Purification of LTA4H
Recombinant human LTA4H (residues 1−611) was expressed in E. coli
BL21-AI/pRARE. An amount of 7.5 g of cell paste was lysed by nitrogen cavitation at 2200 psi for 1 h in a lysis buffer consisting of 50 mM Tris-HCl, pH 8.0, 1 mM PMSF, 0.2 mg/mL lysozyme, and 1.5 U/mL benzonase. The lysate was clarified by centrifugation at 42
000 rpm for 45 min at 4 °C. Clarified lysate was filtered using a 0.2 μM syringe-top filter. Lysate was then applied to 2 × 5 mM HisTrap HP columns (GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 8.0, and 200 mM NaCl. The column was washed with six column volumes of 30 mM imidazole and eluted with a linear gradient of 30−300 mM imidazole in 20 mM Tris-HCl, pH 8.0, containing 200 mM NaCl. The pooled peak fractions were >95% pure by SDS−PAGE at a concentration of 2.7 mg/mL and used directly for crystallization without further concentration or dialysis.
Crystallization and Fragment Screening
LTA4H was crystallized by sitting drop vapor diffusion against a crystallant containing 100 mM imidazole, pH 6.5, 12.67% w/v PEG-8000, 100 mM sodium acetate, and 5 mM YbCl3. Crystal growth was accelerated through the use of a microseeding protocol. Seed stocks were prepared by suspending several crystals in 20 μL of crystallant and crushing them by vortexing in a Seed Bead tube (Hampton Research). Crushed crystals were diluted 1:100 into fresh crystallant and vortexed again to form the final seed stock. Crystallization drops were set up in a Compact Jr. 96-well crystallization plate (Emerald BioSystems) by combining 0.7 μL of LTA4H (2.7 mg/mL) with 0.7 μL of crystallant and 0.2 μL of seed stock over a reservoir volume of 100 μL.
Twenty-five pools of eight compounds from the FOL library were screened against LTA4H by X-ray crystallography. Pool stocks consisted of cocktails of eight fragments, each at 6.25 mM in methanol. Individual FOL compounds are stored in barcode labeled sealed septum vials as 50 mM stocks in pure methanol and stored at −20 °C. Approximately 30% of the fragment stocks have been analyzed over a period of 12 months by mass spectrometry to demonstrate general stability of the compounds in methanol (data not shown). Fragment cocktails in methanol were spotted onto the drop chambers of crystallization plates and the solvent was allowed to evaporate, leaving a precise amount of fragment compounds as a dry powder with no solvent residue that could potentially interfere with crystallization or crystal stability. A volume of crystallization mother liquor equal to the initial volume of fragment cocktail was added to the dry powder, allowing for dissolution of the fragments. Crystals were transferred to the resulting solution and allowed to soak overnight. This technique is versatile, as it is suitable for both cocrystallization and soaking experiments, and adjusting ratios of methanol solution to crystallant solution allows for adjustment of the final fragment concentration in the drop.
Although diversity pooling was conducted to aid in identification of fragments from pool density alone, factors such as low resolution, low occupancy, or competitive binding can lead to uncertainty. Therefore, in cases where crystals exposed to fragment cocktails produce difference electron density indicative of fragment binding, follow-up experiments with individual fragments were performed. During this deconvolution step, individual candidate fragments were soaked into crystals, usually at 10−25 mM. The resulting crystal structures provide unambiguous confirmation of fragment hits, and all structures presented were generated from single molecule crystal soaking experiments.
LTA4H Peptidase Activity Assay
l-Arginine-p-nitroanilide (Arg-pNA) substrate was obtained from Sigma. Stock solution of ARG-pNA (500 mM) was prepared and serially diluted in DMSO. Final peptidase assays were performed in 96-well clear, flat bottom plates (Corning). Each assay plate contained eight positive (no inhibitor) and eight negative (no enzyme) controls. LTA4H (45 μL) and test compounds (5 μL) were preincubated for 10 min followed by addition of substrate (50 μL); final reactions contained 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% DMSO, 0.5 mM Arg-pNA, and 20 nM LTA4H. Control experiments showed that 5% DMSO had no effect by itself on enzyme activity (data not shown). Reaction rates were monitored in a SpectraMax 190 plate reader (Molecular Devices) at room temperature (rt) by measuring the increase in absorbance at 410 nm due to cleavage of the amide bond of the substrate and formation of p-nitroaniline. Reactions were measured for 30 min, and initial rates were determined from the linear portions of the progression curves using SoftMax Pro software (Molecular Devices). Dose−response curves ware calculated by fitting data to a sigmoidal four-parameter logistic equation using Prism (Graphpad).
