Discovery of 1-aryloxyethyl piperazine derivatives as Kv1.5 potassium channel inhibitors (part I)

Accepted Manuscript Discovery of 1-Aryloxyethyl Piperazine Derivatives as Kv1.5 Potassium Channel Inhibitors (Part I) Xiaoke Guo, Xianglei Ma, Qian Yang, Jing Xu, Lu Huang, Jianmin Jia, Jiaojiao Shan, Li Liu, Weilin Chen, Hongxi Chu, Jinlian Wei, Xiaojin Zhang, Haopeng Sun, Yiqun Tang, Qidong You PII:

S0223-5234(14)00289-X

DOI:

10.1016/j.ejmech.2014.03.075

Reference:

EJMECH 6859

To appear in:

European Journal of Medicinal Chemistry

Received Date: 30 December 2013 Revised Date:

22 March 2014

Accepted Date: 27 March 2014

Please cite this article as: X. Guo, X. Ma, Q. Yang, J. Xu, L. Huang, J. Jia, J. Shan, L. Liu, W. Chen, H. Chu, J. Wei, X. Zhang, H. Sun, Y. Tang, Q. You, Discovery of 1-Aryloxyethyl Piperazine Derivatives as Kv1.5 Potassium Channel Inhibitors (Part I), European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.03.075. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights Lead compound 7k was identified by screening as a moderate Kv1.5 inhibitor.

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48 derivatives were designed, synthesized and bio-evaluated.

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Physicochemical, pharmacodynamics and acute toxicity studies were undertaken.

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Compound 8 deserves further evaluations as a promising lead for treatment of AF.

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ACCEPTED MANUSCRIPT Discovery of 1-Aryloxyethyl Piperazine Derivatives as Kv1.5 Potassium Channel Inhibitors (Part I) Xiaoke Guo a, b, Xianglei Ma d, Qian Yang c, Jing Xu d, Lu Huang d, Jianmin Jia a, c, Jiaojiao Shan d, Li

Tang d, *, Qidong You a, b, *

Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,

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a

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Nanjing 210009, China b

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Liu e, Weilin Chen a, b, Hongxi Chu a, b, Jinlian Wei a, b, Xiaojin Zhang a, Haopeng Sun a, b, c, *, Yiqun

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,

China

Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China

d

Research Division of Pharmacology, China Pharmaceutical University, Nanjing 210009, China

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Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing

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210009, China

Corresponding author. Tel. /fax: +86-25-8327-1351; Tel. /fax: +86-25-8327-1070.

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*

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c

E-mail addresses: [email protected] (Haopeng Sun); [email protected] (Qidong You); [email protected] (Yiqun Tang)

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Abstract: Kv1.5 potassium channel is an efficacious and safe therapeutic target for the treatment of atrial fibrillation (AF), the most common arrhythmia that threatens human. Herein, by modifying the

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hit compound 7k from an in-house database, 48 derivatives were synthesized for the assay of their Kv1.5 inhibitory effects by whole cell patch clamp technique. Six compounds which showed better potency than the positive compound dronedarone were selected for the next evaluation of their

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drug-like properties. Compound 8 exhibited balanced solubility and permeability. It also showed

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acceptable pharmacodynamics profile with very low acute toxicity. Taking all these data into account, compound 8 can serve as a promising lead for the development of novel therapeutic agent for the treatment of AF.

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Keywords: Atrial fibrillation; Kv1.5 potassium channel; structure−activity relationship

ACCEPTED MANUSCRIPT 1.

Introduction Atrial fibrillation (AF) is the most common arrhythmia that threatens human beings [1-3]. It can

obviously increase the onset risk of a series of severe diseases, including stroke (2~5 -fold), heart

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failure (3-fold) and dementia (2-fold) [4-5]. It is estimated that the morbidity of AF will at least double

in the next 5 decades [6]. As a result, it is critical to discover efficacious and safe therapeutic agents.

Antiarrhythmic drugs have been used for nearly 100 years and remain the preferred choice for the

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treatment of AF in the clinical trials till now [7]. However, these drugs such as dofetilide [8, 9] and

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sotalol [10], are limited in clinical use because of the production of negative feedback on the ventricular repolarizations, leading to QT interval prolongation and/or the potentially life threatening arrhythmia torsades de pointes (Tdp) [10-12]. Therefore, novel drugs with improved safety are urgently required [1, 14-15].

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The ultra-rapid potassium current (IKur), encoded by Kv1.5 gene, plays a significant role in the repolarization of the atrial action potential [16-18]. Kv1.5 potassium channel is recognized as an ideal target because it is only recorded in the human atrial myocytes while is absent in the ventricular

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myocytes [19-21].With the aim to discover novel Kv1.5 potassium channel inhibitors, an in-house

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database was screened using whole cell patch clamp technique. The hit compound 7k was identified with moderate inhibitory activity against human Kv1.5 potassium channel (hKv1.5 IC50 = 48.36 µM). Considering the alkoxyl chain including two carbon is a common segment in the drugs now on the market (amiodarone, dromedarone and vernakalant), we introduced this fragment into the structure of 7k (Figure 1). A series of resulted compounds were designed and synthesized. Herein we present the optimization, structure−activity relationship (SAR) studies, in vitro and in vivo characterization of the designed compounds. Although some agents with the similar core to our

ACCEPTED MANUSCRIPT series II compounds had earlier been reported in other paper [22] as class III antiarrhythmic drugs, we performed a comprehensive SAR study with more detailed information about the design of this scaffold. Among these derivatives, compound 8, possessing potent Kv1.5 inhibitory activity and satisfactory

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physicochemical properties, was selected for further evaluation. The acceptable pharmacodynamics profile and favorable safety results promoted 8 as a promising lead for treatment of AF.

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Chemistry

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To improve the Kv1.5 inhibitory activity, an alkoxy chain was introduced into the structure of the

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hit 7k. Treatment of thiophene-2-carboxylic acid with SOCl2 gave the thiophene-2-carbonyl chloride (1a). The substituted phenols were treated with BrCH2CH2Br in the presence of K2CO3 to yield the 3a-3r. The 3a-3r was reacted with piperazine hydrobromide (1:1) to give the 4a-4r in 35% - 70% yields. Series I compounds 1~ 11 (Scheme 1) were obtained after reacting 4a-4r with 1a in DMF.

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In order to investigate the SAR, several structural modifications were conducted on the aromatic ring and the linker. Thiophene ring (Ring A) was replaced by benzene or thiazole and the linker was changed from carbonyl to acylamino (Figure 2). The synthesis of these compounds was outlined in

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Scheme 2. Substituent benzoyl chlorides (1b-1e) were prepared as the same procedure of 1a.

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Commercially available aromatic amines were acylated with chloroacetyl chloride, and compounds 2a-2e were obtained in 75% - 95% yields. Series II-IV compounds were (Scheme 2) synthesized from 4a-4r treated with 1b-1e or 2a-2e in DMF.

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Results and Discussion

3.1. IKur blockade Assays The synthesized derivatives were evaluated for their inhibitory activities against Kv1.5 (IKur) potassium channel using whole cell patch clamp technique in a representative HEK 293 cell stably

ACCEPTED MANUSCRIPT expressing hKv1.5 channels [23, 24]. The results for series I compounds were shown in Table 1. On the basis of the structure of 7k, an alkoxy chain (-OCH2CH2-) was introduced to increase the inhibitory activity against Kv1.5 potassium channel. The resulting compound 5 exhibited a higher

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Kv1.5 potency (~8 fold) compared to 7k. Then a series of substituents with varying electronic and steric properties were introduced into the phenyl ring to modulate the Kv1.5 inhibitory activity (1~ 11). The results (Table 1) showed that the molecule without substitution at phenyl exhibited decreased

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activity compared to compounds with steric substituents (e.g. unsubstituted 1 vs methoxyl substituted 4

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or vs chlorine substituted 8, 2- to 50-fold potency decrease), revealing that bulky groups at phenyl ring was preferred for the optimization of the compounds. The biological activities of compounds with substitution at para position were better than those ones substituted at ortho position (2 > 3; 8 > 10). In this series, the most potent compound 8 (IC50 = 0.72 µM) displayed ~3 fold improved potency than the

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positive control dronedarone (IC50 = 2.37 µM).

With the aim to investigate the SARs, thiophene ring (Ring A) was replaced by benzene and the linker was changed from carbonyl to acylamino, leading to series II and III compounds (Table 2 and

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Table 3). No significant variation was observed when ring A was changed from thiophene to benzene

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( IC50 = 37.50 µM, 12 IC50 = 38.78 µM, 21 IC50 = 28.57 µM), demonstrating a structural tolerance at this position. The derivatives with 4-methylphenyl at ring A (18, 29) instead of phenyl (12, 21) resulted in improved activities against Kv1.5. Meanwhile, the compounds with 2-chlorphenyl (20, 32) at ring A exhibited decreased Kv1.5 potency. Compounds with either the electron-withdrawing group, such as chlorine group (13, 24) and cyano group (14, 25), or electron-donating group, such as methyl group (15, 22), at the R2 obviously showed an increased activity against Kv1.5 compared to 12 (or 21) without substituent. Replacing the linker from carbonyl (II series) to acylamino (III series) remained or slightly

ACCEPTED MANUSCRIPT increased the activity (21 vs 12; 26 vs 16; 28 vs 17; 32 vs 20), suggesting that the linker could bear different steric modifications. We further synthesized a series of thiazole derivatives (series IV compounds) to expand the SAR

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analysis. The results (Table 4) showed that replacing the phenyl with thiazolyl maintained (21 vs 34; 24 vs 46) or slightly increased (23 vs 42) the activities. Compounds with small substituents improved activity, which was same with series I-III, but with bulky substituents, such as dimethyl group (37, 38

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and 39), 4-C(Me)3 (40), 2-OCH2CH3 (43), 4-COOC2H5 (47) and naphthyl (48) led to significant loss of

3.2. Physicochemical properties Studies

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activity, indicating that it was not suitable to introduce large groups at phenyl.

After the IKur blockade assay, six compounds that possessed excellent activities against Kv1.5 potassium channel (IC50 < positive control 2.37 µM) were picked out to determine their

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physicochemical properties (pKa, log D7.4 and permeability), in order to identify potential druglike compounds. The pKa and partition coefficient (log D, pH 7.4) were determined according to the methods of Avdeef and Tsinman [25, 26] on a Gemini Profiler instrument (pION) by the

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“goldstandard” Avdeef−Bucher potentiometric titration method [26, 27]. Permeability coefficients

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were determined using MDCK (Madin-Darby canine kidney) and MDR1-MDCK (stably expressing multidrug resistance gene 1) cells. As shown in Table 5, six compounds exhibited acceptable physicochemical properties, especially

compounds 8, 36 and 45, which showed superior permeability and LogD (lipid-water distribution coefficient), the key factors influencing drug absorption [28]. In the permeability experiment, we also investigated whether these compounds were substrates of P-glycoprotein (Pgp) using MDR1-MDCK (stably expressing multidrug resistance gene 1) cell line.

ACCEPTED MANUSCRIPT Efflux ratio was equal to Papp (apparent permeability coefficient) (B-A)/ Papp (A-B), RT is efflux ratio in MDR1-MDCK cell while RW is efflux ratio in MDCK cell. According to the results, all of our compounds were not the substrates of Pgp (R < 1.50) [29, 30].

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Based on the above evaluation, compound 8 with was potent in Kv1.5 inhibitory effect, and was balanced in solubility and permeability. As a result, it was chosen for further in vivo evaluation. 3.3. Effect of 8 on AF model induced by CaCl2-ACh

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An AF model induced by CaCl2-ACh (calcium chloride and acetylcholine) in SD rats was applied

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to evaluate the anti-AF effect of compound 8 as described previously [24], and the results were characterized by the changes of duration of AF and AERP (atrial effective refractory period) before and after treatment with 8. Figure 2A showed that there was no significant difference between 5 groups (8 treatment groups, model and two positive control groups) during the model-creation

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(Day1-Day3). Then duration of AF was significantly reduced when the animals were treated with 8 (Day4-Day7). The effect of 8 (6.45 µM, 2.50 mg/kg) was close to that of sotalol (8.09 µM, 2.50 mg/kg). In addition, the effect of 8 (3.23 µM, 1.25 mg/kg) was better than that of dronedarone (2.25

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µM, 1.25 mg/kg). The AERP of 8-treated group was increased to normal level (Figure 2B), the effect

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was close to sotalol and better than dronedarone. These data showed that compound 8 could reverse the AF induced by CaCl2-ACh in vivo. 3.4. Acute toxicity studies of 8 Acute toxicity of compound 8 was observed by tail intravenous injection (IOCV) on mice (Table

6). LD50 of injection was 73.65 mg/kg, far higher than the effective dose (1.25 mg/kg), indicating that compound 8 possessed a high security in the treatment of AF. 4.

Conclusion

ACCEPTED MANUSCRIPT We have designed and synthesized 48 novel compounds modified from the compound 7k which was obtained through the screening of an in-house database by whole cell patch clamp technique. Structural modifications mainly addressing the aromatic ring, coupling chain and substituents at phenyl

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enabled modulation of Kv1.5 inhibitory potency. Most of the derivatives endowed improved potency compared to 7k and some of them possessed more potent inhibitory activity than dronedarone (positive control). The physicochemical properties of some of the potent compounds were evaluated to consider

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the drug-like property. Among them, compound 8, with outstanding activity, balanced LogD and

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permeability, was selected for further in vivo evaluations. This compound also showed comparable in vivo potency with sotalol and dronedarone and remarkable safety in rats. Based on these results, it can serve as a promising lead for further development of novel agents treating AF. 5.

