A Simple Litmus Test for Aldehyde Oxidase Metabolism of Heteroarenes

Transcription

1 A Simple Litmus Test for Aldehyde Oxidase Metabolism of Heteroarenes Fionn O Hara, Aaron C. Burns, Michael R. Collins, Deepak Dalvie, Martha A. Ornelas, Alfin D. N. Vaz ǁ, Yuta Fujiwara and Phil S. Baran, * Department of Chemistry, The Scripps Research Institute, North Torrey Pines Road, La Jolla, California 92037, United States Pfizer Pharmaceuticals, Chemistry Department, La Jolla Laboratories, Science Center Drive, La Jolla, California 92121, United States Pfizer Pharmaceuticals, Department of Pharmacokinetics, Dynamics and Metabolism, La Jolla Laboratories, Science Center Drive, La Jolla, California 92121, United States ǁ Pfizer Pharmaceuticals, Department of Pharmacokinetics, Dynamics and Metabolism, Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States SUPPORTING INFORMATION Table of Contents Experimental procedure for optimized litmus test... 2 Screening to determine suitable "litmus test" conditions... 3 Full LCMS chromatograms of difluoromethylation reaction for substrates Additional examples of "litmus test" results... 8 General notes on synthetic chemistry and scale up procedures... 9 Characterization data for compounds Procedure for AO activity assay and control on known substrate Experimental data for AO activity assay for compounds AO test results for compounds that had not previously been tested NMR spectra for compounds SI-1

2 Experimental Procedure for Optimized Litmus Test : Reagents: Bis(((difluoromethyl)sulfinyl)oxy)zinc (Zinc difluoromethanesulfinate, DFMS). Aldrich product number: Tert-butyl hydroperoxide (TBHP) (70% aq.) DMSO Trifluoroacetic acid (TFA) Conditions: 1. Test substrate (approx. 5 mg for a MW of ) and DFMS (12 mg) weighed into an LCMS vial. 2. Small (approx.. 3 mm) stir bar added. 3. DMSO (150 µl) added using a micro pipettor, and reaction mixture stirred at RT to obtain a uniform solution. 4. TFA (2 µl) added using a micro pipettor, and reaction mixture stirred at RT. If required, seal the LCMS vial with a screw cap and shake vigorously to ensure mixing of any reagent residues from the side of the vial. 5. TBHP (10 µl of a 70% aq. solution) was added using a micro pipettor while continuing stirring. 6. The vial was sealed with a screw cap and shaken to ensure mixing of residues on the side of the vial. 7. The reaction was stirred at room temperature for 2 h. 8. A small sample was of the reaction mixture was diluted with MeOH and analysed by LCMS (Agilent 6230 TOF LC/MS, 6 min run time: 0.5 ml/min varying from 95:5 to 1:99 water: MeCN over 4 min, and held at 1:99 water: MeCN for 2 min). SI-2

3 9. The TIC chromatogram was analyzed for a characteristic M+50 difluoromethylated product peak. A peak > 10% integral of the starting material (M) peak (i.e. a noticeable product peak) was indicative of a positive result. This result indicates that the heteroarene is likely susceptible to AO, and provides an alert that early testing in an assay with human AO should be considered. This procedure has been designed to be suitable for high throughput testing. Note: altering reaction time, concentrations, reagent quantities etc. will all reduce the predictive capability of the litmus test. In particular, prolonged stirring is likely to result in ambiguous results or false positives. Screening to determine suitable litmus test conditions: Difluoromethylation with DFMS was attempted on test substrates 1-5 under a variety of different conditions, with monitoring by LCMS. Temperature was varied from RT to 80 C, solvents included DMSO and CH 2 Cl 2 or CHCl 3 /water mixtures, and reaction time varied from 2 to 18 h. Reactions were carried out on 5 mg scale (about mmol) with set quantities of DFMS, solvent, TFA and TBHP. Although this did not give a traditional fixed molar ratio, it was felt that on such small scale, it was more important to develop a test robust to such errors in weighing, rather than focus on a level of accuracy that may not be merited by the quality of the balance, and is unlikely to be maintained in everyday use. SI-3

