Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically Resistant L858R/T790M/C797S EGFR Mutant: Targeting of Both Hydrophobic Regions and the Phosphate Binding Site

Article abstract of DOI:10.1021/acs.jmedchem.7b00316

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.

Full text of DOI:10.1021/acs.jmedchem.7b00316

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Article
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 Doering, Marina
Keul, Julian Engel, Hannah Lea Tumbrink, Daniel Rauh, and Stefan A. Laufer
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017
Downloaded from http://pubs.acs.org on June 12, 2017
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Trisubstituted Pyridinylimidazoles as Potent
Inhibitors of the Clinically Resistant
L858R/T790M/C797S EGFR Mutant: Targeting of
Both Hydrophobic Regions and the Phosphate
Binding Site
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1
,‡
2,‡
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Marcel Günther, Jonas Lategahn, Michael Juchum, Eva Döring, Marina Keul, Julian
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1,
Engel, Hannah L. Tumbrink, Daniel Rauh, and Stefan Laufer *
1
Institute of Pharmaceutical Sciences, Pharmaceutical and Medicinal Chemistry, Eberhard Karls
University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
2
Faculty of Chemistry and Chemical Biology, TU Dortmund University, OttoꢀHahnꢀStraße 4a,
4
4227 Dortmund, Germany
KEYWORDS. EGFR, cancer, medicinal chemistry, tyrosine kinase inhibitors, mutation
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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 longꢀterm treatment. The recently discovered EGFRꢀ
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
structureꢀactivity 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 nonꢀcovalent 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
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survival. 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
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nonꢀsmall cell lung cancer (NSCLC) in particular. Mutations in the EGFR kinase domain that
cause an increased and ligandꢀindependent catalytic activity, so called activating mutations, have
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been identified as oncogenic drivers for NSCLC. 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 of patients harbor a single point mutation in exon 21, the L858R mutation
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, 4
or the exon 19 deletion, del746_A750.
Patients bearing one of these activating mutations
generally respond well (50 – 80%) to a therapy based on first generation EGFR inhibitors
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, 6
(gefitinib or erlotinib) and these responders show significant tumor shrinkage in the clinic.
Suddenly, patients who have initially responded well to a TKI therapy develop secondary drug
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resistances during longꢀterm treatment. In approximately 60% of cases, the secondary mutation
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, 9
T790M has been identified in resistant patients.
First generation EGFR inhibitors show low
inhibitory activity for this mutant as well as for the double mutant L858R/T790M, which
indicates that this soꢀcalled 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
effects: significantly increased binding affinity of ATP to this mutant protein and the increased
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spatial requirement of methionine, compared to threonine, causes steric clashes with the 3ꢀ
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chloroꢀ4ꢀfluoroaniline residue of gefitinib. Second generation EGFR inhibitors, e.g., afatinib,
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have been designed to overcome T790M resistance by a covalent interaction with the protein.
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Thus, these compounds bear acrylamide moieties as Michael acceptors that can undergo
Michael addition reactions with the thiol group of a nonꢀcatalytic cysteine (Cys797). This
additional covalent interaction restores inhibitory activity while increasing target residence time.
Both effects result in significant tumor growth inhibition in preꢀclinical settings. However, the
clinical efficacy of second generation EGFR inhibitors was unsatisfying. Since second
generation EGFR inhibitors are nonꢀselective with respect to EGFR mutant inhibition, they are
potent cellular EGFR wildꢀtype (wt) inhibitors as well. As a consequence, these inhibitors
showed dose limiting toxic side effects which have been directly linked to wt inhibition,
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resulting in low tolerable, sub therapeutically concentrations in the clinic.
Third generation
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EGFR inhibitors, e.g., osimertinib
or Nꢀ(3ꢀ((5ꢀChloroꢀ2ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀmethylꢀ1ꢀ
1
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piperazinyl)phenyl)amino)ꢀ4ꢀpyrimidinyl)oxy)phenyl)ꢀ2ꢀpropenamide, 85 (WZ4002 ), have
been designed to overcome T790M resistance by (i) employing novel scaffolds, while (ii)
sparing the wildꢀtype to circumvent toxic sideꢀeffects and (iii) covalent alkylation of the reactive
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cysteine.
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. Since
this amino acid is nonꢀcatalytic and not conserved, it is not surprising that a C797S mutation has
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been identified to be one of the key drivers for osimertinib resistance in the clinic.
