Unique Functional and Structural Properties of the LRRK2 ATP-binding Pocket
Zhiyong Liu1, 2,, Robert A. Galemmo Jr.3, Kyle B. Fraser1, Mark S Moehle1, Saurabh Sen1, Laura A. Volpicelli-Daley1, Lawrence J. DeLucas3, Larry J. Ross3, Jacob Valiyaveettil3, Omar Moukha-Chafiq3, Ashish K. Pathak3, Sam Ananthan3, Hollis Kezar3, E. Lucile White3, Vandana Gupta3, Joseph A. Maddry3, Mark J. Suto3, Andrew B. West1*
From the 1Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, 2Center for Biophysical Sciences and Engineering, Department of Optometry, The University of Alabama at Birmingham, Birmingham, Alabama and 3Drug Discovery Division, Southern Research Institute, Birmingham, Alabama.
*Running title: Analysis of the LRRK2 ATP-Pocket using Small Molecules
To whom correspondence should be addressed: Andrew West, Civitan International Research Building 5th floor, 1719 6th Ave S., Birmingham, AL. Phone: 1-205-996-7697; Fax: 1-205-996-6580; Email:
Keywords: PARK8, Drug Discovery, AlphaScreen, Neurodegeneration
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Cyototoxicity Assays- For HepG2 assays (ATCC HB-8065), compounds or carrier control (DMSO) were diluted in E-MEM with 10% fetal bovine serum (complete growth medium) and dispensed into each well of a 384-well black clear-bottom tissue culture treated plates. Compounds were evaluated over a 10 point, 2 fold dilution series, ranging from 20 to 0.039 µg/ml. Twenty µl of complete growth medium containing 3,000 HepG2 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 72h prior to endpoint detection. The assay plates were equilibrated to room temperature for 10 min and twenty-five µl of Cell Titer Glo reagent (Promega) was added to each well using a WellMate (Matrix, Hudson, NH) and the plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer Envision microplate reader with an integration time of 0.1 s.
For THP-1 cells (ATCC TIB-202), cCompounds and carrier controls were diluted in RPMI 1640 with 10% fetal bovine serum (complete growth medium) to prepare a concentrated dosing solution that was dispensed into 384-well black clear-bottom tissue culture treated plates. Compounds were evaluated over a 10 point, 2 fold dilution series, ranging from 40 to 0.078 µg/ml. Twenty µl of complete growth medium containing 3,000 THP-1 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 72h prior to endpoint detection. The assay plates were equilibrated to room temperature for 10 min and twenty-five µl of Cell Titer Glo reagent (Promega) was added to each well using a WellMate (Matrix, Hudson, NH) and the plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer Envision microplate reader with an integration time of 0.1 s.
Medicinal Chemistry- The exact mass spectral data and % purity were obtained with a Agilent LC-MSTOF electrospray ionization (ESI) using an Agilent 1100 LC equipped with a diode array UV detector and monitored at 254 nm. A gradient elution was performed with MeOH and water both containing 0.1% formic acid as a mobile phase with a Chromolith Fast Gradient, RP-18e, 50-2mm HPLC column. 1HNMR spectra were recorded on a Agilent/Varian MR-400 spectrometer operating at 399.843 MHz or on Agilent/Varian MR-400 spectrometer operating at 399.930 MHz. Chemical shifts in CDCl3 and Me2SO-d6 are expressed in parts per million downfield from tetramethylsilane (TMS) Chemical shifts () listed for multiplets were measured from the approximate centers, and relative integrals of peak areas agreed with those expected for the assigned structures.
Synthesis of 1-16 and 19
Ethyl 2-((6-chloropyridazin-3-yl)thio)acetate [II a]: 6-Chloropyridazine-3-thiol (1 g, 6.83 mmol) was added to an aqueous NaOH solution (300 mg, 7.5 mmol, in 10 mL water), and the mixture was stirred for 10 min before a solution of ethyl chloroacetate (837 mg, 7.35 mmol) in acetone (2 mL) was added dropwise over 10 min. Stirring was continued for 1 h, and then, the side of the flash was scratched with a glass rod to induce crystal formation. The crystalline product was collected by filtration, washed with water, and then dried . The product was recrystallized from EtOH to give 3 (1.03 g, 65%) as a light tan powderHRMS (M+H) calculated for C8H9ClN2O2S+H: m/z 233.0146; found: m/z 233.0149
Ethyl 2-((6-chloropyridazin-3-yl)thio)propanoate [II b]: This compound was prepared by the same procedure used for II a with ethyl 2-chloropropionate substituted for ethyl chloroacetate. The product was recrystallized from EtOH to give the product as a powder ;Yield 70 %; HRMS (M+H) calculated for C9H11ClN2O2S+H: m/z 247.0303; found: m/z 247.0305.
