Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically

Article pubs.acs.org/jmc

Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically Resistant L858R/T790M/C797S EGFR Mutant: Targeting of Both Hydrophobic Regions and the Phosphate Binding Site Marcel Günther,†,∥ Jonas Lategahn,‡,∥ Michael Juchum,† Eva Döring,† Marina Keul,‡ Julian Engel,‡,§ Hannah L. Tumbrink,‡ Daniel Rauh,‡ and Stefan Laufer*,† †

Institute of Pharmaceutical Sciences, Pharmaceutical and Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany ‡ Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 4a, 44227 Dortmund, Germany S Supporting Information *

ABSTRACT: Inhibition of the epidermal growth factor receptor represents one of the most promising strategies in the treatment of lung cancer. Acquired resistance compromises the clinical efficacy of EGFR inhibitors during long-term treatment. The recently discovered EGFR-C797S mutation causes resistance against third-generation EGFR inhibitors. Here we present a rational approach based on extending the inhibition profile of a p38 MAP kinase inhibitor toward mutant EGFR inhibition. We used a privileged scaffold with proven cellular potency as well as in vivo efficacy and low toxicity. Guided by molecular modeling, we synthesized and studied the structure−activity relationship of 40 compounds against clinically relevant EGFR mutants. We successfully improved the cellular EGFR inhibition down to the low nanomolar range with covalently binding inhibitors against a gefitinib resistant T790M mutant cell line. We identified additional noncovalent interactions, which allowed us to develop metabolically stable inhibitors with high activities against the osimertinib resistant L858R/T790M/C797S mutant.

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INTRODUCTION The epidermal growth factor receptor (EGFR), a member of the ERBB receptor tyrosine kinase family, is a transmembrane protein modulating cell growth, proliferation, migration, and cell survival.1 Pathologically increased EGFR activity is closely linked to tumorigenesis and tumor cell growth in general and has been identified as a key driver for the onset and progression of nonsmall cell lung cancer (NSCLC) in particular.2 Mutations in the EGFR kinase domain that cause an increased and ligandindependent catalytic activity, so-called activating mutations, have been identified as oncogenic drivers for NSCLC.3 These activating mutations ultimately lead to an “oncogene addiction”, meaning the survival of these particular tumor cells is highly dependent on the EGFR signaling. Thus, blockage of the EGFR signaling with either tyrosine kinase inhibitors (TKIs) or monoclonal antibodies (mAb) is a validated principal to inhibit cell growth in EGFR mutant NSCLC. Several activating mutations have been identified in patients, however, the majority © 2017 American Chemical Society

of patients harbor a single-point mutation in exon 21, the L858R mutation or the exon 19 deletion, del746_A750.2,4 Patients bearing one of these activating mutations generally respond well (50−80%) to a therapy based on first-generation EGFR inhibitors (gefitinib or erlotinib), and these responders show significant tumor shrinkage in the clinic (Figure 1).5,6 Suddenly, patients who have initially responded well to a TKI therapy develop secondary drug resistances during long-term treatment.7 In approximately 60% of cases, the secondary mutation T790M has been identified in resistant patients.8,9 First-generation EGFR inhibitors show low inhibitory activity for this mutant as well as for the double-mutant L858R/T790M, which indicates that this so-called gatekeeper mutation is the most abundant cause for tumor relapse in patients who initially responded well to first-generation EGFR inhibitors. T790M has mainly two Received: February 27, 2017 Published: June 12, 2017 5613

DOI: 10.1021/acs.jmedchem.7b00316 J. Med. Chem. 2017, 60, 5613−5637

Journal of Medicinal Chemistry

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Figure 1. First-, second-, and third-generation EGFR inhibitors and their binding modes: (A) Two-dimensional binding mode of osimertinib found in the X-ray structure compared to pyridinylimidazoles based on our molecular model. HR, hydrophobic region; PB, phosphate binding site; PFG, pocketfilling group. (B) Difference between the dihedral angle of gefitinib and a vicinally substituted imidazole. (C) Generations of EGFR inhibitors occupying the ATP binding site.

