Discovery of Imidazoquinolines as a Novel Class of Potent, Selective

Article pubs.acs.org/jmc

Discovery of Imidazoquinolines as a Novel Class of Potent, Selective, and in Vivo Efficacious Cancer Osaka Thyroid (COT) Kinase Inhibitors Ralf Glatthar,*,† Aleksandar Stojanovic,† Thomas Troxler,† Henri Mattes,† Henrik Möbitz,† Rene Beerli,† Joachim Blanz,‡ Ernst Gassmann,‡ Peter Drückes,§ Gabriele Fendrich,§ Sascha Gutmann,§ Georg Martiny-Baron,⊥ Fiona Spence,∥ Jeff Hornfeld,∇ John Edmonson Peel,∇ and Helmut Sparrer⊥ †

Global Discovery Chemistry, ‡Analytical Sciences, §Center for Proteomic Chemistry, ∥Preclinical Safety, and ⊥Autoimmunity Transplantation Inflammation, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland S Supporting Information *

ABSTRACT: Cancer Osaka thyroid (COT) kinase is an important regulator of pro-inflammatory cytokines in macrophages. Thus, pharmacologic inhibition of COT should be a valid approach to therapeutically intervene in the pathogenesis of macrophage-driven inflammatory diseases such as rheumatoid arthritis. We report the discovery and chemical optimization of a novel series of COT kinase inhibitors, with unprecedented nanomolar potency for the inhibition of TNFα. Pharmacological profiling in vivo revealed a high metabolism of these compounds in rats which was demonstrated to be predominantly attributed to aldehyde oxidase. Due to the very low activity of hepatic AO in the dog, the selected candidate 32 displayed significant blood exposure in dogs which resulted in a clear prevention of inflammation-driven lameness. Taken together, the described compounds both potently and selectively inhibit COT kinase in primary human cells and ameliorate inflammatory pathologies in vivo, supporting the notion that COT is an appropriate therapeutic target for inflammatory diseases.

1. INTRODUCTION

been located downstream of the IL-17 receptor, with consequences for inflammatory indication and cancers.8,9 The critical location of COT kinase in a key proinflammatory pathway has made it an attractive target for LMW cytokine inhibition therapy. Elevated levels of these

Cancer Osaka thyroid (COT) kinase, also called Tpl2 or MAP3K8, plays an important role in the regulation of the inflammatory response and the progression of some cancers.1 The function of COT kinase is crucial for the secretion of various cytokines and chemokines in macrophages. COT kinase activity is required to process TNFα and IL-1β.2,3 Consequently, COT kinase ko mice are protected in models of endotoxic shock.2 COT kinase forms a heterotrimeric complex with p105, one of the five members of the NFκB transcription factor family, and ABIN-2.4 Signal-induced degradation of p105 to p50 allows COT kinase to dissociate and mediate activation of the Erk pathway and also JNK in fibroblasts or pancreatic cells, possibly via MKK4.5,6 Since COT kinase is downstream of IKK2, the NFκB pathway is not affected. At the same time, all or most ligands activating the canonical NFκB pathway via the family of TNF-receptors (TNF, CD40, etc.), the IL-1/Toll-like receptor (TLR2/3/4/7/9, IL-1R), or NOD2 can activate COT kinase via IKK2. This molecular organization of the signaling pathway coordinates input of pro-inflammatory receptors to the ERK pathway. Growth factor receptors, which are also known to feed into the ERK pathway, use different MAP3 kinases and scaffold proteins to assemble different signaling complexes that allow separation of the COT- and RAF-dependent pathways on the molecular level (Figure 1).7 In addition, COT kinase has © XXXX American Chemical Society

Figure 1. COT kinase signaling pathway. Received: April 19, 2016

A

DOI: 10.1021/acs.jmedchem.6b00598 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 2. Representative compounds of COT kinase inhibitor scaffolds reported in the literature.

cytokines have been clinically implicated as mediators of a number of autoimmune diseases, in particular, the pain and joint destruction characteristic of rheumatoid arthritis.10 Since COT kinase appears to affect primarily macrophage-derived pro-inflammatory targets, all diseases with underlying persistent macrophage activation may be considered as therapeutic opportunities for pharmacological intervention. The spectrum of potential indications may range from acute macrophage activation syndromes (e.g., pancreatitis or gout), to low grade activation of macrophages in adipose tissue leading to type 2 diabetes. In comparison to other MAP kinases for which several inhibitors have advanced to clinical trials over the past years, drug discovery activities on COT inhibition are still in their infancy. In fact, there are only a few reports in the literature describing COT inhibitor scaffolds.11 Representative compounds of these are shown in Figure 2 and include 1,7naphthyridine-3-carbonitrile (1),12 quinoline-carbonitrile (2),13 indazole (3),14 thieno[2,3-c]pyridines (4, 5),15,16 and thieno[3,2-d]pyrimidine (6).17 Whereas most of these inhibitors demonstrate good kinase selectivity profiles combined with a reasonable correlation of enzymatic COT inhibition and functional blockade of TNFα release in primary human monocytes (PBMCs), potency in whole blood assays is significantly reduced, which limits their potential as drug candidates. A general complication that has hampered faster progress in drug development for this target is the difficulty to obtain high-quality protein and crystal structures of COT kinase, which impeded structure-based drug design approaches.18 Only recently, we reported on the successful production of highly pure COT protein and the first X-ray cocrystal structures of COT kinase with the two inhibitors 7 and 8.19 Here we present the discovery, optimization, and characterization of a novel class of potent, selective, and in vivo efficacious COT kinase inhibitors, namely imidazonaphthyridines of type A (Figure 3). In addition, we disclose an X-ray structure of an imidazonaphthyridine cocrystallized with COT kinase showing a very different protein conformation compared to our two previously reported structures. The new structural insights facilitated rational optimization of kinase selectivity and potency of this scaffold. Furthermore, we report on the

Figure 3. COT kinase inhibitors: pyrrolopyridine (7), aminopyridine (8), and imidazonaphthyridine scaffold A.

pharmacokinetic profile of selected compounds in vivo in different species and the metabolic fate of this compound class.

2. CHEMISTRY Compounds of type A were prepared according to the general syntheses described in Schemes 1−3. Nitration of the commercially available hydroxynaphthyridine 9 to 10, followed by a Suzuki coupling, afforded intermediates 11−14. Subsequent chlorination using phosphoryl trichloride followed by conversion of the crude chlorides with the corresponding aniline building blocks 35−37, 40, 44, and 47 (Scheme 2) provided the nitronaphthyridines 15−23. Reduction of the nitro group followed by cyclization of the diamino intermediates with triethyl orthoformate resulted in the final compounds 24−32 in low to moderate yields (Tables 1 and 3). Optically active compounds 33 and 34 were obtained by enantiomer separation of racemic 32 with chiral preparative HPLC. The synthesis of the noncommercial aniline building blocks is described in Scheme 2. Aminobenzothiazole 40 was prepared in one step from commercially available 2chlorobenzo[d]thiazol-6-amine (38) and 2-morpholinoethanamine (39) by an aromatic substitution reaction. For coupling of the sterically hindered 1-morpholinopropan-2-amine (42), the strongly activated nitro derivatives 41 and 45 were needed. B



Article

Scheme 1. General Synthesis of Compounds of Type Aa

a Conditions: (a) HNO3, propanoic acid, 130 °C, 56%; (b) (hetero-)arylboronic acid, Pd(OAc)2, SPhos, K2CO3, solvent, 100−130 °C, 65−94%; (c) 1. POCl3, N(n-Pr)3, DMF, 75 °C, 1 min or POCl3, Et3N, −15 °C to rt. 2. Y-NH2, Et3N, solvent, rt or Y-NH2, HCl, solvent, rt, 18−76%; (d) 1. H2, Pd/C, MeOH, rt or SnCl2, conc. HCl, EtOH, rt or Fe, AcOH, H2O/THF, 60 °C or aq. Na2S2O4, DMF, rt. 2. CH(OEt)3, EtOH 120−150 °C or 1. H2/Pd/C, EtOH, rt. 2. CH(OEt)3, EtOH 120−150 °C, 15−30%. For structures of intermediates 11−23; see the Supporting Information.

Scheme 2. Aniline Building Blocks Y-NH2: (I) Commercial Compounds 35−37 and (II) Synthesis of the Noncommericial Compounds 40, 44, and 47a

Conditions: (a) 5 equiv Et3N, dioxane 130 °C, 80%; (b) 2 equiv K2CO3, DMF, rt, 94%; (c) Ra−Ni, H2 (1 atm), THF, rt 82%; (d) 2 equiv K2CO3, dioxane, 120 °C, 78%; (e) 10% Pd/C, H2 (1 atm), MeOH, rt, 97%.

a

Scheme 3. Synthesis of the Benzimidazole Substituted Derivatives 50−-59a

a Conditions: (a) 1. POCl3, N(n-Pr)3, DMF, 75 °C; 2. 1H-benzo[d]imidazol-6-amine, HCl-dioxane, DMF, rt, 50%; (b) 1. H2, Raney-Ni, MeOH, rt; 2. CH(OEt)3, EtOH, HCl, 145 °C, 55%. (c) (hetero-) arylboronic acid, Pd(dppf)2Cl2, Cs2CO3, DME/H2O/EtOH, 140 °C, 4−49% or piperidine, 160 °C, 67%.

