Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAF V600E Conformation

Transcription

1 Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAF V600E Conformation Michael Grasso 1,2,*, Michelle A. Estrada 2,*, Christian Ventocilla 2, Minu Samanta 3, Jasna Maskimoska 2, Jessie Villanueva 3, Jeffrey D. Winkler 2 and Ronen Marmorstein 1,2 1

2 Figure S1. BRAF V600E potencies of PLX4720 (PLX) and PLX4032 (Vem). BRAF V600E was assayed against Vem and PLX using an ELISA assay. IC 50 values were 119 nm for PLX and 42.8 nm Vem. The difference in these inhibitors may be due to the difference in suppliers (SelleckChem for PLX and Santa Cruz Biotechnology for Vem) as their reported IC 50 values in the literature are comparable. Assay are performed in duplicate with +/- SEM shown Figure S2: Normalized fluorescence data from thermal stability assay. 2

3 Figure S3. Potencies of Vem-BisAmide inhibitors against BRAF WT. BRAF WT was assayed against Vem-BisAmide-1, Vem-BisAmide-2, Vem-BisAmide-3, and Vem control using an ELISA assay. IC 50 values were 2.68 µm for Vem- BisAmide-2, 1.35 µm for Vem-BisAmide-3, 697 nm for Vem, and N/A for Vem- BisAmide-1. Assay were performed in duplicate with +/- SEM shown Figure S4. Simulated annealing omit map of Vem-BisAmide-2 and Q461 from the BRAF V600E / Vem-BisAmide-2 structure. The omit map is shown in 3

4 green and contoured at 2.0 sigma. Density for one Q461 residue within the offstate dimer is observable, however the other is not. Figure S5. Vem-BisAmide-4 assayed against BRAF WT and BRAF V600E. The IC 50 values are: BRAF V600E /Vem-BisAmide-2 (37.8 nm), BRAF WT /Vem-BisAmide- 2 (2.69 µm), BRAF V600E /PLX (116 nm) BRAF WT /PLX (3.91 µm), BRAF V600E /Vem- BisAmide-4 (17.5 nm), and BRAF WT /Vem-BisAmide-4 (529 nm). The assay performed in duplicate with +/- SEM shown 4

5 Figure S6. Thermal stability assay of BRAF proteins in the absence or presence of inhibitors. n=6 with +/- SEM shown. 5

6 Figure S7. AUC sedimentation velocity of Vem-BisAmide-4 and Vem- BisAmide-2 complexed with BRAF V600E/R509H. RMS deviation (Au 200nm ) Residuals-Sedimentation Equilibrium Radius (mm) Figure S8. Analytical ultracentrifugation sedimentation equilibrium residuals for Figure 6D. A graph of residuals for all three speeds (12000 r.p.m, r.p.m, r.p.m) for BRAF V600E/R509H (10 µm) treated with both Vem- BisAmide-2 and PLX is shown. 6

7 General. Solvents used for extraction and purification were HPLC grade from Fisher. Unless otherwise indicated, all reactions were run under an inert atmosphere of argon. Anhydrous tetrahydrofuran, ethyl ether, and toluene were obtained via passage through an activated alumina column. Merck pre-coated silica gel plates (250 mm, 60 F254) were used for analytical TLC. TLC plates were visualized using 254 nm ultraviolet light, with either anisaldehyde or potassium permanganate stains as visualizing agents. Chromatographic purifications were performed on Sorbent Technologies silica gel (particle size microns). 1 H and 13 C NMR spectra were recorded at 500 MHz and 125 MHz, or 360 MHz and 90 MHz, respectively, in CDCl 3, DMSO-d 6, or CD 3 OD on a Bruker AM-500, a DRX-500, or a DMX-360 spectrometer. Chemical shifts are reported relative to internal chloroform (δ 7.26 for 1 H, δ 77.0 for 13 C), DMSO-d 6 (δ 2.50 for 1 H, δ 39.5 for 13 C), or CD 3 OD (δ 3.31 for 1 H, δ 49.0 for 13 C). Infrared spectra were recorded on a NaCl plate using a Perkin-Elmer 1600 series Fourier transform spectrometer. High resolution mass spectra were obtained by Dr. Rakesh Kohli at the University of Pennsylvania Mass Spectrometry Service Center on an Autospec high resolution double-focusing electrospray ionization/chemical ionization spectrometer with either DEC 11/73 or OPUS software data system. 7