imidazole core as potent inhibitors that retain activity toward the cysteine mutant EGFR variant.19 In contrast to third-generation EGFR inhibitors, our compounds occupy a hydrophobic region (HR I) in the back of the ATP binding pocket, whose access is limited by the respective gatekeeper residue. As shown by Engel, Becker, et al., additional interactions between compounds and the ATP binding site reduce dependence on the covalent interaction for inhibitory activities and in consequence yields efficiency against EGFR-C797S.20 Following this concept, we herein present insights into the SAR of trisubstituted pyridinylimidazoles as EGFR inhibitors. With the help of modular and flexible organic synthetic strategies, we gained a deep understanding of a variety of substituents in different positions of this structure class. Following an iterative process from molecular modeling over synthesis to biological testing and back, allowed us to find an ideal attachment point for a Michael acceptor, able to inhibit the enzymatic activity of EGFR-L858R/ T790M in an irreversible manner. We studied the influence of aromatic and aliphatic groups that can occupy HR I, aromatic residues occupying HR II, and different H-bond donors as well as acceptors that increase the inhibitory activity by interactions with the phosphate binding site (PB). In sum, the additional interactions mentioned above separate our newly synthesized compounds from third-generation inhibitors in terms of interaction profile and result in effective inhibition of the osimertinib-resistant L858R/T790M/C797S EGFR mutant variant.

effects: significantly increased binding affinity of ATP to this mutant protein and the increased spatial requirement of methionine, compared to threonine, causes steric clashes with the 3-chloro-4-fluoroaniline residue of gefitinib.10 Secondgeneration EGFR inhibitors, e.g., afatinib, have been designed to overcome T790M resistance by a covalent interaction with the protein.11,12 Thus, these compounds bear acrylamide moieties as Michael acceptors that can undergo Michael addition reactions with the thiol group of a noncatalytic cysteine (Cys797). This additional covalent interaction restores inhibitory activity while increasing target residence time. Both effects result in significant tumor growth inhibition in preclinical settings. However, the clinical efficacy of second-generation EGFR inhibitors was unsatisfying. Because second-generation EGFR inhibitors are nonselective with respect to EGFR mutant inhibition, they are potent cellular EGFR wild-type (wt) inhibitors as well. As a consequence, these inhibitors showed dose limiting toxic side effects which have been directly linked to wt inhibition, resulting in low tolerable, subtherapeutic concentrations in the clinic.13,14 Third-generation EGFR inhibitors, e.g., osimertinib15 or N-(3((5-chloro-2-((2-methoxy-4-(4-methyl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)oxy)phenyl)-2-propenamide, 85 (WZ400216), have been designed to overcome T790M resistance by (i) employing novel scaffolds, while (ii) sparing the wild-type to circumvent toxic side effects and (iii) covalent alkylation of the reactive cysteine.15,17 Because third-generation EGFR inhibitors activity is merely based on their covalent interaction with the protein, these compounds are highly vulnerable for a Cys797 mutation. Because this amino acid is noncatalytic and not conserved, it is not surprising that a C797S mutation has been identified to be one of the key drivers for osimertinib resistance in the clinic.18 We previously reported a small series of EGFR inhibitors based on a trisubstituted

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RESULTS AND DISCUSSION Rational Design of EGFR Inhibitors. Starting from lead compound 1,21 we performed a rational design approach guided by molecular modeling. Docking studies predicted a bidentate hinge binding motif between the 2-aminopyridine moiety of 5614



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Figure 2. Proposed three-dimensional (left) and two-dimensional (right) binding modes of lead compound 1 in PDB 2JIU. Hydrogen bond acceptors are depicted as red arrows and hydrogen bond donors as green arrows. Hydrophobic interactions are depicted as yellow spheres (3D model) or yellow brackets (2D model).

I, we synthesized compounds bearing various alkyl or aryl residues in the imidazole C4 position, having different polarities as well as growing spatial requirements (23−30). Our findings led us to combine the most promising partial structures in a hybrid compound (31a). In previous studies, we examined whether the sulfur atom could be substituted with a carbon atom without any significant change in activity.25 With this approach, we synthesized one compound bearing all four interaction patterns: a Michael acceptor targeting Cys797, a bidentate hinge binding to Met793, aromatic residues occupying HR II as well as HR I, and a polar side chain interacting with the phosphate binding site. Chemistry. Compounds 1−13 were synthesized according to our previously reported synthetic route26 by applying minor changes and optimizations (Scheme 1). Thus, the corresponding commercially available benzoic acid esters were condensed with 2-fluoro-4-picoline using NaHMDS as a strong non-nucleophilic base in good yields. Subsequent nitrosation with sodium nitrite in glacial acetic acid followed by reduction with zinc dust under acidic conditions yielded α-aminoketones 38a−c as the corresponding hydrochloride salts after workup. Cyclization with potassium thiocyanate in refluxing DMF resulted in thiones 39a−c in excellent yields. Key intermediates 40a−c were synthesized by means of nucleophilic aromatic substitution reactions with aniline under acidic conditions in refluxing NMP in good yields. Compounds 1−4 and 10−11 were synthesized by the application of nucleophilic substitution reactions by employing the corresponding alkyl halides and K2CO3 as base in refluxing THF. To introduce aliphatic linkers to attach either a Michael acceptor as the corresponding acrylamide or the generic propionyl amide, imidazole thiones 40a−c were decorated with aliphatic amines protected as phthalimides under similar conditions. Compounds 41a−d were subsequently deprotected via hydrazinolysis using hydrazine hydrate in EtOH at elevated temperature to give primary amines 5 and 12 in decent yields. Amide couplings of 5, 12, and 42e,f with the corresponding carboxylic acids were conducted using TBTU and DIPEA in THF to obtain 6a−9 and 13 in moderate yields. Cyclic alkyl linkers 45a,b and 49 were synthesized according to Scheme 2. Commercially available enantiomerically pure (R)- or (S)-proline was Boc-protected and subjected to reduction with borane in THF, leading to the corresponding alcohols 44a,b. The

