Discovery and Optimization of Isoquinoline Ethyl Ureas as

Article pubs.acs.org/jmc

Discovery and Optimization of Isoquinoline Ethyl Ureas as Antibacterial Agents Philippe Panchaud,* Thierry Bruyère, Anne-Catherine Blumstein, Daniel Bur, Alain Chambovey, Eric A. Ertel, Markus Gude,† Christian Hubschwerlen, Loïc Jacob, Thierry Kimmerlin, Thomas Pfeifer,‡ Lars Prade,§ Peter Seiler, Daniel Ritz, and Georg Rueedi Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland S Supporting Information *

ABSTRACT: Our strategy to combat resistant bacteria consisted of targeting the GyrB/ParE ATP-binding sites located on bacterial DNA gyrase and topoisomerase IV and not utilized by marketed antibiotics. Screening around the minimal ethyl urea binding motif led to the identification of isoquinoline ethyl urea 13 as a promising starting point for fragment evolution. The optimization was guided by structure-based design and focused on antibacterial activity in vitro and in vivo, culminating in the discovery of unprecedented substituents able to interact with conserved residues within the ATP-binding site. A detailed characterization of the lead compound highlighted the potential for treatment of the problematic fluoroquinolone-resistant MRSA, VRE, and S. pneumoniae, and the possibility to offer patients an intravenous-to-oral switch therapy was supported by the identification of a suitable prodrug concept. Eventually, hERG K-channel block was identified as the main limitation of this chemical series, and efforts toward its minimization are reported.

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INTRODUCTION Antimicrobial resistance is a serious threat to public health with bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae becoming increasingly resistant to currently available antibacterial agents.1 The emergence of mutations in bacterial topoisomerases jeopardizing the efficacy of fluoroquinolones2 is an example of such resistance development and highlights the urgent need to develop novel antibacterial agents devoid of cross-resistance to marketed antibiotics. DNA topoisomerases, i.e., DNA gyrase and topoisomerase IV (topo IV), are enzymes essential for cell viability. They are critical in resolving problems of DNA topology by performing reactions of supercoiling/relaxation, decatenation/catenation, and knotting/unknotting in processes such as DNA replication, transcription and recombination.3 In particular, gyrase is unique in its ability to introduce negative supercoils into DNA, while topo IV is responsible for both decatenation of circular chromosomes after replication and relaxation of supercoiled DNA.4,5 Their inhibition by fluoroquinolone antibiotics results in the accumulation of double-strand DNA breaks leading to bacterial cell death.6 Even though resistance to fluoroquinolones has appeared since their introduction on the market, these two multidomain enzymes remain attractive targets due to their conservation across all bacteria.7 Moreover, targeting two enzymes with one © 2017 American Chemical Society

inhibitor is a superior strategy to reduce resistance development compared to single-target approaches.8 Both gyrase and topo IV are homologous heterotetramers consisting of two GyrA and two GyrB subunits, which together form an A2B2 complex for gyrase, or two ParC and two ParE subunits forming a C2E2 complex for topo IV, respectively. This architecture distinguishes the bacterial topoisomerases from their related eukaryotic homodimer counterparts.6 The N-terminal domains of the GyrB and ParE subunits form the ATP-binding site of gyrase and topo IV, respectively. Novobiocin 1 (Figure 1) is a natural product inhibiting the ATPase activity of gyrase and topo IV with an ATP-competitive mechanism of inhibition9 as confirmed by X-ray structures of 1 bound to the ATP site of E. coli GyrB and ParE.10,11 Novobiocin was approved for clinical use in 1964 and was later withdrawn from the market mainly because of the successful development of more effective antibiotics.12 It suggests nevertheless that the GyrB/ParE ATPase domains constitute validated targets for identifying new antibiotics with a mode of inhibition different from the fluoroquinolones that act at the enzyme−DNA interface.13,14 Surprisingly, no other antibiotics targeting the ATPase activity of gyrase or topo IV have been developed successfully so far despite Received: December 14, 2016 Published: April 13, 2017 3755

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Figure 1. Structures of novobiocin 1, benzimidazole urea 2, and schematic representation of the H-bond network involving the conserved Asp73 residue and water molecule.

the high attractiveness of the targets and the major efforts pursued by several pharmaceutical companies and academic laboratories.12,15−18 We decided to investigate new GyrB/ParE inhibitors after compound 2 (Figure 1) had been reported to have potent antibacterial activity.19 Similarly to 1, it interacts with a conserved Asp73 residue (E. coli GyrB numbering) and a structurally conserved water molecule in the ATP-binding sites, but unlike 1, whose carbamate is involved in single H-bonds with Asp73 and this water, the urea moiety in 2 can establish two H-bonds with the aspartate residue while the benzimidazole receives an H-bond from the water molecule. This optimized H-bond network was assumed to lead to increased binding affinity and was already successfully used by several groups.20 Herein, we report on a hitto-lead program followed by lead optimization efforts toward the identification of a safe antibacterial agent for the treatment of Gram-positive and respiratory tract infection (RTI) pathogens via both oral and intravenous routes.

Figure 2. Reaction scheme for producing ethyl ureas 3 from respective amines 4. Estimated IC50s and LEs for E. coli gyrase ATPase inhibition are shown for selected derivatives.

RESULTS AND DISCUSSION Ethyl Urea Screening and Hit Discovery. As only a limited number of 1-ethyl-2-heteroaryl urea derivatives 3 (Figure 2) were commercially available, a synthesis and screening procedure was developed to generate such compounds and to evaluate their biological activity while avoiding extensive purification. To this end, readily available amines of generic formula 4 were dissolved in DMSO, incubated with ethyl isocyanate, and treated with an amine-based resin scavenger. After filtration and centrifugation, the supernatants were analyzed by LC-MS to determine the proportions of unreacted starting material and desired urea product, the latter being the only product detected in most cases. All supernatants were tested for inhibition of E. coli gyrase ATPase activity at 500 and 100 μM, and those displaying interesting inhibition levels were resynthesized and purified in a conventional way. A selection of urea derivatives, their estimated IC50s on ATPase inhibition, and associated ligand efficiencies (LE) are presented in Figure 2. The results proved most interesting for hits devoid of substitution and allowed the comparison with core scaffolds of a few inhibitors published since. For instance, the known benzothiazole scaffold 621,22 was among the most potent molecules with an IC50 of 20 μM. Its benzene ring increased inhibition by at least 10-fold when compared to that of 5. A heteroatom was tolerated in 7, while loss of the aromatic character of the 6-membered ring led to less potent 8 and 9. However, introduction of a lactam moiety in 10 reestablished

activity. The high LE of these low molecular weight compounds highlighted the contribution of the urea unit to binding affinity. A similar trend was observed in the corresponding 6-membered scaffolds starting from 11, which was independently identified as a starting point for leads with potent antibacterial activity in both a fragment screening, using, however, a different assay with gyrase enzyme from other bacterial species,23 and also following a de novo design strategy.24 In our setting, 11 did not show gyrase ATPase inhibition up to 200 μM, but addition of a benzene ring had a positive effect for quinoline 12 and 3-isoquinolinyl urea 13, while 1-isoquinolinyl urea 14 lost activity, likely due to unfavorable intramolecular steric interactions. The positioning of a second nitrogen atom turned out to be critical since it was well tolerated in 15 but detrimental in 16. On the basis of these findings, the fragments with the lowest IC50 were investigated in molecular modeling by replacing the ADPNP ligand in a published E. coli GyrB structure (1EI1). With the particular orientation of its two rings, 3-isoquinolinyl urea 13 was considered the most promising scaffold for a fragment evolution program. Indeed, positions 5 and 6 were ideally positioned to allow exploration of the territory which is occupied by the phosphate groups of ATP, while position 8 provided an exit vector pointing toward the space occupied by the coumarin ring of 1 and the 3-pyridyl substituent of 2. Finally, positions 4 and 1 were considered inappropriate for substitution in order to avoid intramolecular clashes with the urea carbonyl (position 4) or intermolecular repulsion with the enzyme (position 1).

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Scheme 1. Synthesis of 3-Ethyl Urea Isoquinoline Intermediates 21a−ea

Reagents and conditions: (a) 17, MeOH, rt−70 °C, 2−23 h. (b) H2SO4, rt−80 °C, 3−22 h. (c) EtNCO, dioxane, 50 °C, 1−6 days; or EtNCO, pyridine, 65 °C, 42 h.

a

Chemistry. The chemistry strategy chosen for exploring the chemical space around the hit compound 13 consisted of the construction of isoquinoline urea intermediates followed by transition metal-catalyzed cross-coupling reactions to introduce the chemical diversity needed for our investigations. Isoquinolines 20a−e were formed by reacting the corresponding benzyl amines 18a−e with 2,2-diethoxy-ethanimidic acid methyl ester 1725 followed by treatment with sulfuric acid (Scheme 1). Reaction with ethyl isocyanate gave the urea intermediates 21a−e. Monosubstituted derivatives 22 and 23 were obtained by performing a Negishi-type reaction with dimethylzinc or a Suzuki coupling starting from 21a and 21b, respectively (Scheme 2).

Suzuki coupling reactions. A Buchwald−Hartwig amination on 21e with 3-aminopyridine derivatives using XPhos as ligand selectively yielded 34 and 35, which were further functionalized at position 5 to give 36 and 37, respectively. Compound 39 was prepared in a similar way, while an Ullmann-type reaction led to the formation of the corresponding ether 40 on the way to 41. Aliphatic side chains were introduced at position 8 starting with a selective carbonylation reaction on 21e (Scheme 4). Methyl ester 42 was reduced with LAH at low temperature to avoid reduction of the isoquinoline ring. After Suzuki coupling, primary alcohol 44 was activated with CDI and converted into carbamate 45 upon treatment with morpholine. We found that such derivatives would react with boronic acids under Suzuki conditions to give a formal substitution of the carbamate group and allowed accessing derivatives 46 and 47. This reaction appeared to be of quite general scope provided a 4-pyridyl substituent was present at position 5, whereas the corresponding phenyl substituted derivative failed to react under these conditions. This may suggest that these reactions proceeded via a mechanism different from the recently described cross-coupling reaction of benzyl carbamates with arylboronic acids.26 Finally, nucleophilic substitution of the hydroxyl group of 44 with 1,2,3-triazole under Mitsunobu-type conditions delivered 48 after separation of the two regioisomers. Many carbamates and amides were prepared from the versatile amine intermediate 49 (Scheme 5), which was prepared in a one-pot two-step reaction via a Staudinger reduction of the corresponding azide previously formed in situ after treatment of alcohol 43 with DPPA and DBU. Treatment of 49 either with a chloroformate or with an alcohol previously activated with CDI yielded carbamates which were next subjected to Suzuki coupling to introduce the pyridyl substituent in 50 and 51. Analogues 52 and 53 bearing a basic amine were prepared in a similar manner followed by a final Boc deprotection. In the case of the latter, an additional reductive amination was performed to yield 54. The order of the steps was also inverted, performing first the Suzuki coupling to prepare 55 and 56 which could be further reacted in order to provide carbamates 57−64 and amides 65−67. The scope of these transformations was broad, and many derivatives were produced in the form of libraries. The phosphoryloxymethyl prodrug 68 was prepared in a onepot two-step reaction sequence from 50 and di-tert-butyl chloromethyl phosphate in the presence of a stoichiometric amount of sodium iodide followed by hydrolysis of the tert-butyl phosphate with TFA (Scheme 6).27 Finally, inverted carbamates 69−74 were prepared similarly to 45 by treatment of CDI-activated 44 with an amine followed by a deprotection step when necessary (Scheme 7).

Scheme 2. Synthesis of Derivatives 22−28, 30, and 31a

Reagents and conditions: (a) Me2Zn, Pd(dppf)Cl2·DCM, THF, 80 °C, 22 h. (b) py-3-B(OH)2, Pd(PPh3)4, K2CO3, dioxane, 80 °C, 4 h. (c) R5B(OH)2, Pd(PPh3)4, K2CO3, dioxane, 90−110 °C, 2−4 h. (d) bis(neopentyl glycolato)diboron, KOAc, Pd(dppf)Cl2·DCM, DMSO, 90 °C, 2 h. (e) R5Br, Pd(PPh3)4, K2CO3, dioxane, 90−110 °C, 2−4 h. a

Similarly, 24−28 bearing a methyl substituent at position 8 were prepared from 21c. The corresponding boronate ester 29 allowed for the preparation of 30 and 31 using aryl halides as coupling partners since the required boronate esters were not commercially available. The presence of both bromine and chlorine atoms in isoquinolines 21d and 21e allowed one to perform sequential cross-coupling reactions in a selective manner (Scheme 3). Compound 33 was prepared by two successive 3757

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Scheme 3. Synthesis of R8-Heteroaryl Derivatives 33, 36, 37, 39, and 41a

Reagents and conditions: (a) py-4-B(OH)2, Pd(PPh3)4, K2CO3, dioxane, 90 °C, 2 h. (b) py-3-B(OH)2, Pd(PPh3)4, K2CO3, dioxane, 90 °C, 3 h. (c) ArNH2, Pd2(dba)3, XPhos, NaOtBu, dioxane, 90 °C, 2−4 h. (d) 2-R-py-4-B(OH)2, Pd2(dba)3, PCy3, K2CO3, dioxane, 100 °C, 2−3 h. (e) TFA, rt, 2 h. (f) py-3-OH, CuI, N,N-dimethylGly, K3PO4, DMSO, 90 °C, 23 h. a

Scheme 4. Synthesis of R8-Heteroaryl Derivatives 46−48a

Reagents and conditions: (a) CO (5 atm), Pd(dppf)Cl2·DCM, NaOAc, MeOH, 60 °C, 7 h. (b) LAH, THF, −40 °C, 5 h. (c) 2-Me-py-4-B(OH)2, Pd2(dba)3, PCy3, K3PO4, dioxane, 90 °C, 3 h. (d) (1) CDI, DIPEA, DMF, 40 °C, 4 h; (2) morpholine, DIPEA, DCM, rt, 18 h. (e) ArB(OH)2, Pd2(dba)3, PCy3, K3PO4, dioxane, 90 °C, 5−10 h. (f) 1H-1,2,3-triazole, PPh3, DIAD, THF, 0 °C to rt, 3 h.

a

Fragment Evolution. The hit-to-lead program was initiated with the introduction of various chemotypes at positions 5, 6, 7, and 8 of 13. Positions 5 and 8 were soon identified to be most attractive for further investigations (Table 1).

The addition of a methyl group at position 8 led to 22, which displayed slightly improved inhibition of E. coli gyrase ATPase and supercoiling activities as well as E. coli topo IV relaxation. Interestingly, the better inhibition of S. aureus gyrase resulted in a 3758

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Scheme 5. Synthesis of R8-Carbamate and Amide Derivatives 50−54 and 57−67a

a Reagents and conditions: (a) (1) DPPA, DBU, THF, rt, 3 h; (2) PPh3, water, rt, 2 h. (b) R8OCOCl, Et3N, DCM, rt, 18 h. (c) (1) R8OH, CDI, DIPEA, DCM, rt, 3 h; (2) 49, DIPEA, DMF, rt, 18 h. (d) 2-Me-py-4-B(OH)2, Pd2(dba)3, PCy3, K3PO4, dioxane, 90 °C, 2−3 h. (e) HCl, dioxane, rt, 2 h. (f) py-4-B(OH)2, Pd2(dba)3, PCy3, K2CO3, dioxane, 100 °C, 1 h. (g) TFA, DCM, rt, 1 h. (h) (1) pivaldehyde, 1:1 DCE/MeOH, rt, 16 h; (2) NaBH4, rt, 4 h. (i) (1) R8OH, CDI, DIPEA, DCM, rt, 3 h; (2) 55 or 56, DIPEA, NMP, rt, 18 h. (j) TFA, 10:1 THF/water, rt, 1 h. (k) R8COOH, T3P, DIPEA, DMF, rt, 18 h.

