Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human

Article pubs.acs.org/jmc

Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human dCTP Pyrophosphatase 1 Inhibitors Sabin Llona-Minguez,*,†,# Andreas Höglund,†,#,∇ Artin Ghassemian,†,○ Matthieu Desroses,† José Manuel Calderón-Montaño,†,◆ Estefanía Burgos Morón,†,◆ Nicholas C. K. Valerie,† Elisee Wiita,† Ingrid Almlöf,† Tobias Koolmeister,† André Mateus,⊥,¶ Cindy Cazares-Körner,† Kumar Sanjiv,† Evert Homan,† Olga Loseva,† Pawel Baranczewski,⊥ Masoud Darabi,§ Amir Mehdizadeh,§ Shabnam Fayezi,∥ Ann-Sofie Jemth,† Ulrika Warpman Berglund,† Kristmundur Sigmundsson,†,‡,+ Thomas Lundbac̈ k,†,‡,● Annika Jenmalm Jensen,†,‡ Per Artursson,⊥ Martin Scobie,† and Thomas Helleday*,† †

Division of Translational Medicine and Chemical Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 65, Sweden ‡ Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 65, Sweden § Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz 5165665931, Iran ∥ Department of Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1983969411, Iran ⊥ Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Department of Pharmacy, Science for Life Laboratory, Uppsala University, Uppsala 752 37, Sweden S Supporting Information *

ABSTRACT: The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool “housekeeping” enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme.

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INTRODUCTION

up-regulation of the multidrug resistance protein 1 (MDR1) and degradation of the nucleoside’s active form.2,6 Only a handful of dCTPase inhibitors have been reported to date (Figure 1): triptolide 1, a terpenoid epoxide with a broad poly pharmacological profile;7 the photo-cross-linking agent 2, with no reported binding affinity toward dCTPase;8 benzimidazole 3 and triazolothiadiazole 4, the first potent dCTPase inhibitors.9,10 Herein, we report on the discovery and

The human dCTPase, also known as DCTPP1 or XTP3transactivated protein A (XTP3TPA), regulates the cellular nucleotide pool through catabolism of canonical and noncanonical dNTPs.1,2 Recent evidence suggests that dCTPase is implicated in cancer development and progression through nuclear accumulation in multiple cancer histological subtypes,3 up-regulation and promotion of cancer cell growth and stemness, association with poor clinical prognosis,4,5 decreased response to anticancer nucleoside analogues through epigenetic © 2017 American Chemical Society

Received: February 3, 2017 Published: May 16, 2017 4279

DOI: 10.1021/acs.jmedchem.7b00182 J. Med. Chem. 2017, 60, 4279−4292

Journal of Medicinal Chemistry

Article

Figure 1. Structures of known dCTPase inhibitors/binders.

condition c), or as a final step with section B + RHS (8, 9, 10, 14, 17, 23, 24; Scheme 2, condition c). Once the tertbutyloxycarbonyl (BOC)-protected LHS region (32, 35−38) and the BOC-protected section B + RHS region (53−56) were assembled, the BOC groups were removed using HCl in 1,4dioxane conditions (Scheme 1, condition b, and Scheme 2, condition b). The resulting amine hydrochlorides 33 and 39− 42 were coupled with the required RHS regions via an aromatic nucleophilic substitution, to afford compounds 5, 6, 7, 13, 15, 16, 19, and 20 (Scheme 1, condition d), and the amine hydrochlorides 59−62 were coupled with the required sulfonyl chlorides to afford compounds 8, 9, 10, 14, 17, 23, and 24 (Scheme 2, condition c). The LHS regions containing an amino-substituted benzene rings (38, 11, and 12) were prepared via an aromatic nucleophilic substitution on the parent fluoro-benzene precursors 37 (Scheme 1, condition e) and 10 (Scheme 2, condition d). The methyl-substituted pyridine derivative 18 was prepared via a Suzuki coupling on the parent chloro-aryl precursor 17 with methyl boronic acid (Scheme 2, condition e). Compounds 21 and 22 were synthesized following Scheme 3. Hydroxy-piperidine 57 was converted to mesylate 63 and treated with 2-methylbenzene-1-thiol to yield thioether 64. A subsequent mild meta-chloroperoxybenzoic acid (m-CPBA)mediated oxidation delivered a mixture of sulfoxide 21 and sulfone 22. Amine 25 was prepared following Scheme 4. Methyl ester 58 was reduced with NaBH4 to alcohol 65, followed by chlorination with SOCl2 to afford 66 and subsequently reacted with benzylamine to give 25. Sulfone 26 was prepared in three synthetic steps as depicted in Scheme 5. 3,6-Dichloropyridazine 67 was treated with phenylethane-1-thiol, and the resulting thioether 68 was fully oxidized with m-CPBA to the sulfone 69. Compound 26 was obtained after aromatic nucleophilic substitution of 39 with 69. The synthesis of the amide replacement derivatives 27−30 is presented in Scheme 6. Chloropyridazine-3-carboxylic acid 43 was converted to the corresponding acid chloride 70 and reacted with the organocuprate derivate from 4-fluorophenethyl bromide to afford 71. The chloro-aryl intermediate 71 was then reacted with amine 39 via an aromatic nucleophilic substitution to obtain 27. A Wittig reaction between ketone 27 and (methoxymethyl)triphenylphosphonium chloride provided

structure−activity relationships (SAR) of a series of piperazin1-ylpyridazine derivatives as novel and potent dCTPase inhibitors.

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CHEMISTRY Compounds in this series (5−30) can be conveniently divided into left-hand side (LHS) and right-hand side (RHS) regions (Figure 2). The LHS region contains sections A (aromatic ring)

Figure 2. Structure−activity relationship (SAR) summary. Color code: green > yellow > red, inhibitory potency.

and B (piperazine derivative) connected by an amide or a sulfonamide bond. The RHS region contains sections C (pyridazine) and D (aromatic ring) connected by an amide or an alternative bond. The bond between sections A and B (amide 32) and C and D (amides 44−49; esters 50 and 51) was established via a propylphoshonic anhydride-mediated coupling between the corresponding carboxylic acids and amines/alcohols (Scheme 1, condition a). The sulfonamide bond between sections A and B was formed from the corresponding sulfonyl chlorides and amines, either as a first synthetic step to assemble the LHS region (35−37, Scheme 1, 4280



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Scheme 1. Synthesis of Compounds 5, 6, 7, 13, 15, 16, 19, and 20a

Reagents and conditions: (a) amine or alcohol (1.1 equiv), propylphoshonic anhydride (2.2 equiv), Et3N (2.5 equiv), CH2Cl2, 0 °C to rt; 18 h; (b) 4 M HCl in 1,4-dioxane, rt, 18 h; (c) sulfonyl chloride (1.1 equiv), Et3N (2.2 equiv), rt, 18 h; (d) amine (1 equiv), Et3N (2 equiv), 1,4-dioxane, 100 °C, 18 h; (e) amine (5 equiv), DMF, 150 °C, 18 h. a

Scheme 2. Synthesis of Compounds 8, 9, 10, 11, 12, 14, 17, 18, 23, and 24a

Reagents and conditions: (a) amine (1 equiv), Et3N (2 equiv), 1,4-dioxane, 100 °C, 18 h; (b) 4 M HCl in 1,4-dioxane, rt, 18 h; (c) sulfonyl chloride (1.1 equiv), Et3N (2.2 equiv), rt, 18 h; (d) amine (5 equiv), DMF, 150 °C, 18 h; (e) MeB(OH)2 (3 equiv), Pd(dppf)Cl2·CH2Cl2 (0.1 equiv), P(Cy)3 (0.4 equiv), K3PO4 (3.5 equiv), toluene/water, 100 °C, 5 h. a

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Scheme 3. Synthesis of Compounds 21 and 22a

a

Reagents and conditions: (a) mesyl chloride (1.2 equiv), pyridine (1 equiv), CH2Cl2, rt, 18 h; (b) 2-methylbenzene-1-thiol (1.1 equiv), K2CO3 (1.1 equiv), DMF, 60 °C, 18 h; (c) m-CPBA (3 equiv), CH2Cl2, 0 °C to rt, 18 h.

Scheme 4. Synthesis of Compound 25a

Reagents and conditions: (a) NaBH4 (14 equiv), EtOH, 0−40 °C, 1 h; (b) SOCl2 (28 equiv), CH2Cl2, 0 °C to rt, 18 h; (c) benzylamine (11 equiv), Et3N (3 equiv), acetonitrile, rt, 18 h.

a

Scheme 5. Synthesis of Compound 26a

Reagents and conditions: (a) phenylethane-1-thiol (1 equiv), Et3N (1 equiv), DMF, 70 °C, 18 h; (b) m-CPBA (3 equiv), CH2Cl2, 0 °C to rt, 18 h; (c) 39 (1 equiv), Et3N (2 equiv), 1,4-dioxane, 100 °C, 18 h.

a

established that shortening the linker between sections C and D to a one carbon unit brought potency down to the submicromolar range (1-C > 2-C > 0-C) (6, 5, 7; Table 1). Replacement of the carboxamide bond between sections A and B in 6 with a sulfonamide in 8 retained potency but decreased binding efficiency (6 BEI = 14.1 vs 8 BEI = 13.0). This could be improved by changing the trifluoromethyl group to a methyl group in 9 (BEI = 15.0). The RHS region of the molecule was able to accommodate benzene surrogates such as 2-thiophene (13), 4-fluorobenzene (14), or acetylene (16), delivering improved or equivalent potency when compared with their respective matched pairs. Altering the LHS benzene ring with a fluorine (10) or an amine substituent (11, 12) at the 2-position was less tolerated but improved aqueous solubility (visual inspection of biochemical assay buffer solutions, data not shown). An additional lipophilic group at the benzene ring’s 3position (15, 16) or an exchange for a pyridine nitrogen lone pair (17, 18) were favorable structural modifications. Minor modifications to the piperazine ring in section B resulted in a significant drop in activity (carbon/nitrogen swap at the 4position, either as a sulfoxide (21) or a sulfone (22), bridged piperazine (23), or ring expansion (24) (Table 2). The carboxamide motif linking sections C and D could be replaced by a carboxylate (19, 20) to give more potent compounds than their respective matched pairs (9, 12) and, to a lesser extent by

enol ether 72, which was subsequently hydrolyzed to aldehyde 73. A Cannizzaro reaction on 73 delivered diol 28, which after tosylation and deprotonation cyclized to oxetane 29, with concomitant formation of the Grob fragmentation product 30, in a ratio of 1:3.