compound 1 and Met793 when docked into an X-ray structure of EGFR-L858R/T790M (Figure 2). Moreover, in this predicted binding mode, the 4-fluorophenyl residue extended deep into HR I, occupying this hydrophobic pocket. The phenyl ring of the aniline moiety was located in HR II. Because lead compound 1 bears a small methyl residue attached to the sulfur in the imidazole C2 position, it does not interact with the PB in our docking studies. On the basis of this model, we first studied possible substituents in the imidazole C2 position to interact with the PB, thus increasing the inhibitory activity of the resulting compounds. We synthesized compounds bearing different substituents at the imidazole C2 position, keeping the aniline moiety at the pyridine C2 position unchanged. To evaluate the possibility for polar interactions with the phosphate-binding site and to study the structure−activity relationship (SAR), we initially synthesized several derivatives with the capability to form additional hydrogen bonds, both as acceptors as well as donors.22 Our docking model revealed a distance of 7.5 Å between the sulfur atom of lead compound 1 and the sulfur atom of Cys797 with an angle of roughly 90° based on the imidazole carbon−sulfur bond (Figure 2). Hence, we investigated the possibility of attaching a Michael acceptor via a suitable linker to the imidazole C2 position in order to form a covalent bond with the reactive Cys797 side chain (Figure 3a), resulting in compounds (6a and 7−9). To direct the reactive Michael acceptor in close proximity to the cysteine, suitable geometries and distances of an aliphatic linker were of utmost importance. We varied linker lengths as well as three-dimensional geometries by the introduction of rigidized cyclic alkyl linkers (14−16). By the application of covalent docking algorithms (Figure 4), we identified the 3 position of the aniline ring of lead compound 1 to be a suitable attachment point for a Michael acceptor as well and synthesized the respective compounds (17−31). The incorporation of a reactive Michael acceptor at this attachment point displayed a conformationally less flexible geometry, thus arranging the reactive group in a rigidized manner toward Cys797. The concept of reducing conformational flexibility has been shown previously to result in improved inhibitory activity.23,24 We studied the influence of the acrylamides on the inhibitory activity by comparison with the reversible analogues, the respective propionamides. To investigate the role of residues occupying HR 5615



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Scheme 1. Synthesis of Trisubstituted Imidazoles (1−13)a

a Reagents and conditions: (i) NaHMDS, THF, 0 °C to rt, 65−79%. (ii) NaNO2, HOAc, 10 °C, 84−97%. (iii) Zn dust, HOAc, 10 °C, 78−85%. (iv) KSCN, DMF, reflux, 73−84%. (v) aniline, NMP, conc HClaq, reflux, 63−88%. (vi) alkyl halide, K2CO3, THF, reflux, 48−79%. (vii) (n = 2), N-(2bromoethyl)phthalimide, K2CO3, THF, reflux; (n = 3), N-(3-Iodopropyl)phthalimide, K2CO3, THF, reflux, 57−81%. (viii) Hydrazine hydrate, EtOH, 70 °C, 55−65%. (ix) Carboxylic acid, TBTU, DIPEA, THF, 27−63%.

