pronounced anti-proliferative and apoptosis-inducing ...synthesis of indole-derived allocolchicine...
TRANSCRIPT
Synthesis of indole-derived allocolchicine congeners exhibiting pronounced anti-proliferative and apoptosis-inducing properties
Nikolay S. Sitnikov,a,b Alexander V. Sinzov, Diane Allegro,c Pascale Barbier,c Sebastien Combes,c Liliane Abodo Onambele,d Aram Prokop,d Hans-Günther Schmalzb and
Alexey Yu. Fedorova*
aDepartment of Organic Chemistry, Lobachevsky State University of Nizhni Novgorod, Gagarina av. 23, Nizhni Novgorod 603950 (Russia); [email protected]
bDepartment für Chemie, Universität zu Köln, Greinstr. 4, 50939, Köln (Germany).
cLaboratory of Integrative Structural and Chemical Biology, Institut Paoli-Calmettes, Aix-Marseille Université, UM105, F-13009, Marseille (France)
dDepartment of Pediatric Hematology/Oncology, Children’s Hospital Köln, Amsterdamer Str. 59, 50735 Köln (Germany).
SUPPORTING INFORMATION
Electronic Supplementary Material (ESI) for MedChemComm.This journal is © The Royal Society of Chemistry 2015
S2
Content:
1. Synthesis
1.1. General information…………………………………………………………………………………………………………S3
1.2. Synthetic procedures and characterization data for 5a-f, 6, 9, 12, rac-13………………………..S4
2. Biological studies
2.1. Cytotoxicity of 5a-f towards BJAB cells
Detailed assays description………………………………………………………………………………………….S15
Concentration-activity diagrams………………………………………………………………………………….S17
2.2. Tubulin polymerization assay……………………………………………………………………………………………S23
S3
1. Synthesis.
1.1. General information.
All moisture sensitive reactions were carried out under argon atmosphere using Schlenk and
needle/syringe techniques. Glassware was flame dried under vacuum (0.5–1 mbar) and allowed to cool
down under an argon atmosphere. Syringes, needles and transfer cannulas were dried in an oven at
100 °C and were flushed with argon directly prior to use. Solvent preparations were carried according
to described procedures [W. L. F. Armarego, D. D. Perrin, Purification of Laboratory Chemicals, 4th
edition, Butterworth-Heinemann, Oxford, 2000, pp. 63-454]. Flash chromatography was performed
using silica 60 (230-400 mesh) supplied by Merck. 1H NMR spectra were recorded on Bruker DRX 500,
Bruker AV 300 or Bruker DPX 300 instruments. Chemical shifts (δ) are given in ppm relative to the
solvent reference as an internal standard (δ 7.26 ppm for CDCl3; δ 3.31 ppm for CD3OD). 13C NMR
spectra were recorded on Bruker DRX 500 (126 MHz), Bruker AV 300 (75 MHz) or Bruker DPX 300 (75
MHz) instruments with complete proton decoupling. Chemical shifts (δ) are given in ppm relative to
the solvent reference as the internal standard (δ 77.16 ppm for CDCl3; δ 49.00 ppm for CD3OD). The
assignments of 1H NMR are supported by HMBC, HMQC(HSQC), and/or H,H-COSY spectra. Infrared
spectra (FT-IR) were recorded on a Perkin Elmer FT-IR Paragon 1000 spectrometer using Fourier
transform infrared (FTIR) attenuated total reflection (ATR) technique. Absorption bands are given in
wave numbers (ṽ, cm-1). Melting points (M.p.) were measured on a Büchi B-545 melting point
apparatus. Mass spectra (MS) were recorded on a Finnigan Incos 50 Galaxy System or Polaris Q/Trace
GC Ultra (DIP-MS). High resolution mass spectra (HRMS) were recorded on a Finnigan MAT 900 (HR-
EIMS). Compound 7 was synthesized according to the previously reported procedure [N. Sitnikov, J.
Velder, L. Abodo, N. Cuvelier, J.-M. Neudorfl, A. Prokop, G. Krause, A. Y. Fedorov, H.-G. Schmalz, Chem.
Eur. J. 2012, 18, 12096].
