032 Journal of Medicinal Chemistry, 2008, VoL. 51, No. 7 Xie et al Table 2. Inhibition of AChE-Induced AB Aggregation by elution with EtoH/CHCI3 (0.8: 9.2 to 3: 7). The eluent was concen- Bis-(-H-nor-MEPs 5g-i and Reference Compounds trated in vacuo to afford 8(4.58 g, 54%0) Salt forming reaction of chain inhibition (%)at inhibition(%)at 8(1.07 g)was carried out in dry ether by adding dry HCl-ether mpd length(n) 200M and adjusting the pH to 4, which afforded 8. HCl as a white powder 856±44986±59128+04(93NMR①DMO4)942(HAOH,821ors12 98g,79%):mp73-75℃C;[alb-7.10°(c0.286,MeOH;IR )-MEP 0 15.2±0.2nd 90.8±0.2 2±0.116.6±0.5 6.74-6.65(m,3H,ArH),3.49(d,H,J=14.1Hz,N-CH2),3.21 958±0.5 984±0.158±0.3 (d,H,J=14.5Hz,N-CH2),3.08-3.00(m,2H,N-CH2),2.14 4 nd: not determined (m,H,CH2),1.77-155(m,7H,CH2),0.49(t,3H,J= B): MS (ESD)[M +H+220. AB aggregation with ICso values of 16.6 and 5.8 uM, compatible General Procedure for the Synthesis of Bis-(-)-Nor-MEP with that of propidium(IC50=12. 8 uM), which pointed out a promising disease-modifying action. Further pharmacological Compounds 5a, b, e-k. Triethylamine(2 equiv) and a, @-dihaloal study is needed to evaluate their abilities to reverse memory kane(0.5 equiv) were added to a solution of (-)-nor-MEP 8 in impairment in animal models in order to select ideal candidates acetonitrile. The reaction mixture was refluxed for 2-5 h Evapora tion of the solvent gave a residue, which was diluted with saturated for the treatment of AD patients K CO3 solution and extracted with CHCl3. The combined CHCls Experimental Sect extracts were dried (anhydrous Na2SO4) and evaporated under Chemistry. Melting points were taken in glass silica gel Eluting with petroleum ether/EtOAc(1: 2)afforded the and were uncorrected. Specific rotation (alp)was corresponding bis-(-)-nor-MEP compounds 5 as a yellow oil a JASCOP-1020 rotatory apparatus. IR data were on an Addition of dry HCI-ether to a solution of 5 in dry ether and AVATAR 360 FT-IR spectrometer(KBr). NMR data were recorded adjusting the ph to 3-4 gave the final salt 5. 2HCI as powder with a Mercury Plus 400 instrument. Chemical shifts(o)are N N-(1, 2-Ethylene)-bis-(-)-nor-MEP Hydrochloride(5a expressed in parts per million(ppm) relative to tetramethylsilane 2HCD) (-)-nor-MEP 8(1.50 g, 6.85 mmol), acetonitrile(15 mL). triethylamine (1.9 mL, 13.7 mmol), and 1, 2-dibromoethane(0. 297 disappeared after D2O exchange. Mass spectra were measured on mL, 3.43 mmol) were used to produce 5a(0.80 g, 50%). Subsequent an Agilent 1100 series LC/MSD 1946D spectrometer. HRMs salt formation gave 5a.2HCI(0.80 g, 86%0): mp 142-145C; [aID petra were recorded with an lon Spec 4.7 T FTMS instrument. 1.96°(c0.204,MeOH;Rv3176,2935,1599,1447,1229cm he purity of all target compounds(95%) was verified via HPLC. H NMR(DMSO-d6)10.84(br s, 1/2 H, NH*),10.58(br The elution was methanol-0.05 mol/L ammonium acetate sol NH+),9.51,9.49,943,9.34(s,2H,Ar-OH,909brs,12H, djustment of pH to 7. 