Ethyl 2-((6-chloropyridazin-3-yl)thio)butanoate [II c]: This compound was prepared by the same procedure used for II a with ethyl 2-chlorobutanonate substituted for ethyl chloroacetate. Yield 47%; 1H NMR [DMSO-d6] :7.80 (d, 1H, pyridazine, J = 11.1 Hz), 7.78 (d, 1H, pyridazine, J = 11.1 Hz), 4.57 (t, 1H, SCH, J = 6.8 Hz), 4.13 (q, 2H, OCH2, J = 7.1 Hz), 1.85-2.03 (m, 2H, CH2), 1.16 (t, 3H, ester CH3, J = 7.4 Hz), 1.01 (t, 3H, CH3, J = 7.2 Hz); HRMS (M+H) calculated for C10H13ClN2O2S+H: m/z 261.0459; found: m/z 261.0459. LCMS Purity 95.95% /retention time 6.353 min.
Ethyl 2-((6-chloropyridazin-3-yl)thio)pentanoate [II d]: This compound was prepared by the same procedure used for II a with ethyl 2-chloropentanonate substituted for ethyl chloroacetate.The product was recrystallized from EtOH to give the product.Yield 55%.HRMS (M+H) calculated for C11H15ClN2O2S+H: m/z 275.0616; found: m/z 275.0616.
Preparation of 2-((3-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)acetic acid [1]:To thiophene-2-carbohydrazide (215 mg, 1.5 mmol) dissolved in n-BuOH (3 mL) was added ethyl 2-((6-chloropyridazin-3-yl)thio)acetate [II a] (232 mg, 1 mmol). This mixture was heated for 5 h in a 140-145oC oil bath then the reaction mixturewas cooled to room temperature and the solid removed by filtration. The solid filtrate was dissolved with methylene chloride (50 mL), and washed with saturated with NaHCO3 (~15 mL). The combined aqueous layers was washed twice with methylene chloride, then the organic layers were combined, washed with brine, dried [MgSO4], filtered, and evaporated. The resulting solid was recrystallized from EtOH to give the product [208 mg, 65% yield] as a light tan powder. HRMS (M+H) calculated for C13H12N4O2S2+H: m/z 321.0474; found: m/z 321.0473.The ester prepared above (50 mg, 0.156 mmol) was dissolved in 2:1 THF:MeOH was added 100 uL of 2N NaOH, and the whole was stirred at room temperature for 3 h. The reaction mixture was then evaporated to dryness, the residue dissolved in water (5 mL), and the solution acidified with 6M HCl. The resulting precipitate was filtered, washed twice with cold water, and dried.The product was recrystallized from EtOActo give 1 (30 mg, 66% yield) as acolorless solid. 1H NMR [DMSO-d6] : 13.09 (br s, 1H, OH), 8.29 (d, 1H, pyridazine, J = 9.8 Hz), 8.19 (d d, 1H, thienyl, J = 1.2, 3.9 Hz), 7.86 (d d, 1H, benzo, J = 1.2, 5.1 Hz), 7.41 (d, 1H, pyridazine, J = 9.4 Hz), 7.30 (d d, 1H, benzo, J = 3.5, 5.0 Hz), 4.21 (s, 2H, SCH2) ; HRMS (M+H): calculated for C11H8N4O2S2+H: m/z 293.0161; found: m/z 293.0158. LCMS Purity 98% /retention time 5.82 min.