Scheme 6. Synthesis of Phosphoryloxymethyl Prodrug 68a

a

with a similar outcome. The combination of both substituents yielded 24 with significantly increased inhibitory activities against gyrases from both E. coli and S. aureus, resulting in a much lower MIC against S. aureus (2 μg/mL). From this point on, gyrase supercoiling and topo IV relaxation inhibition assays were used to guide the potency optimization, while inhibition of E. coli gyrase ATPase activity served only as a control for the mode of inhibition. Variation of the heteroaryl group at position 5 led to the 4-pyridyl substituent in 25, which displayed further improved inhibition of all four enzymes. In addition, 25 displayed a low MIC against both S. aureus (0.5 μg/mL) and an efflux-deficient E. coli strain (2 μg/mL). However, it remained inactive against a

Reagents and conditions: (a) (1) NaI, DCM, rt, 72 h; (2) TFA, 0 °C, 3 h.

high but measurable MIC of 32 μg/mL against S. aureus ATCC 29213. Introduction of a 3-pyridyl group at position 5 provided 23 Scheme 7. Synthesis of R8-Carbamate Derivatives 69−74a

Reagents and conditions: (a) (1) CDI, DIPEA, DMF, 40 °C, 4 h; (2) R8NH2, DIPEA, DCM, rt−40 °C, 18−70 h. (b) TFA, DCM, rt, 1 h. (c) HCl, dioxane, rt, 2 h. a

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Table 1. Influence of R5 on ATPase Inhibition, Gyrase, and Topo IV Inhibition and Antibacterial Activity

a ATPase inhibition assay. bSupercoiling inhibition assay. cRelaxation inhibition assay. dMinimal inhibitory concentration. eMutant strain with the TolC efflux pump gene knocked-out.

wild-type strain of E. coli (ATCC 25922, vide infra), suggesting that efflux limited the cytoplasmic accumulation of this chemotype in this Gram-negative bacterium. The beneficial effect of a nitrogen atom in the heteroaryl substituent at the position opposite to the attachment to the scaffold could also be observed when comparing pyrimidines 26 and 30. Small substituents were tolerated next to the heteroatom, as shown with 27. Phenyl groups substituted with various H-bond donors and acceptors as well as 5-membered heteroaryls were also examined. However, as exemplified by 28 and 31, they were all less potent than 25 and 27. The importance of the pyridyl-type substituent in general and of the nitrogen atom at the position opposite to the attachment to the scaffold in particular was revealed by an X-ray structure of 27 bound to a 24 kDa fragment of E. coli GyrB (Figure 3). The 4-pyridyl nitrogen interacted with Val120 backbone nitrogen via a water-mediated H-bond. This X-ray structure also displayed the H-bond network between the two urea hydrogens and Asp73 as well as the H-bond donated by the conserved water to the isoquinoline nitrogen. Lead Optimization. With a MIC of 0.5 μg/mL against S. aureus ATCC 29213, 25 demonstrated promising in vitro antibacterial activity. Our initial interest in this lead compound was further strengthened by the results obtained in a neutropenic murine screening model of thigh infection. As oral bioavailability was deemed an important property for a potential drug targeting treatment of Gram-positive and RTI infections, we decided to evaluate oral bioavailability and efficacy concomitantly by dosing the compounds orally. At the same time, we used a reduced experiment time of 6.5 h and administered the compounds twice to avoid missing the efficacy of those with high clearance. In this screening model, 25 showed efficacy against staphylococci after

Figure 3. Crystal structure of 27 (yellow) bound to E. coli GyrB 24 kDa (gray). Residues 77−98 have been omitted for clarity. Residues engaged in key interactions (Asp73 and Val120) are represented in stick format. Hydrogen bonds are displayed as black dotted lines, and water molecules appear as red balls. PDB code: 5MMN.

oral administration (Table 2). Colony forming units (CFU) of S. aureus A-1178 decreased by 1.3 logs in thighs of mice dosed orally with 25 (30 mg/kg, 2q2.75 h) as compared to those treated with vehicle only. In order to further optimize the compounds, we kept the 4-pyridyl residue at position 5 as in 25 or 27 and further improved substituents at position 8. According to the X-ray structure of 27 (Figure 3), the R8 methyl group is pointing in the direction of Arg76, which is involved in a structurally important salt-bridge with Glu50. Our strategy consisted therefore of searching for interactions with this highly conserved arginine residue. 3760

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Table 2. Influence of R8 on Gyrase and Topo IV Inhibition, Antibacterial Activity, and Oral Efficacy

a Supercoiling inhibition assay. bRelaxation inhibition assay. cMinimal inhibitory concentration. dOral efficacy in a neutropenic murine thigh infection model with S. aureus A-1178. Mice were treated orally (30 mg/kg, 2q2.75 h) starting at 1 h post-infection and sacrificed at 6.5 h post-infection. Median Δlog10CFU/thigh of animals treated with compounds compared to vehicle is reported. Vehicle-treated animals showed median Δlog10CFU/ thigh about +3 compared to CFU/thigh at treatment start. end: not determined.

values, but its oral efficacy was still inadequate. Attempts to improve inhibitory activities by appending substituents on the triazole core resulted in marginally improved in vitro and in vivo results (data not shown). These heteroaryls groups could be successfully replaced by smaller carbamate moieties as seen in 50 and 57. These compounds indeed remained potent inhibitors of both gyrases with in vitro antibacterial activity against S. aureus similar to the one observed for 37. Remarkably, these compounds showed improved oral efficacy in the screening model with significant reduction of CFUs compared to that of vehicletreated mice. Given these interesting antibacterial activities in vitro and in vivo, the carbamate series was selected as a new lead family and was subsequently characterized. The binding mode of 58, a close analogue of 57, was determined by X-ray crystallography (Figure 4C and D). Similarly to the 3-pyridyl substituent in 36 (Figure 4A and B), the carbamate was located over Arg76 for a stacking interaction. However, it was also involved in an H-bond interaction with Arg136 similar to that of the coumarin carbonyl of 1. However, in contrast to 1, this interaction appeared to be weak in the case of carbamates since the mutation Arg136His did not significantly impact the inhibitory effect of 57 (Table 4, vide infra).

Appending a heteroaryl directly on the isoquinoline core as in 33 was unfavorable probably due to sterical reasons, while the introduction of a nitrogen atom spacer as in 36 led to improved IC50s and MIC. In the X-ray structure of 36 in the 24 kDa fragment of E. coli GyrB (Figure 4A and B), the 3-pyridyl substituent was located over Arg76 driven by a cation-π or a π-stacking interaction.12,17 The methyl analogue 37 was one of the most active compounds in vitro with a MIC ≤ 0.03 μg/mL against S. aureus ATCC 29213, but it nevertheless did not display improved oral efficacy in vivo as compared to 25 in the screening model. At the enzyme level, 37 showed a potent inhibition of gyrases and a 16- to 60-fold weaker inhibition of topo IV. Compound 39 showed a similar level of enzyme inhibition but increased MIC, presumably due to the presence of a negatively charged carboxylate leading to limited membrane permeability. Modification of the spacer in R8 by exchanging the nitrogen with an oxygen atom as in 41 or with a methylene group as in 46 and 47 resulted in some loss of potency in vitro. Introducing 5-membered heteroaryl groups containing a variety of heteroatoms also had unsatisfactory outcomes. Among all R8 unsubstituted heteroaryls tested, triazole 48 was the best when considering IC50 and MIC 3761

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Figure 4. (A) Crystal structure of 36 (yellow) in E. coli GyrB 24 kDa (gray). Residues 77−98 have been omitted for clarity. Residues engaged in key interactions (Val120, Asp73, Glu50, and Arg76) are represented in stick format. Hydrogen bonds are displayed as black dotted lines, and water molecules appear as red balls. PDB code: 5MMP. (B) Side view of 36. (C) Crystal structure of 58 (yellow) in E. coli GyrB 24 kDa (gray). PDB code: 5MMO. (D) Side view of 58.

Table 3. In Vitro Antibacterial Activity (MIC in μg/mL) of Representative Compounds against Selected Bacterial Strains bacterial strains

25

37

50

57

1

ciprofloxacin

Staphylococcus aureus ATCC 29213 Staphylococcus aureus A-798 (MRSA/QR)a Streptococcus pneumoniae ATCC 49619 Streptococcus pneumoniae A-70 (QR)b Streptococcus pyogenes ATCC 19615 Enterococcus faecalis ATCC 29212 Enterococcus faecium A-949 (VRE/QR)c Moraxella catarrhalis A-894 Haemophilus inf luenzae A-921 Escherichia coli ATCC 25922 Escherichia coli ΔtolCd

0.5 0.5 1 1 4 0.25 1 0.5 4 >16 2

≤0.03 ≤0.03 0.125 0.125 0.125 ≤0.03 0.06 0.25 0.25 >16 0.5

0.06 0.06 0.5 1 0.5 0.25 1 0.5 2 >16 1

≤0.03 ≤0.03 0.5 0.5 0.25 0.25 1 0.25 1 >16 0.5

0.25 0.125 1 1 1 8 2 1 0.125 >16 2

0.25 >16 1 >16 0.25 1 >16 0.03 ≤0.03 ≤0.03 ≤0.03

a

Fluoroquinolone-resistant, mutations in topoisomerase genes: gyrA, S84L; grlA, S80F, and Glu84V. bFluoroquinolone-resistant, mutations in topoisomerase genes: gyrA, S81F; parC, S79F; parE, and I440V. cFluoroquinolone-resistant, mutations in topoisomerase genes: gyrA, E88K; parC, S80R; and vancomycin-resistant (vanA). dMutant strain with the TolC efflux pump gene knocked out.

Antibacterial Spectrum and Inhibition of Enzymes. A few compounds with different substituents at position 8 were characterized in vitro against a panel of representative bacterial strains to assess their antibacterial activity (Table 3). Importantly, the potent antistaphylococcal activity was retained against the ciprofloxacin-resistant strain S. aureus A-798, a clinical isolate of MRSA containing the typical topoisomerase mutations associated with fluoroquinolone resistance. Activity was also retained against fluoroquinoloneresistant (QR) strains of S. pneumoniae and vancomycin-resistant E. faecium (VRE), highlighting the potential of this chemical series to control fluoroquinolone-resistant and other resistant strains. Low MICs were also measured against the fastidious Gram-negative bacteria M. catarrhalis and H. inf luenzae but not

against Enterobacteriaceae exemplified by E. coli. Replacement of the R8 methyl group of 25 with an aminopyridine in 37 led to improved MICs against all bacteria tested. The outcome was different for carbamates 50 and 57, and only MIC against S. aureus and S. pyogenes were significantly improved. With the exception of H. inf luenzae, the compounds tested showed equal or superior potency compared to that of novobiocin 1. None of the compounds showed meaningful activity against the wild-type E. coli strain, most probably due to efflux as evidenced by their good activity against the TolC efflux knockout mutant strain. The differences in MICs observed with these compounds were further investigated by measuring the inhibition of the corresponding enzymes (Table 4). The replacement of the methyl group at position 8 in 25 by an aminopyridine in 37 or a 3762

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Table 4. Inhibitory in Vitro Activities (IC50 in μM) of Representative Compounds against Selected Enzymesa enzyme

25

37

50

57

1

ciprofloxacin

S. aureus gyrase S. aureus gyrase QRb S. aureus topo IV S. aureus topo IV QRc S. pneumoniae gyrase S. pneumoniae gyrase QRd S. pneumoniae topo IV H. inf luenzae gyrase H. inf luenzae topo IV E. coli gyrase E. coli gyrase NovoRe E. coli topo IV human topo IIα

0.03 0.03 8 2 2 2 0.5 0.125 8 0.125 0.125 8 >256

0.008 256

0.008 256

0.008 256

0.125 0.03 128 128 8 0.5 128 0.5 32 2 128 8 >256

128 >256 8 >256 >256 >256 32 8 2 0.5 8 8 >256

a

Supercoiling inhibition assay for gyrases and relaxation inhibition assay for topo IV and human topo IIα. bIsolated from QR clinical isolate with mutation S84L in gyrA. cIsolated from QR clinical isolate with mutations S80F and E84V in grlA. dIsolated from strain A-70 with mutation S81F in gyrA. eR136H in gyrB (generated by site-directed mutagenesis on the WT gene).

carbamate in 50 and 57 led to a 4- to 64-fold improvement on all enzymes tested, which mirrors relatively well the MIC differences. For S. aureus enzymes, all compounds were more potent on gyrase than on topo IV, as previously reported for 1.11 The IC50s were not affected by gyrase and topo IV mutations known to be involved in fluoroquinolone-resistance in S. aureus, in line with the low MICs measured against the MRSA/QR strain. Surprisingly, compounds tended to display an enhanced inhibition of both QR gyrase and topo IV enzymes, but it cannot be excluded that this finding is the consequence of the lower apparent turnover number of the QR enzyme compared to that of the wild-type enzyme. For S. pneumoniae enzymes, a similar observation was made, and in contrast to novobiocin, our urea derivatives showed a slightly more potent inhibition of topo IV than of gyrase. For H. inf luenzae and E. coli, all compounds displayed an inhibition pattern similar to that for S. aureus. The compounds were also evaluated against a mutated E. coli gyrase enzyme, where Arg136 was exchanged for a His residue. This mutant was reported to confer resistance to 1,10 which indeed suffered a 64-fold increase in IC50 in our assay, whereas there was no significant difference in inhibitory activity for 25, 37, 50, and 57. While this was expected for 25 and 37, it suggested that the H-bond interaction of the carbamate moiety with Arg136 observed in the X-ray structure (Figure 4C and D) was not adding to the binding of 57 as much as that for 1 and that the interactions of pyridyl and carbamate substituents with Arg76 were contributing to binding to a similar extent. Finally, all compounds were selective for bacterial topoisomerases since they did not show any inhibition of the human topoisomerase IIα homologue. Solubility and Prodrug Approach. The possibility to offer patients an iv-to-oral switch therapy was considered early in the program, and the in vivo efficacy achieved after oral administration was especially encouraging in this regard. Nevertheless, the development of a formulation adequate for iv infusion was required, and for this purpose, we were targeting a minimal solubility of 10 mg/mL. However, carbamate 50 showed a solubility of 20 μg/mL in phosphate buffer at pH 7.0 and of 340 μg/mL in citrate buffer at pH 4.0. In order to reach markedly higher solubility, we modified the 4-pyridyl moiety with an enzymatically cleavable phosphate group: such types of prodrugs had previously been reported28 and appeared to be readily cleaved in vitro and in vivo. The phosphoryloxymethyl

Figure 5. Chemical structure of prodrug 68 at physiological pH and halflives upon incubation in blood in vitro.

prodrug 68 (Figure 5) was soluble at 12.7 mg/mL in water at pH 6.3 and stable in solution at room temperature for several weeks. In vitro, the disappearance of 68 was fast as shown by the short half-lives measured in human, rat, and mouse blood. In rats, 50 and 68 were given orally (2 and 2.56 mg/kg) and intravenously (0.5 and 0.64 mg/kg) at equimolar doses (Table 5). For both routes, the exposures of 50 were similar irrespective of the form administered, thus indicating a complete conversion of 68 to 50 in vivo. In addition, 68 (12.8 and 38.4 mg/kg, 2q2.75 h, iv) showed superior efficacy as compared to 50 (10 and 30 mg/kg, 2q2.75 h, po) in the neutropenic murine screening model of thigh infection (median Δlog10CFU/ thigh of animal treated with compounds compared to vehicle: −3.6, resp. ≥ −4.8 (CFU below limit of detection) for 68 and −2.5, resp. −3.5 for 50). All of these early observations suggested that this prodrug was an adequate prototype. hERG K-Channel Block. A strong hERG K-channel block (hERG block) was observed in vitro with 50 at 10 μM (Table 6). As could be expected, this block translated into prolongation of the QT interval of the electrocardiogram in vivo (vehiclecorrected prolongation of the Bazett-corrected QT interval in anesthetized guinea pigs using iv infusion: 0% and 6% at 0.4 μM and 1.5 μM free plasma concentration, respectively), which prevented further development of this compound.29 We noticed that the corresponding pyridine des-methyl derivative 59 was slightly less lipophilic and produced less hERG block. We subsequently modulated compound lipophilicity by modifying the substituent on the R8 carbamate. Compared to the methyl carbamate 59, the isopropyl derivative 60 was more lipophilic with increased hERG block, whereas the more polar hydroxyethyl derivative 61 did not block hERG at 10 μM. Unfortunately, 61 showed a significantly reduced antibacterial activity compared to that of 59 or 60 despite similar enzyme inhibition. 3763

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and linezolid caused a dose-dependent reduction of bacterial growth after oral administration. At 40 mg/kg/day, both compounds displayed a minimal effect while net stasis was obtained with 120 mg/kg/day of 50.

Table 5. Pharmacokinetic Parameters of Compound 50 and Its Prodrug Derivative 68 in Rats cmpd 50 administered

prodrug 68 administered

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iv (0.5 po (2 iv (0.64 po (2.56 mg/kg)a mg/kg)b mg/kg)c mg/kg)b cmpd 50 AUC (ng·h/mL) cmpd 50 Cl (mL/min·kg) cmpd 50 t1/2 (h) cmpd 50 Vss (L/kg) cmpd 50 Cmax (ng/mL) cmpd 50 Tmax (h) cmpd 50 F (%) prodrug 68 AUC (ng·h/mL) prodrug 68 Cl (mL/min·kg) prodrug 68 t1/2 (h) prodrug 68 Vss (L/kg)

261 32 0.6 0.65

319

301

CONCLUSION A new series of GyrB/ParE inhibitors based on the ethyl urea binding motif has been described. A focused screening around this moiety followed by molecular modeling led to the selection of the isoquinoline core as a promising starting point. The subsequent fragment evolution program was guided by structure-based design, and the optimization efforts focused both on antibacterial activity in vitro and on oral efficacy in a mouse thigh infection model. These investigations resulted in the identification of carbamates as novel chemotypes interacting with an Arg residue in a salt-bridge conserved in all bacterial type II topoisomerases. These new isoquinoline ethyl urea derivatives displayed antibacterial activity in vitro against fluoroquinoloneresistant Gram-positive and RTI pathogens. Interestingly, inhibition of gyrase or topo IV was stronger depending on the bacteria from which the enzymes were isolated, and we report data where both enzymes are inhibited to a similar extent as well as cases where a 250-fold difference exists despite the relative structural homologies between the ATP-binding sites of all these topoisomerases. More work will be needed to assess the relative contribution of gyrase and topo IV inhibition to the observed effects in these bacteria. The carbamate lead compound 50 showed oral efficacy similar to that of linezolid against S. aureus in the neutropenic murine thigh infection model. The possibility to offer patients a safe iv-to-oral switch therapy was deemed important, and the identification of a suitable phosphate prodrug concept supported the potential for developing an adequate iv formulation. On the other hand, the cardiovascular safety limitation proved more difficult to solve: hERG K-channel block could be reduced by introducing either a charge or a heteroatom at the distal end of the carbamate but only at the expense of both antibacterial activity in vitro and exposure after oral administration, which together compromised efficacy in vivo. At this point, none of the compounds exhibits an interesting pharmacological profile overall to justify progression toward preclinical development, and further optimization efforts are required.

310

0.8 1.1 287 0.25 30 79 130 0.2 0.5

250 0.5 30 BLQd

a

Solution in 30% propylene glycol in water at pH 4.2. bSuspension in 0.5% methyl cellulose in water. cSolution in 5% mannitol in water at pH 7.1. dBLQ: below the limit of quantification.