4 with aqueous ammonia)(70: 30 to NH+),7.22-7.08(m,2H,Ar-H,6.83-6.61(m,6H,Ar-H) The chromatographic condition was a flow rate of 1.0 mL/mi 3.79-3.71(m,H,N-CH2),3.47-3.42(m,H,N-CH2),3.14-3.10 UV detection at 225 nm on a VP-ODS C18(150 mm x 4.6 mm (m,2H,N-CH2),3.03-2.86(m,6H,N-CH2),2.71-269(m,2H, 5 um)column at a temperature of 50C. All reagents were of N-CH2), 2.14-2.08( m, 2H, CH2), 1.84-1 46(m, 14H, CH2), 0.47 commercial quality. Rivastigmine hydrochloride standard was (m, 6H, CH3); MS(ESD) [M+ H] 465.6 HRMS m/z calcd for available from Sunve(Shanghai) Pharmaceutical Co, Ltd C30HasN2O [ M H, 465.3476: found, 465 3463. HPLC: IR (-)-N-Carboethoxy-nor-MEP (7). A stirred suspension of(-)- 4.48 min, 98.2% purity MEP 6(9.83 g, 42.2 mmol) and KHCO3 (74 g, 740 mmol)in sOiling CHCl3(500 mL) was treated with ethyl chloroformate(30.5 2HCD).(-)-nor-MEP8(1.54 g, 7.03 mmol), acetonitrile(15 mL) and the CHCl phase was separated and concentrated in vacuo. The mL, 3. 52 mmol)were used to produce Sb(1.20g.no 3197(r sidue was dissolved in MeOH (400 mL), treated with an aqu salt formation of 5b(0.90 g) gave 5b. 2HCI solution(400 mL) containing 66g of K2CO3, and stirred under N2 165-168C; [a]D-48.38(c 0.228, MeOH); IR at room temperature for 1 h After the MeOh was removed, the 1599, 1447, 1229 cm ; H NMR (DMSO-d6)10. 18, sidue was neutralized with 6 M HCI(126 mL)and extracted with 6/5 H, NH*), 9.58, 9.52, 944, 9.42(, 2H, Ar-OH),8.68,8.59 Et,0(150 mL x 3). The combined Et,O extracts were washed (br s, 4/5 H, NH), 7.20-7.13(m, 2H, Ar-H), 6.90-6. 65(m, 6H with saturated NaCl solution(150 mL) and dried with anhydrous Ar-H),3.92(, 4/5 H,J=14.5 Hz, N-CH2), 3.56(m, 6/5 H, Na2SO4 Evaporation of the solvent under reduced pressure gave 7 N-CH2,3.44-3.21(m, 10H, N-CH2, 2.40(m, 2H, CH2). 11.17 g, 95%)as a yellowish oil Recrystallization of 7(1.2 g) 2.10-1.50(m, 16H, CH2), 0.50(m, 6H, CH3); MS(ESD)[M from ethyl acetate(2 mL) afforded 7 as off-white crystals(0.64g, HI. 4, [M+ 2H] 240.2. HRMS m/z calcd for C31H47N2O2 53%):mp79-81C;alo-61.35°(c0.11,MeOH;HNM M+H+,479.3632; found,479.3641.HPLC:tk=700min, (CDCl3)7.18(t,H,J=7.8Hz,ArH),687-681(m,2H,ArH 6.70(d,H,J=8.0Hz,ArH,6.00(brs,1/2H,Ar-OH,5.57(br N, N-(1, 6-Hexylene)-bis-(-)-nor-MEP Hydrochloride (5e S, 1/2 H, Ar-OH), 4. 17-4.03(m, 2H, -OCH2), 3.94(d, H, N-CH2, 2HCI).(-)-nor-MEP 8(1.96g, 8.95 mmol), acetonitrile(20 mL) J= 14.6 Hz), 3.90-3.85(, 1/2 H, N-CH2CH3), 3.79-3. 73(m, triethy lamine (2.5 mL, 18.0 mmol), and 1, 6-dibromohexane(0.702 1/2 H, N-CH2), 3.52(d, 1/2H, J=14.4 Hz, N-CH2), 3.34(d, mL, 4.48 mmol) were used to produce 5e(0.96g, 41%) Subsequent l/2H,J=14.9Hz,N-CH2),3.06-295(m,H,N 20-2.05 salt formation gave 5e 2HCI (1.06g, 97%0): mp 13 CH3),-478°(c0.175,MeOH);IRv3414,31 2876,2732,1600, 0.56(t,3H,J=7.3H2,CH3);Ms(ESD)[M+ -)-Nor-MEP Hydrochloride(8.HCD). A mixture of 7(11. 17 2H, Ar-OHD),8.43,8.33(br s, 3/4 H, NH+), 7. 