 1,4-disubstituted phenyl ring acting as a rigid linker group, and an eight-carbon chain length aliphatic tail for interaction with the hydrophobic binding pocket of 3472
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
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Scheme 2. Synthesis of Structural analogues of FTY720 Phosphatea
a
Reagents: (i) LiCl, NaBH4, THF/EtOH rt, overnight; (ii) 1 M NaOH, MeOH, reflux, 6 h.
Scheme 3. Synthesis of ω-Fluorinated Analogues of FTY720a
Reagents: (i) (a) (Boc)2O, DMF, 25 °C, 2 h, (b) DMP, PTSA, DMF, 25 °C, 12 h; (ii) (COCl)2, DMSO, TEA, DCM, −78 °C → rt, 1 h; (iii) 26, K2CO3, toluene, 120 °C, 12 h; (iv) hex-5-yn-1-ol or oct-7-yn-1-ol, Pd/C, TPP, CuI, TEA, MeCN, microwave, 80 °C, 90 min; (v) H2, Pd/C, EtOAc, rt, 2 h; (vi) PBSF, Et3N·3HF, EtiPr2N, THF, rt, 2 days for 30 and DAST, DCM, −78 °C → rt, 8 h for 31; (vii) TFA, DCM, rt, 12 h; (viii) triethyl orthoacetate, EtiPr2N, DMF, 120 °C, 2 h; (ix) (a) TBDPA, 0.45 M tetrazole, 30% H2O2, rt, 4 h, (b) HCl/ethanol, 1:10, 50 °C, 6 h. a
S1PRs. After the synthetic discovery of 2 by Fujita22 and development of its immunomodulatory activity,23 a number of other compounds have been synthesized.24−30 2.1. Chemistry. Syntheses of 2 and some structural analogues including radiofluorinated analogues are depicted in Schemes 1−4. The routes for the synthesis of FTY720 (2) and the fluorinated derivative 18, shown in Scheme 1, are analogous to those described by Durand et al.24 Intermediates 8 and 9 were obtained by Friedel−Crafts acylation with bromoacetyl bromide of 6 and 7 followed by nucleophilic substitution of the bromide with diethylacetamidomalonate in the presence of cesium carbonate to afford 10 and 11. Alcohol 11, in turn, was treated with perfluoro-1-butanesulfonyl fluoride (PBSF) in the presence of triethylamine tris-hydrogenfluoride and N,Ndiisopropylethylamine (DIPEA) in DCM to yield the desired fluoride 12.31 Compound 10 was converted to 13 using triethylsilane (Et3SiH) and titanium tetrachloride in DCM.24 This protocol was not successful for 12. In the presence of TiCl4, the terminal fluoride was replaced by chloride. Therefore, 12 was converted to 14 using Et3SiH and palladium(II)-chloride in DCM;32 however, this proceeded with low yield. Compounds 15 and 16 were synthesized by reduction of the corresponding diester 13 with sodium
borohydride and lithium chloride in THF/EtOH or with LiBH4 in THF in the case of 14. Hydrolysis of the amide moiety of compounds 15 and 16 was accomplished with aqueous sodium hydroxide to give 2 and its fluorinated structural analogue 17. Fluorination of diol 2 with DAST in DCM gave the known 1833 in low yield.34 Scheme 2 presents the synthesis of the 4-hydroxy analogue and the ω-fluorinated 4-hydroxy analogue of 2. Compounds 19 and 20 were synthesized by reduction of 10 and 12 using sodium borohydride and lithium chloride in THF/EtOH. Further amide hydrolysis of 19 and 20 with aqueous sodium hydroxide gave 21 and 22. Synthesis of ω-fluorinated exact and shorter-chain analogues of FTY720 starting from commercial triethanolamine (23) is shown in Scheme 3. One-pot protection of 23 using Bocanhydride and 2,2-dimethoxy propane in the presence of a catalytic amount of p-toluenesulfonic acid gave 24, which was further oxidized to aldehyde 25 using Swern oxidation.35 Wittig reaction of 25 with intermediate 26 and potassium carbonate as base in toluene gave a 3:1 mixture of the Z/E-isomeric iodides 27. Sonogashira coupling of 27 with hex-5-yne-1-ol and oct-7yne-1-ol under microwave irradiation led to 28 and 29, respectively.36 Hydrogenation over palladium on carbon in 3473
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
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Scheme 4. Synthetic Scheme for Radiofluorinationa
Reagents: (i) TsCl, TEA, DCM, 0 °C → rt, overnight; (ii) (a) [18F]F−, K222, K2CO3, CH3CN, 84 °C, 10−12 min; (b) 10:1, TFA/H2O, 40 °C, 5 min.
a
Table 1. Lymphopenia Induced in Vivo by Different Compounds as a Readout for S1P1 Activitya compound control 2 (FTY720) 17 34 36
WBC
CD4+
CD8+
B cells
activity
(106/mL)
(106/mL)
(106/mL)
(106/mL)
(%)
± ± ± ± ±
97 95 59 89
8.94 3.69 3.04 4.91 3.93
± ± ± ± ±
0.59 0.63b 0.42b 0.53b 0.39b
0.73 0.02 0.04 0.30 0.08
± ± ± ± ±
0.07 0.002b 0.01b 0.06b 0.01b
0.48 0.04 0.03 0.25 0.14
± ± ± ± ±
0.05 0.01b 0.004b 0.07b 0.03b
4.72 0.61 1.03 2.23 1.05
0.53 0.12b 0.24b 0.30b 0.19b
Data are presented as mean ± SEM from at least three independent experiments per compound. Control, untreated mice. Activity was calculated as percent reduction of peripheral blood CD4+ cell number by each compound. bp < 0.05 compared to control.
a
region at different wavelengths, with the result that concentrations below 20 μg/mL cannot be detected with the UV detector. These missing UV signals were observed for the final solutions of the preparations of [18F]34 and [18F]17, respectively. Therefore, the AS of the [18F]34 and [18F]17 preparations had to be estimated. Under the assumption that the concentration of carrier and radiolabeled compound in the final solution of the radiosyntheses is 20 μg/mL (detection limit) or lower, the AS of the preparations of [18F]34 and [18F]17 is calculated to be ≥0.9 Gbq/μmol. Actually, for similar [18F]-radiolabeling procedures with comparable starting activities, we achieved AS in the range of 10−127 GBq/μmol, which is also a realistic range for the syntheses of [18F]34 and [18F]17.20,38 2.3. Biological Studies. 2.3.1. Biological Activity in Vivo and in Vitro. The synthesized compounds were tested for biological activity toward S1P1 in vivo by measuring their ability to induce peripheral blood lymphopenia as a characteristic of successful downregulation of the S1P1 receptor (Table 1). Compounds 17 and 36 showed excellent in vivo activity, comparable to that of FTY720 (2), as evidenced by the ∼20fold reduction of T-cell counts (Table 1). In contrast, compound 34 was only moderately effective (∼2-fold reduction in lymphocytes). Compounds 18, 21, and 22 were not tested because of insolubility in the desired formulation. The similar effectiveness of 17 and its phosphorylated form 36 is not surprising given the high efficiency of endogenous phosphorylation known for FTY720 that, apparently, also applies to its ω-fluorinated analogue 17. Interestingly, compound 34, which features two fewer carbons in its aliphatic chain than that of 17, has substantially impaired activity in vivo. Subsequently, both the biological activity and the S1P receptor subtype specificity of compound 36 were tested in vitro using Chinese hamster ovary (CHO) cells stably overexpressing the human S1P1 and S1P3 receptors, respectively. The functional readout was phosphorylation of the p44/ 42 mitogen activated kinases (MAPK) after stimulation with compound 36 in direct comparison with that after stimulation by S1P (1). In both CHO-S1P1 and CHO-S1P3, compound 36 showed a robust time-dependent phosphorylation of MAPK,
ethyl acetate at room temperature provided intermediates 30 and 31. The OH functions were converted to fluoride either by reaction with PBSF and Et3N·3HF31 or with DAST in DCM34 to achieve 32 and 33. Then, acid-catalyzed deprotection of Boc and acetonide led to the ω-fluorinated structural analogues of FTY720, 34 and 17. Aminodiol 17 was reacted with triethyl orthoacetate and DIPEA in DMF to afford intermediate 35.37 Finally, 35 was reacted with di-tert-butyl N,N-diethylphosphoramidite (TBDPA) in the presence of tetrazole and hydrogen peroxide in DCM, and the formed phosphate was then hydrolyzed with concentrated hydrochloric acid in ethanol to give the final compound 36.18,37 2.2. Radiochemistry. For the syntheses of [18F]-radiolabeled structural analogues of FTY720, precursors 37 and 38 were synthesized from 30 and 31 by tosylation with tosyl chloride and triethylamine as base in DCM (Scheme 4). Radiosyntheses of [18F]34 and [18F]17 were performed over two steps. In the first step, [18F]32 and [18F]33 were prepared by heating precursors 37 and 38 in acetonitrile with anhydrous potassium [18F]fluoride in the presence of Kryptofix 222 (K222) at 84 °C for 10 min. Without isolation and purification, the obtained intermediates were directly hydrolyzed with TFA/ H2O (10:1) at 40 °C for 5 min to yield [18F]34 and [18F]17, respectively. These products were obtained in radiochemical yields (rcy) of 29.2 ± 3.3% ([18F]34) and 30.2 ± 4.2% ([18F]17) (decay-corrected based on cyclotron-derived [18F]fluoride ions) in 115 ± 12 min ([18F]34) and 115 ± 16 min ([18F]17) from the end of radionuclide production. The target compounds were isolated in ≥99% radiochemical purity after HPLC. A standard procedure to determine specific activities (AS) of radiolabeled compounds is to generate an UV calibration curve of the nonradioactive isotopomere with analytical radio-HPLC, e.g., with concentrations in a range of 1−50 μg/mL. Then, from the UV signal of the radiolabeled compound and carrier (the nonradioactive counterpart) in the HPLC quality control (QC) of the radiochemical preparations, the concentration can be calculated. Finally, from the concentration, the AS can be determined. In this case, the nonradioactive counterparts 34 and 17 showed high detection limits (20 μg/mL) in the UV 3474
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
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Figure 3. Western blots showing phosphorylation of p44/42 MAP kinases after stimulation of CHO cells overexpressing S1P1 or S1P3 with 1 μM 2amino-4-[4-(8-fluorooctyl)phenyl]-2-(hydroxymethyl)butyldihydrogen phosphate (36) and 1 μM S1P.
comparable to that of S1P, indicating its ability to interact with and activate the S1P1 and S1P3 receptors (Figure 3). 2.3.2. In Vitro Stability. The serum stability of radioligand [18F]17 was evaluated (Figure 4) by incubation in human serum at 37 °C for 90 min. Significant radiometabolites or decomposition products could not be observed.
Figure 4. In vitro stability of [18F]17 (tR = 11.15 min) after incubation in human blood serum at 37 °C for 10 min (top), 60 min (middle), and 90 min (bottom) measured using analytical HPLC system B, method B.
