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European Journal of Medicinal Chemistry 130 (2017) 15e25

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Short communication

Design, synthesis and biological evaluation ofaminobenzyloxyarylamide derivatives as selective k opioid receptorantagonists

Junwei Wang a, 1, Qiao Song a, 1, Anhua Xu a, Yu Bao a, Yungen Xu a, b, **, Qihua Zhu a, b, *

a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, Chinab Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China

a r t i c l e i n f o

Article history:Received 10 November 2016Received in revised form8 February 2017Accepted 11 February 2017Available online 16 February 2017

Keywords:k opioid receptor antagonistsAminobenzyloxyarylamidesLY2456302SelectivityAntidepressants

* Corresponding author. Department of Medicinaceutical University, Nanjing 210009, China.** Corresponding author. Department of Medicinaceutical University, Nanjing 210009, China.

E-mail addresses: xyg@cpu.edu.cn (Y. Xu), zhuqihu1 Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ejmech.2017.02.0290223-5234/© 2017 Elsevier Masson SAS. All rights res

a b s t r a c t

Opioid receptors play an important role in both behavioral and mood functions. Based on the structuralmodification of LY2456302, a series of aminobenzyloxyarylamide derivatives were designed and syn-thesized as k opioid receptor antagonists. The k opioid receptor binding ability of these compounds wereevaluated with opioid receptors binding assays. Compounds 1a-d showed high affinity for k opioid re-ceptor. Especially for compound 1c, exhibited a significant Ki value of 15.7 nM for k opioid receptorbinding and a higher selectivity over m and d opioid receptors compared to (±)LY2456302. In addition,compound 1c also showed potent k antagonist activity with k IC50 ¼ 9.32 nM in [35S]GTP-g-S functionalassay. The potential use of the representative compounds as antidepressants was also investigated. Themost potent compound 1c not only exhibited potent antidepressant activity in the mice forced swim-ming test, but also displayed the effect of anti-anxiety in the elevated plus-maze test.

© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

Major depression or depression, characterized by negativemood, reduced motivation and decreased energy, affects nearly 5%of people worldwide each year [1,2]. The first line of drugs for thetreatment of depression is normally selective serotonin reuptakeinhibitors (SSRIs) such as fluoxetine and escitalopram. However,60% of patients treated with these medications fail to achieveremission [3]. The clinical treatment of depression is limited due tothe drawbacks of existing antidepressants such as the slow onsettime, low tolerance and common relapse [4]. Therefore, newmethods of treatment that work through alternative mechanismsare urgent to be developed.

Opioid receptors belong to the super family of G-protein-coupled receptors (GPCRs) which include mu (m), delta (d), kappa(k) and the opioid-like receptor (ORL-1). They are involved in

l Chemistry, China Pharma-

l Chemistry, China Pharma-

a@vip.126.com (Q. Zhu).

erved.

multiple physiological activities [5,6]. The three opioid receptors, m,d and k opioid receptors, regulate major functions including pain,emotional tone, appetite and reward circuitry [7]. Each of thesethree types of opioid receptors has its specific endogenous ligandand exerts different biological effect. The k opioid receptor is closelyassociated to the action of dynorphin (DYN) peptides as specificendogenous ligands. The role of k opioid receptor ligands in themodulation of mood has attracted great interest in recent years[8,9]. The function of cyclic adenosine monophosphate (cAMP)response element binding protein (CREB) can be disrupted byantagonizing k opioid receptor, thus increasing the dopaminerelease, resulting in antidepressant-like effects [10,11]. Recently,accumulating evidence indicates that k opioid receptor antagonists:nor-BNI [12,13] and JDTic [14,15] (Fig. 1), have anxiolytic andantidepressant-like activity in rodents [16]. In addition, the short-acting k opioid receptor antagonists PF-04455242 [17] andLY2456302 [18] (Fig. 1) also show antidepressant-like effects.LY2456302 has passed phase I clinical trial and is currently in phaseII trial for adjunctive treatment of major depressive disorders andfor the treatment of substance abuse disorders [19].

LY2456302 is a structurally-unique, high-affinity and selective k

opioid receptor antagonist with 24- and 175-fold selectivity over mand d opioid receptors, respectively. In contrast to the prototypical k

Fig. 1. Structures of nor-BNI, JDTic, PF-04455242 and LY2456302.

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e2516

opioid receptor antagonists nor-BNI and JDTic, LY2456302 exhibitsrapid absorption, good oral bioavailability (F ¼ 25%, tmax ¼ 1e2 h)and a more typical rate of clearance (T1/2¼ 2e4 h) [20]. This uniquebiopharmacological profile makes it as an attractive lead compoundin the search for new k opioid receptor antagonists. Recent studyindicates that selective k opioid receptor antagonism is a promisingexperimental strategy for the treatment of depression [20]. Selec-tive k opioid receptor antagonists can reduce both ethanol intakeand reinstatement in a number of preclinical paradigms [21,22],while nonselective k opioid antagonists produce neither reliableantidepressant- nor anxiolytic-like effects in animals or humans,which may be caused by the functional opposition between m and k

receptors [10,23]. In this paper, we describe details of our discoveryof potent and selective k opioid receptor antagonists in an amino-benzyloxyarylamide scaffold. We also describe the evaluation ofthese selective k opioid receptor antagonists for potential use asantidepressants.

2. Results and discussion

2.1. Design of selective k opioid receptor antagonists

In order to search novel and efficient antidepressants,LY2456302 which is a selective k opioid receptor antagonist waschosen as the lead compound. According to previous studies[24,25], some modifications have been taken in part I (Fig. 2)including different substitution on the benzene ring, the replace-ment of benzene ring with N-heterocycle, the replacement of pyr-rolidine ring and the chiral isomers of pyrrolidine with other cyclicamine. However, the modifications of part II have been rarely re-ported, with modification only on aromatic ring B. For example, thesubstitution on benzene ring B was changed or the benzene ring

Fig. 2. Process of rational design of aminobenzyloxyary

was replaced with pyridine ring [24,25]. Thus, in this work, wefocus on the modification of part II (Fig. 2). Firstly, fluorine orchlorine was introduced into the ring A to investigate the influenceon activity and selectivity towards k opioid receptor. Secondly, thebenzene ring B was replaced by naphthalene ring and the aromaticring bound to the pyrrolidine was modified according to previousstudy [24] to study the structure-activity relationship.

2.2. Chemistry

The synthetic routes of the proposed aminobenzyloxyarylamidederivatives were illustrated in Schemes 1e3. Treatment of 3a-hwith thionyl chloride produced the corresponding acid chloridewhich reacted with ethanol giving ethyl benzoate derivatives 4a-h.Intermediates 4a-h was coupled with N-Vinylpyrrolidone undersodium-hydrogen condition to form 5a-h which were heated inhydrochloric acid to give 5-phenyl-3,4-dihydro-2H-pyrrole de-rivatives 6a-h. Reduction of 6a-h with sodium borohydride pro-vided 2-phenylpyrrolidine derivatives 7a-h (Scheme 1). Thesynthesis of the designed compounds 1a-d stemmed from thereductive amination of aromatic aldehyde derivatives with 7h.Obtained reductive amination 8a-b was etherified with benzoni-trile derivatives 9a-b to give etherification products 10a-d. Thetarget compounds 1a-d were finally obtained by the hydrolysis of10a-d (Scheme 2). Substitution of bromine with cyano-group on 1-bromo-4-fluoronaphthalene 11 gave intermediate 12. Ether-ification of 12 with 4-hydroxybenzaldehyde afforded ether 13whichwas further hydrolyzed by hydrogen peroxide and gave 4-(4-formylphenoxy)-1-naphthamide 14. Compounds 2a-h were ob-tained by reductive amination of 14 with 2-aryl substituted pyr-rolidine 7a-h (Scheme 3). All the target compounds have beenconfirmed by IR, 1H NMR, 13C NMR and HR-MS.

lamide derivatives as k opioid receptor antagonists.