We then introduced substituents carrying a positive charge and measured logD at pH 7.4 as well as pKa. The pKa values for pyridyl and isoquinoline nitrogens (see general structure above Table 6) varied less than those of the R8 substituents. The influence of an ionizable nitrogen in side chains of inverted carbamates 69−71 illustrated a dichotomy between logD and pKa: increasing lipophilicity (logD and clogP) led to stronger hERG block, whereas the antibacterial activity was governed by the proportion of neutral species since decreasing pKa values improved the MIC. In this respect, 70 displayed an optimal balance of lipophilicity versus ionizability, and we further finetuned logD and pKa by preparing 52 and 72−74. Compound 52 had the lowest pKa (6.5) and a MIC of 0.5 μg/mL against S. aureus but displayed only minor hERG block despite significant lipophilicity, thereby contradicting our first hypothesis. Actually, it appeared that a hydrophilic part at the distal end of the molecule was sufficient to strongly reduce hERG block, ruling out the need for a positive charge as further illustrated by 54, 62, and 63. Neutral hydroxyl or ether groups were further studied, and while the hydroxyl derivative 64 displayed moderate antibacterial activity, the corresponding methyl ether 51 represented a better compromise. Finally, amides 65 and 66 blocked hERG to a much lesser extent but were also less potent against bacteria compared to carbamates 50 and 51. Increasing the lipophilicity at the distal end in amide 67 resulted in a better antibacterial activity but also in an unacceptable hERG block as previously noticed in the carbamate series. Pharmacology. A few compounds with minor hERG block were evaluated for oral efficacy against S. aureus A-1178 in the 6.5 h screening model of thigh infection (Table 7), except for carbamate 52 which was not studied in vivo due to prohibitively high intrinsic clearances measured in mouse and rat liver microsomes. Neither carbamates 51 and 70 nor amide 65 exhibited comparable oral efficacies relative to lead compound 50, which can be explained by the lower free exposure of these compounds in addition to the higher MIC. In the standard 24 h neutropenic murine thigh infection model,31 50 exhibited oral efficacy comparable to that of linezolid, a drug used to treat infections caused by Gram-positive bacteria (Figure 6). The vehicle-treated control exhibited about 1.8 log10 CFU/thigh increase until the end of treatment. Both 50

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EXPERIMENTAL SECTION

Chemistry. All solvents and chemicals were used as purchased without further purification. Analytical TLC characterizations were performed with 0.2 mm plates: Merck, Silica gel 60 F254. Column chromatography (CC) was performed using Brunschwig 60A silica gel (0.032−0.63 mm), SNAP KP-Sil cartridges from Biotage, or EasyVarioFlash cartridges from Merck; elution was performed with EtOAc, Heptane, DCM, MeOH, or mixtures thereof. In the cases of compounds containing a basic function (e.g., amine), 1% of NH4OH (25% aq) was added to the eluent(s). Prep-HPLCs were performed on XBridge Prep C18 columns from Waters. Eluents: A, H2O + 0.5% acidic or basic additive; B, MeCN; gradient, 5% B to 95% B over 5 min. Detection: UV/ vis and/or MS and/or ELSD. Prep-HPLC (acidic conditions): additive in A is HCO2H. Prep-HPLC (basic conditions): additive in A is NH4OH (25% aq). In case an amine-containing compound was purified under acidic conditions, HCl was added prior to evaporation to avoid the possible formation of the formamide and usually yielded the corresponding hydrochloride salt. NMR spectra were recorded on a Varian Mercury 300 (300 MHz) or a Bruker Avance 400 (400 MHz for 1 H; 100 MHz for 13C) spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to deuterated solvent as the internal standard (δH: CDCl3 7.26 ppm, d6-DMSO 2.50 ppm); multiplicities, 3764

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Table 6. Influence of Lipophilicity and Charge on hERG Block and Antibacterial Activity

clogP was calculated using DataWarrior.30 bPercent K+ current inhibition measured at 10 μM. cMinimal inhibitory concentration. dGyrase supercoiling inhibition assay. eTopo IV relaxation inhibition assay.

a

s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, and br = broad signal; coupling constants are given in Hz. LC-HRMS was performed on Waters Acquity UPLC-MS; pump, Waters Acquity Binary, Solvent Manager; MS, SYNAPT G2MS; DAD, Acquity UPLC PDA detector; column, Acquity UPLC CSH C18 1.7 μm 2.1 × 50 mm from Waters, thermostated in the Acquity UPLC Column Manager at 60 °C. Eluents: A, H2O + 0.05% formic acid; B, MeCN + 0.05% formic acid; gradient, 2% B to 98% B over 2.0 min; flow, 0.6 mL/min; detection, UV 214 nm and MS; the retention time tR is given in min. The purity of all final compounds was ≥95%. Other LC-MS data were obtained on Sciex API 2000 with Agilent 1100 Binary Pump with DAD and ELSD; Agilent quadrupole MS 6140 with Agilent 1200 Binary Pump, DAD, and ELSD; Thermo Finnigan MSQ Surveyor MS with Agilent 1100 Binary

Pump, DAD, and ELSD; or Thermo MSQ Plus with Dionex GHP 3200 Binary Pump, DAD, and ELSD. The number of decimals given for the corresponding [M + H+] peak(s) of each tested compound depends upon the accuracy of the LC-MS device actually used. Treatment with Scavengers. In cases where the Pd catalyst was used in the last step before biological testing, the crude product was treated with scavengers before performing the purification: the crude residue (0.1 mmol-scale reaction) was dissolved in 9:1 DCM/MeOH (2.0 mL) and treated with a 1:1 mixture (40 mg) of triamine ethyl sulfide amide silica (PhosphonicS STA3; loading, 0.8 mmol/g; particle size, 60−200 μm; pore diameter, 60 Å) and methyl thiourea ethylsulfide ethyl silica (PhosphonicS MTCf; loading, 0.6 mmol/g; particle size, 60−200 μm; pore diameter, 90 Å). The mixture was shaken at rt overnight and filtered. 3765

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Table 7. Oral Efficacy in the Neutropenic Murine Thigh Infection Screening Model against S. aureus A-1178 for Selected Compounds cmpd

oral efficacya (Δlog10CFU/thigh)

mean calcd AUC24h (μg·h/mL)b

PPB (%)

mean calcd fAUC24h (μg·h/mL)b

MIC (μg/mL)c

50 51 65 70

−3.5 −1.3 0 −0.3

45.3 14.0 0.12 9.2

96.5 95.5 87.7 94.8

1.59 0.63 0.015 0.48

0.06 0.125 0.5 0.5

Mice were treated orally (30 mg/kg, 2q2.75 h) starting at 1 h post-infection and sacrificed at 6.5 h post-infection. Median Δlog10CFU/thigh of animal treated with compounds compared to vehicle is reported. Vehicle-treated animals showed median Δlog10CFU/thigh about +3 compared to CFU/thigh at treatment start. bCalcd AUC24h were calculated from PK experiments in mice dosed po at 30 mg/kg for 50 and 65, and at 100 mg/kg for 51 and 70. cMinimal inhibitory concentration of S. aureus A-1178. a

under reduced pressure gave 19c as a beige solid (37.19 g). MS (ESI, m/z): 329.2 and 331.2 [M + H+ of the two main isotopes]. N-(5-Bromo-2-chloro-benzyl)-2,2-diethoxy-acetamidine (19d). Prepared according to General Procedure 1 from 5-bromo-2-chlorobenzylamine 18d (15.09 g, 68.4 mmol) at rt for 10 h. Concentration under reduced pressure gave 19d as an orange viscous oil (23.95 g). MS (ESI, m/z): 349.2 and 351.0 [M + H+ of the two main isotopes]. N-(2-Bromo-5-chloro-benzyl)-2,2-diethoxy-acetamidine (19e). Prepared according to General Procedure 1 from 18e32 (84.01 g, 381 mmol) at rt for 23 h. Concentration under reduced pressure gave 19e as a yellow oil (129.10 g). MS (ESI, m/z): 348.7 and 350.8 [M + H+ of the two main isotopes]. General Procedure 2 (Isoquinoline Formation). To the crude product of General Procedure 1 was added concd H2SO4 (45 equiv) at 0 °C. The reaction mixture was stirred at rt or at 80 °C and monitored by LC-MS. Upon reaction completion, the reaction mixture was slowly poured into water at 0 °C. The resulting acidic aqueous solution was then treated with a 12 N aq NaOH solution at 0 °C until a pH of 12 was obtained. The resulting suspension was filtered and the cake washed with warm water, collected, and dried in vacuo. Purification of the residue gave the desired product. 8-Bromo-isoquinolin-3-yl-amine (20a). Prepared according to General Procedure 2 from 19a (43.45 g) at rt for 22 h. Purification by CC (heptane/EtOAc 100:0 to 30:70) gave 20a as a yellow solid (11.85 g, 40% yield over 2 steps). 1H NMR (300 MHz, d6-DMSO) δ: 8.92 (s, 1H); 7.55−7.50 (m, 1H); 7.41−7.37 (m, 1H); 7.33−7.26 (m, 1H); 6.61 (s, 1H); 6.18 (br, 2H). MS (ESI, m/z): 223.1 and 225.4 [M + H+ of the two main isotopes]. 5-Bromoisoquinolin-3-amine (20b). Prepared according to General Procedure 2 from 19b (24.47 g) at rt for 22 h. Purification by CC (heptane to heptane/EtOAc 1:1) gave 20b as a brown solid (3.34 g, 13% yield over 2 steps). 1H NMR (300 MHz, d6-DMSO) δ: 8.82 (s, 1H); 7.86−7.75 (m, 2H); 7.04 (dd, J = 8.0, 7.4 Hz, 1H); 6.78−6.73 (m, 1H); 6.27 (br, 2H). MS (ESI, m/z): 223.0 and 225.3 [M + H+ of the two main isotopes]. 5-Bromo-8-methyl-isoquinolin-3-ylamine (20c). Prepared according to General Procedure 2 from 19c (37.19 g, 113 mmol) at rt for 15 h. Purification by CC (DCM/MeOH 100:0 to 97:3) gave 20c as a yellow solid (9.70 g, 39% yield over 2 steps). 1H NMR (300 MHz, d6-DMSO) δ: 8.93 (d, J = 0.6 Hz, 1H); 7.66 (d, J = 7.3 Hz, 1H); 6.84 (dd, J = 7.6, 1.2 Hz, 1H); 6.79 (d, J = 0.9 Hz, 1H); 6.24 (br, 2H); 2.57 (d, J = 0.9 Hz, 3H). MS (ESI, m/z): 237.1 and 239.0 [M + H+ of the two main isotopes]. 5-Bromo-8-chloro-isoquinolin-3-ylamine (20d). Prepared according to General Procedure 2 from 19d (23.95 g) at rt for 22 h. Purification by CC (heptane/EtOAc 10:0 to 0:10) gave 20d as a yellow solid (11.29 g, 64% yield over 2 steps). 1H NMR (300 MHz, d6-DMSO) δ: 9.02 (d, J = 0.9 Hz, 1H); 7.76 (d, J = 7.9 Hz, 1H); 7.14 (d, J = 7.9 Hz, 1H); 6.82 (d, J = 0.6 Hz, 1H); 6.58 (br, 2H). MS (ESI, m/z): 257.2 and 259.1 [M + H+ of the two main isotopes]. 8-Bromo-5-chloro-isoquinolin-3-ylamine (20e). Prepared according to General Procedure 2 from 19e (114.69 g, 328 mmol) at 80 °C for 3 h. Drying the precipitate under reduced pressure gave 20e as a brown solid (75.0 g, 88% yield). 1H NMR (300 MHz, d6-DMSO) δ: 8.97 (s, 1H); 7.52 (d, J = 7.9 Hz, 1H); 7.37 (d, J = 7.9 Hz, 1H); 6.82 (s, 1H);

Figure 6. Oral efficacy in a 24 h neutropenic murine thigh infection model with S. aureus A-1178. Mice were treated orally at 40 and 120 mg/kg (1q22h for linezolid and 4q5.5h for 50) starting at 2 h post-infection and sacrificed at 24 h post-infection. Bars and error bars represent median and interquartile ranges. The scavengers were washed with 9:1 DCM/MeOH, and the filtrate was concentrated under reduced pressure. General Procedure 1 (Addition of Benzyl Amine on Imidate Derivatives). To a solution of the required benzyl amine derivative (10 mmol, 1.0 equiv) in dry MeOH (25 mL) was added a solution of 2,2-diethoxy-ethanimidic acid methyl ester 1725 (1.2 equiv) in dry MeOH (25 mL) at rt under inert atmosphere (N2). The reaction mixture was stirred at rt and monitored by LC-MS. Upon reaction completion, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM, and the organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was dried in vacuo and used without further purification in the next step. N-(2-Bromo-benzyl)-2,2-diethoxy-acetamidine (19a). Prepared according to General Procedure 1 from 2-bromobenzylamine 18a (25.0 g, 134 mmol) at rt for 2 h. Concentration under reduced pressure gave 19a as an orange oil (43.45 g). MS (ESI, m/z): 315.2 and 317.0 [M + H+ of the two main isotopes]. N-(3-Bromobenzyl)-2,2-diethoxyacetimidamide (19b). Prepared according to General Procedure 1 from 3-bromobenzylamine hydrochloride 18b (25.0 g, 112 mmol) at 70 °C for 2 h. Concentration under reduced pressure gave 19b as an orange oil (24.47 g). MS (ESI, m/z): 315.2 and 317.2 [M + H+ of the two main isotopes]. N-(5-Bromo-2-methyl-benzyl)-2,2-diethoxy-acetamidine (19c). Prepared according to General Procedure 1 from 5-bromo-2-methylbenzylamine 18c32 (21.21 g, 106 mmol) at 50 °C for 3 h. Concentration 3766

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6.57 (s, 2H). MS (ESI, m/z): 256.9 and 259.0 [M + H+ of the two main isotopes]. General Procedure 3 (Ethyl Urea Formation). To a solution of the required amine (1.0 mmol, 1.0 equiv) in dry dioxane (10 mL) was added ethyl isocyanate (2.5 equiv). The reaction mixture was stirred at 50 °C and monitored by LC-MS. Upon reaction completion, the reaction mixture was cooled to 10 °C. The precipitate formed was filtered, washed with a minimum amount of dioxane, collected, and dried in vacuo to give the desired product. Additional purification might have been performed to isolate the desired product. 1-(8-Bromo-isoquinolin-3-yl)-3-ethyl-urea (21a). Prepared according to General Procedure 3 from 20a (10.84 g, 49 mmol) and ethyl isocyanate (11.50 mL, 145 mmol) at 50 °C for 5 days. A first batch of product was obtained (6.93 g), and the mother liquors were concentrated under reduced pressure. The residue was purified by CC (DCM/MeOH 100:0 to 95:5) to give a second batch of product (4.68 g). Combining the two batches gave 21a as a yellow solid (11.62 g, 81% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.19 (s, 1H); 9.14 (s, 1H); 8.09 (s, 1H); 7.81 (d, J = 8.5 Hz, 1H); 7.69 (d, J = 7.6 Hz, 1H); 7.51 (dd, J = 8.5, 7.6 Hz, 1H); 6.94 (t, J = 5.6 Hz, 1H); 3.22−3.11 (m, 2H); 1.08 (t, J = 7.3 Hz, 3H). MS (ESI, m/z): 294.4 and 296.6 [M + H+ of the two main isotopes]. 1-(5-Bromoisoquinolin-3-yl)-3-ethylurea (21b). Prepared according to General Procedure 3 from 20b (1.18 g, 5.3 mmol) at 50 °C for 24 h. A first batch of product was obtained (340 mg), and the mother liquors were concentrated under reduced pressure. The residue was purified by CC (heptane to heptane/EtOAc 1:1) to give a second batch of product (1.14 g). Combining the two batches gave 21b as a white solid (1.48 g, 96% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.17 (br, 1H); 9.05 (d, J = 0.5 Hz, 1H); 8.34 (s, 1H); 8.03−7.97 (m, 2H); 7.32 (t, J = 7.7 Hz, 1H); 6.94 (t, J = 5.4 Hz, 1H); 3.23−3.10 (m, 2H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 294.5 and 296.6 [M + H+ of the two main isotopes]. 1-(5-Bromo-8-methylisoquinolin-3-yl)-3-ethylurea (21c). Prepared according to General Procedure 3 from 20c (5.66 g, 23.9 mmol) at 50 °C for 5 days. Drying the precipitate under reduced pressure gave 21c as a yellow solid (6.07 g, 82% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.20 (s, 1H); 9.15 (s, 1H); 8.33 (s, 1H); 7.85 (d, J = 7.6 Hz, 1H); 7.12 (dd, J = 7.6, 0.9 Hz, 1H); 7.01 (t, J = 5.6 Hz, 1H); 3.23−3.12 (m, 2H); 2.66 (s, 3H); 1.08 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 155.0; 150.7; 149.4; 137.1; 136.3; 134.6; 126.3; 125.0; 118.0; 103.1; 34.4; 18.2; 15.9. MS (ESI, m/z): 308.4 and 310.4 [M + H+ of the two main isotopes]. 1-(5-Bromo-8-chloro-isoquinolin-3-yl)-3-ethyl-urea (21d). Prepared according to General Procedure 3 from 20d (5.00 g, 19.4 mmol) at 50 °C for 6 days. Drying the precipitate under reduced pressure gave 21d as a pale yellow solid (4.25 g, 67% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.37 (s, 1H); 9.24 (d, J = 0.6 Hz, 1H); 8.45 (d, J = 0.9 Hz, 1H); 7.97 (d, J = 8.2 Hz, 1H); 7.45 (d, J = 8.2 Hz, 1H); 6.91 (t, J = 5.3 Hz, 1H); 3.23−3.12 (m, 2H); 1.08 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, d6-DMSO) δ: 154.8; 151.7; 148.7; 138.3; 134.9; 131.7; 125.6; 122.4; 119.4; 103.0; 34.4; 15.8. MS (ESI, m/z): 328.3 and 330.1 [M + H+ of the two main isotopes]. 1-(8-Bromo-5-chloro-isoquinolin-3-yl)-3-ethyl-urea (21e). To a solution of 20e (39.9 g, 155 mmol) in dry pyridine (1 L) at rt was added ethyl isocyanate (18.2 mL, 230 mmol). The reaction mixture was stirred at 65 °C for 42 h, and pyridine was removed under reduced pressure. Dioxane (500 mL) was added to the residue, and the suspension was stirred at rt for 1 h and cooled to 10 °C. The precipitate was filtered, washed with a minimum amount of dioxane, and dried to give 21e as a yellow solid (40.5 g, 80% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.38 (s, 1H); 9.18 (s, 1H); 8.42 (s, 1H); 7.73 (d, J = 7.9 Hz, 1H); 7.67 (d, J = 7.9 Hz, 1H); 6.92 (t, J = 5.5 Hz, 1H); 3.22−3.12 (m, 2H); 1.08 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 154.8; 151.5; 151.0; 137.2; 131.7; 129.4; 128.7; 123.4; 121.1; 100.2; 34.4; 15.8. MS (ESI, m/z): 328.1 and 330.2 [M + H+ of the two main isotopes]. General Procedure 4 (Negishi-Type Coupling). To a solution of the appropriate aromatic halide (0.1 mmol, 1.0 equiv) and Pd(dppf)Cl2· DCM (0.01 equiv) in dry THF (0.5 mL) was added dimethylzinc

