Synthesis, Structure-affinity Relationships and Radiolabeling of Selective High-affinity 5-HT₄ Receptor Ligands as Prospective Imaging Probes for PET

Chemistry

We considered several approaches for modifying the structure of 1 to allow carbon-11 to be introduced as a radiolabel into the structure on the carbonyl side of the ester bond, using readily accessible labeling agents such as [¹¹C]methyl iodide¹⁸, [¹¹C]methyl triflate¹⁹ or [¹¹C]cyanide ion²⁰. These strategies included: i) replacing the iodo group in 1 with a group of similar or smaller size, namely nitrile, as in 5 and 11, or methyl as in 7 (Scheme 1), ii) N-methylation, as in 6 and 8–11 (Scheme 1), and iii) replacement of the dioxan ring by a single O-methyl group, as in 12 and 13 (Scheme 2). Ligand 5 was obtained in low but useful yield (19%) by treating 1 with potassium cyanide. The N-methyl compound 6 was obtained in good yield (60%) by treating the primary arylamine 4 with paraformaldehyde and then sodium borohydride. Treatment of 6 with N-halosuccinimides (NXS, X = Cl, Br or I) gave the respective halo derivatives 8–10 in moderate yields (30–56%). Ligand 7 was obtained from 1 in moderate yield (44%) through Pd-catalyzed methylation with tetramethyltin. Ligand 11 was similarly obtained from ligand 10 but in much lower yield (7%). The ring O-methyl derivatives, 12 and 13, were obtained in moderate (32%) and low (8%) overall yields, respectively, from the corresponding methyl esters 12a and 13a in two steps, namely N-protection and trans-esterification (Scheme 2). Analogs of 13, in which either the ester group was replaced with an amido group, as in 14, or in which the length of the N-alkyl group was altered to vary lipophilicity, as in 15 and 16, were also prepared in two steps and moderate overall yields (30–69%) from 4-amino-3-methoxybenzoic acid (Scheme 3).

For the purpose of creating a radioligand that might be labeled with fluorine-18, we succeeded in preparing 17, a 7-fluoro analog of 1, in six steps from 5-fluoro-2-hydroxybenzoic acid (Scheme 4). All steps proceeded in > 75% yield, except the final two-stage esterification, involving Cbz–protection (34%) and trans-esterification (59%). Also, two fluoroalkoxy analogs of 13, the fluoro-methoxy compound 18 and the 2-fluoroethoxy compound 19, were prepared in two steps from methyl 4-amino-3-hydroxybenzoate in moderate overall yields of 37 and 20%, respectively (Scheme 5).

Potential precursors for radiolabeling were also synthesized, including the phenol precursor 20 by demethylation of 13 (42% yield) (Scheme 6), and the nitro compound 21 in three steps from 8-amino-7-nitro-2,3-dihydrobenzo[b][1,4]dioxine carboxylic acid methyl ester (21a) in 42% overall yield (Scheme 7).

Pharmacological Assays and Screen

Assay of compounds 1, 5, 8, 13, 15 and 17–19 for binding to 5-HT₄ receptors in guinea pig striatal membranes showed all these compounds to have sub-nanomolar K_i values (Table 1). However, assay of the same ligand set against human recombinant 5-HT₄ receptors (h5-HT₄), expressed in HEK293T cells, revealed a greater variation in K_i, with only some of the compounds (1, 8, 13 and 17) showing low nanomolar values. This assay was applied to the full range of new ligands and revealed interesting structure-activity information (Table 1).

Table 1.

Binding affinities (1/K_i),^a efficacies and cLogD values of new 5-HT₄ ligands.

Compound	R1	R2	R3	R4	X	n	5-HT4Ki (nM)a	h5-HT4Ki (nM)b	Efficacyc	pEC50	cLogDd
1	I	H	OCH2	OCH2	O	2	0.20	2.2 ± 0.3	Inv. Ag.	7.9	3.20
4	H	H	OCH2	OCH2	O	2		1.4 ± 0.2	Antagonist	-	1.74
5	CN	H	OCH2	OCH2	O	2	0.45	33 ± 5	n.m.	n.m.	2.59
6	H	Me	OCH2	OCH2	O	2		9.1 ± 0.7	Inv.Ag	8.05	2.37
7	Me	H	OCH2	OCH2	O	2		6.3 ± 1	Antagonist	-	2.20
8	Cl	Me	OCH2	OCH2	O	2	0.577	2.0 ± 0.2	Inv. Ag.	7.85	3.27
9	Br	Me	OCH2	OCH2	O	2		4.5 ± 0.4	Inv. Ag.	8.0	3.36
10	I	Me	OCH2	OCH2	O	2		7.4 ± 0.5	Inv. Ag.	7.7	3.41
11	CN	Me	OCH2	OCH2	O	2		37 ± 5	Inv. Ag.	7.8	2.52
12	H	H	H	OMe	O	2		50 ± 7	Ag.	9.75	1.77
13	H	H	OMe	H	O	2	0.738	2.4 ± 0.3	Antagonist	-	2.07
14	H	H	OMe	H	N	2		8,664 ± 857	n.m.	n.m.	0.16
15	H	H	OMe	H	O	1	0.901	17 ± 1	n.m.	n.m.	1.54
16	H	H	OMe	H	O	3		12 ± 1	n.m.	n.m.	2.60
17	F	H	OCH2	OCH2	O	2	0.334	2.1 ± 0.3	Inv. Ag.	8.23	2.42
18	H	H	OCH2F	H	O	2	0.334	17 ± 1	n.m.	n.m.	1.97
19	H	H	O(CH2)2F	H	O	2	0.215	11 ± 1	n.m.	n.m.	2.30
21	NO2	H	OCH2	OCH2	O	2		7.8 ± 0.9	n.m.	n.m.	3.04

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^a

For guinea pig striatal membrane 5-HT₄ receptors. Values are averages of triplicate measurements.

^b

For h5-HT₄ receptors. Binding assay results are averages of triplicate measurements.

^c

In agonist/inverse agonist assay. Ag. = agonist; Inv. Ag. = Inverse agonist; n.m. = not measured.

^d

cLogD was calculated with ACD software; estimated errors, are approximately ± 1.0.

Replacement of the iodine atom in 1 with a nitrile, methyl or nitro group resulted in approximately 15-, 3- and 3.5-fold decrease in binding affinity, respectively, whereas replacement with fluorine retained binding affinity and replacement with hydrogen slightly increased affinity. N-Methylation of 1 resulted in about a 4-fold reduction in binding affinity, while the N-methylation of the corresponding nitrile, 5, resulted in only a marginal reduction in binding affinity. The N-methyl 8-chloro analog 8 showed a binding affinity comparable to that of 1.

Replacement of the dioxan ring in 1 with a 2-methoxy group gave ligand 12 with much reduced affinity whereas replacement with a 3-methoxy group gave ligand 13 with affinity comparable to that of 1. Shortening or lengthening of the N-alkyl chain length in 13 gave ligands of 5- and 7-fold lower affinity, respectively.

Replacement of the ester group in 13 with an amido group drastically reduced binding affinity.

Replacement of the methoxy group in 13 with fluoromethoxy or 2-fluoroethoxy led to 7- and 5-fold reduction in binding affinity, respectively.

A selection of the compounds was also assessed for intrinsic activity (Table 1). The majority of these ligands were found to be quite potent partial or full inverse agonists with pEC₅₀ values in the range 7.70–8.23. The 2-methoxy ligand 12 was found to be a potent full agonist with a pEC₅₀ value of 9.75. In stark contrast, the high-affinity regional isomer 13 was found to be an antagonist. The arylmethyl analog of 1 was also found to be an antagonist, as was the previously known ligand, 4.

Ligand 13 showed greater than 2000-fold selectivity for h5-HT₄ receptors versus twelve other h5-HT receptors and binding sites (Table 2). Ligands 8 and 17 were not quite as selective among h5-HT receptors and binding sites. The lowest selectivity for 8 was 33-fold versus h5-HT_2B receptors and for 17, 80-fold versus the same receptors (Table 2). Ligand 13 also exhibited high selectivity for h5-HT₄ receptors versus a wide range of non-serotonergic receptors and binding sites (Table 3). For 13, the lowest selectivities for h5-HT₄ receptors were versus σ₁ and σ₂ receptors, namely 88- and 62–fold, respectively. Ligands 8 and 17 were also generally selective for h5-HT₄ receptors versus other receptors and binding sites, except that they displayed much lower selectivities versus σ₁ and especially σ₂ receptors (Table 3). Ligand 8 also showed a quite low 22-fold selectivity versus D₄ receptors (Table 3).

Computation of cLogD

The. cLogD values of ligands ranged from 1.74 to 3.27 (Table 1).