Preparation of 2-((3-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanoic acid [2]:2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanoate [2] was prepared from II b by the same method used for 1. The product was recrystallized from EtOAc .Yield 65 %; 1H NMR [DMSO-d6] : 8.28 (d, 1H, pyridazine, J = 9.7 Hz), 8.21 (d d, 1H, thienyl, J = 1.1, 3.7 Hz), 7.86 (d d, 1H, thienyl, J = 1.2, 5.1 Hz), 7.35 (d, 1H, pyridazine, J = 9.7 Hz), 7.31 (d d, 1H, thienyl, J = 3.9, 5.1 Hz), 3.63 (q, 1H, SCH, J = 7.5 Hz), 1.68 (d, 3H, CH3, J = 7.1 Hz); HRMS (M+H) calculated for C12H10N4O2S2+H: m/z 307.0318; found: m/z 307.0321. LCMS Purity 97 % /retention time 6.224 min.
Preparation of 2-((3-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoic acid [3]:2-((3-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoate [3]was prepared from II cby the same method used for 1. The product was recrystallized from ethyl acetate.Yield 65%; 1H NMR [DMSO-d6] : 13.26 (br s, 1H, OH), 8.29 (d, 1H, pyridazine, J = 9.4 Hz), 8.20 (d d, 1H, thienyl, J = 1.1, 3.9 Hz), 7.87 (d d, 1H, thienyl, J = 1.1, 5.0 Hz), 7.37 (d, 1H, pyridazine, J = 9.8 Hz), 7.32 (d d, 1H, thienyl, J = 3.6, 4.7 Hz), 4.57 (t, 1H, SCH, J = 6.6 Hz), 2.04-2.24 (m, 2H, CH2), 1.07 (t, 3H, CH3, J = 7.5 Hz); HRMS (M+H) calculated for C12H10N4O2S2+H: m/z 307.0318; found: m/z 307.0321 LCMS Purity 96.5/retention time 6.46 min.
Preparation of 2-((3-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)pentanoic acid [4]:2-((3-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)pentanoic[4] was prepared from II d by the same method used for 1.The product was recrystallized from EtOAc.Yield 60 %; 1H NMR [DMSO-d 6] : 13.37 (br s, 1H, OH), 8.29 (d, 1H, pyridazine, J = 9.8 Hz), 8.21 (d d, 1H, thienyl, J = 1.1, 3.9 Hz), 7.87 (d d, 1H, thienyl, J = 1.2, 5.1 Hz), 7.37 (d, 1H, pyridazine, J = 9.7 Hz), 7.32 (d d, 1H, thienyl, J = 3.5, 5.0 Hz), 4.58 (t, 1H, SCH, J = 6.8 Hz), 1.95-2.06 (m, 2H, CH2), 1.45-1.56 (m, 2H, CH2), 0.93 (t, 3H, CH3, J = 7.3 Hz); HRMS (M+H) calculated for C14H12N4O2S2+H: m/z 335.0631; found: m/z 335.0633. LCMS Purity 98.5%/retention time 6.682 min.
Preparation of 2-((3-(Thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanamide [5]: Ethanolic ammonia (5 mL) was added to ethyl 2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoate [the ethyl ester intermediate prepared from II c to give 3] (60 mg, 0.188 mmol), and the reaction vessel sealed and the mixture stirred overnight at 90-95oC. The mixture was then evaporated to dryness, the solid residue was filtered, washed 3x with EtOH, and dried to give the desired product 6 as an off-white solid (38 mg, 70%) .1H NMR [DMSO-d6] : 8.26 (d, 1H, pyridazine, J = 9.8 Hz), 8.22 (d d, 1H, thienyl, J = 1.1, 3.9 Hz), 7.90 (br s, 1H, NH2), 7.86 (d d, 1H, thienyl, J = 1.2, 5.1 Hz), 7.36 (br s, 1H, NH2), 7.32 (d, 1H, pyridazine, J = 9.4 Hz), 7.32 (d, 1H, thienyl, J = 1.2 Hz), 4.52 (d d, 1H, SCH, J = 5.5, 7.9 Hz), 1.96-2.14 (m, 2H, CH2), 1.04 (t, 3H, CH3, J = 7.5 Hz); HRMS (M+H) calculated for C13H13N5OS2+H: 320.0634; found: 320.0637. LCMS Purity 94.5 % /retention time 5.96 min.