2.3.3. In Vivo Biodistribution/PET Imaging. Motivated by the in vivo immunosuppressive activity of compounds 17 and 34, 18F-labeled structural analogues (Scheme 4) [18F]34 and [18F]17 were selected for biodistribution experiments in wildtype mice. Whole-body imaging was performed in C57BL/6 wild-type mice (n = 4, male, 13 weeks old) over a 90 min period after intravenous injection of [18F]34 and [18F]17 (400 KBq/g bodyweight in 100 μL of saline). The in vivo imaging studies showed similar tracer kinetics and biodistribution for both [18F]34 (Figure 5a) and [18F]17 (Figure 5b). On the basis of quantitative PET time−activity curves, a rapid clearance from blood within 3 min postinjection (p.i.) followed by a nearly constant blood activity concentration of about ∼4% ID/ mL until the end of the study at 90 min p.i. was confirmed ([18F]34, Figure 6a,b; [18F]17, Figure 6c,d). The decrease in
Figure 5. In vivo PET/CT studies in healthy C57BL/6 mice of (a) [18F]34 and (b) [18F]17 from 0 to 90 min postinjection. PET (color scale) revealed a fast uptake and elimination of the tracer via the liver (L) and the kidneys (K). In addition, tracer uptake was observed in the myocardium (M) and the spleen (not displayed on selected slides).
blood concentration coincides with an intense uptake of both tracers into the liver, with a maximum of 25% ID/mL at about 10 min p.i. and a less pronounced increase of tracer signal in the kidneys up to 20% ID/mL, reflecting the routes of tracer elimination. Furthermore, both [18F]34 and [18F]17 were found to accumulate in lungs and the myocardium. After the 3475
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
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Figure 6. (a) In vivo biodistribution of radioactivity in C57BL/6 WT mice after intravenous injection of [18F]34 (a, b) and [18F]17 (c, d). Time− activity curves illustrate tracer dynamics in selected regions of interests (ROI) for tissues involved in tracer elimination (a, c) and S1P relevant structures or reference tissues (b, d).
initial peak in the lungs early after injection, [18F]34’s activity was decreased to 5% ID/mL at 20 min p.i. with a slight washout to 4% ID/mL until the end of the study. [18F]17’s kinetics in the lungs showed similar characteristics as those of [18F]34, with activity concentrations of 6.4% ID/mL at 20 min p.i. and 4.8% ID/mL at the end of the study. The myocardium presented a biphasic time−activity curve, with an initial perfusion peak and a smaller second maximum at 10 min p.i. and activities of 8% ID/mL for [18F]34 and 8.6% ID/mL for [18F]17. To evaluate organs typically hosting S1P receptor positive immune cells, we analyzed the activity concentration in the spleen and inguinal lymph nodes. In the spleen, an accumulation of [18F]34 and [18F]17 was observed with a maximum of 7% ID/mL at 10 min p.i. and 7.8% ID/mL at 30 min p.i., respectively. In vivo defluorination of the radioligands (bone uptake of [18F]fluoride ions, as observed in our earlier study with an 18Fanalogue of W146, a S1P1 selective antagonist19) was not observed in these imaging studies. In summary, biodistribution studies based on PET revealed rapid clearance of radiotracers [18F]34 and [18F]17 from the blood in wild-type mice within the first 10 min after injection. Besides the predominantly hepatic elimination, an increased uptake/retention of both [18F]34 and [18F]17 was observed in S1P receptor positive tissues such as the lungs and the myocardium as well as in the lymphocyte-containing spleen in healthy mice.
3. CONCLUSIONS Six fluorinated structural analogues of FTY720 (2) were synthesized and tested for in vitro and in vivo activity. The goal was to elaborate their application for PET imaging studies in vivo after introducing 18F. The ω-fluorinated analogue 17 and its phosphorylated form 36 showed excellent biological activity in vivo, suggesting that incorporation of a fluorine atom at the end of the aliphatic chain, which is expected to increase lipophilicity within the hydrophobic receptor pocket, preserves interaction with the S1P receptor. Interestingly, shortening of the chain length from eight to six carbons, as in 34, impaired the activity. Moreover, we have synthesized two [18F]radiolabeled structural analogues of FTY720, [18F]34 and [18F]17, in good radiochemical yields of 29 to 30% in 115 min and >99% radiochemical purity. The in vivo biodistribution studies of [18F]34 and [18F]17 using PET and CT showed rapid clearance of the activity from the blood in C57BL/6 wildtype mice within the first 10 min after injection mainly via the liver. We observed a slightly increased uptake in S1P receptor relevant tissues, namely, the lungs (mainly S1P1) and myocardium (mainly S1P3), as well as in the lymphocytecontaining spleen (mainly S1P4) in the healthy mouse. No accumulation in bones and other organs was observed. Although first imaging results showed positive uptake in S1PR-rich tissues, the uptake, specificity (predosing, blocking studies, S1PR knockout and overexpression models, etc.), and metabolism of [18F]34 and [18F]17 have to be tested and further optimized. This should finally result in a molecular 3476
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
Journal of Medicinal Chemistry
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2), 31.2, 31.1 (C-10, C-15), 29.5 (C-11), 29.4 (C-12), 29.2 (C-13), 25.8 (C-14). HRMS (ESI+): C16H23BrO2 + Na+: calcd, 349.0774; found, 349.0775; (C16H23BrO2)2 + Na+: calcd, 677.1637; found, 677.1624. Diethyl 2-Acetamido-2-[2-(4-octylphenyl)-2-oxoethyl]malonate (10). Intermediate 8 (8.90 g, 19.7 mmol, 1.3 equiv), diethyl 2acetamidomalonate (3.21 g, 14.8 mmol), and cesium carbonate (5.15 g, 15.8 mmol, 1.1 equiv) were suspended in acetonitrile (125 mL) and stirred under reflux for 4 h. After cooling to rt, the reaction mixture was adsorbed on silica gel and purified by column chromatography (10.5 × 8 cm, cyclohexane/ethyl acetate, 4:1 → 2:1) to give the product as a pale yellow oil. Yield: 5.87 g (89%). 1H NMR (400 MHz, CDCl3): δ 7.88 (m, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 7.26 (m, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 7.15 (s, 1H, 2-NH), 4.27 (q, 3JH,H = 7.1 Hz, 4H, 22-CH2, 24-CH2), 4.25 (s, 2H, 4-CH2), 2.66 (m, 2H, 12CH2), 1.97 (s, 3H, 21-CH3), 1.61 (m, 2H, 13-CH2), 1.26−1.33 (m, 1 H, 14-CH2 to 18-CH2), 1.24 (t, 3JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH3), 0.87 (t, 3JH,H = 6.7 Hz, 3H, 19-CH3). 13C NMR (101 MHz, CDCl3): δ 196.5 (C-5), 169.4 (C-20), 167.4 (C-1, C-3), 149.6 (C-6), 133.8 (C9), 128.7 (C-7, C-11), 128.4 (C-8, C-10), 64.0 (C-2), 62.8 (C-22, C24), 42.2 (C-4), 36.0 (C-12), 31.9 (C-17), 31.1 (C-14, C-15), 29.4 (C13), 29.2 (C-16), 22.9 (C-21), 22.7 (C-18), 14.1 (C-19), 13.9 (C-23, C-25). HRMS (ESI+): C25H37NO6 + H+: calcd, 448.2694; found, 448.2689; C25H37NO6 + Na+: calcd, 470.2513; found, 470.2505; (C25H37NO6)2 + Na+: calcd, 917.5134; found, 917.5132. Diethyl 2-Acetamido-2-{2-[4-(8-hydroxyoctyl)phenyl]-2oxoethyl}malonate (11). Synthesis and purification were performed as described for compound 10. Yield: 221 mg (48%). 1H NMR (300 MHz, CDCl3): δ 7.88 (d, 3JH,H = 8.3 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 7.26 (d, 3JH,H = 8.5 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 7.15 (s, 1H, 2-NH), 4.22−4.32 (m, 6H, 4-CH2, 22-CH2, 24-CH2), 3.63 (t, 3JH,H = 6.6 Hz, 2H, 19-CH2), 2.65 (t, 3JH,H = 7.7 Hz, 2H, 12CH2), 1.97 (s, 3H, 21-CH3), 1.51−1.67 (m, 4H, CH2, 13-CH2, 18CH2), 1.28−1.36 (m, 8H, 14-CH2 to 17-CH2), 1.24 (t, 3JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH3). 13C NMR (75 MHz, CDCl3): δ 196.6 (C-5), 169.6 (C-20), 167.4 (C-1, C-3), 149.7 (C-6), 133.9 (C-9), 128.8 (C-7, C-11), 128.5 (C-8, C-10), 64.1 (C-2), 63.1, 63.0 (C-19, C-22, C-24), 42.3 (C-4), 36.1 (C-12), 32.8 (C-18), 31.1 (C-15), 29.5 (C-14), 29.4 (C-13), 29.2 (C-16), 25.8 (C-17), 23.1 (C-21), 14.0 (C-23, C-25). HRMS (ESI+): C25H37NO7 + H+: calcd, 464.2643; found, 464.2639; C25H37NO7 + Na+: calcd, 486.2462; found, 486.2447. Diethyl 2-Acetamido-2-{2-[4-(8-fluorooctyl)phenyl]-2-oxoethyl}malonate (12). Intermediate 11 (166 mg, 0.35 mmol) was dissolved in dry THF (3 mL) in a PTFE vessel. Perfluoro-1-butanesulfonyl fluoride (PBSF, 0.19 mL, 1.06 mmol, 3.0 equiv), triethyl amine trishydrofluoride (0.18 mL, 1.10 mmol, 3.1 equiv), and N,Ndiisopropyl ethyl amine (0.56 mL, 3.24 mmol, 9.0 equiv) were added, and the mixture was stirred at rt for 2 days. The reaction was quenched by addition of saturated sodium bicarbonate solution (5 mL), and the aqueous phase was extracted with DCM (3 × 15 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (16.5 × 3 cm, cyclohexane/ethyl acetate, 2:1) to give the product as a colorless oil. Yield: 132 mg (80%). 1H NMR (300 MHz, CDCl3): δ 7.88 (d, 3JH,H = 8.3 Hz, 2H, 7-CH, 11-CH or 8CH, 10-CH), 7.27 (d, 3JH,H = 8.2 Hz, 2H, 7-CH, 11-CH or 8-CH, 10CH), 7.13 (br s, 1H, 2-NH), 4.43 (dt, 3JH,H = 6.1 Hz, 2JH,F = 47.4 Hz, 2H, 19-CH2), 4.27 (q, 3JH,H = 7.2 Hz, 4H, 22-CH2, 24-CH2), 4.25 (s, 2H, 4-CH2), 2.66 (t, 3JH,H = 7.7 Hz, 2H, 12-CH2), 1.98 (s, 3H, 21CH3), 1.59−1.79 (m, 4H, 13-CH2, 18-CH2), 1.30−1.41 (m, 8H, 14CH2 to 17-CH2), 1.25 (t, 3JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH3). 13C NMR (75 MHz, CDCl3): δ 196.6 (C-5), 169.6 (C-20), 167.5 (C-1, C3), 149.6 (C-6), 133.9 (C-9), 128.9 (C-7, C-11), 128.5 (C-8, C-10), 84.3 (d, 1JC,F = 164.1 Hz, C-19), 64.1 (C-2), 63.0 (C-22, C-24), 42.3 (C-4), 36.1 (C-12), 31.2 (C-13), 30.5 (d, 2JC,F = 19.4 Hz, C-18), 29.4 (C-14, C-16), 29.2 (C-15), 25.3 (d, 3JC,F = 5.5 Hz, C-17), 23.1 (C-21), 14.0 (C-23, C-25). 19F NMR (282 MHz, CDCl3): δ −218.6 (tt, 3JH,F = 25.0 Hz, 2JH,F = 47.4 Hz, 1F). HRMS (ESI+): C25H36FNO6 + H+: calcd, 466.2599; found, 466.2605; C25H36FNO6 + Na+: calcd, 488.2419; found, 488.2422.
imaging tracer with potential applications in cardiovascular, neuroinflammatory, and immune system diseases.
