7,8-dichloro-1-oxo-b-carbolines as a versatile scaffold for the development of potent and selective...
TRANSCRIPT
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Supporting Information
7,8-Dichloro-1-oxo--carbolines as a versatile scaffold for the
development of potent and selective kinase inhibitors with
unusual binding modes
Kilian Huber, Oleg Fedorov, Laurent Brault,Christelle Gasser,
Panagis Filippakopoulos, Alex N.
Bullock, Doriano Fabbro, Jrg Trappe, Jrg Schwaller, Stefan Knapp and Franz Bracher
Page S1 Contents
Page S2 Thermal shift stability assay data
Page S5 Crystallographic data collection and refinement statistics
Page S6 Supplemental Figure S1
Page S7 Materials and methods
Page S8 Additional procedures & experimental data for synthesised compounds
Page S12 References
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Table S1. Thermal shift kinase assay data.
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Values are shown in degrees Celsius. Emptyspaces indicate that no data have been measured. Targetswith significant Tm shift have been coloured in red (>8 degrees) and yellow (>4 degrees), respectively.
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Table S2.Crystallographic data collection and refinement statistics.
Data Collection
Complex PIM1/17 PIM1/20 DAPK3/20
PDB ID 3CXW 3CY2 3BHY
Space group P65 P65 P21
Cell dimensions: a, b, c (), , (deg)
98.24 98.24 81.4590.00 90.00
120.00
98.67 98.67 81.1390.00 90.00
120.00
33.55 90.68 41.4690.00 100.15
90.00
Resolution* () 2.10 (2.18-2.10) 2.01 (2.12-2.01) 1.24 (1.28-1.24)
Unique observations* 26203 (2592) 29963 (4345) 68373 (6829)
Completeness* (%) 100.0 (100.0) 100.0 (100.0) 99.7 (100.0)
Redundancy* 3.8 (3.8) 5.2 (5.1) 3.7 (3.6)
Rmerge* 0.126 (0.724) 0.075 (0.704) 0.079 (0.707)I/ I* 13.9 (1.8) 18.3 (2.0) 14.7 (2.0)
Refinement
Resolution () 2.10 2.01 1.24
Rwork/ Rfree(%) 16.3 / 20.6 16.7 / 19.9 14.3 / 18.7
Number of atoms
(protein/other/water)2199 / 108 / 218 2220 / 105 / 207 2126 / 25 / 273
B-factors (2)(protein/other/water)
24.07 / 29.07 /27.77
31.96 / 38.52 /40.70
19.91 / 29.47 /32.46
r.m.s.d bonds ()r.m.s.d angles (o)
0.0161.477
0.0151.371
0.0161.703
Ramachadran Favoured (%)Allowed (%)Disallowed
(%)
98.191.450.36
98.181.460.36
96.593.030.38
*Values in parentheses correspond to the highest resolution shell.
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Supplemental Figure S1: Detailed view of the interaction of 20 (A) and 17 (B) with PIM1. Main
interacting residues are shown in ball and stick representation. The images have been generated using
PDB codes: 3CXW, 3CY2. Hydrogen bonds are highlighted by dots. In the cocrystal structure of
20/PIM1 (PDB code: 3CY2) the chlorine in position 8 was radiolysed during data collection (arrow) and
has therefore not been included in the model.
