Supporting information
Identification of tetrahydrocarbazoles as novel multifactorial drug candidates in the treatment of Alzheimer’s disease
Kamran Honarnejad, Alexander Daschner , André P. Gehring, Aleksandra Szybinska, Armin Giese, Jacek Kuznicki, Franz Bracher and Jochen Herms
Supplementary figure legends
Fig. S1. The list of commercially available tetrahydrocarbazole analogues
Shown are 38 tested commercially available tetrahydrocarbazoles. Next to their chemical structure and physical properties, the measures for their activity in different assays are presented with their corresponding normalized values for: CCh-evoked calcium release peak; mitochondrial membrane potential (TMRM); and the levels of three different secreted Aβ peptides (measured at 10 µM).
Fig. S2. The list of synthesized tetrahydrocarbazoles and analogues
Shown are 23 strategically synthesized tetrahydrocarbazole analogues. Next to their chemical structure and physical properties, the measures for their activity in different assays are presented with their corresponding normalized values for: CCh-evoked calcium release peak; mitochondrial membrane potential (TMRM); and the levels of three different secreted Aβ peptides (measured at 10 µM).
Fig. S3. The effect of commercially available tetrahydrocarbazole analogues on Aβ production
Relative Aβ38, Aβ40 and Aβ42 levels are decreased after 16 h treatment with commercially available tetrahydrocarbazole analogues at 10 µM in HEK293 cells coexpressing APPsw and PS1-M146L. Sulindac sulfide (50 µM) and DAPT (10 µM), respectively, a γ-secretase modulator and a γ-secretase inhibitor, were used as positive controls. All the values are normalized to the value of DMSO, which is set to 1. (n.s.: non-significant; * P<0.05, ** P<0.01 and *** P<0.001; n=2).
Fig. S4. The effect of commercially available tetrahydrocarbazole analogues on Aβ42/Aβ40 ratio
Relative Aβ42/Aβ40 ratios calculated from figure S2. Treatment with the majority of the lead structure derivatives does not alter the Aβ42/Aβ40 ratio, whereas the positive control Sulindac sulfide (but not DAPT), significantly lowers the Aβ42/Aβ40 ratio. All values are normalized to the value of DMSO, which is set to 1. (n.s.: non-significant; * P<0.05, ** P<0.01 and *** P<0.001; n=2).
Supplementary Materials and Methods
Commercially available compound library and derivatives: The DIVERSet® compound library and further commercially available tetrahydrocarbazole analogues and related structures were obtained from ChemBridge (ChemBridge Corp., San Diego, CA). The database for the library compounds and the tested analogues are available at http://www.chembridge.com and https://www.hit2lead.com
Synthesis of the derivative compounds: The target 2,3,4,9-tetrahydro-1H-carbazol-1-amines were prepared in 1 step from appropriately substituted 2,3,4,9-tetrahydro-1H-carbazol-1-ones by reductive amination with primary amines (or ammonia for gea_99) using the sodium borohydride/magnesium perchlorate reagent, sodium triacetoxyborohydride or sodium cyanoborohydride. The products were isolated as free amines or as hydrochlorides.
Ring homologue gea_139 was obtained in an analogous manner from the seven-membered ketone analogue.
Tertiary amine gea_92 was obtained by N-methylation of gea_85, ketones gea_88 and gea_89 (as a precursor of N9-methylated 1-aminotetrahydrocarbazole 90) by methylation of appropriate 2,3,4,9-tetrahydro-1H-carbazol-1-ones.
