Synthesis of Pyridazine-Based α-helix Mimetics

Transcription

1 Synthesis of Pyridazine-Based α-helix Mimetics Allyn T. Londregan, David W. Piotrowski, Liuqing Wei Pfizer Inc. Medicine Design, Eastern Point Rd., Groton, Connecticut, USA Materials and Methods General procedures for the synthesis of all the intermediates and final compounds, their characterizations X-ray crystal structure (10d) Superposition of CCDC with 10d and 10b MR Spectra

2 S1 Materials and Methods All chemicals, reagents and solvents were purchased from commercial sources when available and used without further purification. Proton nuclear magnetic spectroscopy ( 1 H MR) was recorded with 400 and 500 MHz spectrometers. Chemical shifts are expressed in parts per million downfield from tetramethylsilane. The peak shapes are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet. Mass spectrometry (MS) was performed via atmospheric pressure chemical ionization (APCI) or electron scatter (ES) ionization sources. High resolution mass spectrometry (HRMS) was performed via atmospheric pressure chemical ionization (APCI) or electron scatter ionization (ESI) sources. Silica gel chromatography was performed primarily using a medium pressure system using columns pre-packaged by various commercial vendors. The terms concentrated and evaporated refer to the removal of solvent at reduced pressure on a rotary evaporator with a bath temperature less than 60 C. The abbreviation min and h stand for minutes and hours respectively. The abbreviation rt stands for room temperature. Analytical UPLC-MS Method 1: Column: Waters Acquity HSS T3, C x 50 mm, 1.7 µm; Column T = 60 C Gradient: Initial conditions: A-95%:B-5%; hold at initial from min; Linear Ramp to A- 5%:B-95% over min; hold at A-5%:B-95% from min; return to initial conditions min Mobile Phase A: 0.1% formic acid in water (v/v) Mobile Phase B: 0.1% formic acid in acetonitrile (v/v) Flow rate: 1.25 ml/min Detection: UV-205 nm Analytical UPLC-MS Method 2: Column: Waters Acquity HSS T3, C x 50 mm, 1.7 µm; Column T = 60 C Gradient: Initial conditions: A-95%:B-5%; hold at initial from min; Linear Ramp to A- 5%:B-95% over min; hold at A-5%:B-95% from min; return to initial conditions min Mobile Phase A: 0.1% formic acid in water (v/v)

3 S2 Mobile Phase B: 0.1% formic acid in acetonitrile (v/v) Flow rate: 1.25 ml/min Detection: UV-210 nm LCMS Method 1: Column: Ultimate XB, C 18 3 x 50 mm, 3 um; Column T = 35 C Gradient: Initial conditions: A-99%:B-1%; Linear Ramp to A-95%:B-5% over min; Linear Ramp from A-95%:B-5% to A-100%:B-0% over min; hold at A-100%:B-0% for 6.0 to 8.0 minutes; return to initial conditions from 8.0 to 8.01 min; hold at A-99%:B-1% from min. Mobile Phase A: 0.1% trifluoroacetic acid in water (v/v) Mobile Phase B: 0.1% trifluoroacetic acid in acetonitrile (v/v) Flow rate: 1.2 ml/ min Detection: UV-215 nm LCMS Method 2: Column: Xtimate XB, C x 30 mm, 3 µm; Column T = 50 C Gradient: Initial conditions: A-100%:B-0%; Linear Ramp to A-40%:B-60% over min; hold at A-40%:B-60% from 0.9 to 1.5 min; return to initial conditions from 1.5 to 1.51 min; hold at A-100%:B-0% from min. Mobile Phase A: % trifluoroacetic acid in water (v/v) Mobile Phase B: % trifluoroacetic acid in acetonitrile (v/v) Flow rate: 1.2 ml/min Detection: UV-220 nm Analytical HPLC Method 1: Column: Xbridge, C mm, 3μm; Column T = 50 C Gradient: Initial conditions: A-99%:B-1%; Linear Ramp to A-0%:B-100% over min. Mobile Phase A: % trifluoroacetic acid in water (v/v) Mobile Phase B: % trifluoroacetic acid in acetonitrile (v/v) Flow rate: 0.8 ml/min

