First Photoswitchable Neurotransmitter Transporter Inhibitor: Light


Jul 15, 2014 - First Photoswitchable Neurotransmitter Transporter Inhibitor: Light-Induced Control of γ-Aminobutyric Acid Transporter 1 (GAT1) Activi...
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First Photoswitchable Neurotransmitter Transporter Inhibitor: LightInduced Control of γ‑Aminobutyric Acid Transporter 1 (GAT1) Activity in Mouse Brain Gabriele Quandt,† Georg Höfner,† Jörg Pabel,† Julien Dine,‡ Matthias Eder,‡ and Klaus T. Wanner*,† †

Department für PharmazieZentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, 81377 Munich, Germany ‡ Max-Planck-Institut für Psychiatrie, Kraepelinstrasse 2-10, 80804 Munich, Germany S Supporting Information *

ABSTRACT: Inhibition of mGAT1, the most abundant GABA transporter in the brain, enhances GABA signaling and alleviates symptoms of CNS disorders such as epilepsy assumed to be associated with low GABA levels. We have now developed a potent and subtype selective photoswitchable inhibitor of this transporter, which for the first time extends the photoswitch concept for the light-induced control of ligand affinity to active membrane transporters. The new inhibitor exhibited reduced activity upon irradiation with light, as demonstrated in GABA uptake assays and electrophysiological experiments with brain slices, and might be used as a tool compound for deepening the understanding of mGAT1 function in brain.



INTRODUCTION By using light to control the activity of ligands to study the structure and function of plasma membrane bound transport proteins, as an important class of biological targets, different concepts may be followed. Photoaffinity labeling for example uses a photochemical reaction between a photolabile group of a ligand molecule and a functional group in the protein to covalently link the ligand to the transporter of interest.1−8 Phototriggering makes use of light to suddenly liberate a ligand or substrate of a transporter by removing a photolabile protection group from a suitable precursor molecule.9 Photoswitchable ligands are finally a class of compounds that allow their activity at a target of interest to be controlled in a reversible manner by light. Under irradiation with light of a certain wavelength, such ligands undergo a reversible change of their structure, ideally resulting in a significant change of their affinity toward or intrinsic activity at the target of interest. Photoswitchable ligands, which are also termed photochromic ligands, offer the possibility to induce light sensitivity to biologically relevant targets, thereby allowing temporally and spatially precise control of neuronal activity with light. A photoswitchable μ-opioid receptor agonist,10 nicotinic acetylcholine receptor agonists,11 ionotropic glutamate receptor agonists,12 and photochromic azo-propofols as photochromic potentiators of GABAA receptors13 are examples for applications of this concept. But to the best of our knowledge, no examples for photoswitchable ligands for membrane bound transporter proteins are known so far. © 2014 American Chemical Society

In this article, we report on the successful development of photoswitchable inhibitors of the plasma membrane bound γ‑aminobutyric acid (GABA, 1) transporter GAT1. GAT1 represents one of the four subtypes of GABA transporters that accomplish GABA transport across the plasma membrane. When of murine origin, these transporters are termed mGAT1−4,14 which corresponds to the human GATs GAT1, BGT1, GAT2, and GAT3, respectively.15 Because the biological studies of this work are based on the murine transporters, also the nomenclature will be restricted to this species in the following. The two GABA transporters mGAT1 and mGAT4 are found almost exclusively in the brain.16,17 Of these, especially mGAT1 is widely expressed throughout the brain, particularly along GABAergic pathways, and is therefore believed to be the main transporter in charge of neuronal GABA uptake after a neuronal impulse.18 As low GABA levels are associated with CNS disorders like epilepsy,19 neuropathic pain,20 depression, or anxiety,21 mGAT1 selective inhibitors can potentially alleviate those diseases by increasing the amount of GABA in the synaptic cleft. Actually, with the introduction of tiagabine (3), a selective mGAT1 inhibitor used for the treatment of epilepsy, to the market, mGAT1 has already been validated as a drug target.22,23 By allowing a light-induced control of the inhibitory potency, photoswitchable mGAT1 inhibitors could thus be expected to be a promising tool for Received: June 6, 2014 Published: July 15, 2014 6809

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more in-depth studies of the physiology of this important drug target. Tiagabine (3, pIC50 (mGAT1) = 6.88), SKF 89976A (4, pIC50 (mGAT1) = 6.16), and compound 5 (pIC50 (mGAT1) = 7.00) represent typical mGAT1 inhibitors that nicely illustrate the structural requirements for potent mGAT1 inhibitors (Figure 1).24,25 In general, such inhibitors are composed of a

Figure 1. Potent GABA uptake inhibitors. Figure 2. Photoswitchable target structures.

polar head, which is most often nipecotic acid (2) and a symmetric or asymmetric lipophilic aromatic moiety attached to the former by a four to five atom pure carbon or carbon/ oxygen spacer. Because of the similarities in the gross chemical structure and the steric demand of an azobenzene moiety with the 2benzylphenyl residue in 5,24 we considered this compound the most promising starting point for the development of photoswitchable mGAT1 inhibitors. Thus, we intended to replace the 2-benzylphenyl residue in 5 with an azobenzene group, which should be attached to the linker via different, i.e., the ortho-, meta-, and para-positions (see (E)-6a−c, Figure 2). In addition, also the more bulky naphthylazobenzene derivatives (E)-6d and (E)-6e should be included in this study, as molecular modeling had indicated that these compounds might display similar or even higher potencies at mGAT1 than (E)-6a−c. Considering the structural change these compounds experience when transformed from the stretched (E)-configured into the partly U-shaped (Z)configured isomer upon irradiation, as exemplified for (R)(E)-6e/(R)-(Z)-6e in Scheme 1, it was assumed that with this (E)/(Z)-transformation also the mGAT1 inhibitory potencies should significantly alter. The inhibitory potency of nipecotic acid (2) derived mGAT1 ligands is well-known to reside mainly in the (R)-enantiomers. Hence, selected target compounds were synthesized in enantiopure form.

Scheme 1. Photoisomerization of (R)-6e

The required azo-iodides and -triflates had been obtained either using classical diazo-coupling chemistry or by condensation of nitrosobenzene with the corresponding aniline (see Supporting Information). Selective hydrogenation of the vinylether double bond of 9a−b and (R)-9d−e using Wilkinson’s catalyst led to the corresponding ethers 11a−b and (R)-11d−e (conditions c, Scheme 2). Because 2iodoazobenzene could not be coupled to the olefin via Heck reaction, the ortho-isomer 11c was synthesized using a different approach in which 2-iodonitrobenzene was coupled with 7 to give 9c. Subsequent reduction of the nitro function and the double bond with H2 and Pd/C as catalyst yielded 10 (98%, conditions e, Scheme 2), and subsequent condensation with nitrosobenzene yielded the azo compound 11c (conditions f, Scheme 2). Hydrolysis of the ethyl esters 11a−c and (R)-11d− e yielded finally the carboxylic acids (E)-6a−c and (R)-(E)6d−e (conditions d, Scheme 2). Photoisomerization. UV/vis spectra with in situ irradiation of the sample revealed 350 nm as the optimal wavelength for the photoisomerization of the (E)-isomers (E)-6a−c to the



RESULTS AND DISCUSSION Synthesis. For the synthesis of the target compounds, we followed a synthetic pathway recently developed by us for the construction of related nipecotic acid derivatives.26 Reacting the N-[(vinyloxy)ethyl]nipecotate 7 and (R)-7, respectively, with the corresponding aryl triflates (conditions a, Scheme 2) or iodides (conditions b, Scheme 2) in a chelation controlled Heck reaction27 provided intermediates 9a−b and (R)-9d−e in good yields (63−83%). The β-regioselectivity of this reaction may be attributed to the formation of the intermediary chelate 8.27−31 6810

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Scheme 2. Synthesis of (E)-6a−c and (R)-(E)-6d−ea

For the naphthylazobenzene derivatives (R)-(E)-6d−e, the ideal wavelength for photoisomerization of the (E)- to the (Z)isomers was determined to be 375 nm. As a reliable light source for irradiation at this wavelength (375 nm), in this case a selfbuilt light chamber equipped with LED lamps with an emission maximum at 375 nm was used. At the photostationary state (reached within 30 min), the (Z)-isomers (R)-(Z)-6d and (R)(Z)-6e prevailed over the (E)-isomers with 88% and 87%, respectively (determined by 1H NMR; see Table 1). Back isomerization was, again, achieved through thermal relaxation in the dark, which led to quantitative amounts of (E)-isomer, or by irradiation with visible light (λ = 450 nm), which resulted in 59% (R)-(E)-6d and 58% (R)-(E)-6e (see Table 1). For the thermal relaxation in phosphate buffer (50 mM, pH 7.4) at 25 °C, the half-life amounted to 13 and 19 h for (R)-(Z)-6d and (R)-(Z)-6e, respectively. Biological Evaluation. Uptake inhibition of the pure (E)isomers toward mGAT1−4 was determined following a standardized [3H]GABA uptake assay with HEK cells stably expressing the individual murine GABA transporters that was appropriately adapted for this purpose.33 All five compounds show moderate to good inhibitory potencies toward mGAT1 and only weak potencies toward mGAT2−4, thus being all reasonably subtype selective for this transporter (Table 2). The most effective mGAT1 inhibitors were found to be the naphthylazobenzene derivatives (R)-(E)-6d (pIC50 (mGAT1) = 6.57, Table 2, entry 4) and (R)-(E)-6e (pIC50 (mGAT1) = 6.39, Table 2, entry 5), exhibiting pIC50 values in the range of tiagabine (3, pIC50 (mGAT1) = 6.88). To determine the potency of each compound after photoisomerization, a 10 mM stock solution of the corresponding (E)-isomer, either in D2O or in DMSO-d6 depending on its solubility, was irradiated at 350 or 375 nm prior to biological testing until the photostationary state was reached (30 min). The (E)/(Z)-ratio after irradiation was determined by 1H NMR analysis as described before. After irradiation, 6c (Table 2, entry 3) showed a significantly higher inhibitory potency (pIC50 (mGAT1) = 5.26) than before (pIC50 (mGAT1) = 4.97), while all other (Z)-isomers were found to be the less potent inhibitors than their (E)-isomers. The phenylazobenzene derivatives (E)-6a (Table 2, entry 1) and (E)-6b (Table 2, entry 2) exhibit decreased inhibitory potencies after irradiation ((Z)-6a, pIC50 (mGAT1) = 5.34; (Z)-6b, pIC50 (mGAT1) = 5.38) compared to the pure (E)isomers ((E)-6a, pIC50 (mGAT1) = 5.72, difference statistically significant; (E)-6b, pIC50 (mGAT1) = 5.50, difference statistically not significant). In contrast, the inhibitory potencies of (R)-(E)-6d (Table 2, entry 4) and (R)-(E)-6e (Table 2, entry 5) are significantly lower after irradiation ((R)-(Z)-6d, pIC50 (mGAT1) = 6.10; (R)-(Z)-6e, pIC50 (mGAT1) = 5.78)

a

Reaction conditions: (a) ArOTf, Pd(OAc)2, PPh3, NEt3, DMF, 80 °C; (b) ArI, Pd(OAc)2, K2CO3, NaOAc, LiCl, DMF/H2O = 10:1 (v/ v), 80 °C; (c) H2 (5−10 bar), [Rh(PPh3)3]Cl, MeOH/THF = 1:1 (v/ v), rt; (d) 12 M NaOH, EtOH, 0 °C to rt; (e) 9c, H2 (5 bar), Pd/C, MeOH, rt; (f) nitrosobenzene, AcOH, 85 °C.

