Supplemental Information. Covalent Protein Labeling at Glutamic Acids

Cell Chemical Biology, Volume 24 Supplemental Information Covalent Protein Labeling at Glutamic Acids Pablo Martín-Gago, Eyad K. Fansa, Michael Winzker, Sandip Murarka, Petra Janning, Carsten Schultz-Fademrecht, Matthias Baumann, Alfred Wittinghofer, and Herbert Waldmann

Supplementary Information Supplementary Figures Figure S1. Related to Figure 1. a) Chemical structures of representative compounds with sulfonate esters or isoxazolium salt warheads tested for covalent binding to PDE. b) Chemical structures and binding affinities (K D, determined by competitive fluorescence polarization assay of three independent experiments, see below for details) of the corresponding precursor and hydrolysis product of 16.

Figure S2. Related to Figure 2. Positive ion mode MALDI TOF mass spectra of WT-PDEδ before (a), after 30 min (b, d, f, h) and after 24 h (c, e, g, i) incubation with compounds 12, 13, 14 and 15 (2.4 µm, 1.2 eq), respectively. j) Positive ion mode MALDI TOF mass spectra of WT-PDEδ at different time points after incubation with compound 16 (2.4 µm, 1.2 eq). The reaction was stopped by diluting 1 µm aliquots of the reaction mixture into 75 µm of saturated solution of sinapic acid in H 2O (0.1% TFA) / ACN (2:1).

Figure S3. Mechanism of the enol ester formation in the linear (a, 12-13) and branched (b, 14-16) WRK-derived aliphatic warheads. See also Figure 1. c) Related to Figure 5. Chemical structures of compounds used in the fluorescence quenching based assay. d) Related to Figure 6C. Chemical structures and binding affinities (K D, determined by competitive fluorescence polarization assay of three independent experiments, see below for details) of the corresponding precursors of 12-16 and hydrolysis product of 16. e) Related to Figure 3. Fluorescence polarization monitoring of titration of fluorescein-labeled 25 (25-L*) (10 nm) with increasing concentrations of PDEδ WT (left) or with the mutant PDEδ E88A (right) indicating that 25-L* binds PDEδ E88A with similar affinity than wild type.

Figure S4. Upper row: Related to Figure 2. Best docking pose (Schrödinger, Maestro suite) of the reactive ketenimine derived from derivatives containing a 5-methyl substituted isoxazolium salt linked by two (a) or three (b) methylene units. Lower row: Related to Figure 6A. Crystal structure of inhibitor 15 in complex with PDEδ. Inhibitor 15 binds to PDEδ displaying the expected binding mode for this chemotype. Hydrogen bonds (yellow dotted line) and aromatic interactions (blue) between 15 and PDEδ are shown. Key residues are displayed as sticks.

Figure S5. Related to Figure 3. Upper row: Compound 16 does not react with His tagged PDEδ E88A. Positive ion mode MALDI TOF mass spectra of PDEδ E88A before (a) and after (b) incubation with compound 16 (3 µm, 1.5 eq). After 30 min incubation, addition of PDEδ WT (1 eq) resulted in near complete covalent adduct formation (c). 2 nd, 3 rd and 4 th rows: Positive ion mode MALDI TOF mass spectra of HRas G12D, GST and BSA before (d, f and h, respectively) and after (e, g and i, respectively) 5 h incubation with compound 16 (8 µm, 10 eq).

Figure S6. Related to Figure 4. Positive ion mode MALDI TOF mass spectra of WT-PDEδ and mass spectra of WT-PDEδ/Arl2 (1:1 mixture) before (a, b, respectively) and after incubation with 2.4 µm (1.2 eq) compounds 14, 15 and 16 (c, d, e, respectively). His tagged Arl2 was used in these experiments. f) Flow chart of the chemical stability assay procedure. g) Chemical stability of compound 15 at ph=1, 7.5 and 9 at 37 C. Values represent percentage of remaining material.