General Information: NMR spectra were recorded using a Jeol JNMRGSX 400 or Jeol JNMRGSX 500 (Jeol, Peabody, USA), chemical shifts are given in ppm, coupling constants in Hertz. Mass spectra (electronic ionization, EI, 70eV) were recorded using a Hewlett Packard 5989A Mass Spectrometer with a 59980B Particle Beam LC/MS-interface (Agilent Technologies, Palo Alto, USA). High resolution mass spectra were obtained using a Jeol JMS GCmate II (Jeol, Peabody, USA). IR spectra were recorded as KBr discs on a PerkinElmer FTIR Paragon 1000 (PerkinElmer, Waltham, USA) or Jasco FT/IR410 (Jasco, Easton, USA). Melting points were determined with a Büchi B540 apparatus (Büchi, Flawil, Switzerland) and are uncorrected. Purification of products by flash column chromatography (FCC) was done using Silica gel 60 (Merck, Darmstadt, Germany). HPLC purities were obtained using a HP Agilent 1100 HPLC equipped with a diode array detector (Agilent Technologies, Waldbronn, Germany) and an Agilent Poroshell column (120 EC-C18, 3.0 x 100 mm, 2.7 Micron). Mobile phase consisted of acetonitrile/water/THF/1M NaOH (850:150:1.5:0.3), flow rate was set at 0.7 mL/min at a constant temperature of 45 °C. The chromatographic separations were monitored at 254 nm, using a band width of 4 nm. Injection volume was 10 µL of a dilution of 100 µg/mL (sample in mobile phase).
All chemicals were purchased from Sigma-Aldrich, Fluka and Acros.
Tetrahydrocarbazoles:
(±)-N1-Benzyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (gea_83)
C19H21ClN2; Mr = 312.84 g/mol (free amine: C19H20N2; Mr = 276.38 g/mol)
A suspension of 200 mg (1.08 mmol) 2,3,4,9-tetrahydro-1H-carbazol-1-one 1, 12 mg (0.054 mmol) magnesium perchlorate, and 286 µL (2.62 mmol) benzylamine in 20 mL 1,2-dichlorethane is stirred at room temperature for 8 h, then concentrated to dryness. The residue is dissolved in 20 mL methanol, treated with 78 mg (2.1 mmol) sodium borohydride and stirred for 5 h. Then 20 mL of a saturated sodium bicarbonate solution are added, and the mixture is extracted with ethyl acetate (3 x 15 mL). The combined organic layers are dried over sodium sulfate, concentrated, and the residue purified by FCC (isohexane/ethyl acetate 2:1). The pure product is dissolved in diethyl ether, and HCl gas is passed through the solution until precipitation ceases. The precipitate is collected and dried in vacuo.
Yield: 180 mg (54 %), white powder
Melting range: 187 – 189 °C
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 11.46 (br s, 1H, 9-NH), 9.95 (br s, 1H, 1´-NH), 9.80 (br s, 1H, 1´-NH), 7.74 – 7.63 (m, 2H, 2´´-H, 6´´-H), 7.49 (d, J = 7.6 Hz, 1H, 5-H), 7.45 – 7.38 (m, 4H, 8-H, 3´´-H, 4´´-H, 5´´-H), 7.15 (t, J = 7.6 Hz, 1H, 7-H), 7.02 (t, J = 7.5 Hz, 1H, 6-H), 4.68 (s, 1H, 1-H), 4.27 (t, J = 6.1 Hz, 2H, 1´-H), 2.77 – 2.60 (m, 2H, 4-H), 2.28 – 2.20 (m, 2H, 2-H), 2.15 – 2.06 (m, 1H, 3-H), 1.85 – 1.74 (m, 1H, 3-H).
13C-NMR (100 MHz, DMSO-d6): δ (ppm) = 135.9 (C-8a), 131.9 (C-1´´), 130.1 (C-2´´, C-6´´), 128.7 (C-4´´), 128.4 (C-3´´, C-5´´), 127.5 (C-9a), 125.9 (C-4b), 122.3 (C-7), 118.8 (C-6), 118.5 (C-5), 113.7 (C-4a), 111.3 (C-8), 50.9 (C-1), 47.5 (C-1´), 25.3 (C-2), 20.2 (C-4), 19.8 (C-3).
