- Email: [email protected]
Balancing reactivity and antitumor activity: heteroarylthioacetamide derivatives as potent and time-dependent inhibitors of EGFR
Accepted Manuscript Balancing reactivity and antitumor activity: Heteroarylthio acetamide derivatives as potent and time-dependent inhibitors of EGFR Riccardo Castelli, Nicole Bozza, Andrea Cavazzoni, Mara Bonelli, Federica Vacondio, Francesca Ferlenghi, Donatella Callegari, Claudia Silva, Silvia Rivara, Alessio Lodola, Graziana Digiacomo, Claudia Fumarola, Roberta Alfieri, Pier Giorgio Petronini, Marco Mor PII:
S0223-5234(18)30988-7
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.029
Reference:
EJMECH 10886
To appear in:
European Journal of Medicinal Chemistry
Received Date: 9 July 2018 Revised Date:
8 November 2018
Accepted Date: 9 November 2018
Please cite this article as: R. Castelli, N. Bozza, A. Cavazzoni, M. Bonelli, F. Vacondio, F. Ferlenghi, D. Callegari, C. Silva, S. Rivara, A. Lodola, G. Digiacomo, C. Fumarola, R. Alfieri, P.G. Petronini, M. Mor, Balancing reactivity and antitumor activity: Heteroarylthio acetamide derivatives as potent and time-dependent inhibitors of EGFR, European Journal of Medicinal Chemistry (2018), doi: https:// doi.org/10.1016/j.ejmech.2018.11.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Balancing reactivity and antitumor activity: heteroarylthio acetamide derivatives as potent and
RI PT
time-dependent inhibitors of EGFR
Riccardo Castelli,‡1 Nicole Bozza,‡1 Andrea Cavazzoni,2 Mara Bonelli,2 Federica Vacondio,1 Francesca Ferlenghi,1 Donatella Callegari,1 Claudia Silva,1 Silvia Rivara,1 Alessio Lodola,*1
1
2
Department of Food and Drug, University of Parma, Parma, Italy
Department of Medicine and Surgery, University of Parma, Parma, Italy
These authors contributed equally to this work
*Corresponding Authors
EP
Prof. Alessio Lodola,
TE D
‡
M AN U
Mor*1
SC
Graziana Digiacomo,2 Claudia Fumarola,2 Roberta Alfieri,2 Pier Giorgio Petronini,2 and Marco
Department of Food and Drug, University of Parma, Parma, Italy
&
AC C
E-mail: [email protected]
Prof. Marco Mor,
Department of Food and Drug, University of Parma, Parma, Italy E-mail: [email protected]
1
ACCEPTED MANUSCRIPT
ABSTRACT
Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates
RI PT
Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-
SC
yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by
M AN U
replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a
TE D
time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in
concentration.
KEYWORDS
4-anilinoquinazoline;
warhead;
cysteine;
time-dependent
inhibition;
QM/MM
AC C
EGFR;
EP
gefitinib-resistant H1975 (EGFR L858R/T790M) lung cancer cells at low micromolar
simulations.
Abbreviations
DMF, dimethylformamide; PDDG, Pairwise Distance Directed Gaussian; QM/MM, quantum mechanics/molecular mechanics; SMD, steered molecular dynamics; TBTU, O-(benzotriazol-1yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate; THF, tetrahydrofuran; TS, transition state.
2
ACCEPTED MANUSCRIPT
1. INTRODUCTION
RI PT
Non-small-cell lung cancer (NSCLC) accounts for the 80% of all lung cancers, which represent the leading cause of cancer-related deaths worldwide. NSCLC is featured by a 5-year survival rate lower than 15% [1]. Gefitinib (1) and erlotinib (2) are the first generation of EGFR
SC
inhibitors approved for the treatment of NSCLC [2]. Tumor responsiveness to these drugs has been related to the insurgence of “activating mutations” such as short in-frame deletions in exon
M AN U
19 (del19) or point mutations in exon 21, the latter resulting in arginine replacing leucine at codon 858 (L858R) [3,4]. Gefitinib and erlotinib display a remarkable therapeutic outcome in patients expressing activated forms of EGFR. However, their efficacy in clinical use is limited by the occurrence of resistance. Nearly 60% of patients with EGFR alterations present a single
TE D
aminoacid mutation at the gatekeeper position of the ATP-binding site, from threonine to methionine (T790M) [5]. The presence of this bulkier methionine reduced the potency of first generation inhibitors for EGFR, favoring their competitive displacement by ATP, which is
EP
present at millimolar concentrations inside the cells [6]. This resistance mechanism has been overcome by equipping the scaffold of 4-anilinoquinazoline inhibitors with an acrylamide
AC C
warhead: by virtue of a hetero-Michael addition, the acrylamide moiety is capable of alkylating a cysteine residue (i.e. Cys797) proximal to the hinge region of the EGFR active site [7,8]. The formation of a covalent bond with Cys797 allowed second-generation inhibitors to exert protracted occupancy of EGFR active site, overcoming competition with ATP [9]. Among the second-generation inhibitors that entered into clinical studies, afatinib (compound 3) was approved for the treatment of patients with metastatic NSCLC with EGFR mutations [10]. In patients with acquired resistance to gefitinib (1) through the development of T790M mutation,
3
ACCEPTED MANUSCRIPT
afatinib (3) showed marginal therapeutic benefit. Its clinical use has been restricted by adverse effects occurring in skin and gastrointestinal tract, resulting from the concomitant inhibition of the wild type (WT) form of EGFR [11]. The disclosure of pyrimidine-based inhibitors [12],
RI PT
selective for the T790M mutant over the WT, prompted the search for new candidates to be advanced into the clinic [13]. Osimertinib (4) [14,15], the leader of this third generation of EGFR inhibitors [16], is a 2-aminopyrimidine derivative featuring an acrylamide warhead portion. It
SC
has been approved for patients affected by metastatic EGFR T790M mutation-positive NSCLC who have progressed on after the therapy with first- and second-generation inhibitors [17], and
M AN U
very recently for the first-line treatment of patients with metastatic NSCLC carrying EGFRactivating mutation [18]. However, mutations conferring resistance to osimertinib have been reported [19]. Among the identified mutations, C797S, in which the critical cysteine 797 is replaced by a less nucleophilic serine, directly hampers EGFR alkylation, while loss of T790M
TE D
or appearance of L718Q mutation may affect the accommodation of the inhibitor in the active site [20], preventing the formation of a protein-ligand reactive conformation that leads to target alkylation [21]. These findings indicate that both the acrylamide group and the driving portion
EP
need to be optimized to ensure selective inhibition of activated forms of EGFR (i.e. L858R, or del19) in the presence of novel mutations. Moreover, the presence of a highly electrophilic
AC C
warhead (i.e. as for acrylamide) remains a toxicity burden [22]. A recent work on osimertinib has shown that it inhibits several lysosomal cathepsins when administered to mice [23]. Considering that cathepsins targeted by osimertinib are also expressed by human cells, some concerns have been raised on the protracted use of osimertinib.
4
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 1. Chemical structures of selected EGFR inhibitors.
In this scenario, the availability of warheads able to form a covalent bond with Cys797 [24],
TE D
but less reactive than acrylamide present in EGFR inhibitors, would facilitate the design of novel and selective inhibitors able to overcome resistance in NSCLC [25, 26]. Our previous work, performed installing new warheads on a 4-anilinoquinazoline nucleus, has led to the
EP
identification of long-lasting inhibitors of EGFR having electrophilic groups other than acrylamide [27]. However, epoxide-based inhibitor 5 (Fig. 1) raised safety concerns similar to
AC C
those of acrylamides, while the 2-(pentafluorophenoxy)-acetamide 6 resulted incapable to overcome T790M resistance. Herein, using a 4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline scaffold (compound 7, Scheme 1) featured by a substitution pattern similar to that of the clinically approved drug afatinib (3, Fig. 1), we prepared a series of compounds possessing at the position 6 an “activated” acetamide group, which may potentially alkylate Cys797 by a nucleophilic substitution (SN). Our investigation started with the synthesis of a 4-(3-chloro-4fluoroaniline)-7-ethoxyquinazoline bearing a chloroacetamide at position 6 (compound 8,
5
ACCEPTED MANUSCRIPT
Scheme 1). The chloroacetamide was selected as the reference warhead for our work as this group has been reported to alkylate cysteine both in solution [28] and within the ATP binding site of kinases [29] and it has been already installed on a different 4-anilinoquinazoline to
RI PT
achieve irreversible inhibition of EGFR [30]. As consequent step of our research, we modulated the reactivity of the acetamide portion by replacing the chlorine atom in 8 with (hetero)-aromatic thiols or carboxylate esters that may act as leaving groups in the framework of a SN reaction. Our
from the
necessity
of
having
compounds
with
SC
choice of using the 4-anilinoquinazoline nucleus for the installation of the new warheads arises good
inhibitory potency in
the
M AN U
autophosphorylation assay performed on A549 cells, which expressed WT EGFR. This test was used as a screening method to identify potential irreversible inhibitors of EGFR. Regardless of the chemical nature of the driver portion employed to target the ATP binding site of EGFR, our approach can lead to the identification of soft warheads able to bind the reactive cysteine
2. CHEMISTRY
TE D
(Cys797) of EGFR, while sparing all other thiols present in the cellular environment.
EP
The newly synthesized inhibitors reported in this work (Scheme 1) feature the 6-amino-4-(3-
AC C
chloro-4-fluoroaniline)-7-ethoxyquinazoline core (7) equipped with specific amide-group based warheads obtained by means of acylation of the 6-amino group with a panel of “electrophilic traps”. The synthesis of the anilinoquinazoline scaffold 7 was performed as previously reported with minor adaptations (see Supplementary Data) [31]. The general strategy features the chloroacetamide-functionalized quinazoline core (compound 8) as starting material in the synthesis of most of the compounds of the class. Compound 8 was prepared by reacting the amino group of 7 with the mixed-anhydride obtained from the reaction of the trialkyl ammonium
6
ACCEPTED MANUSCRIPT
salt of chloroacetic acid with pivaloyl chloride. This method proved superior to the direct use of chloroacetyl chloride both in terms of purity of the final compound and overall yield, and allowed the use of tetrahydrofuran as the solvent, being 7 sparingly soluble in either chlorinated
RI PT
solvents and acetonitrile. The obtained chloroacetyl-functionalized anilinoquinazoline 8 was further elaborated by reacting with the appropriate nucleophile to furnish compounds 10, 12, 1422, as depicted in Scheme 1. The general procedure for this subset of compounds features the
AC C
EP
TE D
M AN U
SC
alkylation of the heterocyclic thiol or carboxylate salt by 8 in the presence of a base.
Scheme 1. Reagents and conditions: a) i. chloroacetic acid, diisopropylethylamine, THF; ii. pivaloyl chloride, 30 min; iii. 7, room temperature (yield 83%); b) See Experimental Section for
7
ACCEPTED MANUSCRIPT
specific conditions (yield range: 19-89%); c) 3-pyridylmercaptoacetic acid, TBTU, DMF, triethylamine, room temperature, (for 11, yield: 25%); 3-pyridin-3-yl-propionyl chloride, THF, pyridine, room temperature, (for 13, yield: 39%); d) NaOCH3, THF, CH3OH, room temperature
RI PT
(23, yield: 62%).
The exact conditions for each compound varied extensively depending on the reactivity of
SC
each thiol or carboxylate salt, details are given in the Experimental Section. The synthesis of compounds 11 and 13 had to be devised differently, and was performed condensing 7 and 3acid
in
the
presence
of
O-(benzotriazol-1-yl)-N,N,N’,N’-
M AN U
pyridylmercaptoacetic
tetramethyluronium tetrafluoroborate (TBTU) for 11 and 3-pyridin-3-yl-propionyl chloride for 13 respectively. Compound 23 (the derivative of glycolic acid) was derived from 20 (the acetoxy-derivative) by means of a transesterification reaction performed with a substoichiometric
TE D
amount of sodium methoxide in THF and methanol.
EP
3. RESULTS AND DISCUSSION
3.1. Reactivity with cysteine in solution
AC C
We set to investigate whether our newly prepared warheads could indeed engage in a nucleophilic substitution, and evaluate their potential reactivity in a purely chemical setting, in order to obtain a scale of relative reactivity. Quinazoline derivatives 8-23 were tested for their ability to undergo a nucleophilic substitution in the presence of a large molar excess (1:100) of cysteine at a 10 µM concentration in 50 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS; pH 10) buffer at 37 °C (Table 1). We set the pH of the buffer to 10 in order to have the thiol
8
ACCEPTED MANUSCRIPT
group of cysteine (pKa = 8.5 [32]) predominantly in its thiolate form and thus more prone to react with electrophiles. Consumption of the starting compounds and formation of the corresponding cysteine-
acrylamide
RI PT
conjugate were monitored by LC-MS, adopting our previously reported protocol [33]. An derivative
(N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-
yl)acrylamide, compound 9 [31a]) was included in this study as a benchmark, for its established
SC
reactivity towards thiols. Acrylamide derivatives have high propensity to react with several sites. This results in a high non-specific binding in cells, also at rather low compound concentration
acrylamide group [34].
M AN U
(i.e. 0.06 µM) as reported for [18F]-ML04, a labelled 4-anilinoquinazoline inhibitor bearing a 6This further stress the importance of finding chemical selective
warheads. Compound 8 was included considering the ability of the chloroacetamide group to alkylate cysteines [28, 29]. Chloroacetamide derivatives have been recently shown to exhibit low
TE D
cross-reactivity versus a panel of eleven enzymes, suggesting that chloroacetamide itself could be intrinsically less promiscuous than what initially expected in the presence of biological nucleophiles [35].
EP
Both the acrylamide 9 and the chloroacetamide 8 reacted with a large molar excess of cysteine at pH 10 following pseudo first-order kinetics. LC-UV analysis showed that the disappearance of
AC C
compound 8 paralleled the formation of the cysteine-conjugate (see Supplementary Data, Fig. S1) indicating the absence of competing reactions (e.g. hydrolysis). Compound 8 displayed a half-life time (t1/2) of nearly 60 min (Table 1), resulting 4-fold less reactive than the acrylamide analogue 9 (t1/2 = 15 min). Additionally, stability assays performed at pH 10 confirmed that both compounds were stable for the tested time period. As opposed to 8 and 9, all the other compounds reported in Table 1 reacted with cysteine either very slowly (t1/2 > 1440 min for
9
ACCEPTED MANUSCRIPT
compounds 10, 12, 14-22) or not at all (compounds 11, 13, 23), thereby precluding a quantitative ranking of their relative electrophilicity. To qualitatively rank them, the area of the peak of the cysteine-conjugate was measured by LC-MS after 24 h of reaction time (see Supplementary
RI PT
Data, Fig. S2), and used as a relative descriptor of reactivity. The area of the covalent conjugate given between 8 and cysteine was arbitrarily set at the reference value of 10,000, while the cysteine-conjugate areas for the other compounds were referred to this value. The 2-((1H-
SC
tetrazol-5-yl)thio)acetamide 14 and the 2-((1H-imidazol-2-yl)thio)acetamides 15 and 16 resulted significantly more reactive than the 2-((pyridin-2-yl)thio)acetamide 10, the 2-((pyrimidin-2-
M AN U
yl)thio)acetamide 12 and the 2-((1-methyl-benzimidazol-2-yl)thio)acetamide derivative 17, as well as than its analogues 18 and 19. For 2-((pyridin-3-yl)thio)acetamide 11 and 3-(pyridin-3yl)-propanamide 13 no cysteine-conjugate could be observed. Among the carboxylate esters, 20, 21 and 22, the benzoyloxyacetamide derivative 21 showed a reactivity comparable to that of the
TE D
2-((1H-imidazol-2-yl)thio)acetamide 16, while the acetoxy derivative 20 resulted one of the less reactive compounds of the whole set. Additionally, for esters 20 and 21 significant amounts of the hydrolyzed product (glycolate derivative, compound 23) could be observed after 24 h.
EP
Compound 13, in which the sulfur atom on the acetamide portion is replaced by a methylene unit, expectedly failed to give the conjugate, as well as 11, where the sulfur atom is not in the 2-
AC C
position of a nitrogen-containing heterocycle. Moreover, the hydroxyacetamide 23 did not show any conjugate when incubated with cysteine. As an additional test, we measured the chemical stability of the cysteine-conjugate, obtained in situ by incubating a prototypical acetamide (i.e. 2-chloroacetamide 8) with a large excess of cysteine, under the conditions of the reactivity assay (see above) by LC-MS. The cysteineconjugate was stable for at least 24 hours as evidenced by Fig. S3 of the supplementary data
10
ACCEPTED MANUSCRIPT
section. This result indicates that cysteine alkylation by activated acetamide is an irreversible process.
RI PT
3.2. Simulation of Cys797 alkylation by QM/MM simulations To support the hypothesis of covalent bond formation within EGFR, we evaluated whether the putative warheads could undergo attack by Cys797 via a SN2 reaction pathway by means of a
SC
quantum mechanics/molecular mechanics (QM/MM) approach [36]. The reaction mechanism (depicted in Fig. 2) was simulated for the chloroacetamide 8 and for the two 2-(imidazol-2-
M AN U
ylthio)acetamide derivatives 15 and 16, which emerged as the most promising compounds in the reactivity assay. QM/MM mechanistic modelling is currently providing useful indications for inhibitor design specifically when the formation and breakage of covalent bonds are involved in the inhibitory process [37]. Models of enzyme-ligand non-covalent complexes were built by docking 8, 15 and 16 within the ATP-binding pocket of the X-ray structure of the covalent
TE D
adduct of WT EGFR with a N-(4-anilinoquinazolin-6-yl) acrylamide inhibitor [8]. Previous calculations performed by our group [38] suggest that Cys797 can act as nucleophile in the form of thiolate anion, with the nearby Asp800 as the natural acceptor of the thiol hydrogen. The
EP
Cys797/Asp800 pair was therefore modelled in Cys-S-/Asp-COOH form (as reported in Fig. 2A).
AC C
Among the obtained docking poses, we selected those in which the quinazoline portion of the inhibitors well fitted the hinge region of the ATP binding site, and the angle formed by the sulfur atom of Cys797, the Csp3 of the acetamide fragment and the leaving group atom (i.e., chlorine for 8, sulfur for 15 and 16) was higher than 130°. Such an angle approaches the value of 180° in an ideal transition state (TS) for a SN2 reaction. The docking complexes were solvated and equilibrated by classic molecular dynamics simulations (see Experimental Section for details). The relaxed structures were then employed to model the SN2 reaction by means of a PDDG/PM3
11
ACCEPTED MANUSCRIPT
Hamiltonian [39] coupled to the AMBER99SB potential [40]. The PDDG/PM3 Hamiltonian has been shown to yield energy barriers for SN2 reactions in gas phase similar to those calculated by ab initio methods [41]. In our approach, the substituted acetamide fragment of the inhibitor and
RI PT
the side chains of Cys797 and of Asp800 were treated at PDDG/PM3 level, while the remaining part of the system was described by AMBER99SB force field [42]. To account for solvation and structural flexibility of both EGFR active site and inhibitor, the hybrid PDDG/PM3-
SC
AMBER99SB Hamiltonian was used in steered molecular dynamics (SMD) simulations. To simulate the nucleophilic substitution involving Cys797, we applied the SMD approach
M AN U
implemented in AMBER, applying an adaptive force along a given reaction coordinate (RC) to form or break covalent bonds, similarly to what described in reference [43]. In the case of compound 8, thiolate addition and chlorine expulsion were modeled using the difference of distances [d(Cαwarhead − Clwarhead) - d(Scys797 − Cαwarhead)]. For compounds 15 and 16, cysteine
TE D
thiolate attack and imidazol-2-ylthiolate expulsion were described by the difference [d(Cαwarhead − Swarhead) - d(Scys797 − Cαwarhead)]. While for compound 8 our QM/MM steered-MD approach led to the identification of a stable alkylation product with low free-energy, this was not the case for
EP
compounds 15 and 16 (data not shown). We thus hypothesized that protonation at the imidazole ring could stabilize the imidazolylthio leaving group. In fact, the carboxylic acid of Asp800 was
AC C
close enough to the distal nitrogen at the imidazole ring to protonate it. For compounds 15 and 16, therefore, Cys797 alkylation was modelled starting from the cationic species resulting from protonation of the nitrogen atom at the imidazole ring, with deprotonated carboxylate group of Asp800 (Fig. 2B).
12
RI PT
ACCEPTED MANUSCRIPT
SC
Fig. 2. Reactions modelled by QM/MM simulation for compound 8, with expulsion of a chloride ion (panel A) and for compounds 15 and 16 with expulsion of a protonated imidazolylthiolate
M AN U
group (panel B). In the former case, Asp800 was modelled as carboxylic acid, while in the latter as a carboxylate anion. In panel B, R stands for -CH3 (compound 15) or -H (compound 16). The free-energy profiles calculated for Cys797 alkylation by compounds 8, 15 and 16 are reported in Fig. 3. A single energy barrier separates reactants (R) from products (P). No
TE D
intermediates are identified as stable minima in QM/MM simulations, which is consistent with a SN2 mechanism. The calculated activation energy for Cys797 alkylation, obtained applying the Jarzynski equality [44], (see Experimental section) was 27.0 ± 0.7 kcal/mol for compound 8,
EP
40.4 ± 1.2 kcal/mol for compound 15 and 34.2 ± 0.7 kcal/mol for compound 16. The calculated
AC C
reaction energy was -12.6 ± 0.7 and -10.2 ± 1.2 kcal/mol for 8 and 16 respectively, indicating that Cys797 alkylation by these two compounds has an exergonic character. Conversely, the calculated reaction energy for 15 was 0.9 ± 1.0 kcal/mol, suggesting that for this specific compound the reaction was not energetically favored.
13
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 3. Free-energy curves with uncertainties for Cys797 alkylation by compounds 8 (blue), 15 (orange) and 16 (magenta), obtained by the Jarzynski equality as exponential average of five independent replicas for each EGFR-inhibitor complex, using different snapshots taken from the equilibrating QM/MM MD trajectory. Reaction coordinate is expressed in Å, while the free
TE D
energy is given in kcal/mol.
The geometries identified along the SMD trajectory indicate that the TS structure for the
EP
alkylation of Cys797 by 8 and 16 features a trigonal bipyramidal geometry (Fig. 4A and Fig. 4B). For compound 15 such a geometry is distorted, due to steric hindrance between the methyl
AC C
group of the imidazole ring and the p-loop of the protein (Fig. 4C). Calculations thus indicate that compounds 8 and 16 (but not 15) can easily react with WT EGFR, through alkylation of the Cys797 residue and leading to stable adducts. It is worth mentioning that these calculations were performed contextually to the synthesis of the compounds and their biological testing. These results were in fact critical for the decision to synthesize and test compound 16, which resulted one of the most interesting ones on the gefitinib resistant cell line (vide infra).
14
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 4. Representative TS structures for Cys797 alkylation by compound 8 (panel A, cyan carbon
M AN U
atoms), 16 (panel B, purple carbon atoms) and 15 (panel C, orange carbon atoms). Protein carbon atoms are depicted in white, with EGFR secondary elements depicted as white cartoons.
3.3. EGFR Inhibitory activity on A549 lung cancer cells
TE D
Compounds 8-23 were tested for their ability to inhibit EGFR autophosphorylation in A549 lung cancer cells, harboring WT EGFR. We decided to investigate the effect of the novel warheads on this cell line because the 4-anilinoquinazoline scaffold possesses high affinity for
EP
WT EGFR [13,16]. Gefitinib (1) and the acrylamide derivative (9) were tested as reference inhibitors. All the synthesized compounds inhibited EGFR autophosphorylation, with IC50 values
AC C
ranging from the single digit nM for compounds 8, 11 and 14, to 83 and 197 nM for the 2(benzothiazolyl-2-thio)acetamides 18 and 19, respectively (Table 1). These data confirm that the structural modifications introduced by the warheads on the 4-anilinoquinazoline nucleus allowed to retain the high inhibitory potency of the parent structure. To assess the persistency of EGFR inhibition within cells, test compounds were added to cell cultures at 1 µM concentration and autophosphorylation in A549 cells was measured both 1 h
15
ACCEPTED MANUSCRIPT
after the addition and 8 h after washing of the inhibitor from the extra-cellular medium. Inhibition enduring after compound removal could be ascribed to irreversible inhibition of the kinase domain [45]. 8 h after inhibitor washing, gefinitib (1) gave 46% of inhibition, while
RI PT
acrylamide 9 and chloroacetamide 8 inhibited EGFR autophosphorylation by more than 80%. In the same conditions, compounds 10-12 inhibited EGFR by more than 70%, which is evocative of a covalent interaction with the target Cys797. However, 60% of inhibition observed for
SC
compound 13, which lacks a group able to undergo nucleophilic substitution, suggests that persistent accumulation of the inhibitor within the cells may account for the observed inhibition
M AN U
data, as it had been observed for other EGFR inhibitors [46]. The tetrazolyl (14), imidazolyl (15, 16), benzimidazolyl (17) and benzothiazolyl (18) derivatives also blocked EGFR autophosphorylation 8 h after washing. Within this subset, the two imidazole and the benzimidazole derivatives were able to reach at least 90% of inhibition.
TE D
Finally, we tested the set of ester derivatives 20-22 on A549 cells. Compounds with similar groups were reported in the literature to covalently bind activated cysteines [47]. All these derivatives were able to ensure EGFR inactivation 8 h after dilution. Despite its failure to give
EP
detectable amounts of conjugate in the presence of cysteine at basic pH, also the hydroxyacetamide 23 displayed significant long-term inhibition (77% of inhibition after 8 h).
AC C
Overall, the evaluation of the autophosphorylation levels of EGFR in A549 cells allowed to identify several 4-anilinoquinazoline derivatives giving a percentage of EGFR inhibition close to or even higher than 80% 8 h after inhibitor removal, which may indicate an irreversible binding to the target [48]. A representative set of these compounds was further evaluated in vitro (vide infra), to assess whether this prolonged inhibition can be ascribed to a covalent interaction with the target.
16
ACCEPTED MANUSCRIPT
Table 1. LC-MS reactivity assay in the presence of cysteine and EGFR autophosphorylation
1 (Gefitinib)
R
Reactivity Assay a t1/2 Peak (min) Areab
Autophosphorylation A549 cellsc IC50 (nM)
M AN U
Cpds
SC
RI PT
inhibition for compounds 1, 8–23 in A549 cell line.
% inhibition @ 1µMd 1h 8h
N. T.e
N. T.e
60±25
96 ± 4.0
46 ± 5.0
60 ± 5.0
10000
7.0±5.7
100 ± 0.5
81 ± 2.9
15 ± 3.0
-
15±6.4
96 ± 4.0
99 ± 1.0
> 1440
0.51
70±21
73 ± 4.3
71± 8.1
11
> 1440
N. D.f
0.60±0.14
96 ± 4.0
89 ± 4.6
12
> 1440
0.66
27±18
99 ± 1.0
88 ± 16
13
> 1440
N. D.f
14±1.4
99 ± 0.50
60± 2.1
-
9
AC C
10
EP
TE D
8
17
> 1440
6.5
5.0±1.5
100 ± 6.4
75 ± 4.1
15
> 1440
4.5
11±5.0
97 ± 2.5
95 ± 4.8
16
> 1440
15
27±3.5
99 ± 0.10
90 ± 4.6
17
> 1440
0.38
12±0.71
100 ± 1.0
100 ± 1.0
18
> 1440
0.39
88 ± 0.90
75 ± 0.50
EP
AC C
23
> 1440
1.4
197±18.4
63 ± 0.10
93 ± 0.50
1400 ± 100
0.64
34±11
97 ± 0.40
75 ± 19
> 1440
11
25±12
88 ± 3.1
89 ± 5.7
> 1440
5
44±0.71
97 ± 1.9
97 ± 2.6
> 1440
N. D.f
27±11
90 ± 3.6
77 ± 11
TE D
20
22
83±28
M AN U
19
21
RI PT
14
SC
ACCEPTED MANUSCRIPT
Half-life measured at pH 10 and 37 °C by LC-MS. bArea of Cys-conjugate of compound 8 was arbitrarily set at the value of 10,000 and all the Cys-conjugate areas of remaining compounds were referred to this value. cInhibition of EGFR autophosphorylation in A549 cells was evaluated by Western blot analysis. dPercentage of inhibition at 1 µM concentration was measured after 1 h of treatment and 8 h after washing of the compound from the medium (1 h incubation). e Not tested. f Not detected. a
18
ACCEPTED MANUSCRIPT
3.4. Intracellular dosage in A549 cells of selected inhibitors
RI PT
In order to discriminate whether the observed EGFR inhibition 8 h after washing had to be ascribed to a covalent interaction or to accumulation into A549 cells, intracellular dosages of selected compounds were carried out. Chloroacetamide 8, 2-((1H-tetrazol-5-yl)thio)acetamide
SC
14, 2-((1H-imidazol-2-yl)thio)acetamides 15 and 16 and 2-acetoxyacetamide 20 were selected as representatives for highly (8), moderately (14-16) and weakly reactive (20) compounds,
M AN U
according to their susceptibility to nucleophilic substitution in the presence of free cysteine (Table 1). Intracellular concentrations of these compounds were measured in A549 cells by LCMS under conditions reproducing those of the EGFR autophosphorylation assay, i.e. immediately after 1 h of exposure to the inhibitor (1 µM) or 8 h after washing the inhibitor from
TE D
the extracellular medium (Table 2).