Syntheses of Radioligands

Some ligands, namely ligands 5, 8, 13, 17 and 18, were selected to test their amenability for rapid labeling with a positron-emitter, either carbon-11 or fluorine-18. Ligand [¹¹C]5, a [¹¹C]nitrile, was produced in an average of 26% decay-corrected radiochemical yield (RCY) by Pd-mediated exchange of the iodine atom with no-carrier-added (NCA) [¹¹C]cyanide ion in the presence of potassium carbonate-K 2.2.2 in THF (Scheme 8). Use of potassium dihydrogen phosphate as base in THF or DMSO²¹ gave much lower RCY (Table 4). The N-[¹¹C]methyl ligand, [¹¹C]8, was obtained in 11% RCY by ¹¹C-methylation of 1 with NCA [¹¹C] methyl iodide in the presence of the solid base, Li₃N, under the influence of ultrasound (Scheme 9). Use of Li₂O as solid base resulted in a lower RCY (7%) of [¹¹C]8. The O-[¹¹C]methyl ligand [¹¹C]13 was obtained from the phenol 20 by methylation with either NCA [¹¹C]methyl iodide or [¹¹C]methyl triflate in either 36 or 27% RCY, respectively (Scheme 9). An experiment based on ¹³C/¹¹C co-labeling, followed by ¹³C-NMR,²² confirmed the position of the radiolabel. This radioligand was obtained in moderately high specific radioactivity, namely 2,848 mCi/μmol from [¹¹C]methyl iodide and 2,517 mCi/μmol from [¹¹C]methyl triflate at the end of radiosynthesis.

All attempts to prepare [¹⁸F]17 by substitution of the nitro group in precursor 21 or the N-Boc-protected analog 21c with cyclotron-produced [¹⁸F]fluoride ion were unsuccessful. However, the [¹⁸F]fluoro-di-deutero-methoxy ligand, NCA [¹⁸F]18 was obtained in 13% RCY from [¹⁸F]fluoride ion by treating the phenol 21 with derived [¹⁸F]d₂-fluorobromomethane in acetonitrile with NaOH as base at 100 ºC (Scheme 10). Other tested conditions gave inferior RCYs (Table 5).

Materials

Methyl 3-methoxy-4-nitrobenzoate, 4-(aminomethyl)-1-(n-butyl)-piperidine, and tetra-kis(triphenylphosphine)palladium(0) were purchased from Alfa Aesar (Ward Hill, MA). Chlorofluoromethane and 1-chloro-2-fluoroethane were purchased from SynQuest (Alachua, FL). Other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used as received. Compounds 3 (SB 204070; 1-butylpiperidin-4-yl)methyl 8-amino-7-chloro-2,3-dihydrobenzo[b][1,4]dioxane-5-carboxylate) and the des-chloro analog 4 were synthesized from 2,3-dihydro-benzodioxin by modified literature procedures.^{25, 37, 38}

General Methods

¹H- (400.13 MHz), ¹³C- (100.62 MHz) and ¹⁹F- (376.46 MHz) NMR spectra were recorded at rt on an Avance-400 spectrometer (Bruker, Billerica, MA). Chemical shifts are reported in δ units (ppm) downfield relative to the chemical shift for tetramethylsilane. Abbreviations s, d, t, dd, dt and bs denote singlet, doublet, triplet, doublet of doublet, doublet of triplet and broad singlet. Thin layer chromatography was performed with POLYGRAM^® SIL G/UV₂₅₄ layers (0.2 mm silica gel with fluorescent indicator; Grace Davison Discovery Sciences; Deerfield, IL); compounds were visualized under UV light (λ = 254 nm).

High resolution mass spectra (HRMS) were acquired from the Mass Spectrometry Laboratory, University of Illinois at Urbana-Champaign (Urbana, IL) under electron ionization conditions using a double-focusing high-resolution mass spectrometer (Autospec, Micromass Inc., USA) with samples introduced through a direct insertion probe.

LC-MS analyses of synthesized compounds were performed on an LCQ Deca model instrument (Thermo Fisher Scientific Corp.; Waltham, MA). A gradient or isocratic LC analysis of sample was carried out with binary solvents (A: B; 150 μL/min) composed of water-methanol-acetic acid (90: 10: 0.5 by vol.) (A) and methanol-acetic acid (100: 0.5, v/v) (B) on a Luna C18 column (3 μm, 50 × 2 mm; Phenomenex; Torrance, CA). Following electrospray ionization of the column effluent, ions m/z 150 through 750 were acquired.

Melting points were measured with a Mel-Temp manual apparatus (Electrothermal, Fisher Scientific) and were uncorrected.

γ^-Radioactivity from 11C and 18F was measured using a calibrated dose calibrator (Atomlab 300; Biodex Medical Systems). Radioactivity measurements were corrected for physical decay. All radiochemistry was performed in lead-shielded hot-cells for personnel protection from radiation.

Radioactive products were separated by HPLC on a Gemini or Gemini-NX C18 column (5 μm, 10 × 250 mm; Phenomenex) eluted with 10 mM-NH₄OH-MeCN or 100 mM-HCOONH₄-MeCN at the stated composition and flow rate. Eluates were monitored for radioactivity (pin diode detector; Bioscan) and absorbance at 294 nm (System Gold 166 detector; Beckman).

The purity of each new non-radioactive compound was assessed by reverse phase HPLC under the conditions tabulated in Supporting Information. Each compound was shown to have a chemical purity of > 98%. Radioactive compounds were analyzed with HPLC on a Gemini C18 column (5 μm, 4.6 × 150 mm) or Gemini-NX C18 column (5 μm, 4.6 × 250 mm; Phenomenex) eluted with 10 mM-NH₄OH-MeCN or 100 mM-HCOONH₄-MeCN at the later stated composition and flow rate. Eluates were monitored for radioactivity (pin diode detector; Bioscan). Samples were injected alone, and then co-injected with the reference non-radioactive compound to check for co-elution. RCYs were calculated for labeled products isolated with HPLC.

Computation of cLogP and cLogD

cLogP and cLogD (at pH 7.4) values for ligands were computed with ACD software.

(1-Butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1)

N-Iodosuccinimide (498 mg, 2.21 mmol) was added portion-wise to a solution of 4 (770 mg, 2.21 mmol) in acetic acid (5 mL) at 0 °C and stirred at rt for 2 h. The acetic acid was evaporated off, and the residue was basified with NaHCO₃ solution and extracted twice with CH₂Cl₂. The combined organic layers were evaporated to dryness. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 20 v/v) of the residue gave 1 as a light yellow oil (410 mg, 40%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.25–1.53 (6H, m), 1.74–1.79 (3H, m), 1.94 (2H, t, J = 11.2 Hz), 2.31–2.34 (2H, m), 2.96 (2H, d, J = 11.2 Hz), 4.10 (2H, d, J = 6.4 Hz), 4.30–4.38 (4H, m), 4.54 (2H, bs), 7.82 (1H, s). ¹³C-NMR (CDCl₃): δ 14.09, 20.92, 29.02, 29.17, 35.53, 53.44, 58.89, 63.97, 64.44, 68.86, 71.21, 110.80, 129.77, 133.29, 141.06, 144.75, 164.14.

(1-Butylpiperidin-4-yl)methyl 8-amino-7-cyano-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (5) ³⁹

A mixture of 1 (200 mg, 0.42 mmol), KCN (42 mg, 0.64 mmol), CuI (17 mg, 0.084 mmol) and 1,10-phenanthroline (30 mg, 0.17 mmol) in DMF (500 μL) was stirred at 110 °C in an oven-dried sealed tube under Ar for 42 h. Then the mixture was cooled to rt and filtered through celite. The celite pad was rinsed twice with CH₂Cl₂. The combined filtrates were evaporated to dryness and dissolved in MeOH. Separation by HPLC on an XTerra RP18 column (10 μm, 19 × 250 mm; Waters) eluted with MeOH -aq. NH₄OH (0.025%) (9: 1 v/v) at 10 mL/min) gave 5 as a pale yellow solid (t_R = 14 min; 30 mg, 19%). Mp: 108–110 °C. ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.28–1.50 (6H, m), 1.81–1.84 (3H, m), 2.15 (2H, t, J = 11.4 Hz), 2.51 (2H, m), 3.16 (2H, d, J = 11.6 Hz), 4.12 (2H, d, J = 6.0 Hz), 4.34–4.44 (4H, m), 4.87 (2H, s), 7.67 (1H, s). ¹³C-NMR (CDCl₃): δ 14.09, 20.92, 29.06, 29.23, 35.53, 53.42, 58.89, 63.73, 64.85, 69.13, 88.00, 110.11, 116.81, 128.80, 129.90, 143.32, 147.47, 163.64. LC-MS m/z: [M + H]⁺, 374.2. HRMS: calc’d for C₂₀H₂₇N₃O₄ (M⁺ + H), 374.2080; found, 374.2070.