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RESULTS AND DISCUSSION To identify novel dCTPase inhibitors, we screened a proprietary library (5500 molecules tested at 10 μM compound concentration at the Chemical Biology Consortium Sweden) against the full-length human dCTPase protein using a HTSadapted malachite green assay.9,11 There were 110 preliminary hits identified (2% hit rate, μ + 3σ at 36.9% inhibition, Figure S1). Among the initial hits, compound 5 (Figure 1) inhibited 90% of dCTPase activity at 10 μM and was confirmed to be a low micromolar inhibitor (IC50 = 2.8 μM). Despite the low binding efficiency index (BEI = pIC50/MW) of 5,12 we were attracted by the drug-like properties and modular nature of the piperazin-1-ylpyridazine scaffold13,14 and decided to pursue the SAR study presented in this manuscript (Figure 2 for SAR overview and Tables 1−3 for detailed SAR). For this concise SAR study, it was decided to modify sections A and D while maintaining section C constant and introducing discrete alterations to section B (Figure 2). First, it was 4282



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Scheme 6. Synthesis of Compounds 27, 28, 29, and 30a

a Reagents and conditions: (a) (CO)2Cl2 (2 equiv), DMF (cat.), CH2Cl2, 0 °C to rt, 2 h; (b) 4-fluorophenethyl bromide (1.2 equiv), Mg (1.2 equiv), I2 (cat.), CuCN·2LiCl (1.3 equiv), THF, −40 °C, 1 h; (c) 39 (1 equiv), Et3N (2 equiv), 1,4-dioxane, 100 °C, 18 h; (d) LDA (1.7 equiv), (methoxymethyl)triphenylphosphonium chloride (1.7 equiv), THF, −78 °C to rt, 5 h; (e) H2SO4/THF/water, 45 °C, 2 days; (f) aq CH2O/aq NaOH/THF, rt, 18 h; (g) NaH (1.1 equiv), p-tosyl chloride (2 equiv), THF, −78 °C to rt, 2 h, then NaH (20 equiv), −78 to 50 °C, THF, 1 h.

Table 1. Structure−Activity Relationships: LHS/RHS Variations

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a

R1

W

X

R2

IC50 (μM)a

BEI

2-(trifluoromethyl)phenyl 2-(trifluoromethyl)phenyl 2-(trifluoromethyl)phenyl 2-(trifluoromethyl)phenyl 2-methylphenyl 2-fluorophenyl 2-(dimethylamino)phenyl 2-(4-hydroxypiperidin-1-yl)phenyl 2-methylphenyl 2-methylphenyl 3-chloro-2-methylphenyl 3-chloro-2-methylphenyl 2-chloropyridin-3-yl 2-methylpyridin-3-yl 2-methylphenyl 2-(4-hydroxypiperidin-1-yl)phenyl

CO CO CO SO2 SO2 SO2 SO2 SO2 SO2 SO2 SO2 SO2 SO2 SO2 SO2 SO2

NH NH NH NH NH NH NH NH NH NH NH NH NH NH O O

2-phenylethan-1-yl benzyl phenyl benzyl benzyl benzyl benzyl benzyl thiophen-2-ylmethanyl 4-fluoro-benzyl thiophen-2-ylmethanyl propargyl 4-fluoro-benzyl 4-fluoro-benzyl benzyl propargyl

2.8 0.240 >10 0.250 0.170 0.350 0.502 0.260 0.051 0.065 0.019 0.034 0.025 0.036 0.038 0.041

11.5 14.1 13.0 15.0 14.2 13.1 12.3 15.9 15.3 15.7 17.2 15.5 15.8 16.4 15.2

The 11-point IC50 curves were calculated based on the average of two replicates per data point with standard deviation.

Compound 18 consistently docked with the RHS-benzene ring wedged between Trp47(C) and Trp73(B), thus overlaying with the cytosine base of dCTP, while the LHS-pyridine substituent was oriented toward the solvent, consistent with the SAR observations. Representative poses are shown for 18 (Figure 3B,C. The top-ranked pose suggested H-bond formation between the carboxamide −NH and Tyr102(C), the carboxamide carbonyl and Lys121(D), one of the pyridazine ring nitrogens and Trp73(B), and one of the sulfonamide oxygens and Arg87(B). The positioning and orientation of the RHS 4-fluorophenyl suggested the possibility of face-to-face π−π interactions with Tyr102(C), while additional edge-to-face π−π interactions were possible between the central pyridazine ring and Trp73(B) and between the LHS pyridine ring and

a ketone (27, Table 3). Removal or replacement of the carbonyl group completely abolished activity, as seen with the amine (25), sulfone (26), bis-hydroxymethyl (28), oxetane (29), or alkene (30) derivatives, highlighting the importance of this structural feature. To rationalize the observed SAR, we built a homology model of the human dCTPase enzyme based on available crystal structures of the mouse enzyme (see Molecular Docking, Experimental Section). The resulting model contained four active sites (dimer of dimers), each occupied by dCTP, and residues from three different monomers contributing to each active site. Because the human and mouse dCTPase active site share identical residue sequence, this homology model was deemed a good starting point for docking studies (Figure 3A). 4283



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Table 2. Structure−Activity Relationships: Piperazine Variationsa

a *The 11-point IC50 curves were calculated based on the average of two replicates per data point with standard deviation.

Table 3. Structure−Activity Relationships: RHS Amide Replacementsa

a *The 11-point IC50 curves were calculated based on the average of two replicates per data point with standard deviation

Figure 3. (A) Overall view of the human dCTPase homology model. (B,C) Representative poses of 18 docked into the dCTPase active site, shown from different perspectives. (D) Two-dimensional residue interaction diagram of 18 with the dCTPase active site.

Trp73(B). Furthermore, the LHS pyridine might be involved in a π−cation interactions with Arg87(B) (interaction summary in Figure 3D). Having discovered a number of potent inhibitors (IC50 < 50 nM) with reasonable BEI (≥15), we set to validate a diverse set of compounds as suitable probes for cellular experiments. First, we confirmed target engagement using thermal and proteolytic stabilization assays. Potent inhibitors 13, 18, and 20 increased the melting temperature of the purified dCTPase protein in a differential scanning fluorimetry (DSF) assay, whereas the inactive inhibitors 29 and 30 did not (Table S1).15 Next, we demonstrated that the ligand-mediated thermal stabilization of dCTPase translated to a whole cell thermal shift assay (CETSA), as seen with compound 9 (Figure 4A).16 On the basis of the principle of stabilization to protease digestion upon ligand binding, we also observed that 20 protected dCTPase from Pronase digestion using a whole cell drug affinity responsive target stability (DARTS) assay (Figure 4B).17 Next, we assessed the pharmacological profile of the inhibitors within the dNTPase/nucleoside diphosphate linked to X (NUDIX) protein family. Compounds 9, 18, and 20 displayed >1000-fold selectivity against a panel of relevant enzymes (Supporting Information, Table S2). On the basis of the encouraging intracellular bioavailability (Fic) of all the inhibitors tested (>50% of the compound

concentration was available in the intracellular environment, Supporting Information, Table S3),18 and the results observed in the whole cell CETSA and DARTS assays, it was assumed that this series of piperazin-1-ylpyridazines generally possessed good cellular exposure. Key compounds were then assessed in a cellular efficacy model.9 When leukemia-derived HL60 cells were exposed to compounds 9, 13, or 15 in a combination matrix with 5-azacytidine (5-AzaC) (Supporting Information, Figure S2), a nucleoside analogue of clinical relevance, we observed a synergistic decrease in cell viability (combination index (CI) < 1) (Figure 5A), according to the Chou−Talalay method for synergy quantification.19 Moreover, the combination of a low-cytotoxic dose of 5-AzaC with a nontoxic dose of compounds 14, 18, or 27 caused synergistic lethality in the leukemic cells, as determined by fluorescence-activated cell sorting (FACS) analysis (Figure 5B). Furthermore, compounds 9, 18, and 20 did not show a cytotoxic effect on BJ hTERT normal cells (Supporting Information, Table S4). It is worth noting that compounds 5, 6, and 7 are potent inhibitors of the stearoyl-CoA desaturase-1 (SCD1) published by Zhang et al. at Xenon Pharmaceuticals.13 SCD1 is a key 4284



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Figure 4. Target engagement validation: cellular assays. (A) Western blot images from the CETSA assay. MCF7 cells were incubated for 2 h with 9 (0, 5, 20 μM) and subsequently heated at different temperatures. (B) Western blot images from the DARTS assay. HL60 cells were incubated for 4 h with DMSO (0.1%) or 20 (10 μM) followed by pronase digestion. ND = nondigested sample.

Figure 5. (A) Combination index (CI) plots of compounds 9, 13, and 15 in combination with 5-AzaC. HL60 leukemia-derived cells were incubated for 72 h with different concentrations of the inhibitors and 5-AzaC. Cytotoxic effect was determined by Resazurin cell viability assay. (B) Inhibitors 14, 18, and 27 show synergystic lethality with 5-AzaC. HL60 leukemia-derived cells were incubated for 72 h with the inhibitor (10 μM) and 5-AzaC (2.5 μM). Cell death was determined by flow cytometry analysis.

(74 IC50 = 0.67 μM vs 6 IC50 = 0.75 μM). In comparison, compound 9 was >20-fold less active (IC50 = 19.74 μM), indicating that introducing a sulfonamide motif in this chemotype can reduce SCD1 inhibitory activity.

enzyme in the synthesis of monounsaturated fatty acids, and SCD1 inhibitors have received a great deal of attention as a therapeutic option against diabetes, cancer, and some skin disorders.20,21 Selected dCTPase inhibitors were profiled for their effect on SCD1 activity by examining the ratio of cellular SCD1 products over substrates (16:1/16:0)/(18:1/18:0) (16:0 = palmitic acid, 16:1 = palmitoleic acid, 18:0 = stearic acid, 18:1 = oleic acid), referred as the cumulative SCD1 desaturation index. Unlike kinetic inhibition data obtained using labeled substrates, estimation of SCD1 activity based on the cell’s endogenous desaturation index offers a realistic assessment of inhibitor potency in the cellular context. We confirmed that 6 was equipotent to the Abbott compound 74 (3-[4-(2-chloro-5fluorophenoxy)-1-piperidinyl]-6-(5-methyl-1,3,4-oxadiazol-2yl)-pyridazine, CAY10566, Supporting Information, Figure S2)22 in decreasing the SCD1 activity index in HepG2 cells

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CONCLUSION In summary, we have reported on a new class of potent inhibitors of dCTPase, a novel biological target with clear links to cancer progression. Here we have profiled a diverse set of compounds in biochemical and cellular assays in order to validate this class of piperazin-1-ylpyridazines as dCTPase inhibitors. Several compounds presented in this study inhibit dCTPase enzymatic activity in the low nanomolar range, engage with the biological target in the intracellular environment, and do not interfere with related enzymes. Moreover, this class of inhibitors enhances the cytotoxic effect of the cytidine 4285