(Z)-isomers (Z)-6a−c. The percentage of (Z)-isomers at the photostationary state was determined through 1H NMR analysis of the respective samples after their irradiation in a quartz 1H NMR tube by means of a Rayonet photoreactor.32 At the photostationary state, achieved within 30 min of irradiation (which will also be accomplishable almost instantly, in seconds to milliseconds, with a sufficiently intense light source) at 350 nm, the amount of the (Z)-isomers (Z)-6a−c contained in the irradiated samples was found to be 82−89% (see Table 1). Nearly quantitative back isomerization [(E)-6a−c/(Z)-6a−c ∼ 99:1] could be achieved by irradiation with visible light (λ = 440 nm for 6a and λ = 420 nm for 6b−c) or in the dark through thermal relaxation. For the thermal relaxation of (Z)6a−c in D2O in the dark, the half-life ranged from 2.4 to 6.0 days at 37 °C (see Table 1). Table 1. Photochemical Properties of 6a−c and (R)-6d−e

(E)/(Z) ratio photostat state entry

compound

solvent

λforward (nm)

λback (nm)

(Z)→(E)

(E)→(Z)

t1/2a

1 2 3 4 5

6a 6b 6c (R)-6d (R)-6e

D2O D2O D2O DMSO-d6 DMSO-d6

350 350 350 375 375

440 420 420 450 450

11/89 14/86 18/82 12/88 13/87

99/1 99/1 99/1 59/41 58/42

2.4 db 6.0 db 4.8 db 13.6 hc 19.3 hc

Half-life of thermal relaxation. bDetermined by 1H NMR, solvent: D2O, T = 37 °C, conc 10 mM. cDetermined by UV/vis-spectroscopy, solvent: DMSO-d6/phosphate-buffer (pH 7.4) = 1:200 (v/v), T = 25 °C, conc 50 μM. a

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Table 2. GABA Uptake Inhibition of (E)-6a−c and (E)-(R)-6d−e GABA uptake inhibition (pIC50 ± SEM) entry 1 2 3 4 5

compd

mGAT1

(E)-6a (E)-6b (E)-6c (R)-(E)-6d (R)-(E)-6e

± ± ± ± ±

5.72 5.50 4.97 6.57 6.39

0.02 0.06 0.02 0.01 0.08

mGAT1 (ai)a c,d

5.34 5.38c,e 5.26c,f 6.10g,h 5.78g,i

± ± ± ± ±

mGAT2 j

0.01* 0.05#k 0.02*j 0.02*j 0.03*j

b

mGAT3

95%, as confirmed by reverse phase analytical HPLC or elemental analysis. The purity of all tested compounds was >95% as confirmed by reverse phase analytical HPLC or elemental analysis. General Procedure 1 (GP1): Heck Reaction of Vinyl Ethers. Method A: Pd(OAc)2 (10 mol %) and PPh3 (20 mol %) were dissolved in abs DMF, and the reactants were added in the following order: (1) aryl triflate (1 equiv), (2) NEt3 (1.5 equiv), (3) 6 (1.5 equiv). The reaction mixture was heated to 80 °C and stirred for 15− 48 h. The resulting solution was diluted with CH2Cl2, washed with water and brine. The combined aqueous phases were extracted with CH2Cl2, and the organic extracts were dried over K2CO3, filtered, and concentrated in vacuo. The crude product was purified on silica gel. Method B: The aryl iodide (1 equiv) was dissolved in abs DMF, and the reactants were added in the following order: (1) 6 (2 equiv), (2) Pd(OAc)2 (10 mol %), (3) (1.5 equiv), (4) LiCl (2 equiv), (5) K2CO3 (1.2 equiv), and (6) H2O (DMF/H2O = 10:1 (v/v)). The reaction mixture was heated to 80 °C and stirred for 3−20 h. The workup was analogue method A and the crude product was purified on silica gel. General Procedure 2 (GP2): Selective Hydrogenation of Vinyl Ethers. An (E)/(Z)-mixture of the vinyl ether was dissolved in a mixture of THF and MeOH (v/v = 1:1). The mixture was degassed, [Rh(PPh3)3]Cl (Wilkinson’s catalyst, 5−25 mol %) was added, and hydrogenation was performed with an H2 pressure of 5−10 bar under vigorous stirring. After 3−6 h, the reaction was complete and the homogeneous catalyst could be precipitated by adding Et2O. Subsequent filtration and evaporation of the solvent gave the crude product which was purified on silica gel. General Procedure 3 (GP3): Hydrolysis of Ethyl Esters. The corresponding ethyl ester was dissolved in abs EtOH and cooled to 0 °C. An excess of 12 N NaOH was added and the solution stirred at rt until the reaction was complete. The mixture was diluted with CH2Cl2 and brought to pH 6 with phosphate buffer (0.4 M, pH 6). The organic phase was separated and the aqueous phase extracted with CH2Cl2 (2×). The combined organic phases were dried over MgSO4, and the solvent was removed in vacuo to yield the free carboxylic acid. 1-[2-(2-{4-[(E)-2-Phenyldiazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylic Acid ((E)-6a). The hydrolysis was performed according to GP3. 11a (31 mg, 0.076 mmol) was dissolved in abs 6814

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2864, 1710, 1597, 1479, 1451, 1338, 1367, 1218, 1153, 1116, 953, 927, 775, 755, 688 cm−1. 1H NMR (500 MHz, CD2Cl2, 21.5 °C, TMS): δ = 1.50−1.57 (m, 1H; NCH 2 CH 2 CH 2,ax ), 1.57−1.63 (m, 1H; NCH2CH2,eqCH2), 1.76 (qt, J = 13.0/4.3 Hz, 1H; NCH2CH2,axCH2), 1.87−1.94 (m, 1H; NCH 2 CH 2 CH 2,eq ), 2.11−2.31 (m, 1H; NCH2,axCH2CH2), 2.31−2.37 (m, 1H; NCH2,axCH), 2.57−2.61 (m, 1H; NCH2CH), 2.62−2.70 (m, 2H; NCH2CH2O), 2.87−2.95 (m, 1H; NCH2,eqCH2CH2), 3.04−3.11 (m, 1H; NCH2,eqCH), 3.43 (t, J = 7.0 Hz, 2H; NCH 2 CH 2 OCH 2 CH 2 ), 3.59 (t, J = 5.0 Hz, 2H; NCH2CH2O), 3.68−3.76 (m, 2H; NCH2CH2OCH2), 7.33 (ddd, J = 8.1/5.9/2.6 Hz, 1H; H2CCCHarCHarCHar), 7.40−7.45 (m, 2H; H2CCCHarCHar), 7.50 (t, J = 7.3 Hz, 1H; NNCCH ar CH ar CH ar CH ar CH ar ), 7.55 (t, J = 7.3 Hz, 2H; NNCCH ar CH ar CH ar CH ar CH ar ), 7.67 (d, J = 7.5 Hz, 1H; H2CCCHarCHarCHarCHar), 7.90−7.94 (m, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CD2Cl2, 20.0 °C, TMS): δ = 22.38 (NCH2CH2CH2), 26.64 (NCH2CH2CH2), 32.17 (OCH2CH2Car), 40.58 (NCH2CH), 53.63 (NCH2CH2CH2), 55.39 (NCH2CH), 57.01 (NCH2CH2O), 67.17 (NCH2CH2O), 72.67 (OCH2CH2Car), 115.7 (H2CCarCarHCarHCarHCarH), 123.3 (2C; NNCarCarHCarHCarHCarHCarH), 127.5 (H2CCarCarHCarHCarH), 129.6 (2C; NNCarCarHCarHCarHCarHCarH), 131.4 (NNCCarHCarHCarHCarHCarH), 131.5 (H2CCarCarHCarH), 131.8 (H2CCarCarH), 139.5 (H2CCar), 150.9 (H2CCarCarNN), 153.3 (H2CCarCarNNCar), 176.6 (COOH) ppm. MS (CI, CH5+) m/z (%): 382 (100, [M + H]+), 293 (13), 156 (11), 142 (33). HRMS (EI+): M+ calcd for C22H27N3O3 381.2052, found 381.2051. (3R)-1-[2-(2-{4-[(E)-2-(Naphthalen-1-yl)diazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylic Acid ((R)-(E)-6d). The synthesis was performed according to GP3, using (R)-11d (20.0 mg, 0.044 mmol) and 12 M NaOH (0.040 mL, 0.440 mmol) in abs EtOH (0.500 mL). The reaction was completed after 2.5 h. The product was obtained as orange oil (20.0 mg, quant); [α]20 D = 40.8 (c = 0.1 in CH2Cl2). UV (phosphate buffer, pH 7.4) λmax, nm (ε): 274 (8640), 310 (9660), 374 (12540). IR (KBr): ν̃ = 3425, 3060, 2917, 2865, 1601, 1499, 1449, 1382, 1359, 1114, 842, 808, 776 cm−1. 1H NMR (400 MHz, CD2Cl2, 15.9 °C, TMS): δ = 1.50−1.68 (m, 2H; NCH2CH2,axCH2,ax), 1.72−1.89 (m, 1H; NCH2CH2,eqCH2), 1.89− 1.99 (m, 1H; NCH2CH2CH2,eq), 2.27 (t, J = 11.0 Hz, 1H; NCH2,axCH2CH2), 2.33−2.43 (m, 1H; NCH2,axCH), 2.59−2.66 (m, 1H; NCH2CH), 2.66−2.77 (m, 2H; NCH2CH2O), 3.00 (t, J = 6.6 Hz, 2H; OCH2CH2CCHar), 2.92−3.05 (m, 1H; NCH2,eqCH2CH2), 3.15 (dbr, J = 10.0 Hz, 1H; NCH2,eqCH), 3.61 (t, J = 5.0 Hz, 2H; NCH2CH2O), 3.66−3.79 (m, 2H; OCH2CH2CCHar), 7.45 (d, J = 8.0 Hz, 2H; CH2CCHarCHarCCHarCHar), 7.59 (t, J = 7.8 Hz, 1H; NNCCH ar CH ar CH ar ), 7.61 (ddd, J = 8.1/6.7/1.5 Hz, 1H; NNCCCHarCHarCHar), 7.67 (ddd, J = 8.3/6.9/1.3 Hz, 1H; NNCCCHarCHar), 7.82 (dd, J = 7.6/0.8 Hz, 1H; NNCCHarCHarCHar), 7.96 (d, J = 8.4 Hz, 1H; NNCCCH a r CH a r CH a r CH a r ), 7.99 (d, J = 8.4 Hz, 2H; CH2CCHarCHarCCHar), 7.99−8.04 (m, 1H; NNCCHarCHarCHar), 8.93 (d, J = 8.8 Hz, 1H; NNCCCHar) ppm. 13C NMR (101 MHz, CD2Cl2, 17.5 °C, TMS): δ = 22.42 (NCH2CH2CH2), 26.77 (NCH2CH2CH2), 36.50 (OCH2CH2Car), 40.62 (NCH2CH), 53.68 (NCH2CH2CH2), 55.41 (NCH2CH), 57.04 (NCH2CH2O), 67.50 (NCH2CH2O), 72.04 (OCH2CH2Car), 112.1 (NNCarCarHCarHCarH), 123.5 (2C; CH2CarCarHCarHCarCarH), 123.7 (NNCarCarCarH), 126.1 (NNCarCarHCarHCarH), 126.9 (NNCarCarCarHCarHCarH), 127.2 (NNCarCarCarHCarH), 128.3 (NNCarCarCarHCarHCarHCarH), 130.2 (2C; CH2CarCarHCarHCarCarHCarH), 131.5 (NNCarCarHCarHCarH), 131.7 (NNCarCarCarH), 134.7 (NNCarCarHCarHCarHCar), 143.5 (CH2Car), 148.1 (NNCarCarCarH), 152.2 (CH2CarCarHCarHCar), 176.6 (COOH) ppm. MS (EI, 70 eV) m/z (%): 431 (2, M+), 299 (4), 258 (13), 214 (3). HRMS (EI+): M+ calcd for C26H29N3O3 431.2209; found 431.2210. (3R)-1-[2-(2-{3-[(E)-2-(Naphthalen-1-yl)diazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylic Acid ((R)-(E)-6e). The synthesis was performed according to GP3 with (R)-11e (29.7 mg, 0.064 mmol) in abs EtOH (0.500 mL) and 12 M NaOH (0.060 mL, 0.720 mmol). The reaction was completed after 1 h. The product was obtained as