C



Article

Table 1. Structures and Profiles of the Original HTS Hit 60 and Key Derivatives 61, 24, 50, and 25 during Early Optimization

Compound

60

61

24

50

25

COT IC50 (μM)a Kinase selectivityb cLogPd

2.60 18/36 4.9

0.80 6/42 5.4

0.05 4/58 3.9

0.04 6/72 4.0

0.02 2c/72 4.4

Enzymatic COT kinase assay. bKinase selectivity displayed as number of kinases inhibited with at least IC50 < 5 μM in relation to the total number of kinases tested from the Novartis kinase selectivity panel. For assay description see Supporting Information. cFLT3 (563-D835Y-993): IC50 < 0.34 μM and MKNK2: IC50 < 1.4 μM. dOctanol/water partition coefficient.21 a

Table 2. Structure Activity Relationship in Position 8 of Benzimidazole Derivatives

a

Enzymatic COT kinase assay.

displayed a 16-fold gain in potency as measured in the enzymatic COT assay. Subsequent substituent modification in position 1 resulted in candidates 50 and 25. Compound 50 was used for a wider SAR exploration in position 8. Unfortunately, only little diversity was tolerated, with 3-thiophene remaining the most potent subtstituent in this position (Table 2). The most promising compound from initial scaffold exploration was the 1-quinoline substituted derivative 25, which showed low nanomolar potency on COT (IC50: 0.02 μM) paired with an exquisite kinase selectivity profile across 72 kinases, including MAP kinases involved in TNFα production, such as p38, JNK, and ERK. In order to investigate the selectivity of the scaffold in a cellular context, lead compound 25 was incubated with human epidermoid carcinoma A431 cells (ATCC No.: CRL.1555) followed by activation of the MEK/ERK pathway via either the pro-inflammatory TNF receptor or the COT-independent EGF receptor that signals via the RAS/RAF pathway (Figure 4).22 The A431 cell line allows the pERK readout to be downstream of the two pathways in one cell type. COT-dependency of the TNF-pathway was shown by siRNA knockdown (Supporting Information). As expected, the literature known MEK inhibitor (R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-((2-fluoro-4iodophenyl)amino)benzamide (PD-0325901), which was inactive in our COT biochemical assay (IC50 COT: >25 μM, data not shown), was highly active in both pathway readouts (IC50 EGF: 0.038 μM, IC50 TNF: 10 μM), whereas the levels of pERK were significantly reduced after TNF-stimulation (IC50: 0.061 ± 0.008 μM). We also tested compound 25 in primary human PBMCs post LPS stimulation. The IC50 of 25 for the inhibition of pERK was 0.54 ± 0.14 μM, suggesting an interference of 25 with a target on the pro-inflammatory pathway which nicely correlated with the functional blockade of TNFα-release (IC50: 0.44 ± 0.19 μM), confirming the postulated role of COT kinase on the regulation of TNFα production.2 Importantly, 25 did not affect NFκB pathway signaling, as shown by the absence of inhibition of p65phosphorylation (IC50 > 25 μM), confirming lack of interaction with an upstream target of COT. Although compound 25 nicely matched the desired kinase and pathway selectivity, several other properties, in particular aqueous solubility and activity in a whole blood assay, were not ideal. Furthermore, the thiophene substituent in position 8 was not considered acceptable based on the well documented risk of thiophene-related toxicity.23 Building on available SAR information at this position (Table 2), we switched to the

Figure 4. Pharmacologic profile of compound 25 in PBMCs after LPS stimulation (graph and table) and in A431 after EGF- and TNFstimulation (table).

Table 3. Lead Optimization and in Vitro Profiles of Compounds 25−34

IC50 (μM) Compd

COTa

pERKb

TNFαb

25 26 27 28 29 30 31 32 33 34

0.022 0.096 0.080 0.016 0.009 0.009 0.022 0.006 0.004 0.038

0.54

0.44

0.28 0.12 0.08 0.28 0.06 0.03 0.62

0.27 0.13 0.07 0.29 0.06 0.03 1.25

hWB TNFαc

LogPd

Solubilitye (μM)

hPPBf (%)

1.78

4.2

>99

2.5 1.01 0.60 >10 0.20 0.08

4.6 4.4 3.3 5.1 3.3 3.2

99 >99 98 >99 97

a

Enzymatic COT kinase assay. bPBMCs. cHuman whole blood. dOctanol/water partition coefficient.21 eEquilibrium solubility at pH 6.8/pH 4.0 (assay details in the Supporting Information). fHuman plasma protein binding assessed by rapid quilibrium dialysis (assay details in the Supporting Information). E



Article

conformation in 28 involves an unfavorable dihedral angle, which may explain the potency increase of the α−Me substitution in 29−33. The core and aromatic side chains of 28 engage in tight stacking interactions with P-loop residues Trp132, Ile134, and Val152. The amino group of the benzothiazole hydrogen bonds to a water occupying the Mg coordination site between Asn258 and Asp270. Although the C-terminal part of the pocket resembles a typical active kinase conformation (e.g., pdb code: 1atp), the hinge is distorted due to the flexibility of the gk+2/3 Asp209Gly210 motif. This creates space for a water molecule that hydrogen bonds the 7-N of the core and the buried carbonyl of Glu208 and connects to the two conserved waters hydrogen bonding the DFG motif. Due to its location in a region that is hydrophobic in most other kinases, the polar 7-N acceptor carries a desolvation penalty that may be responsible for the increased selectivity of the naphthyridines over the quinoline analogs. To visualize the untypical hinge conformation, Figure 6 shows the crystal structure of COT in complex with 28 overlaid with its closest phylogenetic neighbor, NIK.

phenyl analog 26, which turned out to be roughly 4-fold less potent (Table 3). The 2-methyl-benzo[d]thiazole substituent in position 1 (compound 27) proved to be an equally potent replacement to the quinoline moiety of analog 26. With the goal to increase the solubility of the scaffold, we investigated whether the 2-methyl group of the benzo[d]thiazole could be used as an exit vector for the introduction of an ionizable moiety. Although the weakly basic 2-morpholinethylamine substituted derivative 28 did only result in an improved solubility at pH 1, the 5-fold gain in enzymatic potency was very encouraging and suggestive of an additional protein− ligand interaction. Compound 28 was the first imidazonaphthyridine that could be cocrystallized with human COT protein and for which an Xray structure (2.7 Å) was solved (Figure 5).

Figure 5. Top (a) and side (b) views of the crystal structure of imidazonaphthyridine (28) (blue sticks) in complex with human COT kinase protein (66−395) at 2.7 Å resolution. Key residues are shown as sticks. The unique P-loop inset (residues 133−144) is colored in red, and polar interactions of 28 with the protein and selected water molecules are shown as red dashed lines. The inset in part b shows a comparison of the P-loop conformations in COT·28 versus a indazole cocrystal structure (green, only β1−β2 and ligand shown, pdb code: 4y85).

Figure 6. Overlay of the X-ray structure of imidazonaphthyridine (28) (blue) with human COT kinase protein (66−395, white) and with the closest related kinase, NIK (MAP3K14, magenta (pdb code: 4dn5)). Key residues are shown as sticks. The hinge in COT is distorted, enabling the typical hydrogen bond pattern with a shifted core of 28.

The X-ray structure of 28 in complex with COT revealed an unusual binding mode and several features that were not expected from the sequence alignment. Strikingly, a unique 15amino acid insert is found before the GxGxxG motif of the Ploop, disrupting the typical β1/β2 β sheet. Another unexpected observation was the frame shift in helix-αC, with Asp178, instead of Glu180, forming the salt bridge with Lys167. COT shares this substitution with MEK4−7 and M3K12/13, as observed in the structures of MEK4 (pdb code: 3alo, DOI: 10.1016/j.bbrc.2010.08.071), MEK6 (pdb code: 3vn9, DOI: 10.1093/jb/mvs023) and MEK7 (pdb code: 2dyl). The P-loop insert wraps around the ligand and creates an unusual ATP pocket shape that bears little resemblance to our previously published COT·indazole cocrystal structure (Figure 5b inset).19 The P-loop conformation in complex with 28 exposes the morpholine ethylamine side chain to solvent, explaining why substitutions in this region are tolerated. This contrasts with the potency loss that we observed upon introduction of solubilizing groups to our previous scaffolds which induced much more buried P-loop pockets. Whereas the morpholine was originally intended as solubilizing group, it was found to interact with the catalytic Lys167 via a water molecule, explaining the potency increase of 28 compared to 27. We assume that pyridine analogues 30, 32, and 33 form a similar water mediated interaction with Lys167 (see the Supporting Information). The aliphatic side chain

The nanomolar enzymatic potency of 28 nicely translated into a potent cellular blockade of pERK (IC50: 0.28 μM) and TNFα (IC50: 0.27 μM) in human PBMCs. Compound 29, bearing an additional α-methyl substituent at the 2morpholinethylamine group, further improved enzymatic activity, potentially a consequence of the less constrained conformation of the aliphatic side chain caused by the α−Me group. However, a significant potency drop for 28 and 29 was observed comparing TNFα release data from human PBMCs and human whole blood, which was hypothesized to relate to the high lipophilicity of the compounds. Reduction of LogP by more than one log unit could be accomplished by replacing the phenyl by a 4-pyridyl substituent in position 8. Resulting compound 30 was the first derivative of the series showing submicromolar activity (IC50: 0.60 μM) in the human whole blood assay, likely a consequence of the improved physicochemical profile. This hypothesis is also supported by comparing 30 to the far more lipophilic and in human whole blood inactive 2-thiophene analog 31. Based on the similar enzymatic potency of the quinoline 26 and benzo[d]thiazole 27, we investigated the feasibility of attaching the aliphatic 1morpholinopropan-2-amine side chain also to the quinoline bicycle. Resulting compound 32 showed a slightly superior profile to the corresponding benzo[d]thiazole analog 30, particularly an increased activity in the human whole blood F



Article

assay (IC50: 0.20 μM) and a further improvement in solubility. Separation of racemic 32 by chiral HPLC afforded the two enantiomers, of which the R-eutomer 33 showed around 10fold higher potency in the COT biochemical assay (IC50: 0.004 μM) compared to the S-eutomer 34 (IC50: 0.038 μM). The Reutomer 33 and racemic 32 were screened against an in-house panel of kinases, confirming a high selectivity, as already observed for compound 25 (Supporting Information). Compound 33 was further subjected to pharmacokinetic profiling in rats. After administration of an intravenous dose of 1 mg/kg, a very high plasma clearance (221 mL·min−1· kg−1) was measured, vastly exceeding the hepatic blood flow. Extension of rat PK profiling to a wider set of compounds resulted in a surprisingly big difference in in vivo clearance for the two close analogs, the 4-pyridyl derivative 30 (297 mL· min−1·kg−1) and corresponding 2-thiophene 31 (40 mL·min−1· kg−1), which was not expected based on the similarly short halflife (90% determined by HPLC/MS). Subsequent structure elucidation by 1H and 13C NMR

Figure 7. Biotransformation of 30 in vitro in rat liver microsomes and in vivo in bile-duct cannulated rats, and relative peak areas of 30 and metabolites detected in the different matrices. aIncubation period. b Sampling time or period in hours post dose. cSum of relative peak areas of metabolites modified only at the side chain. dSum of relative peak areas of metabolites modified at the side chain and the aromatic core.