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Supplementary Tables Table S1, related to STAR Methods. Data collection and refinement statistics for the structure of compound 13 in complex with PDEδ 13 Data collection Space group P 32 2 1 Celldimensions a, b, c (Å) 56.07, 56.07, 115.08 ( ) 90.0, 90.0, 120.0 Resolution (Å) 28.03 (2.20) R sym orr merge 17.5 (75.9) I/σ(I) 8.5 (2.8) Completeness (%) 98.6 (97.3) Redundancy 5.0 (5.1) Refinement Resolution (Å) 2.20 No. reflections 10469 R work / R free 19.7 / 24.3 No. atoms Protein 1212 Ligand 44 Water 87 Bfactors Protein 27.3 Ligand 38.2 Water 31.3 R.m.s. deviations Bond lengths (Å) 0.011 Bond angles ( ) 1.60

Methods S1, related to STAR METHODS Synthesis of 13 and 15 General procedures for reductive amination Reductive amination procedure I: To a solution of the aldehyde (1.0 eq) and MgSO 4 (4 eq, anh) in MeOH (1 ml/mmol) was added the amine (1.0 mmol). After stirring at room temperature for 12 h, the solution was cooled down to 0 C and sodium borohydride (0.5 eq) was added portionwise. The resulting solution was stirred at room temperature for 1 h. After addition of water, methanol was removed under reduced pressure and the resulting aqueous phase was extracted with DCM. The combined extracts were washed with brine, dried over MgSO 4, filtered and concentrated in vacuo to give corresponding compounds. Unless otherwise specified, the secondary amines were used in the next reaction without further purification.

Reductive amination procedure II: A mixture of the aldehyde (1.0 eq), the amine (1.0 eq), sodium triacetoxyborohydride (2.0 eq), and acetic acid (2.0 eq) in 1,2-dichloroethane was stirred at room temperature overnight, diluted with CH 2 Cl 2, washed with water, dried over MgSO 4, filtered and concentrated in vacuo to give corresponding compounds. Unless otherwise specified, the secondary amines were used in the next reaction without further purification. Isoxazole numbering 5-oxohexanenitrile, 33 Synthesized as previously described.(thuong et al., 2012) 1 H NMR (400 MHz, cdcl 3 ) δ 2.58 (t, J = 7 Hz, 2H, CH 2 ), 2.36 (dd, J = 9, 5 Hz, 2H, CH 2 ), 2.11 (s, 3H, CH 3 ), 1.85 (p, J = 7 Hz, 2H, CH 2 ). 7-(dimethylamino)-5-oxohept-6-enenitrile and 4-((dimethylamino)methylene)-5- oxohexanenitrile, 27 5-oxohexanenitrile (3 g, 27 mmol) was treated with dimethylformamide dimethyl acetal (2.1 eq.) and glacial acetic acid (0.3 eq.) and heated at 120 C for 18 hrs. The mixture was evaporated in vacuo to give a -40:60

mixture of 7-(dimethylamino)-5-oxohept-6-enenitrile and 4-((dimethylamino)methylene)- 5-oxohexanenitrile respectively as a brown oil. This mixture was used in the next step without any purification. LC-MS (ESI): calc. for [M+H] + C 9 H 15 N 2 O: 167.12, found: 167.22. 4-(isoxazol-5-yl)butanenitrile and 3-(5-methylisoxazol-4-yl)propanenitrile, 28 A solution of 7-(dimethylamino)-5-oxohept-6-enenitrile, 4- ((dimethylamino)methylene)-5-oxohexanenitrile (4 g, 24 mmol) and hydroxylamine hydrochloride (1 eq.) in ethanol (1.5 ml/mmol) was heated at reflux for 10 h. After cooling to room temperature the mixture was diluted with water and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate and concentrated (98% yield). 1 H NMR (400 MHz, cdcl 3 ) δ 8.17 (d, J = 2 Hz, 1H, CH 3, linear isoxazole), 8.15 (s, 1H, CH 3, branched isoxazole), 6.07 (dt, J = 2, 1 Hz, 1H, CH 4, linear isoxazole), 2.97 (t, J = 7 Hz, 2H, CH 2 ), 2.74 (t, J = 7 Hz, 2H, CH 2 ), 2.54 (td, J = 7, 1 Hz, 2H, CH 2 ), 2.44 2.38 (m, 5H, CH 2 and CH 3 ), 2.09 (p, J = 7 Hz, 2H). 13 C NMR (101 MHz, cdcl 3 ) δ 170.1 (C 5, linear isoxazole), 166.0 (C 5, branched isoxazole), 150.6 (2 CH, C 3 ), 118.9 (2 CN), 111.1 (C 4, branched isoxazole), 101.3 (CH, C 4 linear isoxazole), 25.5 (CH 2 ), 23.6 (CH 2 ), 19.3 (CH 2 ), 18.8 (CH 2 ), 16.8 (CH 2 ), 10.9 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 7 H 9 N 2 O: 137.0637, found: 137.0637.