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General Procedure D for the Synthesis of 32 and 44−51. A propylphoshonic anhydride (T3P) solution (≥50 wt % in EtOAc, 23.1 mmol, 2.2 equiv) was added to a solution of carboxylic acid (2 g, 10.52 mmol, 1equiv), amine (11.57 mmol, 1.1 equiv), and Et3N (26.3 mmol, 2.5 equiv) in CH2Cl2 (10 mL) at 0 °C. The reaction was allowed to warm to room temperature overnight. The mixture was subsequently diluted with CH2Cl2, and the organic layer was sequentially washed with saturated aqueous NaHCO3 and aqueous 2 M HCl and finally dried over Na2SO4. After evaporation, the product was obtained and used in the next step without any further purification. General Procedure E for the Synthesis of 33, 39−42, and 59−62. A 4 M solution of HCl in 1,4-dioxane (10 mL) was added to a solution of BOC-protected amine (3.50 g, 9.86 mmol) in 1,4-dioxane (10 mL) and stirred at room temperature overnight. The solvent was removed by filtration and the residue triturated with diethyl ether to afford the title product. Used in the next step without any further purification. N-Phenethyl-6-(4-(2-(trifluoromethyl)benzoyl)-piperazin-1-yl)pyridazine-3-carboxamide (5). General procedure A. Prepared from 44 (0.1 mmol) and 33. The crude product was purified by flash chromatography (CH2Cl2/MeOH 95/5) to afford the title compound. Yield: 7 mg, 15%. 1H NMR (400 MHz, MeOD4) δ = 7.94 (d, J = 9.5 Hz, 1 H), 7.83 (d, J = 8.0 Hz, 1 H), 7.79−7.72 (m, 1 H), 7.72−7.64 (m, 1 H), 7.52 (d, J = 7.5 Hz, 1 H), 7.32 (d, J = 9.5 Hz, 1 H), 7.30− 7.23 (m, 4 H), 7.22−7.15 (m, 1 H), 4.56 (s, 1 H), 3.99−3.87 (m, 4 H), 3.76−3.71 (m, 2 H), 3.66 (d, J = 14.8 Hz, 2 H), 3.46−3.33 (m, 2 H), 2.92 (t, J = 7.4 Hz, 2 H). LC-MS (ESI+) m/z = 484 [M + H]+. N-Benzyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (6). General procedure A. Prepared from 45 (0.08 mmol) and 33. The crude product was purified by flash chromatography (5% MeOH in DCM) to afford the title compound. Yield: 6 mg, 20%. 1H NMR (400 MHz, CDCl3) δ = 8.20 (t, J = 5.8 Hz, 1 H), 8.08 (d, J = 9.3 Hz, 1 H), 7.77−7.71 (m, 1 H), 7.64 (s, 1 H), 7.60−7.53 (m, 1 H), 7.39−7.31 (m, 6 H), 7.31−7.26 (m, 1 H), 7.00 (d, J = 9.5 Hz, 1 H), 4.66 (d, J = 6.0 Hz, 2 H), 4.09−4.00 (m, 1 H), 3.93−3.83 (m, 2 H), 3.83−3.69 (m, 3 H), 3.37−3.31 (m, 2 H). LCMS (ESI+) m/z = 470 [M + H]+. N-Phenyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (7). General procedure A. Prepared from 46 (0.08 mmol) and 33. The crude product was purified by flash chromatography (2−5% MeOH in DCM) to afford the title compound. Yield: 9 mg, 26%. 1H NMR (400 MHz, CDCl3) δ = 9.82 (s, 1 H), 8.16 (d, J = 9.4 Hz, 1 H), 7.80−7.69 (m, 3 H), 7.65 (d, J = 14.4 Hz, 1 H), 7.58 (d, J = 15.2 Hz, 1 H), 7.36 (s, 1 H), 7.40 (s, 2 H), 7.19−7.10 (m, 1 H), 7.05 (d, J = 9.5 Hz, 1 H), 4.15−4.03 (m, 1 H), 3.98−3.87 (m, 2 H), 3.87−3.71 (m, 3 H), 3.42−3.32 (m, 2 H). LC-MS (ESI+) m/z = 456 [M + H]+. N-Benzyl-6-(4-((2-(trifluoromethyl)phenyl)sulfonyl)-piperazin-1yl)pyridazine-3-carboxamide (8). General procedure B. Prepared from 59 (0.12 mmol) and 2-(trifluoromethyl)benzene-1-sulfonyl chloride. Product purified by flash chromatography (5−80% EtOAc in pentane). Yield: 38 mg, 75%. 1H NMR (400 MHz, CDCl3) δ = 8.21−8.15 (m, 2 H), 8.06 (d, J = 9.5 Hz, 1 H), 7.94−7.90 (m, 1 H), 7.77−7.70 (m, 2 H), 7.37−7.27 (m, 5 H), 6.98 (d, J = 9.5 Hz, 1 H), 4.66 (d, J = 6.0 Hz, 2 H), 3.90−3.81 (m, 4 H), 3.44−3.37 (m, 4 H). LC-MS (ESI+) m/z = 506 [M + H]+. N-Benzyl-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (9). General procedure B. Prepared from 59 (0.12 mmol) and o-tolylsulfonyl chloride. Product purified by flash chromatography (5−80% EtOAc in pentane). Yield: 30 mg, 66%. 1H NMR (400 MHz, CDCl3) δ = 8.18 (t, J = 5.8 Hz, 1 H), 8.06 (d, J = 9.5 Hz, 1 H), 7.92 (dd, J = 8.4, 1.4 Hz, 1 H), 7.52−7.45 (m, 1 H), 7.37−7.28 (m, 6 H), 6.97 (d, J = 9.5 Hz, 1 H), 4.66 (d, J = 6.0 Hz, 2 H), 3.88−3.82 (m, 4 H), 3.34−3.32 (m, 4 H), 2.66 (s, 3 H). LC-MS (ESI+) m/z = 452 [M + H]+. N-Benzyl-6-(4-((2-fluorophenyl)sulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (10). General procedure B. Prepared from 59 (0.4 mmol) and 2-fluorobenzene-1-sulfonyl chloride. Product purified by flash chromatography (20−100% EtOAc in hexane). Yield: 115 mg, 63%. 1H NMR (400 MHz, CDCl3) δ = 8.20 (t, J = 5.9 Hz, 1H), 8.05 (d, J = 9.5 Hz, 1H), 7.89−7.82 (m, 1H), 7.63−7.55 (m,

analogue 5-AzaC in leukemia-derived HL60 cells. Concomitant SCD1 antagonism is to be expected in some piperazin-1ylpyridazines given their structural similarity to known SCD1 inhibitors, although SCD1 affinity can be reduced through subtle structural changes, as seen in 9. The utility of dual nucleotide/lipid metabolism modulators is unknown, and additional work will be required to elucidate the relevance of this polypharmacological profile and will dictate the optimal target potency balance. Currently, there is a need for chemical probes to explore dNTPases/NUDIX hydrolase biology, and the compounds presented in this article represent an exciting starting point toward the development of novel and wellvalidated probes of the dCTPase enzyme.

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EXPERIMENTAL SECTION

Chemistry: General Information for Synthetic Procedures. All commercial reagents and solvents were used without further purification. Compounds 31, 34, 43, 52, and 67 are commercial reagents. Analytical thin-layer chromatography was performed on silica gel 60 F-254 plates (Merck) and visualized under a UV lamp. Flash column chromatography was performed in a Biotage SP4MPLC system using Merck silica gel 60 Å (40−63 mm mesh). 1H NMR NMR spectra were recorded on a Bruker DRX-400. Chemical shifts are expressed in parts per million (ppm) and referenced to the residual solvent peak. Analytical HPLC-MS was performed on an Agilent MSD mass spectrometer connected to an Agilent 1100 system with: method acidic pH, Column ACE 3 C8 (50 mm × 3.0 mm), H2O (+0.1% TFA), and MeCN were used as mobile phases at a flow rate of 1 mL/ min, with a gradient time of 3.0 min; or Method basic pH, Column XTerra MSC18 (50 mm × 3.0 mm), H2O (containing 10 mM NH4HCO3; pH = 10), and MeCN were used as mobile phases at a flow rate of 1 mL/min, with a gradient time of 3.0 min. Preparative HPLC was performed on a Gilson HPLC system. Basic pH: column Xbridge Prep C18, 5 μM CBD (30 mm × 75 mm), H2O (containing 50 mM NH4HCO3; pH = 10), and MeCN were used as mobile phases at a flow rate of 45 mL/min, with a gradient time of 9 min. Acidic pH: column ACE 5 C8 (150 mm × 30 mm), H2O (containing 0.1% TFA), and MeCN were used as mobile phases at a flow rate of 45 mL/min, with a gradient time of 9 min. For HPLC-MS, detection was made by UV using the 180−305 nM range and MS (ESI+). For preparative HPLC, detection was made by UV at 254 or 214 nM. All final compounds were assessed to be >95% pure by HPLC-MS analysis. General Synthetic Procedures. General Procedure A for the Synthesis of 5, 6, 7, 13, 15, 16, 19, 20, and 53−58. Aryl halide (30 mg, 0.1 mmol, 1 equiv), amine (0.1 mmol, 1 equiv), and Et3N (0.2 mmol, 2 equiv) were heated in 1,4-dioxane (1 mL) at 100 °C for 18 h. The resulting solution was concentrated in vacuo, dissolved in CH2Cl2, and washed with brine. The organic layer was concentrated in vacuo, and the desired compound was purified by chromatography (final compounds) or used in the next step without any further purification (intermediate compounds). General Procedure B for the Synthesis of 8, 9, 10, 14, 17, 23, 24, and 35−37. A solution amine (3 mmol, 1 equiv) and Et3N (3.6 mmol, 1.2 equiv) in CH2Cl2 (10 mL) was added to a solution of sulfonyl chloride (570 mg, 3 mmol, 1 equiv) in CH2Cl2 (5 mL) at 0 °C, and the reaction was stirred at room temperature overnight. The resulting mixture was filtered, washed with water, dried with MgSO4, and then passed through a short silica pad. After evaporation of the solvent in vacuo, the desired compound was purified by chromatography (final compounds) or used in the next step without any further purification (intermediate compounds). General Procedure C for the Synthesis of 11, 12, and 38. Aryl halide (15 mg, 0.033 mmol, 1 equiv) and amine (0.165 mmol, 5 equiv) were dissolved in DMF (0.5 mL) and heated at 150 °C overnight in a sealed tube. The desired compound was purified by chromatography (final compounds) or used in the next step without any further purification (intermediate compounds). 4286