techniques revealed that oxidation occurred at position 4 of the 1,3,5,7-tetraazacyclopenta[a]naphthalen moiety (Figure 9). As nitrogen-containing aromatic heterocycles are described in the literature as typical substrates for aldehyde oxidase (AO) mediated metabolism, we hypothesized that AO might also be the responsible enzyme causing the high clearance in this case.24,25 In order to test our hypothesis, 30 was incubated in vitro with rat and human S9 liver homogenate fractions without addition of the cofactor NADPH and screened for metabolite M1. The fast disappearance of 30 with concomitant formation of M1 confirmed the independence of this conversion from cytochrome P450 isoenzymes, since these enzymes require NADPH. In order to further confirm whether the oxidation was AO-mediated, the AO specific inhibitor raloxifene was added to the incubation at two different concentrations. Raloxifene is described as a very potent inhibitor of human AO (IC50 0.003− 0.008 μM) but a less potent inhibitor of rat aldehyde oxidases (IC50 1.1−2.8 μM).25 Indeed, the formation of M1 was only poorly inhibited in the rat liver S9 fraction at a raloxifene concentration of 10 μM (not shown), whereas its formation was dose-dependently inhibited in the human liver S9 fraction. This investigation unambiguously proved that oxidation by AO led to the formation of metabolite M1 (Figure 10). Based on the reported very low activity of AO in the liver of dog, we compared the pharmacokinetic profile of the more potent compound 32 in rats and dogs first in vitro in the liver S9 fraction and in liver microsomes, and subsequently in vivo after intravenous (i.v.) and oral (p.o.) compound administration (Table 4).25 Whereas in rat a high clearance resulting in a very low exposure was confirmed also for derivative 32, the pharmacoG



Article

a positive control (Meloxicam at 0.2 mg/kg), vehicle, and two doses of 32: low dose at 5 mg/kg and high dose at 15 mg/kg. This lameness is acute and short-lived, and responsive to commercially available nonsteroidal anti-inflammatory drugs. The degree of lameness is measured on a Force Plate apparatus. The vertical force applied by the limb when trotting over the Force Plate is considered representative of the degree of lameness. The results are displayed in Figure 11, wherein the time post-treatment required to reach 60% of the prelameness vertical peak force of the affected limb is shown. Here, we show a clear and statistically significant (p < 0.5) benefit for the high dose of 32 (AUC0−10.5h: 4575 ± 2385 nmol· h) over vehicle. There is no statistical difference between the vehicle and the low dose of 32 (AUC0−10.5h: 2057 ± 625 nmol· h); neither is there a statistical difference between the high dose of 32 and the positive control meloxicam (AUC0−10.5h: 13975 ± 1941 nmol·h).

4. CONCLUSIONS The discovery and chemical optimization of a novel series of selective imidazonaphthyridine COT kinase inhibitors has been described. Improvement of the physicochemical profile was absolutely crucial to achieve low nanomolar activity in the human whole blood assay. The X-ray cocrystal structure of an advanced candidate revealed an unusual binding mode, providing a rationale for the excellent kinase selectivity and enzymatic potency. Gained insights can serve as invaluable starting points for structure-based drug design of COT kinase inhibitors and facilitate future drug development. A lack of correlation between in vitro liver microsomal clearance and in vivo rat clearance could be attributed to a dominating aldehyde oxidase mediated metabolism in vivo. Moreover, pharmacodynamic evaluation of compound 32 in dogs showed a clear amelioration of inflammatory events in vivo, which further supports the notion that COT is an appropriate and attractive therapeutic target for inflammatory diseases.

Figure 8. Biotransformation of 31 in vitro in rat liver microsomes and in vivo in bile-duct cannulated rats, and relative peak areas of 31 and metabolites detected in the different matrices. aIncubation period. b Sampling time or period in hours post dose. cSum of relative peak areas of metabolites modified only at the side chain. dSum of relative peak areas of GSH, cysteinylglycine, and cysteine adducts at thiophene.

5. EXPERIMENTAL SECTION 5.1. Chemistry. All reagents and solvents were purchased from commercial suppliers and used without further purification or were prepared according to published procedures. All reactions were performed under inert conditions (argon) unless otherwise stated. 1H NMR spectra were recorded on a Bruker 400 MHz or a Bruker 600 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm) relative to an internal solvent reference. Significant peaks are tabulated in the order multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quintet; m, multiplet; br, broad), coupling constants, and number of protons. Final compounds were purified to ≥95% purity as assessed by analytical liquid chromatography with one of the following methods. 5.1.1. Method A. Waters Acquity UPLC−MS; column HSS T3 1.8 μm, 2.1 mm × 50 mm; A, water +0.05% formic acid +3.75 mM ammonium acetate; B, acetonitrile + 0.04% formic acid; 2−98% B in 1.4 min, 98% B 0.45 min, flow 1.2 mL/min; column temperature 50 °C. 5.1.2. Method B. Waters Acquity UPLC−MS; column HSS T3 1.8 μm, 2.1 mm × 50 mm; A, water +0.05% formic acid + 3.75 mM ammonium acetate; B, acetonitrile + 0.04% formic acid; 5−98% B in 1.4 min, 98% B 0.45 min, flow 1.0 mL/min; column temperature 60 °C. The accurate mass analyses were performed by using electrospray ionization in positive mode after separation by liquid chromatography. Enantiomerically enriched compounds were obtained by chiral preparative chromatography: Gilson PLC 2020; column Chiralcel OD-H 5 μm, 250 × 20 mm; n-heptane:EtOH:MeOH 75:15:10, flow

Figure 9. Chemical structure of the isolated metabolite M1 with key 1 H (red) and 13C (blue) chemical shifts and important HMBC and ROESY correlations.

kinetic profile in dog was significantly better. Clearance from blood was moderate, resulting in a prolonged mean residence time (MRT), terminal elimination half-life, and significant exposure after i.v. and p.o. dosing. Based on the promising PK profile in dog, the efficacy of 32 was evaluated in the dog uric acid-induced synovitis model.26 Preceding in vitro assessment of 32, in uric-acid, stimulated macrophages demonstrated potent inhibition of the formation of the pro-inflammatory cytokine interleukin-1β (IC50: 0.2 μM) by this compound. In the in vivo model, the test compound was examined for its ability to counteract lameness as a result of inflammation and pain induced by injection of 15 mg of uric acid crystals in suspension into one stifle joint (Figure 11). Eight dogs were employed in a four-phase crossover study with H



Article

Figure 10. Turnover of compound 30 (A) and formation of metabolite M1 (B) after incubation with human S9 fractions (without NADPH) in the presence and absence of the aldehyde oxidase inhibitor raloxifene. mL/min. LC-HRMS analyses were performed by using electrospray ionization in positive ion modus after separation by liquid chromatography (Nexera from Shimadzu). The elemental composition was derived from the mass spectra acquired at the high resolution of about 30′000 on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). The high mass accuracy below 1 ppm was obtained by using a lock mass. 5.2. General Procedures. 5.2.1. Chloro-Dehydroxylation of Phenols. 5.2.1.1. Procedure 1. A mixture of the naphthyridine 4-ol (1.0 equiv) and tripropylamine (2.0 equiv) in DMF (0.3 M) was heated to 75 °C. POCl3 (1.0 equiv) was added and the mixture stirred for 1 min at 75 °C and then immediately cooled to 0 °C in an ice bath. Diethyl ether (2.5 mL) was added and the formed suspension stirred at 0 °C for 15 min. The solid was filtered and the filter cake washed with cold diethyl ether and dried. 5.2.1.2. Procedure 2. Triethyl amine (5.0 equiv) was added dropwise to a suspension of the naphthyridine 4-ol (1.0 equiv) and POCl3 (20 equiv) at −15 °C. The mixture was allowed to warm to rt and stirred at rt or until the reaction was complete (typically ca. 30 min). The reaction mixture was concentrated in vacuo and the residue taken up in CH2Cl2 and diluted with H2O at 0 °C. The pH was adjusted to 8 with sat. aq. K2CO3 soln., the layers were separated and the organic layer concentrated under reduced pressure to provide the crude chloride which was taken for the next step without further purification (typically ca. 90% pure). 5.2.2. Suzuki Reaction. 5.2.2.1. Procedure 1. A microwave vial was charged with 6-chloro-3-nitro-1,7-naphthyridin-4-ol (10) (1.0 equiv), boronic acid (1.5 equiv) and K2CO3 (2.1 equiv) in DMA/H2O 3:1 (0.1M) and the mixture was degassed with Argon. Pd(OAc)2 (0.2 equiv) and S-Phos (0.4 equiv) were added, the vial sealed and the reaction mixture heated to 100−120 °C for the indicated time period. The mixture was cooled to rt, filtered through a pad of Celite, the filter cake washed with MeOH (2x) and the filtrate concentrated. Water was added and the pH adjusted to 5 by careful addition of acetic acid. The precipitated solid was filtered, the filter cake washed with H2O (3x) and dried. The solid was triturated with ethyl acetate, filtered and dried to provide the title compound. 5.2.2.2. Procedure 2. A microwave vial was charged with 6-chloro3-nitro-1,7-naphthyridin-4-ol (10) (1.0 equiv), boronic acid (1.2−1.3 equiv) and Cs2CO3 (1.6 equiv) in DME/H2O/EtOH 7:3:2 and the mixture was degassed with Argon. PdCl2(dppf)2 (0.11 equiv) was added, the vial sealed and the reaction mixture heated to 140 °C for the indicated time period. The mixture was cooled to rt, filtered through a pad of Celite, the filter cake washed with EtOH (2x). The reaction mixture was concentrated in vacuo and the residue taken up in H2O, extracted twice with ethyl acetate, dried (Na2SO4), filtered and concentrated under reduced pressure. Title compound was obtained after purification by prep HPLC (Waters; column Sunfire C18 5 μm, 30 mm × 100 mm; A, water +0.1% TFA; B, acetonitrile +0.1% TFA; 15−35% B in 15 min.

Table 4. In Vitro and in Vivo Pharmacokinetic Profiles of Compound 32 in Rat and Dog Compound 32 t1/2 (min), S9−NADPH t1/2 (min), liver microsomes doses i.v./p.o., (mg/kg) CL (mL·min−1kg−1) t1/2 terminal (h) MRT (h) AUCi.v.c (nmol·h) AUCp.o.c (nmol·h) Cmaxc (nM) Tmax (h) F (%)

rata in vitro 120 10 0.1/0.3 25 ± 2 3.2 ± 0.7 2.4 ± 0.1 1′570 ± 130 957 ± 310 61 ± 20 143 ± 97 61 ± 20

a c

n = 3 Sprague−Dawley male rats/group. bn = 3 Beagle dogs/group. Dose-normalized values.