Gefinitib (1) was chosen as a representative of non-covalent inhibitor of WT EGFR. The results previously obtained for the acrylamide derivative PD168393 [48] (compound 24) are also
EP
reported in Table 2 for comparison. Considering the complexity associated with the measurement of intracellular volume, the concentrations reported herein are approximate.
AC C
Gefitinib (1) displayed an intracellular concentration of 271 µM after 1 h, when EGFR autophosphorylation is inhibited almost completely (96%), while 8 h after washing its concentration was approximately 100-times lower (2.4 µM) and autophosphorylation was inhibited by 46% (Table 1). The intracellular concentration of acrylamide 24 was below the limit of detection (LOD) of the LC-MS method both at 1 h and 8 h, as expected for its high propensity to react promiscuously within the cell. This result clearly indicates irreversible inhibition of
19
ACCEPTED MANUSCRIPT
EGFR, while the case of gefitinib pointed out that residual concentrations of a non-covalent inhibitor may cause some inhibition even 8 h after compound removal. With the sole exception of the 2-acetoxyacetamide 20, all other tested compounds (8, 14-16)
RI PT
showed high intracellular concentrations (i.e. > 100 µM) after 1h of incubation. This observation corroborates the assumption of higher stability in the intracellular environment of our new set of inhibitors with respect to acrylamide-based derivatives. However, it does not provide
SC
information regarding the mechanism of action of the tested compounds. EGFR inhibition by compound 20 in A549 cells after 1 h of incubation, when it showed a much lower concentration
M AN U
than other inhibitors, may be ascribed to its hydrolyzed counterpart (compound 23), which possesses the ability to inhibit EGFR autophosphorylation.
A significant drop in the intracellular concentration was observed 8 h after the washing from A549 cells for all the tested compounds. However, only for chloroacetamide 8 and 2-((1H-
TE D
imidazol-2-yl)thio)acetamide 16 sub-micromolar levels were observed, with a 144- and 222-fold reduction compared to the values measured after 1 h, respectively. While not a conclusive evidence of covalent inhibition, it is remarkable that both compounds, having the ability to
EP
alkylate cysteine groups, show long-term inhibition of EGFR autophosphorylation in spite of a
AC C
large decrease of their intracellular concentrations.
Table 2. Intracellular concentrations in A549 cells and inhibitory potency on recombinant WT EGFR for selected anilinoquinazoline derivatives.
20
Cpds
Intracellular concentrationa (µM)
R
RI PT
ACCEPTED MANUSCRIPT
Ratio 1 h/8 h
Kinase Assayb (WT EGFR) IC50 (nM)
8h
271 ± 34
2.4 ± 0.2
112
0.47 ± 0.05
24 (PD168393)
< 0.05c
<0.05c
-
0.27 ± 0.04
8
115 ± 10
0.80 ± 0.10
144
0.87 ± 0.10
14
181 ± 2
5.6 ± 0.1
32
0.53 ± 0.09
179 ± 6
10.2 ± 2.1
18
N.T.
118 ± 10
0.53 ± 0.10
222
0.62 ± 0.08
5.8 ± 0.6
<0.05
-
0.52 ± 0.03
132 ± 10d
1.1 ± 0.1d
120d
0.34 ± 0.05
16
M AN U
AC C
20
TE D
15
-
EP
1 (Gefitinib)
23
SC
1h
A549 cells were incubated with 1 µM solution of test compound for 1 h. Intracellular content was quantified immediately after incubation and 8 h after removal of the compound from the medium as pmol/mg of protein in each sample. Reported µM concentrations were calculated dividing the compound content (in pmol) by an average value of cell volume of 4 µL per mg of protein, estimated by measurements of the equilibrium distribution in A549 cells of 3-O-methylD-[1-3H]glucose using the method of Kletzien et al, [49]. b Measured by LanthaScreen Eu kinase a
21
ACCEPTED MANUSCRIPT
binding assay (see Experimental Section for details). Mean values of three independent experiments ± SD are reported). c Data from Vacondio et al. [45]. d Generated in A549 cells from the hydrolysis of compound 20.
RI PT
3.5. Binding to recombinant WT EGFR and EGFR L858R/790M double mutant The ability of compounds 1, 8, 14-16, 20, 23 and 24 to displace a fluorescent tracer bound to the kinase domain of WT EGFR was assessed by means of a time-resolved fluorescence
SC
resonance energy transfer (TR-FRET) assay [50]. Compared to EGFR autophosphorylation in intact cells, this assay yields inhibitory potency in the absence of cellular transport and/or
M AN U
metabolism. Sub-nanomolar potencies were observed for all the tested compounds, with IC50 values ranging from 0.27 nM for the acrylamide derivative 24 to 0.87 nM for chloroacetamide 8. Thus, the concentrations in the micromolar range, detected for compounds 8, 14, 15, 16 and 23 at 8 h, were significantly higher than IC50 values measured on the isolated enzyme. Therefore, these concentrations could be sufficient to explain the inhibition of EGFR autophosphorylation
TE D
observed at 8 h (75-95%, see Table 1). On the other hand, being the IC50 values obtained in an artificial environment, the TR-FRET assay provides only a relative order of potency among different compounds, while the high intracellular concentration of ATP can compete with
EP
reversible inhibitors at its binding site. In fact, gefitinib (1), a non-covalent inhibitor, only
AC C
inhibited by 46% EGFR autophosphorylation in A549 cells after 8 h, in spite of an intracellular concentration of 2.4 µM. By comparison, 8 and 16 possess similar IC50 values on recombinant WT EGFR, but they inhibited EGFR autophosphorylation in A549 cells, 8 h after washing, significantly more than 1 (81% and 90% for 8 and 16 vs. 46% for 1). Considering that 8 and 16 reached intracellular concentration at 8 h lower than 1 (0.80 µM and 0.53 µM for 8 and 16 vs. 2.4 µM for 1), it is conceivable that these two compounds might inhibit EGFR through a covalent mechanism of action.
22
ACCEPTED MANUSCRIPT
We next tested the ability of 8 and 16 to inhibit EGFR L858R/T790M double mutant using the TR-FRET binding assay (see Experimental section for details). Quinazoline-based inhibitors usually display lower inhibitory potency for this mutant, when compared to WT or EGFR L858R
RI PT
single mutant, due to steric clashes with M790 [9]. The insertion in the anilinoquinazoline core of an electrophilic group, able to covalently bind the target, usually allows the inhibitor to recover activity, but the impact on the measured potency depends on the rate of target alkylation.
SC
Thus, when a slow covalent reaction occurs at the binding site, the observed IC50 value becomes dependent on the time of incubation [51]. Fig. 5 reports the inhibitory potency for gefitinib (1,
M AN U
panel A), 8 (panel B) and 16 (panel C) measured with no pre-incubation (left column) or preincubating the inhibitor for 5 h with EGFR L858R/T790M double mutant (right column). The IC50 value of 1 did not change with the pre-incubation, as expected for a non-covalent inhibitor, while for compounds 8 and 16 a significant reduction in the IC50 values was observed. This
AC C
EP
L858R/T790M enzyme.
TE D
further supports a covalent mechanism of inhibition for these two compounds, also on EGFR
A
23
RI PT
ACCEPTED MANUSCRIPT
C
TE D
M AN U
SC
B
EP
Fig. 5. Effect of the pre-incubation time on the potency (expressed as IC50) of gefitinib, 1 (panel A), compound 8 (panel B) and compound 16 (panel C) on recombinant EGFR L858R/T790M.
AC C
IC50 were measured by means of a time-resolved fluorescence resonance energy transfer (TRFRET) assay using LanthaScreen technology. Values are reported as mean ± standard error of the mean (n = 5). Statistical significance was set at P<0.05. **: P<0.01; ***: P<0.001. TR-FRET experiments on EGFR L858R/T790M with 5 h inhibitor pre-incubation were repeated either in the absence or in the presence of 500 nM ATP. Although the displacement curves for compounds 1, 8 and 16 were barely affected by ATP (see supplementary data, Fig.
24
ACCEPTED MANUSCRIPT
S4), the limited signal-to-noise ratio of these TR-FRET measures precludes a conclusive assessment of the effect of ATP on the inhibitory process for the tested inhibitors.
RI PT
3.6. Selectivity on kinases bearing a conserved cysteine on the ATP front pocket We next tested the ability of compounds 8, 9 and 16 to inhibit a panel of kinases reported to have a cysteine in a position corresponding to that of Cys797 in EGFR [52]. In this investigation,
SC
the following kinases were included: i. ErbB2 and ErbB4 of the Erb family, the same of EGFR, ii. BMX and BTK belonging to the Tek family, iii. JAK3 representative of the Janus kinase
M AN U
family and iv. BLK, member of the Src family. Acrylamide 9 inhibited the activity of nearly all the kinases of the panel at 1 µM, in agreement with previous findings reported in the literature [52]. On the contrary, 2-chloroacetamide 8 and imidazol-2-ylthioacetamide 16, markedly inhibited EGFR and ErbB4 only (i.e. 87% of higher), as they moderately (ErbB2, BTX and
TE D
BLK) or marginally (BMX, JAK3) affected the activity of the other kinases included in the panel. This assay confirms further that the replacement of acrylamide with less reactive warheads ensures higher selectivity.
EP
Table 3. Selectivity of compounds 8, 9 and 16 on a panel of kinases bearing a front-pocket
9
AC C
cysteine corresponding to EGFR Cys797.a
90
92
100
88
87
92
97
16
90
65
87
30
54
0
58
Cpds
EGFR
ErbB2
ErbB4
BMX
BTK
JAK3
BLK
8
98
76
100
38
53
32
62
a
Inhibitory activity determined with an activity-based assay (see methods for details). Percent of inhibition measured at 1 µM. Color code: red, ≥80% inhibition; yellow, 50−80% inhibition; green, ≤50% inhibition. Reported data are the average of two independent experiments.
25
ACCEPTED MANUSCRIPT
3.7. Activity on H1975 lung cancer cells H1975 lung cancer cells are resistant to first generation inhibitors of EGFR due to the presence
RI PT
of the T790M mutation which somehow hampers the interaction between the 4anilinoquinazoline nucleus and ATP-binding site (see above). The introduction in the quinazoline scaffold of a group capable of alkylating Cys797 is expected to ensure a significant
SC
improvement in the antiproliferative potency on H1975 cells compared to gefitinib [16]. With this premise, we examined the ability of the synthesized compounds to block proliferation of
M AN U
H1975 cells and to inhibit EGFR autophosphorylation at two concentrations (0.1 and 1 µM) in the same cell line (Table 4) along with gefitinib (1) and acrylamide 9, which were used as controls as they are expected to be the least and most potent compounds of the series, respectively.
TE D
Compound 1 resulted poorly active on H1975 cells [53] as it inhibited cell growth with an IC50 of 9.1 µM and it did not affect autophosphorylation at 0.1 or 1 µM concentration, confirming its incapability to engage EGFR L858R/T790M mutant. On the contrary, the 9
showed
sub-micromolar
antiproliferative
potency
and
inhibited
EP
acrylamide
autophosphorylation almost completely at both concentrations. The same compound had shown
AC C
an antiproliferative IC50 value of 1.6 µM in a previous experiment [31a]. Among the newly synthesized compounds, the hydroxyacetamide 23, which lacks a potential warhead, did not show any antiproliferative activity up to 10 µM and did not inhibit EGFR L858R/T790M autophosphorylation. Chloroacetamide 8 and the 2-((1H-imidazol-2-yl)thio)acetamide 16 significantly outperformed gefitinib and displayed a pharmacological activity superior (8) or comparable (16) to that of the acrylamide 9. Indeed, compound 8 resulted extremely potent in
26
ACCEPTED MANUSCRIPT
inhibiting H1975 cell proliferation (IC50 of 0.06 µM) and capable of fully abolishing EGFR L858R/T790M autophosphorylation at 0.1 µM. Compound 16 inhibited H1975 growth with an
RI PT
IC50 of 1.4 µM and completely inhibited autophosphorylation at 1 µM concentration. Remarkably, the ability to overcome the effect of T790M mutation in H1975 cells is achieved introducing electrophilic groups featuring moderate (8) or very low reactivity (16) toward free cysteine, when compared to acrylamide.
SC
Table 4. Inhibition of cell proliferation and inhibition of EGFR autophosphorylation in H1975
1 (Gefitinib)
R
EP
Cpds
TE D
M AN U
cells for compounds 1, 8-12 and 14-23.
9.1 ± 1.1
8.0 ± 4.9
18 ± 4.2
8
0.06 ±0.01
100 ± 0.9
100 ± 0.10
9
0.70 ± 0.14
93 ± 0.70
95 ± 0.10
10
3.7 ±0.28
1.5 ± 2.1
42 ± 7.0
AC C
-
H1975 cell line Autophosphorylationb Cell growth a % of inhibition @ Inhibition 0.1 µM 1 µM IC50 (µM)
27
ACCEPTED MANUSCRIPT
3.0 ±0.42
29 ± 10
92 ± 9.9
12
3.6 ±1.8
40 ± 12
87 ± 9.8
14
2.1 ±0.78
16 ± 8.5
96 ± 5.7
15
6.9 ±0.64
45 ± 14
67 ± 8.5
16
1.4 ±0.10
SC
RI PT
11
M AN U
80 ± 7.0
100 ±0.71
9.1±1.0
6.0 ± 4.2
70 ± 6.8
9.8±0.14
29 ± 11
24 ± 7.7
3.2±0.71
30 ± 13
92 ± 0.90
3.4±0.57
27 ± 8.5
68 ±13
8.3±1.8
0.5 ± 0.7
43 ± 4.4
22
6.1±1.4
20 ± 3.9
16 ± 8.0
23
> 10
0.0 ± 0.1
0.0 ± 0.1
17
20
AC C
21
EP
19
TE D
18
a
Concentration able to inhibit 50% of cell proliferation as determined by the MTT assay, after 72 h of incubation with compounds (dose ranging from 0.1 to 10 µM). Osimertinib displays an IC50
28
ACCEPTED MANUSCRIPT
of 0.10 ± 0.1 µM in the employed conditions. b Inhibition of EGFR autophosphorylation in H1975 cells was evaluated by Western blot analysis.
4. CONCLUSION
RI PT
We report a structure-reactivity and structure-activity study aimed to identify novel cysteinetargeting warheads, less reactive than acrylamide, that may be exploited for the design of irreversible kinase inhibitors with improved chemical selectivity. In particular, we focused on the
SC
design and synthesis of 4-anilinoquinazoline derivatives bearing warheads with reduced risk of nonspecific covalent binding to thiols, yet able to exploit the high nucleophilicity of Cys797 of
M AN U
EGFR and to overcome the resistance conferred by the T790M gatekeeper mutation to reversible, anilinoquinazoline-based inhibitors. Starting from the chloroacetamide derivative 8, reduction of reactivity for a series of potential SN2 substrates led to compound 16, a 2-((1Himidazol-2-yl)thio)acetamide derivative, which showed a inhibitory potency on H1975 cell
TE D
growth (IC50 = 1.4 ± 0.10 µM) comparable to that of the reference acrylamide-based inhibitor 9 (IC50 = 0.70 ± 0.10 µM). Considering that 9 and 16 have the same 4-anilino-quinazoline scaffold, the imidazol-2-ylthioacetamide group emerges as a bioisostere of the acrylamide warhead. It
EP
should be pointed out that while compound 16 displays negligible reactivity towards free cysteine in solution and possess higher selectivity than acrylamide 9 toward a panel of selected
AC C
kinases, its imidazol-2-ylthioacetamide warhead is a metabolic soft spot potentially able to give toxic effects in vivo.
Several hints, including hybrid QM/MM simulations and pre-incubation studies on recombinant EGFR L858R/T790M double mutant, support the hypothesis that compound 16 irreversibly inhibits EGFR by reacting covalently with Cys797. By comparison with acrylamide-based inhibitors, we propose that long-lasting inhibition can be achieved by modulating the reactivity
29
ACCEPTED MANUSCRIPT
of the cysteine-targeting moiety, to potentially overcome the low specificity of hetero-Michael acceptors. Reported cases of resistance to covalent EGFR inhibitors by Cys797 mutation discourage the strategy to target this specific residue by covalent binders as a stand-alone
RI PT
approach to provide next-generation antitumor agents. However, the results here reported may help the design of novel drugs targeting specific cysteines with improved selectivity. In particular, it is suggested that heteroarylthio acetamide warheads can lead to irreversible
SC
inhibition of kinases by covalent binding to cysteine residues, and that the low reactivity of such
molecular modelling simulations.
5. EXPERIMENTAL SECTION 5.1. Chemistry
M AN U
a warhead can be counterbalanced by appropriate design of the overall inhibitor through
TE D
Reagents were obtained from commercial suppliers and used without further purification. Solvents were purified and stored according to standard procedures. Anhydrous reactions were conducted under a positive pressure of dry N2. Reactions were monitored by TLC, on Kieselgel
EP
60 F 254 (DC-Alufolien, Merck). Final compounds and intermediates were purified by flash chromatography (SiO2 60, 40−63 µm). Melting points were not corrected and were determined
AC C
with a Gallenkamp melting point apparatus. The 1H NMR spectra were recorded on a Bruker 300 MHz Avance or on a Bruker 400 MHz Avance spectrometer; chemical shifts (δ scale) are reported in parts per million relative to the central peak of the solvent. 1H NMR spectra are reported in the following order: multiplicity, approximate coupling constant (J value) in hertz, and number of protons; signals are characterized as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet), m (multiplet), and br s (broad signal).
30
ACCEPTED MANUSCRIPT
Mass spectra of final compounds were recorded using a Thermo Scientific LTQ Orbitrap XL. All tested compounds were > 95% pure by HPLC/UV analysis. Compound 9 was prepared as reported in reference [31a], while compound 24 has been prepared according to reference [48]. H and 13C NMR spectra for final compounds 8, 10-23 are reported in the Supplementary Data.
RI PT
1
5.1.1. 2-chloro-N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)acetamide
(8).
SC
Chloroacetic acid (355 mg, 3.76 mmol) is dissolved in anhydrous THF (5 ml), DIPEA (650 µl, 3.73 mmol) and pivaloyl chloride (460 µl, 3.73 mmol) are added and the mixture is
M AN U
stirred at room temperature for 30 minutes before adding a solution of 6-amino-4-(3chloro-4-fluoroaniline)-7-ethoxyquinazoline (7, 227 mg, 0.68 mmol) in anhydrous THF (3 ml). After 2 h the suspension is diluted with water and the mixture is extracted with AcOEt. The organic layer is washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column
TE D
chromatography (AcOEt/hexane 4:1), affording the title compound (231 mg, 83%) as a white solid. Mp: 278°C (dec.). 1H NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.74 (s, 1H), 8.87 (d, J = 3.9 Hz, 1H), 8.53 (s, 1H), 8.10 (dd, J = 6.8, 2.6 Hz, 1H), 7.78 (ddd, J =
EP
9.0, 4.3, 2.6 Hz, 1H), 7.42 (t, J = 9.1 Hz, 1H), 7.29 (s, 1H), 4.49 (d, J = 1.5 Hz, 2H), 4.30 13
C NMR (75 MHz, DMSO-d6) δ 165.04,
AC C
(q, J = 6.9 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H).
156.89, 154.84, 154.28, 154.03, 151.63, 149.11, 136.73, 126.59, 123.75, 122.61 (d, J = 6.8 Hz), 118.80, 116.43 (d, J = 21.5 Hz), 115.57, 108.73, 107.37, 64.64, 43.33, 14.24. HRMS (ESI): m/z: calcd for C18H15Cl2FN4O2 [(M+H)+]: 409.06289; found: 409.06291. 5.1.2. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-(pyridin-2ylthio)acetamide (10). 2-mercaptopyridine (174 mg, 1.57 mmol) is added to a suspension of sodium hydroxide (64.5 mg, 1.61 mmol) in THF (3 ml). To the clear solution thus
31
ACCEPTED MANUSCRIPT
obtained, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 61.4 mg, 0.15 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with ethyl acetate. The organic layer is washed with water, saturated aqueous
RI PT
NaHCO3 and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by triturating with AcOEt, affording the title compound (44 mg, 61%) as a white powder. Mp: 225°C (dec.). 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.86 (s, 1H),
SC
8.53 (d, J = 5.3 Hz, 1H), 8.50 (s, 1H), 7.94 (dd, J = 6.7, 2.7 Hz, 1H), 7.68 – 7.59 (m, 2H), 7.37 (d, J = 8.1 Hz, 1H), 7.27 – 7.10 (m, 3H), 4.26 (q, J = 7.0 Hz, 2H), 4.11 (s, 2H), 1.38 13
C NMR (75 MHz, DMSO-d6) δ 167.34, 156.82 (d, J = 3.1 Hz),
M AN U
(t, J = 7.0 Hz, 3H).
154.79, 153.73, 153.29, 149.56, 148.62, 137.12, 136.82, 127.26, 123.69, 122.57 (d, J = 6.8 Hz), 121.94, 120.45, 118.65 (d, J = 18.1 Hz), 116.39 (d, J = 21.7 Hz), 113.46, 108.89, 107.18, 64.60, 34.02, 14.13. HRMS (ESI): m/z: calcd for C23H19ClFN5O2S [(M+H)+]:
TE D
484.10048; found: 484.10038.
5.1.3. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-(pyridin-3ylthio)acetamide (11). 3-pyridylmercaptoacetic acid [54] (142 mg, 0.84 mmol) is dissolved
EP
in DMF (6 ml) and triethylamine (0.12 ml, 0.58 mmol) is added. The solution is cooled to 0°C and TBTU (275 mg, 0.85 mmol) is added followed, after 30 minutes, by 6-amino-4-
AC C
(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (7, 160 mg, 0.48 mmol). The mixture is warmed to 40°C and stirred at the same temperature for 4 h. After cooling to room temperature, the solution is diluted with AcOEt and washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/MeOH 9:1) to afford the title compound (58 mg, 25%) as a white solid. Mp: 210°C. 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.81 (s,
32
ACCEPTED MANUSCRIPT
1H), 8.65 (s, 1H), 8.49 (s, 1H), ), 8.44 (s, 1H), 7.99 – 7.82 (m, 2H), 7.65 – 7.54 (m, 1H), 7.38 (dd, J = 8.3, 4.6 Hz, 1H), 7.24 – 7.10 (m, 2H), 4.30 (q, J = 6.9 Hz, 2H), 4.00 (s, 2H), 1.54 (t, J = 6.9 Hz, 3H).
13
C NMR (75 MHz, CDCl3/CD3OD) δ 167.82, 158.35, 157.14,
RI PT
154.68, 153.89, 150.11, 148.72, 148.19, 138.50, 136.23, 127.66, 125.38, 123.15 (d, J = 6.7 Hz), 121.15 (d, J = 18.5 Hz), 116.90 (d, J = 22.2 Hz), 112.07, 109.78, 106.86, 65.79, 39.07, 14.63, 0.04. HRMS (ESI): m/z: calcd for C23H19ClFN5O2S [(M+H)+]: 484.10048;
SC
found: 484.10043.
5.1.4. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-(pyrimidin-2-
M AN U
ylthio)acetamide (12). 2-mercaptopyrimidine (38 mg, 0.34 mmol) is added to a suspension of sodium hydroxide (14.0 mg, 0.35 mmol) in THF (2 ml). To the clear solution, 6(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 57 mg, 0.14 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with
TE D
ethyl acetate. The organic layer is washed with water, saturated aqueous NaHCO3 and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt), affording the title compound (59
EP
mg, 88%) as a white solid. Mp: 243°C. 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 9.68 (s, 1H), 8.93 (s, 1H), 8.69 (d, J = 4.9 Hz, 2H), 8.50 (s, 1H), 8.08 (dd, J = 6.9, 2.6 Hz,
AC C
1H), 7.85 – 7.65 (m, 1H), 7.39 (t, J = 9.1 Hz, 1H), 7.29 (t, J = 4.9 Hz, 1H), 7.24 (s, 1H), 4.41 – 4.06 (m, 4H), 1.36 (t, J = 6.9 Hz, 3H).
13
C NMR (75 MHz, DMSO-d6) δ 169.98,
166.78, 158.02, 156.81, 154.78, 153.45, 151.56, 148.65, 136.81, 127.19, 123.69, 122.56 (d, J = 6.9 Hz), 118.64 (d, J = 18.5 Hz), 117.74, 116.36 (d, J = 21.6 Hz), 113.83, 108.85, 107.17, 64.59, 35.14, 14.19. HRMS (ESI): m/z: calcd for C22H18ClFN6O2S [(M+H)+]: 485.09573; found: 485.09555.
33
ACCEPTED MANUSCRIPT
5.1.5. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-3-(pyridin-3yl)propanamide (13). 3-pyridin-3-yl-propionic acid (338 mg, 2.24 mmol) is suspended in thionyl chloride (3.3 ml, 45.5 mmol) and DMF (9 µl, 0.12 mmol) is added. The mixture is
RI PT
heated to reflux for 1 h, then cooled to room temperature. The volatile material is evaporated under reduced pressure, the residue is dissolved in anhydrous THF (3 ml) and added to a solution of 6-amino-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (7, 194
SC
mg, 0.58 mmol). Pyridine (230 µl, 2.84 mmol) is added and the mixture is stirred at room temperature for 16 h, then diluted with AcOEt, washed with water and brine. The organic
M AN U
layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (DCM + 5% MeOH(NH3)), affording the title compound (105 mg, 39%) as a white solid. Mp: 241°C. 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.81 (s, 1H), 8.64 – 8.45 (m, 2H), 8.45 – 8.29 (m, 1H), 7.93 (dd, J = 6.6,
TE D
2.6 Hz, 1H), 7.75 (dt, J = 7.9, 2.0 Hz, 1H), 7.62 (ddd, J = 9.0, 4.2, 2.6 Hz, 1H), 7.35 (dd, J = 7.9, 4.9 Hz, 1H), 7.27 – 7.02 (m, 2H), 4.30 (q, J = 7.0 Hz, 2H), 3.14 (t, J = 7.4 Hz, 2H), 2.92 (t, J = 7.3 Hz, 2H), 1.55 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3/CD3OD) δ
EP
158.16, 156.98, 154.39, 149.50, 148.21, 147.45, 137.49, 137.24, 127.88, 125.16, 124.47, 122.87 (d, J = 6.8 Hz), 116.82 (d, J = 22.1 Hz), 112.06, 109.60, 106.56, 65.58, 38.48,
AC C
28.75, 14.51. HRMS (ESI): m/z: calcd for C24H21ClFN5O2 [(M+H)+]: 466.14406; found: 466.14368.
5.1.6. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((1-methyl-1Htetrazol-5-yl)thio)acetamide (14).5-mercapto-1-methyltetrazole (36.2 mg, 0.30 mmol) is added to a suspension of sodium hydroxide (12 mg, 0.30 mmol) in THF (2 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8,
34
ACCEPTED MANUSCRIPT
90 mg, 0.22 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with AcOEt. The organic layer is washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column
RI PT
chromatography (AcOEt), affording the title compound (65 mg, 61%) as a white solid. Mp: 232-234°C.1H NMR (300 MHz, DMSO-d6) δ 9.85 (s, 1H), 9.83 (s, 1H), 8.86 (s, 1H), 8.52 (s, 1H), 8.08 (dd, J = 6.9, 2.6 Hz, 1H), 7.76 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.41 (t, J = 9.1
SC
Hz, 1H), 7.27 (s, 1H), 4.47 (s, 2H), 4.30 (q, J = 7.0 Hz, 2H), 4.00 (s, 3H), 1.46 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.66, 156.85, 154.81, 153.98, 153.42, 151.59,
M AN U
148.94, 136.76, 126.94, 123.71, 122.58 (d, J = 6.9 Hz), 118.65 (d, J = 18.4 Hz), 116.39 (d, J = 21.5 Hz), 114.88, 108.75, 107.27, 64.63, 37.39, 33.70, 14.20. HRMS (ESI): m/z: calcd for C20H18ClFN8O2S [(M+H)+]: 489.10187; found: 489.10161. 5.1.7. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((1-methyl-1H-
TE D
imidazol-2-yl)thio)acetamide (15). 2-mercapto-1-methylimidazole (26 mg, 0.23 mmol) is added to a suspension of sodium hydroxide (9.5 mg, 0.24 mmol) in THF (2 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8,
EP
65 mg, 0.16 mmol) is added and the mixture is stirred at 45°C for 2 h, then cooled to room temperature and diluted with AcOEt. The organic layer is washed with water, saturated
AC C
aqueous NaHCO3 and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt), affording the title compound (24 mg, 31%) as a white solid. Mp: 215-217°C. 1H NMR (300 MHz, DMSO-d6) δ 10.14 (s, 1H), 9.83 (s, 1H), 8.93 (s, 1H), 8.51 (s, 1H), 8.09 (dd, J = 6.9, 2.6 Hz, 1H), 7.77 (ddd, J = 9.1, 4.3, 2.6 Hz, 1H), 7.41 (t, J = 9.1 Hz, 1H), 7.33 – 7.20 (m, 2H), 7.00 (d, J = 1.3 Hz, 1H), 4.29 (q, J = 7.0 Hz, 2H), 4.06 (s, 2H), 3.60 (s, 3H), 1.42 (t, J = 7.0
35
ACCEPTED MANUSCRIPT
Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 167.22, 156.84, 154.76, 153.75, 153.60, 136.83, 128.39, 127.33, 123.67, 123.55, 122.55 (d, J = 6.7 Hz), 116.40 (d, J = 21.5 Hz), 113.85,
[(M+H)+]: 487.11138; found: 487.11123.