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in hexane). Yield: 45 mg, 76%. 1H NMR (400 MHz, CDCl3) δ = 8.59 (dd, J = 4.8, 1.8 Hz, 1 H), 8.43 (dd, J = 7.8, 2.0 Hz, 1 H), 8.19 (br. s, 1 H), 8.08 (d, J = 9.3 Hz, 1 H), 7.45 (dd, J = 7.8, 4.8 Hz, 1 H), 7.29− 7.35 (m, 2 H), 6.95−7.07 (m, 3 H), 4.63 (d, J = 6.1 Hz, 2 H), 3.83− 3.94 (m, 4 H), 3.49−3.57 (m, 4 H). LC-MS (ESI+) m/z = 491 [M + H]+. N-(4-Fluorobenzyl)-6-(4-((2-methylpyridin-3-yl)sulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (18). 17 (36 mg, 0.07 mmol, 1 equiv), methyl boronic acid (0.22 mmol, 3 equiv), Pd(dppf)Cl2·CH2Cl2 complex (0.1 equiv), tricyclohexylphosphine (0.03 mmol, 0.4 equiv), and tripotassium phosphate (0.26 mmol, 3.5 equiv) were suspended in toluene/water mixture (95:5, 5 mL), degassed with N2, and heated at 100 °C for 2 h. Additional amounts of all reagents were added and stirred at 100 °C for 5 h. The reaction mixture was purified by flash chromatography (0−3% MeOH in CH2Cl2). The product was further purified by preparative HPLC acidic pH to yield the title product. Yield: 19 mg, 47%. 1H NMR (400 MHz, DMSO-d6) δ = 9.40 (t, J = 6.4 Hz, 1 H), 8.71 (dd, J = 1.8, 4.8 Hz, 1 H), 8.19 (dd, J = 8.1, 1.8 Hz, 1 H), 7.86 (d, J = 9.6 Hz, 1 H), 7.52−7.46 (m, 1 H), 7.38−7.31 (m, 3 H), 7.16−7.08 (m, 2 H), 4.45 (d, J = 6.4 Hz, 2 H), 3.83 (app dd, J = 5.9, 4.4 Hz, 5 H), 3.26 (app dd, J = 5.9, 4.4 Hz, 4 H), 2.79 (s, 3 H). LC-MS (ESI+) m/z = 471 [M + H]+. Benzyl 6-(4-(o-Tolylsulfonyl)piperazin-1-yl)pyridazine-3-carboxylate (19). General procedure A. Prepared from 50 (0.08 mmol) and 39. Reaction was run in acetonitrile (0.5 mL) and was heated in the microwave oven at 190 °C for 15 min. The resulting solution was concentrated in vacuo and purified by flash chromatography (5−80% EtOAc in hexane) to afford the title compound. Yield: 17 mg, 46%. 1H NMR (400 MHz, CDCl3) δ = 7.93−7.88 (m, 2H), 7.51−7.44 (m, 3H), 7.39−7.29 (m, 5H), 6.85 (d, J = 9.8 Hz, 1H), 5.43 (s, 2H), 3.93− 3.84 (m, 4H), 3.36−3.26 (m, 4H), 2.65 (s, 3H). LC-MS (ESI+) m/z = 453 [M + H]+. Prop-2-yn-1-yl 6-(4-((2-(4-Hydroxypiperidin-1-yl)phenyl)sulfonyl)piperazin-1-yl)pyridazine-3-carboxylate (20). General procedure A. Prepared from 51 (0.08 mmol) and 42. Reaction was run in acetonitrile (0.5 mL) and was heated in the microwave oven at 190 °C for 15 min. The resulting solution was concentrated in vacuo and purified by flash chromatography (50−100% EtOAc in hexane) to afford the title compound. Yield: 11 mg, 30%. 1H NMR (400 MHz, CDCl3) δ = 7.95 (dd, J = 8.0, 1.8 Hz, 1 H), 7.89 (d, J = 9.5 Hz, 1 H), 7.55−7.49 (m, 1 H), 7.34−7.30 (m, 1 H), 7.25−7.19 (m, 1 H), 6.83 (d, J = 9.5 Hz, 1 H), 4.97 (d, J = 2.5 Hz, 2 H), 3.91−3.83 (m, 5 H), 3.31−3.25 (m, 4 H), 3.25−3.18 (m, 2 H), 2.85−2.76 (m, 2 H), 2.50 (t, J = 2.5 Hz, 1 H), 2.04−1.97 (m, 2 H), 1.82−1.71 (m, 2 H). LC-MS (ESI+) m/z = 486 [M + H]+. N-Benzyl-6-(4-(o-tolylsulfinyl)piperidin-1-yl)pyridazine-3-carboxamide (21) and Benzyl-6-(4-(o-tolylsulfonyl)piperidin-1-yl)pyridazine-3-carboxamide (22). 1-(6-(Benzylcarbamoyl)pyridazin3-yl)piperidin-4-yl Methanesulfonate (63). A solution of mesyl chloride (0.576 mmol, 1.2 equiv) in CH2Cl2 (0.5 mL) was added dropwise to a stirred solution of 57 (150 mg, 0.480 mmol, 1 equiv) and pyridine (0.480 mmol, 1 equiv) in CH2Cl2 (1.5 mL) at 0 °C. The mixture was left to warm to rt overnight, then diluted with CH2Cl2. The organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The crude was used in the next step without any further purification. LC-MS (ESI+) m/z = 391 [M + H]+. N-Benzyl-6-(4-(o-tolylthio)piperidin-1-yl)pyridazine-3-carboxamide (64). 63 (81 mg, 0.207 mmol, 1 equiv), 2-methylbenzene-1-thiol (0.228 mmol, 1.1 equiv), and potassium carbonate (0.228 mmol, 1.1 equiv) were suspended in DMF (1 mL) and stirred overnight at 60 °C. The reaction was purified by flash chromatography (20−50% EtOAc in hexane) to yield the title compound. Yield: 80 mg, 92%. 1H NMR (400 MHz, acetone-d6) δ = 8.77−8.65 (m, 1 H), 7.96−7.90 (m, 1 H), 7.80−7.74 (m, 1 H), 7.48−7.38 (m, 4 H), 7.36−7.21 (m, 5 H), 4.77− 4.70 (m, 1 H), 4.69−4.63 (m, 3 H), 3.22−3.03 (m, 3 H), 2.45−2.39 (m, 3 H), 1.95−1.73 (m, 2 H), 1.60−1.51 (m, 1 H). LC-MS (ESI+) m/z = 419 [M + H]+. N-Benzyl-6-(4-(o-tolylsulfinyl)piperidin-1-yl)pyridazine-3-carboxamide (21). To a cooled solution (ice-bath) of 64 (80 mg, 0.191 mmol, 1 equiv) in CH2Cl2 (3 mL) was added m-CPBA (0.382 mmol,

1H), 7.40−7.17 (m, 7H), 7.00 (d, J = 9.5 Hz, 1H), 4.65 (d, J = 5.9 Hz, 2H), 3.92−3.84 (m, 4H), 3.40−3.31 (m, 4H). LC-MS (ESI+) m/z = 456 [M + H]+. N-Benzyl-6-(4-((2-(dimethylamino)phenyl)sulfonyl)piperazin-1yl)pyridazine-3-carboxamide (11). General procedure C. Prepared from 10 (0.033 mmol) and dimethylamine (5.6 M solution in EtOH). The product was purified by preparative HPLC basic pH to afford the desired product. Yield: 4 mg, 24%. 1H NMR (400 MHz, CDCl3) δ = 8.18 (t, J = 5.8 Hz, 1H), 8.04 (d, J = 9.5 Hz, 1H), 7.98 (dd, J = 7.9, 1.6 Hz, 1H), 7.54−7.48 (m, 1H), 7.35−7.27 (m, 6H), 7.24−7.18 (m, 1H), 6.95 (d, J = 9.5 Hz, 1H), 4.65 (d, J = 5.8 Hz, 2H), 3.85−3.78 (m, 4H), 3.36−3.30 (m, 4H), 2.73 (s, 6H). LC-MS (ESI+) m/z = 481 [M + H]+. N-Benzyl-6-(4-((2-(4-hydroxypiperidin-1-yl)phenyl)sulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (12). General procedure C. Prepared from 10 (0.04 mmol) and 4-hydroxypiperidine. The product was purified by preparative HPLC basic pH to afford the desired product. Yield: 11 mg, 47%. 1H NMR (400 MHz, CDCl3) δ = 8.17 (t, J = 5.8 Hz, 1H), 8.03 (d, J = 9.5 Hz, 1H), 7.95 (dd, J = 8.0, 1.8 Hz, 1H), 7.55−7.48 (m, 1H), 7.35−7.26 (m, 6H), 7.25−7.20 (m, 1H), 6.93 (d, J = 9.5 Hz, 1H), 4.64 (d, J = 5.8 Hz, 2H), 3.93−3.84 (m, 1H), 3.83−3.76 (m, 4H), 3.32−3.25 (m, 4H), 3.25−3.16 (m, 2H), 2.86− 2.75 (m, 2H), 2.08−1.97 (m, 2H), 1.82−1.70 (m, 2H). LC-MS (ESI+) m/z = 537 [M + H]+. N-(Thiophen-2-ylmethyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (13). General procedure A. Prepared from 48 (0.08 mmol) and 39. The crude product was purified by flash chromatography (20−80% EtOAc in pentane) to afford the title compound. Yield: 18 mg, 50%. 1H NMR (400 MHz, CDCl3) δ = 8.23−8.16 (m, 1 H), 8.04 (d, J = 9.5 Hz, 1 H), 7.92 (d, J = 7.5 Hz, 1 H), 7.52−7.45 (m, 1 H), 7.38−7.31 (m, 2 H), 7.22 (d, J = 5.0 Hz, 1 H), 7.03 (d, J = 3.3 Hz, 1 H), 6.99−6.92 (m, 2 H), 4.82 (d, J = 5.8 Hz, 2 H), 3.89−3.80 (m, 4 H), 3.36−3.29 (m, 4 H), 2.66 (s, 3 H). LC-MS (ESI+) m/z = 458 [M + H]+. N-(4-Fluorobenzyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (14). General procedure B. Prepared from 62 (0.12 mmol) and o-tolylsulfonyl chloride. Purified by preparative HPLC acidic pH. Yield: 15 mg, 26%. 1H NMR (400 MHz, CDCl3) δ = 8.18 (t, J = 5.8 Hz, 1 H), 8.05 (d, J = 9.6 Hz, 1 H), 7.92 (d, J = 8.1 Hz, 1 H), 7.43−7.55 (m, 1 H), 7.28−7.39 (m, 4 H), 7.02−6.94 (m, 3 H), 4.62 (d, J = 5.8 Hz, 2 H), 3.85 (m, 4 H), 3.33 (m, 4 H), 2.66 (s, 3 H). LC-MS (ESI+) m/z = 470 [M + H]+. 6-(4-((3-Chloro-2-methylphenyl)sulfonyl)piperazin-1-yl)-N-(thiophen-2-ylmethyl)pyridazine-3-carboxamide (15). General procedure A. Prepared from 48 (0.08 mmol) and 40. Reaction was refluxed in nBuOH overnight. The resulting solution was concentrated in vacuo and purified by flash chromatography (20−75% EtOAc in hexane). Fractions containing the product were pooled, concentrated in vacuo, and triturated with Et2O to afford the title compound. Yield: 34 mg, 87%. 1H NMR (400 MHz, DMSO-d6) δ = 9.41 (t, J = 6.3 Hz, 1 H), 7.92−7.84 (m, 2 H), 7.82−7.75 (m, 1 H), 7.46 (t, J = 8.0 Hz, 1 H), 7.41−7.33 (m, 2 H), 7.02−6.98 (m, 1 H), 6.94 (dd, J = 3.4, 5.1 Hz, 1 H), 4.63 (d, J = 6.3 Hz, 2 H), 3.86−3.79 (m, 4 H), 3.29−3.19 (m, 4 H), 2.63 (s, 3 H). LC-MS (ESI+) m/z = 492 [M + H]+. 6-(4-((3-Chloro-2-methylphenyl)sulfonyl)piperazin-1-yl)-N-(prop2-yn-1-yl)pyridazine-3-carboxamide (16). General procedure A. Prepared from 49 (0.08 mmol) and 40. Reaction was refluxed in nBuOH overnight. The resulting solution was concentrated in vacuo and purified by flash chromatography (20−75% EtOAc in hexane). Fractions containing the product were pooled, concentrated in vacuo, and triturated with Et2O to afford the title compound. Yield: 20 mg, 57%. 1H NMR (400 MHz, CDCl3) δ = 8.03 (br s, 1 H), 8.02 (d, J = 9.3 Hz, 1 H), 7.90 (dd, J = 1.3, 8.0 Hz, 1 H), 7.62 (dd, J = 1.3, 8.0 Hz, 1 H), 7.32−7.26 (m, 1 H), 6.97 (d, J = 9.5 Hz, 1 H), 4.26 (dd, J = 2.5, 5.5 Hz, 2 H), 3.89−3.83 (m, 4 H), 3.38−3.32 (m, 4 H), 2.70 (s, 3 H), 2.26 (t, J = 2.5 Hz, 1 H). LC-MS (ESI+) m/z = 434 [M + H]+. 6-(4-((2-Chloropyridin-3-yl)sulfonyl)piperazin-1-yl)-N-(4fluorobenzyl)pyridazine-3-carboxamide (17). General procedure B. Prepared from 62 (0.12 mmol) and 2-chloropyridine-3-sulfonyl chloride. Product purified by flash chromatography (40−95% EtOAc 4287