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Materials and methods
Thermal stability shift assay.Thermal denaturation experiments were carried out in a Mx3005p real-
time PCR machine (Agilent) using a protein concentration of 2 M and an inhibitor concentration of
10 M. Samples were buffered in 10 mM HEPES, pH 7.5, 500 mM NaCl and a 1:1000 dilution of
SyproOrange (Invitrogen, CA). The assay and data evaluation were carried out as described.1
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Additional procedures & experimental data for synthesised compounds
Ethyl 6,7-dichloro-1-methyl-1H-indole-2-carboxylate (14).Under nitrogen, a solution of ethyl 6,7-
dichloro-1H-indole-2-carboxylate (13)2(1.60 g, 6.20 mmol) in anhydrous DMF (10 mL) was added to a
suspension of NaH (372 mg, 9.30 mmol) in DMF (20 mL) and the mixture was heated to 40 C for 1h.After cooling to rt, methyl iodide (880 mg, 9.30 mmol) was added dropwise and the mixture was stirred
for 5h. The mixture was poured into water (50 mL) and extracted with ethyl acetate (330 mL). The
combined organic layers were washed with brine (30 mL), dried over magnesium sulfate, filtered and
the solvent removed. The crude product was recrystallized from ethanol to give 1.02 g (62%) of 14as
white needles. Mp 74 C; 1H NMR (400 MHz, CD2Cl2-d2) 7.49 (d,J= 8.5 Hz, 1 H, 4-H), 7.25 (s, 1 H,
3-H), 7.20 (d, J= 8.5 Hz, 1 H, 5-H), 4.44 (s, 3 H, N-CH3), 4.35 (q, J= 7.1 Hz, 2 H, CH2), 1.39 (t,
J= 7.1 Hz, 3 H, CH2CH3);13C NMR (100 MHz, CD2Cl2-d2) 161.8 (C=O), 136.3 (C-7a), 131.2 (C-2),
130.5 (C-6), 127.7 (C-3a), 123.1 (C-5), 122.0 (C-4), 116.6 (C-7), 111.1 (C-3), 61.4 (CH 2), 35.0
(N-CH3), 14.5 (CH2CH3); MS EI m/z (relative intensity, %) 275 [M++4] (11), 273 [M++2] (63),
271 [M+] (100), 243 (95), 226 (31), 198 (37); Anal. (C12H11Cl2NO2) C, H, N.
Ethyl 6,7-dichloro-3-formyl-1-methyl-1H-indole-2-carboxylate (15).With cooling in an ice bath,
POCl3(1.35 ml, 14.7 mmol)was added dropwise to anhydrous DMF (4.0 ml, 51.8 mmol).A solution of
14(2.67 g, 9.81 mmol)in DMF (5 mL) was added and the mixture was allowed to stir for 30 min at rt
and then heated to 60 C for 4h. The reaction was poured into ice water (100 mL), neutralized with 2M
NaOH and extracted with ethyl acetate (330 mL). The combined organic layers were washed with brine
(30 mL), dried over magnesium sulfate, filtered and the solvent removed. The crude product was
recrystallized from ethanol to give 1.75 g (59%) of 15 as yellow needles. Mp 122 C; 1H NMR
(400 MHz, CD2Cl2-d2) 10.42 (s, 1 H, Ar-CHO), 8.33 (d, J= 8.6 Hz, 1 H, 4-H), 7.39 (d, J= 8.6 Hz,
1 H, 5-H), 4.52 (q,J= 7.1 Hz, 2 H, CH2), 4.36 (s, 3 H, N-CH3), 1.46 (t,J= 7.1 Hz, 3 H, CH2CH3);13C
NMR (100 MHz, CD2Cl2-d2) 187.2 (Ar-CHO), 160.4 (C=O), 137.4 (C-2), 134.7 (C-7a), 131.4 (C-6),
125.8 (C-5), 125.7 (C-3a), 122.3 (C-4), 118.9 (C-3), 116.6 (C-7), 62.8 (CH2), 35.9 (N-CH3), 14.1
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(CH2CH3); MS EI m/z (relative intensity, %) 303 [M++4] (2), 301 [M++2] (12), 299 [M+] (19), 270
(100), 254 (4), 198 (5); Anal. (C13H11Cl2NO3) C, H, N.
6,7-Dichloro-5-methyl-3H-pyridazino[4,5-b]indol-4(5H)-one (16). 13C NMR (100 MHz, DMSO-
d6) 155.9 (C=O), 136.0 (C-5a), 133.0 (C-1), 131.5 (C-4a), 131.2 (C-7), 123.6 (C-8), 121.7 (C-9a),
121.4 (C-9), 117.1 (C-9b), 116.0 (C-6), 34.8 (N-CH3).
(4RS)-7',8'-Dichloro-1,9'-dimethyl-1'-oxo-1',2',4',9'-tetrahydrospiro[piperidine-4,3'-pyrido[3,4-
b]indole]-4'-carbonitrile (17).13C NMR (100 MHz, DMSO-d6, 50 C) 159.3 (C=O), 134.6 (C-8a),
129.2 (C-7), 127.9 (C-9a), 125.0 (C-4b), 122.9 (C-6), 119.9 (C-5), 117.6 (CN), 115.8 (C-8), 112.0
(C-4a), 54.6 (C-4/-3), 50.5 (C-2/-6), 49.7 (C-2/-6), 45.3 (1-CH3), 34.4 (C-3/-5), 34.2 (9-CH3), 33.9
(C-3/-5), 31.6 (C-4); IR max(cm-1) 2235 (CN).
