Targeting Cytotoxic Agents through EGFR-Mediated Covalent Binding and Release

A major drawback of cytotoxic chemotherapy is the lack of selectivity toward noncancerous cells. The targeted delivery of cytotoxic drugs to tumor cells is a longstanding goal in cancer research. We proposed that covalent inhibitors could be adapted to deliver cytotoxic agents, conjugated to the β-position of the Michael acceptor, via an addition–elimination mechanism promoted by covalent binding. Studies on model systems showed that conjugated 5-fluorouracil (5FU) could be released upon thiol addition in relevant time scales. A series of covalent epidermal growth factor receptor (EGFR) inhibitors were synthesized as their 5FU derivatives. Achieving the desired release of 5FU was demonstrated to depend on the electronics and geometry of the compounds. Mass spectrometry and NMR studies demonstrated an anilinoquinazoline acrylate ester conjugate bound to EGFR with the release of 5FU. This work establishes that acrylates can be used to release conjugated molecules upon covalent binding to proteins and could be used to develop targeted therapeutics.


■ INTRODUCTION
−4 While conceptually simple, molecular targeting of cancers requires the identification of a specific mechanism to selectively localize a therapeutic agent within a tumor and for the agent to deliver a cytotoxic effect once it has reached its site of action.Identification of an appropriate mechanism is challenging due to the similarities between tumor and normal cells.
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly mutated in non-small cell lung cancer (NSCLC). 5,6Activating mutations in EGFR such as the exon-19 deletion and the L858R point mutation are key oncogenic drivers, and tumors harboring these mutations have been shown to be amenable to treatment with small molecule EGFR inhibitors. 7The first compounds to be used clinically were the reversible inhibitors gefitinib 1 8 and erlotinib, 9 but subsequently irreversible inhibitors such as dacomitinib 2, 10 osimertinib 3, 11 and poziotinib 4 12 have become prominent and are effective treatments for EGFR-driven NSCLC (Figure 1a).
The irreversible EGFR inhibitors function by targeting cysteine residue C797, which reacts via a conjugate addition to the pendant acrylamides upon binding of the inhibitor to the adenosine triphosphate (ATP) pocket. 13We reasoned that this binding event could be used as a targeting mechanism, and that if a cytotoxic agent was appended to the β-position of the acrylamide, an addition−elimination process could trigger its specific release (Figure 1b).We selected 5-fluorouracil (5FU) as the cytotoxic agent for initial studies since its small size would minimize the perturbation of the physical properties of the targeting inhibitor and would be most likely to be tolerated in the active site.Additionally, 5FU could be linked via the 1nitrogen in a manner that would retain the reactivity of the Michael acceptor and act as a suitable leaving group due to its electron-deficient nature.Modeling of the conjugates of osimertinib 5 and poziotinib 6 in the active site suggested that they should be tolerated (Figure 1c,d).
Accordingly, we set out to investigate the synthesis, binding, and release of 5FU-EGFR inhibitor conjugates to assess their potential to provide a targeted delivery paradigm.During the course of this work, a similar approach, termed covalent liganddirected release (CoLDR) involving the attachment of releasable ligands on the methyl position of α-methacrylamide inhibitors, was disclosed by London 14,15 and Fang. 16Our concept of conjugating inhibitors at the β-position would be complementary to this approach and would offer versatility with regard to the tolerability of the conjugates' target binding and their release kinetics.

Synthesis of Model Release Systems.
To determine the feasibility of selectively releasing a cytotoxic drug as a leaving group via conjugate addition−elimination, model release systems were prepared.The purposes of this were twofold; first, they would provide evidence that the release could be achieved controllably in the presence of a cysteine residue, and second, to investigate the tuning of the respective systems with modifications to the electronic properties of the Michael acceptors.
There were no reported examples in the literature in which a thiol has been able to displace a leaving group from the βposition of an enamide.There were, however, numerous reports of the analogous reaction on a vinyl ketone.The most relevant reaction reported is of N-acetyl cysteine with phenyloxybut-2-enones, in which a detailed kinetic study was described under conditions that closely resemble those encountered in vivo. 17Ester, amide, and ketone activating groups were hypothesized to be the best suited to allow the release of the cytotoxic payload, and so model systems were proposed with each of these functionalities, along with varied electron-donating and -withdrawing groups on the aryl substituent.