(1-Butylpiperidin-4-yl)methyl 8-(methylamino)-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (6) ⁴⁰

A mixture of 4 (2.24 g, 6.44 mmol) and paraformaldehyde (580 mg) in ethanol (58 mL) was heated at 60 °C overnight and then NaBH₄ (245 mg, 6.44 mmol) was added. The mixture was heated at 70 °C for 2 h and then evaporated to dryness. The residue was diluted with water and extracted thrice with CH₂Cl₂. The combined organic layers were dried on MgSO₄ and evaporated to dryness. Silica gel chromatography of the residue (MeOH-CH₂Cl₂, 1: 20 v/v) gave 6 as a colorless oil (1.4 g, 60%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.29–1.35 (2H, m), 1.52–1.53 (4H, m), 1.78–1.82 (3H, m), 1.99 (2H, t, J = 11.2 Hz ), 2.39–2.35 (2H, m), 2.90 (2H, d, J = 5.2 Hz), 3.02 (2H, d, J = 11.2 Hz), 4.10 (2H, d, J = 6.0 Hz), 4.28–4.30 (2H, m), 4.34–4.36 (2H, m), 4.46–4.52 (1H, m), 6.18 (1H, d, J = 8.8 Hz), 7.51 (1H, d, J = 8.8 Hz). ¹³C-NMR (CDCl₃): δ 14.02, 20.85, 28.78, 28.88, 29.85, 35.47, 53.38, 58.75, 63.83, 64.50, 68.18, 100.99, 107.36, 125.60, 129.78, 143.15, 144.11, 165.40. LC-MS m/z: [M + H]⁺, 363.3. HRMS: calc’d for C₂₀H₃₀N₂O₄ (M⁺ + H), 363.2284; found, 363.2281.

(1-Butylpiperidin-4-yl)methyl 8-amino-7-methyl-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (7) ⁴¹

1 (210 mg, 0.44 mmol), N-methyl pyrrolidone (560 μL), Pd₂(dba)₃ (14 mg, 0.015 mmol) and PPh₃ (28 mg, 0.11 mmol) were added to an oven-dried sealed tube. The mixture was heated at 50 °C for 10 min and then CuI (6.0 mg, 0.031mmol) was added. The mixture was stirred for another 10 min and then Me₄Sn (91 μL, 0.65 mmol) was added. The mixture was heated at 70 °C for 48 h. The solvent was evaporated off and, the residue was diluted with water and extracted thrice with CH₂Cl₂. The combined organic layers were dried over MgSO₄ and evaporated to dryness. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 20 v/v) of the residue gave 7 as a pale yellow oil (70 mg, 44%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.25–1.58 (7H, m), 1.72–1.81 (3H, m), 1.78–1.82 (3H, m), 1.95 (2H, t, J = 11.2 Hz), 2.11 (3H, s), 2.31–2.35 (2H, m), 2.98 (2H, d, J = 11.6 Hz), 4.08 (2H, s), 4.10 (2H, d, J = 6.4 Hz), 4.30–4.36 (4H, m), 7.28 (1H, s). ¹³C-NMR (CDCl₃): δ 14.02, 16.48, 20.84, 28.69, 35.39, 53.34, 58.72, 63.87, 64.45, 68.27, 107.79, 113.41, 125.34, 130.17, 138.86, 143.34, 165.46. LC-MS: m/z [M + H]⁺, 363.1. HRMS: calc’d for C₂₀H₃₀N₂O₄ (M⁺ + H), 363.2284; found, 363.2279.

(1-Butylpiperidin-4-yl)methyl 7-chloro-8-(methylamino)-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (8)

N-Chlorosuccinimide (70 mg, 0.52 mmol) was added in portions to a stirred solution of 6 (190 mg, 0.52 mmol) in acetic acid (5 mL) at rt and left for 1 h. The acetic acid was evaporated off and the residue was basified with NaHCO₃ solution and extracted thrice with CH₂Cl₂. The combined organic layers were evaporated to dryness. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 20 v/v) of the residue gave 8 as a pale yellow oil (80 mg, 38%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.29–1.35 (2H, m), 1.44–1.53 (4H, m), 1.75–1.80 (3H, m), 1.98 (2H, t, J = 11.2 Hz), 2.34–2.38 (2H, m), 3.00 (2H, d, J = 11.2 Hz), 3.12 (3H, d, J = 4.8 Hz), 4.10 (2H, d, J = 6.4 Hz), 4.28–4.36 (5H, m), 7.45 (1H, s). ¹³C-NMR (CDCl₃): δ 14.03, 20.84, 28.83, 28.97, 33.82, 35.42, 53.35, 58.76, 63.63, 64.26, 68.72, 109.54, 112.30, 124.79, 133.42, 140.14, 144.06, 164.27. LC-MS: m/z [M + H]⁺, 397.6. HRMS: calc’d for C₂₀H₂₉ClN₂O₄ (M⁺ + H), 397.1894; found, 397.1886.

(1-Butylpiperidin-4-yl)methyl 7-bromo-8-(methylamino)-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (9)

The procedure for the synthesis of 8 was used with a solution of 6 (140 mg, 0.39 mmol) in acetic acid (4 mL) and with N-bromosuccinimide (70 mg, 0.39 mmol) replacing N-chlorosuccinimide, and gave 9 as a pale yellow oil (50 mg, 30%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.29–1.52 (6H, m), 1.73–1.78 (3H, m), 1.92 (2H, dt, J = 2.4, 11.6 Hz ), 2.29–2.33 (2H, m), 2.96 (2H, d, J = 11.2 Hz), 3.10 (3H, d, J = 5.2 Hz), 4.10 (2H, d, J = 6.4 Hz), 4.27–4.37 (5H, m), 7.62 (1H, s). ¹³C-NMR (CDCl₃): δ 14.09, 20.93, 29.11, 29.27, 34.11, 35.58, 53.48, 58.93, 63.60, 64.31, 68.96, 102.04, 110.64, 127.61, 133.67, 141.18, 144.76, 164.21. LC-MS: m/z [M + H]⁺ 441.1. HRMS: calc’d for C₂₀H₂₉⁸⁰BrN₂O₄ (M⁺ + H), 441.1389; found, 441.1368.

(1-Butylpiperidin-4-yl)methyl 7-iodo-8-(methylamino)-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (10)

The procedure for the synthesis of 8 was used with a solution of 6 (195 mg, 0.54 mmol) in acetic acid (4 mL) and with N-iodosuccinimide (122 mg, 0.54 mmol) replacing N-chlorosuccinimide to give 10 as a colorless oil (147 mg, 56%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.19–1.54 (6H, m), 1.72–1.80 (3H, m), 1.96 (2H, t, J = 11.2 Hz), 2.33–2.37 (2H, m), 3.00 (2H, d, J = 11.2 Hz), 3.07 (3H, d, J = 3.2 Hz), 4.05 (1H, bs), 4.09–4.11 (3H, m), 4.28–4.38 (4H, m), 7.84 (1H, s). ¹³C-NMR (CDCl₃): δ 14.06, 20.89, 28.91, 29.06, 34.50, 35.47, 53.39, 58.83, 63.57, 64.35, 68.88, 112.25, 133.12, 133.80, 143.57, 145.67, 164.02. LC-MS: m/z [M + H]⁺, 489.2. HRMS: calc’d for C₂₀H₂₉IN₂O₄ (M⁺ + H), 489.1250; found, 489.1258.

(1-Butylpiperidin-4-yl)methyl 7-cyano-8-(methylamino)-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (11) ³⁹

A mixture of 10 (140 mg, 0.29 mmol), KCN (21 mg, 0.32 mmol), CuI (6.0 mg, 0.029 mmol), 1,10-phenanthroline (11 mg, 0.057 mmol) in DMF (300 μL) was stirred at 110 °C in an oven-dried sealed tube under Ar for 48 h. The mixture was cooled to rt and then filtered through celite. The celite pad was rinsed twice with CH₂Cl₂. The combined filtrates were evaporated to dryness and then dissolved in MeOH. Separation by HPLC on an XTerra RP18 column (10 μm, 19 × 250 mm; Waters) eluted with MeOH-aq. NH₄OH (0.025%) (9: 1 v/v) at 15 mL/min gave 11 (t_R = 10.31 min; 8 mg, 7%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.25–1.52 (7H, m), 1.73–1.79 (3H, m), 1.90–1.96 (2H, t, J = 11.2 Hz), 2.30–2.34 (2H, m), 2.96 (2H, d, J = 11.2 Hz), 3.33 (3H, d, J = 5.2 Hz), 4.09 (2H, d, J = 6.0 Hz), 4.30–4.40 (4H, m), 5.04–5.06 (1H, m), 7.71 (1H, s). ¹³C-NMR (CDCl₃): δ 14.09, 20.92, 29.10, 29.26, 31.63, 35.53, 53.44, 58.91, 63.79, 64.61, 69.07, 85.21, 109.42, 119.52, 130.19, 131.81, 144.86, 145.98, 163.61. LC-MS: m/z [M + H]⁺, 388.2. HRMS: calc’d for C₂₁H₂₉N₃O₄ (M⁺ + H), 388.2236; found, 388.2219.