Experimental Section

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5.1. Chemistry

Starting materials, reagents, and analytical grade solvents were purchased from commercial sources and, unless otherwise noted, were used without further purification. Column and thin-layer

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chromatography (CC and TLC, resp.) were performed on silica gel (200-300 mesh) and silica gel

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GF254 resp., supplied by Qingdao Marine Chemical Factory. Melting points were determined with a Melt-Temp II apparatus and were reported without correction. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR spectrometer using KBr film. The 1H-NMR and 13C-NMR spectra were measured on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane (TMS) as internal standard. Chemical shifts were reported as d values (parts per million) relative to TMS peak. Coupling constants were reported in hertz. The multiplicity was defined by an s (singlet), d (doublet), t (triplet), or m (multiplet). EI-mass spectra were recorded with a Shimadzu GCMS-2010 mass spectrometer.

ACCEPTED MANUSCRIPT High-resolution mass spectra (HR-MS) were recorded on a Water Q-Tof micro mass spectrometer. Purity for final compounds was greater than 95% and was measured by HPLC with Agilent Technologies 1260 infinity C18 4.60 mm × 150 mm column using a mixture of solvent methanol/water

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at the flow rate of 0.5 mL/min and peak detection at 245 nm under UV. For experimental procedures and spectral characterization data see the Supporting Information. 5.2. Whole cell patch clamp assay

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The HEK 293 cell line that stably expressed hKv1.5 potassium channel was a kindly gift from Dr.

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Gui-Rong Li (Department of Medicine and Department of Physiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, SAR, China) [23]. Transfected HEK 293 cells were maintained at 37 °C in Minimal Eagle Medium (MEM) or Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 2 mmol/L

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L-glutamine, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and 0.2 mg/mL geneticin (Invitrogen Corporation, Carlsbad, CA). Cells were passaged weekly and used at ≤ 80% confluence. For electrophysiological recordings, the cells were harvested from the culture dish by

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trypsinization, and then washed twice with standard MEM or DMEM and maintained in culture

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medium at room temperature for later use on the same day. The whole cell membrane currents were recorded by the patch clamp technique, using an EPC-10

double patch clamp amplifier (HEKA, Pfalz, Germany). Recording pipettes, made from borosilicate glass (1.2 mm, o.d.), pulled with a pipette puller (PIP5, HEKA, Germany), had resistances of between 4 and 6MΩ when filled with the pipette solution. Pulse software (HEKA, Pfalz, Germany) was used to generate voltage pulse protocols and to record and analyse data. Standard voltage protocols for IKur: 500-ms depolarizing pulse steps to +60 mV from a holding potential of -80 mV at 0.1 Hz [23]

ACCEPTED MANUSCRIPT Compounds were applied at least 5 min after current stabilization. IC50 value was determined by using cell-by-cell fits with the Hill equation for three cells at least for all concentrations. Data were presented as the mean and standard deviation (x ± SD), n = 3. All experiments were performed at 25 °C.

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5.3. CaCl2-Ach induced AF Model SD rats (250 ± 20 g) were anesthetized with 10% chloral hydrate (intraperitoneal injection, 0.3 mL/kg), followed by iv administration of CaCl2 (10 mg/mL) and acetylcholine (ACh; 66 mg/mL)

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through the caudal vein (1 mL/kg) once a day for 7 days. The ECG was recorded for the complete

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experiment procedure from rat anesthetization to the ECG being restored to normal, approximately 50 min for each rat each day. On Day 4, rats were randomly divided into four groups (n = 10/group): (1) AF model group: repeated prior procedure until day 7, CaCl2 (10 mg/mL, i.v.) and ACh (66 mg/mL, i.v.); (2) and (3) 8 treatment groups (1.25 mg/kg and 2.50 mg/kg, ip) combined with CaCl2 (10 mg/mL)

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and ACh (66 mg/mL) (i.v.) from Day 4 to Day 7; (4) positive control: sotalol hydrochloride (2.50 mg/kg, ip) or dronedarone hydrochloride (1.25 mg/kg, ip) combined with CaCl2 (10 mg/mL, i.v.) and ACh (66 mg/mL, i.v.) from Day 4 to Day 7. Controls were anesthetized with 10% chloral hydrate (3

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mL/kg, ip), followed by physiological saline i.v. for 7 days. The disappearance of the P wave and

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appearance of the f wave was determined as the beginning of AF while the end was designated by disappearance of the f wave and the appearance of the regular P wave. After 7 days of administration, SD rats were anesthetized and the hearts were rapidly excised and placed in ice-cold Krebs-Henseleit buffe. The atrial appendage was separated and pinned to the bottom of a recording chamber perfused with oxygenated Krebs-Henseleit buffer maintained at 37 ºC. Atrial muscles were equilibrated for 40 min and contractile responses recorded for a series of continuous S1–S2 couplings at 10 Hz and 150% threshold voltage. Atrial ERP was measured (BL-420 system of Tai Meng, Cheng Du) using an

ACCEPTED MANUSCRIPT incremental technique, with 1-ms steps at basic drive cycle lengths of 50 ms [23]. Data were analyzed using Student’s t test and presented as the mean (x ± SD), n = 6. 5.4. Acute toxicity

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70 SPF mice were divided into 7 groups (1 for control and 6 for treatment groups) and administrated with distilled water (30 mL/kg) or compound 8 (84.6, 79.8, 75.3, 71, 67 and 63.2 mg/kg). Response of mice (eg. behavior, mental state, excrement and death) was observed in 0-0.5 h

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test, LD50 was calculated by Bliss-Finney method [31].

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(continuously), 0.5-6 h (0.5 h interval), 1-15 day (1 day interval). Data were analyzed using Student’s t

Acknowledgments

The authors thank Dr. Gui-Rong Li (Department of Medicine and Department of Physiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, SAR, China) for

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providing the HEK293 cells in which Kv1.5 channels are stably expressed. This work was supported by the Project Program of Jiang Su Key Laboratory of Drug Design and Optimization and State Key Laboratory of Natural Medicine, the innovation Program for the

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Postgraduates in Jiangsu in 2012, National Major Science and Technology Project of China

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(Innovation and Development of New Drugs. No. 2012ZX09103101-002 and No. 2012ZX09401-005). References

[1]. Dobrev, D.; Carlsson, L.; Nattel, S. Novel molecular targets for atrial fibrillation therapy. Nat. Rev. Drug Discov. 2012, 11, 275-291.

[2]. Kalantarian, S.; Stern, T. A.; Mansour, M.; Ruskin, J. N. Cognitive Impairment Associated With Atrial Fibrillation: A Meta-analysis. Ann. Intern. Med. 2013, 158, 338-346. [3]. Lip, G. Y.; Tse, H. F., Management of atrial fibrillation. Lancet 2007, 370, 604-618.

ACCEPTED MANUSCRIPT [4]. Mulder, B. A.; Schnabel, R. B.; Rienstra, M. Predicting the future in patients with atrial fibrillation: who develops heart failure? Euro. J. Heart. Fail. 2013, 15, 366-367. [5]. Nattel, S.; Carlsson, L. Innovative approaches to anti-arrhythmic drug therapy. Nat. Rev. Drug

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Discov 2006, 5, 1034-1049. [6]. Pamukcu, B.; Lip, G. Y. Dronedarone as a new treatment option for atrial fibrillation patients: pharmacokinetics, pharmacodynamics and clinical practice. Expert Opin. Pharmacother. 2011, 12,

SC

131-140.

M AN U

[7]. Zimetbaum, P. Antiarrhythmic drug therapy for atrial fibrillation. Circulation 2012, 125, 381-389. [8]. Chandra, P.; Rosen, T. S.; Yeom, Z. H.; Lee, K.; Kim, H. Y.; Danilo, P., Jr.; Rosen, M. R., Evaluation of KCB-328, a new IKr blocking antiarrhythmic agent in pacing induced canine atrial fibrillation. Europace 2004, 6, 384-391.

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[9]. Allen LaPointe, N. M.; Chen, A.; Hammill, B.; DeLong, E.; Kramer, J. M.; Califf, R. M. Evaluation of the dofetilide risk-management program. Am. Heart J. 2003, 146, 894-901. [10]. Marill, K. A.; Runge, T. Meta-analysis of the Risk of Torsades de Pointes in patients treated with

EP

intravenous racemic sotalol. Acad. Emerg. Med. 2001, 8, 117-124.

AC C

[11]. Hohnloser, S. H.; van de Loo, A.; Baedeker, F. Efficacy and proarrhythmic hazards of pharmacologic cardioversion of atrial fibrillation: prospective comparison of sotalol versus quinidine. J. Am. Coll. Cardiol. 1995, 26, 852-858.

[12]. Finlay, H. J.; Lloyd, J.; Vaccaro, W.; Kover, A.; Yan, L.; Bhave, G.; Prol, J.; Huynh, T.; Bhandaru, R.; Caringal, Y.; DiMarco, J.; Gan, J.; Harper, T.; Huang, C.; Conder, M. L.; Sun, H.; Levesque, P.; Blanar, M.; Atwal, K.; Wexler, R. Discovery of ((S)-5-(methoxymethyl)-7-(1-methyl-1Hindol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxaz

ACCEPTED MANUSCRIPT ol-5-yl)pyrrolidin-1-yl) methanone as a potent and selective IKur inhibitor. J. Med. Chem. 2012, 55, 3036-3048. [13]. Wu, H. J.; Wu, W.; Sun, H. Y.; Qin, G. W.; Wang, H. B.; Wang, P.; Yalamanchili, H. K.; Wang, J.;

RI PT

Tse, H. F.; Lau, C. P.; Vanhoutte, P. M.; Li, G. R. Acacetin causes a frequency- and use-dependent blockade of hKv1.5 channels by binding to the S6 domain. J. Mol. Cell Cardiol. 2011, 51, 966-973.

SC

[14]. Wilber, D. J.; Pappone, C.; Neuzil, P.; De Paola, A.; Marchlinski, F.; Natale, A.; Macle, L.; Daoud,

M AN U

E. G.; Calkins, H.; Hall, B.; Reddy, V.; Augello, G.; Reynolds, M. R.; Vinekar, C.; Liu, C. Y.; Berry, S. M.; Berry, D. A. Comparison of antiarrhythmic drug therapy and radiofrequency catheter ablation in patients with paroxysmal atrial fibrillation: a randomized controlled trial. JAMA. 2010, 303, 333-340.

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[15]. Elming, H.; Brendorp, B.; Pehrson, S.; Pedersen, O. D.; Kober, L.; Torp-Petersen, C. A benefit-risk assessment of class III antiarrhythmic agents. Expert Opin. Drug Saf. 2004, 3, 559-577.

EP

[16]. Wang, Z.; Fermini, B.; Nattel, S. Sustained depolarization-induced outward current in human

AC C

atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents. Circ. Res. 1993, 73, 1061-1076.

[17]. Van Wagoner, D. R.; Pond, A. L.; McCarthy, P. M.; Trimmer, J. S.; Nerbonne, J. M. Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ. Res. 1997, 80, 772-781. [18]. Roden, D. M. Cellular basis of drug-induced torsades de pointes. Br. J. Pharmacol. 2008, 154, 1502-1507.

ACCEPTED MANUSCRIPT [19]. Li, G. R.; Feng, J.; Yue, L.; Carrier, M.; Nattel, S. Evidence for two components of delayed rectifier K+ current in human ventricular myocytes. Circ. Res. 1996, 78, 689-696. [20]. Tomaselli, G. F.; Beuckelmann, D. J.; Calkins, H. G.; Berger, R. D.; Kessler, P. D.; Lawrence, J. H.;

repolarization. Circulation 1994, 90, 2534-2539.

RI PT

Kass, D.; Feldman, A. M.; Marban, E. Sudden cardiac death in heart failure. The role of abnormal

[21]. Page, R. L.; Roden, D. M. Drug therapy for atrial fibrillation: where do we go from here? Nat. Rev.

SC

Drug Discov. 2005, 4 (11), 899-910.

M AN U

[22]. Kanojia, R. M.; Salata, J. J.; Kauffman, J. Synthesis and class III type antiarrhythmic activity of 4-aroyl (and aryl)-1-aralkylpiperazines. Bioorg. Med. Chem. Lett. 2000, 10 (24), 2819-2823. [23]. Guo, X. K.; Shen, Z.; Yu, P.; Chu, H. X.; Tang, Y. Q.; You, Q. D. Discovery of Benzamide Derivatives as Potent Kv1.5 Inhibitors. J. Chin. Pharmaceut. Sci. 2012, 21, 553-560.

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[24]. Guo, X.; Chen, C. L.; Yang, Q.; Yin, Y. M.; You, Q. D.; Tang, Y. Q. Effects of a Novel Class III Antiarrhythmic Agent, CPUY11018, on Rat Atrial Fibrillation. Drug Dev. Res. 2010, 71, 303-312. [25]. Avdeef, A.; Tsinman, O. Miniaturized Rotating Disk Intrinsic Dissolution Rate Measurement:

EP

Effects of Buffer Capacity in Comparisons to Traditional Wood’s Apparatus. Pharm. Res. 2008,

AC C

25, 2613-2627.