We
previously reported a small series of EGFR inhibitors based on a trisubstituted imidazole core as
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potent inhibitors that retain activity toward the cysteine mutant EGFR variant. In contrast to
third generation EGFR inhibitors, our compounds occupy a hydrophobic region (HR I) in the
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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
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consequence yields efficiency against EGFRꢀC797S. 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 position 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 EGFRꢀ
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 Hꢀbond 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 osimertinibꢀresistant L858R/T790M/C797S EGFR mutant variant.
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Figure 1: First, second and third generation EGFR inhibitors and their binding modes: (A) Twoꢀ
dimensional binding mode of osimertinib found in the xꢀray structure compared to
pyridinylimidazoles based on our molecular model. HR: hydrophobic region, PB: phosphate
binding site, PFG: pocketꢀfilling group. (B) Difference between the dihedral angle of gefitinib
and a vicinally substituted imidazole. (C) Generations of EGFR inhibitors occupying the ATP
binding site.
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RESULTS & DISCUSSION
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1
Rational Design of EGFR inhibitors. Starting from lead compound 1 , we performed a
rational design approach guided by molecular modeling. Docking studies predicted a bidentate
hinge binding motif between the 2ꢀaminopyridine moiety of compound 1 and Met793 when
docked into an Xꢀray structure of EGFRꢀL858R/T790M (Figure 2). Moreover, in this predicted
binding mode the 4ꢀfluorophenyl residue extended deep into HR I, occupying this hydrophobic
pocket. The phenyl ring of the aniline moiety was located in HR II. Since 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. Based on 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.
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Figure 2: Proposed threeꢀdimensional (left) and twoꢀdimensional (right) binding modes of lead
compound 1 in pdb 2JIU. Hydrogen bond acceptors are depicted as red arrows, hydrogen bond
donors as green arrows. Hydrophobic interactions are depicted as yellow spheres (3D model) or
yellow brackets (2D model).
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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 phosphateꢀbinding site and to study the structure activity relationship
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(SAR), we initially synthesized several derivatives with the capability to form additional
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hydrogen bonds, both as acceptors as well as donors. 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 carbonꢀsulfur 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). In order 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
towards Cys797. The concept of reducing conformational flexibility has been shown previously
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to result in improved inhibitory activity.
We studied the influence of the acrylamides on the
inhibitory activity by comparison with the reversible analogs, the respective propionamides. In
order to investigate the role of residues occupying HR I, we synthesized compounds bearing
various alkyl or aryl residues in the imidazole C4 position, having different polarities as well as
growing spatial requirements (23ꢀ30). Our findings led us to combine the most promising partial
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structures in a hybrid compound (31a). In previous studies, we examined whether the sulfur atom
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could be substituted with a carbon atom without any significant change in activity. 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.
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Chemistry. Compounds 1ꢀ13 were synthesized according to our previously reported synthetic
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route applying minor changes and optimizations (Scheme 1). Thus, the corresponding
commercially available benzoic acid esters were condensed with 2ꢀfluoroꢀ4ꢀpicoline using
NaHMDS as a strong nonꢀnucleophilic base in good yields. Subsequent nitrosation with sodium
nitrite in glacial acetic acid followed by reduction with zinc dust under acidic conditions yielded
αꢀaminoketones 38aꢀc as the corresponding hydrochloride salts after workup. Cyclization with
potassium thiocyanate in refluxing DMF resulted in thiones 39aꢀc in excellent yields. Key
intermediates 40aꢀc were synthesized by means of nucleophilic aromatic substitution reactions
with aniline under acidic conditions in refluxing NMP in good yields. Compounds 1ꢀ4 & 10ꢀ11
were synthesized by the application of nucleophilic substitution reactions employing the
corresponding alkyl halides and K CO as base in refluxing THF. To introduce aliphatic linkers
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to attach either a Michael acceptor as the corresponding acryl amide or the generic propionyl
amide, imidazole thiones 40aꢀc were decorated with aliphatic amines protected as phthalimides
under similar conditions. Compounds 41aꢀd were subsequently deprotected via hydrazinolysis
using hydrazine hydrate in EtOH at elevated temperature to give primary amines 5 & 12 in
decent yields. Amide couplings of 5, 12 & 42eꢀf with the corresponding carboxylic acids were
conducted using TBTU and DIPEA in THF to obtain 6aꢀ9 & 13 in moderate yields.
