复旦大学：《药物设计学》课程教学资源（教学研究）基于配体结构的药物设计_Novel bis-（?）-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property

Toxicology and Applied Pharmacology 264(2012)65-72 Contents lists available at sciverse science Direct Toxicology and Applied pharmacology ELSEVIER journalhomepagewww.elsevier.com/locate/ytaap Novel bis-()-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property Wei Zheng a, c, I, Juan Li b, Zhuibai Qiu a, Zheng Xia Wei Li, Lining Yu, Hailin Chen, Jianxing Chen Yan Chen b, Zhuqin Hu b, Wei Zhou b, Biyun Shao Yongyao Cui Qiong Xie a, * Hongzhuan Chen b, *x Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zh ng Road, Shanghal 200032, PR China NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of planned Parenthood Research, 2140 Xietu Road, Shanghai 200032, PR China ARTICLE INFO A BSTRACT The strategy of dual binding site acetylcholinesterase(AChE)inhibition along with metal chelate 30May2012 resent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimers disease(AD). In the present study, two derivatives(ZLA and ZlB) of a potent dual binding site vailable online 25 July 2012 values of 9.63 AM(for ZLA)and 8.64 HM(for ZLB), and prevent AChE-induced amyloid-B(AB)aggregation tazinol derivatives with ICso values of 49.1 HM(for ZLA)and 55.3 uM(for ZLB). In parallel, molecular docking analysis showed ase inhibitor that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore A阝 aggregation they exhibited abilities to complex metal ions such as Cu() and Zn(ll), and inhibit AB aggregation triggered letal chelation by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEls with ment of AD 02012 Elsevier Inc. All rights reserved. Introduction disease-modifying effects. While multiple-medication therapy may have potential disadvantages to AD patients such as ADME interactions Alzheimers disease(AD)is a progressive neurodegenerative disorder, and poor compliance, one-compound-multiple-target strategy may be with extracellular senile plaques formed by aggregates of amyloid-B(AB) more favorable in AD treatment. In recent years, several multifunctional as one of the most prominent neuropathological hallmarks, which may agents capable of hitting different biological targets have been developed, trigger cholinergic dysfunction in brain(Mancuso et al, 2010: Schliebs and their biological profiles seem to be promising( Bajda et al, 2011 nd Arendt, 2011). To date, acetylcholinesterase inhibitors(AChEls), Fernandez-Bachiller et al, 2010: Minarini et al, 2012 such as tacrine, donepezil, galantamine and rivastigmine, are the main Biochemical and neuropathological evidence converge to suggest approved agents for AD therapy. However they do not retard the neuro- that dyshomeostasis of cerebral biometals( such as copper, zinc and degenerative processes, although beneficial in improving cognitive and iron) plays crucial roles in Ap aggregation and neurotoxicity, which behavioral symptoms. The multifactorial nature of AD strongly suggests are central in neurodegeneration related to AD( Duce and Bush, 2010: that combination therapy with several drugs intervening in various path- Hung et al, 2010). In amyloid plaques, elevated concentrations of physiological processes or compounds capable of interacting with sev- Cu(ll)and Zn(il) have been detected by spectroscopic studies, and eral molecule targets involved in the neurotoxic cascade may have in vitro experiments these metals are able to bind to AB and promot its aggregation. Furthermore, redox-active metal ions like copper and ron contribute to production of reactive oxygen species(ROS) and ox idative stress, which are early events of neurod generatIon Therefore Abbreviations:AD,Alzheimer's disease: AB, amyloid-A: AChE, acetylcholinesterase: metal chelation may represent a rational therapeutic approach for xcretion;ROS, reactive oxygen species; ACh, acetylcholine: MEP, meptazinol: LAH, lithi. interdicting AD pathogenesis. Several lipophilic metal chelators such aluminum hydride; THE, tetrahydrofuran: HFIP, 1. 1,1,3,3.3-hexafluoro-2-propanol; as clioquinol and its derivative PBt2 have been studied in clinical trials TPA, diethylenetriaminepentaacetic acid; CCK-8, cell counting kit-8: TcAChE, Torpedo and shown encouraging results in some Ad patients(Faux et al, 2010 s Corresponding author. Fax: +86 21 5198012 Guay,2004) ** Corresponding author. Fax: 86 21 64674721 The recent discovery of the so-called"nonclassical function"of E-mail addresses: yaolieshsmuedu cn(H Chen) xiejoanxqegmaiLcom(Q Xie). AChE has renewed interest in search for novel AChEls with 0041-008X/S- see front matter o 2012 Elsevier Inc. All rights reserved. http://dxdoiorg/10.1016/j.taap.2012.07.018

Novel bis-(−)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property Wei Zheng a,c,1 , Juan Li b,1 , Zhuibai Qiu a , Zheng Xia b , Wei Li a , Lining Yu c , Hailin Chen c , Jianxing Chen c , Yan Chen b , Zhuqin Hu b , Wei Zhou b , Biyun Shao b , Yongyao Cui b , Qiong Xie a, ⁎, Hongzhuan Chen b, ⁎⁎ a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 200032, PR China b Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, PR China c NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032, PR China article info abstract Article history: Received 30 May 2012 Revised 11 July 2012 Accepted 17 July 2012 Available online 25 July 2012 Keywords: Bis-(−)-nor-meptazinol derivatives Acetylcholinesterase inhibitor Aβ aggregation Metal chelation The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may rep￾resent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(−)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal che￾lating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC50 values of 9.63 μM (for ZLA) and 8.64 μM (for ZLB), and prevent AChE-induced amyloid-β (Aβ) aggregation with IC50 values of 49.1 μM (for ZLA) and 55.3 μM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aβ aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treat￾ment of AD. © 2012 Elsevier Inc. All rights reserved. Introduction Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with extracellular senile plaques formed by aggregates of amyloid-β (Aβ) as one of the most prominent neuropathological hallmarks, which may trigger cholinergic dysfunction in brain (Mancuso et al., 2010; Schliebs and Arendt, 2011). To date, acetylcholinesterase inhibitors (AChEIs), such as tacrine, donepezil, galantamine and rivastigmine, are the main approved agents for AD therapy. However, they do not retard the neuro￾degenerative processes, although beneficial in improving cognitive and behavioral symptoms. The multifactorial nature of AD strongly suggests that combination therapy with several drugs intervening in various path￾ophysiological processes or compounds capable of interacting with sev￾eral molecule targets involved in the neurotoxic cascade may have disease-modifying effects. While multiple-medication therapy may have potential disadvantages to AD patients such as ADME interactions