Methyl 4-(benzyloxycarbonylamino)-2-methoxybenzoate (12b) ⁴²

Saturated NaHCO₃ solution (50 mL) and then CbzCl (1.70 mL, 12.1 mmol) were added to a solution of methyl 4-amino-2-methoxybenzoate (12a, 2.0 g, 11 mmol) in THF (50 mL). The mixture was stirred at rt for 4 h and then filtered through celite. The filtrate was acidified to pH < 1 and extracted with EtOAc. After concentration of this solution, the residue was purified by recrystallization from EtOAc-hexane to give 12b as a white solid (3.09 g, 89%). Mp: 132–134 °C. ¹H-NMR (CDCl₃): δ 3.84 (3H, s), 3.87 (3H, s), 5.19 (2H, s), 6.77 (1H, dd, J = 1.6, 8.4 Hz), 6.66, 7.11 (1H, s), 7.35–7.39 (5H, m). 7.79 (1H, d, J = 8.4 Hz). ¹³C-NMR (CDCl₃): δ 51.84, 55.97, 67.30, 101.72, 109.43, 114.05, 128.32, 128.52, 128.68, 133.06, 135.67, 143.19, 152.96, 160.76, 166.07.

(1-Butylpiperidin-4-yl)methyl 4-amino-2-methoxybenzoate (12) ⁴³

A THF solution of n-BuLi (1.6M; 612 μL, 0.98 mmol) was added drop-wise to a solution of (4-butylpiperidin-1-yl)methanol (190 mg, 1.11 mmol) in THF (3 mL) in an oven-dried flask under Ar at 0°C. After stirring the mixture for 10 min, a solution of 12b (350 mg, 1.11 mmol) in THF (3 mL) was added drop-wise. The mixture was stirrred for 2 h, poured into water and extracted with CH₂Cl₂. The combined organic layers were dried on MgSO₄ and evaporated to dryness. Silica gel chromatography of the residue (MeOH-CH₂Cl₂, 1: 15 v/v) gave a white solid (200 mg). MeOH (15 mL) was added to this solid (170 mg) plus Pd/C (10%; 25 mg). The mixture was degassed with H₂ for 30 min, stirred at rt overnight, filtered and evaporated to dryness. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 20 v/v) of the residue gave 12 as a colorless oil (108 mg, 36%). ¹H-NMR (CDCl₃): δ 0.91 (3H, t, J = 7.6 Hz), 1.28–1.53 (6H, m), 1.71–1.80 (3H, m), 1.95 (2H, t, J = 11.2 Hz), 2.31–2.35 (2H, m), 2.97 (2H, d, J = 11.6 Hz), 3.81 (3H, s), 4.09 (2H, d, J = 6.0 Hz), 4.22 (2H, br), 6.18–6.22 (2H, m), 7.72 (1H, d, J = 8.4 Hz). ¹³C-NMR (CDCl₃): δ 14.06, 20.88, 28.94, 29.07, 35.53, 53.45, 55.66, 58.84, 68.33, 97.58, 106.23, 108.78, 134.06, 152.29, 161.87, 165.63. LC-MS: m/z [M + H]⁺_, 321.1. HRMS: calc’d for C₁₈H₂₈N₂O₃ (M⁺ + H), 321.2178; found, 321.2181.

Methyl 4-amino-3-methoxybenzoate (13a)

Pd/C (10%; 1.8 g) and HCOOK (7.4 g, 88 mmol) were added to a solution of methyl 3-methoxy-4-nitrobenzoate (2.0 g, 9.5 mmol) in MeOH (50 mL). The mixture was refluxed at 80 °C for 1 h. The suspension was cooled and filtered through celite. The filtrate was evaporated to dryness. Silica gel chromatography (30% EtOAc in hexane) of the residue gave 13a as a white solid (1.72 g, 100%). Mp: 127–129 °C. Lit. mp 127–128 °C.^{44 1}H-NMR (CDCl₃): δ 3.86 (3H, s), 3.90 (3H, s), 4.22 (2H, br), 4.45 (1H, s), 6.66 (1H, d, J = 8.4 Hz), 7.55 (1H, d, J = 8.4 Hz). ¹³C-NMR (CDCl₃): δ 51.69, 55.58, 111.15, 113.09, 119.41, 124.09, 141.21, 146.10, 167.35.

Methyl 4-(benzyloxycarbonylamino)-3-methoxybenzoate (13b)

As described for 12b from 12a, 13b was obtained from 13a as a white solid (58%). Mp: 95–96 °C. ¹H-NMR (CDCl₃): δ 3.89 (6H, 2s), 5.22 (2H, s), 7.34–7.52 (6H, m), 7.69 (1H, dd, J = 1.6, 8.4 Hz), 8.20 (1H, d, J = 8.4 Hz). ¹³C-NMR (CDCl₃): δ 52.05, 55.90, 67.27, 110.73, 116.92, 123.48, 124.25, 128.43, 128.49, 128.68, 132.06, 135.84, 146.96, 152.93, 166.81.

(1-Butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate (13)

As described for 12 from 12b, compound 13 was obtained from 13b as a white solid in 13% yield. Mp: 162–164 °C. ¹H-NMR (CDCl₃): δ 0.94 (3H, t, J = 7.2 Hz), 1.31–1.39 (2H, m), 1.72–1.80 (2H, m), 1.95–1.99 (5H, br), 2.55 (2H, br), 2.75–2.79 (2H, m), 3.37 (2H, d, J = 11.6 Hz), 3.90 (3H, s), 4.18 (2H, d, J = 4.0 Hz), 4.38 (2H, br), 6.69 (1H, d, J = 8.0 Hz), 7.43 (1H, d, J = 2 Hz), 7.52–7.53 (1H, dd, J = 1.6, 8 Hz); ¹³C-NMR: δ (CDCl₃) 14.09, 20.94, 29.10, 29.25, 35.71, 53.50, 55.63, 58.94, 68.84, 111.21, 113.05, 119.75, 124.02, 141.09, 146.16, 166.86. LC-MS: m/z [M + H]⁺, 321.3. HRMS: calc’d for C₁₈H₂₈N₂O₃ (M⁺ + H), 321.2178; found, 321.2190.

4-Amino-N-((1-butylpiperidin-4-yl)methyl)-3-methoxybenzamide (14) ³⁷

CDI (485 mg, 2.99 mmol) was added in portions to a suspension of 4-amino-3-methoxybenzoic acid (500 mg, 2.99 mmol) in MeCN (30 mL) in an oven-dried flask and then stirred for 2 h at rt. (1-Butylpiperidin-4-yl)methanamine (510 mg, 2.99 mmol) in MeCN (10 mL) was added drop-wise. The above solution was stirred overnight and evaporated to dryness. Silica gel chromatography (from 9% to 33%. MeOH in CH₂Cl₂) of the residue gave an oil which was then dissolved in MeCN and filtered through an Iso-Disc^™ Filter (PTFE 25-4, 25 mm × 0.45 μm). The filtrate was dried to give 14 as a light brown foam-like oil (660 mg, 69%). ¹H-NMR (CDCl₃): δ 0.91 (3H, t, J = 7.2 Hz), 1.26–1.38 (4H, m), 1.42–1.48 (2H, m), 1.57–1.64 (1H, m), 1.88 (2H, dt, J = 2.0, 11.6 Hz), 2.27–2.31 (2H, m), 2.92 (2H, d, J = 11.6 Hz), 3.30 (2H, t, J = 6.4 Hz), 3.86 (3H, s), 4.14 (2H, bs), 6.34 (1H, t, J = 5.6 Hz), 6.63 (1H, d, J = 8.0 Hz), 7.14 (1H, dd, J = 2.0, 8.4 Hz), 7.37 (1H, d, J = 1.6 Hz). ¹³C-NMR (CDCl₃): δ 12.58, 19.41, 27.72, 28.58, 34.79, 43.98, 52.06, 54.08, 57.36, 108.34, 111.67, 118.05, 122.76, 138.15, 145.21, 166.04. LC-MS: m/z [M + H]⁺, 320.2. HRMS: calc’d for C₁₈H₃₀N₃O₂ (M⁺ + H), 320.2338; found, 320.2328.