4 S3 Detection: UV-220 nm Analytical HPLC Method 2: Column: Xbridge, C mm, 5 μm; Column T = 50 C Gradient: Initial conditions: A-99%:B-1%; Linear Ramp to A-95%:B-5% over min; Linear Ramp to A-0%:B-100% over min; return to initial conditions min; hold at A-99%:B-1% from min. Mobile Phase A: % trifluoroacetic acid in water (v/v) Mobile Phase B: % trifluoroacetic acid in acetonitrile (v/v) Flow rate: 0.8 ml/min Detection: UV-210 nm Analytical HPLC Method 3: Column: Xbridge, C mm 5 μm; Column T = 50 C Gradient: Initial conditions: A-90%:B-10%; hold at initial from min; Linear Ramp to A- 0%:B-100% over min; return to initial conditions min; hold at A-90%:B-10% from min. Mobile Phase A: % trifluoroacetic acid in water (v/v) Mobile Phase B: % trifluoroacetic acid in acetonitrile (v/v) Flow rate: 0.8 ml/ min Detection: UV-210 nm Analytical HPLC Method 4: Column: Xbridge, C mm, 5 μm; Column T = 50 C Gradient: Initial conditions: A-95%:B-5%; hold at initial from min; Linear Ramp to A- 0%:B-100% over min; hold at A-0%:B-100% from min; return to initial conditions min; hold at A-95%:B-5% from min. Mobile Phase A: 0.05% ammonium hydroxide in water (v/v) Mobile Phase B: acetonitrile (v/v) Flow rate: 0.8 ml/ min Detection: UV-210 nm

5 S4 Experimental Data Section 1: Synthesis of Compounds and Intermediates Synthesis of library intermediate 7 + CEt 3 HC Ag3 (H 4)2 S 2 8 TFA, H 2 70 o C CEt 6a aq. ah MeH H S1 Amine HATU DIPEA DMF 7 Boc General procedure I. Minisci reaction of 3-chloro-6-carboxypyridazine ethyl ester 3. Ethyl 5-benzyl-6-chloropyridazine-3-carboxylate 6a. A mixture of 3-chloro-6-carboxypyridazine ethyl ester 3 (20 g, mmol), 2-phenylacetic acid (24.81 g, 182 mmol), Ag 3 (1.8 g, mmol), H 2 (200 ml) and TFA (2.45 g, mmol) was heated to 70 o C, and a solution of (H 4 ) 2 S 2 8 (44.0 g, mmol) in H 2 (80 ml) was added dropwise over 20 min. The reaction was stirred for another 50 min at 70 o C, cooled to 0 o C, neutralized with 30 % aq H 4 H, extracted twice with EtAc. The combined organic phases were dried with a 2 S 4, concentrated under reduced pressure and the crude purified by flash chromatography to give compound 6a (10 g, 37%) as a yellow solid. 1 H MR (CD 3, 400 MHz) δ 7.83 (s, 1H), (m, 3H), (m, 2H), 4.50 (q, J=7.2 Hz, 2H), 4.13(s, 2H), 1.45 (t, J=7.2 Hz, 3H). General procedure II. Hydrolysis of the ethyl ester. 5-benzyl-6-chloropyridazine-3- carboxylic acid S1. To a solution of ethyl 5-benzyl-6-chloropyridazine-3-carboxylate 6a (12 g, 43.4 mmol) in MeH (25 ml) was added aq. ah (25 ml, 2 ) at 0 o C. Then the reaction was stirred below 0 o C for 30 min. The reaction was acidified with aq H and extracted twice with EtAc. The combined organic layers were dried over anhydrous a 2 S 4 and concentrated to give the free carboxylic acid S1 that was used without any further purification (9.5 g, 88%) as a yellow solid. 1 H MR (DMS-d 6, 400 MHz) δ (br, 1H), 8.08 (s, 1H), (m, 2H), (m, 3H), 4.25 (s, 2H). General procedure III. Amide formation. tert-butyl 4-(5-benzyl-6-chloropyridazine-3- carbonyl)piperazine-1-carboxylate 7. To a solution of 5-benzyl-6-chloropyridazine-3- carboxylic acid S1 (11 g, mmol) in DMF (50 ml) was added HATU (25.3 g, mmol)

