7,8-Dichloro-1-Oxo-b-carbolines as a Versatile Scaffold for the Development of Potent and Selective...

8/13/2019 7,8-Dichloro-1-Oxo-b-carbolines as a Versatile Scaffold for the Development of Potent and Selective Kinase Inhibito

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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.

7,8-Dichloro-1-Oxo-b-carbolines as a Versatile Scaffold for the Development of Potent and Selective...

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