N-(cyclohexylmethyl)-4-(isoxazol-5-yl)butan-1-amine, 31 or N-(cyclohexylmethyl)-3- (5-methylisoxazol-4-yl)propan-1-amine, 32 To a solution of the nitriles (1 g, 7.3 mmol) in dry DCM (2mL/mmol) was added a solution of DIBAL in toluene (2 eq.) at -78 C under argon. The mixture was then warmed to -40 C. After the solution was stirred for 2 hours, MeOH (0.5 ml) was added to the resulting reaction, then saturated solution of potassium sodium tartrate and HCl (2 M) and the mixture was stirred for 30 minutes. The mixture was diluted with water and extracted with DCM. The combined organic phases were washed with brine and dried over Na 2 SO 4. DCM was removed under reduced pressure and the mixture was subjected to the reductive amination procedure II by using cyclohexylmethanamine (1.05 ml, 8.0 mmol, 1.1 eq), acetic acid (0.88 ml, 2.0 eq) and then sodium triacetoxyborohydride (3.1 g, 14.6 mmol, 2.0 eq). The crude compounds were purified on preparative HPLC using a reversed-phase C18 column to afford the pure compounds: 31 (394 mg, 23% yield, brownish oil) 1 H NMR (400 MHz, cdcl 3 ) δ 8.08 (s, 1H, CH 3 ), 5.95 (s, 1H, CH 4 ), 2.89 (s, 2H, CH 2 ), 2.80 2.64 (m, 4H, 2 CH 2 ), 1.69 (m, 10H), 1.24 1.01 (m, 3H, including CH, Cy), 0.88 (m, 2H, CH 2 ). 13 C NMR (101 MHz, cdcl 3 ) δ 171.5 (C 5 ), 150.5 (C 3 H), 100.7 (C 4 H), 54.1 (CH 2 ), 48.0 (CH 2 ), 35.0 (CH), 30.7 (2 CH 2 ), 26.1 (CH 2 ), 26.0 (CH 2 ), 25.5 (2 CH 2 ), 25.1 (CH 2 ), 24.8 (CH 2 ). HRMS (ESI): calc. for [M+H] + C 14 H 25 N 2 O: 237.1961, found: 237.1954. 32 (245 mg, 14% yield, brownish oil) 1 H NMR (400 MHz, cdcl 3 ) δ 7.97 (s, 1H, CH 3 ), 2.86 (m, 2H, CH 2 ), 2.70 (m, 2H, CH 2 ), 2.34 (m, 2H, CH 2 ), 2.25 (s, 3H, CH 3 ), 1.87 (m, 2H, CH 2 ), 1.65 (m, 6H), 1.10 (m, 3H, including CH, Cy), 0.87 (s, 2H, CH 2 ). 13 C NMR (101 MHz, cdcl 3 ) δ 165.3 (C 5 ), 150.8

