Heterocyclic Analogues of Modafinil as Novel, Atypical Dopamine

Article Cite This: J. Med. Chem. 2017, 60, 9330-9348

pubs.acs.org/jmc

Heterocyclic Analogues of Modafinil as Novel, Atypical Dopamine Transporter Inhibitors Predrag Kalaba,†,◆ Nilima Y. Aher,†,◆ Marija Ilić,† Vladimir Dragačević,† Marcus Wieder,† Andras G. Miklosi,† Martin Zehl,§ Judith Wackerlig,† Alexander Roller,∥ Tetyana Beryozkina,‡ Bojana Radoman,† Sivaprakasam R. Saroja,⊥ Wolfgang Lindner,§ Eduardo Perez Gonzalez,# Vasiliy Bakulev,‡ Johann Jakob Leban,† Harald H. Sitte,∇ Ernst Urban,† Thierry Langer,*,† and Gert Lubec*,○ †

Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstraße 14, 1090 Vienna, Austria Ural Federal University named after the first President of Russia B. N. Yeltsin, 19 Mira St., Yekaterinburg 620002, Russia § Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria ∥ X-ray Structure Analysis Centre, Faculty of Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria ⊥ Department of Pediatrics, Medical University of Vienna, 1090 Vienna, Austria # Laboratory of Fine Organic Chemistry, Department of Chemistry and Biochemistry, Faculty of Science and Technology, University of Sao Paulo State, Roberto Simonsen 305, CEP 19060-900 Presidente Prudente, Sao Paolo, Brazil ∇ Institute of Pharmacology, Centre of Physiology and Pharmacology, Medical University of Vienna, 1090, Vienna, Austria ○ Neuroscience Laboratory, Paracelsus Medical University, 5020 Salzburg, Austria ‡

S Supporting Information *

ABSTRACT: Modafinil is a wake promoting compound with high potential for cognitive enhancement. It is targeting the dopamine transporter (DAT) with moderate selectivity, thereby leading to reuptake inhibition and increased dopamine levels in the synaptic cleft. A series of modafinil analogues have been reported so far, but more target-specific analogues remain to be discovered. It was the aim of this study to synthesize and characterize such analogues and, indeed, a series of compounds were showing higher activities on the DAT and a higher selectivity toward DAT versus serotonin and norepinephrine transporters than modafinil. This was achieved by substituting the amide moiety by five- and six-membered aromatic heterocycles. In vitro studies indicated binding to the cocaine pocket on DAT, although molecular dynamics revealed binding different from that of cocaine. Moreover, no release of dopamine was observed, ruling out amphetamine-like effects. The absence of neurotoxicity of a representative analogue may encourage further preclinical studies of the above-mentioned compounds.

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INTRODUCTION

It is mode of action remained unknown for long time despite of several years of preclinical research.3,5 Using radioligand binding assays, Mignot et al. (1994) have demonstrated that modafinil binds to the dopamine transporter (DAT) and not to any other transporters and receptors tested in their study.6 Zolkowska et al. (2009) performed an in vitro binding study and concluded that the DAT was the exclusive target at which modafinil displayed relevant binding.7 Loland et al. (2012)

Modafinil, i.e., 2-[(diphenylmethyl)sulfinyl]acetamide, (1) (Figure 1) is an FDA-approved drug for treatment of narcolepsy and other sleep disorders.1−3 It is a mild psychostimulant-like agent that increases wakefulness, improves attention, and enhances performance in a variety of cognitive tasks.3,4 It has been shown to reduce jet lag and improve mood among shift workers. It has also been studied as an alternative to amphetamines for military usage and is currently also approved for Air Force missions in the U.S.1,2 © 2017 American Chemical Society

Received: September 6, 2017 Published: November 1, 2017 9330

DOI: 10.1021/acs.jmedchem.7b01313 J. Med. Chem. 2017, 60, 9330−9348

Journal of Medicinal Chemistry

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substituting the amide moiety with five- and six-membered aromatic heterocycles. The most potent inhibitors from this series were used in release studies to show that these compounds are not causing efflux of dopamine (amphetamine-like effect). Furthermore, binding studies were carried out to determine whether these compounds are targeting the same binding pocket as cocaine. Molecular docking was performed using a homology model of the DAT in order to propose the binding site of two compounds that showed significant dopamine-reuptake inhibition in vitro. Subsequently, molecular dynamics (MD) simulations with the highest scoring ligand poses were performed. To mimic the physiological system, the DAT structural model was placed in a double lipid membrane. The obtained binding poses for the compounds from molecular docking and from MD simulations were compared with already established binding sites for the cocaine analogue CFT12 and mephedrone analogues.13 Furthermore, we aimed to demonstrate that a selected compound from the series shows no general in vivo neurotoxicity that may render it suitable for further preclinical development.

Figure 1. Structure of 2-[(diphenylmethyl)sulfinyl]acetamide (modafinil).

showed that R- and S-modafinil bind to the DAT less potently than cocaine, with R-modafinil having an in vitro profile different from cocaine.8 Modafinil’s pharmacological profile can be clearly distinguished from those of other classical psychostimulants.2−4 It binds to DAT in a different way as compared to cocaine, which may lead to its unique pharmacological profile and, unlike amphetamine, modafinil does not cause efflux of dopamine.4 Modafinil has been intensively studied, and many of its derivatives have been synthesized and tested for biological activity. Cao et al. (2010) have synthesized a series of modafinil analogues by adding para-halo-substituents to the aryl rings, modifying the sulfoxide function and replacing the primary amide group with secondary and tertiary amides and amines.9 Okonula-Bakare et al. (2014) synthesized a series of thioacetamide and sulfinylacetamide analogues and further investigated the role of the terminal amide or substituted amine functions on DAT vs serotonin transporter (SERT) and norepinephrine transporter (NET) binding.10 Cao et al. (2016) further extended the SAR of modafinil analogues by chemical modifications of the oxidation states of sulfoxide and the amide functional groups, by halogenating the phenyl rings, and/or by functionalizing the terminal nitrogen with substituted piperazines.11 The aim of the current study was to generate a novel series of modafinil analogues with higher dopamine reuptake inhibition activity and higher selectivity at DAT vs SERT and NET by

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CHEMISTRY Synthesis of novel 2-[(diphenylmethyl)sulfinyl]thiadiazole and [(benzhydrylsulfinyl)methyl]oxadiazole analogues (4a−e) was achieved as depicted in Scheme 1. Nucleophilic substitution of commercially available substituted chloro-methyl-thiodiazoles and oxadiazole analogues with diphenylmethanethiol (2) in the presence of t-BuOK in DMF or diisopropylethylamine (DIPEA) in 1,4-dioxane provided substituted [(benzhydrylsulfenyl)methyl]-thiodiazole and -oxadiazole intermediates (3a−e) in 35−95% yield. The oxidation reaction in CH3COOH with H2O2 gave final products (4a−e) in 30−70% yield. Substituted [(benzhydrylsulfinyl)methyl]-pyridinyl, -isoxazolyl, -pyrimidinyl, -oxadiazolyl, and -thiophenyl analogues were generated via another synthetic route depicted in Scheme 2.

Scheme 1. Synthesis of Compounds 3a−e and 4a−ea

a

Reagents and conditions: (a) DMF, t-BuOK, rt, overnight; (b) 1,4-dioxane, DIPEA, rt, 3 h; (c) CH3COOH, H2O2, rt, 24 h. 9331



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Scheme 2. Synthesis of Compounds 6a−q and 7a−qa

a

Reagents and conditions: (a) MeOH, K2CO3, rt, 48 h; (b) CH3COOH, H2O2, rt, overnight.

Scheme 3. Synthesis of Compounds 10a−f and 11a−fa

a

Reagents and conditions: (a) BF3·Et2O, CH3COOH, rt, overnight; (b) CH3COOH, H2O2, rt, overnight.

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Alkylation of diphenylmethyl-S-isothiuronium hydrobromide (5) with plain or substituted chloro-methyl-heterocycles with K2CO3 in MeOH provided intermediates (6a−q) in 34−97% yield. Oxidation of the sulfenyl moiety gave the desired sulfoxide-containing analogues (7a−q) in 27−90% yield. Unsubstituted and substituted 2-((benzhydrylsulfinyl)methyl)thiophene analogues were obtained as depicted in Scheme 3. Condensation of commercially available benzhydrols (8a−f) with thiophen-2-ylmethanethiol (9) and BF3·Et2O in CH3COOH provided analogues with sulfenyl moiety (10a−f) in 66−94% yield. As in the previous synthetic routes, oxidation of sulfenyl intermediates with H2O2 in CH3COOH generated sulfoxide containing products (11a−f) in 19−85% yield. Compound 11c was separated further into two mixtures, 11g and 11h, both containing two pairs of stereoisomers. Each mixture was screened on Chiralpack IA HPLC column (Daicel Inc., Tokyo, Japan) to separate into individual enantiomers. For compound 11g, this was achieved using 90% hexane:10% 2propanol as the mobile phase. Biological data is not available because they were not soluble in stock solution. Compound 11h was separated by HPLC into compounds 11i and 11j, using 60% hexane:40% ethyl acetate as the mobile phase.

Table 1. Uptake Inhibition Data in DAT WT, NET WT, and SERT WTa IC50 (μM) ± SD

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RESULTS AND DISCUSSION SARs at DAT, NET, and SERT. All final compounds (4a−e, 7a−q, and 11a−f) were evaluated for uptake inhibition at the monoamine transporters (DAT, SERT, and NET) in HEK293 cells stably expressing human isoforms of DAT, NET, and SERT. The inhibition of the uptake of [3H]-dopamine (for DAT), 3 [ H]-1-methyl-4-phenylpyridinium (for NET), and [3H]serotonin (for SERT) was measured as described in detail in the Experimental Procedures. Changes in DAT inhibition and selectivity as the result of modifications compared to modafinil (1) as a parent compound were evaluated; SAR analyses explored the effect of substituting the amide moiety with heterocycles as well as the influence of introducing of halogen atoms and methyl groups on phenyl ring(s) in analogues containing the thiophene part. Results are presented in Table 1. Substitution of the amide moiety with substituted (and in some cases partially reduced) thiadiazoles and oxadiazoles led to a decrease of DAT reuptake inhibition compared to modafinil (4a,b, 4d,e, and 7c), with compound 4c being nonspecific to all three transporters under the same experimental conditions. Modifications with pyridines (7a,b) and 5-cyclopropyl-isoxazole (7e) also led to the decrease of DAT reuptake inhibition. However, when the amide part was replaced with 2-methylpyrimidine-4-ol (7d), an analogue with higher DAT inhibition potency was obtained. Modifications of the substituent pattern on the pyrimidine moiety (7f−j) led to a decrease in DAT reuptake inhibition potency. Substitution with a 2-thiophenyl-group (11b) provided an analogue with higher in vitro activity on DAT (IC50 = 1.4 μM) and higher selectivity compared with modafinil (1). An analogue containing a 3-thiophenyl-group (7k) also showed higher activity on DAT (IC50 = 3.0 μM) than modafinil but in contrast to 11b also displayed some affinity toward NET (IC50 = 57.6 μM). Further modifications of compounds 7k and 11b, by introduction of halogen atoms and methyl groups on the thiophene ring, yielded analogues 7l−q. With the exception of 7n (IC50 = 283.6 μM on DAT), those analogues showed only moderately lower activity than 7k and 11b.

compd

DAT

NET

SERT

1 4a 4b 4c 4d 4e 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 11a 11b 11c 11d 11e 11f 11g 11h 11i 11j

11.5 ± 4.3 38.1 ± 2.3 107.6 ± 1.5 NA 43.7 ± 1.4 69.1 ± 4.1 NA 63.5 ± 6.2 56.7 ± 0.2 3.1 ± 1.7 38.6 ± 11.3 40.4 ± 1.4 109.0 ± 14.4 90.5 ± 9.8 82.6 ± 3.9 155.7 ± 10.0 3.0 ± 0.3 29.3 ± 5.1 4.9 ± 0.3 283.6 ± 66.4 16.6 ± 0.7 12.8 ± 1.0 13.7 ± 1.8 7.2 ± 1.3 1.4 ± 0.4 4.2 ± 0.3 2.0 ± 0.5 4.0 ± 0.6 5.8 ± 1.7 3.7 ± 0.4 2.4 ± 0.8 4.1 ± 0.4 1.5 ± 0.5

283.6 ± 23.7 85.7 ± 33.5 145.8 ± 6.0 NA 71.0 ± 27.2 30.0 ± 8.0 NA NA NA NA NA NA NA 227.5 ± 47.0 767.5 ± 77.9 NA 57.6 ± 21.9 144.2 ± 33.2 188.1 ± 85.6 NA 67.2 ± 32.5 68.4 ± 33.0 NA NA NA NA 152.5 ± 15.6 112.6 ± 24.7 NA NA NA NA NA

NA NA NA NA 209.5 ± 13.4 NA NA NA NA NA NA NA NA NA NA NA NA 59.6 ± 2.7 NA NA 44.7 ± 9.5 57.2 ± 28.1 70.9 ± 38.0 NA NA NA 118.4 ± 42.7 120.5 ± 58.5 NA NA NA NA NA

a

IC50 values represents data from at least three independent experiments, each performed in triplicate. NA: Not active in used concentration range.