LTA4H Hydrolase Activity Assay
substrate was prepared from the methyl ester of LTA4
(BioMol or Cayman Chemicals) by treatment under nitrogen with 100 mol equiv of NaOH in an acetone/H2
O (4:1) solution at rt for 40 min. Stock solutions of LTA4
were kept frozen at −80 °C for a maximum of 1 week prior to use. Recombinant human LTA4
H (10 nM final concentration) was incubated with various concentrations of test compound for 10 min at rt in assay buffer (0.1 M Tris-HCl, 0.1 M NaCl, 5 mg/mL fatty acid free BSA, 10% DMSO, pH 8.0). Immediately before the assay, LTA4
was diluted in assay buffer (without DMSO) and added to the reaction mixture to a final concentration of 1 μM to initiate the enzyme reaction. Samples were incubated for 10 min at rt followed by addition of 2 volumes of chilled quenching buffer (CH3
CN with 1% CH3
COOH and 73 nM LTB4
(BioMol). The samples were then stored at 4 °C for 12 h to complete protein precipitation and centrifuged for 15 min at 1800g
formed was measured in the supernatant by LCMS/MS using LTB4
as an internal standard and an external LTB4
standard (BioMol) for a calibration curve. Briefly, the analyte was separated from LTB4
isomers formed by spontaneous hydrolysis of LTA4
using isocratic elution on an HPLC system (Waters) and analyzed on a tandem quadrupole mass spectrometer (Waters Micromass Quattro Premier). MRM transitions followed on two channels were 335.2 → 195.3 (LTB4
) and 339.2 → 197.3 (LTB4
). On the basis of the measured amounts of LTB4
formed at each inhibitor concentration, a dose−response curve was fitted to the data using a sigmoidal four-parameter function (Prism, Graphpad) and an IC50
value was calculated. Ligand efficiency values were calculated for hydrolase and peptidase assays independently using LE (kcal/(mol·HA)) = −RT
)/HA = −0.59179(ln
)/HA, where HA is the number of heavy atoms (non-hydrogen).
Human Whole Blood LTB4 Assay
Human blood (45 mL) was collected in heparin-containing Vacutainer tubes (Greiner-Bio One) with informed consent. Individual experiments were performed with blood from a single subject. For each sample, 200 μL of blood was dispensed into a prewarmed 96-well plate and 188 μL of RPMI-1640 medium (Invitrogen) containing 20 μg/mL indomethacin (Sigma) was added. An amount of 4 μL of a series of compound dilutions (final DMSO concentration of 1%) was added in triplicate, followed by a 15 min incubation at 37 °C with gentle shaking. Blood samples were stimulated by adding 8 μL of ionomycin (from Streptomyces conglobatus, Calbiochem) to a final concentration of 36 μM. After another incubation at 37 °C for 30 min, samples were centrifuged at 4 °C for 5 min at 1800g. LTB4 concentrations in supernatants were determined using a commercially available enzyme-linked immunosorbent assay (R&D Systems) according to the manufacturer’s instructions. On the basis of the measured amounts of LTB4 formed at each inhibitor concentration, a dose−response curve was fitted to the data using a sigmoidal three-parameter function (XLfit, IDBS), and an IC50 value was calculated. Each assay plate contained eight positive controls (no compound) and eight negative controls (no Ionomycin).