(C 3 H), 112.4 (C 4 ), 54.4 (CH 2 ), 48.0 (CH 2 ), 35.1 (CH), 30.6 (2 CH 2 ), 26.2 (CH 2 ), 25.9 (CH 2 ), 25.4 (2 CH 2 ), 19.8 (CH 2 ), 10.6 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 14 H 25 N 2 O: 237.1961, found: 237.1965. N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1-cyclopentyl-N4-(4-(isoxazol-5- yl)butyl)benzene-1,4-disulfonamide, 19 A solution of N-(cyclohexylmethyl)-4-(isoxazol-5-yl)butan-1-amine (211 mg, 0.89 mmol) and triethylamine (148 ul, 1.07 mmol) in dichloromethane (15 ml/mmol) was treated with a solution of 4-(N- (4-chlorobenzyl)-N-cyclopentylsulfamoyl)benzenesulfonyl chloride (400 mg, 0.89 mmol) in dichloromethane (10 ml/mmol) and stirred at 0 C for 20 minutes. Water was then added and the product was extracted into DCM. The combined extracts were dried over MgSO 4 and filtered. The solvent was evaporated and the mixture was purified by flash column chromatography using silica gel (9:1 cyclohexane/ethyl acetate) to afford the titled compound (497 mg, 86% yield) as a brown solid. 1 H NMR (400 MHz, cdcl 3 ) δ 8.09 (d, J = 2 Hz, 1H, CH 3 ), 7.83 (m, 4H, 4 CH, Ar), 7.23 (s, 4H, 4 CH, Ar), 5.90 (d, J = 2 Hz, 1H, CH 4 ), 4.28 (s, 2H, CH 2 ), 4.20 (dt, J = 18, 9 Hz, 1H, CH, Cp), 3.07 (m, 2H, CH 2 ), 2.87 (d, J = 8 Hz, 2H, CH 2 ), 2.72 (t, J = 7 Hz, 2H), 1.70 1.32 (m, 16H, including CH, Cy), 1.16 (m, 2H), 1.10 (m, 2H), 0.79 (m, 2H). 13 C NMR (101 MHz, cdcl 3 ) δ 172.1 (C 5 ), 150.5 (C 3 H), 144.5 (C), 144.0 (C), 137.1 (C), 133.5 (C), 129.0 (2 CH, Ar), 128.7 (2 CH, Ar), 128.0 (2 CH, Ar), 128.0 (2 CH, Ar), 100.5 (C 4 H), 59.9 (CH, Cp), 55.2 (CH 2 ), 48.7 (CH2), 47.2 (CH2-Ar), 36.6 (CH, Cy), 31.0 (2 CH 2 ), 29.6 (2 CH 2 ), 28.1 (CH 2 ), 26.5 (CH 2 ), 26.2 (CH 2 ), 26.0

(CH 2 ), 25.0 (CH 2 ), 23.5 (2 CH 2 ). HRMS (ESI): calc. for [M+H] + C 32 H 43 ClN 3 O 5 S 2 : 648.2254, found: 648.2266. N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1-cyclopentyl-N4-(3-(5- methylisoxazol-4-yl)propyl)benzene-1,4-disulfonamide, 21 A solution of N-(cyclohexylmethyl)-3-(5-methylisoxazol-4-yl)propan- 1-amine (105 mg, 0.45 mmol) and triethylamine (74 ul, 0.54 mmol) in dichloromethane (15 ml/mmol) was treated with a solution of 4-(N-(4- chlorobenzyl)-n-cyclopentylsulfamoyl)benzenesulfonyl chloride (200 mg, 0.45 mmol) in dichloromethane (10 ml/mmol) and stirred at 0 C for 20 minutes. Water was then added and the product was extracted into DCM. The combined extracts were dried over MgSO 4 and filtered. The solvent was evaporated and the resulting mixture was purified by flash column chromatography using silica gel (9:1 cyclohexane/ethyl acetate) to afford the titled compound (238 mg, 82% yield) as a brown solid. 1 H NMR (400 MHz, cdcl 3 ) δ 7.99 (s, 1H, CH 3 ), 7.82 (dd, J = 25, 8 Hz, 4H, 4 CH, Ar), 7.23 (s, 4H, 4 CH, Ar), 4.28 (s, 2H, CH 2 -Ar), 4.21 (dt, J = 17, 9 Hz, 1H, CH, Cp), 3.01 (m, 2H), 2.86 (d, J = 8 Hz, 2H), 2.31 (t, J = 8 Hz, 2H), 2.28 (s, 3H, CH 3 ), 1.77 1.50 (m, 10H), 1.50 1.31 (m, 5H, including CH from Cy), 1.17 (m, 2H), 1.10 (m, 2H), 0.80 (m, 2H). 13 C NMR (101 MHz, cdcl 3 ) δ 164.9 (C 5 ), 151.0 (C 3 H), 144.7 (C), 143.5 (C), 137.0 (C), 133.5 (C), 128.9 (2 CH, Ar), 128.7 (2 CH, Ar), 128.0 (2 CH, Ar), 128.0 (2 CH, Ar), 113.2 (C 4 ), 59.9 (CH, Cp), 55.7 (CH 2 ), 48.7 (CH 2 ), 47.2 (CH 2 -Ar), 36.9 (CH, Cy), 31.0 (2 CH 2 ), 29.6 (2 CH 2 ), 29.4 (CH 2 ), 26.5 (CH 2 ), 25.9 (2 CH 2 ), 23.5 (2