Figure 11. Time needed for the z peak force of the affected limb to reach 60% of that measured prior to uric acid injection (time to recovery). (Short time to recovery reflects better efficacy.) Vehicle, 2 doses of 32, and a positive control (meloxicam) were tested. 13 mL/min. Optical purity of the compounds was assessed by chiral analytical chromatography: Merck LaChrom HPLC system; Chiralcel OD-H 5 μm, 250 × 4.6 mm; n-heptane:EtOH:MeOH 70:20:10, flow 1 I



Article

5.2.2.2.1. 6-chloro-3-nitro-1,7-naphthyridin-4-ol (10). A solution of 6-chloro-1,7-naphthyridin-4-ol 9 (360 g, 2.0 mol, 1.0 equiv) in propanoic acid (3.60 l) was heated to 130 °C. HNO3 (236 mL, 2.4 mol, 1.2 equiv) was added dropwise at 130 °C. The reaction mixture was kept for 1 h at 130 °C and was then cooled to rt. The precipitated solid was separated by filtration, the filter cake washed with diethyl ether (3 × 2l) and dried to give the title compound 10 (252 g, 56%) as a pale yellow solid which was used without further purification. UPLCMS tR,B: 0.55 min, 225, 227 [M + H], UPLC-purity: 95%; 1H NMR (600 MHz, DMSO-d6) δ 13.54 (br. s, 1H), 9.36 (s, 1H), 8.97 (s, 1H), 8.08 (s, 1H). 5.2.2.2.2. 3-nitro-6-(thiophen-3-yl)-1,7-naphthyridin-4-ol (12). Title compound 12 was obtained as beige solid (1.08 g, 70%, ca. 90% pure) according to the general Suzuki procedure 1 starting from 10 (1.13 g, 5.0 mmol) and thiophen-3-ylboronic acid (1.00 g, 7.5 mmol). UPLC-MS tR,B: 0.73 min, 273 [M + H], UPLC-purity: 89%; 1H NMR (400 MHz, DMSO-d6) δ 13.33 (br. s, 1H), 9.31 (s, 1H), 9.17 (s, 1H), 8.46 (s, 1H), 8.36 (dd, J = 3.0, 1.3 Hz, 1H), 7.87 (dd, J = 5.1, 1.3 Hz, 1H), 7.71 (dd, J = 5.0, 3.0 Hz, 1H). 5.2.2.2.3. 2-(4-((3-nitro-6-(thiophen-3-yl)-1,7-naphthyridin-4-yl)amino)phenyl)acetonitrile (15). The chloride (270 mg, 0.93 mmol, 1.0 equiv), prepared from 12 in 90% yield according to general chlorination procedure 2, was dissolved in anhydrous DMF (6 mL). 4 M HCl in dioxane (28 μL, 0.11 mmol, 0.1 equiv), followed by 2-(4aminophenyl)acetonitrile (185 mg, 1.3 mmol, 1.5 equiv) were added. The reaction mixture was stirred at rt overnight and was then quenched by addition of sat. aq. NaHCO3 soln. to pH 8 and concentrated under reduced pressure. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−10% MeOH) to provide the title compound 15 (274 mg, 75%) as brown solid. UPLC-MS tR,B: 1.08 min, 387 [M + H], UPLC-purity: 98%; 1H NMR (400 MHz, DMSOd6) δ 10.28 (s, 1H), 9.37 (s, 1H), 9.10 (s, 1H), 8.73 (s, 1H), 8.21 (dd, J = 2.6, 1.4 Hz, 1H), 7.81−7.72 (m, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 4.09 (s, 2H). 5.2.2.2.4. 2-(4-(8-(thiophen-3-yl)-1H-imidazo[4,5-c][1,7]naphthyridin-1-yl)phenyl)acetonitrile (24). SnCl2 (402 mg, 2.1 mmol, 3.0 equiv) was added to a solution of 15 (274 mg, 0.71 mmol) in conc. HCl/EtOH 3:2 (10 mL) and the mixture stirred for 1 h at rt. The mixture was cooled to 0 °C and the pH adjusted to 11 with 30% aq. NaOH. The mixture was partiotioned between CH2Cl2 (20 mL) and H2O (20 mL), the layers separated and the organic phase concentrated in vacuo to provide the crude aniline as brown foam (218 mg). The crude material was taken up in triethyl orthoformate (4 mL) and the mixture heated in a sealed microwave vial to 150 °C for 25 min. The reaction mixture was cooled to rt and the volatiles removed in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−10% MeOH), followed by a slurry of the chomatographed material in CH2Cl2/diethyl ether 3:1 (2 mL) to provide 24 (39.0 mg, 14% over 2 steps) as beige solid. UPLC-MS tR,B: 0.88 min, 367 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 9.50 (d, J = 0.8 Hz, 1H), 9.45 (s, 1H), 8.82 (s, 1H), 7.94−7.90 (m, 2H), 7.89 (dd, J = 3.1, 1.3 Hz, 1H), 7.84−7.80 (m, 2H), 7.63 (dd, J = 5.0, 3.0 Hz, 1H), 7.45−7.42 (m, 1H), 7.26 (dd, J = 5.1, 1.3 Hz, 1H), 4.35 (s, 2H). 5.2.2.2.5. 3-Nitro-N-(quinolin-6-yl)-6-(thiophen-3-yl)-1,7-naphthyridin-4-amine (16). The crude chloride (160 mg, 0.55 mmol, 1.0 equiv), prepared from 12 in 76% yield according to general chlorination procedure 1, was dissolved in anhydrous DMF (15 mL) and triethylamine (1.70 mL, 12 mmol, 10.0 equiv) and 6-aminoquinoline (138 mg, 0.96 mmol, 1.8 equiv) were added. The mixture was stirred at rt for 30 min and then poured onto a mixture of ice water and EtOAc/Et2O 1:1 (20 mL). The precipitated solid was filtered, washed with H2O (5 mL) and diethyl ether (2 × 5 mL) and dried under reduced pressure. The title compound 16 was obtained as brown solid (140 mg, 64%). UPLC-MS tR,B: 1.04 min, 400 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, Chloroform-d) δ 10.86 (s, 1H), 9.55 (s, 1H), 9.46 (s, 1H), 9.03 (dd, J = 4.3, 1.7 Hz, 1H), 8.27 (d, J = 8.9 Hz, 1H), 8.17−8.10 (m, 1H), 7.74 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 9.0, 2.5 Hz, 1H), 7.57 (s, 1H), 7.52 (dd, J = 8.3, 4.3 Hz, 1H), 7.46 (dd, J =

3.1, 1.3 Hz, 1H), 7.14 (dd, J = 5.1, 3.0 Hz, 1H), 6.75 (dd, J = 5.1, 1.3 Hz, 1H). 5.2.2.2.6. 1-(quinolin-6-yl)-8-(thiophen-3-yl)-1H-imidazo[4,5-c][1,7]naphthyridine (25). A mixture of 16 (110 mg, 0.28 mmol) and 10% Pd/C (15.0 mg) in THF/EtOH 1:1 (14 mL) was stirred under H2 (1 atm.) for 1.5 h at rt. The reaction mixture was filtered over Celite, the filter cake washed with EtOH (3 × 2 mL) and the filtrate concentrated in vacuo to provide the aniline (106 mg) as brown solid which was used for the next step without further purification. The crude material was taken up in triethyl orthoformate (4 mL) and the mixture heated in a sealed microwave vial to 150 °C for 25 min. The reaction mixture was cooled to rt and the volatiles removed in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−10% MeOH) to provide 25 (38.0 mg, 36% over 2 steps) as beige solid. UPLC-MS tR,B: 0.88 min, 380 [M + H], UPLC-purity: 98%; 1H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 9.49 (s, 1H), 9.16 (dd, J = 4.2, 1.7 Hz, 1H), 8.93 (s, 1H), 8.61−8.57 (m, 2H), 8.44 (d, J = 8.9 Hz, 1H), 8.27 (dd, J = 8.9, 2.4 Hz, 1H), 7.86 (dd, J = 3.1, 1.3 Hz, 1H), 7.77 (dd, J = 8.3, 4.3 Hz, 1H), 7.59 (s, 1H), 7.56 (dd, J = 5.0, 3.0 Hz, 1H), 7.01 (dd, J = 5.0, 1.3 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 154.0, 152.9, 148.0, 147.5, 147.0, 146.9, 141.5, 140.2, 138.7, 137.0, 134.0, 132.1, 131.4, 128.6, 128.5, 128.3, 126.8, 125.4, 124.6, 123.4, 122.3, 109.0; HRMS (ESI) for C22H14N5S [M + H], caluculated 380.0964, found 380.0962. 5.2.2.2.7. 3-Nitro-6-phenyl-1,7-naphthyridin-4-ol (13). A mixture of 6-chloro-1,7-naphthyridin-4-ol (9.10 g, 40 mmol, 1.0 equiv), phenylboronic acid (6.53 g, 52 mmol, 1.3 equiv) and K2CO3 (11.2 g, 80 mmol, 2.0 equiv) in acetonitrile/H2O 3:2 (185 mL) was degassed with argon. The mixture was warmed to 50 °C and Pd(OAc)2 (362 mg, 1.6 mmol, 0.025 equiv) and S-Phos (1.34 g, 3.2 mmol, 0.05 equiv) were added. The reaction mixture was then stirred under reflux for 15 h. The mixture was cooled to 70 °C, filtered through a pad of activated charcoal and Celite and the filtrate concentrated to about 1/3 of its initial volume. The mixture was then warmed to 70 °C and the pH adjusted to 5 by careful addition of acetic acid. Ethyl acetate (20 mL) was added and the mixture left to cool to rt. The precipitated solid was filtered, the filter cake washed with ethyl acetate (2 × 10 mL) and H2O (3 × 20 mL) and dried to provide the title compound 13 (10.0 g, 94%) as gray solid. UPLC-MS tR,B: 0.32 min, 268 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSOd6) δ 13.42 (br. s, 1H), 9.31 (s, 1H), 9.21 (s, 1H), 8.51 (s, 1H), 8.19− 8.11 (m, 2H), 7.57−7.50 (m, 2H), 7.49−7.43 (m, 1H). 5.2.2.2.8. 3-Nitro-6-phenyl-N-(quinolin-6-yl)-1,7-naphthyridin-4amine (17). The chloride (160 mg, 0.56 mmol, 1.0 equiv), prepared from 13 in 80% yield according to the general chlorination procedure 1, was dissolved in absolute DMF (6 mL) and triethylamine (0.78 mL, 5.6 mmol, 10 equiv) and 6-aminoquinoline (161 mg, 1.1 mmol, 2.0 equiv) were added. The mixture was stirred at rt for 30 min and was then quenched by addition of sat. aq. NaHCO3 soln. (20 mL). The reaction mixture was extracted into CH2Cl2 (2 × 20 mL), the combined organics dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−20% MeOH) to provide 17 (90.0 mg, 41%) as a yellow solid. UPLC-MS tR,A: 1.05 min, 394 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.45 (s, 1H), 9.13 (s, 1H), 9.00 (s, 1H), 8.85 (s, 1H), 8.42−8.25 (m, 1H), 8.24− 8.14 (m, 2H), 8.02 (d, J = 9.0 Hz, 1H), 7.71 (s, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.56−7.44 (m, 4H). 5.2.2.2.9. 8-Phenyl-1-(quinolin-6-yl)-1H-imidazo[4,5-c][1,7]naphthyridine (26). A solution of Na2S2O4 (159 mg, 0.92 mmol, 4.0 equiv) in H2O (0.9 mL) was added to a solution of 17 (90.0 mg, 0.23 mmol, 1.0 equiv) in anh. DMF (4 mL). The mixture was stirred at rt for 20 min and the solvent removed in vacuo at 60 °C. The residual yellow solid was taken up in EtOH (1 mL) and triethylorthoformate (5 mL) and the mixture heated in a sealed vial to 150 °C for 1 h. The reaction mixture was cooled to rt and partitioned between ethyl acetate (20 mL) and H2O (10 mL). The layers were separated and the organic layer was washed with brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced J