Scheme 2. Synthetic route of target compounds 1a-d. Regents and conditions: (e) Aldehyde, NaBH3CN, AcOH, MeOH, r.t., 12 h; (f) K2CO3, DMF, N2, 120 �C, 10 h (g) H2O2, DMSO, 10 �Cto r.t., 3 h.

Scheme 1. Synthetic route of intermediates 7a-h. Regents and conditions: (a) SOCl2, EtOH, reflux, 2 h; (b) N-Vinylpyrrolidone, NaH, THF, r.t. to 60 �C, 3 h; (c) HCl, THF, reflux, 12 h;(d) NaBH4, AcOH, MeOH, r.t., 1 h.

Scheme 3. Synthetic route of target compounds 2a-h. Regents and conditions: (h) K4Fe(CN)6, K2CO3, Pd(OAc)2, DMAC, N2, 120 �C, 12 h; (i) 4-Hydroxybenzaldehyde, K2CO3, DMF, N2,120 �C, 12 h; (j) H2O2, DMSO, 10 �C to r.t, 3 h; (k) NaBH3CN, AcOH, MeOH, r.t. 12 h.

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e25 17

2.3. Molecular modeling studies

In order to investigate the potential activity and selectivity ofthe designed compounds against k opioid receptors, modelingstudies of compound 1c in the ligand binding pocket of k opioidreceptor were carried out. The docking model (PDB code: 4DJH)revealed that comparingwith LY2456302, compound 1c could forman additional halogen bond with Tyr139 by the introduction ofchlorine atom (Fig. 3). The s-hole interaction shift the wholemolecule to the left, thus the oxygen atom of diphenyl ether could

form an additional hydrogen bond interaction with HOH1311,which was speculated to improve the selectivity for k opioid re-ceptor. As shown in Fig. 3, the optimum conformation of compound1c is (R)-isomer. We will isolate this isomer in the following workand the binding affinity is expected to be improved.

2.4. In vitro opioid receptor binding and [35S] GTP-g-S bindingassays

In vitro receptor binding affinity of compounds for m, k and

Fig. 3. Docked conformation of compound 1c (A) and LY2456302 (B) in the ligand k opioid receptor binding pocket. The interaction mode was obtained through molecular docking(PDB ID: 4DJH) and depicted using MOE 2013.08. (A) Best docking pose of (R)-isomer of 1c, the carbon atoms and the key residues in the active site of k opioid receptor were coloredin blue and purple red, respectively. (B) Best docking pose of (S)-isomer of LY2456302, the carbon atoms and the key residues were colored in yellow and purple red, respectively.The H-bonds were shown as black dot lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1In vitro receptor binding affinity of compounds 1a-d and (±)LY2456302 for m, k and d opioid receptors with the competitive inhibition ratio.

Compd m Inhibition (%)a k Inhibition (%)b d Inhibition (%)c

1 � 10�5 M 1 � 10�6 M 1 � 10�5 M 1 � 10�6 M 1 � 10�5 M 1 � 10�6 M

1a 99.1 ± 0.6 78.8 ± 0.8 100 ± 0.0 99.3 ± 0.4 87.5 ± 0.6 36.5 ± 1.61b 95.3 ± 0.3 71.0 ± 1.0 100 ± 0.0 98.8 ± 0.1 97.1 ± 0.2 45.5 ± 2.21c 94.2 ± 0.2 51.4 ± 0.7 100 ± 0.0 98.5 ± 0.3 92.4 ± 0.1 54.1 ± 2.31d 96.8 ± 0.5 73.4 ± 0.3 98.7 ± 0.2 95.8 ± 0.5 89.2 ± 0.2 37.4 ± 3.4(±)LY2456302 93.6 ± 0.2 56.1 ± 1.0 100 ± 0.0 97.0 ± 0.3 87.2 ± 0.7 45.5 ± 0.7

a Competitive binding inhibition ratio of m opioid receptor, [3H]-DAMGO was used.b Competitive binding inhibition ratio of k opioid receptor, [3H]-U69,593 was used.c Competitive binding inhibition ratio of d opioid receptor, [3H]-DPDPE was used.

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e2518

d opioid receptors were first evaluated with radioligand bindingassays and (±)LY2456302 was used as the positive control. Asshown in Table 1, the results indicated that compounds 1a-d showed similar affinities as (±)LY2456302 with k opioid receptorinhibition ratio > 90% at the concentration of 1 mM. The results ofcompounds 2a-h were summarized in Table 2. Compounds 2b, 2c,2d and 2h showed moderate binding affinities with k opioid re-ceptor inhibition ratio >50% at the concentration of 10 mM. How-ever, their binding affinities for m and d opioid receptors were weakwith m opioid receptor inhibition ratio <20% and d opioid receptorinhibition ratio <10% at the concentration of 10 mM. Among 2-arylpyrrolidine derivatives, the binding affinity of 3-methyl substitutedcompound 2h was stronger than that of 2-methyl substitutedcompound 2e and 3,5-dimethyl substituted compound 2a(2h > 2e > 2a), and the binding affinities of 3-fluorinated com-pound 2b was stronger than that of 4-fluorinated compound 2g.

Together with compounds 1a-d, compounds 2d and 2h wereselected to determine the Ki values of opioid receptors binding af-finities. As shown in Table 3, compounds 1a-d exhibited potent

binding affinities for k opioid receptor with the Ki values in thesame order of magnitude as (±)LY2456302. Among these de-rivatives, compound 1c showed the strongest binding affinity for kopioid receptor, which was almost as potent as (±)LY2456302.Notably, we found that the selectivity of 1c over m and d opioidreceptors was 14- and 12.2-fold, respectively, which was almosttwice as much as that of (±)LY2456302. The docking results aboveexplained the reason why compound 1c showed higher k opioidreceptor selectivity over m and d opioid receptors compared with(±)LY2456302. The cause of the affinity was not obviouslyimproved may be the chiral problem. The chiral separation of theactive compounds would be continued in the further work basedon the optimum conformation in the molecular docking studies.

Compound 1c was evaluated for in vitro antagonist activity,utilizing inhibition of agonist stimulated [35S]GTP-g-S binding withcloned human opioid receptors expressed in Chinese hamster ovarycells (CHO) cells (m and k) or human embryonic kidney 293(HEK293) cells (d). Results are shown in Table 4. Compound 1cshowed good k antagonist potency and selectivity in functional

Table 2In vitro receptor binding affinity of compounds 2a-h and (±)LY2456302 for m, k and d opioid receptors with the competitive inhibition ratio.

Compd m Inhibition (%)a k Inhibition (%)b d Inhibition (%)c

1 � 10�5 M 1 � 10�6 M 1 � 10�5 M 1 � 10�6 M 1 � 10�5 M 1 � 10�6 M

2a 0 0 23.7 ± 0.8 12.2 ± 2.1 0 02b 0 0 57.9 ± 0.4 27.7 ± 1.0 4.0 ± 0.2 2.3 ± 0.22c 0 0 64.1 ± 2.5 31.4 ± 0.1 0 02d 15.0 ± 2.0 0 80.5 ± 0.0 46.5 ± 1.5 9.0 ± 0.8 02e 0 0 34.0 ± 0.2 13.9 ± 2.7 2.9 ± 0.9 02f 52.2 ± 1.0 11.3 ± 1.2 12.0 ± 0.1 5.1 ± 0.8 0 02g 0 0 39.0 ± 1.5 13.8 ± 0.4 0 02h 16.3 ± 1.1 0 86.9 ± 0.1 39.9 ± 2.1 0 0(±)LY2456302 93.6 ± 0.2 56.1 ± 1.0 100 ± 0.0 97.0 ± 0.3 87.2 ± 0.7 45.5 ± 0.7

a Competitive binding inhibition ratio of m opioid receptor, [3H]-DAMGO was used.b Competitive binding inhibition ratio of k opioid receptor, [3H]-U69,593 was used.c Competitive binding inhibition ratio of d opioid receptor, [3H]-DPDPE was used.