Fragment Selection for the Fragments of Life Library
Approximately 400 candidate fragments representing natural molecules of life with (i) molecular weights less than 350, (ii) fewer than six hydrogen bond donors, and (iii) fewer than seven hydrogen bond acceptors were manually selected from known metabolic pathways. Molecules were selected from eukaryotic, archaebacterial, and eubacterial metabolic pathways and from presumed primordial pathways.89,90
There was no bias toward any particular metabolic pathway; however, molecules of conserved intermediary metabolism were prioritized.(91
) Approximately 300 of the resulting fragments were commercially available, reasonably priced, and acquired. These fragments were tested for solubility (≥50 mM in methanol) to yield a final core set of 218 natural molecules (FOL-Nat). The chemical diversity of the first 218 FOL-Nat fragments was determined using a 210 × 210 principal component analysis (PCA) using Cerius2
(Accelrys). In this analysis 1D, 2D, and 3D descriptors (conformational, cat shape, electronic, quantum chemical, information content, spatial, structural, thermodynamical, topological, and geometrical) were calculated and the 3D distribution of the fragments was visualized in a 3D plot (see Supporting Information
Figure 2). This analysis showed that the fragments were diverse but did not densely cover the desired chemical space. Therefore, in addition to the manual selection, we explored the Available Chemicals Directory (ACD/Lab, version 7, MDL) and the Maybridge Fragment Library (www.maybridge.com
) as a source of additional fragments. We intended to diversify the original set by selecting heteroatom-containing derivatives of natural molecules (FOL-NatD) with fragment-like properties. Toward this goal, a selection of 238
996 compounds were analyzed using modified “rule of three”(11
) parameters to afford 49
877 fragments with (i) molecular weights between 110 and 250 Da, (ii) calculated log of octanol/water partition coefficient (ClogP) of less than or equal to 3.0 (calculated with software available from Daylight Chemical Information Systems, Inc.), (iii) hydrogen bond donors less than or equal to 3, and (iv) hydrogen bond acceptors less than or equal to 6 (calculated with Sybyl, version 6.8, Tripos). Virtual selection of the 49
877 molecules was further subjected to a panel of SMARTS filters(92
) in order to eliminate molecules endowed with problematic functionalities. A partial list of these SMARTS filters included reactive functional groups, lipophilic chains of seven or more carbon atoms, crown ethers, disulfides, excessive (>3) acidic groups, thiols, epoxides, aziridines, hydrazones, thioureas, thiocyanates, benzylic quaternary nitrogens, thioesters cyanamide, four membered lactones, di- and triphosphates, metals, phosphines, phosphonic acids, sulfonic acids, sulfonyl halides, boronic acids, isotopes, salts, metals, more than 3 halogens, more than two nitro groups, and lanthanides. This culling process resulted in 40
489 candidate fragment compounds. Each fragment from the resultant filtering was converted from 2D to 3D projection using CONCORD (Tripos) and used to identify fragments with ≤3 rotatable bonds and calculated PSA values of ≤60 Å2
. The resulting 5606 fragments were again analyzed using the previously described principal component analysis (PCA), and 1016 unique fragments were selected with preference for molecules that could be visually identified to have at least 8 atoms with chemical arrangements that matched that of a known natural molecule of life. From this set, 880 fragments were commercially available and obtained at a reasonable price. These fragments were tested for solubility (>50 mM in methanol), and the resulting 666 fragments (FOL-NatD) were added to the FOL library. PCA analysis showed that the FOL-NatD fragments occupy distinctly different chemical space (see Supporting Information
Figure 2) and that together the FOL-Nat and Fol-NatD fragments combine to form a diverse and relatively dense set of fragments. Finally, we were also intrigued by the possibility of mimicking a protein 3D architecture using biaryl small molecule fragments.59−61,93
In order to identify protein mimetic fragments (FOL-Biaryl), we first identified 566 molecules containing 5−5, 6−5, and 6−6 biaryls connected via a σ-bond to allow for a controlled torsional freedom from the ChemBlock library (www.chemblock.com
). The energy minimized conformations (Tripos force field, Sybyl 6.8) of a selection of these biaryl fragments were overlaid with the α, β, γ turns of a known protein structure (1RTP
.pdb) and shown to have good steric and electronic mimicry of protein structure. The resulting fragments were tested for solubility (>50 mM in methanol), resulting in 445 fragment molecules (FOL-Biaryl). Results from the PCA analysis and physical properties of the fragments [MW, ClogP, total polar surface area, number of hydrogen bond donor/acceptors, rotatable bonds, and rings] of the complete 1329 member FOL library are provided in Supporting Information
(see Supporting Information
Computational and Physical Pooling
When fragments are pooled into structurally diverse cocktails of 4−10 compounds per mix, the throughput of X-ray screening of fragment libraries can be significantly increased.12,94,95
The pooling of fragments was carried out using the same 210 × 210 descriptor space principle component analysis (Cerius2
, Accelrys) in order to select eight diverse fragments per pool. The nature of the fragment (FOL-Nat, FOL-NatD, FOL-Biaryl) was not considered in the pooling strategy. Monte Carlo optimization using “Diversity Metric Function MAXMIN Distance” (Accelrys) with the optimum distance range of 1.97−2.57 Å was used to randomly extract eight diverse fragments. After extraction of eight fragments from the library, the process was repeated until the entire library was computationally pooled into structurally diverse pools each containing eight fragments. Individual fragments, each >95% pure as determined by NMR spectroscopy, were prepared as 50 mM stocks in methanol. Physical pools of eight fragments each were prepared by mixing fragment stocks such that the final concentration of each was ~6 mM. The fragment pools were found to be visibly stable, with no observed precipitation, suggesting that fragment pairs or combinations do not have a propensity to aggregate.