approach.19 First, we α-brominated commercially available ketones with bromine in methylene chloride (for aromatic ketones) or methanol (for aliphatic ketones) to yield 52a−j. For the S-methyl series, we next cyclized these α-bromoketones with S-methyl isothiourea hemisulfate under mild basic conditions to give 53a,c−j in acceptable yields. The following SEM protection initially yielded both corresponding regioisomeres. The regioisomeric mixtures were converted to the less sterically demanding isomer by stirring with a catalytic amount of SEM chloride in acetonitrile at elevated temperature in good yields.29 Electrophilic bromination with NBS in acetonitrile at −30 °C yielded key intermediates 55a,c−j, which were subjected to a high yielding Suzuki cross coupling reaction with an N-protected 2-amino-pyridine-4-boronic acid ester under optimized conditions. After deprotection of the amino pyridines 56a,c−j, the free amines 57a,c−j were obtained in good yields. The subsequent Pd catalyzed aryl amination with predecorated mbromoanilines gave precursors 58a−j and 59−63 that were

following bromination was achieved under Appel conditions using triphenylphosphine and carbon tetrabromide in THF, giving 45a,b in good yields.27 Final compounds 14 and 15 were synthesized by nucleophilic substitution reactions of 45a,b with 40a, followed by Boc-deprotection under acidic conditions and subsequent amide coupling with acrylic acid in acceptable yields. Boc protection of 3-hydroxypyrrolidine (racemic mixture) yielded intermediate 48 that was transformed into the corresponding bromide 49 via Appel reaction using triphenylphosphine and CBr4 in good yields.28 Again, we attached this cyclic side chain to the imidazole thione 40a by nucleophilic substitution reaction under similar conditions as mentioned above. Deprotection followed by amide bond formation yielded final compound 16 as racemic mixture. Because we were not able to produce analogues of 40a−c using substituted anilines for a nucleophilic aromatic substitution with thiones 39a−c, we established a different synthetic route according to Scheme 3 based on a Suzuki cross coupling 5616



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Scheme 2. Synthesis of Trisubstituted Imidazoles with Cyclic Alkyl Linkers (14−16)a

Reagents and conditions: (i) BH3*THF, THF, 0 °C 62−65%; (ii) PPh3, CBr4, rt, 65−71%; (iii) 36a, K2CO3, THF, reflux, 76−80%; (iv) TFA, DCM, rt, 81−88%; (v) acrylic acid, TBTU, DIPEA, THF, 28−37%; (vi) Boc2O, neat, rt; DMAP, 90%; (vii) PPh3, CBr4,THF, 0 °C to rt, 62%; (viii) 36a, K2CO3, THF, reflux, 84%. a

Scheme 3. Synthesis of Compounds 17a,b and 19a−30a

a

Reagents and conditions: (i) Br2, DCM or MeOH, rt, 64−95%; (ii) S-methylisothiourea hemisulfate salt, NaHCO3,THF/H2O, reflux, 15−65%; (iii) NaH, SEM-Cl, THF, −15 °C, quant; (iv) SEM-Cl (5 mol %), MeCN, 80 °C, 50−91%; (v) NBS, MeCN, −30 °C, 21−90%; (vi) corresponding boronic acid or acid ester, Pd(OAc)2/XPhos or Pd2(dba)3/(t-Bu)3P*HBF4, K3PO4, 1,4-dioxane/H2O, reflux, 54−91%; (vii) 5 M NaOHaq, MeOH, 60 °C, 63−92% or NH2OH, DIPEA, ethanol, rt 53−63% or TFA, DCM, rt 88−99%; (viii) (hetero)aryl bromide, Brettphos Pd G1, Cs2CO3, 1,4dioxane/t-BuOH, 130 °C, 61−91%; (ix) TFA, DCM, rt, 55−86%.

deprotected under acidic conditions using TFA to obtain final compounds 17a−b and 19−30 in moderate to excellent yields. For the synthesis of compound 18 (Scheme 4), αbromoketone 52b was converted to the corresponding α-

aminoketone 64 via Delépine reaction and subsequently cyclized with potassium thiocyanate under acidic conditions to give imidazole thione 65 in good yield. We obtained the N-protected intermediate 66 by means of nucleophilic substitution of N-(25617



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Scheme 4. Synthesis of Compound 18a

