Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2029 Scheme 3. Synthesis of 5c, d CH2)n-2 9(,d) been reported for the demethylation of tertiary amines, such as Table 1. Inhibition of AChE and BChE by Bis-(-)-nor-MEPs 5a-k ()MEP, and Rivastigmine employment of azodicarboxylic acid esters, cyanogen bro- ICso(nM) as the reagent, taking both the availability and reaction simplicity ice brain Mice serum selectivity for to account. treating MEP 6 with ethyl chloroformate in BChE the presence of KHCO3 in boiling CHCl3, followed by dealing 5a 43000±20000125±9 the resulting residue with a mild base, afforded a nonbasic 342000±14000132±510.0 carbamate intermediate, (-)-N-carboethoxy nor-MEP 7How 421400±7600104±290.0049 4000±1000192±41 0.048 ever, troubles were encountered in the hydrolysis and decar- se 1220±20 0.098 xylation of the resulting carbamate intermediate 7. Al 5f 7 270±70 102±19 reactions failed under the reported alkaline condition in KOH24 ±19 63±8 nd acidic conditions in hydrobromic acid or 25% sulfuric acid 3.9±1.3 10士3 Finally this transformation was accomplished in 50% sulfuric .5±4.5 74±11 acid under nitrogen for 4 h, and (-)-nor-MEP 8 was obtained 42±20 in a 54% yield(Scheme 1) rivastigmine 5500±15001600±300.29 Alkylation of (-)-nor-MEP 8 with a, o-dihaloalkanes(0.5 (一)MEP 41000±1400015000±40000.37 equiv) in the presence of triethylamine, followed by chromato- graphic purification, easily produced the bis-(-)-nor-MEP of AChE. Mice serum was the source of BChE. AChE was assayed compounds 5a, b e-k in 35-83% yield(Scheme 2). However. spectrophotometrically with acetylthiocholine as substrate in the presence Ikylation with 1, 4-dichlorobutane or 1, 5-dichloropentane failed with butyrylthiocholine as substrate and 1025 M BW284C51 as AChE to furnish the bivalent compounds 5c, d, since the N-4-chlo- inhibitor ICso values were computed by a nonlinear least squares regression robutyl (or 5-chloropentyl)-(-)-nor-MEP intermediate was prone program that also provided an estimate of statistical precision(standard to forming stable intramolecular five-membered or six error of the mean). Selectivity for AChE: ICso for BChE divided by ICso membered ring structures, resulting in failure to link another for AChE. )-nor-MEP unit and leading to the generation of spiro ■AChE◆BChE quaternary ammoniums Structures of two quaternary ammo- niums derived from the R enantiomer were confirmed by X-ray crystallographic diffraction. Eventually, the synthesis of the bis-ligands 5c, d was accomplished by acylation with a,a- Ikanediacyl dihalide(0.5 equiv) to form bis-amides intermedi- ates 9c, d followed by reduction using lithium aluminum hydride (LAH) in tetrahydrofuran(THF)(Scheme 3) The chemical structures of all target compounds or their ynthesized hydrochloride salts were characterized by specific rotation [a]D, IR, H NMR, and HR-ESI, as reported in the 5269只 Experimental Section. The complicated property of NMR data from bis-(-)-nor-MEP hydrochloride salts in DMSO-d6 re- sembled the case of (+)-MEP hydrochloride. It was reasonably explained by conformational switch of the azepane ring and onfigurational inversion of nitrogen AChE Inhibitory Potency and AChE/BChE Selectivity Newly synthesized compounds were tested in vitro for potency 即mh时nr and selectivity as cholinesterase( ChE)inhibitors. Extracts from mice brain and mice serum were used as sources of AChE and the length of the alkylene chain. The optimal chain length BChE, respectively. The results showed that both(-)-MEP and determined experimentally was achieved in compound 5h, with its bis-ligand analogues possessed ChE inhibitory activity. The nine methylene groups between two (-)-nor-MEP units 50 value of(-)-MEP was 41 uM, about 10 times higher than Compared with(-)-MEP (ICso=41 uM)and rivastigmine(ICso that obtained with AChE from bovine erythrocytes, and the=5.5 uM), 5h(ICs0=3.9 nM) showed a 10000-fold and 1400 testing data of the reference drug rivastigmine conformed to fold increase, respectively, in the inhibition of mice brain AChE the previous report 27 The AChE inhibitory potency within the (Table 1). Further decrease or increase of the chain length series of bis-(-)-nor-MEP derivatives was closely related to weakened the AChE inhibition(Figure 3). For instanbeen reported for the demethylation of tertiary amines, such as employment of azodicarboxylic acid esters,21 cyanogen bro￾mide,22 or chloroformates.23,24 Ethyl chloroformate was chosen as the reagent, taking both the availability and reaction simplicity into account. Treating (-)-MEP 6 with ethyl chloroformate in the presence of KHCO3 in boiling CHC13, followed by dealing the resulting