and poor compliance, one-compound-multiple-target strategy may be more favorable in AD treatment. In recent years, several multifunctional agents capable of hitting different biological targets have been developed, and their biological profiles seem to be promising (Bajda et al., 2011; Fernández-Bachiller et al., 2010; Minarini et al., 2012). Biochemical and neuropathological evidence converge to suggest that dyshomeostasis of cerebral biometals (such as copper, zinc and iron) plays crucial roles in Aβ aggregation and neurotoxicity, which are central in neurodegeneration related to AD (Duce and Bush, 2010; Hung et al, 2010). In amyloid plaques, elevated concentrations of Cu(II) and Zn(II) have been detected by spectroscopic studies, and in in vitro experiments these metals are able to bind to Aβ and promote its aggregation. Furthermore, redox-active metal ions like copper and iron contribute to production of reactive oxygen species (ROS) and ox￾idative stress, which are early events of neurodegeneration. Therefore, metal chelation may represent a rational therapeutic approach for interdicting AD pathogenesis. Several lipophilic metal chelators such as clioquinol and its derivative PBT2 have been studied in clinical trials and shown encouraging results in some AD patients (Faux et al., 2010; Guay, 2004). The recent discovery of the so-called “nonclassical function” of AChE has renewed interest in search for novel AChEIs with real disease-modifying potency. It has been reported that AChE might Toxicology and Applied Pharmacology 264 (2012) 65–72 Abbreviations: AD, Alzheimer's disease; Aβ, amyloid-β; AChE, acetylcholinesterase; AChEIs, acetylcholinesterase inhibitors; ADME, absorption, distribution, metabolism and excretion; ROS, reactive oxygen species; ACh, acetylcholine; MEP, meptazinol; LAH, lithi￾um aluminum hydride; THF, tetrahydrofuran; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; DTPA, diethylenetriaminepentaacetic acid; CCK-8, cell counting kit-8; TcAChE, Torpedo californica AChE. ⁎ Corresponding author. Fax: +86 21 51980122. ⁎⁎ Corresponding author. Fax: + 86 21 64674721. E-mail addresses: yaoli@shsmu.edu.cn (H. Chen), xiejoanxq@gmail.com (Q. Xie). 1 These authors contributed equally to this work. 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2012.07.018 Contents lists available at SciVerse ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

W. Zheng et al Toxicology and Applied Pharmacology 264(2012)65-72 erone in inducing AB fibrillogenesis through Material and methods peripheral anionic site with the peptide (Inestrosa et al, 2008: Johnson and Moore, 2006). In light of this, Preparation of N'Na-bis(3-chloropropyl)oxalamide(oxalamide ) Oxalyl AChEls that simultaneously block both the catalytic and peripheral dichloride( 1.27 g 10.0 mmol) in CHCl(10 mL) was added to a solution sites might not only alleviate the cognitive deficit of Ad patients by of 3-chloropropylamine hydrochloride (2.6 g, 20.0 mmol), NaoH (2 g) elevating acetylcholine (ACh) levels, but also act as disease- and H20(4 mL)in CHa(20 mL) atc. The mixture was stirred at modifying agents via delaying amyloid plaque formation. 0C for 20 min, extracted with CHCl3(20 mLx 3)and dried with anhy In our previous study based on(-)-meptazinol(MEP), a com- drous MgSO4 Evaporation of the solvent gave oxalamide(2.40 g, 99.6%) pound exhibiting moderate potency of AChE inhibition(Ennis et al., a yellowish powder Recrystallization of crude oxalamide from ethyl ace- 1986), we developed a homo-bivalent(-)-MEP derivative, bis-(-)- tate afforded oxalamide as white needle-like crystals(1.61 g, 67.1%). nor-MEP(bis-MEP). as an effective drug candidate for Ad therapy Melting points(mp)169-170'C. H NMR(CDCI3 ): Chemical shifts(6) (Xie et al, 2008). This compound has multiple functions, e.g., inhib- 7.65(S, 2H, NHCO), 3.59(t, 4H, J =6 Hz, dI-CH2), 3.51(dd, 4H, JI iting AChE activity and preventing AChE-induced AB aggregation, 7 Hz, h=13 Hz, N-CH2). 2.08-2.02(m, 4H, CH2): MS(ESD): 241.0 with an acceptable safety profile( LD50=313 mg/kg). Computational [M+H+: 263.0 [M+Na]*. analysis and subsequent crystallographic studies have demonstrated that bis-MEP is able to simultaneously interact with both catalytic Synthesis of ZLA. Detailed synthetic procedure for the intermediates nd peripheral anionic sites of the enzyme(Paz et al, 2009: Xie normethyl-MEP (nom-MEP)was described in our previous studies(Xie etal,2008) et al. 2008) Triethylamine(0.8 mL 5.75 mmol) and oxalamide(0.35 g Recently, in a search for new rationally-designed multifunctional 1.45 mmol)were added to a solution of nom-MEP (0.79 g 3.60 mmol) agents against AD, we started from the dual binding site AChEI in acetonitrile. The reaction mixture was refluxed for 20 h and evaporat- bis-MEP, and focused on the spacer as the carrier of a third biological ac- ed. The residue was diluted with saturated Na2 CO solution(10 mL). tivity. As the important role of transition metal ions in AB aggregation, extracted with CHCl3 and then dried with anhydrous Na 2SOa and evapo- etal chelation pharmacophores were introduced into the middle rated The residue was purified by column chromatography on silica gel chain of bis-MEP, with an intention to endow additional metal- using CHCl3/CH3 OH (93: 7)as eluent. Addition of dry HCI-ether to a solu- complexing potency while keeping its other multifunctional pro- tion of the above purified compound (0.53 g, 60.2%)in dry ether and files. A successful modification has been obtained by incorporating adjusting pH to 3-4 gave the hydrochloride salt ZLA(0.37 g, 62.3%)as xalamide functionality as a chelation pharmacophore into the spacer white powder. mp. 154-157 C, specific rotation (a6)=-77(c of bis-tacrine( Bolognesi et al, 2007) Ethylenediamine is a bidentate 0.390, MeOH). H NMR(DMSO-d6): 810.30 and 10. 14(br s, 4/3 H, as Cu(lin-ethylenediamine complex (Inada et al, 1993). Therefore, (br s, 2/3 H, NH). 7.27-7.20(m, 2H, ArH). 6.91-6.75(m, 6H, ArH). we rationally designed two novel bis-MEP derivatives N, N2-bis(3- 3. 89(d, 2/3 H, J=13.6 Hz. N-CH2). 3.57(d, 4/3 H, J=14.1 Hz, ((S)-3-ethyl-3-(3-hydroxyphenyl)azepan-1-yl)propyl)oxalamide hy- N-CH2) 3.42(m, 2H, N-CH2) 3.32-3.18(m, 12H, N-CH2), 2.46-2.41 drochloride(ZLA)and N N-bis(3-(S)-3-ethyl-3-(3-hydroxyphenyl) (m, 1H, CH2), 225-2.13(m, 5H, CH2), 2.08-2.01(m, 2H, CH2),1.98- azepan-1-yl)propyl)-ethane-1, 2-diamine hydrochloride(ZlB)with 1.79(m, 9H, CH2), 1.61-1.48(m, 3H, CH2), 0.57(t, 6H, J=7.3 Hz, CH3) NMR(DMSO-d6): 8 12942,12925,11723, nd Cu(in)or Zn(ll)chela evaluated in the 11689,11410.113.57,113.49,113.30,6368,61.35,5863,57.13,56.58 metal chelation AChE inhibition AChE-induced AB aggregation (MEP bisMEP ZLB