4 Typical procedure: 1. Substrate (5 mg) and DFMS (12 mg) weighed into an LCMS vial. A small (3mm) stir bead was added. 2. Solvent added using a micropipette and reagents stirred to obtain a solution/suspension. 3. TFA (2 µl) added using a micropipette. 4. TBHP (10 µl) added at RT using a micropipette. Stirred at the indicated temperature for the required time. A sand bath was used for uniform heating of small sample vials. 5. A small sample of the reaction mixture was diluted with MeOH and analysed by LCMS (method as described above). The TIC chromatogram was used for analysis in these examples as it gave the sharpest peaks for SM and product for the heterocyclic drugs tested, but other chromatograms such as UV 254 nm could equally be used (although peaks may appear broader and less defined than those illustrated here). SI-4

5 Full width LCMS spectra for substrates 1-13: All major mass peaks are marked; the M and M+50 peaks are indicated. Bis-difluoromethylated products are sometimes observed with a mass of M+100. SI-5

6 SI-6

7 SI-7

8 Additional examples of litmus test results: *AO testing did reveal an oxidation product, but analysis of the fragmentation pattern indicated that oxidation occurred on the phenyl ring, rather than the heteroarene, therefore this oxidation is unlikely to be due to AO activity. Thus, this data point is best viewed as a false positive. SI-8

9 Experimental Details for Synthetic Chemistry General Procedures. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Yields refer to chromatographically and spectroscopically ( 1 H-NMR) homogeneous material, unless otherwise stated. Reactions were monitored by HPLC-MS on a reverse phase column, using acetonitrile/water/0.1% formic acid as the mobile phase. NMR spectra were recorded on Bruker DRX-600, DRX-500 and AMX-400 instruments and are calibrated using residual undeuterated solvent or TMS as an internal reference. The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, heptet = heptet, m = multiplet, br = broad. 19 F- NMR recorded referenced to trichlorofluoromethane; high resolution mass spectra (HRMS) were recorded on an Agilent LC/MSD TOF mass spectrometer by electrospray ionization time of flight reflectron experiments. IR experiments were recorded on a Perkin Elmer Spectrum BX FTIR spectrometer. Melting points were recorded on a Fisher-Johns melting point apparatus and are uncorrected. Standard Procedure for Larger Scale Reactions (typically or 0.25 mmol scale): The substrate (0.125 mmol) and DFMS (74 mg) were weighed and placed in a 3 ml round bottomed vial. DMSO (0.7 ml was added) and the mixture stirred until a uniform solution was obtained. (Occasionally it was not possible to get a solution at room temperature. In these cases the mixture was stirred to obtain a uniform suspension.) TFA (10 µl) was added and stirring continued. The reaction mixture was then cooled in ice as TBHP (52 µl) was added dropwise using a micropipettor. The tube was removed from the ice, sealed and shaken to allow mixing of any residues on the side of the tube. The reaction mixture was then stirred at room temperature, or placed in a heating block at the required temperature and stirred for 2-12 h. (Reaction was typically complete within 2 h.) The reaction mixture was cooled to room temperature and diluted with EtOAc and approx. 10 ml of ethylenediaminetetraacetic acid (EDTA) disodium salt/sodium bicarbonate SI-9