CH 2 ), 19.9 (CH 2 ), 10.9 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 32 H 43 ClN 3 O 5 S 2 : 648.2254, found: 648.2273. 5-(4-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N- (cyclohexylmethyl)phenyl)sulfonamido)butyl)-2-methylisoxazol-2-ium, 13 A solution of N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1- cyclopentyl-n4-(4-(isoxazol-5-yl)butyl)benzene-1,4-disulfonamide in DCM was treated with methyl trifluoromethanesulfonate (1.5 eq), and the reaction was stirred until TLC indicated completion (4 h). The volatiles were removed in vacuo to afford pure compound 11 (quant.). 1 H NMR (700 MHz, DMSO) δ 9.52 (s, 1H, CH 3 ), 8.05 (dd, J = 53, 8 Hz, 4H, 4 CH, Ar), 7.41 (q, J = 8 Hz, 4H, 4 CH, Ar), 7.16 (s, 1H, CH 4 ), 4.41 (s, 2H, CH 2 -Ar), 4.34 (s, 3H, N-Me), 4.25 (m, 1H, CH, Cp), 3.16 (t, J = 7 Hz, 2H), 3.00 (t, J = 7 Hz, 2H), 2.95 (d, J = 7 Hz, 2H), 1.66 (m, 4H), 1.60-1.56 (m, 6H), 1.46 (m, 5H, including CH, Cy), 1.36 (m, 2H), 1.15 (m, 4H), 0.82 (m, 2H). 13 C NMR (176 MHz, DMSO) δ 177.9 (C5, isoxazole), 150.4 (C 3 H, isoxazole), 143.9 (C), 143.5 (C), 138.9 (C), 130.0 (C), 129.1 (2 CH, Ar), 128.7 (2 CH, Ar), 128.6 (2 CH, Ar), 128.4 (2 CH, Ar), 106.8 (C 4 H, isoxazole), 59.6 (CH, Cp), 54.6 (CH 2 ), 48.3 (CH 2 ), 46.6 (CH 2 -Ar), 41.3 (CH 3, Me-N), 36.1 (CH 2 ), 30.6 (CH, Cy), 29.0 (2 CH 2 ), 27.8 (CH 2 ), 26.4 (CH 2 ), 25.9 (2 CH 2 ), 25.7 (2 CH 2 ), 23.8 (CH 2 ), 23.3 (2 CH 2 ). HRMS (ESI): calc. for [M] C 33 H 45 ClN 3 O 5 S + 2 : 662.2484, found: 662.2465.

