p38Î± MAP Kinase I - ACS Publications - American Chemical Society

Article pubs.acs.org/jmc

Design, Synthesis, and Biological Evaluation of Novel Type I1/2 p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the R‑Spine Niklas M. Walter,†,∥ Heike K. Wentsch,†,∥ Mike Bührmann,‡,∥ Silke M. Bauer,† Eva Döring,† Svenja Mayer-Wrangowski,‡ Adrian Sievers-Engler,† Nicole Willemsen-Seegers,§ Guido Zaman,§ Rogier Buijsman,§ Michael Lam ̈ merhofer,† Daniel Rauh,‡ and Stefan A. Laufer*,† †

Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Eberhard-Karls-Universitaet Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany ‡ Faculty of Chemistry and Chemical Biology, Technische Universitaet Dortmund, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany § Netherlands Translational Research Center B.V. (NTRC), Pivot Park, RE1210, Molenstraat 110, 5342 CC Oss, The Netherlands S Supporting Information *

ABSTRACT: We recently reported 1a (skepinone-L) as a type I p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, as a type I inhibitor, it is entirely ATP-competitive and shows just a moderate residence time. Thus, the scope was to develop a new class of advanced compounds maintaining the structural binding features of skepinone-L scaffold like inducing a glycine flip at the hinge region and occupying both hydrophobic regions I and II. Extending this scaffold with suitable residues resulted in an interference with the kinase’s R-Spine. By synthesizing 69 compounds, we could significantly prolong the target residence time with one example to 3663 s, along with an excellent selectivity score of 0.006 and an outstanding potency of 1.0 nM. This new binding mode was validated by cocrystallization, showing all binding interactions typifying type I1/2 binding. Moreover, microsomal studies showed convenient metabolic stability of the most potent, herein reported representatives.

■

INTRODUCTION

accepted for market introduction. This has several reasons, e.g., organ toxicity4,12−14 and insufficient or the drug candidates’ complete loss of efficacy in vivo.14−16 Generally, these inhibitors target the kinase ATP-binding cleft which is located between the C-terminal and an N-terminal subdomain, connected by the so-called hinge region. Protein kinase inhibitors can be divided into different classes with regard to the adopted binding mode within the active site and the addressed conformational state of the enzyme (Figure 1). This categorization is connected to their corresponding selectivity profile, potency range, and binding kinetics.17 Important hallmarks for noncovalent kinase inhibitor assignment are the occupied areas within the active site as well as the DFG motif and the α-helix C orientation. Type I inhibitors represent the majority of kinase inhibitors. They typically bind to the ATP pocket, forming polar interactions with the hinge region by mimicking the cosubstrate adenine structure, thereby being strictly ATP-competitive. Following the described binding mode, these ligands are able to interact with the hinge-flanking hydrophobic region I and/or

Since the first protein kinase inhibitor imatinib was launched in 2002, protein kinases have increasingly attracted attention in drug research and discovery. 518 human genes encoding for protein kinases have been discovered by now,2 and approximately 180 protein kinases are directly associated with human diseases.3 By transferring a phosphoryl group from ATP to their molecular target, protein kinases control essential physiological processes such as regulation of signal transduction cascades. Therefore, kinases play an important role in the generation of inflammations, cancer, autoinflammatory afflictions, and neurodegenerative diseases.4−7 The p38α mitogen-activated protein (MAP) kinase plays a key role in regulating the biosynthesis of proinflammatory cytokines like interleukin 1β, tumor necrosis factor α (TNFα), and the oncogenic activating transcription factor 2 (ATF-2),8 which are capable of inducing severe diseases.9 Thus, p38α has emerged as an important target in medicinal chemistry and pharmaceutical research. A well-established therapeutic approach to address directly kinase-related diseases is based on the inhibition of dysfunctioning or misregulated kinases with small molecule inhibitors.10 Currently, about 32 protein kinase inhibitors have been approved by the FDA.11 However, no inhibitor of the p38α MAP kinase has been 1

© 2017 American Chemical Society

Received: May 19, 2017 Published: August 23, 2017 8027

DOI: 10.1021/acs.jmedchem.7b00745 J. Med. Chem. 2017, 60, 8027−8054

Journal of Medicinal Chemistry

Article

Figure 1. Comparison of type I, type II, and type III kinase inhibitors based on representative cocrystal structures in complex with corresponding p38α MAP kinase inhibitors. (A) 1a (white, type I) addressing the kinase’s active DFG-“in between” conformation, occupying the adenine binding site (PDB code 3QUE). (B) 13b (yellow, type II) bound to the kinase’s inactive DFG-out conformation, occupying both the adenine binding site and the deep pocket (PDB code 1KV2). (C) 13c (green, type III) addressing the kinase’s inactive DFG-out conformation, occupying the deep pocket (PDB code 3HV7). Hydrogen bonds are shown as yellow dots.

region II (HRI and HRII) (Figure 1A).18 Commonly, type I inhibitors address the kinases’ DFG-in conformation which is defined by a rotation of the corresponding Phe169 side chain toward the α-helix C, opening up the ATP-binding cleft. However, this behavior is not clearly observed for one of the most prominent examples for p38α MAP kinase type I inhibitors, 4-(4-(4fluorophenyl)-2-(4-(methylsulfinyl)phenyl)-1H-imidazol-5-yl)pyridine 13a (SB20358019), which rather depicts a DFG-“in between” orientation of the corresponding motif. Type I inhibition generally enables high potency but at the same time intrinsically implicates shortcomings in selectivity due to the highly conserved nature of the active site within the kinase family. To overcome these drawbacks, novel classes of kinase inhibitors arose making use of structurally unique elements of specific kinases. The success of imatinib as a type II inhibitor stimulated the research toward type II binders targeting p38α MAP kinase. 1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-(2-morpholinoethoxy)naphthalen-1-yl)urea 13b (BIRB 76920) is a potent representative that emerged from these efforts and reached phase IIa in clinical studies. Type II inhibitors can be seen as extended type I inhibitors binding to the hinge region and HRI/HRII and beyond that additionally extending to the less conserved so-called deep pocket (Figure 1B). This cavity opens up as a consequence of Phe169 rotation away from the α-helix C, defining the inactive DFG-out conformation. For the p38α type II inhibitor 13b, it has been shown that the formation of polar interactions within the deep pocket results in an increased target residence time (TRT) at the enzyme combined with very good potency.21 However, 13b shows poor selectivity over other kinases and failed in clinical trials.22,23 In contrast, type III inhibitors represent a completely different binding mode than type I and type II inhibitors, as they do not form any hydrogen bonds to the hinge region and thus act entirely ATP-independently. The selectivity of type III inhibitors is comparably high because they address binding sites that are uniquely associated with particular kinases.24 This is typically achieved by targeting allosteric sites in contrast to the ATP binding pocket, particularly the deep pocket (Figure 1C). Hence, as described for 13b fragments like 1-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol5-yl)-3-(naphthalen-1-yl)urea 13c (RL3825), DFG-out conformation is required for the ligands to access the deep pocket in p38α. The discovery of type III inhibitors is yet quite coincidental as detailed knowledge about the three-dimensional structure of a kinase is required for a target-oriented design.25 Concerning the importance of p38α MAP kinase, our group first started its work on selective p38α inhibition based on the improvement of substituted isoxazoles26 and diarylpurinones27

as well as the development of dibenzepinones toward a novel class of p38α MAP kinase inhibitors.28 These efforts resulted in the discovery of (R)-2-((2,4-difluorophenyl)amino)-7-(2,3dihydroxypropoxy)-10,11-dihydro-5H-dibenzo[a,d][7]annulen5-one 1a (skepinone-L29) as an exceptionally potent type I inhibitor and a selective high quality probe for p38α MAP kinase. The excellent selectivity of 1a is achieved by inducing a glycine flip at the hinge region and addressing both HRI and HRII (Figure 1A). The glycine flip in p38α is described by a 180° rotation of Gly110, induced upon binding of specific inhibitors only.30 However, this compound is still a fully ATP-competitive type I binder, consequently prompting us to undertake optimization studies, regarding the combination of the favorable properties of 1a-type templates with features of type II inhibitors, characterized by being nearly ATP-independent due to long TRTs as shown by 13b.20 These attempts resulted in the development of a novel class of p38α inhibitors recently described by Fischer et al.31 and Baur et al.32 Analysis of the accordingly published cocrystal structure of compound (R)-N-(5-((7-(2,3dihydroxypropoxy)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)-2-fluorophenyl)benzamide 1b31 surprisingly revealed a binding mode not entirely following the typical features of DFG-out type II binders as we initially assumed (Figure 2). The reported inhibitor 1b forms hydrogen-bond-mediated contact to the hinge region and induces a glycine flip; the fluorophenyl moiety occupies HRI, while the polar diol moiety targets HRII and displaces water.31 Certainly, the more important interactions are represented by two hydrogen bonds formed between the amide linker of 1b and Glu71 located at the α-helix C and Asp168 of the DFG-motif, respectively. In contrast to our initial design approach of incorporating structural features inspired by type II inhibitors, 1b stabilizes the active DFG-in conformation of the kinase by forming an edge-to-face Ar−Ar interaction between the terminal benzamide moiety and Phe169. Of particular note is that the inhibitor binds to the kinase in DFG-in conformation while showing almost no loss in potency in the presence of increasing concentrations of competing ATP, indicating a particularly high affinity toward the enzyme.31 Thus, the ligand is apparently not able to reach into the deep pocket of the kinase and is consequently not inducing the DFG-out conformation. Surface plasmon resonance (SPR) measurements revealed a contrasting kinetic profile to 13b (1b, kon [M−1 s−1] of 1.10 × 106; koff [s−1] of 1.29 × 10−2; 13b, kon [M−1 s−1] of 4.30 × 103; koff [s−1] of 5.16 × 10−5), demonstrating further differences to type II inhibitors. Moreover, 1b was shown to be less ATP-competitive than 13a,31,33 underlining that 1b is not related to either type I or type II inhibitors. The combination of 8028



Article

Figure 2. Assembly of the R-Spine. Crystal structures of p38α MAP kinase in complex with (A) compound 1b targeting the active DFG-in conformation with assembled R-Spine (green) and (B) 13b addressing the inactive DFG-out conformation with nonassembled R-Spine (white and red surfaces) (PDB codes 1KV2 and 3UVQ). Hydrogen bonds are shown as yellow dots; regions relevant for inhibitor binding are highlighted in the background: HRII (beige), HRI (brown), deep pocket (red).

type I and type II features is coined the definition of type I1/2 inhibition.34 This binding type was discovered and termed in 2010 and is characterized by a unique combination of interactions formed within a kinase active site representing a promising approach to overcome the limitations related to the highly flexible mechanism of kinase inhibition.17 Among others, possible shortcomings of current kinase inhibitors revolve around a missing selectivity over the humane kinome causing cross-reactiveness and poor binding kinetics expressed in a comparably short TRT at the enzyme. The prolongation of the TRT has several advantages as it can help to overcome selectivity deficiencies, increase compound efficacy in vitro and in vivo, and reduce undesired side effects.35,36 It also contributes to sustain the pharmacodynamic effects of the inhibitor after its clearance from the bloodstream.37 Furthermore, a particularly long TRT can also be assigned to a decreased ATP-competition, as it binds tighter to the target, exemplified by (pseudo)irreversible binding of kinase inhibitors.38 One way to effectively prolong TRT of ligands at a protein, besides utilizing structurally unique regions and binding sites for selective targeting, is provided by the conformational stabilization of substructures within the enzyme.39,40 As observed for compound 1b, type I1/2 inhibitors are able to reach toward the DFG motif and stabilize a certain conformational activity state of a kinase.34 In addition, Phe169 of the DFG motif serves as a member of a hydrophobic backbone spine too. Two of those spines were recently discovered in kinases, labeled as the regulatory spine (R-Spine) and the catalytic spine (C-spine).41−43 The R-Spine is assembled by four highly conserved lipophilic amino acids, A1−A4, originating from crucial regions of the kinase and is a signature of every kinase.44 In the case of p38α, A1 refers to His148 of the HRD-motif, A2 represents Phe169 of the DFG-motif, and A3 and A4 are two leucines from α-helix C and β4-strand.45 When the kinase is activated upon diphosphorylation of Thr180 and Tyr182 by MAPKK3/6, the change of charge triggers a chain reaction: Phe169 flips by 180° from DFG-out to DFG-in conformation, thereby leading to an assembly of the R-Spine (Figure 2A). To substantiate the importance of the R-Spine, Bukhtiyarova et al. showed that a mutation of Phe169 in p38α MAP kinase caused a nearly complete loss of catalytic function.46 We hypothesized that skepinone-derived type I1/2 inhibitors as exemplified by the dibenzosuberone compound 1b could putatively serve to gain selectivity via stabilization of the assembled R-Spine. This is caused by the observed

interaction with Phe169 in DFG-in state, supposedly leading to an increased TRT at the kinase and higher affinity due to the additional interaction. Here, we present the structure-based design and chemical synthesis of a focused library of dibenzepinones, following up on the previously reported 1a-derived p38α MAP kinase type I1/2 inhibitor 1b.47 Biochemical evaluation was conducted by means of direct activity-based assays on the isolated kinase domain as well as using a whole blood TNFα release test system. Selected compounds that arose from these measurements were further characterized as representatives for type I1/2 inhibitors regarding TRT and binding mode employing SPR experiments and protein crystallography, respectively. Finally, we aimed to demonstrate the metabolic stability in microsomal studies, paving the way for type I1/2 inhibitors to serve as powerful in vivo probes or potent and selective clinical drug candidates. Structure-Based Design. Starting from the aforementioned findings, we pursued a structure-based design approach to transfer and improve the newly discovered concept of type I1/2 inhibition with respect to p38α MAP kinase. On the basis of the conclusions deduced from the X-ray structure of compound 1b in complex with p38α (Figure 2A), we pursued an optimization program of both dibenzooxepinone and dibenzosuberone scaffolds to improve affinity and activity toward the kinase as well as their influence on metabolic stability and elucidate the impact on type I1/2 inhibition. The developed synthetic strategy should lead to a diverse range of dibenzepinones, which are able to induce a glycine flip due to the rigidized carbonyl group present in these scaffolds and furthermore effectively target HRI and HRII, combined with moieties interfering with the R-Spine by targeting the DFG-motif to increase potency and residence time. All these properties were considered for our conceptual ligands as they proved to be important for selective and potent inhibitor design.31,32 Concerning the glycine flip, this property is used to form additional interactions within the enzyme’s binding site, essential to the hinge region plus gaining selectivity over other kinases that are not able to perform the glycine flip. Only 46 kinases of the human kinase genome are reported to undergo this conformational adaptation upon ligand binding.30 Both HRI and HRII are surrounding the hinge region and allow for selective targeting by specific pharmacophores. The small and solvent-exposed HRII supports the introduction of a wide range of chemical moieties, thus allowing a certain influence on solubility of the inhibitor. A further gain in potency 8029



Article

Chemical synthesis of the proposed type I1/2 inhibitors was realized by initial preparation of the bare scaffolds, suitable for following derivatization. Moieties that were designed to occupy HRI and to interact with the DFG-motif targeting the R-Spine (R1) were synthesized via Buchwald−Hartwig cross-couplings while the implementation of suitable residues for HRII was accomplished by amide coupling (Scheme 2). For the synthesis of the dibenzepinone scaffolds, the commercially available starting material 3-bromo-4-methylbenzoic acid 14 was deprotonated with methylmagnesium bromide, followed by halogen−lithium exchange using n-BuLi (Scheme 2). The organometallic intermediate was quenched with dry ice to obtain 4-methylisophthalic acid 15 in 85% yield.48 This approach is an optimization of the conditions used earlier in the synthesis of 15.31 The subsequent steps (Scheme 2, ii, iii, vi, and vii) for synthesis of the dibenzosuberone scaffold have already been published by Fischer et al.31 For synthesis of the corresponding dibenzooxepinones, the central oxygen was introduced via Wohl−Ziegler bromination of the methyl group in position 6 yielding intermediate 18, followed by Williamson ether synthesis with 3-chlorophenol 19 to yield compound 21. The corresponding acids 22 and 23 were obtained by hydrolysis of the esters using KOH. The resulting acids were subsequently activated with thionyl chloride and treated with dry aluminum chloride to achieve the respective dibenzepinone scaffold 24/25 via intramolecular Friedel−Crafts acylation. This synthesis led to the desired scaffolds with a carboxylic acid in position 3 of the dibenzosuberones and position 9 in the dibenzooxepinones, respectively. This facilitated a fast introduction of a broad variety of aliphatic and functionalized moieties on the A-ring. The chlorine in position 8 of the dibenzosuberone scaffold and in position 3 of the dibenzooxepinone scaffold enabled the introduction of suitable residues via Buchwald−Hartwig cross-coupling for potential DFG-motif interaction. For compounds with a free carboxylic acid function, Buchwald−Hartwig coupling was unsuccessful or accompanied by low yields. Therefore, compounds 24 and 25 were esterified with methanol to generate compounds 26 and 27 or were directly converted with 2-morpholine-4-ylethylamine or methylamine to give compounds 28 and 29. To investigate the interaction potential with the DFG motif, we introduced a simple methylamine moiety in position 7 of the dibenzosuberone scaffold, assumingly preventing any interaction with HRII (2; 3a−c; 4a−d; 5a−d,k; 8d,k; 9a,b; 10a,b; 11a,b; 12). The residues for the proposed DFG-motif interaction were synthesized according to Schemes 3 and 4. Both amides and inverse amides were introduced as linker systems on the B-ring of the dibenzepinone scaffolds (Scheme 1). The inverse amides 37a−d and 38a−d (Scheme 3) were synthesized in good to acceptable yields by the activation with oxalyl chloride or thionyl chloride of the respective benzoic acid derivatives and subsequent conversion with the amine followed by reduction of the nitro group with palladium on activated carbon. The synthesis of the regular amide residues 39; 40a,b; 41a−g; 42a−d; 43; 44; 45a,b (Scheme 4) was conducted by starting with the respective aniline derivatives being converted with the corresponding acid chloride in the presence of sodium hydride yielding up to 70% of the described target compounds. If the corresponding acid chloride was not commercially available, the respective carboxylic acids were activated with thionyl chloride and subsequently underwent nucleophilic substitution with the described amines. The resulting nitro compounds were reduced using hydrogen and palladium on activated carbon or stannous chloride to obtain the desired residues bearing an

can be achieved with suitable residues displacing water molecules in the periphery of the binding site to increase entropy. The access to HRI is limited by the gatekeeper amino acid Thr106 and is mainly addressed by aromatic and hydrophobic interactions. Introduction of various methylated and fluorinated aromatic moieties at this position might contribute to increased binding affinity and inhibitory activity. Following this design approach, we decided to equip our new series of type I1/2 inhibitors with an amide targeting HRII (R1) in contrast to the previously used ether substitution (compound 1b), thereby offering the advantage of a faster synthesis of a diverse set of analogs.31 HRI should be addressed with a conjugated (L1), substituted (R2 and R3) aromatic system. Notably, our designed dibenzepinones should feature the previously described R-Spine stabilization, as we believed it would contribute to prolonged TRTs. Therefore, a suitable hydrophobic moiety was attached via another linker (L2), namely, an amide and an inverse amide, respectively, to potentially form interactions with the DFG-motif and thereby interfere with the R-Spine (Figure 3).

Figure 3. Planned design of type I1/2 dibenzepinone inhibitors interfering with the R-Spine. The core template of 1a is equipped with an additional hydrophobic moiety (HM), reaching into the R-Spine.

■

CHEMISTRY Due to the intended optimization of both the dibenzooxepinone and dibenzosuberone scaffolds, the nomenclature and substitution pattern differ between the two scaffolds. The scheme below illustrates the numbering following IUPAC rules and is valid for the compounds presented in this paper. As listed below, scaffold A is further named dibenzosuberone and scaffold B dibenzooxepinone, together hereinafter referred to as dibenzepinones (Scheme 1). Scheme 1. Numbering Pattern of the Two Scaffolds:a

a

Scaffold A: dibenzooxepinone. Scaffold B: dibenzosuberone. 8030



Article

Scheme 2. Preparation of the Dibenzepinonesa

Reagents and reaction conditions: (i) (1) MeMgBr, THFdry, 0 °C, (2) n-BuLi,-78 °C, (3) dry ice, −40 °C; (ii) H2SO4, MeOH, reflux; (iii) LDA, THFdry, −78 °C; (iv) NBS, AIBN, CCl4, reflux; (v) K2CO3, Me2CO, reflux; (vi) KOH, MeOH/H2O, reflux; (vii) (1) SOCl2, DCM, reflux, (2) AlCl3, room temperature; (viii) H2SO4, MeOH, reflux; (ix) (1) CDI/TBTU, DMF, 50 °C, (2) methylamine/2-morpholin-4-ethan-1-amine, 50 °C; (x) substituted aniline derivative, Cs2CO3, X-Phos, Pd(OAc)2, 1,4-dioxane, t-BuOH, reflux; (xi) KOH, MeOH/H2O, reflux; (xii) CDI/TBTU, corresponding amine, DMF/DCM, 50 °C. For the nature of R1−R6, see Tables 1−6. a

amino function suitable for Buchwald−Hartwig cross-coupling. After reduction, the corresponding anilines could be implemented in position 8 of the dibenzosuberone scaffold and position 3 of the dibenzooxepinone scaffold as coupling partners for the

Buchwald−Hartwig reaction using standard coupling conditions (2; 3a−c; 4a−d; 5a−f,m; 6a,b,f,g,k,l,q; 7a; 8a,b,g; 9a,b; 10a,b; 11a,b; 12).49 Upon coupling, the resulting ester compounds were hydrolyzed prior to introduction via amide 8031



Article

Scheme 3. Synthesis of the Inverse Amides 37a−d and 38a−da

a

Reagents and reaction conditions: (i) C2O2Cl2/SOCl2, triethylamine, DCM, room temperature; (ii) H2, Pd/C, EtOAc, room temperature. For the nature of R1−R3, see Tables 1−6.

Scheme 4. Synthesis of the Regular Amides 39, 40a,b, 41a−g, 42a−d, 43, 44, 45a,ba

a Reagents and reaction conditions: (i) (1) NaH, THFdry, room temperature, (2) acid chloride, 0 °C; (ii) (1) acid, SOCl2, reflux, (2) amine, NaH, THFdry/DMF, room temperature, (3) 1 + 2, 0 °C; (iii) H2, Pd/C, EtOAc, room temperature; (iv) SnCl2, EtOH, reflux. For the nature of R1−R3 see Tables 1−6.

improved activity. This indicated the importance of the additional hydrogen bond interaction to Glu71 of the N-lobe of the enzyme (Figure 2A). With a change of R4 from an isopropyl moiety 3c to a cyclopropyl residue 4d, an aromatic-alike structure, the biological activity increased up to 16-fold.53,54 The decoration of the aniline (C-ring) seemed to be important when using a methyl as substituent. The methyl group in ortho position 4a resulted in an increased inhibitory activity compared to the para-substituted compound 4b, whereas this deviation in activity was not observed for a fluorine substitution (4c vs 4d). In contrast to the ortho-fluorine substitution we assume that the ortho-methyl substitution enforces the aniline ring to be more out of the plane of the tricyclic structure, therefore leading to a better occupied HR I. The most active compound 4a in the kinase assay was also tested regarding its inhibition of TNFα-release in human whole blood; however, it revealed a relatively low IC50 value of 903.2 nM. All dibenzosuberone compounds (scaffold A, X = CH2) depicted in Table 2 showed activities on the enzyme in the low nanomolar range down to 2 nM (5h, 5j, and 5k, Table 2). The chemical rearrangement of the amide structure R4 from amides to reverse amides led to further improvement of the inhibitory activity in both assays. On the basis of the determined potencies of 4a−d (Table 1) compared to 5a−d (Table 2), it is most likely that the regiochemistry of the amide linker bond R4 is relevant for the inhibitory activity. Considering the introduction of a methyl group on the C-ring, the decoration of the aniline, as already noted (4a vs 4b), is important (5a vs 5b) for an adequate potency of the compounds. However, introduction of fluorine on the C-ring of the dibenzosuberone scaffold as R2 and/or as R3 led to increased inhibition compared to a methyl substitution (e.g., 5b vs 5d). These observations led us to the decoration of fluorine as preferred substituents on the C-ring for all following designs. The dibenzooxepinone compounds (scaffold B, X = O) 5m−o showed IC50 values in the low double-digit range on the enzyme level. However, the IC50 value of the dibenzosuberone compound 5i (IC50(p38α) = 5.4 nM) was almost 5-fold higher in comparison to the corresponding dibenzooxepinone compound 5n (IC50(p38α) = 23.8 nM). This factor is even increased to more than 60 in the TNFαrelease assay (5i vs 5n). The most potent compound of Table 2

coupling of suitable residues putatively occupying HRII. On the basis of the analysis of the known crystal structure, we selected positions and substituents for this interaction; polar (poly)alcohols and basic residues seemed to match these criteria.31,50 Activation of the carboxylic acid with CDI or TBTU and conversion with the respective amines led to the test compounds 5g−l,n,o; 6c−e,h−j,m−p,r−v; 7b,c; 8c−f,i−n; 9a.