S4
1.2. Synthetic procedures and characterization data for 5a-f, 6, 9, 12, rac-13
3-(2-Bromo-3,4,5-trimethoxy-6-(1-methyl-1Н-indol-6-yl)phenyl)propanoic acid methyl ester (9)
BrCO2Me
MeO
MeOOMe
CO2Me
Br I
MeOOMe
OMeN
Me
BO
O
NMe
NNMe
MeCO2Me
BrMeO
MeOOMe
CO2Me
Br
MeOOMe
OMe
B O
O
+
Pd(OAc)2 (0.05 equiv.)PPh3, Cs2CO3,toluene, 110oC
Side products formed in the reaction:
51%7 8 9
S1 S2 S3
Chemical Formula: C22H24BrNO5Molecular Weight: 462,33
A mixture of aryl iodide 7 (1.01 g, 2.2 mmol), 1-methylindol-4-boronic acid pinacol ester 8 (0.51 g, 2
mmol), anhydrous Cs2CO3 (1.3 g, 4 mmol), Pd(OAc)2 (22.5 mg, 0.1 mmol) and PPh3 (79 mg, 0.3 mmol) in
degassed dry toluene (4 ml) was stirred at reflux for 24 hours under argon atmosphere. The resulting
mixture was cooled to r.t., filtrated through Celite® with EtOAc (20 ml) and solvents were evaporated.
The product was purified using column chromatography on silica (EtOAc/Cy, 1:5) to give compound 9
(0.67 g, containing S1, 51% yield of 9 (0.47 g) based on 1H NMR integration). This mixture was directly
used in the next step.
Rf = 0.28 (EtOAc/CyH, 1:5).1H NMR (300 MHz, CDCl3, 25 °C): δ = 2.37 – 2.49 (m, 2 H, CH2), 2.84 – 2.93 (m, 2 H, CH2), 3.53 (s, 3 H,
CH3), 3.55 (s, 3 H, CH3), 3.78 (s, 3 H, CH3), 3.95 (s, 3 H, CH3), 3.96 (s, 3 H, CH3), 6.52 (dd, 4J = 1.0 Hz, 3J =
3.1 Hz, 1 H, 3-H), 6.90 (dd, 4J = 1.0 Hz, 3J = 8.1 Hz, 1 H, 4-H), 7.09 (d, 3J = 3.1 Hz, 1 H, 2-H), 7.12 (s, 1
H, 7-H), 7.64 (d, 3J = 8.1 Hz, 1 H, 5-H).
S5
3-(2′-Bromo-3′,4′,5′-trimethoxy-6′-(1′′-methyl-1H-indol-6′′-yl)phenyl)propanoic acid (6)
BrCO2Me
MeO
MeOOMe
NMe
9
BrCO2H
MeO
MeOOMe
NMe
6
LiOH aqTHF/MeOH
95%Chemical Formula: C21H22BrNO5
Molecular Weight: 448,31
To a solution of crude 9 (mixture containing 0.47 g of 9, 1.02 mmol) in THF/MeOH (2:1, 9 mL) was
added 1 N aq. LiOH (3 mL) and the solution was stirred for 1 h at 40 °C. The resulting mixture was
cooled to r.t., acidified to pH = 6 with 1 N aq. HCl, and the organic solvents were evaporated. The
residue was extracted with MTBE (4×10 mL), the organic layers were washed with brine (3 × 10 mL)
and dried over MgSO4 before the solvent was evaporated to yield a brown solid, which was further
purified using column chromatography on silica gel (EtOAc/Cy, 1:1) to yield compound 6 (434 mg, 95%)
as a white solid.
M. p. = 138 °C.
Rf = 0.4 (EtOAc/CyH, 1:1).1H NMR (300 MHz, CDCl3, 25 °C): δ = 2.38-2.57 (m, 2 H, CH2), 2.77-2.94 (m, 2 H, CH2), 3.56 (s, 3 H,
OCH3), 3.75 (s, 3 H, NCH3), 3.96 (s, 6 H, OCH3), 6.51 (dd, 4J = 1.0 Hz, 3J = 3.1 Hz, 1 H, 3-H), 6.90 (dd, 4J =
1.0 Hz, 3J = 8.0 Hz, 1 H, 4-H), 7.06 (d, 3J = 3.1 Hz, 1 H, 2-H), 7.12 (s, 1 H, 7′-H), 7.65 (d, 3J = 8.0 Hz, 1 H,
5-H).13C NMR (75 MHz, CDCl3, 25 °C): δ = 28.7, 32.9, 33.7, 61.02, 61.2 (2С), 101.0, 110.1, 114.8, 120.7, 121.1,
127.8, 129.4, 129.7, 134.4, 134.8, 136.7, 145.7, 150.4, 151.4, 178.6.
IR: ṽ = 906, 940, 966, 1010, 1050, 1078, 1146, 1193, 1233, 1253, 1296, 1319, 1396, 1409, 1443, 1456,
1510, 1616, 1704, 2620, 2826, 2866, 2934, 2973, 3100 сm-1.
MS (ESI): m/z (%) = 281 (3), 448 (100) [M-Н]–.