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4.39 (s, 2 H), 4.28−4.34 (m, 2 H), 3.70−3.77 (m, 4 H), 3.26−3.33 (m, 4 H), 2.64 (s, 3 H). LC-MS (ESI+) m/z = 438 [M + H]+. 3-(Phenethylsulfonyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine (26). 3-Chloro-6-[(2-phenylethyl)sulfanyl]pyridazine (68). 67 (200 mg, 1.3 mmol, 1 equiv), Et3N (1.3 mmol, 1 equiv), and 2-phenylethane-1-thiol (1.3 mmol, 1 equiv) were dissolved in DMF (5 mL) and heated at 70C overnight. Silica was added to the reaction mixture and concentrated in vacuo for flash chromatography purification (0−8% EtOAc in hexane) to obtain the title product. Yield. 337 mg, 82%. LC-MS (ESI+) m/z = 251 [M + H]+. 3-Chloro-6-(phenethylsulfonyl)pyridazine (69). m-CPBA (3.30 mmol, 3 equiv) was added portionwise to an ice-bath cooled solution of 68 (276 mg, 1.10 mmol, 1 equiv) in CH2Cl2 (15 mL), and the reaction was allowed to warm up to rt overnight. The reaction mixture was diluted with CH2Cl2 and washed with satd aq NaHCO3. The organic layer was dry-loaded for flash chromatography (0−30% EtOAc in hexane) to afford the title product. Yield: 230 mg, 74%. LC-MS (ESI+) m/z = 283 [M + H]+. 3-(Phenethylsulfonyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine (26). 69 (20 mg, 0.070 mmol, 1 equiv), 39 (0.085 mmol, 1.2 equiv), and Et3N (0.15 mmol, 2 equiv) were heated in DMF at 130 °C for 3 h. The reaction mixture was dry-loaded on silica for flash chromatography (25−70% EtOAc in hexane). The resulting solid was triturated with Et2O to afford the title product. Yield: 22 mg, 64%. 1H NMR (400 MHz, CDCl3) δ = 7.95−7.90 (m, 1 H), 7.79 (d, J = 9.7 Hz, 1 H), 7.53−7.47 (m, 1 H), 7.39−7.32 (m, 2 H), 7.26−7.20 (m, 2 H), 7.20−7.12 (m, 3 H), 6.90 (d, J = 9.7 Hz, 1 H), 3.94−3.87 (m, 4 H), 3.81−3.74 (m, 2 H), 3.36−3.30 (m, 4 H), 3.14−3.07 (m, 2 H), 2.66 (s, 3 H). LC-MS (ESI+) m/z = 487 [M + H]+. Procedures for the Synthesis of Compounds 27−30. 6Chloropyridazine-3-carbonyl Chloride (70). 43 (200 mg, 1 mmol, 1 equiv) was suspended in CH2Cl2 (10 mL) and cooled to 0 °C. Oxalyl chloride (2 mmol, 2 equiv) was added, followed by addition of one drop of DMF. The reaction was allowed to warm to room temperature and stirred for 2 days. The clear solution was concentrated in vacuo and azeotropped with toluene. Used in the next step without any further purification. LC-MS (ESI+) m/z = 173 [M + H]+ (methyl ester). 1-(6-Chloropyridazin-3-yl)-3-(4-fluorophenyl)propan-1-one (71). A solution of 4-fluorophenethyl bromide (1.51 mmol, 1.2 equiv) in anhydrous THF (2 mL) was added dropwise to the degassed suspension of magnesium turnings (1.51 mmol. 1.2 equiv) and a single crystal of iodine in anhydrous THF (3 mL) at room temperature. Once all the alkyl bromide was added, the suspension was refluxed at 100 °C under N2 for 1 h. The solution was allowed to cool to RT, then cooled to −40 °C on a dry ice/acetonitrile bath. An anhydrous 1 M solution of CuCN2·LiCl (1.64 mmol, 1.3 equiv) was then added dropwise to the cooled Grignard solution for transmetalation. The mixture was allowed to stir for 30 min. 70 (223 mg, 1.26 mmol, 1 equiv) was dissolved in anhydrous THF (5 mL) and added dropwise to the organometallic suspension at −40 °C over 5 min and allowed to stir for 1 h. The reaction mixture was then quenched by addition of satd NH4Cl. The mixture was then diluted with EtOAc, and a thick precipitate was formed. The mixture was filtered via vacuum filtration, and the residue was washed thoroughly with EtOAc. The filtrate was then washed with water and brine. The organic layer was dried with MgSO4, filtered, and loaded on to silica for purification by flash chromatography (0−15% EtOAc in cyclohexane) to obtain the desired product. Yield: 185 mg, 56%. 1H NMR (400 MHz, CDCl3): δ = 8.09 (d, J = 8.8 Hz, 1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.19−7.26 (m, 2 H), 6.93−7.02 (m, 2 H), 3.70 (t, J = 7.6 Hz, 2 H), 3.09 (t, J = 7.6 Hz, 2 H). LC-MS (ESI+) m/z = 265 [M + H]+. 3-(4-Fluorophenyl)-1-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)propan-1-one (27). 27 was prepared from 71 and 39, following general procedure A. The reaction mixture was diluted with EtOAc and washed with satd aq NaHCO3. The organic layer was dryloaded for flash chromatography (10−50% EtOAc in cyclohexane) to afford the title product. Yield: 68 mg, 79%. 1H NMR (CDCl3, 400 MHz) δ = 7.86−7.97 (m, 2 H), 7.46−7.53 (m, 1 H), 7.31−7.40 (m, 2 H), 7.17−7.25 (m, 2 H), 6.87−6.99 (m, 3 H), 3.87−3.95 (m, 4 H),

2 equiv), and the mixture was let to warm up overnight. The mixture was concentrated in vacuo, and after purification by preparative HPLC basic pH, the title compound was isolated. Yield: 29 mg, 35%. 1H NMR (400 MHz, acetone-d6) δ = 8.73 (t, J = 6.1 Hz, 1 H), 7.91 (d, J = 9.5 Hz, 1 H), 7.77−7.73 (m, 1 H), 7.47−7.43 (m, 2 H), 7.42−7.37 (m, 2 H), 7.34−7.28 (m, 4 H), 7.25−7.19 (m, 1 H), 4.75−4.62 (m, 2 H), 4.66 (d, J = 6.3 Hz, 2 H), 3.20−3.01 (m, 3 H), 2.40 (s, 3 H), 1.92− 1.72 (m, 3 H), 1.57−1.49 (m, 1 H). LC-MS (ESI+) m/z = 435 [M + H]+. N-Benzyl-6-(4-(o-Tolylsulfonyl)piperidin-1-yl)pyridazine-3-carboxamide (22). After preparative HPLC basic pH purification of the above-mentioned crude mixture, the title compound was also isolated. Yield: 15 mg, 17%. 1H NMR (400 MHz, acetone-d6) δ = 8.70 (t, J = 6.1 Hz, 1 H), 7.96−7.89 (m, 2 H), 7.67−7.60 (m, 1 H), 7.51−7.47 (m, 2 H), 7.44−7.37 (m, 2 H), 7.37−7.30 (m, 3 H), 7.28−7.22 (m, 1 H), 4.77−4.69 (m, 2 H), 4.66 (d, J = 6.3 Hz, 2 H), 3.68 (tt, J = 3.9, 11.8 Hz, 1 H), 3.20−3.10 (m, 2 H), 2.73 (s, 3 H), 2.04−1.96 (m, 2 H), 1.86−1.76 (m, 2 H). LC-MS (ESI+) m/z = 451 [M + H]+. N-Benzyl-6-(5-(o-tolylsulfonyl)-2,5-diazabicyclo[2.2.1]heptan-2yl)pyridazine-3-carboxamide (23). General procedure B. Prepared from 60 (0.12 mmol) and o-tolylsulfonyl chloride. Product purified by flash chromatography (20−80% EtOAc in hexane). Yield: 42 mg, 68%. 1 H NMR (400 MHz, CDCl3) δ = 8.18 (t, J = 6.0 Hz, 1 H), 8.06 (d, J = 9.5 Hz, 1 H), 7.97−7.91 (m, 1 H), 7.50−7.43 (m, 1 H), 7.39−7.27 (m, 7 H), 6.68 (d, J = 9.3 Hz, 1 H), 5.20−5.18 (m, 1 H), 4.69−4.66 (m, 3 H), 3.69−3.56 (m, 2 H), 3.48−3.39 (m, 2 H), 2.62 (s, 3 H), 2.06 (s, 2 H). LC-MS (ESI+) m/z = 464 [M + H]+. N-Benzyl-6-(4-(o-tolylsulfonyl)-1,4-diazepan-1-yl)pyridazine-3carboxamide (24). General procedure B. Prepared from 61 (0.12 mmol) and o-tolylsulfonyl chloride. Product purified by flash chromatography (20−80% EtOAc in hexane). Yield: 31 mg, 49%. 1 H NMR (400 MHz, CDCl3) δ = 8.18 (t, J = 6.0 Hz, 1 H), 8.04 (d, J = 9.5 Hz, 1 H), 7.81 (d, J = 8.5 Hz, 1 H), 7.46−7.39 (m, 1 H), 7.38− 7.25 (m, 7 H), 6.88 (d, J = 9.5 Hz, 1 H), 4.67 (d, J = 6.0 Hz, 2 H), 4.10−4.07 (m, 2 H), 3.99−3.88 (m, 2 H), 3.59−3.50 (m, 2 H), 3.34− 3.26 (m, 2 H), 2.55 (s, 3 H), 2.19−2.07 (m, 2 H). LC-MS (ESI+) m/z = 466 [M + H]+. N-Benzyl-1-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)methanamine 2,2,2-trifluoroacetate Salt (25). (6-(4-(oTolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)methanol (65). 58 (130 mg, 0.34 mmol, 1 equiv) was dissolved in THF (20 mL) under N2 atmosphere and cooled to 0 °C. NaBH4 (4.8 mmol, 14 equiv) was dissolved absolute EtOH (5 mL) and slowly added to the ester solution at 0 °C. The mixture was stirred at 0 °C for 10 min and then stirred at 40 °C for 1 h. The reaction was quenched by addition of 2 M HCl until H2 gas evolution ceased. The solution was then brought to pH ∼ 8 by addition of satd NaHCO3. Water was added, and the resulting mixture was extracted with EtOAc and dried with MgSO4. The organic layer was dried in vacuo to afford the title product. Yield: 114 mg, 94%. Used in the next step without any further purification. LC-MS (ESI+) m/z = 349 [M + H]+. 3-(Chloromethyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine (66). 65 (34 mg, 0.09 mmol, 1 equiv) was suspended in CH2Cl2 (5 mL) and cooled to 0 °C, to which SOCl2 (2.7 mmol, 28 equiv) was added. The mixture was stirred overnight at room temperature. The resulting solution was washed with satd NaHCO3 and brine and then dried over MgSO4. The organic layer was concentrated in vacuo and azeotroped with toluene to yield the title product. Yield: 36 mg, 87%. Used in the next step without any further purification. LC-MS (ESI+) m/z = 367 [M + H]+. N-Benzyl-1-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)methanamine 2,2,2-Trifluoroacetate Salt (25). 66 (31 mg, 0.084 mmol, 1 equiv), benzylamine (0.93 mmol, 11 equiv), and Et3N (0.25 mmol, 3 equiv) were suspended in acetonitrile (5 mL) and stirred at room temperature overnight. The mixture was concentrated in vacuo and purified by preparative HPLC acidic pH to obtain the title product as the trifluoroacetate salt. Yield: 8 mg, 17%. 1H NMR (400 MHz, CDCl3): δ = 7.90 (dd, J = 8.3, 1.3 Hz, 1 H), 7.63 (d, J = 9.6 Hz, 1 H), 7.47−7.53 (m, 1 H), 7.32−7.45 (m, 7 H), 7.16 (d, J = 9.1 Hz, 1 H), 4288