■

RESULTS AND DISCUSSION Biological Evaluation. The successfully synthesized library of designed type I1/2 inhibitors was evaluated using two different enzyme-linked immunosorbent assays (ELISAs). The effect on kinase activity was tested in an isolated p38α enzyme assay using a monoclonal antiphospho-ATF-2 peroxidase-conjugated antibody and 13a as reference compound.51 The experimental setup of this assay limited the detection range to >3 nM. Additionally, compounds that showed promising IC50 values with respect to p38α MAP kinase were also tested in a human whole blood TNFα-release system.52 Due to various influencing factors, like solubility, plasma protein binding, cell−cell interaction, transporters, and ATP concentration, the results obtained from this human whole blood TNFα-release assay deviated from the results of the isolated enzyme assay. Moreover, results depended on interindividual variations by blood from different donors. Herein, we characterized our synthesized library of diverse derivatives of lead compound 1b designed as type I1/2 inhibitors.31 We further used the latest version of ZINC15 database to virtually test all final compounds for structural elements representing pan-assay interference compounds (PAINS). We did not get any PAIN hit from this database search. First, we compared inhibitors that carried N-isopropyl and N-cyclopropyl substituents (R4), respectively, designed to reach into the R-Spine. Introduction of N-isopropyl residues led to compounds with decreased biological activity on the isolated enzyme (2 and 3a−c) compared to the lead compound 1b (IC50(p38α) = 1.0 nM). The loss of activity could be explained by the isopropyl residue of 2, 3a−c, which is not able to form any charge transfer or π−π-interactions with Phe169 of the DFG-motif and is therefore unable to interfere with the hydrophobic spine of the enzyme. Deletion of the methyl group of the R4 substituent in 2 by a hydrogen (3a) resulted in a 20-fold 8032



Article

Table 1. Biological Data of the Substituted p38α MAP Kinase Dibenzosuberone Inhibitors 2−4d

a

Results from three experiments. bResults from four experiments. cn.d.: not determined.

between a 3-thiophene and a 2-thiophene for R4. The corresponding IC50 value in the kinase assay of 6u was marginally lower than 7b. A comparison of the IC50 values of the different substitution patterns on the C-ring of 6o (R2 = H), 6t (R2 = F), and 7c (R2 = CH3) illustrates the adverse role of a methyl substitution. However, the evaluated TNFα values of the dibenzooxepinones (scaffold B) 7a−c were again comparably high, underlining the advantage of the dibenzosuberones (scaffold A). Next, we investigated the benzamide substitution inspired by the lead compound 1b. The corresponding inhibitors 8a−n exhibited an excellent biochemical activity in the p38α MAP kinase assay below the detection limit of the enzyme assay. This was also observed regarding the determined IC50 values in the TNFα-release assay. Evaluated values in human whole blood testing system represented again the superiority of the dibenzosuberone scaffold compared to the dibenzooxepinones scaffold. The dibenzooxepinones (scaffold A) 8l−n were in fact as potent as the dibenzosuberones (scaffold B) 8a−k in the enzyme assay, but when comparing the dibenzooxepinone compound 8m with its direct dibenzosuberone equivalent 8b, the difference in a whole blood system became evident. Once again, the introduction of an aromatic residue as an amide for proposed interaction with the DFG motif in combination with a fluorine substitution on the C-ring led to very low IC50 values on the enzyme level down to the picomolar range.

was 5i showing a remarkable IC50 value of 25.1 nM in human whole blood (Table 2). All listed compounds in Table 3, apart from compound 6p, showed excellent potencies on the enzyme with IC50 values below the detection limit of the assay (3 nM), which confirmed the assumption that an aromatic or an aromatic-alike structure is needed for interaction with Phe169 of the DFG-motif. The significantly high value of 6p in this series indicates the importance of the free amide hydrogen. There is no significant difference between the dibenzosuberone and the dibenzooxepinone compounds observed on the enzyme level. However, the TNFα values in part provided remarkably higher potencies for the dibenzosuberone compounds when compared to the dibenzooxepinone compounds. Considering the IC50 values of compound 6e vs 6o and 6g vs 6r, the corresponding dibenzosuberone compounds are up to 4-fold better in its whole blood TNFα-release activity. However, the dibenzooxepinone compounds still displayed moderate IC50 values down the low three-digit nanomolar range 6v. Compounds 6b, 6c, 6e, and 6j were the most potent ones in the TNFα-release assay with IC50 values below 40 nM. Another set of dibenzooxepinones was synthesized in order to study the effect of the substitution pattern of the thiophene and the combination of a fluorine substitution with a methyl substitution on the C-ring (Table 4). Obviously, there was no big difference in the inhibitory activity on the isolated enzyme 8033



Article

Table 2. Biological Data of the Substituted p38α MAP Kinase Dibenzosuberone Inhibitors 5a−l and p38α-MAP Kinase Dibenzooxepinone Inhibitors 5m−o

a

Results from three experiments. bResults from four experiments. cn.d.: not determined. dIC50 value below the detection limit of the assay; IC50 < 3 nM.

crystallography, and metabolic studies. These compounds were not chosen primarily based on especially low IC50-value activities determined in both assays. The selected inhibitors showcased suitable representatives by means of the shown structure− activity-relationships and would allow us to make as many conclusions as possible about the other nontested compounds in order to shed a light on the mechanisms and details of type I1/2 inhibition. With this selection, we wanted to further

Substitution of the benzamide structure with different halogen and methoxy groups in ortho- and para-position displayed inhibitors with excellent IC50 values down to picomolar activities on the isolated enzyme. Thus, there was no obvious difference to the unsubstituted benzamide inhibitors depicted in Table 5, 8a−k. To obtain a more detailed insight based on the presented biochemical data, we selected a few compounds for further characterization by means of SPR measurements, X-ray 8034



Article

Table 3. Biological Data of the Substituted p38α MAP Kinase Dibenzosuberone Inhibitors 6a−j and p38α-MAP Kinase Dibenzooxepinone Inhibitors 6k−v

8035



Article

Table 3. continued

a


dibenzosuberone and dibenzooxepinone compounds. Comparing the TRTs of the reference inhibitors with our type I1/2 series showed an improved TRT of the dibenzosuberones 6b, 6e, 6g, 6j, 6l, and 8b (τ = 259−3660 s) compared to the classical type I inhibitors such as 13d (τ = 36 s) and 13a (τ = 4.6 s). The longest TRTs in the dibenzosuberone series were observed for compounds containing an additional fluorine group at position 2 (e.g., 8b and 8e). Worthy of note is that the improved TRT of this series correlated with a high potency in the human whole blood cellular assay (8b, IC50 = 31 nM; 8e, IC50 = 34 nM). The dibenzooxepinone series generally showed a less pronounced improvement in TRT. However, an important exception to this trend was observed regarding 6q, which showed the highest TRT among all tested inhibitors (τ = 4099 s). Binding Mode. To validate our conceptual design concept of type I1/2 kinase inhibitors, we proceeded with the cocrystallization of a small selection from our library. Consequently, the complex structures of 6j and 8m were successfully solved (Figure 4). As conceived, the following compounds complied with the binding mode of our lead compound 1b that was essentially maintained for both cocrystallized compounds due to their analogous scaffolds and substitution patterns.

investigate the impact on binding kinetics, the binding mode, and metabolic stability of the compounds of the regiochemistry of the amide linker R4, the attached moiety for interaction with the R-Spine R4, the different scaffolds A and B, and the metabolic stability of the additional residues for DFG interaction and HRII interaction. Binding Kinetics. The kinetic constants of the aforementioned selected p38α MAP kinase inhibitors were determined by surface plasmon resonance (SPR) measurement (Table 7) to investigate the impact of type I1/2 inhibition on a compound’s binding kinetics.55 Equilibrium dissociation constants (KD) were determined by measuring the association (ka) and dissociation rates (kd) of the individual inhibitors, and binding affinities in the low nanomolar range (1.3−43.5 nM) were obtained. The inhibitors selected from our synthesized library were tested against a small panel of reference compounds consisting of literature-described type I and type II p38α MAP kinase inhibitors. 13b, a prototypical type II inhibitor, showed the lowest KD with 0.123 nM. 3-(3-Bromo-4-((2,4-difluorobenzyl)oxy)6-methyl-2-oxopyridin-1(2H)-yl)-N,4-dimethylbenzamide 13d (PH-79780456), a type I inhibitor and clinical candidate from Pfizer, had an affinity (2.5 nM) similar to most of the

Table 4. Biological Data of the Substituted p38α MAP Kinase Dibenzooxepinone Inhibitors 7a−c

a

Results from three experiments. bResults from four experiments. cn.d.: not determined. dIC50 value below the detection limit of the assay; IC50 < 3 nM. 8036



Article

Table 5. Biological Data of the Substituted p38α MAP Kinase Dibenzosuberone Inhibitors 8a−k and p38α-MAP Kinase Dibenzooxepinone Inhibitors 8l−n

a Results from three experiments. bResults from four experiments. cn.d.: not determined. dIC50 value below the detection limit of the assay; IC50 < 3 nM.

the quality of the compounds to bind the kinase in a DFG-in conformation were also found in both cases. The aromatic thiophene (6j) and phenyl (8m) systems, respectively, were forming an edge-to-face interaction with the side chain of Phe169. Thereby, the kinase R-Spine was stabilized in its assembled form, presumably leading to the by now most efficient interaction enabling very low IC50 values and long TRTs. In Vitro Metabolism. In order to examine if and to what extent the variety of residues influences metabolic stability, the in vitro biotransformation of selected dibenzosuberone and

In agreement with our working hypothesis, upon variation of the scaffold from dibenzosuberone to dibenzooxepinone as well as the solubilizing group on the A-ring reaching into HRII and the fluorination pattern on the C-ring addressing HRI, the crucial main interactions with the kinase were kept. Namely, the formation of two hydrogen bonds to Met109 and Gly110 of the hinge region inducing the glycine flip and an efficient occupancy of HRI and HRII were part of the compliance. Most importantly, two hydrogen bonds from the amide linker to Glu71 of α-helix C and Asp168 of the DFG-motif leading to 8037



Article

Table 6. Biological Data of the Substituted p38α MAP Kinase Dibenzosuberone Inhibitors 9a,b, 10a,b, 11a,b, 12

a


a 1000 nM concentration,31 and compound 6g (τ = 3663 s, Table 7) was screened against a panel of 320 wild type kinases and showed no off-target at a 500 nM concentration.47 This indicates that the additional R-Spine interaction has no negative influence on selectivity. The affinity of type I1/2 toward p38 MAP kinases seems to be specifically increased.

dibenzooxepinone inhibitors was investigated in male rat liver microsomes (RLMs). Furthermore, the hypothesis of the potentially blocked metabolic position of the dibenzosuberone scaffold A with the introduction of an oxygen leading to the dibenzooxepinone scaffold B was investigated. Therefore, the compounds profiled (Table 8) reflected the structural diversity and revealed remarkable results in the RLM assay. All tested substances appear to undergo metabolic degradation and reveal metabolite formation. An exception is represented by 5g not showing any metabolites. The introduction of the cyclopropyl residue as an anilide (5a) led to an increased metabolic stability compared to the reverse amide (4d). In addition, the insertion of a diolamine moiety showed decreased substrate degradation (5g) and a significant suppression of metabolite formation. To our surprise, the insertion of a heteroatom to the scaffold A led to decreased metabolic stability and increased metabolite formation when compared with its dibenzosuberone equivalent (8b vs 8m). In comparison to the corresponding phenylbenzamides (8b, 8m), the introduction of the 2-thiopheneanilide residue (6b, 6g) seems to support the substrate stability (Figure 5). Fortunately, compounds 8b−m were not metabolized to the possible 1,4-iminioquinone-like metabolites. Kinase Selectivity Profiling. The high potency and long TRTs of the compounds encouraged us to investigate the influence of the additional interaction on kinome-wide selectivity. To examine the impact of very potent but yet chemically different compounds, 8b and 6g were chosen as representatives for type I1/2 inhibitors. Compound 8b was screened against a panel of 451 kinases and only showed CSNK1E as off-target at

■

CONCLUSION In summary, we presented the conceptual design and successful synthesis of a new generation of skepinones as exceptionally potent p38α MAP kinase inhibitors. A focused compound library comprising a total number of 69 compounds following the type I1/2 concept was generated. From this set we determined biochemical data imposingly showing effective inhibition of TNFα-release in human whole blood reflected by IC50 values down in the low nanomolar range. We subsequently selected a structurally diverse set of compounds for characterization regarding binding kinetics and binding mode via SPR and X-ray studies, respectively. The corresponding results showed a significant improvement in TRT caused by direct interaction with the kinase’s R-Spine leading to its stabilization, providing explanations for the high observed potency of our ligands. This observed R-Spine interaction even increased the already high selectivity of 1a down to a selectivity score of 0.006 for compound 6g. Furthermore, we could show that despite an extension of the scaffolds, the metabolic stability of the molecules was maintained. This novel class of p38α MAP kinase inhibition opens a new chapter in the book of antiinflammatory drug design and will be further investigated and optimized toward kinase selectivity and drug efficacy. 8038



Article

Table 7. Binding Kinetics of Testing Compounds 6b, 6e, 6g, 6j, 6l, 6o, 6q, 8b, and 8m to Active p38α MAP Kinasea p38α MAP kinase 13a 13d 13b 6b 6e 6g 6j 6l 6o 6q 8b 8m a

inhibitor type

ka [1/M s]

I I II I1/2 I1/2 I1/2 I1/2 I1/2 I1/2 I1/2 I1/2 I1/2

× × × × × × × × × × × ×

1.00 1.08 1.21 5.32 8.32 6.15 5.82 4.11 1.61 1.09 1.23 1.75

7

10 107 104 105 105 104 105 106 106 105 106 106

kd [1/s] 2.18 2.75 1.00 2.08 2.00 2.73 7.53 3.22 1.17 2.44 3.86 3.09

× × × × × × × × × × × ×

−1

10 10−2 10−5 10−3 10−3 10−4 10−4 10−2 10−2 10−4 10−3 10−2

KD [nM]

τ [s]

t1/2 [s]

21.7 2.5 0.123 3.9 2.4 4.4 1.3 7.8 7.3 2.2 3.14 17.7

4.6 36 >105 480 499 3663 1329 31 86 4099 259 32

3.2 25 >69300 333 346 2538 921 22 59 2840 180 22

ka, association rate constant; kd, dissociation rate constant; KD, equilibrium dissociation constant; τ, residence time; t1/2, half-life.

Figure 4. Cocrystal structure of type I1/2 inhibitors in complex with p38α MAP kinase. (A) Detailed binding mode of 6j (cyan) and (B) 8m (yellow) in the kinases active site. Hydrogen bonds shown as yellow dots. (C) Overlay of the inhibitors occupying HRI (brown) and HRII (beige) (PDB codes 5MTY and 5MTX).

■

or from the Institute of Sciences, Department of Pharmaceutical Analytics and Bioanalytics. GC/MS analyses were carried out on a Hewlett-Packard HP 6890 series GC system equipped with a HP-5MS capillary column (0.25 μm film thickness, 30 m × 250 μm) and a HP 5973 mass selective detector (EI ionization). Helium was used as carrier gas in the following temperature program: start at 100 °C and hold for 1 min, then increase to 160 °C and hold for 5 min, then increase to 250 °C and hold for 8 min. High resolution, high accuracy mass spectrometry (ESI-HRMS) was performed on an Sciex (Ontario, Canada) TripleTof 5600+ mass spectrometer with a Duospray source, coupled to a 1290 UHPLC from Agilent (Waldbron, Germany) equipped with an PAL-HTS autosampler from CTC (Zwingen, Switzerland).

EXPERIMENTAL SECTION

Synthesis. All reagents and (anhydrous) solvents are commercially available and were used without further purification. NMR. 1H and 13C NMR spectra were obtained with Bruker Avance 200 or with Bruker Avance 400. The spectra were obtained in the indicated solvent and calibrated against the residual proton peak of the deuterated solvent. Chemical shifts (δ) are reported in parts per million. Mass Spectrometry. Mass spectra were obtained by DC-MS (ESI) and from the Mass Spectrometry Department (FAB-MS; EI-MS), Institute of Organic Chemistry, Eberhard-Karls-Universitaet Tübingen 8039



Article

Table 8. Metabolic Stability of Selected Dibenzosuberone Inhibitors 4d, 5a,g, 6b,g−j, 8b,d and Dibenzooxepinone Inhibitors 6t and 8m after 180 min Incubation with Rat Liver microsomes

HPLC. The purity of all compounds is, unless otherwise stated, >95% and was determined via reverse phase high performance liquid chromatography on Hewlett-Packard HP 1090 series II LC equipped with a UV diode array detector (DAD, detection at 230 and 254 nm). The chromatographic separation was performed on a Phenomenex Luna 5u C8 column (150 mm × 4.6 mm, 5 μm) at 35 °C oven temperature. The injection volume was 5 μL, and the gradient of the used method was the following (flow, 1.5 mL/min), with 0.01 M KH2PO4, pH 2.3 (solvent A), methanol (solvent B): from 40% B to 85% B in 8 min, 85% B for 5 min, from 85% to 40% B in 1 min, 40% B for 2 min, stop time 16 min. Melting Points. Büchi melting point B-545 with thermodynamic correction was used. Melting points were determined after the compound was completely melted. Visualization. For visualization and generation of pharmacophore models, PyMOL molecular graphics system (version 1.3, Schrödinger, LLC) and Schrö dinger release 2015-2 Maestro (version 10.2, Schrödinger, LLC, New York, NY, 2015) were used. General Procedure Ia: Preparation of the Inverse Amide Intermediates 30a−d and 31a−d. The respective carboxylic acid was activated at first. The acid (1.0 equiv) was dissolved in DCM (0.3 M) and catalytic amounts of DMF. Oxalyl chloride (1.0 equiv) was added dropwise, and the mixture was allowed to stir at ambient

For chromatographic separation, samples were dissolved in MeOH. Separation was performed on a Phenomenex (Torrance, CA, USA) Kinetex C18 2.8 μm, 100 mm × 3 mm 100 Å core shell technology column for optimal chromatographic resolution using the following gradient profile: 0−1 min 5% B, 1−10 min 5−100% B, 5−15 100% B. Solvent A was MS-Grade H2O with 0.1% formic acid, and solvent B was Ultra-MS-Grade acetonitrile (Carl Roth, Karlsruhe, Germany) with 0.1% formic acid. Flow was set to 500 μL/min and column temperature to 20 °C. Mass analysis was run in ESI+ mode. Settings were as follows: curtain gas 30 psi, nebulizer gas 40, drying gas 50, source temperature 400 °C, ion source floating voltage +5500 V. Analysis was run as information dependent acquisition; i.e., every cycle consisted of a TOF survey scan from 100 to 2000 m/z, and subsequent fragmentation scans were triggered automatically by acquisition software. To ensure MSMS spectra generation of synthesized compounds, IDA was supplied with an inclusion list containing the respective [M + H+]+ m/z ratios. Collision energy was set to 27 V. TLC. Analyses were performed on fluorescent silica gel 60 F254 plates (Merck) and visualized under UV illumination at 254 and 366 nm. Column Chromatography. Column chromatography was performed on Davisil LC60A 20−45 μm silica from Grace Davison and Geduran Si60 63−200 μm silica from Merck for the precolumn using an Interchim PuriFlash 430 automated flash chromatography system. 8040



Article

Figure 5. Substrate degradation and metabolite formation of compounds 6b, 8b, and 8m. (A) Overview of the analyte decrease during 180 min incubation with RLM. (B) Percentage of all formed metabolites during 180 min incubation. Incubations were carried out in triplicates, and average mean values with standard deviation are shown. temperature for 1 h under argon atmosphere. Afterward, a solution of the respective amine derivative (1.1 equiv) and triethylamine (4.0 equiv) in DCM (0.1 M) was prepared. The activated carboxylic acid was added dropwise and the reaction mixture was stirred at ambient temperature under argon atmosphere until the reaction was completed. Then it was poured into H2O, extracted with DCM, dried over Na2SO4, and the solvent was removed under reduced pressure. The extract was further purified by flash chromatography (silica gel, petroleum ether/ethyl acetate). General Procedure Ib: Amide Synthesis of the Intermediates 32a−d. Sodium hydride (1.0 equiv) was suspended in THFdry (0.3 M), and the respective aniline (1.0 equiv) was added dropwise. The reaction mixture was allowed to stir under argon atmosphere at ambient temperature until the implementation was completed. Afterward, a solution of the cyclopropanecarbonyl chloride (1.0 equiv) in THFdry (0.1 M) was prepared. The acid chloride was added dropwise at 0 °C under argon atmosphere in 30 min. The reaction mixture was allowed to stir until the conversion is completed. The reaction mixture was poured into H2O, extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed in vacuo. The extract was further purified using flash chromatography (silica gel, petroleum ether/ethyl acetate). General Procedure Ic: Amide Synthesis of the Intermediates 33a, 34c, 35b. Sodium hydride (1.0 equiv) was suspended in THFdry (0.1 M), and the respective aniline (1.0 equiv), dissolved in THFdry (0.1 M), was added dropwise. The reaction mixture was allowed to stir under argon atmosphere at ambient temperature until the deprotonation was completed (1−2 h). At the same time, the corresponding acid (1.1 equiv) was dissolved in SOCl2 (10.0 equiv) and was refluxed for 1 h. After cooling to room temperature, the excess SOCl2 was removed in vacuo and the resulting acid chloride was directly dissolved in THFdry. (0.1 M). The acid chloride was added dropwise at 0 °C under argon atmosphere in 30 min. The reaction mixture was allowed to stir until the conversion was completed (3−10 h). The reaction mixture was poured into H2O, extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed under reduced pressure. The extract was further purified using flash chromatography (silica gel, petroleum ether/ethyl acetate). General Procedure Id: Amide Synthesis of the Intermediates 33b, 34, 34d, 36a−g. The aniline (1.0 equiv) was dissolved in THFdry (0.1 M), and sodium hydride (1.0 equiv) was added. After 1 h of stirring, the cyclopropyl acid chloride/benzoyl chloride (1.1 equiv) was added at 0 °C. The reaction mixture was allowed to stir at ambient

temperature. After complete conversion, the reaction mixture was diluted with water, neutralized with diluted HCl, and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by recrystallization in hexane/ethyl acetate. General Procedure II: Nitration 34a and 35a.