Preparation of 1-(Piperidin-1-yl)-2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butan-1-one[11, SRI 29132]: To 2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoic acid [3] (100 mg, 0.34 mmol) dissolved in DMF (3 mL) was added HATU (150 mg, 0.39 mmol), and after 15 minutes stirring, piperidine (40 mg, 0.47 mmol) followed by DIPEA (200 uL). The mixture was stirred at room temperature overnight and then evaporated to dryness. The crude product was purified by flash chromatography (silica, chloroform/methanol (9:1) ) to afford the product as a off -white solid (70 mg, 56% yield). 1H NMR [DMSO-d6] : 8.28 (d, 1H, pyridazine, J = 9.8 Hz), 8.13 (d d, 1H, thienyl, J = 1.2, 4.0 Hz), 7.92 (d d, 1H, thienyl, J = 1.2, 5.1 Hz), 7.36 (app t, 1H, thienyl, J = 3.9 Hz), 7.34 (d, 1H, pyridazine, J = 9.8 Hz), 5.15-5.20 (m, 1H, SCH), 3.36-3.66 (m, 4H, piperidyl NCH2), 1.96-2.17 (m, 2H, CH2), 1.40-1.68 (m, 6H, piperidyl CH2CH2), 1.01 (t, 3H, CH3, J = 7.2 Hz); HRMS (M+H) calculated for C18H21N5OS2+H: m/z 388.1260; found: m/z 388.1268. LCMS Purity 100 % /retention time 6.95 min.
Preparation of N,N-Dimethyl-2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanamide[6]: This reaction was conducted under the same conditions used in the preparation of 11with dimethylamine substituted for piperidine. The desired product was isolated by preparative TLC (20 cm x 20 cm silica gel plates, 1,000 microns thickness, 9:1 CHCl3/MeOH) as a light tan solid. Yield 55 %; 1H NMR [DMSO-d6] : 8.28 (d, 1H, pyridazine, J = 9.8 Hz), 8.12 (d d, 1H, thienyl, J = 1.2, 3.5 Hz), 7.91 (d d, 1H, thienyl, J = 1.2, 5.1 Hz), 7.35 (d d, 1H, thienyl, J = 3.5, 5.1 Hz), 7.43 (d, 1H, pyridazine, J = 9.4 Hz), 5.13 (d d, 1H, SCH, J = 5.8, 7.0 Hz), 3.15 (s, 3H, CH3), 2.91 (s, 3H, CH3), 1.97-2.16 (m, 3H, CH2), 1.01 (t, 3H, CH3, J = 7.2 Hz); HRMS (M+H) calculated for C15H17N5OS2+H: m/z 348.0947; found: m/z 348.0949. LCMS Purity 96 % /retention time 6.22 min.
Preparation of N,N-Dipropyl-2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanamide[7]: This reaction was conducted under the same conditions used in the preparation of 11 with 2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanoic acid [2] substituted for 3and di-n-propylamine substituted for piperidine. The crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to afford the product as a colorless solidYield 52 %; 1H NMR [DMSO-d6] : 8.28 (d, 1H, pyridazine, J = 9.8 Hz), 8.12 (d d, 1H, thienyl, J = 1.2, 3.5 Hz), 7.90 (d d, 1H, thienyl, J = 1.2, 5.1 Hz), 7.34-7.37 (m, 1H, thienyl), 7.34 (d, 1H, pyridazine, J = 9.4 Hz), 5.14 (q, H, SCH, J = 6.7 Hz), 3.20-3.37 (m, 4H, NCH2), 1.71 (d, 3H, CH3, J = 6.6 Hz), 1.52-1.65 (m, 2H, CH2), 1.50 (sextet, 2H, CH2, J = 7.4 Hz), 0.83 (t, 3H, CH3, J = 7.5 Hz), 0.75 (t, 3H, CH3, J = 7.4 Hz); HRMS (M+H) calculated for C18H23N5OS2 +H: m/z 390.1417; found: m/z 390.1408. LCMS Purity 99.6 % /retention time 6.904 min.
Preparation of N-(2-hydroxyethyl)-2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanamide[8]: This reaction was conducted under the same conditions used in the preparation of 11 with 2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanoic acid [2] substituted for 3and 2-(hydroxyl)ethylamine substituted for piperidine. The crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to afford the product as an off-white solid. Yield 55 %; 1H NMR [DMSO-d6]: 8.48 (m, 1H, NH), 8.26 (d, 1H, pyridazine, J = 9.8 Hz), 8.20 (d d, 1H, thienyl, J = 0.8, 3.5 Hz), 7.86 (d d, 1H, thienyl, J = 1.1, 5.0 Hz), 7.32-7.36 (m, 1H, thienyl), 7.32 (d, 1H, pyridazine, J = 9.4 Hz), 4.66-4.73 (m, 2H, SCH and OH), 3.39 (q, 2H, CH2, J = 5.9 Hz), 3.09-3.22 (app heptet, 2H, CH2), 1.66 (d, 3H, CH3, J = 6.6 Hz); HRMS (M+H) calculated for C14H15N5OS2+H: m/z 350.0947; found: m/z 350.0949. LCMS Purity 98 % /retention time 5.674 min.