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e25 19

GTP-g-S binding assays, with k IC50 ¼ 9.32 nM, m IC50 ¼ 144.2 nM,and d IC50 ¼ 15.82 nM. This demonstrates that compound 1cwas anantagonist for each of the types of opioid receptors.

2.5. Biological activity in vivo

2.5.1. Mice forced swimming testCompounds 1b, 1c and 2h were evaluated for in vivo antide-

pressant activity in mice forced swimming model and (±)

Table 3The binding affinities Ki values of 1a-d, 2d, 2h and (±)LY2456302 for opioidreceptors.a

Compd Ki (nM) Selectivity

mb kc dd m/k d/k

1a 32.3 ± 0.1 37.6 ± 0.6 489.4 ± 11.5 0.85 13.01b 62.8 ± 4.7 29.4 ± 0.3 266.6 ± 4.0 2.13 9.01c 220.0 ± 1.9 15.7 ± 0.5 191.4 ± 4.2 14.0 12.21d 27.9 ± 0.4 42.7 ± 1.4 429.3 ± 5.8 0.65 10.12d >10 000 4603 ± 484.1 >10 000 >2.2 >2.22h >10 000 1738 ± 309.5 >10 000 >5.8 >5.8(±)LY2456302 90.9 ± 4.2 11.9 ± 0.09 91.5 ± 8.8 7.6 7.6

a Binding assays were carried out in duplicate.b [3H-DAMGO was used.c [3H]-U69,593 was used.d [3H]-DPDPE was used.

Table 4In vitro [35S]GTP-g-S binding assays for k, m, and d opioid receptors.

Compd m IC50 (nM)a k IC50 (nM)b d IC50 (nM)c m/k d/k

1c 144.2 ± 5.0 9.32 ± 1.09 15.82 ± 3.35 15.5 1.7(±)LY2456302 22.61 ± 6.07 6.68 ± 1.83 95.50 ± 0.33 3.4 14.3

a Mu (m) antagonism was determined in a human [35S] GTP-g-S assay usingDAMGO as the agonist stimulus. The IC50 is reported as the mean ± SEM of at leastthree independent experiments.

b Kappa (k) antagonism was determined in a human [35S] GTP-g-S assay usingU69,593 as the agonist stimulus. The IC50 is reported as the mean ± SEM of at leastthree independent experiments.

c Delta (d) antagonism was determined in a human [35S] GTP-g-S assay usingDPDPE as the agonist stimulus. The IC50 is reported as the mean ± SEM of at leastthree independent experiments.

LY2456302 was used as the positive control. The results indicatedthat all the test compounds could shorten the motionless-time ofmice in the forced swimming test at the dosage of 30mg/kg (Fig. 4).Among them, compounds 1c, 2h and LY2456302 can significantlyshorten the immobility time of mice in the forced swimming testcompared with the normal saline group (P < 0.05), suggesting thatthey have similar antidepressant effect.

2.5.2. Mice elevated plus-maze testBased on the above result, compounds 1c and 2hwere chosen to

evaluate their anti-anxiety effect in the elevated plus-maze test(EPM) which is the international common-used anxiety animalmodel. The results showed that compounds 1c, 2h and (±)LY2456302 can significantly increase the number proportion ofmice going into the open arms [OE/(CE þ OE)] and the retentiontime proportion in the open arms [OT/(OT þ CT)] compared withthe blank control group (P < 0.01) (Fig. 5). Based on these profiles,

Fig. 4. Mice forced swimming test of compounds 2h, 1c, 1b, (±)LY2456302 and vehicle(dosages 30 mg/kg, i.p.). Each bar represents mean ± SEM of immobility time (s) ob-tained from 10 mice (*p < 0.05 vs. vehicle).

Fig. 5. Representative images show typical examples of compounds 2h, 1c, (±)LY2456302 and vehicle (dosages 30 mg/kg, i.p.) mice exploring in the elevated plus maze apparatus.(A) Each bar represents mean ± SEM of the number proportion of mice going into the open arms [OE/(CE þ OE)] obtained from 10 mice (*p < 0.01 vs. vehicle). (B) Each barrepresents mean ± SEM of the retention time proportion in the open arms [OT/(OT þ CT)] obtained from 10 mice (*p < 0.01 vs. vehicle).

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e2520

compounds 1c and 2h showed some anti-anxiety effect.

3. Conclusions

In conclusion, highly selective antagonists for k opioid receptorhave been found from the aminobenzyloxyarylamide scaffold. Allthe compounds were biologically evaluated for their binding af-finities for opioid receptors. Representative compounds wereevaluated in vitro for opioid receptors antagonist potency andin vivo for their antidepressant and anti-anxiety effects. The highestaffinity and selectivity for k receptor was found for compound 1c,with k Ki ¼ 15.7 nM. The selectivity of 1c over m and d opioid re-ceptors was improved nearly 2-fold in comparison to that of (±)LY2456302. Compound 1c also showed good in vitro k antagonistpotency in GTP-g-S functional assays, with k IC50¼ 9.32 nM. Inmiceforced swimming and elevated plus-maze tests, compound 1cshowed good effect on antidepressant and anti-anxiety, whichindicated that it could be a promising candidate for the treatmentof depression.

4. Experimental section

4.1. General procedures

Reactions were monitored by thin-layer chromatography onsilica gel plates (60F-254) visualized under UV light. Melting pointswere determined on a Mel-TEMP II melting point apparatuswithout correction. 1HNMR and 13CNMR spectra were recorded inCDCl3 on a Bruker Avance 300 MHz spectrometer at 300 MHz and75 MHz, respectively. Chemical shifts (d) are reported in parts permillion (ppm) from tetramethylsilane (TMS) using the residualsolvent resonance (CDCl3: 7.26 ppm for 1H NMR, 77.16 ppm for 13CNMR. Multiplicities are abbreviated as follows: s ¼ singlet,d¼ doublet, t¼ traplet, q¼ quartet, m¼multiplet). IR spectrawererecorded on a Nicolet iS10 Avatar FT-IR spectrometer using KBrfilm. MS spectra were recorded on a LC/MSD TOF HR-MS Spectrum.

All chemicals purchased from commercial suppliers were usedas received unless otherwise stated. Reactions and chromatographyfractions were monitored by Merck silica gel 60F-254 glass TLCplates. All solvents were reagent grade and, when necessary, werepurified and dried by standard methods.

4.2. Preparation of the compounds

4.2.1. General procedure for the preparation of intermediates 7a-hTo a solution of substituted benzoic acid 3a-h (0.07 mol) in

absolute ethyl alcohol (100 mL) was slowly added thionyl chloride

(15 mL). The mixture was heated to reflux for 2 h, and then cooledto room temperature. The solvent was removed and the solid res-idue was dissolved in ethyl acetate (100 mL), and washed withsaturated NaHCO3 solution (100 mL � 2). The organic layer wasdried and concentrated to give the intermediates 4a-h as paleyellow oily matter in 94.4e99.4% yield. They were used in the nextstep without additional purification.