4. EXPERIMENTAL SECTION 4.1. Materials and Methods. All substrates, reagents, and solvents for the reactions were of analytical grade and were used without further purification. Concerning reactions under dry conditions, the glassware was heated under vacuum and flushed with argon before use. The reactions were performed under an argon atmosphere. Reactions using microwave irradiation were performed in closed glass vessels using a CEM Discover microwave. To characterize the synthesized compounds, the melting point (if the products were solid at room temperature), NMR, and ESI-MS data were determined. 1 H NMR (300, 400, and 600 MHz), 13C NMR (75, 100, and 150 MHz), and 19F NMR (282 and 564 MHz) spectra were recorded on Bruker spectrometers in CDCl3 or MeOD for 13C NMR with TMS for 1 H NMR, CDCl3 or MeOD, and CFCl3 for 19F NMR as the internal standards. DEPT and two-dimensional NMR techniques (COSY, HSQC, and HMBC) were recorded to assign the signals of some complex structures. All chemical shifts are indicated in the δ scale in parts per million. The numeration of the assigned nuclei is depicted in the Supporting Information. Exact mass analyses were recorded with a Bruker MicroTOF apparatus. All spectroscopic and analytical investigations were performed by staff members of the Organic Chemistry Institute, University of Münster. Thin-layer chromatography (TLC) analyses were performed on silica-coated aluminum foil (silica gel 60 F254) with a 0.02 mm layer thickness. Silica gel (60−120 mesh) was used for column chromatography. The purity of all compounds tested in vitro or in vivo was proved by HPLC to be >95%. A Knauer HPLC system (RP-HPLC Nucleodur 100−10 C18ec column, 250 × 16 mm) was used for the purification of some intermediates. 4.2. Synthesis. 4-Octyl-α-bromoacetophenone (8). Aluminum chloride (4.80 g, 32.0 mmol, 1.0 equiv) and 1-phenyloctane (6.84 g, 32.0 mmol) were dissolved in dry DCM (10 mL) and cooled to −10 °C. A solution of bromoacetyl bromide (3.14 mL, 32.0 mmol, 1.0 equiv) in DCM (12 mL) was added dropwise, and the mixture was allowed to return to rt while stirring overnight. The mixture was slowly poured into ice water (100 mL), and the aqueous phase was extracted with DCM (3 × 50 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The product was obtained as a brown, highly viscous oil and was used in the next reaction without purification. Yield: 8.90 g (62%). 1H NMR (300 MHz, CDCl3): δ 7.90 (m, 2 H, 5-CH, 7-CH), 7.29 (m, 2 H, 4-CH, 8CH), 4.44 (s, 2 H, 2-CH2), 2.67 (m, 2 H, 9-CH2), 1.63 (m, 2 H, 10CH2), 1.22−1.35 (m, 10 H, 11-CH2 to 15-CH2), 0.88 (m, 3 H, 16CH3). 13C NMR (75 MHz, CDCl3): δ 191.1 (C-1), 150.1 (C-3), 131.7 (C-6), 129.2 (C-4, C-8), 129.0 (C-5, C-7), 36.2 (C-9), 32.0 (C-2), 31.2 (C-10), 31.1 (C-14), 29.5 (C-12), 29.4 (C-13), 29.3 (C-11), 22.8 (C-15), 14.2 (C-16). 8-Hydroxyoctyl-α-bromoacetophenone (9). Aluminum chloride (200 mg, 1.50 mmol, 3.0 equiv) was suspended in dry DCM (8 mL) and cooled to 0 °C. 8-Phenyl-1-octanol (0.11 mL, 0.50 mmol) and αbromoacetyl bromide (53 μL, 0.60 mmol, 1.2 equiv) were added dropwise. After 20 min, the mixture was warmed to rt and stirred overnight. The reaction was stopped by pouring the solution into a mixture of ice water (25 mL) and concentrated hydrochloric acid (10 mL). The phases were separated, and the aqueous layer was extracted with DCM (2 × 10 mL). The combined organic phases were washed with saturated sodium bicarbonate solution (1 × 10 mL) and brine (1 × 10 mL) and dried over MgSO4. The solvent was evaporated, and the residue was purified by column chromatography (12 × 4 cm, cyclohexane/ethyl acetate, 8:1) to give the product as a colorless oil. Yield: 120 mg (73%). 1H NMR (300 MHz, CDCl3): δ 7.90 (d, 3JH,H = 8.3 Hz, 2H, 4-CH/8-CH or 5-CH/7-CH), 7.29 (d, 3JH,H = 8.4 Hz, 2H, 4-CH/8-CH or 5-CH/7-CH), 4.45 (s, 2H, 2-CH2), 3.63 (t, 3JH,H = 6.6 Hz, 2H, 16-CH2), 2.67 (t, 3JH,H = 7.7 Hz, 2H, 9-CH2), 1.50−1.66 (m, 4H, 10-CH2, 15-CH2), 1.29−1.39 (m, 8H, 11-CH2 to 14-CH2). 13C NMR (75 MHz, CDCl3): δ 191.1 (C-1), 150.0 (C-3), 131.7 (C-6), 129.2 (C-4, C-8), 129.0 (C-5, C-7), 63.0 (C-16), 36.1 (C-9), 32.8 (C3477
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
Journal of Medicinal Chemistry
Article
Diethyl 2-Acetamido-2-(4-octylphenethyl)malonate (13). To a solution of triethyl silane (3.0 mL, 27.1 mmol, 5.4 equiv) in dry DCM (25 mL) was added a solution of intermediate 10 (2.24 g, 5.0 mmol) in dry DCM (6 mL) dropwise, followed by the addition of titanium tetrachloride (2.0 mL, 19.1 mmol, 2.6 equiv). The reaction mixture was stirred at rt overnight and was then slowly poured into ice water (150 mL). After phase separation, the aqueous layer was extracted with DCM (2 × 70 mL). The organic phases were collected, dried over MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (14 × 4 cm, cyclohexane/ethyl acetate, 4:1) to give the product as a white solid. Yield: 1.87 g, (86%). mp 55−57 °C. 1H NMR (300 MHz, CDCl3): δ 7.06 (m, 4H, 7-CH, 8-CH, 10CH, 11-CH), 6.79 (s, 1H, 2-NH), 4.19 (m, 4H, 22-CH2, 24-CH2), 2.68 (m, 2H, 5-CH2), 2.55 (m, 2H, 12-CH2), 2.45 (m, 2H, 4-CH2), 1.97 (s, 3H, 21-CH3), 1.56 (m, 2H, 13-CH2), 1.23−1.33 (m, 10H, 14CH2 to 18-CH2), 1.24 (t, 3JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH3), 0.86 (m, 3H, 19-CH3). 13C NMR (75 MHz, CDCl3): δ 169.0 (C-20), 168.1 (C-1, C-3), 140.7 (C-6), 137.7 (C-9), 128.4 (C-7, C-11), 128,3 (C-8, C-10), 66.4 (C-2), 62.5 (C-22, C-24), 35.5 (C-12), 33.4 (C-17), 31.9 (C-5), 31.6 (C-4), 29.8 (C-15), 29.7 (C-14), 29.5 (C-13), 29.3(C-16), 23.0 (C-21), 22.7 (C-18), 14.1 (C-19), 14.0 (C-23, C-25). HRMS (ESI+): C25H39NO5 + H+: calcd, 434.2901; found, 434.2904; C25H39NO5 + Na+: calcd, 456.2720; found, 456.2725; (C25H39NO5)2 + Na+: calcd, 889.5549; found, 889.5541. Diethyl 2-Acetamido-2-[4-(8-fluorooctyl)phenethyl]malonate (14). A solution of ketone 12 (63 mg, 136 μmol) in ethanol (4 mL) was treated with triethyl silane (89 μL, 544 μmol, 4.0 equiv) and a catalytic amount of palladium chloride under an argon atmosphere. The reaction mixture was stirred at rt overnight and was subsequently heated at 98 °C for 3 h until completion of the reaction. After cooling to rt, water (3 mL) was added, and the mixture was extracted with DCM (5 × 6 mL). The organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (16 × 3 cm, cyclohexane/ethyl acetate, 2:1) to give the product as a colorless oil. Yield: 35 mg (57%). 1H NMR (300 MHz, CDCl3): δ 7.04−7.09 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 6.76 (s, 1H, 2-NH), 4.43 (dt, 3JH,H = 6.2 Hz, 2JH,F = 47.4 Hz, 2H, 19CH2), 4.20 (m, 4H, 22-CH2, 24-CH2), 2.55 (m, 2H, 12-CH2), 2.41− 2.72 (m, 4H, 4-CH2, 5-CH2), 1.51−1.75 (m, 4H, 13-CH2, 18-CH2), 1.98 (s, 3H, 21-CH3), 1.29−1.35 (m, 8H, 14-CH2 to 17-CH2), 1.24 (t, 3 JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH2). 13C NMR (75 MHz, CDCl3): δ 169.1 (C-20), 168.2 (C-1, C-3), 140.7 (C-6), 137.8 (C-9), 128.5 (C-7, C-11), 128.4 (C-8, C-10), 84.3 (d, 1JC,F = 164.0 Hz, C-19), 66.5 (C-2), 62.7 (C-22, C-24), 35.6 (C-12), 33.5 (C-4), 31.7 (C-5), 30.5 (d, 2JC,F = 19.5 Hz, C-18), 29.8 (C-14, C-15), 29.5 (C-13), 29.3 (C-16), 25.3 (d, 3JC,F = 5.5 Hz, C-17), 23.1 (C-21), 14.1 (C-23, C-25). 19F NMR (282 MHz, CDCl3): δ −218.5 (tt, 3JH,F = 24.9 Hz, 2JH,F = 47.4 Hz, 1F, 19-CH2F). Exact mass (ESI+): C25H38FNO5 + Na+: calcd, 474.2626; found, 474.2621; (C25H38FNO5)2 + Na+: calcd, 925.5360; found, 925.5354. N-[1-Hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl]acetamide (15). To a solution of diester 13 (1.27 g, 2.93 mmol) in THF (30 mL) were added lithium chloride (636 mg, 15.0 mmol, 5.1 equiv) and sodium borohydride (567 mg, 15.0 mmol, 5.1 equiv). The mixture was cooled to 0 °C and treated with ethanol (60 mL). After 30 min at 0 °C, the mixture was allowed to warm to rt and was stirred for 3 days. The mixture was adjusted to pH 4 with 10% citric acid at 0 °C. THF was removed in vacuo, and the residue was extracted with DCM (4 × 30 mL). The combined organic phases were washed with brine (1 × 80 mL), dried over Na2SO4, and concentrated under reduced pressure. The product was purified by column chromatography (10 × 4 cm, cyclohexane/ethyl acetate, 1:2) to give the product as a white solid. Yield: 974 mg (95%). mp 87−88 °C. 1H NMR (400 MHz, CDCl3): δ 7.09 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 6.23 (s, 1H, 2NH), 4.61 (brs, 2H, 1-OH, 3-OH), 3.80 (d, 2JH,H = 11.5 Hz, 2H, 1CH, 3-CH), 3.61 (d, 2JH,H = 11.5 Hz, 2H, 1-CH, 3-CH), 2.52−2.60 (m, 4H, 5-CH2, 12-CH2), 1.96 (m, 2H, 4-CH2), 1.93 (s, 3H, 21-CH3), 1.56 (m, 2H, 13-CH2), 1.20−1.33 (m, 10H, 14-CH2 to 18-CH2), 0.87 (m, 3 H, 19-CH3). 13C NMR (101 MHz, CDCl3): δ 172.0 (C-20), 140.7 (C-6), 138.7 (C-9), 128.6 (C-7, C-11), 128.2 (C-8, C-10), 14.1