S6
1′-Bromo-2′,3′,4′-trimethoxybenzo[5′,6′:4,5]1H-6,7-dihydro-1-oxocyclohepta-[2,3-f]1H-1-
methylindole (12)
N
O
OMeMeO
MeOBr
C21H20BrNO4Mr = 430,2918
12
3
4 5 6
7
1' 2'3'
4'7'
Me
M. p. = 144 °C.
Rf = 0.4 (EtOAc/CyH, 1:1).1H NMR (500 MHz, CDCl3, 25 °C): δ = 2.80 – 2.99 (m, 3 H, СН2), 3.30 (s, 3 H, CH3), 3.38 – 3.44 (m, 1 H,
СН2), 3.84 (s, 3 H, CH3), 3.95 (s, 3 H, CH3), 3.97 (s, 3 H, CH3), 6.60 (d, 3J = 3.1 Hz, 1 H, 3-Н), 7.17 (d, 3J =
3.1 Hz, 1 H, 2-Н), 7.50 (s, 1 H, 4-Н), 7.87 (s, 1 H, 7-Н).13C NMR (126 MHz, CDCl3, 25 °C): δ = 207.1, 151.7, 150.5, 146.6, 138.0, 135.5, 132.6, 131.2, 130.2,
127.9, 126.5, 121.2, 113.3, 111.9, 102.6, 61.6, 61.1, 61.0, 46.4, 33.3, 29.3.
HRMS (ESI): calculated for C21H21BrNO4 [M+H]+ 430.0648, found 430.0650 [M+H]+; calculated for
C21H20BrNO4Na [M+Na]+ 452.0468, found 452.0470 [M+H]+.
S7
1′-Bromo-2′,3′,4′-trimethoxybenzo[5′,6′:4,5]1H-6,7-dihydro-1-hydroxycyclohepta-[2,3-f]1H-1-
methylindole (rac-13)
BrMeO
MeOOMe
NMe
11
i-Bu2AlCl
CH2Cl20oC to r.t.30 min
O
Cl
N
MeO
OMeMeO
OH
Me
Br
rac-1368%
BrCO2H
MeO
MeOOMe
NMe
6
NMe2
ClMe
Me
CH2Cl20oC, 12 h
Chemical Formula: C21H22BrNO4Molecular Weight: 432,31
Compound 6 (0.3 g, 0.67 mmol) was dissolved in anhydrous CH2Cl2 (3 mL) under argon atmosphere.
The solution was cooled to 0 °C and N,N,2-trimethyl-1-chloropropenylamine (98 μL, 0.74 mmol) was
added. The reaction mixture was stirred for 12 h at 0 °C. The resulting solution of acyl chloride 11 was
diluted with CH2Cl2 to 0.03 М (19 mL) and i-Bu2AlCl (0.8 М in heptane, 1.68 mL, 1.34 mmol) was added
via syringe. The reaction temperature was allowed to increase to r.t. and stirring was continued for 30
min before saturated aq. NaHCO3 (5 mL) and brine (5 mL) were added. The organic layer was separated
and the aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The combined organic extracts were
dried over MgSO4 and the solvent was removed under reduced pressure. After column
chromatography on silica gel (EtOAc/CyH, 2:3) product rac-13 (0.2 g, 68%) was obtained as a white
solid.
M. p. = 118 °C.
Rf = 0.4 (EtOAc/CyH, 1:1).1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.87 – 1.67 (m, 1 H, 6-Н), 2.26 – 2.09 (m, 1 H, 5-Н), 2.65 – 2.43 (m,
1 H, 6-Н), 3.09 – 3.17 (m, 1 H, 5-Н), 3.46 (s, 3 H, CH3), 3.82 (s, 3 H, CH3), 3.96 (s, 3 H, CH3), 3.99 (s, 3 H,
CH3), 4.63 (dd, 3J = 10.4 Hz, 3J = 7.0 Hz, 1 H, 7-H), 6.53 (d, 3J = 3.0 Hz, 1 H, 3-Н), 7.11 (d, 3J = 3.0 Hz, 1 H,
2-Н), 7.45 (s, 1 H, 4-Н), 7.89 (s, 1 H, 7-Н).13C NMR (75 MHz, CDCl3, 25 °C): δ = 150.1, 149.9, 146.0, 135.7, 135.1, 133.6, 130.5, 129.7, 128.1,
126.2, 114.3, 113.6, 110.8, 101.0, 70.3, 61.5, 61.0, 60.9, 39.8, 33.1, 29.9.
IR: ṽ = 905, 928, 971, 988, 1013, 1044, 1080, 1133, 1196, 1238, 1296, 1316, 1342, 1399, 1435, 1455,
1483, 1507, 2933, 3429 (broad) сm-1.
MS (EI): m/z (%) = 205 (44), 216 (36), 234 (24), 248 (22), 261 (76), 278 (29), 294 (22), 310 (89), 324 (22),
337 (18), 352 (36), 372 (13), 384 (5), 400 (9), 415 (13), 431 (100) [M+].