6 S5 followed by tert-butyl piperazine-1-carboxylate (9.9 g, mmol) and DIPEA (17.2 g, 133 mmol). The reaction was stirred at rt for 20 min. The reaction was quenched by saturated H 4 (200 ml) and extracted three times with EtAc (200 ml). The combine organic layers were dried with a 2 S 4, concentrated under reduced pressure and the crude purified by flash chromatography to give compound 7 (7 g, 39.0%) as a white solid. 1 H MR (CD 3, 400 MHz) δ 7.51 (s, 1H), (m, 3H), 7.20 (d, J=7.0 Hz, 2H), 4.10 (s, 2H), (m, 2H), (m, 2H), 3.53 (td, J=5.2, 15.6 Hz, 4H), 1.47 (m, 9H). LCMS (method 1) m/z = (M + a) +. HPLC (analytical HPLC method 1) purity = 95 %. Library protocol I. Suzuki coupling-deprotection to 8 Ar/Het Ar/Het Boc Boc H 7 S2 8 Suzuki coupling. To an 8 ml vial containing a dioxane solution of tert-butyl 4-(5-benzyl-6- chloropyridazine-3-carbonyl)piperazine-1-carboxylate 7 (750 µl, 100 µmol, 1 equiv) was added boronic ester (150 µmol, 1 equiv), K 3 P 4 (100 µl, 300 umol, 2.25 M in water, 3 equiv). 2 was bubbled through the mixture for 30 seconds. To the mixture was added Pd(dtbpf) 2 (5 µmol, 0.05 equiv). The vial was capped and shaken at 140 o C until complete by LCMS (typically 16 h). The reaction mixture was cooled to rt, concentrated by Speedvac to give crude intermediate S2. Deprotection. The crude intermediate S2 was dissolved in MeH (0.5 ml), H (1 ml, 1 M in MeH) was added, and the reaction mixture was shaken at 30 o C until complete by LCMS (typically 1 h). The reaction mixture was concentrated by Speedvac and purified by preparative HPLC to give compound 8. Library size = 57. umber of compounds isolated/purified with >80% purity and identity confirmed by LC/MS analysis = 34. Success rate = 59.6%. Explicit details for compounds 8a-8e can be found in Supplementary Table 1.

7 S6 Library protocol II. SAr mediated etherification-deprotection to 9 R R Boc Boc H 7 S3 9 S Ar mediated etherification. To an 8 ml vial containing a THF solution of tert-butyl 4-(5- benzyl-6-chloropyridazine-3-carbonyl)piperazine-1-carboxylate 7 (800 µl, 100 µmol, 1 equiv) was added alcohol (300 µmol, 3 equiv), Cs 2 C 3 (200 µmol, 2 equiv). The vial was capped and shaken at 100 o C until complete by LCMS (typically 16 h). The reaction mixture was cooled to rt, filtered. The filtrate was concentrated by Speedvac to give crude intermediate S3. General procedure IV. Deprotection. The crude intermediate S3 was dissolved in a mixed solution of TFA/DCM (1 ml, v/v = 1/10 or 0.8 ml, v/v = 1/4). The reaction mixture was shaken at 30 o C until complete by LCMS (typically 45 min). The reaction mixture was concentrated by Speedvac and purified by preparative HPLC to give compound 9. Library size = 50. umber of compounds isolated/purified with >80% purity and identity confirmed by LC/MS analysis = 24. Success rate = 48%. Explicit details for selected compounds 9a-9e can be found in Supplementary Table 1. H Characterization data for a representative (5-benzyl-6-(isopentyloxy)pyridazin-3-yl)(piperazin- 1-yl)methanone 9d. 1 H MR (CD 3 D, 400MHz) δ 7.59 (s, 1H), (m, 5H), 4.58 (t, J=6.44 Hz, 2H), (m, 6H), (m, 4H), (m, 3H), 0.97 (d, J=6.24 Hz, 6H). 13 C MR (CD 3 D, 101MHz) δ , , , , , , , , , , 66.40, 44.20, 43.39, 42.95, 39.19, 37.25, 34.69, 24.80, HRMS (ESI) (m/z): calculated for C 21 H 28 4 a 2 [M + a] ; found d