IR (KBr): (cm-1) = 3425, 3257, 2929, 2754, 2577, 2436, 1624, 1586, 1498, 1455, 1438, 1411, 1358, 1330, 1305, 1226, 1156, 1015, 975, 922, 752, 737, 694, 665, 599, 582, 569, 532, 500, 486
MS (CI): m/z (rel. int. in %) = 277 [M++H] (50), 170 (100), 108 (10)
MS (EI): m/z (rel. int. in %) = 276[M+•] (35), 248 (15), 185 (15), 169 (100), 157 (40), 157 (40), 143 (10), 130 (10), 106 (15), 91 (40), 77 (10), 65 (15)
HR-MS (EI): m/z = 276.1632 (calculated for C19H20N2: 276.1627)
HPLC purity: > 99 %
(±)-N1-Benzyl-7,8-dichloro-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (gea_84)
C19H19Cl3N2; Mr = 381.73 g/mol (free amine: C19H18Cl2N2; Mr = 345.27 g/mol)
A suspension of 200 mg (0.787 mmol) 7,8-dichloro-2,3,4,9-tetrahydro-1H-carbazol-1-one 2, 9 mg (0.04 mmol) magnesium perchlorate, and 205 mg (1.92 mmol) benzylamine in 20 mL 1,2-dichlorethane is stirred at room temperature for 8 h, then concentrated to dryness. The residue is dissolved in 20 mL methanol, treated with 57 mg (1.5 mmol) sodium borohydride and stirred for 5 h. Then 20 mL of a saturated sodium bicarbonate solution are added, and the mixture is extracted with ethyl acetate (3 x 15 mL). The combined organic layers are dried over sodium sulfate, concentrated, and the residue purified by FCC (dichloromethane/ethyl acetate/ triethylamine 9:1:0.5). The pure product is dissolved in diethyl ether, and HCl gas is passed through the solution until precipitation ceases. The precipitate is collected and dried in vacuo.
Yield: 125 mg (42 %), pale brown solid
Melting range: 165 – 167 °C
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 11.89 (br s, 1H, 9-NH), 10.12 (br s, 1H, 1´-NH), 9.92 (br s, 1H, 1´-NH), 7.68 (d, J = 6.5 Hz, 2H, 2´´-H, 6´´-H), 7.49 (d, J = 8.4 Hz, 1H, 5-H), 7.42 (m, 3H, 3´´-H, 4´´-H, 5´´-H), 7.22 (d, J = 8.4 Hz, 1H, 6-H), 4.74 (s, 1H, 1-H), 4.26 (m, 2H, 1´H), 2.68 (t, J = 5.4 Hz, 2H, 4-H), 2.37 – 2.25 (m, 1H, 2-H), 2.24 – 2.15 (m, 1H, 2-H), 2.15 – 2.03 (m, 1H, 3-H), 1.84 – 1.70 (m, 1H, 3-H).
13C-NMR (125 MHz, DMSO-d6): δ (ppm) = 134.0 (C-8a), 132.0 (C-1´´), 130.3 (C-2´´, C-6´´), 130.2 (C-9a), 128.9 (C-4´´), 128.6 (C-3´´, C-5´´), 126.3 (C-4b), 124.6 (C-7), 120.8 (C-6), 118.7 (C-5), 115.6 (C-4a), 114.2 (C-8), 50.8 (C-1), 47.3 (C-1´), 24.9 (C-2), 20.2 (C-4), 20.1 (C-3).