act as a pathological chaperone in inducing Aβ fibrillogenesis through direct interactions of its peripheral anionic site with the peptide (Inestrosa et al., 2008; Johnson and Moore, 2006). In light of this, AChEIs that simultaneously block both the catalytic and peripheral sites might not only alleviate the cognitive deficit of AD patients by elevating acetylcholine (ACh) levels, but also act as disease￾modifying agents via delaying amyloid plaque formation. In our previous study based on (−)-meptazinol (MEP), a com￾pound exhibiting moderate potency of AChE inhibition (Ennis et al., 1986), we developed a homo-bivalent (−)-MEP derivative, bis-(−)- nor-MEP (bis-MEP), as an effective drug candidate for AD therapy (Xie et al., 2008). This compound has multiple functions, e.g., inhib￾iting AChE activity and preventing AChE-induced Aβ aggregation, with an acceptable safety profile (LD50=313 mg/kg). Computational analysis and subsequent crystallographic studies have demonstrated that bis-MEP is able to simultaneously interact with both catalytic and peripheral anionic sites of the enzyme (Paz et al., 2009; Xie et al., 2008). Recently, in a search for new rationally-designed multifunctional agents against AD, we started from the dual binding site AChEI bis-MEP, and focused on the spacer as the carrier of a third biological ac￾tivity. As the important role of transition metal ions in Aβ aggregation, metal chelation pharmacophores were introduced into the middle chain of bis-MEP, with an intention to endow additional metal￾complexing potency while keeping its other multifunctional pro- files. A successful modification has been obtained by incorporating oxalamide functionality as a chelation pharmacophore into the spacer of bis-tacrine (Bolognesi et al., 2007). Ethylenediamine is a bidentate ligand which can form stable complexes with various metal ions, such as Cu(II)–ethylenediamine complex (Inada et al, 1993). Therefore, we rationally designed two novel bis-MEP derivatives N1 ,N2 -bis(3- ((S)-3-ethyl-3-(3-hydroxyphenyl)azepan-1-yl)propyl)oxalamide hy￾drochloride (ZLA) and N1 ,N2 -bis(3-((S)-3-ethyl-3-(3-hydroxyphenyl) azepan-1-yl)propyl)-ethane-1,2-diamine hydrochloride (ZLB) with promising profiles of dual binding site AChE inhibition and metal chela￾tion (Fig. 1). Their properties of dual binding site AChE inhibition and Cu(II) or Zn(II) chelation were evaluated in the present study. Material and methods Preparation of N1 ,N2 -bis(3-chloropropyl) oxalamide (oxalamide). Oxalyl dichloride (1.27 g, 10.0 mmol) in CHCl3 (10 mL) was added to a solution of 3-chloropropylamine hydrochloride (2.6 g, 20.0 mmol), NaOH (2 g) and H2O (4 mL) in CHCl3 (20 mL) at 0 °C. The mixture was stirred at 0 °C for 20 min, extracted with CHCl3 (20 mL×3) and dried with anhy￾drous MgSO4. Evaporation of the solvent gave oxalamide (2.40 g, 99.6%) a yellowish powder. Recrystallization of crude oxalamide from ethyl ace￾tate afforded oxalamide as white needle-like crystals (1.61 g, 67.1%). Melting points (mp) 169–170 °C. 1 H NMR (CDCl3): Chemical shifts (δ) 7.65 (s, 2H, NHCO), 3.59 (t, 4H, J=6 Hz, Cl\CH2), 3.51 (dd, 4H, J1= 7 Hz, J2=13 Hz, N\CH2), 2.08–2.02 (m, 4H, CH2); MS (ESI): 241.0 [M+H]+; 263.0 [M+Na]+. Synthesis of ZLA. Detailed synthetic procedure for the intermediates (−)- normethyl-MEP (nom-MEP) was described in our previous studies (Xie et al., 2008). Triethylamine (0.8 mL, 5.75 mmol) and oxalamide (0.35 g, 1.45 mmol) were added to a solution of nom-MEP (0.79 g, 3.60 mmol) in acetonitrile. The reaction mixture was refluxed for 20 h and evaporat￾ed. The residue was diluted with saturated Na2CO3 solution (10 mL), extracted with CHCl3 and then dried with anhydrous Na2SO4 and evapo￾rated. The residue was purified by column chromatography on silica gel using CHCl3/CH3OH (93:7) as eluent. Addition of dry HCl–ether to a solu￾tion of the above purified compound (0.53 g, 60.2%) in dry ether and adjusting pH to 3–4 gave the hydrochloride salt ZLA (0.37 g, 62.3%) as white powder. mp. 154–157 °C, specific rotation ([α]D 20)=−7.7° (c= 0.390, MeOH). 1 H NMR (DMSO-d6): δ10.30 and 10.14 (br s, 4/3 H, NH+), 9.59, 9.50 (s, 2H, ArOH), 8.98–8.96 (m, 2H, NHCO), 8.63 and 8.55 (br s, 2/3 H, NH+), 7.27–7.20 (m, 2H, ArH), 6.91–6.75 (m, 6H, ArH), 3.89 (d, 2/3 H, J=13.6 Hz, N\CH2), 3.57 (d, 4/3 H, J=14.1 Hz, N\CH2), 3.42 (m, 2H, N\CH2), 3.32–3.18 (m, 12H, N\CH2), 2.46–2.41 (m, 1H, CH2), 2.25–2.13 (m, 5H, CH2), 2.08–2.01 (m, 2H, CH2), 1.98– 1.79 (m, 9H, CH2), 1.61–1.48 (m, 3H, CH2), 0.57 (t, 6H, J=7.3 Hz, CH3); MS (ESI): 607.5 [M+H]+, 304.3 [M+2H]2+; 13C NMR (DMSO-d6): δ 160.08, 160.01, 157.55, 157.47, 144.82, 143.64, 129.42, 129.25, 117.23, 116.89, 114.10, 113.57, 113.49, 113.30, 63.68, 61.35, 58.63, 57.13, 56.58, Fig. 1. Design strategy for new multifunctional compounds. 66 W. Zheng et al. / Toxicology and Applied Pharmacology 264 (2012) 65–72

W. Zheng et aL Toxicology and Applied Pharmacology 264(2012)65-72 56.31, 43.70, 43.37, 36.39, 36.20, 3533, 35.26, 34.43, 33.05, 26.33, 24.88, buffer-saturated octanol. Then 1.0 mL sample solution and 1.0 mL 22.84, 22.79. 20.76, 20.46, 8.10, 796: MS(ESI): 607.5 [M+H+, 304.3 octanol-saturated phosphate buffer were pipetted into a centrifuging A+2H2+ ube and shaken for 48 h at 37C, followed by centrifuging for 5 min (2000 rpm), the aqueous phase was separated and analyzed with re- Synthesis of ZlB. A solution of ZLA (0.53 g, 0.87 mmol) in dry tetrahy versed phase-high performance liquid chromatography(RP-HPL drofuran(THF, 20 mL) was added to lithium aluminum hydride The mobile phase was methanol/.02 mol L ammonium acetate solu (0.23 g, 6.05 mmol)in dry ThF(10 mL)at room temperature. The tion(75: 25: pH was adjusted to 7. 4 with aqueous ammonia )at a flow mixture was refluxed for 24 h, and then H20(0.23 mL). 15% NaOH rate of 1.0 mL/min through a VP-ODS column(250 mmx 4.6 mm. (0. 23 mL) and H20(0.69 mL)were added. The mixture was stirred 5 um; Shimadzu, Kyoto, Japan) at 50C. Experiments were conducted for 15 min at room temperature and filtered; the solid material was in triplicate and log P values were calculated. washed with THF and evaporated. The residue was treated with H20(15 mL), and aqueous ammonia was added to adjust the ph to In vitro cholinesterase inhibition assay. Inhibitory potency against mice 9. The mixture was extracted with CHCl3, dried with anhydrous or human-derived AChE of ZLa or ZlB was evaluated by a modified Na2SO4 and concentrated in vacuo to give a residue(0.51 g)as a yel- Ellman's method (Bartolini et al, 2003; Li et al, 2007). Mice forebrain low oil, which was purified using silica graphy homogenates, which were prepared in normal saline(1: 9 w/v)and rith CHCl3/CH3OH (95: 5) as eluent, and final salt used as a source of AChE, were pre-incubated with the tested inhibitors in dry HCI-ether solution to afford ZLB 18.%3mp.132-136°Clal6=-271°( (DMSO-d6):610.26 and 10. 19 and 9.94 and 9.83(br s, 4H, NH+). nitrobenzoic)acid(DTNB ). Then acetylthiocholine iodide(500 M) 9.51. 943(s, 2H, ArOH), 8.77 and 8.26 and 8.17(5, 2H, NH), 7.22- was added as the substrate, and AChE activity was determined by UV 7. 13(m, 2H, ArH), 6.90-6.69(m, 6H, ArH), 3.94(m, 2/3 H, N-CH2), spectrophotometry at 412 nm. The concentration of compounds that 3.57(m, 4/3 H, N-CH2) 3.32-2.80(m, 14H, N-CH2). 