Article

extracted with CH2Cl2 (2 × 10 mL) and the combined organics were washed with H2O (20 mL) and brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude aniline as brown solid (142 mg). The solid was suspended in triethyl orthoformate (2.4 mL), and the mixture heated to 145 °C in a sealed vial for 18 h. The reaction mixture was cooled to rt and the volatiles removed in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−5% MeOH) to provide the title compound 28 (24.0 mg, 16% over 2 steps) as red solid. UPLC-MS tR,A: 0.70 min, 508 [M + H], UPLC-purity: 99%; 1H NMR (600 MHz, DMSO-d6) δ 9.55 (s, 1H), 9.47 (s, 1H), 8.81 (s, 1H), 8.39 (t, J = 5.5 Hz, 1H), 8.25 (d, J = 1.83 Hz, 1H), 7.77−7.74 (m, 2H), 7.72−7.65 (m, 3H), 7.44−7.37 (m, 3H), 3.62 (t, J = 4.50 Hz, 6H), 2.61 (t, J = 6.5 Hz, 2H), 2.50−2.45 (m, 4H); 13C NMR (101 MHz, DMSO-d6) δ 168.5, 154.4, 153.8, 150.7, 147.1, 147.0, 139.9, 138.9, 138.8, 132.6, 132.1, 129.5, 129.3, 128.8, 126.8, 125.2, 122.4, 120.6, 118.7, 109.4, 66.7, 57.5, 53.8, 41.8; HRMS (ESI) for C28H26ON7S [M + H], calculated 508.1914, found 508.1912. 5.2.2.2.14. N2-(1-morpholinopropan-2-yl)-N6-(3-nitro-6-phenyl1,7-naphthyridin-4-yl)benzo[d]thiazole-2,6-diamine (20). A solution of 44 (221 mg, 0.76 mmol, 1.2 equiv) in CH2Cl2 (2 mL) and triethylamine (0.88 mL, 6.30 mmol, 10.0 equiv) were added to a suspension of the chloride (180 mg, 0.63 mmol, 1.0 eq, prepared from 13 in quantitative yield according to the general chlorination procedure 2) in CH2Cl2 (2.7 mL) at rt. The reaction mixture was concentrated under reduced pressure and the residue purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−10% MeOH) to provide 20 as red solid (250 mg, 73%) as red solid. UPLC-MS tR,A: 0.87 min, 542 [M + H], UPLCpurity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.33 (s, 1H), 9.05 (s, 1H), 8.75 (s, 1H), 8.07 (d, J = 7.0 Hz, 2H), 7.96 (s, 1H), 7.56 (s, 1H), 7.52−7.42 (m, 3H), 7.34 (d, J = 8.6 Hz, 1H), 7.14− 6.95 (m, 1H), 4.17−3.94 (m, 1H), 3.61−3.44 (m, 4H), 2.42 (br. s, 4H), 2.30 (dd, J = 12.5, 6.7 Hz, 2H), 1.21 (d, J = 6.5 Hz, 3H). 5.2.2.2.15. N-(1-morpholinopropan-2-yl)-6-(8-phenyl-1Himidazo[4,5-c][1,7]naphthyridin-1-yl)benzo[d]thiazol-2-amine (29). A mixture of 20 (250 mg, 0.46 mmol) and Raney-Ni in MeOH (4 mL) was stirred under H2 (1 atm.) for 5 h at rt. The reaction mixture was filtered over Celite, the filter cake washed with MeOH (3 × 2 mL) and the filtrate concentrated in vacuo to provide the aniline (190 mg) as red solid which was used for the next step without further purification. The crude material was taken up in triethyl orthoformate (2.4 mL) and the mixture heated in a sealed microwave vial to 150 °C for 18 h. The reaction mixture was cooled to rt and the volatiles removed in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2/ MeOH (0−15% MeOH) to provide 29 (160 mg, 83% over 2 steps) as beige solid. UPLC-MS tR,A: 0.73 min, 522 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 9.44 (s, 1H), 8.79 (s, 1H), 8.28 (d, J = 7.8 Hz, 1H), 8.22 (d, 1H), 7.80−7.68 (m, 2H), 7.69−7.59 (m, 3H), 7.39 (m, 3H), 4.30−4.13 (m, 1H) 3.58 (br. s, 4H), 2.60−2.50 (m, 4H), 2.41−2.30 (m, 2H), 1.26 (d, J = 6.0 Hz, 3H). 5.2.2.2.16. 3-Nitro-6-(pyridin-4-yl)-1,7-naphthyridin-4-ol (14). The title compound 14 was obtained from a Suzuki reaction as a pale brown solid (2.18 g, 78%) according to the general Suzuki procedure 1 starting from 10 (2.00 g, 8.9 mmol) and pyridine 4-boronic acid (1.64 g, 13 mmol) with stirring at 120 °C for 1 h. UPLC-MS tR,B: 0.43 min, 269 [M + H], UPLC-purity: 85%; 1H NMR (400 MHz, DMSO-d6) δ 13.50 (br. s, 1H), 9.30 (s, 1H), 9.25 (s, 1H), 8.78−8.72 (m, 2H), 8.70 (s, 1H), 8.20−8.13 (m, 2H). 5.2.2.2.17. N2-(1-morpholinopropan-2-yl)-N6-(3-nitro-6-(pyridin4-yl)-1,7-naphthyridin-4-yl)benzo[d]thiazole-2,6-diamine (21). A suspension of the chloride (125 mg, 0.41 mmol, 1.0 equiv) prepared from 14 in 55% yield according to general chlorination procedure 2 and triethyl amine (0.23 mL, 1.6 mmol, 4.0 equiv) in anhydrous CH2Cl2 (3 mL) was cooled to 0 °C and a solution of 44 (1.40 mg, 0.43 mmol, 1.05 equiv) in anhydrous CH2Cl2 (1 mL) was added dropwise. The layers were separated and the aq, phase was extracted with CH2Cl2 (2 × 10 mL). The combined organics were washed with H2O (10 mL) and brine (10 mL), dried (Na2SO4), filtered and concentrated in vacuo. The residue was triturated with CH2Cl2/diethyl ether 1.1 (10 mL) at

pressure. The residue was triturated with H2O (2 mL), the solid filtered and the filter cake washed with diethyl ether (3 × 2 mL) and dried to provide 26 (25.0 mg, 29%) as a beige solid. UPLC-MS tR,A: 0.89 min, 374 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 9.56 (d, J = 0.9 Hz, 1H), 9.50 (s, 1H), 9.14 (dd, J = 4.3, 1.7 Hz, 1H), 8.94 (s, 1H), 8.62 (d, J = 2.4 Hz, 1H), 8.62−8.55 (m, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.26 (dd, J = 8.9, 2.4 Hz, 1H), 7.76 (dd, J = 8.3, 4.2 Hz, 1H), 7.69 (d, J = 0.9 Hz, 1H), 7.68−7.60 (m, 2H), 7.36−7.26 (m, 3H). 5.2.2.2.10. 2-methyl-N-(3-nitro-6-phenyl-1,7-naphthyridin-4-yl)benzo[d]thiazol-6-amine (18). The chloride (295 mg, 1.0 mmol), prepared from 13 in 80% yield according to the general chlorination procedure 2, was dissolved in absolute DMF (6 mL) and 4 M HCl in dioxane (31 μL, 0.12 mmol, 0.1 equiv), followed by 2-methylbenzo[d]thiazol-6-amine (262 mg, 1.5 mmol, 1.5 equiv). The mixture was stirred at rt overnight and was then quenched by addition of sat. aq. NaHCO3 soln. (2 mL) and concentrated under reduced pressure. The residue was purified by FC on SiO2 eluting with CH2Cl2 containing 3% AcOH and MeOH (0−10% MeOH) to provide 18 (451 mg, 79%, ca. 75% pure) as brown solid. UPLC-MS tR,B: 1.15 min, 413 [M + H], UPLC-purity: 75%; 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 9.45 (s, 1H), 9.12 (s, 1H), 8.94 (s, 1H), 8.23−8.14 (m, 2H), 7.93 (dd, J = 5.4, 3.2 Hz, 2H), 7.59−7.48 (m, 2H), 7.36 (dd, J = 8.8, 2.2 Hz, 1H), 5.78 (br. s, 1H), 2.83 (s, 3H). 5.2.2.2.11. 2-methyl-6-(8-phenyl-1H-imidazo[4,5-c][1,7]naphthyridin-1-yl)benzo[d]thiazole (27). SnCl2 (465 mg, 2.5 mmol, 3.0 equiv) was added to a solution of 18 (451 mg, 0.82 mmol, 75% pure) in conc. HCl/EtOH 3:2 (10 mL) and the mixture stirred for 1 h at rt. The mixture was cooled to 0 °C and the pH adjusted to 11 with 30% aq. NaOH. The mixture was partitioned between CH2Cl2 (20 mL) and H2O (20 mL), the layers were separated and the organic phase was concentrated in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2 containing 3% AcOH and MeOH (0−10% MeOH) to provide the aniline as a yellow solid (213 mg). This material was taken up in triethyl orthoformate (4 mL) and the mixture heated in a sealed microwave vial to 150 °C for 25 min. The reaction mixture was cooled to rt and the volatiles were removed in vacuo. The residue was purified by a slurry in CH2Cl2/diethyl ether 2:1 (4.5 mL) to provide 27 (65.0 mg, 20% over 2 steps) as a pale yellow solid. UPLC-MS tR,B: 0.98 min, 394 [M + H], UPLC-purity: 99%; 1H NMR (400 MHz, DMSO-d6) δ 9.58 (d, J = 0.9 Hz, 1H), 9.50 (s, 1H), 8.89 (s, 1H), 8.69 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 8.5 Hz, 1H), 8.01 (dd, J = 8.5, 2.2 Hz, 1H), 7.75−7.70 (m, 2H), 7.68 (d, J = 0.9 Hz, 1H), 7.43−7.38 (m, 3H), 2.97 (s, 3H). 5.2.2.2.12. N2 -(2-morpholinoethyl)-N 6-(3-nitro-6-phenyl-1,7naphthyridin-4-yl)benzo[d]thiazole-2,6-diamine (19). A solution of 40 (177 mg, 0.64 mmol, 1.0 equiv) in CH2Cl2 (2 mL) was added at rt to a suspension of the chloride (200 mg, 0.64 mmol, 1.0 equiv), prepared from 13 in quantitative yield according to the general chlorination procedure 2) and triethylamine (0.35 mL, 2.5 mmol, 4.0 equiv) in CH2Cl2 (4 mL). The mixture was stirred for 6 h at rt and then partitioned between CH2Cl2 (10 mL) and H2O (10 mL). The layers were separated and the aq. phase was extracted with CH2Cl2 (2 × 10 mL). The combined organics were washed with H2O (10 mL) and brine (10 mL), dried (Na2SO4), filetered and concentrated in vacuo. The residue was triturated with CH2Cl2 (5 mL) to provide 19 (257 mg, 76%) as a red solid. UPLC-MS tR,B: 0.86 min, 528 [M + H], UPLC-purity: 93%; 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.34 (s, 1H), 9.05 (s, 1H), 8.76 (s, 1H), 8.14−8.01 (m, 3H), 7.56 (s, 1H), 7.53−7.41 (m, 3H), 7.36 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 3.58 (t, J = 4.6 Hz, 4H), 3.50 (q, J = 6.0 Hz, 2H), 2.60−2.51 (m, 2H), 2.42 (t, J = 4.5 Hz, 4H). 5.2.2.2.13. N-(2-morpholinoethyl)-6-(8-phenyl-1H-imidazo[4,5c][1,7]naphthyridin-1-yl)benzo[d]thiazol-2-amine (28). Acetic acid (1.0 mL, 18 mmol, 37 equiv), H2O (1.0 mL, 58 mmol, 118 equiv) and Fe (272 mg, 4.9 mmol, 10 equiv) were added to a solution of 19 (84.0 mg, 0.14 mmol, 1.0 equiv) in THF (5 mL). The mixture was heated to 60 °C for 15 min, cooled to rt and partitioned between CH2Cl2 (20 mL) and H2O (20 mL). The pH was adjusted to 11 with sat. aq. K2CO3 soln. and the layers were separated. The aq. phase was K