Concentrated nitric acid (10% V/V) was added dropwise over 20 min to a stirred solution of the corresponding substituted aniline in concentrated sulfuric acid (0.75 M) at −10 °C. The mixture was stirred under 5 °C for 30 min and then poured on ice and the pH was set to 13 with solid NaOH. Temperature was regulated under 80 °C. After cooling to room temperature, the nitrated aniline was extracted three times with diethyl ether. The extract was further purified by flash chromatography (silica gel, petroleum ether/ethyl acetate). General Procedure IIIa: Reduction Using Pd on Activated Carbon Preparing Residues 37a−d, 38a−d, 39, 40a,b, and 41a−g. The nitro compound (1.0 equiv) was dissolved in ethyl acetate (0.3 M), and 10 wt % palladium on activated carbon was added. The resulting suspension was stirred under hydrogen atmosphere at ambient temperature. Afterward, the mixture was filtered and the solvent was removed under reduced pressure. The residue was further purified using flash chromatography (silica gel, petroleum ether/ethyl acetate). General Procedure IIIb: Reduction Using SnCl2 Preparing Residues 42a−d, 43, 44m, and 45a,b. The nitro compound (1.0 equiv) was dissolved in a three-necked flask in ethanol or methanol (0.075 M) and was heated to reflux. The corresponding amount of stannous chloride (5.0 equiv) was added in small portions followed by stirring for 3−6 h. After complete conversion (monitored by TLC and HPLC), sodium hydrogen carbonate was added carefully and the alcohol was removed in a rotary evaporator. The remainder was suspended in ethyl acetate followed by stirring for 15 min and filtering over Celite. After washing generously with ethyl acetate, the solvent was removed in vacuo. The crude product was purified by flash chromatography (silica gel, petroleum ether/ethyl acetate). General Procedure IV: Ester Hydrolysis to Compounds 49−60. For hydrolysis, the ester (1.0 equiv) was dissolved in methanol (0.05 M) 8041



Article

and some water and was heated to 50 °C. After complete solvation, potassium hydroxide (1.0−2.5 equiv) was added and the mixture was refluxed for 4 h. When the reaction was completed (monitored by TLC), the solvent was evaporated in vacuo and the remainder was suspended in water. The aqueous layer was set to pH 3 with HCl, and the acid was extracted three times with EtOAc. The solvent was then removed in vacuo to yield the product. In some cases, the acid had to be purified by flash chromatography (DCM/methanol/formic acid). General Procedure Va: Buchwald−Hartwig Coupling 46, 47, 48, 2, 3a−c, 4a−d, 5a−d,m, 6a,f,k,p,q, 7a, 8a,d,k,l. For preparation of the diarylamine, the respective amine (1.1 equiv) was dissolved under inert gas atmosphere in a three-necked flask. A mixture of 1,4-dioxane and tert-butanol (10:2, 0.075 M) was used. The mixture was heated to 60 °C, and Cs2CO3 (1.5 equiv), Pd(OAc)2 (0.1 equiv), X-Phos ((2-(dicyclohexylphosphino)-2′,4′,6′-triisopropylbiphenyl) (0.3 equiv), and the corresponding chlorodibenzepinone (1.0 equiv) were added. The reaction was refluxed 1−6 h until TLC showed complete conversion. After cooling to room temperature, the suspension was filtered and washed with DCM, methanol, and ethyl acetate. The combined organic layers were removed in vacuo. The remainder was purified using flash chromatography (silica gel, petroleum ether/ ethyl acetate, DCM/methanol, ethyl acetate/petroleum ether/DCM). General Procedure Vb: Buchwald−Hartwig Coupling 9a,b, 10a,b, 11a,b, 12. For preparation of the diarylamine, the respective amine (1.2 equiv), K2CO3 (3.0 equiv), BrettPhos G3 precatalyst (0.02 equiv), and the corresponding chlorodibenzepinone (1.0 equiv) was solved in 1,4-dioxane (0.1−0.2 M) under inert gas atmosphere in a three-necked flask. The reaction was stirred at 90 °C until the reaction was completed. After cooling to room temperature, an ammonium chloride solution was added and the product extracted with ethyl acetate. The combined organic layers were dried over Na2SO4, and the solvent was removed in vacuo before purification using flash chromatography (silica gel, petroleum ether/ethyl acetate, DCM/methanol). General Procedure VI: Amide Coupling 5e−l,n,o, 6b−e, g−j,l−o,r−v, 7b,c, 8b,c,e−j,m,n. For synthesis of the amide, the arylcarboxylic acid (1.0 equiv) was dissolved in DMF (0.02 M) under inert gas atmosphere, and CDI (2.0 equiv) or DIPEA (2.5 equiv) and TBTU (1.5 equiv) were added. The mixture was stirred at 50 °C for 1−3 h. An excess of the corresponding amine (2.5 equiv) was added, and stirring continued for 1−12 h (monitored by TLC and HPLC). Afterward the mixture was poured into water and extracted with ethyl acetate/DCM. The crude product was purified by flash chromatography (silica gel, petroleum ether/ethyl acetate, DCM/methanol). 8-((5-(Isopropyl(methyl)carbamoyl)-2-methylphenyl)amino)-N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (2). Compound 2 was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 70:30) to afford 0.11 g (0.23 mmol, 31.0%) of the title compound 2. Melting point, 168.6 °C. HPLC, 6.62 min. IR (ATR) [cm−1] 3255, 2370, 2076, 1785, 1646, 1554, 1500, 1351, 1261, 1085, 786, 450. 1H NMR (200 MHz, DMSO-d6) δ 8.58−8.49 (m, 1H), 8.37 (s, 1H), 8.34−8.29 (m, 1H), 8.01 (d, J = 8.7 Hz, 1H), 7.95−7.84 (m, 1H), 7.44−7.28 (m, 2H), 7.18 (s, 1H), 7.12−6.98 (m, 1H), 6.83−6.72 (m, 1H), 6.68−6.60 (m, 1H), 4.10−3.83 (m, 1H), 3.15−2.95 (m, 4H), 2.77 (m, 6H), 2.23 (s, 3H), 1.11 (d, J = 6.7 Hz, 6H). 13C NMR (50 MHz, DMSO-d6) δ 190.1, 169.6, 166.0, 149.8, 146.9, 145.4, 144.5, 141.0, 139.0, 138.9, 135.8, 133.56, 132.8, 132.5, 131.3, 130.2, 129.1, 128.9, 126.75, 113.8, 112.5, 39.52, 35.5, 33.8, 26.3, 17.8. ESI-HRMS [M − H]+ calculated: 470.2438. Found: 470.2434. 8-((5-(Isopropylcarbamoyl)-2-methylphenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (3a). Compound 3a was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 30:70) to afford 0.18 g (0.40 mmol, 47.4%) of the title compound 3a. Melting point, 233.1 °C. HPLC, 6.10 min. IR (ATR) [cm−1] 3304, 2938, 2356, 1730, 1644, 1522, 1519, 1398, 1157, 960, 785, 719, 626, 512, 418. 1H NMR (200 MHz, DMSO-d6) δ 8.59−8.48 (m, 1H), 8.39 (s, 1H), 8.36−8.30 (m, 1H), 8.20−8.13 (m, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.90

(dd, J = 7.9, 1.3 Hz, 1H), 7.78−7.72 (m, 1H), 7.64−7.57 (m, 1H), 7.44−7.31 (m, 2H), 6.78−6.67 (m, 1H), 6.60−6.54 (m, 1H), 4.15−3.98 (m, 1H), 3.19−2.97 (m, 4H), 2.78 (d, J = 4.3 Hz, 3H), 2.23 (s, 3H), 1.14 (d, J = 6.6 Hz, 6H). 13C NMR (50 MHz, DMSO-d6) δ 190.2, 165.9, 164.7, 150.2, 145.4, 144.5, 139.0, 138.8, 135.8, 133.6, 133.5, 132.8, 130.8, 130.2, 129.1, 128.9, 126.5, 123.4, 123.0, 113.5, 112.1, 40.9, 40.8, 40.4, 39.9, 39.5, 39.5, 39.1, 38.7, 38.3, 35.5, 33.8, 26.3, 22.3, 17.8. ESI-HRMS [M − H]+ calculated: 456.2282. Found: 456.229. 8-((2-Fluor-5-(isopropylcarbamoyl)phenyl)amino)-N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (3b). Compound 3b was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 30:70) to afford 0.28 g (0.61 mmol, 67%) of the title compound 3b. Melting point, 162.6 °C. HPLC, 6.15 min. IR (ATR) [cm−1] 3312, 2969, 2360, 2342, 1738, 1633, 1575, 1589, 1416, 1354, 1267, 1203, 1115, 1036, 965, 835, 781, 701, 668, 563, 456. 1H NMR (200 MHz, CDCl3) δ 8.74 (s, 1H), 8.60−8.48 (m, 1H), 8.36 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 7.8 Hz, 2H), 7.72−7.60 (m, 1H), 7.45−7.31 (m, 2H), 6.89 (d, J = 8.9 Hz, 1H), 6.76 (s, 1H), 4.19−3.95 (m, 1H), 3.21−3.00 (m, 4H), 2.89−2.71 (m, 3H), 1.19−1.11 (m, 6H). 13C NMR (50 MHz, CDCl3) δ 191.0, 166.3, 164.5, 157.0 (d, J = 249.7 Hz), 148.9, 145.2 (d, J = 31.6 Hz), 139.2, 133.6, 133.2, 132.1 (d, J = 3.1 Hz), 130.7, 129.4 (d, J = 6.4 Hz), 128.8, 128.6, 128.1, 124.0 (d, J = 7.4 Hz), 123.5, 123.5, 116.5 (d, J = 19.9 Hz), 114.9, 113.3, 35.7, 34.2, 26.6, 22.6. ESI-HRMS [M − H]+ calculated: 460.2031. Found: 460.2034. 8-((4-Fluor-3-(isopropylcarbamoyl)phenyl)amino)-N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (3c). Compound 3c was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 20:80) to afford 0.40 g (0.87 mmol, 74.6%) of the title compound 3a. Melting point, 210.3 °C. HPLC, 6.27 min. IR (ATR) [cm−1] 3292, 2976, 2937, 1737, 1651, 1539, 1435, 1327, 1269, 1138, 966, 852, 814, 775, 668, 522, 461, 424. 1 H NMR (200 MHz, DMSO) δ 8.90 (s, 1H), 8.62−8.47 (m, 1H), 8.38−8.32 (m, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.91 (dd, J = 7.9, 1.9 Hz, 1H), 7.48−7.37 (m, 1H), 7.36−7.19 (m, 3H), 7.01−6.89 (m, 1H), 6.87−6.77 (m, 1H), 4.15−3.94 (m, 1H), 3.20−3.01 (m, 4H), 2.85−2.73 (m, 3H), 1.15 (d, J = 6.6 Hz, 6H). 13C NMR (50 MHz, DMSO) δ 190.4, 165.9, 162.6, 154.3 (d, J = 243.7 Hz), 148.4, 145.4, 144.5, 138.9, 137.3 (d, J = 2.6 Hz), 133.6, 132.8, 130.3, 129.0 (d, J = 7.5 Hz), 127.5, 125.4 (d, J = 16.4 Hz), 123.0 (d, J = 8.5 Hz), 120.9, 120.8, 116.9 (d, J = 25.5 Hz), 114.1, 112.7, 41.1, 35.4, 33.8, 26.3, 22.2. ESI-HRMS [M − H]+ calculated: 460.2031. Found: 460.2028. 8-((5-(Cyclopropylcarbamoyl)-2-methylphenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3carboxamide (4a). Compound 4a was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 30:70) to afford 0.09 g (0.20 mmol, 30.0%) of the title compound 3a. Melting point, 235.6 °C. HPLC, 5.79 min. IR (ATR) [cm−1] 3344, 3290, 2920, 2850, 1641, 1560, 1485, 1436, 1404, 1207, 1020, 961, 895, 642, 624, 501, 433. 1H NMR (400 MHz, DMSO-d6) δ 8.59−8.49 (m, 1H), 8.42−8.36 (m, 2H), 8.35−8.25 (m, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.93−7.86 (m, 1H), 7.73 (s, 1H), 7.59−7.52 (m, 1H), 7.43−7.32 (m, 2H), 6.76−6.69 (m, 1H), 6.61−6.53 (m, 1H), 3.15−2.98 (m, 4H), 2.87−2.80 (m, 1H), 2.80−2.73 (m, 3H), 2.23 (s, 3H), 0.71−0.61 (m, 2H), 0.61−0.49 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 190.2, 166.9, 165.9, 150.1, 145.3, 144.4, 139.0, 138.8, 135.8, 133.5, 133.1, 132.8, 130.9, 130.1, 129.1, 128.8, 126.6, 123.3, 122.8, 113.5, 112.1, 35.5, 33.8, 30.6, 26.2, 17.8, 5.6. ESI-HRMS [M − H]+ calculated: 454.2125. Found: 454.2121. 8-((3-(Cyclopropylcarbamoyl)-4-methylphenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3carboxamide (4b). Compound 4b was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 30:70) to afford 0.15 g (0.33 mmol, 34%) of the title compound 4b. Melting point, 229.2 °C. HPLC, 5.54 min. IR (ATR) [cm−1] 3326, 3264, 3091, 1601, 1552, 1492, 1403, 1341, 1216, 1118, 1031, 937, 863, 825, 701, 606, 526, 418. 1 H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.58−8.52 (m, 1H), 8042



Article

8.52−8.45 (m, 1H), 8.35 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.95−7.85 (m, 2H), 7.65−7.58 (m, 1H), 7.42−7.33 (m, 2H), 6.89 (d, J = 8.8 Hz, 1H), 6.75 (s, 1H), 3.17−3.02 (m, 4H), 2.87−2.75 (m, 4H), 0.72−0.64 (m, 2H), 0.58−0.52 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 192.5, 172.8, 167.0, 166.9, 150.4, 145.1, 144.9, 143.1, 138.9, 138.2, 133.1, 133.0, 132.3, 131.7, 131.0, 129.8, 129.3, 123.6, 123.1, 122.8, 120.7, 35.1, 34.00, 26.7, 17.8, 7.6. ESI-HRMS [M − H]+ calculated: 454.2125. Found: 454.2128. 8-((5-(Cyclopropylcarbamoyl)-2-fluorphenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (4c). Compound 4c was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 20:80) to afford 0.36 g (0.79 mmol, 78.3%) of the title compound 4c. Melting point, 146.7 °C. HPLC, 5.85 min. IR (ATR) [cm−1] 3223, 1639, 1574, 1520, 1362, 1273, 1137, 1019, 928, 840, 760, 621, 418. 1H NMR (400 MHz, DMSO) δ 8.76 (s, 1H), 8.58−8.52 (m, 1H), 8.52−8.45 (m, 1H), 8.35 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.95−7.85 (m, 2H), 7.65−7.58 (m, 1H), 7.42−7.33 (m, 2H), 6.89 (d, J = 8.8 Hz, 1H), 6.75 (s, 1H), 3.17−3.02 (m, 4H), 2.87−2.75 (m, 4H), 0.72−0.64 (m, 2H), 0.58−0.52 (m, 2H). 13C NMR (100 MHz, DMSO) δ 190.9, 166.5, 166.2, 156.9 (d, J = 249.6 Hz), 148.7, 145.3, 144.7, 139.0, 133.5, 133.1, 131.6 (d, J = 2.8 Hz), 130.5, 129.2 (d, J = 9.6 Hz), 128.6 (d, J = 12.3 Hz), 128.0, 123.8 (d, J = 8.2 Hz), 123.1, 123.1, 116.3 (d, J = 20.4 Hz), 114.8, 113.2, 40.2, 35.5, 34.0, 3.5, 23.3, 5.9. ESI-HRMS [M − H]+ calculated: 458.1874. Found: 458.1878. 8-((3-(Cyclopropylcarbamoyl)-2-fluorphenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (4d). Compound 4d was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 20:80) to afford 0.33 g (0.72 mmol, 64.0%) of the title compound 4d. Melting point, 226.9 °C. HPLC, 5.92 min. IR (ATR) [cm−1] 3313, 3089, 2956, 1633, 1492, 1327, 1268, 1136, 1036, 927, 786, 702, 612, 453, 414. 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.83 (s, 1H), 8.63−8.48 (m, 1H), 8.34 (s, 1H), 8.08−7.97 (m, 1H), 7.93−7.75 (m, 2H), 7.46−7.28 (m, 1H), 7.32−7.13 (m, 1H), 7.00−6.90 (m, 2H), 6.83 (s, 1H), 3.16−3.00 (m, 4H), 2.85−2.71 (m, 3H), 2.07−2.00 (m, 1H), 0.84−0.72 (m, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 206.3, 145.3, 144.4, 138.8, 137.3, 133.5, 132.8, 131.7, 130.2, 128.9 (d, J = 12.0 Hz), 127.5, 123.2 (d, J = 7.4 Hz), 120.7, 120.7, 117.0, 116.8, 114.1, 112.6, 35.3, 33.8, 26.2, 22.9, 5.7. ESI-HRMS [M − H]+ calculated: 458.1874. Found: 458.1869. 8-((3-(Cyclopropanecarboxamido)-4-fluorophenyl)amino)N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (5a). Compound 5a was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 20:80) to afford 0.39 g (0.85 mmol, 85.0%) of the title compound 5a. Melting point, 193.9 °C. HPLC, 5.90 min. IR (ATR) [cm−1] 3315, 1615, 1538, 1486, 1403, 1317, 1199, 1039, 950, 809, 760, 589, 483, 451. 1H NMR (400 MHz, DMSO) δ 10.02 (s, 1H), 8.83 (s, 1H), 8.63−8.48 (m, 1H), 8.34 (s, 1H), 8.08−7.97 (m, 1H), 7.93−7.75 (m, 2H), 7.46−7.28 (m, 1H), 7.32−7.13 (m, 1H), 7.00−6.90 (m, 2H), 6.83 (s, 1H), 3.16−3.00 (m, 4H), 2.85−2.71 (m, 3H), 2.07−2.00 (m, 1H), 0.84−0.72 (m, 4H). 13 C NMR (100 MHz, DMSO) δ 190.3, 172.2, 170.3, 165.9, 149.9, 148.7 (d, J = 239.9 Hz), 145.3, 144.4, 138.9, 137.0 (d, J = 2.0 Hz), 133.4, 132.8, 130.2, 128.9 (d, J = 19.5 Hz), 127.1, 126.8 (d, J = 12.7 Hz), 116.0 (d, J = 6.7 Hz), 115.8, 115.6 (d, J = 7.8 Hz), 113.9, 112.5, 35.4, 33.8, 26.2, 14.0, 7.5. ESI-HRMS [M − H]+ calculated: 458.1874. Found: 458.1879. 8-((5-(Cyclopropancarboxamido)-2-fluorphenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (5b). Compound 5b was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 20:80) to afford 0.38 g (0.83 mmol, 83.0%) of the title compound 5b. Melting point, 219.8 °C. HPLC, 6.40 min. IR (ATR) [cm−1] 3281, 1602, 1525, 1505, 1503, 1353, 1360, 1194, 1114, 955, 810, 785, 611, 521, 451. 1H NMR (400 MHz, DMSO) δ 10.02 (s, 1H), 8.83 (s, 1H), 8.63−8.48 (m, 1H), 8.34 (s, 1H), 8.08−7.97 (m, 1H), 7.93−7.75 (m, 2H), 7.46−7.28 (m, 1H), 7.32−7.13

(m, 1H), 7.00−6.90 (m, 2H), 6.83 (s, 1H), 3.16−3.00 (m, 4H), 2.85−2.71 (m, 3H), 2.07−2.00 (m, 1H), 0.84−0.72 (m, 4H). 13C NMR (100 MHz, DMSO) δ 190.6, 171.6, 165.9, 150.5 (d, J = 240.9 Hz), 148.5, 145.1, 144.5, 138.9, 136.00 (d, J = 2.3 Hz), 133.2, 132.8, 130.3, 129.0 (d, J = 7.8 Hz), 128.3 (d, J = 12.6 Hz), 127.5, 116.1 (d, J = 20.5 Hz), 114.6, 114.5, 113.7, 113.0, 35.3, 33.7, 26.3, 14.5, 7.1.ESI-HRMS [M − H]+ calculated: 458.1874. Found: 458.1878. 8-((5-(Cyclopropancarboxamido)-2-methylphenyl)amino)N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (5c). Compound 5c was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 30:70) to afford 0.34 g (0.75 mmol, 75.0%) of the title compound 5c. Melting point, 209.7 °C. HPLC, 6.11 min. IR (ATR) [cm−1] 3291, 1633, 1574, 1519, 1403, 1353, 1263, 1196, 1114, 954, 821, 785, 760, 611, 451. 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.59−8.51 (m, 1H), 8.34 (s, 1H), 8.28 (s, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.61 (s, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.32−7.24 (m, 1H), 7.20−7.13 (m, 1H), 6.78 (d, J = 8.9 Hz, 1H), 6.64 (s, 1H), 3.14−2.96 (m, 4H), 2.87−2.71 (m, 3H), 2.14 (s, 3H), 1.80−1.65 (m, 1H), 0.83−0.68 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 190.2, 171.5, 166.1, 150.1, 145.3, 144.4, 139.1, 138.9, 138.0, 133.5, 132.8, 131.0, 130.1, 129.1, 128.8, 126.4, 126.4, 115.3, 114.3, 113.7, 112.4, 35.6, 33.84, 4.4, 17.2, 14.0, 7.0. ESI-HRMS [M − H]+ calculated: 454.2128. Found: 454.2125. 8-((3-(Cyclopropancarboxamido)-4-methylphenyl)amino)N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (5d). Compound 5d was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 30:70) to afford 0.20 g (0.44 mmol, 44.0%) of the title compound 5d. Melting point, 205.9 °C. HPLC, 5.95 min. IR (ATR) [cm−1] 3238, 2358, 1639, 1603, 1525, 1504, 1445, 1395, 1354, 1267, 1113, 1029, 889, 852, 780, 616, 455. 1H NMR (200 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.79 (s, 1H), 8.62−8.47 (m, 1H), 8.38−8.28 (m, 1H), 8.01 (d, J = 8.5 Hz, 1H), 7.95−7.86 (m, 1H), 7.48−7.33 (m, 2H), 7.15 (d, J = 8.5 Hz, 1H), 7.01−6.81 (m, 3H), 3.17−2.97 (m, 4H), 2.84−2.72 (m, 3H), 2.20 (s, 3H), 2.01− 1.81 (m, 1H), 0.84−0.72 (m, 4H). 13C NMR (50 MHz, DMSO-d6) δ 190.5, 172.1, 166.3, 149.2, 145.7, 144.8, 139.3, 139.0, 137.4, 133.9, 133.1, 131.2, 130.6, 129.4, 129.2, 127.1, 124.9, 116.9, 116.7, 114.2, 112.9, 35.8, 34.1, 26.6, 17.6, 14.4, 7.4. ESI-HRMS [M − H]+ calculated: 454.2128. Found: 454.2125. 8-((5-(Cyclopropancarboxamido)-2-fluorophenyl)amino)-N(2-morpholinoethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (5e). Compound 5e was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.10 g (0.18 mmol, 30.0%) of the title compound 5e. Melting point, 148.7 °C. HPLC, 4.50 min. IR (ATR) [cm−1] 3272, 2923, 1634, 1580, 1530, 1435, 1403, 1262, 1114, 1030, 955, 858, 785, 613, 455. 1 H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 8.67 (s, 1H), 8.58−8.44 (m, 1H), 8.07−7.97 (m, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.79−7.68 (m, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.32−7.16 (m, 2H), 6.93−6.85 (m, 1H), 6.77 (s, 1H), 3.57 (s, 4H), 3.44−3.37 (m, 2H), 3.15−3.02 (m, 4H), 2.47 (s, 2H), 2.42 (s, 3H), 1.78−1.67 (m, 1H), 0.81−0.75 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 190.8, 171.7, 165.6, 150.8 (d, J = 240.8 Hz), 148.7, 145.2, 144.7, 139.1, 136.2 (d, J = 2.3 Hz), 133.3, 133.1, 130.6, 129.2, 129.1, 128.5 (d, J = 13.1 Hz), 127.7, 116.3 (d, J = 20.7 Hz), 114.8, 114.0, 114.0, 113.2, 66.3, 57.5, 5.4, 36.7, 35.6, 33.9, 1487, 7.3. ESI-HRMS [M − H]+ calculated: 557.2559. Found: 557.2553. 8-((3-(Cyclopropancarboxamido)-4-fluorophenyl)amino)-N(2-morpholinoethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (5f). Compound 5f was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.07 g (0.13 mmol, 17.0%) of the title compound 5f. Melting point, 203.0 °C. HPLC, 4.59 min. IR (ATR) [cm−1] 2816, 1636, 1577, 1488, 1398, 1354, 1261, 1213, 1143, 1112, 1024, 950, 860, 782, 567, 418. 1H NMR (200 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.82 (s, 1H), 8.51 (t, J = 5.3 Hz, 1H), 8.31 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.94−7.78 (m, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.28−7.12 (m, 1H), 8043