8 S7 Library protocol III. Amination-deprotection to 10 R R' R R' Boc Boc H 7 S4 10 Amination. To an 8 ml vial containing a tert-amyl alcohol solution of tert-butyl 4-(5-benzyl-6- chloropyridazine-3-carbonyl)piperazine-1-carboxylate 7 (300 µl, 100 µmol, 1 equiv) was added amine (150 µmol, 1.5 equiv), DIPEA (200 µmol, 2 equiv). The vial was capped and shaken at 110 o C until complete by LCMS (typically 16 h). The reaction mixture was cooled to rt, then filtered. The filtrate was concentrated by Speedvac. Toluene was added to the residue, concentrated by Speedvac to give crude intermediate S4. Deprotection. Compound 10 was prepared following General Procedure IV starting from S4. Library size = 55. umber of compounds isolated/purified with >80% purity and identity confirmed by LC/MS analysis = 31. Success rate = 56.3%. Explicit details for selected compounds 10a-10e can be found in Supplementary Table 1. 10b H Characterization data for a representative (5-benzyl-6-(3-benzylazetidin-1-yl)pyridazin-3- yl)(piperazin-1-yl)methanone 10b. 1 H MR (CD 3 D, 400MHz) δ (m, 11H), 4.65 (t, J=8.59 Hz, 2H), 4.31 (dd, J=9.17, 5.66 Hz, 2H), 4.17 (s, 2H), (m, 4H), (m, 4H) 3.18 (d, J=5.07 Hz, 1H), 3.00 (d, J=7.80 Hz, 2H). 13 C MR (CD 3 D, 101MHz) δ , , , , , , , , , , , , , , , , , 58.92, 44.10, 43.33, 42.86, 40.32, 39.31, 39.06, 35.92,

9 S8 HRMS (ESI) (m/z): calculated for C 26 H 30 5 [M + H] ; found d H Characterization data for a representative (5-benzyl-6-(benzyl(methyl)amino)pyridazin-3- yl)(piperazin-1-yl)methanone 10d prepared by library protocol III, but H was used for deprotection instead of TFA to get 10d as an H salt. 1 H MR (CD 3 D, 400MHz) δ 7.83 (s, 1H), (m, 6H), 7.33 (d, J=7.4 Hz, 2H), 7.24 (d, J=7.4 Hz, 2H), (m, 2H), 4.33 (s, 2H), (m, 4H), 3.43 (br, 4H), (m, 3H). 13 C MR (CD 3 D, 101MHz) δ 162.8, 158.0, 146.9, 139.5, 136.1, 135.2, 134.7, 129.0, 128.8, 128.7, 127.9, 127.3, 127.1, 57.2, 44.13, 43.38, 42.86, 40.30, 39.1, HRMS (ESI) (m/z): calculated for C 24 H 27 5 [M + H] ; found Synthesis of library intermediate 12 + CEt 3 H HCbz Ag3 (H4)2S 2 8 TFA, H 2 70 o C CEt 11 HCbz Amine DIPEA MP 120 o C CEt S5 HCbz aq. ah MeH S6 H HCbz Amine HATU DIPEA DMF S7 HCbz Boc Pd/C, H 2 MeH 12 H 2 Boc Ethyl 5-((benzyloxycarbonylamino)methyl)-6-chloropyridazine-3-carboxylate 11 was prepared following General Procedure I, as a yellow oil (6.5 g, 24%) starting from 3-chloro-6-