HRMS (EI): calculated for C21H22BrNO4 [M]+ 431.0732; found 431.071.
S8
1′,2′,3′-Trimethoxybenzo[4′,5′:4,5]1H-6,7-dihydro-1-hydroxycyclohepta-[2,3-f]1H-1-methylindole
(rac-5a)
N
MeO
OMeMeO
OH
Merac-13
N
MeO
OMeMeO
OH
Merac-5a
1) t-BuLi (3,05 equiv)THF, -78oC2) MeOH
96%
Br
Chemical Formula: C21H23NO4Molecular Weight: 353,41
Compound 13 (0.23 g, 0.53 mmol) was dissolved in anhydrous THF (7 mL) under argon atmosphere.
The solution was cooled to -78 °C and tert-butyllithium (1.6 М in pentane, 1 mL, 1.62 mmol) was added
dropwise via syringe. The mixture was stirred for 30 min at -78 oC followed by addition of МеОН (0.5
mL). The resulting solution was filtered through Celite® with THF and the solvents were removed under
reduced pressure. The residue was dissolved in ethyl acetate and filtrated through a short pad of silica
gel. After solvent evaporation compound rac-5a (0.18 g, 96%) was obtained as a white solid.
M. p. = 134 °C.
Rf = 0.38 (EtOAc/CyH, 1:1).
Two diastereomers (due to the presence of chiral axis) with a ratio of 8:1 were detected in 1Н NMR.
Major diastereomer:1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.80 – 1.95 (m, 1 H, СН2), 2.22 – 2.66 (m, 3 H, СН2), 3.52 (s, 3 H,
CH3), 3.81 (s, 3 H, CH3), 3.92 (s, 3 H, CH3), 3.94 (s, 3 H, CH3), 4.72 (dd, 3J = 10.5 Hz, 3J = 7.0 Hz, 1 H, 7-H),
6.52 (d, 3J = 3.0 Hz, 1 H, 3-Н), 6.61 (s, 1 Н, 4-Н), 7.09 (d, 3J = 3.0 Hz, 1 H, 2-Н), 7.46 (s, 1 H, 4-Н), 7.88
(s, 1 H, 7-Н).13C NMR (75 MHz, CDCl3, 25 °C): δ = 30.8, 33.1, 41.4, 56.1, 60.9, 61.3, 70.5, 101.0, 107.6, 110.8, 114.2,
125.9, 126.9, 127.7, 129.4, 133.7, 135.9 (2С), 141.1, 150.9, 152.3.
Minor diastereomer:1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.80 – 1.95 (m, 1 H, СН2), 2.22 – 2.66 (m, 3 H, СН2), 3.52 (s, 3 H,
CH3), 3.81 (s, 3 H, CH3), 3.92 (s, 3 H, CH3), 3.94 (s, 3 H, CH3), 4.72 (dd, 3J = 10.5 Hz, 3J = 7.0 Hz, 1 H, 7-H),
6.91 (d, 3J = 3.0 Hz, 1 H, 3-Н), 6.49 (s, 1 Н, 4-Н), 7.09 (d, 3J = 3.0 Hz, 1 H, 2-Н), 7.46 (s, 1 H, 4-Н), 7.55
(s, 1 H, 7-Н).
IR: ṽ = 908, 971, 1009, 1041, 1082, 1101, 1131, 1145, 1192, 1232, 1259, 1311, 1343, 1404, 1428, 1454,
1493, 1509, 1595, 2852, 2927, 3441 (broad) сm-1.
MS (EI): m/z (%) = 191 (6), 206 (6), 219 (3), 234 (6), 262 (3), 277 (17), 289 (5), 304 (7), 320 (21), 335
(100), 353 (10) [M]+.
HRMS (EI): calculated for C21H23NO4 [M]+ 353.1627; found 353.162.
S9
1′,2′,3′-Trimethoxybenzo[4′,5′:4,5]1H-6,7-dihydro-1-oxocyclohepta-[2,3-f]1H-1-methylindole (5b)
N
MeO
OMeMeO
OH
Me
rac-5a
N
MeO
OMeMeO
O
Me
5b
1) Pr4NRuO4 (cat)NMO, molar sievesCH2Cl2/MeCN
r.t., 1h
90%Chemical Formula: C21H21NO4
Molecular Weight: 351,40
Compound rac-5a (30 mg, 0.085 mmol) and N-methylmorpholine-N-oxide (15 mg, 0.128 mmol) was
dissolved in CH2Cl2/MeCN (1 mL, 10:1 v/v) under argon atmosphere. To the resulting solution finely
grounded molar sieves 4 Å (~40 mg) and tetrapropylammonium perruthenate (1.5 mg, 4.3 μmol) were
added. The reaction mixture was stirred at r.t. for 2 h and the solvents were removed under reduced
pressure. After column chromatography on silica gel (EtOAc/CyH, 1:2) compound 5b (27 mg, 90%) was
isolated as a white solid.
