Photochemical synthesis of 3-azabicyclo[3.2.0]heptanes: advanced building blocks for drug discovery

Transcription

1 Photochemical synthesis of 3-azabicyclo[3.2.0]heptanes: advanced building blocks for drug discovery Aleksandr V. Denisenko, a,b Tetiana Druzhenko, c Yevhen Skalenko, a Maryna Samoilenko, b Oleksandr O. Grygorenko, a Sergey Zozulya, d and Pavel K. Mykhailiuk* a a Department of Chemistry, National Taras Shevchenko University of Kyiv, Volodymyrska 64, Kyiv (Ukraine) b Enamine Ltd., Chervonotkatska 78, Kyiv (Ukraine), c Institute of High Technologies, Academician Glushkov av. 4G, Kyiv (Ukraine) d ChemBioCenter, National Taras Shevchenko University of Kyiv, Volodymyrska 64, Kyiv (Ukraine) Table of Contents Measurement of lipophilicity, aqueous solubility and metabolic stability of compounds S-2 X-Ray crystallography S-4 Copies of NMR spectra S-8 S1

2 Measurement of lipophilicity, aqueous solubility and metabolic stability of compounds Metabolic stability is defined as the percentage of parent compound lost over time in the presence of a metabolically active test system. 1. Materials 1.1 Reagents and consumables DMSO (Sigma-Aldrich, Chromasolv Plus, for HPLC, 99.7%) Acetonitrile (Sigma-Aldrich, Chromasolv Plus, for HPLC, 99.9%) Potassium phosphate monobasic (Helicon, Am-O ) Potassium phosphate dibasic (Helicon, Am-O ) Magnesium chloride hexahydrate (Helicon, Am-O ) Microsomes from liver, pooled, male BALB/c mice Glucose-6-phosphate dehydrogenase from baker's yeast, type XV (Sigma-Aldrich, G6378) Glucose-6-phosphate sodium salt (Sigma-Aldrich, G7879) β-nicotinamide adeninedinucleotide-2'-phosphate reduced, tetrasodium salt (Santa Cruz Biotechnology, Inc., sc a) Formic acid (Sigma-Aldrich, 94318) DMSO stock solutions of the tested compound(s) 10mM (+,-) Propranolol hydrochloride (Sigma-Aldrich, P0884) Imipramine hydrochloride (Sigma-Aldrich, I7379) Discovery C18 (50 x 2.1 mm, 5µm) 1.1 ml microtubes in microracks, pipettor tips (Thermo Scientific). 1.2 Equipment Gradient HPLC system VP (Shimadzu) MS/MS detector API 3000 with TurboIonSpray Electrospray module (PE Sciex, USA) Nitrogen generator N2-04-L1466, nitrogen purity 99%+ (Whatman) Environmental Incubator Shaker G24; Digital Refrigerated Incubator/Shaker Innova 4330 (New Brunswick Scientific) Water purification system NANOpure Diamond D11911 (Barnstead) Multichannel pipettors µl, µl, µl (Thermo Scientific) 1.3 Analytical System S2

3 All measurements were performed using Shimadzu VP HPLC system including vacuum degasser, gradient pumps, reverse phase HPLC column, column oven and autosampler. The HPLC system was coupled with tandem mass spectrometer API 3000 (PE Sciex). The TurboIonSpray ion source was used in both positive and negative ion modes. Acquisition and analysis of the data were performed using Analyst software (PE Sciex). 2. Methods Mouse hepatic microsomes were isolated from pooled (50), perfused livers of Balb/c male mice according to the standard protocol (Hill, J.R. in Current Protocols in Pharmacology , Wiley Interscience, 2003). The batch of microsomes was tested for quality control using Imipramine, Propranolol and Verapamil as reference compounds. Microsomal incubations were carried out in 96-well plates in 5 aliquots of 40 µl each (one for each time point). Liver microsomal incubation medium contained PBS (100 mm, ph 7.4), MgCl 2 (3.3 mm), NADPН (3 mm), glucose-6-phosphate (5.3 mm), glucose-6-phosphate dehydrogenase (0.67 units/ml) with 0.42 mg of liver microsomal protein per ml. Control incubations were performed replacing the NADPH-cofactor system with PBS. Test compound (2 µm, final solvent concentration 1.6 %) was incubated with microsomes at 37 C, shaking at 100 rpm. Incubations were performed in duplicates. Five time points over 40 minutes had been analyzed. The reactions were stopped by adding 12 volumes of 90% acetonitrile-water to incubation aliquots, followed by protein sedimentation by centrifuging at 5500 rpm for 3 minutes. Incubations were performed in duplicates. Supernatants were analyzed using the HPLC system coupled with tandem mass spectrometer. The elimination constant (k el ), half-life (t 1/2 ) and intrinsic clearance (Cl int ) were determined in plot of ln(auc) versus time, using linear regression analysis: k el = slope t 1 = k Cl int = t 1/ 2 µ mg lincubation microsomes S3

4 X-Ray crystal structure of compound 13a. Thermal ellipsoids are shown at 15% probability level. S4

5 X-Ray crystal structure of compound 14a. Thermal ellipsoids are shown at 15% probability level. S5

6 X-Ray crystal structure of compound 14b. Thermal ellipsoids are shown at 15% probability level. S6

7 X-Ray crystal structure of compound 20a*HCl. Thermal ellipsoids are shown at 15% probability level. S7

8 Copies of NMR spectra Substance 5 S8

9 Substance 5a S9

10 Substance 6 S10

11 S11

12 Substance 6a S12

13 S13

14 Substance 7 S14

15 S15

16 Substance 7a S16

17 S17

18 Substance 8 S18

19 S19

20 Substance 8a S20

21 S21

22 Substance 9 S22

23 Substance 9a S23

24 Substance 10 S24

25 Substance 10a S25

26 Substance 11 S26

27 Substance 11a S27

28 Substance 12 S28

29 S29

30 Substance 12a S30

31 S31

32 S32

33 S33

34 Substance 13a S34

35 Substance 13b S35

36 Substance 14a S36

37 S37

38 Substance 14b S38

39 S39

40 Substance 15 S40

41 Substance 15a S41

42 Substance 16 S42

43 Substance 16a S43

44 Substance 17 S44

45 Substance 17a S45

46 Substance 18 S46

47 Substance 18a S47

48 Substance 19 S48

49 Substance 19a S49

50 Substance 20 S50

51 Substance (S)-21 S51

52 S52

53 Substance (R)-21 S53

54 S54