8 PLX4032 Dimers: Representative procedure for the synthesis of Vem-X-Vem dimers: To a solution of substrate PLX-Br 1 (0.15 mmol) and tether glycolic boronic ester 2 (0.15 mmol) in THF (3 ml) were added Pd(PPh 3 ) 4 (0.03 mmol) and K 2 CO 3 (1.5 mmol) followed by water (1.5 ml). The mixture was flushed with argon, capped, and heated to 70 C for 18 h. The mixture was cooled to 24 C, then diluted with EtOAc (10 ml) and water (10 ml). The layers were separated, and the aqueous layer was extracted with EtOAc (3 x 10 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, filtered, and concentrated in vacuo. To the resultant crude residue was added NH 3 (0.75 ml, 7N solution in MeOH, 5 mmol). The mixture was heated to 55 C for 18 h. The mixture was cooled to 24 C, concentrated in vacuo, and the resultant residue was purified by flash chromatography (3:97 MeOH:CH 2 Cl 2 ) to afford Vem-X-Vem dimers. 8

9 Vem-1-Vem 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 2H), 9.77 (s, 2H), 8.66 (s, 2H), 8.57 (s, 2H), 8.19 (s, 2H), 7.69 (d, J = 7.7 Hz, 4H), (m, 2H), 7.27 (t, J = 8.7 Hz, 2H), 7.15 (d, J = 7.5 Hz, 4H), 4.42 (s, 4H), 3.11 (t, J = 7.5 Hz, 4H), 1.72 (dq, J = 14.6, 7.3 Hz, 4H), 0.94 (t, J = 7.3 Hz, 6H) ppm; 13 C NMR (125 MHz, DMSO-d 6 ) δ 181.0, 158.5, 155.5, 155.4, 153.7, 151.8, 151.7, 148.9, 144.1, 139.0, 131.9, 131.8, 131.6, 131.1, 129.2, 129.1, 128.7, 128.4, 126.9, 122.3, 118.8, 118.6, 117.9, 116.0, 115.7, 112.8, 112.6, 66.9, 53.9, 17.2, 13.0 ppm; HRMS (ESI) m / z calcd for C 48 H 41 N 6 O 8 F 4 S 2 (M + H) ; Found Vem-2-Vem 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 2H), 9.76 (s, 2H), 8.65 (d, J = 2.2 Hz, 2H), 8.56 (s, 2H), 8.19 (s, 2H), 7.67 (d, J = 8.6 Hz, 4H), 7.57 (td, J = 9.0, 6.0 Hz, 2H), 7.26 (t, J = 8.4 Hz, 2H), 7.10 (d, J = 8.7 Hz, 4H), 4.20 (t, J = 4.5 Hz, 4H), 3.87 (t, J = 4.5 Hz, 4H), 3.11 (t, J = 7.7 Hz, 4H), 1.72 (sextet, J = 7.6 Hz, 4H), 0.94 (t, J = 7.4 Hz, 6H) ppm; 13 C NMR (125 MHz, DMSO-d 6 ) δ 181.2, 158.9, 149.1, 144.4, 139.2, 138.8, 131.9, 131.2, 129.5, 129.3, 128.9, 127.1, , 122.5, 120.0, 118.8, 118.2, 118.1, 116.2, 115.8, 113.1, 112.9, 69.7, 67.9, 54.1, 17.4, 13.2 ppm; HRMS (ESI) m / z calcd for C 50 H 45 N 6 O 9 F 4 S 2 (M + H) ; Found Vem-3-Vem 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 2H), 9.76 (s, 2H), 8.64 (d, J = 2.2 Hz, 2H), 8.56 (s, 2H), 8.19 (s, 2H), 7.65 (d, J = 8.6 Hz, 4H), 7.57 (td, J = 9.0, 5.9 Hz, 2H), 7.26 (t, J = 8.3 Hz, 2H), 7.08 (d, J = 8.7 Hz, 4H), 4.16 (t, J = 4.6 Hz, 4H), 3.79 (t, J = 4.6 Hz, 4H), 3.65 (s, 3H), (m, 4H), 1.72 (dq, J = 15.1, 7.5 Hz, 4H), 0.94 (t, J = 7.4 Hz, 6H) ppm; 13 C NMR (125 MHz, DMSO-d 6 ) δ 181.2, 158.9, 149.2, 144.4, 136.3, 136.1, 131.9, 131.1, 130.0, 129.6, 129.4, 129.0, 9