Reagents and conditions: (i) Br2, DCM, rt, 90%; (ii) urotropine, CHCl3, 50 °C, then conc HClaq, EtOH, rt, 99% (over two steps); (iii) KSCN, AcOH, reflux, 50%; (iv) N-(2-bromoethyl)phthalimide, K2CO3, THF, reflux, 80%; (v) NaH, SEM-Cl, DMF, rt, 84%; (vi) N2H4*H2O, EtOH, 50 °C, 85%; (vii) propionic acid, TBTU, DIPEA, THF, rt, 91%; (viii) NBS, DMF, rt, 98%; (ix) 4-bromo-2-(2,5-dimethyl-1H-pyrrol-1-yl)pyridine, bis(pinacolato)diboron, KOAc, Pd(OAc)2, XPhos, K3PO4, 40%; (x) NH2OH*HCl, trimethylamine, EtOH/H2O, 45 °C, 37%; (xi) N-(3bromophenyl)acrylamide, Cs2CO3, Brettphos Pd G1, 1,4-dioxane/t-BuOH, 51%; (xii) TFA, DCM, rt, 73%. a

Scheme 5. Synthesis of Compounds 32−35a

a

Reagents and conditions: (i) Cs2CO3, Brettphos Pd G1, 1,4-dioxane/t-BuOH, 78−92%; (ii) TFA, DCM, 50−93%.

coupling reaction30 with 4-bromo-2-(2,5-dimethyl-1H-pyrrol-1yl)pyridine gave the dimethylpyrrole protected aminopyridine 71 in acceptable yield. Under very controlled conditions, the dimethylpyrrole protective group could be cleaved in good yield without hydrolysis of the aliphatic amide by the use of hydroxylamine hydrochloride at 45 °C under neutral conditions. Subsequent Pd catalyzed arylamination followed by acidic SEM deprotection gave the final compound 18 in acceptable yield.

bromoethyl)phthalimide under basic conditions. N-Protection of the imidazole core gave the SEM protected precursor 67, which was converted to the free aliphatic amine 68 by hydrazinolysis in excellent yield. Amide bond formation was performed by utilizing propionic acid, TBTU, and DIPEA in THF to obtain the corresponding amide 69 in excellent yield. Subsequent electrophilic bromination of the imidazole core followed by a one-pot, two-step tandem borylation Suzuki cross 5618



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Scheme 6. Synthesis of Compounds 31a and 31ba

Reagents and conditions: (i) SeO2, 1,4-dioxane/H2O, reflux, 73%; (ii) NaH, TIPSCl, THF, 0 °C to rt, quant; (iii) C2O2Cl2, DMSO, DCM, −78 °C to rt, 62%; (iv) NH4OAc, MeOH, rt, 51%; (v) NaH, SEMCl, DMF, 0 °C to rt, 64%; (vi) NBS, MeCN, 0 °C to rt, 86%; (vii) N-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)acetamide, X-Phos/Pd(OAc)2, K3PO4, 1,4-dioxane/H2O, 60 °C, 75%; (viii) 5 N NaOH(aq), 1,4dioxane/MeOH, 60 °C, 63%; (ix) substituted 3-bromoaniline, BrettPhos Pd G I, Cs2CO3, 1,4-dioxane/t-BuOH, 120 °C, 62% (acrylamide)/74% (propionyl chloride); (x) TFA,DCM, rt, 41% (acryloylamide)/ 56% (propionylamide). a

showed a slight gain of activity (81 nM) as well as 3 (83 nM), which is decorated with a hydroxyethyl group. The primary amine 5 showed an IC50 value of 29 nM, which displayed a 4-fold improvement of inhibitory activity when compared with compound 1 (130 nM). However, the morpholine moiety of 4 with an IC50 value of 1246 nM against EGFR-L858R/T790M was not tolerated. Amides 6a, 6b, and 7 were well tolerated and showed IC50 values of 38, 52, and 29 nM, respectively. The introduction of an acrylamide resulted in a slightly higher IC50 value for 6a in the L858R/T790M assay when compared with the free amine 5. In addition, the reversible counterpart 6b showed only a minor decrease of activity with an IC50 value of 52 nM. Supported by kinetic characterization, we identified 6a to be a weak covalent inhibitor of EGFR-L858R/T790M with a slow covalent binding rate of 0.09 min−1 (Table 1). When the amide was diminished to the corresponding ketone (10), we observed a dramatic loss of inhibitory activity (1328 nM). The ideal distance between hydrogen bond donors or acceptors to address the phosphate binding site appeared to be an ethylene linker, while the increased chain lengths of 11, 12, and 13 showed a marked decrease of activity displayed by IC50 values of 746, 240, and 321 nM, respectively. Our concepts to rigidize alkyl linkers (14−16) by cyclization did not result in higher inhibitory activities. For 9, the most potent compound in this series, we observed an IC50 value of 18 nM in the double-mutant EGFR L858R/T790M enzyme assay.