10 S9 carboxypyridazine ethyl ester 3 (15 g, 80.4 mmol) and -carbobenzyloxyglycine (28.65 g, mmol). 1 H MR (CD 3, 400 MHz) δ 8.14 (s, 1H), (m, 5H), (m, 1H), 5.14 (s, 2H), (m, 4H), 1.44 (t, J=7.20 Hz, 3H). UPLC (method 2) m/z = (M + H) +. Ethyl 5-((benzyloxycarbonylamino)methyl)-6-(2-benzylpyrrolidin-1-yl)pyridazine-3- carboxylate S5. To a solution of ethyl 5-((benzyloxycarbonylamino)methyl)-6-chloropyridazine- 3-carboxylate 11 (10.7 g, 30.6 mmol) and 2-benzylpyrrolidine (5.4 g, 33.6 mmol) in MP (50 ml) were added DIPEA (11.8 g, 91.8 mmol). The resulting mixture was stirred at 120 o C for 12 h, cooled to 0 o C, extracted three times with EtAc. The combined organic phases were dried with a 2 S 4, concentrated under reduced pressure. The crude material was recrystallized from 10 % EtAc in petroleum to give compound S5 (13.7 g, 94%) as a brown solid. 1 H MR (CD 3, 400 MHz) δ 8.00 (br, 1H), (m, 10H), 5.17 (m, 4H), 4.86 (br, 1H), (m, 1H), 4.28 (dd, J=16.8, 5.5 Hz, 2H), 3.72 (d, J=7.5 Hz, 2H), 3.49 (br, 1H), 3.17 (d, J=11.0 Hz, 2H), (m, 1H), (m, 2H), (m, 3H). 5-((Methyl)-6-(2-benzylpyrrolidin-1-yl)pyridazine-3-carboxylic acid S6 was prepared following General Procedure II, as a yellow solid (13.7 g, 97%) starting from ethyl 5- ((benzyloxycarbonylamino)methyl)-6-(2-benzylpyrrolidin-1-yl)pyridazine-3-carboxylate S5 (15 g, 31.6 mmol). tert-butyl 4-(5-((benzyloxycarbonylamino)methyl)-6-(2-benzylpyrrolidin-1-yl)pyridazine-3- carbonyl)piperazine-1-carboxylate S7 was prepared following General Procedure III, as a white solid (13.1 g, 74%) starting from 5-((benzyloxycarbonylamino)methyl)-6-(2-benzylpyrrolidin-1- yl)pyridazine-3-carboxylic acid S6 (13.7 g, 28.9 mmol) and tert-butyl piperazine-1-carboxylate (6.8 g, 34.7 mmol). 1 H MR (CD 3, 400 MHz) δ 7.69 (s, 1H), (m, 5H), (m, 2H), (m, 3H), 5.30 (br, 1H), (m, 2H), 4.85 (br, 1H), 4.55 (dd, J=17.2, 6.9 Hz, 1H), 4.21 (dd, J=16.9, 4.9 Hz, 1H), (m, 4H), (m, 4H), (m., 1H), 3.10 (dd, J=13.3, 3.3 Hz, 1H), 2.73 (dd, J=13.3, 8.0 Hz, 1H), (m, 2H), (m, 1H), (m, 2H), 1.48 (s, 9H). tert-butyl 4-(5-(aminomethyl)-6-(2-benzylpyrrolidin-1-yl)pyridazine-3-carbonyl)piperazine- 1-carboxylate 12. To a solution of tert-butyl 4-(5-((benzyloxycarbonylamino)methyl)-6-(2-