10 128.9, 127.1, 118.1, 116.3, 116.2, , 115.8, 113.1, 112.8, 70.6, 69.6, 67.9, 54.1, 17.4, 13.2 ppm; HRMS (ESI) m / z calcd for C 52 H 49 N 6 O 10 F 4 S 2 (M + H) ; Found Vem-4-Vem 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 2H), 9.75 (s, 2H), 8.63 (d, J = 2.2 Hz, 2H), 8.55 (s, 2H), 8.18 (s, 2H), 7.65 (d, J = 8.7 Hz, 4H), (m, 2H), 7.26 (t, J = 8.9 Hz, 2H), 7.07 (d, J = 8.8 Hz, 4H), 4.14 (dd, J = 5.3, 3.8 Hz, 4H), (m, 4H), (m, 8H), (m, 4H), (m, 4H), 0.94 (dd, J = 8.7, 6.1 Hz, 6H) ppm; 13 C NMR (125 MHz, DMSO-d 6 ) δ 181.4, 158.8, 149.2, 144.3, 137.6, 134.0, 131.9, , 130.4, 128.9, 128.7, 118.8, 118.2, 116.2, 115.8, 112.7, 70.6, 70.5, 69.6, 67.9, 54.0, 17.5, 13.3 ppm; HRMS (ESI) m / z calcd for C 54 H 53 N 6 O 11 F 4 S 2 (M + H) ; Found Vem-5-Vem 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 2H), 9.86 (s, 2H), 8.64 (d, J = 1.9 Hz, 2H), 8.56 (s, 2H), 8.19 (s, 2H), 7.64 (d, J = 8.5 Hz, 4H), 7.57 (td, J = 8.8, 6.0 Hz, 2H), 7.26 (t, J = 8.6 Hz, 2H), 7.06 (d, J = 8.6 Hz, 4H), 4.13 (t, J = 4.4 Hz, 4H), 3.75 (t, J = 4.4 Hz, 4H), (m, 12H), 3.10 (t, J = 7.7 Hz, 4H), (m, 4H), 0.94 (t, J = 7.4 Hz, 6H) ppm; 13 C NMR (125 MHz, DMSO-d 6 ) δ 181.2, 159.6, 158.9, 155.7, 153.9, 149.2, 144.3, 139.3, 132.9, 131.9, 131.1, 129.4, 129.3, 128.9, 127.1, 122.7, 122.6, 119.0, 118.1, 116.4, 116.3, 116.2, 116.0, 115.8, 113.0, 112.8, 70.6, 70.4, 69.6, 67.9, 54.1, 17.5, 13.2 ppm ; HRMS (ESI) m / z calcd for C 56 H 57 N 6 O 12 F 4 S 2 (M + H) ; Found Vem-6-Vem 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 2H), 9.76 (s, 2H), 8.64 (d, J = 2.2 Hz, 2H), 8.56 (s, 2H), 8.19 (s, 2H), 7.65 (d, J = 8.7 Hz, 4H), 7.57 (td, J = 9.0, 5.9 Hz, 2H), 7.26 (t, J = 8.7 Hz, 2H), 7.06 (d, J = 8.8 Hz, 4H), 4.12 (t, J = 4.6 Hz, 4H), 10