Preparation of 1-(Pyrrolidin-1-yl)-2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butan-1-one [9]: This reaction was conducted under the same conditions used in the preparation of 11 with pyrrolidine substituted for piperidine. The crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to afford the product. Yield 60 % ; 1H NMR[DMSO-d6] : 8.28 (d, 1H, pyridazine, J = 9.8 Hz), 8.14 (d d, 1H, thienyl, J = 1.2, 3.5 Hz), 7.34-7.37 (m, 1H, thienyl), 7.34 (d, 1H, pyridazine, J = 9.8 Hz), 4.92 (d d, 1H, SCH, J = 5.9, 7.9 Hz), 3.54-3.66 (m, 4H, morpholine NCH2), 3.32-3.41 (m, 4H, morpholine NCH2), 1.98-2.18 (m, 2H, CH2), 1.75-1.95 (m, 4H, morpholine CH2), 1.03 (t, 3H, CH3, J = 7.4 Hz); HRMS (M+H) calculated for C17H19N5OS2.+H: m/z 374.1104; found: m/z 374.1107. LCMS Purity 98 % /retention time 6.463 min.
Preparation of 1-(Piperidin-1-yl)-2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propan-1-one [10]: This reaction was conducted under the same conditions used in the preparation of 11with 2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanoic acid [2] substituted for 3. The crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to afford the product. Yield 60 %; 1H NMR [DMSO-d6]: 8.28 (d, 1H, pyridazine, J = 9.8 Hz), 8.13 (d d, 1H, thienyl, J = 1.1, 3.5 Hz), 7.90 (d d, 1H, thienyl, J = 1.2, 5.1 Hz), 7.34-7.37 (m, 1H, thienyl), 7.34 (d, 1H, pyridazine, J = 9.4 Hz), 5.20 (d d, 1H, SCH, J = 6.6, 7.1 Hz), 3.40-3.63 (m, 4H, piperidyl NCH2), 1.67 (d, 3H, CH3, J = 6.6 Hz), 1.51-1.64 (m, 4H, piperidyl), 1.43-1.51 (m, 2H, piperidyl); HRMS (M+H) calculated for C17H19N5OS2+H: m/z 374.1103; found m/z 374.1113. LCMS Purity 100 % /retention time 6.812 min.
Preparation of 1-Morpholino-2-((3-(thiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butan-1-one [12]: This reaction was conducted under the same conditions used in the preparation of 11 with morpholine substituted for piperidine. The crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to afford the product. Yield 55% ; 1H NMR [DMSO-d6] : 8.28 (d, 1H, pyridazine, J = 9.6 Hz), 8.13 (d d, 1H, thienyl, J = 1.1, 3.6 Hz), 7.91 (d d, 1H, thienyl, J = 1.2, 5.0 Hz), 7.35 (d d, 1H, thienyl, J = 3.7, 5.0 Hz), 7.35 (d, 1H, pyridazine, J = 9.6 Hz), 5.11 (d d, 1H, SCH, J = 6.0, 7.1 Hz), 3.46-3.66 (m, 8H, morpholine CH2), 1.98-2.18 (m, 2H, CH2), 1.02 (t, 3H, CH3, J = 7.4 Hz); HRMS (M+H) calculated for C17H19N5O2S2+H: m/z 390.1053; found m/z 390.1052. LCMS Purity 100% /retention time 6.181 min.
