Inhibition of osimertinib-resistant epidermal growth factor receptor EGFR-T790M/C797S† †Electronic supplementary information (ESI) available. CCDC 1876852. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c9sc03445e

We present inhibitors of drug resistant mutants of EGFR including T790M and C797S. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR to gain insight into their binding mode.


TABLE OF CONTENTS
Scheme S1. Synthetic route for generation of compound 1 ____________________________S3 Scheme S2. Optimized synthetic route, yielding compound 10 and 17 ___________________S4 Scheme S3. Synthetic key step for synthesis of compound 20__________________________S5 Figure S1. 2Fo-Fc maps and Fo-Fc stimulated annealing omit maps of complex crystal structures ________________________________________________________S6 Figure S2 and Table S1. Kinase selectivity profiling of compound 17a ___________________S7 Figure S3. HMBC NMR analysis reveals the structure of the constitution isomers 28a and 28b _______________________________________________________________S8 Figure S4. Mass spectrometry-based analysis of covalent bond formation ________________S9 Table S2. Cellular evaluation of pyrrolopyrimidine EGFR inhibitors on KRAS-mutant cell lines______________________________________________________________S10 Table S3 and Chart S1. Metabolic stability for selected pyrrolopyrimidine inhibitors in mouse liver microsomes _________________________________________________S11 Table S4. Pharmacokinetic parameters of 19h, WZ4002 and osimertinib ________________S12 Table S5. Data collection and refinement statistics of complex crystal structures __________S13 Table S6. Crystal data and structure refinement for 28a ______________________________S14

S11
Metabolic stability in mouse liver microsomes for selected pyrrolopyrimidine-based inhibitors. Chart S1. Relative compound degradation, grouped by (A) low, (B) medium, and (C) high clearance (as indicated in Table S3).    Compound dilutions were generated using the acoustic dispensing system "ECHO 520 Liquid

S22
Cell line authentication has been performed last in August 2017 by STR profiling of 16 alleles.

Viability assay.
Cells were seeded at cell numbers that assure linearity and optimal signal intensity (150-300 cells/well, 25 µL) and cultured for 24 hours in serum-and antibiotics-containing media in humidified chambers at 37 °C / 5% CO 2 . The cells were then treated with EGFR inhibitors in serial dilutions (14 nM to 30 µM) with DMSO and Staurosporine as control and incubated for 96 hours.
Afterwards viability studies were carried out using CellTiter-Glo Assay (Promega, USA) that is a homogeneous method of determining the number of viable cells in culture. It is based on quantification of ATP, indicating the presence of metabolically active cells. For these studies, CellTiter-Glo reagent was prepared according to the instructions of the kit and diluted 1:1 with the complete growth medium suitable for the corresponding cell line. Thereon, reagent and assay plates were equilibrated at room temperature for 20 min. Equal volumes of the reagent were added to the volume of culture medium present in each well (25 µL). The plates were mixed for 2 minutes on an orbital shaker to induce cell lysis. The microplates were then incubated at room temperature for 20 minutes for stabilization of the luminescent signal. Following incubation the luminescence was recorded on an EnVision microplate reader (Perkin Elmer) using 500 ms integration time. The data was then analyzed using the Quattro Research Software Suite for EC 50determination. As quality control the Z'-factor was calculated from 16 positive and negative control values. Only assay results showing a Z'-factor ≥0.5 were used for further analysis. All data points were measured in duplicates for each plate and were replicated in at least two plates.

Western blotting.
Cells were plated in six-well plates on day 1 at 400.000 (H1975) and 500.000 (A431) cells per well and incubated at 37 °C and 5% CO 2 . On day 2 cells were starved over night with media containing 0.5% FBS to be incubated on day 3 for 1 h with EGFR inhibitors following an additional 30 min of EGF stimulation at 50 ng/mL. The media was discarded and cells were washed once with DPBS 1x. Using 100 μL per well of lysis buffer, cells were then scraped off and lysed while rotating
For nano-LC-MS/MS measurements the samples were incubated with 10 mM iodoacetamide for 30 min at RT in the dark, in order to carbamidomethylate all free cysteine residues. Next, 1 μg was digested using the broad specificity protease subtilisin (Sigma Aldrich) in a 1:10 ratio S25 (protease:protein) for 20 min at 56 °C. Digestion was quenched by addition of 1% TFA and 2 pmol were analyzed by nano-LC-MS/MS using a Orbitrap Fusion Lumos mass spectrometer onlinecoupled to an Ultimate 3000 nano RSLC system (both Thermo Scientific).
Peptides were preconcentrated on a 75 μm x 2 cm C18 trapping column for 5 min using 0.1% TFA Sickmann, L. Martens, J. Proteome Res. 2012, 11, 10, 5065-5071) and searches were conducted in a target/decoy mode to calculate a false discovery rate (FDR). Enzyme specificity was set to "none" and cysteine modifications by either carbamidomethylation (+57.02146 Da) or 19h (+510.27432) were set as variable, with a maximum of 3 per peptide. Fragmentation mode was set to ETD to consider c and z ions for scoring. Mass tolerances were set to 10 ppm for precursor S26 masses and 0.02 Da for fragment ions. FDR was adjusted to 1% on peptide spectrum match (PSM) level.