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−78 °C for 15 min, warmed to RT, and stirred for 30 min, then stirred at 50 °C for 30 min. The mixture was concentrated in vacuo, taken up in CH2Cl2, and washed with satd NaHCO3. The organic layer was then dry loaded on to silica and purified by flash chromatography (30−85% EtOAc in cyclohexane) to obtain the product. Yield: 6 mg, 19%. 1H NMR (400 MHz, CDCl3) δ = 7.90−7.98 (m, 1 H), 7.46− 7.52 (m, 1 H), 7.32−7.40 (m, 2 H), 7.13 (d, J = 9.6 Hz, 1 H), 7.01− 7.08 (m, 2 H), 6.87−6.96 (m, 3 H), 5.06 (d, J = 6.1 Hz, 2 H), 4.70 (d, J = 6.1 Hz, 2 H), 3.72−3.81 (m, 4 H), 3.30−3.39 (m, 4 H), 2.68 (s, 3 H), 2.40−2.52 (m, 4 H). LC-MS (ESI+) m/z = 497 [M + H]+. 3-(4-(4-Fluorophenyl)but-1-en-2-yl)-6-(4-(o-tolylsulfonyl)-piperazin-1-yl)pyridazine (30). After flash chromatography purification of the above-mentioned crude mixture, the title compound was also isolated; 12 mg, 42% yield. 1H NMR (400 MHz, CDCl3) δ = 7.93 (d, J = 7.8 Hz, 1 H), 7.45−7.53 (m, 2 H), 7.31−7.40 (m, 2 H), 7.07−7.18 (m, 2 H), 6.85−6.98 (m, 3 H), 5.53 (s, 1 H), 5.20 (s, 1 H), 3.73−3.84 (m, 4 H), 3.33 (t, J = 4.7 Hz, 4 H), 2.97−3.07 (m, 2 H), 2.81−2.91 (m, 2 H), 2.67 (s, 3 H). LC-MS (ESI+) m/z = 467 [M + H]+. tert-Butyl-4-(2-(trifluoromethyl)benzoyl)-piperazine-1-carboxylate (32). General procedure D. Prepared from 31 (10.52 mmol) and 34. Yield: 3.5 g, 94%. 1H NMR (400 MHz, CDCl3) δ = 7.74−7.68 (m, 1H), 7.63−7.57 (m, 1H), 7.56−7.50 (m, 1H), 7.35−7.30 (m, 1H), 3.86−3.79 (m, 1H), 3.76−3.68 (m, 1H), 3.57−3.46 (m, 2H), 3.37− 3.31 (m, 2H), 3.18−3.11 (m, 2H), 1.46 (s, 9H). LC-MS (ESI+) m/z = 359 [M + H]+. Piperazin-1-yl(2-(trifluoromethyl)phenyl)-methanone Hydrochloride (33). General procedure E. Prepared from 32 (9.86 mmol). Yield: 2.81 g, 97%. 1H NMR (400 MHz, DMSO-d6) δ = 9.63 (br s, 2H), 7.87−7.82 (m, 1H), 7.81−7.76 (m, 1H), 7.72−7.66 (m, 1H), 7.65−7.60 (m, 1H), 4.07−3.97 (m, 1H), 3.79−3.69 (m, 1H), 3.41− 3.29 (m, 2H), 3.27−3.17 (m, 1H), 3.16−3.03 (m, 2H), 2.96−2.85 (m, 1H). LC-MS (ESI+) m/z = 259 [M + H]+. tert-Butyl 4-(o-Tolylsulfonyl)piperazine-1-carboxylate (35). General procedure B. Prepared from 34 and o-tolylsulfonyl chloride. Yield: 930 mg, 91%. 1H NMR (400 MHz, CDCl3) δ = 7.94−7.86 (m, 1 H), 7.52−7.44 (m, 1 H), 7.38−7.31 (m, 2 H), 3.55−3.43 (m, 4 H), 3.19− 3.06 (m, 4 H), 2.64 (s, 3 H), 1.44 (s, 9 H). LC-MS (ESI+) m/z = 341 [M + H]+. tert-Butyl 4-((3-Chloro-2-methylphenyl)sulfonyl)piperazine-1carboxylate (36). General procedure B. Prepared from 34 and 3chloro-2-methylbenzene-1-sulfonyl chloride. Yield: 830 mg, 96%. 1H NMR (400 MHz, CDCl3) δ = 7.88 (dd, J = 8.0, 1.0 Hz, 1 H), 7.61 (dd, J = 8.0, 1.0 Hz, 1 H), 7.31−7.25 (m, 1 H), 3.55−3.39 (m, 4 H), 3.23− 3.08 (m, 4 H), 2.69 (s, 3 H), 1.45 (s, 9 H). LC-MS (ESI+) m/z = 376 [M + H]+. tert-Butyl 4-((2-Fluorophenyl)sulfonyl)piperazine-1-carboxylate (37). General procedure B. Prepared from 34 and 2-fluorobenzene1-sulfonyl chloride. Yield: 900 mg, 89%. LC-MS (ESI+) m/z = 345 [M + H]+. tert-Butyl 4-((2-(4-Hydroxypiperidin-1-yl)phenyl)sulfonyl)piperazine-1-carboxylate (38). General procedure C. Prepared from 37 (0.3 mmol) and 4-hydroxypiperidine. The reaction mixture was concentrated in vacuo, and the resulting solid was triturated with water to yield to title compound. Yield: 117 mg, 95%. Used in the next step without further purification. LC-MS (ESI+) m/z = 426 [M + H]+. 1-(o-Tolylsulfonyl)piperazine Hydrochloride (39). General procedure E. Prepared from 35. Yield: 1.45 g, 59%. 1H NMR (400 MHz, DMSO-d6) δ = 9.45 (br s, 2 H), 7.82 (d, J = 7.8 Hz, 1 H), 7.63 (app t, J = 7.5 Hz, 1 H), 7.53−7.40 (m, 2 H), 3.37−3.26 (m, 4 H), 3.16−3.10 (m, 4 H). LC-MS (ESI+) m/z = 241 [M + H]+. 1-((3-Chloro-2-methylphenyl)sulfonyl)piperazine Hydrochloride (40). General procedure E. Prepared from 36. Yield: 2.20 g, 88%. LC-MS (ESI+) m/z = 275 [M + H]+. 1-((2-Fluorophenyl)sulfonyl)piperazine Hydrochloride (41). General procedure E. Prepared from 37. Yield: 2.28 g, 93%. LC-MS (ESI+) m/z = 245 [M + H]+. 1-[2-(Piperazine-1-sulfonyl)phenyl]piperidin-4-ol Hydrochloride (42). General procedure E. Prepared from 38 (0.27 mmol). Yield: 95 mg, 96%. LC-MS (ESI+) m/z = 326 [M + H]+. 6-Chloro-N-phenethylpyridazine-3-carboxamide (44). General procedure D. Prepared from 43 (3.17 mmol) and 2-phenylethan-1-