10 solution (18 g EDTA disodium salt dissolved in 150 ml saturated sodium bicarbonate solution). The mixture was extracted into EtOAc (2 x 10 ml) and the combined organics washed with brine, dried (MgSO 4 ) and concentrated under reduced pressure. The residue was purified by preparative HPLC (Waters CSH C um column, eluting with 10 mm NH 4 OAc aqueous solution, 2.25mL/min, 140 bar). Yields were typically 10-40%. It was not always possible to isolate every regioisomer in sufficient purity for AO testing. Regioisomers 20 and 21 were only isolated in trace quantities. The fluorine peak from TFA salts of the heteroaromatic bases is typically observed at around -77 ppm. Experimental Data: 5-Cyclopropyl-1-(2-(difluoromethyl)quinolin-5-yl)-N,N-diethyl-1H-pyrazole-4- carboxamide (14). Standard procedure was followed, and the product was isolated by preparative HPLC as a light yellow solid; m.p C; R f = 0.53 (100% EtOAc); 1 H-NMR (600 MHz, DMSO-d 6 ) δ 8.32 (d, J = 8.4 Hz, 1H), 8.04 (t, J = 8.4, 7.4 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H), (m, 2H), 7.80 (s, 1H), 7.18 (t, J = 54.7 Hz, 1H), 3.45 (m, 4H), 1.61 (m, 1H), 1.14 (t, J = 6.9 Hz, 6H), 0.58 (m, 2H), 0.52 (m, 2H); 13 C-NMR (151 MHz, DMSO-d 6 ) δ 164.0, (t, J = 25.9 Hz), 146.9, 143.7, 138.2, 135.5, 134.1, 130.6, 130.2, 127.5, 125.7, 118.5, 116.0, (t, J = Hz), 42.9, 38.6, 14.3, 12.6, 6.2, 6.2; 19 F-NMR (376 MHz, DMSO-d 6 ) δ ; IR (solid) ν = 2973, 2936, 1621, 1565, 1474, 1420, 1267, 1077, 1030 cm - 1 ; HRMS (ESI-TOF) calc d for C 21 H 22 F 2 N 4 OH + [M+H + ] ; found SI-10

11 N-((1r,3r)-3-((5-cyano-6-methylpyridin-2-yl)oxy)-2,2,4,4-tetramethylcyclobutyl)-7- (difluoromethyl)imidazo[1,2-a]pyrimidine-3-carboxamide (15). Standard procedure was followed, and the product was isolated by preparative HPLC as a white residue; R f = 0.43 (50% EtOAc/hexanes); 1 H-NMR (600 MHz, DMSO-d 6 ) δ 9.85 (d, J = 7.1 Hz, 1H), 8.81 (s, 1H), 8.06 (d, J = 9.5 Hz, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 7.1 Hz, 1H), 7.05 (t, J = 54.4 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.77 (s, 1H), 4.11 (d, J = 9.5 Hz, 1H), 2.55 (s, 3H), 1.23 (s, 6H), 1.13 (s, 6H); 13 C-NMR (151 MHz, DMSO-d 6 ) δ 174.1, 164.5, 161.2, 159.8, (t, J = 26.4 Hz), 148.3, 142.9, 139.6, 138.1, 117.6, 117.3, (t, J = Hz), 108.8, 106.1, 100.6, 82.3, 57.4, 24.3, 23.2, 23.1; 19 F-NMR (376 MHz, DMSO-d 6 ) δ ; IR (thin film) ν = 3369, 2921, 2852, 1651, 1592, 1544, 1459, 1316, 1102, 1041 cm -1 ; HRMS (ESI-TOF) calc d for C 23 H 24 F 2 N 6 O 2 H + [M+H + ] ; found Bromo-2-(difluoromethyl)-N-(4,5-dihydro-1H-imidazol-2-yl)quinoxalin-6-amine TFA salt (16) and 5-Bromo-3-(difluoromethyl)-N-(4,5-dihydro-1H-imidazol-2-yl)quinoxalin-6-amine TFA salt (17). Standard procedure was followed, and the products were isolated by preparative HPLC. Small quantities of an isomer resulting from substitution on the aryl ring were also isolated, although this material was insufficiently pure for AO testing. Assignment of 16 and 17 used chemical shifts as a guide, but distinguishing between the isomers is subject to some uncertainty. Neither isomer was an AO substrate. The C2-isomer SI-11