M. p. = 153 °C.
Rf = 0.25 (EtOAc/CyH, 1:2).1H NMR (300 MHz, CDCl3, 25 °C): δ = 2.56 – 2.70 (m, 1 H, СН2), 2.79 – 3.17 (m, 3 H, СН2), 3.38 (s, 3 H,
CH3), 3.82 (s, 3 H, CH3), 3.92 (s, 3 H, CH3), 3.93 (s, 3 H, CH3), 6.58 (d, 3J = 2.9 Hz, 1 H, 3-Н), 6.63 (s, 1 Н,
4-Н), 7.14 (d, 3J = 2.9 Hz, 1 H, 2-Н), 7.52 (s, 1 H, 4-Н), 7.90 (s, 1 H, 7-Н).13C NMR (75 MHz, CDCl3, 25 °C): δ = 30.4, 33.1, 47.7, 56.0, 60.9, 61.3, 102.3, 106.9, 111.7, 121.4, 125.5,
127.1, 127.3, 130.8, 132.4, 136.4, 138.2, 141.5, 152.4, 152.6, 207.3.
IR: ṽ = 908, 923, 960, 990, 1005, 1026, 1090, 1105, 1139, 1193, 1238, 1313, 1338, 1350, 1404, 1426,
1450, 1493, 1510, 1594, 1606, 1666 (strong), 2826, 2932, 3093 сm-1.
MS (EI): m/z (%) = 206 (6), 222 (16), 234 (8), 250 (23), 265 (13), 277 (8), 292 (21), 308 (15), 323 (26),
336 (8), 351 (100) [M]+.
HRMS (EI): calculated for C21H21NO4 [M]+ 351.1471; found 351.146.
S10
1′,2′,3′-Trimethoxybenzo[4′,5′:4,5]1H-1-acetoxy-6,7-dihydro-cyclohepta-[2,3-f]1H-1-methylindole
(rac-5c)
N
MeO
OMeMeO
OH
Me
rac-5a
N
MeO
OMeMeO
OAc
Me
rac-5c
EtOLi, EtOAc40oC, 50 mmHg
30 min
98%Chemical Formula: C23H25NO5Molecular Weight: 395,45
Compound rac-5a (30 mg, 0.085 mmol) and EtOLi (5 mg, 0.096 mmol) were dissolved in EtOAc (7 mL).
The reaction mixture was heated to 40 °C at 50 mmHg on a rotavap for 30 minutes (and more EtOAc
was occasionally added once the flask content had reached dryness). The residue was dissolved in
EtOAc and filtered through a short pad of silica gel. After solvent evaporation compound rac-5c (33 mg,
98%) was obtained as a white solid.
M. p. = 96 °C (with decomposition).
Rf = 0.35 (EtOAc/CyH, 1:2).1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.88 – 2.09 (m, 1 H, СН2), 2.19 (s, 3 Н, С(О)СН3), 2.28 – 2.62 (m, 3
H, СН2), 3.49 (s, 3 H, CH3), 3.80 (s, 3 H, CH3), 3.92 (s, 3 H, CH3), 3.96 (s, 3 H, CH3), 5.71 (dd, 3J = 10.7 Hz,
3J = 6.8 Hz, 1 H, 7-H), 6.52 (d, 3J = 2.5 Hz, 1 H, 3-Н), 6.61 (s, 1 Н, 4-Н), 7.09 (d, 3J = 2.5 Hz, 1 H, 2-Н),
7.49 (s, 1 H, 4-Н), 7.68 (s, 1 H, 7-Н).13C NMR (75 MHz, CDCl3, 25 °C): δ = 21.5, 30.4, 33.1, 38.4, 56.2, 61.2, 61.5, 72.6, 100.9, 107.9, 110.9,
114.2, 125.8, 127.1, 127.6, 129.6, 129.8, 135.3, 136.1, 141.3, 151.2, 152.5, 170.1.
MS (EI): m/z (%) = 206 (30), 218 (19), 234 (26), 250 (12), 262 (17), 277 (36), 290 (13), 305 (42), 320
(40), 335 (50), 352 (8), 395 (100) [M]+.
HRMS (EI): calculated for C23H25NO5 [M]+ 395.1732; found 395.171.