Compound 11b was further modified by introducing substitutions on the phenyl rings. Introduction of an electron-donating methyl group on a single phenyl in paraposition yielded compound 11d (IC50 = 2.0 μM on DAT), while introduction of a methyl group in paraposition of both phenyl rings yielded compound 11f (IC50 = 5.8 μM on DAT). Introduction of electron-withdrawing groups, such as chlorine and fluorine in para- positions on both phenyl rings, generated compounds 11a and 11e, respectively, with higher activity on DAT than modafinil (IC50 = 7.2 and 4.0 μM, respectively). Introduction of one bromine in paraposition yielded analogue 11c (IC50 = 4.2 μM on DAT). Compounds 11i and 11j show better activity on DAT than modafinil (IC50 = 4.1 and 1.6 μM, respectively). Absolute configuration was attributed by crystallographic data and was determined to be S,S and R,R for 11i and 11j, respectively. Compounds 7d and 11b, are enantiomeric compounds. Separation of individual enantiomers into R- and S-form could lead to a further gain in binding to DAT. Examination of Amphetamine-Like Releasing Properties. Amphetamines are substrates of DAT which cause the 9333



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Figure 2. A release assay was performed to examine whether compounds 7d and 11b have any ability to release the neurotransmitter from inside the cells, like amphetamine. HEK-DAT cells were grown on PDL-coated coverslips, treated with 0.1 μM [3H]MPP+ (methyl-4-phenylpyridinium) at 37 °C for 20 min and washed with KHB for 40 min in superfusion chambers. Four experiments were compared, and all four started with three fractions under untreated conditions (baseline). The next four fractions were either again untreated or treated with 10 μM monensin. The final five fractions were either treated with 10 μM test compound (7d or 11b) or 10 μM D-amphetamine. Nonlinear regression analysis was carried out by using GraphPad Prism 6. Values are presented as mean ± SD.

Figure 3. Mutually exclusive binding of cocaine in the presence of analogues 7d and 11b. Intact HEK-DAT cells were incubated with the radioligand (∼10 nM [3H]WIN 35,428), the indicated concentrations of 7d or 11b, and increasing concentrations of cocaine in binding buffer. Nonspecific binding was determined in the presence of 30 μM mazindol (control). The binding data from the competition experiments is transformed into a Dixon plot by expressing the reciprocal of [3H]WIN 35,428 bound (measured in femtomole) as a function of the concentration of cocaine at a fixed concentration of 7d or 11b. Data are means ± SD of three experiments performed in triplicate.

release of dopamine from neuronal cells.14 To investigate whether our novel dopamine reuptake inhibitors cause release of dopamine, release assays of two analogues of choice (7d, 11b) have been performed. As depicted in Figure 2, neither 10 μM 7d nor 10 μM 11b did alter [3H]MPP+ release opposed to the positive control (10 μM D-amphetamine). Elevation of intracellular Na+ levels accelerates substrate efflux, hence we applied 10 μM monensin, an ionophore that promotes electroneutral Na+/H+ exchange, to unambiguously confirm the absence of 7d and/or 11binduced substrate release. The observations from HEK-DAT cells therefore clearly indicate that 7d and 11b specifically block DAT without acting as a substrate and can be clearly distinguished from amphetamine. [3H]WIN 35,428 Binding Assays. Dixon plots (in which the reciprocal of bound radioactivity is plotted as a function of one inhibitor’s concentration at a fixed concentration of the second inhibitor) allow reading out whether two inhibitors can occupy the same binding site simultaneously or whether their binding is mutually exclusive.15 Dixon plots showed a nearly parallel shift for 7d in the presence of cocaine, indicating mutually exclusive binding of analogue 7d and cocaine as shown in Figure 3A.15,16 The same result was obtained for compound 11b as depicted in Figure 3B. Molecular Docking and MD Simulation. The highest scored ligand positions are shown in Figure 4. The docked ligand positions of compound 7d and 11b are very similar to each other, and both resemble the putative binding sites of

cocaine and the cocaine analogue CFT12 as well as the binding site for mephedrone analogues.13 Molecular docking is a fast and often reliable tool to investigate possible ligand poses in the binding site of a protein, but receptor flexibility is still a challenge for docking protocols.17 This can be circumvented using MD simulations to evaluate and refine the predicted poses.18,19 While the simulation time of the MD simulations is sufficient to give a basis for a validation of the ligand stability at the specific binding site, it cannot be used to validate the binding and its affinity. Especially motion and/or rearrangement of the protein backbone happen on a much longer time scale and are not regarded in MD simulations at the nanosecond scale.20 Visual inspection and the monitored RMSD values (provided in the Supporting Information) indicate that the systems were stable during the MD simulation (Figure S-1 in Supporting Information). The RMSD analysis shows that the ligand is stable in the binding site, but both 7d and 11b changed their conformation in the binding site during the first 500 ps from the initial, docked position shown in Figure 4A to the conformation shown in Figure 4B. The used docking protocol did not regard the dynamics of the amino acids in the binding site, but it could still reproduce the binding position previously described for mephedrone analogues.13 Using MD simulations the flexibility of all simulated compounds was explicitly regarded, and the binding position of both compounds changed significantly. Especially for a subsequent structure-based molecular modeling approach, 9334



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Figure 4. For compound 7d and 11b the highest scored docking pose (A) and the final pose after the MD simulation (B) is shown. The labeled amino acids are shown explicitly on the protein backbone. The ligands move during the MD simulations from their initial position (A) to their final position (B), however, they were found not to leave the dopamine binding site. The homology model was based on the crystallized Drosophila DAT, deposited in the Protein Data Bank with the PDB-ID 4M48.

R-modafinil with Phe76 is also present during the MD simulations for 7d and 11b, but the interaction with Ser321 is not observed. The reported interaction between R-modafinil and Tyr156 could be observed for 11b but not for 7d. These dissimilarities are most likely a result of the different methods used. Loland et al. utilized induced-fit docking and obtained the interactions based on the docked pose, while herein MD simulation and analysis of the interactions during the simulation were carried out. Neurotoxicity of Compound 11b. To evaluate basic neurotoxicity, the open field, elevated plus maze, rota rod studies, and the forced swim test were carried out. Young male rats, age 10−12 week,s were divided in two groups: 10 compound 11b treated (treated group) and 10 DMSO-treated animals (control group). Rats were handled during 3 days before beginning of the experiment. Ten mg/kg body weight of 11b was administered by intraperitoneal injection 30 min before each behavioral test. As shown in Figure 6, there were no significant differences between treated and untreated animals for the parameter’s total distance traveled, resting time, local and large movements, average velocity, number of crossings of the center, frequency of spontaneous changes of direction, and time spent at the

these proposed binding poses are an important addition to the already established interaction patterns.12 The interaction maps, shown in Figure 5, for both simulated compounds reveal that the sulfoxide group is not involved in any interaction with the protein binding site, but interactions are governed by hydrophobic contacts and π−π and π−cation interactions. Most prominently for 7d, hydrophobic contacts to Phe326 and Phe76 as well as Tyr156 are shown. Hydrogen bond interactions with Ala480, Arg85, and Asp476 occur also throughout the simulation. In addition to the hydrophobic contact, Arg85 has a stable π−cation interaction with 7d. The interaction pattern for 11b shows hydrophobic interactions between Phe326, Phe76, Phe320, Arg85, and Tyr156. There are no hydrogen bond interactions present during the simulation other than hydrophobic interactions there are weak aromatic interactions and a π−cation interaction between Arg85 and 11b. Both compounds have similar hydrophobic interaction partners (Phe326, Phe76, Tyr156) and π−cation interactions with Arg85. Comparing the interaction pattern to R- and S-modafinil as described by Loland et al.,8 some similarities and differences are striking. The reported interaction of the amide moiety of S- and 9335



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Figure 5. Different interactions between parts of the ligand and amino acids are shown as heatmap ranging from dark violet (lowest interactions), green (medium interactions) to bright yellow (highest interactions). Rows indicate part of the ligands and columns the different amino acids. The first letters before the column indicate the type of interaction, H for hydrophobic interaction, HBA for hydrogen bond acceptor, HBD for hydrogen bond donor, AR for aromatic interaction, and PI for positively ionizable interaction.

(amphetamine-like properties). Binding studies have demonstrated that analogues 7d and 11b are competing for binding with cocaine on DAT. This result was further supported by a docking study of the compounds to a homology model of human DAT. While molecular docking experiments support the finding that 7d and 11b are binding into the same putative binding pocket of cocaine, molecular dynamics simulations indicate a different binding spot to that of cocaine, i.e., different amino acids are involved in binding of 7d and 11b within the pocket. As compound 11b showed the highest reuptake inhibition, it was therefore the compound of choice for neurotoxicity studies. Results indicate no general neurotoxicity of 11b. Therefore, by introducing heterocyclic modifications to the modafinil skeleton, a novel series of atypical DAT inhibitors has been synthesized. Several compounds showed improved activity and specificity compared to modafinil, which together with absence of neurotoxicity for at least one tested compound, makes them suitable for further development.

margin. Analogue results were obtained in the elevated plus maze test, where no significant differences between treated and untreated rats were observed regarding the parameters time spent in the left open arm, left closed arm, total distance traveled, distance traveled in open and closed arms, time spent in the open and closed arm, and the resting time (Figure 7). Likewise, the 11b-treated group did not show significantly altered behavior in the rotarod (Figure 8) and forced swim test (Figure 9) compared to the control group. An observational battery was used to check all important neurological functions as listed in Table 2. There were no significant differences between controls and the group treated with 11b. Behavioral assays did not show any behavioral/toxic changes following daily intraperitoneal administrations of 11b at an intraperitoneal dose of 10 mg/kg body weight. This makes 11b a promising compound for further development, and indeed, first behavioral studies of this compound class showed cognitive enhancement that challenged synthesis and further development.21

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EXPERIMENTAL PROCEDURES

Synthesis. 1H and 13C NMR spectra were recorded on a Bruker Avance 500 NMR spectrometer (UltraShield) using a 5 mm switchable probe (PA BBO 500SB BBF-H-D-05-Z, 1H, BB = 19F and 31P, 15N) with z axis gradients and automatic tuning and matching accessory (Bruker BioSpin). The resonance frequency for 1H NMR was 500.13 MHz and for 13C NMR 125.75 MHz. All measurements were performed for a solution in fully deuterated chloroform or DMSO at 298 K. Standard 1D and gradient-enhanced (ge) 2D experiments, like double quantum filtered (DQF) COSY, NOESY, HSQC, and HMBC, were used as supplied by the manufacturer. Chemical shifts are referenced internally to the residual, nondeuterated

CONCLUSION In the current study, a novel series of modafinil analogues containing five- and six-membered aromatic heterocycles have been synthesized. Analogues were tested for dopamine reuptake inhibition, and several compounds showed similar or improved activity and selectivity (DAT vs NET and SERT) compared to modafinil. Compounds 7d and 11b have been selected for further testing. When tested in dopamine release assay, both compounds caused no efflux of dopamine 9336



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Figure 6. Open field study: Total distance traveled, resting time, local and large movements, average velocity, number of crossings of the center, frequency of spontaneous changes of direction, and time spent at the margin are measured as parameters between control and 11b-treated group. solvent signal for chloroform 1H (δ 7.26 ppm) or DMSO 1H (δ 2.50 ppm) and to the carbon signal of the solvent for chloroform 13C (δ 77.00 ppm) or DMSO 13C (δ 39.57 ppm). HRESIMS spectra were obtained on a maXis HD ESI-Qq-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Samples were dissolved to 20 μg/mL in MeOH and directly infused into the ESI source at a flow rate of 3 μL/min with a syringe pump. The ESI ion source was operated as follows: capillary voltage, 0.9 to 4.0 kV (individually optimized); nebulizer, 0.4 bar (N2); dry gas flow, 4 L/ min (N2); dry temperature, 200 °C. Mass spectra were recorded in the range of m/z 50−1550 in the positive-ion mode. The sum formulas were determined using Bruker Compass DataAnalysis 4.2 based on the mass accuracy (Δm/z ≤ 2 ppm) and isotopic pattern matching (SmartFormula algorithm).