To a solution of NaH (5.6 g, 0.14 mol, 60%) in anhydrous THF(250 mL) was added 4a-h (0.07 mol) under the mechanical agita-tion. After heating to 60 �C, N-Vinylpyrrolidone (0.07 mol) wasadded by dropwise and the mixture was heated to 72 �C for 3 h.Then the reaction mixture was cooled to room temperature, thenpoured into ice water (500 mL), extracted with ethyl acetate(100 � 3 mL), and washed with saturated NH4Cl solution (300 mL)and brine (300 mL). The organic layer was dried and concentratedto give the intermediates 5a-h as brown oily matter in 82.3e94.2%yield, which was used in the next step without additionalpurification.

A solution of 5a-h (0.06 mol) in THF (15 mL) was added to the5 N HCl (60 mL) under reflux. After the reaction mixture wasrefluxed for 3e5 h, then the reaction mixture was cooled to roomtemperature and concentrated in vacuo to remove the solvent THF.The pH was adjusted to 11 with saturated NaOH solution, thenextracted with ethyl acetate (100 mL � 3), and washed with brine(150 mL � 2). The organic layer was dried and concentrated to givethe intermediates 6a-h as brown oily matter in 82.8e87.2% yield,which was used in the next step without additional purification.

To a solution of 6a-h (0.05 mol) in methanol (80 mL) was addedacetic acid two drops, and then NaBH4 (3.8 g, 0.1mol) was addedslowly under the condition of ice salt bath. After reaction at roomtemperature for 3 h, the solvent was removed and the residue wasdissolved in water. The pH was adjusted to 1 with 6 N HCl, washedwith methyl tertiary butyl ether (50 mL � 3), and then the pH wasadjusted to 12 with NaOH solution. The mixturewas extracted withdichloromethane (100 mL � 3), and washed with brine(150 mL � 2). The organic layer was dried and concentrated to givethe brown oil, which was dissolved in acetone to form oxalic acidsalt. Intermediates 7a-h were obtained as white solid in42.3e55.6% yield.

4.2.2. General procedure for the preparation of compounds 1a-dTo a solution of 7h (0.8 g, 4.8 mmol) in methanol (20 mL) was

added substituted benzaldehyde (4.8 mmol) and acetic acid(0.8 mL). After stirring at room temperature for 1 h, NaBH3CN(0.38 g, 9.6 mmol) was added in batches under the condition of icebath. After reaction at room temperature for 12 h, the solvent wasremoved and water (20 mL) was added. The pH was adjusted to

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e25 21

9e10 with saturated Na2CO3 solution, then extracted with ethylacetate (50 mL � 3), and washed with brine (100 mL � 2). Theorganic layer was dried and concentrated to give the intermediates8a-b as light yellow oil in 81.1e83.4% yield.

To a solution of 8a-b (1.58mmol) in anhydrous DMF (10mL)wasadded K2CO3 powder (0.44 g, 3.17 mmol). After stirring at roomtemperature for 0.5 h, intermediate 9a-b (1.58 mmol) was addedand the mixture was heated to 140 �C for 10 h under nitrogenprotection. After cooling to room temperature, water (50 mL) wasadded, then the mixture was extracted with ethyl acetate(50 mL � 3) and washed with water (100 mL � 2) and brine(100 mL � 2). The organic layer was dried and concentrated, theresidue was dissolved in DMSO (10 mL), and then 30% H2O2 (3 mL)was added in at 10 �C. After stirring at room temperature for 3 h,water (50 mL) was added and then the mixture was extracted withethyl acetate (30 mL � 3), and washed with water (50 mL � 2) andbrine (50 mL � 2). The organic layer was dried and concentrated,and the pure products 1a-d were obtained with further purified bycolumn chromatograph on silica gel (petroleum ether/ethylacetate ¼ 5/1) as white solid in 38.2e45.5% yield.

4.2.2.1. 4-[2-Chloro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]meth-ylphenoxyl] benzamide (1a). Yield: 42.1%. White solid.M.p.:146e147 �C. 1H NMR (300 MHz, CDCl3) d (ppm): 7.77 (d,J ¼ 8.8 Hz, 2H, ArH), 7.40 (s, 1H, ArH), 7.18 (d, J ¼ 8.3 Hz, 1H, ArH),7.10e6.96 (m, 3H, ArH), 6.91 (d, J ¼ 8.7 Hz, 3H, ArH), 6.23e5.51 (br,2H), 3.79 (d, J¼ 13.1 Hz, 1H, 1/2CH2), 3.28 (t, J¼ 7.9 Hz,1H, CH), 3.14(t, J ¼ 7.8 Hz, 1H, 1/2CH2), 3.04 (d, J ¼ 13.2 Hz, 1H, 1/2CH2), 2.33 (s,6H, 2CH3), 2.23e2.14 (m, 2H, CH2), 1.98e1.69 (m, 3H, CH2, 1/2CH2).13C NMR (75 MHz, CDCl3) d (ppm): 168.32, 160.13, 148.92, 142.88,137.90, 137.48, 130.50, 128.90, 128.34, 127.94, 127.12, 125.79, 124.87,121.44, 115.97, 76.99, 76.56, 76.14, 69.39, 56.91, 53.17, 34.54, 21.83,20.88. HRMS (ESI):m/z [MþH]þ. Calcd for C26H27ClN2O2: 435.1839;Found: 435.1841. IR (cm�1): 3391.98, 3184.63, 2967.84, 2911.73,2793.11, 1645.07, 1609.17, 1580.04, 1509.50, 1488.85, 1419.49,1401.87, 1364.10, 1299.40, 1259.32, 1213.34, 1171.14, 1147.14, 1115.57,1058.30, 900.92, 845.06, 794.84, 688.13, 641.34, 622.12, 547.10,482.50.

4.2.2.2. 4-[2-Fluoro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]methyl phenoxyl] benzamide (1b). Yield: 38.2%. White solid.M.p.:122e124 �C. 1H NMR (300 MHz, CDCl3) d (ppm): 7.77 (d,J¼ 8.7 Hz, 2H, ArH), 7.17 (d, J¼ 11.7 Hz, 1H, ArH), 7.10e6.98 (m, 4H),6.99e6.84 (m, 3H, ArH), 5.91 (br, 2H), 3.81 (d, J ¼ 13.3 Hz, 1H, 1/2CH2), 3.29 (t, J¼ 7.9 Hz,1H, CH), 3.14 (t, J¼ 7.7 Hz,1H,1/2CH2), 3.03(d, J ¼ 13.3 Hz, 1H, 1/2CH2), 2.33 (s, 6H, 2CH3), 2.24e2.10 (m, 2H,CH2), 1.98e1.64 (m, 3H, CH2, 1/2CH2). 13C NMR (75 MHz, CDCl3)d (ppm): 168.51, 160.41, 155.4, 152.11, 142.92, 137.48, 128.87, 128.36,127.15, 124.84, 124.36, 121.90, 116.93, 116.69, 115.63, 77.01, 76.59,76.16, 69.34, 57.01, 53.13, 34.54, 21.85, 20.89. HRMS (ESI): m/z[MþH]þ. Calcd for C26H27FN2O2: 419.2135; Found: 419.2136. IR(cm�1): 3392.39, 3191.20, 2966.28, 2914.71, 2787.19, 1647.79,1604.00, 1578.12, 1505.66, 1417.67, 1396.24, 1279.23, 1224.83,1167.19, 1111.29, 951.01, 862.85, 847.45, 703.50, 620.15.