Compounds 32−35 were synthesized via Pd catalyzed arylamination with the appropriate bromoarenes followed by acidic SEM deprotection with TFA in DCM (Scheme 5). Compounds 31a,b (Scheme 6) were synthesized starting from commercially available p-fluoroacetophenone, which was converted to keto aldehyde 76 using selenium dioxide in a mixture of 1,4-dioxane and water under refluxing conditions in good yield. Cyclization with O-TIPS protected aldehyde 78 and ammonium acetate resulted in imidazole 79 in moderate yield. N-Protection of the imidazole core with SEM chloride followed by electrophilic bromination with NBS led to key intermediate 81 in good yields. Again, we used our previously optimized Suzuki cross coupling reaction with N-acetyl protected 2-aminopyridine-4-boronic acid pinacol ester for the C−C bond formation to obtain intermediate 82 in good yield. Subsequent Pd catalyzed arylamination with predecorated m-bromoanilines gave the SEM-protected precursors 84a,b in decent yields. The final SEM deprotection was achieved under acidic conditions (TFA in DCM) to give compounds 31a,b in moderate yields. Biological Evaluation. To evaluate the effect of substituents extending into the phosphate binding site, we characterized compounds 1−5 and 11−12 in an activity based enzyme assay against EGFR-wt, -L858R, as well as -L858R/T790M. We found increased activities against EGFR-L858R/T790M for compounds bearing an ethylene linker with a terminal heteroatom. Compound 2, bearing a dimethyl amide group in this position 5619



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Table 1. Determination of Inhibitory Activities against EGFR Wild-Type (wt), L858R, and L858R/T790M Mutants (Series I)

with the Asp855 side chain of the DFG motif stabilizes the flexible linker chain into an orientation directed away from Cys797. This results in a higher inhibitory activity in general but prevents a covalent interaction of the Michael acceptor. It is noteworthy that both docking poses yielded high docking scores and showed identical hinge binding patterns. Next, we attached the reactive Michael acceptor to the hinge binding pyridine core via arylamination. We assumed that this

With 7, we showed that the 4-fluoro substituent did not influence the biological activities. However, the additional 3-CF3 group of 8 was not tolerated and led to a dramatic decrease of activity (IC50 value = 646 nM). Figure 3 shows the different proposed binding modes of 6a modeled into an X-ray crystal structure of the L858R/T790M mutant applying covalent docking (A) and induced-fit docking (B). As depicted in Figure 3, the additional hydrogen bond of 6a 5620



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Figure 3. Different outcomes of modeling studies of 6a using covalent docking (A) and induced-fit docking (B) (PDB 2JIU). Covalent docking indicates a bond formation with Cys797 and bidentate hinge binding, while induced-fit docking indicates an additional hydrogen bond to Asp855, preventing the Michael acceptor from covalent binding.

Table 2. Determination of Inhibitory Activities against EGFR Wild-Type (wt), L858R, and L858R/T790M (Series 2)

approach toward a less flexible geometry would give a better preorientation of the Michael acceptor in close proximity to Cys797. Our initial approach included the decoration of the aniline residue of 1 in the meta position with a suitable Michael

acceptor. We anticipated that polar interactions would not direct the Michael acceptor-bearing group away from Cys797 in this series. With this approach, we were able to synthesize covalent inhibitors of EGFR mutants that showed subnanomolar IC50 5621