The purity of the compounds was determined by HPLC either on an UltiMate 3000 series system equipped with VWD detector (Dionex/Thermo Fisher Scientific, Germering, Germany) or on LC2010A HT liquid chromatograph device (Shimadzu Corporation, Tokyo, Japan). Separation was carried out on an Acclaim 120 C18, 2.1 mm × 150 mm, 3 μm HPLC column (Thermo Fisher Scientific) using LC-MS-grade water and acetonitrile as mobile phases A and B, respectively. The sample components were separated and eluted with a linear gradient from 10% to 90% B in 25 min followed by an isocratic column cleaning and re-equilibration step. The flow rate was 0.2 mL/ min and the column oven temperature was set to 25 °C. The purity was determined from the UV chromatogram (254 nm) as the ratio of the peak area of the compound to the total peak area (i.e., the sum of the areas of all peaks that were not present in the solvent blank). On the basis of the HPLC data, all final compounds are ≥95% pure. 9337



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Figure 7. Elevated plus maze study: Time spent in the left open arm, left closed arm, total distance traveled, distance traveled in open and closed arms, time spend in the open and closed arm, and the resting time between groups are measured as determining parameters. There is no significant difference between control and treated group.

Figure 8. Rotarod study of 11b: Times spent on the revolving rod were measured for the control and 11b-treated group. There is no significant difference between control and treated group.

Figure 9. Forced swim test study: Immobility times in the water were measured. There is no significant difference between control and treated group. 9338



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HRESIMS m/z 353.0393 [M + Na]+ (calcd for C16H14N2NaO2S2+, 353.0389, Δ = −1.0 ppm). 1H NMR (500 MHz, CDCl3) δ = 10.17 (br s, 1H), 7.44−7.39 (m, 10H), 5.10 (s, 1H), 3.89 (d, J = 15 Hz, 1H), 3.74 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 172.2 (CO), 145.1 (C), 134.3 (C), 133.6 (C), 129.6 (2CH), 129.5 (2CH), 128.9 (2CH + 2p-CH), 128.8 (2CH), 71.7 (CH), 51.5 (CH2). 5-((Benzhydrylsulfinyl)methyl)-2-methoxy-2,3-dihydro-1,3,4-thiadiazole (4b). H2O2 (27.6%, 0.07 mL) was added to the solution of 3b (6.1 mmol, 0.2 g) in glacial acetic acid (10 mL). The reaction mixture was stirred at room temperature during 24 h. Two new compounds were detected on TLC. The solvent was evaporated in vacuum until dryness. The residue was diluted with water, and the precipitate was filtered off and purified by column chromatography on silica gel (CH2Cl2) to give 0.07 g of compound 4b as white solid (yield 33%); mp 127−129 °C; retention time 18.61 min; purity 98.8%. HRESIMS m/z 367.0549 [M + Na]+ (calcd for C17H16N2NaO2S2+, 367.0545, Δ = −1.1 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.44−7.38 (m, 10H), 5.10 (s, 1H), 3.89 (d, J = 15 Hz, 1H), 3.7 (d, J = 15 Hz, 1H), 3.56 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 169.8 (C), 142.1 (C), 134.6 (C), 133.6 (C), 129.5 (4CH), 128.9 (2CH), 128.8 (4CH + 2p-CH), 71.9 (CH), 52.0 (CH2), 34.3 (CH3). 2-((Benzhydrylsulfinyl)methyl)-5-methyl-1,3,4-thiadiazole (4c). H2O2 (27.6%, 0.04 mL) was added to the solution of 3c (3.65 mmol, 1.14 g) in glacial acetic acid (15 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness, and the residue was diluted with water and filtered off to separate the precipitate, which was purified by column chromatography on silica gel (CH2Cl2) to give 0.85 g of 4c as white solid (yield 71%); mp 155−156 °C; retention time 17.14 min; purity 99.2%. HRESIMS m/z 351.0601 [M + Na] + (calcd for C17H16N2NaOS2+, 351.0596, Δ = −1.4 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.43−7.34 (m, 10H), 5.0 (s, 1H), 4.32 (d, J = 15 Hz, 1H), 4.11 (d, J = 15 Hz, 1H), 2.83 (3H). 13C NMR (125 MHz, CDCl3) δ = 167.53 (C), 158.2 (C), 134.3 (C), 133.7 (C), 129.5 (4CH), 129.1 (2CH), 128.8 (2CH + 2p-CH), 128.5 (2CH), 70.4 (CH), 48.0 (CH2), 15.7 (CH3). 5-((Benzhydrylsulfinyl)methyl)-3-(trifluoromethyl)-1,2,4-oxadiazole (4d). H2O2 (27.6%, 0.78 mL) was added to the solution of 3d (7.05 mmol, 2.47 g) in glacial acetic acid (20 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness. The residue was diluted with water and extracted with CH2Cl2 then evaporated and purified by column chromatography on silica gel (CH2Cl2) to give 2.11 g of a white solid (yield 81%) of 4d; mp 90−92 °C; retention time 22.72 min; purity 99.6%. HRESIMS m/z 389.0542 [M + Na]+ (calcd for C17H13F3N2NaO2S+, 389.0542, Δ = 0.1 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.50−7.39 (m, 10H), 5.31 (s, 1H), 4.29 (d, J = 15 Hz, 1H), 4.01 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 173.7 (C), 161.9 (q, 2J(13C, 19F) = 40.6 Hz, C), 133.9 (C), 133.2 (C), 129.7 (2CH), 129.4 (2CH), 129.1 (CH), 129.0 (2 m-CH + p-CH), 117.6 (q, 1 13 J( C, 19F) = 270.8 Hz, CF3), 72.5 (CH), 46.0 (CH2). 5-(Benzhydrylsulfinylmethyl)-4-isopropyl-1,2,3-thiadiazole (4e). To the solution of 3e (2.08 g, 6.10 mmol) in glacial HOAc (10 mL), the solution of 27.6% H2O2 in water (6.10 mmol, 0.75 mL) was added. The reaction mixture was stirred at room temperature for 24 h and evaporated in vacuum until dryness. The resulting oil was dissolved in EtOAc and washed with water (3 × 50 mL). The organic layer was dried over Na2SO4 and evaporated until dryness. The crude product was purified with column chromatography on silica gel (EtOAc/hexanes 1:3 →1:1 → EtOAc). Pure product 4e was isolated as a white solid (yield 65%, 1.41 g); mp 107−108 °C; retention time 21.98 min; purity 99.9%. HRESIMS m/z 379.0910 [M + Na]+ (calcd for C19H20N2NaOS2+, 379.0909, Δ = −0.2 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.42−7.38 (m, 10H), 4.14 (d, J = 15 Hz, 1H), 3.96 (d, J = 15 Hz, 1H), 2.75 (m, 1H), 1.46 (d, J = 7 Hz, 3H), 1.28 (d, J = 7 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ = 167.2 (C), 136.1 (C), 134.4 (C), 133.7 (C), 129.7 (2CH), 129.2 (2CH), 129.0 (CH), 128.9 (2CH), 128.8 (CH), 128.4 (2CH), 71.0 (CH), 45.8 (CH2), 27.6 (CH), 23.4 (CH3), 22.4 (CH3).

Table 2. Results of the Neurological Observational Battery Study parameter body position locomotor activity transfer arousal piloerection limb rotation pelvic elevation tail elevation finger approach touch escape visual placing grip strength pinna reflex toe pinch limb tone tail pinch

control group 1.8 2.600 3.100 2.100 3.300 3.800 2.200 1.400 1.200 1.900 2.400 1.200 1.800 3.300 0.8000

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4216 0.9661 0.3162 0.3162 0.4830 0.6325 0.6325 1.350 1.033 0.3162 1.265 1.033 0.6325 0.6749 0.7888

treated group

P value (t test)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5566 >0.9999 0.5566 0.5566 0.7147 0.5566 0.1744 >0.9999 >0.9999 0.29 0.3833 0.6601 0.7164 0.5676 0.7915

1.9 2.600 3.200 2.200 3.400 3.600 1.800 1.400 1.200 1.700 3.000 1.400 1.600 3.500 0.9000

0.3162 1.350 0.4216 0.4216 0.6992 0.8433 0.6325 1.350 1.033 0.4830 1.700 0.9661 1.578 0.8498 0.8756

Melting points were measured on Leica Galen III apparatus (Leica Biosystems, Germany). 5-((Benzhydrylthio)methyl)-1,3,4-thiadiazol-2(3H)-one (3a) and 5-((Benzhydrylthio)methyl)-2-methoxy-2,3-dihydro-1,3,4-thiadiazole (3b). To a solution of diphenylmethanethiol (1.35 g, 6.75 mmol) in DMF (15 mL) was added t-BuOK (1.34 g) and 2-(chloromethyl)-5methoxy-1, 3, 4-thiadiazole (0.89, 5.4 mmol), and the reaction mixture was stirred at room temperature overnight. Then, saturated aqueous NH4Cl solution was added, and the reaction mixture was extracted with EtOAc. After removal of the solvent, the residue was purified by silica gel column chromatography (hexane:CH2Cl2 1:1), affording 0.20 g of compound 3b (yield 11%) and 0.60 g of compound 3a (yield 35%). 2-((Benzhydrylthio)methyl)-5-methyl-1,3,4-thiadiazole (3c). To a solution of diphenylmethanethiol (2.00 g, 10.0 mmol) in DMF (20 mL) was added t-BuOK (1.0 g) and 2-(chloromethyl)-5-methyl-1,3,4thiadiazole (1.22 g, 8.2 mmol), and the reaction mixture was stirred overnight at room temperature. Then, a saturated aqueous NH4Cl solution was added and the reaction mixture was extracted with EtOAc. After removal of the solvent, the residue was purified by column chromatography on silica gel (hexane:CH2Cl2, 1:1), to give 1.14 g of compound 3c (yield 44%). 5-((Benzhydrylthio)methyl)-3-(trifluoromethyl)-1,2,4-oxadiazole (3d). To a solution of diphenylmethanethiol (1.50 g, 7.5 mmol) in dioxane (20 mL) was added DIPEA (1.4 mL) and 5-(chloromethyl)-3(trifluoromethyl)-1,2,4-oxadiazole (1.33 g, 7.1 mmol), and the reaction mixture was stirred at room temperature overnight. Then, the reaction mixture was quenched with water and extracted with EtOAc. After removal of the solvent, the residue was purified by flash chromatography on silica gel (CH2Cl2) to give 2.47 g of compound 3d (yield >95%). 5-(Benzhydrylthiomethyl)-4-isopropyl-1,2,3-thiadiazole (3e). To the solution of diphenylmethanethiol (1.87 g, 9.30 mmol) in anhydrous 1,4-dioxane (5 mL), 5-(chloromethyl)-4-isopropyl-1,2,3thiadiazole (1.31 g, 7.44 mmol) and (1.54 mL) DIPEA (1.2 g, 9.30 mmol) were added dropwise. The resulting mixture was stirred at room temperature for 3 h and evaporated in vacuum until dryness. The crude product was purified by column chromatography on silica gel (EtOAc:hexane, 1:20), affording 1.91 g of the colorless crystals (yield 47%). 5-((Benzhydrylthio)methyl)-2-methoxy-2,3-dihydro-1,3,4-thiadiazole (4a). H2O2 (27.6%, 1.46 mL) was added to the solution of 3a (1.5 g) in glacial acetic acid (35 mL). The reaction mixture was stirred at room temperature during 24 h. The solvent was evaporated in vacuum until dryness. The residue was diluted with water, and the precipitate was filtered and purified by flash chromatography on silica gel (hexane:EtOAc, 3:1) to give 1.0 g of compound 4a as white solid (yield 64%); mp 142−145 °C; retention time 16.96 min; purity 95.5%. 9339