Synthesis of the desired esters began with the corresponding phenol coupled with propiolic acid to give propargyl esters 7− 10, followed by the DABCO-promoted addition of 5FU to afford conjugates 11−14 (Scheme 1a).Synthesis of the analogous nitro-containing acrylate was not feasible through this route but was prepared by the initial addition of 5FU to methyl propiolate to give ester 15, followed by hydrolysis to the corresponding acid 16 and coupling with 4-nitrophenol to give conjugate 17 (Scheme 1b).
Aqueous solubility of the corresponding amides was too low to allow the NMR experiments (see below); therefore, an analogue containing a solubilizing bis-ethylene glycol chain was prepared (Scheme 1c).Etherification of 4-nitrophenol gave bisethyleneglycol 18 followed by reduction to aniline 19 to enable coupling with propiolic acid to give propargyl amide 20.DABCO-promoted addition with 5FU gave conjugate 21.
Synthesis of the enone began with installing TMS acetylene to the starting phenylacetyl chloride to give ketone 22, followed by removal of the TMS protecting group using TBAF in methanol to give then ynone 23.5FU was added in the presence of DABCO resulting in target enone 24 (Scheme 1d).
19 F NMR Cytotoxic Release Studies.Reaction of the model systems with N-acetyl-L-cysteine methyl ester was assessed by 19 F NMR. Observation of a 19 F NMR signal corresponding to 5FU indicated that the release of the cytotoxic had occurred.Ketone 24 showed the formation of 5FU with a half-life of approximately 10 h (Figure 2a).PEGylated amide 21 showed no detectable release within the time scale of the experiment (Figure 2b).
While it is likely that the rate of elimination would be increased within the context of binding to the target protein, these studies indicated that the rate of release was dependent on the electronic properties of the Michael acceptor.Consistent with this, the corresponding methoxy-substituted acrylate (with electronics intermediate between 21 and 24) showed a small amount of release after 24 h (half-life ∼36 h; Figure 2c and Table 1).For acrylates, the rate showed an approximately linear Hammett dependency on the electronics of the para substituent (ρ = 1.82; Figure 2d).Importantly, the range of half-lives could be modulated between 36 h (−OMe) and 0.1 h (−NO 2 ), indicating that it should be possible to achieve the desired release kinetics in compounds of this type.
Formation of EGFR Inhibitor-Cytotoxic Hybrids.Having established that the desired release was achievable in a relevant time scale, we synthesized the corresponding elaborated EGFR inhibitor-5FU hybrids.Initially, we focused on pyridinylpyrazolopyrimidine-based inhibitors, such as 30 (Scheme 2).This template had been shown previously to give potent EGFR inhibitors. 18ster analogues were prepared with a pyrimidinyl indole EGFR inhibitor motif, also precedented to give potent EGFR inhibitors, including osimertinib (Scheme 3). 11,19arget molecule 30 was prepared in 6 overall steps from 2,4,5-trichloropyrimidine, commencing with the Heck-type coupling with butyl vinyl ether to give enol-ether 25 (Scheme 2).This compound was then reacted with 1-aminopyridinium iodide to give pyrazolopyrimidine 26, which underwent S N Ar reaction with 2-methoxy-5-nitroaniline-5-nitroaniline to give anilinopyrimidine 27.Nitro reduction gave aniline 28, which was coupled with propiolic acid to give propiolamide 29.The desired conjugate was prepared by the DABCO-mediated addition of 5FU to give 30.
Synthesis of 2-fluoro-4-methoxyacrylate derivative 35 started from 2-fluoro-4-methoxyphenol, which was esterified to give acetate 31 (Scheme 3).The acetate underwent nitration with the desired regiochemistry to give 32, which underwent     The corresponding 4-nitroacrylamide was prepared from 3aminophenol by acetamide protection to give 36, which underwent nitration to give 37 (Scheme 3).Acetamide hydrolysis revealed the aniline, which underwent S N Ar reaction to give phenol 38.Compound 38 was esterified with 16 to afford conjugate 39.The enone analogue of 39 proved too reactive leading to difficulties in the final purification and so was not profiled further.