4.2.2.3. 4-[2-Chloro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]meth-ylphenoxyl]-3-fluorobenzamide (1c). Yield: 45.5%. White solid.M.p.:68e70 �C. 1H NMR (300 MHz, CDCl3) d (ppm): 7.67 (dd,J ¼ 11.2, 2.1 Hz, 1H, ArH), 7.53e7.43 (m, 1H, ArH), 7.22e7.13 (m, 1H,ArH), 7.10e6.95 (m, 4H, ArH), 6.91e6.80 (m, 2H, ArH), 5.90 (br, 2H),3.81 (d, J ¼ 13.3 Hz, 1H, 1/2CH2), 3.35e3.26 (m, 1H, CH), 3.15e3.01(m, 2H, CH2), 2.32 (s, 6H, 2CH3), 2.26e2.15 (m, 2H, CH2), 2.00e1.70(m, 3H, CH2, 1/2CH2). 13C NMR (75 MHz, CDCl3) d (ppm): 154.85,153.51, 151.55, 150.20, 137.50, 128.39, 124.84, 124.32, 123.25, 123.20,120.83, 117.12, 116.96, 116.72, 116.12, 115.86, 114.50, 76.96, 76.54,

76.12, 69.35, 56.92, 53.09, 34.47, 21.82, 20.87. HRMS (ESI): m/z[MþH]þ. Calcd for C26H26ClFN2O2: 453.1745; Found: 453.1742. IR(cm�1): 3411.14, 3200.42, 2965.59, 2916.15, 2872.37, 2788.93,1663.46, 1618.21, 1578.28, 1511.59, 1489.05, 1438.41, 1382.50,1277.29, 1231.61, 1201.20, 1153.73, 1095.54, 1054.84, 934.02, 897.91,846.71, 756.54, 704.05, 614.80.

4.2.2.4. 4-[2-Fluoro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]meth-ylphenoxyl]-3-fluorobenzamide (1d). Yield: 43.8%. White solid.M.p.:70e72 �C. 1H NMR (300MHz, CDCl3) d (ppm): 7.70 (dd, J¼ 11.1,2.1 Hz,1H, ArH), 7.54e7.47 (m,1H, ArH), 7.42 (d, J¼ 1.8 Hz,1H, ArH),7.19 (dd, J¼ 8.3,1.7 Hz,1H, ArH), 7.07 (s, 2H, ArH), 7.00e6.87 (m, 2H,ArH), 6.79 (t, J ¼ 8.3 Hz, 1H, ArH), 5.91 (br, 2H), 3.81 (d, J ¼ 13.2 Hz,1H,1/2CH2), 3.30 (t, J¼ 7.9 Hz,1H, CH), 3.15e2.97 (m, 2H, CH2), 2.34(s, 6H, 2CH3), 2.29e2.15 (m, 2H, CH2), 2.02e1.68 (m, 3H, CH2, 1/2CH2). 13C NMR (75MHz, DMSO-d6) d (ppm): 165.91,153.23,149.96,149.11, 146.12, 145.97, 143.30, 137.94, 137.17, 130.59, 130.52, 130.14,128.51, 128.36, 124.98, 124.73, 123.76, 120.31, 118.30, 116.36, 116.11,68.81, 56.30, 53.00, 34.74, 21.94, 20.92. HRMS (ESI): m/z [MþH]þ.Calcd for C26H26F2N2O2: 437.2041; Found: 437.2040. IR (cm�1):3411.19, 3200.95, 2965.54, 2915.44, 2788.75, 1662.82, 1618.36,1587.04, 1489.02, 1437.98, 1382.56, 1277.13, 1231.45, 1200.92,1139.32, 1095.95, 1054.96, 934.24, 898.46, 846.87, 763.69, 703.61,615.50.

4.2.3. General procedure for the preparation of compounds 2a-hTo a solution of 1-bromo-4-fluoronaphthalene 11 (0.5 g,

2.22 mmol) and K4Fe(CN)6 (0.23 g, 0.62 mmol) in DMAc (20 mL)was added anhydrous Na2CO3 (0.24 g, 1.69 mmol) and Pd(OAc)2(0.02 g, 0.1 mmol). The mixturewas heated to 120 �C for 12 h undernitrogen protection, then the reaction mixture was cooled to roomtemperature. Water (50 mL) was added and then the mixture wasextracted with ethyl acetate (50 mL � 3), and washed with brine(100 mL � 2). The organic layer was dried and concentrated to givethe intermediate 12 (0.34 g) as off-white solid in 88.4% yield.

To a solution of 4-hydroxybenzaldehyde (4.78 g, 39.1 mmol) inDMF (100 mL) was added K2CO3 powder (10.8 g, 78.3 mmol). Afterstirring at room temperature for 0.5 h, intermediate 12 (6.7 g,39.1 mmol) was added and the mixture was heated to 140 �C for12 h under nitrogen protection. After cooling to room temperature,the reaction mixture was filtered and washed with ethyl acetate(50 mL � 3), then the filtrate was washed with water (100 mL � 2)and brine (100 mL � 2). The organic layer was dried and concen-trated to give the crude product, which was recrystallized inethanol-water mixed solvent to give intermediate 13 (8.78 g) aswhite solid in 81.6% yield.

To a solution of 13 (8.26 g, 30.22 mmol) in DMSO (100 mL) wasadded K2CO3 powder (2.1 g, 15.2 mmol), and then 30% H2O2(13.7 mL) was added at 10 �C. After stirring at room temperature for3 h, water (300 mL) was added and then the mixture was extractedwith ethyl acetate (100 mL � 3), and washed with water(200 mL � 2) and brine (200 mL � 2). The organic layer was driedand concentrated to give the intermediate 14 (7.6 g) as light yellowsolid in 81.4% yield.

To a solution of 14 (0.5 g, 1.72 mmol) in the mixture of absolutemethanol (20 mL) and anhydrous dichloromethane (10 mL) wasadded 7a-h (1.72 mmol). After stirring at room temperature for0.5 h, acetic acid (0.8 mL) and NaBH3CN (0.22 g, 3.44 mmol) wasadded. After reaction at room temperature for 8 h, the solvent wasremoved and water (30 mL) was added. The pH was adjusted to9e10 with saturated Na2CO3 solution, then extracted with ethylacetate (50 mL � 3), and washed with brine (100 mL � 2). Theorganic layer was dried and concentrated, and the pure products2a-h were obtained with further purified by column chromato-graph on silica gel (dichloromethane/ethyl acetate ¼ 5/1) as white

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e2522

solid in 58.7e81.2% yield.

4.2.3.1. 4-[4-[2-(3,5-Dimethylphenyl)pyrrolidin-1-yl]methyl-phenoxyl]-1-naphthamide (2a). Yield: 81.2%. White solid.M.p.:164e166 �C. 1H NMR (300 MHz, CDCl3) d (ppm): 8.54 (d,J ¼ 8.6 Hz, 1H, ArH), 8.39 (d, J ¼ 7.8 Hz, 1H, ArH), 7.67e7.57 (m, 3H,ArH), 7.32 (d, J ¼ 8.3 Hz, 2H, ArH), 7.10 (s, 2H, ArH), 7.04 (d,J ¼ 8.4 Hz, 2H, ArH), 6.92 (s, 1H, ArH), 6.78 (d, J ¼ 7.9 Hz, 1H, ArH),5.88 (br, 2H, NH2), 3.87 (d, J ¼ 13.0 Hz, 1H, 1/2CH2), 3.31 (t,J ¼ 8.0 Hz, 1H, CH), 3.15 (t, J ¼ 8.1 Hz, 1H, 1/2CH2), 3.06 (d,J ¼ 12.8 Hz, 1H, 1/2CH2), 2.35 (s, 6H, 2CH3), 2.27e2.13 (m, 2H, CH2),1.99e1.67 (m, 3H, 1/2CH2, CH2). 13C NMR (75 MHz, CDCl3) d (ppm):170.95, 155.91, 154.61, 143.13, 137.42, 135.40, 131.31, 129.87, 128.24,127.54, 126.91, 126.08, 125.88, 125.74, 125.03, 124.84, 121.88, 118.99,109.28, 69.28, 57.23, 53.01, 34.61, 21.78, 20.90. HRMS (ESI): m/z[MþH]þ. Calcd for C30H30N2O2: 451.23 846; Found: 451.2384. IR(cm�1): 3380.46, 3187.62, 2964.48, 2920.45, 2779.77, 1644.93,1614.12, 1585.52, 1504.30, 1464.24, 1428.47, 1365.01, 1321.52,1264.12, 1242.33, 1217.22, 1161.26, 1116.02, 1098.52, 1053.21,1018.78, 989.32, 847.38, 830.81, 769.17, 706.10, 647.10, 530.19,515.19, 515.08, 419.27.