Preparation of 2-((3-(5-Chlorothiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)-1-(piperidin-1-yl)butan-1-one [13]
2-((3-(5-Chlorothiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoic acid: To 5-chlorothiophene-2-carbohydrazide (215 mg, 1.25 mmol) dissolved in n-BuOH (3 mL) was added ethyl 2-((6-chloropyridazin-3-yl)thio)butanoate[II c](261 mg, 1 mmol). This mixture was heated for 5 h in a 140-145oC oil bath then the reaction mixture was cooled to room temperature and the solid removed by filtration. The solid filtrate was dissolved with methylene chloride (50 mL), and washed with saturated with NaHCO3 (~15 mL). The combined aqueous layers was washed twice with methylene chloride, then the organic layers were combined, washed with brine, dried [MgSO4], filtered, and evaporated. The resulting solid was recrystallized from EtOH to give ester [200 mg, 56% yield] as a light tan powder.
The ester prepared above (200 mg, 0.52 mmol) was dissolved in 2:1 THF:MeOH was added 400 uL of 2N NaOH, and the whole was stirred at room temperature for 3 h. The reaction mixture was then evaporated to dryness, the residue dissolved in water (5 mL), and the solution acidified with 6M HCl. The resulting precipitate was filtered, washed twice with cold water, and dried to give 2 (120 mg, 65% yield) as an off-white solid: 1H NMR [DMSO-d6] : 8.29 (d, 1H, pyridazine, J = 9.8 Hz), 8.03 (d, 1H, thienyl, J = 3.9 Hz), 7.39 (d, 1H, pyridazine, J = 9.8 Hz), 7.35 (d, 1H, thienyl, J = 4.3 Hz), 4.52 (t, 1H, SCH, J = 6.7 Hz), 2.06 (quint, 2H, CH2, J = 7.2 Hz), 1.07 (t, 3H, CH3, J = 7.3 Hz); HRMS (M+H) calculated for C13H11ClN4O2S2+H: m/z 355.0085; found: m/z 355.0090. LCMSPurity 90 % /retention time 6.95 min.
To 2-((3-(5-chlorothiophen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoic acid (100 mg, 0.28 mmol) dissolved in DMF (3 mL) was added HATU (150 mg, 0.39 mmol), and after 15 minutes stirring, piperidine (40 mg, 0.47 mmol) followed by DIPEA (200 uL). The mixture was stirred at room temperature overnight and then evaporated to dryness. The desired product [13] was isolated by preparative TLC (20 cm x 20 cm silica gel plates, 1,000 microns thickness, 9:1 CHCl3/MeOH) as a light tan solid (66 mg, 55% yield); 1H NMR [DMSO-d6] : 8.29 (d, 1H, pyridazine, J = 9.4 Hz), 8.13 (d, 1H, thienyl, J = 3.9 Hz), 7.28-7.44 (m, 1H, thienyl), 7.36 (d, 1H, pyridazine, J = 9.7 Hz), 5.17 (t, 1H, SCH, J = 6.2 Hz), 3.62-3.73 (m, 2H, piperidyl NCH2), 3.30-3.56 (m, 2H, piperidyl NCH2), 1.96-2.17 (m, 2H, CH2), 1.37-1.70 (m, 6H, piperidyl CH2CH2), 1.01 (t, 3H, CH3, J = 7.2 Hz); HRMS (M+H) calculated for C18H20ClN5OS2+H: m/z 422.0871; found: 422.0874. LCMS Purity 90 % /retention time 7.152 min.
Preparation of 1-(Piperidin-1-yl)-2-((3-(thiazol-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butan-1-one[14]
2-((3-(thiazol-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanoic acid: This acid was prepared by the method used for 13. The esterethyl 2-((3-(thiazol-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propanoate was prepared by reaction of thiazole-2-carbohydrazide with ethyl 2-((6-chloropyridazin-3-yl)thio)butanoateII c (LRMS (M+H): m/z 350.1) then saponifiedto give the title acid.The product was recrystallized from EtOAc.Yield 60 %; 1H NMR [DMSO-d6] : 8.39 (d, 1H, pyridazine, J = 9.7 Hz), 8.20 (d, 1H, thiazole, J = 3.1 Hz), 8.15 (d, 1H, thiazole, J = 3.1 Hz), 7.47 (d, 1H, pyridazine, J = 9.8 Hz), 4.74 (q, 2H, SCH, J = 7.2 Hz), 1.72 (d, 3H, CH3, J = 7.4 Hz); HRMS (M+H) calculated for C13H11ClN4O2S2+H: m/z 355.0085; found: m/z 355.0090.