[26]. Zhang, X. J.; Li, X.; Sun, H. P.; Wang, X. J.; Zhao, L.; Gao, Y.; Liu, X. R.; Zhang, S. L.; Wang, Y. Y.; Yang, Y. R.; Zeng, S.; Guo, Q. L.; You, Q. D. Garcinia Xanthones as Orally Active Antitumor Agents. J. Med. Chem. 2013, 56, 276-292.

[27]. Avdeef, A.; Bucher, J. J. Accurate measurements of the concentration of hydrogen ions with a glass electrode: calibrations using the Prideaux and other universal buffer solutions and a computer-controlled automatic titrator. Anal. Chem. 1978, 50, 2137-2142.

ACCEPTED MANUSCRIPT [28]. Wohnsland, F.; Faller, B. High-Throughput Permeability pH Profile and High-Throughput Alkane/Water log P with Artificial Membranes. J. Med. Chem. 2001, 44, 923-930. [29]. Liu, Y.; Zeng, S. Advances in the MDCK-MDR1 cell model and its applications to screen drug

RI PT

permeability. Acta Pharmacol. Sin. 2008, 43, 559-564. [30]. Doan, K. M. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L. J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W. Passive Permeability and P-Glycoprotein-Mediated Efflux

SC

Differentiate Central Nervous System (CNS) and Non-CNS Marketed Drugs. J. Pharmacol. Exp.

M AN U

Ther. 2002, 303, 1029-1037.

[31]. Wang, A. P., Wu, H., Zhang, S. Y. Acute toxicity test. In Preclinical safety evaluation and practice of drugs, 1st, ed.; Yuan, B. J., Wang, Z. J.; Military Medical Science Press: Beijing, 1997,

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EP

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pp 23-42.

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Figure Captions Figure 1. Design of target compounds by comparison of the hit 7k with market drugs.

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Figure 2. A: Effect of compound 8 on duration of AF; B: Effect of compound 8 on AERP. (AERP = Atrial Effective Refractory Period; Data are expressed as mean ± SD, n = 6; * P < 0.05, ** P < 0.01 vs

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control)

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Figure 1.

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ACCEPTED MANUSCRIPT

Figure 2.

ACCEPTED MANUSCRIPT Table 1. Kv1.5 inhibition profile of series I compounds

R

IC50 (hKv1.5) µM a

Comp.

R

IC50 (hKv1.5) µM a

7k

-

48.36 ± 7.42

6

4-CN

10.17 ± 1.05

1

H

37.50 ± 4.38

7

4-F

2

4-Me

1.52 ± 0.29

8

4-Cl

3

2-Me

13.53 ± 1.61

9

4-Br

4

4-OMe

21.92 ± 3.53

10

2-Cl

5

4-OCF3

6.44 ± 0.77

0.72 ± 0.13

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12.18 ± 1.56 9.74 ± 1.02

11

2,4-diCl

1.48 ± 0.13

2.37 ± 0.13

Data are expressed as mean ± SD, n = 3.

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a

12.71 ± 2.14

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Dronedarone

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Comp.

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Table 2. Kv1.5 inhibition profile of series II compounds

R1

R2

IC50 (hKv1.5) µM a

Comp.

R1

12

H

H

38.78 ± 5.37

17

4-OMe

4-Cl

5.50 ± 1.08

13

H

4-Cl

3.97 ± 0.58

18

4-Me

H

7.14 ± 1.35

14

H

4-CN

4.23 ± 0.79

19

4-Me

4-Cl

4.51 ± 0.52

15

H

4-Me

25.27 ± 4.21

20

4-Cl

H

56.75 ± 9.81

16

4-OMe

H

16.07 ± 3.93

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Comp.

a

Data are expressed as mean ± SD, n = 3.

R2

IC50 (hKv1.5) µM a

ACCEPTED MANUSCRIPT Table 3. Kv1.5 inhibition profile of series III compounds

R2

IC50 (hKv1.5) µM a

Comp.

R1

R2

21

H

H

28.57 ± 4.29

28

4-OMe

4-Cl

2.62 ± 0.31

22

H

2-Me

11.82 ± 1.76

29

4-Me

H

16.11 ± 1.97

23

H

4-OCF3

7.33 ± 1.14

30

4-Me

4-CN

4.49 ± 0.75

24

H

4-Cl

4.38 ± 0.72

31

25

H

4-CN

9.10 ± 1.30

32

26

4-OMe

H

10.13 ± 1.19

27

4-OMe

4-CN

14.46 ± 2.04

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SC 4-Me

4-Cl

5.02 ± 0.69

4-Cl

H

29.42 ± 5.11

4-Cl

3.59 ± 0.47

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Data are expressed as mean ± SD, n = 3.

33

IC50 (hKv1.5) µM a

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R1

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a

Comp.

4-Cl

ACCEPTED MANUSCRIPT

Table 4. Kv1.5 inhibition profile of series IV compounds

R

IC50 (hKv1.5) µM a

Comp.

R

34

H

29.34 ± 6.32

41

4-OMe

35

4-Me

8.93 ± 1.18

42

4-OCF3

36

2-Me

1.24 ± 0.16

43

2-OCH2CH3

37

2,4-diMe

17.16 ± 1.94

44

2-Cl

38

3,4-diMe

50.24 ± 8.23

45

4-CN

0.97 ± 0.08

39

2,6-diMe

85.93 ± 10.07

46

4-Cl

5.18 ± 0.72

40

4-C(Me)3

63.18 ± 10.48

47

4-COOC2H5

32.1% b

9.13 ± 1.34 2.29 ± 0.26 11.9% b

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48

3.39 ± 0.51

68.03 ± 10.29

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Data are expressed as mean ± SD, n = 3; b Percent inhibition of current at 11 µM.

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a

IC50 (hKv1.5) µM a

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Comp.

ACCEPTED MANUSCRIPT

Table 5. Physicochemical properties of compounds MDCK

pKa

Log D7.4

Papp (nm/sec) b

Papp (nm/sec) b RW

A-B

RT

c

B-A

A-B

6.40

3.24

311 ± 50

200 ± 14

0.64

328 ± 6

256 ± 7

0.78

1.22

8

6.35

1.94

219 ± 44

213 ± 11

0.97

193 ± 48

161 ± 6

0.84

0.87

11

5.23

3.73

191 ± 33

170 ± 6

0.89

315 ± 54

391 ± 42

1.24

1.39

36

7.11

2.45

342 ± 42

322 ± 35

1.09

323 ± 32

289 ± 21

0.92

0.84

42

6.74

3.94

169 ± 18

45

6.37

1.72

167 ± 2

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2

122 ± 8

1.50

196 ± 21

163 ± 6

1.42

0.95

148 ± 21

1.14

198 ± 16

189 ± 2

1.14

1.00

ND for Not Determined; b Data are expressed as mean ± SD, n = 3; c RW (or RT) = Papp (A-B)/ Papp (B-A).

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a

B-A

R = RT/RW

b

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Comp.

MDR1-MDCK

ACCEPTED MANUSCRIPT

Table 6. Determination of LD50 of compound 8 on mice by IOCV IOCV (mg/kg)

LD50

73.65

95% confidence interval

70.17-77.50

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Compound 8

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ACCEPTED MANUSCRIPT

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Scheme 1. Reagents and conditions: i) BrCH2CH2Br, K2CO3, reflux; ii) (1) piperazine, EtOH, HBr

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(40% aqueous solution), (2) HCl, (3) NaOH; iii) SOCl2, CH2Cl2, reflux; iv) K2CO3, DMF, 80 °C.

Scheme 2. Reagents and conditions: i) SOCl2, CH2Cl2, reflux; ii) ClCH2COCl, TEA, DMF, 5 °C; iii) K2CO3, DMF, 80 °C.

Supporting Information

ACCEPTED MANUSCRIPT

Discovery of 1-Aryloxyethyl Piperazine Derivatives as Kv1.5 Potassium Channel Inhibitors (Part I) Xiaoke Guo a, b, Xianglei Ma d, Qian Yang c, Jing Xu d, Lu Huang d, Jianmin Jia a, c, Jiaojiao Shan d, Li

Tang d, *, Qidong You a, b, *

Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,

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a

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Nanjing 210009, China b

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Liu e, Weilin Chen a, b, Hongxi Chu a, b, Jinlian Wei a, b, Xiaojin Zhang a, Haopeng Sun a, b, c, *, Yiqun

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,

China

Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China

d

Research Division of Pharmacology, China Pharmaceutical University, Nanjing 210009, China

e

Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing

Experimental section

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1.

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210009, China




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c

General Procedure for the Synthesis of Intermediates 1a-1e

SOCl2 (0.30 mol) was added to a solution of aromatic acid (0.06 mol) in CH2Cl2 (80 mL), the

mixture was stirred and refluxed for 4 h, then evaporated until dryness to give compounds 1a-1e with colorless oily liquid which was immediately applied for further reaction as a crude product after quick weighing. 2.

General Procedure for the Synthesis of Intermediates 2a-2e

Supporting Information

ACCEPTED MANUSCRIPT

To a solution of commercially available aromatic amines (0.02 mol) and TEA (0.02 mol) in DMF (40 mL) was added chloroacetyl chloride (0.024 mol) in DMF (10 mL). The mixture was stirred at r.t. for 2 h, taken up in water (300 mL) and filtered to obtain the compounds 2a-2e in 75.91-95.37% yields. General Procedure for the Synthesis of Intermediates 3a-3r

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3.

K2CO3 (0.02 mol) was suspended in BrCH2CH2Br (30 mL, 0.06 mol), and dropwise added different substituted phenols (0.015 mol) in CH3CN (10 mL) at 50 °C, then the mixture was refluxed for 24 h

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and filtered, the filtrate was concentrated and purified by CC (column chromatography) over silica gel

4.

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(eluent, PE:EA = 10:1) to give the white solid 3a-3r in 40.28-74.32 % yields. General Procedure for the Synthesis of Intermediates 4a-4r

HBr (aq. 40%, 0.02 mol and 2.60 mL) was dropwise added to a solution of piperazidine (0.02 mol) in EtOH (50 mL) at r.t., and then 3a-3r (0.01 mol) in EtOH (50 mL) was added. The mixture was

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refluxed for 48 h, filtered and concentrated. The residue was dissolved in water, acidified with 10% HCl aq. to pH = 5-6 and washed with CH2Cl2, water layer was basified by 10-50 % NaOH till pH > 9 and extracted with CH2Cl2, dried over NaSO4 and evaporated until dryness to obtain the compounds

Thiophen-2-yl(4-(2-phenoxyethyl)piperazin-1-yl)methanone hydrochloride (1)

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5.

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4a-4r with colorless oil in 35.24-70.98 % yields.

Compound 1a (0.015 mol) was dropwise added to a solution of 4a (0.01 mol) and TEA (0.02 mol)

in dry CH2Cl2 under ice-salt bath, the mixture was stirred at r.t. for 8 h and washed with water (× 3), saturation sodium bicarbonate solution (× 2) and saturation sodium chloride solution (× 2), the organic layer was concentrated and recrystallized in PE: EA = 3 : 2, and then the residue was acidulated with HCl in EtOH to obtain the title compound 1 as white solid (28.71%, m.p. = 258-259 °C) IR (KBr): 2577, 2511, 2425, 1606, 1464, 1426, 1243, 847, 758, 734 cm-1; 1H-NMR (300

MHz, D2O)

Supporting Information

ACCEPTED MANUSCRIPT

δ 7.39 (d, 1H, J = 4.86 Hz, Ar-H), 7.14 (d, 1H, J = 3.48 Hz, Ar-H), 7.06 (t, 2H, J = 7.89 Hz, Ar-H), 6.84 (t, 1H, J = 4.20 Hz, Ar-H), 6.79-6.70 (m, 3H, Ar-H), 4.10 (t, 2H, J = 4.56 Hz, -CH2CH2O-), 3.75 (brs, 4H, -N(CH2CH2)2N-), 3.36 (t, J = 4.68, -CH2CH2O-), 3.22 (brs, 4H, -N(CH2CH2)2N-); 13C-NMR

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(75MHz, CDCl3, basic group) δ 163.0, 156.7, 136.5, 128.9, 128.3, 128.1, 126.2, 125.4, 115.4, 65.7, 56.5, 53.1, 44.9; MS (EI) m/z 316 [M+]; Anal. (C17H21ClN2O2S.1/2H2O) C, H, N. Calcd for: 56.42, 6.13, 7.74; Found: 56.72, 5.89, 7.59; HPLC purity: 99.44%, retention time: 4.69 min (methanol/water

Thiophen-2-yl(4-(2-(p-tolyloxy)ethyl)piperazin-1-yl)methanone (2)

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80:20, v/v).