Microsomal stability (phase I) assay.
Metabolic stability under oxidative conditions was measured in liver microsomes from different species by LC/MS-based measuring of depletion of compound at a concentration of 3 µM over time up to 50 minutes (HLM) or 60 minutes (MLM) at 37 °C. Based on the compound half-life t 1/2 , the in vitro intrinsic clearance CL int was calculated.

Plasma stability.
Plasma stability was measured by LC/MS-based determination of the percentage of remaining compounds at a concentration of 5 µM after incubation in plasma obtained from different species for 1 h at 37 °C.

Plasma protein binding.
Assessment of plasma protein binding was measured by equilibrium dialysis by incubating plasma with the compound of interest at a concentration of 5 µM for 6 h at 37 °C followed by LC/MSbased determination of final compound concentrations.

PAMPA (Parallel artificial membrane permeability assay).
The compound was diluted from a 10 mM stock in DMSO to a final concentration of 500 µM in 50 mM Hepes buffer pH 7.4 and transferred onto a transwell membrane covered with a membrane-forming solution of 10% 1,2-Dioleyl-sn-glycero-3-phosphocholine (Sigma Aldrich) and 0.5% (w/v) cholesterol (Sigma Aldrich) in dodecane. Following an incubation of 16 hours at room temperature in a wet chamber, the optical density of the solution in the receiver well was measured between 250 and 500 nm in intervals of 10 nm. The percent flux was calculated from the AUC between 250 and 500 nm and normalized to the absorption of the compound following a 16 hour incubation in a parallel transwell containing a membrane covered with 50% MeOH in 50 mM Hepes buffer pH 7.4 (M. Kansy, F. Senner, K. Gubernator, J Med Chem 1998, 41, 1007-1010).

Caco-2 assay.
For Caco-2 cell assay, a 10 mM DMSO stock of the compound was diluted to a final concentration of 5 μM in HBSS buffer pH 7.4 and incubated for 2 hours at 37 °C and 5% CO 2 on a monolayer of Caco-2 cells (ATCC) that had been grown on a transwell membrane (Millipore, Schwalbach, Germany) for 21 days. The compound concentration was measured in the receiver as well as the donor well. Apparent permeability (P app ) from either the apical to basolateral direction or vice versa was calculated by the equation: P app = 1/AC 0 (dQ/dt), where A is the membrane surface area, C 0 is the donor drug concentration at t = 0, and dQ/dt is the amount of drug transported within the given time period of 2 hours.

In vivo pharmacokinetics.
To determine the pharmacokinetics of 19h, totally 36 male RjOrl:SWISS (CD1) mice, age 8−10 weeks, body weight (bw) between 29 and 31 g, were purchased from Janvier, Saint-Berthevin Cedex, France. Mice were fed ad libitum with Allein-Futter für Ratten-/Mäusehaltung from Sniff Special Diets GmbH, Germany. They had free access to water and were kept in a 12 h day/night rhythm. All experiments were approved by the local authorities. Pharmacokinetics were studied for the oral (PO), intraperitoneal (IP), and intravenous (IV) route of administration. For the oral and S28 intraperitoneal routes, a stock solution of 2 mg/mL of 19h in a solution consisting of 10% of DMSO and to 90% of an aqueous solution of 30% 2-hydroxypropyl-β-cyclodextrin was prepared. To reach doses of 20 mg/kg bw, 300 μL were administered per 30 g of mouse bw. For the intravenous route of administration, a 2 mg/mL DMSO solution was prepared and 30 μL were applied to a 30 g mouse to reach a concentration of 2 mg/kg bw. Mice were sacrificed 5, 15, 45, and 135 min after administration of the test compound, and approximately 100 μL of blood was taken from the left heart chamber. Three mice were analyzed per time point and route of administration (PO, IP and IV), respectively. Immediately after sampling, blood was centrifuged at 16200g for 10 min at 4 °C and plasma was stored at -80 °C until LC/MS analysis of 19h. Prior to LC-MS/MS analysis, plasma proteins were precipitated with acetonitrile containing an internal standard, and samples were filtered. A calibration curve was obtained from spiked blank plasma samples. 19h was measured using a Shimadzu UPLC system connected to a QTrap 4000 hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex). The regression equation of the calibration curve was used to calculate plasma concentrations. Pharmacokinetic parameters were calculated using the PKSolver software2.