22-7.13.g2 Na]+3142,[M+K+330.2 1/2H,NH),9.80(brs,3/4H,NH+,956,9.53,9 g, 38.38 mmol) in 50% H,SOA(120 mL) was refluxed under N2 Ar-H). 6.87-6.76(m, 4H, Ar-H). 6.71-6.66(m, 2H, Ar-H), 3.84 for 4 h. The solution was treated with aqueous ammonia(180 mL), (m, 3/4 H. N-CH2), 3.55(m, 5/4 H, N-CH2). 3.34-3.09(m, 10H, dj usted to pH 9, and extracted with CHCl3(200 mL x 3). The N-CH2, 2.38(m, H, CH2), 2. 12-1.73(m, 15H, CH2), 1.57-1.4 mbined CHCla extracts were washed with saturated NaCl solution(m, 4H, CH2). 1.35-1.25(m, 4H, CH, ), 0.50(t, 6H,J=7.4 Hz (200 mL) and dried with anhydrous Na2SO4. The solvent was CH3): MS(ESD)[M+ H 521.7, [M+ 2H]- 261.4 HRMS m/z removed in vacuo, and the oily residue underwent chromatography calcd for C34HS3N2O2 [M H, 521.4102: found, 521.4086 on a column of 170 g of silica gel (200-300 mesh) and gradient HPLC: IR =810 min, 97.6% purityA
aggregation with IC50 values of 16.6 and 5.8 µM, compatible with that of propidium (IC50 ) 12.8 µM), which pointed out a promising disease-modifying action. Further pharmacological study is needed to evaluate their abilities to reverse memory impairment in animal models in order to select ideal candidates for the treatment of AD patients. Experimental Section Chemistry. Melting points were taken in glass capillary tubes and were uncorrected. Specific rotation ([R]D) was determined on a JASCOP-1020 rotatory apparatus. IR data were taken on an AVATAR 360 FT-IR spectrometer (KBr). NMR data were recorded with a Mercury Plus 400 instrument. Chemical shifts (δ) are expressed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. All signals of active hydrogen disappeared after D2O exchange. Mass spectra were measured on an Agilent 1100 series LC/MSD 1946D spectrometer. HRMS spectra were recorded with an IonSpec 4.7 T FTMS instrument. The purity of all target compounds (>95%) was verified via HPLC. The elution was methanol-0.05 mol/L ammonium acetate solution (adjustment of pH to 7.4 with aqueous ammonia) (70:30 to 80:20). The chromatographic condition was a flow rate of 1.0 mL/min with UV detection at 225 nm on a VP-ODS C18 (150 mm × 4.6 mm, 5 µm) column at a temperature of 50 °C. All reagents were of commercial quality. Rivastigmine hydrochloride standard was available from Sunve (Shanghai) Pharmaceutical Co., Ltd. (-)-N-Carboethoxy-nor-MEP (7). A stirred suspension of (-)- MEP 6 (9.83 g, 42.2 mmol) and KHCO3 (74 g, 740 mmol) in boiling CHCl3 (500 mL) was treated with ethyl chloroformate (30.5 mL, 320 mmol) and refluxed for 1 h. H2O (350 mL) was added, and the CHCl3 phase was separated and concentrated in vacuo. The residue was dissolved in MeOH (400 mL), treated with an aqueous solution (400 mL) containing 66 g of K2CO3, and stirred under N2 at room temperature for 1 h. After the MeOH was removed, the residue was neutralized with 6 M HCl (126 mL) and extracted with Et2O (150 mL × 3). The combined Et2O extracts were washed with saturated NaCl solution (150 mL) and dried with anhydrous Na2SO4. Evaporation of