RI PT
108.83, 107.18, 64.56, 37.44, 32.85, 14.22. HRMS (ESI): m/z: calcd for C22H20ClFN6O2S
5.1.8. 2-((1H-imidazol-2-yl)thio)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6yl)acetamide (16). 2-mercaptoimidazole (231 mg, 2.31 mmol) and 6-(chloroacetamido)-4-
SC
(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 94 mg, 0.23 mmol) are dissolved in THF (6 ml) and triethylamine (320 µl, 2.30 mmol) is added. The mixture is stirred at 50°C
M AN U
for 2 h, then cooled to room temperature, diluted with AcOEt and washed with water, saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is crystallized from EtOH/water 7:3, affording the title compound (97 mg, 89%) as a white solid. Mp: 243°C. 1H NMR (300
TE D
MHz, CDCl3/CD3OD) δ 8.75 (s, 1H), 8.38 (s, 1H), 7.83 (dd, J = 6.6, 2.6 Hz, 1H), 7.54 – 7.46 (m, 1H), 7.17 – 7.03 (m, 2H), 6.99 (s, 2H), 4.21 (q, J = 7.0 Hz, 2H), 3.89 (s, 2H), 1.44 (t, J = 7.0 Hz, 3H).
13
C NMR (75 MHz, DMSO-d6) δ 167.50, 156.84, 154.77, 153.73,
EP
153.56, 151.56, 148.68, 139.06, 136.89, 127.40, 123.64, 122.51 (d, J = 7.0 Hz), 118.78, 116.39 (d, J = 21.5 Hz), 113.71, 108.86, 107.13, 64.57, 36.99, 14.22. HRMS (ESI): m/z:
AC C
calcd for C21H18ClFN6O2S [(M+H)+]: 473.09573; found: 473.09570. 5.1.9. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((1-methyl-1Hbenzo[d]imidazol-2-yl)thio)acetamide (17). 1-methylbenzimidazole (95 mg, 0.58 mmol) is added to a suspension of sodium hydroxide (39 mg, 0.97 mmol) in THF (4 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 78 mg, 0.19 mmol) is added and the mixture is stirred at room temperature for 1 h, then
36
ACCEPTED MANUSCRIPT
diluted with AcOEt and washed with water, saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (DCM + 1.5% MeOH(NH3)),
RI PT
affording the title compound (56 mg, 54%) as a white solid. Mp: 226°C. 1H NMR (300 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.82 (s, 1H), 8.93 (s, 1H), 8.50 (s, 1H), 8.08 (dd, J = 6.9, 2.6 Hz, 1H), 7.76 (ddd, J = 9.1, 4.3, 2.6 Hz, 1H), 7.60 – 7.49 (m, 2H), 7.41 (t, J = 9.1 Hz,
6.9 Hz, 3H).
13
SC
1H), 7.26 – 7.17 (m, 3H), 4.46 (s, 2H), 4.22 (q, J = 7.0 Hz, 2H), 3.75 (s, 3H), 1.28 (t, J = C NMR (75 MHz, DMSO-d6) δ 166.59, 156.82, 154.77, 153.78, 153.59,
M AN U
151.55, 151.15, 148.74, 142.55, 136.95, 136.81, 127.15, 123.65, 122.52 (d, J = 6.8 Hz), 121.70 (d, J = 10.9 Hz), 117.49, 116.38 (d, J = 21.7 Hz), 114.25, 109.59, 108.80, 107.19, 64.52, 36.08, 30.00, 13.95. HRMS (ESI): m/z: calcd for C26H22ClFN6O2S [(M+H)+]: 537.12703; found: 537.12658.
TE D
5.1.10. 2-(benzo[d]thiazol-2-ylthio)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin6-yl)acetamide (18). 2-mercaptobenzothiazole (25 mg, 0.15 mmol) is added to a suspension of sodium hydroxide (6.0 mg, 0.15 mmol) in THF (2 ml). To the clear solution,
EP
6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 61 mg, 0.15 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with
AC C
AcOEt and washed with water, saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/hexane 4:1), followed by crystallization from 1,2-dichloroethane, affording the title compound (15 mg, 19%) as white slender needles. Mp: 203-204°C. 1H NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.82 (s, 1H), 8.89 (s, 1H), 8.50 (s, 1H), 8.07 - 8.01 (m, 2H), 7.86 (d, J = 8.0 Hz, 1H), 7.74 (ddd, J = 9.1, 4.4, 2.6 Hz,
37
ACCEPTED MANUSCRIPT
1H), 7.48 (td, J = 8.2, 7.7, 1.4 Hz, 1H), 7.44 – 7.33 (m, 2H), 7.25 (s, 1H), 4.54 (s, 2H), 4.25 (q, J = 7.0 Hz, 2H), 1.36 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 166.07 (d, J = 7.2 Hz), 156.97, 154.94, 153.93, 152.57, 151.72, 148.92, 136.80 (d, J = 3.0 Hz), 134.95,
RI PT
127.12, 126.60, 124.79, 123.86, 122.74 (d, J = 6.9 Hz), 122.02, 121.24, 118.76 (d, J = 18.6 Hz), 116.50 (d, J = 21.5 Hz), 114.70, 108.85, 107.32, 64.72, 37.34, 14.19. HRMS (ESI): m/z: calcd for C25H19ClFN5O2S2 [(M+H)+]: 540.07255; found: 540.07226.
methoxybenzo[d]thiazol-2-yl)thio)acetamide
(19).
SC
5.1.11.N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((5-
2-mercapto-5-methoxybenzothiazole
M AN U
(118.5 mg, 0.60 mmol) is added to a suspension of sodium hydroxide (24.7 mg, 0.62 mmol) in THF (4 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4fluoroaniline)-7-ethoxyquinazoline (8, 86 mg, 0.21 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with AcOEt and washed with water,
TE D
saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/hexane 4:1), followed by crystallization from 1,2-dichloroethane,
EP
affording the title compound (23 mg, 19%) as a white crystalline solid. Mp: 202-203°C. 1H NMR (300 MHz, DMSO-d6) δ 9.83 (d, J = 3.2 Hz, 2H), 8.90 (s, 1H), 8.51 (s, 1H), 8.08
AC C
(dd, J = 6.9, 2.6 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.76 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.49 – 7.33 (m, 2H), 7.27 (s, 1H), 7.02 (dd, J = 8.8, 2.5 Hz, 1H), 4.53 (s, 2H), 4.27 (q, J = 6.9 Hz, 2H), 3.82 (s, 3H), 1.38 (t, J = 6.9 Hz, 3H).
13
C NMR (75 MHz, DMSO-d6) δ
166.95, 165.85, 158.74, 156.85, 154.80, 153.83, 151.58, 148.92, 136.77 (d, J = 3.2 Hz), 127.01, 126.42, 123.73, 122.60 (d, J = 6.8 Hz), 122.21, 118.63 (d, J = 18.3 Hz), 116.39 (d, J = 21.6 Hz), 114.66, 113.83, 108.78, 107.32, 104.59, 64.60, 55.52, 37.25, 14.13. HRMS
38
ACCEPTED MANUSCRIPT
(ESI): m/z: calcd for C26H21ClFN5O5S2 [(M+H)+]: 570.08311; found: 570.08297. 5.1.12.2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)amino)-2-oxoethyl acetate (20). 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8,
RI PT
150 mg, 0.37 mmol) and sodium acetate (30 mg, 0.37 mmol) are dissolved in DMF (4 ml). The mixture is stirred at reflux for 3 h. The solution is cooled to room temperature and diluted with water. The obtained precipitate is filtered, affording the title compound (141
SC
mg, 89%) as a white solid. The compound could be used without any further purification. Mp: 221°C. 1H NMR (300 MHz, DMSO-d6) δ 9.83 (s, 1H), 9.53 (s, 1H), 8.86 (s, 1H), 8.52
M AN U
(s, 1H), 8.10 (dd, J = 6.9, 2.6 Hz, 1H), 7.78 (ddd, J = 9.1, 4.4, 2.7 Hz, 1H), 7.40 (t, J = 9.1 Hz, 1H), 7.26 (s, 1H), 4.81 (s, 2H), 4.28 (q, J = 6.9 Hz, 2H), 2.16 (s, 3H), 1.46 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 169.84, 165.88, 156.82, 154.79, 154.16, 153.91, 151.57, 149.03, 136.88, 126.58, 123.65, 122.51 (d, J = 6.9 Hz), 118.66 (d, J = 18.2 Hz),
TE D
116.39 (d, J = 21.5 Hz), 115.19, 108.83, 107.30, 64.60, 62.55, 20.47, 14.24. HRMS (ESI): m/z: calcd for C20H18ClFN4O4 [(M+H)+]: 433.10734; found: 433.10699. 5.1.13.2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)amino)-2-oxoethyl
EP
benzoate (21). 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 70 mg, 0.17 mmol) and benzoic acid (21 mg, 0.17 mmol) are dissolved in DMF (3 ml).
AC C
Triethylamine (24 µl, 0.17 mmol) is added and the mixture is stirred at reflux for 3 h. The solution is cooled to room temperature and diluted with water. The obtained precipitate is filtered, affording the title compound (75 mg, 88%) as a white solid. The compound is pure enough for biological testing. Mp: 243°C. 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.93 (s, 1H), 8.53 (s, 1H), 8.20 (s, 1H), 8.19 – 8.14 (m, 1H), 7.95 (dd, J = 6.7, 2.7 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.63 (ddd, J = 8.9, 4.1, 2.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.25 – 7.11
39
ACCEPTED MANUSCRIPT
(m, 2H), 5.08 (s, 2H), 4.25 (q, J = 7.0 Hz, 2H), 1.38 (t, J = 6.9 Hz, 3H).
13
C NMR (75
MHz, CDCl3/CD3OD) δ 166.50, 165.63, 158.05, 156.90, 154.61, 153.64, 152.96, 148.48, 135.86, 134.54, 130.18, 129.15, 126.80, 125.12, 122.78 (d, J = 7.0 Hz), 121.02 (d, J = 18.4
RI PT
Hz), 116.72 (d, J = 22.0 Hz), 111.04, 109.61, 106.77, 65.61, 63.73, 14.46. HRMS (ESI): m/z: calcd for C25H20ClFN4O4 [(M+H)+]: 495.12299; found: 495.12263.
5.1.14.2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)amino)-2-oxoethyl 2,6(22).
6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-
SC
dimethylbenzoate
ethoxyquinazoline (8, 65 mg, 0.16 mmol) and 2,6-dimethoxy benzoic acid (70.5 mg, 0.47
M AN U
mmol) are dissolved in DMF (4 ml). Triethylamine (100 µl, 0.72 mmol) is added and the mixture is stirred at 100°C for 30 minutes. The solution is cooled to room temperature and diluted with water. The obtained precipitate is filtered, affording the title compound (49 mg, 47%) as a white solid. Mp: 240°C (dec.). 1H NMR (300 MHz, DMSO-d6) δ 9.85 (s,
TE D
1H), 9.67 (s, 1H), 8.86 (s, 1H), 8.53 (s, 1H), 8.09 (dd, J = 6.8, 2.6 Hz, 1H), 7.76 (ddd, J = 9.1, 4.4, 2.7 Hz, 1H), 7.42 (t, J = 9.1 Hz, 1H), 7.34 – 7.19 (m, 2H), 7.12 (d, J = 7.6 Hz, 2H), 5.09 (s, 2H), 4.29 (q, J = 7.0 Hz, 2H), 2.35 (s, 6H), 1.41 (t, J = 6.9 Hz, 3H). 13C NMR
EP
(75 MHz, DMSO-d6) δ 168.45, 165.69, 156.89, 154.83, 154.24, 153.97, 151.61, 149.11, 136.78 (d, J = 2.9 Hz), 134.82, 132.88, 129.68, 127.57, 126.57, 123.73, 122.61 (d, J = 7.0
AC C
Hz), 118.67 (d, J = 18.2 Hz), 116.43 (d, J = 21.6 Hz), 108.81, 107.39, 64.61, 63.13, 19.41, 14.16. HRMS (ESI): m/z: calcd for C27H24ClFN4O4 [(M+H)+]: 523.15429; found: 523.15387.
5.1.15.N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-hydroxyacetamide (23). 6-(2-acetoyloxyacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (20, 93 mg, 0.22 mmol) is dissolved in a mixture of THF (3 ml) and MeOH (30 µl, 0.8 mmol).
40
ACCEPTED MANUSCRIPT
Sodium (3 mg, 0.13 mmol) is added and the mixture is stirred at room temperature for 30 minutes, quenched by addition of HCl 1M until neutralization and diluted with AcOEt. The organic layer is washed with water and brine, dried over Na2SO4 and concentrated under
RI PT
reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/hexane 4:1) to afford the title compound (52 mg, 62%) as a white solid. Mp: 261°C. 1H NMR (300 MHz, DMSO-d6) δ 9.87 (s, 1H), 9.48 (s, 1H), 9.08 (s, 1H), 8.52 (s,
SC
1H), 8.08 (dd, J = 7.0, 2.6 Hz, 1H), 7.76 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.42 (t, J = 9.1 Hz, 1H), 7.31 (s, 1H), 6.25 (t, J = 5.6 Hz, 1H), 4.33 (q, J = 6.9 Hz, 2H), 4.09 (d, J = 5.6 Hz, 13
C NMR (75 MHz, DMSO-d6) δ 170.42, 156.88, 154.78,
M AN U
2H), 1.46 (t, J = 6.9 Hz, 3H).
153.62, 152.71, 151.56, 148.48, 136.87, 126.73, 123.73, 122.62 (d, J = 6.9 Hz), 118.63 (d, J = 18.6 Hz), 116.38 (d, J = 21.5 Hz), 111.60, 109.09, 107.23, 64.77, 61.75, 14.28. HRMS (ESI): m/z: calcd for C18H16ClFN4O3 [(M-H)-]: 389.08112; found: 389.08215.
5.2.1.
TE D
5.2. Analytical Chemistry
Reactivity assay. Compounds of 8-23 were added (10 µM) to freshly-prepared 1 mM
cysteine solutions in 50 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS; pH 10), ionic
strength
for
KCl
addition.
The
formation
of
the
conjugate
EP
0.15M
quinazolinylacetamide (S-(2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-
AC C
yl)amino)-2-oxoethyl)cysteine) was measured after 24 h of incubation at 37 °C by LC-MS in multiple reaction monitoring (MRM) mode with the following parent-product ion transitions: m/z 494.1 [M+H]+ → m/z 305.1 + m/z 333.1 + m/z 407.2 (Tube Lens (TL) 95 V; Collision Energies (CE) 35, 35, 25 eV, respectively). To quantify the amount of conjugate produced, the area of the peak in the chromatogram was referred to the arbitrarily-set value of 10,000 for the conjugate between compound 8 and cysteine. To
41
ACCEPTED MANUSCRIPT
monitor the time course of the LC-UV peak area of 8 and of the cysteine conjugate (See Supplementary Data, Fig. S1), a Shimadzu HPLC gradient system (Shimadzu Corp., Kyoto, Japan) consisting of two LC-10ADvp solvent delivery modules, a SPD-10Avp UV-
RI PT
VIS detector set at λ=254 nm, equipped with a reversed-phase C18 column (Supelcosil LC18-DB, 5 µm, 150 x 4.6 mm i.d.; Supelco, Bellefonte, USA) was employed.
5.2.2. Stability of the cysteine-conjugate. Chloroacetamide 8 was incubated in the presence of a
SC
1:100 molar excess of cysteine under nitrogen atmosphere at pH 10 and 37 °C for 24 h. The area ratio of cysteine-conjugate vs internal standard and the area ratio of 8 vs internal
M AN U
standard were monitored over time by LC-MS in MRM mode with the parent-product ion transitions: 8: m/z 409.1 [M+H]+ → m/z 381.0 + m/z 363.0 + m/z 305.0 (TL 106 V; CE 24, 28, 34 eV); cysteine-conjugate: m/z 494.1 [M+H]+ → m/z 305.1 + m/z 333.1 + m/z 407.2 ; TL = 95 V; CE = 35, 35, 25 eV, respectively.
TE D
5.2.3. Determination of intracellular concentrations. We applied a protocol previously developed by our group [45]. A549 Cells were plated at 1,000,000 cells/dish (25 cm2) density. After 24 h, test compounds (1, 8, 14-16, 20, 23) were added to the culture medium (final
EP
concentration: 1 µM; final DMSO concentration: 0.1% v/v). Cells were incubated for 1 h at
AC C
37 °C and tested either immediately after or 8 h after removal of the compound from the extracellular medium by washing the cells for three times with 1 mL aliquots of fresh medium. Compounds were then extracted from cells with absolute EtOH (1.1 mL at 4 °C), cell extracts were centrifuged (4 °C, 10,000 g, 5 min) and collected. A fixed volume of ethanolic extract was evaporated to dryness, dissolved in LC eluent and injected into the LC-MS system for quantitative measurement (vide infra). Cell proteins were quantified after solubilization in NaOH 0.5 N (2 mL/25 cm2 dish) by the Bradford method [55].
42
ACCEPTED MANUSCRIPT
Calibration curves were prepared by spiking into control matrices the stock solutions of test compounds, freshly prepared in DMSO to yield final concentrations in the 1000-1 nM concentration range. Chromatographic separation was achieved on a Phenomenex Synergi
RI PT
Fusion C18 column (100 × 2.1 mm i.d., 3 µm particle size; Phenomenex, USA). Mobile phases consisted of water (eluent A) and acetonitrile (eluent B), both added with formic acid at 0.1% v/v and at a flow rate of 350 µL min-1. A linear gradient elution was set up:
SC
T(0 min): 95% A: 5% B; T(1 min): 95% A: 5% B; T(6 min) 5%A:95% B; T(8 min) 5%A:95% B returning to initial conditions after 1 min, followed by 3 min re-equilibration
M AN U
time. Mass spectrometric detection was performed on a Thermo TSQ Quantum Access Max triple quadrupole mass spectrometer (Thermo, San Jose, CA, USA) equipped with a heated electrospray ionization (H-ESI) source. Mass spectrometric analyses were done in positive ion (ESI+) and in multiple reaction monitoring (MRM) mode. H-ESI interface
TE D
parameters were set as follows: probe middle (D) position; capillary temperature 270 °C; spray voltage 3.5 kV. Nitrogen was used as nebulizing gas at the following pressure: sheath gas: 35 psi; auxiliary gas 15 arbitrary units (a.u.). Argon was used as the collision
EP
gas at a pressure of approximately 1.5 mtorr (1 torr = 133.3 Pa). For quantitative analysis, the following parent→product ion transitions were selected: 1: m/z 447.2 [M+H]+ → m/z
AC C
128.2 + m/z 320.1 + m/z 360.1 (Tube Lens (TL) 81 V; Collision Energies (CE) 23, 28, 23 eV, respectively); 8: m/z 409.1 [M+H]+ → m/z 381.0 + m/z 363.0 + m/z 305.0 (TL 106 V; CE 24, 28, 34 eV); 14: m/z 489.1 [M+H]+ → m/z 405.0 + m/z 333.1 + m/z 330.9 (TL 115 V; CE 19, 25, 42 eV); 15: m/z 487.1 [M+H]+ → m/z 405.1 + m/z 317.1 + m/z 155.1 (TL 135 V; CE 25, 39, 22 eV); 16: m/z 473.1 [M+H]+ → m/z 405.1 + m/z 363.0 + m/z 305.1 (TL 106 V; CE 24, 28, 34 eV); 20: m/z 433.1 [M+H]+ → m/z 345.1 + m/z 317.0 + m/z
43
ACCEPTED MANUSCRIPT
305.0 (TL 106 V; CE 26, 37, 33 eV); 23: m/z 391.1 [M+H]+ → m/z 345.1 + m/z 314.8 + m/z 305.0 (TL 105 V; CE 25, 33, 29 eV), with a scan time of 0.1 s per transition. Data acquisition was performed by Xcalibur1.3 software (Thermo, USA). Peak integration and
RI PT
calibration were performed using LCQuan software (Thermo, USA). 5.3. Pharmacology
5.3.1. Cell culture. The human NSCLC cell lines A549 and H1975 were cultured as
SC
recommended. Medium was supplemented with L-glutamine (2 mmol/L) and fetal calf serum (FCS) (10%) and cells were maintained under standard cell culture conditions at 37
M AN U
°C in a water-saturated atmosphere of 5% CO2 in air. A549 cell line was from the American Type Culture Collection (Manassas, VA, USA) and H1975 was kindly provided by Dr E. Giovannetti (Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands).
TE D
5.3.2. Antibodies and reagents. Medium was from Euroclone, FCS was purchased from GibcoBRL (Grand Island, NY, USA). Monoclonal anti-EGFR and polyclonal anti-phosphoEGFR (Tyr1068) antibodies were from Cell Signaling Technology (Beverly, MA, USA).
EP
Horseradish peroxidase-conjugated (HRP) secondary antibodies were from Pierce. The enhanced chemiluminescence system (ECL) was from Millipore (Millipore, MA, USA).
AC C
Reagents for electrophoresis were obtained from BIO-RAD Laboratories, blotting reagents from Millipore.
5.3.3. Western blot analysis. Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [56]. Briefly, 40-50 of µg proteins from lysates were resolved by 7.5% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were then incubated with primary antibody, washed and then
44
ACCEPTED MANUSCRIPT
incubated with HRP-anti-rabbit antibody. Immunoreactive bands were visualized using an enhanced chemiluminescence system. The chemiluminescent signal was acquired by CDiGit® Blot Scanner and the spots were quantified by Image Studio™ Software, LI-COR
RI PT
Biotechnology (Lincoln, NE).
5.3.4. Autophosphorylation assay. Inhibition of EGFR autophosphorylation was determined as
antibodies by Western blot analysis.
SC
previously described [27] using specific anti-phosphotyrosine and anti-total EGFR
5.3.5. Cell growth inhibition. Cell viability was assessed after 3 days of treatment by tetrazolium
M AN U
dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Sigma, Dorset, UK] assay as previously described [56]. Representative results of at least three independent experiments were used for evaluation of dose-response curves, calculated from experimental points using Graph Pad Prism software. The concentration that inhibits 50%
TE D
(IC50) was extrapolated from the dose-response curves.
5.3.6. Kinase binding assay. Invitrogen LanthaScreen® Eu Kinase Binding Assay, based on TRFRET, was employed for inhibitor binding assays (Thermo Fisher Scientific, Madison, WI,
EP
USA). Briefly, 5 nM recombinant GST-EGFR (WT, Part Number PV3872 or L858R T790M, Part Number PV4879), 2 nM anti-GST Eu Antibody and kinase tracer 199 (5 nM
AC C
for WT EGFR and 20 nM for EGFR L858R T790M) were incubated in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35) in the absence or in the presence of different concentrations of EGFR inhibitors (range:10 µM-100 pM) for 1 h at room temperature in a black, low-volume 384-well plate (Corning Inc., USA). For preincubation experiments, inhibitors gefitinib (1), 8 and 16 were pre-incubated for 5 h before addition of tracer and Anti-GST Eu antibody. In one set of experiments, after 5 h of
45
ACCEPTED MANUSCRIPT
inhibitor pre-incubation, 0.5 µM of ATP were added along with tracer and Anti-GST Eu antibody. At the end of incubation period, plate was read and the ratio of emission values at 665 nm, due to the ternary complex formation among GST-EGFR, Anti-GST-Eu-
RI PT
antibody, and tracer, and at 620 nm, due to Anti-GST-Eu antibody, was plotted against inhibitor concentrations (on a log scale) to generate the binding curve and calculate IC50 values. A Tecan Spark 10M plate reader (Tecan Italia Srl, Italy) was employed for data
SC
acquisition. Instrument settings: excitation filter: 340 nm (35 nm bandpass); emission filters: 665 nm (5 nm bandpass); 620 nm (10 nm bandpass); delay time: 100 µs; integration
M AN U
time: 200 µs; gain: optimal. Reported are means together with their SEM (n=3-5). GraphPad Prism 6.01 (GraphPad Inc, USA) was employed in the LanthaScreen® TRFRET assay for nonlinear fitting of experimental data to get final IC50 values. For preincubation assays, follow-up analyses were conducted using Student’s t test. Statistical
TE D
significance was set at p<0.05.
5.3.7. Selectivity Assay. Inhibitory activity for selected compounds (8, 9 and 16) was measured at 1 µM in a set of human kinases (EGFR, ErbB2, ErbB4, BMX, BTK, JAK3, BLK) having a
EP
cysteine at the front pocket of the ATP binding site. The kinase activity of each enzyme was measured in the presence of an ATP concentration equal to the Km value, detecting the
AC C
FRET signal generated by the kinase-catalyzed phosphorylation of a reference peptide. (Z’-LYTE assay, Thermo Fisher Scientific, Madison, WI, USA). Compounds were tested in 1% DMSO solution. 5.4. Molecular Modelling 5.4.1. Model building and MD simulations. Non-covalent complexes of EGFR bound to compounds 8, 15 and 16 were built starting from the 2J5E.pdb crystal structure by docking
46
ACCEPTED MANUSCRIPT
simulations using Glide 6.1 [57]. Among the obtained docking poses, we selected those in which the quinazoline portion of the inhibitors well fitted the hinge region of the ATP binding site, the distance between Csp3 of acetamide was at less than 4Å from the sulfur
RI PT
atom of Cys797, and the angle formed by the sulfur atom of Cys797, the Csp3 of acetamide and the leaving group atom (i.e., chlorine for 8, sulfur for 15 and 16) was higher than 130°. A similar approach has been recently used to identify reasonable poses for EGFR covalent
SC
inhibitors [27, 58]. The resulting complexes were immersed in a box of TIP3P water molecules [59] and neutralized with 3 Cl- ions by using the tleap tool implemented in
M AN U
AMBER11 [60] The total system size amounted to 47179, 47187 and 47190 atoms for complexes involving 8, 15 and 16, respectively. The resulting systems were energyminimized and gradually heated to 300 K in the NVT ensemble. A short (1 ns) equilibration at pressure of 1 atm in the NPT ensemble was performed for each system before running the
TE D
production phase. The systems were then submitted to MD simulations (NVT) for 10 ns applying the AMBER99SB force field [42] for the protein and the generalized Amber force field (GAFF) [61] for the ligand. The pmemd module of AMBER11 was used to perform
EP
these tasks. Electrostatic and Van der Waals interactions were computed within a cut-off of 10 Å, while long-range electrostatic interactions were treated using the particle mesh Ewald
AC C
(PME) [62] with 128x128x128 grid points. The covalent bonds involving hydrogen atoms were constrained with the SHAKE method and a time-step of 2 fs was applied. 5.4.2. Application of the QM/MM potential. Combined QM/MM methods are effective in the study of enzyme catalysis, but given the sizes typically employed and sampling requirements, studies are often performed using semi-empirical models. This kind of QM/MM methodology has been widely applied to simulate enzymatic reactions and
47
ACCEPTED MANUSCRIPT
reported to give reasonable descriptions of reaction geometries and energetics [63]. Herein we use the Pairwise Distance Directed Gaussian modification of PM3 (PDDG/PM3) implemented in AMBER11 given its acceptable accuracy and computational performance.
RI PT
PDDG/PM3 adds a Gaussian function to the PM3 core repulsion functions to better describe chemical properties (including reactivity) of organic functional groups [39]. The MM part was treated with the AMBER99SB force field [42]. Side chain atoms of Cys797, Asp800
SC
and the substituted acetamide portions of 8, 15 and 16 were treated with the PDDG/PM3. All the other atoms of the system were described with AMBER99SB force field. The
M AN U
resulting QM systems were composed by 22, 30, 33 atoms for systems involving compound 8, 15 and 16, respectively. These included three link atoms for each complex, two placed along the C-C bond connecting Cβ of Cys797 and Asp800 to their backbone Cα, and one along the C-N bond connecting the acetamide portion to the quinazoline nucleus. The
TE D
adjust_q function of AMBER was applied to conserve the total charge of the system. During QM/MM MD simulations, all the atoms of the system (including hydrogens) were free to move, and a time step of 0.2 fs was used to integrate the equation of motion. The SHAKE
EP
option was turned off for the QM region during all QM/MM simulations. A PME approach was used to treat the QM/MM long-range electrostatic interactions.