Synthetic procedures.
Unless otherwise noted, all commercially available compounds, reagents, solvents and anhydrous solvents were used as provided without further purification. 1 H and 13 C NMR spectra were recorded on a Bruker Avance DRX 300, DRX 400 or DRX 500 spectrometer or 400 MHz AVANCE-III HD and 600 MHz AVANCE-III HD (Bruker BioSpin GmbH) equipped with a 5 mm helium cooled broadband BBFO cryo probe from Bruker BioSpin GmbH 500 MHz DD2-500 (Agilent Technologies). Chemical shifts (δ) are reported in parts per million (ppm) and coupling constants (J) are expressed in Hertz (Hz). 1 H and 13 C spectra are referenced to the residual solvent signal DMSO-d 6 (2.50 or 39.52 ppm), CDCl 3 (7.26 or 77.16 ppm) or CD 3 OD (1.94 or 49.00 ppm, respectively). High-resolution electrospray ionization mass spectra (ESI-FTMS) were recorded on a Thermo LTQ Orbitrap (high-resolution mass spectrometer from Thermo Electron) coupled to an "Accela" HPLC system supplied with a "Hypersil GOLD" column (Thermo Electron).
Microwave reactions were performed using the Monowave 300 reactor by Anton Paar. Analytical TLC was carried out on Merck 60 F254 aluminum-backed silica gel plates and monitored by UV at λ = 254 and 366 nm. Compounds were purified by column chromatography using Baker silica gel (40-70 µm particle size) or Flash Chromatography on a Biotage Isolera One using Biotage SNAP, SNAP Ultra, ZIP Sphere or ZIP KP-Sil columns (25, 10, 5 or 120 g, respectively) or Grace Reverleris C18 40 µm columns (4 or 12 g) and monitored by UV at λ = 254 and 280 nm.
Preparative HPLC was conducted on an Agilent HPLC system (1200 series) with a VP 125/21 Nucleodur C18 column from Macherey-Nagel and monitored by UV at λ = 210, 254 and 280 nm.

N-[3-(4-Methoxy-6-phenyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl]acrylamide (1a)
To (1.5 mL) at 0 °C, after which TFA was added (0.5 mL) and the reaction stirred at rt until full conversion (1 h). The reaction mixture was added to aqueous solution of NaHCO 3 , NaCl added and extracted with CH 2 Cl 2 . The combined organic layers were dried over Na 2 SO 4 and the solvent removed in vacuo. Purification by preparative HPLC yielded the desired product as a white solid.

S37
NaCl added and extracted with EtOAc. The combined organic layers were dried over Na 2 SO 4 and the solvent removed in vacuo. Purification by column chromatography yielded the desired product as a white solid.

3-(4-methoxy-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-5-yl)aniline
A suspension of compound 14 (1.6 g, 4.0 mmol, 1.0 eq.), iron (1.1 g, 20.0 mmol, 5.0 eq.) and NH 4 Cl (2.1 g, 40.0 mmol, 10 eq.) in a mixture of EtOH:H 2 O (1:1) was sonicated at 60 °C for 3 h, S39 during which time the reaction mixture became a chocolate brown color. The reaction mixture was cooled to rt, filtered through Celite and washed with MeOH. After removal of the solvent in vacuo, the mixture was diluted with CH 2 Cl 2 and water and the organic layer was separated. The aqueous layer was extracted with aliquots of CH 2 Cl 2 and the combined organic layers were then washed with a saturated solution of NaCl, dried over MgSO 4 and filtered. After removal of the solvent in vacuo, the crude product was adsorbed onto silica and purified by flash chromatography, which afforded the pure compound as a light-brown semi-solid.
Purification by column chromatography yielded the desired product as white solid.
Purification by column chromatography yielded the desired product as a yellow oil.