Incubation of compound 30 with EGFR protein and subsequent analysis by mass spectrometry showed that an adduct formed and was confirmed by protein digestion and peptide mapping to be the result of reaction with C797, confirming that the 5FU conjugate was sufficiently reactive and tolerated sterically within the active site (Figure S1).However, only the adduct was observed and there was no evidence of the release of 5FU.Both acrylates 35 and 39 also showed evidence of adduct formation by mass spectrometry, again without evidence of the release of 5FU in both cases (Figures S2 and  S3).Interestingly, methoxy derivative 35 showed more extensive labeling than the more electron-deficient (and therefore more reactive) nitro derivative 39.Hence, despite the addition being feasible, this was not sufficient to achieve the desired subsequent elimination in the environment of the bound state.
Anilinoquinazoline-Based EGFR Inhibitor-Cytotoxic Hybrids.The anilinoquinazoline-derived covalent inhibitors, exemplified by afatinib 2 and poziotinib 4, provided an alternative scaffold for a cytotoxic conjugate with different geometries, which we considered worth exploring to assess if this facilitated the required elimination.Accordingly, we prepared a series of covalent acrylamide anilinoquinazoline- 5FU conjugates with varying geometries in the ring linking the warhead to the quinazoline core via 4-, 5-, and 6-membered rings 48−51 (Scheme 4).Synthesis was performed by amide coupling from known intermediates 40−43 20,21 by amide coupling with propiolic acid to give propiolates 44−47 followed by the addition of 5FU to the ynamide Michael acceptor.
We also prepared the acrylate ester analogue 53.The acrylate ester, 53, was conveniently available via a single-step esterification of intermediate phenol 52 with carboxylic acid 16 (Scheme 5).Acrylamide, 57, was also synthesized due to the potential metabolic instability of acrylate ester 53, accessed in one-pot via the acyl chloride derivative of carboxylic acid 16 and aniline 58 (Scheme 6).
Incubation of acrylamide 48 with EGFR and subsequent mass spectrometry analysis again indicated the covalent adduct formation without evidence of the release of 5FU (Figure S4).The other acrylamide conjugates 49−51 did not show any evidence of adduct formation (Figure S5) despite showing potent EGFR inhibition (Table S1).In contrast to all previous compounds, protein mass spectrometry of acrylate 53 after incubation with WT EGFR revealed two new major adducts corresponding to the initial adduct (mass increase of 502 Da) and the lower mass peak equating to the loss of 5FU from the complex (increase of 372 Da relative to apo protein) (Figure 3a).This provided clear evidence that the desired release of 5FU is feasible for this compound.
Acrylamide analogue 57 demonstrated a clear adduct with WT EGFR (mass increase of 501 Da), with a negligible release of 5FU (mass increase of 371 Da relative to apo protein), suggesting a much slower retro-Michael addition (Figure 3b).
To confirm the release of 5FU, the reactions of 53 and 57 with thiol nucleophiles N-acetyl cysteine and glutathione were studied by 19 F NMR.As expected, on reaction with N-acetyl cysteine, 57 showed no evidence of 5FU release even after 24 h.However, 53 was shown to rapidly release 5FU, with a halflife of ca. 5 min and a complete release of 5FU after 1 hour (Figure 4a).This level of reactivity is comparable to that observed with model nitro ester 17.Reaction with glutathione proceeded in a similar manner, although at a slightly slower rate, with some levels of 53 remaining even after 1 h (Figure 4b).
Biological Evaluation of the Inhibitor-Cytotoxic Hybrids.The acrylate-5FU conjugates 53 and 57 were profiled in biological assays for EGFR activity alongside dacomitinib 2 and its corresponding acrylate analogue 56 (Scheme S1).Both 2 and 56 were potent inhibitors of EGFR in vitro (Figure 5a).Conjugate 53 was slightly less potent but retained activity (pIC 50 6.8), while conjugate 57 was slightly more potent (pIC 50 7.3).
In A431 cells, conjugate 53 showed a marked inhibition of EGFR phosphorylation at 10 μM but not at 1 μM (Figure 5b), with a pIC 50 7.8.This level of inhibition was better than ester 56 (pIC 50 5.3), despite demonstrating some inhibition of EGFR phosphorylation at 1 μM.Whereas 2 was more potent as expected (pIC 50 8.0), 57 showed little inhibition of EGFR phosphorylation even at 10 μM and a pIC 50 of 5.6.In 72 h of growth inhibition assays, conjugate 53 showed a potent antiproliferative effect (pGI 50 5.3; Figure 5c).This was a marked increase in potency compared to ester 56 (pGI 50 < 5.0).Disappointingly, 57 showed no antiproliferative effects, despite the strong antiproliferative effects demonstrated by its corresponding analogue 2 (pGI 50 7.1).