AC C
5.4.3. Steered-MD simulation. The equilibrated complexes were employed to model the SN2 reaction with QM/MM steered-MD. In the case of compound 8, thiolate addition and chlorine expulsion were modeled using the difference of distances [d(Cαwarhead − Clwarhead) d(Scys797 − Cαwarhead)] as reaction coordinate. For compounds 15 and 16 cysteine thiolate attack and imidazol-2-ylthiolate expulsion were described by [d(Cαwarhead − Swarhead) d(Scys797 − Cαwarhead)] difference of distances. QM/MM steered-MD simulations were carried
48
ACCEPTED MANUSCRIPT
out by sequentially pulling the systems along the reaction coordinate using a force constant of 300 kcal/mol Å-2 at a constant velocity of 0.1 Å/ps. In the case of compound 8 the system was sequentially pulled along the reaction coordinate to the target value of 1.5 Å, while for
RI PT
compounds 15 and 16 the target value of the reaction coordinate was set to 2.5 Å. All QM/MM steered-MD simulation were performed at 300 K in NVT conditions, using the sander. MPI module of AMBER11 in combination with PLUMED 1.3 [64]. The work
SC
performed on the system (W) during the QM/MM steered-MD simulations was calculated by numerical integration of the exerted force (Fex) (eq. 1, where dx = v dt). The free-energy
[
〈
]
M AN U
profiles reported in Fig. 3 were calculated applying the Jarzynski’s equality (eq. 2, [44]) =
〉=〈
〉
(1) (2)
In details, for each point of the reaction coordinate, the free energy (∆F) is expressed as the
TE D
exponential average of the work (W) calculated for five independent SMD replicas. The error associated to the free-energy estimation was calculated from eq. 3 [44]. 〉−〈 〉
(3)
EP
= 〈
ACKNOWLEDGMENTS
AC C
This work was supported by Associazione Augusto per la Vita (Novellara, Reggio Emilia) and Associazione Marta Nurizzo, Brugherio (MI) to P.G. Petronini. This research benefits from the HPC (High Performance Computing) facility of the University of Parma, Italy http://www.hpc.unipr.it.
REFERENCES
49
ACCEPTED MANUSCRIPT
[1] Pallis, A. G.; Serfass, L.; Dziadziuszko, R.; van Meerbeeck, J. P.; Fennell, D.; Lacombe, D.; Welch, J.; Gridelli, C. Targeted Therapies in the Treatment of Advanced/Metastatic NSCLC.
RI PT
Eur. J. Cancer 2009, 45, 2473−2487. [2] Carmi, C.; Mor, M.; Petronini, P. G.; Alfieri, R. Clinical Perspectives for Irreversible Tyrosine Kinase Inhibitors in Cancer. Biochem. Pharmacol. 2012, 84, 1388−1399.
SC
[3] (a) Lynch, T. J.; Bell, D. W.; Sordella, R.; Gurubhagavatula, S.; Okimoto, R. A.;
M AN U
Brannigan, B. W.; Harris, P. L.; Haserlat, S. M.; Supko, J. G.; Haluska, F. G.; Louis, D. N.; Christiani, D. C.; Settleman, J.; Haber, D. A. Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib. N. Engl. J. Med. 2004, 350, 2129−2139. (b) Paez, J. G.; Jänne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.; Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T. J.; Naoki, K.; Sasaki, H.;
TE D
Fujii, Y.; Eck, M. J.; Sellers, W. R.; Johnson, B. E.; Meyerson, M. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy. Science 2004, 304, 1497– 1500. (c) Pao, W.; Miller, V.; Zakowski, M.; Doherty, J.; Politi, K.; Sarkaria, I.; Singh, B.;
EP
Heelan, R.; Rusch, V.; Fulton, L.; Mardis, E.; Kupfer, D.; Wilson, R.; Kris, M.; Varmus, H. EGF Receptor Gene Mutations Are Common in Lung Cancers from “Never Smokers″ and Are
AC C
Associated with Sensitivity of Tumors to Gefitinib and Erlotinib. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 13306– 13311
[4] (a) Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal Growth Factor Receptor Mutations in Lung Cancer. Nat. Rev. Cancer 2007, 7, 169−181. Pao, W.; Chmielecki, J. (b) Rational, Biologically Based Treatment of EGFR-Mutant Non-Small-Cell Lung Cancer. Nat. Rev. Cancer 2010, 10, 760−774.
50
ACCEPTED MANUSCRIPT
[5] Engelman, J. A.; Janne, P. A. Mechanisms of Acquired Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer. Clin. Cancer Res.
RI PT
2008, 14, 2895−2899. [6] Yun, C.-H.; Mengwasser, K. E.; Toms, A. V.; Woo, M. S.; Greulich, H.; Wong, K.-K.; Meyerson, M.; Eck, M. J. The T790M Mutation in EGFR Kinase Causes Drug Resistance by
SC
Increasing the Affinity for ATP. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2070−2075.
M AN U
[7] Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.; Greis, K. D.; Hicks, J. L.; Hook, K. E.; Keller, P. R.; Leopold, W. R.; Loo, J. A.; McNamara, D. J.; Nelson, J. M.; Sherwood, V.; Smaill, J. B.; Trumpp-Kallmeyer, S.; Dobrusin, E. M. Specific, Irreversible Inactivation of the Epidermal Growth Factor Receptor and ErbB2, by a New Class of Tyrosine Kinase Inhibitor.
TE D
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12022–12027.
[8] Blair, J. A.; Rauh, D.; Kung, C.; Yun, C.-H.; Fan, Q.-W.; Rode, H.; Zhang, C.; Eck, M. J.; Weiss, W. A.; Shokat, K. M. Structure Guided Development of Affinity Probes for Tyrosine
EP
Kinases Using Chemical Genetics. Nat. Chem. Biol. 2007, 3, 229–238. [9] Carmi, C.; Lodola, A.; Rivara, S.; Vacondio, F.; Cavazzoni, A.; Alfieri, R. R.; Ardizzoni,
AC C
A.; Petronini, P. G.; Mor, M. Epidermal Growth Factor Receptor Irreversible Inhibitors: Chemical Exploration of the Cysteine-Trap Portion. Mini-Rev. Med. Chem. 2011, 11, 1019− 1030.
[10] Hirsh, V. Afatinib (BIBW 2992) Development in Non-Small-Cell Lung Cancer. Future Oncol. 2011, 7, 817−825.
51
ACCEPTED MANUSCRIPT
[11] Katakami, N.; Atagi, S.; Goto, K.; Hida, T.; Horai, T.; Inoue, A.; Ichinose, Y.; Koboyashi, K.; Takeda, K.; Kiura, K.; Nishio, K.; Seki, Y.; Ebisawa, R.; Shahidi, M.; Yamamoto, N. LUX-
RI PT
Lung 4: a Phase II Trial of Afatinib in Patients with Advanced Non-Small-Cell Lung Cancer Who Progressed During Prior Treatment with Erlotinib, Gefitinib, or Both. J. Clin. Oncol. 2013, 31, 3335−3341.
SC
[12] Zhou, W.; Ercan, D.; Chen, L.; Yun, C.-H.; Li, D.; Capelletti, M; Cortot, A. B.; Chierac, L.; Iacob, R. E.; Padera, R.; Engen, J. R.; Wong, K.-K.; Eck, M. J.; Gray, N. S.; Janne, P. Novel
M AN U
Mutant-Selective EGFR Kinase Inhibitors Against EGFR T790M. Nature 2009, 462, 1070−1074.
[13] Cheng, H.; Nair, S. K.; Murray, B.W. Recent Progress on Third Generation Covalent
TE D
EGFR Inhibitors. Bioorg. Med. Chem. Lett. 2016, 26, 1861–1868.
[14] Ramalingam, S. S.; Yang, J. C.; Lee, C. K.; Kurata, T.; Kim, D. W.; John, T.; Nogami, N.; Ohe, Y.; Mann, H.; Rukazenkov, Y.; Ghiorghiu, S.; Stetson, D.; Markovets, A.; Barrett, J. C.;
EP
Thress, K. S.; Jänne, P. A. Osimertinib As First-Line Treatment of EGFR Mutation-Positive Advanced Non-Small-Cell Lung Cancer. J. Clin. Oncol. 2017, doi: 10.1200/JCO.2017.74.7576.
AC C
[15] Finlay, M. R. V.; Anderton, M.; Ashton, S.; Ballard, P.; Bethel, P. A.; Box, M. R.; Bradbury, R. H.; Brown, S. J.; Butterworth, S.; Campbell, A.; Chorley, C.; Colclough, N.; Cross, D. A. E.; Currie, G. S.; Grist, M.; Hassall, L.; Hill, G. B.; James, D.; James, M.; Kemmitt, P.; Klinowska, T.; Lamont, G.; Lamont, S. G.; Martin, N.; McFarland, H. L.; Mellor, M. J.; Orme, J. P.; Perkins, D.; Perkins, P.; Richmond, G.; Smith, P.; Ward, R. A.; Waring, M. J.; Whittaker, D.; Wells, S.; Wrigley, G. L. Discovery of a Potent and Selective EGFR Inhibitor (AZD9291) of
52
ACCEPTED MANUSCRIPT
Both Sensitizing and T790M Resistance Mutations That Spares the Wild Type Form of the Receptor. J. Med. Chem. 2014, 57, 8249−8267.
RI PT
[16] Song, Z.; Ge, Y.; Wang, C.; Huang, S.; Shu, X.; Liu, K.; Zhou, Y.; Ma, X. Challenges and Perspectives on the Development of Small-Molecule EGFR Inhibitors against T790M-Mediated Resistance in Non-Small- Cell Lung Cancer. J. Med. Chem. 2016, 59, 6580–6594.
SC
[17] Cross, D. A.; Ashton, S. E.; Ghiorghiu, S.; Eberlein, C.; Nebhan, C. A.; Spitzler, P. J.;
M AN U
Orme, J. P.; Finlay, M. R.; Ward, R. A.; Mellor, M. J.; Hughes, G.; Rahi, A.; Jacobs, V. N.; Red Brewer, M.; Ichihara, E.; Sun, J.; Jin, H.; Ballard, P.; Al-Kadhimi, K.; Rowlinson, R.; Klinowska, T.; Richmond, G. H.; Cantarini, M.; Kim, D. W.; Ranson, M. R.; Pao, W. AZD9291, an Irreversible EGFR TKI, Overcomes T790M-Mediated Resistance to EGFR Inhibitors in Lung
TE D
Cancer. Cancer Discovery 2014, 4, 1046−1061.
[18] Soria, J. C.; Ohe, Y.; Vansteenkiste, J.; Reungwetwattana, T.; Chewaskulyong, B.; Lee, K. H.; Dechaphunkul, A.; Imamura, F.; Nogami, N.; Kurata, T.; Okamoto, I.; Zhou, C.; Cho, B. C.;
EP
Cheng, Y.; Cho, E. K.; Voon, P. J.; Planchard, D.; Su, W. C.; Gray, J. E.; Lee, S. M.; Hodge, R.; Marotti, M.; Rukazenkov, Y.; Ramalingam, S. S. Osimertinib in Untreated EGFR-Mutated
AC C
Advanced Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2018, 378, 113-125. [19] Engel, J.; Lategahn, J.; Rauh, D. Hope and Disappointment: Covalent Inhibitors to Overcome Drug Resistance in Non-Small Cell Lung Cancer. ACS Med. Chem. Lett. 2016, 7, 2−5.
[20] (a) Thress, K. S.; Paweletz, C. P.; Felip, E.; Cho, B. C.; Stetson, D.; Dougherty, B.; Lai, Z.; Markovets, A.; Vivancos, A.; Kuang, Y.; Ercan, D.;Matthews, S.E.; Cantarini,M.; Barrett,
53
ACCEPTED MANUSCRIPT
J.C.; Janne, P. A.; Oxnard, G. R. Acquired EGFR C797S Mutation Mediates Resistance to AZD9291 in Non-Small Cell Lung Cancer Harboring EGFR T790M. Nat. Med. 2015, 21,
RI PT
560−562. (b) Ercan, D.; Choi, H. G.; Yun, C. H.; Capelletti, M.; Xie, T.; Eck, M. J.; Gray, N. S.; Jänne, P. A. EGFR Mutations and Resistance to Irreversible Pyrimidine-Based EGFR Inhibitors. Clin. Cancer Res. 2015, 21, 3913-3923. (c) Bersanelli, M.; Minari, R.; Bordi, P.; Gnetti, L.;
SC
Bozzetti, C.; Squadrilli, A.; Lagrasta, C. A.; Bottarelli, L.; Osipova, G.; Capelletto, E.; Mor, M.; Tiseo, M. L718Q Mutation as New Mechanism of Acquired Resistance to AZD9291 in EGFR-
M AN U
Mutated NSCLC. J. Thorac. Oncol. 2016; 11, e121-3.
[21] Callegari, D.; Ranaghan, K. E.; Woods, C. J.; Minari, R.; Tiseo, M.; Mor, M.; Mulholland A. J. Lodola A. L718Q Mutant EGFR Escapes Covalent Inhibition by Stabilizing a NonReactive Conformation of The Lung Cancer Drug Osimertinib. Chem. Sci. 2018, 9, 2740-2749.
TE D
[22] Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf.
EP
Model. 2017, 57, 3124-3137.
[23] Niessen, S.; Dix, M. M.; Barbas, S.; Potter, Z. E.; Lu, S.; Brodsky, O.; Planken, S.;
AC C
Behenna, D.; Almaden, C.; Gajiwala, K. S.; Ryan, K.; Ferre, R.; Lazear, M. R.; Hayward, M. M.; Kath, J. C.; Cravatt, B. F. Proteome-Wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors. Cell Chem Biol 2017, 24, 1388-1400. [24] Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem. Biol. 2013, 20, 146159.
54
ACCEPTED MANUSCRIPT
[25] Truong, T. H. Ung, P. M.; Palde, P. B.; Paulsen, C. E.; Schlessinger, A.; Carroll, K. S. Molecular Basis for Redox Activation of Epidermal Growth Factor Receptor Kinase. Cell Chem.
RI PT
Biol. 2016, 23, 837-848. [26] Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W.
SC
Covalent EGFR Inhibitor Analysis Reveals Importance of Reversible Interactions to Potency and
M AN U
Mechanisms of Drug Resistance. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 173−178. [27] Carmi, C.; Cavazzoni, A.; Vezzosi, S.; Bordi, F.; Vacondio, F.; Silva, C.; Rivara, S.; Lodola, A.; Alfieri, R. R.; La Monica, S.; Galetti, M.; Ardizzoni, A.; Petronini, P. G.; Mor, M. Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the
TE D
Cysteine-Trap Portion. J. Med. Chem. 2010, 53, 2038−2050.
[28] Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.;
EP
Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and
AC C
Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014, 57, 10072−10079. [29] Kung, A.; Chen, Y. C.; Schimpl, M.; Ni, F.; Zhu, J.; Turner, M.; Molina, H.; Overman, R.; Zhang, C. Development of Specific, Irreversible Inhibitors for a Receptor Tyrosine Kinase EphB3. J. Am. Chem. Soc. 2016, 138, 10554-10560.
55
ACCEPTED MANUSCRIPT
[30] Mishani, E.; Abourbeh, G.; Jacobson, O.; Dissoki, S.; Daniel, R. B.; Rozen, G.; Shaul, M.; Levitzki, A. High-affinity Epidermal Growth Factor Receptor (EGFR) Irreversible Inhibitors
RI PT
with DiminIshed Chemical Reactivities as Positron Emission Tomography (PET)-Imaging Agent Candidates of EGFR Overexpressing Tumors. J. Med. Chem. 2005, 48, 5337–5348.
[31] (a) Carmi, C.; Galvani, E.; Vacondio, F.; Rivara, S.; Lodola, A.; Russo, S.; Aiello, S.;
SC
Bordi, F.; Costantino, G.; Cavazzoni, A.; Alfieri, R.R.; Ardizzoni, A.; Petronini, P.G.; Mor, M. Irreversible Inhibition of Epidermal Growth Factor Receptor Activity by 3-Aminopropanamides.
M AN U
J. Med. Chem. 2012, 55, 2251–2264. (b) Mao, Y.; Liu, Z.; Yang, X.; Xia, X.; Zhang, R.; Li, J.; Jiang, X.; Xie, K.; Zheng, J.; Zhang, H.; Suo, J.; Shen, J. A New and Improved Process for N-(4Chloro-3-cyano-7-ethoxyquinolin-6-yl)acetammide. Org. Process Res. Dev. 2012, 16, 19701973.
TE D
[32] Benesch, R. E.; Benesch, R. The acid strength of the −SH group in cysteine and related compounds. J. Am. Chem. Soc. 1955, 77, 5877− 5881.
EP
[33] Berteotti, A.; Vacondio, F.; Lodola, A.; Bassi, M.; Silva, C.; Mor, M.; Cavalli, A. Predicting the Reactivity of Nitrile-Carrying Compounds with Cysteine: A Combined
AC C
Computational and Experimental study. ACS Med. Chem. Lett. 2014, 5, 501−505. [34] Abourbeh, G.; Dissoki, S.; Jacobson, O.; Litchi, A.; Ben Daniel, R.; Laki, D.; Levitzki, A.; Mishani, E. Evaluation of Radiolabeled ML04, a Putative Irreversible Inhibitor of Epidermal Growth Factor Receptor, as a Bioprobe for PET Imaging of EGFR-overexpressing Tumors. Nucl. Med. Biol. 2007 34, 55-70.
56
ACCEPTED MANUSCRIPT
[35] Jost C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and Selectivity in Covalent Enzyme Inhibition: a Systematic Study of Electrophilic Fragments. J Med Chem. 2014,
RI PT
57, 7590-7599. [36] (a) Field, M. J.; Bash, P. A.; Karplus, M. A Combined Quantum Mechanical and Molecular Mechanical Potential for Molecular Dynamics Simulations. J. Comput. Chem. 1990,
SC
11, 700− 733. (b) Gao, J. Methods and Applications of Combined Quantum Mechanical and Molecular Mechanical Potentials. In Reviews in Computational Chemistry; Lipkowitz, K. B.,
M AN U
Boyd, D. R., Eds.; VCH Publishers: New York, 1996; Vol. 7, pp 119−185. (c) Warshel, A. Computer Simulations of Enzyme Catalysis: Methods, Progress, and Insights. Annu. Rev. Biophys. Biomol. Struct. 2005, 32, 425−443.
[37] (a) Schirmeister, T.; Kesselring, J.; Jung, S.; Schneider, T.; Weickert, A.; Becker, J.; Lee,
TE D
W.; Bamberger, D.; Wich, P.; Distler, U.; Tenzer, Stefan; Johe, Patrick; Hellmich, U. A.; Engels, B. Quantum Chemical-Based Protocol for the Rational Design of Covalent Inhibitors. J. Am. Chem. Soc. 2016, 138, 8332−8335. (b)
Lodola, A.; Capoferri, L.; Rivara, S.; Tarzia, G.;
EP
Piomelli, D.; Mulholland, A.; Mor, M. Quantum Mechanics/Molecular Mechanics Modeling of Fatty Acid Amide Hydrolase Reactivation Distinguishes Substrate from Irreversible Covalent
AC C
Inhibitors. J. Med. Chem. 2013, 56, 2500−2512. (c) Van der Kamp, M. W.; Mc Geagh, J. D.; Mulholland, A. J. Lethal Synthesis” of Fluorocitrate by Citrate Synthase Explained through QM/MM Modeling. Angew. Chem., Int. Ed. 2012, 50, 10349−10351. (d) Lodola, A.; De Vivo, M. The Increasing Role of QM/MM in Drug Discovery. Adv. Protein Chem. Struct. Biol. 2012, 87, 337−362.
57
ACCEPTED MANUSCRIPT
[38] Capoferri, L.; Lodola, A.; Rivara, S.; Mor, M. Quantum Mechanics/Molecular Mechanics Modeling of Covalent Addition between EGFR–Cysteine 797 and N-(4-Anilinoquinazolin-6-yl)
RI PT
Acrylamide. J. Chem. Inf. Model. 2015, 55, 589–599. [39] Repasky, M.; Chandrasekhar, J.; Jorgensen, W. PDDG/PM3 and PDDG/MNDO: Improved semiempirical methods. J. Comput. Chem. 2002, 23, 1601–1622.
SC
[40] Walker, R. C.; Crowley, M. F.; Case, D. A. The Implementation of a Fast and Accurate
M AN U
QM/MM Potential Method in Amber. J. Comput. Chem. 2008, 29,1019-1031. [41] Vayner, G.; Houk, K. N.; Jorgensen, W. L.; Brauman, J. I. Steric Retardation of SN2 Reactions in the Gas Phase and Solution. J. Am. Chem. Soc. 2004, 126, 9054– 9058. [42] Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Comparison
TE D
of Multiple Amber Force Fields and Development of Improved Protein Backbone Parameters. Proteins 2006, 65, 712−725.
[43] (a) Crespo, A.; Martí, M. A.; Estrin, D. A.; Roitberg, A. E. Multiple-Steering QM−MM
EP
Calculation of the Free Energy Profile in Chorismate Mutase. J. Am. Chem. Soc. 2005, 127,
AC C
6940-6941. (b) Lodola, A.; Branduardi, D.; Capoferri, L.; De Vivo, M.; Mor, M.; Piomelli, D. A Catalytic Mechanism for Cysteine N-terminal Nucleophile Hydrolases, as Revealed by Free energy simulations. PLoS One 2012, 7, e32397. [44] (a) Jarzynski, C. Nonequilibrium Equality for Free Energy Differences. Phys. Rev. Lett. 1997, 78, 2690−2693. (b) Park, S.; Khalili-Araghi, F.; Tajkhorshid, E.; Schulten, K. Free Energy Calculation from Steered Molecular Dynamics Simulations using Jarzynski’s Equality. J. Chem. Phys. 2003, 119, 3559.
58
ACCEPTED MANUSCRIPT
[45] Vacondio, F.; Carmi, C.; Galvani, E.; Bassi, M.; Silva, C.; Lodola, A.; Rivara, S.; Cavazzoni, A.; Alfieri, R. R.; Petronini, P. G.; Mor M. Long-lasting Inhibition of EGFR
Inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 5290-5284.
RI PT
Autophosphorylation in A549 Tumor Cells by Intracellular Accumulation of Non-Covalent
[46] Galetti, M.; Alfieri, R. R.; Cavazzoni, A.; La Monica, S.; Bonelli, M.; Fumarola, C.;
SC
Mozzoni, P.; De Palma, G.; Andreoli, R.; Mutti, A.; Mor, M.; Tiseo, M.; Ardizzoni, A.; Petronini, P. G. Functional Characterization of Gefitinib Uptake in Non-Small Cell Lung Cancer
M AN U
Cell Lines. Biochem. Pharmacol. 2010, 80, 179−187.
[47] Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine and Threonine Proteases. Chem. Rev. 2002, 102, 4369-4750.
TE D
[48] Smaill, J.B.; Palmer, B.D.; Rewcastle, G.W.; Denny, W.A.; McNamara, D.J.; Dobrusin, E.M.; Bridges, A.J.; Zhou, H.; Showalter, H.D.H.; Winters, R.T.; Leopold, W.R.; Fry, D.W.; Nelson, J.M.; Slintak, V.; Elliot, W.L.; Roberts, B.J.; Vincent, P.W.; Patmore, S.J. Tyrosine
EP
Kinase Inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(Phenylamino)pyrido[d]pyrimidine Acrylamides as Irreversible Inhibitors of the ATP Binding Site of the Epidermal Growth Factor
AC C
Receptor. J. Med. Chem. 1999, 42, 1803− 1815. [49] Kletzien, R. F.; Pariza, M. W.; Becker, J. E.; Potter, V. R. A Method Using 3-O-methylD-glucose and Phloretin for the Determination of Intracellular Water Space of Cells in Monolayer culture. Anal. Biochem. 1975, 68, 537-544. [50] Lanthascreen® Eu Kinase Binding Assay, ThermoFisher Scientific Inc., USA.
59
ACCEPTED MANUSCRIPT
[51] Barf, T.; Kaptein, A. Irreversible Protein Kinase Inhibitors: Balancing the Benefits and Risks. J. Med. Chem. 2012, 55, 6243− 6262.
RI PT
[52] Hur, W.; Velentza, A.; Kim, S.; Flatauer, L.; Jiang, X.; Valente, D.; Mason, D. E.; Suzuki, M.; Larson, B.; Zhang, J.; Zagorska, A.; Di Donato, M.; Nagle, A.; Warmuth, M.; Balk, S. P.; Peters, E. C.; Gray, N. S. Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase.
SC
Bioorg. Med. Chem. Lett. 2008, 18, 5916−5919.
M AN U
[53] Van Schaeybroeck, S.; Kyula, J.; Kelly, D. M.; Karaiskou-McCaul, A.; Stokesberry, S. A.; Van Cutsem, E.; Longley, D. B.; Johnston, P. G. Chemotherapy-Induced Epidermal Growth Factor Receptor Activation Determines Response to Combined Gefitinib/Chemotherapy Treatment in Non-Small Cell Lung Cancer Cells. Mol. Cancer Ther. 2006, 5, 1154-1165.
TE D
[54] Tschitschibabin, A. E.; Woroshtzow, N. N. Ueber einige Derivate des α,β-Pyridinthioindoxyls. Chem. Ber. 1933, 66, 364-368.
[55] Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram
AC C
248–254.
EP
Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72,
[56] Cavazzoni, A.; Petronini, P. G.; Galetti, M.; Roz, L.; Andriani, F.; Carbognani, P.; Rusca, M.; Fumarola, C.; Alfieri, R.; Sozzi, G. Dose-Dependent Effect of FHIT-Inducible Expression in Calu-1 Lung Cancer Cell Line. Oncogene 2004, 23, 8439–8446. [57] Glide, Version 6.1, Schrodinger, LLC, New York, NY, 2013.
60
ACCEPTED MANUSCRIPT
[58] Tarozzi, A.; Marchetti, C.; Nicolini, B.; D'Amico, M.; Ticchi, N.; Pruccoli, L.; Tumiatti, V.; Simoni, E.; Lodola, A.; Mor, M.; Milelli, A.; Minarini, A. Combined Inhibition of the
RI PT
EGFR/AKT Pathways by a Novel Conjugate of Quinazoline with Isothiocyanate. Eur. J. Med. Chem. 2016, 117, 283-291.
[59] Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L.
SC
Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983,
M AN U
79, 926−935.
[60] Case, D. A.; Darden, T. A.; Cheatham III, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; Roberts, B. P.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Kolossváry, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Wu, X.; Brozell,
S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, J.; Hsieh, M.-J.; Cui, G.; Roe, D. R.;
TE D
Mathews, D. H.; Seetin, M. G.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER11, University of California, San Francisco.
EP
[61] Wang, J. M.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and testing of a general Amber force field. J. Comput. Chem. 2004, 25, 1157−1174.
AC C
[62] Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald - an NLog(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089−10092. [63] (a) Lodola, A.; Mor, M.; Hermann, J. C.; Tarzia, G.; Piomelli, D.; Mulholland, A. J. QM/MM Modelling of Oleamide Hydrolysis in Fatty Acid Amide Hydrolase (FAAH) Reveals a New Mechanism of Nucleophile Activation. Chem. Commun. 2005, 4399−4401. (b) TubertBrohman, I.; Acevedo, O.; Jorgensen, W. L. Elucidation of Hydrolysis Mechanisms for Fatty
61
ACCEPTED MANUSCRIPT
Acid Amide Hydrolase and its Lys142Ala Variant via QM/MM Simulations. J. Am. Chem. Soc. 2006, 128, 16904−16913. (c) Lodola, A.; Mor, M.; Sirirak, J.; Mulholland, A. J. Insights into the
RI PT
Mechanism and Inhibition of Fatty Acid Amide Hydrolase from Quantum Mechanics/Molecular Mechanics (QM/MM) Modelling. Biochem. Soc. Trans. 2009, 37, 363−367. (d) Acevedo, O.; Jorgensen, W. L. Advances in Quantum and Molecular Mechanical (QM/MM) Simulations for
SC
Organic and Enzymatic Reactions. Acc. Chem. Res. 2010, 43, 142-151. Lima, M. C.; (e) Seabra, G. M. Reaction Mechanism of the Dengue Virus Serine Protease: a QM/MM Study. Phys Chem
M AN U
Chem Phys. 2016, 18, 30288-30296.