4-(3-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N- (cyclohexylmethyl)phenyl)sulfonamido)propyl)-2,5-dimethylisoxazol-2-ium, 15 A solution of N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1- cyclopentyl-n4-(3-(5-methylisoxazol-4-yl)propyl)benzene-1,4- disulfonamide in DCM was treated with methyl trifluoromethanesulfonate (1.5 eq), and the reaction was stirred until TLC indicated completion (4 h). The volatiles were evaporated under vacuum to afford pure compound 11 (quant.). 1 H NMR (700 MHz, DMSO) δ 9.51 (s, 1H, CH 3 ), 8.05 (dd, J = 59, 8 Hz, 4H, 4 CH, Ar), 7.40 (dd, J = 19, 8 Hz, 4H, 4 CH, Ar), 4.40 (s, 2H, CH 2 -Ar), 4.31 (s, 3H, Me-N), 4.25 (m, 1H, CH, Cp), 3.17 (t, J = 7 Hz, 2H), 2.95 (d, J = 7 Hz, 2H), 2.58 (s, 3H, Me), 1.76 (m, 2H), 1.62 (m, 6H), 1.56 (m, 1H, CH, Cy), 1.46 (m, 4H), 1.35 (m, 2H), 1.22 1.09 (m, 4H), 0.82 (m, 2H). 13 C NMR (176 MHz, DMSO) δ 171.4 (C 5, isoxazole), 149.9 (C 3 H, isoxazole), 144.0 (C), 143.2 (C), 138.9 (C), 132.0 (C), 129.1 (2 CH, Ar), 128.7 (2 CH, Ar), 128.6 (2 CH, Ar), 128.5 (2 CH, Ar), 119.5 (C 4, isoxazole), 59.6 (CH, Cp), 54.9 (CH 2 ), 48.4 (CH 2 ), 46.6 (CH 2 -Ar), 41.2 (CH 3, N-Me), 36.2 (CH, Cy), 30.6 (2 CH 2 ), 29.0 (2 CH 2 ), 28.5 (CH 2 ), 26.4 (CH 2 ), 25.7 (2 CH 2 ), 23.3 (2 CH 2 ), 19.0 (CH 2 ), 11.2 (CH 3 ). HRMS (ESI): calc. for [M] C 33 H 45 ClN 3 O 5 S + 2 : 662.2484, found: 662.24837.

Synthesis of 12 2-(isoxazol-5-yl)acetonitrile, 35 KCN (0.9 g, 1.2 eq., 13.7 mmol) was carefully added to a stirred solution of 4-(chloromethyl)-5-methylisoxazole (1.5 g, 11.4 mmol) in ACN/water (20 ml, 5:15) at room temperature. The mixture was stirred overnight at 50 C. Water was then added and the product was extracted with AcOEt (5 x 20 ml). The organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Purification of the resulting mixture by flash column chromatography using silica gel (7:3 cyclohexane/ethyl acetate) afforded the titled compound as a yellowish oil (1.27 g, 91% yield). 1 H NMR (400 MHz, cdcl 3 ) δ 8.20 (d, J = 2 Hz, 1H, CH 3 ), 6.30 (bs, 1H, CH 4 ), 3.87 (s, 2H, CH 2 ). 13 C NMR (101 MHz, cdcl 3 ) δ 160.7 (C 5 ), 150.9 (C 3 H),

114.1 (CN), 103.2 (C 4 H), 16.7 (CH 2 ). LC-MS (ESI): calc. for [M+H] + C 5 H 5 N 2 O: 109.03, found: 109.14. N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1-cyclopentyl-N4-(2-(isoxazol-5- yl)ethyl)benzene-1,4-disulfonamide, 18 DIBAL-H (1 M solution in toluene, 3.2 ml, 3.2 mmol, 1.5 eq) was added dropwise at -40 C to a solution of 2-(isoxazol-5-yl)acetonitrile (230 mg, 2.13 mmol) in CH 2 Cl 2 (4 ml/mmol), and the mixture was stirred for 3 h. The reaction mixture was then quenched with MeOH (0.1 ml) and a solution of Rochelle s salt (6 ml). The mixture was warmed to ambient temperature, stirred for 1 h and diluted with CH 2 Cl 2. The organic layer was separated, then washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The complex mixture was then subjected to a reductive amination (procedure II) to obtain N-(cyclohexylmethyl)-2-(isoxazol-5-yl)ethan-1-amine. The complex mixture was dissolved in DCM and treated with triethylamine (2 eq) in dichloromethane (15 ml/mmol). A solution of 4-(N-(4-chlorobenzyl)-Ncyclopentylsulfamoyl)benzenesulfonyl chloride (1 eq) in dichloromethane (10 ml/mmol) was added and the mixture was stirred at 0 C for 20 minutes. Water was added and the product was extracted into DCM. The combined extracts were dried over MgSO 4 and filtered. The solvent was evaporated and purification of the resulting mixture by flash column chromatography using silica gel (9:1 cyclohexane/ethyl acetate) yielded compound 18 (277 mg, 21% yield, 3 steps) as a brown solid. 1 H NMR (600 MHz, CDCl 3 ) δ 8.10 (d, J = 1.4 Hz, 1H, CH 3 ), 7.85 (q, J = 9 Hz, 4H, 4 CH, Ar), 7.22 (m, 8H), 6.01 (d, J