S11
1′,2′,3′-Trimethoxybenzo[4′,5′:4,5]1H-1-azido-6,7-dihydro-cyclohepta-[2,3-f]1H-1-methylindole (rac-
5d)
N
MeO
OMeMeO
N3
MeN
MeO
OMeMeO
OH
Me
rac-5a rac-5d
Zn(N3)2*2PyPPh3, DIAD,toluene, r.t.
91%Chemical Formula: C21H22N4O3Molecular Weight: 378,42
To a suspension of alcohol rac-5a (120 mg, 0.34 mmol), Zn(N3)2·2Py (58 mg, 0.188 mmol), and
triphenylphosphine (94 mg, 0.357 mmol) in dry toluene (6 mL) diisopropyl azodicarboxylate (74 μL,
0.357 mmol) was added dropwise under argon atmosphere. After stirring at r.t. for 5 h the reaction
mixture was diluted with EtOAc, filtered through Celite® and the solvents were removed under
reduced pressure. Column chromatography (EtOAc/Cy, 1:5) gave rac-5d (117 mg, 91%) as a white solid.
M. p. = 88 °C.
Rf = 0.33 (EtOAc/CyH, 1:3).
Two diastereomers (due to the presence of chiral axis) with a ratio of 5:1 were detected in 1Н NMR.
Major diastereomer:1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.88 – 2.07 (m, 1 H, СН2), 2.17 – 2.62 (m, 3 H, СН2), 3.57 (s, 3 H,
CH3), 3.82 (s, 3 H, CH3), 3.93 (s, 3 H, CH3), 3.97 (s, 3 H, CH3), 4.57 (dd, 3J = 11.3 Hz, 3J = 6.3 Hz, 1 H, 7-H),
6.55 (d, 3J = 2.9 Hz, 1 H, 3-Н), 6.63 (s, 1 Н, 4-Н), 7.11 (d, 3J = 2.9 Hz, 1 H, 2-Н), 7.49 (s, 1 H, 4-Н), 7.81
(s, 1 H, 7-Н).13C NMR (75 MHz, CDCl3, 25 °C): δ = 30.7, 33.0, 39.0, 56.2, 61.0, 61.3, 61.8, 101.2, 107.7, 111.1, 115.6,
125.8, 127.5, 127.8, 129.0, 129.7, 135.1, 136.1, 141.4, 151.1, 152.6.
Minor diastereomer:1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.88 – 2.07 (m, 1 H, СН2), 2.17 – 2.62 (m, 3 H, СН2), 3.57 (s, 3 H,
CH3), 3.83 (s, 3 H, CH3), 3.92 (s, 3 H, CH3), 3.98 (s, 3 H, CH3), 4.88 – 4.93 (m, 1 H, 7-H), 6.51 (d, 3J = 3.0
Hz, 1 H, 3-Н), 6.61 (s, 1 Н, 4-Н), 7.12 (d, 3J = 3.0 Hz, 1 H, 2-Н), 7.51 (s, 1 H, 4-Н), 7.62 (s, 1 H, 7-Н).
IR: ṽ = 907, 971, 1008, 1041, 1080, 1102, 1132, 1148, 1192, 1232, 1315, 1342, 1404, 1428, 1450, 1492,
1508, 1594, 1674, 1748, 2094 (strong), 2932 сm-1.
MS (EI): m/z (%) = 193 (19), 206 (18), 221 (16), 234 (10), 249 (15), 263 (15), 277 (14), 291 (29), 307 (49),
320 (9), 335 (100), 350 (19), 378 (31) [M]+.
HRMS (EI): calculated for C21H22N4O3 [M]+ 378.1692; found 378.168.
S12
1′,2′,3′-Trimethoxybenzo[4′,5′:4,5]1H-1-amino-6,7-dihydro-cyclohepta-[2,3-f]1H-1-methylindole (rac-
5e)
N
MeO
OMeMeO
NH2
MeN
MeO
OMeMeO
N3
Me
rac-5d rac-5e93%
Chemical Formula: C21H24N2O3Molecular Weight: 352,43
LiAlH4THF, r.t., 24 h
To the solution of rac-5d (100 mg, 0.27 mmol) in anhydrous THF (2 mL) under argon atmosphere LiAlH4
(1 M in THF, 0.4 mL, 0.4 mmol) was added dropwise via syringe. The reaction mixture was stirred for 24
h at r.t., then the excess of hydride was quenched by dropwise addition of MeOH (1 mL) at 0 oC. The
solvents were removed under reduced pressure. After column chromatography on silica gel
(MeOH/CH2Cl2, 1:10) compound rac-5e (87 mg, 93%) was isolated as a white solid.