Article

[(Diphenylmethyl)sulfanyl]methanimideamide (5). Diphenylmethanol (30.9 g, 168 mmol) and thiourea (14.7 g, 194 mmol) were mixed in a 1 L three-neck round bottomed flask and dissolved in 150 mL of MeOH. The reaction mixture was refluxed for 0.5 h and 48% HBr (87.4 mL, 772 mmol) was added dropwise over 1 h and the reaction mixture was stirred under reflux for additional 2.5 h. After cooling to the normal temperature, MeOH was removed in vacuum. A pale-yellow product was obtained and suspended in 100 mL of DCM under stirring for 0.5 h at room temperature. Then solution was filtered and washed with cold DCM. The solid residue was suspended in 100 mL of water and stirred for additional 0.5 h at room temperature. Finally, the product was filtered under reduced pressure, washed with cold water, and dried under high vacuum to give 38.4 g of a white, powdered solid (yield 71%). 2-((Benzhydrylthio)methyl)-3,4-dimetohoxypyridine (6a). In a round-bottom flask 3.33 g (10.30 mmol) of 5 was dissolved in 75 mL of MeOH. Afterward, 2.24 g (10.3 mmol) of 2-(chloromethyl)3,4-dimethoxypyridine and 6.9 g (5.0 equiv) of potassium carbonate were also added to the solution. Then, the reaction mixture was left under stirring for 24 h at room temperature. The MeOH was evaporated, and the residual mixture was diluted in water. Then, the reaction products were extracted with EtOAc and dried over anhydrous Na2SO4. Then, the solvent was removed by rotary evaporation. The crude product was purified by flash column chromatography on silica gel (5% MeOH in DCM), affording 2.60 g of the isolated solid (yield 59%). 4-((Benzhydrylthio)methyl)pyridine (6b). In a 250 mL round bottomed flask, 3.23 g (10 mmol) of 5 is dissolved in 100 mL of MeOH. Afterward, 2.53 g (10 mmol) of 4-(bromomethyl)pyridine hydrochloride and 6.9 g (5 equiv) of potassium carbonate were added to the mixture. The mixture was left to stir at room temperature for 24 h. MeOH was evaporated followed of addition of water. Reaction mixture was extracted with EtOAc, and organic extracts were dried over anhydrous Na2SO4 and filtered off, and EtOAc was removed by rotary evaporation. The crude product was purified by flash column chromatography on silica gel (5% MeOH in DCM), affording 2.0 g of the solid product (yield 68%). 5-((Benzhydrylthio)methyl)-3-methyl-1,2,4-oxadiazole (6c). To a suspension of thiouronium salt 5 (2 g, 6.2 mmol) in MeOH (30 mL) was added solid K2CO3 (5 equiv, 4.28 g) and 5-(chloromethyl)-3methyl-1,2,4-oxadiazole (0.83 g, 6.2 mmol) in several portions. The reaction mixture was stirred at room temperature for 24 h. MeOH was evaporated and water was added, then dilute reaction mixture was extracted with CH2Cl2 and purified by flash chromatography on silica gel (CH2Cl2:petroleum ether, 1:1), to give 1.32 g of compound 6c (yield 72%). 6-((Benzhydrylthio)methyl)-2-methylpyrimidin-4-ol (6d). To a suspension of thiouronium salt 5 (3.0 g, 9.3 mmol) in MeOH (30 mL) was added solid K2CO3 (5 equiv, 6.42 g) and 6-(chloromethyl)-2methylpyrimidin-4-ol (1.47, 9.3 mmol) in several portions. The reaction mixture was stirred at room temperature for 24 h. Then, the MeOH was evaporated, and residual was diluted with water and extracted with EtOAc. Organic layer was separated and acidified with 12 M HCl solution under stirring. The precipitate was filtered to give 2.2 g of compound 6d (yield 73%). 3-((Benzhydrylthio)methyl)-5-cyclopropylisoxazole (6e). To a suspension of thiouronium salt 5 (3.0 g, 9.3 mmol) in MeOH (30 mL) was added solid K2CO3 (5 equiv, 6.42 g) and 3-(chloromethyl)-5cyclopropylisoxazole (1.46 g, 9.3 mmol) in portions. The reaction mixture was stirred at room temperature for 24 h. Then, the MeOH was evaporated and residual was diluted with water. The product was extracted with CH2Cl2 and purified by flash chromatography on silica gel (CH2Cl2:petroleum ether, 3:5), to give 2.89 g of the isolated 6e (yield 97%). 2-((Benzhydrylthio)methyl)-6-(trifluoromethyl)pyrimidin-4-ol (6f). To a suspension of thiouronium salt 5 (303 mg, 0.93 mmol) in MeOH (30 mL) was added solid K2CO3 (5 equiv, 0.64 g) and 2(chloromethyl)-6-(trifluoromethyl)pyrimidin-4-ol (200 mg, 0.93 mmol) in several portions. The reaction mixture was stirred at room temperature for 24 h. The MeOH was evaporated and the residual

products were diluted with water and aqueous solution was extracted with CHCl3 and then acidified with 12 M HCl. The precipitate was filtered to give 0.22 g of isolated compound 6f (yield 63%). 2-((Benzhydrylthio)methyl)-6-methylpyrimidin-4-ol (6g). Same procedure as for 6f. Final isolated product gave 178 mg of 6g (yield 59%). 2-((Benzhydrylthio)methyl)-4-methoxy-6-methylpyrimidine (6h). Same procedure as for 6f. Reaction mixture was extracted with CH2Cl2, and extracts were purified by flash chromatography on silica gel (CH2Cl2), to give 0.25 g of isolated 6h (yield 80%). 4-((Benzhydrylthio)methyl)-2,6-dimethylpyrimidine (6i). The synthesis was carried out by the same procedure used for 6h, affording 0.26 g of the isolated 6i (yield 88%). 2-((Benzhydrylthio)methyl)-5-methylpyrimidine (6j). To a suspension of thiouronium salt 5 (0.35 mg, 1.08 mmol) in MeOH (30 mL) was added solid K2CO3 (5 equiv, 0.74 g) and 2-(chloromethyl)-5methylpyrimidine hydrochloride (0.194 g, 1.08 mmol) in several portions. The reaction mixture was stirred at room temperature for 24 h. The MeOH was evaporated, and the residual products were extracted with CH2Cl2 and purified by flash chromatography on silica gel (CH2Cl2) to give 0.30 mg of isolated 6j (yield 90%). 3-((Benzhydrylthio)methyl)thiophene (6k). To a suspension of thiouronium salt 5 (4.9 g, 15.0 mmol) in MeOH (50 mL) was added solid K2CO3 (5 equiv, 10.35 g) and 3-(chloromethyl)-thiophene (2.0 g, 15.0 mmol) in several portions. The reaction mixture was stirred at room temperature for 24 h. The MeOH was evaporated, and the residual products were extracted with CH2Cl2 and purified by flash chromatography on silica gel (CH2Cl2), affording 3.26 g of isolated 6k (yield 73%). 3-((Benzhydrylthio)methyl)-2-bromothiophene (6l). To a suspension of thiouronium salt 5 (0.18 g, 0.6 mmol) in MeOH (25 mL) was added solid K2CO3 (5 equiv, 0.385 g) and 3-(chloromethyl)-thiophene (0.211 g, 0.6 mmol) in several portions. The reaction mixture was stirred at room temperature for 24 h. The MeOH was evaporated and the residual products were extracted with EtOAc and purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, 1:1), affording 0.15 g of isolated 6l (yield 67%). 2-((Benzhydrylthio)methyl)-3-chlorothiophene (6m). To a suspension of thiouronium salt 5 (0.96 g, 2.9 mmol) in MeOH (50 mL) was added solid K2CO3 (5 equiv, 2.06 g) and 3-chloro-2(chloromethyl) thiophene (0.50 g, 2.9 mmol) in several portions. The reaction mixture was stirred at room temperature for 24 h. The MeOH was evaporated and the residual products were extracted with EtOAc and purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, 4:1), to give 0.33 g of isolated 6m (yield 33%). 4-((Benzhydrylthio)methyl)-2,3-dimethylthiophene (6n). To a suspension of thiouronium salt 5 (0.582 g, 1.8 mmol) in MeOH (25 mL) was added solid K2CO3 (5 equiv, 1.24 g) and 4(chloromethyl)-2,3-dimethylthiophene (0.289 g, 1.8 mmol) in several portions. The reaction mixture was stirred at room temperature overnight. The MeOH was evaporated and the residual products were extracted with EtOAc and concentrated under reduced pressure to give 0.53 g of isolated 6n (yield 89%). 4-((Benzhydrylthio)methyl)-2-bromothiophene (6o). To a suspension of thiouronium salt 5 (0. 306 g, 0.9 mmol) in MeOH (30 mL) was added solid K2CO3 (5 equiv, 0.62 g) and 2-bromo-4(chloromethyl) thiophene (0.211 g, 0.9 mmol) in portions. The reaction mixture was stirred at room temperature overnight. The MeOH was evaporated, and the residual products were extracted with EtOAc and concentrated under reduced pressure to give 0.30 g of isolated 6o (yield 89%). 2-((Benzhydrylthio)methyl)-5-chlorothiophene (6p). To a suspension of thiouronium salt 5 (0.71 g, 2.2 mmol) in MeOH (30 mL) was added solid K2CO3 (5 equiv, 1.51 g) and 2-chloro-5-(chloromethyl) thiophene (0.30 mg, 1.8 mmol) in portions. The reaction mixture was stirred at room temperature overnight. The MeOH was evaporated and the residual products were extracted with EtOAc and concentrated under reduced pressure to give 0. 50 mg of isolated 6p (yield 84%). 2-((Benzhydrylthio)methyl)-4-bromothiophene (6q). To a suspension of thiouronium salt 5 (0.73 g, 2.3 mmol) in MeOH (30 mL) was 9340



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purity 99.4%. HRESIMS m/z 361.0984 [M + Na]+ (calcd for C19H18N2NaO2S+, 361.0981, Δ = −0.7 ppm). 1H NMR (500 MHz, CDCl3) δ = 12.9 (br s, OH), 7.52−7.35 (m, 10H), 6.25 (s, 1H), 5.19 (s, 1H), 3.71 (d, J = 15 Hz, 1H), 3.57 (d, J = 15 Hz, 1H), 2.45 (s, 3H). 13 C NMR (125 MHz, CDCl3) δ = 164.6 (C), 159.1 (C), 159.0 (C), 135.6 (C), 134.2 (C), 129.6 (2CH), 128.8 (2CH), 128.7 (2CH), 128.5 (2CH), 113.7 (CH), 71.7 (CH), 57.9 (CH2), 21.7 (CH3). 3-[(Benzhydrylsulfinyl)methyl]-5-cyclopropylisoxazole (7e). H2O2 (27.6%, 0.8 mL) was added to the solution of 6e (2.8 g, 8.7 mmol) in glasial acetic acid (20 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness. The residue was diluted with water and extracted with CH2Cl2 then organic extracts were evaporated and the residual product was purified by column chromatography on silica gel (EtOAc:petroleum ether 1:3), affording 1.76 g of the isolated 7e as white powder (yield 60%); mp 68−70 °C; retention time 21.28 min; purity 99.3%. HRESIMS m/z 360.1030 [M + Na]+ (calcd for C20H19NNaO2S+, 360.1029, Δ = −0.4 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.48−7.35 (m, 10H), 6.05 (s, 1H), 4.99 (s, 1H), 3.91 (d, J = 15 Hz, 1H), 3.63 (d, J = 15 Hz, 1H), 2.04 (m, 1H), 1.08 (m, 2H), 0.99 (m, 2H). 13C NMR (125 MHz, CDCl3) δ = 175.8 (C), 155.0 (C), 135.1 (C), 133.8 (C), 129.7 (2CH), 129.3 (2CH), 128.9 (2CH), 128.7 (2CH), 128.5 (CH), 128.4 (CH), 100.4 (CH), 70.4 (CH), 45.7 (CH2), 8.7 (CH2), 8.6 (CH2), 8.2 (CH). 2-[(Benzhydrylsulfinyl)methyl]-6-(trifluoromethyl)pyrimidin-4-ol (7f). H2O2 (27.6%, 0.052 mL) was added to the solution of 6f (0.197 mg, 0.55 mmol) in glacial acetic acid (4 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness. The product was crystallized from ethanol to give 0.136 g of 7f. Additional portions of the product were obtained from the filtrate using column chromatography on silica gel (petroleum ether:EtOAc 3:1), to give 41 mg, yielding 86% of 7f as white powder; mp 173−174 °C; retention time 3.16 min; purity 99.4%. HRESIMS m/z 415.0696 [M + Na] + (calcd for C19H15F3N2NaO2S+, 415.0699, Δ = 0.6 ppm). 1H NMR (500 MHz, CDCl3) δ = 11.95 (br s, OH), 7.47−7.37 (m, 10H), 6.76 (s, 1H), 5.37 (s, 1H), 3.94 (d, J = 15 Hz, 1H), 3.62 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 161.6 (C), 155.2 (C), 152.5 (q, 2J(13C,19F) = 35.4 Hz, C), 133.8 (C), 133.5 (C), 129 5 (2 o-CH + 2-m CH), 129.0 (2 o-CH + 2 m-CH + p-CH), 128.8 (CH), 120.4 (q, 2J(13C,19F) = 35.4 Hz, CF3), 113.2 (CH), 71.1 (CH), 51.2 (CH2). 2-[(Benzhydrylsulfinyl)methyl]-6-methylpyrimidin-4-ol (7g). H2O2 (27.6%, 0.052 mL) was added to the solution of 6g (0.178 mg, 0.55 mmol) in glacial acetic acid (4 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness. The product was crystallized from EtOH to yield 114 mg of 7g. Additional portions of the product were obtained from the filtrate using column chromatography on silica gel (petroleum ether:EtOAc, 3:1) to give 40 mg, yielding 83% of the isolated 7g as white powder; mp 150−152 °C; retention time 14.99 min; purity 98.2%. HRESIMS m/z 361.0981 [M + Na]+ (calcd for C19H18N2NaO2S+, 361.0981, Δ = 0.0 ppm). 1H NMR (500 MHz, CDCl3) δ = 12.1 (br s, OH), 7.46−7.35 (m, 10H), 6.21 (s, 1H), 5.27 (s, 1H), 3.82 (d, J = 15 Hz, 1H), 3.65 (d, J = 15 Hz, 1H), 2.30 (s, 3H). 13 C NMR (125 MHz, CDCl3) δ = 165.3 (C), 163.3 (C), 152.5 (C), 134.5 (C), 133.7 (C), 129.6 (2CH), 129.4 (2CH), 128.9 (2 o-CH + 2 m-CH), 128.8 (CH), 128.7 CH), 112.3 (CH), 71.9 (CH), 53.3 (CH2), 24.0 (CH3). 2-[(Benzhydrylsulfinyl)methyl]-4-methoxy-6-methylpyrimidine (7h). H2O2 (27.6%, 0.074 mL) was added to the solution of 6h (0.250 mg,0.74 mmol) in glacial acetic acid (4 mL). The reaction mixture was stirred at room temperature during 24 h. The solvent was evaporated in vacuum until dryness. The residue was diluted with water and extracted with CH2Cl2 then organic extracts were evaporated and the residual product was purified by column chromatography (petroleum ether:EtOAc 3:1, EtOAc), affording 0.197 g of the isolated 7h as white powder (yield 75%); mp 128 °C; retention time 19.5 min; purity 99.9%. HRESIMS m/z 375.1136 [M + Na] + (calcd for C20H20N2NaO2S+, 375.1138, Δ = 0.5 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.54−7.30 (m, 10H), 6.45 (s, 1H), 5.38 (s, 1H), 4.06 (d, J