Article

167.2, 149.0, 145.1, 144.6, 138.9, 133.2, 132.6, 130.6, 129.5, 128.8, 127.3, 124.0 (dd, J = 12.9, 2.8 Hz), 122.9 (dd, J = 12.6, 3.5 Hz), 119.5, 113.9, 112.4, 104.8 (t, J = 25.1 Hz), 35.3, 33.7, 13.9, 7.4. ESI-HRMS [M − H]+ calculated: 462.1624 found: 462.1639. 8-((5-(Cyclopropanecarboxamido)-2,4-difluorophenyl)amino)-N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (5k). Compound 5k was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.03 mmol, 42.3%) of the title compound 5k. Melting point, 240.0 °C. HPLC, 6.65 min. IR (ATR) [cm−1] 3408, 3311, 3051, 1669, 1563, 1435, 1360, 1218, 1106, 1030, 931, 826, 719, 679, 625, 503, 462. 1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.62 (s, 1H), 8.59−8.53 (m, 1H), 8.31 (s, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.95−7.85 (m, 2H), 7.49−7.39 (m, 2H), 6.78 (d, J = 10.0 Hz, 1H), 6.65 (s, 1H), 3.15−3.02 (m, 4H), 2.81−2.73 (m, 3H), 2.01−1.91 (m, 1H), 0.83−0.76 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 190.6, 172.3, 166.0, 149.1, 145.2, 144.5, 138.9, 133.3, 132.8, 130.3, 129.0, 129.0, 127.3, 124.1, 123.0, 119.6, 113.9, 112.4, 104.8, 35.4, 33.8, 26.3, 14.0, 7.5. ESI-HRMS [M − H]+ calculated: 475.1707 found: 475.1727. 8-((5-(Cyclopropancarboxamido)-2,4-difluorphenyl)amino)N-(2,3-dihydroxypropyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-carboxamid (5l). Compound 5l was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.05 g (0.10 mmol, 44.9%) of the title compound 5h. Melting point, 149.0 °C. HPLC, 7.45 min. IR (ATR) [cm−1] 2359, 2934, 1665, 1568, 1504, 1404, 1316, 1354, 1201, 1158, 1112, 1052, 923, 786, 706, 576, 456. 1H NMR (400 MHz, DMSO-d6) δ 8.40−8.35 (m, 2H), 8.11−8.02 (m, 2H), 7.97−7.85 (m, 4H), 7.42−7.32 (m, 2H), 7.13 (t, J = 10.4 Hz, 2H), 6.89−6.81 (m, 2H), 6.72 (s, 2H), 4.36−4.27 (m, 2H), 4.13−4.03 (m, 2H), 3.81−3.67 (m, 2H), 3.57−3.48 (m, 4H), 3.20−3.05 (m, 8H), 1.92−1.80 (m, 2H), 0.99−0.92 (m, 4H), 0.92−0.80 (m, 5H). 13C NMR (100 MHz, DMSO-d6) δ 193.3, 169.8, 150.9, 147.2, 147.0, 140.8, 134.9, 134.0, 131.7, 130.6, 130.2, 129.1, 126.1 (dd, J = 11.1, 4.2 Hz), 123.8 (dd, J = 12.2, 3.3 Hz), 121.0, 115.3, 114.0, 110.6, 105.4 (t, J = 25.1 Hz), 76.0, 68.3, 43.5, 37.0, 35.6, 15.3, 8.3. ESIHRMS [M − H]+ calculated: 536.1991. Found: 536.2005. Methyl 3-((3-(Cyclopropanecarboxamido)-4-fluorophenyl)amino)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxylate (5m). Compound 5m was prepared according to general procedure Va to afford 0.04 g (0.08 mmol, 28.8%) of the title compound 5m. Melting point, 237.9 °C. HPLC, 7.00 min. IR (ATR) [cm−1] 3254, 2921, 1724, 1607, 1556, 1505, 1434, 1262, 1122, 1093. 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.88 (s, 1H), 8.37 (s, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.72 (dd, J = 18.0, 6.9 Hz, 2H), 7.24 (dd, J = 23.3, 13.2 Hz, 2H), 6.76 (d, J = 8.7 Hz, 1H), 6.44 (s, 1H), 5.29 (s, 2H), 3.89 (s, 3H), 1.77−1.67 (m, 1H), 0.78 (d, J = 5.7 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.0, 171.6, 165.4, 162.8, 151.3, 149.4, 140.6, 140.1, 136.1, 133.4, 132.6, 130.1, 129.8, 128.8, 127.7, 116.6, 116.1, 115.2, 114.1, 110.6, 102.6, 72.4, 52.4, 14.5, 7.1. ESI-HRMS [M − H]+ calculated: 461.1507. Found: 461.1511. 3-((3-(Cyclopropanecarboxamido)-4-fluorophenyl)amino)N-(2-hydroxyethyl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine9-carboxamide (5n). Compound 5n was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 90:10) to afford 0.05 g (0.12 mmol, 87.7%) of the title compound 5n. Melting point, 217.2 °C. IR (ATR) [cm−1] 3271, 2945, 2854, 1633, 1606, 1525, 1383, 1257, 1229, 1124, 1031, 955. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (d, J = 43.4 Hz, 1H), 8.85 (s, 1H), 8.66 (s, 1H), 8.31 (s, 1H), 8.05 (t, J = 8.5 Hz, 2H), 7.95 (s, 1H), 7.73 (t, J = 17.5 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.37−7.12 (m, 2H), 6.83−6.65 (m, 1H), 6.45 (s, 1H), 5.29 (d, J = 25.6 Hz, 2H), 4.76 (t, J = 5.2 Hz, 1H), 3.53 (d, J = 5.6 Hz, 2H), 1.73 (d, J = 5.1 Hz, 1H), 1.19 (dd, J = 26.2, 6.5 Hz, 2H), 1.05 (t, J = 7.0 Hz, 1H), 0.79 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.8, 171.6, 165.4, 162.7, 162.3, 151.2, 149.4, 139.9, 138.3, 136.1, 135.1, 133.2, 130.9, 128.2, 127.8, 127.7, 116.8, 116.3, 116.1, 115.1,

7.00−6.85 (m, 2H), 6.85−6.72 (m, 1H), 3.61−3.51 (m, 4H), 3.44−3.34 (m, 2H), 3.19−2.96 (m, 4H), 2.45 (s, 2H), 2.43−2.34 (m, 4H), 0.81−0.73 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 190.3, 172.2, 165.4, 151.2 (d, J = 223.1 Hz), 148.7, 146.6, 145.3, 144.5 (d, J = 1.9 Hz), 138.9, 137.0, 133.4, 132.8, 130.3, 129.0, 128.8, 127.0, 126.8 (d, J = 12.4 Hz), 116.1 (d, J = 9.6 Hz), 115.7 (d, J = 20.8 Hz), 113.9, 112.5, 66.1, 57.3, 53.2, 36.5, 35.4, 33.7, 14.0, 7.4. ESI-HRMS [M − H]+ calculated: 557.2559. Found: 557.2558. 8-((3-(Cyclopropanecarboxamido)-4-fluorophenyl)amino)N-(2,3-dihydroxypropyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (5g). Compound 5g was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.06 mmol, 23.0%) of the title compound 5g. Melting point, 163.9 °C. HPLC, 3.19 min. IR (ATR) [cm−1] 3302, 2929, 1601, 1531, 1487, 1402, 1354, 1263, 1215, 1113, 953, 859, 784, 760, 521, 454. 1H NMR (200 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.82 (s, 1H), 8.51 (t, J = 5.3 Hz, 1H), 8.31 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.94−7.78 (m, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.28−7.12 (m, 1H), 7.00−6.85 (m, 2H), 6.85−6.72 (m, 1H), 3.61−3.51 (m, 4H), 3.44−3.34 (m, 2H), 3.19−2.96 (m, 4H), 2.45 (s, 2H), 2.43−2.34 (m, 4H), 0.81−0.73 (m, 4H). 13C NMR (50 MHz, DMSO-d6) δ 190.3, 172.2, 165.9, 148.8, 145.3, 144.5, 138.9, 137.0 (d, J = 2.4 Hz), 133.4, 132.8, 130.39, 129.2, 128.8, 127.1, 126.8 (d, J = 12.6 Hz), 116.1 (d, J = 7.8 Hz), 115.8, 115.6 (d, J = 7.8 Hz).114.0, 112.5, 70.4, 63.9, 43.0, 35.5, 33.8, 14.0, 7.5. ESIHRMS [M − H]+ calculated: 518.2086. Found: 518.2081. 8-((5-(Cyclopropanecarboxamido)-2,4-difluorophenyl)amino)-N-(2-morpholinoethyl)-5-oxo-10,11-dihydro-5Hdibenzo[a,d][7]annulene-3-carboxamide (5h). Compound 5h was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/ methanol 95:05) to afford 0.08 g (0.14 mmol, 55.6%) of the title compound 5h. Melting point, 120.4 °C. HPLC, 4.58 min. IR (ATR) [cm−1] 3015, 2696, 1739, 1637, 1538, 1436, 1365, 1216, 1112, 859, 785, 527 1H NMR (400 MHz, Acetone-d6) δ 10.05 (s, 1H), 8.62 (s, 1H), 8.53 (t, J = 5.6 Hz, 1H), 8.35−8.28 (m, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.96−7.88 (m, 2H), 7.48−7.37 (m, 2H), 6.79 (d, J = 8.9 Hz, 1H), 6.66 (s, 1H), 3.59−3.53 (m, 4H), 3.41−3.37 (m, 2H), 3.14−3.01 (m, 4H), 2.48−2.44 (m, 2H), 2.41 (s, 4H), 1.98−1.94 (m, 1H), 0.83−0.77 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 190.5, 172.2, 165.4, 149.1, 145.2, 144.5, 138.9, 133.2, 132.9, 130.4, 129.0, 128.9, 127.2, 124.0 (dd, J = 12.4, 3.2 Hz), 122.9 (dd, J = 12.6, 3.5 Hz), 119.5, 113.9, 112.4, 104.8 (t, J = 25.3 Hz), 66.2, 57.3, 53.3, 36.5, 35.4, 33.7, 13.9, 7.4. ESI-HRMS [M − H]+ calculated: 575.2464 found: 575.2459. 8-((5-(Cyclopropanecarboxamido)-2,4-difluorophenyl)amino)-N-(2-hydroxyethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (5i). Compound 5i was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.05 g (0.10 mmol, 92.6%) of the title compound 5i. Melting point, 130.2 °C. HPLC, 6.32 min. IR (ATR) [cm−1] 3271, 1633, 1603, 1537, 1403, 1266, 1193, 1114, 1062, 857, 758, 516 1H NMR (200 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.61 (s, 1H), 8.57−8.49 (m, 1H), 8.39−8.26 (m, 1H), 8.07−7.85 (m, 3H), 7.51−7.34 (m, 2H), 6.84−6.72 (m, 1H), 6.67−6.56 (m, 1H), 4.80−4.66 (m, 1H), 3.58−3.45 (m, 3H), 3.38−3.36 (m, 2H), 3.20−2.94 (m, 4H), 2.03−1.85 (m, 1H), 0.85−0.70 (m, 4H). ESI-HRMS [M − H]+ calculated: 506.1886 found: 506.1892. 8-((5-(Cyclopropanecarboxamido)-2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (5j). Compound 5j was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.09 mmol, 57.3%) of the title compound 5j. Melting point, 243.4 °C. HPLC, 6.20 min. IR (ATR) [cm−1] 3324, 3270, 3184, 1662, 1580, 1496, 1404, 1388, 1314, 1275, 1221, 1119, 1061, 863, 784, 617, 529. 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.61 (s, 1H), 8.35 (s, 1H), 8.07 (s, 1H), 8.01−7.88 (m, 3H), 7.52−7.34 (m, 3H), 6.78 (d, J = 9.0 Hz, 1H), 6.66 (s, 1H), 3.17−3.01 (m, 4H), 2.02−1.92 (m, 1H), 0.84−0.73 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 190.5, 172.2, 8044



Article

114.1, 110.5, 102.6, 72.4, 59.7, 14.5, 7.1. ESI-HRMS [M − H]+ calculated: 494.1886. Found: 494.1881. 3-((3-(Cyclopropanecarboxamido)-4-fluorophenyl)amino)N-(1,3-dihydroxypropan-2-yl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (5o). Compound 5o was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 90:10) to afford 0.036 g (0.14 mmol, 53,0%) of the title compound 5o. Melting point, 234.1 °C. HPLC, 4.75 min. 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.84 (s, 1H), 8.31 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.05 (dd, J = 14.2, 8.4 Hz, 2H), 7.74 (d, J = 5.9 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.24 (dd, J = 23.0, 12.9 Hz, 2H), 6.76 (d, J = 8.8 Hz, 1H), 6.44 (s, 1H), 5.26 (s, 2H), 4.68 (s, 2H), 3.98 (dd, J = 12.9, 6.0 Hz, 1H), 3.52 (d, J = 4.3 Hz, 4H), 1.72 (dd, J = 12.0, 6.0 Hz, 1H), 1.23 (s, 1H), 0.78 (d, J = 5.8 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.9, 171.6, 165.3, 162.7, 151.2, 139.9, 138.2, 136.0, 135.3, 133.1, 131.1, 128.1, 127.9, 116.8, 116.3, 116.1, 115.2, 114.1, 110.4, 102.6, 72.4, 60.4, 54.0, 14.5, 7.1. FAB (m/z): 520.3 [M − H]+. Methyl 8-((4-Fluoro-3-(thiophene-2-carboxamido)phenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxylate (6a). Compound 6a was prepared according to general procedure Va to afford 0.06 g (0.12 mmol, 60%) of the title compound 6a. Melting point, 98.3 °C. HPLC, 7.55 min. IR (ATR) [cm−1] 3330, 2916, 1601, 1555, 1515, 1435, 1414, 1383, 1353, 1217, 1109, 954, 881, 853, 736, 701, 520. 1H NMR (200 MHz, CDCl3) δ 8.66−8.60 (m, 1H), 8.19 (dd, J = 6.9, 2.7 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 8.07−7.96 (m, 2H), 7.66 (dd, J = 3.8, 1.1 Hz, 1H), 7.56 (dd, J = 5.0, 1.1 Hz, 1H), 7.15−7.07 (m, 1H), 7.05−6.96 (m, 1H), 6.93−6.82 (m, 2H), 6.71 (d, J = 2.2 Hz, 1H), 6.45 (s, 1H), 3.90 (s, 3H), 3.22−2.94 (m, 4H). 13 C NMR (50 MHz, CDCl3) δ 191.4, 166.7, 159.9, 148.7 (d, J = 240.2 Hz), 148.4, 146.6, 145.4, 139.7, 138.7, 137.2 (d, J = 2.8 Hz), 134.2, 132.4 (d, J = 9.7 Hz), 131.6, 129.0, 129.0, 128.8, 128.6, 128.1, 126.8 (d, J = 11.3 Hz), 116.8 (d, J = 7.4 Hz), 115.5 (d, J = 20.4 Hz), 114.9, 114.7, 113.4, 77.2, 52.2, 35.9, 34.8. ESI-HRMS [M + H]+ calculated: 501.1279. Found: 501.1277. N-(2-Fluoro-5-((7-((2-morpholinoethyl)carbamoyl)-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)phenyl)thiophene-2-carboxamide (6b). Compound 6b was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.05 mmol, 64.0%) of the title compound 6b. Melting point, °C. HPLC, 6,93 min, IR (ATR) [cm−1] 3290, 2920, 1633, 1530, 1504, 1442, 1416, 1258, 1213, 1144, 1111, 1068, 913, 854, 719, 493, 524, 450, 426. 1H NMR (400 MHz, acetone-d6) δ 9.17 (s, 1H), 8.34 (s, 2H), 8.13−7.85 (m, 10H), 7.85−7.70 (m, 3H), 7.41−7.30 (m, 2H), 7.23−7.11 (m, 4H), 7.08−6.97 (m, 4H), 6.93 (s, 2H), 3.57 (s, 9H), 3.53−3.46 (m, 4H), 3.20−3.08 (m, 8H), 2.59−2.51 (m, 4H), 2.43 (s, 8H), 2.00−1.99 (m, 8H). 13C NMR (100 MHz, acetone-d6) δ 205.7, 191.2, 166.6, 146.4, 145.7, 143.2, 140.4, 140.3, 138.5, 138.5, 138.5, 134.57, 134.4, 132.5, 131.3, 129.9, 129.8, 129.2, 128.8, 116.7, 116.5, 115.3, 115.2, 113.9, 113.9, 109.8, 67.5, 58.4, 54.5, 37.5, 36.7, 35.2. ESI-HRMS [M − H]+ calculated: 599.2117. Found: 599.2123. N-(2-Fluoro-5-((7-((2-hydroxyethyl)carbamoyl)-5-oxo-10,11dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)phenyl)thiophene-2-carboxamide (6c). Compound 6c was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 96:04) to afford 0.03 g (0.05 mmol, 33.0%) of the title compound 6c. Melting point, 106.2 °C. HPLC, 5.70 min IR (ATR) [cm−1] 1715, 1652, 1530, 1436, 1348, 1261, 1067, 965, 857, 844, 828, 751, 696, 534, 478, 441. 1H NMR (200 MHz, acetone-d6) δ 9.24 (s, 1H), 8.41 (d, J = 1.6 Hz, 1H), 8.15−8.06 (m, 2H), 8.06−7.92 (m, 4H), 7.81 (d, J = 4.9 Hz, 1H), 7.42−7.33 (m, 1H), 7.28−7.16 (m, 2H), 7.16−6.89 (m, 4H), 3.77−3.66 (m, 2H), 3.59−3.49 (m, 2H), 3.24−3.07 (m, 4H). 13C NMR (50 MHz, acetone-d6) δ 206.3, 191.3, 167.4, 161.0, 151.0 (d, J = 240.9 Hz), 148.6, 146.5, 145.9, 140.4, 140.3, 138.5 (d, J = 2.7 Hz), 134.7, 134.3, 132.7, 131.4, 130.0, 130.0 (d, J = 13.6 Hz), 129.1, 128.9, 128.5, 127.5 (d, J = 12.6 Hz), 118.3 (d, J = 7.3 Hz), 117.6, 117.6, 116.7 (d, J = 20.9 Hz), 115.3, 113.9, 62.0, 43.7, 36.8, 35.3. ESI-HRMS [M − H]+ calculated: 530.1543. Found: 530.1544.

N-(5-((7-Carbamoyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)-2-fluorophenyl)thiophene-2-carboxamide (6d). Compound 6d was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.04 mmol, 68.0%) of the title compound 6d (0.025 g, 68%). Melting point, 65.4 °C. HPLC, 5.73 min. IR (ATR) [cm−1] 3305, 2920, 2850, 1651, 1600, 1576, 1525, 1416, 1394, 1260, 1168, 1109, 922, 853, 768, 451. 1 H NMR (400 MHz, acetone-d6) δ 9.24 (s, 1H), 8.49−8.43 (m, 1H), 8.15−8.05 (m, 2H), 8.05−7.97 (m, 3H), 7.83−7.77 (m, 1H), 7.70−7.61 (m, 1H), 7.41−7.36 (m, 1H), 7.24−7.16 (m, 2H), 7.11−7.01 (m, 2H), 6.96 (s, 1H), 6.72 (s, 1H), 3.23−3.09 (m, 4H). 13C NMR (100 MHz, acetone-d6) δ 191.3, 168.5, 160.9, 150.9 (d, J = 240.8 Hz), 149.8, 146.4, 146.0, 140.4, 140.4, 138.5 (d, J = 2.2 Hz), 134.6, 133.8, 132.6, 131.6, 130.6, 130.0, 129.8, 129.2, 128.8, 127.5 (d, J = 12.7 Hz), 118.28 (d, J = 7.3 Hz), 117.6, 116.6 (d, J = 21.1 Hz), 115.3, 113.9, 36.7, 35.3, 29.8. ESI-HRMS [M − H]+ calculated: 486.1287. Found: 486.1282. N-(5-((7-((2,3-Dihydroxypropyl)carbamoyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)-2fluorophenyl)thiophene-2-carboxamide (6e). Compound 6e was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.05 mmol, 49.0%) of the title compound 6e. Melting point, 67.3 °C. HPLC, 5.73 min. IR (ATR) [cm−1] 3303, 2921, 2852, 1731, 1632, 1575, 1586, 1353, 1213, 1035, 853, 719, 520. 1 H NMR (400 MHz, acetone-d6) δ 9.22 (s, 1H), 8.43 (s, 1H), 8.15−7.92 (m, 6H), 7.81 (s, 1H), 7.40 (s, 1H), 7.20 (s, 2H), 7.11−6.94 (m, 3H), 3.86−3.71 (m, 2H), 3.68−3.52 (m, 4H), 3.25−3.11 (m, 4H). 13 C NMR (100 MHz, acetone-d6) δ 190.3, 167.2, 160.0, 150.0 (d, J = 240.9 Hz), 148.9, 145.5, 145.1, 139.5, 139.4, 137.6 (d, J = 2.4 Hz), 133.7, 133.0, 131.6, 130.5, 129.2, 129.0, 129.0, 128.2, 127.9, 126.6 (d, J = 12.4 Hz), 117.4 (d, J = 7.4 Hz), 116.6, 115.7 (d, J = 20.9 Hz), 114.4, 113.0, 71.3, 63.8, 43.0, 35.8, 34.3. ESI-HRMS [M − H]+ calculated: 530.1543. Found: 530.1544. Methyl 8-((2,4-Difluoro-5-(thiophene-2-carboxamido)phenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxylate (6f). Compound 6f was prepared according to general procedure Va to afford 0.12 g (0.23 mmol, 69.9%) of the title compound 6f. Melting point, 97.4 °C. HPLC, 7.53 min. IR (ATR) [cm−1] 3307, 1947, 1633, 162, 1582, 1530, 1434, 1416, 1353, 1192, 1120, 1036, 949, 786, 719, 649, 509, 456. 1H NMR (200 MHz, CDCl3) δ 8.64 (d, J = 1.8 Hz, 1H), 8.52−8.37 (m, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.05 (dd, J = 7.7, 1.8 Hz, 1H), 7.89−7.80 (m, 1H), 7.70−7.54 (m, 2H), 7.30 (s, 1H), 7.20−7.07 (m, 1H), 7.07−6.86 (m, 2H), 6.83−6.74 (m, 1H), 6.00 (s, 1H), 3.92 (s, 3H), 3.32−3.11 (m, 4H). 13C NMR (50 MHz, CDCl3) δ 207.2, 191.7, 166.7, 160.0, 150.7 (dd, J = 244.5, 9.3 Hz), 148.5 (dd, J = 244.3, 12.2 Hz), 145.3, 139.6, 138.5, 134.1, 132.6, 132.3, 131.6, 129.3, 129.1 (d, J = 2.4 Hz), 128.8, 128.1, 125.1 (dd, J = 12.2, 3.5 Hz), 122.37 (dd, J = 11.4, 3.8 Hz), 120.1, 117.9, 116.2, 115.1, 113.7, 104.3 (t, J = 24.7 Hz), 52.2, 35.8, 34.9. ESI-HRMS [M + H]+ calculated: 519.1185. Found: 519.1189. N-(2,4-Difluoro-5-((7-((2-morpholinoethyl)carbamoyl)-5oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)phenyl)thiophene-2-carboxamide (6g). Compound 6g was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol and 1%NH3 95:05) to afford 0.03 g (0.05 mmol, 84.5%) of the title compound 6g. Melting point, 104.7 °C. HPLC, 4.18 min. IR (ATR) [cm−1] 3271, 2921, 2851, 1698, 1633, 1530, 1434, 1353, 1260, 1207, 1067, 914, 859, 786, 719, 622, 507, 457. 1H NMR (400 MHz, CDCl3) δ 8.46 (t, J = 8.4 Hz, 1H), 8.30 (d, J = 1.4 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.01−7.92 (m, 1H), 7.88 (s, 1H), 7.62 (dd, J = 28.8, 4.1 Hz, 2H), 7.30 (d, J = 7.9 Hz, 1H), 7.17−7.12 (m, 1H), 7.05−6.96 (m, 1H), 6.96−6.87 (m, 2H), 6.81 (s, 1H), 6.06 (s, 1H), 3.86−3.69 (m, 4H), 3.63−3.52 (m, 2H), 3.22−3.08 (m, 4H), 2.63−2.59 (m, 2H), 2.51 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 191.7, 166.9, 159.9, 150.6 (dd, J = 248.3, 8.5 Hz), 147.4, 147.0 (d, J = 11.1 Hz), 145.5, 145.3, 139.2, 138.6, 134.3, 133.2, 131.6, 131.4, 129.5, 129.1, 128.6, 128.1, 125.3 (dd, J = 11.7, 3.1 Hz), 122.7 (dd, J = 11.1, 3.7 Hz), 115.7, 115.3, 114.0, 104.2 (d, J = 24.5 Hz), 104.1, 77.2, 67.1, 57.2, 8045