(1.2 M in toluene, 1.6 equiv) at rt under inert atmosphere (N2). The reaction mixture was purged with N2 for 5 min, stirred at 80 °C, and monitored by LC-MS. Upon reaction completion, the reaction mixture was cooled down to rt and diluted with 9:1 DCM/MeOH and water. The two layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification of the residue gave the desired product. 1-Ethyl-3-(8-methylisoquinolin-3-yl)urea (22). Prepared according to General Procedure 4 from 21a (50 mg, 0.17 mmol) at 80 °C for 22 h. Purification by prep-HPLC (basic) gave 22 as a white solid (5 mg, 14% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.13 (s, 1H); 9.01 (s, 1H); 7.95 (s, 1H); 7.56 (d, J = 8.5 Hz, 1H); 7.52−7.44 (m, 1H); 7.21−7.15 (m, 1H); 7.15−7.07 (m, 1H); 3.22−3.11 (m, 2H); 2.67 (s, 3H); 1.08 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C13H15N3O 229.1215, found 230.1291 [M + H+]; tR = 1.01 min. General Procedure 5 (Suzuki Coupling with Pd(PPh3)4). To the aromatic halide (0.1 mmol, 1.0 equiv), the required boronic acid (1.5− 2.0 equiv) and Pd(PPh3)4 (0.1 equiv) were added dioxane (0.8 mL) and an aq 1 N K2CO3 solution (0.2−0.3 mL, 2.0 equiv for 1.5 equiv boronic acid, resp. 3.0 equiv for 2.0 equiv boronic acid) at rt under inert atmosphere (N2). The reaction mixture was purged with N2 for 5 min, stirred at 80 °C−110 °C, and monitored by LC-MS. Upon reaction completion, the reaction mixture was diluted with DCM and a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with a saturated aq NaHCO3 solution, water, and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the residue gave the desired product. 1-Ethyl-3-(5-(pyridin-3-yl)isoquinolin-3-yl)urea (23). Prepared according to General Procedure 5 from 21b (30 mg, 0.1 mmol) and pyridine-3-boronic acid (25.2 mg, 0.2 mmol) at 80 °C for 4 h. Purification by CC (DCM/MeOH 100:0 to 90:10) gave 23 as a white solid (12 mg, 40% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.10 (s, 1H); 9.00 (s, 1H); 8.70−8.64 (m, 2H); 8.09−8.00 (m, 2H); 7.94−7.90 (m, 1H); 7.65−7.45 (m, 3H); 6.98 (t, J = 5.5 Hz, 1H); 3.17−3.01 (m, 2H); 1.03 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C17H16N4O 292.1324, found 293.1404 [M + H+]; tR = 0.83 min. 1-Ethyl-3-(8-methyl-5-(pyridin-3-yl)isoquinolin-3-yl)urea (24). Prepared according to General Procedure 5 from 21c (38.5 mg, 0.125 mmol) and pyridine-3-boronic acid (30.7 mg, 0.25 mmol) at 110 °C for 4 h. Purification by prep-HPLC (acidic) gave 24 as a yellow solid (6 mg, 15% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.22 (d, J = 0.8 Hz, 1H); 9.04 (s, 1H); 8.67−8.61 (m, 2H); 8.01 (d, J = 0.5 Hz, 1H); 7.90−7.84 (m, 1H); 7.58−7.51 (m, 1H); 7.48 (d, J = 7.2 Hz, 1H); 7.30 (dd, J = 7.2, 0.8 Hz, 1H); 7.14−7.07 (m, 1H); 3.17−3.02 (m, 2H); 2.73 (s, 3H); 1.03 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C18H18N4O 306.1481, found 307.1559 [M + H+]; tR = 0.90 min. 1-Ethyl-3-(8-methyl-5-(pyridin-4-yl)isoquinolin-3-yl)urea (25). Prepared according to General Procedure 5 from 21c (60 mg, 0.195 mmol) and pyridine-4-boronic acid (35.9 mg, 0.29 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 98:2) gave 25 as a yellow solid (51 mg, 86% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.22 (s, 1H); 9.03 (s, 1H); 8.70 (d, J = 5.9 Hz, 2H); 8.06 (s, 1H); 7.51−7.46 (m, 3H); 7.30 (d, J = 7.1 Hz, 1H); 7.07 (t, J = 5.0 Hz, 1H); 3.16−3.05 (m, 2H); 2.73 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 155.0; 150.3 (2CH); 150.0; 149.0; 147.3; 136.9; 135.9; 133.7; 131.8; 125.3; 125.1 (2CH); 124.1; 101.8; 34.3; 18.6; 15.9. LC-HRMS (ESI, m/z): calcd for C18H18N4O 306.1481, found 307.1557 [M + H+]; tR = 0.80 min. 1-Ethyl-3-(8-methyl-5-(pyrimidin-5-yl)isoquinolin-3-yl)urea (26). Prepared according to General Procedure 5 from 21c (38.5 mg, 0.125 mmol) and pyrimidine-5-boronic acid (31 mg, 0.25 mmol) at 110 °C for 4 h. Purification by prep-HPLC (acidic) gave 26 as a yellow solid (7 mg, 17% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.27 (s, 1H); 9.23 (s, 1H); 9.05 (s, 1H); 8.92 (s, 2H); 7.96 (s, 1H); 7.57 (d, J = 7.4 Hz, 1H); 7.34 (d, J = 7.4 Hz, 1H); 7.16−7.08 (m, 1H); 3.18−3.02 (m, 2H); 2.97 (s, 3H); 1.03 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C17H17N5O 307.1433, found 308.1509 [M + H+]; tR = 0.99 min. 3767

DOI: 10.1021/acs.jmedchem.6b01834 J. Med. Chem. 2017, 60, 3755−3775

Journal of Medicinal Chemistry

Article

1-Ethyl-3-(8-(pyridin-3-yl)-5-(pyridin-4-yl)isoquinolin-3-yl)urea (33). Prepared according to General Procedure 5 from 32 (60 mg, 0.184 mmol) and pyridine-3-boronic acid (45.1 g, 0.367 mmol) at 90 °C for 3 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 97:3) gave 33 as a pale yellow solid (43 mg, 63% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.11 (br, 1H); 8.84 (s, 1H); 8.78−8.70 (m, 4H); 8.15 (s, 1H); 8.06−8.00 (m, 1H); 7.70 (d, J = 7.3 Hz, 1H); 7.63−7.58 (m, 1H); 7.57−7.54 (m, 2H); 7.45 (d, J = 7.3 Hz, 1H); 7.06−6.97 (m, 1H); 3.15− 3.04 (m, 2H); 1.02 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C22H19N5O 369.1589, found 370.1668 [M + H+]; tR = 0.58 min. General Procedure 6 (Buchwald−Hartwig Amination). To the aromatic halide (0.1 mmol, 1.0 equiv), the required amine derivative (1.5 equiv), Pd2(dba)3 (0.05 equiv), XPhos (0.1 equiv), and NaOtBu (1.2 equiv) was added dry dioxane (0.5 mL) at rt under inert atmosphere (N2). The resulting reaction mixture was purged with N2 for 5 min, stirred at 90 °C, and monitored by LC-MS. Upon reaction completion, the reaction mixture was diluted with DCM and a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with a saturated aq NaHCO3 solution, water, and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the residue gave the desired product. 1-[5-Chloro-8-(pyridin-3-ylamino)-isoquinolin-3-yl]-3-ethyl-urea (34). Prepared according to General Procedure 6 from 21e (1.50 g, 4.6 mmol) and 3-aminopyridine (644 mg, 6.8 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 96:4) gave 34 as a yellow solid (1.26 g, 81% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.34 (s, 1H); 9.23 (s, 1H); 8.81 (s, 1H); 8.47 (d, J = 2.6 Hz, 1H); 8.29 (s, 1H); 8.14 (dd, J = 4.6, 1.3 Hz, 1H); 7.61 (d, J = 8.3 Hz, 1H); 7.55 (ddd, J = 8.3, 2.7, 1.4 Hz, 1H); 7.29 (dd, J = 8.4, 4.6 Hz, 1H); 7.03 (d, J = 8.3 Hz, 1H); 6.99 (t, J = 5.5 Hz, 1H); 3.22−3.12 (m, 2H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 342.1 [M + H+]. 1-[5-Chloro-8-(6-methyl-pyridin-3-ylamino)-isoquinolin-3-yl]-3ethyl-urea (35). Prepared according to General Procedure 6 from 21e (200 mg, 0.609 mmol) and 5-aminopicoline (98.7 mg, 0.913 mmol) at 90 °C for 2 h, and using BrettPhos as a ligand instead. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 95:5) gave 35 as a yellow solid (86 mg, 40% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.35 (s, 1H); 9.20 (s, 1H); 8.69 (s, 1H); 8.35 (d, J = 2.5 Hz, 1H); 8.27 (s, 1H); 7.56 (d, J = 8.3 Hz, 1H); 7.50 (dd, J = 8.3, 2.8 Hz, 1H); 7.18 (d, J = 8.4 Hz, 1H); 6.99 (t, J = 5.6 Hz, 1H); 6.88 (d, J = 8.3 Hz, 1H); 3.22−3.13 (m, 2H); 2.41 (s, 3H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 356.0 [M + H+]. General Procedure 7 (Suzuki Coupling with PCy3/K2CO3). To the aromatic halide (0.1 mmol, 1.0 equiv), the required boronic acid (1.1−2.0 equiv), Pd2(dba)3 (0.1 equiv), and PCy3 (0.2 equiv) were added dioxane (0.8 mL) and an aq 1 N K2CO3 solution (0.2 mL, 2.0 equiv) at rt under inert atmosphere (N2). The reaction mixture was purged with N2 for 5 min, stirred at 100 °C, and monitored by LC-MS. Upon reaction completion, the reaction mixture was diluted with DCM and a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with a saturated aq NaHCO3 solution, water, and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the residue gave the desired product. 1-Ethyl-3-[5-pyridin-4-yl-8-(pyridin-3-ylamino)-isoquinolin-3-yl]urea (36). Prepared according to General Procedure 7 from 34 (100 mg, 0.293 mmol) and pyridine-4-boronic acid (72 mg, 0.585 mmol) at 100 °C for 2 h. Purification by prep-HPLC (basic conditions) gave 36 as a yellow solid (65 mg, 58% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.39 (d, J = 0.4 Hz, 1H); 9.07 (s, 1H); 8.90 (s, 1H); 8.70−8.64 (m, 2H); 8.52 (d, J = 2.6 Hz, 1H); 8.17 (dd, J = 4.6, 1.3 Hz, 1H); 8.11 (s, 1H); 7.62 (ddd, J = 8.3, 2.7, 1.4 Hz, 1H); 7.51−7.46 (m, 3H); 7.32 (ddd, J = 8.2, 4.7, 0.4 Hz, 1H); 7.15 (d, J = 8.0 Hz, 1H); 7.03 (t, J = 5.6 Hz, 1H); 3.17− 3.06 (m, 2H); 1.04 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 155.0; 150.5; 150.3 (2CH); 147.5; 147.4; 142.8; 141.9; 141.8; 140.0; 137.0; 133.1; 127.4; 126.1; 125.0 (2CH); 124.4; 117.7; 109.2; 101.4; 34.3; 15.9. LC-HRMS (ESI, m/z): calcd for C22H20N6O 384.1699, found 385.1772 [M + H+]; tR = 0.56 min.

1-Ethyl-3-[8-methyl-5-(2-methyl-pyridin-4-yl)-isoquinolin-3-yl]urea (27). Prepared according to General Procedure 5 from 21c (100 mg, 0.324 mmol) and 2-methylpyridine-4-boronic acid (88.9 mg, 0.649 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 96:4) gave 27 as a beige solid (44 mg, 43% yield). 1 H NMR (300 MHz, d6-DMSO) δ: 9.22 (s, 1H); 9.03 (s, 1H); 8.55 (d, J = 5.1 Hz, 1H); 8.02 (s, 1H); 7.47 (d, J = 7.2 Hz, 1H); 7.33 (s, 1H); 7.31−7.23 (m, 2H); 7.16 (t, J = 5.4 Hz, 1H); 3.17−3.06 (m, 2H); 2.73 (s, 3H); 2.54 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C19H20N4O 320.1637, found 321.1714 [M + H+]; tR = 0.74 min. 1-(5-(4-Cyanophenyl)-8-methylisoquinolin-3-yl)-3-ethylurea (28). Prepared according to General Procedure 5 from 21c (38.5 mg, 0.125 mmol) and 4-cyanophenylboronic acid (36.7 mg, 0.25 mmol) at 110 °C for 4 h. Purification by prep-HPLC (acidic) gave 28 as a beige solid (12 mg, 24% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.24 (s, 1H); 9.06 (s, 1H); 8.06−7.98 (m, 3H); 7.68 (d, J = 8.2 Hz, 2H); 7.50 (d, J = 7.2 Hz, 1H); 7.32 (d, J = 7.3 Hz, 1H); 7.10−7.01 (m, 1H); 3.17−3.06 (m, 2H); 2.75 (s, 3H); 1.05 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C20H18N4O 330.1481, found 331.1563 [M + H+]; t R = 1.26 min. 1-[5-(5,5-Dimethyl-[1,3,2]dioxaborinan-2-yl)-8-methyl-isoquinolin-3-yl]-3-ethyl-urea (29). To a mixture of 21c (679 mg, 2.2 mmol), bis(neopentyl glycolato)diboron (597 mg, 2.65 mmol), KOAc (649 mg, 6.6 mmol), and Pd(dppf)Cl2·DCM (180 mg, 0.22 mmol) was added degassed DMSO (15.0 mL) at rt under inert atmosphere (N2). The resulting reaction mixture was stirred at 90 °C for 2 h. The reaction mixture was concentrated under reduced pressure and the residue diluted with 9:1 DCM/MeOH, and a saturated aq NH4Cl solution was added. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. Purification by CC (DCM/MeOH 100:0 to 96:4) followed by trituration in EtOAc gave 29 as a yellow solid (544 mg, 72% yield). 1 H NMR (300 MHz, d6-DMSO) δ: 9.12 (d, J = 0.5 Hz, 1H); 9.03 (s, 1H); 8.67 (s, 1H); 7.90 (d, J = 7.0 Hz, 1H); 7.53 (t, J = 5.2 Hz, 1H); 7.16 (dd, J = 7.0, 0.7 Hz, 1H); 3.82 (s, 4H); 3.24−3.13 (m, 2H); 2.67 (s, 3H); 1.09 (t, J = 7.2 Hz, 3H); 1.00 (s, 6H). MS (ESI, m/z): 274.0 [M + H+ of the corresponding boronic acid]. 1-Ethyl-3-(8-methyl-5-pyrimidin-4-yl-isoquinolin-3-yl)-urea (30). Prepared according to General Procedure 5 from 29 (210 mg, 0.32 mmol) and 4-bromo-pyrimidine hydrobromide (154 mg, 0.64 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH 100:0 to 96:4) gave 30 as a yellow solid (40 mg, 40% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.33 (d, J = 1.3 Hz, 1H); 9.25 (d, J = 0.8 Hz, 1H); 9.06 (s, 1H); 8.93 (d, J = 5.2 Hz, 1H); 8.37 (d, J = 0.7 Hz, 1H); 7.79 (dd, J = 5.2, 1.4 Hz, 1H); 7.76 (d, J = 7.3 Hz, 1H); 7.34 (dd, J = 7.3, 0.9 Hz, 1H); 7.16 (t, J = 5.4 Hz, 1H); 3.18−3.07 (m, 2H); 2.75 (d, J = 0.4 Hz, 3H); 1.05 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C17H17N5O 307.1433, found 308.1514 [M + H+]; tR = 0.97 min. 1-Ethyl-3-[8-methyl-5-(1-methyl-1H-pyrazol-4-yl)-isoquinolin-3yl]-urea Hydrochloride (31). Prepared according to General Procedure 5 from 29 (34.1 mg, 0.1 mmol) and 4-bromo-1-methyl-1H-pyrazole (32.2 mg, 0.2 mmoL) at 110 °C for 4 h. Purification by prep-HPLC (acidic conditions) and treatment with HCl gave 31 as an amorphous solid (7.6 mg, 25% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.70 (br, 1H); 9.22 (s, 1H); 8.27 (s, 1H); 8.05 (s, 1H); 7.74 (s, 1H); 7.58 (d, J = 7.2 Hz, 1H); 7.48−7.25 (m, 1H); 7.30 (d, J = 7.2 Hz, 1H); 3.95 (s, 3H); 3.19 (q, J = 7.1 Hz, 2H); 2.70 (s, 3H); 1.10 (t, J = 7.1 Hz, 3H); N+H missing. LC-HRMS (ESI, m/z): calcd for C17H19N5O 309.159, found 310.1668 [M + H+]; tR = 1.04 min. 1-(8-Chloro-5-(pyridin-4-yl)isoquinolin-3-yl)-3-ethylurea (32). Prepared according to General Procedure 5 from 21d (3.00 g, 9.13 mmol) and pyridine-4-boronic acid (2.24 g, 18.3 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 96:4) gave 32 as a pale yellow solid (2.19 g, 74% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.32 (s, 1H); 9.20 (br, 1H); 8.75−8.70 (m, 2H); 8.15 (s, 1H); 7.62 (d, J = 7.6 Hz, 1H); 7.59 (d, J = 7.6 Hz, 1H); 7.53−7.47 (m, 2H); 6.92 (t, J = 5.4 Hz, 1H); 3.17−3.03 (m, 2H); 1.03 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 327.2 [M + H+]. 3768