These data provide clear evidence that 53 binds to EGFR in a cellular setting.The reduced potency of ester 56 for the inhibition of EGFR phosphorylation and growth inhibition relative to its in vitro potency is likely a consequence of its increased reactivity and competitive reaction with glutathione in a cellular environment. 15he increased growth inhibition potency of conjugate 53 relative to 56 is likely in part a consequence of the reduced reactivity of the warhead from introducing the 5FU substituent to the acrylate.However, the improved growth effects of 53 may also imply an additional effect from the release of 5FU.
In growth inhibition assays, an increase in potency is seen for 2 combined with 5FU compared to either 2 or 5FU alone (Figure 6a) and for 56 in combination with 5FU compared to 56 alone but comparable to 5FU alone (Figure 6b).It was expected that this would mimic the effect of the proposed release of 5FU from 53 or 57, with the observed increase in potency providing additional evidence that the growth effects of 53 compared to those of 56 come from the release of 5FU.
Acrylate conjugate 53 was incubated in cell media to demonstrate that the release of 5FU must be catalyzed by EGFR and does not simply occur in the media.For 25 h, some hydrolysis did occur; however, it was the ester bond that was hydrolyzed: there was no observation of the release of 5FU (Figure S6).Given the significantly slower release of 5FU for acrylamide 57, a suitable bioisosteric replacement of an ester with comparable electronic properties would be necessary for compounds with in vivo stability.

■ DISCUSSION
Protein mass spectrometry of EGFR after incubation with 53 shows a conclusive proof of covalent binding to EGFR with the release of 5FU in vitro.Study of the reaction of 53 with acetyl cysteine by NMR shows that 5FU is released in relevant time scales.Inhibition of EGFR phosphorylation in A431 cells shows that 53 engages EGFR in a cellular setting at appropriate concentrations.Together, these data provide evidence that the compound can both engage EGFR and release 5FU in tumor cells.
Our results suggest that obtaining the desired release profile requires appropriate electronics of the Michael acceptor system to enable both the initial addition and the release of the cytotoxic in a suitable time scale, without being so reactive as to be unstable.For 5FU, the acrylate ester system strikes the appropriate balance of reactivity.We would expect this to be transferrable to other conjugates with similar electronic properties to uracil but, in general, it is likely to depend on the codependent effects of the electron-donating ability of the conjugate that controls the initial addition and its leaving group ability that influences the rate of release.The observation of stable adducts without the release of 5FU for a number of analogues suggests that the elimination process is less facile and that protonation of the intermediate enolate species can be more favorable than the elimination.
It is perhaps surprising that the desired elimination of 5FU occurred with quinazoline 53 but not with the analogous pyrimidine-derived acrylates 35 and 39.We would postulate that the electronic properties of these compounds are similar and that the difference likely arises due to their bound geometries.To investigate this, we performed molecular modeling of the intermediate enolate adducts of 39 and 53 using QM-optimized ligand poses that were covalently docked into the appropriate crystal structure (pdb 6JX4 for 39 and 4G5J for 53) (osimertinib template) before a final refinement of the structures.In order to preserve the optimized poses from the QM simulation, each ligand was restricted to move only 0.3 Å during the covalent docking process (Figure 7).This showed that in both cases, the intermediate enolate has the potential to be stabilized by a hydrogen bond between the ester oxygen and the C797 backbone NH.In the case of anilinoquinazoline 53, modeling showed the potential additional intramolecular hydrogen bond between the aniline NH and the uracil carbonyl.Hence, it is possible that the release profiles depend on the presence of secondary interactions that stabilize the transition state leading to elimination.