Article

53.6, 36.4, 36.0, 34.8. ESI-HRMS [M − H]+ calculated: 617.2029. Found: 617.2018. N-(5-((7-Carbamoyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)-2,4-difluorophenyl)thiophene-2-carboxamide (6h). Compound 6h was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.06 mmol, 84.5%) of the title compound 6h. Melting point, 139.6 °C. HPLC, 5.75 min. IR (ATR) [cm−1] 3293, 2920, 1652, 1580, 1525, 1416, 1397, 1353, 1265, 1110, 1037, 974, 857, 767, 603, 508, 445. 1H NMR (400 MHz, acetone-d6) δ 9.28 (s, 1H), 8.45 (s, 1H), 8.16−8.05 (m, 2H), 8.03−7.93 (m, 2H), 7.85−7.76 (m, 2H), 7.71−7.50 (m, 1H), 7.43−7.36 (m, 1H), 7.30−7.18 (m, 2H), 7.01−6.95 (m, 1H), 6.90 (s, 1H), 6.72−6.52 (m, 1H), 3.22−3.11 (m, 4H). 13C NMR (100 MHz, acetone-d6) δ 201.3, 178.4, 170.8, 159.4, 155.9, 155.7, 149.9, 149.8, 143.3, 142.2, 141.3, 140.3, 139.7, 139.5, 139.0, 138.5, 135.5 (dd, J = 12.2, 3.0 Hz), 135.4, 135.4, 132.8, 131.0, 125.0, 123.54, 115.1 (t, J = 25.0 Hz), 114.9, 46.2, 44.8, 39.5. ESI-HRMS [M − H]+ calculated: 504.1188. Found: 504.118. N-(5-((7-((2,3-Dihydroxypropyl)carbamoyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)-2,4-difluorophenyl)thiophene-2-carboxamide (6i). Compound 6i was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.05 mmol, 90.3%) of the title compound 6i. Melting point, 68.1 °C. HPLC, 5.43 min. IR (ATR) [cm−1] 3123, 3019, 2911, 2696, 1735, 1627, 1538, 1445, 1353, 1260, 1146, 1054, 930, 825, 736, 617, 418. 1H NMR (400 MHz, acetone-d6) δ 9.28 (s, 1H), 8.45 (s, 1H), 8.16−8.05 (m, 2H), 8.03−7.93 (m, 2H), 7.85−7.76 (m, 2H), 7.71−7.50 (m, 1H), 7.43−7.36 (m, 1H), 7.30−7.18 (m, 2H), 7.01−6.95 (m, 1H), 6.90 (s, 1H), 6.72−6.52 (m, 1H), 3.22−3.11 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 190.9, 166.2, 160.4, 149.3, 145.6, 144.9, 139.2, 139.1, 133.7, 133.2, 132.5, 130.8, 130.1, 129.6, 129.2, 128.5, 127.7, 124.6, 122.8, 122.0, 114.3, 112.8, 70.7, 64.3, 43.4, 35.7, 34.1. ESI-HRMS [M − H]+ calculated: 578.1556. Found: 578.1551. N-(2,4-Difluoro-5-((7-((2-hydroxyethyl)carbamoyl)-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulen-2-yl)amino)phenyl)thiophene-2-carboxamide (6j). Compound 6j was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.06 mmol, 75.0%) of the title compound 6j. Melting point, 220.4 °C. HPLC, 5.65 min. IR (ATR) [cm−1] 3043, 2935, 1623, 1577, 1507, 1433, 1356, 1267, 1199, 1119, 1088, 978, 826, 763, 678, 566, 446, 413. 1H NMR (400 MHz, acetone-d6) δ 9.32 (s, 1H), 8.44−8.35 (m, 1H), 8.12−8.02 (m, 2H), 8.02−7.93 (m, 3H), 7.85−7.77 (m, 2H), 7.37 (d, J = 7.9 Hz, 1H), 7.29−7.18 (m, 2H), 7.01−6.93 (m, 1H), 6.88 (s, 1H), 3.76−3.69 (m, 2H), 3.59−3.51 (m, 2H), 3.21−3.09 (m, 4H). 13C NMR (100 MHz, acetone-d6) δ 191.6, 167.4, 161.0, 149.7, 146.2, 145.8, 140.2, 140.2, 134.4, 134.2, 132.6, 131.4, 130.1, 130.0, 129.9, 129.6, 128.8, 125.9 (dd, J = 12.8, 3.3 Hz), 123.3 (dd, J = 12.6, 3.7 Hz), 121.1, 121.0, 115.5, 114.0, 105.5 (dd, J = 42.9, 17.9 Hz), 62.0, 43.7, 36.6, 35.2. ESI-HRMS [M − H]+ calculated: 548.1450. Found: 548.1459. Methyl 3-((4-Fluoro-3-(thiophene-2-carboxamido)phenyl)amino)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxylate (6k). Compound 6k was prepared according to general procedure Va to afford 0.10 g (0.20 mmol, 40.0%) of the title compound 6k. Melting point, 184.5 °C. HPLC, 7.44 min. IR (ATR) [cm−1] 3301, 2925, 2852, 1573, 1526, 1241, 1116, 851, 715, 529. 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.09 (s, 1H), 8.37 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.04 (dd, J = 12.3, 6.0 Hz, 2H), 7.88 (d, J = 4.6 Hz, 1H), 7.68 (t, J = 10.5 Hz, 1H), 7.48 (dd, J = 10.8, 6.5 Hz, 1H), 7.30 (t, J = 9.5 Hz, 1H), 7.26−7.19 (m, 1H), 7.11−7.03 (m, 1H), 6.80 (d, J = 9.0 Hz, 1H), 6.56 (s, 1H), 5.29 (s, 2H), 4.56 (s, 1H), 3.89 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 208.3, 185.8, 165.4, 163.0, 159.9, 151.4, 140.6, 140.1, 138.9, 133.6, 132.5, 132.1, 130.2, 129.8, 129.7, 128.8, 128.1, 118.9, 118.7, 116.5, 116.3, 110.4, 101.8, 72.4, 29.6. ESI-HRMS [M + H]+ calculated: 503.1071. Found: 503.1071.

3-((4-Fluoro-3-(thiophene-2-carboxamido)phenyl)amino)-N(2-morpholinoethyl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (6l). Compound 6l was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.024 g (0.06 mmol, 69,6%) of the title compound 6l. HPLC, 4.39 min. 1 H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 9.07 (s, 1H), 8.57 (d, J = 49.7 Hz, 1H), 8.28 (s, 1H), 8.03 (s, 3H), 7.88 (s, 1H), 7.60 (t, J = 20.3 Hz, 1H), 7.48 (s, 1H), 7.37−7.18 (m, 2H), 7.07 (s, 1H), 6.80 (d, J = 7.4 Hz, 1H), 6.56 (s, 1H), 5.26 (s, 2H), 3.56 (s, 4H), 3.40 (s, 2H), 2.48−2.44 (m, 2H), 2.41 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.6, 165.2, 162.9, 159.96, 151.3, 140.0, 138.9, 138.4, 136.6, 135.1, 133.4, 132.1, 130.8, 129.7, 128.2, 128.1, 127.7, 118.8, 118.7, 116.6, 116.3, 110.3, 101.8, 72.4, 66.2, 57.2, 53.2, 36.6. ESIHRMS [M − H]+ calculated: 601.1915. Found: 601.1911. 3-((4-Fluoro-3-(thiophene-2-carboxamido)phenyl)amino)-N(2-hydroxyethyl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9carboxamide (6m). Compound 6m was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.010 g (0.07 mmol, 26.3%) of the title compound 5n. Melting point, 201.8 °C. HPLC, 5.45 min. IR (ATR) [cm−1] 3016, 2924, 2853, 1738, 1634, 1568, 1441, 1416, 1365, 1217. 1H NMR (400 MHz, DMSO-d6) δ 10.14 (d, J = 21.9 Hz, 1H), 9.07 (s, 1H), 8.65 (s, 1H), 8.30 (s, 1H), 8.05 (d, J = 8.7 Hz, 3H), 7.86 (d, J = 19.2 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.48 (s, 1H), 7.28 (dd, J = 21.3, 11.9 Hz, 2H), 7.03 (d, J = 40.7 Hz, 1H), 6.77 (t, J = 14.5 Hz, 1H), 6.53 (d, J = 23.6 Hz, 1H), 5.26 (s, 2H), 4.82 (d, J = 49.5 Hz, 1H), 3.52 (s, 2H), 1.35−1.18 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 186.6, 162.9, 160.0, 139.9, 138.9, 138.3, 135.1, 133.4, 132.1, 130.9, 129.7, 128.1, 127.8, 118.7, 116.5, 110.3, 101.8, 72.4, 59.6, 42.3. ESI-HRMS [M − H]+ calculated: 532.1337. Found: 532.1342. 3-((4-Fluoro-3-(thiophene-2-carboxamido)phenyl)amino)11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (6n). Compound 6n was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.02 g (0.04 mmol, 40.6%) of the title compound 6n. HPLC, 6.02 min. 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 9.07 (s, 2H), 8.63 (t, J = 5.3 Hz, 1H), 8.27 (s, 1H), 8.04 (t, J = 7.8 Hz, 2H), 7.97 (d, J = 7.7 Hz, 2H), 7.61 (t, J = 7.2 Hz, 2H), 7.53 (t, J = 7.3 Hz, 3H), 7.29 (t, J = 9.5 Hz, 1H), 7.12−7.00 (m, 1H), 6.80 (d, J = 9.0 Hz, 1H), 6.56 (s, 1H), 5.25 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 186.6, 186.6, 165.50, 165.5, 165.2, 162.9, 162.9, 151.3, 150.1, 140.0, 138.4, 135.1, 133.9, 133.4, 131.9, 130.8, 128.4, 128.2, 127.8, 127.7, 126.3, 118.6, 116.5, 110.2, 101.7, 72.4, 66.2, 57.2, 53.2, 36.6. ESI-HRMS [M − H]+ calculated: 488.1075. Found: 488.1045. N-(2,3-Dihydroxypropyl)-3-((4-fluoro-3-(thiophene-2carboxamido)phenyl)amino)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (6o). Compound 6o was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0,05 mmol, 87.0%) of the title compound 6o. Melting point, 173.8 °C. HPLC, 5.16 min. IR (ATR) [cm−1] 3289, 2922, 1633, 1537, 1531, 1455, 1385, 1260, 1120. 1H NMR (400 MHz, DMSO-d6) δ 10.17 (d, J = 15.8 Hz, 1H), 9.04 (s, 1H), 8.59 (s, 1H), 8.30 (s, 1H), 8.13−8.00 (m, 3H), 7.88 (d, J = 3.9 Hz, 1H), 7.69−7.53 (m, 1H), 7.45 (d, J = 22.4 Hz, 1H), 7.35−7.19 (m, 2H), 7.13−7.00 (m, 1H), 6.80 (d, J = 7.2 Hz, 1H), 6.52 (d, J = 32.4 Hz, 1H), 5.27 (s, 2H), 4.81 (s, 1H), 4.57 (d, J = 3.8 Hz, 1H), 3.66 (s, 1H), 3.23 (d, J = 4.0 Hz, 2H), 1.21 (ddd, J = 40.1, 30.9, 11.3 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 186.6, 165.6, 162.9, 160.0, 151.3, 139.9, 138.9, 138.4, 136.6, 135.1, 133.4, 132.0, 130.9, 129.7, 128.1, 127.9, 118.7, 116.6, 110.3, 101.8, 72.4, 70.3, 64.0, 43.1. ESI-HRMS [M − H]+ calculated: 562.1443. Found: 562.1443. N-(2-Fluoro-5-((9-(morpholine-4-carbonyl)-11-oxo-6,11dihydrodibenzo[b,e]oxepin-3-yl)amino)phenyl)thiophene-2carboxamide (6p). Compound 6p was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.05 mmol, 57.0%) of the title compound 6p. Melting point, 195.1 °C. HPLC, 8046



Article

6.05 min. IR (ATR) [cm−1] 3293, 2917, 2852, 1573, 1519, 1435, 1269, 1111, 1026, 846, 714, 573. 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.06 (s, 1H), 8.04 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 4.7 Hz, 1H), 7.79 (s, 1H), 7.63 (dd, J = 14.6, 7.6 Hz, 2H), 7.49 (d, J = 6.2 Hz, 1H), 7.35−7.18 (m, 2H), 7.06 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 8.9 Hz, 1H), 6.56 (s, 1H), 5.25 (s, 2H), 4.35 (s, 2H), 3.71−3.39 (m, 8H). 13C NMR (100 MHz, DMSO-d6) δ 185.5, 167.1, 162.0, 158.9, 150.2, 138.9, 137.8, 135.9, 135.4, 135.0, 132.4, 130.9, 129.5, 128.5, 127.1, 126.9, 126.3, 124.6, 117.3, 115.2, 109.0, 100.3, 70.8, 64.4, 60.3. ESIHRMS [M − H]+ calculated: 558.1493. Found: 558.1498. Methyl 3-((2,4-difluoro-5-(thiophene-2-carboxamido)phenyl)amino)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9carboxylate (6q). Compound 6q was prepared according to general procedure Va to afford 0.25 g (0.48 mmol, 61.1%) of the title compound 6q. Melting point, 194.0 °C. HPLC, 6.10 min. IR (ATR) [cm−1] 2357, 2341, 1715, 1607, 1574, 1525, 1508, 1417, 1244, 1117, 1087. 1H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H), 8.82 (d, J = 60.2 Hz, 1H), 8.37 (s, 1H), 8.08 (d, J = 50.2 Hz, 3H), 7.87 (s, 1H), 7.78−7.40 (m, 3H), 7.22 (s, 1H), 6.72 (s, 1H), 6.31 (d, J = 45.6 Hz, 1H), 5.29 (s, 2H), 3.88 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 186.0, 165.4, 162.9, 160.0, 151.8, 140.6, 140.0, 138.7, 133.5, 132.6, 132.1, 130.1, 129.8, 129.8, 128.8, 128.1, 123.9, 122.8, 121.9, 116.6, 110.1, 105.6, 105.3, 105.1, 102.0, 72.4, 52.4. ESI-HRMS [M + H]+ calculated: 521.0977. Found: 521.0967. 3-((2,4-Difluoro-5-(thiophene-2-carboxamido)phenyl)amino)-N-(2-morpholinoethyl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (6r). Compound 6r was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.02 g (0.03 mmol, 62.8%) of the title compound 6r. HPLC, 4.49 min. 1 H NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.86 (s, 1H), 8.64 (s, 1H), 8.28 (s, 1H), 8.14−7.95 (m, 3H), 7.88 (d, J = 3.6 Hz, 1H), 7.68−7.49 (m, 3H), 7.19 (d, J = 36.2 Hz, 1H), 6.67 (t, J = 26.2 Hz, 1H), 6.36 (s, 1H), 5.26 (s, 2H), 3.57 (s, 4H), 3.40 (s, 2H), 2.48−2.45 (m, 2H), 2.41 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.8, 165.1, 162.8, 160.0, 151.6, 139.9, 138.7, 138.4, 135.1, 133.3, 132.1, 130.9, 129.7, 128.2, 128.1, 127.7, 122.8, 116.7, 109.9, 102.0, 72.4, 66.2, 57.2, 53.2, 36.6. ESI-HRMS [M − H]+ calculated: 619.1821. Found: 619.1911. 3-((2,4-Difluoro-5-(thiophene-2-carboxamido)phenyl)amino)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (6s). Compound 6s was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.02 g (0.04 mmol, 60.1%) of the title compound 6s. HPLC, 5.44 min. 1H NMR (400 MHz, DMSO-d6) δ 10.23 (d, J = 19.4 Hz, 1H), 8.85 (s, 1H), 8.30 (s, 1H), 8.16 (s, 1H), 8.11−7.97 (m, 3H), 7.87 (d, J = 4.9 Hz, 1H), 7.66−7.42 (m, 4H), 7.23 (dd, J = 5.8, 2.8 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 6.35 (s, 1H), 5.25 (s, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 186.8, 166.9, 162.8, 160.0, 151.6, 139.9, 138.7, 138.5, 134.8, 133.3, 132.1, 131.1, 129.7, 128.2, 122.8, 116.7, 110.0, 102.0, 72.4. ESI-HRMS [M − H]+ calculated: 506.0981. Found: 506.0984. 3-((2,4-Difluoro-5-(thiophene-2-carboxamido)phenyl)amino)-N-(2,3-dihydroxypropyl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (6t). Compound 6t was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.01 g (0.02 mmol, 43.7%) of the title compound 6t. HPLC, 5.14 min. 1 H NMR (400 MHz, MeOD) δ 8.34 (s, 1H), 8.25 (s, 1H), 8.11 (dd, J = 9.0, 3.1 Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.76 (dd, J = 18.0, 9.8 Hz, 1H), 7.60 (dd, J = 12.4, 11.2 Hz, 1H), 7.53 (dd, J = 5.0, 2.8 Hz, 2H), 7.28−7.12 (m, 1H), 6.72 (d, J = 8.9 Hz, 1H), 6.45 (s, 1H), 5.20 (d, J = 2.4 Hz, 2H), 3.64 (s, 2H), 3.56 (dd, J = 5.0, 2.0 Hz, 2H), 3.43 (dd, J = 12.8, 6.3 Hz, 1H). 13C NMR (100 MHz, MeOD) δ 187.9, 168.2, 163.7, 152.3, 140.4, 139.3, 136.5, 135.0, 133.4, 130.7, 129.68, 127.9, 126.5, 126.3, 122.1, 117.2, 110.1, 104.5, 102.3, 72.8, 70.7, 63.8, 42.8. ESI-HRMS [M − H]+ calculated: 580.1348. Found: 580.1337. 3-((2,4-Difluoro-5-(thiophene-2-carboxamido)phenyl)amino)-N-(1,3-dihydroxypropan-2-yl)-11-oxo-6,11dihydrodibenzo[b,e]oxepine-9-carboxamide (6u). Compound 6u was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/

methanol 95:05) to afford 0.01 g (0.02 mmol, 87.4%) of the title compound 6u. HPLC, 5.23 min: 1H NMR (400 MHz, MeOD) δ 8.35 (s, 1H), 8.10 (dd, J = 8.9, 4.9 Hz, 1H), 8.07−7.99 (m, 1H), 7.89 (s, 1H), 7.78−7.66 (m, 2H), 7.56−7.47 (m, 1H), 7.27−7.13 (m, 2H), 6.71 (d, J = 8.8 Hz, 1H), 6.44 (s, 1H), 5.19 (d, J = 4.8 Hz, 2H), 4.24−4.11 (m, 1H), 3.75 (d, J = 4.2 Hz, 4H). 13C NMR (100 MHz, MeOD) δ 187.9, 167.9, 167.9, 163.7, 163.7, 161.7, 161.7, 152.2, 140.3, 139.2, 138.1, 135.2, 133.4, 131.5, 130.8, 129.5, 128.0, 127.8, 127.6, 122.1, 117.2, 110.0, 104.5, 102.3, 72.8, 60.7, 53.9. ESI-HRMS [M − H]+ calculated: 580.1348. Found: 580.1355. 3-((2,4-Difluoro-5-(thiophene-2-carboxamido)phenyl)amino)-N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-11oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (6v). Compound 6v was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.05 mmol, 69.2%) of the title compound 6v. HPLC, 5.10 min. 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.85 (s, 1H), 8.21 (s, 1H), 8.04 (d, J = 8.9 Hz, 1H), 8.00 (s, 2H), 7.88 (d, J = 4.9 Hz, 1H), 7.64−7.43 (m, 4H), 7.23 (dd, J = 6.0, 2.7 Hz, 1H), 6.68 (t, J = 14.5 Hz, 1H), 6.32 (d, J = 24.1 Hz, 1H), 5.26 (s, 2H), 4.74 (s, 3H), 3.70 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 186.8, 166.3, 162.8, 160.0, 151.6, 139.8, 138.7, 138.3, 135.8, 133.3, 132.1, 129.7, 128.1, 127.9, 116.7, 109.9, 102.0, 72.4, 66.1, 62.8, 60.2, 53.1. ESI-HRMS [M − H]+ calculated: 610.1454. Found: 610.1456. Methyl 3-((4-Fluoro-2-methyl-5-(thiophene-3-carboxamido)phenyl)amino)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine9-carboxylate (7a). Compound 7a was prepared according to general procedure Va to afford 0.16 mmol (0.31 mmol, 38.4%) of the title compound 7a. Melting point, 154.4 °C. HPLC, 7.95 min. IR (ATR) [cm−1] 3296, 2953, 1716, 1607, 1568, 1558, 1520, 1247, 1118, 1002. 1H NMR (400 MHz, DMSO-d6) δ (s, 1H), 8.56 (s, 1H), 8.36 (d, J = 6.0 Hz, 2H), 8.08 (dd, J = 45.2, 7.9 Hz, 2H), 7.74−7.53 (m, 3H), 7.47 (d, J = 6.5 Hz, 1H), 7.33−7.21 (m, 1H), 6.62 (d, J = 8.1 Hz, 1H), 6.20 (s, 1H), 5.26 (s, 2H), 3.89 (s, 3H), 2.19 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 183.6, 163.3, 160.9, 158.7, 151.1, 149.4, 138.5, 138.0, 134.8, 131.94, 131.5, 130.3, 130.1, 130.1, 128.0, 128.0, 127.7, 126.6, 125.0, 124.8, 121.5, 121.4, 121.1, 115.6, 115.4, 113.7, 107.6, 98.8, 70.3, 62.7, 50.2. ESI-HRMS [M + H]+ calculated: 517.1228. Found: 517.1218. 3-((2,4-Difluoro-5-(thiophene-3-carboxamido)phenyl)amino)N-(1,3-dihydroxypropan-2-yl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (7b). Compound 7b was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.07 mmol, 33.0%) of the title compound 7b. Melting point, 171.3 °C. HPLC, 5.14 min. IR (ATR) [cm−1] 3271, 2921, 2852, 1606, 1531, 1471, 1416, 1254, 1121, 1033. 1H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.85 (s, 1H), 8.35 (d, J = 15.0 Hz, 1H), 8.30 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.05 (dd, J = 12.0, 8.8 Hz, 2H), 7.71−7.44 (m, 5H), 6.71 (d, J = 8.8 Hz, 1H), 6.36 (s, 1H), 5.29 (d, J = 23.4 Hz, 2H), 4.67 (s, 1H), 3.98 (d, J = 6.1 Hz, 1H), 3.52 (d, J = 5.1 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.9, 165.3, 162.7, 160.9, 151.7, 139.9, 138.2, 136.7, 135.3, 133.3, 131.1, 130.3, 128.1, 127.9, 127.1 123.9, 122.7, 116.7, 109.9, 105.23, 102.0, 72.4, 60.4, 54.0. ESI-HRMS [M − H]+ calculated: 580.1348. Found: 580.1304. N-(2,3-Dihydroxypropyl)-3-((4-fluoro-2-methyl-5-(thiophene3-carboxamido)phenyl)amino)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (7c). Compound 7c was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.01 g (0.02 mmol, 78.6%) of the title compound 7c. HPLC, 5.45 min. 1H NMR (400 MHz, MeOD) δ 8.32 (d, J = 17.6 Hz, 1H), 8.21 (d, J = 12.0 Hz, 1H), 8.08 (dd, J = 9.0, 2.4 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 6.8 Hz, 2H), 7.51 (dd, J = 9.6, 4.8 Hz, 2H), 7.15 (d, J = 11.0 Hz, 1H), 6.59 (d, J = 9.0 Hz, 1H), 6.22 (d, J = 2.1 Hz, 1H), 5.17 (s, 2H), 4.59 (s, 3H), 3.90−3.79 (m, 1H), 3.57−3.53 (m, 2H), 3.43 (td, J = 13.5, 5.6 Hz, 2H). 13C NMR (100 MHz, MeOD) δ 187.8, 172.1, 168.2, 164.0, 156.4, 154.1, 139.4, 135.0, 133.6, 130.7, 129.5, 128.0, 126.5, 126.3, 123.4, 117.2, 109.6, 101.2, 72.8, 70.7, 8047