(q, C-19), 65.5 (C-1, C-3), 61.4 (C-2), 35.5 (C-12), 34.3 (C-17), 31.9 (C-5), 31.6 (C-4), 29.5 (C-15), 29.4 (C-14), 29.3 (C-13), 29.2 (C16), 23.9 (C-21), 22.7 (C-18). HMRS (ESI+): C21H35NO3 + H+: calcd, 350.2690; found, 350.2693; C21H35NO3 + Na+: calcd, 372.2509; found, 372.2512; (C21H35NO3)2 + Na+: calcd, 721.5126; found, 721.5127. N-{4-[4-(8-Fluorooctyl)phenyl]-1-hydroxy-2-(hydroxymethyl)butan-2-yl}acetamide (16). Diester 14 (48 mg, 106 μmol) was dissolved in THF (3 mL) and cooled to 0 °C. Lithium borohydride (4 M solution in THF, 0.11 mL, 0.44 mmol, 4.2 equiv) was added to the reaction mixture followed by ethanol (6 mL). The reaction mixture was warmed to rt after 30 min and stirred overnight. The reaction was diluted with 20% potassium sodium tartrate solution (4 mL), and the aqueous phase was extracted with DCM (4 × 6 mL). The combined organic layers were dried over Na2SO4, and the solvent was removed under reduced pressure. Column chromatography (8 × 3 cm, DCM/ methanol, 20:1) gave the desired product as an oil. Yield: 13 mg (33%). 1H NMR (400 MHz, CD3OD, CDCl3): δ 7.02−7.18 (m, 4 H, 7-CH, 8-CH, 10-CH, 11-CH), 5.35 (s, 1H, 2-NH), 4.44 (dt, 3JH,H = 6.2 Hz, 2JH,F = 47.4 Hz, 2H, 19-CH2), 3.76 (d, 2JH,H = 11.5 Hz, 2H, 1CH, 3-CH), 3.64 (d, 2JH,H = 11.5 Hz, 2H, 1-CH, 3-CH), 2.51−2.68 (m, 6H, 4-CH2, 5-CH2, 12-CH2), 1.99 (s, 3H, 21-CH3), 1.54−1.76 (m, 4H, 13-CH2, 18-CH2), 1.26−1.44 (m, 8H, 14-CH2 to 17-CH2). 13C NMR (101 MHz, CD3OD, CDCl3): δ 172.4 (C-20), 140.2 (C-6), 138.9 (C-9), 128.0 (C-7, C-11), 128.2 (C-8, C-10), 84.1 (d, 1JC,F = 163.5 Hz, C-19), 64.4 (C-1, C-3), 61.2 (C-2), 35.3 (C-12), 33.7 (C-4), 31.3 (C-5), 30.2 (d, 2JC,F = 19.3 Hz, C-18), 29.2 (C-14, C-15), 29.0 (C-13, C-16), 24.9 (d, 3JC,F = 5.4 Hz, C-17), 23.0 (q, C-21). 19F NMR (282 MHz, CDCl3): δ −218.4 (tt, 3JH,F = 25.0 Hz, 2JH,F = 47.4 Hz, 1F, 19-CH2F). HRMS (ESI+): C21H34FNO3 + Na+: calcd, 390.2415; found, 390.2415. 2-Amino-2-(4-octylphenethyl)propane-1,3-diol (FTY720) (2). Protected aminodiol 15 (349 mg, 1.0 mmol) was dissolved in methanol (20 mL) and treated with 1 M sodium hydroxide solution (1.2 mL, 1.2 mmol, 1.2 equiv). The reaction mixture was refluxed for 5 h. After cooling to rt, the mixture was diluted with 1 M sodium hydroxide solution (15 mL), and the aqueous phase was extracted with DCM (5 × 20 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The product was crystallized from ethyl acetate to give a white solid. Yield: 252 mg (82%). mp 127 °C. 1H NMR (300 MHz, CD3OD, CDCl3): δ 7.09 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 3.54 (d, 2JH,H = 11.0 Hz, 2H, 1-CH2, 3-CH2), 3.48 (d, 2JH,H = 10.9 Hz, 2H, 1-CH2, 3-CH2), 2.51−2.65 (m, 4H, 5-CH2, 12-CH2), 1.69 (m, 2H, 4-CH2), 1.57 (m, 2H, 13-CH2), 1.22−1.37 (m, 10H, 14-CH2 to 18CH2), 0.87 (m, 3H, 19-CH3). 13C NMR (75 MHz, CD3OD, CDCl3): δ 140.9 (C-6), 140.1 (C-9), 129.0 (C-7, C-11), 128.7 (C-8, C-10), 66.2 (C-1, C-3), 56.4 (C-2), 37.0 (C-12), 36.1 (C-17), 32.5 (C-4), 32.2 (C-5), 30.1 (C-13), 29.9 (C-15), 29.5 (C-14, C-16), 23.2 (C-18), 14.3 (C-19). HMRS (ESI+): C19H33NO2 + H+: calcd, 308.2584; found, 308.2585; C19H33NO2 + Na+: calcd, 330.2404; found, 330.2408; (C19H33NO2)2 + Na+: calcd, 637.4915; found, 637.4917. 2-Amino-2-[4-(8-fluorooctyl)phenethyl]propane-1,3-diol (17). Synthesis and purification were performed as described for compound 2. Yield: 5 mg (44%). 1H NMR (300 MHz, MeOD): δ 7.14 (m, 4H, 7CH to 10-CH), 4.41 (dt, 2JH,F = 47.6 Hz, 3JH,H = 6.1 Hz, 2H, 19-CH2), 3.70 (s, 4H, 3-CH2, 4-CH2), 2.62 (m, 4H, 5-CH2, 12-CH2), 1.94 (m, 2H, 1-CH2), 1.66 (m, 4H, 13-CH2, 18-CH2), 1.36 (m, 8H, 14-CH2 to 17-CH2). 13C NMR (75 MHz, MeOD): δ 141.9 (C-6), 139.5 (C-11), 129.6 (C-7, C-8), 129.1 (C-9, C-10), 82.8 (d, 1JC,F = 163.6 Hz, C-19), 62.6 (C-3, C-4), 61.9 (C-2), 36.4 (C-12), 34.8 (C-5), 32.7 (C-13), 31.5 (d, 2JC,F = 19.6 Hz, C-18), 30.5 (C-16), 30.3 (C-15), 30.2 (C-1), 29.6 (C-14), 26.3 (d, 3JC,F = 5.3 Hz, C-17). 19F NMR (282 MHz, MeOD): δ −220.0 (tt, 2JH,F = 47.4 Hz, 3JH,F = 24.9 Hz, 19-F). HRMS (ESI+): C19H32NO2F + Na+: calcd, 348.2315; found, 348.2310; C19H32NO2F + H+: calcd, 326.2495; found, 326.2490. 2-Amino-2-(fluoromethyl)-4-(4-octylphenyl)butan-1-ol (18). Compound 2 (310 mg, 1.00 mmol) was suspended in dry DCM (10 mL) and cooled to −78 °C. Diethylaminosulfur trifluoride (DAST, 0.13 mL, 1.00 mmol, 1.0 equiv) was slowly added to this suspension, and the mixture was allowed to warm to rt and stirred 3478
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
Journal of Medicinal Chemistry
Article
rt. The aqueous phase was extracted with DCM (4 × 30 mL), and the organic layers were dried over Na2SO4. The solvent was evaporated, and the product was purified by column chromatography (DCM/ methanol, 4:1) to give the product as colorless, waxy solid. Yield: 503 mg (78%). mp 74−76 °C. 1H NMR (400 MHz, CDCl3): δ 7.15 (d, 3 JH,H = 7.8 Hz, 2H, 7-CH/11-CH or 8-CH/10-CH), 7.04 (d, 3JH,H = 7.7 Hz, 2H, 7-CH/11-CH or 8-CH/10-CH), 4.83 (d, 3JH,H = 10.3 Hz, 1H, 5-CH), 4.03 (br s, 5H, 1-OH, 3-OH, 5-OH, 2-NH2), 3.28−3.58 (m, 4H, 1-CH2, 3-CH2), 2.50 (m, 2H, 12-CH2), 1.47−1.70 (m, 4H, 4CH2, 13-CH2), 1.16−1.34 (m, 10H, 14-CH2 to 18-CH2), 0.87 (t, 3JH,H = 6.7 Hz, 3H, 19-CH3). 13C NMR (101 MHz, CDCl3): δ 142.3 (C-6), 142.1 (C-9), 128.5 (C-7, C-11), 125.6 (C-8, C-10), 70.4 (C-5), 67.2 (C-1), 65.8 (C-3), 56.5 (C-2), 43.8 (C-4), 35.8 (C-12), 32.0 (C-16), 31.7 (C-15), 29.6 (C-13, C-17), 29.5 (C-14), 22.8 (C-18), 14.2 (C19). HRMS (ESI+): C19H33NO3 + H+: calcd, 324.2533; found, 324.2550; C19H33NO3 + Na+: calcd, 346.2353; found, 346.2355. 3-Amino-1-[4-(8-fluorooctyl)phenyl]-3-(hydroxymethyl)butane1,4-diol (22). Amide 20 (20 mg, 0.05 mmol) was dissolved in methanol (3 mL), treated with 1 M sodium hydroxide solution (0.09 mL, 0.09 mmol, 1.7 equiv), and heated at 120 °C for 6 h in a pressure vessel. The mixture was allowed to cool to rt and stirred overnight. Then the reaction mixture was diluted with 1 M sodium hydroxide solution (5 mL), and the aqueous phase was extracted with DCM (5 × 8 mL). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by gradient HPLC (RP-HPLC Nucleodur 100−10 C18ec column (250 × 16 mm), acetonitrile/water (0.1% TFA)) with a Knauer HPLC system. Afterward, the obtained TFA salt was dissolved in methanol (1 mL) and 1 M sodium hydroxide solution (3 mL). The mixture was extracted with DCM (5 × 5 mL), and the organic phases were dried over Na2SO4 and concentrated. The product was dried in high vacuum and obtained as a highly viscous oil. Yield: 12 mg (68%). 1H NMR (300 MHz, CDCl3): δ 7.23 (d, 3JH,H = 7.8 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 7.11 (d, 3 JH,H = 7.7 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 4.94 (d, 3JH,H = 10.2 Hz, 1H, 5-CH), 4.43 (dt, 3JH,H = 6.2 Hz, 2JH,F = 47.4 Hz, 2H, 19CH2), 3.42−3.79 (m, 4H, 1-CH2, 3-CH2), 2.55 (t, 3JH,H = 7.8 Hz, 2H, 12-CH2), 1.49−1.82, 1.21−1.46 (m, 14H, 4-CH2, 13-CH2 to 18-CH2). 13 C NMR (101 MHz, CDCl3): δ 142.3 (C-6), 142.2 (C-9), 128.6 (C7, C-11), 125.6 (C-8, C-10), 84.4 (d, 1JC,F = 163.9 Hz, C-19), 70.7 (C5), 68.1(C-1), 66.4 (C-3), 56.6 (C-2), 43.8 (C-4), 35.7 (C-12), 30.5 (d, 2JC,F = 19.4 Hz, C-18), 31.6 (C-13), 29.8 (C-16), 29.5 (C-14), 29.3 (C-15), 25.3 (d, 3JC,F = 5.6 Hz, C-17). 19F NMR (282 MHz, CDCl3): δ −218.5 (tt, 3JH,F = 24.9 Hz, 2JH,F = 47.4 Hz, 1F, 19-CH2F). HRMS (ESI+): C19H32FNO3 + H+: calcd, 342.2439; found, 342.2437; C19H32FNO3 + Na+: calcd, 364.2258; found, 364.2255. tert-Butyl [5-(Hydroxymethyl)-2,2-dimethyl-1,3-dioxane-5-yl]carbamate (24).34 Compound 23 (7 g, 57 mmol, 1 equiv) was suspended in DMF (100 mL), and Boc anhydride (13.87 g, 63 mmol, 1.1 equiv) was added. The reaction mixture was stirred at 25 °C for 2 h. Then, 2,2-dimethoxypropane (8.51 mL, 69 mmol, 1.2 equiv) and ptoluenesulfonic acid (0.55 g, 2.9 mmol, 0.05 equiv) were added, and the resulting mixture was stirred at 25 °C for 12 h. The reaction was quenched by addition of diethyl ether (100 mL). The organic phase was washed with saturated sodium carbonate solution (1 × 50 mL) and brine (1 × 50 mL). The separated organic layer was dried over MgSO4, and solvent was removed under reduced pressure. The product was obtained as a white solid and used without further purification. Yield: 13.0 g (87%). mp 100 °C. 1H NMR (400 MHz, CDCl3): δ 5.33 (s, 1H, NH), 4.33 (s, 1H, OH), 3.83 (q, 3JH,H = 11.7 Hz, 4H, 3-CH2, 4-CH2), 3.73 (d, 3JH,H = 6.5 Hz, 2H, 1-CH2), 1.46 (m, 15H, 5-CH3, 6-CH3, 8-CH3, 9-CH3, 10-CH3). 13C NMR (101 MHz, CDCl3): δ 156.4 (C-11), 98.7 (C-7), 80.4 (C-12), 64.6 (C-1), 64.3 (C3, C-4), 53.2 (C-2), 28.3 (C-8, C-9, C-10), 26.7 (C-6), 20.2 (C-5). HRMS (ESI+): C12H23NO5 + Na+: calcd, 284.1468; found,: 284.1468. tert-Butyl (5-Formyl-2.2-dimethyl-1,3-dioxan-5-yl)carbamate (25).34 A solution of oxalyl chloride (1.31 mL, 15.3 mmol, 2 equiv) in DCM (25 mL) was cooled to −78 °C, treated with a solution of DMSO (1.63 mL, 22.9 mmol, 1.1 equiv) in DCM (10 mL), and allowed to stir for 15 min. After that, a solution of 24 (2 g, 7.66 mmol) in DCM (20 mL) was added slowly dropwise, and stirring was