The ligand docking into LTA4H was carried out with the Surflex interface implemented in Sybyl 7.2 (Tripos Inc., St. Louis, MO). The Surflex-Dock engine uses an empirical scoring function and a patented search engine to dock ligands into a protein’s binding site.(96
) The PDB ID 3CHI file was converted to a mole2 file, and hydrogens were added. The B
-values were replaced by the AMBER charges using Sybyl 7.2. The bound ligand of 3CHI
was removed from the binding site. The Surflex-Dock protomol, a computational representation of the intended binding site, was generated using the probes CH4
, −N−H, and −C=O. The protomol directed the placement of the ligand during the docking process. LTA4H inhibitors were drawn in ISIS Draw and converted to 3D structures with CONCORD of Sybyl 7.2 (Tripos Inc., St. Louis, MO). All of the inhibitors were properly typed with hydrogens, and a 3D structure data file was created for docking. Each ligand was then fragmented which reduced the conformational space to be explored. The fragments were then aligned to the protomol probes. All the fragments were scored,(97
) and the highest scoring fragment was kept as the head fragment. The tail fragments were selected on the basis of the similar principle, and gradual attachment was carried out to build the docked ligand. The poses were refined, eliminating those with excessive penetration into the protein. Thirty poses for each of the ligand were generated and visualized in the active site based on their Surflex scores. The best fitting ligand poses were chosen for further comparative structural analysis.
All reagents and anhydrous solvents were obtained from commercial sources and used without further purification unless otherwise noted. NMR spectra were recorded at 400 or 500 MHz (Varian Instruments) in the solvent indicated, and TMS was used as an internal reference. ACDLabs NMR software was used to process FIDs to generate spectral parameters (δ (ppm)/Hz). Coupling constants (J) are given in Hz. Mass spectra were obtained using either APCI or electrospray ionization (PE-SCIEX single-quad or Agilent mixed-mode units). Elemental analyses were carried out by Galbraith Laboratories, Inc. (Knoxville, TN) or Midwest Microlab, LLC (Indianapolis, IN). Column chromatography was carried out in the solvents indicated with silica gel (MP EcoChrom, 32-63D, 60 Å). The HPLC method was as follows. Compounds were eluted using a gradient of 90/10 to 10/90 A/B over 40 min at a flow rate of 1.0 mL/min, where solvent A was 0.05% TFA in 100% H2O and solvent B was 0.05% TFA in 100% acetonitrile. For HPLC data (final products), peak area percent and retention time (tR in min) are provided. The following compounds were obtained from commercial sources: compound 4 from Matrix, compounds 5, 7, 8, 9, and 10 from Maybridge, compound 6 from VWR, and compounds 11, 12, and 13 from Aldrich.
To a solution of 25 (148 mg, 0.387 mmol) in methanol (3 mL) was added HCl (1 M in diethyl ether, 6 mL). The mixture was stirred at rt for 3 h. The solvent was removed in vacuo to provide the title compound 14 as a hydrochloride salt (128 mg, 93%). 1H NMR (400 MHz, DMSO-d6) δ 1.60−1.73 (m, 1H), 1.87−2.12 (m, 3H), 3.17−3.21 (m, 2H), 3.84−3.87 (m, 2H), 4.19−4.23 (dd, 1H J1 = 3.6 Hz, J2 = 10.8 Hz), 4.11−4.16 (m, 1H), 4.44 (s, 2H), 7.0 (d, 2H J = 8.8 Hz), 7.29−7.38 (m, 5H), 7.48−7.49 (m, 2H) 9.1 (s, 1H), 9.8 (s, 1H). LCMS: 97%; APCI+m/z 283 (M + 1). Anal. (C18H22N2O·2HCl) C, H, N.