added solid K2CO3 (5 equiv, 1.59 g) and 4-bromo-2-(chloromethyl) thiophene (0.40 g, 1.9 mmol) in portions. The reaction mixture was stirred at room temperature overnight. The MeOH was evaporated and the residual products were extracted with EtOAc and concentrated under reduced pressure to give 0.61 mg of isolated 6p (yield 85%). 2-((Benzhydrylsulfinyl)methyl)-3,4-dimethoxypyridine (7a). In a 100 mL round-bottom flask, 1.917 g (5.45 mmol) of 6a was dissolved in 10 mL of glacial acetic acid. Then 0.625 mL (5.51 mmol) of 30% H2O2 was dropped into the solution and the reaction mixture was stirred for 12 h at room temperature. Then the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. Reaction products were extracted with EtOAC, and the organic extracts were collected, combined and dried over anhydrous Na2SO4. Then the organic solution was concentrated and the residual material was purified by flash column chromatography on silica gel (5% MeOH in DCM), to yield 1.49 g of the isolated 7a as white powder (yield 78%); mp 131−132 °C; retention time 18.81 min; purity 95.3%. HRESIMS m/z 390.1133 [M + Na]+ (calcd for C21H21NNaO3S+, 390.1134, Δ = 0.3 ppm). 1H NMR (500 MHz, CDCl3) δ = 8.25 (d, J = 5 Hz, 1H), 7.53−7.31 (m, 10H), 6.80 (d, J = 5 Hz, 1H), 5.31 (s, 1H), 4.06 (d, J = 15 Hz, 1H), 4.0 (d, J = 15 Hz, 1H), 3.90 (s, 3H), 3.80 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 158.6 (C), 146.0 (CH), 145.8 (C), 145.3 (C), 136.6 (C), 134.3 (C), 130.0 (2CH), 129.0 (2CH + 2 mCH), 128.5 (2 m-CH), 128.1 (CH), 128.0 (CH), 107.3 (CH), 70.4 (CH), 61.5 (CH3), 55.7 (CH3), 51.5 (CH3), 52.5 (CH2). 4-((Benzhydrylsulfinyl)methyl)pyridine (7b). In a 100 mL roundbottom flask, (2.0 g, 6.87 mmol) of 6b was dissolved in 10 mL of glacial acetic acid. Then 30% H2O2 (0.79 mL, 6.97 mmol)) was dropped into the solution, and the reaction mixture was stirred for 12 h at room temperature. Then, the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate. Organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated, and residual was purified by flash column chromatography on silica gel (5% MeOH in DCM) affording 1.9 g of the isolated 7b as yellow crystals (yield 90%); mp 139−140 °C; retention time 17.06 min; purity 98.2%. HRESIMS m/z 330.0919 [M + Na]+ (calcd for C19H17NNaOS+, 330.0923, Δ = 1.3 ppm). 1H NMR (500 MHz, CDCl3) δ = 8.59 (m, 2H), 7.42−7.35 (m, 10H), 7.15 (m, 2H), 4.76 (s, 1H), 3.86 (d, J = 15 Hz, 1H), 3.67 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 149.6 (2CH), 139.8 (C), 135.10 (C), 134.2 (C), 129.5 (2CH), 129.3 (2CH), 128.8 (2CH), 128.7 (CH), 128.6 (2CH + p-CH), 125.3 (2CH), 71.4 (CH), 55.1 (CH2). 5-[(Benzhydrylsulfinyl)methyl]-3-methyl-1,2,4-oxadiazole (7c). H2O2 (27.6%, 0.4 mL) was added to the solution of 6c (1.32 g, 4.46 mmol) in glacial acetic acid (15 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness. The residue was diluted with water and extracted with CH2Cl2 then organic extracts were evaporated and the solid product was purified by column chromatography on silica gel (hexane, hexane:CH2Cl2 1:1, CH2Cl2, CH2Cl2:EtOAc 1:1), affording 1.20 g of the isolated 7c as white crystals (yield 86%); mp 109−110 °C; retention time 18.88 min; purity 97.5%. HRESIMS m/z 351.0568 [M + Na]+ (calcd for C17H16KN2O2S+, 351.0564, Δ = −1.3 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.51−7.37 (m, 9H), 5.29 (s, 1H), 4.11 (d, J = 15 Hz, 1H), 3.90 (d, J = 15 Hz, 1H), 2.44 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 170.4 (C), 167.8 (C), 134.7 (C), 133.3 (C), 129.6 (2CH), 129.5 (2CH), 128.9 (2CH), 128.7 (2CH + 2 p-CH), 71.5 (CH), 45.9 (CH2), 11.7 (CH3). 6-[(Benzhydrylsulfinyl)methyl]-2-methylpyrimidin-4-ol (7d). H2O2 (30%, 0.55 mL) was added to the solution of 6d (1.93 g, 6.0 mmol) in glacial acetic acid (20 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness. The residue was diluted with water and extracted with CH2Cl2 then organic extracts were evaporated and the residual product was purified by column chromatography on silica gel (hexane, hexane:CH2Cl2 1:1, CH2Cl2), affording a solid product that was finally crystallized from EtOH to give 1.40 g of the isolated 7d as white crystals (yield 69%); mp 181−182 °C; retention time 14.56 min; 9341



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= 15 Hz, 1H), 3.95 (d, J = 15 Hz, 1H), 3.97 (s, 3H), 2.42 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 170.0 (C), 167.6 (C), 161.2 (C), 136.3 (C), 133.9 (C), 130.0 (2CH), 129.0 (2CH), 128.8 (2CH), 128.6 (2CH), 128.3 (CH), 128.0 (9CH), 105.3 (CH), 70.4 (CH), 59.1 (CH2), 53.9 (s, 3H), 23.7 (CH3). 4-[(Benzhydrylsulfinyl)methyl]-2,6-dimethylpyrimidine (7i). H2O2 (27.6%, 0.080 mL) was added to the solution of 6i (0.261 mg,0.81 mmol) in glacial acetic acid (4 mL). The reaction mixture was stirred during 24 h at room temperature. The solvent was evaporated in vacuum until dryness. The residue was diluted with water and extracted with CH2Cl2 then organic extracts were evaporated and the residual product was purified by column chromatography on silica gel (petroleum ether:EtOAc 3:1), to yield 0.20 g of yellow oil 7i (yield 73%); retention time 16.77 min; purity 98.8%. HRESIMS m/z 359.1185 [M + Na]+ (calcd for C20H20N2NaOS+, 359.1189, Δ = 1.0 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.51−7.35 (m, 10H), 6.96 (s, 1H), 5.16 (s, 1H), 3.88 (d, J = 15 Hz, 1H), 3.68 (d, J = 15 Hz, 1H), 2.71 (s, 3H), 2.46 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 167.8 (C), 167.5 (C), 159.3 (C), 135.5 (C), 134.0 (C), 129.6 (2CH), 129.2 (2CH), 128.9 (2CH), 128.7 (2CH), 128.4 (2CH), 119.2 (CH), 71.3 CH), 57.3 (CH2), 26.0 (CH3), 24.1 (CH3). 2-[(Benzhydrylsulfinyl)methyl]-5-methylpyrimidine (7j). H2O2 (27.6%, 0.080 mL) was added to the solution of 6j (0.255 mg, 0.83 mmol) in glacial acetic acid (4 mL). The reaction mixture was stirred at room temperature during 24 h. The solvent was evaporated in vacuum until dryness. The residue was diluted with water, extracted with CH2Cl2, evaporated, and purified by column chromatography on silica gel (petroleum ether:EtOAc 3:1, EtOAc), to give 0.153 g of the isolated 7j as white crystals (yield 57%); mp 138−140 °C; retention time 17.38 min; purity 99.9%. HRESIMS m/z 345.1030 [M + Na]+ (calcd for C19H18N2NaOS+, 345.1032, Δ = 0.5 ppm). 1H NMR (500 MHz, CDCl3) δ = 8.5 (s, 2H), 7.52−7.32 (m, 10H), 5.30 (s, 1H), 4.14 (d, J = 15 Hz, 1H), 4.05 (d, J = 15 Hz, 1H), 2.31(s, 3H). 13C NMR (125 MHz, CDCl3) δ = 159.6 (C), 157.6 (CH), 136.0 (C), 134.1 (C), 129.9 (2CH), 129.2 (C), 129.1 (2CH), 128.9 (2CH), 128.6 (2CH), 128.3 (CH), 128.1 (CH), 70.8 (CH), 58.7 (CH2), 15.4 (CH3). 3-((Benzhydrylsulfinyl)methyl)thiophene (7k). H2O2 (30%, 1.96 mL) was added to the solution of 6k (17 mmol, 5.26 g) in glacial acetic acid (35 mL). The reaction mixture was stirred during 24 h at room temperature. Then the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate, and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure and residual product was purified by flash column chromatography on silica gel (5% MeOH in DCM) to yield 1.49 g of the isolated 7k as pale-yellow powder (yield 28%); mp 92−96 °C; retention time 3.52 min; purity 97.4%. HRESIMS m/z 335.0535 [M + Na]+ (calcd for C18H16NaOS2+, 335.0535, Δ = −0.0 ppm). 1H NMR (500 MHz, CDCl3) δ 7.42−7.33 (m, 11H), 7.18 (m, 1H), 7.02 (m, 1H), 4.66 (s, 1H), 3.94 (d, J = 15 Hz, 1H), 3.73 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 135.8 (C), 134.5 (C), 129.7 (C), 129.5 (2CH), 129.2 (2CH), 128.8 (CH), 128.7 (2CH), 128.6 (2CH), 128.3 (2CH), 126.2 (CH), 125.2 (CH), 69.7 (CH), 50.4 (CH2) 3-((Benzhydrylsulfinyl)methyl)-2-bromothiophene (7l). H2O2 (30%, 0.04 mL) was added to the solution of 6l (0.15 g, 0.4 mmol) in glacial acetic acid (10 mL). The reaction mixture was stirred during 24 h at room temperature. Then the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate, and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure, and residual was purified by flash column chromatography on silica gel (hexane:EtOAc, 1:1). Product was dried in high vacuum to give 0.05 g of isolated 7l was obtained as brownish powder (yield 32%); mp 99− 102 °C; retention time 23.01 min; purity 95.0%. HRESIMS m/z 412.9644 [M + Na]+ (calcd for C18H15BrNaOS2+, 412.9640, Δ = −0.9 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.44−7.26 (m, 10H), 7.00 (d, J = 10 Hz), 7.02 (m, 1H), 4.87 (s, 1H), 3.90 (d, J = 15 Hz, 1H), 3.72 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 135.5 (C),