the solvent under reduced pressure gave 7 (11.17 g, 95%) as a yellowish oil. Recrystallization of 7 (1.2 g) from ethyl acetate (2 mL) afforded 7 as off-white crystals (0.64 g, 53%): mp 79-81 °C; [R]D -61.35° (c 0.11, MeOH); 1 H NMR (CDCl3) 7.18 (t, H, J ) 7.8 Hz, ArH), 6.87–6.81 (m, 2H, ArH), 6.70 (d, H, J ) 8.0 Hz, ArH), 6.00 (br s, 1/2 H, Ar-OH), 5.57 (br s, 1/2 H, Ar-OH), 4.17–4.03 (m, 2H, -OCH2), 3.94 (d, H, N-CH2, J ) 14.6 Hz), 3.90–3.85 (m, 1/2 H, N-CH2CH3), 3.79–3.73 (m, 1/2 H, N-CH2), 3.52 (d, 1/2H, J ) 14.4 Hz, N-CH2), 3.34 (d, 1/2 H, J ) 14.9 Hz, N-CH2), 3.06–2.95 (m, H, N-CH2), 2.20–2.05 (m, H, CH2), 1.77–1.58 (m, 7H, CH2), 1.24 (m, 3H, -OCH2CH3), 0.56 (t, 3H, J ) 7.3 Hz, CH3); MS (ESI) [M + H]+ 292.2, [M + Na]+ 314.2, [M + K]+ 330.2. (-)-Nor-MEP Hydrochloride (8 ·HCl). A mixture of 7 (11.17 g, 38.38 mmol) in 50% H2SO4 (120 mL) was refluxed under N2 for 4 h. The solution was treated with aqueous ammonia (180 mL), adjusted to pH 9, and extracted with CHCl3 (200 mL × 3). The combined CHCl3 extracts were washed with saturated NaCl solution (200 mL) and dried with anhydrous Na2SO4. The solvent was removed in vacuo, and the oily residue underwent chromatography on a column of 170 g of silica gel (200-300 mesh) and gradient elution with EtOH/CHCl3 (0.8:9.2 to 3:7). The eluent was concen￾trated in vacuo to afford 8 (4.58 g, 54%). Salt forming reaction of 8 (1.07 g) was carried out in dry ether by adding dry HCl-ether and adjusting the pH to 4, which afforded 8 ·HCl as a white powder (0.98 g, 79%): mp 73-75 °C; [R]D -7.10° (c 0.286, MeOH); IR ν 3257, 2932, 1614, 1588, 1481, 1432, 1321, 1276, 1237, 1211 cm-1 ; 1 H NMR (DMSO-d6) 9.42 (s, H, Ar-OH), 8.82 (br s, 1/2 H, NH+), 8.24 (br s, 1/2 H, NH+), 7.16 (t, H, J ) 7.8 Hz, ArH), 6.74–6.65 (m, 3H, ArH), 3.49 (d, H, J ) 14.1 Hz, N-CH2), 3.21 (d, H, J ) 14.5 Hz, N-CH2), 3.08–3.00 (m, 2H, N-CH2), 2.14 (m, H, CH2), 1.77–1.55 (m, 7H, CH2), 0.49 (t, 3H, J ) 7.4 Hz, CH3); MS (ESI) [M + H]+ 220.1. HPLC: tR ) 1.86 min, 98.5% purity. General Procedure for the Synthesis of Bis-(-)-Nor-MEP Compounds 5a,b,e-k. Triethylamine (2 equiv) and R,ω-dihaloal￾kane (0.5 equiv) were added to a solution of (-)-nor-MEP 8 in acetonitrile. The reaction mixture was refluxed for 2-5 h. Evapora￾tion of the solvent gave a residue, which was diluted with saturated K2CO3 solution and extracted with CHCl3. The combined CHCl3 extracts were dried (anhydrous Na2SO4) and evaporated under reduced pressure. The residue was purified by chromatography on silica gel. Eluting with petroleum ether/EtOAc (1:2) afforded the corresponding bis-(-)-nor-MEP compounds 5 as a yellow oil. Addition of dry HCl-ether to a solution of 5 in dry ether and adjusting the pH to 3-4 gave the final salt 5 · 2HCl as powder. N,N′-(1′,2′-Ethylene)-bis-(-)-nor-MEP Hydrochloride (5a · 2HCl). (-)-nor-MEP 8 (1.50 g, 6.85 mmol), acetonitrile (15 mL), triethylamine (1.9 mL, 13.7 mmol), and 1,2-dibromoethane (0.297 mL, 3.43 