DOI: 10.1021/acs.jmedchem.6b01834 J. Med. Chem. 2017, 60, 3755−3775

Journal of Medicinal Chemistry

Article

(300 MHz, d6-DMSO) δ: 9.36 (s, 1H); 9.16 (s, 1H); 8.71 (d, J = 5.9 Hz, 2H); 8.59 (d, J = 2.8 Hz, 1H); 8.48 (d, J = 4.6 Hz, 1H); 8.17 (s, 1H); 7.71−7.65 (m, 1H); 7.55 (d, J = 8.0 Hz, 1H); 7.54−7.47 (m, 3H); 6.97 (t, J = 5.6 Hz, 1H); 6.77 (d, J = 8.0 Hz, 1H); 3.17−3.05 (m, 2H); 1.04 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 154.9; 154.8; 152.9; 151.7; 150.4 (2CH); 146.8; 146.5; 146.4; 142.5; 137.2; 132.8; 130.7; 127.8; 125.4; 125.0 (2CH); 117.5; 109.9; 101.5; 34.3; 15.8. LC-HRMS (ESI, m/z): calcd for C22H19N5O2 385.1539, found 386.1621 [M + H+]; tR = 0.79 min. 5-Chloro-3-(3-ethyl-ureido)-isoquinoline-8-carboxylic Acid Methyl Ester (42). To a solution of 21e (9.99 g, 30.4 mmol) in MeOH (150 mL) were added Pd(dppf)Cl2·DCM (500 mg, 2 mol %) and NaOAc (3.0 g, 36.5 mmol). The reaction mixture was stirred under CO atmosphere (3 atm) at 60 °C for 7 h. The reaction mixture was concentrated under reduced pressure, and the residue dissolved in EtOAc (1 L). The organic layer was washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure. Trituration of the residue in MeOH (100 mL) gave 42 as a yellow solid (5.93 g, 63% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.80 (d, J = 0.9 Hz, 1H); 9.32 (s, 1H); 8.48 (d, J = 0.9 Hz, 1H); 7.92 (s, 2H); 6.94 (t, J = 5.5 Hz, 1H); 3.94 (s, 3H); 3.23−3.12 (m, 2H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 308.0 [M + H+]. 1-(5-Chloro-8-hydroxymethyl-isoquinolin-3-yl)-3-ethyl-urea (43). To a solution of 42 (13.69 g, 44.5 mmol) in dry THF (1 L) at −50 °C was added a 2 M solution of LAH (66 mL, 132 mmol) over 30 min. The reaction mixture was stirred at −40 °C for 5 h, and water (70 mL) was added and the reaction mixture stirred overnight at rt. It was concentrated under reduced pressure, and the residue suspended in 1 M HCl (500 mL). The solid was filtered off, washed with water, and dried under reduced pressure. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 95:5) followed by recrystallization from dioxane (200 mL) gave 43 as a yellow solid (8.17 g, 66% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.25 (s, 1H); 9.21 (s, 1H); 8.36 (s, 1H); 7.76 (d, J = 7.7 Hz, 1H); 7.36 (d, J = 7.5 Hz, 1H); 7.00 (t, J = 4.8 Hz, 1H); 5.45 (t, J = 5.2 Hz, 1H); 4.98 (d, J = 5.2 Hz, 2H); 3.23−3.11 (m, 2H); 1.08 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 155.0, 150.3, 148.9, 139.4, 135.8, 130.7, 128.3, 123.4, 123.3, 100.6, 60.6, 34.4, 15.8. MS (ESI, m/z): 280.1 [M + H+]. General Procedure 8 (Suzuki Coupling with PCy3/K3PO4). To the aromatic halide (1.0 mmol, 1.0 equiv), the required boronic acid (1.0−2.0 equiv), Pd2(dba)3 (0.05 equiv), and PCy3 (0.12 equiv) were added degassed dioxane (3.3 mL) and a degassed aq 1 N K3PO4 solution (1.7 mL, 1.7 equiv) at rt under inert atmosphere (N2). The reaction mixture was stirred at 90 °C and monitored by LC-MS. Upon reaction completion, the reaction mixture was either directly concentrated to give the crude product or diluted with 9:1 DCM/MeOH and a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with a saturated aq NaHCO3 solution, water, and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the residue gave the desired product. 1-Ethyl-3-(8-(hydroxymethyl)-5-(2-methylpyridin-4-yl)isoquinolin-3-yl)urea (44). Prepared according to General Procedure 8 from 43 (2.20 g, 7.86 mmol) and 2-methylpyridine-4-boronic acid (1.62 g, 11.8 mmol) at 90 °C for 3 h. Purification by CC (DCM/MeOH 100:0 to 90:10) gave 44 as a yellow solid (2.13 g, 81% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.27 (s, 1H); 9.03 (s, 1H); 8.55 (d, J = 5.1 Hz, 1H); 8.02 (s, 1H); 7.54 (d, J = 7.0 Hz, 1H); 7.47 (d, J = 7.0 Hz, 1H); 7.34 (d, J = 0.8 Hz, 1H); 7.26 (dd, J = 0.8, 4.9 Hz, 1H); 7.13 (t, J = 5.6 Hz, 1H); 5.45 (t, J = 5.6 Hz, 1H); 5.03 (d, J = 5.5 Hz, 2H); 3.18−3.03 (m, 2H); 2.54 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 337.1 [M + H+]. General Procedure 9 (Carbamate Formation from Hydroxyl Derivative with CDI). To a solution of the required alcohol (0.1 mmol, 1.0 equiv) in dry DMF (0.3 mL) were added CDI (1.2 equiv) and DIPEA (1.3 equiv) at rt under inert atmosphere (N2). The reaction mixture was stirred at 40 °C for 4 h, and a solution of the amine (1.5 equiv) and DIPEA (1.5 equiv) in dry DCM (0.2 mL) was added. The reaction mixture was stirred at rt overnight and then concentrated to dryness. Purification of the residue gave the desired product.

1-Ethyl-3-[5-(2-methyl-pyridin-4-yl)-8-(6-methyl-pyridin-3-ylamino)-isoquinolin-3-yl]-urea (37). Starting according to General Procedure 7 from 35 (83 mg, 0.233 mmol) and 2-methylpyridine-4boronic acid (63.9 mg, 0.467 mmol) at 100 °C for 3 h. Purification by prep-HPLC (basic conditions) gave 37 as a yellow solid (64 mg, 67% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.40 (s, 1H); 9.06 (s, 1H); 8.76 (s, 1H); 8.51 (d, J = 5.1 Hz, 1H); 8.40 (d, J = 2.6 Hz, 1H); 8.05 (s, 1H); 7.54 (dd, J = 8.3, 2.7 Hz, 1H); 7.42 (d, J = 8.0 Hz, 1H); 7.31 (s, 1H); 7.24 (dd, J = 5.0, 1.2 Hz, 1H); 7.20 (d, J = 8.4 Hz, 1H); 7.13 (t, J = 5.2 Hz, 1H); 6.99 (d, J = 8.0 Hz, 1H); 3.18−3.06 (m, 2H); 2.53 (s, 3H); 2.42 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C24H24N6O 412.2012, found 413.2087 [M + H+]; tR = 0.55 min. 3-[5-Chloro-3-(3-ethyl-ureido)-isoquinolin-8-ylamino]-benzoic Acid tert-Butyl Ester (38a). Prepared according to General Procedure 6 from 21e (2.00 g, 6.09 mmol) and tert-butyl 3-aminobenzoate (1.79 g, 9.23 mmol) at 90 °C for 4 h using BrettPhos as ligand instead. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 98:2) gave 38a as a yellow solid (609 mg, 23% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.32 (s, 1H); 9.22 (s, 1H); 8.85 (s, 1H); 8.29 (s, 1H); 7.69 (s, 1H); 7.62 (d, J = 8.3 Hz, 1H); 7.50−7.34 (m, 3H); 7.05 (d, J = 8.3 Hz, 1H); 6.98 (t, J = 5.7 Hz, 1H); 3.23−3.09 (m, 2H); 1.52 (s, 9H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 440.9 [M + H+]. 3-[3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylamino]-benzoic Acid tert-Butyl Ester (38). Prepared according to General Procedure 7 from 38a (300 mg, 0.68 mmol) and 2-methylpyridine-4-boronic acid (186 mg, 1.36 mmol) at 100 °C for 3 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 95:5) gave 38 as a yellow solid (283 mg, 84% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.36 (s, 1H); 9.06 (s, 1H); 8.92 (s, 1H); 8.52 (d, J = 5.1 Hz, 1H); 8.06 (s, 1H); 7.76−7.72 (m, 1H); 7.51−7.37 (m, 4H); 7.34 (s, 1H); 7.27 (dd, J = 5.2, 1.4 Hz, 1H); 7.16 (d, J = 7.9 Hz, 1H); 7.14− 7.07 (m, 1H); 3.17−3.07 (m, 2H); 2.53 (s, 3H); 1.53 (s, 9H); 1.04 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 498.1 [M + H+]. 3-[3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylamino]-benzoic Acid (39). Compound 38 (280 mg, 0.563 mmol) was dissolved in TFA (3.5 mL) and stirred at rt for 2 h, and the reaction mixture was concentrated under reduced pressure. Purification by prepHPLC (basic conditions) gave 39 as a yellow solid (217 mg; 87% yield). 1 H NMR (300 MHz, d6-DMSO) δ: 9.37 (s, 1H); 9.06 (s, 1H); 8.90 (s, 1H); 8.52 (d, J = 5.2 Hz, 1H); 8.06 (s, 1H); 7.81−7.77 (m, 1H); 7.52−7.38 (m, 4H); 7.35 (s, 1H); 7.27 (dd, J = 5.1, 1.4 Hz, 1H); 7.19 (d, J = 7.9 Hz, 1H); 7.14−7.10 (m, 1H); 3.18−3.06 (m, 2H); 2.53 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H); COOH proton missing. LC-HRMS (ESI, m/z): calcd for C25H23N5O3 441.1801, found 442.1881 [M + H+]; t R = 0.76 min. 1-[5-Chloro-8-(pyridin-3-yloxy)-isoquinolin-3-yl]-3-ethyl-urea (40). To 21e (200 mg, 0.609 mmol), 3-hydroxypyridine (87 mg, 0.913 mmol), CuI (11.6 mg, 0.061 mmol), K3PO4 (258 mg, 1.22 mmol), and N,N-dimethylglycine (12.9 mg, 0.122 mmol) was added dry DMSO (1.4 mL) at rt under inert atmosphere (N2). The reaction mixture was purged with N2 for 5 min and stirred at 90 °C for 5 h. 3-Hydroxypyridine (87 mg, 0.913 mmol), CuI (11.6 mg, 0.061 mmol), K3PO4 (258 mg, 1.22 mmol), and N,N-dimethylglycine (12.9 mg, 0.122 mmol) were added again and the reaction mixture further stirred at 90 °C for 18 h. The reaction mixture was cooled down to rt and diluted with 9:1 DCM/ MeOH and a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification by CC (Heptane/EtOAc 100:0 to 50:50) gave 40 as a yellow solid (144 mg, 69% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.32 (s, 1H); 9.30 (s, 1H); 8.55 (d, J = 2.7 Hz, 1H); 8.46 (dd, J = 4.5, 1.0 Hz, 1H); 8.40 (s, 1H); 7.72 (d, J = 8.2 Hz, 1H); 7.66−7.60 (m, 1H); 7.48 (dd, J = 8.4, 4.6 Hz, 1H); 6.95 (t, J = 5.5 Hz, 1H); 6.69 (d, J = 8.3 Hz, 1H); 3.23−3.12 (m, 2H); 1.08 (t, J = 7.1 Hz, 3H). MS (ESI, m/z): 343.0 [M + H+]. 1-Ethyl-3-[5-pyridin-4-yl-8-(pyridin-3-yloxy)-isoquinolin-3-yl]urea (41). Prepared according to General Procedure 7 from 40 (50 mg, 0.146 mmol) and pyridine-4-boronic acid (35.9 mg, 0.292 mmol) at 100 °C for 2 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 97:3) gave 41 as a yellow solid (33 mg, 60% yield). 1H NMR 3769

DOI: 10.1021/acs.jmedchem.6b01834 J. Med. Chem. 2017, 60, 3755−3775

Journal of Medicinal Chemistry

Article

(3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl Morpholine-4-carboxylate (45). Prepared according to General Procedure 9 from 44 (750 mg, 2.23 mmol) and morpholine (0.29 mL, 3.34 mmol). Et2O (10 mL) was added to the reaction mixture, and the precipitate was filtered off, washed, and dried in vacuo to give 45 as a yellow solid (850 mg, 84% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.27 (s, 1H); 9.10 (s, 1H); 8.60 (d, J = 5.0 Hz, 1H); 8.09 (s, 1H); 7.59 (d, J = 7.3 Hz, 1H); 7.51 (d, J = 7.3 Hz, 1H); 7.38 (d, J = 1.2 Hz, 1H); 7.31 (dd, J = 1.2, 5.0 Hz, 1H); 7.08 (t, J = 5.5 Hz, 1H); 5.66 (s, 2H); 3.66−3.48 (m, 4H); 3.44−3.36 (m, 4H); 3.19−3.08 (m, 2H); 2.57 (s, 3H); 1.06 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C24H27N5O4 449.2063, found 450.2145 [M + H+]; tR = 0.73 min. 1-Ethyl-3-(5-(2-methylpyridin-4-yl)-8-(pyridin-3-ylmethyl)isoquinolin-3-yl)urea Hydrochloride (46). Prepared according to General Procedure 8 from 45 (32 mg, 0.07 mmol) and 3-pyridineboronic acid (13 mg, 0.11 mmol) at 90 °C for 5 h. Purification by prepHPLC (basic conditions) followed by HCl treatment gave 46 as a yellow solid (16 mg, 59% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.44 (s, 1H); 9.35 (s, 1H); 9.02 (s, 1H); 8.94 (d, J = 6.1 Hz, 1H); 8.84 (d, J = 5.5 Hz, 1H); 8.50 (d, J = 8.2 Hz, 1H); 8.18 (s, 1H); 8.15 (s, 1H); 8.09− 7.97 (m, 2H); 7.79 (d, J = 7.3 Hz, 1H); 7.53 (d, J = 7.4 Hz, 1H); 7.40− 7.16 (m, 1H); 4.85 (s, 2H); 3.11 (q, J = 7.1 Hz, 2H); 2.85 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H); N+H missing. 13C NMR (100 MHz, d6-DMSO) δ: 156.0; 155.2; 154.3; 150.6; 149.0; 146.3; 141.7; 141.2; 140.5 (2CH); 139.2; 135.9; 133.0; 132.5; 128.8; 127.6; 126.2; 125.6; 122.9; 101.5; 34.3; 33.8; 19.7; 15.8. LC-HRMS (ESI, m/z): calcd for C24H23N5O 397.1903, found 398.1984 [M + H+]; tR = 0.59 min. 3-((3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl)benzoic Acid (47). Prepared according to General Procedure 8 from 45 (840 mg, 1.87 mmol) and 3-carboxyphenylboronic acid (620 mg, 3.74 mmol) at 90 °C for 10 h. However, an additional portion of boronic acid, catalyst, and ligand were added after 3 and 7 h. Purification by CC (DCM/MeOH 100:0 to 90:10 to DCM/MeOH + 1% NH4OH 90:10 to 80:20) gave 47 as a yellow solid (481 mg, 58% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.26 (s, 1H); 8.98 (s, 1H); 8.57−8.52 (m, 1H); 8.03 (s, 1H); 7.84 (s, 1H); 7.76 (d, J = 7.5 Hz, 1H); 7.58−7.51 (m, 2H); 7.45−7.32 (m, 3H); 7.31−7.25 (m, 1H); 7.16−7.10 (m, 1H); 4.60 (s, 2H); 3.13−3.04 (m, 2H); 2.54 (s, 3H); 1.02 (t, J = 7.1 Hz, 3H); COOH proton missing. LC-HRMS (ESI, m/z): calcd for C26H24N4O3 440.1848, found 441.1929 [M + H+]; tR = 0.82 min. 1-(8-((1H-1,2,3-Triazol-1-yl)methyl)-5-(2-methylpyridin-4-yl)isoquinolin-3-yl)-3-ethylurea (48). To a suspension of 44 (85 mg, 0.25 mmol) and PPh3 (100 mg, 0.38 mmol) in THF (2.6 mL) at rt was added 1H-1,2,3-triazole (0.02 mL, 0.34 mmol). The reaction mixture was cooled to 0 °C, and DIAD (0.08 mL, 0.38 mmol) was added. The reaction mixture was stirred for 1 h at 0 °C and 1.5 h at rt and became homogeneous during that time. The reaction mixture was concentrated to dryness, and the residue was diluted with 9:1 DCM/ MeOH and a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 96:4) and by prep-HPLC (acidic conditions) gave 48 as a yellow solid (29 mg, 30% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.41 (s, 1H); 9.07 (s, 1H); 8.56 (d, J = 5.0 Hz, 1H); 8.24 (s, 1H); 8.05 (s, 1H); 7.76 (s, 1H); 7.56 (d, J = 7.3 Hz, 1H); 7.33 (s, 1H); 7.26 (d, J = 4.5 Hz, 1H); 7.22 (d, J = 7.3 Hz, 1H); 7.11−7.02 (m, 1H); 6.22 (s, 2H); 3.16−3.04 (m, 2H); 2.54 (s, 3H); 1.03 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 158.8; 154.9; 150.2; 149.5; 148.3; 147.1; 136.5; 136.3; 134.1 (2CH); 131.4; 125.9; 125.1; 124.2; 122.5; 122.2; 102.2; 49.9; 34.3; 24.6; 15.8. LC-HRMS (ESI, m/z): calcd for C21H21N7O 387.1808, found 388.1880 [M + H+]; t R = 0.63 min. 1-[8-(Aminomethyl)-5-chloro-isoquinolin-3-yl]-3-ethyl-urea (49). To a suspension of 43 (4.0 g, 14.3 mmol) in dry THF (30 mL) in a round-bottomed flask under inert atmosphere (N2) at rt were added DPPA (3.7 mL, 17.2 mmol) and DBU (2.56 mL, 17.2 mmol). The reaction mixture was stirred at rt for 3 h and became a solution during that time. The reaction mixture was treated with water (8 mL) and PPh3 (4.8 g, 18.3 mmol), further stirred at rt for 2 h, and concentrated under