To a solution of 5-hydroxyindole 11 (446 mg, 3.35 mmol) in anhydrous acetone (20 mL) was added potassium carbonate (1.36 g, 9.8 mmol) and 1-(2-chloroethyl)pyrrolidine hydrochloride (1.28 g, 7.5 mmol). The reaction was heated to reflux for 16 h. After the mixture was cooled to rt, a solution of tetrabutylammonium bromide (200 mg, 0.6 mmol) in DMF (10 mL) was added and the mixture was heated to 55 °C for 16 h. The solvent was removed in vacuo, and the resulting residue was partitioned between EtOAc (25 mL) and water (50 mL). The organic layer was separated, dried with anhydrous MgSO4, and concentrated in vacuo to an oil. The oil was purified by silica gel flash chromatography (~100 g of SiO2, 0−5% MeOH/CH2Cl2, gradient) to provide the title compound 15 (145 mg, 19%). 1H NMR (400 MHz, DMSO-d6) δ 1.68 (dt, J = 6.57, 3.15 Hz, 4 H), 2.53 (br s, 4 H), 2.78 (t, J = 6.04 Hz, 2 H), 4.03 (t, J = 6.04 Hz, 2 H), 6.31 (br s, 1 H), 6.71 (dd, J = 8.72, 2.42 Hz, 1 H), 7.03 (d, J = 2.15 Hz, 1 H), 7.23−7.29 (m, 2 H), 10.88 (br s, 1 H). LCMS (APCI+): mass calcd for C14H18N2O, 230.3; m/z found 231 (M + 1), 99%. HPLC: 98.9%, tR = 10.32 min.
1-[2-(1H-Indol-5-yloxy)ethyl]piperidine-4-carboxylic Acid (16)
To a solution of 29 (145 mg, 0.46 mmol) in 5% EtOH/H2O (0.1 mL) was added 50% aqueous NaOH (0.07 mL, 1.15 mmol). The mixture was stirred for 16 h at rt. The reaction mixture was concentrated to ~1/2 volume and neutralized (pH ~7) with 1 M HCl. The solution was washed with 3 × 20 mL of EtOAc. The remaining aqueous layer was concentrated in vacuo to give a crude residue, which was purified by preparatory thin layer chromatography (C-18, 1:2 CH3CN/H2O) to provide the title compound 16 (16.7 mg, 13%). LCMS (APCI+): mass calcd for C16H20N2O3, 288.3; m/z found 289 (M + 1), 99%. 1H NMR (400 MHz, MeOH-d4) δ 2.07 (br s, 4 H), 2.39 (br s, 1 H), 2.99 (br s, 2 H), 3.40 (t, J = 4.70 Hz, 2 H), 3.48 (d, 2 H), 4.30 (t, J = 5.03 Hz, 2 H), 6.37 (d, J = 2.95 Hz, 1 H), 6.83 (dd, J = 8.72, 2.28 Hz, 1 H), 7.14 (d, J = 2.28 Hz, 1 H), 7.20 (d, J = 2.95 Hz, 1 H), 7.29 (d, J = 8.72 Hz, 1 H). HPLC: 95.9%, tR = 10.25 min.
To a solution of 27 (500 mg, 1.7 mmol) in MeOH (4 mL) was added sodium borohydride (280 mg, 7.4 mmol) portionwise over 5 min. The mixture was stirred at rt for 16 h and then warmed to 40 °C for 4 h. The mixture was concentrated in vacuo, and the resulting residue was partitioned between 50 mL of saturated NH4Cl (aq) and EtOAc (25 mL). The organic layer was washed with water (25 mL) and brine (25 mL), then dried over Na2SO4, and concentrated in vacuo. The crude material was purified by preparatory thin layer chromatography (SiO2, 10% 7 N NH3 in MeOH/CH2Cl2 (1:20), isocratic) to provide the title compound 17 (18 mg, 4%). LCMS (APCI+): mass calcd for C18H22N2O2, 298.3; m/z found 299 (M + 1), 99%. 1H NMR (400 MHz, DMSO-d6) δ 1.67 (t, J = 3.09 Hz, 4 H), 2.76 (t, J = 5.84 Hz, 2 H), 4.02 (t, J = 5.90 Hz, 2 H), 5.65 (d, J = 3.76 Hz, 1 H), 6.00 (d, J = 4.03 Hz, 1 H), 6.87 (d, J = 8.59 Hz, 2 H), 7.26 (d, J = 8.59 Hz, 2 H), 7.34 (d, J = 5.77 Hz, 2 H), 8.47 (d, J = 5.77 Hz, 2 H). HPLC: 97.7%, tR = 3.19 min.