(4RS)-7',8'-Dichloro-1-ethyl-9'-methyl-1'-oxo-1',2',4',9'-tetrahydrospiro[piperidine-4,3'-pyrido-
[3,4-b]indole]-4'-carbonitrile (18). 13C NMR (125 MHz, DMSO-d6, 50 C) 159.3 (C=O), 134.6
(C-8a), 129.2 (C-7), 127.9 (C-9a), 125.0 (C-4b), 122.9 (C-6), 119.9 (C-5), 117.6 (CN), 115.8
(C-8), 112.0 (C-4a), 55.2 (C-4/-3), 51.1 (CH2CH3), 48.0 (C-2/-6), 47.3 (C-2/-6), 34.5 (C-3/-5), 34.2
(9-CH3), 34.0 (C-3/-5), 31.5 (C-4), 12.1 (CH2CH3); IR max(cm-1) 2243 (CN).
(4RS)-tert-Butyl 7',8'-dichloro-4'-cyano-9'-methyl-1'-oxo-1',2',4',9'-tetrahydrospiro[piperidine-
4,3'-pyrido[3,4-b]indole]-1-carboxylate (19). 13C NMR (125 MHz, CDCl3, 50 C) 159.8 (C-1),
154.5 (Boc-C=O), 135.9 (C-8a), 131.7 (C-7), 126.8 (C-9a), 124.8 (C-4b), 124.0 (C-6), 118.6 (C-5),
117.4 (C-8), 116.0 (CN), 111.4 (C-4a), 80.5 (C (CH3)3), 55.9 (C-4/3), 39.4 (C-2/6), 39.1 (C-2/6), 35.2
(C-4), 34.8 (9-CH3), 34.8 (C-3/5), 33.9 (C-3/5); IR max(cm-1) 2237 (CN).
(4RS)-7',8'-Dichloro-9'-methyl-1'-oxo-1',2',4',9'-tetrahydrospiro[piperidine-4,3'-pyrido[3,4-
b]indole]-4'-carbonitrile (20). 13C NMR (125 MHz, DMSO-d6) 159.2 (C=O), 134.4 (C-8a), 129.0
(C-7), 127.7 (C-9a), 125.0 (C-4b), 122.9 (C-6), 120.0 (C-5), 117.8 (CN), 115.7 (C-8), 111.9
(C-4a), 55.7 (C-4/-3), 41.5 (C-2/-6), 40.7 (C-2/-6), 35.5 (C-3/-5), 35.1 (C-3/-5), 34.1 (9-CH 3), 31.4
(C-4); IR max(cm-1) 2233 (CN).
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Elemental Analyses
Compound Calcd. Found.
9 C: 59.68 H: 4.73 N: 11.60 C: 59.34 H: 4.78 N: 11.41
14 C: 52.96 H: 4.07 N: 5.15 C: 52.78 H: 3.87 N: 5.11
15 C: 52.02 H: 3.69 N: 4.67 C: 51.81 H: 3.59 N: 4.70
16 C: 47.68 H: 2.91 N: 15.16 C: 47.95 H: 2.49 N: 13.80
17 C: 57.30 H: 4.81 N: 14.85 C: 57.44 H: 4.75 N: 14.69
18 C: 58.32 H: 5.15 N: 14.32 C: 58.37 H: 5.07 N: 14.29
19 C: 57.03 H: 5.22 N: 12.09 C: 56.86 H: 5.14 N: 12.02
20 C: 56.21 H: 4.44 N: 15.42 C: 56.10 H: 4.27 N: 15.12
22 C: 58.63 H: 4.34 N: 12.07 C: 58.15 H: 4.73 N: 12.04
23 C: 55.23 H: 5.25 N: 12.88 C: 55.23 H: 5.29 N: 12.95
24 C: 55.45 H: 5.20 N: 11.41 C: 55.35 H: 5.09 N: 11.16
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References
1. Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.; Marsden, B. D.; Knapp, S.Structural Basis of Inhibitor Specificity of the Human Protooncogene Proviral Insertion Site in MoloneyMurine Leukemia Virus (PIM-1) Kinase.J. Med. Chem. 2005,48, 7604-7614.2. Huber, K.; Kast, O.; Bracher, F. A Versatile Synthesis of 3-Substituted 4-Cyano-1,2,3,4-
tetrahydro-1-oxo-
-carbolines.Synthesis 2010
, 3849-3854.