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Cyclic alkyl linkers 45aꢀb & 49 were synthesized according to Scheme 2. Commercially
available enantiomerically pure (R)ꢀ or (S)ꢀproline was Bocꢀprotected and subjected to reduction
with borane in THF, leading to the corresponding alcohols 44aꢀb. The following bromination
was achieved under Appel conditions using triphenylphosphine and carbon tetrabromide in THF
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giving 45aꢀb in good yields. Final compounds 14 & 15 were synthesized by nucleophilic
substitution reactions of 45aꢀb with 40a followed by Bocꢀdeprotection under acidic conditions
and subsequent amide coupling with acrylic acid in acceptable yields. Boc protection of 3ꢀ
hydroxypyrrolidine (racemic mixture) yielded intermediate 48 that was transformed into the
corresponding bromide 49 via Appel reaction using triphenylphosphine and CBr in good
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yields. 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.
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Scheme 1: Synthesis of trisubstituted imidazoles (1ꢀ13)a
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a
Reagents and conditions: (i) NaHMDS, THF, 0 °C – rt, 65–
79%; (ii) NaNO , HOAc, 10 °C, 84–97%; (iii) Zn dust, HOAc, 10 °C, 78–85%.; (iv) KSCN,
DMF, reflux, 73–84%; (v) aniline, NMP, conc. HCl , reflux, 63–88%; (vi) alkyl halide, K CO ,
THF, reflux, 48–79%; (vii) (n=2): Nꢀ(2ꢀBromoethyl)phthalimide, K CO , THF, reflux, (n=3): Nꢀ
3ꢀIodopropyl)phthalimide, K CO , THF, reflux, 57ꢀ81%; (viii) hydrazine hydrate, EtOH, 70 °C,
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5ꢀ65%; (ix) carboxylic acid, TBTU, DIPEA, THF, 27–63%.
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Scheme 2: Synthesis of trisubstituted imidazoles with cyclic alkyl linkers (14ꢀ16)
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a
Reagents and conditions: (i) BH *THF, THF, 0 °C 62 ꢀ 65%, (ii) PPh , CBr , rt, 65 ꢀ 71%;
3
3
4
(iii), 36a, K CO , THF, reflux, 76ꢀ80%; (iv) TFA, DCM, rt, 81ꢀ88%; (v) acrylic acid, TBTU,
DIPEA, THF, 28ꢀ37%; (vi) Boc O, neat, rt; DMAP, 90% (vii) PPh , CBr ,THF, 0 °Cꢀrt, 62%;
2
3
2
3
4
(
viii) 36a, K CO , THF, reflux, 84%.
2
3
Because we were not able to produce analogs of 40aꢀc using substituted anilines for a
nucleophilic aromatic substitution with thiones 39aꢀc, we established a different synthetic route
1
9
according to Scheme 3 based on a Suzuki cross coupling approach. First, we αꢀbrominated
commercially available ketones with bromine in methylene chloride (for aromatic ketones) or
methanol (for aliphatic ketones) to yield 52aꢀj. For the Sꢀmethyl series, we next cyclized these αꢀ
bromoketones with Sꢀmethyl isothiourea hemisulfate under mild basic conditions to give
5
3a & cꢀj in acceptable yields. The following SEMꢀprotection 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
2
9
temperature in good yields. Electrophilic bromination with NBS in acetonitrile at ꢀ30 °C
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yielded key intermediates 55a & cꢀj which were subjected to a high yielding Suzuki cross
coupling reaction with an Nꢀprotected 2ꢀaminoꢀpyridineꢀ4ꢀboronic acid ester under optimized
conditions. After deprotection of the amino pyridines 56a & cꢀj, the free amines 57a & cꢀj were
obtained in good yields. The subsequent Pd catalyzed aryl amination with preꢀdecorated mꢀ
bromoanilines gave precursors 58aꢀj & 59ꢀ63 that were deprotected under acidic conditions
using TFA to obtain final compounds 17aꢀb & 19ꢀ30 in moderate to excellent yields.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Scheme 3: Synthesis of compounds 17aꢀb & 19aꢀ30
a
Reagents and conditions: (i) Br , DCM or MeOH, rt, 64ꢀ95%; (ii) Sꢀmethylisothiourea
2
hemisulfate salt, NaHCO ,THF/H O, reflux, 15ꢀ65%; (iii) NaH, SEMꢀCl, THF, ꢀ15 °C,
3
2
quant;(iv) SEMꢀCl (5mol%), MeCN, 80 °C, 50ꢀ91%; (v) NBS, MeCN, ꢀ30 °C, 21ꢀ90%;
vi),corresponding boronic acid or acid ester, Pd(OAc) / XPhos or Pd (dba) / (tꢀBu) P*HBF ,
(
2
2
3
3
4
K PO , 1,4ꢀdioxane/H O, reflux, 54–91%; (vii) 5 M NaOH , MeOH, 60 °C, 63ꢀ92% or NH OH,
3
4
2
aq
2
DIPEA, ethanol, rt 53ꢀ63%or TFA, DCM, rt 88ꢀ99%; (viii) (hetero)arylbromide, Brettphos Pd
G1, Cs CO , 1,4ꢀdioxane/tꢀBuOH, 130 °C, 61ꢀ91%;(ix) TFA, DCM, rt, 55ꢀ86%.