11 S10 benzylpyrrolidin-1-yl)pyridazine-3-carbonyl)piperazine-1-carboxylate S7 (13.1 g, 21.3 mmol) in MeH (800 ml) was added Pd/C (3 g). The resulting mixture was purged with H 2 at 50 psi and stirred at rt for 12 h. The reaction was filtrated and concentrated in vacuum to give 12 (9.2 g, 90%) as a white solid. 1 H MR (CD 3, 400 MHz) δ 7.82 (s, 1H), (m, 5H), 4.96 (d, J=3.3 Hz, 1H), (m, 5H), (m, 1H), (m, 4H), (m, 1H), 3.23 (dd, J=13.3, 3.3 Hz, 1H), 2.84 (dd, J=13.1, 8.3 Hz, 1H), (m, 1H), (m, 1H), (m, 3H), 1.59 (s, 9H), (m, 2H). LCMS (method 2) m/z = (M + H) +. Library protocol IV. Acylation-deprotection to 13 H 2 H R H R Boc Boc H 12 S8 13 Acylation. To an 8 ml vial containing acid (120 µmol, 1.2 equiv) was added tert-butyl 4-(5- (aminomethyl)-6-(2-benzylpyrrolidin-1-yl)pyridazine-3-carbonyl)piperazine-1-carboxylate 12 (500 µl, 100 µmol, 0.2 M in DMF, 1 equiv), TEA (300 µmol, 4 equiv) and HATU (400 µl, 120 µmol, 0.3 M in DMF, 1.2 equiv). The vial was capped and shaken at 30 o C until complete by LCMS (typically 16 h). The reaction mixture was cooled to rt, concentrated by Speedvac, purified by preparative HPLC to give intermediate S8. Deprotection. Compound 13 was prepared following General Procedure IV starting from S8. Library size = 98. umber of compounds isolated/purified with >80% purity and identity confirmed by LC/MS analysis = 87. Success rate = 88.8%. Explicit details for selected compounds 13a-13j can be found in Supplementary Table 1.

12 S11 Synthesis of library intermediate 14 H 2 Br 2 ahc 3 MeH H 2 Br BnBr, ah THF H Br MeI, ah THF Br 4 5 S Bromo-6-chloropyridazin-3-amine 5. To a solution of 6-chloropyridazin-3-amine 4 (25 g, mmol) in MeH (350 ml) was added sodium bicarbonate (32.56 g, mmol). Bromine (30.97 g, mmol) was added dropwise over 30 min. The reaction suspension was stirred at rt under 2 overnight. The suspension was filtered through Celite and rinsed by MeH. The filtrate was concentrated under reduced pressure to give a brown solid. It was then partitioned between MTBE/aq ahc 3 /a 2 S 2 3. The separated organic phase was washed with more aq ahc 3 /a 2 S 2 3, then brine. The aqueous phase was extracted twice by MTBE. The combined organic phases were dried with a 2 S 4, concentrated under reduced pressure to yield compound 5 (35 g, 88%) as a brown solid that was used without any further purification. -Benzyl-4-bromo-6-chloropyridazin-3-amine S9. A solution of 4-bromo-6-chloropyridazin-3- amine 5 (35 g, 170 mmol) in THF (500 ml) at 0 o C was treated with sodium hydride (7.4 g, 190 mmol). The reaction was warmed to rt and stirred for 30 min. A solution of benzyl bromide (28.77 g, 170 mmol) was added to the reaction. The reaction was heated to 40 o C for 4 h. The reaction was cooled, poured into saturated ammonium chloride, extracted with EtAc. The combined organic phases were dried with a 2 S 4, concentrated under reduced pressure. The crude was purified by flash chromatography to give compound S9 (13.5 g, 27%) as a yellow solid. 1 H MR (CD 3, 400 MHz) δ 7.49 (s, 1H), (m, 5H), 5.35 (br, 1H), 4.78 (d, J=5.5 Hz, 2H). UPLC (method 2) m/z = (M + H) +. -Benzyl-4-bromo-6-chloro--methylpyridazin-3-amine 14. A solution of -benzyl-4-bromo- 6-chloropyridazin-3-amine S9 (13.5 g, mmol) in THF (200 ml) at 0 o C was treated with sodium hydride (2.17 g, mmol). The reaction was warmed to rt and stirred for 30 min. Methyl iodide (19.25 g, mmol) was added to the reaction. The reaction was heated to 40 o C for 4 h. The reaction was cooled poured into saturated ammonium chloride, and extracted with EtAc. The combined organic phases were dried with a 2 S 4, then concentrated under reduced