4.2.3.2. 4-[4-[2-(3-fluorophenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2b). Yield: 76.2%. White solid. M.p.:116e118 �C. 1HNMR (300 MHz, CDCl3) d (ppm): 8.54 (d, J ¼ 8.5 Hz, 1H, ArH), 8.38(d, J ¼ 8.4 Hz, 1H, ArH), 7.75e7.52 (m, 3H, ArH), 7.32 (d, J ¼ 8.4 Hz,3H, ArH), 7.23 (d, J ¼ 7.6 Hz, 2H, ArH), 7.04 (d, J ¼ 8.5 Hz, 2H, ArH),6.96 (t, J¼ 8.2 Hz,1H, ArH), 6.77 (d, J¼ 7.9 Hz,1H, ArH), 5.91 (br, 2H,NH2), 3.84 (d, J ¼ 12.9 Hz, 1H, 1/2CH2), 3.42 (t, J ¼ 8.1 Hz, 1H, CH),3.15 (t, J ¼ 11.4 Hz, 2H, 1/2CH2), 2.40e2.13 (m, 2H, CH2), 2.00e1.64(m, 3H, CH2, 1/2CH2). 13C NMR (75 MHz, CDCl3) d (ppm): 171.11,164.32, 161.07, 155.81, 154.68, 146.53, 135.14, 131.31, 129.69, 129.33,127.53, 127.01, 125.07, 125.88, 125.74, 125.04, 122.68, 121.86, 119.01,113.45, 109.33, 68.63, 57.16, 53.00, 34.75, 21.95. HRMS (ESI): m/z[MþH]þ. Calcd for C28H25FN2O2: 441.1978; Found: 441.1979. IR(cm�1): 3386.48, 3181.29, 2964.51, 2799.19, 1641.12, 1588.32,1502.76, 1462.52, 1447.77, 1428.50, 1322.91, 1361.55, 1266.66,1242.45, 1241.56, 1160.27, 1137.38, 1094.06, 1054.79, 1015.69,988.92, 864.35, 838.06, 785.44, 768.39, 741.41, 712.44, 695.12,650.46, 613.25, 512.80, 464.66.

4.2.3.3. 4-[4-[2-(3-Chlorophenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2c). Yield: 71.7%. White solid. M.p.:148e149 �C. 1HNMR (300 MHz, CDCl3) d (ppm): 8.54 (d, J ¼ 8.3 Hz, 1H, ArH), 8.38(d, J ¼ 8.0 Hz, 1H, ArH), 7.72e7.52 (m, 3H, ArH), 7.41 (d, J ¼ 8.5 Hz,2H, ArH), 7.32 (d, J ¼ 8.4 Hz, 3H, ArH), 7.03 (d, J ¼ 8.4 Hz, 2H, ArH),6.76 (d, J ¼ 7.9 Hz, 1H, ArH), 5.90 (br, 2H, NH2), 3.80 (d, J ¼ 13.1 Hz,1H, 1/2CH2), 3.39 (t, J ¼ 8.0 Hz, 1H, CH), 3.20e3.08 (m, 2H, CH2),2.31e2.12 (m, 2H, CH2), 2.01e1.66 (m, 3H, CH2, 1/2CH2). 13C NMR(75 MHz, CDCl3) d (ppm): 171.09, 155.81, 154.69, 142.08, 135.13,131.99, 131.31, 129.69, 128.39, 128.09, 127.54, 127.01, 126.07, 125.89,125.78, 125.04, 121.86, 118.99, 109.34, 68.47, 57.11, 53.05, 34.81,21.92. HRMS (ESI): m/z [MþH]þ. Calcd for C28H25ClN2O2: 457.1683;Found: 457.1680. IR (cm�1): 3380.49, 3181.36, 2963.70, 2803.77,1641.70, 1611.22, 1588.66, 1502.19, 1462.70, 1428.56, 1412.96,1362.03, 1323.21, 1266.30, 1243.73, 1215.28, 1159.57, 1118.37,1088.90, 1054.90, 1013.85, 989.30, 824.93, 767.90, 713.15, 662.99,606.18, 536.97, 513.66.

4.2.3.4. 4-[4-[2-(4-Methoxylphenyl)pyrrolidin-1-yl]methyl-phenoxyl]-1-naphthamide (2d). Yield: 68.3%. White solid.M.p.:120e122 �C. 1H NMR (300 MHz, CDCl3) d (ppm): 8.53 (d,J ¼ 8.6 Hz, 1H, ArH), 8.38 (d, J ¼ 8.3 Hz, 1H, ArH), 7.72e7.49 (m, 3H,ArH), 7.38 (d, J ¼ 8.1 Hz, 2H, ArH), 7.32e7.26 (m, 2H, ArH), 7.01 (d,J ¼ 8.5 Hz, 2H, ArH), 6.90 (d, J ¼ 8.6 Hz, 2H, ArH), 6.74 (d, J ¼ 8.0 Hz,

1H, ArH), 5.91 (br, 2H, NH2), 3.88e3.81 (m, 4H, 1/2CH2, CH3),3.38e3.30 (m, 1H, CH), 3.22e2.97 (m, 2H, CH2), 2.33e2.12 (m, 2H,CH2), 2.01e1.66 (m, 3H, 1/2CH2, CH2). 13C NMR (75 MHz, CDCl3)d (ppm): 171.18, 158.21,155.85,154.57,135.48, 135.19,131.30,129.76,128.12, 127.50, 126.98, 126.98, 126.04, 125.85, 125.76, 125.05, 121.86,119.00, 113.35, 109.27, 68.68, 57.01, 54.79, 52.95, 34.66, 21.72. HRMS(ESI): m/z [MþH]þ. Calcd for C29H28N2O3: 453.2178; Found:453.2179. IR (cm�1): 3380.20, 3193.37, 2950.87, 2904.06, 2807.96,1731.86, 1641.80, 1612.90, 1580.69, 1512.02, 1503.30, 1464.57,1428.32, 1362.20, 1319.76, 1299.70, 1263.85, 1240.47, 1214.34,1158.97, 1119.81, 1106.34, 1051,52, 1033.59, 851.34, 827.35, 813.07,767.44, 736.22, 671.13, 649.36, 544.04, 530.84, 514.99.