EtOH (0.5 mL), and 12 N NaOH (0.1 mL, 1.2 mmol) was added. The reaction was complete after 3 h. The product was yielded as orange oil (27.7 mg, 96%). UV (phosphate buffer, pH 7.4): λmax, nm (ε): 231 (13120), 326 (19620), 423 (1740). IR (film): ν̃ = 3064, 2918, 2850, 1710, 1601, 1444, 1365, 1144, 1114, 1070, 1015, 858, 167, 688 cm−1. 1 H NMR (500 MHz, D2O + NaOD, pH 14, 20.9 °C): δ = 1.05−1.19 (m, 1H; NCH2CH2CH2,ax), 1.24−1.36 (m, 1H; NCH2CH2,axCH2), 1.38−1.45 (m, 1H; NCH 2 CH 2,eq CH 2 ), 1.58−1.67 (m, 1H; NCH2,axCH2CH2), 1.75−1.82 (m, 1H; NCH2CH2CH2,eq), 1.92 (t, J = 11.5 Hz, 1H; NCH2,axCH), 2.19−2.29 (m, 2H; NCH2CH, NCH2CH2O), 2.35−2.45 (m, 1H; NCH2CH2O), 2.51 (d, J = 11.0 Hz, 1H; NCH 2 , e q CH 2 CH 2 ), 2.59 (t, J = 6.5 Hz, 2H; NCH2CH2OCH2CH2), 2.84 (d, J = 10.0 Hz, 1H; NCH2,eqCH), 3.26−3.41 (m, 4H; NCH2CH2OCH2), 7.02 (d, J = 8.0 Hz, 2H; H2CCCHarCHarCCHarCHar), 7.14−7.21 (m, 3H; NNCCH a r CH a r CH a r CH a r ), 7.50 (d, J = 8.5 Hz, 2H; H2CCCHarCHarCCHarCHar), 7.54−7.60 (m, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, D2O + NaOD, pH 14, 20.1 °C): δ = 22.39 (NCH2CH2CH2), 25.82 (NCH2CH2CH2), 33.43 (OCH2CH2Car), 42.92 (NCH2CH), 51.14 (NCH2CH2CH2), 54.82 (NCH2CH), 54.96 (NCH2CH2O), 65.94 (NCH2CH2O), 69.28 (OCH2CH2Car), 120.8 (2C; NNCarCarHCarHCarHCarHCarH), 121.0 (2C; H2CCarCarHCarHCarCarH), 127.5 (2C; NNCarCarHCarHCarHCarH), 128.0 (2C; H2CCarCarHCarHCarCarHCarH), 129.5 (NNCarCarHCarHCarH), 141.1 (H2CCar), 148.9 (H2CCarCarHCarHCarNN), 150.3 (NNCarCarHCarHCarH), 181.3 (COOH) ppm. MS (CI, CH5+) m/z (%): 382 (13, [M + H]+), 177 (100), 115 (18), 113 (14). HRMS (EI+): M+ calcd for C22H27N3O3 381.2052, found 381.2050. 1-[2-(2-{3-[(E)-2-Phenyldiazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylic Acid ((E)-6b). The synthesis was performed according to GP3. 11b (75.7 mg, 0.185 mmol) was dissolved in abs EtOH (0.8 mL), and 12 N NaOH (0.1 mL, 1.2 mmol) was added. The reaction was complete after 4 h of stirring. The product was obtained as orange oil (69.3 mg, 98%). UV (phosphate buffer, pH 7.4) λmax, nm (ε): 231 (12072), 321 (16662), 424 (1332). IR (film): ν̃ = 3059, 2926, 2864, 1708, 1597, 1467, 1448, 1364, 1148, 1116, 1019, 924, 864, 795, 766, 695 cm−1. 1H NMR (500 MHz, CD2Cl2, 21.4 °C, TMS): δ = 1.50−1.57 (m, 1H; NCH 2 CH 2 CH 2,ax ), 1.57−1.64 (m, 1H; NCH2CH2,eqCH2), 1.77 (qt, J = 13.2/4.3 Hz, 1H; NCH2CH2,axCH2), 1.87−1.95 (m, 1H; NCH 2 CH 2 CH 2,eq ), 2.22−2.32 (m, 1H; NCH2,axCH2CH2), 2.32−2.42 (m, 1H; NCH2,axCH), 2.57−2.62 (m, 1H; NCH2CH), 2.65−2.77 (m, 2H; NCH2CH2O), 2.93−2.98 (m, 1H; NCH2,eqCH2CH2), 2.99 (t, J = 6.8 Hz, 2H; NCH2CH2OCH2CH2), 3.12 (dbr, J = 10.0 Hz, 1H; NCH2,eqCH), 3.61 (t, J = 5.3 Hz, 2H; NCH2CH2O), 3.68−3.78 (m, 2H; NCH2CH2OCH2), 7.38 (dt, J = 7.5/1.5 Hz, 1H; H2CCCHarCHar), 7.45 (t, J = 7.8 Hz, 1H, H2CCCHarCHar), 7.49 (t, J = 7.3 Hz, 1H; NNCCHarCHarCHarCHar), 7.53 (t, J = 7.3 Hz, 2H; NNCCHarCHarCHarCHar), 7.76 (ddd, J = 7.8/ 1.8/1.3 Hz, 1H; H2CCCHarCHarCHar), 7.79 (t, J = 1.5 Hz, 1H; H2CCCHarCNN), 7.88−7.93 (m, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CD2Cl2, 21.8 °C, TMS): δ = 22.41 (NCH2CH2CH2), 26.69 (NCH2CH2CH2), 36.40 (OCH2CH2Car), 40.62 (NCH2CH), 53.68 (NCH2CH2CH2), 55.44 (NCH2CH), 57.06 (NCH2CH2O), 67.44 (NCH2CH2O), 72.13 (OCH2CH2Car), 121.3 (H2CCarCarHCarHCarH), 123.1 (2C; NNCarCarHCarHCarHCarHCarH), 123.5 (H2CCarCarHCarNN), 129.4 (H2CCarCarHCarH), 129.5 (2C; NNCarCarHCarHCarHCarH), 131.4 (NNCarCarHCarHCarHCarH), 132.2 (H2CCarCarHCarH), 140.9 (H2CCar), 153.1 (2 C, CarNNCar), 176.6 (COOH) ppm. MS (CI, CH5+) m/z (%): 383 (24), 382 (100, [M + H]+), 293 (19), 264 (14), 220 (16), 142 (40), 130 (39), 128 (15). HRMS (EI+): M+ calcd for C22H27N3O3 381.2052, found 381.2043. 1-[2-(2-{2-[(E)-2-Phenyldiazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylic Acid ((E)-6c). The hydrolysis was performed following GP3. 11c (108 mg, 0.26 mmol) was dissolved in abs EtOH (1.00 mL), and 12 N NaOH (0.20 mL, 2.4 mmol) was added. The reaction was completed after 2.5 h. The product was obtained as orange oil (82.1 mg, 83%). UV (phosphate buffer, pH 7.4) λmax, nm (ε): 229 (13500), 324 (18580), 429 (2000). IR (film): ν̃ = 3060, 2939, 6815

dx.doi.org/10.1021/jm5008566 | J. Med. Chem. 2014, 57, 6809−6821

Journal of Medicinal Chemistry

Article

orange oil (28.7 mg, quant); [α]20 D = 13.9 (c = 0.13 in CH2Cl2). UV (phosphate buffer, pH 7.4) λmax, nm (ε): 272 (8340), 292 (7260), 372 (7740). IR (KBr): ν̃ = 3053, 2928, 2863, 1711, 1600, 1508, 1468, 1441, 1387, 1219, 1200, 1116, 1013, 803, 772, 755 cm−1. 1H NMR (500 MHz, CD2Cl2, 22.2 °C, TMS): δ = 1.49−1.63 (m, 2H; NCH2CH2,axCH2,ax), 1.78 (qt, J = 13.0/4.3 Hz, 1H; NCH2CH2,eqCH2), 1.90 (d, J = 12.5 Hz, 1H; NCH2CH2CH2,eq), 2.22−2.32 (m, 1H; NCH2,axCH2CH2), 2.34−2.41 (m, 1H; NCH2,axCH), 2.57−2.62 (m, 1H; NCH2CH), 2.65−2.76 (m, 2H; NCH2CH2O), 2.93−3.01 (m, 1H; NCH2,eqCH2CH2), 3.04 (t, J = 6.8 Hz, 2H; OCH2CH2C), 3.07−3.16 (m, 1H; NCH2,eqCH), 3.62 (t, J = 5.3 Hz, 2H; NCH2CH2O), 3.72− 3.80 (m, 2H; OCH2CH2C), 7.42 (dt, J = 7.5/1.5 Hz, 1H; OCH2CH2CCHarCHar), 7.51 (t, J = 7.8 Hz, 1H; OCH2CH2CCHarCHar), 7.60 (t, J = 8.0 Hz, 1H; NNCCHarCHarCHarCCHarCHar), 7.61 (ddd, J = 8.3/6.8/1.8 Hz, 1H; NNCCCHarCHarCHarCHar), 7.68 (ddd, J = 8.4/6.9/1.4 Hz, 1H; NNCCCHarCH arCHarCHar), 7.83 (dd, J = 7.5/1.5 Hz, 1H; NNCCHarCHarCHarCCHarCHar), 7.90 (dt, J = 7.9/1.6 Hz, 1H; OCH 2 CH 2 CCH a r CH a r CH a r ), 7.93 (t, J = 1.8 Hz, 1H; OCH2CH2CCHarCNN), 7.96 (d, J = 8.5 Hz, 1H; NNCCCH a r CH a r CH a r CH a r ), 8.02 (d, J = 8.0 Hz, 1H; NNCCH ar CH ar CH arCCH ar CH ar ), 8.94 (d, J = 8.5 Hz, 1H; NNCCCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CD2Cl2, 23.9 °C, TMS): δ = 22.61 (NCH2CH2CH2), 26.91 (NCH2CH2CH2), 36.60 (OCH2CH2Car), 40.83 (NCH2CH), 53.86 (NCH2CH2CH2), 55.60 (NCH2CH), 57.24 (NCH2CH2O), 67.70 (NCH2CH2O), 72.31 (OCH2CH2Car), 112.4 (NNCarCarHCarHCarHCarCarHCarH), 121.7 (OCH2CH2CarCarHCarHCarH), 123.9 (OCH2CH2CarCarHCarNN), 124.0 (NNCarCarCarHCarHCarHCarH), 126.2 (NNCarCarHCarHCarHCarCarHCarH), 127.1 127.4 (NNCarCarCarHCarHCarHCarH), 128.5 (NNCarCarCarHCarHCarHCarH), (NNCarCarCarHCarHCarHCarH), 129.7 (OCH2CH2CarCarHCarH), 131.8 (NNCarCarCarHCHarCHarCarHCarCarH), 131.8 (NNCarCarHCarHCarHCarCarHCarH), 132.5 134.9 (OCH2CH2CarCarHCarH), (NNCarCarHCarHCarHCarCarHCarH), 141.2 (OCH2CH2Car), 148.2 (NNCarCarCarH), 153.9 (OCH2CH2CarCarHCarNN), 176.7 (COOH) ppm. MS (ESI+) m/z (%): 432.23 ([M + H]+). HRMS (ESI+): M+ calcd for C26H29N3O3 431.2209, found 431.2179. (R)-N-[(Vinyloxy)ethy]nipecotate ((R)-7). The compound was synthesized in analogy to the racemic compound 726 using (R)-ethyl nipecotate (1.30 g, 1.27 mL, 8.25 mmol) and 2-(vinyloxy)ethyl tosylate (1.00 g, 4.13 mmol) and obtained as colorless oil (762 mg, 80%); [α]20 D = −18.4 (c = 0.11 in CH2Cl2). All spectroscopic data except those for the optical rotation were in accord with those reported for the racemic compound. C12H21NO3 (227.31): Calcd C 63.41, H 9.31, N 6.16. Found C 63.22, H 9.32, N 6.55. Ethyl 1-(2-{[(E)-2-{4-[(E)-2-Phenyldiazen-1-yl]phenyl}ethenyl]oxy}ethyl)piperidine-3-carboxylate (9a, (E)-Isomer) and Ethyl 1-(2{[(Z)-2-{4-[(E)-2-Phenyldiazen-1-yl]phenyl}ethenyl]oxy}ethyl)piperidine-3-carboxylate (9a, (Z)-Isomer). The synthesis was performed in analogy to GP1 (method A). Pd(OAc)2 (79 mg, 0.35 mmol) and PPh3 (0.18 g, 0.70 mmol) were dissolved in abs DMF (14 mL). Phenylazophenyl triflate (1.2 g, 3.5 mmol), NEt3 (0.54 mg, 0.73 mL, 5.3 mmol), and 7 (1.2 g, 5.3 mmol) were added. The reaction time was 16 h. Workup and purification on silica gel (cyclohexane/ MTBE = 2.5:1 + 1% NEt3) gave 7a (1.13 g, 80%) as a mixture of 9a, (E)- and 9a, (Z)-isomer ((E)/(Z) = 1:1.3). The isomers could be separated chromatographically. (E)-isomer: 639 mg (43%); orange oil. IR (film): ν̃ = 3038, 2940, 2791, 1729, 1648, 1633, 1596, 1464, 1438, 1313, 1224, 1153, 1102, 1029, 930, 851, 767, 688 cm−1. 1H NMR (500 MHz, CDCl3, 20.7 °C, TMS): δ = 1.26 (t, J = 7.0 Hz, 3H; CH3), 1.46 (qd, J = 11.8/4.0 Hz, 1H; NCH2CHCH2,ax), 1.58−1.67 (m, 1H; NCH2CH2,axCH2), 1.74 (dt, J = 13.5/3.8 Hz, 1H; NCH2CH2,eqCH2), 1.96 (ddbr, J = 13.0/4.0 Hz, 1H; NCH2CHCH2,eq), 2.11 (td, J = 11.1/ 2.8 Hz, 1H; NCH2,axCH2CH2), 2.28 (t, J = 10.8 Hz, 1H; NCH2,axCH), 2.58−2.63 (m, 1H; NCH2CH), 2.71−2.79 (m, 2H; NCH2CH2O), 2.85 (dbr, J = 11.5 Hz, 1H; NCH2,eqCH2CH2), 3.07 (dbr, J = 9.0 Hz, 1H; NCH2,eqCH), 5.90 (d, J = 13.0 Hz, 1H; OCHCH), 7.17 (d, J =