2
3
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
For the synthesis of compound 18 (Scheme 4), αꢀbromoketone 47b was converted to the
corresponding αꢀaminoketone 64 via Delépine reaction and subsequently cyclized with
potassium thiocyanate under acidic conditions to give imidazole thione 65 in good yield. We
obtained the Nꢀprotected intermediate 66 by means of nucleophilic substitution of Nꢀ(2ꢀ
bromoethyl)phthalimide under basic conditions. NꢀProtection 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 oneꢀpot, twoꢀstep
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
0
tandem borylation Suzuki cross coupling reaction with 4ꢀbromoꢀ2ꢀ(2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ1ꢀ
yl)pyridine gave the dimethylpyrrole protected aminopyridine 70 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 SEMꢀ
deprotection gave the final compound 18 in acceptable yield.
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9
a
Scheme 4: Synthesis of compound 18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (i) Br , DCM, rt, 90%; (ii) urotropine, CHCl , 50 °C, then conc.
aq
2
3
HCl , EtOH, rt, 99% (over two steps); (iii) KSCN, AcOH, reflux, 50%; (iv) Nꢀ(2ꢀ
bromoethyl)phthalimide, K CO , THF, reflux, 80%; (v) NaH, SEMꢀCl, DMF, rt, 84%; (vi)
2
3
N H *H O, EtOH, 50 °C, 85%; (vii) propionic acid, TBTU, DIPEA, THF, rt, 91%; (viii) NBS,
2
4
2
DMF, rt, 98%; (ix) 4ꢀbromoꢀ2ꢀ(2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ1ꢀyl)pyridine, bis(pinacolato)diboron,
KOAc, Pd(OAc) , XPhos, K PO , 40%; (x) NH OH*HCl, trimethylamine, EtOH/H O, 45 °C,
37%; (xi) Nꢀ(3ꢀbromophenyl)acrylamide, Cs CO , Brettphos Pd G1, 1,4ꢀdioxane/tꢀBuOH, 51%;
2
3
4
2
2
2
3
(xii) TFA, DCM, rt, 73%.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compounds 32 – 35 were synthesized via Pd catalyzed aryla mination with the appropriate
bromo arenes followed by acidic SEM deprotection with TFA in DCM (Scheme 6).
a
Scheme 6: Synthesis of compounds 32 – 35
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
F
N
Br
R1
N
i)
ii)
S
S
32 - 35
N
N
N
SEM
R2
N
SEM
NH2
HN
R1
1
2
7
4a R = F, R = NO
2
1
2
74b R = N-methylpiperazine, R = NO
2
R2
1
R = N-methylpiperazine
74c
2
R = acryloylamino
75a-d
1
R = morpholine
74d
2
R = acryloylamino
a
Reagents and conditions: (i) Cs CO , Brettphos Pd G1, 1,4ꢀdioxane/tꢀBuOH, 78 ꢀ 92%; (ii)
2
3
TFA, DCM, 50 – 93%
Compounds 31aꢀb (Scheme 7) were synthesized starting from commercially available pꢀ
fluoroacetophenone which was converted to keto aldehyde 76 using selenium dioxide in a
mixture of 1,4ꢀdioxane and water under refluxing conditions in good yield. Cyclization with Oꢀ
TIPS protected aldehyde 78 and ammonium acetate resulted in imidazole 79 in moderate yield.