reduced pressure. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) followed by trituration from EtOAc (250 mL) gave 49 as a white solid (2.93 g, 74% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.30 (d, J = 0.8 Hz, 1H); 9.18 (s, 1H); 8.33 (d, J = 0.7 Hz, 1H); 7.73 (d, J = 7.7 Hz, 1H); 7.37 (d, J = 7.7 Hz, 1H); 7.03 (t, J = 5.6 Hz, 1H); 4.20 (s, 2H); 3.22−3.12 (m, 2H); 1.92 (br, 2H); 1.08 (t, J = 7.2 Hz, 3H). 13 C NMR (100 MHz, d6-DMSO) δ: 155.1, 150.2, 149.0, 141.1, 135.8, 130.8, 127.8, 123.6, 123.5, 100.6, 42.4, 34.4, 15.9. MS (ESI, m/z): 279.0 [M + H+]. General Procedure 10 (Carbamate Formation from Amino Derivative with Chloroformate). A solution of the amine (0.1 mmol, 1.0 equiv) in dry DCM (1.0 mL) was treated with Et3N (1.2 equiv) and the required chloroformate (1.5 equiv) at 0 °C under inert atmosphere (N2). The reaction mixture was stirred at rt overnight and then diluted with DCM and water. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the residue gave the desired product. [5-Chloro-3-(3-ethyl-ureido)-isoquinolin-8-ylmethyl]-carbamic Acid Methyl Ester (50a). Prepared according to General Procedure 10 from 49 (2.93 g, 10.5 mmol) and methyl chloroformate (1.23 mL, 15.8 mmol). The crude product was triturated in 2:1 Et2O/EtOAc (300 mL), filtered, and dried in vacuo to give 50a as a yellow solid (1.91 g, 54% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.27 (s, 1H); 9.22 (s, 1H); 8.37 (s, 1H); 7.81 (t, J = 6.2 Hz, 1H); 7.76 (d, J = 7.6 Hz, 1H); 7.23 (d, J = 7.7 Hz, 1H); 6.99 (t, J = 5.0 Hz, 1H); 4.67 (d, J = 5.9 Hz, 2H); 3.54 (s, 3H); 3.23−3.11 (m, 2H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 337.0 [M + H+]. [3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylmethyl]-carbamic Acid Methyl Ester (50). Prepared according to General Procedure 8 from 50a (842 mg, 2.5 mmol) and 2-methylpyridine4-boronic acid (514 mg, 3.75 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 95:5) gave 50 as a yellow solid (886 mg, 90% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.30 (s, 1H); 9.04 (s, 1H); 8.56 (d, J = 5.0 Hz, 1H); 8.03 (s, 1H); 7.84 (t, J = 5.9 Hz, 1H); 7.54 (d, J = 7.3 Hz, 1H); 7.38−7.32 (m, 2H); 7.26 (dd, J = 5.1, 1.3 Hz, 1H); 7.11 (t, J = 5.9 Hz, 1H); 4.73 (d, J = 5.9 Hz, 2H); 3.56 (s, 3H); 3.17−3.05 (m, 2H); 2.55 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 158.8; 157.3; 155.0; 149.9; 149.5; 148.3; 147.4; 137.5; 136.2; 135.1; 131.4; 124.3; 123.7; 122.7; 122.2; 102.2; 52.0; 41.5; 34.3; 24.6; 15.9. LC-HRMS (ESI, m/z): calcd for C21H23N5O3 393.1801, found 394.1879 [M + H+]; tR = 0.66 min. General Procedure 11 (Carbamate Formation from Amino Derivative with CDI). To a solution of CDI (2.0 equiv) in dry DCM (0.5 mL) were added DIPEA (2.0 equiv) and the required alcohol (2.0 equiv) at rt under inert atmosphere (N2). The reaction mixture was stirred at rt for 3 h, and a solution of the amine (0.1 mmol, 1.0 equiv) and DIPEA (1.0 equiv) in dry NMP (0.5 mL) was added. The reaction mixture was stirred at rt overnight. A 2 M solution of dimethylamine in THF (10 equiv) was added, and the reaction mixture was further stirred at rt for 2 h. It was then concentrated under reduced pressure, and purification of the residue gave the desired product. [5-Chloro-3-(3-ethyl-ureido)-isoquinolin-8-ylmethyl]-carbamic Acid 2-Methoxy-ethyl Ester (51a). Prepared according to General Procedure 11 from 49 (1.00 g, 3.59 mmol) and 2-methoxyethanol (0.6 mL, 7.6 mmol). However, DMF was used instead of NMP. Purification by CC (DCM/MeOH 100:0 to 95:5) gave 51a as a white solid (1.08 g, 79% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.28 (d, J = 0.7 Hz, 1H); 9.21 (s, 1H); 8.38 (d, J = 0.5 Hz, 1H); 7.95−7.84 (m, 1H); 7.76 (d, J = 7.7 Hz, 1H); 7.23 (d, J = 7.7 Hz, 1H); 7.04−6.95 (m, 1H); 4.66 (d, J = 6.0 Hz, 2H); 4.11−4.03 (m, 2H); 3.51−3.43 (m, 2H); 3.22 (s, 3H); 3.20−3.11 (m, 2H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 381.1 [M + H+]. [3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylmethyl]-carbamic Acid 2-Methoxy-ethyl Ester (51). Prepared according to General Procedure 8 from 51a (230 mg, 0.57 mmol) and 2-methylpyridine-4-boronic acid (117 mg, 0.86 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH 100:0 to 90:10) gave 51 as a beige solid (209 mg, 83% yield). 1H NMR (300 MHz, d6-DMSO) 3770

DOI: 10.1021/acs.jmedchem.6b01834 J. Med. Chem. 2017, 60, 3755−3775

Journal of Medicinal Chemistry

Article

δ: 9.30 (s, 1H); 9.04 (s, 1H); 8.55 (d, J = 5.0 Hz, 1H); 8.03 (s, 1H); 7.99−7.86 (m, 1H); 7.54 (d, J = 7.3 Hz, 1H); 7.38−7.31 (m, 2H); 7.29− 7.21 (m, 1H); 7.16−7.04 (m, 1H); 4.72 (d, J = 5.9 Hz, 2H); 4.12−4.04 (m, 2H); 3.52−3.43 (m, 2H); 3.23 (s, 3H); 3.18−3.04 (m, 2H); 2.54 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 158.8; 156.8; 155.0; 149.9; 149.5; 148.3; 147.4; 137.5; 136.2; 135.1; 131.3; 124.3; 123.7; 122.7; 122.2; 102.2; 70.8; 63.7; 58.5; 41.5; 34.3; 24.6; 15.9. LC-HRMS (ESI, m/z): calcd for C23H27N5O4 437.2063, found 438.2147 [M + H+]; tR = 0.65 min. tert-Butyl 4-((((5-Chloro-3-(3-ethylureido)isoquinolin-8-yl)methyl)carbamoyl)oxy)-3,3-difluoropiperidine-1-carboxylate (52a). Prepared according to General Procedure 11 from 49 (60 mg, 0.215 mmol) and tert-butyl 3,3-difluoro-4-hydroxypiperidine-1-carboxylate (102 mg, 0.43 mmol). However, DMF was used instead of NMP. Purification by CC (DCM/MeOH 100:0 to 95:5) gave 52a as a white solid (34 mg, 29% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.29 (s, 1H); 9.27 (s, 1H); 8.41 (s, 1H); 8.23 (t, J = 5.9 Hz, 1H); 7.80 (d, J = 7.7 Hz, 1H); 7.25 (d, J = 7.7 Hz, 1H); 7.00 (t, J = 5.3 Hz, 1H); 5.16−5.04 (m, 1H); 4.73 (d, J = 5.9 Hz, 2H); 3.99−3.85 (m, 1H); 3.74−3.45 (m, 3H); 3.25−3.12 (m, 2H); 1.98−1.87 (m, 1H); 1.70−1.57 (m, 1H); 1.40 (s, 9H); 1.10 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 542.1 [M + H+]. tert-Butyl 4-((((3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl)carbamoyl)oxy)-3,3-difluoropiperidine-1carboxylate (52b). Prepared according to General Procedure 8 from 52a (34 mg, 0.063 mmol) and 2-methylpyridine-4-boronic acid (13 mg, 0.094 mmol) at 90 °C for 3 h. Purification by CC (DCM/MeOH 100:0 to 95:5) gave 52b as a white solid (29 mg, 77% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.32 (s, 1H); 9.09 (s, 1H); 8.59 (d, J = 5.0 Hz, 1H); 8.26 (t, J = 5.9 Hz, 1H); 8.07 (s, 1H); 7.58 (d, J = 7.2 Hz, 1H); 7.40−7.34 (m, 2H); 7.29 (d, J = 4.5 Hz, 1H); 7.15−7.08 (m, 1H); 5.18− 5.07 (m, 1H); 4.84−4.71 (m, 2H); 3.98−3.87 (m, 1H); 3.74−3.66 (m, 1H); 3.65−3.50 (br, 1H); 3.30−3.19 (m, 1H); 3.17−3.09 (m, 2H); 2.57 (s, 3H); 1.98−1.91 (m, 1H); 1.71−1.62 (m, 1H); 1.41 (s, 9H); 1.06 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 599.1 [M + H+]. General Procedure 12 (NBoc Deprotection with HCl in Dioxane). A solution of the protected amine (0.1 mmol, 1.0 equiv) in 4 M solution of HCl in dioxane (1.0 mL, 40 equiv) was stirred at rt for 2 h and concentrated under reduced pressure. Purification of the residue gave the desired product. 3,3-Difluoropiperidin-4-yl ((3-(3-Ethylureido)-5-(2-methylpyridin4-yl)isoquinolin-8-yl)methyl)carbamate (52). Prepared according to General Procedure 12 from 52b (29 mg, 0.048 mmol). Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 52 as a beige solid (7 mg, 29% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.32 (s, 1H); 9.10 (s, 1H); 8.58 (d, J = 5.1 Hz, 1H); 8.21 (t, J = 6.0 Hz, 1H); 8.07 (s, 1H); 7.57 (d, J = 7.3 Hz, 1H); 7.38−7.34 (m, 2H); 7.29 (d, J = 5.0 Hz, 1H); 7.15−7.08 (m, 1H); 5.04−4.95 (m, 1H); 4.77 (d, J = 5.8 Hz, 2H); 3.18−3.10 (m, 2H); 3.10−3.01 (m, 1H); 2.90−2.78 (m, 2H); 2.67−2.58 (m, 1H); 2.56 (s, 3H); 1.93−1.85 (m, 1H); 1.68−1.57 (m, 1H); 1.06 (t, J = 7.2 Hz, 3H); piperidine NH missing. LC-HRMS (ESI, m/z): calcd for C25H28N6O3F2 498.2191, found 499.2271 [M + H+]; tR = 0.53 min. tert-Butyl (2-((((5-Chloro-3-(3-ethylureido)isoquinolin-8-yl)methyl)carbamoyl)oxy)ethyl)carbamate (53a). Prepared according to General Procedure 11 from 49 (150 mg, 0.538 mmol) and N-(tertbutoxycarbonyl)ethanolamine (0.17 mL, 1.08 mmol). However, DMF was used instead of NMP. In addition, another portion of activated alcohol was added after 3 d, and the reaction mixture was further stirred at rt for 18 h. Purification by CC (DCM/MeOH 100:0 to 90:10) gave 53a as a white solid (152 mg, 61% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.28 (s, 1H); 9.20 (s, 1H); 8.37 (d, J = 0.5 Hz, 1H); 7.84− 7.77 (m, 1H); 7.75 (d, J = 7.7 Hz, 1H); 7.25 (d, J = 7.7 Hz, 1H); 6.99 (t, J = 5.5 Hz, 1H); 6.88−6.80 (m, 1H); 4.66 (d, J = 5.9 Hz, 2H); 3.93 (t, J = 5.6 Hz, 2H); 3.22−3.07 (m, 4H); 1.34 (s, 9H); 1.08 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 466.1 [M + H+]. tert-Butyl (2-((((3-(3-Ethylureido)-5-(pyridin-4-yl)isoquinolin-8-yl)methyl)carbamoyl)oxy)ethyl)carbamate (53b). Prepared according to General Procedure 7 from 53a (150 mg, 0.322 mmol) and pyridine-4boronic acid (48 mg, 0.354 mmol) at 100 °C for 1 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 53b as a yellow solid (123 mg, 75% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.31

(s, 1H); 9.05 (s, 1H); 8.73−8.67 (m, 2H); 8.07 (s, 1H); 7.91−7.84 (m, 1H); 7.55 (d, J = 7.3 Hz, 1H); 7.49−7.45 (m, 2H); 7.40−7.35 (m, 1H); 7.08−7.01 (m, 1H); 6.91−6.84 (m, 1H); 4.75−4.69 (m, 2H); 3.98−3.90 (m, 2H); 3.17−3.05 (m, 4H); 1.34 (s, 9H); 1.03 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 509.2 [M + H+]. General Procedure 13 (NBoc Deprotection with TFA). To a solution of the protected amine (0.1 mmol, 1.0 equiv) in dry DCM (1.0 mL) was added TFA (40 equiv). The reaction mixture was stirred at rt for 1 h and concentrated under reduced pressure. Purification of the residue gave the desired product. 2-Aminoethyl ((3-(3-Ethylureido)-5-(pyridin-4-yl)isoquinolin-8-yl)methyl)carbamate (53). Prepared according to General Procedure 13 from 53b (120 mg, 0.236 mmol). Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 53 as a white solid (85 mg, 88% yield). 1 H NMR (300 MHz, d6-DMSO) δ: 9.31 (s, 1H); 9.05 (s, 1H); 8.71 (d, J = 5.6 Hz, 2H); 8.08 (s, 1H); 7.88−7.80 (m, 1H); 7.57 (d, J = 7.3 Hz, 1H); 7.48 (d, J = 5.7 Hz, 2H); 7.37 (d, J = 7.4 Hz, 1H); 7.07−6.99 (m, 1H); 4.77−4.69 (m, 2H); 3.97−3.88 (m, 2H); 3.15−3.03 (m, 2H); 2.77−2.67 (m, 2H); 1.03 (t, J = 7.2 Hz, 3H); NH2 missing. MS (ESI, m/z): 409.1 [M + H+]. 2-(Neopentylamino)ethyl ((3-(3-Ethylureido)-5-(pyridin-4-yl)isoquinolin-8-yl)methyl)carbamate (54). To a solution of 53 (40 mg, 0.098 mmol) in 1:1 DCE/MeOH (1.0 mL) was added pivaldehyde (0.010 mL, 0.098 mmol), and the reaction mixture was stirred at rt overnight. NaBH4 (5.92 mg, 0.156 mmol) was added cautiously to the reaction mixture which was further stirred at rt for 4 h. It was then diluted with DCM and treated with a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 54 as a white solid (26 mg, 55% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.31 (s, 1H); 9.04 (s, 1H); 8.71 (d, J = 5.9 Hz, 2H); 8.07 (s, 1H); 7.91−7.84 (m, 1H); 7.55 (d, J = 7.2 Hz, 1H); 7.47 (d, J = 5.7 Hz, 2H); 7.35 (d, J = 7.3 Hz, 1H); 7.08−7.00 (m, 1H); 4.75−4.68 (m, 2H); 4.05−3.96 (m, 2H); 3.27 (s, 1H); 3.15−3.04 (m, 2H); 2.74− 2.65 (m, 2H); 2.24 (s, 2H); 1.03 (t, J = 7.2 Hz, 3H); 0.81 (s, 9H). LC-HRMS (ESI, m/z): calcd for C26H34N6O3 478.2692, found 479.2769 [M + H+]; tR = 0.58 min. 1-[8-(Aminomethyl)-5-(2-methyl-pyridin-4-yl)-isoquinolin-3-yl]-3ethyl-urea (55). Prepared according to General Procedure 8 from 49 (500 mg, 1.79 mmol) and 2-methylpyridine-4-boronic acid (368 mg, 2.69 mmol) at 90 °C for 2 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 55 as a yellow solid (478 mg, 80% yield). 1 H NMR (300 MHz, d6-DMSO) δ: 9.33 (d, J = 0.5 Hz, 1H); 9.02 (s, 1H); 8.55 (d, J = 5.1 Hz, 1H); 8.00 (s, 1H); 7.55−7.45 (m, 2H); 7.33 (s, 1H); 7.26 (dd, J = 5.1, 1.4 Hz, 1H); 7.18 (t, J = 5.4 Hz, 1H); 4.26 (s, 2H); 3.17−3.06 (m, 2H); 2.55 (s, 3H); 1.95 (br, 2H); 1.04 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 336.1 [M + H+]. 1-[8-(Aminomethyl)-5-(pyridin-4-yl)-isoquinolin-3-yl]-3-ethylurea (56). Prepared according to General Procedure 7 from 49 (2.30 g, 8.23 mmol) and pyridine-4-boronic acid (1.11 g, 9.06 mmol) at 100 °C for 1 h. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 56 as a yellow solid (1.93 g, 73% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.34 (d, J = 0.8 Hz, 1H); 9.01 (s, 1H); 8.72− 8.68 (m, 2H); 8.05 (d, J = 0.7 Hz, 1H); 7.57−7.53 (m, 1H); 7.51−7.46 (m, 3H); 7.09 (t, J = 5.5 Hz, 1H); 4.27 (s, 2H); 3.16−3.05 (m, 2H); 1.96 (br, 2H); 1.04 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 322.1 [M + H+]. [3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylmethyl]-carbamic Acid Prop-2-ynyl Ester (57). Prepared according to General Procedure 10 from 55 (125 mg, 0.37 mmol) and propargyl chloroformate (0.05 mL, 0.49 mmol). Purification by CC (DCM/ MeOH 100:0 to 95:5) and prep-HPLC (basic conditions) gave 57 as a beige solid (48 mg, 31% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.29 (s, 1H); 9.06 (s, 1H); 8.56 (d, J = 5.0 Hz, 1H); 8.09 (t, J = 5.6 Hz, 1H); 8.04 (s, 1H); 7.55 (d, J = 7.3 Hz, 1H); 7.38−7.32 (m, 2H); 7.26 (d, J = 5.3 Hz, 1H); 7.10 (t, J = 5.3 Hz, 1H); 4.78−4.71 (m, 2H); 4.65 (d, J = 2.4 Hz, 2H); 3.48 (t, J = 2.4 Hz, 1H); 3.16−3.05 (m, 2H); 2.54 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C23H23N5O3 417.1801, found 418.1878 [M + H+]; tR = 0.71 min. 3771