overnight. The reaction was neutralized with saturated sodium bicarbonate solution (30 mL) at −10 °C. The phases were separated, and the aqueous phase was extracted with DCM (3 × 20 mL). The combined organic layers were dried over MgSO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (20 × 3 cm, DCM/methanol, 4:1) and subsequently by gradient HPLC (RP-HPLC Nucleodur 100−10 C18ec column (250 × 16 mm), acetonitrile/water (0.1% TFA)). The obtained TFA salt was dissolved in methanol (1 mL) and 1 M sodium hydroxide solution (3 mL), and the mixture was extracted with DCM (5 × 5 mL). The organic phases were dried over Na2SO4 and concentrated. The product was dried in high vacuum and obtained as a highly viscous oil. Yield: 5 mg (5%). 1H NMR (300 MHz, CDCl3): δ 7.01−7.11 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 4.38 (d, 2JH,F = 47.3 Hz, 2H, 3-CH2), 3.52 (m, 2H, 1-CH2), 2.47−2.68 (m, 4H, 5-CH2, 12-CH2), 1.81 (m, 2H, 4-CH2), 1.56 (m, 2H, 13-CH2), 1.16−1.37 (m, 10H, 14-CH2 to 18-CH2), 0.87 (t, 3JH,H = 6.7 Hz, 3H, 19-CH3). 13C NMR (75 MHz, CDCl3): δ 140.9 (C-6), 138.7 (C-9), 128.7 (C-7, C-11), 128.2 (C-8, C-10), 86.1 (d, 1JC,F = 173.3 Hz, C-3), 65.2 (d, 3JC,F = 4.1 Hz, C-1), 56.1 (d, 2JC,F = 17.2 Hz, C-2), 35.7 (t, C-12), 34.8 (C-5), 32.0 (C-4), 31.7, 29.6 (C-15), 29.5 (C-16), 29.4 (C-13, C17), 29.3 (C-14), 23.5 (C-18), 14.2 (C-19). 19F NMR (282 MHz, CDCl3): δ −230.3 (t, 2JH,F = 47.3 Hz, 1F, 3-CH2F). HRMS (ESI+): C19H32FNO + H+: calcd,310.2541; found, 310.2542; C 19 H32 FNO + Na +: calcd, 332.2360; found, 332.2379. N-[1,4-Dihydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2yl]acetamide (19). Lithium chloride (424 mg, 10.0 mmol, 5.0 equiv) and sodium borohydride (378 mg, 10.0 mmol, 5.0 equiv) were added to a solution of diester 10 (930 mg, 2.08 mmol) in THF (8 mL). The mixture was cooled to 0 °C and treated with ethanol (16 mL). After 45 min, the reaction was warmed to rt and stirred overnight. The reaction mixture was adjusted to pH 4 with 10% citric acid at 0 °C, and THF was removed in vacuo. The residue was extracted with DCM (4 × 10 mL). The organic layers were washed with brine (1 × 15 mL), dried over Na2SO4, and evaporated. The product was purified by column chromatography (20 × 3 cm, DCM/methanol, 20:1) to give the product as a white solid. Yield: 586 mg (77%). mp 123 °C. 1H NMR (300 MHz, CDCl3): δ 7.26 (d, 3JH,H = 8.1 Hz, 2H, 7-CH/11-CH or 8CH/10-CH), 7.13 (d, 3JH,H = 8.1 Hz, 2H, 7-CH/11-CH or 8-CH/10CH), 4.85 (m, 1H, 5-CH), 3.59−3.91 (m, 4H, 1-CH2, 3-CH2), 2.58 (m, 2H, 12-CH2), 2.00 (m, 2H, 4-CH2), 2.00 (s, 3H, 21-CH3), 1.59 (m, 2H, 13-CH2), 1.21−1.36 (m, 10H, 14-CH2 to 18-CH2), 0.87 (m, 3H, 19-CH3). 13C NMR (101 MHz, CD3OD, CDCl3): δ 172.3 (C20), 141.9 (C-6), 141.8 (C-9), 128.1 (C-7, C-11), 125.2 (C-8, C-10), 69.7 (C-5), 65.0 (C-1), 63.7 (C-3), 61.1 (C-2), 41.2 (C-4), 35.3 (C12), 31.6 (C-16), 31.3 (C-15), 29.2 (C-13, C-17), 29.0 (C-14), 23.0 (C-21), 22.4 (C-18), 13.7 (C-19). HRMS (ESI+): C21H35NO4 + Na+: calcd, 388.2458; found, 388.2463; (C21H35NO4)2 + Na+: calcd, 753.5024; found, 753.5032. N-{4-[4-(8-Fluorooctyl)phenyl]-1,4-dihydroxy-2-(hydroxymethyl)butan-2-yl}-acetamide (20). Synthesis and purification were performed as described for compound 19. Yield: 21 mg (48%). 1H NMR (300 MHz, CDCl3): δ 7.09−7.27 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 5.30 (1 H, 2-NH), 4.86 (d, 3JH,H = 10.5 Hz, 1H, 5-CH), 4.43 (dt, 3JH,H = 6.2 Hz, 2JH,F = 47.4 Hz, 2H, 19-CH2), 3.42−3.80 (m, 4H, 1-CH2, 3-CH2), 2.58 (m, 2H, 12-CH2), 2.31 (m, 2H, 4-CH2), 2.02 (s, 3H, 21-CH3), 1.53−1.85 and 1.24−1.43 (m, 12H, 13-CH2 to 18CH2). 13C NMR (75 MHz, CDCl3): δ 172.0 (C-20), 142.9 (C-6), 141.6 (C-9), 128.8 (C-7, C-11), 125.6 (C-8, C-10), 84.4 (d, 1JC,F = 163.9 Hz, C-19), 71.2 (C-5), 66.4 (C-1), 65.1 (C-3), 61.5 (C-2), 41.4 (C-4), 35.7 (C-12), 30.5 (d, 2JC,F = 19.3 Hz, C-18), 31.6 (C-16), 29.5 (C-13), 29.3 (C-14, C-15), 25.3 (d, 3JC,F = 5.4 Hz, C-17), 24.0 (C-21). 19 F NMR (282 MHz, CDCl3): δ −218.5 (tt, 3JH,F = 25.0 Hz, 2JH,F = 47.4 Hz, 1F, 19-CH2F). HRMS (ESI+): C21H34FNO4 + Na+: calcd, 406.2364; found, 406.2358. 3-Amino-3-(hydroxymethyl)-1-(4-octylphenyl)butane-1,4-diol (21). Amide 19 (1.37 g, 2.00 mmol) was dissolved in methanol (30 mL) and treated with 1 M sodium hydroxide solution (2.4 mL, 2.40 mmol, 1.2 equiv). The reaction mixture was refluxed for 6 h and then diluted with 1 M sodium hydroxide solution (20 mL) after cooling to 3479
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
Journal of Medicinal Chemistry
Article
Hz, 3JH,H = 0.2 Hz, 2H, 28-CH2), 2.42 (t, 3JH,H = 6.9 Hz, 2H, 23-CH2), 1.66−1.57 (m, 4H, 24-CH2, 27-CH2), 1.50−1.42 (m, 4H, 25-CH2, 26CH2), 1.38 (s, 12H, 5-CH3, 8-CH3, 9-CH3, 10-CH3), 1.32 (s, 3H, 6CH3). 13C NMR (75 MHz, CDCl3): δ 154.4 (C-11), 136.7 (C-14), 131.6 (C-15), 131.0 (C-18, C-19), 129.8 (C-1), 128.5 (C-16, C-17), 126.2 (C-20), 98.1 (C-7), 90.6 (C-21), 87.3 (C-22), 80.4 (C-12), 65.8 (C-28), 62.9 (C-3, C-4), 52.4 (C-2), 32.6 (C-27), 28.6 (single peak with high intensity, C-24, C-25), 28.34 (C-8, C-9), 28.38 (C-10), 25.2 (single peak with high intensity, C-5, C-6), 19.3 (C-26), 18.8 (C-23). Minor E-Isomer. 1H NMR (300 MHz, CDCl3): δ 7.32 (dd, 3JH,H = 8.3 Hz, 1.9 Hz, 2H, 18-CH, 19-CH), 7.18 (d, 3JH,H = 8.1 Hz, 2H, 16CH, 17-CH), 6.49 (d, 3JH,H = 16.4 Hz, 1H, 1-CH), 6.20 (d, 3JH,H = 16.4 Hz, 1H, 14-CH), 5.18 (s, 1H, NH), 3.93−3.83 (m, 2H, 3-CH, 4CH), 3.73 (d, 2JH,H = 12.2 Hz, 4JH,H = 1.4 Hz, 2H, 3-CH, 4-CH), 3.66 (td, 3JH,H = 6.6 Hz, 3JH,H = 0.2 Hz, 2H, 28-CH2), 2.42 (t, 3JH,H = 6.9 Hz, 2H, 23-CH2), 1.66−1.57 (m, 4H, 24-CH2, 27-CH2), 1.50−1.42 (m, 4H, 25-CH2, 26-CH2), 1.38 (s, 12H, 5-CH3, 8-CH3, 9-CH3, 10CH3), 1.32 (s, 3H, 6-CH3). 13C NMR (75 MHz, CDCl3): δ 154.8 (C11), 136.7 (C-14), 135.6 (C-20), 132.6 (C-15), 131.8 (single peak, C18, C-19), 129.8 (C-1), 122.7 (single peak, C-16, C-17), 98.2 (C-7), 91.0 (C-21), 87.3 (C-22), 79.4 (C-12), 65.8 (C-28), 62.9 (C-3, C-4), 52.9 (C-2), 32.6 (C-27), 28.6 (C-25), 28.38 (C-10), 28.34 (C-8, C-9), 27.8 (C-24), 25.2 (single peak with high intensity, C-5, C-6), 19.4 (C26), 19.2 (C-23). HRMS (ESI+): C27H39NO5+Na+: calcd, 480.2726; found, 480.2720. tert-Butyl {5-[4-(6-Hydroxyhexyl)phenethyl]-2,2-dimethyl-1,3-dioxan-5-yl}carbamate (30). Ultrapure hydrogen gas was generated with a Nitrox UHP-40H hydrogen generator (Domnick Hunter, England). Alkyne 28 (310 mg, 0.65 mmol) was dissolved in ethyl acetate (20 mL) in a pressure vessel, to which Pd/C (31 mg, 10 mol %) was added. Then, the flask was flushed with hydrogen (2 atm pressure), and the reaction mixture was stirred vigorously at rt for 2 h. The reaction was stopped by releasing hydrogen gas, and the mixture was filtered over Celite. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (4.5 × 3 cm, cyclohexane/ethyl acetate, 4:1) to give the product as a white solid. Yield: 240 mg (76%). mp 57 °C. 1H NMR (300 MHz, CDCl3): δ 7.12−7.04 (m, 4H, 16-CH, 17-CH, 18-CH, 19-CH), 4.98 (bs, 1H, 13-NH), 3.89 (d, 2JH,H = 11.8 Hz, 2H, 3-CH, 4-CH), 3.67 (d, 2JH,H = 12.1 Hz, 1.1 Hz, 2H, 3-CH, 4-CH), 3.63 (t, 3JH,H = 6.6 Hz, 2H, 26CH2), 2.60−2.49 (m, 4H, 14-CH2, 21-CH2), 1.97 (t, 3JH,H = 8.5 Hz, 2H, 1-CH2), 1.70−1.50 (m, 6H, 22-CH2, 23-CH2, 25-CH2), 1.47 (s, 9H, 8-CH3, 9-CH3, 10-CH3), 1.45−1.40 (m, 6H, 5-CH3, 6-CH3), 1.36 (dt, 3JH,H = 6.9 Hz, 4.5 Hz, 3H, 24-CH2, −OH). 13C NMR (75 MHz, CDCl3): δ 154.8 (C-11), 140.2 (C-15), 139.1 (C-20), 128.4 (single peak with high intensity, C-16, C-17), 128.2 (single peak with high intensity, C-18, C-19), 98.3 (C-7), 80.1 (C-12), 66.3 (C-26), 62.9 (single peak with high intensity C-3, C-4), 51.7 (C-2), 35.4 (C-21), 32.6 (C-25), 31.4 (C-22), 29.0 (single peak C-14, C-23), 28.6 (C-1), 28.4 (single peak with high intensity C-8, C-9, C-10), 27.4 (C-6), 25.5 (C-24), 19.6 (C-5). HRMS (ESI+): C25H41NO5+Na+: calcd, 458.2882; found, 458.2877. tert-Butyl {5-[4-(8-Hydroxyoctyl)phenethyl]-2,2-dimethyl-1,3-dioxan-5-yl}carbamate (31). Synthesis and purification of product 31 were performed as described for compound 30. Yield: 100 mg (65%). mp 61 °C. 1H NMR (300 MHz, CDCl3): δ 7.09 (d, 3JH,H = 3.9 Hz, 4H, 16-CH, 17-CH, 18-CH, 19-CH), 5.00 (brs, 1H, NH), 3.89 (m, 2H, 3-CH, 4-CH), 3.64 (m, 4H, 28-CH2, 3-CH, 4-CH), 2.55 (m, 4H, 14-CH2, 21-CH2), 1.93 (m, 2H, 1-CH2), 1.51−1.65 (m, 6H, 22-CH2, 26-CH2, 27-CH2), 1.47 (s, 6H, 5-CH3, 6-CH3), 1.41−1.46 (m, 7H, 23CH2, 24-CH2, 25-CH2, OH), 1.31 (s, 9H, 8-CH3, 9-CH3, 10-CH3). 13 C NMR (75 MHz, CDCl3): δ 140.4 (C-15), 139.0 (C-20), 128.4 (single peak high intensity, C-16, C-17), 128.1 (single peak high intensity, C-18, C-19), 98.3 (C-7), 77.2 (C-12), 66.3 (C-28), 63.0 (single peak high intensity, C-3, C-4), 51.6 (C-2), 35.4 (C-21), 32.7 (C-27), 31.5 (C-22), 30.9 (C-25), 29.4 (C-14), 29.3 (C-1), 29.2 (C24), 28.6 (C-23), 28.4 (single peak high intensity, C-8, C-9), 28.3 (C10), 25.6 (single peak high intensity, C-26, C-6), 19.6 (C-5). HRMS (ESI+): C27H45NO5 + H+: calcd, 464.3376; found, 464.3371, C27H45NO5 + Na+: calcd, 486.3195; found, 486.3190.