Article

63.8, 42.8, 29.3, 16.3. ESI-HRMS [M − H]+ calculated: 517.1228. Found: 517.1218. Methyl 8-((3-benzamido-4-fluorophenyl)amino)-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxylate (8a). Compound 8a was prepared according to general procedure Va to afford 0.18 g (0.36 mmol, 56.0%) of the title compound 8a. Melting point, 216.9 °C. HPLC, 8.32 min. IR (ATR) [cm−1] 3324, 1718, 1600, 1555, 1405, 1353, 1256, 1111, 954, 832, 801, 706, 520. 1H NMR (400 MHz, DMSO) δ 1H NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 8.92 (s, 1H), 8.46 (s, 1H), 8.09−7.94 (m, 4H), 7.62−7.44 (m, 5H), 7.35−7.16 (m, 1H), 7.15−7.04 (m, 1H), 6.98 (d, J = 8.6 Hz, 1H), 6.87 (s, 1H), 3.87 (s, 3H), 3.23−3.07 (m, 4H). 13C NMR (100 MHz, DMSO) δ 189.7, 165.8, 165.6, 148.9, 147.1, 145.6, 145.6, 139.1, 137.1, 134.0, 133.8, 132.1, 132.0, 131.4, 129.7, 128.5, 128.1, 127.9, 127.0, 126.5, 118.3, 118.3, 116.6, 116.2, 114.0, 113.9, 112.7, 52.3, 35.3, 34.1. ESI-HRMS [M + H]+ calculated: 495.1715. Found: 495.1712. 8-((3-Benzamido-4-fluorophenyl)amino)-N-(2-morpholinoethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (8b). Compound 8b was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.01 g (0.02 mmol, 87.0%) of the title compound 8b. Melting point, 185.3 °C. HPLC, 5.12 min. IR (ATR) [cm−1] 3306, 2919, 1582, 1639, 1486, 1400, 1317, 1217, 1106, 1001, 834, 870, 834, 790, 677, 601, 473. 1 H NMR (200 MHz, DMSO-d6) δ 10. One (s, 1H), 8.89 (s, 1H), 8.45−8.61 (m, 1H), 8.32 (d, 1H), 7.86−8.06 (m, 4H), 7.47−7.62 (m, 4H), 6.93−7.44 (m, 4H), 6.86 (s, 1H), 3.49−3.62 (m, 4H), 3.24−3.48 (m, 2H), 2.99−3.21 (m, 4H), 2.27−2.62 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 190.3, 165.5, 150.9 (d, J = 241.4 Hz), 148.7, 144.5, 138.9, 137.1, 137.0, 134.0, 133.5, 131.8, 130.4, 129.1, 128.9, 128.4, 127.8, 127.2, 126.2 (d, J = 13.8 Hz), 118.2, 118.1 (d, J = 6.5 Hz). ESI-HRMS [M − H]+ calculated: 593.2559. Found: 593.2555. 8-((3-Benzamido-4-fluorophenyl)amino)-N-methyl-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (8c). Compound 8c was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.02 g (0.42 mmol, 65.0%) of the title compound 8c. Melting point, 166.4 °C. HPLC, 6.28 min. IR (ATR) [cm−1] 3303, 1654, 1577, 1447, 1354, 1214, 973, 706. 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.91 (s, 1H), 8.62−8.48 (m, 1H), 8.33 (s, 1H), 8.04−7.88 (m, 4H), 7.63−7.51 (m, 4H), 7.43−7.37 (m, 1H), 7.30−7.23 (m, 1H), 7.09−7.04 (m, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.87 (s, 1H), 3.18−3.03 (m, 4H), 2.83−2.74 (m, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 190.3, 172.3, 165.9, 165.5, 154.3, 150.9 (d, J = 242.2 Hz), 148.7, 148.7, 145.4, 144.5, 138.9, 137.1, 134.0, 133.5, 132.8, 131.8, 130.2, 129.0 (d, J = 15.5 Hz), 128.4, 127.8, 127.2, 126.2 (d, J = 13.2 Hz), 118.1 (d, J = 10.2 Hz), 116.3 (d, J = 21.4 Hz), 113.9, 112.6, 35.44, 33.8, 26.2. ESI-HRMS [M − H]+ calculated: 480.1718. Found: 480.1682. 8-((3-Benzamido-4-fluorophenyl)amino)-N-methyl-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (8d). Compound 8d was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.09 g (0.18 mmol, 78.0%) of the title compound 8d. Melting point, 135.5 °C. HPLC, 6.49 min. IR (ATR) [cm−1] 3291, 1601, 1575, 1525, 1505, 1398, 1354, 1261, 1213, 1114, 1207, 964, 785, 761, 704, 610, 452. 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.91 (s, 1H), 8.62−8.48 (m, 1H), 8.33 (s, 1H), 8.04−7.88 (m, 4H), 7.63−7.51 (m, 4H), 7.43−7.37 (m, 1H), 7.30−7.23 (m, 1H), 7.09−7.04 (m, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.87 (s, 1H), 3.18−3.03 (m, 4H), 2.83−2.74 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.3, 172.3, 165.9, 165.5, 154.3, 150.9 (d, J = 242.2 Hz), 148.8, 148.7, 145.4, 144.5, 138.9, 137.1, 134.0, 133.5, 132.8, 131.8, 130.2, 129.0 (d, J = 15.5 Hz), 128.4, 127.8, 127.2, 126.3 (d, J = 13.2 Hz), 118.1 (d, J = 10.2 Hz), 116.3 (d, J = 21.4 Hz), 114.0, 112.6, 35.4, 33.8, 26.2. ESI-HRMS [M − H]+ calculated: 494.1874. Found: 494.1871. 8-((3-Benzamido-4-fluorophenyl)amino)-N-(2,3-dihydroxypropyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (8e). Compound 8e was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.02 g (0.54 mmol,

43.7%) of the title compound 8e. Melting point, 107.4 °C. HPLC, 6.01 min. IR (ATR) [cm−1] 3296, 2921, 1600, 1520, 1445, 1353, 1112, 785, 572. 1H NMR (400 MHz, DMSO-d6) δ 1H NMR (400 MHz, DMSO) δ 10.14 (s, 1H), 8.90 (s, 1H), 8.58−8.46 (m, 1H), 8.33 (s, 1H), 8.07−7.85 (m, 6H), 7.66−7.58 (m, 1H), 7.56−7.49 (m, 4H), 7.43−7.39 (m, 1H), 7.01−6.96 (m, 1H), 6.87 (s, 1H), 4.90−4.81 (m, 1H), 4.69−4.59 (m, 1H), 3.20−3.17 (m, 1H), 3.12−3.05 (m, 4H), 2.88 (s, 2H), 2.72 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 190.5, 166.1, 165.7, 162.5, 151.1 (d, J = 242.5 Hz), 148.8, 145.5, 144.7, 139.0, 137.1 (d, J = 2.4 Hz), 134.0, 133.6, 132.9, 132.0, 130.5, 129.3, 129.0, 128.5, 127.9, 127.3, 126.3 (d, J = 13.4 Hz), 118.3, 116.4 (d, J = 21.3 Hz), 114.1, 112.7, 70.5, 64.0, 43.1, 39.5, 35.9, 35.5. ESI-HRMS [M − H]+ calculated: 554.2086. Found: 554.2074. 8-((3-Benzamido-4-fluorophenyl)amino)-N-methyl-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (8f). Compound 8f was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.08 mmol, 64.0%) of the title compound 8f. Melting point, 108.0 °C. HPLC, 6.24 min. IR (ATR) [cm−1] 3292, 2917, 1675, 1560, 1517, 1448, 1354, 1213, 785, 609. 1H NMR (400 MHz, DMSO-d6) δ 1H NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 8.89 (s, 1H), 8.57−8.52 (m, 1H), 8.35 (s, 1H), 8.04−7.90 (m, 4H), 7.65−7.58 (m, 1H), 7.57−7.49 (m, 3H), 7.41 (d, J = 7.8 Hz, 1H), 7.32−7.22 (m, 1H), 7.13−7.05 (m, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.87 (s, 1H), 4.77−4.68 (m, 1H), 3.50−3.46 (m, 2H), 3.44−3.38 (m, 2H), 3.16−3.05 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 190.4, 165.7, 165.5, 150.1 (d, J = 252.4 Hz), 148.7, 145.4, 144.5, 138.9, 137.0, 134.0, 133.5, 132.9, 131.8, 130.4, 129.2, 128.8, 128.4, 127.8, 127.2, 126.2 (d, J = 13.0 Hz), 121.2, 118.1 (d, J = 10.1 Hz), 116.3 (d, J = 21.3 Hz), 114.0, 112.6, 59.7, 42.2, 35.5, 33.8. ESI-HRMS [M − H]+ calculated: 524.1980. Found: 524.1986. 8-((5-Benzamido-2,4-difluorophenyl)amino)-N-(2-morpholinoethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (8g). Compound 8g was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.1 g (0.08 mmol, 65.4%) of the title compound 8g. Melting point, 121.5 °C. HPLC, 5.26 min. IR (ATR) [cm−1] 1643, 1602, 1531, 1437, 1354, 1264, 1114, 860, 709, 626, 531, 455. 1H NMR (400 MHz, DMSO-d6) δ 1H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 8.69 (s, 1H), 8.53 (t, J = 5.6 Hz, 1H), 8.37−8.28 (m, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.99−7.93 (m, 2H), 7.94−7.87 (m, 1H), 7.67−7.58 (m, 2H), 7.57−7.47 (m, 3H), 7.41 (d, J = 8.0 Hz, 1H), 6.85−6.81 (m, 1H), 6.71 (s, 1H), 3.59−3.54 (m, 4H), 3.44−3.38 (m, 2H), 3.16−3.04 (m, 4H), 2.48−2.43 (m, 2H), 2.43−2.37 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 13C NMR (101 MHz, DMSO) δ 190.5, 190.5, 165.5, 165.4, 165.4, 149.0, 145.2, 144.5, 138.9, 133.7, 133.7, 133.3, 132.8, 131.9, 131.9, 130.4, 129.0, 129.0, 128.9, 128.4, 128.4, 127.7, 127.7, 127.3, 122.2, 113.9, 112.4, 66.2, 57.3, 53.2, 36.5, 35.4, 33.7. ESI-HRMS [M − H]+ calculated 611.1650 found 611.1653 [M + H]+. 8-((5-Benzamido-2,4-difluorophenyl)amino)-N-(2,3-dihydroxypropyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (8h). Compound 8h was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.05 mmol, 88.0%) of the title compound 8h. Melting point, 204.5 °C. HPLC, 6.91 min. IR (ATR) [cm−1] 3322, 1685, 1641, 1544, 1517, 1363, 1283, 1217, 1116, 923, 863, 784, 686, 515. 1H NMR (400 MHz, DMSO-d6) δ 1H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 8.69 (s, 1H), 8.51 (s, 1H), 8.34 (s, 1H), 8.06−7.93 (m, 3H), 7.67−7.45 (m, 5H), 7.45−7.37 (m, 1H), 6.88−6.78 (m, 1H), 6.71 (s, 1H), 4.92−4.72 (m, 1H), 4.76−4.46 (m, 1H), 3.69−3.58 (m, 2H), 3.30−3.17 (m, 2H), 3.16−2.98 (m, 4H). ESI-HRMS [M + H]+ calculated: 572.1992. Found: 572.1957. 8-((5-Benzamido-2,4-difluorophenyl)amino)-N-(2-hydroxyethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (8i). Compound 8i was prepared according to general procedure Va. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.74 mmol, 93.0%) of the title compound 8i. Melting point, 188,8 °C. HPLC, 6.82 min. IR (ATR) [cm−1] 3288, 1635, 1577, 1521, 1400, 1355, 1266, 8048



Article

1116, 857, 708. 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.68 (s, 2H), 8.54 (t, J = 5.5 Hz, 2H), 8.40−8.24 (m, 2H), 8.03−7.99 (m, 2H), 7.99−7.95 (m, 4H), 7.94−7.91 (m, 2H), 7.64−7.58 (m, 4H), 7.56−7.45 (m, 7H), 7.43−7.39 (m, 2H), 6.83 (d, J = 8.8 Hz, 2H), 6.70 (s, 2H), 4.73 (t, J = 5.6 Hz, 3H), 3.50 (s, 4H), 3.14−3.06 (m, 8H). ESI-HRMS [M + H]+ calculated: 542,1885. Found: 542,1885. 8-((5-Benzamido-2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (8j). Compound 8j was prepared according to general procedure V. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.02 g (0.04 mmol, 50.1%) of the title compound 8j. Melting point, 106.0. HPLC, 6.91 min. IR (ATR) [cm−1] 3288, 2940, 1772, 1696, 1602, 1560, 1508, 1436, 1263, 1263, 1113, 1028, 955, 858, 761, 689. 1H NMR (400 MHz, DMSO-d6) δ 8.67−8.57 (m, 11H), 8.38 (s, 7H), 8.25−8.15 (m, 12H), 8.06−7.99 (m, 25H), 7.92−7.83 (m, 26H), 7.67−7.59 (m, 16H), 7.58−7.50 (m, 33H), 7.42−7.34 (m, 22H), 7.13−6.95 (m, 24H), 6.49 (s, 6H), 3.25 (dd, J = 23.0, 7.4 Hz, 40H). 13C NMR (100 MHz, DMSO-d6) δ 190.5, 167.2, 165.5, 148.9, 145.1, 144.6, 138.8, 133.7, 133.2, 132.6, 131.8, 130.6, 129.5, 128.8, 128.4, 128.1, 127.7, 127.3, 124.2 (dd, J = 12.8, 3.7 Hz), 122.2, 113.8, 112.4, 105.1 (t, J = 25.7 Hz), 35.3, 33.7. ESI-HRMS [M + H]+ calculated: 498.1624. Found: 498.1583. 8-((5-Benzamido-2,4-difluorophenyl)amino)-N-(2,3-dihydroxypropyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (8k). Compound 8k was prepared according to general procedure V. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.08 mmol, 41.2%) of the title compound 8k. Melting point, 230.0 °C. HPLC, 7.29 min. IR (ATR) [cm−1] 3294, 3048, 1737, 1629, 1532, 1434, 1267, 1117, 1029, 900, 827, 709, 650, 631. 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.66 (s, 1H), 8.62−8.50 (m, 1H), 8.30−8.23 (m, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 7.3 Hz, 2H), 7.90−7.82 (m, 1H), 7.59−7.47 (m, 4H), 7.47−7.37 (m, 2H), 6.83−6.75 (m, 1H), 6.69 (s, 1H), 3.12−2.99 (m, 4H), 2.77 (d, J = 4.5 Hz, 3H). ESI-HRMS [M + H]+ calculated: 512.1780. Found: 512.1788. Methyl 3-((3-Benzamido-4-fluorophenyl)amino)-11-oxo6,11-dihydrodibenzo[b,e]oxepine-9-carboxylate (8l). Compound 8l was prepared according to general procedure Va to afford 0.23 g (0.46 mmol, 72.9%) of the title compound 8l. HPLC, 7.51 min. 1 H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.87 (s, 1H), 9.10 (s, 1H), 8.36 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.04 (t, J = 12.8 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.57−7.46 (m, 5H), 7.29 (t, J = 9.2 Hz, 1H), 7.13−7.00 (m, 1H), 6.94 (dd, J = 22.1, 12.9 Hz, 1H), 6.81 (d, J = 7.0 Hz, 2H), 6.57 (s, 1H), 6.40 (dd, J = 17.7, 13.7 Hz, 1H), 5.26 (s, 2H), 3.84 (d, J = 30.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 185.89, 165.6, 165.5, 165.3, 163.0, 152.5, 151.5, 150.1, 149.04, 146.7, 144.9, 140.5, 140.0, 136.4, 134.1, 133.8, 133.6, 132.5, 131.9, 131.6, 130.2, 129.8, 128.7, 128.4, 128.4, 127.7, 127.6, 126.3, 126.2, 125.5, 125.4, 118.9, 118.8, 118.7, 116.4, 116.3, 115.5, 115.3, 111.8, 111.5, 111.5, 110.4, 101.7, 72.4, 66.3. ESI-HRMS [M + H]+ calculated: 497.1507. Found: 497.1510. 3-((3-Benzamido-4-fluorophenyl)amino)-N-(2-morpholinoethyl)-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxamide (8m). Compound 8m was prepared according to general procedure VI. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.07 mmol, 40.6%) of the title compound 8m. HPLC, 5.27 min. 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 9.07 (s, 1H), 8.63 (t, J = 5.3 Hz, 1H), 8.27 (s, 1H), 8.04 (t, J = 7.8 Hz, 2H), 7.97 (d, J = 7.7 Hz, 2H), 7.61 (t, J = 7.2 Hz, 2H), 7.53 (t, J = 7.3 Hz, 3H), 7.29 (t, J = 9.5 Hz, 1H), 7.12−7.00 (m, 1H), 6.80 (d, J = 9.0 Hz, 1H), 6.56 (s, 1H), 5.25 (s, 2H), 3.64−3.50 (m, 6H), 2.46 (d, J = 6.7 Hz, 2H), 2.41 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.6, 186.6, 165.5, 165.5, 165.2, 162.9, 162.9, 151.3, 150.1, 140.0, 138.9, 135.1, 133.9, 133.4, 131.9, 130.8, 128.4, 128.2, 127.8, 127.7, 126.4, 118.6, 116.5, 110.2, 101.7, 72.4, 66.2, 57.2, 53.2, 36.6. ESI-HRMS [M − H]+ calculated: 595.2351. Found: 595.2352. 3-((3-Benzamido-4-fluorophenyl)amino)-11-oxo-6,11dihydrodibenzo[b,e]oxepine-9-carboxamide (8n). Compound 8n was prepared according to general procedure VI. The compound

was purified by flash chromatography (silica gel, gradient DCM/ methanol 95:05) to afford 0.02 g (0.04 mmol, 40.6%) of the title compound 8n. HPLC, 6.02 min. 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 9.07 (s, 1H), 8.63 (t, J = 5.3 Hz, 1H), 8.27 (s, 1H), 8.04 (t, J = 7.8 Hz, 2H), 7.97 (d, J = 7.7 Hz, 2H), 7.61 (t, J = 7.2 Hz, 2H), 7.53 (t, J = 7.3 Hz, 3H), 7.29 (t, J = 9.5 Hz, 1H), 7.12−7.00 (m, 1H), 6.80 (d, J = 9.0 Hz, 1H), 6.56 (s, 1H), 5.25 (s, 2H), 3.64−3.50 (m, 6H), 2.46 (d, J = 6.7 Hz, 2H), 2.41 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 186.6, 186.6, 165.5, 165.5, 165.2, 162.9, 162.9, 151.3, 150.1, 140.0, 138.4, 135.1, 133.9, 133.4, 131.9, 130.8, 128.4, 128.2, 127.8, 127.7, 126.4, 118.6, 116.5, 110.2, 101.7, 72.4, 66.2, 57.2, 53.2, 36.6. ESI-HRMS [M − H]+ calculated: 482.1511. Found: 482.1510. 8-((5-(4-Chlorobenzamido)-2,4-difluorophenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (9a). Compound 9a was prepared according to general procedure Vb. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.06 g (0.11 mmol, 62.9%) of the title compound 9a. Melting point, 257.0 °C. HPLC, 7.32 min. IR (ATR) [cm−1] 3406, 3288, 3043, 1650, 1557, 1486, 1358, 1262, 1178, 1116, 1030, 900, 826, 784, 648, 566, 450. 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.67 (s, 1H), 8.53 (s, 1H), 8.33 (s, 1H), 8.05−7.88 (m, 5H), 7.67−7.58 (m, 2H), 7.57−7.47 (m, 4H), 7.43−7.38 (m, 1H), 6.87−6.80 (m, 1H), 6.71 (s, 1H), 3.17− 2.97 (m, 5H), 2.79 (d, J = 4.3 Hz, 3H). 13C NMR (100 MHz, DMSOd6) δ 201.0, 190.5, 165.9, 165.5, 152.5 (dd, J = 246.9, 11.1 Hz), 151.7 (dd, J = 246.7, 11.1 Hz), 149.0, 145.1, 144.4, 138.8, 133.7, 133.3, 132.8, 131.8, 130.2, 128.9 (d, J = 11.1 Hz), 128.4, 127.7, 127.4, 124.3 (dd, J = 12.8, 3.5 Hz), 122.3 (dd, J = 13.8, 2.7 Hz), 122.2, 113.8, 112.4, 105.1 (t, J = 25.6 Hz), 35.4, 33.7, 26.2. ESI-HRMS [M-Cl]− calculated: 510.1635. Found: 510.1642 8-((5-(3-Chlorobenzamido)-2,4-difluorophenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (9b). Compound 9b was prepared according to general procedure Vb. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.08 g (0.14 mmol, 85.0%) of the title compound 9b. Melting point, 234,0 °C. HPLC, 7.34 min. IR (ATR) [cm−1] 3408, 3311, 3051, 1669, 1563, 1435, 1360, 1218, 1106, 1030, 931, 826, 719, 679, 625, 503, 462. 1 H NMR (400 MHz, acetone-d6) δ 8.35 (s, 1H), 8.15−8.02 (m, 2H), 8.02−7.89 (m, 3H), 7.82 (s, 1H), 7.62−7.42 (m, 3H), 7.37−7.32 (m, 1H), 7.27−7.17 (m, 1H), 6.98−6.89 (m, 1H), 6.87 (s, 1H), 4.05−3.92 (m, 1H), 3.21−3.04 (m, 4H), 2.87 (d, J = 9.9 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.5, 179.3, 165.9, 165.5, 164.2, 149.0, 145.2, 144.4, 138.8, 135.7, 133.7, 133.3, 133.2, 132.8, 131.9, 131.7, 130.4, 130.2, 129.0, 128.9, 128.4, 127.7, 127.5, 127.4, 126.5, 122.2, 113.8, 112.4, 105.2, 35.3, 33.7, 26.2. ESI-HRMS [M + Na]+ calculated: 568.1210. Found: 568.1215 8-((2,4-Difluoro-5-(4-fluorobenzamido)phenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (10a). Compound 10a was prepared according to general procedure Vb. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.08 mmol, 39.8%) of the title compound 10a. Melting point, 100.3 °C. HPLC, 7.53 min. IR (ATR) [cm−1] 3040, 1732, 1626, 1601, 1525, 1434, 1360, 1266, 1163, 1086, 904, 851, 784, 627, 470. 1H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 8.71 (s, 1H), 8.65−8.45 (m, 1H), 8.13−7.89 (m, 4H), 7.73−7.00 (m, 9H), 6.96−6.82 (m, 1H), 6.74 (s, 1H), 3.38 (s, 4H), 3.26−3.00 (m, 5H). 13C NMR (100 MHz, DMSO-d6) δ 191.0, 166.4, 165.0, 150.0, 145.7, 145.0, 139.3, 133.8, 133.3, 131.1, 131.0, 130.7, 130.4, 129.5, 129.4, 127.9, 122.7, 116.0, 115.8, 114.4, 112.9, 105.7, 35.8, 34.3, 26.7. ESI-HRMS [M + Na]+ calculated: 552.1506. Found: 552.1513 8-((2,4-Difluoro-5-(3-fluorobenzamido)phenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (10b). Compound 10b was prepared according to general procedure Vb. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.04 g (0.08 mmol, 45.3%) of the title compound 10b. Melting point, 244,1 °C. HPLC, 7.62 min. IR (ATR) [cm−1] 3405, 3313, 3044, 1625, 1557, 1519, 1485, 1406, 1358, 1217, 1178, 968, 804, 719, 679, 568, 456. 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.68 (s, 1H), 8049