2.41-2.01(m, produced 50% inhibition of the AChE activity(ICso) was calculated by 8H, CH2), 1.98-1.67(m, 9H, CH2). 1.53-1.47(m, 3H, CH2), 0.51(t, nonlinear regression of the response-concentration (log)curve. As 6H,J=7.4 Hz, CH3): MS(ESI): 579.4 [M+H+, 290.4 [M+2H2+; for determination of inhibition of human AChE activity, AChE stock So- C NMR(DMSO-d6): 6157.55, 157.45, 144.77 and 144.70, 143.84, lution was prepared by dissolving human recombinant AChE lyophi 129.56, 129. 29, 117.34, 117.03, 113.94, 113.60, 113.50, 113.31, 63.99 lized powder (Sigma, St Louis, MO, USA)in 0. 1 M phosphate buffer and 63.85, 61.33, 58.59, 56.23, 55.97, 55.88, 43.98, 43.82, 43.43. (pH 8.0)containing 0. 1% Triton X-100. The assay solution consisted 40.00. 36.16, 35.39, 35.09, 35.07, 33.14, 32.98, 26.38 and 26.34. of a 0.1 M phosphate buffer pH 8.0, with the addition of 340 HM 25.18, 21.22, 20.72 and 20.31, 19.93 and 19.49, 18.50, 10.91, 8.12 DTNB, 0.5 unit/mL human recombinant AChE and 550 HM substrate and801;Ms(ES):5794M+H+,2904M+2H]2+ (acetylthiocholine iodide). Butyrylcholinesterase(BChE; derived from mice sera. 1: 19 w/v in normal saline)inhibition was similarly carried Molecular docking To confirm that substitution of the aliphatic spacer out using butyrylthiocholine iodide(500 HM)as the substrate Results of bis-MEP with metal chelation pharmacophores does not negatively are reported as the mean SEM of ICso obtained from at least three affect the ability for the new compounds to interact with AChE, molec ular docking simulations were performed on an R14000 SGI Fuel work station with the software package SYBYL 6.9(Tripos, St Louis, MO, Inhibition of AChE-induced As aggregation. According to descriptions USA). Standard parameters were used unless otherwise indicated. The of the previous studies(Bartolini et al. 2003), aliquots of 2 HL AB1 crystal structure of Torpedo califomica AChE (TCAChE) was obtained 40 peptide(Invitrogen, Carlsbad, CA. USA), lyophilized from 1 mg, from the Protein Data Bank(PDB code 1EA5 ) Heteroatoms and water mL 1, 1. 1,3, 3. 3-hexafluoro-2-propanol(HFIP: Sigma) solution(HFIP molecules in the proteins were removed, and hydrogen atoms were was used as a solvent to ensure that AChE-induced AB aggregation subsequently added. Bis-MEP derivatives were drawn in ISIS/ Base started with an AB solution mainly random coil or a-helix structure, (SDF)to form a small focused library. The 2D structures of the library (DMSO), were incubated for 48 h at room temperature in 0. 215 M so- vere subsequently converted into 3D structures with CORINA, still in dium phosphate buffer(pH 8.0)at a final concentration of 230 HM. SDF format. The 3D structures were then read into SYBYL Molecular For co-incubation experiments, aliquots(16 uL) of human recombi SpreadSheet table for further treatment, such as energy minimization nant AChE (with the molar ratio of AB/AChE as 100: 1: Sigma) and for 100 steps with Tripos force field and Gasteiger-Marsili charges for AChE in the presence of 2 uL of the tested inhibitors at various con- each molecule. These structures were put into databases and then writ- centrations were added. The final volume of each vial was 20 HL. ten to MOL2 files as input. Molecular docking was carried out using Each assay was run in duplicate. GOLD 3.0(CCDC, Cambridge, UK, 2005)to generate an ensemble of To quantify amyloid fibril formation, the thioflavin T fluorescence docked conformations for the ligand. The active site was defined as all method was then applied. After incubation, the samples containing Ap toms within a radius of 25 A around Tyr1210- of TcAChE This en- AB plus AChE, or aB plus achE in the presence of the test inhibitors arged binding pocket was chosen, as a smaller one might neither ac- were diluted with 50 mM glycine-NaoH buffer (pH 8.5) containing commodate a large bis-ligand nor include both the catalytic and 1.5 uM thioflavin T(Sigma) to a final volume of 2.0 mL Fluorescence peripheral sites of AChE Because of the high flexibility of the ligand, was monitored by PE LS45 spectrophotometer(Perkin Elmer, Waltham, which contains many rotatable bonds, 600 genetic algorithm(GA) MA, USA), with excitation at 446 nm and emission at 490 nm. A time runs were performed rather than the default of 10 For each GA run, scan of fluorescence was performed, and the intensity values reached at the default GA settings were used, except that early termination was the plateau(around 300 s)were averaged after subtracting the back prohibited and pyramidal nitrogen inversion was allowed. ground fluorescence from 1.5 uM thioflavin T and AChE. The percent inhi- bition of the AChE-induced aggregation due to the presence of increasing Log P determination Partition coefficients of ZLA and ZlB were deter- concentrations of test compounds was calculated by the following formu- mined by shake-flask method in the octanol/ phosphate buffer solu- la: 100-(IFi/IFo x 100), where IFi and IFo were the fluorescence intensi tion at pH 4, 5. 6, 7. 7. 4 or 8. The phosphate buffer and octanol ties obtained for AB plus AChE in the presence and in the absence of the were saturated with each other prior to partitioning by mixing inhibitors, respectively, after subtracting the fluorescence of respective and allowing the phases to separate overnight. A stock solution blanks. Inhibition curves and linear regression parameters were obtained for each compound was prepared at 0. 1 mg/mL in phosphate for each compound, and the iCso was extrapolated

56.31, 43.70, 43.37, 36.39, 36.20, 35.33, 35.26, 34.43, 33.05, 26.33, 24.88, 22.84, 22.79, 20.76, 20.46, 8.10, 7.96; MS (ESI): 607.5 [M+H]+, 304.3 [M+2H]2+. Synthesis of ZLB. A solution of ZLA (0.53 g, 0.87 mmol) in dry tetrahy￾drofuran (THF, 20 mL) was added to lithium aluminum hydride (0.23 g, 6.05 mmol) in dry THF (10 mL) at room temperature. The mixture was refluxed for 24 h, and then H2O (0.23 mL), 15% NaOH (0.23 mL) and H2O (0.69 mL) were added. The mixture was stirred for 15 min at room temperature and filtered; the solid material was washed with THF and evaporated. The residue was treated with H2O (15 mL), and aqueous ammonia was added to adjust the pH to 9. The mixture was extracted with CHCl3, dried with anhydrous Na2SO4 and concentrated in vacuo to give a residue (0.51 g) as a yel￾low oil, which was purified using silica gel column chromatography with CHCl3/CH3OH (95:5) as eluent, and then converted to final salt in dry HCl–ether solution to afford ZLB as white powder (115 mg, 18.1%). mp. 132–136 °C, [α]D 20=−27.1° (c=0.240, MeOH). 