1-(Piperidin-1-yl)-2-((3-(thiazol-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butan-1-one[14]:The amide was obtained from the above acid by the same method used for 13;The desired product was isolated by preparative TLC (20 cm x 20 cm silica gel plates, 1,000 microns thickness, 9:1 CHCl3/MeOH) as an off-white solid. Yield 55% ; 1H NMR [DMSO-d6] :8.37 (d, 1H, pyridazine, J = 9.7 Hz), 8.20 (d, 1H, thiazole, J = 3.1 Hz), 8.16 (d, 1H, thiazole, J = 3.1 Hz), 7.44 (d, 1H, pyridazine, J = 9.3 Hz), 5.28-5.33 (m, 1H, SCH), 3.36-3.68 (m, 4H, piperidyl NCH2), 1.94-2.16 (m, 2H, CH2), 1.42-1.66 (m, 6H, piperidyl CH2CH2), 0.99 (t, 3H, CH3, J = 7.3 Hz); HRMS (M+H) calculated for C17H20N6OS2+H: m/z 389.1213; found: m/z 388.1216. HPLC Purity 97% /retention time 6.1 min.
Preparation of 1-(Piperidin-1-yl)-2-((3-(thiazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propan-1-one [15]
Ethyl 2-((3-(thiazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoate: The starting ester was prepared by the same method used for 13 by reaction of thiazole-4-carbohydrazide with ethyl 2-((6-chloropyridazin-3-yl)thio)propanoateII b(LRMS (M+H): m/z 336.1). The title acid was obtained by saponification as detailed in 13.
1-(Piperidin-1-yl)-2-((3-(thiazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)propan-1-one [15]:The amide was obtained from the above acid by the same method used for 13; The crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to afford 15as a colorless solid.Yield 55 %; 1H NMR [DMSO-d6] : 9.37 (d, 1H, thiazole, J = 1.9 Hz), 8.68 (d, 1H, thiazole, J = 1.6 Hz), 7.36 (d, 1H, pyridazine, J = 9.3 Hz), 5.17 (q, 1H, SCH, J = 6.8 Hz), 3.25-3.60 (m, 4H, piperidyl NCH2), 1.61 (d, 3H, CH3, J = 7.0 Hz), 1.39-1.64 (m, 6H, piperidinyl CH2); HRMS (M+H) calculated for C16H18N6OS2+H: m/z 375.1056; found: m/z 375.1066.
Preparation of 2-((3-(1H-Pyrazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)-1-(piperidin-1-yl)propan-1-one [16]:This compound was prepared by the same method used for 13.The crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to give 16as a off-white solid.Yield 55 %; 1H NMR [DMSO-d6] :8.22 (d, 1H, pyridazine, J = 9.8 Hz), 8.01 (br s, 1H, pyrazole CH), 7.30 (d, 1H, pyridazine, J = 9.7 Hz), 7.08 (br s, 1H, pyrazole CH), 5.16 (q, 1H, SCH, J = 6.8 Hz), 3.30-3.62 (m, 4H, piperidyl NCH2), 1.60 (d, 3H, CH3, J = 7.0 Hz), 1.41-1.68 (m, 6H, piperidinyl CH2); HRMS (M+H) calculated for C16H19N7OS+H: m/z 358.1445; found: m/z 358.1438. HPLC Purity 100 % /retention time 5.82 min.
Preparation of 2-((3-(4-Fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)-1-(piperidin-1-yl)butan-1-one[19]
Ethyl 2-((3-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)butanoate: This compound was prepared by the same method used for 13 by reaction of 4-fluorobenzohydrazide with ethyl 2-((6-chloropyridazin-3-yl)thio)butanoate IIc. The product was recrystallized from EtOH. Yield 60 %; 1H NMR [DMSO-d6] : 8.36-8.42 (m, 2H, Ph), 8.31 (d, 1H, pyridazine, J = 9.8 Hz), 7.42-7.48 (m, 2H, Ph), 7.39 (d, 1H, pyridazine, J = 9.8 Hz), 4.53 (t, 1H, SCH, J = 6.8 Hz), 4.08-4.16 (dq, 1H, OCH2, J = 7.1 Hz and 11.0 Hz), 3.95-4.04 (d q, 2H, OCH2, J = 7.0 Hz and 10.9 Hz), 1.98-2.09 (m, 2H, CH2), 1.06 (t, 3H, CH3, J = 7.0 Hz), 1.04 (t, 3H, CH3, J = 9.5 Hz); HRMS (M+H) calculated for C17H17FN4O2S+H: m/z 361.1129; found: m/z 361.1129. HPLC Purity 95.2%/retention time 7.118 min.