4b (0.01 mol) and TEA (0.02 mol) was dissolved in dry CH2Cl2 and cooled to 0 °C and 1a (0.015 mol) was added dropwise, then the solution was warmed to r.t. After being stirred for 8 h, the mixture was washed with water (× 3), saturation sodium bicarbonate solution (× 2) and saturation sodium

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chloride solution (× 2), the organic layer was concentrated and recrystallized in PE: EA = 3 : 2 to yield compound 2 as white solid (52.58 %, m.p. = 84-86 °C). IR(KBr): 3850, 2943, 2832, 1601, 1521, 1513, 1455, 1427, 1248, 996, 803, 737 cm-1; 1H-NMR

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(300 MHz, CDCl3) δ 7.33-7.30 (m, 1H, Ar-H), 7.27-7.23 (m, 1H, Ar-H), 7.09-7.02 (m, 3H, Ar-H),

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6.82-6.78 (m, 2H, Ar-H), 4.10 (t, 2H, J = 5.73 Hz, -CH2CH2O-), 3.77 (t, 4H, J = 5.10, -N(CH2CH2)2N-), 2.83 (t, 2H, J = 5.79 Hz, -CH2CH2O-), 2.62 (t, 4H, J = 5.13, -N(CH2CH2)2N-), 2.28 (s, 3H, -CH3); MS (EI) m/z 330 [M+]; Anal. (C18H22N2O2S) C, H, N. Calcd for: 65.43, 6.71, 8.48; Found: 65.39, 6.95, 8.42; HPLC purity: 99.08%, retention time: 6.41 min (methanol/water 80:20, v/v). 7.

Thiophen-2-yl(4-(2-(o-tolyloxy)ethyl)piperazin-1-yl)methanone hydrochloride (3) Compound 3 was synthesized from 4c and 1a with the procedure described for compound 1 (yellow

solid, 45.51%). m.p. = 207-209 °C.

Supporting Information

ACCEPTED MANUSCRIPT

IR(KBr): 3438, 3053, 2918, 2411, 2389, 1607, 1464, 1429, 1265, 988, 734 cm-1; 1H-NMR (300 MHz, D2O) δ 7.68 (d, 1H, J = 4.80 Hz, Ar-H), 7.44 (d, 1H, J = 3.33 Hz, Ar-H), 7.24-7.13 (m, 2H, Ar-H), 6.92-6.86 (m, 3H, Ar-H), 4.37 (t, 2H, J = 4.59 Hz, -CH2CH2O-), 4.05 (s, 4H, -N(CH2CH2)2N-),

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3.60 (t, 2H, J = 4.53 Hz, -CH2CH2O-), 3.47 (s, 4H, -N(CH2CH2)2N-), 2.26 (s, 3H, -CH3); MS (EI) m/z 330 [M+]; Anal. (C18H23ClN2O2S) C, H, N. Calcd for: 58.94, 6.28, 7.65; Found: 59.02, 6.33, 8.09. 8.

Thiophen-2-yl(4-(2-(4-methoxyphenoxy)ethyl)piperazin-1-yl)methanone (4)

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Compound 4 was synthesized from 4d and 1a with the procedure described for compound 2

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(yellow solid, 59.72%). m.p. = 82-84 °C.

IR(KBr): 3438, 3089, 2940, 2832, 1600, 1514, 1434, 1232, 1051, 823, 737 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.45 (d, 1H, J = 5.16 Hz, Ar-H), 7.29-7.26 (m, 1H, Ar-H), 7.06-7.02 (m, 1H, Ar-H), 6.84-6.79 (m, 4H, Ar-H), 4.08 (t, 2H, J = 5.43 Hz, -CH2CH2O-), 3.79-3.72 (m, 7H, -OCH3,

13

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-N(CH2CH2)2N-), 2.83 (t, 2H, J = 5.49 Hz, -CH2CH2O-), 2.63 (t, 4H, J = 4.80 Hz, -N(CH2CH2)2N-); C-NMR (75MHz, CDCl3) δ 162.9, 153.5, 152.3, 136.6, 128.3, 128.0, 126.1, 115.1, 114.2, 66.1, 56.7,

55.2, 53.0, 45.0; MS (EI) m/z 346 [M+]; Anal. (C18H22N2O3S) C, H, N. Calcd for: 62.40, 6.40, 8.09;

Thiophen-2-yl(4-(2-(4-(trifluoromethoxy)phenoxy)ethyl)piperazin-1-yl)methanone

AC C

9.

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Found: 62.29, 6.59, 7.90; HPLC purity: 98.10%, retention time: 4.87 min (methanol/water 80:20, v/v).

hydrochloride (5)

Compound 5 was synthesized from 4e and 1a with the procedure described for compound 1 (white

solid, 73.39%). m.p. = 208-210 °C. IR(KBr): 3050, 2932, 2500, 2386, 1608, 1521, 1508, 1463, 1427, 1260, 1200, 1160, 987, 846, 728 cm-1; 1H-NMR (300 MHz, D2O) δ 7.45 (d, 1H, J = 5.25 Hz, Ar-H), 7.25-7.21 (m, 1H, Ar-H), 7.10-7.06 (m, 2H, Ar-H), 6.92-6.89 (m, 1H, Ar-H), 6.82-6.78 (m, 2H, Ar-H), 4.17 (t, 2H, J = 4.83 Hz,

Supporting Information

ACCEPTED MANUSCRIPT

-CH2CH2O-), 3.43 (t, 2H, J = 4.83 Hz, -CH2CH2O-), 3.31-3.29 (m, 8H, -N(CH2CH2)2N-); MS (EI) m/z 400 ([M+]); Anal. (C18H20ClF3N2O3S) C, H, N. Calcd for: 49.49, 4.61, 6.41; Found: 49.29, 4.75, 6.40; HPLC purity: 98.21%, retention time: 7.09 min (methanol/water 80:20, v/v). 4-(2-(4-(thiophene-2-carbonyl)piperazin-1-yl)ethoxy)benzonitrile (6)

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10.

Compound 6 was synthesized from 4f and 1a with the procedure described for compound 2 (white solid, 27.14%). m.p. = 131-134 °C.

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IR(KBr): 3452, 3096, 2826, 2221, 1596, 1509, 1442, 1453, 1257, 1175, 1001, 735 cm-1; 1H-NMR

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(300 MHz, CDCl3) δ 7.62-7.57 (m, 2H, Ar-H), 7.47-7.44 (m, 1H, Ar-H), 7.30-7.28 (m, 1H, Ar-H), 7.06-7.03 (m, 1H, Ar-H), 6.98-6.93 (m, 2H, Ar-H), 4.17 (t, 2H, J = 4.77 Hz, -CH2CH2O-), 3.78 (t, 4H, J = 4.83 Hz, -N(CH2CH2)2N-), 2.88 (t, 2H, J = 4.83 Hz, -CH2CH2O-), 2.63 (t, 4H, J = 4.62 Hz, -N(CH2CH2)2N-); 13C-NMR (75MHz, CDCl3) δ 162.9, 161.4, 136.4, 133.4, 128.3, 128.1, 126.2, 118.6,

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114.8, 103.6, 65.8, 56.2, 53.0, 44.9; MS (EI) m/z 341 ([M+]); Anal. (C18H19N3O2S) C, H, N. Calcd for: 63.32, 5.61, 12.31; Found: 62.78, 5.72, 11.80; HPLC purity: 98.30%, retention time: 4.02 min (methanol/water 80:20, v/v).

Thiophen-2-yl(4-(2-(4-fluorophenoxy)ethyl)piperazin-1-yl)methanone hydrochloride (7)

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11.

AC C

Compound 7 was synthesized from 4g and 1a with the procedure described for compound 1 (white solid, 42.35%). m.p. = 228-230 °C. IR(KBr): 3464, 3417, 2411, 1608, 1507, 1214, 838, 735 cm-1; 1H-NMR (300 MHz, CDCl3, base) δ

7.38-7.36 (m, 1H, Ar-H), 7.22-7.19 (m, 1H, Ar-H), 6.98-6.85 (m, 3H, Ar-H), 6.80-6.74 (m, 2H, Ar-H), 4.03 (t, 2H, J = 4.83 Hz, -CH2CH2O-), 3.71 (t, 4H, J = 4.50 Hz, -N(CH2CH2)2N-), 2.77 (t, 2H, J = 4.77 Hz, -CH2CH2O-), 2.56 (t, 4H, J = 4.86 Hz, -N(CH2CH2)2N-); MS (EI) m/z 334 [M+]; Anal. (C17H20ClFN2O2S) C, H, N. Calcd for: 55.05, 5.44, 7.55; Found: 55.05, 5.45, 7.55; HPLC purity:

Supporting Information

ACCEPTED MANUSCRIPT

99.55%, retention time: 4.63 min (methanol/water 80:20, v/v). 12.

Thiophen-2-yl(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)methanone (8) Compound 8 was synthesized from 4h and 1a with the procedure described for compound 2 (gray

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solid, 75.60%). m.p. = 75-77 °C. IR(KBr): 3431, 3096, 2923, 2784, 1599, 1523, 1493, 1429, 1264, 1248, 997, 831, 818, 736 cm-1; 1

H-NMR (300 MHz, CDCl3) δ 7.52 (d, 1H, J = 4.26 Hz, Ar-H), 7.30-7.22 (m, 3H, Ar-H), 7.06-7.03 (dd,

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1H, J1 = 3.66 Hz, J2 = 1.29 Hz, Ar-H), 6.86-6.81 (m, 2H, Ar-H), 4.11 (t, 2H, J = 4.83 Hz, -CH2CH2O-),

Hz, -N(CH2CH2)2N-);

13

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3.81 (t, 4H, J = 4.32 Hz, -N(CH2CH2)2N-), 2.87 (t, 2H, J = 4.74 Hz, -CH2CH2O-), 2.65 (t, 4H, J = 4.56 C-NMR (75MHz, CDCl3) δ 162.5, 156.3, 136.1, 130.3, 129.7, 129.3, 127.2,

125.0, 116.6, 62.6, 54.5, 51.2, 41.8; MS (EI) m/z 350 [M+]; Anal. (C17H19ClN2O2S) C, H, N. Calcd for: 58.19, 5.46, 7.98; Found: 58.27, 5.42, 8.32; HPLC purity: 99.81%, retention time: 9.15 min

13.

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(methanol/20mM monopotassium phosphate 70:30, v/v, containing 0.2% triethylamine). Thiophen-2-yl(4-(2-(4-bromophenoxy)ethyl)piperazin-1-yl)methanone (9) Compound 9 was synthesized from 4i and 1a with the procedure described for compound 2 (yellow

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solid, 37.84%). m.p. = 82-83 °C.

1

AC C

IR(KBr): 3412, 3093, 2944, 2784, 1610, 1600, 1500, 1456, 1428, 1248, 997, 817, 736 cm-1; H-NMR (300 MHz, CDCl3) δ 7.46-7.44 (m, 1H, Ar-H), 7.36-7.32 (m, 2H, Ar-H), 7.29-7.28 (m, 1H,

Ar-H), 7.06-7.01 (m, 1H, Ar-H), 6.80-6.77 (m, 2H, Ar-H), 4.08 (t, 2H, J = 5.31 Hz, -CH2CH2O-), 3.78 (t, 4H, J = 4.83 Hz, -N(CH2CH2)2N-), 2.84 (t, 2H, J = 5.46 Hz, -CH2CH2O-), 2.62 (t, 4H, J = 4.83 Hz, -N(CH2CH2)2N-); MS (EI) m/z 395 [M+]; Anal. (C17H19BrN2O2S) C, H, N. Calcd for: 51.65, 4.84, 7.09; Found: 51.40, 4.82, 6.86; HPLC purity: 99.46%, retention time: 7.00 min (methanol/water 80:20, v/v). 14.

Thiophen-2-yl(4-(2-(2-chlorophenoxy)ethyl)piperazin-1-yl)methanone hydrochloride (10)

Supporting Information

ACCEPTED MANUSCRIPT

Compound 10 was synthesized from 4j and 1a with the procedure described for compound 1 (white solid, 78.05%). m.p. = 204-206 °C. IR(KBr): 3440, 2925, 2374, 2353, 1612, 1465, 1428, 1284, 1244, 989, 759, 723 cm-1; 1H-NMR

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(300 MHz, D2O) δ 7.68 (d, 1H, J = 5.13 Hz, Ar-H), 7.46-7.43 (m, 2H, Ar-H), 7.34-7.28 (m, 1H, Ar-H), 7.15-7.09 (m, 2H, Ar-H), 7.06-7.00 (m, 1H, Ar-H), 4.47 (t, 2H, J = 4.56 Hz, -CH2CH2O-), 4.06 (s, 4H, -N(CH2CH2)2N-), 3.71 (t, 2H, J = 4.51 Hz, -CH2CH2O-), 3.58 (brs, 4H, -N(CH2CH2)2N-); MS (EI) m/z

Thiophen-2-yl(4-(2-(2,4-dichlorophenoxy)ethyl)piperazin-1-yl)methanone (11)

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15.

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350 [M+]; Anal. (C17H20Cl2N2O2S) C, H, N. Calcd for: 52.72, 5.20, 7.23; Found: 52.66, 5.20, 7.52.

Compound 11 was synthesized from 4k and 1a with the procedure described for compound 2 (yellow solid, 53.52%). m.p. = 67-68 °C.

IR(KBr): 3440, 3096, 2940, 2826, 1615, 1484, 1455, 1433, 1292, 1059, 1004, 803, 714 cm-1; H-NMR (300 MHz, CDCl3) δ 7.46-7.44 (m, 1H, Ar-H), 7.36 (d, 1H, J = 2.73 Hz, Ar-H), 7.30-7.28 (m,

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1

1H, Ar-H), 7.20-7.16 (m, 1H, Ar-H), 7.06-7.03 (m, 1H, Ar-H), 6.52 (d, 1H, J = 8.73 Hz, Ar-H), 4.16 (t, 2H, J = 5.07 Hz, -CH2CH2O-), 3.78 (t, 4H, J = 4.50 Hz, -N(CH2CH2)2N-), 2.91 (t, 2H, J = 5.16 Hz,

EP

-CH2CH2O-), 2.68 (t, 4H, J = 4.47 Hz, -N(CH2CH2)2N-); MS (EI) m/z 386 [M+]; Anal.