Article

8.63−8.50 (m, 1H), 8.32 (s, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 6.1 Hz, 1H), 7.85−7.72 (m, 2H), 7.65−7.55 (m, 2H), 7.55−7.45 (m, 2H), 7.41 (d, J = 7.9 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 6.71 (s, 1H), 3.16−3.00 (m, 4H), 2.78 (d, J = 4.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.5, 166.0, 164.3, 163.1, 160.7, 148.9, 145.2, 144.5, 138.9, 136.1 (d, J = 7.1 Hz), 133.3, 132.8, 130.7 (d, J = 8.0 Hz), 130.7, 129.0 (d, J = 8.9 Hz), 127.4, 124.4 (dd, J = 14.4, 4.1 Hz), 124.0 (d, J = 2.7 Hz), 122.3, 122.0 (dd, J = 12.8, 3.4 Hz), 118.9, 118.7, 114.6 (d, J = 23.0 Hz), 113.9, 112.5, 105.2, 35.3, 33.8, 26.3. ESI-HRMS [M + Na]+ calculated: 552.1506. Found: 552.1506 8-((2,4-Difluoro-5-(4-methoxybenzamido)phenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (11a). Compound 11a was prepared according to general procedure Vb. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.06 g (0.11 mmol, 62.9%) of the title compound 11a. Melting point, 269,2 °C. HPLC, 7.40 min. IR (ATR) [cm−1] 3242, 3041, 1626, 1526, 1431, 1359, 1255, 1118, 1029, 903, 826, 768, 607, 502. 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.67 (s, 1H), 8.59−8.51 (m, 1H), 8.39−8.28 (m, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.93−7.86 (m, 1H), 7.60 (t, J = 8.3 Hz, 1H), 7.48 (t, J = 10.4 Hz, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.6 Hz, 1H), 6.70 (s, 1H), 3.83 (s, 3H), 3.13−3.04 (m, 4H), 2.78 (d, J = 4.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 206.9, 190.5, 165.9, 164.9, 162.1, 152.5 (dd, J = 246.5, 2.6 Hz), 151.8 (dd, J = 247.3, 2.2 Hz), 149.0, 145.2, 144.4, 138.8, 133.3, 132.8, 130.2, 129.7, 129.0, 128.9, 127.3, 125.8, 124.2 (dd, J = 12.5, 2.8 Hz), 122.5 (dd, J = 12.8, 3.2 Hz), 122.3, 113.7 (d, J = 19.4 Hz), 112.4, 105.1 (t, J = 25.0 Hz), 55.4, 35.3, 33.8, 26.2. ESI-HRMS [M + Na]+ calculated: 564.1705. Found: 564.1709 8-((2,4-Difluoro-5-(3-methoxybenzamido)phenyl)amino)-Nmethyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3carboxamide (11b). Compound 11b was prepared according to general procedure Vb. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.06 g (0.12 mmol, 68.9%) of the title compound 11b. Melting point, 120.4 °C. HPLC, 7.40 min. IR (ATR) [cm−1] 3405, 3313, 3044, 1625, 1557, 1519, 1485, 1406, 1358, 1217, 1178, 968, 804, 719, 679, 568, 456. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.64−8.50 (m, 1H), 8.36 (s, 1H), 8.13−7.89 (m, 2H), 7.71−7.38 (m, 6H), 7.29−7.16 (m, 1H), 6.93−6.81 (m, 1H), 6.74 (s, 1H), 3.86 (s, 3H), 3.19 (s, 1H), 3.19−3.01 (m, 4H), 2.01 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.90, 172.38, 170.71, 166.30, 165.62, 159.60, 149.38, 145.63, 144.88, 139.26, 135.50, 133.73, 133.14, 130.67, 130.02, 129.45, 129.33, 127.73, 122.79, 122.71, 122.51, 122.44, 120.40, 118.16, 114.27, 113.28, 112.81, 55.73, 35.77, 34.15, 21.12. ESI-HRMS [M + Na]+ calculated: 564.1705. Found: 564.1707 8-((2,4-Difluoro-5-(4-(trifluoromethyl)benzamido)phenyl)amino)-N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (12). Compound 12 was prepared according to general procedure Vb. The compound was purified by flash chromatography (silica gel, gradient DCM/methanol 95:05) to afford 0.03 g (0.05 mmol, 86.2%) of the title compound 12. Melting point, 257,1 °C. HPLC, 8.43 min. IR (ATR) [cm−1] 3043, 1656, 1576, 1531, 1360, 1265, 1165, 1123, 902, 878, 813, 759, 655, 492, 453. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.70 (s, 1H), 8.57−8.53 (m, 1H), 8.32 (d, J = 9.5 Hz, 2H), 8.27 (d, J = 7.8 Hz, 1H), 8.04−7.95 (m, 2H), 7.91 (d, J = 8.7 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.66 (t, J = 8.3 Hz, 1H), 7.53 (t, J = 10.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 6.85 (d, J = 9.5 Hz, 1H), 6.72 (s, 1H), 3.18−3.03 (m, 4H), 2.78 (d, J = 4.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.5, 165.9, 164.2, 152.7 (dd, J = 246.9, 11.2 Hz), 151.6 (dd, J = 245.6, 9.9 Hz), 148.9, 145.1, 144.4, 138.8, 134.7, 133.3, 132.8, 131.9, 130.2, 129.6 (d, J = 38.9 Hz), 128.95 (d, J = 12.1 Hz), 128.4 (d, J = 3.2 Hz), 127.4, 124.5, 124.5, 124.4 (dd, J = 8.7, 3.5 Hz), 122.2, 121.9 (dd, J = 13.1, 3.5 Hz), 113.9, 112.5, 105.2 (t, J = 25.0 Hz), 35.3, 33.8, 26.2. ESI-HRMS [M + Na]+ calculated: 602.1474. Found: 602.1476 4-Methylisophthalic Acid48 (15). 3-Bromo-4-methylbenzoic acid 14 (10.0 g, 46.50 mmol) was suspended in 50 mL of THFdry in a three-neck flask under argon atmosphere. At 0 °C, a 3.0 M solution of

methylmagnesium bromide (17.70 mL, 51.15 mmol) in diethyl ether was added dropwise and was stirred for 2 h. The resulting solution was cooled to −65 °C and then n-butyllithium in a 1.6 M hexane solution (35.05 mL, 93.00 mmol) was added carefully, followed by stirring. The temperature of the reaction solution was elevated to −40 °C, and an amount of approximately 25.0 g of dry ice was added. The flask was sealed and stirred overnight. When the reaction was completed, 20% sodium hydroxide solution was added and the reaction mixture was transferred to a separating funnel. The aqueous phase was washed three times with ethyl acetate. Afterward the solution was ice cooled and acidified with concentrated hydrochloric acid whereupon the product precipitated. The precipitate was filtered and collected, and 4-methylisophthalic acid 15 was obtained as a white solid in 87.4% yield (7.32 g, 40.66 mmol). 1H NMR (200 MHz, DMSO-d6) δ 8.36 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 2.11−2.03 (m, 3H). 13 C NMR (50 MHz, DMSO-d6) δ 167.9, 166.6, 144.3, 132.2, 132.1, 131.2, 130.6, 128.5, 21.2. ESI-MS m/z = 179.0 [M − H]−. Dimethyl 4-Methylisophthalate (16). 3-Bromo-4-methylbenzoic acid 15 and catalytic amounts of H2SO4conc were dissolved in methanol and stirred under reflux. The implementation was controlled with TLC. When the reaction was completed, the mixture was allowed to cool to room temperature, diluted with water, and extracted with ethyl acetate. The organic phase was evaporated under reduced pressure and the residue was purified by column chromatography (silica gel, gradient petroleum ether/ethyl acetate, 95:5 in 60 min to 90:10) affording the desired compound (1.63 g, 7.83 mmol, 94%). HPLC, 6.41 min. IR (ATR) [cm−1] 3051, 2984, 2934, 2843, 1715, 1444, 1315, 1065, 995, 845, 757. 1H NMR (200 MHz, DMSO-d6) δ 8.35 (d, J = 1.9 Hz, 1H), 8.00 (dd, J = 8.0, 1.9 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 3.86 (d, J = 1.1 Hz, 6H), 2.57 (s, 3H). 13C NMR (50 MHz, DMSO-d6) δ 166.3, 165.4, 144.8, 132.3 (s, J = 7.0 Hz), 132.3, 130.7, 129.7, 127.5, 52.2 (d, J = 8.1 Hz), 21.1. ESI-MS m/z = 209.1 [M + H]+. 3-Chlorobenzyliodide (17). 3-Chlorobenzyl chloride (4.1 g, 25 mmol) was dissolved in 18 mL of acetone, and 6.50 g of sodium iodide was added. The reaction mixture was refluxed for 2 h under argon atmosphere. When the reaction was completed, the mixture was allowed to cool to room temperature, diluted with water, and extracted with ethyl acetate. The extracts were combined and the solvent was removed under reduced pressure to give a brown viscous liquid. This compound was further used directly without any purification in the next step, and the purity was below 95% (6.15 g of crude product, 96%). GC−MS 7.11 min; 254/252 [M + H]+. Dimethyl 4-(Bromomethyl)isophthalate (18). Dimethyl 4-methylisophthalate 16 (2.5 g, 12.01 mmol) was dissolved in a singleneck flask in a few milliliters of CCl4 and N-bromosuccinimide (2.35 g, 13.21 mmol), and catalytic amounts of azodiisobutyrodinitrile (AIBN) were added and heated to 70 °C. The reaction mixture was refluxed for 6 h, and the accruing succinimide precipitates on the surface. Afterward the succinimide was separated by filtration and the organic layer was evaporated in vacuo to afford the reaction mixture as a yellow oil. The reaction mixture was further purified by flash chromatography (SiO2, petroleum ether/ethyl acetate 5:1) to obtain dimethyl 4-(bromomethyl)isophthalate as a yellow oil, which crystallizes by cooling (3.11 g, 90.2%). Melting point, 74.1 °C. HPLC, 6.86 min. IR (ATR) [cm−1] 3080, 3007, 2945, 2847, 1439, 1403, 1287, 1128, 1070, 799, 603. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 5.06 (s, 2H), 3.90 (d, J = 8.1 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 165.6, 165.0, 143.6, 132.9, 132.5, 131.2, 129.8, 129.4, 52.5 (d, J = 10.1 Hz), 30.8. GC−MS 10.43 min; m/z 287.1. FAB m/z = 289.0 [M + H]+. Dimethyl 4-(3-Chlorophenethyl)isophthalate (20). n-BuLi (18.0 mL, 45.0 mmol) in a 2.5 M hexane solution was added dropwise in 15 min to diisopropylamine (6.35 mL, 45.2 mmol), dissolved in THF, at a temperature of 0 °C. The reaction mixture was stirred for 15 min. The solution was cooled to −78 °C, and dimethyl 4-methylisophthalate 16 (5.0 g, 24.0 mmol), dissolved in THF, was added dropwise. The solution was stirred for another 1 h. Subsequently the 3-chlorobenzyliodide 17 was added dropwise within 30 min. The reaction mixture was allowed to warm to room temperature, diluted with saturated ammonium chloride solution, and extracted with ethyl 8050



Article

for further 20 min. The solution was then poured onto ice and extracted with DCM. The combined organic layers were washed with water. The solvent was dried over Na2SO4 and evaporated under reduced pressure to yield the product 24 (3.0 g, 10.46 mmol, 98%). HPLC, 8.31 min. 1H NMR (400 MHz, DMSO-d6) δ 13.66 (s, 1H), 8.44 (s, 2H), 8.06−7.99 (m, 2H), 7.93−7.86 (m, 2H), 7.50−7.38 (m, 6H), 3.27−3.12 (m, 8H). 13C NMR (100 MHz, DMSO-d6) δ 192.4, 166.5, 146.8, 144.3, 137.56, 137.4, 136.3, 132.9, 132.33, 131.34, 130.3, 129.3, 129.2, 126.8, 33.9, 33.4. EI-MS m/z = 285.0 [M − H]−. 3-Chloro-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxylic Acid (25). 4-((3-Chlorophenoxy)methyl)isophthalic acid 23 (0.4 g, 1.3 mmol) was dissolved in 25 mL of dry DCM and 2.5 mL of dry DMF and was heated to reflux. Thionyl chloride (0.24 mL, 7.17 mmol) was added dropwise and stirred for 1 h. The reaction mixture was allowed to cool to approximately 40 °C, and aluminum chloride (0.97 g, 7.17 mmol) was added in small portions and was stirred for 1 h. After the reaction showed completeness, the mixture was poured on ice and was stirred for another hour. The reaction mixture was transferred to a separation funnel, and the organic layer was washed sequentially with a 10% solution of hydrochloric acid, dried over Na2SO4, and evaporated to dryness. The resulting solid of 25 could be suspended in cold DCM and be filtered to achieve higher purity (0.21 g, 55.8%). Melting point, 245.6 °C. HPLC, 8.66 min. IR (ATR) [cm−1] 2984, 2814, 2533, 2361, 1681, 1596, 1486, 1414, 1251, 1040, 869, 829, 753, 599, 678. 1H NMR (400 MHz, DMSO-d6) δ 13.71−12.82 (s, 1H), 8.31 (s, 1H), 8.13 (d, J = 9.1 Hz, 2H), 7.72 (d, J = 7.8 Hz, 1H), 7.36−7.15 (m, 2H), 5.42 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 167.23, 166.3, 159.1, 142.6, 133.78, 132.6, 131.28, 131.0, 130.2, 129.6, 128.1, 121.0, 114.9, 113.7, 67.8. FAB m/z = 288.8 [M − H]−. Methyl 8-Chlor-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxylate (26). 8-Chloro-5-oxo-10,11-dihydro-5Hdibenzo[a,d][7]annulene-3-carboxylic acid 24 (0.6 g, 2.09 mmol) was dissolved in 20 mL of methanol. Catalytic amounts of concentrated HCl were added and refluxed while stirring. When the reaction was completed, the mixture was allowed to cool to room temperature, diluted with 20% NaOH solution, and extracted with ethyl acetate. The extracts were combined and dried over Na2SO4. The solvent was removed under reduced pressure to give the desired compound 26 (0.62 g, 2.06 mmol) in 99% yield. HPLC, 8.61. 1H NMR (200 MHz, d6-DMSO) δ 8.44 (s, 1H), 8.08−7.98 (m, 1H), 7.94−7.82 (m, 1H), 7.53−7.37 (m, 3H), 3.86 (s, 3H), 3.29−3.11 (m, 4H). 13C NMR (50 MHz, DMSO-d6) δ 192.2, 165.5, 147.3, 144.3, 137.6, 137.5, 136.1, 132.7, 132.3, 131.2, 130.5, 129.2, 128.1, 126.8, 52.3, 33.8, 33.2. ESI-MS m/z = 301.3 [M + H]+. Methyl 3-Chloro-11-oxo-6,11-dihydrodibenzo[b,e]oxepine9-carboxylate (27). 3-Chloro-11-oxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxylic acid 25 (2.0 g, 6.93 mmol) was dissolved in a single-neck flask in 75 mL of methanol while 1 mL of conc sulfuric acid was added. The solution was refluxed for 6 h until the reaction showed completeness. After cooling to room temperature, the excess methanol was evaporated in vacuo. The residue was redissolved in ethyl acetate and washed three times with a 20% sodium hydroxide solution. The organic layer was dried over Na2SO4, and the solvent was then removed in vacuo to obtain methyl 3-chloro-11-oxo-6,11dihydrodibenzo[b,e]oxepine-9-carboxylate (27) as a white powder. (2.01 g, 6.64 mmol, 95.8%). Melting point, 271.4 °C. HPLC, 8.62 min. IR (ATR) [cm−1] 2982, 2962, 2925, 1639, 1594, 1567, 1376, 1263, 1196, 1017, 761, 644. 1H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.18−8.08 (m, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 7.1 Hz, 2H), 5.44 (s, 3H), 3.89 (s, 3H). FAB m/z = 303.1 [M + H]+. 8-Chloro-N-methyl-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (28). 8-Chloro-5-oxo-10,11-dihydro5H-dibenzo[a,d][7]annulene-3-carboxylic acid 24 (1.0 g, 3.49 mmol) was dissolved in DCM and catalytic amounts of DMF. Oxalyl chloride (0.66 g, 5.19 mmol) was added and stirred at ambient temperature for 1 h under argon atmosphere. Afterward, a solution of methylamine and trietylamine in DCM is prepared. The activated carboxylic acid was added dropwise, and the reaction mixture was stirred at ambient temperature under argon atmosphere overnight. Then the solution

acetate. The extracts were combined and dried over Na2SO4. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (silica gel, gradient petroleum ether/ethyl acetate, 95:5 to 90:10 in 1 h) to afford the desired compound 20 (3.92 g, 11.78 mmol, 50%). HPLC, 9.23 min. 1H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.32−7.22 (m, 3H), 7.15 (d, J = 7.2 Hz, 1H), 3.86 (s, 6H), 3.25−3.17 (m, 2H), 2.87−2.77 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 166.3, 165.3, 147.8, 143.64, 132.9, 132.2, 131.8, 130.9, 130.0, 129.5, 128.1, 127.7, 127.0, 125.9, 52.3, 52.2, 36.4, 35.3. ESI-MS m/z = 331.1 [M − H]−. Dimethyl 4-((3-Chlorophenoxy)methyl)isophthalate (21). A suspension of potassium carbonate (1.64 g, 11.88 mmol) and 3-chlorophenol (1.53 g, 11.88 mmol) in 50 mL of acetone in a singleneck flask was heated to 50 °C. A solution of dimethyl 4-(bromomethyl)isophthalate 18 (3.1 g, 10.80 mmol) in acetone was added, and the reaction mixture was refluxed for 6 h. When the reaction was finished, the solvent was removed in vacuo and the residue was redissolved in ethyl acetate. The organic layer was washed with a 20% sodium hydroxide solution, dried over Na2SO4, and evaporated in vacuo. The remaining oil was purified with flash chromatography (SiO2, petroleum ether/ethyl acetate 4:1) to obtain dimethyl 4-((3-chlorophenoxy)methyl)isophthalate 21 as a white solid (2.87 g, 79.4%). Melting point, 111.6 °C. HPLC, 9.44 min. IR (ATR) [cm−1] 3072, 2994, 2949, 2839, 1715, 1590, 1433, 1300, 1231, 1085, 879, 751, 677. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.18 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.14−6.91 (m, 3H), 5.52 (s, 2H), 3.88 (d, J = 4.4 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 166.2, 165.5, 159.3, 143.5, 134.1, 133.2, 131.3 (d, J = 10.4 Hz), 129.4, 128.9 (d, J = 17.0 Hz), 121.4, 115.2, 114.1, 67.9, 52.8. FAB m/z = 337.1 [M + H]+. 4-(3-Chlorophenethyl)isophthalic Acid (22). Dimethyl 4-(3chlorophenethyl)isophthalate 20 (0.7 g, 2.1 mmol) was dissolved in 20 mL of methanol and a little portion of H2O. KOH (0.24 g, 4.2 mmol) was added and refluxed for 6 h while stirring. When the reaction was completed, the mixture was allowed to cool to room temperature, diluted with 1 N HCl solution (pH value 1), and extracted with ethyl acetate. The extracts were combined, washed with Na2CO3 solution, and dried over Na2SO4. The solvent was removed under reduced pressure to give a white solid (0.59 g, 1.94 mmol, 92%). HPLC, 7.52 min. 1 H NMR (400 MHz, DMSO-d6) δ 13.17 (s, 2H), 8.41 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.34−7.15 (m, 4H), 3.30−3.22 (m, 2H), 2.90−2.81 (m, 2H). 13C NMR (100 MHz, DMSOd6) δ 167.9, 166.5, 147.4, 144.0, 132.9, 132.1, 131.4, 131.4, 130.5, 130.0, 128.9, 128.2, 127.0, 125.9, 36.5, 35.6. EI-MS m/z = 303.4 [M − H]−. 4-((3-Chlorophenoxy)methyl)isophthalic Acid (23). For saponification, dimethyl 4-((3-chlorophenoxy)methyl)isophthalate 21 (2.0 g, 5.97 mmol) was dissolved in sufficient methanol and heated to 50 °C. After complete solvation, potassium hydroxide (0.84 g, 14.94 mmol) was added and refluxed for 4 h. When the reaction was completed, the solvent was evaporated in vacuo, and the residue was suspended in water. The aqueous layer got ice cooled and acidified with a 10% solution of hydrochloric acid whereupon the product precipitated. The white powder of 4-((3-chlorophenoxy)methyl)isophthalic acid 23 was obtained by filtration (1.81 g, 98.8%). Melting point, 243.4 °C. HPLC, 7.95 min. IR (ATR) [cm−1] 2908, 2851, 2639, 2524, 1681, 1596, 1431, 1249, 869, 752, 676, 493. 1H NMR (400 MHz, DMSO-d6) δ 13.25 (s, 2H), 8.47 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.38−7.24 (m, 1H), 7.06 (d, J = 12.5 Hz, 1H), 7.01 (t, J = 7.4 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 5.53 (s, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 167.3, 166.3, 159.1, 142.6, 133.7, 132.6, 131.23, 130.9, 130.2, 129.5, 128.0, 120.9, 114.9, 113.7, 67.8. FAB m/z = 306.9 [M − H]−. 8-Chloro-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene3-carboxylic Acid (24). 4-(3-Chlorophenethyl)isophthalic acid 22 (3.25 g, 10.67 mmol) was suspended in 150 mL of DCM and refluxed while stirring. Then SOCl2 (6.98 g, 58.67 mmol) was added dropwise. The reaction mixture was refluxed until a clear solution is obtained. Otherwise, 1−2 drops of DMF were added after 3 h. The solution was cooled to 0 °C, and AlCl3 (7.83 g, 58.72 mmol) was added and stirred 8051



Article

DMSO-d6) δ 8.76−8.68 (m, 1H), 8.38−8.23 (m, 1H), 7.70−7.35 (m, 7H). 13C NMR (50 MHz, DMSO-d6) δ 171.9, 144.4, 144.3, 133.7, 132.8, 129.0, 128.6, 128.4, 126.6, 126.1, 117.9, 112.4. ESI-MS m/z = 283.3 [M + Na]+. 2,4-Difluoro-5-nitroaniline (34a). Compound 34a was synthesized according to general procedure II. The residue was not further purified (2.41g, 89.4%). HPLC, 5.85 min. 1H NMR (400 MHz, DMSO-d6) δ 7.57−7.32 (m, 2H), 5.65 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 155.9, 149.2, 144.2, 135.0, 111.5, 107.0. FAB (m/z) m/z = 175.2 [M + H]+. 3-Amino-N-cyclopropyl-N,4-dimethylbenzamide (37a). Compound 37a was prepared according to general procedure IIIa to afford 0.9 g (4.36 mmol, 96.9%) of the title compound 37a. HPLC, 4.03 min. 1H NMR (200 MHz, DMSO-d6) δ 6.94 (d, J = 7.4 Hz, 1H), 6.56 (s, 1H), 6.40 (d, J = 7.4 Hz, 1H), 4.97 (s, 2H), 2.74 (s, 3H), 2.06 (s, 3H), 1.08 (d, J = 6.7 Hz, 6H). 13C NMR (50 MHz, DMSO-d6) δ 170.6, 146.5, 135.6, 129.6, 121.9, 113.7, 111.6, 42.1, 19.7, 17.2. EI-MS m/z = 205.3 [M − H]−. N-(5-Amino-2-fluorophenyl)cyclopropanecarboxamide (42a). Compound 42a was prepared according to general procedure IIIb to afford 0.43 g (2.21 mmol, 82.73%) of the title compound 42a. HPLC, 1.91 min. 1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 7.21−7.07 (m, 1H), 6.94−6.78 (m, 1H), 6.32−6.20 (m, 1H), 4.92 (s, 2H), 2.05−1.85 (m, 1H), 0.76 (d, J = 6.8 Hz, 4H). 13C NMR (50 MHz, DMSO-d6) δ 171.7, 147.0−144.4 (m), 144.9 (d, J = 1.2 Hz), 126.1 (d, J = 12.5 Hz), 115.0 (d, J = 20.2 Hz), 109.4 (d, J = 5.4 Hz), 19.9, 7.1. EI-MS m/z = 192.8 [M − H]−. Methyl 8-((5-(Cyclopropanecarboxamido)-2,4difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxylate (46). Compound 46 was prepared according to general procedure Va to afford 0.28 g (0.59 mmol, 96%) of the title compound 46. HPLC, 8.21 min. 1H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.65 (s, 1H), 8.44 (d, J = 1.8 Hz, 1H), 8.03−7.97 (m, 2H), 7.94−7.86 (m, 1H), 7.50−7.40 (m, 2H), 6.81−6.74 (m, 1H), 6.65 (s, 1H), 3.87 (s, 3H), 3.17−3.01 (m, 4H), 2.00−1.93 (m, 1H), 0.80 (s, 2H), 0.78 (s, 2H). ESI-MS = 475.1 [M − H]−. Methyl 8-((3-(Cyclopropylcarboxamido)-4-fluorphenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3carboxylate (47). Compound 47 was prepared according to general procedure Va to afford 0.33 g (0.72 mmol, 72.2%) of the title compound 47. HPLC, 7.59 min. 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.85 (s, 1H), 8.45 (d, J = 2.0 Hz, 1H), 8.07−7.95 (m, 2H), 7.92−7.80 (m, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.32−7.15 (m, 1H), 7.04−6.90 (m, 2H), 6.87−6.76 (m, 1H), 5.75 (s, 1H), 3.87 (s, 3H), 3.24−3.00 (m, 4H), 2.15−2.01 (m, 1H), 0.92−0.72 (m, 4H). ESI-MS = 457.3 [M − H]−. Methyl 8-((5-Benzamido-2,4-difluorophenyl)amino)-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxylate (48). Compound 48 was prepared according to general procedure Va to afford 0.13 g (0.25 mmol, 42.0%) of the title compound 48. HPLC, min. 1H NMR (400 MHz, DMSO-d6) δ 8.62−8.54 (m, 1H), 8.45 (t, J = 8.4 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 8.02−7.93 (m, 2H), 7.84−7.77 (m, 2H), 7.56−7.39 (m, 3H), 7.30−7.08 (m, 2H), 6.96−6.88 (m, 1H), 6.87−6.79 (m, 1H), 6.80−6.72 (m, 1H), 6.00 (s, 1H), 3.84 (s, 3H), 3.15−3.03 (m, 4H). ESI-MS = 510.8 [M − H]−.