11 3.74 (t, J = 4.5 Hz, 4H), (m, 16H), (m, 4H), 1.72 (sextet, J = 7.5 Hz, 4H), 0.94 (t, J = 7.4 Hz, 6H) ppm; 13 C NMR (125 MHz, DMSO-d 6 ) δ 181.2, 158.9, 149.2, 144.4, 139.2, 131.9, 131.1, 128.9, 127.1, 122.5, 118.1, 116.2, 115.8, 113.0, 112.9, 70.6, 70.44, 70.42, 70.40, 69.5, 67.9, 54.1, 17.5, 13.2 ppm; HRMS (ESI) m / z calcd for C 58 H 61 N 6 O 13 F 4 S 2 (M + H) ; Found General method for the sythesis of aryl amides: Diglycolic acid (1 eq.) was suspended in toluene (0.1 M) at 24 C. Thionyl chloride (1.1 equivs.) and then DMF (cat.) were added sequentially to the resuling suspension, which was heated to reflux for 30 mins. The resulting diacid chloride was concentrated under reduced pressure. The residue was then redissolved with toluene and concentrated three times to afford the crude diacid chloride which was used without further purification. The crude diacid chloride was then dissolved in toluene and stirred at 0 C. The desired bromoamine (2 eq.), and triethylamine (2.2 eq) were added sequentially. The resulting mixture was stirred for 3 hours at 24 C. The resulting solid was filtered and washed with 1N HCl and then 1N NaOH. The solid was then dried in a dessicator and used without further purification. 2,2'-oxybis(N-(4-bromophenyl)acetamide) White solid; Yield =1. 19 g (72% yield); 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.52 (d, J = 6.9 Hz, 2H), 4.26 (s, 2H) ppm; 13 C NMR (126 MHz, DMSO) δ , , , , , , , ppm; IR (thin film): ν = 3219, 1679, 1660, 1610 cm -1 ; HRMS (ESI) m/z: [M + Na] + Calcd for C 16 H 14 Br 2 N 2 O ; Found ,2'-oxybis(N-(4-bromobenzyl)acetamide) White solid; Yield = 1.1 g (68% yield); 1 H NMR (500 MHz, Acetone-d 6 ) δ 8.02 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 4.39 (d, J = 6.4 Hz, 2H), 4.11 (s, 2H) ppm; 13 C NMR (126 MHz, DMSO) δ , , , , , 70.85, ppm; IR (thin film): ν = 3255, 1650, 1537, 1486 cm -1 ; HRMS (ESI) m/z: [M + Na] + Calcd for C 18 H 18 Br 2 N 2 O ; Found ,2'-oxybis(N-(4-bromophenethyl)acetamide) 11

12 White solid; Yield = 1.34 g (72% yield); 1 H NMR (500 MHz, Acetone-d 6 ) δ 7.51 (s, 1H), 7.45 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 3.95 (s, 2H), 3.45 (q, J = 6.7 Hz, 2H) ppm; 13 C NMR (126 MHz, Acetone) δ , , , , , 70.72, 39.66, ppm; IR (thin film): ν = 3294, 1647, 1543, 1487 cm -1 ; HRMS (ESI) m/z: [M + Na] + Calcd for C 20 H 22 Br 2 N 2 O ; Found

13 General method for the synthesis of boronic esters: Bromide (1 eq.), KOAc (4.0 eq.), (PPh 3 ) 2 PdCl 2 (0.05 eq) and (Bpin) 2 (2.1 eq.) were added to a sealed tube fitted with a stir bar. The flask was evacuated and filled with argon 5 times and then degassed 1,4-dioxane (0.09 M) was added to the flask. The reaction was then warmed to 100 C for 18 hours, after which the reaction was cooled to 24 C and then diluted with EtOAc. The suspension was then filtered through a pad of celite. The filtrate was concentrated under reduced pressure and the solid was purified by silica gel column chromatography. (EtOAc/Hexanes) 2,2'-oxybis(N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)phenyl)acetamide) White foam; Yield = 305 mg (83% yield); 1 H NMR (500 MHz, Chloroform-d) δ 8.43 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 4.21 (s, 2H), 1.34 (s, 12H) ppm. 13 C NMR (126 MHz, CDCl 3 ) δ , , , , 83.96, 71.85, ppm. IR (thin film): ν = 3276, 2979, 1682, 1362 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 28 H 38 B 2 N 2 O ; Found