Article

rt. The solid was filtered and dried to provide 21 (153 mg, 68%) as an orange solid. UPLC-MS tR,B: 0.75 min, 543 [M + H], UPLC-purity: 99%; 1H NMR (600 MHz, DMSO-d6) δ 10.49 (s, 1H), 9.44 (s, 1H), 9.12 (s, 1H), 9.02 (s, 1H), 8.74 (d, J = 5.1 Hz, 2H), 8.09−8.05 (m, 2H), 8.04 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 2.3 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.10 (dd, J = 8.6, 2.3 Hz, 1H), 4.15−4.03 (m, 1H), 3.57 (t, J = 4.8 Hz, 4H), 2.55−2.48 (m, 1H), 2.44 (s, 4H), 2.31 (dd, J = 12.4, 6.7 Hz, 1H), 1.23 (d, J = 6.4 Hz, 3H). 5.2.2.2.18. N-(1-morpholinopropan-2-yl)-6-(8-(pyridin-4-yl)-1Himidazo[4,5-c][1,7]naphthyridin-1-yl)benzo[d]thiazol-2-amine (30). Acetic acid (0.60 mL, 10 mmol, 37 equiv), H2O (0.60 mL, 33 mmol, 118 equiv) and Fe (157 mg, 2.8 mmol, 10 equiv) were added to a solution of 21 (153 mg, 0.28 mmol, 1.0 equiv) in THF (3 mL). The reaction mixture was heated to 60 °C for 20 min, cooled to rt and stirred for additional 2 h for the completion of the reaction. The mixture was partitioned between CH2Cl2 (20 mL) and H2O (20 mL). The pH was adjusted to 11 with sat. aq. K2CO3 soln. and the layers were separated. The aq. phase was extracted with CH2Cl2 (2 × 10 mL) and the combined organics washed with H2O (20 mL) and brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude aniline as a brown solid (155 mg). The solid was suspended in triethyl orthoformate (2.4 mL), and the mixture heated to 145 °C in a sealed vial for 30 min. The reaction mixture was cooled to rt and the volatiles removed in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0− 10% MeOH) to provide 30 (84.0 mg, 51% over 2 steps) as a pale brown solid. UPLC-MS tR,B: 0.61 min, 523 [M + H], UPLC-purity: 97%; 1H NMR (600 MHz, DMSO-d6) δ 9.61 (s, 1H), 9.53 (s, 1H), 8.85 (s, 1H), 8.65−8.60 (m, 2H), 8.32 (d, J = 7.9 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 7.84 (s, 1H), 7.71−7.63 (m, 4H), 4.22 (br. s, 1H), 3.58 (br. s, 4H), 2.60−2.41 (m, 4H), 2.41−2.35 (m, 2H), 1.32−1.25 (m, 3H); 13C NMR (101 MHz, DMSO-d6) δ 168.0, 154.6, 154.2, 150.8, 147.9, 147.8, 147.3, 145.8, 140.0, 139.6, 132.7, 131.9, 128.6, 125.2, 122.2, 120.9, 120.5, 118.7, 111.0, 66.7, 64.0, 54.2, 48.2, 19.5; HRMS (ESI) for C28H27ON8S [M + H] calculated 523.2023, found 523.2025. 5.2.2.2.19. 3-nitro-6-(thiophen-2-yl)-1,7-naphthyridin-4-ol (11). Title compound 11 was obtained as a yellow solid (283 mg, 78%) by Suzuki reaction according to the general Suzuki procedure 1 starting from 10 (400 mg, 1.8 mmol) and thiophen-2-ylboronic acid (340 mg, 2.7 mmol) with stirring at 120 °C for 1 h. UPLC-MS tR,B: 0.71 min, 274 [M + H], UPLC-purity: 90%; 1H NMR (600 MHz, DMSO-d6) δ 13.48 (br. s, 1H), 9.27 (s, 1H), 9.09 (s, 1H), 8.46 (s, 1H), 7.97 (d, J = 3.7 Hz, 1H), 7.70 (d, J = 4.9 Hz, 1H), 7.21 (dd, J = 5.0, 3.7 Hz, 1H). 5.2.2.2.20. N2-(1-morpholinopropan-2-yl)-N6-(3-nitro-6-(thiophen-2-yl)-1,7-naphthyridin-4-yl)benzo[d]thiazole-2,6-diamine (22). A suspension of the chloride 85.0 mg (0.26 mmol, 1.0 eq., 90% pure), prepared from 11 in 76% yield according to the general chlorination procedure 2, and triethyl amine (147 μL, 2.3 mmol, 4.0 equiv) in CH2Cl2 (1.5 mL) was cooled to 0 °C. A solution of 44 (85.0 mg, 0.26 mmol) was added and the resulting red solution was stirred for 1 h at rt. The reaction mixture was partitioned between CH2Cl2 (15 mL) and H2O (15 mL), the layers were separated and the organic layer washed with H2O (10 mL) and brine (10 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−5% MeOH) to provide the title compound 22 as an orange solid (84.0 mg, 54%). UPLC-MS tR,B: 0.89 min, 548 [M+H, UPLC-purity: 93%]; 1H NMR (600 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.25 (s, 1H), 9.02 (s, 1H), 8.72 (s, 1H), 8.00 (s, 1H), 7.70 (d, J = 5.0 Hz, 1H), 7.66 (d, J = 3.6 Hz, 1H), 7.58 (s, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.21 (dd, J = 5.0, 3.7 Hz, 1H), 7.12−7.05 (m, 1H), 4.15−4.03 (m, 1H), 3.56 (t, J = 4.6 Hz, 4H), 2.47−2.37 (m, 5H), 2.31 (dd, J = 12.4, 6.6 Hz, 1H), 1.22 (d, J = 6.4 Hz, 3H). 5.2.2.2.21. N-(1-morpholinopropan-2-yl)-6-(8-(thiophen-2-yl)1H-imidazo[4,5-c][1,7]naphthyridin-1-yl)benzo[d]thiazol-2-amine (31). Acetic acid (0.30 mL, 5.3 mmol, 37 equiv), H2O (0.30 mL, 16 mmol, 118 equiv) and Fe (80.0 mg, 1.4 mmol, 10 equiv) were added to a solution of 22 (84.0 mg, 0.14 mmol, 1.0 equiv) in THF (1.5 mL). The mixture was heated to 60 °C for 30 min, cooled to rt and partitioned between CH2Cl2 (10 mL) and H2O (10 mL). The pH was