AC C

(C17H18Cl2N2O2S) C, H, N. Calcd for: 52.79, 4.71, 7.27; Found: 53.13, 4.76, 7.31; HPLC purity: 98.69%, retention time: 9.42 min (methanol/water 80:20, v/v). 16.

Phenyl(4-(2-phenoxyethyl)piperazin-1-yl)methanone (12)

Compound 12 was synthesized from 4a and 1b with the procedure described for compound 2

(white solid, 93.97%). m.p. = 93-94 °C. IR(KBr): 3051, 2944, 2926, 2795, 1628, 1278, 1177, 1156, 1041, 1018, 1003, 782, 761, 695, 555 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.41-7.28 (m, 7H, Ar-H), 6.98-6.89 (m, 3H, Ar-H), 4.12 (t, 2H, J =

Supporting Information

ACCEPTED MANUSCRIPT

5.43 Hz, -CH2CH2O-), 3.82-3.40 (m, 4H, -N(CH2CH2)2N-), 2.85 (t, 2H, J = 5.43 Hz, -CH2CH2O-), 2.60 (m, 4H, -N(CH2CH2)2N-); 13C-NMR (75MHz, CDCl3) δ 169.8, 158.1, 135.3, 129.1, 129.0, 128.0, 126.6, 120.4, 114.1, 65.4, 56.6, 53.0, 47.3, 41.6; MS (EI) m/z 310 [M+]; Anal. (C19H22N2O2) C, H, N. Calcd

RI PT

for: 73.52, 7.14, 9.03; Found: 73.06, 6.84, 8.58; HPLC purity: 99.05%, retention time: 5.23 min (methanol/water 80:20, v/v). 17.

Phenyl(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)methanone (13)

SC

Compound 13 was synthesized from 4h and 1b with the procedure described for compound 2

M AN U

(white solid, 65.75%). m.p. = 81-82 °C.

IR(KBr): 3030, 3004, 2927, 2787, 1627, 1596, 1492, 1451, 1246, 1135, 1058, 1016, 972, 728, 697, 508 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.43-7.38 (m, 4H, Ar-H), 7.25-6.81 (m, 5H, Ar-H), 4.08 (t, 2H, J = 4.80 Hz, -CH2CH2O-), 3.46-3.82 (m, 4H, -N(CH2CH2)2N-), 2.84 (t, 2H, J = 4.83 Hz, -CH2CH2O-),

TE D

2.65-2.52 (m, 4H, -N(CH2CH2)2N-); MS (EI) m/z 344 [M+]; Anal. (C19H21ClN2O2) C, H, N. Calcd for: 66.81, 6.14, 8.12; Found: 66.41, 6.12, 7.99; HPLC purity: 98.11%, retention time: 6.88 min (methanol/water 80:20, v/v).

4-(2-(4-benzoylpiperazin-1-yl)ethoxy)benzonitrile (14)

EP

18.

AC C

Compound 14 was synthesized from 4f and 1b with the procedure described for compound 2 (white solid, 58.21%). m.p. = 83-84 °C. IR(KBr): 2922, 2796, 2221, 1628, 1590, 1451, 1253, 1170, 1044, 1016, 840, 792, 548 cm-1;

1

H-NMR (300 MHz, CDCl3) δ 7.61-7.41 (m, 7H, Ar-H), 6.96-6.93 (m, 2H, Ar-H), 4.15 (t, 2H, J = 5.13

Hz, -CH2CH2O-), 3.61-3.46 (m, 4H, -N(CH2CH2)2N-), 2.87 (t, 2H, J = 4.86 Hz, -CH2CH2O-), 2.65-2.54 (m, 4H, -N(CH2CH2)2N-); MS (EI) m/z 335[M+]; Anal. (C20H21N3O2) C, H, N. Calcd for: 71.62, 6.31, 12.53; Found: 71.48, 6.23, 12.44; HPLC purity: 98.98%, retention time: 4.15 min

Supporting Information

ACCEPTED MANUSCRIPT

(methanol/water 80:20, v/v). 19.

Phenyl(4-(2-(4-methoxyphenoxy)ethyl)piperazin-1-yl)methanone hydrochloride (15) Compound 15 was synthesized from 4d and 1b with the procedure described for compound 1

RI PT

(white solid, 35.51%). m.p. = 190-191 °C. IR(KBr): 3445, 3129, 2708, 2437, 1630, 1512, 1461, 1443, 1430, 1400, 1287, 1231, 1183, 1035, 996, 828, 729, 714, 574, 523 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.37-7.31 (m, 5H, Ar-H), 6.75 (s, 4H,

SC

Ar-H), 3.98 (t, 2H, J = 5.52 Hz, -CH2CH2O-), 3.74 (brs, 2H, -N(CH2CH2)2N-), 3.68 (s, 3H, -OCH3),

M AN U

3.37 (brs, 2H, -N(CH2CH2)2N-), 2.74 (t, 2H, J = 5.49 Hz, -CH2CH2O-), 2.50 (brs, 4H, -N(CH2CH2)2N-); MS (EI) m/z 340[M+]; HPLC purity: 96.80%, retention time: 4.77 min (methanol/water 80:20, v/v). 20.

(4-methoxyphenyl)(4-(2-phenoxyethyl)piperazin-1-yl)methanone (16) Compound 16 was synthesized from 4a and 1c with the procedure described for compound 2 (white

TE D

solid, 76.90%). m.p. = 82-83 °C.

IR(KBr): 3000, 2935, 2838, 1627, 1513, 1498, 1418, 1371, 1251, 1175, 1041, 1006, 976, 842, 758, 694, 511 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.40-7.29 (m, 4H, Ar-H), 6.98-6.90 (m, 5H, Ar-H), 4.12

EP

(t, 2H, J = 5.31 Hz, -CH2CH2O-), 3.83 (s, 3H, -OCH3), 3.80-3.30 (brs, 4H, -N(CH2CH2)2N-), 2.86 (t, 13

C-NMR (75MHz, CDCl3) δ

AC C

2H, J = 5.31 Hz, -CH2CH2O-), 2.70-2.40 (brs, 4H, -N(CH2CH2)2N-);

169.7, 160.3, 158.1, 129.0, 128.6, 127.4, 120.4, 114.1, 113.2, 65.3, 56.6, 54.8, 53.1; MS (EI) m/z 340 [M+]; Anal. (C20H24N2O3) C, H, N. Calcd for: 70.56, 7.11, 8.23; Found: 70.56, 6.82, 8.03; HPLC purity: 97.62%, retention time: 5.67 min (methanol/water 80:20, v/v). 21.

(4-methoxyphenyl)(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)methanone (17) Compound 17 was synthesized from 4h and 1c with the procedure described for compound 2

(white solid, 52.08%). m.p. = 82-83 °C.

Supporting Information

ACCEPTED MANUSCRIPT

IR(KBr): 2944, 2835, 1627, 1489, 1429, 1302, 1279, 1243, 1173, 1112, 1003, 840, 762, 635, 587, 512 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.40-7.37 (m, 2H, Ar-H), 7.24-7.21 (m, 2H, Ar-H), 6.92-6.81 (m, 4H, Ar-H), 4.08 (t, 2H, J = 4.31 Hz, -CH2CH2O-), 3.83 (s, 3H, -OCH3), 3.66 (brs, 4H,

RI PT

-N(CH2CH2)2N-), 2.59 (brs, 4H, -N(CH2CH2)2N-), 2.84 (t, 2H, J = 4.53 Hz, -CH2CH2O-); MS (EI) m/z 374 [M+]; Anal. (C20H23N2O3) C, H, N. Calcd for: 64.08, 6.18, 7.47; Found: 64.24, 6.16, 7.19; HPLC purity: 96.90%, retention time: 7.10 min (methanol/water 80:20, v/v). (p-Tolyl) (4-(2-phenoxyethyl)piperazin-1-yl)methanone (18)

SC

22.

(white solid, 49.31%). m.p. = 69-70 °C.

M AN U

Compound 18 was synthesized from 4a and 1d with the procedure described for compound 2

IR(KBr): 2933, 2788, 1618, 1500, 1454, 1295, 1242, 1136, 1080, 1047, 1012, 948, 827, 753, 690, 607 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.32-7.28 (m, 6H, Ar-H), 7.22-6.88 (m, 3H, Ar-H), 4.13 (t, 2H,

TE D

J = 5.13 Hz, -CH2CH2O-), 3.90-3.45 (m, 4H, -N(CH2CH2)2N-), 2.86 (t, 2H, J = 5.22 Hz, -CH2CH2O-), 2.63-2.45 (brs, 4H, -N(CH2CH2)2N-), 2.38 (s, 3H, -CH3); MS (EI) m/z 324 [M+]; Anal. (C20H24N2O2) C, H, N. Calcd for: 74.04, 7.46, 8.64; Found: 73.82, 7.27, 8.50; HPLC purity: 99.13%, retention time: 6.66

(p-Tolyl)(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)methanone (19)

AC C

23.

EP

min (methanol/water 80:20, v/v).

Compound 19 was synthesized from 4h and 1d with the procedure described for compound 2

(white solid, 50.85%). m.p. = 84-85 °C. IR(KBr): 2943, 2803, 1623, 1495, 1466, 1435, 1306, 1250, 1233, 1212, 1002, 826, 794, 670, 506

cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.32-7.29 (m, 3H, Ar-H), 7.24-7.21 (m, 3H, Ar-H), 6.82 (d, 2H, J = 8.79 Hz, Ar-H), 4.08 (t, 2H, J = 5.16 Hz, -CH2CH2O-), 3.78 (brs, 2H, -N(CH2CH2)2N-), 3.48 (brs, 2H, -N(CH2CH2)2N-), 2.83 (t, 2H, J = 5.34 Hz, -CH2CH2O-), 2.58 (brs, 4H, -N(CH2CH2)2N-), 2.38 (s, 3H,

Supporting Information

ACCEPTED MANUSCRIPT

-CH3); 13C-NMR (75MHz, CDCl3) δ 169.9, 156.8, 139.3, 132.2, 128.8, 128.5, 126.7, 125.3, 115.4, 65.8, 56.5, 53.1, 47.3, 41.8, 20.9; HR-MS (ESI) m/z 359 ([M + H]+); Anal. (C20H23ClN2O2) C, H, N. Calcd for: 66.94, 6.46, 7.81; Found: 67.18, 6.42, 7.63; HPLC purity: 98.85%, retention time: 8.55 min

24.

RI PT

(methanol/water 80:20, v/v). (2-Chlorophenyl)(4-(2-phenoxyethyl)piperazin-1-yl)methanone (20)

Compound 20 was synthesized from 4h and 1e with the procedure described for compound 2

SC

(yellow solid, 35.3%). m.p. = 54-55 °C.

M AN U

IR(KBr): 3051, 2936, 2829, 1628, 1595, 1478, 1459, 1295, 1233, 1169, 1039, 1005, 769, 743, 694 cm-1; 1H-NMR (300 MHz, CDCl3) δ 7.41-7.28 (m, 5H, Ar-H), 6.98-6.88 (m, 3H, Ar-H), 4.12 (t, 3H, J = 5.46 Hz, -CH2CH2O-), 3.92-3.22 (m, 4H, -N(CH2CH2)2N-), 2.86 (t, 2H, J = 5.61 Hz, -CH2CH2O-), 2.70-2.51 (m, 4H, -N(CH2CH2)2N-); MS (EI) m/z 344 [M+]; HPLC purity: 96.28%, retention time: 5.99

25.

TE D

min (methanol/water 80:20, v/v).

N-phenyl-2-(4-(2-phenoxyethyl)piperazin-1-yl)acetamide (21) Compound 21 was synthesized from 4a and 2b with the procedure described for compound 2

EP

(white solid, 55.31%). m.p. = 111-115 °C.

AC C

IR(KBr): 3300, 2828, 2804, 1689, 1598, 1521, 1244, 1161, 950, 756, 692, 518, 508 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.11 (s, 1H, -NHCO-), 7.58-7.56 (m, 2H, Ar-H), 7.36-7.26 (m, 4H, Ar-H), 7.11 (t, 1H, J = 7.35 Hz, Ar-H), 7.01-6.84 (m, 3H, Ar-H), 4.13 (t, 2H, J = 5.64 Hz,-CH2CH2-O-), 3.03(s, 2H, -CH2CO-), 2.87 (t, 2H, J = 5.67 Hz, -CH2CH2O-), 2.69 (s, 8H, -N(CH2CH2)2N-); 13C-NMR (75MHz, CDCl3) δ 167.8, 158.2, 137.2, 129.0, 128.5, 124.3, 120.4, 118.9, 114.1, 65.5, 61.5, 56.6, 53.3, 53.0; MS (EI) m/z 339

[M+]; Anal. (C20H25N3O2) C, H, N. Calcd for: 70.77, 7.42, 12.38; Found: 70.69, 7.27,

12.37; HPLC purity: 98.40%, retention time: 6.72 min (methanol/water 80:20, v/v).

Supporting Information

26.

ACCEPTED MANUSCRIPT

N-phenyl-2-(4-(2-(o-tolyloxy)ethyl)piperazin-1-yl)acetamide (22) Compound 22 was synthesized from 4c and 2b with the procedure described for compound 2

(white solid, 86.40%). m.p. = 128-130 °C.