In a pressure resistant vial, a suspension of 26 (250 mg, 0.5 mmol), thiophene-3-boronic acid (130 mg, 1 mmol), palladium(II) acetate (20 mg, 0.1 mmol), and triphenylphosphine (60 mg, 0.25 mmol) in DME (10 mL) was stirred, and a mixture of K2CO3 (500 mg, 3 mmol) in EtOH (1 mL) and water (1 mL) was added at rt. The tube was sealed, and the mixture was allowed to stir at rt for 30 min, and then it was heated to 90 °C for 16 h. The mixture was cooled to rt and poured into ice−water (200 mL). The resulting material was extracted with EtOAc (250 mL). The organic layers were washed with water (100 mL) and brine (100 mL), then dried over anhydrous Na2SO4. The solvent was removed in vacuo to obtain the crude product. Purification (~75 g of SiO2, 0−20% EtOAc/hexanes, gradient) provided the desired product, BOC-protected intermediate (130 mg, 60%), which was used as such for the next step. To this material (50 mg, 0.1 mmol) dissolved in dioxane (2 mL) was added HCl (4 M in dioxane, 2 mL) at 0 °C. The mixture was allowed to warm to rt and was stirred for 16 h. The solvent was reduced to 1 mL, and Et2O (15 mL) was added to form a precipitate. The resulting solid was filtered and dried in vacuo to provide the title compound 18 as a hydrochloride salt (30 mg, 80%). 1H NMR (400 MHz, DMSO-d6) δ 1.91−2.31 (m, 4H), 3.26−3.40 (m, 2H), 4.05−4.10 (m, 1H), 4.20−4.25 (m, 1H), 4.46 (dd, 1H, J1 = 10.8 Hz, J2 = 3.6 Hz), 7.16 (d, J = 9.2 Hz, 2H), 7.53−7.57 (m, 2H), 7.78−7.87 (m, 7H). LCMS (APCI+): mass calcd for C22H21NO2S, 363.5; m/z found 364.7 (M + 1), 98%.
Nitrobenzene (45 mL) was cooled in an ice bath and treated portionwise with AlCl3 (13.5 g, 101 mmol, 1.15 equiv) and followed by addition of 4-iodobenzoic acid chloride 21 (25 g, 94 mmol, 1.07 equiv) in nitrobenzene (25 mL) while maintaining a maximum of 10 °C. The reaction mixture was stirred at 0 °C for 10 min, whereupon anisole (9.5 g, 88 mmol, 1 equiv) was added dropwise in such a manner that the temperature didn′t exceed 10 °C. The solution was left to warm to rt overnight. The yellow suspension was poured into ice−water (750 mL). The precipitate was collected by filtration and washed with water (3 × 25 mL). Residue was dissolved in CH2Cl2 (1 L), washed sequentially with aqueous NaHCO3 (2 × 150 mL), dried over anhydrous MgSO4, and concentrated in vacuo to provide the title product 22 (26.7 g, 90%). 1H NMR (400 MHz, CDCl3) δ 3.89 (s, 3H), 6.96 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H). MS (APCI+): mass calcd for C14H11IO2, 338.2; m/z found 339.3 (M + 1).
To a solution of 22 (1.7 g, 14 mmol) in CH2Cl2 (20 mL) was added 1 M BBr3 in CH2Cl2 (15 mL, 15 mmol) at −78 °C. The resulting mixture was allowed to warm to rt and was stirred for 6 h. The mixture was poured onto 50 mL of ice−water and extracted with CH2Cl2 (2 × 100 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL) and dried over anhydrous Na2SO4. The solvent was removed in vacuo to obtain the crude product which was purified by recrystallization from acetone−EtOAc−hexane (~1:2:1) to provide the desired product 23 as a white solid (1.4 g, 85%). 1H NMR (400 MHz, CDCl3) δ 6.76 (d, J = 8.5 Hz, 2 H), 6.92 (d, J = 8.1 Hz, 2 H), 7.03 (d, J = 8.3 Hz, 2 H), 7.60 (d, J = 8.2 Hz, 2 H).