continued for a further 15 min. Triethylamine (5.37 mL, 38.3 mmol, 5 equiv) was added to the reaction mixture, and stirring was continued for a further 15 min. The mixture was allowed to warm to rt and stirred for 30 min. The reaction was quenched with water and extracted with DCM (2 × 20 mL). Then, the organic layer was washed with 1% H2SO4 (1 × 30 mL) and brine (2 × 20 mL), dried over MgSO4, and concentrated in vacuo to obtain the crude product, which was further purified by column chromatography (cyclohexane/ethyl acetate, 4:1) to obtain the desired pure product as a white solid. Yield: 1.71 g (86%). mp 118 °C. 1H NMR (300 MHz, CDCl3): δ 9.63 (s, 1H, 1-CHO), 5.58 (s, 1H, NH), 4.04 (m, 4H, 3-CH2, 4-CH2), 1.47 (s, 15H, 5-CH3, 6-CH3, 8-CH3, 9-CH3, 10-CH3). 13C NMR (75 MHz, CDCl3): δ 199.2 (C-1), 155.4 (C-11), 98.7 (C-7), 80.9 (C-12), 62.6 (C-3, C-4), 59.8 (C-2), 28.2 (C-8, C-9, C-10), 27.3 (C-6), 19.5 (C-5). HRMS (ESI+): C12H21NO5 + Na+: calcd, 282.1317; found,: 282.1312. tert-Butyl [5-(4-Iodostyryl)-2,2-dimethyl-1,3-dioxan-5-yl]carbamate (27). Potassium carbonate (1.26 g, 9.2 mmol, 4 equiv), was flame-dried in a pressure tube. After cooling to rt, 25 (0.6 g, 2.3 mmol, 1 equiv) dissolved in toluene (7 mL) and 26 (2.58 g, 4.6 mmol, 2 equiv) were added, and the mixture was heated at 120 °C for 12 h. Then, the reaction mixture was cooled to rt, absorbed on silica gel, and purified by column chromatography (12 × 2 cm, cyclohexane/ethyl acetate, 4:1) to give the Z/E-isomeric products (dr 3:1) as a white solid. Yield: 1.02 g (96%). mp 107 °C. Z-isomer: 1H NMR (300 MHz, CDCl3): δ 7.66−7.57 (m, 2H, 18-CH, 19-CH), 7.05−6.94 (m, 2H, 16CH, 17-CH), 6.58 (d, 3JH,H = 12.7 Hz, 1H, 14-CH), 5.58 (d, 3JH,H = 12.6 Hz, 1H, 1-CH), 5.14 (s, 1H, 13-NH), 3.87 (d, 3JH,H = 5.2 Hz, 2H, 3-CH, 4-CH), 3.74 (dt, 3JH,H = 12.2 Hz, 1.4 Hz, 2H, 3-CH, 4-CH), 1.40−1.32 (m, 15H, 5-CH3, 6-CH3, 8-CH3, 9-CH3, 10-CH3). 13C NMR (75 MHz, CDCl3): δ 154.3 (C-11), 137.5 (C-14), 136.9 (C-18, C-19), 131.2 (C-1), 130.5 (C-16, C-17), 128.1 (C-15), 98.3 (C-20), 98.1 (C-7), 79.4 (C-12), 65.8 (C-3, C-4), 52.4 (C-2), 28.3 (C-8, C-9, C-10), 18.7 (C-5, C-6). HRMS (ESI+): C19H26INO4+Na+: calcd, 482.0804; found, 482.0799. tert-Butyl {5-[4-(6-Hydroxyhex-1-yn-1-yl)styryl]-2,2-dimethyl-1,3dioxan-5-yl}carbamate (28). Intermediate 27 (0.15 g, 0.27 mmol, 1 equiv) was dissolved in acetonitrile (2 mL) and treated with triphenyphosphine (15 mg, 10 mol %), Pd/C (15 mg, 10 mol %), copper iodide (7.5 mg, 5 mol %), and triethylamine (0.14 mL, 0.98 mmol, 3 equiv). The mixture was stirred for 20 min, hex-5-yn-1-ol (53 μL, 0.49 mmol, 1.5 equiv) was added, and the mixture was stirred at 25 °C for 1 h. Finally, the reaction mixture was heated under microwave irradiation at 80 °C for 90 min. After cooling to rt, the reaction mixture was diluted with ethyl acetate (2 mL) and filtered through Celite. The organic layer was concentrated, and the crude compound was purified by column chromatography (13.5 × 3 cm, cyclohexane/ ethyl acetate, 3:2) to get the Z/E-isomeric products (dr 3:1) as a slightly yellowish liquid. Yield: 120 mg, (85%). Z-isomer: 1H NMR (300 MHz, CDCl3): δ 7.29−7.35 (m, 2H, 18-CH, 19-CH), 7.15−7.21 (m, 2H, 16-CH, 17-CH), 6.64 (d, 3JH,H = 12.6 Hz, 1H, 14-CH), 5.58 (d, 3JH,H = 12.7 Hz, 1H, 1-CH), 5.18 (brs, 1H, NH), 3.88 (d, 2JH,H = 11.7 Hz, 2H, 3-CH2), 3.67−3.79 (m, 4H, 4-CH2, 26-CH2), 2.47 (t, 3 JH,H = 6.5 Hz, 2H, 23-CH2), 1.65−1.82 (m, 4H, 24-CH2, 25-CH2), 1.43 (s, 6H, 5-CH3, 6-CH3), 1.38 (s, 9H, 8-CH3, 9-CH3, 10-CH3), 1.32 (bs, 1H, OH). 13C NMR (75 MHz, CDCl3): δ 164.7 (C-11), 136.8 (C-14), 131.8 (C-1), 131.7 (C-15), 131.0 (single peak with high intensity, C-18, C-19), 128.5 (single peak with high intensity, C-16, C17), 125.2 (C-20), 98.1 (C-7), 90.2 (C-21), 80.7 (C-22), 77.2 (C-12), 65.8 (C-26), 62.4 (C-3, C-4), 52.4 (C-2), 31.8 (C-25), 28.39 (C-10), 28.35 (C-8, C-9), 26.9 (C-24), 24.9 (C-23), 19.2 (C-6), 18.7 (C-5). HRMS (ESI+): C25H35NO5+Na+: calcd, 452.2413; found, 452.2407 tert-Butyl {5-[4-(8-Hydroxyoct-1-yn-1-yl)styryl]-2,2-dimethyl-1,3dioxan-5-yl}carbamate (29). Synthesis and purification of compounds 29 (dr 3:1) were performed as described for compound 28. Yield: 95 mg (85%). Major Z-Isomer. 1H NMR (300 MHz, CDCl3): δ 7.32 (dd, 3JH,H = 8.3 Hz, 1.9 Hz, 2H, 18-CH, 19-CH), 7.18 (d, 3JH,H = 8.1 Hz, 2H, 16CH, 17-CH), 6.64 (d, 3JH,H = 12.7 Hz, 1H, 1-CH), 5.58 (d, 3JH,H = 12.7 Hz, 1H, 14-CH), 5.18 (s, 1H, NH), 3.93−3.83 (m, 2H, 3-CH, 4CH), 3.73 (d, 2JH,H = 12.2 Hz, 2H, 3-CH, 4-CH), 3.66 (td, 3JH,H = 6.6 3480
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
Journal of Medicinal Chemistry
Article
10H, 4-CH2, 13-CH2 to 16-CH2). 13C NMR (75 MHz, CDCl3, CD3OD): δ 140.1, 139.0 (C-6, C-9), 128.3 (C-7, C-11), 128.0 (C-8, C-10), 84.1 (d, 1JC,F = 163.5 Hz, C-17), 65.5 (single peak, C-1, C-3), 56.1 (C-2), 35.2 (C-12), 31.2 (C-13), 30.2 (d, 2JC,F = 19.3 Hz, C-16), 28.7 (single peak, C-4, C-5, C-14), 24.9 (d, 3JC,F = 5.4 Hz, C-15). 19F NMR (282 MHz, CDCl3, CD3OD): δ −218.5 (tt, 3JH,F = 25.1 Hz, 2JH,F = 47.4 Hz, 1F, 17-CH2F). HRMS (ESI+): C17H28FNO2 + H+: calcd, 298.2177; found, 298.2182; C17H28FNO2 + Na+: calcd, 320.1996; found, 320.2001. {4-[4-(8-Fluorooctyl)phenethyl]-2-methyl-4,5-dihydro-oxazol-4yl}methanol (35). A solution of diol 17 (100 mg, 0.307 mmol, 1 equiv), N,N-diisopropylethyl amine (0.080 mL, 0.461 mmol, 1.5 equiv), and triethyl orthoacetate (0.084 mL, 0.461 mmol, 1.5 equiv) in DMF (2 mL) was stirred at 120 °C for 2 h. The solution was extracted with ethyl acetate (2 × 5 mL). The combined organic layer was washed with brine (5 mL), dried over MgSO4, and concentrated. The crude product was purified by column chromatography (12 × 2 cm, cyclohexane/ethyl acetate, 5:1) to give the product as a yellowish oil. Yield: 70 mg (58%). 1H NMR (400 MHz, CDCl3): δ 7.09 (s, 4H, 7CH to 10-CH), 4.43 (dt, 2JH,F = 47.4 Hz, 3JH,H = 6.2 Hz, 2H, 19-CH2), 4.26 (d, 2JH,H = 11.4 Hz, 1H, 3-CH), 4.09 (d, 2JH,H = 11.4 Hz, 1H, 3CH), 3.71 (d, 2JH,H = 8.5 Hz, 1H, 4-CH), 3.45 (d, 2JH,H = 8.5 Hz, 1H, 4-CH), 2.56 (m, 4H, 5-CH2, 12-CH2), 2.05 (s, 3H, 21-CH3), 1.89 (ddd, 3JH,H = 13.7 Hz, 11.5 Hz, 5.8 Hz, 1H, 1-CH), 1.50−1.81 (m, 5H, 13-CH2, 18-CH2, 1-CH), 1.33 (m, 8H, 14-CH2 to 17-CH2). 13C NMR (101 MHz, CDCl3): δ 140.4 (C-6), 138.7 (C-11), 128.4 (single peak with high intensity, C-7, C-8), 128.1 (single peak with high intensity, C-9, C-10), 109.9 (C-20), 84.2 (d, 1JC,F = 163.5 Hz, C-19), 77.2 (C-2), 67.1 (C-3, C-4), 38.0 (C-12), 35.4 (C-5), 31.4 (C-13), 29.36 (C-15), 29.29 (C-18), 29.2 (C-1), 29.16 (single peak, C-17, C-16), 25.1 (C14), 13.9 (C-21). 19F NMR (282 MHz, CDCl3): δ −218.0 (tt, 2JH,F = 47.3 Hz, 3JH, F = 24.9 Hz). HRMS (ESI+): C21H32NO2F + Na+: calcd, 372.2315; found, 372.2309; C19H32NO2F + H+: calcd, 350.2495; found, 350.2490. 2-Amino-4-[4-(8-fluorooctyl)phenyl]-2-(hydroxymethyl)butyl dihydrogen phosphate (36). Alcohol 35 (300 mg, 0.859 mmol) was dissolved in DCM (7 mL), and 0.45 M tetrazole in MeCN (0.6 mL, 2.57 mmol, 3 equiv) was added. The reaction was stirred for 15 min at rt, and then N,N-diethyl-di-tert-butyl-phosphoramidite (0.48 mL, 1.71 mmol, 2 equiv) was added. The mixture was stirred overnight, a 30% solution of hydrogen peroxide (2 mL) was added, and the mixture was further stirred for 4 h. The organic solution was washed with Na2S2O3 solution (5 mL) and saturated NaHCO3 solution (5 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was passed through a short column (DCM/methanol/acetic acid, 79:20:1) to remove some impurities. After concentration, the residue was dissolved in conc. HCl/ethanol, 1:10 (2 mL), and the solution was stirred at 50 °C for 6 h. Water (1 mL) was added, and the mixture was stirred at rt for 1 h to form a precipitate. The precipitate was collected by filtration and washed with ethyl acetate to obtain the fluffy off-white product. Yield: 36 mg (10%). mp 219 °C. 1H NMR (600 MHz, MeOD): δ 7.14 (d, 3JH,H = 7.9 Hz, 2H, 9-CH, 10-CH), 7.09 (d, 3JH,H = 8.0 Hz, 2H, 7-CH, 8-CH), 4.39 (dt, 2JH,F = 47.6 Hz, 3JH,H = 6.1 Hz, 2H, 19-CH2), 4.00 (m, 2H, 3-CH, 4-CH), 3.69 (m, 2H, 3-CH, 4-CH), 2.63 (m, 2H, 5-CH2), 2.56 (t, 3JH,H = 7.7 Hz, 2H, 12-CH2), 1.96 (m, 2H, 1-CH2), 1.55−1.71 (m, 4H, 13-CH2, 18-CH2), 1.35 (m, 8H, 14CH2 to 17-CH2). 