[64] Bonomi, M.; Branduardi, D.; Bussi, G.; Camilloni, C.; Provasi, D.; Raiteri, P.; Donadio, D.; Marinelli, F.; Pietrucci, F.; Broglia, R. A.; Parrinello, M. PLUMED: A Portable Plugin for Free-Energy Calculations with Molecular Dynamics. Comput. Phys. Commun. 2009, 180,
Graphical Abstract
AC C
EP
TE D
1961−1972
62
ACCEPTED MANUSCRIPT Highlights A series of N-(4-(phenylamino)quinazolin-6-yl)acetamides was synthetized
•
Imidazol-2-ylthioacetamide 16 causes long-lasting inhibition of EGFR
•
16 shows negligible reactivity with cysteine in solution
•
Biochemical assays suggest the formation of a covalent bond between 16 and Cys797
•
Compound 16 inhibits proliferation of H1975 cells in the low micromolar range
AC C
EP
TE D
M AN U
SC
RI PT
•
S0223-5234(18)30988-7
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.029
Reference:
EJMECH 10886
To appear in:
European Journal of Medicinal Chemistry
Received Date: 9 July 2018 Revised Date:
8 November 2018
Accepted Date: 9 November 2018
Please cite this article as: R. Castelli, N. Bozza, A. Cavazzoni, M. Bonelli, F. Vacondio, F. Ferlenghi, D. Callegari, C. Silva, S. Rivara, A. Lodola, G. Digiacomo, C. Fumarola, R. Alfieri, P.G. Petronini, M. Mor, Balancing reactivity and antitumor activity: Heteroarylthio acetamide derivatives as potent and time-dependent inhibitors of EGFR, European Journal of Medicinal Chemistry (2018), doi: https:// doi.org/10.1016/j.ejmech.2018.11.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Balancing reactivity and antitumor activity: heteroarylthio acetamide derivatives as potent and
RI PT
time-dependent inhibitors of EGFR
Riccardo Castelli,‡1 Nicole Bozza,‡1 Andrea Cavazzoni,2 Mara Bonelli,2 Federica Vacondio,1 Francesca Ferlenghi,1 Donatella Callegari,1 Claudia Silva,1 Silvia Rivara,1 Alessio Lodola,*1
1
2
Department of Food and Drug, University of Parma, Parma, Italy
Department of Medicine and Surgery, University of Parma, Parma, Italy
These authors contributed equally to this work
*Corresponding Authors
EP
Prof. Alessio Lodola,
TE D
‡
M AN U
Mor*1
SC
Graziana Digiacomo,2 Claudia Fumarola,2 Roberta Alfieri,2 Pier Giorgio Petronini,2 and Marco
Department of Food and Drug, University of Parma, Parma, Italy
&
AC C
E-mail: [email protected]
Prof. Marco Mor,
Department of Food and Drug, University of Parma, Parma, Italy E-mail: [email protected]
1
ACCEPTED MANUSCRIPT
ABSTRACT
Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates
RI PT
Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-
SC
yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by
M AN U
replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a
TE D
time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in
concentration.
KEYWORDS
4-anilinoquinazoline;
warhead;
cysteine;
time-dependent
inhibition;
QM/MM
AC C
EGFR;
EP
gefitinib-resistant H1975 (EGFR L858R/T790M) lung cancer cells at low micromolar
simulations.
Abbreviations
DMF, dimethylformamide; PDDG, Pairwise Distance Directed Gaussian; QM/MM, quantum mechanics/molecular mechanics; SMD, steered molecular dynamics; TBTU, O-(benzotriazol-1yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate; THF, tetrahydrofuran; TS, transition state.
2
ACCEPTED MANUSCRIPT
1. INTRODUCTION
RI PT
Non-small-cell lung cancer (NSCLC) accounts for the 80% of all lung cancers, which represent the leading cause of cancer-related deaths worldwide. NSCLC is featured by a 5-year survival rate lower than 15% [1]. Gefitinib (1) and erlotinib (2) are the first generation of EGFR
SC
inhibitors approved for the treatment of NSCLC [2]. Tumor responsiveness to these drugs has been related to the insurgence of “activating mutations” such as short in-frame deletions in exon
M AN U
19 (del19) or point mutations in exon 21, the latter resulting in arginine replacing leucine at codon 858 (L858R) [3,4]. Gefitinib and erlotinib display a remarkable therapeutic outcome in patients expressing activated forms of EGFR. However, their efficacy in clinical use is limited by the occurrence of resistance. Nearly 60% of patients with EGFR alterations present a single
TE D
aminoacid mutation at the gatekeeper position of the ATP-binding site, from threonine to methionine (T790M) [5]. The presence of this bulkier methionine reduced the potency of first generation inhibitors for EGFR, favoring their competitive displacement by ATP, which is
EP
present at millimolar concentrations inside the cells [6]. This resistance mechanism has been overcome by equipping the scaffold of 4-anilinoquinazoline inhibitors with an acrylamide
AC C
warhead: by virtue of a hetero-Michael addition, the acrylamide moiety is capable of alkylating a cysteine residue (i.e. Cys797) proximal to the hinge region of the EGFR active site [7,8]. The formation of a covalent bond with Cys797 allowed second-generation inhibitors to exert protracted occupancy of EGFR active site, overcoming competition with ATP [9]. Among the second-generation inhibitors that entered into clinical studies, afatinib (compound 3) was approved for the treatment of patients with metastatic NSCLC with EGFR mutations [10]. In patients with acquired resistance to gefitinib (1) through the development of T790M mutation,
3
ACCEPTED MANUSCRIPT
afatinib (3) showed marginal therapeutic benefit. Its clinical use has been restricted by adverse effects occurring in skin and gastrointestinal tract, resulting from the concomitant inhibition of the wild type (WT) form of EGFR [11]. The disclosure of pyrimidine-based inhibitors [12],
RI PT
selective for the T790M mutant over the WT, prompted the search for new candidates to be advanced into the clinic [13]. Osimertinib (4) [14,15], the leader of this third generation of EGFR inhibitors [16], is a 2-aminopyrimidine derivative featuring an acrylamide warhead portion. It
SC
has been approved for patients affected by metastatic EGFR T790M mutation-positive NSCLC who have progressed on after the therapy with first- and second-generation inhibitors [17], and
M AN U
very recently for the first-line treatment of patients with metastatic NSCLC carrying EGFRactivating mutation [18]. However, mutations conferring resistance to osimertinib have been reported [19]. Among the identified mutations, C797S, in which the critical cysteine 797 is replaced by a less nucleophilic serine, directly hampers EGFR alkylation, while loss of T790M
TE D
or appearance of L718Q mutation may affect the accommodation of the inhibitor in the active site [20], preventing the formation of a protein-ligand reactive conformation that leads to target alkylation [21]. These findings indicate that both the acrylamide group and the driving portion
EP
need to be optimized to ensure selective inhibition of activated forms of EGFR (i.e. L858R, or del19) in the presence of novel mutations. Moreover, the presence of a highly electrophilic
AC C
warhead (i.e. as for acrylamide) remains a toxicity burden [22]. A recent work on osimertinib has shown that it inhibits several lysosomal cathepsins when administered to mice [23]. Considering that cathepsins targeted by osimertinib are also expressed by human cells, some concerns have been raised on the protracted use of osimertinib.
4
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 1. Chemical structures of selected EGFR inhibitors.
In this scenario, the availability of warheads able to form a covalent bond with Cys797 [24],
TE D
but less reactive than acrylamide present in EGFR inhibitors, would facilitate the design of novel and selective inhibitors able to overcome resistance in NSCLC [25, 26]. Our previous work, performed installing new warheads on a 4-anilinoquinazoline nucleus, has led to the
EP
identification of long-lasting inhibitors of EGFR having electrophilic groups other than acrylamide [27]. However, epoxide-based inhibitor 5 (Fig. 1) raised safety concerns similar to
AC C
those of acrylamides, while the 2-(pentafluorophenoxy)-acetamide 6 resulted incapable to overcome T790M resistance. Herein, using a 4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline scaffold (compound 7, Scheme 1) featured by a substitution pattern similar to that of the clinically approved drug afatinib (3, Fig. 1), we prepared a series of compounds possessing at the position 6 an “activated” acetamide group, which may potentially alkylate Cys797 by a nucleophilic substitution (SN). Our investigation started with the synthesis of a 4-(3-chloro-4fluoroaniline)-7-ethoxyquinazoline bearing a chloroacetamide at position 6 (compound 8,
5
ACCEPTED MANUSCRIPT
Scheme 1). The chloroacetamide was selected as the reference warhead for our work as this group has been reported to alkylate cysteine both in solution [28] and within the ATP binding site of kinases [29] and it has been already installed on a different 4-anilinoquinazoline to
RI PT
achieve irreversible inhibition of EGFR [30]. As consequent step of our research, we modulated the reactivity of the acetamide portion by replacing the chlorine atom in 8 with (hetero)-aromatic thiols or carboxylate esters that may act as leaving groups in the framework of a SN reaction. Our
from the
necessity
of
having
compounds
with
SC
choice of using the 4-anilinoquinazoline nucleus for the installation of the new warheads arises good
inhibitory potency in
the
M AN U
autophosphorylation assay performed on A549 cells, which expressed WT EGFR. This test was used as a screening method to identify potential irreversible inhibitors of EGFR. Regardless of the chemical nature of the driver portion employed to target the ATP binding site of EGFR, our approach can lead to the identification of soft warheads able to bind the reactive cysteine
2. CHEMISTRY
TE D
(Cys797) of EGFR, while sparing all other thiols present in the cellular environment.
EP
The newly synthesized inhibitors reported in this work (Scheme 1) feature the 6-amino-4-(3-
AC C
chloro-4-fluoroaniline)-7-ethoxyquinazoline core (7) equipped with specific amide-group based warheads obtained by means of acylation of the 6-amino group with a panel of “electrophilic traps”. The synthesis of the anilinoquinazoline scaffold 7 was performed as previously reported with minor adaptations (see Supplementary Data) [31]. The general strategy features the chloroacetamide-functionalized quinazoline core (compound 8) as starting material in the synthesis of most of the compounds of the class. Compound 8 was prepared by reacting the amino group of 7 with the mixed-anhydride obtained from the reaction of the trialkyl ammonium
6
ACCEPTED MANUSCRIPT
salt of chloroacetic acid with pivaloyl chloride. This method proved superior to the direct use of chloroacetyl chloride both in terms of purity of the final compound and overall yield, and allowed the use of tetrahydrofuran as the solvent, being 7 sparingly soluble in either chlorinated
RI PT
solvents and acetonitrile. The obtained chloroacetyl-functionalized anilinoquinazoline 8 was further elaborated by reacting with the appropriate nucleophile to furnish compounds 10, 12, 1422, as depicted in Scheme 1. The general procedure for this subset of compounds features the
AC C
EP
TE D
M AN U
SC
alkylation of the heterocyclic thiol or carboxylate salt by 8 in the presence of a base.
Scheme 1. Reagents and conditions: a) i. chloroacetic acid, diisopropylethylamine, THF; ii. pivaloyl chloride, 30 min; iii. 7, room temperature (yield 83%); b) See Experimental Section for
7
ACCEPTED MANUSCRIPT
specific conditions (yield range: 19-89%); c) 3-pyridylmercaptoacetic acid, TBTU, DMF, triethylamine, room temperature, (for 11, yield: 25%); 3-pyridin-3-yl-propionyl chloride, THF, pyridine, room temperature, (for 13, yield: 39%); d) NaOCH3, THF, CH3OH, room temperature
RI PT
(23, yield: 62%).
The exact conditions for each compound varied extensively depending on the reactivity of
SC
each thiol or carboxylate salt, details are given in the Experimental Section. The synthesis of compounds 11 and 13 had to be devised differently, and was performed condensing 7 and 3acid
in
the
presence
of
O-(benzotriazol-1-yl)-N,N,N’,N’-
M AN U
pyridylmercaptoacetic
tetramethyluronium tetrafluoroborate (TBTU) for 11 and 3-pyridin-3-yl-propionyl chloride for 13 respectively. Compound 23 (the derivative of glycolic acid) was derived from 20 (the acetoxy-derivative) by means of a transesterification reaction performed with a substoichiometric
TE D
amount of sodium methoxide in THF and methanol.
EP
3. RESULTS AND DISCUSSION
3.1. Reactivity with cysteine in solution
AC C
We set to investigate whether our newly prepared warheads could indeed engage in a nucleophilic substitution, and evaluate their potential reactivity in a purely chemical setting, in order to obtain a scale of relative reactivity. Quinazoline derivatives 8-23 were tested for their ability to undergo a nucleophilic substitution in the presence of a large molar excess (1:100) of cysteine at a 10 µM concentration in 50 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS; pH 10) buffer at 37 °C (Table 1). We set the pH of the buffer to 10 in order to have the thiol
8
ACCEPTED MANUSCRIPT
group of cysteine (pKa = 8.5 [32]) predominantly in its thiolate form and thus more prone to react with electrophiles. Consumption of the starting compounds and formation of the corresponding cysteine-
acrylamide
RI PT
conjugate were monitored by LC-MS, adopting our previously reported protocol [33]. An derivative
(N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-
yl)acrylamide, compound 9 [31a]) was included in this study as a benchmark, for its established
SC
reactivity towards thiols. Acrylamide derivatives have high propensity to react with several sites. This results in a high non-specific binding in cells, also at rather low compound concentration
acrylamide group [34].
M AN U
(i.e. 0.06 µM) as reported for [18F]-ML04, a labelled 4-anilinoquinazoline inhibitor bearing a 6This further stress the importance of finding chemical selective
warheads. Compound 8 was included considering the ability of the chloroacetamide group to alkylate cysteines [28, 29]. Chloroacetamide derivatives have been recently shown to exhibit low
TE D
cross-reactivity versus a panel of eleven enzymes, suggesting that chloroacetamide itself could be intrinsically less promiscuous than what initially expected in the presence of biological nucleophiles [35].
EP
Both the acrylamide 9 and the chloroacetamide 8 reacted with a large molar excess of cysteine at pH 10 following pseudo first-order kinetics. LC-UV analysis showed that the disappearance of
AC C
compound 8 paralleled the formation of the cysteine-conjugate (see Supplementary Data, Fig. S1) indicating the absence of competing reactions (e.g. hydrolysis). Compound 8 displayed a half-life time (t1/2) of nearly 60 min (Table 1), resulting 4-fold less reactive than the acrylamide analogue 9 (t1/2 = 15 min). Additionally, stability assays performed at pH 10 confirmed that both compounds were stable for the tested time period. As opposed to 8 and 9, all the other compounds reported in Table 1 reacted with cysteine either very slowly (t1/2 > 1440 min for
9
ACCEPTED MANUSCRIPT
compounds 10, 12, 14-22) or not at all (compounds 11, 13, 23), thereby precluding a quantitative ranking of their relative electrophilicity. To qualitatively rank them, the area of the peak of the cysteine-conjugate was measured by LC-MS after 24 h of reaction time (see Supplementary
RI PT
Data, Fig. S2), and used as a relative descriptor of reactivity. The area of the covalent conjugate given between 8 and cysteine was arbitrarily set at the reference value of 10,000, while the cysteine-conjugate areas for the other compounds were referred to this value. The 2-((1H-
SC
tetrazol-5-yl)thio)acetamide 14 and the 2-((1H-imidazol-2-yl)thio)acetamides 15 and 16 resulted significantly more reactive than the 2-((pyridin-2-yl)thio)acetamide 10, the 2-((pyrimidin-2-
M AN U
yl)thio)acetamide 12 and the 2-((1-methyl-benzimidazol-2-yl)thio)acetamide derivative 17, as well as than its analogues 18 and 19. For 2-((pyridin-3-yl)thio)acetamide 11 and 3-(pyridin-3yl)-propanamide 13 no cysteine-conjugate could be observed. Among the carboxylate esters, 20, 21 and 22, the benzoyloxyacetamide derivative 21 showed a reactivity comparable to that of the
TE D
2-((1H-imidazol-2-yl)thio)acetamide 16, while the acetoxy derivative 20 resulted one of the less reactive compounds of the whole set. Additionally, for esters 20 and 21 significant amounts of the hydrolyzed product (glycolate derivative, compound 23) could be observed after 24 h.
EP
Compound 13, in which the sulfur atom on the acetamide portion is replaced by a methylene unit, expectedly failed to give the conjugate, as well as 11, where the sulfur atom is not in the 2-
AC C
position of a nitrogen-containing heterocycle. Moreover, the hydroxyacetamide 23 did not show any conjugate when incubated with cysteine. As an additional test, we measured the chemical stability of the cysteine-conjugate, obtained in situ by incubating a prototypical acetamide (i.e. 2-chloroacetamide 8) with a large excess of cysteine, under the conditions of the reactivity assay (see above) by LC-MS. The cysteineconjugate was stable for at least 24 hours as evidenced by Fig. S3 of the supplementary data
10
ACCEPTED MANUSCRIPT
section. This result indicates that cysteine alkylation by activated acetamide is an irreversible process.
RI PT
3.2. Simulation of Cys797 alkylation by QM/MM simulations To support the hypothesis of covalent bond formation within EGFR, we evaluated whether the putative warheads could undergo attack by Cys797 via a SN2 reaction pathway by means of a
SC
quantum mechanics/molecular mechanics (QM/MM) approach [36]. The reaction mechanism (depicted in Fig. 2) was simulated for the chloroacetamide 8 and for the two 2-(imidazol-2-
M AN U
ylthio)acetamide derivatives 15 and 16, which emerged as the most promising compounds in the reactivity assay. QM/MM mechanistic modelling is currently providing useful indications for inhibitor design specifically when the formation and breakage of covalent bonds are involved in the inhibitory process [37]. Models of enzyme-ligand non-covalent complexes were built by docking 8, 15 and 16 within the ATP-binding pocket of the X-ray structure of the covalent
TE D
adduct of WT EGFR with a N-(4-anilinoquinazolin-6-yl) acrylamide inhibitor [8]. Previous calculations performed by our group [38] suggest that Cys797 can act as nucleophile in the form of thiolate anion, with the nearby Asp800 as the natural acceptor of the thiol hydrogen. The
EP
Cys797/Asp800 pair was therefore modelled in Cys-S-/Asp-COOH form (as reported in Fig. 2A).
AC C
Among the obtained docking poses, we selected those in which the quinazoline portion of the inhibitors well fitted the hinge region of the ATP binding site, and the angle formed by the sulfur atom of Cys797, the Csp3 of the acetamide fragment and the leaving group atom (i.e., chlorine for 8, sulfur for 15 and 16) was higher than 130°. Such an angle approaches the value of 180° in an ideal transition state (TS) for a SN2 reaction. The docking complexes were solvated and equilibrated by classic molecular dynamics simulations (see Experimental Section for details). The relaxed structures were then employed to model the SN2 reaction by means of a PDDG/PM3
11
ACCEPTED MANUSCRIPT
Hamiltonian [39] coupled to the AMBER99SB potential [40]. The PDDG/PM3 Hamiltonian has been shown to yield energy barriers for SN2 reactions in gas phase similar to those calculated by ab initio methods [41]. In our approach, the substituted acetamide fragment of the inhibitor and
RI PT
the side chains of Cys797 and of Asp800 were treated at PDDG/PM3 level, while the remaining part of the system was described by AMBER99SB force field [42]. To account for solvation and structural flexibility of both EGFR active site and inhibitor, the hybrid PDDG/PM3-
SC
AMBER99SB Hamiltonian was used in steered molecular dynamics (SMD) simulations. To simulate the nucleophilic substitution involving Cys797, we applied the SMD approach
M AN U
implemented in AMBER, applying an adaptive force along a given reaction coordinate (RC) to form or break covalent bonds, similarly to what described in reference [43]. In the case of compound 8, thiolate addition and chlorine expulsion were modeled using the difference of distances [d(Cαwarhead − Clwarhead) - d(Scys797 − Cαwarhead)]. For compounds 15 and 16, cysteine
TE D
thiolate attack and imidazol-2-ylthiolate expulsion were described by the difference [d(Cαwarhead − Swarhead) - d(Scys797 − Cαwarhead)]. While for compound 8 our QM/MM steered-MD approach led to the identification of a stable alkylation product with low free-energy, this was not the case for
EP
compounds 15 and 16 (data not shown). We thus hypothesized that protonation at the imidazole ring could stabilize the imidazolylthio leaving group. In fact, the carboxylic acid of Asp800 was
AC C
close enough to the distal nitrogen at the imidazole ring to protonate it. For compounds 15 and 16, therefore, Cys797 alkylation was modelled starting from the cationic species resulting from protonation of the nitrogen atom at the imidazole ring, with deprotonated carboxylate group of Asp800 (Fig. 2B).
12
RI PT
ACCEPTED MANUSCRIPT
SC
Fig. 2. Reactions modelled by QM/MM simulation for compound 8, with expulsion of a chloride ion (panel A) and for compounds 15 and 16 with expulsion of a protonated imidazolylthiolate
M AN U
group (panel B). In the former case, Asp800 was modelled as carboxylic acid, while in the latter as a carboxylate anion. In panel B, R stands for -CH3 (compound 15) or -H (compound 16). The free-energy profiles calculated for Cys797 alkylation by compounds 8, 15 and 16 are reported in Fig. 3. A single energy barrier separates reactants (R) from products (P). No
TE D
intermediates are identified as stable minima in QM/MM simulations, which is consistent with a SN2 mechanism. The calculated activation energy for Cys797 alkylation, obtained applying the Jarzynski equality [44], (see Experimental section) was 27.0 ± 0.7 kcal/mol for compound 8,
EP
40.4 ± 1.2 kcal/mol for compound 15 and 34.2 ± 0.7 kcal/mol for compound 16. The calculated
AC C
reaction energy was -12.6 ± 0.7 and -10.2 ± 1.2 kcal/mol for 8 and 16 respectively, indicating that Cys797 alkylation by these two compounds has an exergonic character. Conversely, the calculated reaction energy for 15 was 0.9 ± 1.0 kcal/mol, suggesting that for this specific compound the reaction was not energetically favored.
13
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 3. Free-energy curves with uncertainties for Cys797 alkylation by compounds 8 (blue), 15 (orange) and 16 (magenta), obtained by the Jarzynski equality as exponential average of five independent replicas for each EGFR-inhibitor complex, using different snapshots taken from the equilibrating QM/MM MD trajectory. Reaction coordinate is expressed in Å, while the free
TE D
energy is given in kcal/mol.
The geometries identified along the SMD trajectory indicate that the TS structure for the
EP
alkylation of Cys797 by 8 and 16 features a trigonal bipyramidal geometry (Fig. 4A and Fig. 4B). For compound 15 such a geometry is distorted, due to steric hindrance between the methyl
AC C
group of the imidazole ring and the p-loop of the protein (Fig. 4C). Calculations thus indicate that compounds 8 and 16 (but not 15) can easily react with WT EGFR, through alkylation of the Cys797 residue and leading to stable adducts. It is worth mentioning that these calculations were performed contextually to the synthesis of the compounds and their biological testing. These results were in fact critical for the decision to synthesize and test compound 16, which resulted one of the most interesting ones on the gefitinib resistant cell line (vide infra).
14
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 4. Representative TS structures for Cys797 alkylation by compound 8 (panel A, cyan carbon
M AN U
atoms), 16 (panel B, purple carbon atoms) and 15 (panel C, orange carbon atoms). Protein carbon atoms are depicted in white, with EGFR secondary elements depicted as white cartoons.
3.3. EGFR Inhibitory activity on A549 lung cancer cells
TE D
Compounds 8-23 were tested for their ability to inhibit EGFR autophosphorylation in A549 lung cancer cells, harboring WT EGFR. We decided to investigate the effect of the novel warheads on this cell line because the 4-anilinoquinazoline scaffold possesses high affinity for
EP
WT EGFR [13,16]. Gefitinib (1) and the acrylamide derivative (9) were tested as reference inhibitors. All the synthesized compounds inhibited EGFR autophosphorylation, with IC50 values
AC C
ranging from the single digit nM for compounds 8, 11 and 14, to 83 and 197 nM for the 2(benzothiazolyl-2-thio)acetamides 18 and 19, respectively (Table 1). These data confirm that the structural modifications introduced by the warheads on the 4-anilinoquinazoline nucleus allowed to retain the high inhibitory potency of the parent structure. To assess the persistency of EGFR inhibition within cells, test compounds were added to cell cultures at 1 µM concentration and autophosphorylation in A549 cells was measured both 1 h
15
ACCEPTED MANUSCRIPT
after the addition and 8 h after washing of the inhibitor from the extra-cellular medium. Inhibition enduring after compound removal could be ascribed to irreversible inhibition of the kinase domain [45]. 8 h after inhibitor washing, gefinitib (1) gave 46% of inhibition, while
RI PT
acrylamide 9 and chloroacetamide 8 inhibited EGFR autophosphorylation by more than 80%. In the same conditions, compounds 10-12 inhibited EGFR by more than 70%, which is evocative of a covalent interaction with the target Cys797. However, 60% of inhibition observed for
SC
compound 13, which lacks a group able to undergo nucleophilic substitution, suggests that persistent accumulation of the inhibitor within the cells may account for the observed inhibition
M AN U
data, as it had been observed for other EGFR inhibitors [46]. The tetrazolyl (14), imidazolyl (15, 16), benzimidazolyl (17) and benzothiazolyl (18) derivatives also blocked EGFR autophosphorylation 8 h after washing. Within this subset, the two imidazole and the benzimidazole derivatives were able to reach at least 90% of inhibition.
TE D
Finally, we tested the set of ester derivatives 20-22 on A549 cells. Compounds with similar groups were reported in the literature to covalently bind activated cysteines [47]. All these derivatives were able to ensure EGFR inactivation 8 h after dilution. Despite its failure to give
EP
detectable amounts of conjugate in the presence of cysteine at basic pH, also the hydroxyacetamide 23 displayed significant long-term inhibition (77% of inhibition after 8 h).
AC C
Overall, the evaluation of the autophosphorylation levels of EGFR in A549 cells allowed to identify several 4-anilinoquinazoline derivatives giving a percentage of EGFR inhibition close to or even higher than 80% 8 h after inhibitor removal, which may indicate an irreversible binding to the target [48]. A representative set of these compounds was further evaluated in vitro (vide infra), to assess whether this prolonged inhibition can be ascribed to a covalent interaction with the target.
16
ACCEPTED MANUSCRIPT
Table 1. LC-MS reactivity assay in the presence of cysteine and EGFR autophosphorylation
1 (Gefitinib)
R
Reactivity Assay a t1/2 Peak (min) Areab
Autophosphorylation A549 cellsc IC50 (nM)
M AN U
Cpds
SC
RI PT
inhibition for compounds 1, 8–23 in A549 cell line.
% inhibition @ 1µMd 1h 8h
N. T.e
N. T.e
60±25
96 ± 4.0
46 ± 5.0
60 ± 5.0
10000
7.0±5.7
100 ± 0.5
81 ± 2.9
15 ± 3.0
-
15±6.4
96 ± 4.0
99 ± 1.0
> 1440
0.51
70±21
73 ± 4.3
71± 8.1
11
> 1440
N. D.f
0.60±0.14
96 ± 4.0
89 ± 4.6
12
> 1440
0.66
27±18
99 ± 1.0
88 ± 16
13
> 1440
N. D.f
14±1.4
99 ± 0.50
60± 2.1
-
9
AC C
10
EP
TE D
8
17
> 1440
6.5
5.0±1.5
100 ± 6.4
75 ± 4.1
15
> 1440
4.5
11±5.0
97 ± 2.5
95 ± 4.8
16
> 1440
15
27±3.5
99 ± 0.10
90 ± 4.6
17
> 1440
0.38
12±0.71
100 ± 1.0
100 ± 1.0
18
> 1440
0.39
88 ± 0.90
75 ± 0.50
EP
AC C
23
> 1440
1.4
197±18.4
63 ± 0.10
93 ± 0.50
1400 ± 100
0.64
34±11
97 ± 0.40
75 ± 19
> 1440
11
25±12
88 ± 3.1
89 ± 5.7
> 1440
5
44±0.71
97 ± 1.9
97 ± 2.6
> 1440
N. D.f
27±11
90 ± 3.6
77 ± 11
TE D
20
22
83±28
M AN U
19
21
RI PT
14
SC
ACCEPTED MANUSCRIPT
Half-life measured at pH 10 and 37 °C by LC-MS. bArea of Cys-conjugate of compound 8 was arbitrarily set at the value of 10,000 and all the Cys-conjugate areas of remaining compounds were referred to this value. cInhibition of EGFR autophosphorylation in A549 cells was evaluated by Western blot analysis. dPercentage of inhibition at 1 µM concentration was measured after 1 h of treatment and 8 h after washing of the compound from the medium (1 h incubation). e Not tested. f Not detected. a
18
ACCEPTED MANUSCRIPT
3.4. Intracellular dosage in A549 cells of selected inhibitors
RI PT
In order to discriminate whether the observed EGFR inhibition 8 h after washing had to be ascribed to a covalent interaction or to accumulation into A549 cells, intracellular dosages of selected compounds were carried out. Chloroacetamide 8, 2-((1H-tetrazol-5-yl)thio)acetamide
SC
14, 2-((1H-imidazol-2-yl)thio)acetamides 15 and 16 and 2-acetoxyacetamide 20 were selected as representatives for highly (8), moderately (14-16) and weakly reactive (20) compounds,
M AN U
according to their susceptibility to nucleophilic substitution in the presence of free cysteine (Table 1). Intracellular concentrations of these compounds were measured in A549 cells by LCMS under conditions reproducing those of the EGFR autophosphorylation assay, i.e. immediately after 1 h of exposure to the inhibitor (1 µM) or 8 h after washing the inhibitor from
TE D
the extracellular medium (Table 2).
Gefinitib (1) was chosen as a representative of non-covalent inhibitor of WT EGFR. The results previously obtained for the acrylamide derivative PD168393 [48] (compound 24) are also
EP
reported in Table 2 for comparison. Considering the complexity associated with the measurement of intracellular volume, the concentrations reported herein are approximate.