14 2,2'-oxybis(N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzyl)acetamide) White foam; Yield = 229 mg (63% yield); 1 H NMR (500 MHz, Chloroform-d) δ 7.77 (d, J = 9.2 Hz, 0H), 7.25 (d, J = 7.2 Hz, 2H), 4.45 (d, J = 6.1 Hz, 2H), 4.02 (d, J = 2.0 Hz, 2H) ppm. 13 C NMR (126 MHz, CDCl 3 ) δ , , , , , 84.02, 71.28, 43.14, ppm. IR (thin film): ν = 3314, 2978, 1656, 1361 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 30 H 42 B 2 N 2 O ; Found ,2'-oxybis(N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)phenethyl)acetamide) White foam; Yield = 233 mg (65% yield); 1 H NMR (500 MHz, Chloroform-d) δ 7.75 (d, J = 7.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 3.93 (s, 2H), 3.56 (q, J = 6.9 Hz, 2H), 2.84 (t, J = 6.9 Hz, 2H), 1.32 (s, 12H) ppm. 13 C NMR (126 MHz, CDCl 3 ) δ , , , , 83.94, 71.08, 39.97, 35.90, ppm. IR (thin film): ν = 3314, 2978, 1659, 1370 cm -1 ; HRMS (ESI) m/z: [M + Na] + Calcd for C 32 H 46 B 2 N 2 O ; Found General method for the synthesis of PLX dimers: Boronic ester (1 eq), PLX-Br 1 (2.35 eq), KOAc (4.07 eq) and (PPh 3 ) 2 PdCl 2 (0.05 eq) were added to a sealed tube fitted with a magnetic stir bar. The flask was evacuated and purged with argon five times and then was added degassed 1,4- dioxane (0.16 M). The reaction was warmed to 90 C for 18 h. The reaction was then cooled to 24 C, diluted with EtOAc and filtered through a pad of celite. The filtrate was the concentrated and used in the next step without further purification. The resulting residue was diluted with DMA (0.096 M) and 7N NH 3 in MeOH (10 eq.) was added. The resulting solution was then warmed to 50 C for 18 h under an argon atmosphere. The reaction was cooled to 24 C and was concentrated under reduced pressure. The resulting residue was redissolved in EtOAc, and the organic solution was washed with water and brine. The organic layer was then dried over Na 2 SO 4, filtered and concentrated. The crude solid was then purified by preparative thin layer chromatography (MeOH/DCM) and then triturated with Et 2 O to afford pure products. Vem-BisAmide-3 14

15 Pale Yellow Foam; Yield = 59 mg (38% yield); 1 H NMR (500 MHz, Chloroform-d) δ 8.61 (s, 1H), 8.37 (s, 1H), (m, 3H), (m, 3H), 6.82 (t, J = 8.5 Hz, 1H), 4.10 (s, 2H), (m, 2H), 1.65 (dt, J = 17.9, 7.4 Hz, 2H), (m, 3H) ppm; 13 C NMR (126 MHz, MeOD) δ , , , , , , , , , , , , , , , , , , , , , , , 71.77, 54.80, 17.55, ppm; IR (thin film): ν = 2925, 1625, 1521, 1483 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 50 H 42 F 4 N 8 O 9 S ; Found