(1-Propylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate (15)

CDI (485 mg, 2.99 mmol) was added in portions to a suspension of 4-amino-3-methoxybenzoic acid (500 mg, 2.99 mmol) in MeCN (15 mL) in an oven-dried flask and stirred for 30 min at rt. Solvent was then evaporated off and the residue dissolved in anhydrous THF (7 mL). In another oven-dried flask, (1-propylpiperidin-4-yl)methanol (319 mg, 2.03 mmol) was dissolved in anhydrous THF (7 mL) and cooled (ice-bath). A THF solution of n-BuLi (1.6M, 1.27 mL, 2.03 mmol) was added drop-wise to this solution and stirred for 10 min. The solution of CDI-activated acid was then added, stirred overnight and evaporated to dryness. The residue was diluted with water and extracted thrice with CH₂Cl₂. The combined organic layers were dried on MgSO₄ and evaporated to dryess. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 30 v/v) of the residue gave 15 as a yellow oil (312 mg, 50%). ¹H-NMR (CDCl₃): δ 0.89 (3H, t, J = 7.2 Hz), 1.37–1.56 (4H, m), 1.72–1.78 (2H, m), 1.94 (2H, dt, J = 2.0, 12.0 Hz), 2.25–2.29 (2H, m), 2.94 (2H, d, J = 11.6 Hz), 3.86 (3H, s), 4.13 (2H, d, J = 6.0 Hz), 4.36 (2H, bs), 6.63 (1H, d, J = 8.4 Hz), 7.44 (1H, d, J = 2.0 Hz), 7.54 (1H, dd, J = 1.6, 8.0 Hz). ¹³C-NMR (CDCl₃): δ 12.05, 20.15, 29.06, 35.67, 53.40, 55.50, 61.09, 68.76, 111.08, 112.90, 119.35, 124.03, 141.31, 146.03, 166.83. LC-MS: m/z [M + H]⁺_, 307.2. HRMS: calc’d for C₁₇H₂₆N₂O₃ (M⁺ + H), 307.2022; found, 307.2022.

(1-Pentylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate (16)

As described for 15, compound 16 was obtained from 4-amino-3-methoxybenzoic acid and 1-pentylpiperidin-4-yl)methanol as a pale yellow oil (200 mg, 30%). ¹H-NMR (CDCl₃): δ 0.89 (3H, t, J = 7.2 Hz), 1.24–1.52 (8H, m), 1.77–1.79 (3H, m), 1.89–1.95 (2H, dt, J = 2.4, 12 Hz), 1.95–2.32 (2H, m), 2.96 (2H, d, J = 11.6 Hz), 3.89 (3H, s), 4.13 (2H, d, J = 6.4 Hz), 4.26 (2H, bs), 6.65 (1H, d, J = 8 Hz), 7.45 (1H, d, J = 1.6 Hz), 7.55 (1H, dd, J = 2, 8.4 Hz). ¹³C-NMR (CDCl₃): δ 14.06, 22.64, 26.78, 29.10, 29.94, 35.71, 53.49, 55.59, 59.23, 68.82, 111.16, 113.00, 119.61, 124.03, 141.17, 146.12, 166.86. LC-MS: m/z [M + H]⁺ 335.2. HRMS: calc’d for C₁₉H₃₀N₂O₃(M⁺_,+ H), 335.2335; found, 335.2325.

3-Bromo-5-fluoro-2-hydroxybenzoic acid (17a)

5-Fluoro-2-hydroxybenzoic acid (20 g, 0.13 mol) and N-bromosuccinimide (23 g, 0.13 mol) were added to acetic acid (200 mL). The mixture was heated at 80 °C for 24 h. After evaporation of all acetic acid, the residue was recrystalized from EtOAc and hexane to give 17a (27 g, 90%). Mp 233–235 °C. Lit. mp 233°C.^{45 1}H-NMR (MeOD): δ 7.48–7.50 (2H, br). ¹³C-NMR (MeOD): δ 111.91 (d, J = 10.0 Hz), 114.92 (d, J = 7.0 Hz), 116.22 (d, J = 24.0 Hz), 127.13 (d, J = 26.0 Hz), 155.70 (d, J = 239 Hz), 156.56, 172.24.

5-Fluoro-2,3-dihydroxybenzoic acid (17b) ⁴⁶

NaOH solution (2.5M; 300 mL) was stirred under an aspirator for 2 h. Then CuSO₄ (250 mg, 1.57 mmol) was added and the solution further stirred for 1h. To the filtrate of this solution was added 17a (22 g, 0.094 mol) and the mixture was refluxed overnight. The reaction mixture was cooled, acidified with 37% HCl to pH < 2 and then evaporated to dryness. The residue was dissoved in MeOH and filtered through celite. The combined filtrates were evaporated to dryness. Silica gel chromatography (30% EtOAc in hexane with 1% HOAc) of the residue gave 17b as a white solid (12 g, 75%). Mp 184–186 °C. ¹H-NMR (MeOD): δ 6.58 (1H, dd, J = 3.2, 9.6 Hz), 7.01 (1H, dd, J = 3.0, 9.0 Hz). ¹³C-NMR (MeOD): δ 105.80 (d, J = 24.0 Hz), 109.44 (d, J = 26.0 Hz), 113.48 (d, J = 9.0 Hz), 148.36, 148.50 (d, J = 9.0 Hz), 156.24 (d, J = 234 Hz), 173.03. ¹⁹F-NMR (CDCl₃): δ −125.39. HRMS: calc’d for C₇H₅FO₄ (M⁺ + H), 172.01719; found, 172.01717.

Methyl 7-fluoro-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (17c) ⁴⁷

TMSCHN₂ (2.0M, 45 mL, 90 mmol) was added in portions to a solution of 17b (6.0 g, 35 mmol) in MeOH (150 mL) and Et₂O (150 mL). After 20 min the solvent was evaporated off. Silica gel chromatography (15% EtOAc in hexane) of the residue gave the methyl ester (5.6 g, 93%). A mixture of this ester (3.9 g, 20 mmol) and Cs₂CO₃ (16.2 g, 49.8 mmol) in DMF (40 mL) was stirred at rt for 0.5 h. Then 1,2-dibromoethane (5.07 g, 27.0 mmol) was added and the mixture stirred at 80 °C for 16 h. The mixture was cooled to rt and filtered through celite, which was then rinsed twice with DMF. The combined DMF rinses were evaporated to dryness under high vacuum to give a dark red residue, which after silica gel chromatography (20% EtOAc in hexane with 1% HOAc) gave 17c as a white solid (3.37 g, 77%). Mp 108–110 °C. ¹H-NMR (MeOD): δ 3.89 (3H, s), 4.28–4.34 (4H, m), 6.77 (1H, dd, J = 3.2, 8.8 Hz), 7.11 (1H, dd, J = 3.2, 8.8 Hz). ¹³C-NMR (MeOD): δ 52.27, 64.10, 64.30, 108.71 (d, J = 26.0 Hz), 109.73 (d, J = 24.0 Hz), 120.07 (d, J = 9.0 Hz), 140.65, 144.69 (d, J = 12.0 Hz), 155.69 (d, J = 238 Hz), 164.99. ¹⁹F-NMR (CDCl₃): δ −121.14. HRMS: calc’d for C₁₀H₉FO₄ (M⁺ + H), 213.0563; found, 213.0561.

Methyl 7-fluoro-8-nitro-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (17d)

17c (3.4 g, 15.9 mol) was added in portions to a flask cooled between −50 and −60 °C and containing HNO₃ (90%; 30 mL). After 16 min, the reaction mixture was warmed gradually and water was added. The precipitate was filtered off and washed with water to give 17d as a yellow solid (3.58 g, 88%). 17d was used in the next step without further purification. Mp 133–135 °C. ¹H-NMR (CDCl₃): δ 3.92 (3H, s), 4.33–4.40 (4H, m), 6.83 (1H, d, J = 11.2 Hz). ¹³C-NMR (CDCl₃): δ 53.59, 64.22, 64.80, 107.12 (d, J = 25.0 Hz), 119.75 (d, J = 1.0 Hz), 137.57 (d, J = 3.0 Hz), 148.50 (d, J = 12.0 Hz), 150.10 (d, J = 257 Hz), 162.83 (d, J = 3.0 Hz). ¹⁹F-NMR (CDCl₃): δ −124.76. HRMS: calc’d for C₁₀H₈FNO₆ (M⁺ + Na), 280.0233; found, 280.0225.