4-(Piperidin-1-yl)but-2-enoic acid hydrochloride
Methyl 4-(piperidin-1-yl)but-2-enoate (1.3 g, 6.8 mmol, 1.0 eq.) was dissolved in an aqueous solution of HCl (1 M, 15 mL) and heated to reflux until full conversion. To this was added toluene, the layers separated and the organic layer concentrated to dryness in vacuo. The desired product was isolated without further purification as a white solid.
After 2 h, this mixture was added at 0 °C to a solution of compound 16 (50.0 mg, 0.1 mmol, 1 eq.) in THF (1.0 mL) and N-methylpyrrolidinone (0.5 mL). After completion of the reaction, aqueous solution of NaOH was added, the layers separated and the aqueous layer extracted with 10%
The combined organic layers were dried over Na 2 SO 4 and the solvent removed in vacuo.
The reaction mixture was then diluted with water and EtOAc and the organic layer was separated.
The aqueous layer was extracted with aliquots of EtOAc and the combined organic layers were then washed with a saturated solution of NaHCO 3 , a saturated solution of NaCl, dried over MgSO 4 S53 and filtered. After removal of the solvent in vacuo, the crude product was adsorbed onto silica and purified by flash chromatography. Pure fractions were evaporated to dryness to afford the desired compound as a yellow semi-solid.  48, 152.93, 151.54, 150.48, 141.04, 133.65, 127.00, 125.77, 123.73, 116.80, 115.70, 103.29, 76.52, 76.07, 73.34, 66.82, 59.60, 54.13, 17.87, 17.12
The reaction flask was covered in foil and allowed to stir in darkness at rt overnight (16 h). The reaction mixture developed an orange-brown color, which reverted to colorless upon addition of a sufficient amount of aqueous Na 2 S 2 O 3 (10 mol%). The reaction mixture was then diluted with CH 2 Cl 2 and water and the organic layer was separated. The aqueous layer was extracted with S55 aliquots of CH 2 Cl 2 and the combined organic layers were then washed with a saturated solution of NaCl, dried over MgSO 4 and filtered. After removal of the solvent in vacuo, the crude product was adsorbed onto silica and purified by flash chromatography. Pure fractions were evaporated to dryness to afford the desired compound as a yellow solid.  24, 152.79, 151.72, 150.85, 140.48, 135.73, 127.85, 125.17, 115.80, 114.32, 111.75, 104.32, 76.31, 76.02, 72.12, 66.94, 59.62, 54.13, 17.85, 17.

7H-pyrrolo[2,3-d]pyrimidin-4-ol (26)
A suspension of compound 25 (1.3 g, 2.3 mmol, 1.0 eq.), NH 4 Cl (1.1 g, 20.9 mmol, 9.0 eq.) and iron (388 mg, 7.0 mmol, 3.0 eq.) in a mixture of EtOH:H 2 O (4:1, 21 mL) was stirred at 80 °C for 3 h. The reaction mixture was cooled to rt, filtered through Celite and concentrated in vacuo. The residue was diluted with CH 2 Cl 2 and water and the organic layer was separated. The aqueous layer was extracted with aliquots of CH 2 Cl 2 , the combined organic layers dried over Na 2 SO 4 and the solvent removed in vacuo. The desired product was isolated without further purification as a white solid.

General method: Mitsunobu derivatization
To a solution of compound 27a or 27b (1.0 eq.) in THF (30 mM) was added PPh 3 (5.0 eq.) and the respective alcohol (5.0 eq.) and the reaction mixture sonicated for 5 min. Subsequently, DIAD (5.0 eq.) was added and the resulting mixture sonicated until full conversion at 40 °C. To this was added water and the mixture was extracted with 5% MeOH in CH 2 Cl 2 . The combined organic layers were dried over Na 2 SO 4 and the solvent removed in vacuo. Purification by flash chromatography using a C18 column yielded the intermediate as a light yellow solid. This was subsequently taken up in CH 2 Cl 2 (30 mM) and TFA (2 mL) was added. The reaction mixture was then stirred at rt until full conversion (3 h). After removal of the solvent in vacuo, the crude residue was re-dissolved in THF (30 mM) and an aqueous solution of NaOH (1 M, 2 mL) was added after which the reaction was allowed to stir for a further 1 h. To this was added CH 2 Cl 2 and the mixture was extracted with aliquots of 5% MeOH in CH 2 Cl 2 . The combined organic layers were dried over Na 2 SO 4 and the solvent removed in vacuo. Purification by column chromatography or preparative