13C NMR (151 MHz, MeOD): δ 140.4 (C-6), 138.0 (C-11), 128.1 (single peak with high intensity C-7, C-8), 127.7 (single peak with high intensity C-9, C-10), 83.4 (d, 1JC,F = 163.7 Hz, C-19), 64.3 (C-4), 61.0 (C-3), 59.9 (C-2), 35.0 (C-12), 33.4 (C-5), 31.2 (13), 30.1 (d, 2JC,F = 19.6 Hz, C-18), 29.0 (C-16), 28.9 (C-15), 28.7 (C-14), 28.1 (C-1), 24.8 (d, 3JC,F = 5.3 Hz, C-17). 19F NMR (564 MHz, MeOD): δ −220.0 (tt, 2JH,F = 47.5 Hz, 3JH,F = 24.9 Hz, 1F, 19-F). 31P NMR (243 MHz, MeOD): δ 0.859 (s). HRMS (ESI+): C19H33NO5FP + Na+: calcd, 428.1978; found, 428.1973. 7-[4-(2-{5-[(tert-Butoxycarbonyl)amino]-2,2-dimethyl-1,3-dioxan-5-yl}ethyl)heptyl]-4-methylbenzenesulfonate (37). Alcohol 30 (100 mg, 0.22 mmol, 1 equiv) was dissolved in dry DCM (10 mL). The solution was cooled to 0 °C, and then triethylamine (64 μL, 0.445 mmol, 2 equiv) was added dropwise followed by the addition of tosyl
tert-Butyl N-{5-[4-(6-Fluorohexyl)phenethyl]-2,2-dimethyl-1,3-dioxan-5-yl}-carbamate (32). Alcohol 30 (161 mg, 0.37 mmol) was dissolved in dry THF (5 mL) and treated with PBSF (0.14 mL, 0.78 mmol, 2.1 equiv), triethylamine trishydrofluoride (0.12 mL, 0.73 mmol, 2.0 equiv), and N,N-diisopropylethyl amine (0.39 mL, 2.22 mmol, 6.0 equiv). The reaction mixture was stirred at rt for 2 days and quenched by addition of saturated sodium bicarbonate solution (5 mL). The aqueous phase was extracted with DCM (3 × 15 mL), and the combined organic layer was dried over MgSO4. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (9.5 × 3 cm, cyclohexane/ethyl acetate, 2:1) to give the product as a white solid. Yield: 72 mg (45%). mp 88−90 °C. 1H NMR (300 MHz, CDCl3): δ 7.08 (s, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 4.99 (brs, 1H, 2-NH), 4.42 (dt, 3JH,H = 6.1 Hz, 2JH,F = 47.4 Hz, 2H, 17-CH2), 3.89 (d, 2JH,H = 11.7 Hz, 2H, 1-CH, 3-CH), 3.68 (d, 2 JH,H = 11.7 Hz, 2H, 1-CH, 3-CH), 2.49−2.60 (m, 4H, 5-CH2, 12CH2), 1.97 (m, 2H, 4-CH2), 1.47 (s, 9H, 23-CH3 to 25-CH3), 1.35− 1.44, 1.56−1.76 (m, 14H, 13-CH2 to 16-CH2, 19-CH3, 20-CH3). 13C NMR (75 MHz, CDCl3): δ 155.0 (C-21), 140.3, 139.3 (C-6, C-9), 128.5 (single peak, C-7, C-11), 128.3 (single peak, C-8, C-10), 98.5 (C-18), 84.2 (d, 1JC,F = 164.1 Hz, C-17), 79.4 (C-22), 66.4 (C-1, C-3), 51.8 (C-2), 35.5 (C-12), 31.5 (C-13), 30.4 (d, 2JC,F = 19.4 Hz, C-16), 29.0 (C-4, C-14), 28.8 (C-5), 28.6 (single peak, C-23, C-24, C-25), 25.2 (d, 3JC,F = 5.4 Hz, C-15), 19.8 (single peak, C-19, C-20). 19F NMR (282 MHz, CDCl3): δ −218.6 (tt, 3JH,F = 23.7 Hz, 2JH,F = 47.6 Hz, 1F, 17-CH2F). HRMS (ESI+): C25H40FNO4 + H+: calcd, 438.3014; found, 438.3014; C25H40FNO4 + Na+: calcd, 460.2834; found, 460.2832. tert-Butyl {5-[4-(8-Fluorooctyl)phenethyl]-2,2-dimethyl-1,3-dioxan-5-yl}carbamate (33). A stirred solution of alcohol 31 (800 mg, 1.72 mmol, 1 equiv) in dry DCM (10 mL) was cooled to −78 °C, under nitrogen, and treated with DAST (0.456 mL, 3.45 mmol, 2 equiv). The flask was stoppered, and the mixture was kept at rt for 8 h. Then, the reaction was quenched with ice, neutralized with 10% aqueous NaHCO3, and extracted with DCM (2 × 5 mL). The extract was washed with water and brine, dried over MgSO4, and concentrated. The product was purified by column chromatography (15 × 3.5 cm, cyclohexane/ethyl acetate, 5:1) to give the product as a white solid. Yield: 700 mg (87%). mp 63 °C. 1H NMR (300 MHz, CDCl3): δ 7.04−7.13 (m, 4H, 16-CH to 19-CH), 4.97 (brs, 1H, NH), 4.43 (dt, 2JH,F = 47.4 Hz, 3JH,H = 6.2 Hz, 2H, 28-CH2), 3.90 (d, 2JH,H = 11.8 Hz, 2H, 3-CH, 4-CH), 3.67 (d, 2JH,H = 11.8 Hz, 2H, 3-CH, 4CH), 2.47−2.62 (ddd, 3JH,H = 8.8 Hz, 7.1 Hz, 5.0 Hz, 4H, 14-CH2, 21CH2), 1.91−2.03 (m, 2H, 1-CH2), 1.52−1.67 (m, 4H, 22-CH2, 27CH2), 1.47 (s, 9H, 8-CH3, 9-CH3, 10-CH3), 1.40−1.45 (m, 8H, 23CH2 to 26-CH2), 1.32 (s, 6H, 5-CH3, 6-CH3). 13C NMR (75 MHz, CDCl3): δ 156.4 (C-11), 140.5 (C-15), 138.7 (C-20), 128.52 (C-17), 128.42 (C-16), 128.19 (C-19), 128.13 (C-18), 98.3 (C-7), 84.2 (d, 1 JC,F = 163.9 Hz, C-28), 80.1 (C-12), 66.6 (C-3), 59.3 (C-4), 51.7 (C2), 35.5 (C-21), 31.5 (C-22), 30.3 (d, 2JC,F = 9.4 Hz, C-27), 29.3 (C24, C-14), 29.19 (C- 23), 29.16 (C-1), 29.09 (C-26), 28.4 (C-8), 28.3 (single peak high intensity, C-9, C-10, C-25), 25.17 (C-6), 25.10 (C5). 19F NMR (282 MHz, CDCl3): δ −218.0 (tt, 2JH,F = 47.4 Hz, 3JH,F = 24.9 Hz, 28-F). HRMS (ESI+): C27H44NO4F + Na+: calcd, 488.31; found, 488.30. 2-Amino-2-[4-(6-fluorohexyl)phenethyl]propane-1,3-diol (34). According to ref 25, intermediate 32 (72 mg, 0.16 mmol) was dissolved in a mixture of DCM, TFA, and water (v/v 2:2:1; 2.5 mL). The reaction mixture was allowed to warm to rt and stirred further for 12 h. Then, the solvent was removed under reduced pressure, and the residue was diluted with water (2 mL) and neutralized with a saturated NaHCO3 solution. The aqueous phase was extracted with DCM (4 × 8 mL). The combined organic phase was washed with brine (1 × 8 mL) and dried over Na2SO4. The solvent was evaporated, and the residue was crystallized from ethyl acetate to yield white crystals. Yield: 38 mg (81%). mp 108 °C (ethyl acetate). 1H NMR (300 MHz, CDCl3, CD3OD): δ 6.97−7.10 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 4.34 (dt, 3JH,H = 6.1 Hz, 2JH,F = 47.4 Hz, 2H, 17-CH2), 3.48 (d, 2JH,H = 10.9 Hz, 2H, 1-CH, 3-CH), 3.40 (d, 2JH,H = 11.1 Hz, 2H, 1-CH, 3CH), 2.43−2.60 (m, 4H, 5-CH2, 12-CH2), 1.12−1.40, 1.46−1.70 (m, 3481
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
Journal of Medicinal Chemistry
Article
Siemens) by irradiation of a 2.8 mL water target using 10 MeV proton beams on 97.0% enriched [18O]H2O by the 18O(p,n)18F nuclear reaction. 4.3.2. Synthesis of [18F]34 and [18F]17. The same synthetic protocol was used for both compounds. In a computer controlled TRACERLab FxFDG Synthesizer, a batch of aqueous [18F]fluoride ions (213−2918 MBq) from the cyclotron target was passed through anion exchange resin (Sep-Pak Light Waters Accell Plus QMA cartridge, preconditioned with 1 M K2CO3 (5 mL) and water for injection (10 mL)). [18F]Fluoride ions were eluted from the resin with a mixture of 40 μL of 1 M K2CO3, 200 μL of water for injection, and 800 μL of DNA-grade CH3CN containing 20 mg (53 μmol) of Kryptofix 222 (K222) in the reactor. Subsequently, the aqueous K(K222) [18F]F solution was carefully evaporated to dryness in vacuo. Then, precursor (5.0−5.5 mg, 8.1−9.3 μmol) in dry acetonitrile (1 mL) was added, and the reaction mixture was heated at 84 °C for 10−12 min. Then, the solvent was evaporated, and a solution of 1.1 mL TFA/H2O (10:1) was added at 40 °C and allowed to stir for 5 min. After that, the solvent was evaporated to dryness, water (10 mL) was added, and the mixture was passed through a Waters Sep-Pak C18 Light cartridge (preconditioned with 10 mL of ethanol and 10 mL of water). The cartridge was washed with water (10 mL) and eluted with MeOH (0.5 mL). After addition of water (0.5 mL), the raw product solution was purified by gradient-radio-HPLC system A (method A). The product fraction of compound [18F]34 (retention time tR = 16.63 min) and [18F]17 (tR = 18.15 min) was evaporated to dryness in vacuo and formulated in 1.0 mL of water for injection/EtOH (9:1, (v/v)). Products [18F]34 and [18F]17 were obtained in radiochemical yields (rcy) of 29.2 ± 3.3% (n = 5) and 30.2 ± 4.2% (n = 3) (decay corrected based on cyclotron-derived [18F]fluoride ions) in 115 ± 12 min ([18F]34) and 115 ± 16 min ([18F]17) from the end of the radionuclide production. The target compounds were isolated in >99% radiochemical purity (rcp). Radiochemical purities of [18F]34 (tR ([18F]34) = 10.82 min) and [18F]17 (tR ([18F]17) = 11.15 min) were determined by analytical radio-HPLC B (method B). 