8-Amino-7-fluoro-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylic acid (17e)

Pd/C (10%, 340 mg) and potassium formate (1.4 g, 17 mmol) were added to a solution of 17d (500 mg, 1.94 mmol) in MeOH (10 mL). The mixture was refluxed at 80 °C for 2 h, and then cooled and filtered through celite. 37% HCl was added to the filtrate until no CO₂ was released. The white solid was filtered off and the filtrate evaporated to dryness. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 30 v/v) of the residue gave 17e as light brown solid (350 mg, 85%). Mp 174–176°C. ¹H-NMR (MeOD): δ 4.08–4.10 (2H, m), 4.18–4.21 (2H, m), 6.67 (1H, d, J = 11.6 Hz). ¹³C-NMR (MeOD): δ 65.06, 66.29, 106.06 (d, J = 4.0 Hz), 109.21 (d, J = 23.0 Hz), 134.30 (d, J = 11.0 Hz), 134.44, 140.54 (d, J = 3.0 Hz), 147.01 (d, J = 232 Hz), 169.25. ¹⁹F-NMR (MeOD): δ −143.14. HRMS: calc’d for C₉H₈FNO₄ (M⁺ + H), 214.0516; found, 214.0510.

(1-Butylpiperidin-4-yl)methyl 8-amino-7-fluoro-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (17)

Saturated NaHCO₃ solution (5 mL) and THF (20 mL), followed by CbzCl (662 mg, 3.87 mmol) were added to a flask containing 17e (750 mg, 3.52 mmol). After stirring the mixture overnight, the THF was evaporated off. The solution was diluted with water, acidified with 37% HCl to pH <1 and extracted thrice with EtOAc. The combined organic layers were dried on MgSO₄ and evaporated to dryness. Silica gel chromatography (30% EtOAc in hexane) of the residue gave Cbz-protected 17e as a white solid (420 mg, 34%).

Cbz-protected 17e (420 mg, 1.21 mmol), CDI (196 mg, 1.21 mmol) and MeCN (30 mL) were added to an oven-dried flask under Ar. The mixture was stirred for 2 h and evaporated to dryness. The residue was dissolved in THF (10 mL). In another oven-dried flask, a solution of (4-butylpiperidin-1-yl)methanol (207 mg, 1.21 mmol) in THF (10 mL) was added drop-wise to a THF solution of n-BuLi (1.6M; 760 μL, 1.21 mmol) under Ar at 0 °C and stirred at this temperature for 10 min. Then the activated acid in THF was added drop-wise to the prepared lithium alkoxide solution. The mixture was warmed to rt and stirred overnight. The reaction mixture was evaporated to remove THF, diluted with water and extracted with CH₂Cl₂. The residue was purified by silica gel chromatography (MeOH-CH₂Cl₂, 1: 15 v/v) to give an oil. Pd/C (10%; 25 mg) was then added to a solution of the oil in MeOH (15 mL). The suspension was degassed for 30 min with H₂ and then stirred at rt overnight under H₂. The mixture was filtered through celite and evaporated to dryness. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 15 v/v) of the residue gave 17 as a yellow solid (260 mg, 59%). Mp 160–162 °C. ¹H-NMR (CDCl₃): δ 0.95 (3H, t, J = 7.4 Hz), 1.33–1.39 (2H, m), 1.70–1.95 (7H, m), 2.39–2.42 (2H, m), 2.67–2.71 (2H, m), 3.31 (2H, d, J = 11.6 Hz), 4.19–4.27 (6H, m), 4.88 (2H, s), 6.71 (1H, d, J = 11.6 Hz). ¹³C-NMR (CDCl₃): δ 13.74, 20.46, 26.97, 34.35, 52.56, 57.84, 63.84, 64.56, 68.14, 105.60 (d, J = 4.0 Hz), 108.19 (d, J = 23.0 Hz), 132.20 (d, J = 15.0 Hz), 133.43 (d, J = 11.0 Hz), 139.81 (d, J = 3.0 Hz), 145.24 (d, J = 232 Hz), 166.72 (d, J = 4 Hz). ¹⁹F-NMR (CDCl₃): δ −141.99. LC-MS: m/z [M + H]⁺_, 367.1. HRMS: calc’d for C₁₉H₂₇FN₂O₄ (M⁺ + H), 367.2033; found, 367.2033.

Methyl 4-amino-3-(fluoromethoxy)benzoate (18a) ⁴⁸

Chlorofluoromethane was bubbled into a tube (30-mL) containing anhydrous DMF (10 mL), methyl 4-amino-3-hydroxybenzoate (500 mg, 2.99 mmol) and Cs₂CO₃ (1.95 g, 5.98 mmol) for 14 min at −70 ºC. The tube was then sealed and slowly warmed to rt. The mixture was stirred for 5 d then filtered through celite which was then rinsed thrice with EtOAc. The combined rinses were evaporated to dryness. Silica gel chromatography (20% EtOAc in hexane) of the residue gave 18a as a pale yellow solid (368 mg, 62%). Mp: 69–70 °C. ¹H-NMR (CDCl₃): δ 3.84 (3H, s), 4.46 (2H, br), 5.71 (2H, d, J = 54.4 Hz), 6.69 (1H, d, J = 8 Hz), 7.63 (1H, dd, J = 8.4, 1.6 Hz), 7.68 (1H, s). ¹³C-NMR (CDCl₃): δ 51.73, 100.22, 102.40, 114.25, 116.72, 119.23, 126.79, 142.44, 142.92, 142.95, 166.90. ¹⁹F-NMR (CDCl₃): δ −147.56. LC-MS: m/z [M + H]⁺ 200.1. HRMS: calc’d for C₉H₁₁FNO₃ (M⁺ + H), 200.0723; found, 200.0723.

(1-Butylpiperidin-4-yl)methyl 4-amino-3-(fluoromethoxy)benzoate (18)

n-BuLi (1.6M; 1.6 mmol; 1 mL) was added drop-wise to a solution of (4-butylpiperidin-1-yl)methanol (280 mg, 1.64 mmol) in THF (2 mL) contained in an oven-dried flask under Ar at 0 °C. The solution was stirred for 10 min and 18a (160 mg, 0.80 mmol) in THF (2 mL) was added drop-wise. The mixture was stirred overnight, poured into water and extracted with CH₂Cl₂. The combined organic layers were dried on MgSO₄ and evaporated to dryness. Silica gel chromatography (MeOH-CH₂Cl₂, 1: 20 v/v) of the residue gave 18 as a pale yellow oil (160 mg, 59 %). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.6 Hz), 1.43–1.53 (6H, m), 1.75–1.80 (3H, m), 1.91–1.98 (2H, t, J = 12.0 Hz), 2.33 (2H, t, J = 8 Hz), 2.98 (2H, d, J = 11.6 Hz), 4.14 (2H, d, J = 6.0 Hz), 4.28 (2H, br), 5.76 (2H, d, J = 54.4 Hz), 6.71 (1H, d, J = 8.0 Hz), 7.67 (1H, dd, J = 2.0, 8.4 Hz), 7.70 (1H, s). ¹³C-NMR (CDCl₃): δ 12.81, 19.65, 27.73, 27.89, 34.37, 52.18, 57.63, 67.66, 100.17 (d, J = 219 Hz), 113.04, 115.81, 118.78, 125.58, 140.86, 141.85 (d, J = 2.0 Hz), 165.00. ¹⁹F-NMR (CDCl₃): δ −147.50. LC-MS m/z [M + H]⁺, 214.1. HRMS: calc’d for C₁₈H₂₈FN₂O₃ (M⁺ + H), 339.2084; found, 339.2088.

Methyl 4-amino-3-(2-fluoroethoxy)benzoate (19a)

1-Chloro-2-fluoroethane (760 mg, 8.97 mmol) was added to a tube (15-mL) containing anhydrous DMF (5 mL), methyl 4-amino-3-hydroxybenzoate (500 mg, 2.99 mmol) and Cs₂CO₃ (1.95 g, 5.98 mmol). The tube was sealed and stirred for 88 h. The mixture was then filtered through celite which was rinsed thrice with EtOAc. The combined filtrate and rinses were evaporated to dryness. Silica gel chromatography (20% EtOAc in hexane) of the residue gave 19a as a white solid (270 mg, 42%). Mp. 86–87 °C. ¹H-NMR (CDCl₃): δ 3.84 (3H, s), 4.18–4.27 (2H, dt, J = 28.4, 4 Hz), 4.39 (2H, br), 4.65–4.79 (2H, dt, J = 47.2, 3.7 Hz), 6.66 (1H, d, J = 8 Hz), 7.43 (1H, d, J = 1.6 Hz), 7.55 (1H, dd, J = 8.4, 1.6 Hz). ¹⁹F-NMR (CDCl₃): δ −147.56. LC-MS: m/z [M + H]⁺, 214.1. HRMS: calc’d for C₁₀H₁₃FNO₃ (M⁺ + H), 214.0879; found, 214.0876.