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To study the influence of the 4-fluorophenyl moiety in more detail, we synthesized compounds with various substituents in the imidazole 4-position (Table 4). To determine the SARs and the possibility of reduction of molecular size, we decorated the imidazole scaffold with several aromatic as well as aliphatic moieties with increasing sizes, hydrophobicity, and steric demands. We kept the optimized methoxy aniline acrylamide of 19a in this series unchanged to retain the geometry for a covalent interaction that produced highly active compounds in series 3 (Table 4). The exchange of the 4-fluorophenyl moiety with thiophene (24) and even with naphthalene (23) was well tolerated and led to compounds with high activities in all three tested enzyme assays. Compound 25a, which lacks a substituent in this position, showed high inhibitory activities with an IC50 value of 8.6 nM against the L858R/T790M mutant. However, the influence of the acrylamide on the inhibitory activity was much more pronounced for this compound. Thus, the reversible counterpart 25b showed a tremendous loss of activity. This result indicated the crucial role of the aromatic residue in 19b in this position, leading to high inhibitory activities independent of the covalent interaction of the acrylamide. Aliphatic residues showed an increasing trend of activity with growing ring size and spatial requirements. Hence, we observed an IC50 value of 4.5 nM against the L858R/T790M mutant for 29, bearing a cyclopentyl group in this position. However, the sterically more demanding tert-butyl group was not tolerated. In general, we could not obtain high activities for compounds bearing aromatic residues by substitution with alkyl moieties even though the most potent compound in this series, 29, showed a single-digit IC50 value in the L858R/T790M double-mutant enzyme assay. Combining the identified crucial features for efficient inhibiton, targeting the hydrophobic region next to the gatekeeper side chain as well as the phosphate binding site yielded compounds 31a,b. The covalent compound 31a, as well as the reversible counterpart 31b showed intense inhibition in the biochemical setting of below 1 nM, substantiating this approach. Because the reversible counterpart 31b showed no drop of activity when compared with the irreversible inhibitor 31a, T790M resistance could be mainly overcome by reversible binding affinities. This was further strengthened by a very low Ki value of 0.10 nM determined in EGFR-L858R/T790M, while the rate of covalent bond formation was 0.13 min−1. Thus, the high activity of 31a, decorated with a hydroxypropyl substituent in the imidazole 2position, was less dependent on a covalent interaction and mutation of Cys797 should not lead to resistances against this inhibitor. As depicted in Figure 4, our modeling studies of 31a with EGFR-T790M predicted a bidentate hinge binding of the aminopyridine and hydrophobic interactions of the fluorophenyl moiety, the aniline ring and the pyridine ring. Additionally, a polar contact between the terminal hydroxyl group of 31a with the Asp855 side chain could be seen. Most of our synthesized compounds showed very low aqueous solubility, thus we introduced solubilizing groups at the acrylamide-bearing aniline ring to increase aqueous solubility (Table 5). Both compounds 34 and 35 were barely soluble in a physiological phosphate buffer but showed high activities in the tested enzyme assays. Because substituents in this position are exposed to the solvent according to our docking model (Figure 4), we expected derivatization to be highly tolerated. Cellular Evaluation. To further investigate the potential for growth inhibition of the new sets of pyridinylimidazoles, we tested all compounds against the gefitinib-resistant H1975 cell line as well as against A431, derived from metastatic lung cancer

values against all three kinase variants (Table 2). 17a showed high inhibitory activities in the isolated enzyme assay of EGFR wild-type and the L858R mutant with IC50 values below 1 nM and an IC50 value of 3.3 nM when tested against the doublemutant L858R/T790M. However, the reversible analogue 17b showed a loss of inhibitory activities for all three kinases, indicating covalent binding mode of 17a. To further look into this, both compounds were incubated with EGFR-T790M and the mixture analyzed by mass spectrometry. Compound 17a showed an increase in mass as compared to a DMSO treated control sample, corresponding to covalent alkylation of the kinase. In contrast, we did not observe covalent labeling for the reversible counterpart 17b in this setting (Supporting Information, Figure S1). We next decorated the phenylenediamine moiety of 17a with an additional methoxy group in the ortho position as a gentle steric hindrance for the free torsion of the N−C bond. We expected that the methoxy group would direct the benzene ring, carrying the Michael acceptor into a greater out-of-plane conformation, providing a highly targeted orientation of the Michael acceptor toward Cys797. On the basis of this rational concept, we were able to generate 19a, a highly potent EGFR inhibitor displaying IC50 values below 1 nM against all three kinase variants. The covalent bond formation with EGFR-T790M was again shown by means of MS analysis (Supporting Information, Figure S1), and we furthermore characterized the binding kinetics. The rate of covalent bond formation was determined to be moderate, with a kinact = 0.16 min−1 in the context of EGFR-L858R/T790M, but we observed high reversible binding as represented by the Ki value of 1.2 nM (Table 3). Compound 19b, the reversible analogue, displayed Table 3. Determination of Kinetic Parameters Ki, kinact, and kinact/Ki for 6a, 19a, 31a, and osimertinib EGFR

Ki [nM]

kinact [min−1]

kinact/Ki [μM−1 s−1]