1

RI PT

IR(KBr): 3439, 3180, 2926, 2818, 1664, 1596, 1538, 1311, 1243, 1170, 765, 749, 692 cm-1; H-NMR (300 MHz, CDCl3) δ 9.11 (s, 1H, -NH-CO-), 7.57 (d, 2H, J = 7.80 Hz, Ar-H), 7.34 (t, 2H, J =

7.68 Hz, Ar-H), 7.18-7.09 (m, 3H, Ar-H), 6.96-6.80 (m, 2H, Ar-H), 4.14 (t, 2H, J = 5.46 Hz,

SC

-CH2CH2O-), 3.00 (s, 2H, -CH2CO-), 2.90 (t, 2H, J = 5.46 Hz, -CH2CH2O-), 2.69 (s, 8H,

M AN U

-N(CH2CH2)2N-), 2.23 (s, 3H, -CH3); 13C-NMR (75MHz, CDCl3) δ 167.8, 156.4, 137.2, 130.2, 128.5, 126.3, 123.7, 120.1, 118.9, 110.6, 65.9, 61.5, 56.7, 53.3, 53.1, 15.9; MS (EI) m/z 353 [M+]; Anal. (C21H27N3O2) C, H, N. Calcd for: 71.36, 7.70, 11.87; Found: 70.82, 7.78, 12.31; HPLC purity: 96.78%, retention time: 9.03 min (methanol/water 80:20, v/v).

N-phenyl-2-(4-(2-(4-(trifluoromethoxy)phenoxy)ethyl)piperazin-1-yl)acetamide (23)

TE D

27.

Compound 23 was synthesized from 4e and 2b with the procedure described for compound 2 (yellow solid, 56.67%). m.p. = 85-87 °C.

EP

IR(KBr): 3180, 3038, 2940, 2815, 1659, 1598, 1547, 1511, 1270, 1162, 1105, 1022, 819, 800, 767,

AC C

694 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.11 (s, 1H, -NHCO-), 7.57 (d, 2H, J = 7.92 Hz, Ar-H), 7.37-7.31 (m, 2H, Ar-H), 7.20-7.09 (m, 3H, Ar-H), 6.90 (d, 2H, J = 8.90 Hz, Ar-H), 4.14 (brs, 2H, -CH2CH2O-), 3.20 (s, 2H,-COCH2-), 2.91 (brs, 2H, -CH2CH2O-), 2.73 (s, 8H, -N(CH2CH2)2N-); MS (EI) m/z 423 [M+]; Anal. (C21H24F3N3O3.H2O) C, H, N. Calcd for: 59.57, 5.71, 9.92; Found: 59.62, 5.74, 9.65. HPLC purity: 96.52%, retention time: 9.39 min (methanol/water 80:20, v/v). 28.

N-phenyl-2-(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)acetamide (24) Compound 24 was synthesized from 4h and 2b with the procedure described for compound 2

Supporting Information

ACCEPTED MANUSCRIPT

(white solid, 90.37 %). m.p. = 134-136 °C. IR(KBr): 3330, 2810, 1674, 1599, 1529, 1488, 1465, 1455, 1271, 1165, 959, 821, 762, 694, 669, 511 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.11(s, 1H, -NHCO-), 7.57 (d, 2H, J = 7.98 Hz, Ar-H),

RI PT

7.37-7.31 (m, 2H, Ar-H), 7.14-7.09 (m, 2H, Ar-H), 6.91 (s, 1H, Ar-H), 6.84 (d, 2H, J = 8.85 Hz, Ar-H), 4.09 (t, 2H, J = 5.70 Hz, -CH2CH2O-), 3.14 (s, 2H, -CH2CO-), 2.84 (t, 2H, J = 5.55 Hz, -CH2CH2O-), 2.67 (s, 8H, -N(CH2CH2)2N-); MS (EI) m/z 373 [M+]; Anal. (C20H24ClN3O2) C, H, N. Calcd for: 64.25,

SC

6.47, 11.24; Found: 64.49, 6.56, 11.01; HPLC purity: 97.83%, retention time: 8.41 min

29.

M AN U

(methanol/water 80:20, v/v).

N-phenyl-2-(4-(2-(4-cyanophenoxy)ethyl)piperazin-1-yl)acetamide (25) Compound 25 was synthesized from 4f and 2b with the procedure described for compound 2

(yellow solid, 91.85%). m.p. = 109-110 °C.

TE D

IR(KBr): 3323, 2817, 2221, 1671, 1604, 1528, 1506, 1441, 1302, 1255, 1172, 1164, 1131, 1017, 955, 831, 766, 699, 549 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.11 (s, 1H, -NHCO-), 7.60-7.55 (m, 3H, Ar-H), 7.37-7.32 (m, 2H, Ar-H), 7.14-7.09 (m, 2H, Ar-H), 6.96 (d, 2H, J = 8.82 Hz, Ar-H), 4.16 (t, 2H,

EP

J = 5.43 Hz, -CH2CH2O-), 3.15 (s, 2H, -COCH2-), 2.87 (t, 2H, J = 5.31 Hz, -CH2CH2O-), 2.68 (s, 8H,

AC C

-N(CH2CH2)2N-); MS (EI) m/z 364 [M+]; Anal. (C21H24N4O2) C, H, N. Calcd for: 69.21, 6.64, 15.37; Found: 68.90, 6.80, 15.12. 30.

N-phenyl-2-(4-(2-(4-methoxyphenoxy)ethyl)piperazin-1-yl)acetamide (26)

Compound 26 was synthesized from 4d and 2b with the procedure described for compound 2

(white solid, 79.54%). m.p. = 83-85 °C. IR(KBr): 3180, 3046, 2943, 2815, 1655, 1542, 1512, 1465, 1418, 1266, 1244, 1169, 1046, 1029, 1017, 831, 754, 691, 519 cm-1; 1H-NMR (300 MHz, CDCl3) δ 8.99 (s, 1H, -NHCO-), 7.48 (d, 2H, J =

Supporting Information

ACCEPTED MANUSCRIPT

8.73 Hz, Ar-H), 7.30 (d, 2H, J = 7.80 Hz, Ar-H), 6.98-6.86 (m, 5H, Ar-H), 4.14 (brs, 2H, -CH2CH2O-), 3.80 (s, 3H, -OCH3), 3.14 (s, 2H, -CH2CO-), 2.88 (brs, 2H, -CH2CH2O-), 2.70 (s, 8H, -N(CH2CH2)2N-); MS (EI) m/z 369 [M+]; Anal. (C21H27N3O3) C, H, N. Calcd for: 68.27, 7.37, 11.37; Found: 68.08, 7.37,

31.

RI PT

11.26; HPLC purity: 98.94%, retention time: 6.21 min (methanol/water 80:20, v/v). N-(4-methoxyphenyl)-2-(4-(2-(4-cyanophenoxy)ethyl)piperazin-1-yl)acetamide (27)

Compound 27 was synthesized from 4f and 2c with the procedure described for compound 2 (white

SC

solid, 60.41%). m.p. = 150-151 °C.

M AN U

IR(KBr): 3322, 2957, 2817, 2223, 1690, 1603, 1529, 1509, 1304, 1258, 1245, 1171, 1028, 1012, 839, 548 cm-1; 1H-NMR (300 MHz, CDCl3) δ 8.95(s, 1H, -NHCO-), 7.59 (d, 2H, J = 8.73 Hz, Ar-H), 7.47 (d, 2H, J = 8.80 Hz, Ar-H), 6.96 (d, 2H, J = 8.73 Hz, Ar-H), 6.87 (d, 2H, J = 8.80 Hz, Ar-H), 4.11 (brs, 2H, -CH2CH2O-), 3.80 (s, 3H, -OCH3), 3.14 (s, 2H, -CH2CO-), 2.87 (brs, 2H, -CH2CH2O-), 2.68

TE D

(s, 8H, -N(CH2CH2)2N-); 13C-NMR (75MHz, CDCl3) δ 167.4, 161.5, 155.8, 133.5, 130.3, 120.6, 118.6, 114.8, 113.7, 103.6, 66.0, 61.4, 56.2, 55.0, 53.2, 52.9; MS (EI) m/z 394 [M+]; Anal. (C22H26N4O3) C, H, N. Calcd for: 66.99, 6.64, 14.20; Found: 66.27, 6.55, 14.19; HPLC purity: 99.25%, retention time: 4.44

N-(4-methoxyphenyl)-2-(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)acetamide (28)

AC C

32.

EP

min (methanol/water 80:20, v/v).

Compound 28 was synthesized from 4h and 2c with the procedure described for compound 2

(white solid, 86.78%). m.p. = 104-106 °C. IR(KBr): 3301, 2953, 2821, 1681, 1595, 1524, 1491, 1458, 1427, 1298, 1241, 1164, 1132, 1015

950, 827, 669, 511 cm-1; 1H-NMR (300 MHz, CDCl3) δ 8.97 (s, 1H, -NHCO-), 7.47 (d, 2H, J = 8.94 Hz, Ar-H), 7.23 (d, 2H, J = 8.97 Hz, Ar-H), 6.89-6.82 (m, 4H, Ar-H), 4.09 (t, 2H, J = 5.37Hz, -CH2CH2O-), 3.80 (s, 3H, -OCH3), 3.14 (s, 2H, -CH2CO-), 2.86 (t, 2H, J = 5.01 Hz, -CH2CH2O-), 2.68 (s, 8H,

Supporting Information

ACCEPTED MANUSCRIPT

-N(CH2CH2)2N-); MS (EI) m/z 403 [M+]; Anal. (C21H26ClN3O3) C, H, N. Calcd for: 62.45, 6.49, 10.40; Found: 62.30, 6.46, 10.22; HPLC purity: 99.35%, retention time: 7.77 min (methanol/water 80:20, v/v). 33.

N-(p-tolyl)-2-(4-(2-phenoxyethyl)piperazin-1-yl)acetamide (29)

RI PT

Compound 29 was synthesized from 4a and 2d with the procedure described for compound 2 (white solid, 98.53%). m.p. = 58-60 °C.

IR(KBr): 3295, 2952, 2827, 1688, 1597, 1523, 1455, 1404, 1339, 1298, 1246, 1132, 1047, 952, 819,

SC

754, 692, 511 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.03 (s, 1H, -NHCO-), 7.45 (d, 2H, J = 7.32 Hz,

M AN U

Ar-H), 7.30 (d, 2H, J = 8.94 Hz, Ar-H), 7.13 (d, 2H, J = 7.50 Hz, Ar-H), 6.98-6.91 (m, 3H, Ar-H), 4.14 (brs, 2H, -CH2CH2O-), 3.14 (s, 2H, -COCH2-), 2.87 (brs, 2H, -CH2CH2O-), 2.69 (s, 8H, -N(CH2CH2)2N-), 2.32 (s, 3H, -CH3); MS (EI) m/z 353 [M+]; Anal. (C21H27N3O2) C, H, N. Calcd for: 71.36, 7.70, 11.89; Found: 71.21, 7.60, 11.65; HPLC purity: 98.28%, retention time: 8.14 min

34.

TE D

(methanol/water 80:20, v/v).

N-(p-tolyl)-2-(4-(2-(4-cyanophenoxy)ethyl)piperazin-1-yl)acetamide (30) Compound 30 was synthesized from 4f and 2d with the procedure described for compound 2 (white

EP

solid, 94.36%). m.p. = 120-122 °C.

AC C

IR(KBr): 3323, 2944, 2826, 2218, 1669, 1603, 1525, 1477, 1404, 1303, 1257, 1174, 1161, 1023, 1013, 837, 820, 549, 510 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.00 (s, 1H,-NHCO-), 7.58 (d, 2H, J = 8.73 Hz, Ar-H), 7.44 (d, 2H, J = 8.22 Hz, Ar-H), 7.14 (d, 2H, J = 8.10 Hz, Ar-H), 6.76 (d, 2H, J = 8.76 Hz, Ar-H), 4.17 (brs, 2H, -CH2CH2O-), 3.14 (s, 2H, -CH2CO-), 2.88 (brs, 2H, -CH2CH2O-), 2.53(s, 8H, -N(CH2CH2)2N-), 2.32 (s, 3H, -CH3); 13C-NMR (75MHz, CDCl3) δ 167.5, 161.5, 134.6, 133.5, 133.3, 129.0, 119.0, 118.6, 114.8, 103.6, 66.0, 61.4, 56.2, 53.3, 52.9, 20.4; MS (EI) m/z 378 [M+]; Anal. (C22H26N4O2) C, H, N. Calcd for: 69.82, 6.92, 14.80; Found: 69.81, 7.01, 14.68; HPLC purity: 99.08%,

Supporting Information

ACCEPTED MANUSCRIPT

retention time: 5.48 min (methanol/water 80:20, v/v). 35.