NꢀProtection 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 Nꢀacetyl protected 2ꢀaminopyridineꢀ4ꢀboronic acid pinacol
ester for the CꢀC bond formation to obtain intermediate 82 in good yield. Subsequent Pd
catalyzed arylamination with preꢀdecorated mꢀbromoanilines gave the SEMꢀprotected precursors
8
4aꢀb in decent yields. The final SEMꢀdeprotection was achieved under acidic conditions (TFA
in DCM) to give compounds 31aꢀb in moderate yields.
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a
Scheme 7: Synthesis of compounds 31a and 31b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (i) SeO , 1,4ꢀdioxane/H2O, reflux, 73 %; (ii) NaH, TIPSCl, THF, 0
2
°
C ꢀ rt, quant.; (iii) C O Cl , DMSO, DCM, ꢀ78 °C ꢀ rt, 62 %; (iv) NH OAc, MeOH, rt, 51 %;(v)
NaH, SEMCl, DMF, 0 °C ꢀ rt, 64 %; (vi) NBS, MeCN, 0 °C ꢀ rt, 86 %; (vii) Nꢀ(4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridinꢀ2ꢀyl)acetamide, XꢀPhos/Pd(OAc) , K PO , 1,4ꢀ
dioxane/H O, 60 °C, 75 %; (viii) 5 N NaOH(aq), 1,4ꢀdioxane/MeOH, 60 °C, 63 %; (ix)
substituted 3ꢀbromoaniline, BrettPhos Pd G I., Cs CO , 1,4ꢀdioxane/tꢀBuOH, 120 °C, 62 %
2 2 2 4
2
3
4
2
2
3
(
(
acrylamide)/ 74 % (propionyl chloride); (x) TFA,DCM, rt, 41 % (acryloylamide)/ 56 %
propionylamide).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Biological Evaluation
In order to evaluate the effect of substituents extending into the phosphate binding site, we
characterized compounds 1ꢀ5 and 11ꢀ12 in an activity based enzyme assay against EGFRꢀwt, ꢀ
L858R, as well as ꢀL858R/T790M. We found increased activities against EGFRꢀL858R/T790M
for compounds bearing an ethylene linker with a terminal heteroatom. Compound 2, bearing a
dimethyl amide group in this position 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 IC value
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
of 29 nM, which displayed a 4ꢀfold improvement of inhibitory activity when compared with
compound 1 (130 nM). However the morpholine moiety of 4 with an IC value of 1246 nM
50
against EGFRꢀL858R/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 IC value for 6a in the L858R/T790M assay, when compared with
5
0
the free amine 5. In addition, the reversible counterpart 6b showed only a minor decrease of
activity with an IC value of 52 nM. Supported by kinetic characterization, we identified 6a to
5
0
be a weak covalent inhibitor of EGFRꢀL858R/T790M with a slow covalent binding rate of
ꢀ1
0
.09 min (Table 3). 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 IC values of 746, 240 and 321 nM, respectively.
50
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Page 19 of 98
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9
Table 1: Determination of inhibitory activities against EGFR wildꢀtype (wt), L858R and
L858R/T790M mutants (series I).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EGFR IC [nM]
5
0
1
2
Compound
R
R
wt
L858R
L858R/T790M
1
2
4ꢀFꢀ
4ꢀFꢀ
4ꢀFꢀ
4ꢀFꢀ
4ꢀFꢀ
4ꢀFꢀ
4ꢀFꢀ
H
24 ± 3
2.5 ± 0.3
130 ± 25
9.0 ± 2.4
11 ± 3
1.0 ± 0.1
1.1 ± 0.1
75 ± 57
81 ± 8
83 ± 3
3
4
183 ± 160
4.8 ± 0.7
1.6 ± 0.5
33 ± 13
1246 ± 192
29 ±12
5
2.6 ± 1.1
2.2 ± 0.6
3.2 ± 2.3
1.7 ± 0.9
144 ± 52
1.9 ± 1.2
76 ± 41
6a
6b
7
38 ± 10
52 ± 26
29 ± 6
12 ± 5
8
3ꢀCF
3
ꢀ4ꢀFꢀ
45 ± 37
646 ± 50
18 ± 2
9
4ꢀFꢀ
4ꢀFꢀ
4ꢀFꢀ
2.7 ± 1.0
185 ± 138
109 ± 40
1
0
1
1328 ± 886
746 ± 345
1
40 ± 24
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
4ꢀFꢀ
4ꢀFꢀ
4ꢀF
55 ± 35
16 ± 3
28 ± 3
19 ± 5
12 ± 11
22 ± 4
240 ± 104
13
321 ± 35
76 ± 12
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14
15
16
3.1 ± 0.9
1.9 ± 0.4
25 ± 5
4ꢀFꢀ
4ꢀFꢀ
66 ± 14
N
O
217 ± 58
417 ± 167
H
gefitinib
afatinib
< 1
< 1
< 1
< 1
185 ± 98
< 1
8
5
11 ± 7
1.6 ± 0.2
< 1
< 1
osimertinib
1.3 ± 0.5
< 1
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 IC value of 18 nM in
5
0
the double mutant EGFR L858R/T790M enzyme assay. With 7, we showed that the 4ꢀfluoro
substituent did not influence the biological activities. However, the additional 3ꢀCF group of 8
3
was not tolerated and led to a dramatic decrease of activity (IC value = 646 nM).