IR (KBr): (cm-1) = 3424, 3221, 2938, 2782, 2417, 2363, 1618, 1575, 1499, 1456, 1443, 1361, 1323, 1225, 1157, 1122, 972, 913, 793, 745, 696, 579, 556, 488
MS (CI): m/z (rel. int. in %) = 347 [M++H] (25), 345 (15), 239 (100), 198 (30), 120 (10), 108 (25)
MS (EI): m/z (rel. int. in %) = 349 (2), 347 (25), 345[M+•] (15), 317 (25), 254 (15), 242 (3), 240 (40), 238 (55), 226 (25), 202 (20), 190 (20), 167 (10), 106 (40), 91 (100), 77 (10), 65 (20), 51 (10)
HR-MS (EI): m/z = 344.0865 (calculated for C19H18Cl2N2: 344.0847)
HPLC purity: 99 %
(±)-N1-Benzyl-6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (gea_85)
C19H20BrClN2;Mr = 391.73 g/mol (free amine: C19H19BrN2; Mr = 355.27 g/mol)
A suspension of 209 mg (0.791 mmol) 6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one3, 9 mg (0.04 mmol) magnesium perchlorate, and 205 mg (1.92 mmol) benzylamine in 20 mL 1,2-dichlorethane is stirred at room temperature for 8 h, then concentrated to dryness. The residue is dissolved in 20 mL methanol, treated with 57 mg (1.5 mmol) sodium borohydride and stirred for 5 h. Then 20 mL of a saturated sodium bicarbonate solution are added, and the mixture is extracted with ethyl acetate (3 x 15 mL). The combined organic layers are dried over sodium sulfate, concentrated, and the residue purified by FCC (isohexan/ethyl acetate 2:1). The pure product is dissolved in diethyl ether, and HCl gas is passed through the solution until precipitation ceases. The precipitate is collected and dried in vacuo.
Yield: 70 mg (23 %), white solid
Melting range: 204 – 206 °C (ref. 4: no melting range given)
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 11.69 (br s, 1H, 9-NH), 9.94 (br s, 1H, 1´-NH), 9.80 (br s, 1H, 1´-NH), 7.73 – 7.61 (m, 3H, 5-H, 2´´-H, 6´´-H), 7.47 – 7.36 (m, 4H, 8-H, 3´´-H, 4´´-H, 5´´-H), 7.25 (dd, J = 8.6 Hz, 1.8 Hz, 1H, 7-H), 4.68 (s, 1H, 1-H), 4.35 – 4.21 (m, 2H, 1´-H), 2.75 – 2.59 (m, 2H, 4-H), 2.28 – 2.18 (m, 2H, 2-H), 2.16 – 2.02 (m, 1H, 3-H), 1.85 – 1.71 (m, 1H, 3-H).
13C-NMR (100 MHz, DMSO-d6): δ (ppm) = 134.5 (C-8a), 131.8 (C-1´´), 130.1 (C-2´´, C-6´´), 129.3 (C-9a), 128.8 (C-4´´), 128.4 (C-3´´, C-5´´), 127.7 (C-4b), 124.7 (C-7), 120.9 (C-5), 113.5 (C-4a), 113.4 (C-8), 111.3 (C-6), 50.8 (C-1), 47.6 (C-1´), 25.2 (C-2), 20.0 (C-4), 19.5 (C-3).
IR (KBr): (cm-1) = 3424, 3228, 2938, 2754, 2578, 2411, 1629, 1589, 1560, 1499, 1447, 1409, 1317, 1278, 1238, 1226, 1151, 1049, 1015, 973, 913, 891, 855, 802, 749, 696, 669, 640, 596, 539, 487
MS (CI): m/z (rel. int. in %) = 357 [M++H] (85), 355 (100), 276 (20), 250 (100), 248 (85), 169 (10), 120 (10), 108 (45)
MS (EI): m/z (rel. int. in %) = 356[M+•] (35), 354 (35), 328 (30), 326 (30), 249 (100), 247 (90), 237 (20), 235 (20), 168 (70), 106 (15), 91 (65), 65 (10)
HR-MS (EI): m/z = 354.0747 (calculated for C19H19BrN2: 354.0732)
HPLC purity: > 99 %
(±)-N1-Benzyl-6-iodo-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (gea_86)
C19H20ClIN2; Mr = 438.73 g/mol (free amine: C19H19IN2; Mr = 402.27 g/mol)
A suspension of 246 mg (0.791 mmol) 6-iodo-2,3,4,9-tetrahydro-1H-carbazol-1-one5, 9 mg (0.04 mmol) magnesium perchlorate, and 205 mg (1.92 mmol) benzylamine in 20 mL 1,2-dichlorethane is stirred at room temperature for 8 h, then concentrated to dryness. the residue is dissolved in 20 mL methanol, treated with 57 mg (1.5 mmol) sodium borohydride and stirred for 5 h. Then 20 mL of a saturated sodium bicarbonate solution are added, and the mixture is extracted with ethyl acetate (3 x 15 mL). The combined organic layers are dried over sodium sulfate, concentrated, and the residue purified by FCC (dichloromethane/ethyl acetate 2:1). The pure product is dissolved in diethyl ether, and HCl gas is passed through the solution until precipitation ceases. The precipitate is collected and dried in vacuo.