4.2.3.5. 4-[4-[2-(2-Methylphenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2e). Yield: 75.8%. White solid. M.p.:160e162 �C. 1HNMR (300 MHz, CDCl3) d (ppm): 8.54 (d, J ¼ 8.5 Hz, 1H, ArH), 8.39(d, J ¼ 8.1 Hz, 1H, ArH), 7.66 (d, J ¼ 7.4 Hz, 1H, ArH), 7.65e7.51 (m,3H, ArH), 7.33 (d, J ¼ 8.4 Hz, 2H, ArH), 7.25e7.19 (m, 1H, ArH), 7.14(d, J ¼ 3.9 Hz, 2H, ArH), 7.02 (d, J ¼ 8.5 Hz, 2H, ArH), 6.76 (d,J ¼ 8.0 Hz, 1H, ArH), 5.92 (br, 2H, NH2), 3.87 (d, J ¼ 13.3 Hz, 1H, 1/2CH2), 3.62 (t, J¼ 8.4 Hz,1H, CH), 3.21e3.12 (m,1H,1/2CH2), 3.07 (d,J ¼ 12.7 Hz, 1H, CH2), 2.37 (s, 3H, CH3), 2.36e2.23 (m, 2H, CH2),2.01e1.75 (m, 2H, CH2), 1.74e1.60 (m, 1H, 1/2CH2). 13C NMR(75 MHz, CDCl3) d (ppm): 171.17, 155.86, 154.61, 141.35, 135.48,135.41, 131.31, 129.72, 127.53, 126.99, 126.07, 125.87, 125.80, 125.77,125.05, 121.88, 119.03, 109.30, 64.97, 57.35, 52.98, 32.67, 22.00,18.95. HRMS (ESI): m/z [MþH]þ. Calcd for C29H28N2O2: 437.2229;Found: 437.2229. IR (cm�1): 3365.73, 3179.23, 2951.42, 2905.54,2806.40, 2775.12, 1646.83, 1614.36, 1584.65, 1465.05, 1430.75,1367.22,1321.02,1241.64,1265.18,1216.11,1161.65,1122.47,1094.42,1053.26, 987.87, 857.68 815.94, 768.48, 753.23, 726.40, 712.32,684.38, 515.16.

4.2.3.6. 4-[4-[2-(Pyridine-3-yl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2f). Yield: 58.7%. White solid. M.p.:168e170 �C. 1HNMR (300 MHz, CDCl3) d (ppm): 8.65 (s, 1H, ArH), 8.54e8.44 (m,2H, ArH), 8.34 (d, J ¼ 8.1 Hz, 1H, ArH), 7.88e7.55 (m, 1H, ArH),7.68e7.48 (m, 3H, ArH), 7.29 (s, 3H, ArH), 7.00 (d, J ¼ 8.5 Hz, 2H,ArH), 6.74 (d, J ¼ 7.9 Hz, 1H, ArH), 5.92 (br, 2H, NH2), 3.77 (d,J ¼ 13.1 Hz, 1H, 1/2CH2), 3.50e3.38 (m, 1H, CH), 3.24e3.12 (m, 2H,CH2), 2.35e2.16 (m, 2H, CH2), 2.00e1.62 (m, 3H, 1/2CH2, CH2). 13CNMR (75 MHz, CDCl3) d (ppm): 170.76, 154.77, 149.12, 148.15,138.89, 134.89, 134.50, 131.32, 129.67, 128.35, 127.56, 126.99, 126.07,125.90, 125.72, 125.04, 123.14, 121.84, 119.00, 109.37, 66.58, 57.13,53.17, 34.78, 22.06. HRMS (ESI):m/z [MþH]þ. Calcd for C27H25N3O2:424.2025; Found: 424.2023. IR (cm�1): 3351.44, 3159.19, 2967.57,2804.48, 1667.44, 1614.80, 1579.34, 1504.63, 1463.49, 1428.41,1407.80, 1362.18, 1320.17, 1262.67, 1230.67, 1206.39, 1162.78,1097.27, 1050.32, 1027.51, 1018.04, 978.53, 866.86, 843.66, 832.34,798.44, 773.71, 713.24, 639.28, 600.12, 513.91.

4.2.3.7. 4-[4-[2-(4-Fluorophenyl)pyrrolidin-1-yl]methylphenoxy]-1-naphthamide (2g). Yield: 66.2%. White solid. M.p.:160e162 �C. 1HNMR (300 MHz, CDCl3) d (ppm): 8.54 (d, J ¼ 8.7 Hz, 1H, ArH),8.38e8.30 (m, 1H, ArH), 7.69e7.52 (m, 3H, ArH), 7.50e7.36 (m, 2H,ArH), 7.32e7.24 (m, 2H, ArH), 7.09e6.97 (m, 4H, ArH), 6.80e6.70(m, 1H, ArH), 5.91 (br, 2H, NH2), 3.88e3.70 (m, 1H, 1/2CH2),3.45e3.29 (m, 1H, CH), 3.22e3.01 (m, 2H, CH2), 2.33e2.12 (m, 2H,CH2), 2.04e1.64 (m, 3H, 1/2CH2, CH2). 13C NMR (75 MHz, CDCl3)d (ppm): 170.91, 163.05, 159.81, 155.87, 154.64, 139.03, 135.25,131.31, 129.68, 128.50, 128.40, 127.55, 126.94, 126.07, 125.89, 125.74,125.03, 121.86, 119.00, 114.85, 114.57, 109.29, 68.49, 57.07, 53.04,34.84, 21.82. HRMS (ESI): m/z [MþH]þ. Calcd for C28H25FN2O2:441.1978; Found: 441.1978. IR (cm�1): 3388.51, 3183.94, 2965.35,2806.55, 1641.05, 1611.26, 1588.39, 1503.26, 1462.94, 1428.44,

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e25 23

1362.46, 1323.09, 1266.64, 1242.70, 1215.96, 1159.62, 1095.05,1054.78, 1015.02, 833.39, 768.44, 512.16.

4.2.3.8. 4-[4-[2-(3-Methylphenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2h). Yield: 72.1%. White solid. M.p.:158e159 �C. 1HNMR (300MHz, CDCl3) d (ppm): 8.51 (d, J¼ 8.1 Hz,1H, ArH), 8.35 (d,J¼ 8.2 Hz,1H, ArH), 7.72e7.49 (m, 3H, ArH), 7.37e7.22 (m, 6H, ArH),7.08 (d, J ¼ 6.4 Hz, 1H, ArH), 7.01 (d, J ¼ 8.5 Hz, 2H, ArH), 6.75 (d,J ¼ 7.9 Hz, 1H, ArH), 5.97 (br, 2H, NH2), 3.85 (d, J ¼ 12.4 Hz, 1H, 1/2CH2), 3.43e3.24 (m,1H, CH), 3.33e3.31 (m,1H,1/2CH2), 3.25e2.96(m, 2H, CH2), 2.37 (s, 3H, CH3), 2.31e2.10 (m, 2H, CH2), 1.98e1.69(m, 3H, 1/2CH2, CH2). 13C NMR (75 MHz, CDCl3) d (ppm): 171.11,155.87, 154.62, 143.24, 137.51, 135.42, 131.32, 129.81, 127.88, 127.77,127.52, 127.33, 126.99, 126.08, 125.87, 125.75, 125.05, 124.14, 121.88,118.98, 109.31, 69.26, 57.17, 53.02, 34.69, 21.85, 21.04. HRMS (ESI):m/z [MþH]þ. Calcd for C29H28N2O2: 437.2229; Found: 437.2229. IR(cm�1): 3385.50, 3185.50, 2951.39, 2783.35, 1641.40, 1613.01,1582.72, 1504.14, 1427.01, 1409.71, 1358.42, 1263.21, 1319.48,1238.06, 1216.91, 1160.87, 1121.85, 1096.97, 1051.48, 779.99, 701.08,629.75, 606.39.

4.3. Docking studies

The molecular modeling study was performed using the pub-lished crystal structure of k opioid receptor in complex with JDTic(Protein Data Bank code: 4DJH) [26] and the structure was editoraccording to provide a monomer of the protein and protonated useGOLD 5.1. The ligand was then removed to leave the receptorcomplex, which was used for the subsequent docking studies. Forpreparation of ligand structures, the selected analogs were energyminimized using MOE 2013.08 with a MMFF94� forcefield usinggas phase calculations and a cutoff of 0.01. Charges were then fixedusing an MMFF94 forcefield. For computational docking, GOLD 5.1software was used in combination with ChemScore scoring func-tion. The active site was defined as being any volumewithin 10 Å ofthe scaffold of ligand A in its crystal pose in 4DJH. The number ofgenetic algorithm (GA) runwas set to 10, and scoring of the dockedposes was performed with the ChemScore scoring function. EachGOLD runwas saved and the strongest scoring binding pose of eachligand was compared to that of the reference ligand positionobserved in the crystal structure. The best output poses of the li-gands generated were analyzed on the basis of ChemScore, feasi-bility of hydride transfer process, and H-bonding to the enzyme.The best poses were visualized with MOE 2013.08.