M. p. = 92 °C.
Rf = 0.23 (MeOH/CH2Cl2, 1:10).1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.64 – 1.78 (m, 1 H, СН2), 2.20 – 2.52 (m, 3 H, СН2), 3.53 (s, 3 H,
CH3), 3.80 (s, 3 H, CH3), 3.91 (s, 3 H, CH3), 3.95 (s, 3 H, CH3), 3.91 – 3.97 (m, 1 H, 7-H), 6.51 (d, 3J = 2.8
Hz, 1 H, 3-Н), 6.60 (s, 1 Н, 4-Н), 7.07 (d, 3J = 2.8 Hz, 1 H, 2-Н), 7.45 (s, 1 H, 4-Н), 7.80 (s, 1 H, 7-Н).13C NMR (75 MHz, CDCl3, 25 °C): δ = 31.5, 33.0, 42.5, 51.1, 56.1, 60.9, 61.3, 100.9, 107.5, 110.9, 114.3,
126.3, 127.7, 128.2, 129.3, 134.3, 135.7, 136.1, 141.0, 150.9, 152.3.
IR: ṽ = 916, 966, 993, 1006, 1043, 1100, 1143, 1193, 1234, 1311, 1343, 1404, 1456, 1490, 1570, 1594,
1733 (strong), 2840, 2930, 3353 (broad) cm-1.
MS (EI): m/z (%) = 206 (22), 218 (9), 234 (12), 249 (7), 262 (11), 277 (21), 291 (12), 305 (18), 321 (22),
335 (50), 352 (100) [M]+.
HRMS (EI): calculated for C21H24N2O3 [M]+ 352.1787; found 352.176.
S13
1′,2′,3′-Trimethoxybenzo[4′,5′:4,5]1H-1-acetamido-6,7-dihydro-cyclohepta-[2,3-f]1H-1-methylindole
(rac-5f)
N
MeO
OMeMeO
NHAc
MeN
MeO
OMeMeO
NH2
Me
rac-5e rac-5f94%
Chemical Formula: C23H26N2O4Molecular Weight: 394,46
Ac2O, pyridineCH2Cl2
0oC, 10 min
Compound rac-5e (60 mg, 0.17 mmol) was dissolved in СН2Cl2 (1 mL) under argon atmosphere. The
solution was cooled to 0 С and pyridine (38 μL, 0.47 mmol) and acetic anhydride (29 μL, 0.31 mmol)
were sequentially added. The mixture was stirred for 10 min at 0 С followed by addition of EtOH (1
mL). The solvents were removed under reduced pressure. After column chromatography on silica gel
(EtOAc/MeOH, 20:1) compound rac-5f (63 mg, 94%) was isolated as a pale-brown solid.
M. p. = 138 °C.
Rf = 0.45 (EtOAc/MeOH, 20:1).1H NMR (500 MHz, CD3OD, 25 °C): δ = 1.84 – 1.95 (m, 1 H, 6-H), 2.03 (s, 3 Н, С(О)СН3), 2.17 – 2.31 (m, 2
H, 5-Н), 2.43 – 2.54 (m, 1 H, 6-Н), 3.45 (s, 3 H, CH3), 3.79 (s, 3 H, CH3), 3.89 (s, 6 H, CH3), 4.79 (dd, 3J =
11.8 Hz, 3J = 5.8 Hz, 1 H, 7-H), 6.42 (dd, 3J = 3.1 Hz, 4J = 0.8 Hz, 1 H, 3-Н), 6.60 (s, 1 Н, 4-Н), 7.07 (d, 3J =
2.8 Hz, 1 H, 2-Н), 7.45 (s, 1 H, 4-Н), 7.80 (s, 1 H, 7-Н).13C NMR (126 MHz, CD3OD, 25 °C): δ = 22.7, 31.7, 32.9, 40.2, 50.5, 56.6, 61.4, 61.6, 101.6, 108.9, 111.6,
115.2, 127.7, 129.0, 129.3, 130.7, 132.2, 136.9, 137.3, 142.3, 152.1, 153.7, 172.3.
IR: ṽ = 907, 976, 1008, 1043, 1080, 1102, 1138, 1146, 1193, 1234, 1276, 1314, 1348, 1404, 1450, 1493,
1540, 1593, 1650, 2846, 2931, 3286 cm-1.
MS (EI): m/z (%) = 206 (19), 218 (13), 234 (19), 250 (8), 262 (14), 277 (33), 291 (22), 304 (35), 320 (54),
335 (100), 351 (6), 394 (90) [M]+.
HRMS (EI): calculated for C23H26N2O4 [M]+ 394.1892; found 394.188.
S14
2. Biological studies.
2.1. Cytotoxicity of 5a-f towards BJAB tumor cells
Compound preparation:
The compounds were dissolved in DMSO to give a 40 mM stock solution.
Cell lines:
The following cell line was employed: BJAB (Burkitt like lymphoma): construct BJAB/mock.