5
0
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Figure 3 shows the different proposed binding modes of 6a modeled into an Xꢀray crystal
structure of the L858R/T790M mutant applying covalent docking (A) and inducedꢀfit docking
(
B). As depicted in Figure 3, the additional hydrogen bond of 6a with the Asp855 side chain of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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.
A
B
Figure 3: Different outcomes of modeling studies of 6a using covalent docking (A) and inducedꢀ
fit docking (B) (PDBꢀcode: 2JIU). Covalent docking indicates a bond formation with Cys797
and bidentate hinge binding, while inducedꢀfit docking indicates an additional hydrogen bond to
Asp855, preventing the Michaelꢀacceptor from covalent binding.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2: Determination of inhibitory activities against EGFR wildꢀtype (wt), ꢀL858R and ꢀ
L858R/T790M (series 2)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EGFR IC [nM]
5
0
1
2
3
Cpd
R
R
R
wt
L858R
L858R/T790M
1
7a
CH
3
3
< 1
< 1
3.3 ± 1.5
17b
CH
22 ± 11
< 1
6.8 ± 7.8
< 1
93 ± 37
2.5 ± 1.1
< 1
1
8
1
9a
CH
3
3
< 1
< 1
1
9b
CH
< 1
< 1
< 1
< 1
5.0 ±0.9
3.1 ± 0.5
89 ± 32
20
CH
3
3
2
1
2
CH
16 ± 8
4.9 ± 2.0
2
CH
3
164 ± 99
46 ± 32
597 ± 279
gefitinib
afatinib
< 1
< 1
< 1
< 1
185 ± 98
< 1
8
5
11 ± 7
1.6 ± 0.2
< 1
< 1
osimertinib
1.3 ± 0.5
< 1
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Page 23 of 98
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9
Next, we attached the reactive Michael acceptor to the hinge binding pyridine core via
arylamination. We assumed that this approach towards a less flexible geometry would give a
better preꢀorientation 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 acceptorꢀbearing
group away from Cys797 in this series. With this approach we were able to synthesize covalent
inhibitors of EGFR mutants that showed subꢀnanomolar IC50 values against all three kinase
variants (Table 2). 17a showed high inhibitory activities in the isolated enzyme assay of EGFR
wildꢀtype and the L858R mutant with IC values below 1 nM and an IC value of 3.3 nM when
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
0
50
tested against the double mutant L858R/T790M. However, the reversible analogue 17b showed a
loss of inhibitory activities for all three kinases indicating covalent binding mode of 17a. In order
to further look into this, both compounds were incubated with EGFRꢀT790M 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 (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 NꢀC bond. We expected that
the methoxy group would direct the benzene ring, carrying the Michael acceptor into a greater
outꢀofꢀplane conformation providing a highly targeted orientation of the Michael acceptor
toward Cys797. Based on this rational concept, we were able to generate 19a, a highly potent
EGFR inhibitor displaying IC values below 1 nM against all three kinase variants. The covalent
50
bond formation with EGFRꢀT790M was again shown by means of MSꢀanalysis (Figure S1) and
we furthermore characterized the binding kinetics. The rate of covalent bond formation was
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ1
determined to be moderate with a kinact = 0.16 min in the context of EGFRꢀL858R/T790M but
we observed high reversible binding as represented by the K value of 1.2 nM (Table 3).
i
Compound 19b, the reversible analogue, displayed subnanomolar IC values in the wildꢀtype
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EGFR as well as in the L858R assay, but failed to overcome T790M resistance, as indicated by
an IC value of 5.0 nM in the L858R/T790M assay. However, although 19b is a reversible
5
0
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.