1 H NMR (DMSO-d6): δ10.26 and 10.19 and 9.94 and 9.83 (br s, 4H, NH+), 9.51, 9.43 (s, 2H, ArOH), 8.77 and 8.26 and 8.17 (s, 2H, NH), 7.22– 7.13 (m, 2H, ArH), 6.90–6.69 (m, 6H, ArH), 3.94 (m, 2/3 H, N\CH2), 3.57 (m, 4/3 H, N\CH2), 3.32–2.80 (m, 14H, N\CH2), 2.41–2.01 (m, 8H, CH2), 1.98–1.67 (m, 9H, CH2), 1.53–1.47 (m, 3H, CH2), 0.51 (t, 6H, J= 7.4 Hz, CH3); MS (ESI): 579.4 [M+H]+, 290.4 [M+2H]2+; 13C NMR (DMSO-d6): δ157.55, 157.45, 144.77 and 144.70, 143.84, 129.56, 129.29, 117.34, 117.03, 113.94, 113.60, 113.50, 113.31, 63.99 and 63.85, 61.33, 58.59, 56.23, 55.97, 55.88, 43.98, 43.82, 43.43, 40.00, 36.16, 35.39, 35.09, 35.07, 33.14, 32.98, 26.38 and 26.34, 25.18, 21.22, 20.72 and 20.31, 19.93 and 19.49, 18.50, 10.91, 8.12 and 8.01; MS (ESI): 579.4 [M+H]+, 290.4 [M+2H]2+. Molecular docking. To confirm that substitution of the aliphatic spacer of bis-MEP with metal chelation pharmacophores does not negatively affect the ability for the new compounds to interact with AChE, molec￾ular docking simulations were performed on an R14000 SGI Fuel work￾station with the software package SYBYL 6.9 (Tripos, St. Louis, MO, USA). Standard parameters were used unless otherwise indicated. The crystal structure of Torpedo californica AChE (TcAChE) was obtained from the Protein Data Bank (PDB code 1EA5). Heteroatoms and water molecules in the proteins were removed, and hydrogen atoms were subsequently added. Bis-MEP derivatives were drawn in ISIS/Base with ISIS/Draw from MDL and exported into a 2D structure data file (SDF) to form a small focused library. The 2D structures of the library were subsequently converted into 3D structures with CORINA, still in SDF format. The 3D structures were then read into SYBYL Molecular SpreadSheet table for further treatment, such as energy minimization for 100 steps with Tripos force field and Gasteiger–Marsili charges for each molecule. These structures were put into databases and then writ￾ten to MOL2 files as input. Molecular docking was carried out using GOLD 3.0 (CCDC, Cambridge, UK, 2005) to generate an ensemble of docked conformations for the ligand. The active site was defined as all atoms within a radius of 25 Å around Tyr121O_ of TcAChE. This en￾larged binding pocket was chosen, as a smaller one might neither ac￾commodate a large bis-ligand nor include both the catalytic and peripheral sites of AChE. Because of the high flexibility of the ligand, which contains many rotatable bonds, 600 genetic algorithm (GA) runs were performed rather than the default of 10. For each GA run, the default GA settings were used, except that early termination was prohibited and pyramidal nitrogen inversion was allowed. Log P determination. Partition coefficients of ZLA and ZLB were deter￾mined by shake-flask method in the octanol/phosphate buffer solu￾tion at pH 4, 5, 6, 7, 7.4 or 8. The phosphate buffer and octanol were saturated with each other prior to partitioning by mixing and allowing the phases to separate overnight. A stock solution for each compound was prepared at 0.1 mg/mL in phosphate buffer-saturated octanol. Then 1.0 mL sample solution and 1.0 mL octanol-saturated phosphate buffer were pipetted into a centrifuging tube and shaken for 48 h at 37 °C, followed by centrifuging for 5 min (2000 rpm), the aqueous phase was separated and analyzed with re￾versed phase-high performance liquid chromatography (RP-HPLC). The mobile phase was methanol/0.02 mol/L ammonium acetate solu￾tion (75:25; pH was adjusted to 7.4 with aqueous ammonia) at a flow rate of 1.0 mL/min through a VP-ODS column (250 mm×4.6 mm, 5 μm; Shimadzu, Kyoto, Japan) at 50 °C. Experiments were conducted in triplicate and log P values were calculated. In vitro cholinesterase inhibition assay. Inhibitory potency against mice or human-derived AChE of ZLA or ZLB was evaluated by a modified Ellman's method (Bartolini et al., 2003; Li et al., 2007). Mice forebrain homogenates, which were prepared in normal saline (1:9 w/v) and used as a source of AChE, were pre-incubated with the tested inhibitors at various concentrations for 20 min at 37 °C in 0.05 M phosphate buffered solution (pH 7.2), containing 250 μM 5,5′-dithio-bis(2- nitrobenzoic) acid (DTNB). Then acetylthiocholine iodide (500 μM) was added as the substrate, and AChE activity was determined by UV spectrophotometry at 412 nm. The concentration of compounds that produced 50% inhibition of the AChE activity (IC50) was calculated by nonlinear regression of the response–concentration (log) curve. As for determination of inhibition of human AChE activity, AChE stock so￾lution was prepared by dissolving human recombinant AChE lyophi￾lized powder (Sigma, St. Louis, MO, USA) in 0.1 M phosphate buffer (pH 8.0) containing 0.1% Triton X-100. The assay solution consisted of a 0.1 M phosphate buffer pH 8.0, with the addition of 340 μM DTNB, 0.5 unit/mL human recombinant AChE and 550 μM substrate (acetylthiocholine iodide). Butyrylcholinesterase (BChE; derived from mice sera, 1:19 w/v in normal saline) inhibition was similarly carried out using butyrylthiocholine iodide (500 μM) as the substrate. Results are reported as the mean±SEM of IC50 obtained from at least three independent measures. Inhibition of AChE-induced Aß aggregation. According to descriptions of the previous studies (Bartolini et al., 2003), aliquots of 2 μL Aβ1– 40 peptide (Invitrogen, Carlsbad, CA. USA), lyophilized from 1 mg/ mL 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Sigma) solution (HFIP was used as a solvent to ensure that AChE-induced Aβ aggregation started with an Aβ solution mainly random coil or α-helix structure, which is poorly amyloidogenic) and dissolved in dimethyl sulfoxide (DMSO), were incubated for 48 h at room temperature in 0.215 M so￾dium phosphate buffer (pH 8.0) at a final concentration of 230 μM. For co-incubation experiments, aliquots (16 μL) of human recombi￾nant AChE (with the molar ratio of Aβ/AChE as 100:1; Sigma) and AChE in the presence of 2 μL of the tested inhibitors at various con￾centrations were added. The final volume of each vial was 20 μL. Each assay was run in duplicate. To quantify amyloid fibril formation, the thioflavin T fluorescence method was then applied. After incubation, the samples containing Aβ, Aβ plus AChE, or Aβ plus AChE in the presence of the test inhibitors were diluted with 50 mM glycine–NaOH buffer (pH 8.5) containing 1.5 μM thioflavin T (Sigma) to a final volume of 2.0 mL. Fluorescence was monitored by PE LS45 spectrophotometer (Perkin Elmer, Waltham, MA, USA), with excitation at 446 nm and emission at 490 nm. A time scan of fluorescence was performed, and the intensity values reached at the plateau (around 300 s) were averaged after subtracting the back￾ground fluorescence from 1.5 μM thioflavin T and AChE. The percent inhi￾bition of the AChE-induced aggregation due to the presence of increasing concentrations of test compounds was calculated by the following formu￾la: 100−(IFi / IFo×100), where IFi and IFo were the fluorescence intensi￾ties obtained for Aβ plus AChE in the presence and in the absence of the inhibitors, respectively, after subtracting the fluorescence of respective blanks. Inhibition curves and linear regression parameters were obtained for each compound, and the IC50 was extrapolated. W. Zheng et al. / Toxicology and Applied Pharmacology 264 (2012) 65–72 67