N-(p-tolyl)-2-(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)acetamide (31) Compound 31 was synthesized from 4h and 2d with the procedure described for compound 2

RI PT

(white solid, 87.74%). m.p. = 135-137 °C. IR(KBr): 3286, 2949, 2765, 1684, 1597, 1492, 1404, 1369, 1245, 1166, 1095, 1045, 906, 824, 726, 669 , 509, 472 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.02 (s, 1H, -NHCO-), 7.45 (d, 2H, J = 8.31 Hz,

SC

Ar-H), 7.24 (d, 2H, J = 8.97 Hz, Ar-H), 7.14 (d, 2H, J = 8.19 Hz, Ar-H), 6.83 (d, 2H, J = 8.91 Hz,

M AN U

Ar-H), 4.09 (t, 2H, J = 5.55 Hz, -CH2CH2O-), 3.14 (s, 2H, -CH2CO-), 2.85 (t, 2H, J = 5.37 Hz, -CH2CH2O-), 2.67 (s, 8H, -N(CH2CH2)2N-), 2.32 (s, 3H, -CH3); MS (EI) m/z 387 [M+]; Anal. (C21H26ClN3O2) C, H, N. Calcd for: 65.02, 6.76, 10.83; Found: 64.86, 6.74, 10.76; HPLC purity: 99.72%, retention time: 10.65 min (methanol/water 80:20, v/v).

N-(2-chlorophenyl)-2-(4-(2-(4-cyanophenoxy)ethyl)piperazin-1-yl)acetamide (32)

TE D

36.

Compound 32 was synthesized from 4f and 2e with the procedure described for compound 2 (white solid, 86.16%). m.p. = 155-156 °C.

EP

IR(KBr): 3201, 2835, 2221, 1605, 1594, 1519, 1441, 1331, 1263, 1174, 1027, 807, 766, 714, 548,

AC C

514 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.93 (s, 1H, -NHCO-), 8.48-8.45 (m, 1H, Ar-H), 7.58 (d, 2H, J = 8.76 Hz, Ar-H), 7.39-7.37 (m, 1H, Ar-H), 7.29 (d, 1H, J = 7.38 Hz, Ar-H), 7.07-7.02 (m, 1H, Ar-H), 6.96 (d, 2H, J = 8.79 Hz, Ar-H), 4.17 (s, 2H, -CH2CH2O-), 3.20 (s, 2H, -CH2CO-), 2.88 (brs, 2H, -CH2CH2O-), 2.72 (s, 8H, -N(CH2CH2)2N-); MS (EI) m/z 398 [M+]; Anal. (C21H23ClN4O2) C, H, N. Calcd for: 63.23, 5.81, 14.05; Found: 63.20, 5.80, 13.76; HPLC purity: 99.33%, retention time: 7.33 min (methanol/water 80:20, v/v). 37.

N-(2-chlorophenyl)-2-(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)acetamide (33)

Supporting Information

ACCEPTED MANUSCRIPT

Compound 33 was synthesized from 4h and 2e with the procedure described for compound 2 (white solid, 84.15%). m.p. = 91-93 °C. IR(KBr): 3266, 2932, 2877, 1690, 1594, 1515, 1470, 1441, 1380, 1247, 1164, 1130, 1035, 987, 835,

RI PT

765, 675, 641 cm-1; 1H-NMR (300 MHz, CDCl3) δ 9.95 (s, 1H, -NHCO-), 8.48-8.45 (m, 1H, Ar-H), 7.39-7.37 (m, 1H, Ar-H), 7.30-7.28 (m, 1H, Ar-H), 7.26-7.22 (m, 2H, Ar-H), 7.06-7.02 (m, 1H, Ar-H), 6.83 (d, 2H, J = 8.79 Hz, Ar-H), 4.09 (t, 2H, J = 5.49 Hz, -CH2CH2O-), 3.19 (s, 2H, -CH2CO-), 2.85 (t,

SC

2H, J = 5.37 Hz,-CH2CH2O-), 2.71 (s, 8H, -N(CH2CH2)2N-); MS (EI) m/z 407 [M+]; Anal.

M AN U

(C20H23ClN3O2) C, H, N. Calcd for: 58.53, 5.68, 10.29; Found: 58.58, 5.62, 10.07; HPLC purity: 99.32%, retention time: 16.78 min (methanol/water 80:20, v/v). 38.

N-(thiazol-2-yl)-2-(4-(2-phenoxyethyl)piperazin-1-yl)acetamide dihydrochloride(34) Compound 34 was synthesized from 4a and 2a with the procedure described for compound 1

TE D

(white solid, 41.93%). m.p. = 226-228 °C.

IR(KBr): 3142, 2922, 2568, 2294, 1694, 1593, 1566, 1455, 1290, 1240, 758 cm-1; 1H-NMR (300 MHz, D2O) δ 7.46-7.44 (m, 1H, Ar-H), 7.35-7.29 (m, 2H, Ar-H), 7.22-7.19 (m, 1H, Ar-H), 7.03-6.95

EP

(m, 4H, Ar-H), 4.35 (t, 2H, J = 4.50 Hz, -CH2CH2O-), 3.85-3.58 (m, 8H, -N(CH2CH2)2N-, -CH2CH2O-,

AC C

-CH2CO-), 3.25 (brs, 4H, -N(CH2CH2)2N-); MS (EI) m/z 346 [M+]; Anal. (C17H24Cl2N4O2S) C, H, N. Calcd for: 48.69, 5.77, 13.36; Found: 48.29, 6.15, 12.97; HPLC purity: 98.40%, retention time: 3.47 min (methanol/water 80:20, v/v). 39.

N-(thiazol-2-yl)-2-(4-(2-(p-tolyloxy)ethyl)piperazin-1-yl)acetamide (35)

Compound 35 was synthesized from 4b and 2a with the procedure described for compound 2 (white solid, 43.37%). m.p. = 103-105 °C. IR(KBr): 3459, 3174, 3068, 2925, 2811, 1695, 1577, 1513, 1456, 1286, 1239, 1167, 806, 735 cm-1;

Supporting Information

1

ACCEPTED MANUSCRIPT

H-NMR (300 MHz, CDCl3) δ 10.32 (s, 1H, -NHCO-), 7.46 (d, 1H, J = 3.63 Hz, Ar-H), 7.09 (s, 1H,

Ar-H), 7.06 (s, 1H, Ar-H), 6.99 (d, 1H, J = 3.66 Hz, Ar-H), 6.83-6.78 (m, 2H, Ar-H), 4.08 (t, 2H, J = 5.76 Hz, -CH2CH2O-), 3.25 (s, 2H, -CH2CO-), 2.83 (t, 2H, J = 5.73 Hz, -CH2CH2O-), 2.68 (s, 8H,

RI PT

-N(CH2CH2)2N-), 2.28 (s, 3H, -CH3); MS (EI) m/z 360 [M+]; Anal. (C18H24N4O2S) C, H, N. Calcd for: 59.97, 6.71, 15.54; Found: 60.28, 6.99, 15.91; HPLC purity: 98.16%, retention time: 6.28 min (methanol/water 80:20, v/v).

N-(thiazol-2-yl)-2-(4-(2-(o-tolyloxy)ethyl)piperazin-1-yl)acetamide (36)

SC

40.

solid, 73.48%). m.p. = 123-125 °C.

M AN U

Compound 36 was synthesized from 4c and 2a with the procedure described for compound 2 (white

IR(KBr): 2949, 2802, 1601, 1296, 1245, 1164, 1121, 1051, 1038, 752 cm-1; 1H-NMR (300 MHz, CDCl3) δ 10.33 (s, 1H, Ar-H), 7.46 (d, 1H, J = 3.66 Hz), 7.17-7.12 (m, 2H, Ar-H), 7.00 (d, 1H, J = 3.69

TE D

Hz), 6.86-6.79 (m, 2H, Ar-H), 4.12 (t, 2H, J = 5.73 Hz, -CH2CH2O-), 3.26 (s, 2H, -CH2CO-), 2.88 (t, 2H, J = 5.82 Hz, -CH2CH2O-), 2.69 (s, 8H, -N(CH2CH2)2N-), 2.28 (s, 3H, -CH3); 13C-NMR (75MHz, CDCl3) δ 167.8, 157.4, 156.4, 137.1, 130.2, 129.0, 126.3, 120.0, 114.1, 113.2, 110.5, 65.9, 60.5, 56.7,

EP

53.2, 53.0, 15.8; MS (EI) m/z 360 [M+]; Anal. (C18H24N4O2S) C, H, N. Calcd for: 59.97, 6.71, 15.54;

AC C

Found: 60.05, 6.67, 15.83; HPLC purity: 96.42%, retention time: 6.75 min (methanol/water 80:20, v/v). 41.

N-(thiazol-2-yl)-2-(4-(2-(2,4-dimethylphenoxy)ethyl)piperazin-1-yl)acetamide dihydrochloride (37)

Compound 37 was synthesized from 4l and 2a with the procedure described for compound 1 (white

solid, 48.58%). m.p. = 215-217 °C. IR(KBr): 3416, 2918, 2619, 2399, 2351, 1730, 1573, 1505, 1253, 1229, 1133, 1062, 813, 760 cm-1; 1

H-NMR (300 MHz, D2O) δ 7.49 (d, 1H, J = 3.90 Hz, Ar-H), 7.25 (d, 1H, J = 3.93 Hz, Ar-H),

Supporting Information

ACCEPTED MANUSCRIPT

7.04-7.00 (m, 2H, Ar-H), 6.86-6.82 (m, 1H, Ar-H), 4.34 (t, 2H, J = 4.23 Hz, -CH2CH2O-), 3.93 (s, 2H, -CH2CO-), 3.78-3.68 (m, 8H, -N(CH2CH2)2N-), 3.35 (t, 2H, J = 4.26 Hz, -CH2CH2O-), 2.18 (s, 3H, -CH3), 2.13 (s, 3H, -CH3); MS (EI) m/z 374 [M+]; Anal. (C19H28Cl2N4O2S) C, H, N. Calcd for: 51.00,

42.

RI PT

6.31, 12.52; Found: 51.07, 6.46, 12.53. N-(thiazol-2-yl)-2-(4-(2-(3,4-dimethylphenoxy)ethyl)piperazin-1-yl)acetamide dihydrochloride (38)

SC

Compound 38 was synthesized from 4m and 2a with the procedure described for compound 1

M AN U

(white solid, 21.21%). m.p. = 213-215 °C.

IR(KBr): 3466, 2954, 1694, 1566, 1449, 1193, 1165, 1022, 944, 777 cm-1; 1 H-NMR (300 MHz, CDCl3) δ 7.46 (d, 1H, J = 3.66 Hz, Ar-H), 7.04-6.99 (m, 2H, Ar-H), 6.72-6.62 (m, 2H, Ar-H), 4.08 (t, 2H, J = 5.73 Hz, -CH2CH2O-), 3.25 (s, 2H, -CH2CO-), 2.87 (t, 2H, J = 5.76 Hz, -CH2CH2O-), 2.68 (brs,

TE D

8H, -N(CH2CH2)2N-), 2.23 (s, 3H, -CH3), 2.19 (s, 3H, -CH3); MS (EI) m/z 374 [M+]; Anal. (C19H28Cl2N4O2S) C, H, N. Calcd for: 51.00, 6.31, 12.52; Found: 51.20, 6.12, 12.21; HPLC purity: 98.10%, retention time: 7.75 min (methanol/water 80:20, v/v). N-(thiazol-2-yl)-2-(4-(2-(2,6-dimethylphenoxy)ethyl)piperazin-1-yl)acetamide

EP

43.

AC C

dihydrochloride (39)

Compound 39 was synthesized from 4n and 2a with the procedure described for compound 1

(white solid, 27.63%). m.p. = 238-240 °C. IR(KBr): 3460, 3125, 1982, 2641, 2356, 1695, 1562, 1446, 1319, 1293, 1194, 1020, 935, 776 cm-1;

1

H-NMR (300 MHz, DMSO) δ 7.53 (d, 1H, J = 3.63 Hz, Ar-H), 7.30 (d, 1H, J = 3.69 Hz, Ar-H),

7.06-6.92 (m, 3H, Ar-H), 4.19 (s, 2H, -CH2CH2O-), 4.02 (s, 2H, -CH2CO-), 3.62 (t, 2H, J = 5.76 Hz, -CH2CH2O-), 3.51 (brs, 8H, -N(CH2CH2)2N-), 2.27 (s, 3H, -CH3), 2.08 (s, 3H, -CH3); MS (EI) m/z 374

Supporting Information

ACCEPTED MANUSCRIPT

[M+]; Anal. (C19H28Cl2N4O2S) C, H, N. Calcd for: 51.00, 6.31, 12.52; Found: 51.00, 6.67, 12.70; HPLC purity: 98.33%, retention time: 4.85 min (methanol/water 80:20, v/v). 44.

N-(thiazol-2-yl)-2-(4-(2-(4-(tert-butyl)phenoxy)ethyl)piperazin-1-yl)acetamide (40)

RI PT

Compound 40 was synthesized from 4o and 2a with the procedure described for compound 2 (yellow solid, 46.85%). m.p. = 111-113 °C.

IR(KBr): 3423, 3189, 2954, 2826, 1716, 1577, 1517, 1456, 1250, 1164, 1051, 966, 834, 731 cm-1; H-NMR (300 MHz, CDCl3) δ 10.32 (s, 1H, -NHCO-), 7.46 (d, 1H, J = 3.63 Hz, Ar-H), 7.33-7.28 (m,

SC

1

M AN U

2H, Ar-H), 7.00 (d, 1H, J = 3.66 Hz, Ar-H), 6.87-6.82 (m, 2H, Ar-H), 4.09 (t, 2H, J = 5.76 Hz, -CH2CH2O-), 3.26 (s, 2H, -CH2CO-), 2.96 (t, 2H, J = 5.73 Hz, -CH2CH2O-), 2.68 (brs, 8H, -N(CH2CH2)2N-), 1.30 (s, 9H, -C(CH3)3);

13

C-NMR (75MHz, CDCl3) δ 167.8, 157.0, 155.9, 143.0,

137.1, 125.7, 113.6, 113.2, 65.4, 60.5, 56.6, 53.1, 52.9, 33.6, 31.0; MS (EI) m/z 402 [M+]; HR-MS (ESI)

45.