was poured into water, extracted several times with DCM, dried over Na2SO4, and evaporated under reduced pressure. The extract was further purified by column chromatography (silica gel, gradient ethyl acetate/petroleum ether, 50:70 in 40 min to 50:50) to afford the desired compound (0.92 g, 3.22 mmol, 92.1%). HPLC, 6.78 min. 1H NMR (200 MHz, DMSO-d6) δ 8.63−8.54 (m, 1H), 8.38−8.34 (m, 1H), 8.00−7.87 (m, 2H), 7.52−7.42 (m, 3H), 3.20 (s, 4H), 2.83−2.76 (m, 3H). FAB m/z = 300.0 [M + H]+. 8-Chloro-N-(2-morpholinethyl)-5-oxo-10,11-dihydro-5Hdibenzo[a,d][7]annulene-3-carboxamide (29). 8-Chloro-5-oxo10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxylic acid 24 (0.20 g, 0.70 mmol) was dissolved in 2 mL of THFdry. TBTU (0.29 g, 0.91 mmol) and DIPEA (0.27 g, 2.09 mmol) were added to the solution. After activation, 2-morpholinoethan-1-amine (0.09 g, 0.70 mmol) was added. When the reaction was completed, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layers were combined and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (silica gel, gradient petroleum ether/ethyl acetate, 95:5 to 90:10 in 1 h) to give the desired compound (0,75 g, 1.88 mmol, 89.3%). HPLC, 5.19 min. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.35 (s, 1H), 7.94 (dd, J = 20.5, 8.0 Hz, 2H), 7.51 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 3.61−3.53 (m, 4H), 3.42−3.34 (m, 4H), 3.25−3.16 (m, 4H), 2.45−2.36 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 192.9, 165.2, 144.8, 144.3, 137.5, 137.3, 136.4, 133.0, 132.2, 131.1, 129.8, 129.3, 129.0, 126.7, 66.2, 57.3, 53.2, 36.6. N-Isopropyl-N,4-dimethyl-3-nitrobenzamide (30a). Compound 30a was prepared according to general procedure Ia. The residue was purified by flash chromatographie (silica gel, gradient petroleum ether/ethyl acetate 70:30) to afford 1.10 g (4.6 mmol, 84.62%) of the title compound 30a. HPLC, 5.53 min. 1H NMR (200 MHz, DMSO) δ 6.94 (d, J = 7.4 Hz, 1H), 6.56 (s, 1H), 6.40 (d, J = 7.4 Hz, 1H), 4.97 (s, 2H), 2.74 (s, 3H), 2.50 (s, 1H), 2.06 (s, 3H), 1.08 (d, J = 6.7 Hz, 6H). 13C NMR (50 MHz, DMSO-d6) δ 165.2, 148.7, 135.8, 133.3, 132.9, 131.6, 123.0, 61.9, 23.1, 19.5, 5.7. EI-MS m/z = 235.0 [M − H]−. N-Cyclopropyl-4-methyl-3-nitrobenzamide (31a). Compound 31a was prepared according to general procedure Ia. The residue was purified by flash chromatography (silica gel, gradient petroleum ether/ ethyl acetate 60:40) to afford 1.17g (5.32 mmol, 91.05%) of the title compound 31a. HPLC, 4.49 min. 1H NMR (200 MHz, DMSO-d6) δ 8.78−8.60 (m, 1H), 8.41 (d, J = 1.8 Hz, 1H), 8.05 (dd, J = 8.1, 1.8 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 2.95−2.79 (m, 1H), 2.55 (s, 3H), 0.77−0.65 (m, 2H), 0.63−0.53 (m, 2H). 13C NMR (50 MHz, DMSO-d6) δ 165.1, 148.7, 135.7, 133.3, 132.9, 131.6, 123.0, 23.1, 19.45, 5.7. EI-MS m/z = 219.3 [M − H]−. N-(2-Fluoro-5-nitrophenyl)cyclopropanecarboxamide (32a). Compound 32a was prepared according to general procedure Ib. The residue was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 60:40) to afford 0.62 g (2.77 mmol, 88.11%) of the title compound 32a. HPLC, 4.41 min. 1H NMR (200 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.11−8.89 (m, 1H), 8.07−7.87 (m, 1H), 7.68−7.42 (m, 1H), 2.19−1.95 (m, 1H), 0.98−0.72 (m, 4H). 13 C NMR (50 MHz, DMSO-d6) δ 173.3, 156.3 (d, J = 255.5 Hz), 144.0 (d, J = 2.7 Hz), 127.9 (d, J = 13.2 Hz), 120.2 (d, J = 9.5 Hz), 118.2 (d, J = 4.3 Hz), 116.9 (d, J = 22.5 Hz), 14.5, 8.3. EI-MS m/z = 222.6 [M − H]−. N-(2-Fluoro-5-nitrophenyl)thiophene-2-carboxamide (33a). Compound 33a was synthesized according to general procedure Ic. The residue was purified by flash chromatography (silica gel, gradient petroleum ether/ethyl acetate 80:20) to afford 1.51 g (5.72 mmol, 41.6%) of the title compound 33a. Melting point, 86.1 °C. HPLC, 6.34 min. IR (ATR) [cm−1] 3080, 1673, 1528, 1492, 1406, 1345, 1237, 797, 718, 656. 1H NMR 400 MHz, DMSO-d6) δ 11.58 (s, 1H), 9.74 (dd, J = 13.3, 4.1 Hz, 2H), 9.08−8.92 (m, 2H), 8.92−8.74 (m, 2H). FAB m/z = 265 [M + H]−. N-(2-Fluoro-5-nitrophenyl)benzamide (33b). Compound 36 was synthesized according to general procedure Id. The crude product was purified by recrystallization in hexan/ethyl acetate to afford 1.96 g (7.7 mmol, 53.4%) of the desired compound 36. 1H NMR (200 MHz,

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00745. SMILES and some data (CSV) Experimental and analytical data, including HPLC purity, (HR)MS, and NMR data of compounds 2−60; additional characterization of binding kinetics; all general methods; experimental section for materials and intermediates; X-ray crystallography; metabolic stability determination in vitro (PDF) 8052



Article

Accession Codes

(11) Blue Ridge Institute for Medical Research. USFDA approved protein kinase inhibitors. http://www.brimr.org/PKI/PKIs.htm (accessed June 19, 2017). (12) Kumar, S.; Boehm, J.; Lee, J. C. P38 map kinases: Key signalling molecules as therapeutic targets for inflammatory diseases. Nat. Rev. Drug Discovery 2003, 2, 717−726. (13) Dambach, D. M. Potential adverse effects associated with inhibition of p38alpha/beta map kinases. Curr. Top. Med. Chem. 2005, 5, 929−939. (14) Cohen, S. B.; Cheng, T. T.; Chindalore, V.; Damjanov, N.; Burgos-Vargas, R.; Delora, P.; Zimany, K.; Travers, H.; Caulfield, J. P. Evaluation of the efficacy and safety of pamapimod, a p38 map kinase inhibitor, in a double-blind, methotrexate-controlled study of patients with active rheumatoid arthritis. Arthritis Rheum. 2009, 60, 335−344. (15) Schreiber, S.; Feagan, B.; D’Haens, G.; Colombel, J. F.; Geboes, K.; Yurcov, M.; Isakov, V.; Golovenko, O.; Bernstein, C. N.; Ludwig, D.; Winter, T.; Meier, U.; Yong, C.; Steffgen, J.; BIRB 796 Study Group.. Oral p38 mitogen-activated protein kinase inhibition with birb 796 for active Crohn’s disease: a randomized, double-blind, placebocontrolled trial. Clin. Gastroenterol. Hepatol. 2006, 4, 325−334. (16) Genovese, M. C.; Cohen, S. B.; Wofsy, D.; Weinblatt, M. E.; Firestein, G. S.; Brahn, E.; Strand, V.; Baker, D. G.; Tong, S. E. A 24week, randomized, double-blind, placebo-controlled, parallel group study of the efficacy of oral scio-469, a p38 mitogen-activated protein kinase inhibitor, in patients with active rheumatoid arthritis. J. Rheumatol. 2011, 38, 846−854. (17) Zuccotto, F.; Ardini, E.; Casale, E.; Angiolini, M. Through the “gatekeeper door”: Exploiting the active kinase conformation. J. Med. Chem. 2010, 53, 2681−2694. (18) Traxler, P. Tyrosine kinase inhibitors in cancer treatment (part ii). Expert Opin. Ther. Pat. 1998, 8, 1599−1625. (19) Young, P. R.; McLaughlin, M. M.; Kumar, S.; Kassis, S.; Doyle, M. L.; McNulty, D.; Gallagher, T. F.; Fisher, S.; McDonnell, P. C.; Carr, S. A.; Huddleston, M. J.; Seibel, G.; Porter, T. G.; Livi, G. P.; Adams, J. L.; Lee, J. C. Pyridinyl imidazole inhibitors of p38 mitogenactivated protein kinase bind in the atp site. J. Biol. Chem. 1997, 272, 12116−12121. (20) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.; Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.; Regan, J. Inhibition of p38 map kinase by utilizing a novel allosteric binding site. Nat. Struct. Biol. 2002, 9, 268−272. (21) Cusack, K. P.; Wang, Y.; Hoemann, M. Z.; Marjanovic, J.; Heym, R. G.; Vasudevan, A. Design strategies to address kinetics of drug binding and residence time. Bioorg. Med. Chem. Lett. 2015, 25, 2019−2027. (22) Fabian, M. A.; Biggs, W. H., 3rd; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J. M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 2005, 23, 329−336. (23) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127−132. (24) Eswaran, J.; Knapp, S. Insights into protein kinase regulation and inhibition by large scale structural comparison. Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804, 429−432. (25) Kluter, S.; Grutter, C.; Naqvi, T.; Rabiller, M.; Simard, J. R.; Pawar, V.; Getlik, M.; Rauh, D. Displacement assay for the detection of stabilizers of inactive kinase conformations. J. Med. Chem. 2010, 53, 357−367. (26) Laufer, S. A.; Margutti, S.; Fritz, M. D. Substituted isoxazoles as potent inhibitors of p38 map kinase. ChemMedChem 2006, 1, 197− 207.

Cocrystal structures of the human p38α MAP kinase in complex with 6j and 8m, respectively, are deposited under the accession codes 5MTY and 5MTX in the Protein Data Bank. Authors will release the atomic coordinates and experimental data upon article publication.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 7071 2972459. E-mail: stefan.laufer@uni-tuebingen. de. ORCID

Daniel Rauh: 0000-0002-1970-7642 Stefan A. Laufer: 0000-0001-6952-1486 Author Contributions ∥

N.M.W., H.K.W., and M.B. contributed equally to this publication.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Katharina Bauer, Silke Bauer, and Daniela Müller for the skillful technical assistance in compound testing and Michael Forster for synthesis of the precatalysts.

■

ABBREVIATIONS USED MAP, mitogen activated protein; R-Spine, regulatory spine; TRT, target residence time; ATF, activating transcription factor; HR, hydrophobic region; C-Spine, catalytic spine; BTK, Bruton’s tyrosine kinase; IUPAC, International Union of Pure and Applied Chemistry; CDI, 1,1′-carbonyldiimidazole; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; SEM, standard error of the mean; SPR, surface plasmon resonance; RLM, rat liver microsome

■

REFERENCES

(1) Capdeville, R.; Buchdunger, E.; Zimmermann, J.; Matter, A. Glivec (sti571, imatinib), a rationally developed, targeted anticancer drug. Nat. Rev. Drug Discovery 2002, 1, 493−502. (2) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. The protein kinase complement of the human genome. Science 2002, 298, 1912−1934. (3) Cell Signaling Technology Inc. Kinase-disease associations. https://www.cellsignal.com/common/content/content.jsp?id= science-tables-kinase-disease (accessed June 19, 2017). (4) Zhang, J.; Shen, B.; Lin, A. Novel strategies for inhibition of the p38 mapk pathway. Trends Pharmacol. Sci. 2007, 28, 286−295. (5) Schett, G.; Zwerina, J.; Firestein, G. The p38 mitogen-activated protein kinase (mapk) pathway in rheumatoid arthritis. Ann. Rheum. Dis. 2008, 67, 909−916. (6) Tsai, C. J.; Nussinov, R. The molecular basis of targeting protein kinases in cancer therapeutics. Semin. Cancer Biol. 2013, 23, 235−242. (7) Wagey, R. T.; Krieger, C. Abnormalities of protein kinases in neurodegenerative diseases. Prog. Drug Res. 1998, 51, 133−183. (8) Vlahopoulos, S. A.; Logotheti, S.; Mikas, D.; Giarika, A.; Gorgoulis, V.; Zoumpourlis, V. The role of atf-2 in oncogenesis. BioEssays 2008, 30, 314−327. (9) Cuenda, A.; Rousseau, S. P38 map-kinases pathway regulation, function and role in human diseases. Biochim. Biophys. Acta, Mol. Cell Res. 2007, 1773, 1358−1375. (10) Fabbro, D.; Cowan-Jacob, S. W.; Mobitz, H.; Martiny-Baron, G. Targeting cancer with small-molecular-weight kinase inhibitors. Methods Mol. Biol. 2012, 795, 1−34. 8053



Article

(27) Hauser, D. R.; Scior, T.; Domeyer, D. M.; Kammerer, B.; Laufer, S. A. Synthesis, biological testing, and binding mode prediction of 6,9diarylpurin-8-ones as p38 map kinase inhibitors. J. Med. Chem. 2007, 50, 2060−2066. (28) Laufer, S. A.; Ahrens, G. M.; Karcher, S. C.; Hering, J. S.; Niess, R. Design, synthesis, and biological evaluation of phenylaminosubstituted 6,11-dihydro-dibenzo[b,e]oxepin-11-ones and dibenzo[a,d]cycloheptan-5-ones: Novel p38 map kinase inhibitors. J. Med. Chem. 2006, 49, 7912−7915. (29) Koeberle, S. C.; Romir, J.; Fischer, S.; Koeberle, A.; Schattel, V.; Albrecht, W.; Grutter, C.; Werz, O.; Rauh, D.; Stehle, T.; Laufer, S. A. Skepinone-l is a selective p38 mitogen-activated protein kinase inhibitor. Nat. Chem. Biol. 2012, 8, 141−143. (30) Martz, K. E.; Dorn, A.; Baur, B.; Schattel, V.; Goettert, M. I.; Mayer-Wrangowski, S. C.; Rauh, D.; Laufer, S. A. Targeting the hinge glycine flip and the activation loop: Novel approach to potent p38α inhibitors. J. Med. Chem. 2012, 55, 7862−7874. (31) Fischer, S.; Wentsch, H. K.; Mayer-Wrangowski, S. C.; Zimmermann, M.; Bauer, S. M.; Storch, K.; Niess, R.; Koeberle, S. C.; Grutter, C.; Boeckler, F. M.; Rauh, D.; Laufer, S. A. Dibenzosuberones as p38 mitogen-activated protein kinase inhibitors with low atp competitiveness and outstanding whole blood activity. J. Med. Chem. 2013, 56, 241−253. (32) Baur, B.; Storch, K.; Martz, K. E.; Goettert, M. I.; Richters, A.; Rauh, D.; Laufer, S. A. Metabolically stable dibenzo[b,e]oxepin11(6h)-ones as highly selective p38 map kinase inhibitors: Optimizing anti-cytokine activity in human whole blood. J. Med. Chem. 2013, 56, 8561−8578. (33) Thurmond, R. L.; Wadsworth, S. A.; Schafer, P. H.; Zivin, R. A.; Siekierka, J. J. Kinetics of small molecule inhibitor binding to p38 kinase. Eur. J. Biochem. 2001, 268, 5747−5754. (34) Unzue, A.; Dong, J.; Lafleur, K.; Zhao, H.; Frugier, E.; Caflisch, A.; Nevado, C. Pyrrolo[3,2-b]quinoxaline derivatives as types i1/2 and ii eph tyrosine kinase inhibitors: Structure-based design, synthesis, and in vivo validation. J. Med. Chem. 2014, 57, 6834−6844. (35) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug-target residence time and its implications for lead optimization. Nat. Rev. Drug Discovery 2006, 5, 730−739. (36) Hothersall, J. D.; Brown, A. J.; Dale, I.; Rawlins, P. Can residence time offer a useful strategy to target agonist drugs for sustained gpcr responses? Drug Discovery Today 2016, 21, 90−96. (37) Dahl, G.; Akerud, T. Pharmacokinetics and the drug-target residence time concept. Drug Discovery Today 2013, 18, 697−707. (38) Tummino, P. J.; Copeland, R. A. Residence time of receptorligand complexes and its effect on biological function. Biochemistry 2008, 47, 5481−5492. (39) Basavapathruni, A.; Jin, L.; Daigle, S. R.; Majer, C. R.; Therkelsen, C. A.; Wigle, T. J.; Kuntz, K. W.; Chesworth, R.; Pollock, R. M.; Scott, M. P.; Moyer, M. P.; Richon, V. M.; Copeland, R. A.; Olhava, E. J. Conformational adaptation drives potent, selective and durable inhibition of the human protein methyltransferase dot1l. Chem. Biol. Drug Des. 2012, 80, 971−980. (40) Chang, A.; Schiebel, J.; Yu, W.; Bommineni, G. R.; Pan, P.; Baxter, M. V.; Khanna, A.; Sotriffer, C. A.; Kisker, C.; Tonge, P. J. Rational optimization of drug-target residence time: Insights from inhibitor binding to the staphylococcus aureus fabi enzyme-product complex. Biochemistry 2013, 52, 4217−4228. (41) Meharena, H. S.; Chang, P.; Keshwani, M. M.; Oruganty, K.; Nene, A. K.; Kannan, N.; Taylor, S. S.; Kornev, A. P. Deciphering the structural basis of eukaryotic protein kinase regulation. PLoS Biol. 2013, 11, e1001680. (42) Hu, J.; Ahuja, L. G.; Meharena, H. S.; Kannan, N.; Kornev, A. P.; Taylor, S. S.; Shaw, A. S. Kinase regulation by hydrophobic spine assembly in cancer. Mol. Cell. Biol. 2015, 35, 264−276. (43) Yurtsever, Z.; Scheaffer, S. M.; Romero, A. G.; Holtzman, M. J.; Brett, T. J. The crystal structure of phosphorylated mapk13 reveals common structural features and differences in p38 mapk family activation. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2015, 71, 790− 799.

(44) Kornev, A. P.; Taylor, S. S.; Ten Eyck, L. F. A helix scaffold for the assembly of active protein kinases. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14377−14382. (45) Francis, D. M.; Rozycki, B.; Koveal, D.; Hummer, G.; Page, R.; Peti, W. Structural basis of p38 alpha regulation by hematopoietic tyrosine phosphatase. Nat. Chem. Biol. 2011, 7, 916−924. (46) Bukhtiyarova, M.; Karpusas, M.; Northrop, K.; Namboodiri, H. V.; Springman, E. B. Mutagenesis of p38alpha map kinase establishes key roles of phe169 in function and structural dynamics and reveals a novel dfg-out state. Biochemistry 2007, 46, 5687−5696. (47) Wentsch, H. K.; Walter, N. M.; Buhrmann, M.; MayerWrangowski, S.; Rauh, D.; Zaman, G. J. R.; Willemsen-Seegers, N.; Buijsman, R. C.; Henning, M.; Dauch, D.; Zender, L.; Laufer, S. Optimized target residence time: Type i1/2 inhibitors for p38alpha map kinase with improved binding kinetics through direct interaction with the r-spine. Angew. Chem., Int. Ed. 2017, 56, 5363−5367. (48) Seo, Y. Novel isoindolinone derivatives having inhibitory activity against t-type calcium channel and method for preparation. U.S. Patent US 2010/0004286 A1, 2010. (49) Surry, D. S.; Buchwald, S. L. Dialkylbiaryl phosphines in pdcatalyzed amination: A user’s guide. Chem. Sci. 2011, 2, 27−50. (50) Hill, R. J.; Dabbagh, K.; Phippard, D.; Li, C.; Suttmann, R. T.; Welch, M.; Papp, E.; Song, K. W.; Chang, K. C.; Leaffer, D.; Kim, Y. N.; Roberts, R. T.; Zabka, T. S.; Aud, D.; Dal Porto, J.; Manning, A. M.; Peng, S. L.; Goldstein, D. M.; Wong, B. R. Pamapimod, a novel p38 mitogen-activated protein kinase inhibitor: Preclinical analysis of efficacy and selectivity. J. Pharmacol. Exp. Ther. 2008, 327, 610−619. (51) Goettert, M.; Graeser, R.; Laufer, S. A. Optimization of a nonradioactive immunosorbent assay for p38alpha mitogen-activated protein kinase activity. Anal. Biochem. 2010, 406, 233−234. (52) Bauer, S. M.; Kubiak, J. M.; Rothbauer, U.; Laufer, S. From enzyme to whole blood: Sequential screening procedure for identification and evaluation of p38 mapk inhibitors. Methods Mol. Biol. 2016, 1360, 123−148. (53) Liu, C.; Lin, J.; Wrobleski, S. T.; Lin, S.; Hynes, J.; Wu, H.; Dyckman, A. J.; Li, T.; Wityak, J.; Gillooly, K. M.; Pitt, S.; Shen, D. R.; Zhang, R. F.; McIntyre, K. W.; Salter-Cid, L.; Shuster, D. J.; Zhang, H.; Marathe, P. H.; Doweyko, A. M.; Sack, J. S.; Kiefer, S. E.; Kish, K. F.; Newitt, J. A.; McKinnon, M.; Dodd, J. H.; Barrish, J. C.; Schieven, G. L.; Leftheris, K. Discovery of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-n-propylpyrrolo[1,2-f][ 1,2,4]triazine-6-carboxamide (bms-582949), a clinical p38alpha map kinase inhibitor for the treatment of inflammatory diseases. J. Med. Chem. 2010, 53, 6629−6639. (54) Chin, N. X.; Neu, H. C. Ciprofloxacin, a quinolone carboxylic acid compound active against aerobic and anaerobic bacteria. Antimicrob. Agents Chemother. 1984, 25, 319−326. (55) Willemsen-Seegers, N.; Uitdehaag, J. C.; Prinsen, M. B.; de Vetter, J. R.; de Man, J.; Sawa, M.; Kawase, Y.; Buijsman, R. C.; Zaman, G. J. Compound selectivity and target residence time of kinase inhibitors studied with surface plasmon resonance. J. Mol. Biol. 2017, 429, 574−586. (56) Xing, L.; Devadas, B.; Devraj, R. V.; Selness, S. R.; Shieh, H.; Walker, J. K.; Mao, M.; Messing, D.; Samas, B.; Yang, J. Z.; Anderson, G. D.; Webb, E. G.; Monahan, J. B. Discovery and characterization of atropisomer ph-797804, a p38 map kinase inhibitor, as a clinical drug candidate. ChemMedChem 2012, 7, 273−280.

8054


p38Î± MAP Kinase I - ACS Publications - American Chemical Society

Recommend Documents