4.4. Chemicals and reagents for in vitro assays

Membranes from Chinese hamster ovary (CHO) cells expressingthe human k and m opioid receptor and membranes from humanembryonic kidney (HEK) 293 cells expressing the human d opioidreceptor were obtained from Shanghai Institute of Materia Medica,Chinese Academy of Sciences. [3H]-DAMGO, [3H]-DPDPE and [3H]-U69,593 were purchased from Perkin Elmer Italia. DAMGO, DPDPEand U50488 were purchased from Sigma Aldrich. Scintillation re-agents PPO and POPOP were purchased from Sigma Aldrich. Triswas purchased from Amresco company.

4.5. Radioligand binding methods

Radioligand binding assays were carried out according to pre-viously reported procedures [27,28]. Briefly, radioligand displace-ment studies with [3H]-U69,593, [3H]-DAMGO and [3H]-DPDPEwere carried out using membranes prepared from CHO cellsexpressing cloned human k and m opioid receptors or HEK293 cellsexpressing the cloned d opioid receptor. Membranes were

incubated with the appropriate concentration of [3H]-U69,593 or[3H]-DAMGO or [3H]-DPDPE in Tris-HCl buffer at 25 �C for 60min inthe absence or presence of naloxone (1 mM) (for m and d receptors)or U69,593 (10 mM) (for k receptor). The binding reaction wasstopped by rapid filtration under vacuum through glass-fiber filters(Whatman GF/B) using a Brandell 36-sample harvester (Millipore)and thereafter the filters were washed with 4 � 5 mL ice-cold50 mM Tris-HCl buffer (pH 7.4). Filter-bound radioactivity wascounted in a liquid scintillation counter (Pri-carb 2910; Perki-nElmer) using 4 mL of scintillation fluid (Packard Ultima).Displacement curves were carried out using serial dilutions rangingfrom 100 mM to 0.1 nM of the compounds. Compounds were dis-solved in DMSO. Concentrations causing 50% inhibition (IC50) of[3H]-ligand binding were determined from 11-point concentrationresponse curves in assay buffer. All receptor binding experimentswere performed in triplicate and results were confirmed in at leastfour independent experiments. Data from radioligand inhibitionexperiments were analyzed by nonlinear regression analysis of aSigmoid Curve using GraphPad Prism program. Ki values werecalculated from the obtained IC50 values by means of the equationof Cheng and Prusoff [29]. Values of 0.68 nM, 0.92 nM and 1.34 nMfor the dissociation constants of [3H]-U69,593, [3H]-DAMGO and[3H]-DPDPE were used respectively.

4.6. GTP-g-S antagonist functional assays

In vitro [35S] GTP-g-S binding experiments were conducted aspreviously described [24]. Reagent dispensing into plates wasconducted with BeckmanMultimek 96-channel robots and TitertekMultidrops. Stock solutions of test compound or naltrexone werediluted in buffer (50 HEPES, pH 7.4, 5 mMMgCl2) and pipetted intoa 96-well polypropylene plate. Then 11 point concentration curvesthat bracketed the IC50 (a concentration range of 0.01 nMe10 mM)were prepared in this plate using a Tecan Evo liquid handler.Membranes were thawed and diluted with assay buffer (20 mMHEPES, pH 7.4, 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 1 mMdithiothreitol) containing 200 mM GDP and homogenized using aPolytron. A GTP-g-35S mixture (0.5 nM final) was prepared for eachreceptor with the addition of an agonist specific for each opioidreceptor. For k, U69,693 was added (300 nM final assay concen-tration), for m, DAMGOwas added (1 mM final), and for d, DPDPEwasadded (30 nM final). The final agonist concentrations are equal toconcentrations that stimulate approximately 80% of the maximumGTP-g-S binding in the absence of antagonist. Finally, 50 mL of eachcompound dilution, 50 mL of GTP-g-35S/agonist solution, 50 mL ofdiluted membranes (15e30 mg protein), and 50 mL of WGA SPAbeads (1 mg) prepared in assay buffer were added to the assay platein the order listed. Following an incubation of 2 h (d) or 4 h (k and m)at room temperature, the assay plates were placed at 4 �C overnightto allow the SPA beads to settle to the bottom of the well. Radio-activity in the plates was determined using a Trilux Microbetascintillation counter (Perkin-Elmer) and reported in counts perminute (CPM). For total GTP-g-S binding, assay buffer was used inplace of added compound. For nonspecific GTP-g-S binding, thefollowing antagonists were used: k (NorBNI at 10 mM final), m(naltrexone at 10 mM final), and d (naltriben at 10 mM final). Datawere analyzed using four-parameter (curve maximum, curveminimum, IC50, Hill slope) nonlinear regression routines (XLFitversion 4.0; ActivityBase, IDBS), where IC50 is the concentration ofadded compound that results in 50% inhibition of GTP-g-35Sbinding.

4.7. Mice forced swimming test

Male ICR mice weighing 20e25 g were housed in plastic cages

J. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 15e2524

with 10e12 mice/cage in a vivarium for at least 7 days. 50 micewere divided into 5 groups randomly and removed from the vi-varium to the testing area in their home cages, and allowed to adaptto the new environment for at least 15 min before testing. Thegroups were administrated with compounds 1b, 1c, 2h, LY2456302and normal saline as vehicle, respectively (30 mg/kg i.p., 30 minbefore the test) [19]. The forced swim test was performed using theoriginal method described by Porsolt et al. [30]. Briefly, mice wereplaced individually in clear plastic cylinders (10 cm in diameter,25 cm in height) filled to 6 cm with 25 �C water for 6 min. Theduration of immobility was recorded during the last 4 min of a 6-min trial. A mouse was regarded as immobile when floatingmotionless or making only those movements necessary to keep itshead above the water.

4.8. Mice elevated plus-maze test

Elevated plus-maze test apparatus consisted of two open arms(length 30 � width 5 cm) and two closed arms of the same size,along with a semi-transparent wall (height 15 cm) and centralplatform (length 5 �width 5 cm). These arms and central platformwere elevated 50 cm above the floor. Male ICR mice weighing18e22 g were housed in plastic cages with 10e12 mice/cage in avivarium for at least 3 days. 40 mice were divided into 4 groupsrandomly and removed from the vivarium to the testing area andallowed to adapt to the new environment for at least 1 h beforetesting. The mice elevated plus-maze test was performed accordingto published procedures [31]. The groups were administrated withcompounds 1c, 2h, LY2456302 and normal saline as vehicle,respectively (30 mg/kg i.p., 30 min before the test). Thirty-minutesafter the drug administration, each mouse was placed in the centralplatform, facing one of the open arms. During a 5 min test session,mouse behavior was recorded using EthoVision XT. The number ofentries into the open (OE) and closed (CE) arms, and the time spentin the open (OT) and closed (CT) arms were scored. The numberproportion into the open arms [OE/(CE þ OE)] and the retentiontime proportion in the open arms [OT/(OT þ CT)] were calculatedfrom the above indicators.

Acknowledgments

This work was supported by Excellent Science and TechnologyInnovation Team Projects of Jiangsu Province Universities in 2015,China.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2017.02.029.

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