13.0 Hz, 1H; OCHCH), 7.35 (d, J = 8.5 Hz, 2H; NNCCHarCHarCCHar), 7.44 (t, J = 7.3 Hz, 1H; NNCCHarCHarCHar), 7.51 (t, J = 7.5 Hz, 2H; NNCHarCHarCHarCHar), 7.85 (d, J = 8.5 Hz, 2H; NNCH ar CH ar CCH ar CH ar ), 7.90 (d, J = 7.5 Hz, 2H; NNCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CDCl3, 22.4 °C, TMS): δ = 12.59 (CH3), 22.92 (NCH2CH2CH2), 25.18 (NCH2CH2CH2), 40.17 (NCH2CH), 52.46 (NCH2CH2CH2), 54.14 (NCH 2 CH), 55.86 (NCH 2 CH 2 O), 58.72 (CH 2 CH 3 ), 65.92 (NCH2CH2O), 103.9 (OCHCH), 121.1 (2C; NNCarCarHCarHCarHCarHCarH), 121.8 (2C; NNCarCarHCarHCarCarHCarH), 123.8 (2C; NNCarCarHCarHCarCarH), 127.4 (2C; NNCarCarHCarHCarHCarH), 129.0 (NNCarCarHCarHCarH), 138.1 (OCHCHCar), 147.9 (OCHCH), 149.0 (NNCarCarHCarHCar), 151.2 (NNCarCarHCarHCarH), 172.5 (COOEt) ppm. MS (CI, CH5+) m/z (%): 408 (49, [M + H]+), 184 (100), 170 (61). HRMS (EI+): M+ calcd for C24H29N3O3 407.2209, found 407.2203. (Z)-isomer: 491 mg (34%); orange oil. IR (film): ν̃ = 3040, 2939, 2811, 1729, 1644, 1596, 1465, 1438, 1369, 1313, 1264, 1225, 1155, 1096, 1030, 853, 761, 688, 668 cm−1. 1H NMR (500 MHz, CDCl3, 22.7 °C, TMS): δ = 1.22 (t, J = 7.2 Hz, 3H; CH3), 1.43−1.52 (m, 1H; NCH2CH2CH2,ax), 1.54−1.65 (m, 1H; NCH2CH2,axCH2), 1.74 (dt, J = 13.2/3.7 Hz, 1H; NCH2CH2,eqCH2), 1.92−1.96 (m, 1H; NCH2CH2CH2,eq), 2.19 (td, J = 10.9/2.7 Hz, 1H; NCH2,axCH2CH2), 2.36 (t, J = 10.6 Hz, 1H; NCH2,axCH), 2.56−2.60 (m, 1H; NCH2CH), 2.76−2.79 (m, 2H; NCH2CH2O), 2.83 (dbr, J = 10.8 Hz, 1H; NCH2,eqCH2CH2), 3.06 (dbr, J = 9.2 Hz, 1H; NCH2,eqCH), 4.10 (t, J = 5.8 Hz, 2H; NCH2CH2OCHCH), 4.09−4.14 (m, 2H; CH2CH3), 5.31 (d, J = 7.0 Hz, 1H; OCHCH), 6.32 (d, J = 7.0 Hz, 1H; OCHCH), 7.45 (t, J = 7.3 Hz, 1H; NNCCHarCHarCHar), 7.51 (t, J = 7.5 Hz, 2H; NNCCH a r CH a r CH a r CH a r ), 7.72 (d, J = 8.5 Hz, 2H; NNCCHarCHarCCHar), 7.86 (d, J = 8.5 Hz, 2H; NNCCH a r CH a r CCH a r CH a r ), 7.90 (d, J = 8.0 Hz, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CDCl3, 21.8 °C, TMS): δ = 14.20 (CH3), 24.63 (NCH2CH2CH2), 26.67 (NCH2CH2CH2), 41.89 (NCH2CH), 54.17 (NCH2CH2CH2), 55.87 (NCH 2 CH), 57.92 (NCH 2 CH 2 O), 60.37 (CH 2 CH 3 ), 71.84 (NCH2CH2O), 105.3 (OCHCH), 122.69 (2C; NNCarCarHCarHCarHCarHCarH), 123.0 (2C; NNCarCarHCarHCarCarHCarH), 128.7 (2C; NNCarCarHCarHCarCarH), 129.0 (2C; NNCarCarHCarHCarHCarH), 130.6 (NNC ar C ar HC ar HC ar H), 139.2 (NNC ar C ar HC ar HC ar ), 148.3 (OCHCH), 150.3 (NNCarCarHCarHCar), 152.9 (NNCarCarHCarHCarH), 174.0 (COOEt) ppm. MS (CI, CH5+) m/z (%): 408 (92, [M + H]+), 319 (8), 184 (100), 170 (41). HRMS (EI +): M+ calcd for C24H29N3O3 407.2209, found 407.2235. C24H29N3O3 (407.52): Calcd C 70.74, H 7.17, N 10.31. Found C 71.01, H 7.48, N 9.98. Ethyl 1-(2-{[(E)-2-{3-[(E)-2-Phenyldiazen-1-yl]phenyl}ethenyl]oxy}ethyl)piperidine-3-carboxylate (9b, (E)-Isomer) and Ethyl 1-(2{[(Z)-2-{3-[(E)-2-Phenyldiazen-1-yl]phenyl}ethenyl]oxy}ethyl)piperidine-3-carboxylate (9b, (Z)-Isomer). The synthesis was performed according to GP1 (method B) with 3-iodoazobenzene (616 mg, 2.0 mmol), which was dissolved in DMF (8.0 mL). 7 (909 mg, 4.0 mmol), Pd(OAc)2 (45 mg, 0.2 mmol), NaOAc (197 mg, 2.4 mmol), LiCl (170 mg, 4.0 mmol), K2CO3 (332 mg, 2.4 mmol), and dest H2O (0.87 mL) were added. The reaction time was 20 h. After workup, the crude product was purified on silica gel (cyclohexane/ MTBE = 2.5:1 + 1% NEt3). The product was obtained as 9b, (E)- and 9b, (Z)-isomer ((E)/(Z) = 1:3) in an overall yield of 582.4 mg (72%). The isomers were separated chromatographically. (E)-isomer: 114 mg (14%); orange oil. IR (film): ν̃ = 3059, 2940, 2791, 1729, 1638, 1467, 1313, 1175, 1155, 1029, 925, 789, 765, 692 cm−1. 1H NMR (500 MHz, CDCl3, 20.2 °C, TMS): δ = 1.25 (t, J = 7.3 Hz, 3H; CH3), 1.46 (qd, J = 11.9/4.0 Hz, 1H; NCH2 CH 2 CH 2,ax ), 1.59−1.67 (m, 1H; NCH2CH2,axCH2), 1.74 (dt, J = 13.5/3.5 Hz, 1H; NCH2CH2,eqCH2), 1.97 (dd, J = 13.0/3.5 Hz, 1H; NCH2CH2CH2,eq), 2.11 (td, J = 11.3/ 2.8 Hz, 1H; NCH2,axCH2CH2), 2.27 (t, J = 10.8 Hz, 1H; NCH2,axCH), 2.61 (tt, J = 11.0/3.8 Hz, 1H; NCH2CH), 2.72−2.79 (m, 2H; NCH2CH2O), 2.86 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH2CH2), 3.08 (dbr, J = 9.5 Hz, 1H; NCH2,eqCH), 4.00 (t, J = 5.8 Hz, 2H; 6816