Yield: 146 mg (42 %), pale yellow solid
Melting range: 191 – 193 °C (dec.)
1H-NMR (500 MHz, DMSO-d6): δ (ppm) = 11.62 (br s, 1H, 9-NH), 9.89 (br s, 1H, 1´-NH), 9.75 (br s, 1H, 1´-NH), 7.86 (s, 1H, 5-H), 7.67 (d, J = 7.2 Hz, 2H, 2´´-H, 6´´-H), 7.48 – 7.35 (m, 4H, 7-H, 3´´-H, 4´´-H, 5´´-H), 7.28 (d, J = 8.5 Hz, 1H, 8-H), 4.67 (s, 1H, 1-H), 4.36 – 4.20 (m, 2H, 1´-H), 2.76 – 2.60 (m, 2H, 4-H), 2.28 – 2.16 (m, 2H, 2-H), 2.16 – 2.04 (m, 1H, 3-H), 1.86 – 1.72 (m, 1H, 3-H).
13C-NMR (100 MHz, DMSO-d6): δ (ppm) = 134.9 (C-8a), 131.8 (C-3´), 130.1 (C-7, C-2´´, C-6´´), 128.8 (C-9a, C-4´´), 128.6 (C-4b), 128.4 (C-3´´, C-5´´), 127.0 (C-5), 113.9 (C-8), 113.2 (C-4a), 82.4 (C-6), 50.7 (C-1), 47.6 (C-2´), 25.2 (C-2), 20.0 (C-4), 19.5 (C-3).
IR (KBr): (cm-1) = 3424, 3238, 2934, 2752, 2577, 2409, 2361, 1629, 1589, 1498, 1455, 1441, 1408, 1313, 1276, 1226, 1194, 1150, 1041, 1014, 972, 888, 867, 799, 758, 747, 695, 668, 630, 593, 537, 500, 486
MS (CI): m/z (rel. int. in %) = 403 [M++H] (100), 296 (100)
MS (EI): m/z (rel. int. in %) = 402[M+•] (30), 374 (10), 295 (100), 283 (15), 167 (25), 106 (15), 91 (35)
HR-MS (EI): m/z = 402.0604 (calculated for C19H19IN2: 402.0593)
HPLC purity: > 99 %
(±)-6-Bromo-N1-cyclohexyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine (gea_87)
C18H23BrN2; Mr = 347.29 g/mol
A suspension of 417 mg (1.58 mmol) 6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one3, 20 mg (0.12 mmol) magnesium perchlorate, and 556 µL (4.86 mmol) cyclohexylamine in 30 mL 1,2-dichlorethane is stirred at room temperature for 72 h, then concentrated to dryness. The residue is dissolved in 30 mL methanol, treated with171 mg (4.50 mmol) sodium borohydride and stirred for 5 h. Then 20 mL of a saturated sodium bicarbonate solution are added, and the mixture is extracted with ethyl acetate (3 x 15 mL). The combined organic layers are dried over sodium sulfate, concentrated, and the residue purified by FCC (dichloromethane/ethyl acetate 1:1).