12 (16) was isolated as a yellow solid; decomposed > 250 C; R f = 0.18 (10% MeOH/EtOAc); 1 H- NMR (600 MHz, CDCl 3 ) δ 9.05 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H), 6.90 (t, J = 54.5 Hz, 1H), 3.67 (s, 4H); 13 C-NMR (151 MHz, CDCl 3 ) δ 158.2, 149.6, (t, J = 27.5 Hz), 141.6, 140.5, 140.2, 130.7, 129.2, 117.4, (t, J = Hz), 42.8; 19 F-NMR (376 MHz, CDCl 3 ) δ -76.2, ; IR (thin film) ν = 3251, 1646, 1592, 1490, 1419, 1347, 1094, 1041 cm -1 ; HRMS (ESI-TOF) calc d for C 12 H 10 BrF 2 N 5 H + [M+H + ] ; found The C3-isomer (17) was isolated as a yellow solid; decomposed > 250 C; R f = 0.25 (10% MeOH/EtOAc); 1 H-NMR (600 MHz, MeOD) δ 9.08 (s, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 9.0 Hz, 1H), 6.98 (t, J = 54.6 Hz, 1H), 3.56 (s, 4H); 13 C-NMR (151 MHz, MeOD) δ 170.3, 160.9, 154.1, (t, J = 26.4 Hz), 143.8, (t, J = 2.8 Hz), 140.2, 131.8, 129.7, 117.3, (t, J = Hz), 43.5; 19 F-NMR (376 MHz, MeOD) δ -77.1, ; IR (thin film) ν = 3185, 2925, 1640, 1591, 1478, 1363, 1277, 1092, 1038 cm -1 ; HRMS (ESI-TOF) calc d for C 12 H 10 BrF 2 N 5 H + [M+H + ] ; found (Difluoromethyl)-7-(3-(2-oxopiperidin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile (18). Standard procedure was followed, and the product was isolated by preparative HPLC as a yellow solid m.p C; R f = 0.45 (100% EtOAc); 1 H-NMR (600 MHz, MeOD) δ 8.97 (s, 1H), 8.03 (d, J = 6.2 Hz, 1H), 7.79 (s, 1H), 7.62 (t, J = 8.1 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.18 (t, J = 54.0 Hz, 1H), 3.67 (t, J = 5.7 Hz, 2H), 2.41 (t, J = 6.5 Hz, 2H), (m, 4H); 13 C-NMR (151 MHz, MeOD) δ 169.1, (t, J = 26.2 Hz), 150.4, 149.0, 148.5, 143.8, 130.1, 129.6, 129.0, 127.7, 113.1, (t, J = Hz), 106.9, 82.5, 50.8, 32.6, 23.0, 20.9; 19 F-NMR (376 MHz, CDCl 3 ) δ ; IR (thin film) ν = 2923, 2854, 2230, 1715, 1616, 1557, 1308, 1102, 1046, 800 cm -1 ; HRMS (ESI-TOF) calc d for C 18 H 15 N 5 OH + [M+H + ] ; found SI-12