dx.doi.org/10.1021/jm5008566 | J. Med. Chem. 2014, 57, 6809−6821

Journal of Medicinal Chemistry

Article

NCH2CH2O), 4.13 (q, J = 7.2 Hz, 2H; CH2CH3), 5.93 (d, J = 13.0 Hz, 1H; OCHCH), 7.13 (d, J = 13.0 Hz, 1H; OCHCH), 7.32 (dt, J = 7.5/1.5 Hz, 1H; OCHCHCCHarCHar), 7.41 (t, J = 7.8 Hz, 1H; OCHCHCCH a r CH a r ), 7.47 (tt, J = 7.3/1.8 Hz, 1H; NNCCHarCHarCHar), 7.51−7.54 (m, 2H; NNCCHarCHarCHarCHar), 7.69 (ddd, J = 7.8/1.7/1.0 Hz, 1H; OCHCHCCHarCHarCHar), 7.78 (t, J = 1.8 Hz, 1H; OCHCHCCHarCNN), 7.90−7.93 (m, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CDCl3, 21.2 °C, TMS): δ = 14.23 (CH3), 24.56 (NCH2CH2CH2), 26.84 (NCH2CH2CH2), 41.80 (NCH2CH), 54.09 (NCH2CH2CH2), 55.79 (NCH 2 CH), 57.51 (NCH 2 CH 2 O), 60.36 (CH 2 CH 3 ), 67.38 (NCH2CH2O), 105.4 (OCHCH), 119.2 (OCHCHCarCarH), 120.2 (OCHCHCarCarHCarHCarH), 122.8 (2C; NNCarCarHCarHCarHCarHCarH), 127.4 (OCHCHCarCarHCarH), 129.1 (2C; NNC a r C a r HC a r HC ar HC a r H), 129.3 (OCHCHCarCarHCarH), 131.0 (NNCarCarHCarHCarHCarH), 137.6 (OCHCHCar), 148.8 (OCHCH), 152.7 (NNCarCarHCarHCarHCarH), 153.0 (OCHCHCarCarHCarNN), 174.1 (COOEt) ppm. MS (CI, CH5+) m/z (%): 409 (27), 408 (100, [M + H]+), 184 (18), 170 (27). HRMS (EI +): M+ calcd for C24H29N3O3 407.2209, found 407.2229. C24H29N3O3 (407.52): Calcd C 70.74, H 7.17, N 10.31. Found C 70.78, H 7.25, N 10.37. (Z)-isomer: 161 mg (19%); orange oil. IR (film): ν̃ = 3037, 2941, 2808, 1730, 1650, 1468, 1446, 1368, 1308, 1154, 1098, 1030, 801, 766, 692 cm−1. 1H NMR (500 MHz, CDCl3, 20.4 °C, TMS): δ = 1.21 (t, J = 7.3 Hz, 3H; CH3), 1.43 (qd, J = 11.8/4.0 Hz, 1H; NCH2CH2CH2,ax), 1.54−1.62 (m, 1H; NCH2CH2,axCH2), 1.68−1.74 (m, 1H; NCH2CH2,eqCH2), 1.91 (dd, J = 12.8/3.8 Hz, 1H; NCH2CH2CH2,eq), 2.19 (td, J = 11.0/2.5 Hz, 1H; NCH2,axCH2CH2), 2.36 (t, J = 10.5 Hz, 1H; NCH2,axCH), 2.57 (tt, J = 10.3/3.8 Hz, 1H; NCH2CH), 2.74−2.83 (m, 2H; NCH2CH2O), 2.85 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH2CH2), 3.05 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH), 4.06−4.12 (m, 4H; CH2CH3, NCH2CH2O), 5.33 (d, J = 7.0 Hz, 1H; OCHCH), 6.30 (d, J = 7.0 Hz, 1H; OCHCH), 7.43 (t, J = 7.8 Hz, 1H; OCHCHCCHarCHar), 7.45−7.48 (m, 1H; NNCCHarCHarCHarCHar), 7.50−7.53 (m, 2H; NNCCHarCHarCHarCHar), 7.69 (ddd, J = 8.0/2.0/ 1.0 Hz, 1H; OCHCHCCHarCHar), 7.73 (dt, J = 8.0/1.4 Hz, 1H; OCHCHCCHarCHarCHar), 7.90−7.92 (m, 2H; NNCCHarCHarCHarCHarCHar), 8.13 (t, J = 2.0 Hz, 1H; OCHCHCCHarCNN) ppm. 13C NMR (125 MHz, CDCl3, 20.2 °C, TMS): δ = 14.18 (CH3), 24.63 (NCH2CH2CH2), 26.69 (NCH2CH2CH2), 41.88 (NCH2CH), 54.14 (NCH2CH2CH2), 55.85 (NCH2CH), 57.93 (NCH2CH2O), 60.32 (CH2CH3), 71.63 (NCH2CH2O), 105.2 (OCHCH), 119.6 (OCHCHC a r C a r HC a r H), 122.8 (2C; NNCarCarHCarHCarHCarHCarH), 123.1 (OCHCHCarCarHCarNN), 128.8 (OCHCHCarCarHCarH), 129.0 (2C; NNCarCarHCarHCarHCarH), 130.7 (OCHCHCarCarHCarHCarH), 130.8 (NNC ar C ar HC ar HC ar HC ar H), 147.5 (OCHCH), 152.7 (CarNNCar), 174.1 (COOEt) ppm. MS (CI, CH5+) m/z (%): 409 (28), 408 (100, [M + H]+), 184 (17), 170 (45). HRMS (EI+): M+ calcd for C24H29N3O3 407.2209, found 407.2218. Ethyl 1-(2-{[(E)-2-(2-Nitrophenyl)ethenyl]oxy}ethyl)piperidine-3carboxylate (9c, (E)-Isomer) and Ethyl 1-(2-{[(Z)-2-(2-Nitrophenyl)ethenyl]oxy}ethyl)piperidine-3-carboxylate (9c, (Z)-Isomer). The synthesis was performed according to GP1 (method B). 1-Iodo-2nitrobenzene (0.75 mg, 3.0 mmol, CAS 609-73-4) was dissolved in abs DMF (12 mL), and 7 (1.4 g, 6.0 mmol), Pd(OAc)2 (67 mg, 0.30 mmol), NaOAc (0.30 g, 3.6 mmol), LiCl (0.25 g, 6.0 mmol), K2CO3 (0.50 g, 3.6 mmol), and dest water (1.2 mL) were added. The reaction time was 3 h. After workup, the crude product was purified on silica gel (cyclohexane/MTBE = 2.5:1 + 1% NEt3), and the product was obtained as 9c, (E)- and 9c, (Z)-isomer ((E)/(Z) = 1.5:1) in an overall yield of 657.2 mg (63%). The isomers were not separated. (E)and (Z)-isomer; red oil. IR (film): ν̃ = 3071, 2942, 2794, 1728, 1639, 1605, 1521, 1467, 1345, 1311, 1272, 1231, 1176, 1156, 948, 743 cm−1. MS (CI, CH5+) m/z (%): 350 (21), 349 (100, [M + H]+), 184 (22), 170 (18). HRMS (EI+): M+ calcd for C18H24N2O5 348.1685, found 348.1701. C18H24N2O5 (348.40): Calcd C 62.05, H 6.94, N 8.04. Dound C 61.79, H 6.96, N 8.07. NMR signals of the individual isomers could be assigned from the NMR of the mixture of the isomers. (E)isomer. 1H NMR (500 MHz, CDCl3, 21.5 °C, TMS): δ = 1.26 (t, J =

7.0 Hz, 3H; CH3), 1.42−1.50 (m, 1H; NCH2CH2CH2,ax), 1.53−1.67 (m, 1H; NCH2CH2,axCH2), 1.70−1.77 (m, 1H; NCH2CH2,eqCH2), 2.10 (td, J = 11.0/3.0 Hz, 1H; NCH2,axCH2CH2), 2.27 (t, J = 10.8 Hz, 1H; NCH2,axCH), 2.60 (tt, J = 10.8/3.9 Hz, 1H; NCH2CH), 2.74− 2.77 (m, 2H; NCH2CH2O), 2.85 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH), 3.06 (dbr, J = 10.5 Hz, 1H; NCH2,eqCH2CH2), 4.01 (t, J = 5.5 Hz, 2H; NCH2CH2O), 4.13 (q, J = 7.2 Hz, 2H; CH2CH3), 6.40 (d, J = 12.5 Hz, 1H; OCHCH), 7.05 (d, J = 13.0 Hz, 1H; OCHCH), 7.27 (ddd, J = 8.3/6.8/1.5 Hz, 1H; O2NCCHarCHar), 7.45 (dd, J = 8.0/2.0 Hz, 1H; O2NCCCHar), 7.47−7.50 (m, 1H; O2NCCHarCHarCHar), 7.90 (dd, J = 8.3/1.3 Hz, 1H; O2NCCHar) ppm. 13C NMR (125 MHz, CDCl3, 19.5 °C, TMS): δ = 14.22 (CH3), 24.53 (NCH2CH2CH2), 26.82 (NCH2CH2CH2), 41.78 (NCH2CH), 54.07 (NCH2CH2CH2), 55.79 (NCH 2 CH), 57.34 (NCH 2 CH 2 O), 60.37 (CH 2 CH 3 ), 67.21 (NCH2CH2O), 101.1 (OCHCH), 125.0 (O2NCarCarH), 126.2 (O2NCarCarHCarH), 127.1 (O2NCarCarCarH), 132.0 (O2NCarCarCarH), 133.0 (O2NCarCarHCarHCarH), 147.3 (O2NCar), 151.6 (OCHCH), 174.1 (COOEt) ppm. MS (CI, CH5+) m/z (%): 350 (21), 349 (100, [M + H]+), 184 (22), 170 (18). HRMS (EI+): M+ calcd for C18H24N2O5 348.1685, found 348.1701. C18H24N2O5 (348.40): Calcd C 62.05, H 6.94, N 8.04. Found C 61.79, H 6.96, N 8.07. (Z)-isomer. 1 H NMR (500 MHz, CDCl3, 21.5 °C, TMS): δ = 1.23 (t, J = 7.0 Hz, 3H; CH3), 1.42−1.50 (m, 1H; NCH2CH2CH2,ax), 1.53−1.67 (m, 1H; NCH2CH2,axCH2), 1.70−1.77 (m, 1H; NCH2CH2,eqCH2), 1.91−1.98 (m, 1H; NCH2CH2CH2,eq), 2.16 (td, J = 10.8/2.8 Hz, 1H; NCH2,axCH2CH2), 2.34 (t, J = 10.5 Hz, 1H; NCH2,axCH), 2.55 (tt, J = 10.0/3.9 Hz, 1H; NCH2CH), 2.71 (t, J = 5.8 Hz, 2H; NCH2CH2O), 2.77 (dbr, J = 11.6 Hz, 1H; NCH2,eqCH2CH2), 3.00 (dbr, J = 10.0 Hz, 1H; NCH2,eqCH), 4.06 (t, J = 6.0 Hz, 2H; NCH2CH2O), 4.11 (qd, J = 7.1/1.9 Hz, 2H; CH2CH3), 5.70 (d, J = 7.5 Hz, 1H; OCHCH), 6.39 (d, J = 7.0 Hz, 1H; OCHCH), 7.24 (ddd, J = 8.3/7.3/1.3 Hz, 1H; O2NCCHarCHar), 7.48−7.51 (m, 1H; O2NCCHarCHarCHar), 7.80 (dd, J = 8.3/1.3 Hz, 1H; O2NCCHar), 8.12 (dd, J = 8.3/1.3 Hz, 1H; O2NCCCHar) ppm. 13C NMR (125 MHz, CDCl 3 , 19.5 °C, TMS): δ = 14.22 (CH 3 ), 24.61 (NCH2CH2CH2), 26.66 (NCH2CH2CH2), 41.85 (NCH2CH), 54.11 (NCH2CH2CH2), 55.79 (NCH2CH), 57.82 (NCH2CH2O), 60.37 (CH 2 CH 3 ), 71.78 (NCH 2 CH 2 O), 98.95 (OCHCH), 124.2 (O2NCarCarH), 126.02 (O2NCarCarHCarH), 129.9 (O2NCarCarCarH), 131.1 (O2NCarCarCarH), 132.2 (O2NCarCarHCarHCarH), 147.6 (O2NCar), 149.8 (OCHCH), 174.0 (COOEt) ppm. Ethyl (3R)-1-(2-{[(E)-2-{4-[(E)-2-(Naphthalen-1-yl)diazen-1-yl]phenyl}ethenyl]oxy}ethyl)piperidine-3-carboxylate ((R)-9d, (E)-Isomer) and Ethyl (3R)-1-(2-{[(Z)-2-{4-[(E)-2-(Naphthalen-1-yl)diazen1-yl]phenyl}ethenyl]oxy}ethyl)piperidine-3-carboxylate ((R)-9d, (Z)Isomer). The synthesis was performed following to GP1 (method A), with s-4 (380 mg, 1.00 mmol), (R)-7 (340 mg, 1.50 mmol), Pd(OAc)2 (22.0 mg, 0.100 mmol), NEt3 (152 mg, 0.210 mL, 1.50 mmol), and PPh3 (45.0 mg, 0.200 mmol) in DMF (4.00 mL). After a reaction time of 20 h, the crude product was purified via FCC (cyclohexane/MTBE = 2.5:1 + 1% NEt3), which yielded the product as (R)-9d, (E)- and (R)-9d, (Z)-isomer ((E)/(Z) = 1:1.2) in an overall yield of 356 mg (78%). The isomers could be separated via FCC (Et2O + 1% NEt3). (E)-isomer: 173 mg (38%); orange oil; [α]20 D = −17.82 (c = 0.11 in CH2Cl2). IR (film): ν̃ = 3050, 2940, 2868, 2791, 1728, 1648, 1633, 1595, 1463, 1370, 1311, 1221, 1156, 1139, 1030, 851, 802, 773 cm−1. 1 H NMR (500 MHz, CDCl3, 21.1 °C, TMS): δ = 1.26 (t, J = 7.3 Hz, 3H; CH3), 1.47 (qd, J = 11.8/3.8 Hz, 1H; NCH2CH2CH2,ax), 1.58− 1.68 (m, 1H; NCH2CH2,axCH2), 1.75 (dt, J = 13.5/3.6 Hz, 1H; NCH2CH2,eqCH2), 1.93−2.00 (m, 1H; NCH2CH2CH2,eq), 2.12 (td, J = 11.0/2.5 Hz, 1H; NCH2,axCH2CH2), 2.29 (t, J = 10.5 Hz, 1H; NCH2,axCH), 2.61 (tt, J = 10.8/3.9 Hz, 1H; NCH2CH), 2.71−2.80 (m, 2H; NCH2CH2O), 2.86 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH2CH2), 3.07 (dbr, J = 9.5 Hz, 1H; NCH2,eqCH), 4.02 (t, J = 5.5 Hz, 2H; NCH2CH2O), 4.14 (q, J = 7.0 Hz, 2H; CH2CH3), 5.92 (d, J = 13.0 Hz, 1H; OCHCH), 7.20 (d, J = 13.0 Hz, 1H; OCHCH), 7.39 (d, J = 8.5 Hz, 2H; OCHCHCCHarCHarCCHarCHar), 7.56 (t, J = 7.8 Hz, 1H; NNCCHarCHarCHar), 7.56−7.60 (m, 1H; NNCCCHarCHarCHar), 7.64 (ddd, J = 8.4/6.9/1.4 Hz, 1H; NNCCCHarCHar), 7.81 (dd, J = 7.5/1.0 Hz, 1H; NNCCHarCHarCHar), 7.92 (d, J = 8.0 Hz, 1H; 6817