■ CONCLUSIONS
Our results provide strong evidence that acrylate ester systems can be used as a delivery vector to release 5FU in cells via an addition−elimination reaction with EGFR.While the increases in growth inhibition observed with this combination are modest, 5FU is relatively weakly active as a cytotoxic agent.This approach may provide a general method for specific targeting agents, such as cytotoxic chemotherapy, to cancer  Following incubation, intact protein masses were determined using an Agilent 6530 Accurate Mass dual AJS/ESI Q-TOF instrument coupled to an Agilent 1260 Infinity II LC system. 1 μL of purified protein (∼1 mg/mL) was injected onto an MS Pac DS-10 Desalter cartridge ((Thermo Fisher Scientific), PN: 089170, 2.1 × 10 mm) for desalting and reversed phase separation at 70 °C.The mobile phase was 0.1% (v/v) formic acid in LC-MS grade water (A) and LC-MS grade acetonitrile (B) with separation performed over 7.5 min.Sample desalting was achieved at 30% B for 2 min at 1 mL/min before reducing the flow rate to 0.2 mL/min for 2 min.Protein elution was achieved at 100% B for 0.5 min and 1 mL/min before reequilibration at 30% for 1 min.Proteins were detected in positive ion mode using electrospray ionization with a nebulizer pressure of 45 psig, a drying gas flow of 5 L/min, and a source gas temperature of 325 °C.A sheath gas temperature of 400 °C, a gas flow of 11 L/min, a capillary voltage of 3500 V, and a nozzle voltage of 2000 V were also used.Mass spectra were acquired using MassHunter Acquisition software (version B.08.00) over a mass range of 100−3000 m/z at a rate of 1 spectra/s and 1000 ms/spectrum in the standard mass range (3200 m/z) at 2 GHz.The instrument had been calibrated over the selected mass range prior to analysis.
In Vitro TR-FRET Assay.Compounds (dissolved in 10 mM in DMSO) were dispensed into black low-volume 384-well assay plates (Corning) over a final concentration range of 100 000, 30 000, 10 000, 3000, 1000, 300, 100, 30, 10, and 3 nM using an Echo 550 (Labcyte).Positive control compound and DMSO as a negative control were dispensed into the first and last wells, respectively.Each  buffer was added.This solution was vortexed, centrifuged, then placed at 98 °C for 5 min, and then centrifuged to remove the residue.Migration was performed using a 4−20% Criterion TGX Precast Midi Protein Gel, 18 well, 30 μL (Cat.No. 5671094) placed in a Criterion running tank (Cat.No. 1656001) and also using PowerPac HC highcurrent power supply (Bio-Rad) electrodes.Nitrocellulose membrane used was an Amersham Protran Premium 0.45 Nitrocellulose membrane (Cat.No. 15269794) and Ponceau S Stain (Sigma-Aldrich Cat.No. p7170).
Preparation of 1 × Tris Glycine SDS Running Buffer.To deuterated water (900 mL, 50 mol) was added 100 mL of the 10 × tris glycine SDS running buffer.It was stored at room temperature.
Preparation of 10 × TBS Solution.Tris-HCl (48.5 g) and NaCl (160 g, 2.70 mol) were added to deuterated water (1.6 L, 88.9 mol) and stirred until dissolved.The solution was then adjusted to pH 7.6 with NaOH, and then the volume was made up to 2 L with additional deuterated water.It was stored at room temperature.
Preparation of Primary Antibody Solution.Primary antibodies raised to EGFR (CST 4267) or pEGFR (CST 3777) were diluted (1:1000) in 5% BSA w/v.Loading control antibodies were prepared in 5% milk w/v with 1 × TBS/T (0.1 v/v) solution at a determined optimum concentration.
Preparation of Secondary Antibody Solution.Secondary HRPconjugated antibodies were diluted in 5% milk with w/v in 1 × TBS/ T (0.1 v/v) solution at a determined optimum concentration.
Detection.Proteins were detected using the Clarity ECL Western blotting substrate (Bio-Rad).
HTRF Method (A431).Cells were plated at 20 000 cells per well in 96-well plates and placed at 37 °C with 5% CO 2 .Once adhered (after 24 h), cells were treated with compounds dissolved in DMSO at a final concentration of 0.1% DMSO in media.Compounds were diluted in media and then added to cells for 2.5 h in duplicate; 100 ng/mL of EGF (Thermo Fisher, PHG0311) was added to all compound-treated cells as well as control for 30 min.Compound and media were removed from the cells, and then 50 μL of HTRF lysis buffer was added (lysis buffer was diluted from 4× stock to 1× in deionized (DI) water, with 1% blocking agent also added).Cells were lysed on a plate shaker (2000 rpm) for 30 min at room temperature.pEGFR expression was monitored using the Cisbio Phospho-EGFR (Tyr1068) cellular kit (64EG1PEH).Total EGFR expression was monitored using the Cisbio Total EGFR cellular kit (64NG1PEH) as per the manufacturer's instructions.Fluorescence emission was read at two different wavelengths (665 and 620 nm) on a PheraStar.Results were calculated as the ratio of pEGFR/total EGFR and then the percentage of 0 μM control.