134.5 (C), 130.9 (C), 129.4 (2CH), 129.3 (2CH), 128.9 CH), 128.7 (2CH), 128.4 (2CH), 126.3 (CH), 113.3 (CH), 71.8 (CH), 51.0 (CH2). 2-((Benzhydrylsulfinyl)methyl)-3-chlorothiophene (7m). H2O2 (30%, 0.11 mL) was added to the solution of 6m (0.33 mg, 0.9 mmol) in glacial acetic acid (15 mL). The reaction mixture was stirred at room temperature during 24 h. Then the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate, and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure and residual product was purified by flash column chromatography on silica gel (hexane:EtOAc, 1:1) and dried in a high vacuum to give 0.115 g of 7m as gray powder (yield 37%); mp 108−110 °C; retention time 27.6 min; purity 98.6%. HRESIMS m/z 369.0144 [M + Na]+ (calcd for C18H15ClNaOS2+, 369.0145, Δ = 0.3 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.48−7.36 (m, 10H), 7.30 (d, J = 5 Hz, 1H), 6.95 (d, J = 5 Hz, 1H), 4.90 (s, 1H), 4.12 (d, J = 15 Hz, 1H), 3.84 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 135.2 (C), 134.2 (C), 129.5 (2CH), 129.3 (2CH), 128.7 (2 o-CH + 2 mCH), 128.4 (2CH), 127.5 (CH), 126.1 (CH), 125.9 (C), 124.6 (C), 71.0 (CH), 49.1 (CH2). 4-((Benzhydrylsulfinyl)methyl)-2,3-dimethylthiophene (7n). H2O2 (30%, 0.17 mL) was added to the solution of 6n (0.531 g 1.6 mmol) in glacial acetic acid (20 mL). The reaction mixture was stirred at room temperature during 24 h. Then, the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate, and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure, and residual was purified by flash column chromatography on silica gel (5% MeOH in DCM). Product was dried in high vacuum to give 0.14 g of 7n as brownish crystals (yield 27%); mp 114−118 °C; retention. time 23.82 min; purity 96.7%. HRESIMS 363.0853 [M + Na]+ (calcd for C20H20NaOS2+, 363.0848, Δ = −1.4 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.50−7.34 (m, 10H), 6.92 (s, 1H), 4.83 (s, 1H), 3.82 (d, J = 15 Hz, 1H), 3.66 (d, J = 15 Hz, 1H), 2.32 (s, 3H), 1.93 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 134.6 (C), 134.2 (C), 132.0 (C), 132.3 (CH), 130.4 (C), 129.6 (2CH), 129.2 (2CH), 128.8 (2CH), 128.7 (2CH), 128.3 (2CH), 121.4 (CH), 70.5 (CH), 51.5 (CH2), 13.6 (CH3), 12.2 (CH3). 4-((Benzhydrylsulfinyl)methyl)-2-bromothiophene (7o). H2O2 (30%, 0.05 mL) was added to the solution of 6o (0.20 g, 0.5 mmol) in glacial acetic acid (15 mL). The reaction mixture was stirred during 24 h at room temperature. Then the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate, and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure, and residual was purified by flash column chromatography on silica gel (2.5% MeOH in DCM). Product was dried in high vacuum to give 0.90 g of 7o was obtained as brown powder (yield 43%); mp 91−94 °C; retention time 4.21 min; purity 98.9%. HRESIMS 412.9646 [M + Na]+ (calcd for C18H15BrNaOS2+, 412.9640, Δ = −1.6 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.44− 7.36 (m, 10H), 7.06 (s, 1H), 6.95 (s, 1H), 4.70 (s, 1H), 3.85 (d, J = 15 Hz, 1H), 3.62 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 135.5 (C), 134.5 (C), 131.3 (CH), 130.6 (C), 129.4 (2CH), 129.3 (2CH), 128.8 (2CH), 128.6 (2CH), 128.5 (CH), 128.4 (CH), 126.7 (CH), 112.9 (C), 70.4 (CH), 50.4 (CH2). 2-((Benzhydrylsulfinyl)methyl)-5-chlorothiophene (7p). H2O2 (30%, 0.18 mL) was added to the solution of 6p (0.60 g, 1.8 mmol) in glacial acetic acid (20 mL). The reaction mixture was stirred at room temperature during 24 h. Then, the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate, and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure, and residual was purified by flash column chromatography on silica gel (1% MeOH in DCM). Product was dried in high vacuum 9342



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to give 0.74 g of 7p as dark-yellow powder (yield 43%); mp 89−92 °C; retention time 23.33 min; purity 99.5%. HRESIMS 369.0146 [M + Na]+ (calcd for C18H15ClNaOS2+, 369.0145, Δ = −0.2 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.44−7.36 (m, 10H), 6.85 (m, 1H), 6.70 (m, 1H), 4.77 (s, 1H), 3.99 (d, J = 15 Hz, 1H), 3.75 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 135.1 (C), 134.1 (C), 131.0 (C), 129.5 (2CH), 129.4 (2CH), 128.8 (4CH), 128.6 (CH), 128.5 (2CH), 126.1 (CH), 69.7 (CH), 50.1 (CH2). 2-((Benzhydrylsulfinyl)methyl)-4-bromothiophene (7q). H2O2 (30%, 0.2 mL) was added to the solution of 6q (0.71 g, 1.9 mmol) in glacial acetic acid (20 mL). The reaction mixture was stirred at room temperature during 24 h. Then the reaction mixture was neutralized with cold 5% sodium bicarbonate solution. The aqueous neutral solution was extracted (3 × 50 mL) with ethyl acetate, and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated under reduced pressure, and residual was purified by flash column chromatography on silica gel (1% MeOH in DCM). Product was dried in high vacuum to give 0.22 g of 7q as yellow powder (yield 30%); mp 89−91 °C; retention time 23.44 min; purity 98.6%. HRESIMS 412.9638 [M + Na]+ (calcd for C18H15BrNaOS2+, 412.9640, Δ = 0.4 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.44−7.36 (m, 10H), 7.22 (s, 1H), 6.84 (s, 1H), 4.76 (s, 1H), 4.03 (d, J = 15 Hz, 1H), 3.80 (d, J = 15 Hz, 1H). 13 C NMR (125 MHz, CDCl3) δ = 135.1 (C), 134.1 (C), 132.0 (C), 131.3 (CH), 129.5 (2CH), 129.4 (2CH), 128.8 (2CH), 128.7 (2CH), 128.6 (CH), 128.5 (CH), 124.1 (CH), 109.9 (C), 70.1 (CH), 50.1 (CH2). 2-(((Bis(4-chlorophenyl)methyl)thio)methyl)thiophene (10a). Bis(4-chlorophenyl) methanol (0.96 g, 3.8 mmol) and (0.5 g, 3.8 mmol) of thiophen-2-ylmethanethiol were dissolved in glacial acetic acid. Then 1.1 equiv (1.1 mL, 4.2 mmol) of BF3·Et2O was added to a solution, and reaction mixture was left under stirring overnight at room temperature. Reaction mixture was then poured over ice, a small amount of water was added, and acetic acid was neutralized with addition of solid NaHCO3. Product was then extracted (3×) with 50 mL of ethyl acetate. Organic layers were collected, combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Crude product was then purified by flash column chromatography on silica gel (2.5% MeOH in DCM) affording 1.23 g of the oily product (yield 89%). 2-((Benzhydrylthio)methyl)thiophene (10b). Diphenylmethanol (1.4 g, 7.67 mmol) and thiophen-2-yl-methanethiol (1 g, 7.67 mmol) were dissolved in glacial acetic acid and 1.1 equiv (2.2 mL, 8.45 mmol) of BF3·Et2O was added to the solution and the reaction mixture was left under stirring overnight at room temperature. The reaction mixture was then poured over ice and a small amount of water was added to dilute the excess of acetic acid, then the resulting solution was neutralized with addition of solid NaHCO3. Product was extracted (3 × 50 mL) with ethyl acetate and the organic extracts were collected, combined, and dried over anhydrous Na2SO4. The organic solution was concentrated, and the crude residual material was purified via flash column chromatography on silica gel (2.5% MeOH in DCM), to give 2.07 g of the oily product (yield 91%). 2-((((4-Bromophenyl)(phenyl)methyl)thio)methyl)thiophene (10c). 4-Bromobenzhydrol (2.02 g, 7.67 mmol) and thiophen-2-ylmethanethiol (1.0 g, 7.67 mmol) were dissolved in glacial acetic acid. Then 1.1 equiv (2.2 mL, 8.45 mmol) of BF3·Et2O was added to the solution and the reaction mixture was left under stirring overnight at room temperature. Reaction mixture was then poured over ice, a small amount (1−2 mL) of water was added, and acetic acid excess was neutralized with addition of solid NaHCO3. Product was then extracted (3 × 15 mL) with ethyl acetate. Organic layers were collected, combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Crude product was purified via flash column chromatography on silica gel (2.5% MeOH in DCM) affording 2.66 g of the oily product (yield 92%). 2-(((Phenyl(p-tolyl)methyl)thio)methyl)thiophene (10d). 4-Methylbenzhydrol (1.52 g, 7.67 mmol) and thiophen-2-yl-methanethiol 1 g (7.67 mmol) of were dissolved in glacial acetic acid. Then 1.1 equiv (2.2 mL, 8.45 mmol) of BF3·Et2O was added to a solution and the

reaction mixture was left under stirring overnight at room temperature. Reaction mixture was then poured over ice, a small amount (1−2 mL) of water was added, and the acetic acid excess was neutralized with addition of solid NaHCO3. Product was then extracted (3 × 15 mL) with ethyl acetate. Organic layers were collected, combined, dried over anhydrous Na2SO4, filtered off, and concentrated under reduced pressure. Crude product was purified by flash column chromatography on silica gel (2.5% MeOH in DCM), affording 1.9 g of the oily product (yield 79%). 2-(((Bis(4-fluorophenyl)methyl)thio)methyl)thiophene (10e). Bis(4-fluorophenyl) methanol (0.84 g, 3.8 mmol) and (0.5 g, 3.8 mmol) of thiophen-2-ylmethanethiol were dissolved in glacial acetic acid. Then 1.1 equiv (1.1 mL, 4.2 mmol) of BF3·Et2O was added to a solution and the reaction mixture was left under stirring overnight at room temperature. Reaction mixture was then poured over ice, a small amount (1−2 mL) of water was added, and acetic acid excess was neutralized with addition of solid NaHCO3. Product was then extracted (3 × 15 mL) of ethyl acetate. Organic layers were collected, combined, dried over anhydrous Na2SO4, filtered off, and concentrated under reduced pressure. Crude product was purified by flash column chromatography on silica gel (2.5% MeOH in DCM) affording 0.82 g of the oily product (yield 65%). 2-(((Di-p-tolylmethyl)thio)methyl)thiophene (10f). Di-p-tolylmethanol (0.6 g, 2.8 mmol) and thiophen-2-yl-methanethiol (0.37 g, 2.8 mmol) were dissolved in glacial acetic acid. Then 1.1 equiv (0.8 mL, 3.1 mmol) of BF3·Et2O was added to solution and the reaction mixture was left under stirring overnight at room temperature. Reaction mixture was then poured over ice, and a small amount of water (1−2 mL) was added, and acetic acid excess was neutralized with addition of solid NaHCO3. Product was then extracted (3 × 50 mL) with ethyl acetate. Organic extracts were collected, combined, and dried over anhydrous Na2SO4, Organic solution was concentrated under reduced pressure, and the crude product was purified via flash column chromatography on silica gel (2.5% MeOH in DCM) affording 0.72 g of the oily product (yield 86%). 2-(((Bis(4-chlorophenyl)methyl)sulfinyl)methyl)thiophene (11a). H2O2 (30%, 1.1 mL) was added to the solution of 10a (1.23 g, 3.4 mmol) in glacial acetic acid (50 mL). The reaction mixture was stirred during 24 h at room temperature. Acid was neutralized with 5% sodium bicarbonate and ice, and the product was extracted with EtOAc, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Then, product was purified by flash column chromatography on silica gel (hexane:EtOAC, 1:1) and dried in high vacuum to give 0.74 g of 11a as yellow crystals (yield 57%); mp 112−114 °C; retention time 24.24 min; purity 98.7%. HRESIMS 402.9756 [M + Na]+ (calcd for C18H14Cl2NaOS2+, 402.9755, Δ = −0.1 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.40−7.35 (m, 9H), 7.06 (m, 1H), 6.96 (m, 1H), 4.67 (s, 1h), 4.09 (d, J = 15 Hz, 1H), 3.90 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 134.8 (C), 134.6 (C), 133.6 (C), 131.9 (C), 130.9 (2CH), 130.1 (2CH), 129.7 (C), 129.6 (2CH), 129.1 (CH), 128.9 (2CH), 127.4 (CH), 127.0 (CH), 66.9 (CH), 50.3 (CH2). 2-((Benzhydrylsulfinyl)methyl)thiophene (11b). H2O2 (30%, 0.8 mL) was added to the solution of 10b (2.07 g, 7.0 mmol) in glacial acetic acid (50 mL). The reaction mixture was stirred overnight at room temperature. Acid was neutralized with 5% sodium bicarbonate and ice, and the product was extracted with EtOAc, dried over Na2SO4, and concentrated under reduced pressure. Then, product was purified by flash column chromatography on silica gel (2.5% MeOH in DCM) and dried in high vacuum to give 1.49 g of 11b as yellow powder (yield 68%); mp 116−119 °C; retention time 21.45 min; purity 99.5%. HRESIMS 335.0537 [M + Na] + (calcd for C18H16NaOS2+, 335.0535, Δ = −0.6 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.42−7.34 (m, 11H), 7.05 (m, 1H), 6.96 (m, 1H), 4.75 (s, 1H), 4.10 (d, J = 15 Hz, 1H), 3.88 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 135.5 (C), 134.1 (C), 130.2 (C), 129.6 (2CH), 129.3 (2CH), 129.0 (CH), 128.7 (2 o-CH + 2 m-CH), 128.4 (2CH), 127.3 (CH), 126.8 (CH), 69.2 (CH), 50.1 (CH2). 2-((((4-Bromophenyl)(phenyl)methyl)sulfinyl)methyl)thiophene (11c). H2O2 (30%, 0.82 mL) was added to the solution of 10c (7.1 9343