dx.doi.org/10.1021/jm5008566 | J. Med. Chem. 2014, 57, 6809−6821

Journal of Medicinal Chemistry

Article

NNCCCH a r CH a r CH a r CH a r ), 7.96 (d, J = 7.0 Hz, 1H; NNCCHarCHarCHar), 7.98 (d, J = 8.5 Hz, 2H; OCHCHCCHarCHarCCHar), 8.92 (d, J = 8.5 Hz, 1H; NNCCCHar) ppm. 13C NMR (125 MHz, CDCl3, 19.2 °C, TMS): δ = 14.23 (CH3), 24.57 (NCH2CH2CH2), 26.83 (NCH2CH2CH2), 41.79 (NCH2CH), 54.10 (NCH2CH2CH2), 55.77 (NCH2CH), 57.51 (NCH2CH2O), 60.38 (CH 2 CH 3 ), 67.49 (NCH 2 CH 2 O), 105.5 (OCHCH), 111.7 (NNC ar C ar HC ar HC ar H), 123.5 (NNC ar C ar C ar H), 123.8 (2C; OCHCHCarCarHCarHCarCarH), 125.5 (2C; OCHCHCarCarHCarHCarCarHCarH), 125.7 (NNCarCarHCarHCarH), 126.4 (NNC arC arC ar HC arHCar H), 126.7 (NNC ar CarC arHCarH), 127.9 (NNCarCarCarHCarHCarHCarH), 130.9 (NNCarCarHCarHCarH), 131.3 (NNCarCarCarH), 134.3 (NNCarCarHCarHCarHCar), 139.8 (OCHCHCar), 147.9 (NNCarCarCarH), 149.6 (OCHCH), 151.3 (OCHCHCarCarHCarHCar), 174.1 (COOEt) ppm. MS (CI, CH5+) m/z (%): 458 (100, [M + H]+), 184(41), 170 (24). HRMS (EI+): M+ calcd for C28H31N3O3, 457.2365, found 457.2365. (Z)-isomer: 77 mg (17%); orange oil; [α]20 D = −1.79 (c = 0.11 in CH2Cl2). IR (film): ν̃ = 3050, 2940, 2811, 1729, 1643, 1595, 1306, 1222, 1156, 1097, 1030, 852, 804, 774 cm−1. 1H NMR (500 MHz, CDCl3, 19.8 °C, TMS): δ = 1.22 (t, J = 7.0 Hz, 3H; CH 3 ), 1.48 (qd, J = 11.8/3.7 Hz, 1H; NCH2CH2CH2,ax), 1.5−1.65 (m, 1H; NCH2CH2,axCH2), 1.75 (dt, J = 13.5/3.8 Hz, 1H; NCH2 CH 2,eq CH 2), 1.90−1.98 (m, 1H; NCH2CH2CH2,eq), 2.19 (td, J = 10.8/2.8 Hz, 1H; NCH2,axCH2CH2), 2.38 (t, J = 10.5 Hz, 1H; NCH2,axCH), 2.59 (tt, J = 10.3/3.8 Hz, 1H; NCH2CH), 2.74−2.82 (m, 2H; NCH2CH2O), 2.83 (dbr, J = 10.5 Hz, 1H; NCH2,eqCH2CH2), 3.07 (dbr, J = 8.5 Hz, 1H; NCH2,eqCH), 4.11 (t, J = 6.0 Hz, 2H; NCH2CH2O), 4.07−4.17 (m, 2H; CH2CH3), 5.33 (d, J = 7.0 Hz, 1H; OCHCH), 6.34 (d, J = 7.0 Hz, 1H; OCHCH), 7.56 (t, J = 7.8 Hz, 1H; NNCCHarCHarCHar), 7.56−7.60 (m, 1H; NNCCCHarCHarCH ar), 7.64 (ddd, J = 8.3/6.8/1.5 Hz, 1H; NNCCCH ar CH ar ), 7.76 (d, J = 8.5 Hz, 2H; OCHCHCCH a r CH a r CCH a r CH a r ), 7.81 (dd, J = 7.5/1.0 Hz, 1H; NNCCHarCHarCHar), 7.92 (d, J = 8.0 Hz, 1H; NNCCH a r CH a r CH a r CH a r ), 7.96 (d, J = 8.0 Hz , 1H; NNCCHarCHarCHar), 8.00 (d, J = 8.5 Hz, 2H; OCHCHCCHarCHarCCHar), 8.94 (d, J = 8.0 Hz, 1H; NNCCCHar) ppm. 13C NMR (125 MHz, CDCl3, 19.9 °C, TMS): δ = 14.20 (CH3), 24.65 (NCH2CH2CH2), 26.69 (NCH2CH2CH2), 41.91 (NCH2CH), 54.18 (NCH2CH2CH2), 55.89 (NCH2CH), 57.94 (NCH2CH2O), 60.37 (CH 2 CH 3 ), 71.91 (NCH 2 CH 2 O), 105.3 (OCHCH), 111.7 (NNCarCarHCarHCarH), 123.3 (2C; OCHCHCarCarHCarHCarCarH), 123.5 (NNC arC ar CarH), 125.7 (NNC ar C ar HCarHC arH),, 126.4 (NNC arC arC ar HC arHCar H), 126.7 (NNC ar CarC arHCarH), 127.9 (NNCarCarCarHCarHCarHCarH), 128.8 (2C; OCHCHCarCarHCarHCarCarHCarH), 130.9 (NNCarCarHCarHCarH), 131.3 (NNCarCarCarH), 134.3 (NNCarCarHCarHCarHCar), 139.3 (OCHCHCar), 148.0 (NNCarCarCarH), 148.5 (OCHCH), 150.9 (OCHCHCarCarHCarHCar), 174.1 (COOEt) ppm. MS (CI, CH5+) m/z (%): 458 (100, [M + H]+), 184 (86), 170 (68). HRMS (EI+): M+ calcd for C28H31N3O3 457.2365; found 457.2381. Ethyl (3R)-1-(2-{[(Z)-2-{3-[(E)-2-(Naphthalen-1-yl)diazenyl]phenyl}ethenyl]oxy}ethyl)piperidine-3-carboxylate ((R)-9e, (Z)-Isomer). The synthesis was performed according to GP1 (method B). s-5 (179 mg, 0.500 mmol) was dissolved in abs DMF (2.00 mL), and (R)7 (227 mg, 1.00 mmol), Pd(OAc)2 (11.2 mg, 0.050 mmol), NaOAc (49.2 mg, 0.600 mmol), LiCl (42.4 mg, 1.00 mmol), K2CO3 (82.9 mg, 0.600 mmol), and dest H2O (0.220 mL) were added. After a reaction time of 17 h and workup, the crude product was purified by FCC (cyclohexane/MTBE = 2.5:1 + 1% NEt3) to yield (R)-9a, (E)- and (R)-9a, (Z)-isomer as a mixture ((E)/(Z) = 17:83) in a yield of 189 mg (83%). The (Z)-isomer could be isolated as orange−red oil (121 mg, 53%) using FCC (Et2O + 1% NEt3); [α]20 D = −0.7 (c = 0.1 in CH2Cl2). IR (film): ν̃ = 3053, 2939, 2808, 1728, 1649, 1508, 1464, 1439, 1370, 1310, 1266, 1222, 1179, 1155, 1097, 1030, 803, 773, 689 cm−1. 1H NMR (500 MHz, CDCl3, 22.8 °C, TMS): δ = 1.20 (t, J = 7.3 Hz, 3H; CH3), 1.37−1.49 (m, 1H; NCH2CH2CH2,ax), 1.51−1.62 (m, 1H; NCH 2 CH 2,ax CH 2 ), 1.69 (dt, J = 13.0/3.8 Hz, 1H; NCH2CH2,eqCH2), 1.85−1.93 (m, 1H; NCH2CH2CH2,eq), 2.19 (td, J = 11.1/2.8 Hz, 1H; NCH2,axCH2CH2), 2.36 (t, J = 10.5 Hz, 1H;

NCH2,axCH), 2.52−2.61 (m, 1H; NCH2CH), 2.75−2.88 (m, 3H; NCH2CH2O and NCH2,eqCH2CH2), 3.05 (d, J = 10.0 Hz, 1H; NCH2,eqCH), 4.03−4.15 (m, 4H; NCH2CH2O and CH2CH3), 5.37 (d, J = 7.0 Hz, 1H; OCHCH), 6.32 (d, J = 7.0 Hz, 1H; OCHCH), 7.47 (t, J = 7.8 Hz, 1H; OCHCHCCHarCHar), 7.57 (t, J = 7.8 Hz, 1H; NNCCHarCHarCHarCCHarCHar), 7.58 (ddd, J = 8.1/7.0/1.4 Hz, 1H; NNCCCHarCHarCHarCHar), 7.64 (ddd, J = 8.3/6.8/1.5 Hz, 1H; NNCCCHar CH ar CH arCH ar ), 7.74 (dt, J = 8.0/1.4 Hz, 1H; OCHCHCCH a r CH a r ), 7.80 (dd, J = 7.5/1.0 Hz, 1H; NNCCHarCHarCHarCCHarCHar), 7.83 (ddd, J = 8.0/2.0/1.0 Hz, 1H; OCHCHCCH ar CH ar CH ar ), 7.93 (d, J = 8.5 Hz, 1H; NNCCCH a r CH a r CH a r CH a r ), 7.95 (d, J = 8.0 Hz, 1H; NNCCCHarCHarCHarCHarCCHar), 8.27 (t, J = 1.8 Hz, 1H; OCHCHCCHarCNN), 8.94 (d, J = 8.0 Hz, 1H; NNCCCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CDCl3, 24.4 °C, TMS): δ = 14.18 (CH3), 24.61 (NCH2CH2CH2), 26.67 (NCH2CH2CH2), 41.87 (NCH2CH), 54.17 (NCH2CH2CH2), 55.89 (NCH2CH), 57.94 (NCH2CH2O), 60.32 (NCH2CH2O), 71.70 105.2 (OCHCH), 111.8 (CH2CH3), (NNCarCarHCarHCarHCarCarHCarH), 120.1 (OCHCHC ar C ar HC ar HC ar H), 123.3 (OCHCHC ar C ar HC ar NN), 123.6 125.7 (NNCarCarCarHCarHCarHCarH), (NNCarCarHCarHCarHCarCarHCarH), 126.4 126.7 (NNCarCarCarHCarHCarHCarH), 127.9 (NNCarCarCarHCarHCarHCarH), (NNC arC arC arHCar HC arHC arH), 128.9 (OCHCHC arC arHC arH), 131.1 130.8 (OCHCHCarCarHCarH), (NNCarCarHCarHCarHCarCarHCarH), 131.3 (NNCarCarCarH), 134.3 (NNCarCarCarCarH), 137.0 (OCHCHCar), 147.6 (OCHCH), 147.9 (NNCarCarCarH), 153.3 (OCHCHCarCarHCarNN), 174.0 (COOEt) ppm. MS (CI, CH5+) m/z (%): 458 (100, [M + H]+), 184 (50), 170 (40). HRMS (EI+): M+ calcd for C28H31N3O3 457.2365, found 457.2394. Ethyl-1-{2-[2-(2-Aminophenyl)ethoxy]ethyl}piperidine-3-carboxylate (10). 9c was dissolved in MeOH (HPLC grade, 3 mL), and the mixture was degassed. Pd/C (10% Pd, 15.5 mg, 0.015 mmol) was added, and the reaction mixture was hydrogenated for 1 h under 5 bar H2 pressure and vigorous stirring. After completion of the reaction, the catalyst was filtrated through a PET syringe filter and the solvent was evaporated. The crude product was purified on silica gel (Et2O + 1% NEt3) to yield the product as colorless oil (91.3 mg, 98%). IR (film): ν̃ = 3438, 3357, 2940, 2865, 2803, 1728, 1628, 1499, 1456, 1313, 1275, 1225, 1154, 1110, 748 cm−1. 1H NMR (500 MHz, CDCl3, 22.1 °C, TMS): δ = 1.25 (t, J = 7.3 Hz, 3H; CH3), 1.41 (qd, J = 11.9/3.8 Hz, 1H; NCH2CH2CH2,ax), 1.51−1.60 (m, 1H; NCH2CH2,axCH2), 1.69 (dt, J = 13.5/3.5 Hz, 1H; NCH2CH2,eqCH2), 1.91−1.94 (m, 1H; NCH2CH2CH2,eq), 2.02 (td, J = 11.1/2.7 Hz, 1H; NCH2,axCH2CH2), 2.19 (t, J = 10.8 Hz, 1H; NCH2,axCH), 2.53 (tt, J = 11.0/4.0 Hz, 1H; NCH2CH), 2.57 (t, J = 6.0 Hz, 2H; NCH2CH2O), 2.76 (dbr, J = 11.5 Hz, 1H; NCH 2 , e q CH 2 CH 2 ), 2.80 (t, J = 6.3 Hz, 2H; NCH2CH2OCH2CH2), 3.00 (dbr, J = 10.0 Hz, 1H; NCH2,eqCH), 3.55 (t, J = 5.8 Hz, 2H; NCH2CH2O), 3.68 (t, J = 6.3 Hz, 2H; NCH2CH2OCH2), 4.01 (s, 2H; NH2), 4.13 (q, J = 7.2 Hz, 2H; CH2CH3), 6.67 (dd, J = 7.8/1.3 Hz, 1H; H2NCCHar), 6.71 (td, J = 7.4/1.2 Hz, 1H; H2NCCHarCHarCHar), 7.02 (dd, J = 7.5/1.5 Hz, 1H; H 2 NCCH ar CH ar CH ar CH ar ), 7.03 (td, J = 7.6/1.7 Hz, 1H; H2NCCHarCHar) ppm. 13C NMR (125 MHz, CDCl3, 21.4 °C, TMS): δ = 14.22 (CH 3 ), 24.60 (NCH 2 CH 2 CH 2 ), 26.86 (NCH2CH2CH2), 32.61 (NCH2CH2OCH2CH2), 41.85 (NCH2CH), 54.05 (NCH2CH2CH2), 55.84 (NCH2CH), 58.12 (NCH2CH2O), 60.31 (CH2CH3), 68.90 (NCH2CH2O), 72.14 (NCH2CH2OCH2), 116.0 (H 2 NC ar C ar H), 118.6 (H 2 NC ar C ar HC ar HC ar H), 125.0 (OCH2CH2Car), 127.4 (H2NCarCarHCarH), 130.4 (H2NCarCarHCarHCarHCarH), 145.7 (CarNH2), 174.2 (COOEt) ppm. (CI, CH5+) m/z (%): 322 (21), 321 (100, [M + H]+), 183 (11), 170 (12). HRMS (EI+): M+ calcd for C18H28N2O3 320.2100, found 320.2099. Ethyl 1-[2-(2-{4-[(E)-2-Phenyldiazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylate (11a). The synthesis was performed according to GP2. 7a (102 mg, 0.250 mmol) was dissolved in 6818