4.4. Biological Studies. 4.4.1. In Vivo Testing. The compounds were dissolved to a concentration of 0.2 mg/mL in 0.9% NaCl and injected intraperitoneally at 1.25 mg/kg body weight. Twenty four hours later, blood was drawn from the retroorbital plexus in 17.8 mM EDTA as an anticoagulant, and lymphocytes were analyzed by flow cytometry (Gallios, Beckman Coulter)35 using antibodies to CD4, CD8, CD45, and B220 (Becton Dickinson) after lysis of red blood cells with BD PharmLyse (Becton Dickinson) according to the manufacturer’s instructions. 4.4.2. In Vitro Testing. Chinese hamster ovary (CHO) cells overexpressing the human S1P1 and S1P3 receptors were serumstarved overnight and stimulated with compounds and S1P side-byside in the same experiment for the indicated times. Cells were then lysed, and samples were separated on a 12% SDS-PAGE gel. After transfer to PVDF membranes, western blotting was performed with an antibody to the dually phosphorylated p44/42 MAP kinase (Cell Signaling), and signals were visualized by ECL. 4.4.3. In Vitro Stability. An aliquot of formulated [18F]17 (20 μL, 8.7 MBq) was added to a sample of human serum (200 μL), and the mixture was incubated at 37 °C. Samples of 20 μL each were taken after periods of 10, 30, 60, and 90 min and quenched in MeOH/ CH2Cl2 (1:1 (v/v), 100 μL) followed by centrifugation for ≥5 min. The supernatant was analyzed by analytical radio-HPLC B [method B, tR ([18F]17) = 11.15 min]. 4.4.4. Animals. Adult C57BL/6 mice (age 13 weeks, 21−26 g body weight, male) were anaesthetised by isoflurane/O2, and one lateral tail vein was cannulated using a 27 G needle connected to 15 cm polyethylene catheter tubing. [18F]34 or [18F]17 (400 kBq/g bodyweight) was injected as a bolus (100 μL of compound flushed with 100 μL of saline; syringe pump speed, 300 μL/min) via the tail vein, and subsequent PET scanning was performed. All experiments performed in the study were in accordance with the German Law on the Care and Use of Laboratory Animals and approved by the local authorizing agency of North Rhine-Westphalia.
chloride (63 mg, 0.334 mmol, 1.5 equiv) in one portion slowly. The reaction mixture was allowed to warm to rt and was stirred overnight. After completion of the reaction, the mixture was diluted with water (10 mL) and extracted with DCM (2 × 10 mL). The combined organic layer was washed with brine (1 × 10 mL) and water (2 × 5 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by column chromatography (15 × 2 cm, cyclohexane/ethyl acetate, 5:1) to get the product as a pure white solid. Yield: 75 mg (55%). mp 75 °C. 1H NMR (400 MHz, CDCl3): δ 7.83−7.73 (m, 2H, 28-CH, 29-CH), 7.38−7.30 (m, 2H, 30-CH,31CH), 7.12−7.01 (m, 4H, 16-CH, 17-CH, 18-CH, 19-CH), 4.98 (s, 1H, 13-NH), 4.01 (t, 3JH,H = 6.5 Hz, 2H, 26-CH2), 3.90 (d, 3JH,H = 11.8 Hz, 2H, 3-CH, 4-CH), 3.68 (d, 2JH,H = 11.9 Hz, 2H, 3-CH, 4-CH), 2.57−2.48 (m, 4H, 14-CH2, 21-CH2), 2.44 (s, 3H, 33-CH3), 1.96 (t, 3 JH,H = 8.5 Hz, 2H, 1-CH2), 1.60−1.67 (m, 2H, 25-CH2), 1.50−1.57 (m, 2H, 22-CH2), 1.47 (s, 9H, 8-CH3, 9-CH3, 10-CH3), 1.42 (m, 6H, 5-CH3, 6-CH3), 1.38−1.20 (m, 4H, 23-CH2, 24-CH2). 13C NMR (101 MHz, CDCl3): δ 154.8 (C-11), 144.6 (C-32), 140.0 (C-15), 139.2 (C20), 133.2 (C-27), 129.7 (single peak with high intensity C-30, C-31), 128.3 (single peak with high intensity C-16, C-17), 128.2 (single peak with high intensity C-18, C-19), 127.8 (single peak with high intensity C-28, C-29), 98.3 (C-7), 79.3 (C-12), 70.6 (C-26), 66.3 (single peak, C-3, C-4), 51.6 (C-2), 35.3 (C-21), 33.6 (C-25), 31.2 (C-22), 28.7 (C14), 28.6 (C-1), 28.5 (C-24), 28.4 (single peak with high intensity, C23, C-9, C-10), 27.4 (C-8), 25.2 (C-6), 21.6 (C-33), 19.6 (C-5). HRMS (ESI+): C32H47NO7S+Na+: calcd, 612.2971; found,. 612.2965. 8-[4-(2-{5-[(tert-Butoxycarbonyl)amino]-2,2-dimethyl-1,3-dioxan-5-yl}ethyl)phenyl]octyl-4-methylbenzenesulfonate (38). Synthesis and purification of 38 were performed as described for compound 37. Yield: 69 mg (59%). 1H NMR (300 MHz, CDCl3): δ 7.83−7.74 (m, 2H, 32-CH, 33-CH), 7.37−7.30 (m, 2H, 30-CH, 31-CH), 7.13− 7.03 (m, 4H, 16-CH, 17-CH, 18-CH, 19-CH), 4.98 (s, 1H, NH), 4.01 (t, 3JH,H = 6.5 Hz, 2H, 28-CH2), 3.90 (d, 3JH,H = 11.8 Hz, 2H, 3-CH, 4CH), 3.68 (d, 3JH,H = 11.7 Hz, 2H, 3-CH, 4-CH), 2.60−2.48 (m, 4H, 14-CH2, 20-CH2), 2.44 (s, 3H, 35-CH3), 2.02−1.91 (m, 2H, 1-CH2), 1.65−1.59 (m, 2H, 22-CH2), 1.47 (s, 9H, 8-CH3, 9-CH3, 10-CH3), 1.42 (m, 8H, 5-CH3, 6-CH3, 27-CH2), 1.26 (m, 8H, 23-CH2, 24-CH2, 25-CH2, 26-CH2). 13C NMR (75 MHz, CDCl3): δ 154.8 (C-11), 144.6 (C-34), 140.3 (C-15), 139.1 (C-20), 133.1 (C-29), 129.7 (single peak with high intensity, C-32, C-33), 128.4 (single peak with high intensity, C-16, C-17), 128.2 (single peak with high intensity, C-18, C19), 127.8 (single peak with high intensity, C-30, C-31), 98.3 (C-7), 77.2 (C-12), 70.6 (C-28), 66.3 (C-3, C-4), 51.6 (C-2), 35.4 (C-21), 31.4 (C-22), 29.2 (C-24), 29.1 (C-25), 28.8 (C-14), 28.7 (C-27), 28.6 (C-1), 28.4 (single peak with high intensity, C-8, C-9, C-10), 27.4 (C23), 26.9 (C-26), 25.3 (C-5), 21.6 (C-6), 19.7 (C-35). HRMS (ESI+): C34H51NO7S + Na+: calcd, 640.3284; found, 640.3278. 4.3. Radiolabeling Experiments. 4.3.1. General Methods. The radiosynthesis was carried out on a modified PET tracer radiosynthesiser (TRACERLab FxFDG, GE Healthcare). The recorded data were processed by TRACERLab Fx software (GE Healthcare). Separation and purification of the radiosynthesized compounds were performed on the following semipreparative radio-HPLC system A: K500 and K-501 pump, K-2000 UV detector (Herbert Knauer GmbH), NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec), and an ACE 5 AQ column (250 mm × 10 mm). Method A started with a linear gradient from 10 to 90% CH3CN in water (0.1% TFA) over 30 min, holding for 5 min, and followed by a linear gradient from 90 to 10% CH3CN in water (0.1% TFA) over 5 min, with λ = 254 nm and a flow rate of 5.5 mL min−1. Radiochemical purities were determined using the analytical radio-HPLC system B: two Smartline 1000 pumps and a Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γdetector (Raytest Isotopenmessgeräte GmbH), and a Nucleosil 100-5 C-18 column (250 mm × 4 mm). Method B started with a linear gradient from 10 to 90% CH3CN in water (0.1% TFA) over 15 min, followed by a linear gradient from 90 to 10% CH3CN in water (0.1% TFA) over 3 min, with λ = 254 nm and a flow rate of 1.0 mL min−1. The recorded data of both HPLC systems were processed by GINA Star software (Raytest Isotopenmessgeräte GmbH). No-carrier-added aqueous [18F]fluoride was produced on a RDS 111e cyclotron (CTI3482
DOI: 10.1021/jm502021d J. Med. Chem. 2015, 58, 3471−3484
Journal of Medicinal Chemistry
Article
4.4.5. Small Animal PET Scanning. PET experiments were carried out using a submillimeter high-resolution (0.7 mm full width at halfmaximum) small animal scanner (32 module quadHIDAC, Oxford Positron Systems Ltd., Oxford, UK) with uniform spatial resolution (