Cell Culture:
BJAB cells were cultivated in RPMI 1640 medium (GIBCO, Invitrogen, Karlsruhe, Germany),
supplemented with 10% [vol/vol] heat inactivated fetal calf serum (FCS), 0.56 g/L L-glutamine, 100,000
I.U. penicillin and 0.1 g/L streptomycin. Cells were passaged two times per week. 24 h before assay,
cells were adjusted to 3x105 cells/mL to achieve standardized growth conditions. For proliferation and
apoptosis assays cells were diluted to 1x105 cells/mL immediately before treatment with the respective
compounds.
LDH-release assay: Measurement of unspecific cytotoxicity:
The cytotoxicity of the different compounds was measured by the release of lactate dehydrogenase
(LDH) using a Cytotoxicity Detection Kit from Roche Diagnostics (Mannheim, Germany). The maximum
LDH activity that could be released by the cells was determined by cell lysis using 0.1% Triton X-100 in
culture medium and set as 100 % cell death.
Proliferation assay: Determination of cell concentration and cell viability:
Cell viability was determined by using a CASYR Cell Counter + Analyzer System of Innovatis (Reutlingen,
Germany). Settings were specifically defined for the requirements of the cells used. With this system
the cell concentration can be analyzed simultaneously in three different size ranges: thus cell debris,
dead cells, and viable cells could be determined in one measurement. Cells were seeded at a density of
1x105 cells/mL and treated with different concentrations of allocolchicine analogues; non-treated cells
served as controls. After a 24 h incubation period, cells were re-suspended completely and 100 μL of
each well was diluted in 10 mL CASYRton (ready-to-use isotonic saline solution) for immediate
automated counting. The proliferation inhibition was displayed as IC50 that implies a proliferation
inhibition of 50% compared to the density of 2x105cells/mL which was set as a proliferation inhibition
of 100%.
S15
Apoptosis assays: Measurement of DNA fragmentation:
Apoptotic cell death was determined by a modified cell cycle-analysis, which detects DNA
fragmentation on the single cell level as described. Cells were seeded at a density of 1x105 cells/mL and
treated with different concentrations of allocolchicine analogues. After a 72 h incubation period at a
temperature of 37 °C, cells were collected by centrifugation at 1500 rpm for 5 min, washed with PBS at
4 °C and fixed in PBS/2% (v/v) formaldehyde on ice for 30 min. After fixation, cells were pelleted,
incubated with ethanol/PBS (2:1, v/v) for 15 min, pelleted and re-suspended in PBS containing
40μg/mL RNase. RNA was digested for 30 min at a temperature of 37 °C, after which the cells were
pelleted again and finally re-suspended in PBS containing 50μg/mL propidium iodide. Nuclear DNA
fragmentation was quantified by flow cytometric determination of hypodiploid DNA. Data were
collected and analyzed using a FACScalibur (Becton Dickinson, Heidelberg, Germany) equipped with
CellQuest analysis software. Data are given in percent hypoploidy (subG1), which reflects the number
of apoptotic cells. Specific apoptosis was calculated by subtracting background apoptosis, observed in
control cells, from total apoptosis seen in the treated cells. Apoptosis rate was displayed as AC50 that
implies an apoptosis of 50%.
S22
2.2. Tubulin polymerization inhibition
Preparation of lamb brain tubulin. Tubulin was purified from lamb brain by ammonium sulfate
fractionation and ion-exchange chromatography. The pure protein was stored in liquid nitrogen and
prepared as described [(1) Weisenberg, R.C.; Borisy, G.G.; Taylor, E.W. Biochemistry, 1968, 7, 4466-
4479; (2) Andreu, J.M., Gorbunopff, M.J., Lee, J.C. Timasheff, S.N. Biochemistry, 1987, 23, 1742-1752;
(3) Barbier, P., Gregoire, C., Devred, F., Sarrazin, M., Peyrot, V., Biochemistry, 2001, 40, 13510-13519].
Protein concentrations were determined spectrophotometrically with a Perkin Elmer
spectrophotometer Lambda 800 and an extinction coefficient at 275 nm of 1.07 L.g-1.cm-1 in 0.5% SDS
in neutral aqueous buffer or 1.09 L.g-1.cm-1 in 6 M guanidine hydrochloride.
Tubulin Polymerization. Microtubule assembly was performed in 20 mM sodium phosphate buffer, 1
mM EGTA, 10 mM MgCl2, and 3.4 M glycerol, 1 mM GTP pH 6.5. The reaction was started by warming
the samples at 37 oC and the mass of polymer formed was monitored by turbidimetry at 350 nm with a
POLARstar BMG Labtech spectrophotometer using 96-well plate. Samples containing the compound
and controls had less than 1% residual Me2SO.