Table 3: Determination of kinetic parameters K , k
and kinact/K for 6a, 19a, 31a and
i
i
inact
osimertinib.
ꢀ
1
ꢀ1 ꢀ1
Compound
EGFR
wt
K [nM]
kinact [min ]
0.09 ± 0.004
0.04 ± 0.00
0.09 ± 0.01
0.16 ± 0.05
0.03 ± 0.01
0.16 ± 0.07
0.16 ± 0.06
0.10 ± 0.03
0.13 ± 0.003
0.43 ± 0.11
0.30 ± 0.01
kinact/K [µM s ]
i
i
1.6 ± 0.04
0.38 ± 0.00
6.0 ± 0.8
0.94 ± 0.06
0.58 ± 0.02
0.25 ± 0.00
2.8 ± 0.8
1.8 ± 0.9
2.2 ± 0.6
15 ± 4
6a
L858R
L858R/T790M
wt
0.35 ± 0.01
0.10 ± 0.01
1.2 ± 0.5
1
3
9a
1a
L858R
L858R/T790M
wt
0.07 ± 0.03
0.03 ± 0.003
0.10 ± 0.01
14 ± 2.3
L858R
13 ± 3
L858R/T790M
wt
11 ± 2
0.52 ± 0.05
3.2 ± 0.5
osimertinib
L858R
1.6 ± 0.3
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L858R/T790M
1.5 ± 0.1
0.33 ± 0.06
3.8 ± 0.4
Table 4: Determination of inhibitory activities against EGFR wildꢀtype (wt), ꢀL858R and ꢀ
L858R/T790M (series 3)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
3
EGFR IC [nM]
Cpd
R
R
R
50
wt
L858R L858R/T7
2
3
4
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
3
3
3
3
3
3
3
3
3
< 1
< 1
< 1
1.1 ± 0.0
< 1
2
< 1
25a
H
H
17 ± 3
5.6 ± 0.9 8.6 ± 1.1
360 ± 19 3511 ± 698
1
767 ±
25b
310
26
32 ± 3
2.3 ± 0.3
< 1
10 ± 2
< 1
33 ± 5
27
7.4 ± 0.9
6.8 ± 0.6
4.5 ± 0.5
485 ± 47
28
29
30
< 1
< 1
< 1
57 ± 4
16 ± 5
31a
< 1
< 1
< 1
< 1
< 1
< 1
31b
gefitinib
afatinib
< 1
< 1
< 1
< 1
185 ± 98
< 1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
5
11 ± 7
1.6 ± 0.2 1.3 ± 0.5
< 1
< 1
< 1
osimertinib
To study the influence of the 4ꢀfluorophenyl moiety in more detail, we synthesized compounds
with various substituents in the imidazole 4ꢀposition (Table 4). In order 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 4ꢀfluorophenyl 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 IC value of 8.6 nM against the L858R/T790M mutant. However,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
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 tertꢀbutyl 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 IC value in the L858R/T790M double mutant enzyme assay.
50
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 31aꢀ
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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
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
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41
42
43
44
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47
48
49
50
51
52
53
54
55
56
57
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was further strengthened by a very low K value of 0.10 nM determined in EGFRꢀ
i
ꢀ1
L858R/T790M, while the rate of covalent bond formation was 0.13 min . Thus, the high activity
of 31a, decorated with a hydroxypropyl substituent in the imidazole 2ꢀposition, 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 EGFRꢀT790M
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.
Figure 4: Left: threeꢀdimensional binding mode of 31a in a docking study with EGFR T790M
(pdbꢀcode: 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
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
as red arrows. Hydrophobic interactions are depicted as yellow spheres. Most of our synthesized
compounds showed very low aqueous solubility, thus we introduced solubilizing groups at the
acrylamideꢀbearing aniline ring to increase aqueous solubility (Table 5).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5: Determination of inhibitory activities against EGFR wt, L858R and L858R/T790M
(series 4)
EGFR IC [nM]
Cpd
9a
R
50
wt
L858R
L858R/T790M
1
< 1
< 1
< 1
3
2
55 ± 11
586 ± 144
12 ± 1
2
65 ± 66
3
3
2.1 ± 1.1
< 1
3
4
5
< 1
< 1
< 1
< 1
< 1
< 1
3
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