Growth Inhibition in Adherent Cell Lines.A431 cells purchased from ECACC, Cat.85090402, were plated on day 0 in a 96-well plate at a density known to allow for exponential growth over 72 h in DMEM (Sigma-Aldrich, Cat No. D5796) supplemented with 10% FBS (Gibco, Cat No. 10270-106).On day 1, the compounds were diluted to the required concentration in DMEM +10% FBS media, ensuring that the final DMSO concentration was 0.1% once added to cells.The cells were then incubated for 72 h at 37 °C with 5% CO 2 .Cells were then fixed by adding 50% (wt/vol) TCA to each well of the plate and left at 4 °C for 1 h.The plates were then washed thoroughly with water, 100 μL of 0.4% SRB solution was added to the wells, and left at room temperature for 30 min.The plates were rinsed with 1% AcOH and then left to air-dry in a drying cabinet for 1 h.Once dry, 100 μL of 10 mM tris pH 10.5 was added to each well and the plates were placed on a plate shaker for 10 min.The absorbance was read at 570 nm using a FluoStar Omega plate reader.
Computational Methods.Pyrimidine System.An X-ray structure of 3 bound to EGFR wild-type protein was used as a start point for the evaluation of the potential positions on the molecule for the addition of a cytotoxic payload (PDB Code 4ZAU).For reasons that are not completely clear, the covalent bond between the inhibitor and protein was not formed in this X-ray structure.The X-ray structure was prepared for molecular modeling using the protein preparation wizard in Maestro (Schrodinger) to add hydrogens and confirm protonation and tautomer states of amino acids.Initially, the covalent bond was formed between the inhibitor and protein using the builder functionality and the local region was then optimized using the default forcefield (OPLS3).Visual inspection identified the position of the molecule to be most suitable for the addition of the payload and was manually built onto the molecule, followed by an additional round of optimization of the local atoms.The final model gives encouragement that the payload could be accommodated in this position during the formation of the covalent bond.
Quinazoline System.An X-ray structure of afatinib bound to EGFR wild-type protein was used as a start point for the evaluation of the potential positions on the molecule for the addition of a cytotoxic payload (PDB Code 4G5J).The X-ray structure was prepared for molecular modeling using the protein preparation wizard in Maestro (Schrodinger) to add hydrogens and confirm protonation and tautomer states of amino acids.The bound inhibitor was converted into the target molecule using the builder functionality and then allowed to optimize in the pocket.Visual inspection identified the position of the molecule to be most suitable for the addition of the payload and was manually built onto the molecule, followed by an additional round of optimization of the local atoms.The final model gives encouragement that the payload could be accommodated in this position during the formation of the covalent bond.
Intermediate Enolates.Modeling was performed using Maestro v2021-2.Ligand structures were initially prepared as the enolate form, wherein they were optimized in Jaguar using a B3-LYP functional and a 6-31G** basis set.Proteins were prepared using the Protein preparation Workflow in Maestro.The QM-optimized ligand poses were then covalently docked into 4G5J (AQZ template) and 6JX4 (osimertinib template) before a final refinement of the structures was performed using Prime.In order to preserve the optimized poses from the QM simulation, each ligand was restricted to move only 0.3 Å during the covalent docking process.
General Chemical Methods.Chemicals and Solvents.All commercial reagents were purchased from reputable chemical companies.The chemicals were of the highest available purity.Unless otherwise stated, chemicals were used as supplied without further purification.Anhydrous solvents were obtained from either Sigma-Aldrich or Acros and were stored under nitrogen.Petrol refers to the fraction with a boiling point between 40 and 60 °C.