(R)-2-(Toluene-4-sulfonyloxymethyl)pyrrolidine-1-carboxylic Acid tert-Butyl Ester (24)
To a stirred solution of (R)-2-hydroxymethylpyrrolidine-1-carboxylic acid tert-butyl ester (12.0 g, 60 mmol) in pyridine (100 mL) at 5 °C is added p-toluenesulfonyl chloride (11.6 g, 60 mmol). The mixture is allowed to warm to rt and is stirred for 16 h. The solvent is removed in vacuo, and the resulting residue was partitioned between EtOAc (500 mL) and water (250 mL). The organic layer was washed with brine (2 × 125 mL) and dried over anhydrous Na2SO4. The solvent was removed in vacuo to obtain the product 24, which was used for the next step without further purification (18.9 g, 90%). 1H NMR (400 MHz, CDCl3) δ 1.39 (d, J = 15.3 Hz, 9 H), 1.65 (br s, 2 H), 1.75−1.85 (m, 1 H), 1.87−1.98 (m, 2 H), 2.40 (d, J = 8.9 Hz, 1 H), 2.45 (s, 3 H), 3.30 (m, J = 6.2 Hz, 1 H), 3.81−4.02 (m, 1 H), 4.06−4.12 (m, 1 H), 7.35 (d, J = 4.2 Hz, 2 H), 7.78 (d, J = 8.2 Hz, 2 H).
(R)-2-(4-Benzylaminophenoxymethyl)pyrrolidine-1-carboxylic Acid tert-Butyl Ester (25)
To a solution of 4-benzylaminophenol (200 mg, 1 mmol) in DMF (3 mL) at 0−5 °C was added NaH (60% in mineral oil, 48 mg, 1.2 mmol) at 0−5 °C. The mixture was stirred at rt for 15 min. A solution of 24 (356 mg, 1 mmol) in DMF (2 mL) was added to the above mixture at 0−5 °C. The reaction mixture was warmed to rt and then heated to 90 °C for 15 h. The resulting mixture was concentrated in vacuo and partitioned between 25 mL of saturated NaHCO3 (aq) and 25 mL of EtOAc. The organic layer was separated, dried over anhydrous MgSO4, and concentrated in vacuo to obtain ~400 mg of crude product, which was purified by flash chromatography (~50 g of SiO2, 12% EtOAc/hexanes, isocratic) to provide the desired product 25 (169 mg, 44%). 1H NMR (400 MHz, CDCl3) δ 1.47 (s, 9 H), 1.78−2.10 (m, 4 H), 3.26−3.47 (m, 2 H), 3.64−3.76 (m, 1 H), 3.76−3.90 (m, 1 H), 3.99−4.18 (m, 2 H), 4.29 (s, 2 H), 6.60 (d, J = 8.7 Hz, 2 H), 6.80 (d, J = 8.1 Hz, 2 H), 7.29−7.41 (m, 5 H).
(R)-2-[4-(4-Iodobenzoyl)phenoxymethyl]pyrrolidine-1-carboxylic Acid tert-Butyl Ester (26)
To a suspension of NaH (60% in mineral oil, 60 mg, 1.5 mmol) in DMF (10 mL) at 0 °C was added 23 (324 mg, 1 mmol). The mixture was allowed to warm to rt and was stirred for 30 min. The mixture was again cooled to 0 °C, and 24 (400 mg, 1.1 mmol) in 1 mL DMF was added. The resulting mixture was allowed to warm to rt, stirred for 30 min, and heated to 95 °C for 16 h. The reaction was cooled to rt and poured into ice−water (100 mL), and the mixture was stirred for 30 min. The resulting solid was filtered and dried in vacuo to provide the desired product 26 (280 mg, 60%). 1H NMR (400 MHz, CDCl3) δ 0.81−0.92 (m, 2 H), 1.27 (s, 2 H), 1.49 (br s, 8 H), 1.56 (s, 7 H), 2.01−2.09 (m, 1 H), 6.96−7.05 (m, 2 H), 7.48 (d, J = 8.2 Hz, 2 H), 7.78 (d, J = 8.7 Hz, 1 H), 7.85 (d, J = 8.1 Hz, 1 H).