DOI: 10.1021/acs.jmedchem.6b01834 J. Med. Chem. 2017, 60, 3755−3775

Journal of Medicinal Chemistry

Article

[3-(3-Ethyl-ureido)-5-pyridin-4-yl-isoquinolin-8-ylmethyl]-carbamic Acid Methyl Ester (59). Prepared according to General Procedure 10 from 56 (119 mg, 0.37 mmol) and methyl chloroformate (0.043 mL, 0.55 mmol). Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 59 as an amorphous solid (95 mg, 68% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.30 (s, 1H); 9.05 (s, 1H); 8.71 (dd, J = 1.4, 4.5 Hz, 2H); 8.08 (s, 1H); 7.85 (t, J = 5.7 Hz, 1H); 7.56 (d, J = 7.3 Hz, 1H); 7.48 (dd, J = 1.5, 4.5 Hz, 2H); 7.36 (d, J = 7.3 Hz, 1H); 7.06 (t, J = 5.4 Hz, 1H); 4.73 (d, J = 5.9 Hz, 2H); 3.56 (s, 3H); 3.17−2.96 (m, 2H); 1.03 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C20H21N5O3 379.1644, found 380.1731 [M + H+]; tR = 0.67 min. [3-(3-Ethyl-ureido)-5-pyridin-4-yl-isoquinolin-8-ylmethyl]-carbamic Acid Isopropyl Ester (60). Prepared according to General Procedure 11 from 56 (32 mg, 0.1 mmol) and 2-propanol (12 mg, 0.2 mmol). Purification by prep-HPLC (acidic conditions) gave 60 as an amorphous solid (11 mg, 27% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.33 (s, 1H); 9.09 (s, 1H); 8.76−8.72 (m, 2H); 8.11 (s, 1H); 7.80 (t, J = 5.9 Hz, 1H); 7.59 (d, J = 7.3 Hz, 1H); 7.53−7.49 (m, 2H); 7.37 (d, J = 7.3 Hz, 1H); 7.07−7.01 (m, 1H); 4.85−4.75 (m, 1H); 4.74 (d, J = 5.9 Hz, 2H); 3.17−3.08 (m, 2H); 1.19 (d, J = 6.2 Hz, 6H); 1.05 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C22H25N5O3 407.1957, found 408.2041 [M + H+]; tR = 0.83 min. [3-(3-Ethyl-ureido)-5-pyridin-4-yl-isoquinolin-8-ylmethyl]-carbamic Acid 2-(tert-Butyl-dimethyl-silanyloxy)-ethyl Ester (61a). Prepared according to General Procedure 11 from 56 (120 mg, 0.373 mmol) and 2-((tert-butyldimethylsilyl)oxy)ethanol (138 mg, 0.784 mmol). However, DMF was used instead of NMP, and the reaction mixture was stirred 4 d at rt. Purification by CC (DCM/MeOH 100:0 to 90:10) gave 61a as a yellow solid (170 mg, 87% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.32 (s, 1H); 9.03 (s, 1H); 8.74−8.68 (m, 2H); 8.06 (s, 1H); 7.93−7.82 (m, 1H); 7.55 (d, J = 7.3 Hz, 1H); 7.50− 7.43 (m, 2H); 7.36 (d, J = 7.3 Hz, 1H); 7.12−7.01 (m, 1H); 4.72 (d, J = 6.0 Hz, 2H); 4.05−3.97 (m, 2H); 3.77−3.67 (m, 2H); 3.18−3.03 (m, 2H); 1.03 (t, J = 7.2 Hz, 3H); 0.81 (s, 9H); 0.00 (s, 6H). MS (ESI, m/z): 524.3 [M + H+]. General Procedure 14 (OTBS Deprotection with TFA). To a solution of the TBS protected alcohol (0.1 mmol, 1.0 equiv) in 10:1 THF/water (1.0 mL) at 0 °C was added TFA (20 equiv). The reaction mixture was stirred at rt for 1 h and concentrated under reduced pressure. It was diluted with DCM and treated with a saturated aq NaHCO3 solution. The layers were separated, and the aqueous layer was extracted with 9:1 DCM/MeOH (3×). The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the residue gave the desired product. [3-(3-Ethyl-ureido)-5-pyridin-4-yl-isoquinolin-8-ylmethyl]-carbamic Acid 2-Hydroxy-ethyl Ester (61). Prepared according to General Procedure 14 from 61a (196 mg, 0.373 mmol). Purification by CC (DCM/MeOH 100:0 to 90:10) followed by prep-HPLC (basic conditions) gave 61 as a white solid (89 mg, 58% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.32 (s, 1H); 9.04 (s, 1H); 8.74−8.67 (m, 2H); 8.07 (s, 1H); 7.91−7.79 (m, 1H); 7.56 (d, J = 7.3 Hz, 1H); 7.51− 7.45 (m, 2H); 7.37 (d, J = 7.4 Hz, 1H); 7.07−6.99 (m, 1H); 4.77−4.66 (m, 3H); 3.99 (t, J = 5.0 Hz, 2H); 3.53 (q, J = 5.3 Hz, 2H); 3.17−3.04 (m, 2H); 1.03 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C21H23N5O4 409.1750, found 410.1824 [M + H+]; tR = 0.60 min. tert-Butyl (2-((((3-(3-Ethylureido)-5-(pyridin-4-yl)isoquinolin-8-yl)methyl)carbamoyl)oxy)ethyl) (methyl)carbamate (62a). Prepared according to General Procedure 11 from 56 (200 mg, 0.554 mmol) and tert-butyl 2-hydroxyethyl(methyl)carbamate (194 mg, 1.11 mmol). Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) followed by trituration from Et2O (3 mL) gave 62a as a white solid (147 mg, 51% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.34 (s, 1H); 9.08 (s, 1H); 8.73 (d, J = 5.6 Hz, 2H); 8.10 (s, 1H); 7.95 (br, 1H); 7.58 (d, J = 7.2 Hz, 1H); 7.50 (d, J = 5.6 Hz, 2H); 7.39 (d, J = 7.2 Hz, 1H); 7.07 (t, J = 4.9 Hz, 1H); 4.75 (d, J = 5.8 Hz, 2H); 4.12−4.06 (m, 2H); 3.38 (t, J = 5.5 Hz, 2H); 3.16−3.08 (m, 2H); 2.85−2.77 (m, 3H); 1.40− 1.30 (m, 9H); 1.05 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 523.2 [M + H+]. 2-(Methylamino)ethyl ((3-(3-Ethylureido)-5-(pyridin-4-yl)isoquinolin-8-yl)methyl)carbamate (62). Prepared according to General Procedure 12 from 62a (147 mg, 0.281 mmol). Purification

by prep-HPLC (basic conditions) gave 62 as a white solid (58 mg, 49% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.33 (s, 1H); 9.08 (s, 1H); 8.76−8.70 (m, 2H); 8.10 (s, 1H); 7.89 (t, J = 5.9 Hz, 1H); 7.59 (d, J = 7.3 Hz, 1H); 7.51 (m, 2H); 7.38 (d, J = 7.3 Hz, 1H); 7.09−7.03 (m, 1H); 4.75 (d, J = 5.9 Hz, 2H); 4.02 (t, J = 5.8 Hz, 2H); 3.17−3.08 (m, 2H); 2.67 (t, J = 5.8 Hz, 2H); 2.28 (s, 3H); 1.05 (t, J = 7.2 Hz, 3H); side chain NH missing. 13C NMR (100 MHz, d6-DMSO) δ: 157.0; 155.0:150.4 (2CH); 150.1; 148.4; 147.2; 137.7; 136.1; 134.9; 131.5; 125.1 (2CH); 123.7; 122.7; 102.0; 64.0; 50.8; 41.5; 36.5; 34.3; 15.9. LC-HRMS (ESI, m/z): calcd for C22H26N6O3 422.2066, found 423.2141 [M + H+]; t R = 0.49 min. [3-(3-Ethyl-ureido)-5-pyridin-4-yl-isoquinolin-8-ylmethyl]-carbamic Acid 2-Morpholin-4-yl-ethyl Ester Hydrochloride (63). Prepared according to General Procedure 11 from 56 (32 mg, 0.1 mmol) and 4-(2-hydroxyethyl)morpholine (26 mg, 0.2 mmol). Purification by prep-HPLC (acidic conditions) followed by HCl treatment gave 63 as an amorphous solid (36 mg, 75% yield). 1H NMR (400 MHz, d6DMSO) δ: 11.41 (br, 1H); 9.37 (s, 1H); 9.23 (s, 1 H); 8.99 (d, J = 6.4 Hz, 2H); 8.17 (s, 1 H); 8.17−8.11 (m, 1H); 8.00 (d, J = 6.2 Hz, 2H); 7.72 (d, J = 7.3 Hz, 1H); 7.47 (d, J = 7.4 Hz, 1H); 7.18−7.09 (m, 1H); 4.81 (d, J = 5.7 Hz, 2H); 4.43 (t, J = 4.9 Hz, 2H); 4.04−3.76 (m, 4H); 3.51−3.32 (m, 4H); 3.26−3.05 (m, 4H); 1.05 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C25H30N6O4 478.2328, found 479.2409 [M + H+]; tR = 0.51 min. 2-((tert-Butyldimethylsilyl)oxy)ethyl ((3-(3-Ethylureido)-5-(2methylpyridin-4-yl)isoquinolin-8-yl)methyl)carbamate (64a). Prepared according to General Procedure 11 from 55 (195 mg, 0.581 mmol) and 2-(tert-butyldimethylsiloxy)ethanol (215 mg, 1.22 mmol). However, DMF was used instead of NMP, and the reaction mixture was stirred at rt for 4 d. Purification by CC (DCM/MeOH 100:0 to 95:5) gave 64a as a white solid (375 mg, unpure). MS (ESI, m/z): 538.3 [M + H+]. 2-Hydroxyethyl ((3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl)carbamate (64). Prepared according to General Procedure 14 from 64a (375 mg, unpure). Purification by CC (DCM/MeOH 100:0 to 90:10) gave 64 as a white solid (108 mg, 44% yield over 2 steps). 1H NMR (300 MHz, d6-DMSO) δ: 9.31 (s, 1H); 9.04 (s, 1H); 8.56 (d, J = 5.0 Hz, 1H); 8.03 (s, 1H); 7.88−7.83 (m, 1H); 7.54 (d, J = 7.3 Hz, 1H); 7.39−7.32 (m, 2H); 7.26 (dd, J = 0.6, 5.0 Hz, 1H); 7.15−7.07 (m, 1H); 4.76−4.67 (m, 3H); 4.02−3.95 (m, 2H); 3.57−3.50 (m, 2H); 3.16−3.05 (m, 2H); 2.54 (s, 3H); 1.04 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C22H25N5O4 423.1906, found 424.1982 [M + H+]; tR = 0.59 min. General Procedure 15 (Amide Formation). To a solution of the amine (0.1 mmol, 1.0 equiv) in dry DMF (0.5 mL) were added dropwise the required acid (1.5 equiv), DIPEA (3.0 equiv), and a 50 wt % solution of T3P in EtOAc (1.2 equiv) at rt under inert atmosphere (N2). The reaction mixture was stirred at rt overnight, and the reaction mixture was concentrated under reduced pressure. Purification of the residue gave the desired product. N-[3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylmethyl]-acetamide (65). Prepared according to General Procedure 15 from 55 (70 mg, 0.209 mmol) and acetic acid (0.018 mL, 0.313 mmol). Purification by CC (DCM/MeOH 100:0 to 90:10) gave 65 as a pale yellow solid (55 mg, 69% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.26 (d, J = 0.6 Hz, 1H); 9.02 (s, 1H); 8.56 (dd, J = 5.0, 0.6 Hz, 1H); 8.45 (t, J = 5.8 Hz, 1H); 8.03 (s, 1H); 7.55−7.52 (m, 1H); 7.36 (d, J = 7.3 Hz, 1H); 7.33 (br, 1H); 7.26 (dd, J = 5.2, 1.2 Hz, 1H); 7.12 (t, J = 5.3 Hz, 1H); 4.77 (d, J = 5.7 Hz, 2H); 3.16−3.05 (m, 2H); 2.54 (s, 3H); 1.88 (s, 3H); 1.03 (t, J = 7.2 Hz, 3H). LC-HRMS (ESI, m/z): calcd for C21H23N5O2 377.1852, found 378.1934 [M + H+]; tR = 0.57 min. N-[3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylmethyl]-3-methoxy-propionamide (66). Prepared according to General Procedure 15 from 55 (70 mg, 0.209 mmol) and 3-methoxypropionic acid (0.030 mL, 0.313 mmol). Purification by two successive CCs (DCM/MeOH 100:0 to 90:10) gave 66 as a white solid (59 mg, 67% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.25 (d, J = 0.4 Hz, 1H); 9.03 (s, 1H); 8.56 (d, J = 5.0 Hz, 1H); 8.48 (t, J = 5.8 Hz, 1H); 8.03 (s, 1H); 7.56−7.52 (m, 1H); 7.37 (d, J = 7.3 Hz, 1H); 7.33 (br, 1H); 7.26 (dd, J = 5.1, 1.2 Hz, 1H); 7.13 (t, J = 5.3 Hz, 1H); 4.80 (d, J = 5.7 Hz, 2H); 3772