AC C
Gefitinib (1) displayed an intracellular concentration of 271 µM after 1 h, when EGFR autophosphorylation is inhibited almost completely (96%), while 8 h after washing its concentration was approximately 100-times lower (2.4 µM) and autophosphorylation was inhibited by 46% (Table 1). The intracellular concentration of acrylamide 24 was below the limit of detection (LOD) of the LC-MS method both at 1 h and 8 h, as expected for its high propensity to react promiscuously within the cell. This result clearly indicates irreversible inhibition of
19
ACCEPTED MANUSCRIPT
EGFR, while the case of gefitinib pointed out that residual concentrations of a non-covalent inhibitor may cause some inhibition even 8 h after compound removal. With the sole exception of the 2-acetoxyacetamide 20, all other tested compounds (8, 14-16)
RI PT
showed high intracellular concentrations (i.e. > 100 µM) after 1h of incubation. This observation corroborates the assumption of higher stability in the intracellular environment of our new set of inhibitors with respect to acrylamide-based derivatives. However, it does not provide
SC
information regarding the mechanism of action of the tested compounds. EGFR inhibition by compound 20 in A549 cells after 1 h of incubation, when it showed a much lower concentration
M AN U
than other inhibitors, may be ascribed to its hydrolyzed counterpart (compound 23), which possesses the ability to inhibit EGFR autophosphorylation.
A significant drop in the intracellular concentration was observed 8 h after the washing from A549 cells for all the tested compounds. However, only for chloroacetamide 8 and 2-((1H-
TE D
imidazol-2-yl)thio)acetamide 16 sub-micromolar levels were observed, with a 144- and 222-fold reduction compared to the values measured after 1 h, respectively. While not a conclusive evidence of covalent inhibition, it is remarkable that both compounds, having the ability to
EP
alkylate cysteine groups, show long-term inhibition of EGFR autophosphorylation in spite of a
AC C
large decrease of their intracellular concentrations.
Table 2. Intracellular concentrations in A549 cells and inhibitory potency on recombinant WT EGFR for selected anilinoquinazoline derivatives.
20
Cpds
Intracellular concentrationa (µM)
R
RI PT
ACCEPTED MANUSCRIPT
Ratio 1 h/8 h
Kinase Assayb (WT EGFR) IC50 (nM)
8h
271 ± 34
2.4 ± 0.2
112
0.47 ± 0.05
24 (PD168393)
< 0.05c
<0.05c
-
0.27 ± 0.04
8
115 ± 10
0.80 ± 0.10
144
0.87 ± 0.10
14
181 ± 2
5.6 ± 0.1
32
0.53 ± 0.09
179 ± 6
10.2 ± 2.1
18
N.T.
118 ± 10
0.53 ± 0.10
222
0.62 ± 0.08
5.8 ± 0.6
<0.05
-
0.52 ± 0.03
132 ± 10d
1.1 ± 0.1d
120d
0.34 ± 0.05
16
M AN U
AC C
20
TE D
15
-
EP
1 (Gefitinib)
23
SC
1h
A549 cells were incubated with 1 µM solution of test compound for 1 h. Intracellular content was quantified immediately after incubation and 8 h after removal of the compound from the medium as pmol/mg of protein in each sample. Reported µM concentrations were calculated dividing the compound content (in pmol) by an average value of cell volume of 4 µL per mg of protein, estimated by measurements of the equilibrium distribution in A549 cells of 3-O-methylD-[1-3H]glucose using the method of Kletzien et al, [49]. b Measured by LanthaScreen Eu kinase a
21
ACCEPTED MANUSCRIPT
binding assay (see Experimental Section for details). Mean values of three independent experiments ± SD are reported). c Data from Vacondio et al. [45]. d Generated in A549 cells from the hydrolysis of compound 20.
RI PT
3.5. Binding to recombinant WT EGFR and EGFR L858R/790M double mutant The ability of compounds 1, 8, 14-16, 20, 23 and 24 to displace a fluorescent tracer bound to the kinase domain of WT EGFR was assessed by means of a time-resolved fluorescence
SC
resonance energy transfer (TR-FRET) assay [50]. Compared to EGFR autophosphorylation in intact cells, this assay yields inhibitory potency in the absence of cellular transport and/or
M AN U
metabolism. Sub-nanomolar potencies were observed for all the tested compounds, with IC50 values ranging from 0.27 nM for the acrylamide derivative 24 to 0.87 nM for chloroacetamide 8. Thus, the concentrations in the micromolar range, detected for compounds 8, 14, 15, 16 and 23 at 8 h, were significantly higher than IC50 values measured on the isolated enzyme. Therefore, these concentrations could be sufficient to explain the inhibition of EGFR autophosphorylation
TE D
observed at 8 h (75-95%, see Table 1). On the other hand, being the IC50 values obtained in an artificial environment, the TR-FRET assay provides only a relative order of potency among different compounds, while the high intracellular concentration of ATP can compete with
EP
reversible inhibitors at its binding site. In fact, gefitinib (1), a non-covalent inhibitor, only
AC C
inhibited by 46% EGFR autophosphorylation in A549 cells after 8 h, in spite of an intracellular concentration of 2.4 µM. By comparison, 8 and 16 possess similar IC50 values on recombinant WT EGFR, but they inhibited EGFR autophosphorylation in A549 cells, 8 h after washing, significantly more than 1 (81% and 90% for 8 and 16 vs. 46% for 1). Considering that 8 and 16 reached intracellular concentration at 8 h lower than 1 (0.80 µM and 0.53 µM for 8 and 16 vs. 2.4 µM for 1), it is conceivable that these two compounds might inhibit EGFR through a covalent mechanism of action.
22
ACCEPTED MANUSCRIPT
We next tested the ability of 8 and 16 to inhibit EGFR L858R/T790M double mutant using the TR-FRET binding assay (see Experimental section for details). Quinazoline-based inhibitors usually display lower inhibitory potency for this mutant, when compared to WT or EGFR L858R
RI PT
single mutant, due to steric clashes with M790 [9]. The insertion in the anilinoquinazoline core of an electrophilic group, able to covalently bind the target, usually allows the inhibitor to recover activity, but the impact on the measured potency depends on the rate of target alkylation.
SC
Thus, when a slow covalent reaction occurs at the binding site, the observed IC50 value becomes dependent on the time of incubation [51]. Fig. 5 reports the inhibitory potency for gefitinib (1,
M AN U
panel A), 8 (panel B) and 16 (panel C) measured with no pre-incubation (left column) or preincubating the inhibitor for 5 h with EGFR L858R/T790M double mutant (right column). The IC50 value of 1 did not change with the pre-incubation, as expected for a non-covalent inhibitor, while for compounds 8 and 16 a significant reduction in the IC50 values was observed. This
AC C
EP
L858R/T790M enzyme.
TE D
further supports a covalent mechanism of inhibition for these two compounds, also on EGFR
A
23
RI PT
ACCEPTED MANUSCRIPT
C
TE D
M AN U
SC
B
EP
Fig. 5. Effect of the pre-incubation time on the potency (expressed as IC50) of gefitinib, 1 (panel A), compound 8 (panel B) and compound 16 (panel C) on recombinant EGFR L858R/T790M.
AC C
IC50 were measured by means of a time-resolved fluorescence resonance energy transfer (TRFRET) assay using LanthaScreen technology. Values are reported as mean ± standard error of the mean (n = 5). Statistical significance was set at P<0.05. **: P<0.01; ***: P<0.001. TR-FRET experiments on EGFR L858R/T790M with 5 h inhibitor pre-incubation were repeated either in the absence or in the presence of 500 nM ATP. Although the displacement curves for compounds 1, 8 and 16 were barely affected by ATP (see supplementary data, Fig.
24
ACCEPTED MANUSCRIPT
S4), the limited signal-to-noise ratio of these TR-FRET measures precludes a conclusive assessment of the effect of ATP on the inhibitory process for the tested inhibitors.
RI PT
3.6. Selectivity on kinases bearing a conserved cysteine on the ATP front pocket We next tested the ability of compounds 8, 9 and 16 to inhibit a panel of kinases reported to have a cysteine in a position corresponding to that of Cys797 in EGFR [52]. In this investigation,
SC
the following kinases were included: i. ErbB2 and ErbB4 of the Erb family, the same of EGFR, ii. BMX and BTK belonging to the Tek family, iii. JAK3 representative of the Janus kinase
M AN U
family and iv. BLK, member of the Src family. Acrylamide 9 inhibited the activity of nearly all the kinases of the panel at 1 µM, in agreement with previous findings reported in the literature [52]. On the contrary, 2-chloroacetamide 8 and imidazol-2-ylthioacetamide 16, markedly inhibited EGFR and ErbB4 only (i.e. 87% of higher), as they moderately (ErbB2, BTX and
TE D
BLK) or marginally (BMX, JAK3) affected the activity of the other kinases included in the panel. This assay confirms further that the replacement of acrylamide with less reactive warheads ensures higher selectivity.
EP
Table 3. Selectivity of compounds 8, 9 and 16 on a panel of kinases bearing a front-pocket
9
AC C
cysteine corresponding to EGFR Cys797.a
90
92
100
88
87
92
97
16
90
65
87
30
54
0
58
Cpds
EGFR
ErbB2
ErbB4
BMX
BTK
JAK3
BLK
8
98
76
100
38
53
32
62
a
Inhibitory activity determined with an activity-based assay (see methods for details). Percent of inhibition measured at 1 µM. Color code: red, ≥80% inhibition; yellow, 50−80% inhibition; green, ≤50% inhibition. Reported data are the average of two independent experiments.
25
ACCEPTED MANUSCRIPT
3.7. Activity on H1975 lung cancer cells H1975 lung cancer cells are resistant to first generation inhibitors of EGFR due to the presence
RI PT
of the T790M mutation which somehow hampers the interaction between the 4anilinoquinazoline nucleus and ATP-binding site (see above). The introduction in the quinazoline scaffold of a group capable of alkylating Cys797 is expected to ensure a significant
SC
improvement in the antiproliferative potency on H1975 cells compared to gefitinib [16]. With this premise, we examined the ability of the synthesized compounds to block proliferation of
M AN U
H1975 cells and to inhibit EGFR autophosphorylation at two concentrations (0.1 and 1 µM) in the same cell line (Table 4) along with gefitinib (1) and acrylamide 9, which were used as controls as they are expected to be the least and most potent compounds of the series, respectively.
TE D
Compound 1 resulted poorly active on H1975 cells [53] as it inhibited cell growth with an IC50 of 9.1 µM and it did not affect autophosphorylation at 0.1 or 1 µM concentration, confirming its incapability to engage EGFR L858R/T790M mutant. On the contrary, the 9
showed
sub-micromolar
antiproliferative
potency
and
inhibited
EP
acrylamide
autophosphorylation almost completely at both concentrations. The same compound had shown
AC C
an antiproliferative IC50 value of 1.6 µM in a previous experiment [31a]. Among the newly synthesized compounds, the hydroxyacetamide 23, which lacks a potential warhead, did not show any antiproliferative activity up to 10 µM and did not inhibit EGFR L858R/T790M autophosphorylation. Chloroacetamide 8 and the 2-((1H-imidazol-2-yl)thio)acetamide 16 significantly outperformed gefitinib and displayed a pharmacological activity superior (8) or comparable (16) to that of the acrylamide 9. Indeed, compound 8 resulted extremely potent in
26
ACCEPTED MANUSCRIPT
inhibiting H1975 cell proliferation (IC50 of 0.06 µM) and capable of fully abolishing EGFR L858R/T790M autophosphorylation at 0.1 µM. Compound 16 inhibited H1975 growth with an
RI PT
IC50 of 1.4 µM and completely inhibited autophosphorylation at 1 µM concentration. Remarkably, the ability to overcome the effect of T790M mutation in H1975 cells is achieved introducing electrophilic groups featuring moderate (8) or very low reactivity (16) toward free cysteine, when compared to acrylamide.
SC
Table 4. Inhibition of cell proliferation and inhibition of EGFR autophosphorylation in H1975
1 (Gefitinib)
R
EP
Cpds
TE D
M AN U
cells for compounds 1, 8-12 and 14-23.
9.1 ± 1.1
8.0 ± 4.9
18 ± 4.2
8
0.06 ±0.01
100 ± 0.9
100 ± 0.10
9
0.70 ± 0.14
93 ± 0.70
95 ± 0.10
10
3.7 ±0.28
1.5 ± 2.1
42 ± 7.0
AC C
-
H1975 cell line Autophosphorylationb Cell growth a % of inhibition @ Inhibition 0.1 µM 1 µM IC50 (µM)
27
ACCEPTED MANUSCRIPT
3.0 ±0.42
29 ± 10
92 ± 9.9
12
3.6 ±1.8
40 ± 12
87 ± 9.8
14
2.1 ±0.78
16 ± 8.5
96 ± 5.7
15
6.9 ±0.64
45 ± 14
67 ± 8.5
16
1.4 ±0.10
SC
RI PT
11
M AN U
80 ± 7.0
100 ±0.71
9.1±1.0
6.0 ± 4.2
70 ± 6.8
9.8±0.14
29 ± 11
24 ± 7.7
3.2±0.71
30 ± 13
92 ± 0.90
3.4±0.57
27 ± 8.5
68 ±13
8.3±1.8
0.5 ± 0.7
43 ± 4.4
22
6.1±1.4
20 ± 3.9
16 ± 8.0
23
> 10
0.0 ± 0.1
0.0 ± 0.1
17
20
AC C
21
EP
19
TE D
18
a
Concentration able to inhibit 50% of cell proliferation as determined by the MTT assay, after 72 h of incubation with compounds (dose ranging from 0.1 to 10 µM). Osimertinib displays an IC50
28
ACCEPTED MANUSCRIPT
of 0.10 ± 0.1 µM in the employed conditions. b Inhibition of EGFR autophosphorylation in H1975 cells was evaluated by Western blot analysis.
4. CONCLUSION
RI PT
We report a structure-reactivity and structure-activity study aimed to identify novel cysteinetargeting warheads, less reactive than acrylamide, that may be exploited for the design of irreversible kinase inhibitors with improved chemical selectivity. In particular, we focused on the
SC
design and synthesis of 4-anilinoquinazoline derivatives bearing warheads with reduced risk of nonspecific covalent binding to thiols, yet able to exploit the high nucleophilicity of Cys797 of
M AN U
EGFR and to overcome the resistance conferred by the T790M gatekeeper mutation to reversible, anilinoquinazoline-based inhibitors. Starting from the chloroacetamide derivative 8, reduction of reactivity for a series of potential SN2 substrates led to compound 16, a 2-((1Himidazol-2-yl)thio)acetamide derivative, which showed a inhibitory potency on H1975 cell
TE D
growth (IC50 = 1.4 ± 0.10 µM) comparable to that of the reference acrylamide-based inhibitor 9 (IC50 = 0.70 ± 0.10 µM). Considering that 9 and 16 have the same 4-anilino-quinazoline scaffold, the imidazol-2-ylthioacetamide group emerges as a bioisostere of the acrylamide warhead. It
EP
should be pointed out that while compound 16 displays negligible reactivity towards free cysteine in solution and possess higher selectivity than acrylamide 9 toward a panel of selected
AC C
kinases, its imidazol-2-ylthioacetamide warhead is a metabolic soft spot potentially able to give toxic effects in vivo.
Several hints, including hybrid QM/MM simulations and pre-incubation studies on recombinant EGFR L858R/T790M double mutant, support the hypothesis that compound 16 irreversibly inhibits EGFR by reacting covalently with Cys797. By comparison with acrylamide-based inhibitors, we propose that long-lasting inhibition can be achieved by modulating the reactivity
29
ACCEPTED MANUSCRIPT
of the cysteine-targeting moiety, to potentially overcome the low specificity of hetero-Michael acceptors. Reported cases of resistance to covalent EGFR inhibitors by Cys797 mutation discourage the strategy to target this specific residue by covalent binders as a stand-alone
RI PT
approach to provide next-generation antitumor agents. However, the results here reported may help the design of novel drugs targeting specific cysteines with improved selectivity. In particular, it is suggested that heteroarylthio acetamide warheads can lead to irreversible
SC
inhibition of kinases by covalent binding to cysteine residues, and that the low reactivity of such
molecular modelling simulations.
5. EXPERIMENTAL SECTION 5.1. Chemistry
M AN U
a warhead can be counterbalanced by appropriate design of the overall inhibitor through
TE D
Reagents were obtained from commercial suppliers and used without further purification. Solvents were purified and stored according to standard procedures. Anhydrous reactions were conducted under a positive pressure of dry N2. Reactions were monitored by TLC, on Kieselgel
EP
60 F 254 (DC-Alufolien, Merck). Final compounds and intermediates were purified by flash chromatography (SiO2 60, 40−63 µm). Melting points were not corrected and were determined
AC C
with a Gallenkamp melting point apparatus. The 1H NMR spectra were recorded on a Bruker 300 MHz Avance or on a Bruker 400 MHz Avance spectrometer; chemical shifts (δ scale) are reported in parts per million relative to the central peak of the solvent. 1H NMR spectra are reported in the following order: multiplicity, approximate coupling constant (J value) in hertz, and number of protons; signals are characterized as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet), m (multiplet), and br s (broad signal).
30
ACCEPTED MANUSCRIPT
Mass spectra of final compounds were recorded using a Thermo Scientific LTQ Orbitrap XL. All tested compounds were > 95% pure by HPLC/UV analysis. Compound 9 was prepared as reported in reference [31a], while compound 24 has been prepared according to reference [48]. H and 13C NMR spectra for final compounds 8, 10-23 are reported in the Supplementary Data.
RI PT
1
5.1.1. 2-chloro-N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)acetamide
(8).
SC
Chloroacetic acid (355 mg, 3.76 mmol) is dissolved in anhydrous THF (5 ml), DIPEA (650 µl, 3.73 mmol) and pivaloyl chloride (460 µl, 3.73 mmol) are added and the mixture is
M AN U
stirred at room temperature for 30 minutes before adding a solution of 6-amino-4-(3chloro-4-fluoroaniline)-7-ethoxyquinazoline (7, 227 mg, 0.68 mmol) in anhydrous THF (3 ml). After 2 h the suspension is diluted with water and the mixture is extracted with AcOEt. The organic layer is washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column
TE D
chromatography (AcOEt/hexane 4:1), affording the title compound (231 mg, 83%) as a white solid. Mp: 278°C (dec.). 1H NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.74 (s, 1H), 8.87 (d, J = 3.9 Hz, 1H), 8.53 (s, 1H), 8.10 (dd, J = 6.8, 2.6 Hz, 1H), 7.78 (ddd, J =
EP
9.0, 4.3, 2.6 Hz, 1H), 7.42 (t, J = 9.1 Hz, 1H), 7.29 (s, 1H), 4.49 (d, J = 1.5 Hz, 2H), 4.30 13
C NMR (75 MHz, DMSO-d6) δ 165.04,
AC C
(q, J = 6.9 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H).
156.89, 154.84, 154.28, 154.03, 151.63, 149.11, 136.73, 126.59, 123.75, 122.61 (d, J = 6.8 Hz), 118.80, 116.43 (d, J = 21.5 Hz), 115.57, 108.73, 107.37, 64.64, 43.33, 14.24. HRMS (ESI): m/z: calcd for C18H15Cl2FN4O2 [(M+H)+]: 409.06289; found: 409.06291. 5.1.2. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-(pyridin-2ylthio)acetamide (10). 2-mercaptopyridine (174 mg, 1.57 mmol) is added to a suspension of sodium hydroxide (64.5 mg, 1.61 mmol) in THF (3 ml). To the clear solution thus
31
ACCEPTED MANUSCRIPT
obtained, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 61.4 mg, 0.15 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with ethyl acetate. The organic layer is washed with water, saturated aqueous
RI PT
NaHCO3 and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by triturating with AcOEt, affording the title compound (44 mg, 61%) as a white powder. Mp: 225°C (dec.). 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.86 (s, 1H),
SC
8.53 (d, J = 5.3 Hz, 1H), 8.50 (s, 1H), 7.94 (dd, J = 6.7, 2.7 Hz, 1H), 7.68 – 7.59 (m, 2H), 7.37 (d, J = 8.1 Hz, 1H), 7.27 – 7.10 (m, 3H), 4.26 (q, J = 7.0 Hz, 2H), 4.11 (s, 2H), 1.38 13
C NMR (75 MHz, DMSO-d6) δ 167.34, 156.82 (d, J = 3.1 Hz),
M AN U
(t, J = 7.0 Hz, 3H).
154.79, 153.73, 153.29, 149.56, 148.62, 137.12, 136.82, 127.26, 123.69, 122.57 (d, J = 6.8 Hz), 121.94, 120.45, 118.65 (d, J = 18.1 Hz), 116.39 (d, J = 21.7 Hz), 113.46, 108.89, 107.18, 64.60, 34.02, 14.13. HRMS (ESI): m/z: calcd for C23H19ClFN5O2S [(M+H)+]:
TE D
484.10048; found: 484.10038.
5.1.3. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-(pyridin-3ylthio)acetamide (11). 3-pyridylmercaptoacetic acid [54] (142 mg, 0.84 mmol) is dissolved
EP
in DMF (6 ml) and triethylamine (0.12 ml, 0.58 mmol) is added. The solution is cooled to 0°C and TBTU (275 mg, 0.85 mmol) is added followed, after 30 minutes, by 6-amino-4-
AC C
(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (7, 160 mg, 0.48 mmol). The mixture is warmed to 40°C and stirred at the same temperature for 4 h. After cooling to room temperature, the solution is diluted with AcOEt and washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/MeOH 9:1) to afford the title compound (58 mg, 25%) as a white solid. Mp: 210°C. 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.81 (s,
32
ACCEPTED MANUSCRIPT
1H), 8.65 (s, 1H), 8.49 (s, 1H), ), 8.44 (s, 1H), 7.99 – 7.82 (m, 2H), 7.65 – 7.54 (m, 1H), 7.38 (dd, J = 8.3, 4.6 Hz, 1H), 7.24 – 7.10 (m, 2H), 4.30 (q, J = 6.9 Hz, 2H), 4.00 (s, 2H), 1.54 (t, J = 6.9 Hz, 3H).
13
C NMR (75 MHz, CDCl3/CD3OD) δ 167.82, 158.35, 157.14,
RI PT
154.68, 153.89, 150.11, 148.72, 148.19, 138.50, 136.23, 127.66, 125.38, 123.15 (d, J = 6.7 Hz), 121.15 (d, J = 18.5 Hz), 116.90 (d, J = 22.2 Hz), 112.07, 109.78, 106.86, 65.79, 39.07, 14.63, 0.04. HRMS (ESI): m/z: calcd for C23H19ClFN5O2S [(M+H)+]: 484.10048;
SC
found: 484.10043.
5.1.4. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-(pyrimidin-2-
M AN U
ylthio)acetamide (12). 2-mercaptopyrimidine (38 mg, 0.34 mmol) is added to a suspension of sodium hydroxide (14.0 mg, 0.35 mmol) in THF (2 ml). To the clear solution, 6(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 57 mg, 0.14 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with
TE D
ethyl acetate. The organic layer is washed with water, saturated aqueous NaHCO3 and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt), affording the title compound (59
EP
mg, 88%) as a white solid. Mp: 243°C. 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 9.68 (s, 1H), 8.93 (s, 1H), 8.69 (d, J = 4.9 Hz, 2H), 8.50 (s, 1H), 8.08 (dd, J = 6.9, 2.6 Hz,
AC C
1H), 7.85 – 7.65 (m, 1H), 7.39 (t, J = 9.1 Hz, 1H), 7.29 (t, J = 4.9 Hz, 1H), 7.24 (s, 1H), 4.41 – 4.06 (m, 4H), 1.36 (t, J = 6.9 Hz, 3H).
13
C NMR (75 MHz, DMSO-d6) δ 169.98,
166.78, 158.02, 156.81, 154.78, 153.45, 151.56, 148.65, 136.81, 127.19, 123.69, 122.56 (d, J = 6.9 Hz), 118.64 (d, J = 18.5 Hz), 117.74, 116.36 (d, J = 21.6 Hz), 113.83, 108.85, 107.17, 64.59, 35.14, 14.19. HRMS (ESI): m/z: calcd for C22H18ClFN6O2S [(M+H)+]: 485.09573; found: 485.09555.
33
ACCEPTED MANUSCRIPT
5.1.5. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-3-(pyridin-3yl)propanamide (13). 3-pyridin-3-yl-propionic acid (338 mg, 2.24 mmol) is suspended in thionyl chloride (3.3 ml, 45.5 mmol) and DMF (9 µl, 0.12 mmol) is added. The mixture is
RI PT
heated to reflux for 1 h, then cooled to room temperature. The volatile material is evaporated under reduced pressure, the residue is dissolved in anhydrous THF (3 ml) and added to a solution of 6-amino-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (7, 194
SC
mg, 0.58 mmol). Pyridine (230 µl, 2.84 mmol) is added and the mixture is stirred at room temperature for 16 h, then diluted with AcOEt, washed with water and brine. The organic
M AN U
layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (DCM + 5% MeOH(NH3)), affording the title compound (105 mg, 39%) as a white solid. Mp: 241°C. 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.81 (s, 1H), 8.64 – 8.45 (m, 2H), 8.45 – 8.29 (m, 1H), 7.93 (dd, J = 6.6,
TE D
2.6 Hz, 1H), 7.75 (dt, J = 7.9, 2.0 Hz, 1H), 7.62 (ddd, J = 9.0, 4.2, 2.6 Hz, 1H), 7.35 (dd, J = 7.9, 4.9 Hz, 1H), 7.27 – 7.02 (m, 2H), 4.30 (q, J = 7.0 Hz, 2H), 3.14 (t, J = 7.4 Hz, 2H), 2.92 (t, J = 7.3 Hz, 2H), 1.55 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3/CD3OD) δ
EP
158.16, 156.98, 154.39, 149.50, 148.21, 147.45, 137.49, 137.24, 127.88, 125.16, 124.47, 122.87 (d, J = 6.8 Hz), 116.82 (d, J = 22.1 Hz), 112.06, 109.60, 106.56, 65.58, 38.48,
AC C
28.75, 14.51. HRMS (ESI): m/z: calcd for C24H21ClFN5O2 [(M+H)+]: 466.14406; found: 466.14368.
5.1.6. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((1-methyl-1Htetrazol-5-yl)thio)acetamide (14).5-mercapto-1-methyltetrazole (36.2 mg, 0.30 mmol) is added to a suspension of sodium hydroxide (12 mg, 0.30 mmol) in THF (2 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8,
34
ACCEPTED MANUSCRIPT
90 mg, 0.22 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with AcOEt. The organic layer is washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column
RI PT
chromatography (AcOEt), affording the title compound (65 mg, 61%) as a white solid. Mp: 232-234°C.1H NMR (300 MHz, DMSO-d6) δ 9.85 (s, 1H), 9.83 (s, 1H), 8.86 (s, 1H), 8.52 (s, 1H), 8.08 (dd, J = 6.9, 2.6 Hz, 1H), 7.76 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.41 (t, J = 9.1
SC
Hz, 1H), 7.27 (s, 1H), 4.47 (s, 2H), 4.30 (q, J = 7.0 Hz, 2H), 4.00 (s, 3H), 1.46 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.66, 156.85, 154.81, 153.98, 153.42, 151.59,
M AN U
148.94, 136.76, 126.94, 123.71, 122.58 (d, J = 6.9 Hz), 118.65 (d, J = 18.4 Hz), 116.39 (d, J = 21.5 Hz), 114.88, 108.75, 107.27, 64.63, 37.39, 33.70, 14.20. HRMS (ESI): m/z: calcd for C20H18ClFN8O2S [(M+H)+]: 489.10187; found: 489.10161. 5.1.7. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((1-methyl-1H-
TE D
imidazol-2-yl)thio)acetamide (15). 2-mercapto-1-methylimidazole (26 mg, 0.23 mmol) is added to a suspension of sodium hydroxide (9.5 mg, 0.24 mmol) in THF (2 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8,
EP
65 mg, 0.16 mmol) is added and the mixture is stirred at 45°C for 2 h, then cooled to room temperature and diluted with AcOEt. The organic layer is washed with water, saturated
AC C
aqueous NaHCO3 and brine, dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt), affording the title compound (24 mg, 31%) as a white solid. Mp: 215-217°C. 1H NMR (300 MHz, DMSO-d6) δ 10.14 (s, 1H), 9.83 (s, 1H), 8.93 (s, 1H), 8.51 (s, 1H), 8.09 (dd, J = 6.9, 2.6 Hz, 1H), 7.77 (ddd, J = 9.1, 4.3, 2.6 Hz, 1H), 7.41 (t, J = 9.1 Hz, 1H), 7.33 – 7.20 (m, 2H), 7.00 (d, J = 1.3 Hz, 1H), 4.29 (q, J = 7.0 Hz, 2H), 4.06 (s, 2H), 3.60 (s, 3H), 1.42 (t, J = 7.0
35
ACCEPTED MANUSCRIPT
Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 167.22, 156.84, 154.76, 153.75, 153.60, 136.83, 128.39, 127.33, 123.67, 123.55, 122.55 (d, J = 6.7 Hz), 116.40 (d, J = 21.5 Hz), 113.85,
[(M+H)+]: 487.11138; found: 487.11123.