(1-Butylpiperidin-4-yl)methyl 4-amino-3-(2-fluoroethoxy)benzoate (19)

As described for 18 from 18a, compound 19 was obtained from 19a as a white solid in 48% yield. Mp 72–74 °C. ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.6 Hz), 1.27–1.53 (6H, m), 1.74–1.79 (3H, m), 1.94 (2H, t, J =12.0 Hz), 2.33 (2H, t, J = 7.6 Hz), 2.97 (2H, d, J = 11.2 Hz), 4.13 (2H, d, J = 6 Hz), 4.24–4.33 (2H, dt, J = 28.4, 4.0 Hz), 4.36 (2H, br), 4.69–4.83 (2H, dt, J = 47.2, 4.0 Hz), 6.67 (1H, d, J = 8.0 Hz), 7.45 (1H, d, J = 2.0 Hz), 7.57 (1H, dd, J = 1.6, 8.4 Hz). ¹³C-NMR (CDCl₃): δ 14.07, 20.89, 29.03, 29.17, 35.65, 53.45, 58.88, 67.92 (d, J = 20.0 Hz), 68.82, 81.77 (d, J = 170 Hz), 112.94, 113.39, 119.45, 124.79, 141.70, 144.70, 166.67. ¹⁹F-NMR (CDCl₃): δ −147.49. LC-MS m/z [M + H]⁺_, 353.2. HRMS: calc’d for C₁₉H₃₀FN₂O₃ (M⁺ + H), 353.2240; found, 353.2238.

(1-Butylpiperidin-4-yl)methyl 4-amino-3-hydroxybenzoate (20) ^49,50

AlCl₃ (311 mg, 2.34 mmol) and NaI (351 mg, 2.34 mmol) were added to a solution of 13 (500 mg, 1.56 mmol) in MeCN (10 mL). The mixture was refluxed overnight and extracted thrice with EtOAc. The combined organic layers were dried on MgSO₄ and evaporated to dryness. Silica gel chromatography (MeOH/CH₂Cl₂, 1:20 v/v) of the residue gave 20 as an orange-brown solid (200 mg, 42%). Mp: 210–212 °C. ¹H-NMR (CDCl₃): δ 0.98 (3H, t, J = 7.6 Hz), 1.31–1.43 (2H, m), 1.51–1.63 (4H, m), 1.87–1.93 (3H, m), 2.28 (2H, t, J = 11.2 Hz), 2.54–2.58 (2H, m), 3.17 (2H, d, J = 12.0 Hz), 4.14 (2H, d, J = 5.6 Hz), 6.68 (1H, d, J = 8.0 Hz), 7.34 (1H, d, J = 2.0 Hz), 7.38–7.41 (1H, dd, J = 1.6, 8.0 Hz); ¹³C-NMR (CDCl₃): δ 13.96, 21.05, 27.46, 34.81, 53.45, 58.09, 68.05, 114.47, 116.05, 119.06, 124.34, 143.61, 144.84, 168.55. LC-MS: m/z [M + H]⁺_, 307.2. HRMS: calc’d for C₁₇H₂₆N₂O₃ (M⁺ + H), 307.2022; found, 307.2025.

(1-Butylpiperidin-4-yl)methyl 8-amino-7-nitro-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (21)

A mixture of 21a⁵¹(200 mg, 0.79 mmol), (Boc)₂O (515 mg, 2.36 mmol) and DMAP (44 mg, 0.039 mmol) in CH₂Cl₂ (15 mL) was refluxed at 50 °C for 1 h, then quenched with water and extracted thrice with CH₂Cl₂. The combined organic layers were dried on MgSO₄ and then condensed to a crude product, which after silica gel chromatography (30% EtOAc in hexane) gave di-Boc-protected 21a as a yellow solid (21b; 343 mg, 95%). ¹H-NMR (CDCl₃): δ 1.41 (18H, s), 3.94 (3H, s), 4.37–4.49 (4H, m), 8.30 (1H, s). ¹³C-NMR (CDCl₃): δ 27.78, 52.63, 63.77, 64.80, 83.66, 118.16, 120.06, 126.55, 138.19, 140.71, 148.53, 149.49, 163.61.

A THF solution of n-BuLi (1.6M; 0.95 mL, 1.52 mmol) was added drop-wise to a solution of (4-butylpiperidin-1-yl)methanol (260 mg, 1.52 mmol) in THF (2 mL) in an oven-dried flask, under Ar at 0 °C. After stirring this solution for 10 min, a solution of 21b (343 mg, 0.76 mmol) in THF (4 mL) was added dropwise. The mixture was stirred overnight, poured into water and extracted with CH₂Cl₂. The combined organic layers were dried on MgSO₄ and evaporated to dryness. Silica gel chromatography (MeOH/CH₂Cl₂, 1: 15 v/v) of the residue gave mono-Boc-protected 21 as a white solid (21c; 186 mg, 50%). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.29–1.49 (15H, m), 1.78 (3H, t, J =12.4 Hz), 1.93 (2H, t, J =10.0 Hz), 2.31 (2H, t, J = 7.6 Hz), 2.96 (2H, d, J = 11.6 Hz), 4.16 (2H, d, J= 6.4 Hz), 4.39–4.48 (4H, m), 7.20 (1H, br), 8.15 (1H, s). ¹³C-NMR (CDCl₃) δ 13.06, 19.88, 27.05, 28.01, 28.24, 34.43, 52.34, 57.85, 62.79, 63.70, 68.82, 81.43, 119.75, 124.57, 134.69, 135.44, 146.92, 150.51, 162.21.

21c (100 mg, 0.20 mmol) was stirred overnight in a dioxane solution of HCl (4M). The mixture was evaporated to dryness, diluted with water and then neutralized to pH 7 with aq. NH₄OH (1M). The solution was extracted thrice with CH₂Cl₂. The organic layers were combined and dried on MgSO₄. Evaporation of solvent gave 21 as a yellow solid (71 mg, 89%). Mp 108–110 °C (n = 2). ¹H-NMR (CDCl₃): δ 0.92 (3H, t, J = 7.2 Hz), 1.29–1.51 (7H, m), 1.79 (3H, d, J = 8.4 Hz), 1.92 (2H, t, J = 11.6 Hz), 2.33 (2H, t, J = 7.6 Hz), 2.98 (2H, d, J = 11.6 Hz), 4.14 (2H, d, J = 6.4 Hz), 4.38–4.46 (4H, m), 8.45 (1H, s). ¹³C-NMR (CDCl₃): δ 14.06, 20.89, 28.99, 29.16, 35.47, 53.38, 58.85, 63.82, 64.84, 69.28, 108.62, 123.12, 125.47, 130.17, 139.42, 147.16, 163.70. LC-MS: m/z [M + H]⁺, 394.2. HRMS: calc’d for C₁₉H₂₈N₃O₆ (M⁺ + H), 394.1978; found, 394.1972.

Pharmacological Assay and Screen

Some ligands (1, 5, 8, 13, 15, 17–19) were evaluated at Caliper Life Sciences (Hanover, MD) for binding to 5-HT₄ receptors in guinea pig striatal membranes at 37 °C with [³H]GR 113808 as reference radioligand.⁵²

All new ligands (2, 3 and 5–21) plus the already known ligands 1 and 4 were submitted to the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) for assessment of binding affinity to human recombinant 5-HT₄ receptors (reference radioligand, [³H]GR 113808) and a wide range of other receptors and binding sites (5-HT_{1A-1E, 2A-2C, 3, 5A, 6, 7}, α_{1A, 1B, 1D, 2A-2C}, BZP (rat brain site), β_1–3, σ_1,2, D1-5, DAT, DOR, GABAA, H1-4, KOR, M1-5, MOR, NET and SERT). Selected ligands (1, 4, 6–13 and 17) were also assessed for agonist/partial agonist activity in a GloSensor L9 assay for cAMP response and for antagonist activity. Detailed assay protocols are available at the NIMH-PDSP web site (http://pdsp.cwru.edu).

Production of NCA [¹¹C]carbon dioxide

No-carrier-added (NCA) [¹¹C]carbon dioxide was produced with a PEtrace cyclotron (GE; Milwaukee, WI) according to the ¹⁴N(p,α)¹¹C reaction²⁰ by irradiation of nitrogen gas (300 psi) containing 1% oxygen with a proton beam (16.5 MeV, 45 DA) for either 20 or 40 min. A 40-min irradiation produced about 2.0 Ci of [¹¹C]carbon dioxide.

Production of NCA [¹¹C]hydrogen cyanide²⁰

A PETrace Radiotracer Production System was used to produce this labeling agent, as follows. Cyclotron-produced NCA [¹¹C]carbon dioxide was trapped on molecular sieve (13 Å, 80–100 mesh; 0.55g) at 40 °C, while residual [¹³N]nitrogen was directed to waste. The [¹¹C]carbon dioxide was then released with a stream of nitrogen (250 mL/min), mixed with a stream of hydrogen (30 mL/min) and passed through a heated (400 °C) glass tube (10 mm × 200 mm) containing nickel catalyst (Ni-3266 Engelhardt). The effluent containing the generated [¹¹C]methane was passed through an OXY-TRAP® (PNr.-4001R; Alltech), mixed with anhydrous ammonia (research grade, 20 ml/min) and then passed over a wad of platinum wire (d 0.127 mm; 2.6 g) at 920 °C. The generated [¹¹C]hydrogen cyanide was delivered to a hot-cell for subsequent radiochemistry in a mixture of hydrogen, ammonia, and nitrogen carrier gas at 300 mL/min.