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for C18H15BrNaOS2+, 412.9640, Δ = 0.3 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.54−7.36 (m, 7H), 7.34 (m, 1H), 7.31 (m, 2H), 7.05 (m, 1H), 6,96 (m, 1H), 4.69 (s, 1H), 4.09 (d, J = 15 Hz, 1H), 3.89 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 134.8 (C), 133.4 (C), 132.4 (2CH), 130.4 (2CH), 130.1 (C), 129.6 (2CH), 129.0 (CH), 128.8 (2CH), 128.6 (CH), 127.4 (CH), 126.9 (CH), 122.6 (C), 68.0 (CH), 50.3 (CH2). 2-((((4-Bromophenyl)(phenyl)methyl)sulfinyl)methyl)thiophene (11h). Same as 11g. HRESIMS 412.9643 [M + Na]+ (calcd for C18H15BrNaOS2+, 412.9640, Δ = −0.9 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.54−7.31 (m, 10H), 7.05 (m, 1H), 6.96 (m, 1H), 4.69 (s, 1H), 4.09 (d, J = 15 Hz, 1H), 3.89 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 134.8 (C), 133.4 (C), 132.3 (2CH), 130.4 (2CH), 130.1 (C), 129.6 (2CH), 129.0 (CH), 128.8 (2CH), 128.6 (CH), 127.4 (CH), 126.9 (CH), 122.5 (C), 68.0 (CH), 50.3 (CH2). 2-((((4-Bromophenyl)(phenyl)methyl)sulfinyl)methyl)thiophene (11i). From 11h, by separation on Chiralpack IA column using 60% hexane/40% ethyl acetate as the mobile phase, two individual enantiomers were obtained: 11i (less retained enantiomer) and 11j (more retained enantiomer), respectively; retention time (chiral column) 17.4 and 29.7 min for 11i and 11j, respectively. Yield >95%. HRESIMS 412.9640 [M + Na]+ (calcd for C18H15BrNaOS2+, 412.9640, Δ = 0.0 ppm). 1H NMR (500 MHz, CDCl3) δ 7.49−7.38 (m, 7H), 7.34 (m, 1H), 7.29 (m, 2H), 7.05 (m, 1H), 6,96 (m, 1H), 4.70 (s, 1H), 4.09 (d, J = 15 Hz, 1H), 3.89 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 135.0 (C), 133.2 (C), 131.8 (2CH), 131.3 (2CH), 129.9 (C), 129.4 (2CH), 129.1 (CH), 128.8 (2CH), 128.7 (CH), 127.3 (CH), 126.9 (CH), 122.8 (C), 68.4 (CH), 50.1 (CH2). Retention time 28.28 min; relative peak area 98.4%; CCDC code 1569748. 2-((((4-Bromophenyl)(phenyl)methyl)sulfinyl)methyl)thiophene (11j). Same as 11i; retention time (chiral column) 17.4 and 29.7 min for 11i and 11j, respectively. Yield >95%. HRESIMS 412.9640 [M + Na]+ (calcd for C18H15BrNaOS2+, 412.9640, Δ = 0.0 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.49−7.38 (m, 7H), 7.34 (m, 1H), 7.29 (m,2H), 7.05 (m, 1H), 6,95 (m,1H), 4.70 (s, 1H), 4.09 (d, J = 15 Hz, 1H), 3.89 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 135.0 (C), 133.2 (C), 131.8 (2CH), 131.3 (2CH), 129.9 (C), 129.4 (2CH), 129.1 (CH), 128.8 (2CH), 128.7 (CH), 127.3 (CH), 126.9 (CH), 122.8 (C), 68.4 (CH), 50.1 (CH2). Retention time 28.30 min; relative peak area 97.1%; CCDC code 1569749. X-ray Measurement. The X-ray intensity data were measured on Bruker X8 APEX2 diffractometer equipped with a multilayer monochromator, a Mo Kα INCOATEC micro focus sealed tube, and a Kryoflex cooling device. The structures were solved by direct methods and refined by full-matrix least-squares techniques. Nonhydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were inserted at calculated positions and refined with a riding model. The following software was used: Bruker SAINT software package22 using a narrow-frame algorithm for frame integration, SADABS23 for absorption correction, OLEX224 for structure solution, refinement, molecular diagrams, and graphical userinterface, Shelxle25 for refinement and graphical user-interface, SHELXS-201326 for structure solution, SHELXL-201327 for refinement, and Platon28 for symmetry check. Experimental data and CCDC codes can be found in Supporting Information. Crystal data, data collection parameters, and structure refinement details as well as crystal structures visualized are available in Supporting Information. DAT, NET, SERT Reuptake Inhibition Assay. Assays were performed as previously described by Sucic et al.29 Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin, and fetal calf serum (FCS) were purchased from Sigma-Aldrich Handels GmbH (Austria). [3H]5-HT (hydroxytryptamine creatinine sulfate; 5-[1,2-3H[N]]; 27.8 Ci/mmol), [ 3 H]DA (dihydroxyphenylethylamine; 3,4-[ring2,5,6-3[H]]-dopamine; 36.6 Ci/mmol), and [3H]MPP+ (methyl-4phenylpyridinium iodide; 1-[methyl-3H]; 80 Ci/mmol) were purchased from PerkinElmer, Boston, MA. HEK293 cells stably expressing human isoforms of the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT) were used for reuptake inhibition assays. All cell

mmol, 2.66 g) in glacial acetic acid (50 mL). The reaction mixture was stirred at room temperature overnight. Acid was neutralized with 5% sodium bicarbonate and ice. Then product was extracted with EtOAc, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Product was purified via flash column chromatography on silica gel (5% MeOH in DCM) and dried in high vacuum to give 2.36 g of 11c as brown solid (yield 85%); mp 95−98 °C; retention time 23.71 min; purity 98.8%. HRESIMS 412.9644 [M + Na]+ (calcd for C18H15BrNaOS2+, 412.9640, Δ = −1.1 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.54−7.31 (m, 10H), 7.05 (m, 1H), 6.96 (m, 1H), 4.69 (s, 1H), 4.09 (d, J = 15 Hz, 1H), 3.89 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 134.8 (C), 133.4 (C), 132.3 (2CH), 130.4 (2CH), 130.1 (C), 129.6 (2CH), 129.0 (CH), 128.8 (2CH), 128.6 (CH), 127.4 (CH), 126.9 (CH), 122.5 (C), 68.0 (CH), 50.3 (CH2). 2-(((Phenyl(p-tolyl)methyl)sulfinyl)methyl)thiophene (11d). H2O2 (30%, 0.7 mL) was added to the solution of 10d (1.9 g, 6.1 mmol) in glacial acetic acid (40 mL). The reaction mixture was stirred overnight at room temperature. Acid was neutralized with 5% sodium bicarbonate and ice. Product was extracted with EtOAc, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Then, product was purified via flash column chromatography on silica gel (2.5% MeOH in DCM) and dried in high vacuum to give 0.64 g of 11d as orange powder (yield 33%); mp 82−86 °C; retention time 22.55 min; purity 96.2%. HRESIMS 349.0690 [M + Na]+ (calcd for C19H18NaOS2+, 349.0691, Δ = 0.3 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.32−6.96 (m, 12H), 4.70 (1H), 4.08 (d, J = 15 Hz, 1H), 3.87 (d, J = 15 Hz, 1H), 2.36 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 138.2 (C), 138.1 (C), 132.6 (C), 131.3 (C), 130.5 (C), 129.9 (2CH), 129.4 (2CH), 129.3 (2CH), 128.9 (CH), 128.6 (CH), 127.2 (CH), 126.6 (CH), 68.8 (CH), 50.0 (CH2) 21.1 (CH3). 2-(((Bis(4-fluorophenyl)methyl)sulfinyl)methyl)thiophene (11e). H2O2 (30%, 0.25 mL) was added to the solution of 10e (0.82 g, 2.5 mmol) in glacial acetic acid (20 mL). The reaction mixture was stirred overnight at room temperature. Acid was neutralized with 5% sodium bicarbonate and ice. Product was extracted with EtOAc, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Then product was purified by flash column chromatography on silica gel (hexane:EtOAC, 1:1) and dried in high vacuum to give 0.40 g of 11e as yellow powder (yield 47.0%); mp 103−106 °C; retention time 21.22 min; purity 98.5%. HRESIMS 371.0349 [M + Na]+ (calcd for C18H14F2NaOS2+, 371.0346, Δ = −0.7 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.38−9.96 (m, 11H), 4.70 (s, 1H), 4.08 (d, J = 15 Hz, 1H), 3.88 (d, J = 15 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ = 162, 9 (q, 1J(13C,19F) = 246,9 Hz, C), 162.6 (q, 1J(13C,19F) = 247,6 Hz, C), 131.3 (q, 3J(13C,19F) = 8.2 Hz, 2CH), 131.1 (q, 4J(13C,19F) = 3.1 Hz, C), 130.5 (q, 3J(13C,19F) = 8.1 Hz, 2CH), 129.8 (C), 129.5 (q, 4 13 19 J( C, F) = 2.9 Hz, C), 127.4 (CH), 127.0 (CH), 116.4 (q, 2 13 19 J( C, F) = 21.6 Hz, CH), 115.7(q, 2J(13C,19F) = 21.7 Hz, CH), 66.8 (CH), 50.0 (CH2). 2-(((Di-p-tolylmethyl)sulfinyl)methyl)thiophene (11f). H2O2 (30%, 0.32 mL) was added to the solution of 10f (0. 92 mg, 3.1 mmol) in glacial acetic acid (20 mL). The reaction mixture was stirred at room temperature overnight and neutralized with 5% sodium bicarbonate solution and ice. The reaction mixture was extracted with EtOAc, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Then, product was purified by flash column chromatography on silica gel (1% MeOH in DCM) and dried in high vacuum to give 0.20 g of 11f (yield 19%); mp 113−115 °C; retention time 24.40 min; purity 99.9%. HRESIMS 363.0848 [M + Na]+ (calcd for C20H20NaOS2+, 363.0848, Δ = −0.0 ppm). 1H NMR (500 MHz, CDCl3) δ = 7.32− 7.17 (m, 9H), 7.04 (m, 1H), 6.96 (m, 1H), 4.70 (s, 1H), 4.08 (d, J = 15 Hz, 1H), 3.87 (d, J = 15 Hz, 1H), 2.36 (s, 3H), 2.32 (s, 3H). 13C NMR (125 MHz, CDCl3) δ = 138.1 (2C), 132.7 (C), 131.3 (C), 130.5 (C), 129.9 (2CH), 129.4 (2CH), 129.3 (2CH), 128.9 (CH), 128.6 (CH), 127.2 (CH), 126.6 (CH), 68.8 (CH), 50.0 (CH2), 21.1 (2CH3). 2-((((4-Bromophenyl)(phenyl)methyl)sulfinyl)methyl)thiophene (11g). From 11c by separation on silica gel column (mobile phase EtOH/toluene = 60/40), two mixtures of stereoisomer compounds were obtained: 11g and 11h. HRESIMS 412.9639 [M + Na]+ (calcd 9344