TE D

m/z 403 ([M + H]+); HPLC purity: 99.49%, retention time: 11.80 min (methanol/water 80:20, v/v). N-(thiazol-2-yl)-2-(4-(2-(4-methoxyphenoxy)ethyl)piperazin-1-yl)acetamide (41) Compound 41 was synthesized from 4d and 2a with the procedure described for compound 2

EP

(yellow solid, 66.51%). m.p. = 102-103 °C.

AC C

IR(KBr): 3162, 2995, 2861, 1705, 1510, 1454, 1242, 1164, 1022, 955, 825, 704 cm-1; 1H-NMR (300 MHz, CDCl3) δ 10.32 (s, 1H, -NHCO-), 7.46 (d, 1H, J = 3.30 Hz, Ar-H), 7.00 (d, 1H, J = 3.24 Hz, Ar-H), 6.87-6.81 (m, 4H, Ar-H), 4.06 (t, 2H, J = 5.76 Hz, -CH2CH2O-), 3.77 (s, 3H, -OCH3), 3.26 (s, 2H, -CH2CO-), 2.82 (t, 2H, J = 5.73 Hz, -CH2CH2O-), 2.68 (brs, 8H, -N(CH2CH2)2N-); MS (EI) m/z 376 [M+]; Anal. (C18H24N4O3S) C, H, N. Calcd for: 57.43, 6.43, 14.88; Found: 57.83, 6.28, 14.84; HPLC purity: 97.89%, retention time: 4.82 min (methanol/water 80:20, v/v). 46.

N-(thiazol-2-yl)-2-(4-(2-(4-(trifluoromethoxy)phenoxy)ethyl)piperazin-1-yl)acetamide

Supporting Information

ACCEPTED MANUSCRIPT

dihydrochloride (42) Compound 42 was synthesized from 4e and 1b with the procedure described for compound 1 (white solid, 73.44%). m.p. = 225-226 °C.

RI PT

IR(KBr): 3452, 3160, 2982, 1693, 1564, 1507, 1282, 1111, 848, 784, 620 cm-1; 1H-NMR (300 MHz, D2O) δ 7.46 (d, 1H, J = 3.90 Hz, Ar-H), 7.28-7.21 (m, 3H, Ar-H), 7.03 (d, 2H, J = 8.73 Hz, Ar-H), 4.38 (t, 2H, J = 4.32 Hz, -CH2CH2O-), 3.72 (s, 2H, -CH2CO-), 3.64 (t, 2H, J = 4.26 Hz, -CH2CH2O-), 3.55

SC

(s, 4H, -N(CH2CH2)2N-), 3.15 (s, 4H, -N(CH2CH2)2N-); MS (EI) m/z 430 [M+]; Anal.

M AN U

(C18H23Cl2F3N4O3S) C, H, N. Calcd for: 42.92, 4.60, 11.13; Found: 42.43, 4.68, 10.85; HPLC purity: 98.00%, retention time: 5.50 min (methanol/water 80:20, v/v). 47.

N-(thiazol-2-yl)-2-(4-(2-(4-ethoxyphenoxy)ethyl)piperazin-1-yl)acetamide (43) Compound 43 was synthesized from 4p and 2a with the procedure described for compound 2 (yello

TE D

solid, 78.51%). m.p. = 90-92 °C.

IR(KBr): 3438, 3260, 2947, 2826, 1694, 1522, 1250, 1168, 1126, 1008, 780, 728 cm-1; 1H-NMR (300 MHz, CDCl3) δ 10.33 (s, 1H, -NHCO-), 7.45 (d, 1H, J = 3.63 Hz, Ar-H), 6.99 (d, 1H, J = 3.24 Hz,

EP

Ar-H), 6.89 (s, 4H, Ar-H), 4.14 (t, 2H, J = 5.76 Hz, -CH2CH2O-), 4.05 (q, 2H, J = 6.90 Hz, -OCH2CH3),

AC C

3.25 (s, 2H, -CH2CO-), 2.87 (t, 2H, J = 5.73 Hz, -CH2CH2O-), 2.68 (brs, 8H, -N(CH2CH2)2N-), 1.42 (t, 3H, J = 6.96 Hz, -OCH2CH3); MS (EI) m/z 390 [M+]; Anal. (C19H26N4O3S) C, H, N. Calcd for: 58.44, 6.71, 14.35; Found: 58.63, 7.01, 14.11; HPLC purity: 97.92%, retention time: 5.22 min (methanol/water 80:20, v/v). 48.

N-(thiazol-2-yl)-2-(4-(2-(2-chlorophenoxy)ethyl)piperazin-1-yl)acetamide dihydrochloride (44) Compound 44 was synthesized from 4j and 2a with the procedure described for compound 2 (white

Supporting Information

ACCEPTED MANUSCRIPT

solid, 45.09%). m.p. = 218-220 °C. IR(KBr): 3409, 2996, 2420, 2206, 1697, 1565, 1488, 1446, 1250, 1072, 973, 756 cm-1; 1H-NMR (300 MHz, D2O) δ 7.46-6.98 (m, 6H, Ar-H), 4.54 (brs, 2H, -OCH2CH2-), 3.86 (s, 2H, -CH2CO-),

RI PT

3.76-3.57 (m, 8H, -N(CH2CH2)2N-), 3.28 (brs, 2H, -OCH2CH2-); MS (EI) m/z 380 [M+]; Anal. (C17H23Cl3N4O2S) C, H, N. Calcd for: 44.88, 4.62, 12.32; Found: 45.20, 4.89, 11.99. 49.

N-(thiazol-2-yl)-2-(4-(2-(4-cyanophenoxy)ethyl)piperazin-1-yl)acetamide dihydrochloride

SC

(45)

solid, 52.63%). m.p. = 185-187 °C.

M AN U

Compound 45 was synthesized from 4f and 2a with the procedure described for compound 1 (white

IR(KBr): 3174, 3103, 2840, 2254, 2235, 1603, 1562, 1506, 1257, 1162, 1030, 845 cm-1; 1H-NMR (300 MHz, DMSO) δ 7.83 (d, 2H, J = 8.73 Hz, Ar-H), 7.54 (d, 1H, J = 3.54 Hz, Ar-H), 7.33 (d, 1H, J =

TE D

3.54 Hz, Ar-H), 7.22 (d, 2H, J = 8.76 Hz, Ar-H), 4.58 (brs, 2H, -OCH2CH2-), 4.33 (s, 2H, -CH2CO-), 3.73 (brs, 10H, -N(CH2CH2)2N-, -OCH2CH2-); 13C-NMR (75MHz, D2O) δ 168.8, 160.4, 158.0, 136.3, 134.1, 119.5, 114.9, 114.3, 103.3, 61.2, 58.2, 54.7, 51.3, 48.8, 48.4; MS (EI) m/z 371 [M+]; Anal. (Base,

EP

C19H26N4O3S) C, H, N. Calcd for: 58.20, 5.70, 18.85; Found: 58.42, 5.77, 18.58; HPLC purity: 99.08%,

AC C

retention time: 23.20 min (methanol/50mM ammonium acetate, gradient elution). 50.

N-(thiazol-2-yl)-2-(4-(2-(4-chlorophenoxy)ethyl)piperazin-1-yl)acetamide (46)

Compound 46 was synthesized from 4h and 2a with the procedure described for compound 2

(white solid, 40.79%). m.p. = 125-126 °C. 1

H-NMR (300 MHz, CDCl3) δ 10.32 (s, 1H, -NHCO-), 7.46 (d, 1H, J = 3.45 Hz, Ar-H), 7.25-7.21

(m, 2H, Ar-H), 7.00 (d, 1H, J = 3.12 Hz, Ar-H), 6.85-6.81 (m, 2H, Ar-H), 4.07 (t, 2H, J = 5.61 Hz, -CH2CH2O-), 3.26 (s, 2H, -CH2CO-), 2.84 (t, 2H, J = 5.64 Hz, -CH2CH2O-), 2.68-2.64 (m, 8H,

Supporting Information

ACCEPTED MANUSCRIPT

-N(CH2CH2)2N-); 13C-NMR (75MHz, CDCl3) δ 167.8, 157.0, 156.8, 137.1, 128.8, 125.3, 115.4, 113.3, 65.8, 60.5, 56.4, 53.1, 53.0; MS (EI) m/z 380 [M+]; Anal. (C17H21ClN4O2S) C, H, N. Calcd for: 53.61, 5.56, 14.71; Found: 54.01, 5.49, 14.48; HPLC purity: 96.65%, retention time: 5.50 min

51.

RI PT

(methanol/water 80:20, v/v). Ethyl 4-(2-(4-(2-oxo-2-(thiazol-2-ylamino)ethyl)piperazin-1-yl)ethoxy)benzoate (47)

Compound 47 was synthesized from 4q and 2a with the procedure described for compound 2

SC

(white solid, 64.61%). m.p. = 119-121 °C.

M AN U

IR(KBr): 3466, 3167, 2960, 2833, 1610, 1565, 1275, 1257, 1218, 1175, 1015, 720 cm-1; 1H-NMR (300 MHz, CDCl3) δ 10.34 (s, 1H, -NHCO-), 7.78-7.72 (m, 3H, Ar-H), 7.47-742 (m, 2H, Ar-H), 7.34 (t, 1H, J = 8.13 Hz, Ar-H), 7.18-7.15 (m, 2H, Ar-H), 7.00 (d, 1H, J = 3.60 Hz, Ar-H), 4.25 (t, 2H, J = 5.46 Hz, -CH2CH2O-), 3.27 (s, 2H, -CH2CO-), 2.94 (t, 2H, J = 5.34 Hz, -CH2CH2O-), 2.72 (brs, 8H,

TE D

-N(CH2CH2)2N-); MS (EI) m/z 396 [M+]; Anal. (C21H24N4O2S) C, H, N. Calcd for: 63.61, 6.10, 14.13; Found: 63.32, 6.32, 13.72; HPLC purity: 96.65%, retention time: 5.50 min (methanol/water 80:20, v/v). 52.

N-(thiazol-2-yl)-2-(4-(2-(naphthalen-2-yloxy)ethyl)piperazin-1-yl)acetamide

EP

dihydrochloride (48)

AC C

Compound 48 was synthesized from 4r and 2a with the procedure described for compound 1 (white solid, 44.57%). m.p. = 116-118 °C. IR(KBr): 3416, 2680, 2394, 1749, 1567, 1281, 1253, 1174, 1115, 768 cm-1; 1H-NMR (300 MHz,

CDCl3) δ 10.33 (s, 1H, -NHCO-), 8.00 (d, 2H, J = 9.00 Hz, Ar-H), 7.46 (d, 1H, J = 3.93 Hz, Ar-H), 7.00 (d, 1H, J = 3.60 Hz, Ar-H), 6.92 (d, 2H, J = 9.00 Hz, Ar-H), 4.35 (q, 2H, J = 7.20 Hz, -OCH2CH3), 4.16 (t, 2H, J = 5.46 Hz, -CH2CH2O-), 3.26 (s, 2H, -CH2CO-), 2.87 (t, 2H, J = 5.70 Hz, -CH2CH2O-), 2.69 (brs, 8H, -N(CH2CH2)2N-), 1.38 (t, 3H, J = 7.23 Hz, -OCH2CH3); 13C-NMR (75MHz, CDCl3) δ

Supporting Information

ACCEPTED MANUSCRIPT

167.9, 157.1, 156.2, 137.1, 134.0, 128.9, 128.5, 127.2, 126.3, 125.9, 123.2, 118.5, 113.3, 106.3, 65.4, 60.5, 56.6, 53.1, 52.9; HR-MS (ESI) m/z 419 ([M + H]+); HPLC purity: 97.88%, retention time: 7.56

AC C

EP

TE D

M AN U

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RI PT

min (methanol/water 80:20, v/v).

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Compound 4: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

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1

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Compound 6: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

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1

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ACCEPTED MANUSCRIPT

Compound 8: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

RI PT

1

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ACCEPTED MANUSCRIPT

Compound 12: H-NMR, CDCl3

EP

C-NMR, CDCl3

AC C

13

TE D

M AN U

SC

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1

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ACCEPTED MANUSCRIPT

Compound 16: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

RI PT

1

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ACCEPTED MANUSCRIPT

Compound 21: H-NMR, CDCl3

EP

C-NMR, CDCl3

AC C

13

TE D

M AN U

SC

RI PT

1

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ACCEPTED MANUSCRIPT

Compound 22: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

RI PT

1

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ACCEPTED MANUSCRIPT

Compound 27: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

RI PT

1

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ACCEPTED MANUSCRIPT

Compound 30: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

RI PT

1

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ACCEPTED MANUSCRIPT

Compound 36: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

RI PT

1

Supporting Information

ACCEPTED MANUSCRIPT

Compound 40: H-NMR, CDCl3

EP

TE D

C-NMR, CDCl3

AC C

13

M AN U

SC

RI PT

1

Supporting Information

ACCEPTED MANUSCRIPT

Compound 45: H-NMR, D2O

EP

TE D

C-NMR, D2O

AC C

13

M AN U

SC

RI PT

1