The ester was saponified under the same conditions used for 13to give the acid (LRMS (M+H): m/z 333.4).
2-((3-(4-Fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)thio)-1-(piperidin-1-yl)butan-1-one[19]:This compound was prepared by the method used for 13;the crude product was purified by flash chromatography ( silica, chloroform/methanol 9:1 ) to afford the 19as a colorless solidYield 52 %1H NMR [DMSO-d6] : 8.35-8.40 (m, 2H, Ph), 8.29 (d, 1H, pyridazine, J = 9.8 Hz), 7.44-7.51 (m, 2H, Ph), 7.35 (d, 1H, pyridazine, J = 9.7 Hz), 5.01 (d d, 1H, SCH, J = 5.4, 7.4 Hz), 3.49-3.58 and 3.33-3.45 (m, 4H, NCH2), 1.93-2.13 (m, 2H, CH2), 1.50-1.66 (m, 2H, piperidyl), 1.38-1.50 (m, 4H, piperidyl), 0.96 (t, 3H, CH3, J = 7.2 Hz); HRMS (M+H) calculated for C20H22FN5OS+H: m/z 400.1602; found: m/z 400.1603.
Synthesis of 17, 18 and 20
6-Chloro-3-(2-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine [IVa]: 6-Chloro-3-hydrazinopyridazine III (6.9 mmol) was suspended in dioxane (45 mL). Triethylamine (1.1 equiv), and 2-fluorobenzoyl chloride (1.1 equiv) in dioxane (10 ml) were added dropwise over 5-10 min at room temperature. The reaction mixture was stirred at the same temperature for 30-50 min (monitored by TLC) then the dioxane was removed by rotary evaporation. The residue was refluxed in phosphorus oxychloride (40 ml) for 3-4 hours and the solvent was evaporated. The residue triturated several times with ethyl acetate then ice-cold water (50 ml) was added. The aqueous layer was neutralized to pH 7 with saturated aqueous sodium hydrogen carbonate, and the resulting suspended solid was collected by filtration, washed twice with water and hexane, then dried under vacuum at 50 oC; 1H NMR (400 MHz, Me2SO-d6): 8.57 (d, 1H, pyridazine, J = 9.3 Hz), 7.85-7.89 (td, 1H, Ph, J = 1.6 Hz and 7.4 Hz), 7.68-7.72 (m, 1H, Ph), 7.58 (d, 1H, pyridazine, J = 9.4 Hz), 7.45-7.53 (m, 2H, Ph); HRMS (M+H) calculated for C11H6ClFN4+H: m/z 249.0337; found: m/z 249.0339.
6-Chloro-3-(3-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine [IVb]: The title compound was prepared by the same method used for IVa with 2-fluorobenzoyl chloride replaced with 3-fluorobenzoyl chloride; 1H NMR (400 MHz, Me2SO-d6): 8.57 (d, 1H, pyridazine, J = 9.8 Hz), 8.17-8.2 (m, 1H, Ph), 8.08-8.12 (m, 1H, Ph), 7.68-7.73 (m, 1H, Ph), 7.6 (d, 1H, pyridazine, J = 9.4 Hz), 7.43-7.48 (m, 1H, Ph). FABMS (M+H) calculated for C11H6ClFN4+H: m/z 249.0337; found: m/z 249.0342.
6-Chloro-3-(4-trifluoromethylphenyl)-[1,2,4]triazolo[4,3-b]pyridazine [IVc]: The title compound was prepared by the same method used for IVa with 2-fluorobenzoyl chloride replaced with 4-trifluoromethylbenzoyl chloride; 1H NMR (400 MHz, Me2SO-d6): 8.6 (d, 1H, pyridazine, J = 9.4 Hz), 8.55 (d, 2H, Ph, J = 7.9 Hz), 8.03 (d, 2H, Ph, J = 8.3 Hz), 7.62 (d, 1H, pyridazine, J = 9.4 Hz). HRMS (M+H) calculated for C12H6ClF3N4.H: m/z 299.0306; found: m/z 299.0310.