W. Zheng et al Toxicology and Applied Pharmacology 264(2012)65-72 Metal-chelating properties. The ability for Cor ZLA, ZLB or bis-MEP to chelate biometals such as Cu(ll) was studied by Uv-vis Baum and Ng, 2004). The absorption spectra of ZLA, ZLB or bis-MEP, alone (in methanol) or in the presence of CuCl Trp 279 with a molar ratio of 1: 1), were recorded at a duration of 30 min at room temperature in a 1 cm quartz cell using Multiscan MK3 spectro- Tyr 70 photometer(Thermo, Waltham, MA, USA). Additionally, the ratio of metal ion in the complex was determined by molar ratio (Bolognesi et al., 2007). Fixed concentrations of the com- 25 HM)was mixed with ascending doses of CuCl2 (12-40 HM)and the UV-vis absorption spectra were recorded. Tyr 334 \Tyr 121 Aggregation assay by sedimentation. AB1-40, lyophilized from 1 mg/ mL HFIP solution, was dissolved in DMSO to get a 500 uM stock solu- tion, and was brought to 10 HM in 20 mM Hepes buffer, 150 mM Nacl alone or with metal ions(20 HM), in the presence or absence of the tested compounds(100 HM). The reaction mixture(100 uL)was in- cubated for 15 min at 37C, and then centrifuged at 13,000xg for 15 min to sediment aggregated proteins. Peptide concentration in the supernatant was determined by the bradford method using the commercial protein assay Coomassie Brilliant Blue solution(Thermo) Trp 84 His 440 and was represented as a percentage of recovery relative to the con- trol without metal ions and tested compounds(Atwood et al, 1998: Raman et al., 2005). As Ad is complicated by cerebral acidosis with a pH of 6.6(Yates et al, 1990). 20 mM Hepes buffer, 150 mM Nacl Fig. 2. Binding modes of bis-MEP ZLA and ZLB (green, was set to pH 6.6 with hydrochloric acid in this assay ZLA and zlB ar 79 and Tyr 70, while the spacer does not seem to be detrimental to the interactions with the enzyme. Turbidometric assay. Turbidity measurements, also as an assay for Worthy to note, some interactions could be identified for ZLa and ZLB with Tyr121. al- gregation, were performed according to the method described in the though they are not strong enough to increase the AChE inhibitory potency revious studies(Atwood et al, 1998). The stock solution of AB1-40 (500 HM) was brought to 10 HM in 20 mM Hepes buffer, 150 mM Nacl(pH 6.6)alone or with metal ions(20 HM), in the presence absence of the tested compounds(100 HM). The reaction mixture Data analysis and statistics. Values are exp (200 uL)was incubated for 30 min at 37., and absorbance Comparisons among means were performed ANOVA (405 nm) was measured using a Varioskan Flash spectrophotometr and post hoc by Dunnett test. Differences values less than microplate reader(Thermo). Automatic 30-s plate agitation mode 0.05 were considered statistically significant. was selected for the plate reader to evenly suspend the aggregates in the wells before all readings Results Molecular docking studies of zla and ZLB Determination of cell viability. The human neuroblastoma cell line SH-SY5Y cells(American Type Culture Collection, Manassas, VA, USA) The results of molecular docking demonstrate that compounds were cultured in MEM/F-12 (1: 1)medium(Invitrogen )supplemented ZLA and ZLB are able to interact with both the catalytic and peripheral 100 ug/mL streptomycin in a humidified atmosphere containing 5% the nonylene spacer with oxalamide or ethylenediamine may reduce CO2 at 37C. Cells were plated at 5x10 cells /well(100 HL)into AChE binding affinity as compared with their prototype bis-MEP And 96-well plates and allowed to adhere and grow. When cells reached although the derivatives could establish favorable interactions with the required confluence, they were placed into serum-free medium some mid-gorge residues, the increased polarity of ZLA and ZLB may and treated with ZLA or ZLB. Twenty-four hours later the survival of result in a major energy penalty during their desolvation cells was determined by Cell Counting Kit-8(CCK-8: Dojindo, Japan) ssay. Briefly, after incubation with 10 uL of CCK-8(5 mg/mL) at 37c Log P of ZLA and ZlB or 2 h, the absorbance was measured with a test wavelength of 570 nm and a reference wavelength of 655 nm. The absorbance of con- The partition coefficients of ZLA and ZLB in the octanol/phosphate trol cells was set to 100%, and the percentage of viable cells collected buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively )were from each treatment was calculated relative to the control group. determined by the classical shake-flask method using RP-HPLC. As Table 1 pH-dependent partition coefficients(log P) of ZLA and ZLB pH 7.4 0.18±0.001 1.11±0001 0.38±0001 0.39±0.0 1.78±0003 07±0005 422±0.003 artition coefficients of ZLA and ZLB in the octanol /buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively) were determined by the classical shake-flask method. The represent the mean+ SEM of three independent experiments

Metal-chelating properties. The ability for compounds ZLA, ZLB or bis-MEP to chelate biometals such as Cu(II) was studied by UV–vis spectrometry (Baum and Ng, 2004). The absorption spectra of ZLA, ZLB or bis-MEP, alone (in methanol) or in the presence of CuCl2 (with a molar ratio of 1:1), were recorded at a duration of 30 min at room temperature in a 1 cm quartz cell using Multiscan MK3 spectro￾photometer (Thermo, Waltham, MA, USA). Additionally, the ratio of ligand/metal ion in the complex was determined by molar ratio method (Bolognesi et al., 2007). Fixed concentrations of the com￾pounds (25 μM) was mixed with ascending doses of CuCl2 (12–40 μM) and the UV–vis absorption spectra were recorded. Aggregation assay by sedimentation. Aβ1–40, lyophilized from 1 mg/ mL HFIP solution, was dissolved in DMSO to get a 500 μM stock solu￾tion, and was brought to 10 μM in 20 mM Hepes buffer, 150 mM NaCl alone or with metal ions (20 μM), in the presence or absence of the tested compounds (100 μM). The reaction mixture (100 μL) was in￾cubated for 15 min at 37 °C, and then centrifuged at 13,000×g for 15 min to sediment aggregated proteins. Peptide concentration in the supernatant was determined by the Bradford method using the commercial protein assay Coomassie Brilliant Blue solution (Thermo) and was represented as a percentage of recovery relative to the con￾trol without metal ions and tested compounds (Atwood et al., 1998; Raman et al., 2005). As AD is complicated by cerebral acidosis with a pH of 6.6 (Yates et al., 1990), 20 mM Hepes buffer, 150 mM NaCl was set to pH 6.6 with hydrochloric acid in this assay. Turbidometric assay. Turbidity measurements, also as an assay for ag￾gregation, were performed according to the method described in the previous studies (Atwood et al., 1998). The stock solution of Aβ1–40 (500 μM) was brought to 10 μM in 20 mM Hepes buffer, 150 mM NaCl (pH 6.6) alone or with metal ions (20 μM), in the presence or absence of the tested compounds (100 μM). The reaction mixture (200 μL) was incubated for 30 min at 37 °C, and absorbance (405 nm) was measured using a Varioskan Flash spectrophotometric microplate reader (Thermo). Automatic 30-s plate agitation mode was selected for the plate reader to evenly suspend the aggregates in the wells before all readings. Determination of cell viability. The human neuroblastoma cell line SH-SY5Y cells (American Type Culture Collection, Manassas, VA, USA) were cultured in MEM/F-12 (1:1) medium (Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were plated at 5×104 cells/well (100 μL) into 96-well plates and allowed to adhere and grow. When cells reached the required confluence, they were placed into serum-free medium and treated with ZLA or ZLB. Twenty-four hours later the survival of cells was determined by Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. Briefly, after incubation with 10 μL of CCK-8 (5 mg/mL) at 37 °C for 2 h, the absorbance was measured with a test wavelength of 570 nm and a reference wavelength of 655 nm. The absorbance of con￾trol cells was set to 100%, and the percentage of viable cells collected from each treatment was calculated relative to the control group. Data analysis and statistics. Values are expressed as mean± SEM. Comparisons among means were performed using one-way ANOVA and post hoc by Dunnett test. Differences with P values less than 0.05 were considered statistically significant. Results Molecular docking studies of ZLA and ZLB The results of molecular docking demonstrate that compounds ZLA and ZLB are able to interact with both the catalytic and peripheral anionic sites of AChE (Fig. 2). It is worth noting that replacement of the nonylene spacer with oxalamide or ethylenediamine may reduce AChE binding affinity as compared with their prototype bis-MEP. And, although the derivatives could establish favorable interactions with some mid-gorge residues, the increased polarity of ZLA and ZLB may result in a major energy penalty during their desolvation. Log P of ZLA and ZLB The partition coefficients of ZLA and ZLB in the octanol/phosphate buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively) were determined by the classical shake-flask method using RP-HPLC. As Fig. 2. Binding modes of bis-MEP, ZLA and ZLB (green, purple and yellow, respectively) at the TcAChE gorge. ZLA and ZLB are able to properly contact with both sites of the en￾zyme. The two MEP moieties establish π–π stacking with Trp84, Trp279 and Tyr70, while the spacer does not seem to be detrimental to the interactions with the enzyme. Worthy to note, some interactions could be identified for ZLA and ZLB with Tyr121, al￾though they are not strong enough to increase the AChE inhibitory potency. Table 1 pH-dependent partition coefficients (log P) of ZLA and ZLB. Compounds Log P pH 4 pH 5 pH 6 pH 7 pH 7.4 pH 8 ZLA 0.18±0.001 0.22±0.002 1.11±0.001 2.69±0.003 3.71±0.003 4.06±0.006 ZLB 0.38±0.001 0.39±0.001 1.78±0.003 3.07±0.005 4.00±0.004 4.22±0.003 The partition coefficients of ZLA and ZLB in the octanol/buffer solution at different pH (4, 5, 6, 7, 7.4 and 8 respectively) were determined by the classical shake-flask method. The data represent the mean± SEM of three independent experiments. 68 W. Zheng et al. / Toxicology and Applied Pharmacology 264 (2012) 65–72

W. Zheng et aL Toxicology and Applied Pharmacology 264(2012)65-72 Table 2 Table 3 Inhibition of ZLA and ZlB on AChE or BChE activity Inhibition of ZLA and ZLB on human AChE-induced AB aggregation. Compounds ICso+ SEM (uM) Selectivity for Compounds Cs0±SEM Inhibition (6) Inhibition(%) Human recombinant Mice AChE Mice BChE 491±5.0 556±4.7 9.63±1.64 81±0.361.33±026 Propidium iodide 119±04 2±4 .3±5.3 8.64±1.58 Rivastigmine 43±0832.13±0470.39 se. AB, amyloid-B. Human recombinant AChE-induced AB AChE, acetylcholinesterase. BChE, butyrylcholinesterase. Mice forebrain homogenates ibrillogenesis was determined using thioflavin t fluorescence. The final concentratie of AB1-40 was 230 LM, and the molar ratio of AB1-40/AchE was equal to 100/1. The prepared in normal saline were used as a source of AChE. Mice sera were the source data represent the mean+ SEM of three independent experiments f BChE AChE or BChE activity was assayed spectrophotometrically at 412 nm. IC alues represent the concentration of inhibitors required to decrease enzyme activity by 50% and are the mean+ SEM of three independent measurements. Table 1 shows, the lipophilicity of the two compounds increased with Metal-chelating properties of ZLA and ZlB the rise of pH values With log P values of 3.71 and 4.00 respective at the physiological pH 7.4, ZLA and Zlb are sufficiently lipophilic to When ZLA or ZLB in methanol solution were mixed with equal pass the blood brain barrier(BBB molar of CuClz, a spectral change was observed, which was not de- pendent on time after mixing and appeared to be attributed to com- AChE/ BChE inhibitory activity of ZLA and ZLB plex formation between the compounds and Cu(ll)(Figs. 5A, B). In the case of the Cu(ll)-ZLA complex, Cu(Il)binding led to a decrease in absorption at 200-208 nm and a bathochromic shift with a maxi- To determine the potential value of newly synthesized com- mum peak at 220 nm resulting from charge transfer processes be- pounds for the treatment of AD, their inhibitory potency for AChE or tween the coordinated oxalamide and metal. The spectra of Cu(ll)- BChE was assayed, with rivastigmine as a reference compound. As ZLB complex showed an increase in molar absorbance, whereas Table 2 shows, ZLA and ZLB exhibited smaller ICso values for mice AChE or BChE inhibition or similar ICso values for human aChe activ- bis-MEP mixed with CuCl produced no significant spectral change Fig. 5C), indicating that the metal binding is due to specific interac ity inhibition compared with rivastigmine, suggesting that their po- tions of the spacer moiety in these bis-MEP derivatives with metal tency for cholinesterase inhibition is comparable to rivastigmine The inhibition-concentration curves of zla and zlb on human ache ns. The absorption spectra of mixtures of ZLA with ascending con activity are demonstrated in Fig 3. centrations of CuCl2(12-40 uM)are shown in Fig. 6. It was observed that the spectra reached to maximal intensity at 1: 1 molar ratio, suggesting a 1: 1 of stoichiometry of the complex. Inhibition of AchE-induced AB aggregation by ZLA and ZLB Inhibition of metal ion-induced AB aggregation by ZLA and ZLB It has been established that the peripheral anionic site mediates AChE-triggered AB aggregation(Inestrosa et al, 2008: Johnson and To further assess the metal-complexing property we subsequently Moore, 2006). In the present study, the inhibition and ICso values of observed the effects of ZLa or ZLB on AB aggregation triggered by ZLA or ZLB on human recombinant AChE-induced AB fibrillogenesis, Cu(ll)or Zn(Il) by sedimentation and turbidometry, which gave consis- in comparison with the selective peripheral site-binding inhibitor tent results(Figs. 7A, B). Similar with the results of previous studies ropidium iodine, was determined by means of a thioflavin T-based(Atwood et al, 1998; Raman et al, 2005), Cu(ln) and Zn(In) induced fluorometric assay(Bartolini et al, 2003). As Table 3 shows, both markedly aggregation of AB, manifested as decreased percentage of re ZLA and ZLB inhibited AChE-promoted AB aggregation, with IC5o covery and increased turbidity. ZLA or ZLB(at a concentration of values of 49.1 and 55.3 HM respectively. At the dose of 100 HM, 100 uM) dramatically suppressed metal ion-induced aggregation of these compounds could inhibit fibrillogenesis by nearly 50% Similar the peptide as measured by both sedimentation and turbidometry with the results of the previous studies (Bartolini et al., 2003).