adjusted to 11 with sat. aq. K2CO3 soln. and the layers were separated. The aq. phase was extracted with CH2Cl2 (2 × 10 mL) and the combined organics washed with H2O (10 mL) and brine (10 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude aniline as brown solid (72 mg). The solid was suspended in triethyl orthoformate (1.5 mL), and the mixture heated to 145 °C in a sealed vial. The reaction mixture was cooled to rt and the volatiles were removed in vacuo. The residue was purified by FC on SiO2 to provide the title compound 31 (22.0 mg, 29% over 2 steps) as a beige solid. UPLC-MS tR,B: 0.73 min, 528 [M + H], UPLC-purity: 99%; 1H NMR (600 MHz, DMSO-d6) δ 9.43 (d, J = 0.9 Hz, 1H), 9.42 (s, 1H), 8.79 (s, 1H), 8.28 (s, 1H), 8.19 (d, J = 2.1 Hz, 1H), 7.70−7.64 (m, 2H), 7.63 (dd, J = 5.0, 1.3 Hz, 1H), 7.58 (s, 1H), 7.12−7.08 (m, 2H), 4.29−4.14 (m, 1H), 3.58 (s, 4H), 2.58−2.42 (m, 5H), 2.42−2.35 (m, 1H), 1.33−1.22 (m, 3H); 13C NMR (101 MHz, DMSO-d6) δ 168.03, 154.6, 153.8, 147.1, 146.8, 146.4, 144.4, 140.0, 138.8, 132.4, 131.9, 129.1, 128.9, 128.6, 125.1, 124.5, 122.4, 120.4, 118.5, 107.4, 66.8, 64.04, 54.2, 48.2, 19.5. HRMS (ESI) for C27H26ON7S2 [M + H] calculated 528.1635, found 528.1636. 5.2.2.2.22. N2-(1-morpholinopropan-2-yl)-N6-(3-nitro-6-(pyridin4-yl)-1,7-naphthyridin-4-yl)quinoline-2,6-diamine (23). To a solution of the chloride 134 mg (0.77 mmol, 1.0 eq., 94% pure), prepared from 14 according to the general chlorination procedure 2, in DMF (6 mL), were added 47 (219 mg, 0.99 mmol) and 4 M HCl in dioxane (23 μL, 0.090 mmol). The reaction mixture was stirred overnight at rt, quenched with aq. sat NaHCO3 and concentrated in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0− 10% MeOH) to provide the title compound 23 as an orange solid (163 mg, 40%). UPLC-MS tR,B: 0.68 min, 537 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 9.47 (s, 1H), 9.15 (s, 1H), 9.08 (d, J = 1.5 Hz, 1H), 8.74 (d, J = 5.2 Hz, 2H), 8.09 (d, J = 5.7 Hz, 2H), 7.81 (d, J = 8.9 Hz, 1H), 7.58−7.45 (m, 2H), 7.37 (d, J = 8.9 Hz, 1H), 6.94 (br. s, 1H), 6.81 (d, J = 8.9 Hz, 1H), 4.49− 4.27 (m, 1H), 3.59 (br. s, 4H), 2.53−2.44 (m, 5H), 2.36−2.22 (m, 1H), 1.24 (d, J = 6.5 Hz, 3H). 5.2.2.2.23. N-(1-morpholinopropan-2-yl)-6-(8-(pyridin-4-yl)-1Himidazo[4,5-c][1,7]naphthyridin-1-yl)quinolin-2-amine (32). SnCl2 dihydrate (210 mg, 0.91 mmol, 3.0 equiv) was added to a solution of 23 (163 mg, 0.30 mmol) in conc. HCl (5 mL) and the mixture was stirred for 2.5 h at rt. The mixture was cooled to 0 °C and the pH adjusted to 11 with 30% aq. NaOH. The mixture was partitioned between CH2Cl2 (20 mL) and H2O (20 mL), the layers were separated and the organic phase was dried over Na2SO4 and concentrated in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2 containing 1% NH4OH and MeOH (0−15% MeOH) to provide the aniline as yellow solid (111 mg). This material was suspended in triethyl orthoformate (4 mL) and the mixture was heated in a sealed microwave vial to 150 °C for 1.5 h. The reaction mixture was cooled to rt and the volatiles were removed in vacuo. The residue was slurried in CH2Cl2/diethyl ether 2:3 (12.5 mL) for 1 h at rt, the solid was filtered and the filter cake was washed with CH2Cl2/ diethyl ether 2:3 (2 × 2 mL). This material was further purified by FC on SiO2 eluting with CH2Cl2/MeOH (0−20% MeOH) to provide 32 (49.0 mg, 42% over 2 steps) as a pale yellow solid. UPLC-MS tR,B: 0.56 min, 517 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSOd6) δ 9.62 (d, J = 1.4 Hz, 1H), 9.56 (s, 1H), 8.90 (s, 1H), 8.60−8.54 (m, 2H), 8.17 (d, J = 2.5 Hz, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.89 (d, J = 2.5 Hz, 1H), 7.86−7.79 (m, 2H), 7.64−7.55 (m, 2H), 7.26 (d, J = 7.7 Hz, 1H), 6.97 (d, J = 9.0 Hz, 1H), 4.51 (d, J = 9.4 Hz, 1H), 3.66− 3.56 (m, 4H), 2.63−2.53 (m, 5H), 2.36 (dd, J = 11.8, 6.7 Hz, 1H), 1.30 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 158.1, 154.2, 150.7, 149.1, 148.0, 147.3, 145.7, 140.1, 139.6, 136.6, 132.6, 128.6, 127.9, 127.4, 126.3, 123.2, 122.2, 120.8 × 2, 115.4, 111.0, 66.7, 64.5, 54.3, 43.4, 19.6; HRMS (ESI) for C30H29ON8 [M + H] calculated 517.2459, found 517.2458. 5.2.2.2.24. (R)-N-(1-morpholinopropan-2-yl)-6-(8-(pyridin-4-yl)1H-imidazo[4,5-c][1,7]naphthyridin-1-yl)quinolin-2-amine (33). The title compound (47.0 mg, 93%, 99.5% ee) was obtained as a colorless solid by chiral preparative HPLC of racemic 32 (100 mg). tR, chiral: 18.7 min; UPLC-MS tR,B: 0.59 min, 517 [M + H], UPLCL



Article

purity: 99%; 1H NMR (400 MHz, Chloroform-d) δ 9.64 (s, 1H), 9.51 (s, 1H), 8.58−8.51 (m, 2H), 8.27 (s, 1H), 7.96 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.81 (d, J = 2.6 Hz, 2H), 7.69 (dd, J = 8.7, 2.5 Hz, 1H), 7.61−7.55 (m, 2H), 6.83 (d, J = 8.9 Hz, 1H), 5.55 (br. s, 1H), 4.35 (br. s, 1H), 3.79−3.63 (m, 4H), 2.68−2.57 (m, 3H), 2.56− 2.44 (m, 3H), 1.43 (d, J = 6.2 Hz, 3H). The absolute configuration of 33 was unambiguously assigned by independent synthesis starting from (R)-1-morpholinopropan-2-amine (prepared in 3 steps from commercially available (R)-tert-butyl (1hydroxypropan-2-yl)carbamate according to literature procedure and comparing the chiral HPLC retention times.27 5.2.2.2.25. (S)-N-(1-morpholinopropan-2-yl)-6-(8-(pyridin-4-yl)1H-imidazo[4,5-c][1,7]naphthyridin-1-yl)quinolin-2-amine (34). The title compound (48 mg, 94%, 99.7% ee) was obtained as colorless solid by chiral preparative HPLC of racemic 32 (100 mg). tR, chiral: 23.9 min; UPLC-MS tR,B: 0.59 min, 517 [M + H], UPLCpurity: 98%; 1H NMR (400 MHz, Chloroform-d) δ 9.68 (d, J = 0.7 Hz, 1H), 9.53 (s, 1H), 8.60−8.52 (m, 2H), 8.28 (s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.83 (d, J = 2.4 Hz, 2H), 7.70 (dd, J = 8.8, 2.4 Hz, 1H), 7.61 (dd, J = 4.6, 1.5 Hz 2H), 6.85 (d, J = 8.9 Hz, 1H), 5.69 (br. s, 1H), 4.41 (br. s, 1H), 3.85−3.60 (m, 4H), 2.69 (br. s, 3H), 2.63−2.45 (m, 3H), 1.44 (d, J = 6.2 Hz, 3H). 5.2.2.2.26. N2-(2-morpholinoethyl)benzo[d]thiazole-2,6-diamine (40). A solution of 2-chlorobenzo[d]thiazol-6-amine (250 mg, 1.35 mmol), 2-morpholinoethanamine (881 mg, 6.8 mmol, 5 equiv) and triethylamine (0.94 mL, 6.8 mmol) in abs. dioxane (2.4 mL) was heated in a sealed microwave vial for 24 h at 130 °C. The reaction mixture was cooled to rt, concentrated under reduced pressure and the residue partitioned between H2O (20 mL) and ethyl acetate (20 mL). The organic layer was separated, dried (Na2SO4), filtered and concentrated in vacuo. The crude product was purified by flash chromatography on SiO2 eluting with CH2Cl2/MeOH (0−10% MeOH) to provide the title compound (300 mg, 80%) as a pale yellow solid. UPLC-MS tR,A: 0.32 min, 279 [M + H], UPLC-purity 100%; 1H NMR (400 MHz, DMSO-d6) δ 7.41 (t, J = 5.5 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 6.48 (dd, J = 8.4, 2.3 Hz, 1H), 4.76 (s, 2H), 3.56 (t, J = 4.6 Hz, 4H), 3.39 (q, J = 6.4 Hz, 2H), 2.55−2.44 (m, 2H), 2.39 (t, J = 4.7 Hz, 4H). 5.2.2.2.27. N 2 -(1-morpholinopropan-2-yl)-6-nitrobenzo[d]thiazol-2-amine (43). 1-Morpholinopropan-2-amine (5.38 g, 37 mmol) were added to a mixture of 2-chloro-6-nitrobenzo[d]thiazole (4.00 g, 19 mmol) and K2CO3 (5.15 g, 37 mmol, 2.0 equiv) in abs. DMF (130 mL). The mixture was stirred at rt for 1h. The reaction mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure. The residue was purified by FC on SiO2 eluting with ethyl acetate/cyclohexane 1:1 to provide 40 (5.65 g, 94%) as yellow solid. UPLC-MS tR,A: 0.63 min, 323 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 2.7 Hz, 1H), 8.08 (dd, J = 8.9, 2.5 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 4.14 (br. s, 1H), 3.51 (t, J = 4.7 Hz, 4H), 2.50−2.35 (m, 6H), 2.31 (dd, J = 12.4, 6.3 Hz, 1H), 1.20 (d, J = 6.5 Hz, 3H). 5.2.2.2.28. N2-(1-morpholinopropan-2-yl)benzo[d]thiazole-2,6diamine (44). Raney-Ni (399 mg) was added to a suspension of N2-(1-morpholinopropan-2-yl)-6-nitrobenzo[d]thiazol-2-amine 40 (1.50 g, 4.6 mmol) in MeOH (8 mL) and the mixture was stirred under H2 (1 atm.) at rt for 18 h. The reaction mixture was filtered through a pad of Celite, the filter cake washed with MeOH (2 × 3 mL) and the filtrate concentrated in vacuo. The residue was purified by FC on SiO2 eluting with CH2Cl2/MeOH (0 to 10% MeOH) to give 41 (1.04 g, 76%) as an orange solid. UPLC-MS tR,A: 0.35 min, 293 [M + H], UPLC-purity: 98%; 1H NMR (400 MHz, DMSO-d6) δ 7.33 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 6.48 (dd, J = 8.5, 2.2 Hz, 1H), 4.77 (s, 1H), 4.02−3.90 (m, 1H), 3.53 (t, J = 4.5 Hz, 4H), 2.47−2.33 (m, 6H), 2.24 (dd, J = 12.3, 6.9 Hz, 1H), 1.15 (d, J = 6.4 Hz, 3H). 5.2.2.2.29. N-(1-morpholinopropan-2-yl)-6-nitroquinolin-2amine (46). A mixture of 2-chloro-6-nitro-quinoline (10.0 g, 48 mmol), 1-morpholinopropan-2-amine (9.33 g, 65 mmol, 1.3 equiv) and K2CO3 (16.6 g, 0.12 mol, 2.5 equiv) in anhydrous dioxane (120 mL) was stirred under reflux for 20 h. The reaction mixture was cooled