Production of NCA [¹¹C]methyl iodide

NCA [¹¹C]methyl iodide was produced from NCA [¹¹C]carbon dioxide (~ 2.0 Ci) via reduction to [¹¹C]methane and then vapour phase iodination¹⁸, either in a TRACERlab FX C Pro module (GE; Milwaukee, WI) for the synthesis of [¹¹C]8 or a MeI MicroLab apparatus (GE; Milwaukee, WI)for the synthesis of [ ¹¹C]13.

Production of NCA [¹¹C]methyl triflate

A quartz column (i.d. 2.6 mm, length 26 cm) was packed around its center with a 6 cm length of AgOTf/Graphac (50: 50 w/w/) held in place at each end with glass wool. NCA [¹¹C]methyl triflate (~ 350 mCi) was produced by passing [¹¹C]methyl iodide in helium gas (17 mL/min) into the heated (180 °C) column.¹⁹

Production of NCA [¹⁸F]d₂-fluoromethyl bromide⁵³

Cyclotron-produced [¹⁸F]fluoride ion (~ 150 mCi) in [¹⁸O]water was delivered into a glass vial containing K 2.2.2 (5.0 mg, 13.3 μmol) and potassium carbonate (0.50 mg, 3.6 μmol) in MeCN-H₂O (0.1 mL, 9: 1 v/v). This solution was transferred to a modified version of a TRACERlab FX_F-N module and diluted with MeCN (1 mL). The mixture was evaporated to dryness at 90 ºC under reduced pressure with a nitrogen flow. MeCN (2 mL) was again added and then evaporated to dryness. The vessel was sealed and then CD₂Br₂ (100 μL) in MeCN (1.0 mL) was added to the dry [¹⁸F]fluoride ion-K 2.2.2-K⁺ complex which was then heated at 95 ºC for 15 min. The reaction vessel was then cooled to 35 ºC. Nitrogen gas was used to transfer the volatile [¹⁸F]d₂-fluoromethyl bromide through a series of four silica gel cartridges (SepPak Plus) and then into a V-vial (1-mL) having a crimp-sealed silicon-Teflon septum cap. The RCY of [¹⁸F]d₂-fluoromethyl bromide was typically about 28%.⁵³

Radiosynthesis of [¹¹C]5

NCA [¹¹C]hydrogen cyanide (~ 200 mCi) was trapped in a V-vial (5-mL) containing THF (500 μL), precursor (1, ~ 1.0 mg), Pd(PPh₃)₄ (1.5–2.0 mg), base [K₂CO₃ (~ 2.0 mg) plus K 2.2.2 (~ 5.0 mg), or usually KH₂PO₄ (~ 2.0 mg) only]. The reaction mixture was heated at 80 ºC for 5 min. HPLC mobile phase (3 mL) was added to the V-vial and [¹¹C]5 isolated with radio-HPLC on a Gemini C18 column (5 μm, 10 × 250 mm; MeCN-aq. NH₄OH (10 mM; 3: 2 v/v; 6 mL/min; t_R = 7.2 min). The identity of [¹¹C]5 was confirmed by analytical radio-HPLC on a Gemini C18 column (5 μm, 4.6 × 150 mm) eluted at 1 mL/min with MeCN-10 mM NH₄OH (16: 9 v/v; t_R = 5.8 min) and also by LC-MS of associated carrier. The radiochemical purity of [¹¹C]5 was > 99%. The decay-corrected radiochemical yield (RCY) from [¹¹C]hydrogen cyanide was 26% (n = 3).

Radiosynthesis of [¹¹C]8

[¹¹C]Methyl iodide (~ 15 mCi) was trapped in a capped fluoro-polymer custom-made reaction vial (1.5-mL) containing anhydrous DMF (300 μL), precursor (3, ~ 1.0 mg) and base (Li₃N or Li₂O; ~ 5.0 mg). The mixture was sonicated in an ultrasound apparatus (UIS250L, Hielscher Ultrasonics, Germany) for 10 min and then filtered through an Iso-Disc^TM filter (PFTE13-4, 13 mm × 0.45 μm) which was then rinsed twice with DMF. The combined filtrates were diluted with water and then the [¹¹C]8 was isolated with radio-HPLC (MeCN-HCOONH₄; 2: 3 v/v; 3 mL/min; t_R = 13.1 min). The identity of [¹¹C]8 was confirmed by analytical radio-HPLC on a Gemini-NX C18 column (5 μm, 4.6 × 250 mm) eluted with MeCN-100 mM HCOONH₄ (16: 9 v/v) at 1 mL/min (t_R = 7.0 min) and also by LC-MS of associated carrier. The RCYs from [¹¹C]methyl iodide were 7% (Li₂O) and 11% (Li₃N).

Radiosynthesis of [¹¹C]13 from [¹¹C]methyl triflate

[¹¹C]Methyl triflate (30–20 mCi) was trapped in a tapered bottom vial (0.9-mL) containing 20 (0.15–0.8 mg), 0.5M NaOH (3 eq.) and MeCN (300 μL). The mixture was heated at 80 ºC for 5 min, diluted with HPLC mobile phase (500 μL), and then the [¹¹C]13 (t_R = 8.7 min) isolated with radio-HPLC (MeCN-HCOONH₄; 2: 3 v/v; 4 mL/min). The identity of [¹¹C]13 was confirmed by analytical radio-HPLC on a Gemini-NX C18 column (5 μm, 4.6 × 250 mm) eluted with MeCN-100 mM HCOONH₄ (2: 3 v/v) at 1 mL/min (t_R = 4.4 min) and also by LC-MS of associated carrier. The RCY was 27% (n = 12) from [¹¹C]methyl triflate and the radiochemical purity > 99%. The absorbance detector response of the analytical HPLC system was calibrated for mass of carrier ligand 13. This allowed the mass of 13 in measured samples of [¹¹C]13 to be determined, and the specific radioactivity to be calculated. The specific radioactivity was 2,517 mCi/μmol at end of synthesis (EOS).

Radiosynthesis of [¹¹C]13 from [¹¹C]methyl iodide

[¹¹C]Methyl iodide (30–550 mCi) was trapped in a tapered bottom vial (0.9-mL) containing 20 (~ 1.0 mg, 3.2 μmol), 1.0M (n-Bu)₄NOH (3 eq., 10 μL, 3.2 μmol) and DMF (300 μL). The mixture was heated at 80 ºC for 5 min and then diluted with water (500 μL). [¹¹C]13 was separated and analyzed as described above. The RCY of [¹¹C]13 from [¹¹C]iodomethane was 36% (n = 6) and the radiochemical purity > 99%. The specific radioactivity was 2,848 mCi/μmol at EOS.

The following experiment²² was performed to confirm the position of radiolabel in [¹¹C]13. [¹¹C]Methyl iodide (~ 47 mCi) was trapped in a solution of 20 (~ 1.0 mg) in DMF (300 μL). Then [¹³C]methyl iodide (10 μL from 47.7 mM stock solution in DMF) was added. [¹¹C]/[¹³C]13 was isolated by HPLC, as described above. A sample of the collected radioactive fraction was then analyzed by HPLC and LC-MS. The radioactive fraction was then evaporated to dryness, dissolved in CDCl₃ and, analyzed by ¹³C-NMR{DEPT 135}.

Radiosynthesis of [¹⁸F]18

Phenol 20 (~ 0.15 mg, 0.49 umol), MeCN (300 μL) and 0.5M NaOH (1.5 μmol, 3 μL) were added to a V-vial (1-mL). [¹⁸F]FCD₂Br (~ 20 mCi) was transferred to the solution under computer control from a TRACERlab FX_F-N module. Radioactivity transfer was monitored by two external radioactivitydetectors (Bioscan) and was stopped when radioactivity maximized. The mixture was heated at 100 ºC for 15 min and then diluted with water (700 μL). [¹⁸F]18 was isolated with HPLC (MeCN-HCOONH₄; 2: 3 v/v; 3 mL/min; t_R = 10.1 min). The identity of [¹⁸F]18 was confirmed by analytical HPLC on a Gemini-NX C18 column (5 μm, 4.6 × 250 mm) eluted with MeCN-100 mM HCOONH₄ (1: 1 v/v) at 1 mL/min (t_R = 4.2 min) and also by LC-MS. The RCY of [¹⁸F]18 was 13% (n = 1) from [¹⁸F]fluoride ion and the radiochemical purity > 99%.