RI PT
108.83, 107.18, 64.56, 37.44, 32.85, 14.22. HRMS (ESI): m/z: calcd for C22H20ClFN6O2S
5.1.8. 2-((1H-imidazol-2-yl)thio)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6yl)acetamide (16). 2-mercaptoimidazole (231 mg, 2.31 mmol) and 6-(chloroacetamido)-4-
SC
(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 94 mg, 0.23 mmol) are dissolved in THF (6 ml) and triethylamine (320 µl, 2.30 mmol) is added. The mixture is stirred at 50°C
M AN U
for 2 h, then cooled to room temperature, diluted with AcOEt and washed with water, saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is crystallized from EtOH/water 7:3, affording the title compound (97 mg, 89%) as a white solid. Mp: 243°C. 1H NMR (300
TE D
MHz, CDCl3/CD3OD) δ 8.75 (s, 1H), 8.38 (s, 1H), 7.83 (dd, J = 6.6, 2.6 Hz, 1H), 7.54 – 7.46 (m, 1H), 7.17 – 7.03 (m, 2H), 6.99 (s, 2H), 4.21 (q, J = 7.0 Hz, 2H), 3.89 (s, 2H), 1.44 (t, J = 7.0 Hz, 3H).
13
C NMR (75 MHz, DMSO-d6) δ 167.50, 156.84, 154.77, 153.73,
EP
153.56, 151.56, 148.68, 139.06, 136.89, 127.40, 123.64, 122.51 (d, J = 7.0 Hz), 118.78, 116.39 (d, J = 21.5 Hz), 113.71, 108.86, 107.13, 64.57, 36.99, 14.22. HRMS (ESI): m/z:
AC C
calcd for C21H18ClFN6O2S [(M+H)+]: 473.09573; found: 473.09570. 5.1.9. N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((1-methyl-1Hbenzo[d]imidazol-2-yl)thio)acetamide (17). 1-methylbenzimidazole (95 mg, 0.58 mmol) is added to a suspension of sodium hydroxide (39 mg, 0.97 mmol) in THF (4 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 78 mg, 0.19 mmol) is added and the mixture is stirred at room temperature for 1 h, then
36
ACCEPTED MANUSCRIPT
diluted with AcOEt and washed with water, saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (DCM + 1.5% MeOH(NH3)),
RI PT
affording the title compound (56 mg, 54%) as a white solid. Mp: 226°C. 1H NMR (300 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.82 (s, 1H), 8.93 (s, 1H), 8.50 (s, 1H), 8.08 (dd, J = 6.9, 2.6 Hz, 1H), 7.76 (ddd, J = 9.1, 4.3, 2.6 Hz, 1H), 7.60 – 7.49 (m, 2H), 7.41 (t, J = 9.1 Hz,
6.9 Hz, 3H).
13
SC
1H), 7.26 – 7.17 (m, 3H), 4.46 (s, 2H), 4.22 (q, J = 7.0 Hz, 2H), 3.75 (s, 3H), 1.28 (t, J = C NMR (75 MHz, DMSO-d6) δ 166.59, 156.82, 154.77, 153.78, 153.59,
M AN U
151.55, 151.15, 148.74, 142.55, 136.95, 136.81, 127.15, 123.65, 122.52 (d, J = 6.8 Hz), 121.70 (d, J = 10.9 Hz), 117.49, 116.38 (d, J = 21.7 Hz), 114.25, 109.59, 108.80, 107.19, 64.52, 36.08, 30.00, 13.95. HRMS (ESI): m/z: calcd for C26H22ClFN6O2S [(M+H)+]: 537.12703; found: 537.12658.
TE D
5.1.10. 2-(benzo[d]thiazol-2-ylthio)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin6-yl)acetamide (18). 2-mercaptobenzothiazole (25 mg, 0.15 mmol) is added to a suspension of sodium hydroxide (6.0 mg, 0.15 mmol) in THF (2 ml). To the clear solution,
EP
6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 61 mg, 0.15 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with
AC C
AcOEt and washed with water, saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/hexane 4:1), followed by crystallization from 1,2-dichloroethane, affording the title compound (15 mg, 19%) as white slender needles. Mp: 203-204°C. 1H NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.82 (s, 1H), 8.89 (s, 1H), 8.50 (s, 1H), 8.07 - 8.01 (m, 2H), 7.86 (d, J = 8.0 Hz, 1H), 7.74 (ddd, J = 9.1, 4.4, 2.6 Hz,
37
ACCEPTED MANUSCRIPT
1H), 7.48 (td, J = 8.2, 7.7, 1.4 Hz, 1H), 7.44 – 7.33 (m, 2H), 7.25 (s, 1H), 4.54 (s, 2H), 4.25 (q, J = 7.0 Hz, 2H), 1.36 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 166.07 (d, J = 7.2 Hz), 156.97, 154.94, 153.93, 152.57, 151.72, 148.92, 136.80 (d, J = 3.0 Hz), 134.95,
RI PT
127.12, 126.60, 124.79, 123.86, 122.74 (d, J = 6.9 Hz), 122.02, 121.24, 118.76 (d, J = 18.6 Hz), 116.50 (d, J = 21.5 Hz), 114.70, 108.85, 107.32, 64.72, 37.34, 14.19. HRMS (ESI): m/z: calcd for C25H19ClFN5O2S2 [(M+H)+]: 540.07255; found: 540.07226.
methoxybenzo[d]thiazol-2-yl)thio)acetamide
(19).
SC
5.1.11.N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-((5-
2-mercapto-5-methoxybenzothiazole
M AN U
(118.5 mg, 0.60 mmol) is added to a suspension of sodium hydroxide (24.7 mg, 0.62 mmol) in THF (4 ml). To the clear solution, 6-(chloroacetamido)-4-(3-chloro-4fluoroaniline)-7-ethoxyquinazoline (8, 86 mg, 0.21 mmol) is added and the mixture is stirred at room temperature for 1 h, then diluted with AcOEt and washed with water,
TE D
saturated aqueous NaHCO3 and brine. The organic layer is dried over Na2SO4 and concentrated under reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/hexane 4:1), followed by crystallization from 1,2-dichloroethane,
EP
affording the title compound (23 mg, 19%) as a white crystalline solid. Mp: 202-203°C. 1H NMR (300 MHz, DMSO-d6) δ 9.83 (d, J = 3.2 Hz, 2H), 8.90 (s, 1H), 8.51 (s, 1H), 8.08
AC C
(dd, J = 6.9, 2.6 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.76 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.49 – 7.33 (m, 2H), 7.27 (s, 1H), 7.02 (dd, J = 8.8, 2.5 Hz, 1H), 4.53 (s, 2H), 4.27 (q, J = 6.9 Hz, 2H), 3.82 (s, 3H), 1.38 (t, J = 6.9 Hz, 3H).
13
C NMR (75 MHz, DMSO-d6) δ
166.95, 165.85, 158.74, 156.85, 154.80, 153.83, 151.58, 148.92, 136.77 (d, J = 3.2 Hz), 127.01, 126.42, 123.73, 122.60 (d, J = 6.8 Hz), 122.21, 118.63 (d, J = 18.3 Hz), 116.39 (d, J = 21.6 Hz), 114.66, 113.83, 108.78, 107.32, 104.59, 64.60, 55.52, 37.25, 14.13. HRMS
38
ACCEPTED MANUSCRIPT
(ESI): m/z: calcd for C26H21ClFN5O5S2 [(M+H)+]: 570.08311; found: 570.08297. 5.1.12.2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)amino)-2-oxoethyl acetate (20). 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8,
RI PT
150 mg, 0.37 mmol) and sodium acetate (30 mg, 0.37 mmol) are dissolved in DMF (4 ml). The mixture is stirred at reflux for 3 h. The solution is cooled to room temperature and diluted with water. The obtained precipitate is filtered, affording the title compound (141
SC
mg, 89%) as a white solid. The compound could be used without any further purification. Mp: 221°C. 1H NMR (300 MHz, DMSO-d6) δ 9.83 (s, 1H), 9.53 (s, 1H), 8.86 (s, 1H), 8.52
M AN U
(s, 1H), 8.10 (dd, J = 6.9, 2.6 Hz, 1H), 7.78 (ddd, J = 9.1, 4.4, 2.7 Hz, 1H), 7.40 (t, J = 9.1 Hz, 1H), 7.26 (s, 1H), 4.81 (s, 2H), 4.28 (q, J = 6.9 Hz, 2H), 2.16 (s, 3H), 1.46 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 169.84, 165.88, 156.82, 154.79, 154.16, 153.91, 151.57, 149.03, 136.88, 126.58, 123.65, 122.51 (d, J = 6.9 Hz), 118.66 (d, J = 18.2 Hz),
TE D
116.39 (d, J = 21.5 Hz), 115.19, 108.83, 107.30, 64.60, 62.55, 20.47, 14.24. HRMS (ESI): m/z: calcd for C20H18ClFN4O4 [(M+H)+]: 433.10734; found: 433.10699. 5.1.13.2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)amino)-2-oxoethyl
EP
benzoate (21). 6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (8, 70 mg, 0.17 mmol) and benzoic acid (21 mg, 0.17 mmol) are dissolved in DMF (3 ml).
AC C
Triethylamine (24 µl, 0.17 mmol) is added and the mixture is stirred at reflux for 3 h. The solution is cooled to room temperature and diluted with water. The obtained precipitate is filtered, affording the title compound (75 mg, 88%) as a white solid. The compound is pure enough for biological testing. Mp: 243°C. 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.93 (s, 1H), 8.53 (s, 1H), 8.20 (s, 1H), 8.19 – 8.14 (m, 1H), 7.95 (dd, J = 6.7, 2.7 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.63 (ddd, J = 8.9, 4.1, 2.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.25 – 7.11
39
ACCEPTED MANUSCRIPT
(m, 2H), 5.08 (s, 2H), 4.25 (q, J = 7.0 Hz, 2H), 1.38 (t, J = 6.9 Hz, 3H).
13
C NMR (75
MHz, CDCl3/CD3OD) δ 166.50, 165.63, 158.05, 156.90, 154.61, 153.64, 152.96, 148.48, 135.86, 134.54, 130.18, 129.15, 126.80, 125.12, 122.78 (d, J = 7.0 Hz), 121.02 (d, J = 18.4
RI PT
Hz), 116.72 (d, J = 22.0 Hz), 111.04, 109.61, 106.77, 65.61, 63.73, 14.46. HRMS (ESI): m/z: calcd for C25H20ClFN4O4 [(M+H)+]: 495.12299; found: 495.12263.
5.1.14.2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)amino)-2-oxoethyl 2,6(22).
6-(chloroacetamido)-4-(3-chloro-4-fluoroaniline)-7-
SC
dimethylbenzoate
ethoxyquinazoline (8, 65 mg, 0.16 mmol) and 2,6-dimethoxy benzoic acid (70.5 mg, 0.47
M AN U
mmol) are dissolved in DMF (4 ml). Triethylamine (100 µl, 0.72 mmol) is added and the mixture is stirred at 100°C for 30 minutes. The solution is cooled to room temperature and diluted with water. The obtained precipitate is filtered, affording the title compound (49 mg, 47%) as a white solid. Mp: 240°C (dec.). 1H NMR (300 MHz, DMSO-d6) δ 9.85 (s,
TE D
1H), 9.67 (s, 1H), 8.86 (s, 1H), 8.53 (s, 1H), 8.09 (dd, J = 6.8, 2.6 Hz, 1H), 7.76 (ddd, J = 9.1, 4.4, 2.7 Hz, 1H), 7.42 (t, J = 9.1 Hz, 1H), 7.34 – 7.19 (m, 2H), 7.12 (d, J = 7.6 Hz, 2H), 5.09 (s, 2H), 4.29 (q, J = 7.0 Hz, 2H), 2.35 (s, 6H), 1.41 (t, J = 6.9 Hz, 3H). 13C NMR
EP
(75 MHz, DMSO-d6) δ 168.45, 165.69, 156.89, 154.83, 154.24, 153.97, 151.61, 149.11, 136.78 (d, J = 2.9 Hz), 134.82, 132.88, 129.68, 127.57, 126.57, 123.73, 122.61 (d, J = 7.0
AC C
Hz), 118.67 (d, J = 18.2 Hz), 116.43 (d, J = 21.6 Hz), 108.81, 107.39, 64.61, 63.13, 19.41, 14.16. HRMS (ESI): m/z: calcd for C27H24ClFN4O4 [(M+H)+]: 523.15429; found: 523.15387.
5.1.15.N-(4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-yl)-2-hydroxyacetamide (23). 6-(2-acetoyloxyacetamido)-4-(3-chloro-4-fluoroaniline)-7-ethoxyquinazoline (20, 93 mg, 0.22 mmol) is dissolved in a mixture of THF (3 ml) and MeOH (30 µl, 0.8 mmol).
40
ACCEPTED MANUSCRIPT
Sodium (3 mg, 0.13 mmol) is added and the mixture is stirred at room temperature for 30 minutes, quenched by addition of HCl 1M until neutralization and diluted with AcOEt. The organic layer is washed with water and brine, dried over Na2SO4 and concentrated under
RI PT
reduced pressure. The solid residue is purified by silica gel column chromatography (AcOEt/hexane 4:1) to afford the title compound (52 mg, 62%) as a white solid. Mp: 261°C. 1H NMR (300 MHz, DMSO-d6) δ 9.87 (s, 1H), 9.48 (s, 1H), 9.08 (s, 1H), 8.52 (s,
SC
1H), 8.08 (dd, J = 7.0, 2.6 Hz, 1H), 7.76 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.42 (t, J = 9.1 Hz, 1H), 7.31 (s, 1H), 6.25 (t, J = 5.6 Hz, 1H), 4.33 (q, J = 6.9 Hz, 2H), 4.09 (d, J = 5.6 Hz, 13
C NMR (75 MHz, DMSO-d6) δ 170.42, 156.88, 154.78,
M AN U
2H), 1.46 (t, J = 6.9 Hz, 3H).
153.62, 152.71, 151.56, 148.48, 136.87, 126.73, 123.73, 122.62 (d, J = 6.9 Hz), 118.63 (d, J = 18.6 Hz), 116.38 (d, J = 21.5 Hz), 111.60, 109.09, 107.23, 64.77, 61.75, 14.28. HRMS (ESI): m/z: calcd for C18H16ClFN4O3 [(M-H)-]: 389.08112; found: 389.08215.
5.2.1.
TE D
5.2. Analytical Chemistry
Reactivity assay. Compounds of 8-23 were added (10 µM) to freshly-prepared 1 mM
cysteine solutions in 50 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS; pH 10), ionic
strength
for
KCl
addition.
The
formation
of
the
conjugate
EP
0.15M
quinazolinylacetamide (S-(2-((4-((3-chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-6-
AC C
yl)amino)-2-oxoethyl)cysteine) was measured after 24 h of incubation at 37 °C by LC-MS in multiple reaction monitoring (MRM) mode with the following parent-product ion transitions: m/z 494.1 [M+H]+ → m/z 305.1 + m/z 333.1 + m/z 407.2 (Tube Lens (TL) 95 V; Collision Energies (CE) 35, 35, 25 eV, respectively). To quantify the amount of conjugate produced, the area of the peak in the chromatogram was referred to the arbitrarily-set value of 10,000 for the conjugate between compound 8 and cysteine. To
41
ACCEPTED MANUSCRIPT
monitor the time course of the LC-UV peak area of 8 and of the cysteine conjugate (See Supplementary Data, Fig. S1), a Shimadzu HPLC gradient system (Shimadzu Corp., Kyoto, Japan) consisting of two LC-10ADvp solvent delivery modules, a SPD-10Avp UV-
RI PT
VIS detector set at λ=254 nm, equipped with a reversed-phase C18 column (Supelcosil LC18-DB, 5 µm, 150 x 4.6 mm i.d.; Supelco, Bellefonte, USA) was employed.
5.2.2. Stability of the cysteine-conjugate. Chloroacetamide 8 was incubated in the presence of a
SC
1:100 molar excess of cysteine under nitrogen atmosphere at pH 10 and 37 °C for 24 h. The area ratio of cysteine-conjugate vs internal standard and the area ratio of 8 vs internal
M AN U
standard were monitored over time by LC-MS in MRM mode with the parent-product ion transitions: 8: m/z 409.1 [M+H]+ → m/z 381.0 + m/z 363.0 + m/z 305.0 (TL 106 V; CE 24, 28, 34 eV); cysteine-conjugate: m/z 494.1 [M+H]+ → m/z 305.1 + m/z 333.1 + m/z 407.2 ; TL = 95 V; CE = 35, 35, 25 eV, respectively.
TE D
5.2.3. Determination of intracellular concentrations. We applied a protocol previously developed by our group [45]. A549 Cells were plated at 1,000,000 cells/dish (25 cm2) density. After 24 h, test compounds (1, 8, 14-16, 20, 23) were added to the culture medium (final
EP
concentration: 1 µM; final DMSO concentration: 0.1% v/v). Cells were incubated for 1 h at
AC C
37 °C and tested either immediately after or 8 h after removal of the compound from the extracellular medium by washing the cells for three times with 1 mL aliquots of fresh medium. Compounds were then extracted from cells with absolute EtOH (1.1 mL at 4 °C), cell extracts were centrifuged (4 °C, 10,000 g, 5 min) and collected. A fixed volume of ethanolic extract was evaporated to dryness, dissolved in LC eluent and injected into the LC-MS system for quantitative measurement (vide infra). Cell proteins were quantified after solubilization in NaOH 0.5 N (2 mL/25 cm2 dish) by the Bradford method [55].
42
ACCEPTED MANUSCRIPT
Calibration curves were prepared by spiking into control matrices the stock solutions of test compounds, freshly prepared in DMSO to yield final concentrations in the 1000-1 nM concentration range. Chromatographic separation was achieved on a Phenomenex Synergi
RI PT
Fusion C18 column (100 × 2.1 mm i.d., 3 µm particle size; Phenomenex, USA). Mobile phases consisted of water (eluent A) and acetonitrile (eluent B), both added with formic acid at 0.1% v/v and at a flow rate of 350 µL min-1. A linear gradient elution was set up:
SC
T(0 min): 95% A: 5% B; T(1 min): 95% A: 5% B; T(6 min) 5%A:95% B; T(8 min) 5%A:95% B returning to initial conditions after 1 min, followed by 3 min re-equilibration
M AN U
time. Mass spectrometric detection was performed on a Thermo TSQ Quantum Access Max triple quadrupole mass spectrometer (Thermo, San Jose, CA, USA) equipped with a heated electrospray ionization (H-ESI) source. Mass spectrometric analyses were done in positive ion (ESI+) and in multiple reaction monitoring (MRM) mode. H-ESI interface
TE D
parameters were set as follows: probe middle (D) position; capillary temperature 270 °C; spray voltage 3.5 kV. Nitrogen was used as nebulizing gas at the following pressure: sheath gas: 35 psi; auxiliary gas 15 arbitrary units (a.u.). Argon was used as the collision
EP
gas at a pressure of approximately 1.5 mtorr (1 torr = 133.3 Pa). For quantitative analysis, the following parent→product ion transitions were selected: 1: m/z 447.2 [M+H]+ → m/z
AC C
128.2 + m/z 320.1 + m/z 360.1 (Tube Lens (TL) 81 V; Collision Energies (CE) 23, 28, 23 eV, respectively); 8: m/z 409.1 [M+H]+ → m/z 381.0 + m/z 363.0 + m/z 305.0 (TL 106 V; CE 24, 28, 34 eV); 14: m/z 489.1 [M+H]+ → m/z 405.0 + m/z 333.1 + m/z 330.9 (TL 115 V; CE 19, 25, 42 eV); 15: m/z 487.1 [M+H]+ → m/z 405.1 + m/z 317.1 + m/z 155.1 (TL 135 V; CE 25, 39, 22 eV); 16: m/z 473.1 [M+H]+ → m/z 405.1 + m/z 363.0 + m/z 305.1 (TL 106 V; CE 24, 28, 34 eV); 20: m/z 433.1 [M+H]+ → m/z 345.1 + m/z 317.0 + m/z
43
ACCEPTED MANUSCRIPT
305.0 (TL 106 V; CE 26, 37, 33 eV); 23: m/z 391.1 [M+H]+ → m/z 345.1 + m/z 314.8 + m/z 305.0 (TL 105 V; CE 25, 33, 29 eV), with a scan time of 0.1 s per transition. Data acquisition was performed by Xcalibur1.3 software (Thermo, USA). Peak integration and
RI PT
calibration were performed using LCQuan software (Thermo, USA). 5.3. Pharmacology
5.3.1. Cell culture. The human NSCLC cell lines A549 and H1975 were cultured as
SC
recommended. Medium was supplemented with L-glutamine (2 mmol/L) and fetal calf serum (FCS) (10%) and cells were maintained under standard cell culture conditions at 37
M AN U
°C in a water-saturated atmosphere of 5% CO2 in air. A549 cell line was from the American Type Culture Collection (Manassas, VA, USA) and H1975 was kindly provided by Dr E. Giovannetti (Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands).
TE D
5.3.2. Antibodies and reagents. Medium was from Euroclone, FCS was purchased from GibcoBRL (Grand Island, NY, USA). Monoclonal anti-EGFR and polyclonal anti-phosphoEGFR (Tyr1068) antibodies were from Cell Signaling Technology (Beverly, MA, USA).
EP
Horseradish peroxidase-conjugated (HRP) secondary antibodies were from Pierce. The enhanced chemiluminescence system (ECL) was from Millipore (Millipore, MA, USA).
AC C
Reagents for electrophoresis were obtained from BIO-RAD Laboratories, blotting reagents from Millipore.
5.3.3. Western blot analysis. Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [56]. Briefly, 40-50 of µg proteins from lysates were resolved by 7.5% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were then incubated with primary antibody, washed and then
44
ACCEPTED MANUSCRIPT
incubated with HRP-anti-rabbit antibody. Immunoreactive bands were visualized using an enhanced chemiluminescence system. The chemiluminescent signal was acquired by CDiGit® Blot Scanner and the spots were quantified by Image Studio™ Software, LI-COR
RI PT
Biotechnology (Lincoln, NE).
5.3.4. Autophosphorylation assay. Inhibition of EGFR autophosphorylation was determined as
antibodies by Western blot analysis.
SC
previously described [27] using specific anti-phosphotyrosine and anti-total EGFR
5.3.5. Cell growth inhibition. Cell viability was assessed after 3 days of treatment by tetrazolium
M AN U
dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Sigma, Dorset, UK] assay as previously described [56]. Representative results of at least three independent experiments were used for evaluation of dose-response curves, calculated from experimental points using Graph Pad Prism software. The concentration that inhibits 50%
TE D
(IC50) was extrapolated from the dose-response curves.
5.3.6. Kinase binding assay. Invitrogen LanthaScreen® Eu Kinase Binding Assay, based on TRFRET, was employed for inhibitor binding assays (Thermo Fisher Scientific, Madison, WI,
EP
USA). Briefly, 5 nM recombinant GST-EGFR (WT, Part Number PV3872 or L858R T790M, Part Number PV4879), 2 nM anti-GST Eu Antibody and kinase tracer 199 (5 nM
AC C
for WT EGFR and 20 nM for EGFR L858R T790M) were incubated in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35) in the absence or in the presence of different concentrations of EGFR inhibitors (range:10 µM-100 pM) for 1 h at room temperature in a black, low-volume 384-well plate (Corning Inc., USA). For preincubation experiments, inhibitors gefitinib (1), 8 and 16 were pre-incubated for 5 h before addition of tracer and Anti-GST Eu antibody. In one set of experiments, after 5 h of
45
ACCEPTED MANUSCRIPT
inhibitor pre-incubation, 0.5 µM of ATP were added along with tracer and Anti-GST Eu antibody. At the end of incubation period, plate was read and the ratio of emission values at 665 nm, due to the ternary complex formation among GST-EGFR, Anti-GST-Eu-
RI PT
antibody, and tracer, and at 620 nm, due to Anti-GST-Eu antibody, was plotted against inhibitor concentrations (on a log scale) to generate the binding curve and calculate IC50 values. A Tecan Spark 10M plate reader (Tecan Italia Srl, Italy) was employed for data
SC
acquisition. Instrument settings: excitation filter: 340 nm (35 nm bandpass); emission filters: 665 nm (5 nm bandpass); 620 nm (10 nm bandpass); delay time: 100 µs; integration
M AN U
time: 200 µs; gain: optimal. Reported are means together with their SEM (n=3-5). GraphPad Prism 6.01 (GraphPad Inc, USA) was employed in the LanthaScreen® TRFRET assay for nonlinear fitting of experimental data to get final IC50 values. For preincubation assays, follow-up analyses were conducted using Student’s t test. Statistical
TE D
significance was set at p<0.05.
5.3.7. Selectivity Assay. Inhibitory activity for selected compounds (8, 9 and 16) was measured at 1 µM in a set of human kinases (EGFR, ErbB2, ErbB4, BMX, BTK, JAK3, BLK) having a
EP
cysteine at the front pocket of the ATP binding site. The kinase activity of each enzyme was measured in the presence of an ATP concentration equal to the Km value, detecting the
AC C
FRET signal generated by the kinase-catalyzed phosphorylation of a reference peptide. (Z’-LYTE assay, Thermo Fisher Scientific, Madison, WI, USA). Compounds were tested in 1% DMSO solution. 5.4. Molecular Modelling 5.4.1. Model building and MD simulations. Non-covalent complexes of EGFR bound to compounds 8, 15 and 16 were built starting from the 2J5E.pdb crystal structure by docking
46
ACCEPTED MANUSCRIPT
simulations using Glide 6.1 [57]. Among the obtained docking poses, we selected those in which the quinazoline portion of the inhibitors well fitted the hinge region of the ATP binding site, the distance between Csp3 of acetamide was at less than 4Å from the sulfur
RI PT
atom of Cys797, and the angle formed by the sulfur atom of Cys797, the Csp3 of acetamide and the leaving group atom (i.e., chlorine for 8, sulfur for 15 and 16) was higher than 130°. A similar approach has been recently used to identify reasonable poses for EGFR covalent
SC
inhibitors [27, 58]. The resulting complexes were immersed in a box of TIP3P water molecules [59] and neutralized with 3 Cl- ions by using the tleap tool implemented in
M AN U
AMBER11 [60] The total system size amounted to 47179, 47187 and 47190 atoms for complexes involving 8, 15 and 16, respectively. The resulting systems were energyminimized and gradually heated to 300 K in the NVT ensemble. A short (1 ns) equilibration at pressure of 1 atm in the NPT ensemble was performed for each system before running the
TE D
production phase. The systems were then submitted to MD simulations (NVT) for 10 ns applying the AMBER99SB force field [42] for the protein and the generalized Amber force field (GAFF) [61] for the ligand. The pmemd module of AMBER11 was used to perform
EP
these tasks. Electrostatic and Van der Waals interactions were computed within a cut-off of 10 Å, while long-range electrostatic interactions were treated using the particle mesh Ewald
AC C
(PME) [62] with 128x128x128 grid points. The covalent bonds involving hydrogen atoms were constrained with the SHAKE method and a time-step of 2 fs was applied. 5.4.2. Application of the QM/MM potential. Combined QM/MM methods are effective in the study of enzyme catalysis, but given the sizes typically employed and sampling requirements, studies are often performed using semi-empirical models. This kind of QM/MM methodology has been widely applied to simulate enzymatic reactions and
47
ACCEPTED MANUSCRIPT
reported to give reasonable descriptions of reaction geometries and energetics [63]. Herein we use the Pairwise Distance Directed Gaussian modification of PM3 (PDDG/PM3) implemented in AMBER11 given its acceptable accuracy and computational performance.
RI PT
PDDG/PM3 adds a Gaussian function to the PM3 core repulsion functions to better describe chemical properties (including reactivity) of organic functional groups [39]. The MM part was treated with the AMBER99SB force field [42]. Side chain atoms of Cys797, Asp800
SC
and the substituted acetamide portions of 8, 15 and 16 were treated with the PDDG/PM3. All the other atoms of the system were described with AMBER99SB force field. The
M AN U
resulting QM systems were composed by 22, 30, 33 atoms for systems involving compound 8, 15 and 16, respectively. These included three link atoms for each complex, two placed along the C-C bond connecting Cβ of Cys797 and Asp800 to their backbone Cα, and one along the C-N bond connecting the acetamide portion to the quinazoline nucleus. The
TE D
adjust_q function of AMBER was applied to conserve the total charge of the system. During QM/MM MD simulations, all the atoms of the system (including hydrogens) were free to move, and a time step of 0.2 fs was used to integrate the equation of motion. The SHAKE
EP
option was turned off for the QM region during all QM/MM simulations. A PME approach was used to treat the QM/MM long-range electrostatic interactions.