16 Vem-BisAmide-2 White Foam; Yield = 74 mg (29% yield); 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.67 (s, 2H), 8.60 (s, 1H), 8.21 (s, 1H), 7.70 (d, J = 7.2 Hz, 2H), 7.57 (td, J = 9.4, 5.3 Hz, 1H), 7.42 (d, J = 7.5 Hz, 2H), 7.26 (t, J = 8.8 Hz, 1H), 4.43 (d, J = 6.3 Hz, 2H), 4.09 (s, 2H), (m, 2H), (m, 2H), (m, 3H). 13 C NMR (126 MHz, DMSO) δ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 70.34, 53.41, 41.36, 16.87, ppm; IR (thin film): ν = 3095, 1634, 1520, 1484 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 52 H 46 F 4 N 8 O 9 S ; Found Vem-BisAmide-1 White foam; Yield = 20.3 mg (34% yield); 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.67 (d, J = 2.4 Hz, 1H), 8.59 (s, 1H), (m, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.56 (td, J = 9.0, 5.9 Hz, 1H), 7.34 (d, J = 8.2 Hz, 2H), 7.24 (t, J = 8.7 Hz, 1H), 3.93 (s, 2H), 3.40 (q, J = 7.8, 6.9 Hz, 2H), (m, 2H), 2.81 (t, J = 7.6 Hz, 2H), (m, 2H), 0.93 (t, J = 7.4 Hz, 3H) ppm; 13 C NMR (126 MHz, DMSO) δ , , , , , , , , , , , , , , , , , , , , , , , , 70.66, 53.83, 35.17, 17.22, ppm; IR (thin film): ν = 2923, 1634, 1520, 1484 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 54 H 50 F 4 N 8 O 9 S ; Found Method for the synthesis of Vem-BisAmide-4: 2-(2-(benzylamino)-2-oxoethoxy)acetic acid Diglycolic anhydride (2 g, 17.2 mmols) was dissolved in THF (21 ml) then benzylamine (1.88 ml, 17.2 mmols) was added dropwise. The reaction was then warmed to 50 C for 18 hours and the reaction was allowed to cool to 24 C. The solvent was then evaporated and the resulting white solid was washed with DCM and Et 2 O. The product was used without further purification. 16

17 White solid; Yield = 2.28 g (59% yield); 1 H NMR (500 MHz, Chloroform-d) δ (m, 2H), (m, 2H), 7.27 (d, J = 2.3 Hz, 1H), 4.50 (dd, J = 6.0, 2.3 Hz, 2H), 4.18 (dd, J = 5.9, 2.2 Hz, 4H); 13 C NMR (126 MHz, Acetone) δ , , , , , , 70.79, 68.46, 42.06, 41.94; IR (thin film): 3374, 1717, 1623, 1581 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 11 H 13 NO ; N-benzyl-2-(2-((4-bromobenzyl)amino)-2-oxoethoxy)acetamide To a suspension of 2 g of the crude product (8.9 mmol) in toluene (89 ml) was added oxalyl chloride (0.99 ml, 11.6 mmol) followed by three drops of DMF. The reaction was monitored by TLC (5%MeOH/dichloromethane). After consumption of starting material the solvent was removed under reduced pressure. To the resulting yellow solid was added 20 ml of toluene and the resulting solution was concentrated under reduced pressure. This was repeated once. The resulting yellow oil was dissolved in toluene (89 ml) and was cooled to 0 C. Triethyl amine (1.62 ml, 11.6 mmol) was added to the reaction, followed by 4- bromobenzyl amine (1.24 ml, 9.8 mmol). The reaction was allowed to warm to 24 C and stirred for three hours. The resulting solid was filtered and washed with 1M HCl, 1M NaOH, and then cooled Et 2 O. The white solid was used without further purification. White solid; Yield = 2.39 g (68% yield); 1 H NMR (500 MHz, Chloroform-d) δ 7.45 (dd, J = 8.1, 1.5 Hz, 2H), (m, 4H), 7.28 (d, J = 1.7 Hz, 1H), (m, 2H), 6.71 (s, 1H), 6.57 (s, 1H), 4.49 (d, J = 5.8 Hz, 2H), 4.43 (d, J = 6.1 Hz, 2H), 4.10 (d, J = 1.7 Hz, 4H); 13 C NMR (126 MHz, CDCl 3 ) δ , , , , , , , , , , , , 70.65, 41.91, 41.36; IR (thin film): 3216, 1647, 1531, 1485 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 18 H 19 BrN 2 O ; Found N-benzyl-2-(2-oxo-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2 yl)benzyl)amino) ethoxy)acetamide The above mentioned diamide bromide (250 mg, 0.64 mmol) was added to a sealed tube along with (Bpin) 2 (170 mg, 0.65 mmol), KOAc (129 mg, 1.3 mmol), and Pd(PPh 3 ) 4 (66 mg, mmol). The flask was sealed and back filled with 17

