2028 Journal of Medicinal Chemistry, 2008, VoL. 51, No. 7 Xie et al Me○ CH2) OMe 1 bis-tacrine 2 bis-galanthamine 3bs5-amin56,7,8 (CH2im N(CH2)N(CH2)m) M Figure 2. Structures of reported homobivalent AChE inhibitors and title compounds 5 Scheme 1. Synthesis of (-)-nor-MEP 8 7 8 huperzine B 4(Figure 2, m=2, n=10)(Figure 2)have also Scheme 2. Synthesis of 5a, b,e-k been reported. Our group has been interested in the study of meptazinol (MEP),a racemic marketed opioid analgesic with low addic tion liability, and its()-enantiomer, which has demonstrated moderate inhibition of AChE. We established an approach to the resolution of MEP in acceptable yields and determined the absolute configurations of(-)-MEP and(+)-MEP as S and R, respectively, by X-ray crystal structures. 17 Continuing with our 5(a-b, e- previous research to find new AChE inhibitors through the Reagents and conditions: (i)a, a-dihaloalkane(0.5 equiv), triethylamine molecular modeling of(-)-MEP derivatives, we describe here (2 equiv), acetonitrile, reflux, 2-5h, 35-83% the design, synthesis, pharmacological evaluation, and molecular docking of a series of homobivalent(-)-N-demethylmeptazinols (bis-(-)-nor-MEPs)5. Two identical (-)-nor-MEP units are Newly synthesized compounds were tested in vitro for AChE ind BChE inhibitory potency, and their selectivity for AChE connected by alkylene linkers of different lengths via the was calculated. A molecular docking study was performed on ondary amino groups in compound 5(Figure 2) A suitable length of the alkylene linker, together with ar mouse AChE (mAChE) and human bChe (hBChE) to il luminate the binding modes of the most potent compound 5h appropriate point of the coupling position, guarantees that bis- with both enzymes. Because of the unavailability of crystal ligand will simultaneously bind to the catalytic and peripheral lographic data of mouse BChE, hBChE was used instead sites of the enzyme. According to our predicted binding mode because of a high sequence identity. The ability of compounds of(-)-MEP in AChE active site, the azepane ring is locate 5g, 5h, and 5i to inhibit the AChE-induced AB aggregation in the middle of the gorge whereas the phenolic group is oriented compared with propidium iodine and the reference compound ()-MEP derivatives were designed as bis-(-)-nor-MEPs ()-MEP, was assessed by means of a thioflavin T-based linking, via a point in the azepane ring instead of the phenolic fluorometric assay. Cell viability was tested by MTT assay group(Figure 1). As to the chain length(number of methylene nun er in the case of tacrine- based bivalent ligands was 7.However, that might not be the case in our Results and Discussion study. To find the most potent compound in our series and Chemistry. The synthetic methodology employed for the discuss the effect of linker length on inhibitory potency, preparation of bis-(--nor-MEP derivatives 5 is illustrated in compounds possessing methylene spacers varying from 2 to 12 Schemes 1-3. Key step in this route is the N-demethylation of were synthesized (-)-MEP 6 producing(-)-nor-MEP 8. A few methods havehuperzine B 4 (Figure 2, m ) 2, n ) 10)12 (Figure 2) have also been reported. Our group has been interested in the study of meptazinol (MEP),15 a racemic marketed opioid analgesic with low addic￾tion liability, and its (-)-enantiomer, which has demonstrated moderate inhibition of AChE.16 We established an approach to the resolution of MEP in acceptable yields and determined the absolute configurations of (-)-MEP and (+)-MEP as S and R, respectively, by X-ray crystal structures.17 Continuing with our previous research to find new AChE inhibitors through the molecular modeling of (-)-MEP derivatives,18 we describe here the design, synthesis, pharmacological evaluation, and molecular docking of a series of homobivalent (-)-N-demethylmeptazinols (bis-(-)-nor-MEPs) 5. Two identical (-)-nor-MEP units are connected by alkylene linkers of different lengths via the secondary amino groups in compound 5 (Figure 2). A suitable length of the alkylene linker, together with an appropriate point of the coupling position, guarantees that bis￾ligand will simultaneously bind to the catalytic and peripheral sites of the enzyme. According to our predicted binding mode of (-)-MEP in AChE active site,18 the azepane ring is located in the middle of the gorge whereas the phenolic group is oriented down into the catalytic site at the bottom. Therefore, bivalent (-)-MEP derivatives were designed as bis-(-)-nor-MEPs linking, via a point in the azepane ring instead of the phenolic group (Figure 1). As to the chain length (number of methylene units), the optimal number in the case of tacrine-based bivalent ligands was 7.9,19 However, that might not be the case in our study. To find the most potent compound in our series and discuss the effect of linker length on inhibitory potency, compounds possessing methylene spacers varying from 2 to 12 were synthesized. Newly synthesized compounds were tested in vitro for AChE and BChE inhibitory potency, and their selectivity for AChE was calculated. A molecular docking study was performed on mouse AChE (mAChE) and human BChE (hBChE) to il￾luminate the binding modes of the most potent compound 5h with both enzymes. Because of the unavailability of crystal￾lographic data of mouse BChE, hBChE was used instead because of a high sequence identity. The ability of compounds 5g, 5h, and 5i to inhibit the AChE-induced A
aggregation, compared with propidium iodine and the reference compound (-)-MEP, was assessed by means of a thioflavin T-based fluorometric assay.20 Cell viability was tested by MTT assay for 5h and 5i. Results and Discussion Chemistry. The synthetic methodology employed for the preparation of bis-(-)-nor-MEP derivatives 5 is illustrated in Schemes 1-3. Key step in this route is the N-demethylation of (-)-MEP 6 producing (-)-nor-MEP 8. A few methods have Figure 2. Structures of reported homobivalent AChE inhibitors and title compounds 5. Scheme 1. Synthesis of (-)-nor-MEP 8a a Reagents and conditions: (i) (a) ClCOOEt, KHCO3, CHCl3, reflux, 1 h; (b) K2CO3 (aq), MeOH, N2, room temp, 1 h, 95%; (ii) 50% H2SO4, N2, reflux, 4 h, 54%. Scheme 2. Synthesis of 5a,b,e-ka a Reagents and conditions: (i) R,ω-dihaloalkane (0.5 equiv), triethylamine (2 equiv), acetonitrile, reflux, 2-5 h, 35-83%. 2028 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 Xie et al