dx.doi.org/10.1021/jm5008566 | J. Med. Chem. 2014, 57, 6809−6821

Journal of Medicinal Chemistry

Article

THF/MeOH (2.00 mL, v/v = 1:1), Wilkinson’s catalyst (58.0 mg, 0.062 mmol, 25 mol %) was added, and the mixture was stirred for 3 h. After workup, the crude product was purified on silica gel (Et2O) and yielded as orange oil (71 mg, 69%). IR (film): ν̃ = 2939, 2862, 1729, 1603, 1466, 1444, 1303, 1154, 1112, 1030, 837, 767, 688 cm−1. 1H NMR (500 MHz, CD2Cl2, 21.0 °C, TMS): δ = 1.22 (t, J = 7.0 Hz, 3H; CH3), 1.39 (qd, J = 11.8/3.8 Hz, 1H; NCH2CH2CH2,ax), 1.50−1.60 (m, 1H; NCH2CH2,axCH2), 1.64−1.70 (m, 1H; NCH2CH2,eqCH2), 1.87−1.90 (m, 1H; NCH2CH2CH2,eq), 2.04 (tbr, J = 10.0 Hz, 1H; NCH2,axCH2CH2), 2.21 (t, J = 10.3 Hz, 1H; NCH2,axCH), 2.49−2.54 (m, 1H; NCH2CH), 2.56 (t, J = 5.3 Hz, 2H; NCH2CH2O), 2.76 (dbr, J = 10.5 Hz, 1H; NCH2,eqCH2CH2), 2.95 (t, J = 7.0 Hz, 2H; NCH2CH2OCH2CH2), 2.99 (dbr, J = 10.5 Hz, 1H; NCH2,eqCH), 3.57 (t, J = 5.8 Hz, 2H; NCH2CH2O), 3.67 (t, J = 6.8 Hz, 2H; NCH2CH2OCH2CH2), 4.08 (q, J = 7.2 Hz, 2H; CH2CH3), 7.40 (d, J = 8.0 Hz, 2H; H2CCCHarCHarCCHarCHar), 7.48 (t, J = 7.3 Hz, 1H; NNCCHarCHarCHar), 7.53 (t, J = 7.3 Hz, 2H; NNCCH a r CH a r CH a r CH a r ), 7.85 (d, J = 8.5 Hz , 2H; H2CCCHarCHarCCHar), 7.90 (d, J = 7.0 Hz, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CD2Cl2, 23.7 °C, TMS): δ = 14.22 (CH3), 24.50 (NCH2CH2CH2), 26.83 (NCH2CH2CH2), 36.19 (OCH2CH2Car), 41.75 (NCH2CH), 54.07 (NCH2CH2CH2), 55.82 (NCH2CH), 58.03 (NCH2CH2O), 60.33 (CH2CH3), 68.66 (NCH2CH2O), 71.71 (OCH2CH2Car), 122.8 (2C; NNCarCarHCarHCarHCarHCarH), 122.9 (2C; H2CCarCarHCarHCarCarH), 129.1 (2C; NNCarCarHCarHCarHCarH), 129.6 (2C; H2CCarCarHCarHCarCarHCarH), 130.8 (NNCarCarHCarHCarH), 142.6 (H2CCar), 151.3 (NNCarCarHCarHCarCH2), 152.7 (NNCarCarHCarHCarH), 174.2 (COOEt) ppm. (CI, CH5+) m/z (%): 411 (33), 410 (84, [M + H]+), 171 (10), 170 (100). HRMS (EI+): M+ calcd for C24H31N3O3 409.2365, found 409.2354. Ethyl 1-[2-(2-{3-[(E)-2-Phenyldiazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylate (11b). The synthesis was performed following GP2. 7b (134.5 mg, 0.33 mmol) was dissolved in THF/ MeOH (2 mL, v/v = 1:1), Wilkinson’s catalyst (15.3 mg, 0.017 mmol, 5 mol %) was added, and the mixture was stirred for 6 h. After workup, the crude product was purified on silica gel (Et2O) and obtained as orange oil (84.6 mg, 63%). IR (film): ν̃ = 3061, 2939, 2863, 1729, 1600, 1467, 1447, 1369, 1308, 1225, 1153, 1113, 1030, 792, 768, 694 cm−1. 1H NMR (500 MHz, CDCl3, 21.6 °C, TMS): δ = 1.24 (t, J = 7.0 Hz, 3H; CH3), 1.40 (qd, J = 12.1/3.8 Hz, 1H; NCH2CH2CH2,ax), 1.52−1.61 (m, 1H; NCH 2 CH 2,ax CH 2 ), 1.64−1.69 (m, 1H; NCH2CH2,eqCH2), 1.88−1.95 (m, 1H; NCH2CH2CH2,eq), 2.01 (td, J = 11.3/2.8 Hz, 1H; NCH2,axCH2CH2), 2.20 (t, J = 10.8 Hz, 1H; NCH2,axCH), 2.56 (tt, J = 11.0/3.2 Hz, 1H; NCH2CH), 2.53−2.65 (m, 2H; NCH2CH2O), 2.80 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH2CH2), 3.00 (t, J = 7.0 Hz, 2H; NCH2CH2OCH2CH2), 3.02 (dbr, J = 11.5 Hz, 1H; NCH2,eqCH), 3.57−3.63 (m, 2H; NCH2CH2O), 3.73 (t, J = 7.0 Hz, 2H; NCH2CH2OCH2), 4.11 (q, J = 7.0 Hz, 2H; CH2CH3), 7.35 (dt, J = 7.5/1.5 Hz, 1H; OCH2CH2CCHarCHar), 7.44 (t, J = 7.5 Hz, 1H; OCH 2 CH 2 CCH a r CH a r ), 7.47 (t, J = 7.3 Hz, 1H; NNCCHarCHarCHar), 7.52 (t, J = 7.3 Hz, 2H; NNCCHarCHarCHarCHar), 7.76−7.79 (m, 1H; H2CCCHarCHarCHar), 7.78 (t, J = 1.0 Hz, 1H; H2CCCHarCNN), 7.91 (d, J = 7.0 Hz, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CDCl3, 22.1 °C, TMS): δ = 14.22 (CH3), 24.56 (NCH2CH2CH2), 26.87 (NCH2CH2CH2), 36.13 (OCH2CH2Car), 41.83 (NCH2CH), 54.07 (NCH2CH2CH2), 55.88 (NCH2CH), 58.06 (NCH2CH2O), 60.27 (CH2CH3), 68.72 (NCH2CH2O), 71.82 (OCH2CH2Car), 121.0 (H2CCarCarHCarHCarH), 122.8 (2C; NNCarCarHCarHCarHCarHCarH), 123.1 (H2CCarCarHCarNN), 129.0 (H2CCarCarHCarH), 129.1 (2C; NNCarCarHCarHCarHCarH), 130.9 (NNCarCarHCarHCarHCarH), 131.7 (H2CCarCarHCarH), 140.2 (H2CCar), 152.7 (NNCarCarHCarHCarHCarH), 152.8 (H2CCarCarHCarNN), 174.2 (COOEt) ppm. (CI, CH5+) m/z (%): 411 (24), 410 (100, [M + H]+), 85 (12), 83 (12). HRMS (EI+): M+ calcd for C24H31N3O3 409.2365, found 409.2373. Ethyl 1-[2-(2-{2-[(E)-2-Phenyldiazen-1-yl]phenyl}ethoxy)ethyl]piperidine-3-carboxylate (11c). 10 (150 mg, 0.47 mmol) was added