DOI: 10.1021/acs.jmedchem.6b01834 J. Med. Chem. 2017, 60, 3755−3775

Journal of Medicinal Chemistry

Article

3.56 (t, J = 6.3 Hz, 2H); 3.21 (s, 3H); 3.17−3.06 (m, 2H); 2.55 (s, 3H); 2.39 (t, J = 6.2 Hz, 2H); 1.04 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6-DMSO) δ: 170.6; 158.8; 155.0; 149.9; 149.5; 148.5; 147.4; 137.3; 136.2; 135.2; 131.3; 124.3; 124.1; 122.8; 122.2; 102.1; 68.8; 58.4; 36.5; 34.3; 24.6; 15.9. LC-HRMS (ESI, m/z): calcd for C23H27N5O3 421.2114, found 422.2193 [M + H+]; tR = 0.60 min. Cyclopropanecarboxylic Acid [3-(3-Ethyl-ureido)-5-(2-methyl-pyridin-4-yl)-isoquinolin-8-ylmethyl]-amide (67). Prepared according to General Procedure 15 from 55 (70 mg, 0.209 mmol) and cyclopropanecarboxylic acid (0.025 mL, 0.313 mmol). Purification by CC (DCM/MeOH 100:0 to 90:10) gave 67 as a colorless solid (50 mg, 59% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.25 (d, J = 0.7 Hz, 1H); 9.04 (s, 1H); 8.67 (t, J = 5.7 Hz, 1H); 8.56 (d, J = 5.0 Hz, 1H); 8.04 (s, 1H); 7.58−7.52 (m, 1H); 7.37 (d, J = 7.3 Hz, 1H); 7.33 (br, 1H); 7.26 (dd, J = 5.1, 1.3 Hz, 1H); 7.09 (t, J = 5.4 Hz, 1H); 4.81 (d, J = 5.7 Hz, 2H); 3.16− 3.05 (m, 2H); 2.55 (s, 3H); 1.67−1.56 (m, 1H); 1.04 (t, J = 7.2 Hz, 3H); 0.76−0.62 (m, 4H). LC-HRMS (ESI, m/z): calcd for C23H25N5O2 403.2008, found 404.2084 [M + H+]; tR = 0.64 min. (4-(3-(3-Ethylureido)-8-(((methoxycarbonyl)amino)methyl)isoquinolin-5-yl)-2-methylpyridin-1-ium-1-yl)methyl Hydrogen Phosphate (68). To a yellow suspension of 50 (590 mg, 1.5 mmol) and sodium iodide (480 mg, 3.2 mmol) in DCM (15 mL) at rt under inert atmosphere (N2) was added di-tert-butyl chloromethyl phosphate (4.14 g, 16 mmol). The reaction mixture was stirred at rt for 72 h and then cooled down to 0 °C. TFA (2.9 mL, 37.5 mmol) was added dropwise, and the reaction mixture was stirred at 0 °C for 2 h. Additional TFA (1.5 mL, 19 mmol) was added, the reaction mixture further stirred at 0 °C for 1 h, and then concentrated under reduced pressure. The residue was taken up in water and 9:1 DCM/MeOH and cooled down to 0 °C. Aqueous 8 M NaOH was added until pH 11, and the two layers were separated. The aqueous layer was washed twice with 9:1 DCM/ MeOH and then neutralized with aq 2 M HCl until pH 7. The aqueous layer was then concentrated until about 20 mL was remaining. Purification by prep-HPLC (acidic conditions) gave 68 as a yellow solid (115 mg, 15% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.42 (s, 1H); 9.39−9.36 (m, 1H); 9.18 (d, J = 6.5 Hz, 1H); 8.21 (d, J = 0.6 Hz, 1H); 8.16−8.07 (m, 2H); 7.92−7.89 (m, 1H); 7.76 (d, J = 7.5 Hz, 1H); 7.42−7.38 (m, 2H); 6.10 (d, J = 12.7 Hz, 2H); 4.76 (d, J = 5.8 Hz, 2H); 3.56 (s, 3H); 3.18−3.19 (m, 2H); 2.95 (s, 3H); 1.05 (t, J = 7.2 Hz, 3H); POH proton missing. MS (ESI, m/z): 504.1 [M + H+]. (3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl Piperazine-1-carboxylate (69). Prepared according to General Procedure 9 from 44 (47 mg, 0.138 mmol) and piperazine (18 mg, 0.207 mmol). Purification by two successive CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 69 as a white solid (30 mg, 49% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.23 (d, J = 0.7 Hz, 1H); 9.06 (s, 1H); 8.57 (d, J = 5.3 Hz, 1H); 8.06 (d, J = 0.5 Hz, 1H); 7.56 (d, J = 7.3 Hz, 1H); 7.47 (d, J = 7.3 Hz, 1H); 7.36 (s, 1H); 7.28 (dd, J = 1.3, 5.0 Hz, 1H); 7.07−7.03 (m, 1H); 5.61 (s, 2H); 3.33−3.25 (m, 4H); 3.16−3.05 (m, 2H); 2.66−2.58 (m, 4H); 2.55 (s, 3H); 1.04 (t, J = 7.3 Hz, 3H); piperazine NH missing. LC-HRMS (ESI, m/z): calcd for C24H28N6O3 448.2223, found 449.2288 [M + H+]; tR = 0.50 min. (3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl 4-Methylpiperazine-1-carboxylate (70). Prepared according to General Procedure 9 from 44 (100 mg, 0.297 mmol) and 1-methylpiperazine (0.050 mL, 0.446 mmol). Purification by CC (DCM/MeOH 100:0 to 90:10) gave 70 as a white solid (110 mg, 80% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.23 (s, 1H); 9.06 (s, 1H); 8.58 (s, 1H); 8.06 (s, 1H); 7.56 (d, J = 7.3 Hz, 1H); 7.47 (d, J = 7.3 Hz, 1H); 7.35 (d, J = 0.4 Hz, 1H); 7.28 (dd, J = 1.2, 4.9 Hz, 1H); 7.08−7.04 (m, 1H); 5.61 (s, 2H); 3.39−3.34 (m, 4H); 3.15−3.06 (m, 2H); 2.55 (s, 3H); 2.26−2.20 (m, 4H); 2.14 (s, 3H); 1.03 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, d6DMSO) δ: 158.8; 155.0; 154.7; 150.0; 149.5; 148.4; 147.2; 136.3; 136.2; 134.7; 131.3; 124.9; 124.3; 122.8; 122.2; 102.2; 64.2; 54.7 (2CH); 46.2 (2CH); 43.9; 34.3; 24.6; 15.9. LC-HRMS (ESI, m/z): calcd for C25H30N6O3 462.2379, found 463.2458 [M + H+]; tR = 0.51 min. (3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl 4-(Cyclopropylmethyl)piperazine-1-carboxylate hydrochloride (71). Prepared according to General Procedure 9 from 44 (25 mg, 0.075 mmol) and 1-(cyclopropylmethyl)piperazine (16 mg, 0.113 mmol).

Purification by prep-HPLC (basic conditions) followed by HCl treatment gave 71 as a white solid (20 mg, 52% yield). 1H NMR (400 MHz, d6-DMSO) δ: 10.90 (br, 1H); 9.34 (s, 1H); 9.25 (s, 1H); 8.91 (d, J = 5.9 Hz, 1H); 8.17 (s, 1H); 8.03 (s, 1H), 7.98−7.90 (m, 1H); 7.74 (d, J = 7.3 Hz, 1H); 7.60 (d, J = 7.4 Hz, 1H); 7.19−7.06 (m, 1H); 5.71 (s, 2H); 4.15−4.04 (m, 2H); 3.73−3.21 (m, 4H); 3.17−3.07 (m, 2H); 3.07−2.93 (m, 4H); 2.80 (s, 3H); 1.14−1.05 (m, 1H); 1.06 (t, J = 7.2 Hz, 3H); 0.68−0.58 (m, 2H); 0.42−0.34 (m, 2H). LC-HRMS (ESI, m/z): calcd for C28H34N6O3 502.2692, found 503.2769 [M + H+]; t R = 0.54 min. tert-Butyl 4-((((3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methoxy)carbonyl)amino)piperidine-1-carboxylate (72a). Prepared according to General Procedure 9 from 44 (50 mg, 0.149 mmol) and 1-N-Boc-4-aminopiperidine (46 mg, 0.223 mmol). Et2O (3 mL) was added to the reaction mixture, and the precipitate was filtered off, washed, and dried in vacuo to give 72a as a white solid (55 mg, 66% yield). 1H NMR (300 MHz, d6-DMSO) δ: 9.23 (s, 1H); 9.07 (s, 1H); 8.57 (d, J = 5.0 Hz, 1H); 8.06 (s, 1H); 7.57 (d, J = 7.3 Hz, 1H); 7.47 (d, J = 7.3 Hz, 1H); 7.37−7.32 (m, 2H); 7.30−7.26 (d, J = 5.0 Hz, 1H); 7.13−7.05 (m, 1H); 5.57 (s, 2H); 3.86−3.77 (m, 2H); 3.57−3.43 (m, 1H); 3.17−3.07 (m, 2H); 2.88−2.71 (m, 2H); 2.55 (s, 3H); 1.77−1.66 (m, 2H); 1.37 (s, 9H); 1.31−1.15 (m, 2H); 1.04 (t, J = 7.2 Hz, 3H). MS (ESI, m/z): 563.4 [M + H+]. (3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl Piperidin-4-ylcarbamate (72). Prepared according to General Procedure 13 from 72a (55 mg, 0.098 mmol). Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 72 as a white solid (36 mg, 80% yield). 1H NMR (400 MHz, d6-DMSO) δ: 9.24 (s, 1H); 9.12 (s, 1H); 8.59 (d, J = 5.1 Hz, 1H); 8.08 (s, 1H); 7.59 (d, J = 7.3 Hz, 1H); 7.49 (d, J = 7.3 Hz, 1H); 7.41−7.31 (m, 2H); 7.30 (d, J = 4.6 Hz, 1H); 7.15−7.05 (m, 1H); 5.58 (s, 2H); 3.48−3.22 (m, 3H); 3.17−3.05 (m, 2H); 3.00−2.78 (m, 2H); 2.57 (s, 3H); 1.78−1.64 (m, 2H); 1.43− 1.19 (m, 2H); 1.06 (t, J = 7.2 Hz, 3H); piperidine NH missing. 13C NMR (100 MHz, d6-DMSO) δ: 158.8; 155.5; 155.0; 150.0; 149.5; 148.5; 147.2; 136.2 (2CH); 135.1; 131.2; 124.9; 124.3; 122.8; 122.2; 102.1; 62.9; 48.6; 45.0 (2CH); 34.3; 32.9 (2CH); 24.6; 15.9. LC-HRMS (ESI, m/z): calcd for C25H30N6O3 462.2379, found 463.2462 [M + H+]; t R = 0.52 min. tert-Butyl (cis)-4-((((3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methoxy)carbonyl)amino)-3-fluoropiperidine-1carboxylate (73a). Prepared according to General Procedure 9 from 44 (50 mg, 0.149 mmol) and cis-4-amino-1-Boc-3-fluoropiperidine (49 mg, 0.223 mmol). However, after overnight at rt, the reaction mixture was further stirred at 40 °C for 70 h. Et2O (3 mL) was added to the reaction mixture, and the precipitate was filtered off, washed, and dried in vacuo to give 73a as a white solid (53 mg) which was used without further purification. (3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl ((cis)-3-Fluoropiperidin-4-yl)carbamate (73). Prepared according to General Procedure 13 from 73a (50 mg, 0.085 mmol). Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 73 as a beige solid (26 mg, 39% yield over 2 steps). 1H NMR (300 MHz, d6-DMSO) δ: 9.24 (s, 1H); 9.08 (s, 1H); 8.57 (d, J = 5.0 Hz, 1H); 8.06 (s, 1H); 7.60−7.55 (m, 1H); 7.54−7.46 (m, 2H); 7.36 (s, 1H); 7.31− 7.26 (m, 1H); 7.13−7.06 (m, 1H); 5.59 (s, 2H); 4.66−4.44 (m, 1H); 3.71−3.48 (m, 1H); 3.18−3.06 (m, 2H); 3.06−2.97 (m, 1H); 2.91−2.81 (m, 1H); 2.77−2.54 (m, 1H); 2.55 (s, 3H); 2.05−1.90 (m, 1H); 1.64− 1.41 (m, 2H); 1.04 (t, J = 7.1 Hz, 3H); piperidine NH missing. LCHRMS (ESI, m/z): calcd for C25H29N6O3F 480.2285, found 481.2357 [M + H+]; tR = 0.51 min. tert-Butyl (trans)-4-((((3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methoxy)carbonyl)amino)-3-fluoropiperidine-1carboxylate (74a). Prepared according to General Procedure 9 from 44 (50 mg, 0.149 mmol) and trans-4-amino-1-Boc-3-fluoropiperidine (49 mg, 0.223 mmol). However, after overnight at rt, trans-4-amino-1Boc-3-fluoropiperidine (49 mg, 0.223 mmol) and DIPEA (0.039 mL, 0.223 mmol) were added again, and the reaction mixture was further stirred at 40 °C for 70 h. Et2O (3 mL) was added to the reaction mixture, and the precipitate was filtered off, washed, and dried in vacuo to give 74a as a white solid (42 mg) which was used without further purification. 3773

DOI: 10.1021/acs.jmedchem.6b01834 J. Med. Chem. 2017, 60, 3755−3775

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(3-(3-Ethylureido)-5-(2-methylpyridin-4-yl)isoquinolin-8-yl)methyl ((trans)-3-Fluoropiperidin-4-yl)carbamate (74). Prepared according to General Procedure 12 from 74a (42 mg, 0.073 mmol). However, Et2O (3 mL) was added to the reaction mixture at the end of the reaction, and the precipitate was triturated, filtered, collected, and dried. Purification by CC (DCM/MeOH + 1% NH4OH 100:0 to 90:10) gave 74 as a beige solid (20 mg, 27% yield over 2 steps). 1H NMR (300 MHz, d6-DMSO) δ: 9.25 (s, 1H); 9.13 (s, 1H); 8.64−8.56 (m, 1H); 8.08 (s, 1H); 7.65−7.54 (m, 2H); 7.53−7.46 (m, 1H); 7.38 (s, 1H); 7.34− 7.26 (m, 1H); 7.15−7.06 (m, 1H); 5.60 (s, 2H); 4.33−4.08 (m, 1H); 3.61−3.46 (m, 1H); 3.23−3.06 (m, 3H); 2.86−2.72 (m, 1H); 2.57 (s, 3H); 2.54−2.30 (m, 1H); 1.82−1.73 (m, 1H); 1.39−1.21 (m, 2H); 1.07−1.04 (m, 3H); piperidine NH missing. LC-HRMS (ESI, m/z): calcd for C25H29N6O3F 480.2285, found 481.2370 [M + H+]; tR = 0.52 min.

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formulation and preclinical galenics groups for their continuous support with routine in vitro and in vivo measurements, and Dr. Roman Fisera, Dr. Attila Latika, Dr. Henrich Brath, and Dr. Juraj Rehak from SYNKOLA Ltd. for building block synthesis.

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ABBREVIATIONS USED BrettPhos, 2-(dicyclohexylphosphino)-3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl; CC, column chromatography; CDI, 1,1′carbonyldiimidazole; CFU, colony forming unit; DIAD, diisopropyl azodicarboxylate; DIPEA, N,N-diisopropylethylamine; DPPA, diphenyl phosphoryl azide; dppf, 1,1′-ferrocenediylbis(diphenylphosphine); ELSD, evaporative light scattering detector; MRSA, methicillin-resistant Staphylococcus aureus; RIA, relaxation inhibition assay; RTI, respiratory tract infection; SCIA, supercoiling inhibition assay; T3P, propylphosphonic anhydride solution; VRE, vancomycin-resistant enterococci; XPhos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01834. Experimental details on cloning of DNA gyrase and topo IV genes, protein production and purification, expressed sequence for the GyrB24 (E. coli) crystallography construct, crystallization conditions, data collection and refinement statistics, gyrase and topo IV supercoiling and relaxation inhibition assay, ATPase inhibition assay, MIC testing, hKV11.1 (“hERG”) assay, logD and pKa determination, neutropenic murine thigh infection model, pharmacokinetics in the mouse and in the rat, conversion of 68 in vitro in blood, and procedures for synthesis of 5−16 (PDF) Molecular formula strings (CSV)

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(1) CDC. Antibiotic Resistance Threats in the United States; Centers for Disease Control and Prevention: Atlanta, GA, 2013. https://www.cdc. gov/drugresistance/pdf/ar-threats-2013-508.pdf (accessed Dec 5, 2016). (2) Hooper, D. C.; Jacoby, G. A. Topoisomerase inhibitors: Fluoroquinolone mechanisms of action and resistance. Cold Spring Harbor Perspect. Med. 2016, 6, a025320. (3) Liu, L. F.; Liu, C.-C.; Alberts, B. M. Type II DNA topoisomerases: Enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break. Cell 1980, 19, 697−707. (4) Sissi, C.; Palumbo, M. In front of and behind the replication fork: Bacterial type IIa topoisomerases. Cell. Mol. Life Sci. 2010, 67, 2001− 2024. (5) Bush, N. G.; Evans-Roberts, K.; Maxwell, A. DNA topoisomerases. EcoSal Plus 2015, 6, 6. (6) Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 2010, 17, 421−433. (7) Collin, F.; Karkare, S.; Maxwell, A. Exploiting bacterial DNA gyrase as a drug target: Current state and perspectives. Appl. Microbiol. Biotechnol. 2011, 92, 479−497. (8) East, S. P.; Silver, L. L. Multitarget ligands in antibacterial research: Progress and opportunities. Expert Opin. Drug Discovery 2013, 8, 143− 156. (9) Sugino, A.; Higgins, N. P.; Brown, P. O.; Peebles, C. L.; Cozzarelli, N. R. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc. Natl. Acad. Sci. U. S. A. 1978, 75, 4838−4842. (10) Holdgate, G. A.; Tunnicliffe, A.; Ward, W. H. J.; Weston, S. A.; Rosenbrock, G.; Barth, P. T.; Taylor, I. W. F.; Pauptit, R. A.; Timms, D. The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: A thermodynamic and crystallographic study. Biochemistry 1997, 36, 9663−9673. (11) Bellon, S.; Parsons, J. D.; Wei, Y.; Hayakawa, K.; Swenson, L. L.; Charifson, P. S.; Lippke, J. A.; Aldape, R.; Gross, C. H. Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons): A single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase. Antimicrob. Agents Chemother. 2004, 48, 1856−1864. (12) Bisacchi, G. S.; Manchester, J. I. A new-class antibacterial - almost. Lessons in drug discovery and development: A critical analysis of more than 50 years of effort toward ATPase inhibitors of DNA gyrase and topoisomerase IV. ACS Infect. Dis. 2015, 1, 4−41. (13) Maxwell, A. DNA gyrase as a drug target. Trends Microbiol. 1997, 5, 102−109. (14) Drlica, K.; Malik, M. Fluoroquinolones: Action and resistance. Curr. Top. Med. Chem. 2003, 3, 249−282.

Accession Codes

We will release the atomic coordinates and experimental data upon article publication. PDB code for E. coli GyrB with bound 27 is 5MMN; for E. coli GyrB with bound 36 is 5MMP; and for E. coli GyrB with bound 58 is 5MMO.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +41 61 565 65 65. E-mail: philippe.panchaud@actelion. com. ORCID

Philippe Panchaud: 0000-0001-7823-7639 Present Addresses †

M.G.: Metrohm Schweiz AG, Areal Bleiche West, CH-4800 Zofingen, Switzerland. ‡ T.P.: ADME & More, Märktweg 30, D-79576 Weil am Rhein, Germany. § L.P.: SBCL, CH-3000 Bern, Switzerland. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Aengus Mac Sweeney and Dr. Naomi TidtenLuksch for preparation and submission of files to the PDB, Dr. Carmela Gnerre for helpful discussions, Eser Ihlan and Viktor Ribic for syntheses of libraries, Dr. Hans Locher, Jonathan Delers, and Daniela Sabato from the microbiology team, Marina Dos Santos, Marta Gerber, Stéphanie Kraemer, and Stefan Weiser for IC50 measurements, Michel Enderlin-Paput and Maria Weiss for efficacy experiments, Alexander Hasler and Romain Sube for hERG measurements, the DMPK as well as the pre3774

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