13 S12 pressure. The crude was purified by flash chromatography to give compound 14 (11.5 g, 81%) as a yellow solid. 1 H MR (CD 3, 400 MHz) δ 7.64 (s, 1H), (m, 5H), 4.65 (s, 2H), 3.02 (s, 3H). 13 C MR (CD 3, 101MHz) δ 160.7, 148.5, 137.2, 133.8, 128.6, 127.9, 127.5, , HRMS (ESI) (m/z): calculated for C 12 H 12 Br 3 [M + H] ; found Library protocol V. Etherification-amino carbonylation-deprotection to 15 Br R R R 14 S10 S11 Boc 15 H Etherification. To an 8 ml vial containing a dioxane solution of alcohol (600 µl, 180 µmol, 1.29 equiv) was added ah (270 µmol, 1.93 equiv). The vial was capped and shaken at 30 o C for 1 h. To the mixture was added -benzyl-4-bromo-6-chloro--methylpyridazin-3-amine 14 (500 µl, 140 µmol, 1 equiv) was. The vial was capped and shaken at 30 o C until complete by LCMS (typically 16 h). The reaction mixture was concentrated by Speedvac and purified by preparative HPLC to give intermediate S10. General procedure V. Amino carbonylation. To an 8 ml vial containing S10 (100 µmol, 1.0 equiv) was added tert-butyl piperazine-1-carboxylate (56 mg, 300 µmol, 3.0 equiv), K 3 P 4 (64 mg, 300 µmol, 3.0 equiv), DPPP (4.1 mg, 10 µmol, 0.1 equiv), TEA (42 µl, 300 µmol, 3.0 equiv), Pd(Ac) 2 (2.3 mg, 10 µmol, 0.1 equiv), DMF (750 µl). The vial was capped and shaken under 1 atm C at 110 o C until complete by LCMS (typically 16 h). The reaction mixture was concentrated by Speedvac and purified by preparative HPLC to give intermediate S11. Deprotection. Compound 15 was prepared following General Procedure IV starting from S11 without purification. Library size = 122. umber of compounds isolated/purified with >80% purity and identity confirmed by LC/MS analysis = 83. Success rate = 68%. Explicit details for selected compounds 15a-15e can be found in Supplementary Table 1.

14 S13 Library protocol VI. Suzuki coupling-amino carbonylation-deprotection to 16 Br Ar/Het Ar/Het Ar/Het 14 S12 S13 Boc 16 H Suzuki coupling. To an 8 ml vial containing a dioxane solution of boronic ester (1000 µl, 150 µmol, 1.11 equiv) was added -benzyl-4-bromo-6-chloro--methylpyridazin-3-amine 14 (500 µl, 135 µmol, 0.15 M in dioxane, 1 equiv), Cs 2 C 3 (88 mg, 270 µmol, 2,0 equiv) and H 2 (200 µl). 2 was bubbled through the mixture for 30 seconds. To the mixture was added Pd(dppf) 2 CH 2 2 (5.5 mg, 6.75 µmol, 0.05 eq). The vial was capped and shaken at 100 o C until complete by LCMS (typically 16 h). The reaction mixture was concentrated by Speedvac and purified by preparative HPLC to give intermediate S12. Amino carbonylation. Compound S13 was prepared following General Procedure V starting from S12 (100 µmol). Deprotection. Compound 16 was prepared following General Procedure IV starting from S13 without purification. Library size = 86. umber of compounds isolated/purified with >80% purity and identity confirmed by LC/MS analysis = 61. Success rate = 70.9%. Explicit details for selected compound 16a-16e can be found in Supplementary Table 1.

15 S14 Library protocol VII. Amination-amino carbonylation-deprotection to 17 Br R R' R R' R R' 14 S14 S15 Boc 17 H Amination. To an 8 ml vial containing amine (280 µmol, 2.0 equiv) was added -benzyl-4- bromo-6-chloro--methylpyridazin-3-amine 14 (500 µl, 140 µmol, 0.28 M in DMS, 1 equiv), DIPEA (35 µl, 280 µmol, 2.0 equiv). The vial was capped and shaken at 110 o C until complete by LCMS (typically 16 h). The reaction mixture was concentrated by Speedvac and purified by preparative HPLC to give intermediate S14. Amino carbonylation. Compound S15 was prepared following General Procedure V starting from S14 (100 µmol). Deprotection. Compound 17 was prepared following General Procedure IV starting from S15 without purification Library size = 128. umber of compounds isolated/purified with >80% purity and identity confirmed by LC/MS analysis = 94. Success rate = 73.4%. Explicit details for selected compounds 17a-17e can be found in Supplementary Table 1.