(P<0.01 versus Cu(ll)+ AB or Zn(ll)+AB). These findings, in parallel propidium iodine, used as a positive reference, exhibited more potent with those of spectroscopic analyses, indicate that ZLA or ZLB may effec- inhibition of AChE-promoted AB aggregation(Table 3). The inhibi- tively chelate with biometal ions such as Cu(ll)or Zn(I), and inhibit tion-concentration curves of ZLa and ZLB on human AChE-triggered metal ion-promoted AB aggregation. ZLA and ZLB, at 100 uM, did not fibrillogenesis are shown in Fig. 4. exhibit notable effect on the aggregation of AB(data not shown). 0.0 15 Log[ZA](μM Log[ZB]μM) Fig. 3. Inhibition-concentration curves of ZLA and ZLB on human acetylcholinesterase(AChE)activity in vitro. Each point represents the mean+ SEM of three independent

Table 1 shows, the lipophilicity of the two compounds increased with the rise of pH values. With log P values of 3.71 and 4.00 respectively at the physiological pH 7.4, ZLA and ZLB are sufficiently lipophilic to pass the blood brain barrier (BBB). AChE/BChE inhibitory activity of ZLA and ZLB To determine the potential value of newly synthesized com￾pounds for the treatment of AD, their inhibitory potency for AChE or BChE was assayed, with rivastigmine as a reference compound. As Table 2 shows, ZLA and ZLB exhibited smaller IC50 values for mice AChE or BChE inhibition or similar IC50 values for human AChE activ￾ity inhibition compared with rivastigmine, suggesting that their po￾tency for cholinesterase inhibition is comparable to rivastigmine. The inhibition–concentration curves of ZLA and ZLB on human AChE activity are demonstrated in Fig. 3. Inhibition of AChE-induced Aβ aggregation by ZLA and ZLB It has been established that the peripheral anionic site mediates AChE-triggered Aβ aggregation (Inestrosa et al., 2008; Johnson and Moore, 2006). In the present study, the inhibition and IC50 values of ZLA or ZLB on human recombinant AChE-induced Aβ fibrillogenesis, in comparison with the selective peripheral site-binding inhibitor propidium iodine, was determined by means of a thioflavin T-based fluorometric assay (Bartolini et al., 2003). As Table 3 shows, both ZLA and ZLB inhibited AChE-promoted Aβ aggregation, with IC50 values of 49.1 and 55.3 μM respectively. At the dose of 100 μM, these compounds could inhibit fibrillogenesis by nearly 50%. Similar with the results of the previous studies (Bartolini et al., 2003), propidium iodine, used as a positive reference, exhibited more potent inhibition of AChE-promoted Aβ aggregation (Table 3). The inhibi￾tion–concentration curves of ZLA and ZLB on human AChE-triggered fibrillogenesis are shown in Fig. 4. Metal-chelating properties of ZLA and ZLB When ZLA or ZLB in methanol solution were mixed with equal molar of CuCl2, a spectral change was observed, which was not de￾pendent on time after mixing and appeared to be attributed to com￾plex formation between the compounds and Cu(II) (Figs. 5A, B). In the case of the Cu(II)–ZLA complex, Cu(II) binding led to a decrease in absorption at 200–208 nm and a bathochromic shift with a maxi￾mum peak at 220 nm resulting from charge transfer processes be￾tween the coordinated oxalamide and metal. The spectra of Cu(II)– ZLB complex showed an increase in molar absorbance, whereas bis-MEP mixed with CuCl2 produced no significant spectral change (Fig. 5C), indicating that the metal binding is due to specific interac￾tions of the spacer moiety in these bis-MEP derivatives with metal ions. The absorption spectra of mixtures of ZLA with ascending con￾centrations of CuCl2 (12–40 μM) are shown in Fig. 6. It was observed that the spectra reached to maximal intensity at 1:1 molar ratio, suggesting a 1:1 of stoichiometry of the complex. Inhibition of metal ion-induced Aβ aggregation by ZLA and ZLB To further assess the metal-complexing property, we subsequently observed the effects of ZLA or ZLB on Aβ aggregation triggered by Cu(II) or Zn(II) by sedimentation and turbidometry, which gave consis￾tent results (Figs. 7A, B). Similar with the results of previous studies (Atwood et al., 1998; Raman et al., 2005), Cu(II) and Zn(II) induced markedly aggregation of Aβ, manifested as decreased percentage of re￾covery and increased turbidity. ZLA or ZLB (at a concentration of 100 μM) dramatically suppressed metal ion-induced aggregation of the peptide as measured by both sedimentation and turbidometry (Pb0.01 versus Cu(II)+Aβ or Zn(II)+Aβ). These findings, in parallel with those of spectroscopic analyses, indicate that ZLA or ZLB may effec￾tively chelate with biometal ions such as Cu(II) or Zn(II), and inhibit metal ion-promoted Aβ aggregation. ZLA and ZLB, at 100 μM, did not exhibit notable effect on the aggregation of Aβ (data not shown). Table 2 Inhibition of ZLA and ZLB on AChE or BChE activity. Compounds IC50± SEM (μM) Selectivity for mice AChE Human recombinant AChE Mice AChE Mice BChE ZLA 9.63±1.64 1.81±0.36 1.33±0.26 0.73 ZLB 8.64±1.58 1.54±0.27 1.72±0.30 1.12 Rivastigmine 8.45±1.47 5.43±0.83 2.13±0.47 0.39 AChE, acetylcholinesterase. BChE, butyrylcholinesterase. Mice forebrain homogenates prepared in normal saline were used as a source of AChE. Mice sera were the source of BChE. AChE or BChE activity was assayed spectrophotometrically at 412 nm. IC50 values represent the concentration of inhibitors required to decrease enzyme activity by 50% and are the mean± SEM of three independent measurements. Fig. 3. Inhibition–concentration curves of ZLA and ZLB on human acetylcholinesterase (AChE) activity in vitro. Each point represents the mean± SEM of three independent experiments. Table 3 Inhibition of ZLA and ZLB on human AChE-induced Aβ aggregation. Compounds IC50± SEM (μM) Inhibition (%) at 100 μM Inhibition (%) at 200 μM ZLA 49.1±5.0 53.2±4.3 55.6±4.7 ZLB 55.3±5.8 45.3±4.2 48.4±4.3 Propidium iodide 11.9±0.4 80.2±4.9 94.3±5.3 AChE, acetylcholinesterase. Aβ, amyloid-β. Human recombinant AChE-induced Aβ fibrillogenesis was determined using thioflavin T fluorescence. The final concentration of Aβ1–40 was 230 μM, and the molar ratio of Aβ1–40/AChE was equal to 100/1. The data represent the mean± SEM of three independent experiments. W. Zheng et al. / Toxicology and Applied Pharmacology 264 (2012) 65–72 69