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lines were seeded in 96-well plates precoated with poly-D-lysine (PDL) (5 × 104 cells/well) 24 h prior to the experiment. Each well was washed with 0.1 mL of Krebs−HEPES buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2·2H2O, 2 mM MgCl2·6H2O, 5 mM D-(+)-glucose monohydrate, pH 7.3). Cells were preincubated 5 min in KHB containing increasing concentrations (0.001 μM to 1 mM) of test compound. Compounds were dissolved first in 99.9% dimethyl sulfoxide (DMSO) and subsequently diluted in KHB. Cells were incubated in KHB containing increasing concentrations of compound with addition of 0.2 μM [3H]-dopamine (for HEK-DAT), 0.05 μM [3H]MPP+ (for HEK-NET), and 0.4 μM [3H]5-HT (for HEK-SERT). Incubation times were 1 min for HEK-DAT and HEKSERT and 3 min for HEK-NET. For determination of unspecific uptake in HEK-DAT and HEK-NET, 10 μM mazindol was used and 10 μM paroxetine was used for HEK-SERT. After incubation at room temperature, reactions were stopped by the addition of 0.1 mL of icecold KHB. Finally, cells were lysed with 0.3 mL of 1% SDS and released radioactivity was measured by a liquid scintillation counter (Tricarb-2300TR, PerkinElmer). Nonlinear regression analysis was carried out for reuptake assays. All calculations were performed using GraphPad Prism version 6.00 for Windows, GraphPad Software,2 San Diego, CA, USA. Dopamine Release Assay. Monensin sodium salt and Damphetamine hemisulfate salt were purchased from Sigma-Aldrich Co. For release studies, HEK-DAT cells were grown overnight on 5 mm diameter PDL-coated round glass coverslips placed in a 96-well plate. Cells were incubated with 0.1 μM [3H]MPP+ at 37 °C for 20 min. The coverslips were transferred into superfusion chambers, and excess radioactivity was washed out with KHB for 40 min (0.7 mL/min) at 25 °C to obtain stable baselines as described before.16 The experiment was started with the collection of fractions (2 min) as depicted in Figure 2. During the experiments, the buffer was switched either to monensin or remained at control buffer after the collection of three baseline fractions for another four fractions. Subsequently, 7d (or 11b) or D-amphetamine was added for another five fractions as indicated in Figure 2. Finally, cells were lysed in 1% SDS and the released radioactivity was quantified by liquid scintillation counting. Nonlinear regression analysis was carried out for release assays. All calculations were performed using GraphPad Prism version 6.00 for Windows, GraphPad Software,2 San Diego, CA, USA. [3H]WIN 35,428 Binding Assays. Zinc chloride 0.1 M solution was purchased from Sigma-Aldrich Co. WIN, 35,428, [N-methyl-3H] (84.0 Ci/mmol) was purchased from PerkinElmer, Boston, USA. For binding studies, HEK-DAT cells were grown overnight in 48well plates precoated with PDL (0.1 × 106 cells/well) 24 h prior to the experiment. Each well was washed out with 0.5 mL of binding buffer (10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2·2H2O, 2 mM MgCl2·6H2O, 10 μM ZnCl2, 5 mM D-(+)-glucose monohydrate, pH 7,3). Cells were incubated with the radioligand (∼10 nM [3H]WIN 35,428), the indicated concentrations of 7d and 11b, and increasing concentrations of cocaine (0.001 μM to 1 μM). The reaction volume was 0.1 mL. Nonspecific binding was determined in the presence of 30 μM mazindol. Binding was allowed to proceed for 30 min at 25 °C and terminated by washing the cells two times with 0.5 mL of binding buffer. Cells were lysed in 1% SDS, and the radioactivity was quantified by liquid scintillation counting. Homology Modeling. The protein homology model was kindly provided by the group of Gerhard Ecker from the University of Vienna. The homology model was based on the crystallized Drosophila DAT, as described in Saha et al.,13 in the outward facing conformation deposited in the Protein Data Bank with the PDB-ID 4M48. The DAT structure was in complex with nortriptylin.30 Molecular Docking. Molecular docking was performed using Autodock Vina 1.1.2.31 The ligands were prepared using the Maestro Modeling1 suite, and double bonds and hydrogen atoms were assigned as shown in Figure B. Flexible bonds were added to the ligands using the flexible bond detection of AutoDockTools 1.5.6.32 For the molecular docking run, the standard parameters were used; protein flexibility was not regarded and the exhaustiveness level was set to 16. Waters were removed and the binding site was defined as the center of

mass of nortriptyline. The search space was restricted to the protein structure of the homology model. Docking analysis was performed using compound 7d and 11b. Autodock Vina used the AMBER force field to evaluate the interaction and energy of the different poses. MD Simulations. The protein−ligand systems were oriented and optimized using the position of proteins in membrane (PMM 2.0) method provided by the Orientation of Proteins in Membranes (OPM) web service.33 The obtained orientation of the protein−ligand system were subsequently used. The CHARMM-GUI web interface34 and the membrane builder35 were used to set up the membrane− protein−ligand systems. The protein was placed in a bilayer containing 600 phosphatidylcholine (POPC). The membrane spanned an area of 150 × 150 Å2, leading to a rectangle with 150 × 150 × 105 Å3. The membrane−protein−ligand systems were solvated using TIP3P water molecules. MD simulations were carried out using CHARMM,36 utilizing the CHARMM/OpenMM coupling.37,38 Parameters and molecular topologies for the ligands were generated based on the CGenFF force field39 using the ParamChem server (cgenff. paramchem.org). The membrane−protein−ligand complexes were solvated in rectangular boxes of TIP3P water.40 Ions were added to compensate for any net charge of the solute and to set the ion concentration to 0.15 M KCl. Electrostatic interactions were computed using the particle-mesh-Ewald method.41 SHAKE was used to keep all bonds involving hydrogen atoms rigid. After initial equilibration using the equilibration steps provided by CHARMMGUI for membrane−protein systems, the two complexes were simulated using Langevin dynamics at 303.15 K; the pressure was kept around 1 atm by a Monte Carlo barostat. The system containing 7d was simulated for 80 ns and the system containing 11b for 75 ns. The stability of the simulations was monitored by computing rootmean-square deviations for the protein and ligand, using the MDAnalysis package,42 as well as visual inspection of the trajectories. Additionally, the interactions of the ligand (ionic interactions, hydrophobic interactions, and aromatic and hydrogen bond interactions) with the protein were analyzed during the MD simulations. This was done by generating a structure-based pharmacophore model at every saved coordinate set of the MD simulation and subsequently analyzing the frequency of the features. Structure-based pharmacophore models were generated using an inhouse developed open-source chemoinformatics toolkit (https:// github.com/aglanger/cdpkit). The maximum possible frequency value corresponds to the length of the simulation, i.e., for 80 ns the maximum number a specific feature can appear is 8000. An interaction matrix for the different MD simulations was generated and shown in Figure 5. The columns of the interaction matrix indicate all amino acid residues that are involved in a pharmacophore feature at some point during the MD simulations, the rows designate moieties of the ligand and the color in the matrix indicate how often a specific amino acid was involved in a specific pharmacophore feature: the colors range from violet (zero interaction) to bright yellow (interaction at every time step), with green as intermediate. To distinguish between different interaction types between ligand and protein, the first letters of the column and row names indicate the interaction type: H for hydrophobic interactions, HBD for hydrogen bond donors, HBA for hydrogen bond acceptors, AR for aromatic interactions, and PI for positive ionizable interactions. The amino acid residue numbers correspond to the residue number in the PDB file and are consistent with earlier publication in the field (e.g., Loland et al).8 Ligand atoms were divided in three ring regions for each ligand (ringA1, ringA2, and ringB). The ligand ring naming corresponds to Supporting Information, Figure S-3. The interaction map was generated using the Python package matplotlib.43 Using this work-flow, it is possible to analyze the number of interaction partners and also their statistical frequency and identify amino acids that are important for binding. Animals. Sixty male Sprague−Dawley rats, aged between 12 and 14 weeks, were used in all experiments. They were bred and maintained in cages made of Makrolon and filled with autoclaved woodchips in the CoreUnit of Biomedical Research, Division of Laboratory Animal 9345



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Science and Genetics, Medical University of Vienna. Food and water in bottles was available ad libitum. The room was illuminated with artificial light at an intensity of about 200 lx in 2 m from 5 a.m. to 7 p.m.. Experiments were carried out between 8 a.m. and 2 p.m. All procedures were carried out according to the guidelines of the European Communities Council Directive of 24 November 1986 (86/ 609/EEC) and Ethics committee, Medical University of Vienna, and were approved by Federal Ministry of Education, Science and Culture, Austria (BMWFW-66.009/0114-WF/II/3b/2014). All efforts were made to minimize animal suffering and to reduce the number of animals used. Behavioral Studies. To study the behavioral effects of 11b treatment, multiple experiments were carried out by treating rats (10 per group) with an intraperitoneal dose of 11b 10 mg/kg body weight for 10 days and thereafter on the days of each test evaluation. The sequence of tests used was as follows: open field, elevated plus maze, rota rod, neurological observational battery, and forced swim test. Simultaneously a group of 10 rats with DMSO treatment was studied as vehicle control. Open Field (OF). Rats were observed by a video monitoring system consisting of a video camcorder coupled to the computational tracking system in an arena (100 cm × 100 cm long, with 40 cm high walls) for 10 min. Each rat was placed in the center, and the following parameters were measured: (a) path length, (b) resting time, (c) percent local movements, (d) percent large movements, (e) speed, (f) times crossing the center, and (g) reversals.41 Elevated Plus Maze (EPM). Rats were observed for anxiety-like behavior. The EPM was made out of black PVC and consisted of four arms (each 50 cm long and 10 cm wide) arranged in the shape of a plus sign and elevated 70 cm above the floor. Two opposite arranged arms were open, and the other two arms had 40 cm walls. All arms were interconnected by a 10 cm × 10 cm wide central area. Rats were observed with a video camcorder coupled to a computational tracking system in an arena. Rats were placed on the central area. Following parameters were recorded: (a) the time spent in closed and open arms, (b) number of entries into the closed and open arms, and (c) path length in closed and open arms. An entry was defined as having the rat placing all four legs into the box.44 Rota Rod. The rota rod (Rota Rod “Economex”, Columbus Instruments, Ohio, USA) tests balance and coordination and is comprised of a rotating drum which accelerates from 4 to 40 rpm over the course of 5 min. The time at which each rat fell from the drum was recorded. Each rat received three pretraining trials. Subsequently, each rat completed three more consecutive trials and the longest time on the drum was used for analysis.45 Neurological Observational Battery. The procedure follows the set up by Irwin.46 A battery of tests was applied to reveal defects in gait or posture, changes in muscle tone, grip strength, visual acuity, and temperature. To complete the assessment, vitally important reflexes were scored. In addition, during the manipulations, incidences of abnormal behavior, fear, irritability, aggression, excitability, salivation, and lacrimation were recorded.44 Forced Swim Test (FST). The test was performed as described previously.47 The procedure consisted of two sessions, the pretest session and the test session, using the same apparatus and conditions (diameter 18 cm, height 40 cm, containing 23 cm of water maintained at 25 °C). During the pretest session, rats were forced to swim for 15 min; 24 h later, rats were placed in the same apparatus for 5 min, which wa designated as a test session. The duration of immobility during 5 min was recorded.

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resolution mass spectrometry spectra, 1H and 13C NMR spectra, HPLC-determined purity data (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

*For G.L.: phone, ++43-676-569-48-16; E-mail, gert.lubec@ lubeclab.com. *For T.L.: E-mail, [email protected]. ORCID

Marcus Wieder: 0000-0003-2631-8415 Vasiliy Bakulev: 0000-0002-3312-7783 Gert Lubec: 0000-0002-6333-9461 Author Contributions ◆

Predrag Kalaba and Nilima Y. Aher have contributed equally to this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are indebted to Dr. Hubert Gstach and Prof. Thomas Erker from the University of Vienna (Austria) for allowing us to use the space and instruments in their laboratories and Marion Holey for her guidance to perform the uptake inhibition assays. Furthermore, we are very grateful to Prof. Gerhard Ecker from the University of Vienna (Austria) for providing us the protein homology model and to FAPESP by conceding a postdoctoral grant (2016/10149-0) to Prof. Eduardo R. P. Gonzalez.

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ABBREVIATIONS USED NET, norepinephrine transporter; DIPEA, potassium tertbutoxide; BF3·Et2O, boron trifluoride diethyl etherate; [3H]MPP+, methyl-4-phenylpyridinium; KHB, Krebs−Henseleit buffer; PDL, poly D-lysine; CDCl3, deuterated chloroform; DMSO-d6, deuterated dimethyl sulfoxide

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01313. MD simulation data, structure of CE-103, reuptake inhibition curves, X-ray measurements data, high9346



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Heterocyclic Analogues of Modafinil as Novel, Atypical Dopamine

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