3.55−3.64 (t, J = 7.6 Hz, 2 H), 3.29−3.37 (m, 4 H), 3.05 (t, J = 7.6 Hz, 2 H), 2.66 (s, 3 H). LC-MS (ESI+) m/z = 469 [M + H]+. 3-(4-(4-Fluorophenyl)-1-methoxybut-1-en-2-yl)-6-(4-(otolylsulfonyl)piperazin-1-yl)pyridazine (72). A 1.8 M solution of lithium diisopropylamide (0.675 mmol, 1.7 equiv) in anhydrous THF was added dropwise under N2 atmosphere to a solution of (methoxymethyl)triphenylphosphanium chloride (0.675 mmol, 1.7 equiv) in anhydrous THF (5 mL) cooled to −78 °C. The mixture was stirred at −78 °C for 30 min, then allowed to warm to room temperature and stir for a further 30 min. The bright-orange mixture was then cooled back down to −78 °C, and a solution of 27 (186 mg, 0.40 mmol, 1 equiv) in anhydrous THF (5 mL) was added dropwise. The mixture was stirred at −78 °C for 1 h, allowed to warm to room temperature, and stirred at room temperature for a further 4 h. The solution was dry loaded on to silica for purification by flash chromatography (20−50% EtOAc in cyclohexane), and two isomers were isolated (161 mg, 82% combined yield of two isomers). Isomer A (less polar by TLC): 1H NMR (400 MHz, CDCl3): δ = 7.89−7.95 (m, 2 H), 7.45−7.51 (m, 1 H), 7.31−7.37 (m, 2 H), 7.04−7.10 (m, 2 H), 6.88−6.95 (m, 2 H), 6.85 (d, J = 9.6 Hz, 1 H), 6.08 (s, 1 H), 3.72− 3.78 (m, 4 H), 3.63 (s, 3 H), 3.28−3.35 (m, 4 H), 2.79 (apparent s, 4 H), 2.67 (s, 3 H). LC-MS (ESI+) m/z = 497 [M + H]+. Isomer B (more polar by TLC): 1H NMR (400 MHz, CDCl3) δ = 7.93 (dd, J = 8.6, 1.3 Hz, 1 H), 7.46−7.52 (m, 1 H), 7.32−7.38 (m, 2 H), 7.11−7.19 (m, 3 H), 6.91 (t, J = 8.8 Hz, 2 H), 6.80−6.85 (m, 2 H), 3.69−3.75 (m, 4 H), 3.67 (s, 3 H), 3.29−3.35 (m, 4 H), 2.84−2.92 (m, 2 H), 2.71−2.77 (m, 2 H), 2.67 (s, 3 H). LC-MS (ESI+) m/z = 469 [M + H]+. 4-(4-Fluorophenyl)-2-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)butanal (73). 72 (mixture of isomers) (161 mg, 0.324 mmol, 1 equiv) was dissolved in THF (8 mL) and diluted with water (11 mL). The solution was cooled to 0 °C, and conc H2SO4 (5 mL) was added slowly. The solution was allowed to stir at 45 °C for 2 days. The mixture was then neutralized to pH 8 with 6 M and then satd NaHCO3. The mixture was then extracted with CH2Cl2, and the combined organic layer was dried over MgSO4 and concentrated in vacuo to yield the title compound. Yield: 156 mg, 99%. Used in the next step without any further purification. LC-MS (ESI+) m/z = 487 [M + H]+ (aldehyde). LC-MS (ESI+) m/z = 501 [M + H]+ (aldehyde hydrate). 2-(4-Fluorophenethyl)-2-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)propane-1,3-diol (28). 73 (156 mg, 0.323 mmol, 1 equiv) was dissolved in THF (10 mL). To this was added 10 mL of 37% aqueous formaldehyde followed by 6 M NaOH (2.5 mL). The reaction was stirred at room temperature overnight and subsequently quenched with 2 M HCl and extracted with CH2Cl2. The organic layer was concentrated under vacuum, redissolved in THF:H2O:6 M NaOH (10 mL:10 mL:2.5 mL), and stirred at 40 °C for 30 min. The mixture was diluted with methanol and brine and extracted repeatedly with 10:90 MeOH:CH2Cl2. The combined organic layer was dried over MgSO4, filtered, dry loaded on silica, and purified by flash chromatography (60% EtOAc in cyclohexane to 4% MeOH in EtOAc) to afford the title product; yield 112 mg, 67%). 1H NMR (400 MHz, CDCl3) δ = 7.90−7.95 (m, 1 H), 7.46−7.52 (m, 1 H), 7.31− 7.39 (m, 3 H), 6.92−6.99 (m, 3 H), 6.83−6.91 (m, 2 H), 4.30 (d, J = 11.1 Hz, 2 H), 3.97 (d, J = 11.4 Hz, 2 H), 3.69−3.78 (m, 4 H), 3.59 (br s, 2 H), 3.27−3.36 (m, 4 H), 2.67 (s, 3 H), 2.28−2.39 (m, 2 H), 1.79−1.88 (m, 2 H). LC-MS (ESI+) m/z = 515 [M + H]+. 3-(3-(4-Fluorophenethyl)oxetan-3-yl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine (29). A solution of 28 (30 mg, 0.058 mmol, 1 equiv) in 1 mL of THF was added dropwise to a suspension of NaH (0.064 mmol, 1.1 equiv) in 1 mL o THF at 0 °C. The mixture was stirred at 0 °C for 5 min, then allowed to warm to room temperature and stirred for 30 min. The mixture was then cooled down to −78 °C, and a solution of p-tosyl chloride (0.058 mmol 1 equiv) in 1 mL of THF was added dropwise. The mixture was stirred at −78 °C for 5 min and allowed to warm to R, and stirred for 2 h. Additional p-tosyl chloride and NaH were added at −78 °C. After 5 h, the reaction mixture was diluted with 20 mL of dry THF, cooled to −78 °C, and NaH (1.16 mmol, 20 equiv) was added. The mixture was stirred at 4289



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amine. Yield: 145 mg, 17%. 1H NMR (400 MHz, CDCl3) δ = 8.27 (d, J = 8.8 Hz, 1 H), 8.13−8.05 (m, 1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.35− 7.28 (m, 2 H), 7.26−7.22 (m, 3 H), 3.82−3.75 (m, 2 H), 2.96 (t, J = 7.2 Hz, 2 H). N-Benzyl-6-chloropyridazine-3-carboxamide (45). General procedure D. Prepared from 43 (3.17 mmol) and benzylamine. Yield: 210 mg, 26%. 1H NMR (400 MHz, CDCl3) δ = 8.37 (br s, 1 H), 8.30 (d, J = 8.8 Hz, 1 H), 7.69 (d, J = 8.8 Hz, 1 H), 7.40−7.26 (m, 6 H), 4.70 (d, J = 6.3 Hz, 2 H). LC-MS (ESI+) m/z = 248 [M + H]+. 6-Chloro-N-phenylpyridazine-3-carboxamide (46). General procedure D. Prepared from 43 (3.17 mmol) and aniline. Yield: 460 mg, 62%. 1H NMR (400 MHz, CDCl3) δ = 8.39 (br s, 1H), 8.31 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.38−7.26 (m, 5H), 4.70 (d, J = 6.0 Hz, 2H) 6-Chloro-N-(4-fluorobenzyl)pyridazine-3-carboxamide (47). General procedure D. Prepared from 43 (3.17 mmol) and 4-fluorobenzylamine. Yield: 460 mg, 55%. 1H NMR (400 MHz, CDCl3) δ = 8.36 (br s, 1 H), 8.31 (d, J = 8.8 Hz, 1 H), 7.71 (d, J = 8.8 Hz, 1 H), 7.31−7.39 (m, 2 H), 7.01−7.09 (m, 2 H), 4.68 (d, J = 6.1 Hz, 2 H). LC-MS (ESI +) m/z = 266 [M + H]+. 6-Chloro-N-(thiophen-2-ylmethyl)pyridazine-3-carboxamide (48). General procedure D. Prepared from 43 (3.17 mmol) and thiophen-2-ylmethanamine. Yield: 650 mg, 81%. 1H NMR (400 MHz, CDCl3) δ = 9.09−8.98 (br s, 1 H), 8.60−8.53 (m, 1 H), 8.28 (d, J = 8.8 Hz, 1 H), 7.72−7.64 (m, 2 H), 7.32 (d, J = 8.0 Hz, 1 H), 7.24− 7.17 (m, 1 H), 4.82 (d, J = 5.5 Hz, 2 H). LC-MS (ESI+) m/z = 254 [M + H]+. 6-Chloro-N-(prop-2-yn-1-yl)pyridazine-3-carboxamide (49). General procedure D. Prepared from 43 (3.17 mmol) and propargylamine. Yield: 466 mg, 75%. 1H NMR (400 MHz, CDCl3) δ = 8.29 (d, J = 8.8 Hz, 1 H), 8.25−8.17 (br s, 1 H), 7.71 (d, J = 8.8 Hz, 1 H), 4.33 (dd, J = 5.5, 2.5 Hz, 2 H), 2.31 (t, J = 2.5 Hz, 1 H). LC-MS (ESI+) m/z = 196 [M + H]+. Benzyl 6-Chloropyridazine-3-carboxylate (50). General procedure D. Prepared from 43 (3.17 mmol) and benzyl alcohol. Reaction was run in EtOAc and heated at 100 °C for 30 min in the microwave oven. Same workup as in general procedure. Yield: 270 mg, 34%. LC-MS (ESI+) m/z = 249 [M + H]+. Prop-2-yn-1-yl 6-chloropyridazine-3-carboxylate (51). General procedure D. Prepared from 43 (3.17 mmol) and propargyl alcohol. Reaction was run in EtOAc and heated at 100 °C for 30 min in the microwave oven. Same workup as in general procedure. Yield: 370 mg, 60%. LC-MS (ESI+) m/z = 197 [M + H]+. tert-Butyl 4-(6-(Benzylcarbamoyl)pyridazin-3-yl)piperazine-1carboxylate (53). General procedure A. Prepared from and 45 (8 mmol) and 34. The crude product was triturated with Et2O to obtain the title compound. Yield: 2.8 g, 87%. Used in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ = 8.24 (t, J = 5.7 Hz, 1 H), 8.07 (d, J = 9.5 Hz, 1 H), 7.39−7.24 (m, 6 H), 6.99 (d, J = 9.5 Hz, 1 H), 4.68 (d, J = 6.0 Hz, 2 H), 3.80−3.71 (m, 4 H), 3.63− 3.54 (m, 4 H), 1.52−1.47 (m, 9 H). LC-MS (ESI+) m/z = 398 [M + H]+. (1S,4S)-tert-Butyl-5-(6-(benzylcarbamoyl)pyridazin-3-yl)-2,5diazabicyclo[2.2.1]heptane-2-carboxylate (54). General procedure A. Prepared from 45 (2 mmol) and (1S,4S)-(−)-2-Boc-2,5diazabicyclo[2.2.1]heptane. The crude product was triturated with Et2O to obtain the title compound. Yield: 362 mg, 44%. Used in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ = 8.20 (t, J = 5.9 Hz, 1 H), 8.04 (d, J = 9.3 Hz, 1 H), 7.40−7.20 (m, 5 H), 6.69 (d, J = 9.3 Hz, 1 H), 4.75−4.72 (m, 0.5 H), 4.67 (d, J = 6.0 Hz, 2 H), 4.62−4.60 (m, 0.5 H), 3.70−3.54 (m, 1 H), 3.54−3.34 (m, 3 H), 2.06−1.95 (m, 2 H), 1.47 (s, 4 H), 1.42 (s, 9 H). LC-MS (ESI+) m/z = 410 [M + H]+. tert-Butyl 4-(6-(Benzylcarbamoyl)pyridazin-3-yl)-1,4-diazepane1-carboxylate (55). General procedure A. Prepared from 45 (2 mmol) and tert-butyl 1,4-diazepane-1-carboxylate. The crude product was triturated with Et2O to obtain the title compound. Yield: 391 mg, 47%. Used in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ = 8.16 (t, J = 5.9 Hz, 1 H), 7.97 (d, J = 9.5 Hz, 1 H), 7.33−7.17 (m, 5 H), 6.83 (d, J = 9.5 Hz, 1 H), 4.61 (d, J = 5.9 Hz, 2