16 S15 Supplementary Table 1. MS and HPLC purity for additional analogs Cmpd m/z t R (min) HPLC purity (%) HPLC method 8a b c d e a b c d e a b c d e a b c d e f g h i j a b c d e a b c d e a b c d e

17 S16 X-ray crystal structure of (5-benzyl-6-(benzyl(methyl)amino)pyridazin- 3-yl)(piperazin-1-yl)methanone hydrochloride (10d, CCDC ) Figure S1. RTEP diagram of (5-benzyl-6-(benzyl(methyl)amino)pyridazin-3-yl)(piperazin- 1-yl)methanone hydrochloride with displacement parameters drawn at 50% probability. Data collection was performed on a Bruker APEX diffractometer at -150 o C. Data collection consisted of omega and phi scans. The structure was solved by direct methods using SHELX software suite in the space group P2 1 /c. The structure was subsequently refined by the full-matrix least squares method. All non-hydrogen atoms were found and refined using anisotropic displacement parameters. During refinement, a disordered solvent molecule was found. Examination of the packing plot revealed the solvent molecules to be inside of an unbroken channel along the c axis. A disorder model for the (EtAc) solvent model was tested, but did not refine satisfactory. The solvent molecule was approximately half occupied. The SQUEZE command was used in the PLAT software to remove the residual electron density from the difference map and refinement continued regularly. The hydrogen atoms located on nitrogen were found from the Fourier difference map and refined with distances restrained. The hydrogen atoms bonded to the water molecule were placed in relevant positions in the hydrogen bonding network. The coordinates of these hydrogen atoms were constrained. The remaining hydrogen atoms were placed in calculated positions and were

18 S17 allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms. The final R-index was 5.5%. A final difference Fourier revealed no missing or misplaced electron density. Pertinent crystal, data collection and refinement are summarized in Table S2. Table S2. Crystal data and structure refinement for (5-benzyl-6- (benzyl(methyl)amino)pyridazin-3-yl)(piperazin-1-yl)methanone hydrochloride (10d) Empirical formula C 24 H (H) 2 Formula weight Temperature 296(2) K Wavelength Å Crystal system Monoclinic Space group P2(1)/c Unit cell dimensions a = (7) Å α= 90. b = (2) Å β= (4). c = (6) Å γ = 90. Volume (3) Å 3 Z 4 Density (calculated) Mg/m 3 Absorption coefficient mm -1 F(000) 1040 Crystal size 0.31 x 0.10 x 0.05 mm 3 Theta range for data collection 2.91 to Index ranges -13<=h<=13, -35<=k<=28, -9<=l<=9 Reflections collected Independent reflections 4945 [R(int) = ] Completeness to theta = % Absorption correction Empirical Max. and min. transmission and Refinement method Full-matrix least-squares on F 2

19 S18 Data / restraints / parameters 4945 / 3 / 308 Goodness-of-fit on F Final R indices [I>2sigma(I)] R1 = , wr2 = R indices (all data) R1 = , wr2 = Largest diff. peak and hole and e.å -3 Figure S2: Superposition of X-ray crystal structure 10d with CCDC d = green, CCDC = yellow 1 CCDC is compound 11a from Maity, P.; König, B. rg. Lett. 2008, 10,

20 S19 Figure S3: Superposition of X-ray crystal structure CCDC with minimized 10b 10b = orange, CCDC = yellow

21 S20 CEt 6a CH S1

22 S21 7 Boc

23 S22 9d H

24 S23 10b H

25 S24 10d H

26 S25 HCbz CEt 11 HCbz S5 Et

27 S26 HCbz S7 Boc H 2 12 Boc

28 S27 H Br S9 Br 14

29 S28