18 argon five times. 1,4-Dioxane (7 ml) was added and the reaction was stirred for 18 hours at 90 C. The reaction was cooled to 24 C and then concentrated under reduced pressure. The crude mixture was purified by silica gel column chromatography (3:1 Hexanes:EtOAc to 19:1 DCM:MeOH). White foam; Yield = 150 mg (53% yield); 1 H NMR (500 MHz, Chloroform-d) δ (m, 2H), (m, 7H), 6.64 (s, 2H), 4.49 (td, J = 5.7, 2.4 Hz, 4H), 4.10 (t, J = 2.4 Hz, 4H); 13 C NMR (126 MHz, CDCl 3 ) δ , , , , , , , , 84.03, 71.08, 43.05, 43.01, 25.01; IR (thin film): 3345, 1651, 1612, 1541 cm -1 ; HRMS (ESI) m/z: [M + H] + Calcd for C 24 H 31 BN 2 O ; Found Vem-BisAmide-4 The abovementioned boronic ester (80 mg, mmol) was added to a sealed tube along with PLX-Br 1 (114.9 mg, mmol), Na 2 CO 3 (77mg, mmol) and (PPh 3 ) 2 PdCl 2 (1.2 mg, mmol). The flask was evacuated and purged with argon five times and then degassed toluene (0.82 ml), water (0.82 ml), and 1,4-dioxane (0.82 ml) were added. The reaction was warmed to 90 C for 18 hours. The reaction was then cooled, diluted with EtOAc and filtered through a pad of celite. The filtrate was then concentrated and used in the next step without further purification. The resulting residue was diluted with DMA (1.17 ml) and 7N NH 3 in MeOH (0.167 ml) was added. The resulting yellow solution was then warmed to 50 C for 18 hours under argon. The reaction was cooled to 24 C and was concentrated under reduced pressure. The crude residue was then purified by flash chromatography (97:3 DCM:MeOH to 95:5 DCM:MeOH) to afford pure product. White solid; Yield = 71.6 mg (57% yield); 1 H NMR (500 MHz, Chloroform-d) δ (d, J = 3.1 Hz, 1H), 9.74 (s, 1H), (m, 4H), 8.22 (d, J = 3.1 Hz, 1H), (m, 3H), 7.58 (td, J = 9.0, 5.9 Hz, 1H), (m, 2H), (m, 6H), 4.42 (d, J = 6.0 Hz, 2H), 4.36 (d, J = 6.4 Hz, 2H), 4.07 (d, J = 2.6 Hz, 4H), (m, 2H), (m, 2H), 0.96 (t, J = 7.5 Hz, 4H). 13 C NMR (126 MHz, DMSO) δ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 70.30, 53.40, 41.53, 41.29, 16.85, 12.63, 0.14; IR (thin film): 18

19 1650, 1593, 1517, 1489 cm -1 ; HRMS (ESI) m/z: [M - H] - Calcd for C 35 H 33 F 2 N 5 O 6 S ; Found References 1. Hildbrand, S.; Mair, H.; Nikolaev, R.; Ren, Y.; Wright, A. J. Process for the Manufacture of Pharmaceutically Active Compounds. U.S. Patent WO , Feb 3, Bono, F. ; Guillo, N.; Maffrand, J. P.; Fons, P.; Olsen, J. A.; Gilles, A. Preparation of FGF receptor agonist dimeric compounds, particularly bisindolizines, bisimidazo[1,5-a]pyridines and their derivatives. FR Patent WO