H), 3.88−3.68 (m, 4 H), 3.57 (app t, J = 5.4 Hz, 2 H), 3.32 (app t, J = 5.5 Hz, 1 H), 3.23 (app t, J = 5.9 Hz, 1 H), 1.97−1.88 (m, 2 H), 1.34 (s, 9 H). LC-MS (ESI+) m/z = 412 [M + H]+. tert-Butyl 4-(6-((4-Fluorobenzyl)carbamoyl)pyridazin-3-yl)piperazine-1-carboxylate (56). General procedure A. Prepared from 47 (1 mmol) and 34. The crude product was triturated with Et2O to obtain the title compound (yield: 504 mg, 100%). Used in the next step without further purification. LC-MS (ESI+) m/z = 416 [M + H]+. N-Benzyl-6-(4-hydroxypiperidin-1-yl)pyridazine-3-carboxamide (57). General procedure A. Prepared from 45 (0.56 mmol) and piperidin-4-ol. Reaction was refluxed in n-BuOH overnight. The resulting solution was concentrated in vacuo, dissolved in CH2Cl2, washed with brine, and dried with Na2SO4. The organic layer was concentrated in vacuo to afford the title compound. Yield: 147 mg, 83%. Used in the next step without further purification. LC-MS (ESI+) m/z = 313 [M + H]+. Methyl 6-(4-(o-Tolylsulfonyl)piperazin-1-yl)pyridazine-3-carboxylate (58). General procedure A. Prepared from 52 (1 mmol) and 39. Reaction was refluxed in DMF for 2 h. Solid material crashed out of solution upon cooling to 0 °C and was subsequently triturated with water to provide the title compound. Yield: 287 mg, 77%. Used in the next step without further purification. LC-MS (ESI+) m/z = 377 [M + H]+. N-Benzyl-6-(piperazin-1-yl)pyridazine-3-carboxamide Hydrochloride (59). General procedure E. Prepared from 53 (7 mmol). Yield: 2.25 g, 96%. 1H NMR (400 MHz, DMSO-d6) δ = 9.61 (br s, 2H), 9.43 (t, J = 6.3 Hz, 1H), 7.95 (d, J = 9.5 Hz, 1H), 7.49 (d, J = 9.5 Hz, 1H), 7.36−7.28 (m, 4H), 7.27−7.20 (m, 1H), 4.50 (d, J = 6.3 Hz, 2H), 4.04−3.93 (m, 4H), 3.27−3.16 (m, 4H). LC-MS (ESI+) m/z = 298 [M + H]+. (1S,4S)-N-Benzyl-6-(2,5-diazabicyclo[2.2.1]heptan-2-yl)pyridazine-3-carboxamide Hydrochloride (60). General procedure E. Prepared from 54 (0.8 mmol). Yield: 229 mg, 86%. 1H NMR (400 MHz, DMSO-d6) δ = 9.36 (app t, J = 6.5 Hz, 2 H), 7.97 (d, J = 9.4 Hz, 1 H), 7.39−7.13 (m, 6 H), 5.20−5.08 (m, 1 H), 4.57−4.53 (m, 1 H), 4.51 (d, J = 6.4 Hz, 2 H), 3.94−3.81 (m, 1 H), 3.76−3.67 (m, 1 H), 3.39−3.20 (m, 2 H), 2.24−2.13 (m, 1 H), 2.08−1.99 (m, 1 H). LCMS (ESI+) m/z = 310 [M + H]+. N-Benzyl-6-(1,4-diazepan-1-yl)pyridazine-3-carboxamide Hydrochloride (61). General procedure E. Prepared from 55 (0.8 mmol). Yield: 238 mg, 89%. 1H NMR (400 MHz, DMSO-d6) δ = 9.38 (app t, J = 6.3 Hz, 2 H), 7.92 (d, J = 9.5 Hz, 1 H), 7.38 (d, J = 9.5 Hz, 1 H), 7.34−7.28 (m, 4 H), 7.26−7.18 (m, 1 H), 4.50 (d, J = 6.3 Hz, 2 H), 4.15−4.05 (m, 2 H), 3.87−3.77 (m, 2 H), 3.33−3.24 (m, 2 H), 3.22− 3.12 (m, 2 H), 2.17−2.08 (m, 2 H). LC-MS (ESI+) m/z = 312 [M + H]+. N-(4-Fluorobenzyl)-6-(piperazin-1-yl)pyridazine-3-carboxamide Hydrochloride (62). General procedure E. Prepared from 56 (1.2 mmol). Yield: 426 mg, 100%. LC-MS (ESI+) m/z = 316 [M + H]+. Differential Scanning Fluorimetry (DSF). The assay buffer in which dCTPase, inhibitors, and dye were diluted consisted of 100 mM Tris-acetate pH 8.0, 100 mM KCl, 0.005% Tween 20, and 1 mM DTT. The assay concentrations used were 33 μM inhibitor, 20 μM recombinant human dCTPase, and Sypro Orange 5×; 1% final concentration of DMSO. The assay volumes were 20 μL in 96-well QPCR plates. A BioRad Q-PCR instrument was used to ramp the temperature from 20 to 100 °C at 1 °C/min and analyzing fluorescence intensity at each step. The Bio-Rad software was used to calculate the Tm (negative) of each well of the plate. DARTS Assay.17 HL60 cells were treated with 0.1% DMSO or 10 μM inhibitor in 0.1% DMSO for 4 h, media was aspirated, and the cells were washed with ice-cold PBS. The cells were lysed in the mammalian protein extraction lysis buffer M-PER (Thermo Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche) and 1× Halt phosphatase inhibitor cocktail (Thermo Scientific) on ice for 10 min. After centrifugation at 16000g at 4 °C, the supernatant (cell lysate) was transferred to a new tube and kept on ice. Then 10× TN buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl) was added to the lysate to make a final concentration of 1× TN buffer. Protein concentration of the cell lysate was determined by Bradford method 4290



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*For T.H.: phone, 0046852480000; E-mail, thomas.helleday@ scilifelab.se.

with Coomassie Plus protein assay reagent and BSA as a protein standard (Thermo Scientific). Pronase stock solution (10 mg/mL) was diluted in TN buffer to achieve the final ratio of enzyme to total protein 1:200 and 1:100. The cell lysate was split into aliquots, and 1 μL of the range of Pronase solutions was added and incubated with shaking at room temperature for 30 min. For the nondigested (ND) sample, 1 μL of TN buffer was added instead of protease. Each digestion reaction was stopped by adding of 4× Laemmli loading buffer with 200 mM DTT and heating at 95° for 5 min. The samples were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Bio-Rad) using Trans-Blot Turbo transfer system (BioRad). A blot was first probed with rabbit anti-dCTPase antibody (produced in house), followed by incubation with antirabbit-HRP secondary antibody, and protein bands were visualized using SuperSignal West Femto chemiluminescence substrate (Thermo Scientific). Next, the blot was stripped with Restore Plus Western Blot Stripping buffer (Thermo Scientific) and probed with rabbit antiGAPDH Ab (sc 257782), followed by incubation with donkey antirabbit IRDye 800CW Ab (Li-COR), and images were taken with Odyssey Fc imager (Li-COR). GAPDH was used as a loading and digestion control. SCD1 Index Assay. HepG2 cell line were grown in L-glutamine rich RPMI-1640 (Life Technologies) supplemented with 10% FBS (Gibco). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2:air. Cells were seeded in duplicate sets of 6well plates at 1 × 106 per well. The cells were treated with either vehicle DMSO (0.1%) or 74 (Cayman Chemicals) or inhibitors in 2% FBS-supplemented media. Following 72 h incubation, culture medium was removed and the cell monolayer was washed three times with cold PBS and collected for cellular fatty acid measurement. Cells were collected into glass vials, and total lipids were esterified with MeOH catalyzed by acetyl chloride.23 Fatty acid methyl ester derivatives formed from isolated cellular lipids were separated on a highly polar biscyanopropyl polysiloxane capillary column (TR-CN100 60 mm × 0.25 mm × 0.2 μm film thickness, Teknokroma). The gas chromatograph was a Buck Scientific model 610 equipped with a split flow injector and a hydrogen flame ionization detector. Helium was employed as the carrier gas. The oven temperature program was from 190 to 210 °C with 1 °C/min, then isothermal continued for 10 min. Tridecanoic acid (13:0) (Sigma Chemicals) was used as internal standard. Peak retention times were identified by injecting known fatty acid methyl ester standards (Sigma Chemicals). PeakSimple software was used for data acquisition and processing. Other Assays. Antibodies, purification of human recombinant dCTPase, malachite green screening assay, NTPase/NUDIX hydrolases selectivity assays, cell culture, CETSA assay, viability assays/ combination index, and flow cytometry analysis were performed as described previously.9,24 Intracellular bioavailability (Fic) and molecular docking were performed as described previously.10,25

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ORCID

Sabin Llona-Minguez: 0000-0003-3187-722X Nicholas C. K. Valerie: 0000-0002-9423-964X Present Addresses ∇

For A. Höglund: Sprint BioScience AB, Huddinge, Sweden. For A. Ghassemian: Faculty of Medicine, McGill University, Montreal, Canada. ◆ For J. M. Calderón-Montaño, E. B. Morón: Faculty of Pharmacy, University of Seville, Seville, Spain. ¶ For A. Mateus: European Molecular Biology Laboratory, Heidelberg, Germany. + For K. Sigmundsson: Duke-NUS Graduate Medical School, Singapore. ● For T. Lundbäck: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden. ○

Author Contributions #

S.Ll.-M. and A.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Dr. Adam Throup and Maghsod Shaaker for insightful manuscript and scientific discussions. This project is primarily supported by The Knut and Alice Wallenberg Foundation. Further support was received from the Felix Mindus Foundation for leukemia research, the Swedish Research Council, the European Research Council, Göran Gustafsson Foundation, Swedish Cancer Society, the Swedish Children’s Cancer Foundation, the Swedish Pain Relief Foundation, and the Torsten and Ragnar Sö d erberg Foundation. Chemical Biology Consortium Sweden was supported by the Swedish Research Council. We also acknowledge the Faculty of Medicine at Tabriz University of Medical Sciences for providing research facilities and ChemAxon (http://www.chemaxon.com) for technical support. We are grateful to the Protein Science Facility at Karolinska Institutet for purification of proteins.

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ABBREVIATIONS USED 5-AzaC, 5-azacytidine; dCTP, deoxycytidine triphosphate; dNTP, deoxynucleoside triphosphate; NUDIX, nucleoside diphosphate linked to X; XTP3TPA, XTP3-transactivated protein A

ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00182. HTS output overview, chemical structures of 5-Aza and 74, DSF assay data, NTPase/NUDIX selectivity data, intracellular bioavailability data, BJ hTERT cell viability data (PDF) Homology model (PDB) Molecular formula strings (CSV)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*For S.LL.-M.: phone, 0046852480000; E-mail, sabin.llona. [email protected]. 4291



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