13 1-(4-(Difluoromethyl)-6,7-dimethoxyphthalazin-1-yl)piperidin-4-yl ethylcarbamate TFA salt (19). Standard procedure was followed, and the product was isolated as the trifluoroacetate salt by preparative HPLC as a cream colored powder; m.p C; R f = 0.67 (100% EtOAc); 1 H-NMR (600 MHz, MeOD) δ 7.59 (s, 1H), 7.40 (s, 1H), 7.05 (t, J = 53.8 Hz, 1H), (m, 1H), 4.06 (s, 3H), 4.03 (s, 3H), (m, 2H), (m, 2H), 3.15 (q, J = 7.2 Hz, 2H), (m, 2H), (m, 2H), 1.12 (t, J = 7.5 Hz, 3H); 13 C-NMR (151 MHz, MeOD) δ 162.6, 158.3, 155.5, 155.2, (t, J = 27.0 Hz), 123.1, 119.7, (t, J = Hz), 104.4, (t, J = 3.2 Hz), 71.0, 56.7, 56.7, 36.5, 32.2, 15.4; 19 F-NMR (376 MHz, MeOD) δ -77.0, ; IR (solid) ν = 3276, 2929, 2834, 1698, 1608, 1516, 1453, 1427, 1245, 1214, 1024, 841 cm -1 ; HRMS (ESI-TOF) calc d for C 19 H 24 F 2 N 2 O 4 H + [M+H + ] ; found (Difluoromethyl)-6-((6-(1-methyl-1H-pyrazol-3-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3- yl)thio)quinoline (20). Standard procedure was followed, and the product was isolated by preparative HPLC in trace quantities as a white solid; 1 H-NMR (400 MHz, CDCl 3 ) δ 9.00 (d, J = 4.2 Hz, 1H), 8.37 (s, 1H), (m, 2H), 7.91 (m, 2H), 7.84 (dd, J = 9.0, 1.9 Hz, 1H), SI-13

14 7.59 (d, J = 4.7 Hz, 1H), 7.34 (d, J = 9.7 Hz, 1H), 7.10 (t, J = 54.3 Hz, 1H), 3.96 (s, 3H); HRMS (ESI-TOF) calc d for C 19 H 13 F 2 N 7 SH + [M+H + ] ; found (Difluoromethyl)-6-((6-(1-methyl-1H-pyrazol-3-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3- yl)thio)quinoline (21). Standard procedure was followed, and the product was isolated by preparative HPLC in trace quantities as a white solid; 1 H NMR (400 MHz, CDCl 3 ) δ 8.23 (d, J = 8.7 Hz, 1H), 8.12 (d, J = 9.6 Hz, 1H), (m, 2H), 7.95 (s, 1H), (m, 2H), 7.73 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 9.7 Hz, 1H), 6.74 (t, J = 55.1 Hz, 1H), 3.97 (s, 3H); HRMS (ESI-TOF) calc d for C 19 H 13 F 2 N 7 SH + [M+H + ] ; found Amino-1-((2-(difluoromethyl)-6-methylpyridin-3-yl)methyl)-6-(trifluoromethyl)-1Himidazo[4,5-c]pyridin-2(3H)-one (22). Standard procedure was followed, and the product was isolated by preparative HPLC as an off-white solid; decomposed > 250 C; R f = 0.69 (100% EtOAc); 1 H-NMR (600 MHz, DMSO-d 6 ) δ 7.40 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.21 (t, J = 53.8 Hz, 1H), 6.92 (s, 1H), 6.41 (s, 2H), 5.22 (s, 2H), 2.48 (s, 3H); 13 C-NMR (151 MHz, DMSO-d 6 ) δ 156.9, 154.4, (d, J = 23.5 Hz), 144.3, (q, J = 33.6 Hz), 136.5, 135.2, 128.2, 125.7, (q, J = Hz), (t, J = Hz), 112.7, 93.2 (q, J = 3.2 Hz), 23.4; 19 F-NMR (376 MHz, DMSO-d 6 ) δ -65.0, ; IR (thin film) ν = 3183, 2918, SI-14