AC C
5.4.3. Steered-MD simulation. The equilibrated complexes were employed to model the SN2 reaction with QM/MM steered-MD. In the case of compound 8, thiolate addition and chlorine expulsion were modeled using the difference of distances [d(Cαwarhead − Clwarhead) d(Scys797 − Cαwarhead)] as reaction coordinate. For compounds 15 and 16 cysteine thiolate attack and imidazol-2-ylthiolate expulsion were described by [d(Cαwarhead − Swarhead) d(Scys797 − Cαwarhead)] difference of distances. QM/MM steered-MD simulations were carried
48
ACCEPTED MANUSCRIPT
out by sequentially pulling the systems along the reaction coordinate using a force constant of 300 kcal/mol Å-2 at a constant velocity of 0.1 Å/ps. In the case of compound 8 the system was sequentially pulled along the reaction coordinate to the target value of 1.5 Å, while for
RI PT
compounds 15 and 16 the target value of the reaction coordinate was set to 2.5 Å. All QM/MM steered-MD simulation were performed at 300 K in NVT conditions, using the sander. MPI module of AMBER11 in combination with PLUMED 1.3 [64]. The work
SC
performed on the system (W) during the QM/MM steered-MD simulations was calculated by numerical integration of the exerted force (Fex) (eq. 1, where dx = v dt). The free-energy
[
〈
]
M AN U
profiles reported in Fig. 3 were calculated applying the Jarzynski’s equality (eq. 2, [44]) =
〉=〈
〉
(1) (2)
In details, for each point of the reaction coordinate, the free energy (∆F) is expressed as the
TE D
exponential average of the work (W) calculated for five independent SMD replicas. The error associated to the free-energy estimation was calculated from eq. 3 [44]. 〉−〈 〉
(3)
EP
= 〈
ACKNOWLEDGMENTS
AC C
This work was supported by Associazione Augusto per la Vita (Novellara, Reggio Emilia) and Associazione Marta Nurizzo, Brugherio (MI) to P.G. Petronini. This research benefits from the HPC (High Performance Computing) facility of the University of Parma, Italy http://www.hpc.unipr.it.
REFERENCES
49
ACCEPTED MANUSCRIPT
[1] Pallis, A. G.; Serfass, L.; Dziadziuszko, R.; van Meerbeeck, J. P.; Fennell, D.; Lacombe, D.; Welch, J.; Gridelli, C. Targeted Therapies in the Treatment of Advanced/Metastatic NSCLC.
RI PT
Eur. J. Cancer 2009, 45, 2473−2487. [2] Carmi, C.; Mor, M.; Petronini, P. G.; Alfieri, R. Clinical Perspectives for Irreversible Tyrosine Kinase Inhibitors in Cancer. Biochem. Pharmacol. 2012, 84, 1388−1399.
SC
[3] (a) Lynch, T. J.; Bell, D. W.; Sordella, R.; Gurubhagavatula, S.; Okimoto, R. A.;
M AN U
Brannigan, B. W.; Harris, P. L.; Haserlat, S. M.; Supko, J. G.; Haluska, F. G.; Louis, D. N.; Christiani, D. C.; Settleman, J.; Haber, D. A. Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib. N. Engl. J. Med. 2004, 350, 2129−2139. (b) Paez, J. G.; Jänne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.; Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T. J.; Naoki, K.; Sasaki, H.;
TE D
Fujii, Y.; Eck, M. J.; Sellers, W. R.; Johnson, B. E.; Meyerson, M. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy. Science 2004, 304, 1497– 1500. (c) Pao, W.; Miller, V.; Zakowski, M.; Doherty, J.; Politi, K.; Sarkaria, I.; Singh, B.;
EP
Heelan, R.; Rusch, V.; Fulton, L.; Mardis, E.; Kupfer, D.; Wilson, R.; Kris, M.; Varmus, H. EGF Receptor Gene Mutations Are Common in Lung Cancers from “Never Smokers″ and Are
AC C
Associated with Sensitivity of Tumors to Gefitinib and Erlotinib. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 13306– 13311
[4] (a) Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal Growth Factor Receptor Mutations in Lung Cancer. Nat. Rev. Cancer 2007, 7, 169−181. Pao, W.; Chmielecki, J. (b) Rational, Biologically Based Treatment of EGFR-Mutant Non-Small-Cell Lung Cancer. Nat. Rev. Cancer 2010, 10, 760−774.
50
ACCEPTED MANUSCRIPT
[5] Engelman, J. A.; Janne, P. A. Mechanisms of Acquired Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer. Clin. Cancer Res.
RI PT
2008, 14, 2895−2899. [6] Yun, C.-H.; Mengwasser, K. E.; Toms, A. V.; Woo, M. S.; Greulich, H.; Wong, K.-K.; Meyerson, M.; Eck, M. J. The T790M Mutation in EGFR Kinase Causes Drug Resistance by
SC
Increasing the Affinity for ATP. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2070−2075.
M AN U
[7] Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.; Greis, K. D.; Hicks, J. L.; Hook, K. E.; Keller, P. R.; Leopold, W. R.; Loo, J. A.; McNamara, D. J.; Nelson, J. M.; Sherwood, V.; Smaill, J. B.; Trumpp-Kallmeyer, S.; Dobrusin, E. M. Specific, Irreversible Inactivation of the Epidermal Growth Factor Receptor and ErbB2, by a New Class of Tyrosine Kinase Inhibitor.
TE D
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12022–12027.
[8] Blair, J. A.; Rauh, D.; Kung, C.; Yun, C.-H.; Fan, Q.-W.; Rode, H.; Zhang, C.; Eck, M. J.; Weiss, W. A.; Shokat, K. M. Structure Guided Development of Affinity Probes for Tyrosine
EP
Kinases Using Chemical Genetics. Nat. Chem. Biol. 2007, 3, 229–238. [9] Carmi, C.; Lodola, A.; Rivara, S.; Vacondio, F.; Cavazzoni, A.; Alfieri, R. R.; Ardizzoni,
AC C
A.; Petronini, P. G.; Mor, M. Epidermal Growth Factor Receptor Irreversible Inhibitors: Chemical Exploration of the Cysteine-Trap Portion. Mini-Rev. Med. Chem. 2011, 11, 1019− 1030.
[10] Hirsh, V. Afatinib (BIBW 2992) Development in Non-Small-Cell Lung Cancer. Future Oncol. 2011, 7, 817−825.
51
ACCEPTED MANUSCRIPT
[11] Katakami, N.; Atagi, S.; Goto, K.; Hida, T.; Horai, T.; Inoue, A.; Ichinose, Y.; Koboyashi, K.; Takeda, K.; Kiura, K.; Nishio, K.; Seki, Y.; Ebisawa, R.; Shahidi, M.; Yamamoto, N. LUX-
RI PT
Lung 4: a Phase II Trial of Afatinib in Patients with Advanced Non-Small-Cell Lung Cancer Who Progressed During Prior Treatment with Erlotinib, Gefitinib, or Both. J. Clin. Oncol. 2013, 31, 3335−3341.
SC
[12] Zhou, W.; Ercan, D.; Chen, L.; Yun, C.-H.; Li, D.; Capelletti, M; Cortot, A. B.; Chierac, L.; Iacob, R. E.; Padera, R.; Engen, J. R.; Wong, K.-K.; Eck, M. J.; Gray, N. S.; Janne, P. Novel
M AN U
Mutant-Selective EGFR Kinase Inhibitors Against EGFR T790M. Nature 2009, 462, 1070−1074.
[13] Cheng, H.; Nair, S. K.; Murray, B.W. Recent Progress on Third Generation Covalent
TE D
EGFR Inhibitors. Bioorg. Med. Chem. Lett. 2016, 26, 1861–1868.
[14] Ramalingam, S. S.; Yang, J. C.; Lee, C. K.; Kurata, T.; Kim, D. W.; John, T.; Nogami, N.; Ohe, Y.; Mann, H.; Rukazenkov, Y.; Ghiorghiu, S.; Stetson, D.; Markovets, A.; Barrett, J. C.;
EP
Thress, K. S.; Jänne, P. A. Osimertinib As First-Line Treatment of EGFR Mutation-Positive Advanced Non-Small-Cell Lung Cancer. J. Clin. Oncol. 2017, doi: 10.1200/JCO.2017.74.7576.
AC C
[15] Finlay, M. R. V.; Anderton, M.; Ashton, S.; Ballard, P.; Bethel, P. A.; Box, M. R.; Bradbury, R. H.; Brown, S. J.; Butterworth, S.; Campbell, A.; Chorley, C.; Colclough, N.; Cross, D. A. E.; Currie, G. S.; Grist, M.; Hassall, L.; Hill, G. B.; James, D.; James, M.; Kemmitt, P.; Klinowska, T.; Lamont, G.; Lamont, S. G.; Martin, N.; McFarland, H. L.; Mellor, M. J.; Orme, J. P.; Perkins, D.; Perkins, P.; Richmond, G.; Smith, P.; Ward, R. A.; Waring, M. J.; Whittaker, D.; Wells, S.; Wrigley, G. L. Discovery of a Potent and Selective EGFR Inhibitor (AZD9291) of
52
ACCEPTED MANUSCRIPT
Both Sensitizing and T790M Resistance Mutations That Spares the Wild Type Form of the Receptor. J. Med. Chem. 2014, 57, 8249−8267.
RI PT
[16] Song, Z.; Ge, Y.; Wang, C.; Huang, S.; Shu, X.; Liu, K.; Zhou, Y.; Ma, X. Challenges and Perspectives on the Development of Small-Molecule EGFR Inhibitors against T790M-Mediated Resistance in Non-Small- Cell Lung Cancer. J. Med. Chem. 2016, 59, 6580–6594.
SC
[17] Cross, D. A.; Ashton, S. E.; Ghiorghiu, S.; Eberlein, C.; Nebhan, C. A.; Spitzler, P. J.;
M AN U
Orme, J. P.; Finlay, M. R.; Ward, R. A.; Mellor, M. J.; Hughes, G.; Rahi, A.; Jacobs, V. N.; Red Brewer, M.; Ichihara, E.; Sun, J.; Jin, H.; Ballard, P.; Al-Kadhimi, K.; Rowlinson, R.; Klinowska, T.; Richmond, G. H.; Cantarini, M.; Kim, D. W.; Ranson, M. R.; Pao, W. AZD9291, an Irreversible EGFR TKI, Overcomes T790M-Mediated Resistance to EGFR Inhibitors in Lung
TE D
Cancer. Cancer Discovery 2014, 4, 1046−1061.
[18] Soria, J. C.; Ohe, Y.; Vansteenkiste, J.; Reungwetwattana, T.; Chewaskulyong, B.; Lee, K. H.; Dechaphunkul, A.; Imamura, F.; Nogami, N.; Kurata, T.; Okamoto, I.; Zhou, C.; Cho, B. C.;
EP
Cheng, Y.; Cho, E. K.; Voon, P. J.; Planchard, D.; Su, W. C.; Gray, J. E.; Lee, S. M.; Hodge, R.; Marotti, M.; Rukazenkov, Y.; Ramalingam, S. S. Osimertinib in Untreated EGFR-Mutated
AC C
Advanced Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2018, 378, 113-125. [19] Engel, J.; Lategahn, J.; Rauh, D. Hope and Disappointment: Covalent Inhibitors to Overcome Drug Resistance in Non-Small Cell Lung Cancer. ACS Med. Chem. Lett. 2016, 7, 2−5.
[20] (a) Thress, K. S.; Paweletz, C. P.; Felip, E.; Cho, B. C.; Stetson, D.; Dougherty, B.; Lai, Z.; Markovets, A.; Vivancos, A.; Kuang, Y.; Ercan, D.;Matthews, S.E.; Cantarini,M.; Barrett,
53
ACCEPTED MANUSCRIPT
J.C.; Janne, P. A.; Oxnard, G. R. Acquired EGFR C797S Mutation Mediates Resistance to AZD9291 in Non-Small Cell Lung Cancer Harboring EGFR T790M. Nat. Med. 2015, 21,
RI PT
560−562. (b) Ercan, D.; Choi, H. G.; Yun, C. H.; Capelletti, M.; Xie, T.; Eck, M. J.; Gray, N. S.; Jänne, P. A. EGFR Mutations and Resistance to Irreversible Pyrimidine-Based EGFR Inhibitors. Clin. Cancer Res. 2015, 21, 3913-3923. (c) Bersanelli, M.; Minari, R.; Bordi, P.; Gnetti, L.;
SC
Bozzetti, C.; Squadrilli, A.; Lagrasta, C. A.; Bottarelli, L.; Osipova, G.; Capelletto, E.; Mor, M.; Tiseo, M. L718Q Mutation as New Mechanism of Acquired Resistance to AZD9291 in EGFR-
M AN U
Mutated NSCLC. J. Thorac. Oncol. 2016; 11, e121-3.
[21] Callegari, D.; Ranaghan, K. E.; Woods, C. J.; Minari, R.; Tiseo, M.; Mor, M.; Mulholland A. J. Lodola A. L718Q Mutant EGFR Escapes Covalent Inhibition by Stabilizing a NonReactive Conformation of The Lung Cancer Drug Osimertinib. Chem. Sci. 2018, 9, 2740-2749.
TE D
[22] Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf.
EP
Model. 2017, 57, 3124-3137.
[23] Niessen, S.; Dix, M. M.; Barbas, S.; Potter, Z. E.; Lu, S.; Brodsky, O.; Planken, S.;
AC C
Behenna, D.; Almaden, C.; Gajiwala, K. S.; Ryan, K.; Ferre, R.; Lazear, M. R.; Hayward, M. M.; Kath, J. C.; Cravatt, B. F. Proteome-Wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors. Cell Chem Biol 2017, 24, 1388-1400. [24] Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem. Biol. 2013, 20, 146159.
54
ACCEPTED MANUSCRIPT
[25] Truong, T. H. Ung, P. M.; Palde, P. B.; Paulsen, C. E.; Schlessinger, A.; Carroll, K. S. Molecular Basis for Redox Activation of Epidermal Growth Factor Receptor Kinase. Cell Chem.
RI PT
Biol. 2016, 23, 837-848. [26] Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W.
SC
Covalent EGFR Inhibitor Analysis Reveals Importance of Reversible Interactions to Potency and
M AN U
Mechanisms of Drug Resistance. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 173−178. [27] Carmi, C.; Cavazzoni, A.; Vezzosi, S.; Bordi, F.; Vacondio, F.; Silva, C.; Rivara, S.; Lodola, A.; Alfieri, R. R.; La Monica, S.; Galetti, M.; Ardizzoni, A.; Petronini, P. G.; Mor, M. Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the
TE D
Cysteine-Trap Portion. J. Med. Chem. 2010, 53, 2038−2050.
[28] Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.;
EP
Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and
AC C
Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014, 57, 10072−10079. [29] Kung, A.; Chen, Y. C.; Schimpl, M.; Ni, F.; Zhu, J.; Turner, M.; Molina, H.; Overman, R.; Zhang, C. Development of Specific, Irreversible Inhibitors for a Receptor Tyrosine Kinase EphB3. J. Am. Chem. Soc. 2016, 138, 10554-10560.
55
ACCEPTED MANUSCRIPT
[30] Mishani, E.; Abourbeh, G.; Jacobson, O.; Dissoki, S.; Daniel, R. B.; Rozen, G.; Shaul, M.; Levitzki, A. High-affinity Epidermal Growth Factor Receptor (EGFR) Irreversible Inhibitors
RI PT
with DiminIshed Chemical Reactivities as Positron Emission Tomography (PET)-Imaging Agent Candidates of EGFR Overexpressing Tumors. J. Med. Chem. 2005, 48, 5337–5348.
[31] (a) Carmi, C.; Galvani, E.; Vacondio, F.; Rivara, S.; Lodola, A.; Russo, S.; Aiello, S.;
SC
Bordi, F.; Costantino, G.; Cavazzoni, A.; Alfieri, R.R.; Ardizzoni, A.; Petronini, P.G.; Mor, M. Irreversible Inhibition of Epidermal Growth Factor Receptor Activity by 3-Aminopropanamides.
M AN U
J. Med. Chem. 2012, 55, 2251–2264. (b) Mao, Y.; Liu, Z.; Yang, X.; Xia, X.; Zhang, R.; Li, J.; Jiang, X.; Xie, K.; Zheng, J.; Zhang, H.; Suo, J.; Shen, J. A New and Improved Process for N-(4Chloro-3-cyano-7-ethoxyquinolin-6-yl)acetammide. Org. Process Res. Dev. 2012, 16, 19701973.
TE D
[32] Benesch, R. E.; Benesch, R. The acid strength of the −SH group in cysteine and related compounds. J. Am. Chem. Soc. 1955, 77, 5877− 5881.
EP
[33] Berteotti, A.; Vacondio, F.; Lodola, A.; Bassi, M.; Silva, C.; Mor, M.; Cavalli, A. Predicting the Reactivity of Nitrile-Carrying Compounds with Cysteine: A Combined
AC C
Computational and Experimental study. ACS Med. Chem. Lett. 2014, 5, 501−505. [34] Abourbeh, G.; Dissoki, S.; Jacobson, O.; Litchi, A.; Ben Daniel, R.; Laki, D.; Levitzki, A.; Mishani, E. Evaluation of Radiolabeled ML04, a Putative Irreversible Inhibitor of Epidermal Growth Factor Receptor, as a Bioprobe for PET Imaging of EGFR-overexpressing Tumors. Nucl. Med. Biol. 2007 34, 55-70.
56
ACCEPTED MANUSCRIPT
[35] Jost C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and Selectivity in Covalent Enzyme Inhibition: a Systematic Study of Electrophilic Fragments. J Med Chem. 2014,
RI PT
57, 7590-7599. [36] (a) Field, M. J.; Bash, P. A.; Karplus, M. A Combined Quantum Mechanical and Molecular Mechanical Potential for Molecular Dynamics Simulations. J. Comput. Chem. 1990,
SC
11, 700− 733. (b) Gao, J. Methods and Applications of Combined Quantum Mechanical and Molecular Mechanical Potentials. In Reviews in Computational Chemistry; Lipkowitz, K. B.,
M AN U
Boyd, D. R., Eds.; VCH Publishers: New York, 1996; Vol. 7, pp 119−185. (c) Warshel, A. Computer Simulations of Enzyme Catalysis: Methods, Progress, and Insights. Annu. Rev. Biophys. Biomol. Struct. 2005, 32, 425−443.
[37] (a) Schirmeister, T.; Kesselring, J.; Jung, S.; Schneider, T.; Weickert, A.; Becker, J.; Lee,
TE D
W.; Bamberger, D.; Wich, P.; Distler, U.; Tenzer, Stefan; Johe, Patrick; Hellmich, U. A.; Engels, B. Quantum Chemical-Based Protocol for the Rational Design of Covalent Inhibitors. J. Am. Chem. Soc. 2016, 138, 8332−8335. (b)
Lodola, A.; Capoferri, L.; Rivara, S.; Tarzia, G.;
EP
Piomelli, D.; Mulholland, A.; Mor, M. Quantum Mechanics/Molecular Mechanics Modeling of Fatty Acid Amide Hydrolase Reactivation Distinguishes Substrate from Irreversible Covalent
AC C
Inhibitors. J. Med. Chem. 2013, 56, 2500−2512. (c) Van der Kamp, M. W.; Mc Geagh, J. D.; Mulholland, A. J. Lethal Synthesis” of Fluorocitrate by Citrate Synthase Explained through QM/MM Modeling. Angew. Chem., Int. Ed. 2012, 50, 10349−10351. (d) Lodola, A.; De Vivo, M. The Increasing Role of QM/MM in Drug Discovery. Adv. Protein Chem. Struct. Biol. 2012, 87, 337−362.
57
ACCEPTED MANUSCRIPT
[38] Capoferri, L.; Lodola, A.; Rivara, S.; Mor, M. Quantum Mechanics/Molecular Mechanics Modeling of Covalent Addition between EGFR–Cysteine 797 and N-(4-Anilinoquinazolin-6-yl)
RI PT
Acrylamide. J. Chem. Inf. Model. 2015, 55, 589–599. [39] Repasky, M.; Chandrasekhar, J.; Jorgensen, W. PDDG/PM3 and PDDG/MNDO: Improved semiempirical methods. J. Comput. Chem. 2002, 23, 1601–1622.
SC
[40] Walker, R. C.; Crowley, M. F.; Case, D. A. The Implementation of a Fast and Accurate
M AN U
QM/MM Potential Method in Amber. J. Comput. Chem. 2008, 29,1019-1031. [41] Vayner, G.; Houk, K. N.; Jorgensen, W. L.; Brauman, J. I. Steric Retardation of SN2 Reactions in the Gas Phase and Solution. J. Am. Chem. Soc. 2004, 126, 9054– 9058. [42] Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Comparison
TE D
of Multiple Amber Force Fields and Development of Improved Protein Backbone Parameters. Proteins 2006, 65, 712−725.
[43] (a) Crespo, A.; Martí, M. A.; Estrin, D. A.; Roitberg, A. E. Multiple-Steering QM−MM
EP
Calculation of the Free Energy Profile in Chorismate Mutase. J. Am. Chem. Soc. 2005, 127,
AC C
6940-6941. (b) Lodola, A.; Branduardi, D.; Capoferri, L.; De Vivo, M.; Mor, M.; Piomelli, D. A Catalytic Mechanism for Cysteine N-terminal Nucleophile Hydrolases, as Revealed by Free energy simulations. PLoS One 2012, 7, e32397. [44] (a) Jarzynski, C. Nonequilibrium Equality for Free Energy Differences. Phys. Rev. Lett. 1997, 78, 2690−2693. (b) Park, S.; Khalili-Araghi, F.; Tajkhorshid, E.; Schulten, K. Free Energy Calculation from Steered Molecular Dynamics Simulations using Jarzynski’s Equality. J. Chem. Phys. 2003, 119, 3559.
58
ACCEPTED MANUSCRIPT
[45] Vacondio, F.; Carmi, C.; Galvani, E.; Bassi, M.; Silva, C.; Lodola, A.; Rivara, S.; Cavazzoni, A.; Alfieri, R. R.; Petronini, P. G.; Mor M. Long-lasting Inhibition of EGFR
Inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 5290-5284.
RI PT
Autophosphorylation in A549 Tumor Cells by Intracellular Accumulation of Non-Covalent
[46] Galetti, M.; Alfieri, R. R.; Cavazzoni, A.; La Monica, S.; Bonelli, M.; Fumarola, C.;
SC
Mozzoni, P.; De Palma, G.; Andreoli, R.; Mutti, A.; Mor, M.; Tiseo, M.; Ardizzoni, A.; Petronini, P. G. Functional Characterization of Gefitinib Uptake in Non-Small Cell Lung Cancer
M AN U
Cell Lines. Biochem. Pharmacol. 2010, 80, 179−187.
[47] Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine and Threonine Proteases. Chem. Rev. 2002, 102, 4369-4750.
TE D
[48] Smaill, J.B.; Palmer, B.D.; Rewcastle, G.W.; Denny, W.A.; McNamara, D.J.; Dobrusin, E.M.; Bridges, A.J.; Zhou, H.; Showalter, H.D.H.; Winters, R.T.; Leopold, W.R.; Fry, D.W.; Nelson, J.M.; Slintak, V.; Elliot, W.L.; Roberts, B.J.; Vincent, P.W.; Patmore, S.J. Tyrosine
EP
Kinase Inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(Phenylamino)pyrido[d]pyrimidine Acrylamides as Irreversible Inhibitors of the ATP Binding Site of the Epidermal Growth Factor
AC C
Receptor. J. Med. Chem. 1999, 42, 1803− 1815. [49] Kletzien, R. F.; Pariza, M. W.; Becker, J. E.; Potter, V. R. A Method Using 3-O-methylD-glucose and Phloretin for the Determination of Intracellular Water Space of Cells in Monolayer culture. Anal. Biochem. 1975, 68, 537-544. [50] Lanthascreen® Eu Kinase Binding Assay, ThermoFisher Scientific Inc., USA.
59
ACCEPTED MANUSCRIPT
[51] Barf, T.; Kaptein, A. Irreversible Protein Kinase Inhibitors: Balancing the Benefits and Risks. J. Med. Chem. 2012, 55, 6243− 6262.
RI PT
[52] Hur, W.; Velentza, A.; Kim, S.; Flatauer, L.; Jiang, X.; Valente, D.; Mason, D. E.; Suzuki, M.; Larson, B.; Zhang, J.; Zagorska, A.; Di Donato, M.; Nagle, A.; Warmuth, M.; Balk, S. P.; Peters, E. C.; Gray, N. S. Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase.
SC
Bioorg. Med. Chem. Lett. 2008, 18, 5916−5919.
M AN U
[53] Van Schaeybroeck, S.; Kyula, J.; Kelly, D. M.; Karaiskou-McCaul, A.; Stokesberry, S. A.; Van Cutsem, E.; Longley, D. B.; Johnston, P. G. Chemotherapy-Induced Epidermal Growth Factor Receptor Activation Determines Response to Combined Gefitinib/Chemotherapy Treatment in Non-Small Cell Lung Cancer Cells. Mol. Cancer Ther. 2006, 5, 1154-1165.
TE D
[54] Tschitschibabin, A. E.; Woroshtzow, N. N. Ueber einige Derivate des α,β-Pyridinthioindoxyls. Chem. Ber. 1933, 66, 364-368.
[55] Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram
AC C
248–254.
EP
Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72,
[56] Cavazzoni, A.; Petronini, P. G.; Galetti, M.; Roz, L.; Andriani, F.; Carbognani, P.; Rusca, M.; Fumarola, C.; Alfieri, R.; Sozzi, G. Dose-Dependent Effect of FHIT-Inducible Expression in Calu-1 Lung Cancer Cell Line. Oncogene 2004, 23, 8439–8446. [57] Glide, Version 6.1, Schrodinger, LLC, New York, NY, 2013.
60
ACCEPTED MANUSCRIPT
[58] Tarozzi, A.; Marchetti, C.; Nicolini, B.; D'Amico, M.; Ticchi, N.; Pruccoli, L.; Tumiatti, V.; Simoni, E.; Lodola, A.; Mor, M.; Milelli, A.; Minarini, A. Combined Inhibition of the
RI PT
EGFR/AKT Pathways by a Novel Conjugate of Quinazoline with Isothiocyanate. Eur. J. Med. Chem. 2016, 117, 283-291.
[59] Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L.
SC
Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983,
M AN U
79, 926−935.
[60] Case, D. A.; Darden, T. A.; Cheatham III, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; Roberts, B. P.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Kolossváry, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Wu, X.; Brozell,
S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, J.; Hsieh, M.-J.; Cui, G.; Roe, D. R.;
TE D
Mathews, D. H.; Seetin, M. G.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER11, University of California, San Francisco.
EP
[61] Wang, J. M.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and testing of a general Amber force field. J. Comput. Chem. 2004, 25, 1157−1174.
AC C
[62] Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald - an NLog(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089−10092. [63] (a) Lodola, A.; Mor, M.; Hermann, J. C.; Tarzia, G.; Piomelli, D.; Mulholland, A. J. QM/MM Modelling of Oleamide Hydrolysis in Fatty Acid Amide Hydrolase (FAAH) Reveals a New Mechanism of Nucleophile Activation. Chem. Commun. 2005, 4399−4401. (b) TubertBrohman, I.; Acevedo, O.; Jorgensen, W. L. Elucidation of Hydrolysis Mechanisms for Fatty
61
ACCEPTED MANUSCRIPT
Acid Amide Hydrolase and its Lys142Ala Variant via QM/MM Simulations. J. Am. Chem. Soc. 2006, 128, 16904−16913. (c) Lodola, A.; Mor, M.; Sirirak, J.; Mulholland, A. J. Insights into the
RI PT
Mechanism and Inhibition of Fatty Acid Amide Hydrolase from Quantum Mechanics/Molecular Mechanics (QM/MM) Modelling. Biochem. Soc. Trans. 2009, 37, 363−367. (d) Acevedo, O.; Jorgensen, W. L. Advances in Quantum and Molecular Mechanical (QM/MM) Simulations for
SC
Organic and Enzymatic Reactions. Acc. Chem. Res. 2010, 43, 142-151. Lima, M. C.; (e) Seabra, G. M. Reaction Mechanism of the Dengue Virus Serine Protease: a QM/MM Study. Phys Chem
M AN U
Chem Phys. 2016, 18, 30288-30296.
[64] Bonomi, M.; Branduardi, D.; Bussi, G.; Camilloni, C.; Provasi, D.; Raiteri, P.; Donadio, D.; Marinelli, F.; Pietrucci, F.; Broglia, R. A.; Parrinello, M. PLUMED: A Portable Plugin for Free-Energy Calculations with Molecular Dynamics. Comput. Phys. Commun. 2009, 180,
Graphical Abstract
AC C
EP
TE D
1961−1972
62
ACCEPTED MANUSCRIPT Highlights A series of N-(4-(phenylamino)quinazolin-6-yl)acetamides was synthetized
•
Imidazol-2-ylthioacetamide 16 causes long-lasting inhibition of EGFR
•
16 shows negligible reactivity with cysteine in solution
•
Biochemical assays suggest the formation of a covalent bond between 16 and Cys797
•
Compound 16 inhibits proliferation of H1975 cells in the low micromolar range
AC C
EP
TE D
M AN U
SC
RI PT
•