mmol) were used to produce 5a (0.80 g, 50%). Subsequent salt formation gave 5a · 2HCl (0.80 g, 86%): mp 142-145 °C; [R]D -1.96° (c 0.204, MeOH); IR ν 3176, 2935, 1599, 1447, 1229 cm-1 ; 1 H NMR (DMSO-d6) 10.84 (br s, 1/2 H, NH+), 10.58 (br s, H, NH+), 9.51, 9.49, 9.43, 9.34(s, 2H, Ar-OH), 9.09 (br s, 1/2 H, NH+), 7.22–7.08 (m, 2H, Ar-H), 6.83–6.61 (m, 6H, Ar-H), 3.79–3.71 (m, H, N-CH2), 3.47–3.42 (m, H, N-CH2), 3.14–3.10 (m, 2H, N-CH2), 3.03–2.86 (m, 6H, N-CH2), 2.71–2.69 (m, 2H, N-CH2), 2.14–2.08 (m, 2H, CH2), 1.84–1.46 (m, 14H, CH2), 0.47 (m, 6H, CH3); MS (ESI) [M + H]+ 465.6. HRMS m/z calcd for C30H45N2O2 [M + H]+, 465.3476; found, 465.3463. HPLC: tR ) 4.48 min, 98.2% purity. N,N′-(1′,3′-Propylene)-bis-(-)-nor-MEP Hydrochloride (5b · 2HCl). (-)-nor-MEP 8 (1.54 g, 7.03 mmol), acetonitrile (15 mL), triethylamine (2.0 mL, 14.4 mmol), and 1,3-dibromopropane (0.357 mL, 3.52 mmol) were used to produce 5b (1.20 g, 71%). Subsequent salt formation of 5b (0.90 g) gave 5b · 2HCl (0.90 g, 87%): mp 165-168 °C; [R]D -48.38° (c 0.228, MeOH); IR ν 3176, 2935, 1599, 1447, 1229 cm-1 ; 1 H NMR (DMSO-d6) 10.18, 9.97 (br s, 6/5 H, NH+), 9.58, 9.52, 9.44, 9.42 (s, 2H, Ar-OH), 8.68, 8.59 (br s, 4/5 H, NH+), 7.20–7.13 (m, 2H, Ar-H), 6.90–6.65 (m, 6H, Ar-H), 3.92 (d, 4/5 H, J ) 14.5 Hz, N-CH2), 3.56 (m, 6/5 H, N-CH2), 3.44–3.21 (m, 10H, N-CH2), 2.40 (m, 2H, CH2), 2.10–1.50 (m, 16H, CH2), 0.50 (m, 6H, CH3); MS (ESI) [M + H]+ 479.4, [M + 2H]2+ 240.2. HRMS m/z calcd for C31H47N2O2 [M + H]+, 479.3632; found, 479.3641. HPLC: tR ) 7.00 min, 99.1% purity. N,N′-(1′,6′-Hexylene)-bis-(-)-nor-MEP Hydrochloride (5e · 2HCl). (-)-nor-MEP 8 (1.96 g, 8.95 mmol), acetonitrile (20 mL), triethylamine (2.5 mL, 18.0 mmol), and 1,6-dibromohexane (0.702 mL, 4.48 mmol) were used to produce 5e (0.96 g, 41%). Subsequent salt formation gave 5e · 2HCl (1.06 g, 97%): mp 135-138 °C; [R]D -47.8° (c 0.175, MeOH); IR ν 3414, 3176, 2938, 2876, 2732, 1600, 1585, 1447, 1269, 1232 cm–1; 1 H NMR (DMSO-d6) 10.05 (br s, 1/2 H, NH+), 9.80 (br s, 3/4 H, NH+), 9.56, 9.53, 9.44, 9.42 (s, 2H, Ar-OH), 8.43, 8.33 (br s, 3/4 H, NH+), 7.22–7.13 (m, 2H, Ar-H), 6.87–6.76 (m, 4H, Ar-H), 6.71–6.66 (m, 2H, Ar-H), 3.84 (m, 3/4 H, N-CH2), 3.55 (m, 5/4 H, N-CH2), 3.34–3.09 (m, 10H, N-CH2), 2.38 (m, H, CH2), 2.12–1.73 (m, 15H, CH2), 1.57–1.44 (m, 4H, CH2), 1.35–1.25 (m, 4H, CH2), 0.50 (t, 6H, J ) 7.4 Hz, CH3); MS (ESI) [M + H]+ 521.7, [M + 2H]2+ 261.4. HRMS m/z calcd for C34H53N2O2 [M + H]+, 521.4102; found, 521.4086. HPLC: tR ) 8.10 min, 97.6% purity. Table 2. Inhibition of AChE-Induced A
Aggregation by Bis-(-)-nor-MEPs 5g-i and Reference Compounds compd chain length (n) inhibition (%) at 100 µM inhibition (%) at 200 µM IC50 (µM) propidium iodine 85.6 ( 4.4 98.6 ( 5.9 12.8 ( 0.4 (-)-MEP nda 0 nda 5g 8 0 15.2 ( 0.2 nda 5h 9 90.8 ( 0.2 99.2 ( 0.1 16.6 ( 0.5 5i 10 95.8 ( 0.5 98.4 ( 0.1 5.8 ( 0.3 a nd: not determined. 2032 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 Xie et al