6a

wt L858R L858R/ T790M

1.6 ± 0.04 0.38 ± 0.00 6.0 ± 0.8

0.09 ± 0.004 0.04 ± 0.00 0.09 ± 0.01

0.94 ± 0.06 0.58 ± 0.02 0.25 ± 0.00

19a

wt L858R L858R/ T790M

0.35 ± 0.01 0.10 ± 0.01 1.2 ± 0.5

0.16 ± 0.05 0.03 ± 0.01 0.16 ± 0.07

2.8 ± 0.8 1.8 ± 0.9 2.2 ± 0.6

31a

wt L858R L858R/ T790M

0.07 ± 0.03 0.03 ± 0.003 0.10 ± 0.01

0.16 ± 0.06 0.10 ± 0.03 0.13 ± 0.003

15 ± 4 13 ± 3 11 ± 2

osimertinib

wt L858R L858R/ T790M

14 ± 2.3 1.6 ± 0.3 1.5 ± 0.1

0.43 ± 0.11 0.30 ± 0.01 0.33 ± 0.06

0.52 ± 0.05 3.2 ± 0.5 3.8 ± 0.4

compd

subnanomolar IC50 values in the wild-type EGFR as well as in the L858R assay but failed to overcome T790M resistance, as indicated by an IC50 value of 5.0 nM in the L858R/T790M assay. However, although 19b is a reversible binder, this compound showed high inhibitory activities in all three kinase assays. These findings support our initial hypothesis that addressing the hydrophobic regions of EGFR can yield potent reversible L858R/T790M inhibitors. 5622



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Table 4. Determination of Inhibitory Activities against EGFR Wild-Type (wt), L858R, and L858R/T790M (Series 3)

Figure 4. Left: Three-dimensional binding mode of 31a in a docking study with EGFR T790M (PDB 2JIU). Right: Pharmacophore model produced with the computer software ligandscout 4.11. Hydrogen bond acceptors are depicted as green arrows; hydrogen bond donors are depicted as red arrows. Hydrophobic interactions are depicted as yellow spheres.

cell line (EGFR-L858R/T790M) down to the low micromolar range. Thus, 13 showed a 17-fold gain in activity when compared with 1. However, this compound showed no selectivity over wildtype in this cellular setting. For 9, which showed an IC50 value of 18 nM in the isolated enzyme assay, we determined an EC50 value of >5 μM. We determined moderate activity (2.4 μM) for 6a against the H1975 cell line with a high selectivity over A431 (12

cells (Table 6). While the gefitinib-resistant H1975 cell line harbors the L858R and the T790M mutation, A431 cells overexpress wild-type EGFR. Growth inhibition of the A431 cell line is an indicator for wild-type inhibition-associated toxic side effects of the tested compounds. With compounds of series 1 (2−16), we were able to markedly improve the cellular activity against the gefitinib-resistant H1975 5623



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in the biochemical assays, we determined high selectivity over the A431 cell line (4.1 μM). Notably, compound 19a showed comparable inhibitory activity and an improved selectivity profile over wt in this cellular setting as compared to the gold standard osimertinib. The reversible counterpart 19b showed only poor activity (4 μM) in the cellular assay, indicating the crucial role of the irreversible inhibition in terms of cellular activity. The lack of the methoxy group in 17a resulted in a 10-fold higher EC50 value of 0.19 μM as compared to 19a. The substitution of the 4-fluoro phenyl ring with aliphatic residues (25−30) resulted in a dramatic loss of cellular activity in general. The presence of aliphatic groups may contribute to lower inhibitory activities as well as reduced cell permeability of these compounds, but this line of thought was not further investigated. Among the compounds with a solubilizing group, we observed only moderate activity of about 1 μM in H1975 cells, which could probably be further optimized by repositioning of the methylpiperazine moiety. Biochemical Evaluation against Osimertinib-Resistant EGFR-L858R/T790M/C797S Variant. Because we observed a high reversible binding character of our compounds as reflected in the Ki value of 1.2 nM for compound 19a and 0.10 nM for compound 31a, and because some of our reversible binding compounds (especially 31b) showed high inhibitory activities in the L858R/T790M assay, we assumed that they might inhibit a cysteine mutated EGFR variant as well (Table 7). To verify this hypothesis, we tested selected compounds in an activity based radiolabeled 33P(ATP) EGFR-L858R/T790M/C797S triplemutant kinase assay. This particular EGFR mutation is the leading cause for osimertinib resistance, as observed in the clinical setting, and besides an allosteric inhibitor,31 no highly active compounds for this target have been reported in the literature even though they represent a high unmet need. Indeed, compound 19a revealed a considerable activity of 90 nM, which could be further improved by the introduction of

Table 5. Determination of Inhibitory Activities against EGFR wt, L858R, and L858R/T790M (Series 4)

μM). Interestingly, 7 and 8 showed similar EC50 values in this cellular setting even though we determined a 22-fold lower IC50 value for 8 in the isolated enzyme assay. High activity in the H1975 (L858R/T790M) growth inhibition assay could be observed for 19a, showing an EC50 value of

Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically

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