Chromatography.Thin-layer chromatography (TLC) utilized to monitor the reaction progress was conducted on plates precoated with silica gel (Merck 60F254).The eluent was as stated (where this consisted of more than one solvent; the ratio is stated as volume/ volume), and visualization was either by short-wave (254 nm) ultraviolet light."Flash" MPLC was carried out on a prepaced silica columns.Semipreparative HPLC was carried out on an Agilent instrument passing through a Waters XSelect column, employing a C 18  19 × 150 nm, 3.5 Å column (eluent: (acidic) 0.1% formic acid (aq)/MeCN, (basic) 0.1% NH 3 (aq)/MeCN), using a UV detector at 254 nm and a flow rate of 20 mL/min.Analytical Techniques.Melting points were determined using a VWR Stuart SMP40 apparatus and are uncorrected.Optical rotations were recorded on a PolAAr 3001 Automatic Polarimeter (Optical Activity Ltd., Cambridgeshire, U.K.); units of [α] D are given in 10 −1 deg cm 2 g −1 .LC-MS was carried out on a Waters Acquity UPLC system with PDA and ELSD operating in positive and negative ion electrospray modes, employing an Acquity UPLC BEH C18, 1.7 mm, 2.1 mm × 50 mm column with 0.1% formic acid and water− acetonitrile (5−95%) for gradient elution.FTIR spectra were recorded on an Agilent Cary 630 FTIR spectrometer as a neat sample.Bond stretch frequencies are reported as br (broad), s (sharp), m (medium), or w (weak) based on their relative intensities.UV spectra were obtained using a U-2001 Hitachi spectrophotometer with the sample dissolved in ethanol. 1 H, 13 C, 15 N, and 19 F nuclear magnetic resonance (NMR) spectra were obtained as either CDCl 3 , CD 3 OD, or DMSO-d 6 solutions and recorded at 500, 126, 700, and 470 MHz, respectively, on either a Bruker Avance III 500 or 700 spectrometer.Chemical shifts are quoted in parts per million (δ) referenced to the appropriate deuterated solvent employed.Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet), br (broad), or combinations thereof.Coupling constant values are given in Hz.Homonuclear and heteronuclear two-dimensional NMR experiments were used where appropriate to facilitate the assignment of chemical shifts.The numbering system used in the assignment of aromatic carbons and hydrogens is done according to the IUPAC nomenclature.All final compounds are >95% pure by HPLC analysis.
General Procedure A. To a solution of amine (1.0 equiv) in DMF (0.5 mmol/mL) were added acid (1.0 equiv), DMAP (0.4 equiv), and HATU (1.0 equiv) and allowed to stir at room temperature overnight.Once consumption of starting materials was observed, the reaction mixture was extracted into EtOAc, washed with water and brine, dried over MgSO 4 , and then concentrated in vacuo.
General Procedure B. To a solution of acid (1.0 equiv) in DCM (1.0 mmol/mL) at 0 °C was added a 1 M solution of DCC in DCM and stirred for 5 min before the addition of phenol (0.2 equiv) and DMAP (0.2 equiv).The reaction mixture was stirred for 4 h, with the resulting suspension filtered, and the filtrate was concentrated in vacuo.
General Procedure C. To a solution of 5FU (0.9 equiv) in DMF (1.0 mmol/mL) was added DABCO (0.5 equiv) and allowed to stir for 5 min before the addition of alkyne.The reaction mixture was then left to stir at 40 °C for 2 h.The solvent was then removed in vacuo.

Figure 1 .
Figure 1.(a) Examples of established EGFR inhibitors; (b) depictions of proposed 5FU conjugates with the covalent binding/release process in the EGFR active site; models of bound conformations of (c) pyrimidine-and (d) quinazoline-based conjugates in the EGFR active site based on PDB structures 4ZAU and 4G5J, respectively.

Scheme 1 .
Scheme 1. Synthesis of Enamide and Acrylate Model Systems a
reduction to aniline 33.S N Ar reaction of 33 with 3-(2chloropyrimidin-4-yl)-1-methyl-1H-indole proceeded with concomitant acetate hydrolysis to give the phenol precursor 34.The synthesis of the desired conjugate achieved the DCCmediated coupling of 34 with 16.

Scheme 3 .
Scheme 3. Synthetic Route for the Formation of the Anilinopyrimidine Acrylates a

Scheme 4 .
Scheme 4. Synthesis of the Anilinoquinazoline Amides a

Figure 3 .
Figure 3. Protein mass spectrometry spectra after incubation of WT protein with (a) 53 and (b) 57.
Figure 6.(a) Concentration response curves of the A431 cell viability in response to inhibitor 2, 5FU and equimolar 2 and 5FU (72 h); (b) concentration response curves of the A431 cell viability in response to inhibitor 56, 5FU and equimolar 56 and 5FU (72 h).

Figure 7 .
Figure 7. Molecular modeling of intermediate enolates arising from the conjugate addition for (a) pyrimidine 39 based on the pdb structure 6JX4; (b) quinazoline 53 based on the pdb structure 4G5J.