A solution of (4-fluorophenyl)pyridin-4-yl-methanone 6 (1.5 g, 7.5 mmol) in anhydrous DMSO (45 mL) was cooled to 0 °C, and potassium tert-butoxide (1.0 g, 8.9 mmol) and 1-(2-hydroxyethyl)pyrrolidine (0.95 mL, 8.1 mmol) were added. The resulting mixture was warmed to rt and then heated to 90 °C for 16 h. The reaction mixture was poured into ice−water (100 mL) and extracted with 3 × 25 mL of EtOAc. The organic layer was washed with water (50 mL) and brine (50 mL), then dried over anhydrous MgSO4 and concentrated in vacuo. The crude mixture was purified by flash chromatography (~150 g of SiO2, 0−7.5% 7 N NH3 in 1:20 MeOH/CH2Cl2 mixture, gradient) to provide the title compound 27 (1.2 g, 54%). 1H NMR (400 MHz, MeOH-d4) δ 1.86 (dt, J = 6.54, 3.24 Hz, 4 H), 2.73 (br s, 4 H), 3.00 (t, J = 5.50 Hz, 2 H), 4.26 (t, J = 5.50 Hz, 2 H), 7.11 (d, J = 8.86 Hz, 2 H), 7.64 (d, J = 5.90 Hz, 2 H), 7.84 (d, J = 8.86 Hz, 2 H), 8.74 (d, J = 6.04 Hz, 2 H). LCMS (ESI+): mass calcd for C18H20N2O2, 296.4; m/z found 297.7 (M + 1).
To a solution of 5-hydroxyindole 11 (2.7 g, 20 mmol) in 2-butanone (50 mL) was added potassium carbonate (5.5 g, 40 mmol) and 1-bromo-2-chloroethane (2.0 mL, 24 mmol). The suspension was stirred under argon and heated to reflux for 60 h. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The resulting residue was purified by flash chromatography (~200 g of SiO2, CH2Cl2/hexanes, 1:2) to provide 28 (650 mg, 17%). 1H NMR (400 MHz, CDCl3) δ 3.84 (t, J = 6.0 Hz, 2 H), 4.29 (t, J = 6.0 Hz, 2 H), 6.50 (br s, 1 H), 6.91 (dd, J = 8.8, 2.3 Hz, 1 H), 7.15 (d, J = 2.1 Hz, 1 H), 7.21 (t, J = 2.7 Hz, 1 H), 7.31 (d, J = 8.9 Hz, 1 H).
1-[2-(1H-Indol-5-yloxy)ethyl]piperidiine-4-carboxylic Acid Ethyl Ester (29)
To a stirred mixture of 28 (150 mg, 0.75 mmol), potassium carbonate (207 mg, 1.5 mmol), and potassium iodide (46 mg, 0.28 mmol) in DMF (6 mL) was added piperidine-4-carboxylic acid ethyl ester (0.23 mL, 1.5 mmol). The reaction mixture was heated to 90 °C for 16 h. After cooling to rt, the mixture was diluted with water (6 mL) and stirred for 2 h. The supernatant liquid was decanted from the resulting residue, which was purified by flash chromatography (~25 g of SiO2, 0−3% MeOH/CH2Cl2, gradient) to provide 29 (145 mg, 57%). 1H NMR (400 MHz, CDCl3) δ 1.26 (t, J = 7.2 Hz, 3 H), 1.75−1.88 (m, 2 H), 1.89−1.97 (m, 2 H), 2.15−2.25 (m, 2 H), 2.25−2.36 (m, 1 H), 2.83 (t, J = 6.0 Hz, 2 H), 3.01 (d, J = 11.7 Hz, 2 H), 4.10−4.20 (m, 4 H), 6.48 (br s, 1 H), 6.88 (dd, J = 8.8, 2.3 Hz, 1 H), 7.12 (d, J = 2.0 Hz, 1 H), 7.19 (t, J = 2.7 Hz, 1 H), 7.30 (d, J = 1.0 Hz, 1 H), 8.07 (br. s, 1 H).