to rt and partitioned between ethyl acetate (200 mL) and H2O (200 mL) and the layers were separated. The aq. phase was extracted with ethyl acetate (1 × 200 mL) and the combined organics were washed with sat. aq. NaHCO3 soln. (1 × 200 mL) and brine (1 × 200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude material was recrystallized from ethyl acetate/diethyl ether/ petrolether to give the title compound (11.9 g, 78%) as a pale yellow solid. UPLC-MS tR,A: 0.64 min, 317 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 8.64 (d, J = 2.7 Hz, 1H), 8.20 (dd, J = 9.2, 2.7 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.51 (d, J = 9.2 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 4.48−4.38 (m, 1H), 3.53 (t, J = 4.6 Hz, 4H), 2.53−2.41 (m, 5H), 2.28 (dd, J = 12.2, 6.9 Hz, 1H), 1.21 (d, J = 6.5 Hz, 3H). 5.2.2.2.30. N2-(1-morpholinopropan-2-yl)quinoline-2,6-diamine (47). 10% Pd/C (801 mg, 0.02 equiv) was added to a solution of N-(1-morpholinopropan-2-yl)-6-nitroquinolin-2-amine (46) (11.9 g, 4.6 mmol) in MeOH/THF 1:1 (250 mL) and the mixture was stirred under H2 (1 atm.) at rt for 2.5 h. The reaction mixture was filtered through a pad of Celite, the filter cake washed with MeOH (2 × 20 mL) and the filtrate concentrated in vacuo to give the title compound as a yellow foam which was used without further purification. UPLCMS tR,A: 0.35 min, 287 [M + H], UPLC-purity: 97%; 1H NMR (400 MHz, DMSO-d6) δ 7.54 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 6.88 (dd, J = 8.8, 2.5 Hz, 1H), 6.64 (d, J = 2.5 Hz, 1H), 6.60 (d, J = 8.9 Hz, 1H), 6.21 (d, J = 7.5 Hz, 1H), 4.88 (s, 2H), 4.22 (p, J = 6.8 Hz, 1H), 3.55 (t, J = 4.7 Hz, 4H), 2.53−2.34 (m, 5H), 2.21 (dd, J = 12.0, 7.4 Hz, 1H), 1.16 (d, J = 6.4 Hz, 3H). 5.2.2.2.31. N-(1H-benzo[d]imidazol-6-yl)-6-chloro-3-nitro-1,7naphthyridin-4-amine (48). The crude chloride (5.50 g, 24.4 mmol, 1.0 equiv), prepared from 10 in 76% yield according to general chlorination procedure 1, was dissolved in anhydrous DMF (275 mL) and 4 N HCl in dioxanes (0.64 μL, 24.4 mmol, 1.0 equiv) and 5aminobenzimidazole (4.96 g, 37.2 mmol, 1.5 equiv) were added. The mixture was stirred at rt for 2.5 h and then poured onto a mixture of ice water and EtOAc/Et2O 1:1 (200 mL). The precipitated solid was filtered, washed with H2O (50 mL) and diethyl ether (2 × 50 mL) and dried under reduced pressure. The title compound 48 was obtained as brown solid (4.19 g, 50%). UPLC-MS tR,B: 0.66 min, 341 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 12.46 (br, s, 1H), 10.39 (s, 1H), 9.15 (s, 1H), 9.07 (s, 1H), 8.46 (br, s, 1H), 8.22 (s, 1H), 7.56 (br, s, 1H), 7.36 (br, s, 1H), 7.01 (s, 1H). 5.2.2.2.32. 1-(1H-benzo[d]imidazol-6-yl)-8-chloro-1H-imidazo[4,5-c][1,7]naphthyridine (49). Raney-Ni (1.0 g) was added to a suspension of N-(1H-benzo[d]imidazol-6-yl)-6-chloro-3-nitro-1,7naphthyridin-4-amine (48) (3.92 g, 11.5 mmol) in MeOH (250 mL) and the mixture was stirred under H2 (1 atm.) at rt for 18 h. The reaction mixture was filtered through a pad of Celite, the filter cake washed with MeOH (2 × 60 mL) and the filtrate concentrated in vacuo to provide the aniline (3.13 g) as brown solid which was used for the next step without further purification. The crude material was taken up in EtOH (60 mL) and triethyl orthoformate (16.1 mL, 97 mmol, 10 equiv) and conc. HCl (0.83 mL, 1.0 equiv) were added before the mixture was heated in a sealed microwave vial to 145 °C for 1 h. The reaction mixture was cooled to rt and the volatiles removed in vacuo. Resulting solid was taken up in MeOH (20 mL) and pH adjusted to 12 using ammonia. Resulting solution was evaporated under reduced pressure to provide title compound 49 (1.71 g, 46% over 2 steps) as beige solid. UPLC-MS tR,A: 0.58 min, 321 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 12.91 (br, s, 1H), 9.49 (s, 1H), 9.31 (s, 1H), 8.79 (s, 1H), 8.48 (s, 1H), 8.05 (s, 1H), 7.89 (d, J = 1.6, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.08 (s, 1H). 5.2.2.2.33. 1-(1H-benzo[d]imidazol-6-yl)-8-(thiophen-3-yl)-1Himidazo[4,5-c][1,7]naphthyridine (50). Title compound 50 was obtained as a yellow solid (51.0 mg, 30%) according to the general Suzuki procedure 2 starting from 49 (150 mg, 0.47 mmol) and thiophen-2-ylboronic acid (78.0 mg, 0.61 mmol) with stirring for 1.5 h. UPLC-MS tR,B: 0.77 min, 369 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.43 (s, 1H), 9.08 (s, 1H), 8.80 (s, 1H), 8.26 (s, 1H), 8.09 (d, J = 8.6 Hz, 1H), 7.82 (m, 2H), 7.55 (m, 1H), 7.44 (s, 1H), 7.06 (d, J = 6.3 Hz, 1H). M



Article

NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 9.37 (s, 1H), 8.95 (s, 1H), 8.76 (s, 1H), 8.19 (s 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.03 (s, 1H), 6.80 (s, 1H), 2.12 (s, 2H), 2.48 (br, s, 2H), 1.51 (br, s, 4H). 5.2.2.2.42. 1-(1H-benzo[d]imidazol-6-yl)-8-(piperidin-1-yl)-1Himidazo[4,5-c][1,7]naphthyridine (59). 1-(1H-benzo[d]imidazol-6yl)-8-chloro-1H-imidazo[4,5-c][1,7]naphthyridine (49) (80.0 mg, 0.249 mmol) and piperidine (7 mL) were stirred at 160 °C for 6 h. The mixture was cooled to rt, filtered through a pad of Celite, the filter cake washed with EtOH (2x). The reaction mixture cooled to rt and concentrated in vacuo. Title compound 59 was obtained as a beige solid (61.9 mg, 67%) after purification by prep HPLC (Waters; column Sunfire C18 5 μm, 30 mm × 100 mm; A, water +0.1% TFA; B, acetonitrile +0.1% TFA; 5−25% B in 16 min. UPLC-MS tR,B: 0.70 min, 370 [M + H], UPLC-purity: 100%; 1H NMR (400 MHz, DMSO-d6) δ 12.85 (br, s, 1H), 9.03 (s, 2H), 8.62 (s, 1H), 8.44 (s, 1H), 7.98 (s, 1H), 7.85 (s 1H), 7.51 (d, J = 8.6 Hz, 1H), 6.03 (s, 1H), 3.13 (s, 4H), 1.47 (s, 2H), 1.29 (s, 4H). 5.3. Biology. 5.3.1. Protein Production, Crystallization, and Structure Elucidation. All chemicals, unless stated otherwise, were bought from Sigma-Aldrich. The kinase domain of human COT encompassing amino acids 66 to 395 was overexpressed in Sf21 cells and purified according to procedures described.19 COT.28 complex was concentrated to 3.5 mg/mL using an Amicon Ultra centrifugal filter with 10K molecular weight cutoff (Millipore) before crystallization by vapor diffusion. Hanging drops consisting of an equal ratio of complex to reservoir solution (1 μL) were set up and equilibrated against 500 μL reservoir solution containing 0.1 M BisTris pH 7, 1.7 M ammonium sulfate, 0.01 M proline in a 24-well VDX plate (Hampton Research) at room temperature. Crystals grew within 2 days and were quickly dipped into a reservoir solution containing 10% glycerol before flash-freezing them in liquid nitrogen. Diffraction data were collected at 100 K at the Swiss Light Source beamline X10SA at 1.0 Å wavelength. Diffraction images were recorded on a Pilatus 6 M pixel detector (Dectris) and processed and scaled with XDS and XSCALE respectively (see Table 1 in the Supporting Information).28 The structure was solved by molecular replacement with Phaser using 4y85.pdb as search model.29 The structure was built using COOT and refined with BUSTER to Rwork =23.3% and Rfree =25.8%. 96.6% of all the residues in the structure are in the favored and 0.3% in the outlier regions of the Ramachandran plot.30,31 PyMOL was used for structural visualization and figure preparation.32 5.3.2. Biotransformation and Pharmacokinetic Investigations. The origin of the chemical and reagents used as well as the experimental conditions used for the analyses of the samples for the investigation of the biotransformation, the isolation of a metabolite as well as for the pharmacokinetic parameters are presented in the Supporting Information. 5.3.2.1. Animals. Male wild-type Sprague−Dawley rats (Iffa Credo, France) were kept under standard cages and conditions according to Swiss Animal Welfare guidelines (12h light/dark cycles, room temperature at 22−24 °C, humidity at least 45% but

Discovery of Imidazoquinolines as a Novel Class of Potent, Selective

Recommend Documents