residue with a mild base, afforded a nonbasic carbamate intermediate, (-)-N-carboethoxy nor-MEP 7. How￾ever, troubles were encountered in the hydrolysis and decar￾boxylation of the resulting carbamate intermediate 7. All reactions failed under the reported alkaline condition in KOH24 and acidic conditions in hydrobromic acid or 25% sulfuric acid. Finally this transformation was accomplished in 50% sulfuric acid under nitrogen for 4 h, and (-)-nor-MEP 8 was obtained in a 54% yield (Scheme 1). Alkylation of (-)-nor-MEP 8 with R,ω-dihaloalkanes (0.5 equiv) in the presence of triethylamine, followed by chromato￾graphic purification, easily produced the bis-(-)-nor-MEP compounds 5a,b,e-k in 35-83% yield (Scheme 2). However, alkylation with 1,4-dichlorobutane or 1,5-dichloropentane failed to furnish the bivalent compounds 5c,d, since the N-4-chlo￾robutyl (or 5-chloropentyl)-(-)-nor-MEP intermediate was prone to forming stable intramolecular five-membered or six￾membered ring structures, resulting in failure to link another (-)-nor-MEP unit and leading to the generation of spiro quaternary ammoniums. Structures of two quaternary ammo￾niums derived from the R enantiomer were confirmed by X-ray crystallographic diffraction.25 Eventually, the synthesis of the bis-ligands 5c,d was accomplished by acylation with R,ω- alkanediacyl dihalide (0.5 equiv) to form bis-amides intermedi￾ates 9c,d followed by reduction using lithium aluminum hydride (LAH) in tetrahydrofuran (THF) (Scheme 3). The chemical structures of all target compounds or their synthesized hydrochloride salts were characterized by specific rotation [R]D, IR, 1 H NMR, and HR-ESI, as reported in the Experimental Section. The complicated property of NMR data from bis-(-)-nor-MEP hydrochloride salts in DMSO-d6 re￾sembled the case of (+)-MEP hydrochloride.26 It was reasonably explained by conformational switch of the azepane ring and configurational inversion of nitrogen. AChE Inhibitory Potency and AChE/BChE Selectivity. Newly synthesized compounds were tested in vitro for potency and selectivity as cholinesterase (ChE) inhibitors. Extracts from mice brain and mice serum were used as sources of AChE and BChE, respectively. The results showed that both (-)-MEP and its bis-ligand analogues possessed ChE inhibitory activity. The IC50 value of (-)-MEP was 41 µM, about 10 times higher than that obtained with AChE from bovine erythrocytes,16 and the testing data of the reference drug rivastigmine conformed to the previous report.27 The AChE inhibitory potency within the series of bis-(-)-nor-MEP derivatives was closely related to the length of the alkylene chain. The optimal chain length determined experimentally was achieved in compound 5h, with nine methylene groups between two (-)-nor-MEP units. Compared with (-)-MEP (IC50 ) 41 µM) and rivastigmine (IC50 ) 5.5 µM), 5h (IC50 ) 3.9 nM) showed a 10000-fold and 1400- fold increase, respectively, in the inhibition of mice brain AChE (Table 1). Further decrease or increase of the chain length weakened the AChE inhibition (Figure 3). For instance, the Scheme 3. Synthesis of 5c,da a Reagents and conditions: (i) R,ω-alkanediacyl dihalide (0.5 equiv), triethylamine (2 equiv), dry CH2Cl2, 0 °C, 15 min, 38-41%; (ii) lithium aluminum hydride (LAH), dry THF, reflux, 1 h, 31-36%. Table 1. Inhibition of AChE and BChE by Bis-(-)-nor-MEPs 5a-k, (-)-MEP, and Rivastigminea IC50 (nM) compd chain length (n) mice brain AChE Mice serum BChE selectivity for AChEb 5a 2 43000 ( 20000 125 ( 9 0.0029 5b 3 42000 ( 14000 132 ( 51 0.0031 5c 4 21400 ( 7600 104 ( 29 0.0049 5d 5 4000 ( 1000 192 ( 41 0.048 5e 6 1220 ( 20 119 ( 20 0.098 5f 7 270 ( 70 102 ( 19 0.38 5g 8 79 ( 19 63 ( 8 0.80 5 h 9 3.9 ( 1.3 10 ( 3 2.6 5i 10 9.5 ( 4.5 17 ( 6 1.8 5j 11 24 ( 8 74 ( 11 3.1 5k 12 42 ( 20 100 ( 55 2.4 rivastigmine 5500 ( 1500 1600 ( 30 0.29 (-)-MEP 41000 ( 14000 15000 ( 4000 0.37 a Mice brain homogenate prepared in normal saline was used as a source of AChE. Mice serum was the source of BChE. AChE was assayed spectrophotometrically with acetylthiocholine as substrate in the presence of 1024 M ethopropazine as BChE inhibitor. BChE was assayed similarly with butyrylthiocholine as substrate and 1025 M BW284C51 as AChE inhibitor. IC50 values were computed by a nonlinear least squares regression program that also provided an estimate of statistical precision (standard error of the mean). b Selectivity for AChE: IC50 for BChE divided by IC50 for AChE. Figure 3. Correlation between ChE inhibitory potency (-log(IC50)) and the alkylene chain length (n) in compounds 5. Bis-(-)-nor-meptazinols as Inhibitors Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2029