at rt to a solution of nitrosobenzene (65 mg, 0.61 mmol) in AcOH (1.0 mL). The green solution and was heated to 85 °C for 6 h and slowly turned dark-red during this time. It was diluted with toluene (3.0 mL) and subsequently washed with water, sat NaHCO3 solution, and brine. The organic phase was dried over MgSO4, and the solvent was removed in vacuo to give the crude product which was purified on silica gel (MTBE). The product was obtained as an orange oil (134.1 mg, 70%). IR (film): ν̃ = 3060, 2939, 2862, 1730, 1478, 1467, 1452, 1369, 1309, 1154, 1113, 1031, 774, 739, 688 cm−1. 1H NMR (500 MHz, CDCl3, 19.2 °C, TMS): δ = 1.23 (t, J = 7.3 Hz, 3H; CH3), 1.39 (qd, J = 11.9/4.1 Hz, 1H; NCH2CH2CH2,ax), 1.51−1.61 (m, 1H; NCH2CH2,axCH2), 1.63−1.70 (m, 1H; NCH2CH2eqCH2), 1.89−1.95 (m, 1H; NCH2CH2CH2,eq), 1.98 (td, J = 11.3/2.5 Hz, 1H; NCH2,axCH2CH2), 2.17 (t, J = 10.8 Hz, 1H; NCH2,axCH), 2.52− 2.65 (m, 2H; NCH2CH, NCH2CH2O), 2.78 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH2CH2), 3.01 (dbr, J = 9.0 Hz, 1H; NCH2,eqCH), 3.44 (t, J = 7.3 Hz, 2H; NCH2CH2OCH2CH2), 3.58 (t, J = 6.0 Hz, 2H; NCH2CH2O), 3.73 (t, J = 7.3 Hz, 2H; NCH2CH2OCH2), 4.11 (q, J = 7.2 Hz, 2H; CH2CH3), 7.31 (ddd, J = 8.1/5.6/2.6 Hz, 1H; H2CCCHarCHarCHar), 7.38−7.43 (m, 2H; H2CCCHarCHar), 7.47 (t, J = 7.3 Hz, 1H; NNCCHarCHarCHarCHarCHar), 7.52 (t, J = 7.5 Hz, 2H; NNCCHarCHarCHarCHarCHar), 7.67 (d, J = 7.5 Hz, 1H; H 2 CCCH a r CH a r CH a r CH a r ), 7.92 (d, J = 7.0 Hz, 2H; NNCCHarCHarCHarCHarCHar) ppm. 13C NMR (125 MHz, CDCl3, 20.3 °C, TMS): δ = 14.21 (CH3), 24.53 (NCH2CH2CH2), 26.88 (NCH2CH2CH2), 31.95 (OCH2CH2Car), 41.81 (NCH2CH), 54.01 (NCH2CH2CH2), 55.86 (NCH2CH), 58.05 (NCH2CH2O), 60.28 (CH2CH3), 68.50 (NCH2CH2O), 72.29 (OCH2CH2Car), 115.4 (H2CCarCarCarH), 123.0 (2C; NNCarCarHCarHCarHCarHCarH), 127.1 (H2CCarCarHCarHCarH), 129.1 (2C; NNCarCarHCarHCarHCarHCarH), 130.9 (NNCarCarHCarHCarHCarHCarH), 131.1 (H2CCarCarHCarH), 131.2 (H2CCarCarH), 138.8 (H2CCar), 150.5 (H2CCarCarNN), 152.9 (H2CCarCarNNCar), 174.2 (COOEt) ppm. MS (CI, CH5+) m/z (%): 410 (100, [M + H]+), 184 (11), 170 (29). HRMS (EI+): M+ calcd for C24H31N3O3 409.2365, found 409.2362. Ethyl (3R)-1-[2-(2-{4-[(E)-2-(Naphthalen-1-yl)diazen-1-yl]phenyl}ethoxy)ethyl]-piperidine-3-carboxylate ((R)-11d). The synthesis was performed following GP2. (R)-(E)/(Z)-9d (53.9 mg, 0.120 mmol) and Wilkinson’s catalyst (11.0 mg, 0.012 mmol) were dissolved in THF/MeOH (1 mL, v/v = 1:1) and hydrogenated 5.5 h at 10 bar H2 pressure. The crude product was purified on silica (Et2O + 1%NEt3) to yield the product as orange oil (33.7 mg, 61%); [α]20 D = 10.4 (c = 0.1 in CH2Cl2). IR (film): ν̃ = 3052, 2939, 2863, 2801, 1730, 1602, 1500, 1466, 1388, 1308, 1155, 1112, 1031, 841, 803, 773 cm−1. 1H NMR (500 MHz, CDCl3, 20.4 °C, TMS): δ = 1.24 (t, J = 7.3 Hz, 3H; CH3), 1.41 (qd, J = 12.0/3.9 Hz, 1H; NCH2CH2CH2,ax), 1.53−1.63 (m, 1H; NCH2CH2,axCH2), 1.70 (dt, J = 13.5/3.5 Hz, 1H; NCH2CH2,eqCH2), 1.90−1.97 (m, 1H; NCH2CH2CH2,eq), 2.03 (td, J = 11.1/2.7 Hz, 1H; NCH2,axCH2CH2), 2.21 (t, J = 10.8 Hz, 1H; NCH2,axCH), 2.58 (tt, J = 11.0/3.5 Hz, 1H; NCH2CH), 2.59−2.66 (m, 2H; NCH2CH2O), 2.81 (dbr, J = 11.0 Hz, 1H; NCH2,eqCH2CH2), 3.00 (t, J = 7.0 Hz, 2H; OCH2CH2CCHar), 3.05 (dbr, J = 12.0 Hz, 1H; NCH2,eqCH), 3.61 (t, J = 5.8 Hz, 2H; NCH 2 CH 2 O), 3.73 (t, J = 7.0 Hz, 2H; OCH2CH2CCHar), 4.12 (q, J = 7.2 Hz, 2H; CH2CH3), 7.42 (d, J = 8.0 Hz, 2H; CH2CCHarCHarCCHarCHar), 7.57 (t, J = 8.0 Hz, 1H; NNCCHarCHarCHar), 7.57−7.61 (m, 1H; NNCCCHarCHarCHar), 7.65 (ddd, J = 8.4/6.9/1.4 Hz, 1H; NNCCCHarCHar), 7.80 (dd, J = 7.5/1.0 Hz, 1H; NNCCHarCHarCHar), 7.93 (d, J = 8.0 Hz, 1H; NNCCCH a r CH a r CH a r CH a r ), 7.98 (d, J = 8.0 Hz, 1H; NNCCHarCHarCHar), 7.98 (d, J = 8.5 Hz, 2H; CH2CCHarCHarCCHar), 8.92 (d, J = 8.5 Hz, 1H; NNCCCHar) ppm. 13C NMR (125 MHz, CDCl3, 19.4 °C, TMS): δ = 14.43 (CH3), 24.79 (NCH 2 CH 2 CH 2 ), 27.10 (NCH 2 CH 2 CH 2 ), 36.43 (OCH2CH2Car), 42.06 (NCH2CH), 54.32 (NCH2CH2CH2), 56.09 (NCH 2 CH), 58.28 (NCH 2 CH 2 O), 60.52 (CH 2 CH 3 ), 68.98 (NCH2CH2O), 71.93 (CH2Car), 112.0 (NNCarCarHCarHCarH), 123.4 (2C; CH2CarCarHCarHCarCarH), 123.7 (NNCarCarCarH), 125.9 (NNCarCarHCarHCarH), 126.6 (NNCarCarCarHCarHCarH), 127.0 (NNCarCarCarHCarH), 128.1 (NNCarCarCarHCarHCarHCarH), 129.9 (2C; CH2CarCarHCarHCarCarHCarH), 131.3 (NNCarCarHCarHCarH), 6819

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Journal of Medicinal Chemistry



131.5 (NNCarCarCarH), 134.5 (NNCarCarHCarHCarHCar), 143.0 (CH2Car), 148.1 (NNCarCarCarH), 152.0 (CH2CarCarHCarHCar), 174.5 (COOEt) ppm. MS (CI, CH5+) m/z (%): 460 (100, [M + H]+), 170 (49). HRMS (EI+): M+ calcd for C28H33N3O3 459.2522, found 459.2506. Ethyl (3R)-1-[2-(2-{3-[(E)-2-(Naphthalen-1-yl)diazen-1-yl]phenyl}ethoxy)ethyl]-piperidine-3-carboxylate ((R)-11e). The synthesis was performed according to GP2. (R)-(E)/(Z)-9e (70 mg, 0.15 mmol) was dissolved in THF/MeOH (1.0 mL, v/v = 1:1), and 10 mol % Wilkinson’s catalyst (14 mg, 0.015 mmol) was added. The reaction mixture was hydrogenated for 6 h under 10 bar H2 pressure, and the crude product was purified on silica gel (Et2O + 1% NEt3) to yield the product as orange oil (29.7 mg, 43%); [α]20 D = −9.95 (c = 0.11 in CH2Cl2). IR (film): ν̃ = 3356, 3054, 2921, 2851, 2359, 1728, 1632, 1469, 1309, 1221, 1153, 1111, 1030, 803, 772, 755, 693 cm−1. 1H NMR (500 MHz, CDCl3, 22.4 °C, TMS): δ = 1.23 (t, J = 7.3 Hz, 3H; CH3), 1.39 (qd, J = 12.1/4.3 Hz, 1H; NCH2CH2CH2,ax), 1.51−1.62 (m, 1H; NCH2CH2,axCH2), 1.67 (dt, J = 13.5/3.6 Hz, 1H; NCH2CH2,eqCH2), 1.86−1.96 (m, 1H; NCH2CH2CH2,eq), 2.02 (td, J = 11.3/2.8 Hz, 1H; NCH2,axCH), 2.20 (t, J = 10.5 Hz, 1H; NCH2,axCH2CH2), 2.51−2.61 (m, 1H; NCH2CH), 2.58−2.69 (m, 2H; NCH2CH2O), 2.80 (d, J = 11.5 Hz, 1H; NCH2,eqCH), 3.00−3.07 (m, 1H; NCH2,eqCH2CH2), 3.04 (t, J = 7.0 Hz, 2H; OCH2CH2C), 3.62 (t, J = 6.0 Hz, 2H; NCH2CH2O), 3.76 (t, J = 7.0 Hz, 2H; OCH2CH2C), 4.11 (q, J = 7.2 Hz, 2H; CH2CH3), 7.39 (d, J = 7.0 Hz, 1H; CH2CCHarCHar), 7.48 (t, J = 7.5 Hz, 1H; CH2CCHarCHar), 7.57 (t, J = 7.8 Hz, 1H; CH2CCHarCNNCHarCHar), 7.59 (ddd, J = 8.3/6.8/1.5 Hz, 1H; NNCCCHarCHarCHar), 7.66 (ddd, J = 8.4/6.9/1.4 Hz, 1H; NNCCCHarCHar), 7.81 (dd, J = 7.5/1.0 Hz, 1H; CH2CCHarNNCCHar), 7.88−7.92 (m, 1H; CH2CCHarCHarCHar), 7.91 (s, 1H; CH2 CCH arCNN), 7.93 (d, J = 8.5 Hz, 1H; NNCCCH a r CH a r CH a r CH a r ), 7.99 (d, J = 8.0 Hz, 1H; CH2CCHarCNNCCHarCHarCHar), 8.93 (d, J = 8.93 Hz, 1H; NNCCCHar) ppm. 13C NMR (125 MHz, CDCl3, 23.8 °C, TMS): δ = 14.25 (CH3), 24.57 (NCH2CH2CH2), 26.87 (NCH2CH2CH2), 36.19 (OCH2CH2Car), 41.84 (NCH2CH), 54.09 (NCH2CH2CH2), 55.91 (NCH2CH), 58.08 (NCH2CH2O), 60.27 (CH2CH3), 68.79 (NCH2CH2O), 71.85 (OCH2CH2Car), 111.9 (CH2CarCarHCarNNCarCarH), 121.2 (CH2CarCarHCarHCarH), 123.5 (NNCarCarCarH), 123.6 (CH2CarCarHCarNN), 125.6 (CH2CarCarHCarNNCarCarHCarH), 126.5 (NNCarCarCarHCarHCarH), 126.8 (NNCarCarCarHCarH), 127.9 (NNCarCarCarHCarHCarHCarH), 131.3 129.1 (CH2CarCarHCarH), (CH2CarC arHC arNNC arC arHC arHC arH), 131.3 (NNCarCarCarH), 131.8 (CH 2 C ar C ar HC ar H), 134.3 (NNC ar C ar C ar C ar H), 140.3 (CH2Car), 147.8 (NNCarCarCarH), 153.3 (CH2CarCarHCarNN), 174.2 (COOEt) ppm. MS (APCI) m/z (%): 460 ([M + H]+). HRMS (EI+): M+ calcd for C28H33N3O3 459.2522, found 460.2591.



ABBREVIATIONS USED ar, aromatic proton; ax, axial proton; BGT1, betaine-γ-amino butyric acid transporter 1; BIM, bicuculline methiodide; FCC, flash column chromatography; GAT, γ-amino butyric acid transporter; GP, general procedure; LED, light-emitting diode; mGAT, murine γ-amino butyric acid transporter; pIC50, negative logarithm of the half-maximum inhibitory concentration



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S Supporting Information *

Syntheses of the uses iodides and triflates, UV/vis-spectra of compounds 6a−6c, (R)-6d, and (R)-6e, NMR spectra of all synthesized compounds. This material is available free of charge via the Internet at http://pubs.acs.org.



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*Phone: +49 (0)89 2180 77249. Fax: + 49 (0)89 2180 77247. E-mail: [email protected] Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We kindly thank Prof. Dr. D. Trauner and his group for their support with technical equipment. 6820

dx.doi.org/10.1021/jm5008566 | J. Med. Chem. 2014, 57, 6809−6821

Journal of Medicinal Chemistry

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dx.doi.org/10.1021/jm5008566 | J. Med. Chem. 2014, 57, 6809−6821