15 2341, 1695, 1657, 1625, 1462, 1307, 1241, 1169, 1122, 1094, 1023 cm -1 ; HRMS (ESI-TOF) calc d for C 15 H 12 F 2 N 5 OH + [M+H + ] ; found Amino-1-((4-(difluoromethyl)-6-methylpyridin-3-yl)methyl)-6-(trifluoromethyl)-1Himidazo[4,5-c]pyridin-2(3H)-one (23). Standard procedure was followed, and the product was isolated by preparative HPLC as a white solid; decomposed > 250 C; R f = 0.41 (100% EtOAc); 1 H-NMR (600 MHz, MeOD) δ 8.32 (s, 1H), 7.54 (s, 1H), 7.22 (t, J = 54.1 Hz, 1H), 6.94 (s, 1H), 5.27 (s, 2H), 2.58 (s, 3H); 13 C-NMR (151 MHz, MeOD) δ 160.7, 156.3, 149.7, 145.6, (t, J = 22.4 Hz), (q, J = 34.3 Hz), 137.1, (t, J = 4.0 Hz), 124.6, (q, J = Hz), (t, J = 7.3 Hz), (t, J = Hz), 113.7, 94.9 (d, J = 3.5 Hz), 40.3, 23.7; 19 F-NMR (376 MHz, MeOD) δ -68.7, ; IR (solid) ν = 3363, 2923, 1711, 1658, 1613, 1567, 1463, 1332, 1306, 1119, 1030 cm -1 ; HRMS (ESI-TOF) calc d for C 15 H 12 F 2 N 5 OH + [M+H + ] ; found SI-15

16 Procedure for AO activity assay: To a 1.0 ml reaction buffered with 50 mm potassium phosphate ph 7.4 and containing 10 µm test compound is added 50 µl of a partially purified human aldehyde oxidase preparation (Sharma, R.; Strelevitz, T. J.; Gao, H.; Clark, A. J.; Schildknegt, K.; Obach, R. S.; Ripp, S. L.; Spracklin, D. K.; Tremaine, L. M.; Vaz, A. D. N. Drug Metab. Dispos. 2012, 40, ). The reaction is incubated at 37 C. At 30 minutes an aliquot (450 µl) is removed and added to 4 ml of acetonitrile. A second aliquot (450 µl) of the reaction mixture is added to the acetonitrile quench at 60 minutes. The acetonitrile solution is then centrifuged at 2800 rpm for 10 minutes and the supernatant decanted and vacuum evaporated. The residue is resuspended in 250 µl of water:acetonitrile 95:5 and analyzed by LC/MS. Identification and characterization of the AO product is done by MS fragment pattern analysis. LCMS chromatograms of the tested compounds are shown below. The UV chromatogram was examined for the presence of an M+16 peak of >20% of the parent peak, which is indicative of an AO oxidation product. Where an AO product was observed, MS fragment pattern analysis was used to determine the site of oxidation oxidation on the heteroarene is likely to be due to AO activity. The UV traces and the extracted chromatograms for M and M+16 are shown. SI-16

17 AO activity assay control using a known AO substrate Carbazeran (4): SI-17

18 Experimental data for AO activity assay for compounds 14-23: Compound 14 Molecule 14 is not a substrate for h-ao. There is trace oxidized product (m/z 401) but this is at an extremely low intensity relative to the parent peak (<< 20% of the parent). SI-18

19 Compound 15 Molecule 15 is not a substrate for AO. Only trace quantities (< 20% of parent UV peak) of oxidized product are observed. SI-19

20 Compound 16 SI-20

21 Compound 17 SI-21

22 Compound 18 SI-22

23 Compound 19 Molecule 19 is not a substrate for AO. Only trace (<< 20 % of parent peak) quantities of oxidation product formed. SI-23

24 Compound 20 Compound 20 is a h-ao substrate SI-24

25 Compound 21 Molecule 21 is not an h-ao substrate. SI-25

26 Compound 22 SI-26

27 Compound 23 SI-27

28 AO activity test results for compounds that had not previously been tested: Molecule 12: sorafenib Not an AO substrate. SI-28

29 Molecule SI-1: SI-29

30 Molecule SI-4: saquinavir Not an AO substrate. SI-30

31 SI-31

32 SI-32

33 SI-33

34 SI-34

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36 SI-36

37 SI-37

38 SI-38

39 SI-39

40 SI-40

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42 SI-42

43 SI-43

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47 SI-47

48 SI-48

49 SI-49

50 SI-50

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56 SI-56