= 1.3 Hz, 1H, CH 4 ), 4.28 (s, 4H, CH 2 -Ar), 4.22 (m, 1H, CH, Cp), 3.37 (m, 2H), 3.04 (m, 2H), 2.89 (d, J = 7 Hz, 2H), 1.66 (m, 2H), 1.55 (m, 5H, including CH, Cy), 1.47 (m, 2H), 1.33 (m, 2H), 1.30 1.14 (m, 4H), 1.14 1.03 (m, 2H), 0.79 (m, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.5 (C 5 ), 149.4 (C 3 H), 143.6 (C), 142.0 (C), 135.7 (C), 132.2 (C), 127.7 (2 CH, Ar), 127.5 (2 CH, Ar), 126.9 (2 CH, Ar), 126.8 (2 CH, Ar), 100.5 (C 4 H), 58.7 (CH, Cp), 54.7 (CH 2 ), 46.0 (CH 2 ), 45.9 (CH 2 -Ar), 35.2 (CH, Cy), 29.7 (2 CH 2 ), 28.4 (2 CH 2 ), 26.0 (CH 2 ), 25.2 (CH 2 ), 24.6 (2 CH 2 ), 22.2 (2 CH 2 ). HRMS (ESI): calc. for [M+H] + C 30 H 39 ClN 3 O 5 S 2 : 620.941, found: 620.1937. 5-(2-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N- (cyclohexylmethyl)phenyl)sulfonamido)ethyl)-2-methylisoxazol-2- ium, 12 A solution of N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1- cyclopentyl-n4-(2-(isoxazol-5-yl)ethyl)benzene-1,4-disulfonamide in DCM was treated with methyl trifluoromethanesulfonate (1.5 eq), and the mixture was stirred until TLC indicated completion (4 h). The volatiles were evaporated in vaccuo to afford pure compound 12 (quant.). 1 H NMR (600 MHz, CDCl 3 ) δ 9.16 (s, 1H, CH 3 ), 7.83 (dd, J = 28, 8 Hz, 4H, 4 CH, Ar), 7.21 (m, 4H, 4 CH, Ar), 6.93 (s, 1H, C 4 H), 4.31 (s, 3H, N-Me), 4.24 (s, 2H, CH 2 -Ar), 4.19 (m, 1H, Cp), 3.42 (m, 2H), 3.23 (m, 2H), 2.89 (d, J = 7 Hz, 2H), 1.60 (m, 2H), 1.53 (m, 5H, including CH, Cy), 1.42 (m, 2H), 1.35 (m, 2H), 1.23 1.12 (m, 4H), 1.06 (m, 2H), 0.77 (m, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 174.5 (C 5 ), 149.2 (C 3 H), 142.0 (C), 141.5 (C), 136.1 (C), 132.4 (C), 127.9 (2 CH, Ar), 127.7 (2 CH, Ar), 127.3 (2

CH, Ar), 127.2 (2 CH, Ar), 107.2 (C 4 H), 59.0 (CH, Cp), 55.2 (CH 2 ), 46.2 (CH 2 ), 45.8 (CH 2 -Ar), 40.6 (CH 3, N-Me), 35.6 (2 CH 2 ), 29.8 (CH, Cy), 28.6 (2 CH 2 ), 26.9 (CH 2 ), 25.4 (CH 2 ), 24.7 (2 CH 2 ), 22.5 (2 CH 2 ). HRMS (ESI): calc. for [M] C 31 H 41 ClN 3 O 5 S + 2 : 634.2171, found: 634.2178. Synthesis of 14 and 16 (5-methylisoxazol-4-yl)methanol, 38 LiAlH 4 (1.9 g, 51 mmol) was added to a mixture of 5-methylisoxazole-4- carboxylic acid (5 g, 39 mmol) in THF (50 ml) at 0 C. The reaction was stirred overnight at room temperature, cooled to 0 C and quenched with a saturated solution of Rochelle s salt (20 ml). THF was evaporated under reduced pressure and the residue was redisolved and extracted in AcOEt. The combined organic extracts were

washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the product as yellow oil (3.2 g, 72% yield). 1 H NMR (400 MHz, cdcl 3 ) δ 8.14 (s, 1H), 4.45 (s, 2H), 2.36 (s, 3H). 13 C NMR (101 MHz, cdcl 3 ) δ 166.3 (C 5 ), 150.9 (C 3 H), 114.5 (C 4 ), 54.5 (CH2), 11.0 (CH3). HRMS (ESI): calc. for [M+H] + C 5 H 7 NO 2 : 114.0477, found: 114.0432. 4-(chloromethyl)-5-methylisoxazole, 39 Thionyl chloride (1.4 ml, 1.3 eq., 18.4 mmol) in DCM (10 ml) was added drop wise to the solution of the alcohol (1.6 g, 14.1 mmol) in anhydrous DCM (20 ml) at 0 C. After completion of addition the reaction mixture was stirred at room temperature overnight, quenched with water (20 ml) and then extracted with DCM (3x60 ml). Combined organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the product as yellow oil (1.6 g, 88% yield). 1 H NMR (400 MHz, cdcl 3 ) δ 8.14 (s, 1H), 4.35 (s, 2H), 2.38 (s, 3H). 13 C NMR (101 MHz, cdcl 3 ) δ 166.2 (C 5 ), 151.1 (C 3 H), 111.5 (C 4 ), 61.4 (CH 2 ), 11.0 (CH 3 ). LC-MS (ESI): calc. for [M+H] + C 5 H 6 ClNO: 132.01, found: 131.97. 2-(5-methylisoxazol-4-yl)acetonitrile, 40 KCN (0.9 g, 1.2 eq., 13.7 mmol) was carefully added to a stirred solution of 4-(chloromethyl)-5-methylisoxazole (1.5 g, 11.4 mmol) in ACN/water (20 ml, 5:15) at room temperature. The mixture was then stirred overnight at 50 C. Water was then added and the product was extracted with AcOEt (5 x 20 ml). The organic layers

were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Purification of the resulting mixture by flash column chromatography using silica gel (7:3 cyclohexane/ethyl acetate) afforded the titled compound as a yellowish oil (1.27 g, 91% yield). 1 H NMR (400 MHz, cdcl 3 ) δ 8.18 (s, 1H), 3.49 (m, 2H), 2.45 (m, 3H). 13 C NMR (101 MHz, cdcl 3 ) δ 166.5 (C 5 ), 150.4 (C 3 H), 116.3 (CN), 104.2 (C 4 ), 12.2 (CH 2 ), 11.0 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 6 H 6 N 2 O: 123.0480, found: 123.0422. 2-(5-methylisoxazol-4-yl)acetaldehyde, 41 To a solution of the nitrile in dry DCM (2mL/mmol) was added a solution of DIBAL in toluene at -78 C under argon. The mixture was stirred for 4h at - 40 degrees. After that, to the resulting reaction mixture was added MeOH, then saturated solution of potassium sodium tartrate and HCl 1M and the mixture was stirred for 30 minutes. The mixture was diluted with water and extracted with DCM ( 3). The combined organic phases were washed with brine and dried over Na 2 SO 4. DCM was removed under reduced pressure. This compound was used in the next step without any purification. 1 H NMR (400 MHz, cdcl 3 ) δ 9.66 (t, J = 1.5 Hz, 1H), 8.08 (s, 1H), 3.45 (m, 2H), 2.30 (s, 3H). 13 C NMR (101 MHz, cdcl 3 ) δ 196.9 (CHO), 166.6 (C 5 ), 151.5 (C 3 H), 105.2 (C 4 ), 37.77 (CH 2 ), 11.0 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 6 H 8 NO 2 : 126.0477, found: 126.0450.

N-(cyclohexylmethyl)-2-(5-methylisoxazol-4-yl)ethan-1-amine, 42 The title compound (255 mg, 72% yield) was prepared according to the reductive amination procedure II by using a mixture of the aldehyde (200 mg, 1.6 mmol), the amine (200 mg, 1.7 mmol), sodium triacetoxyborohydride (678 mg, 3.2 mmol), and acetic acid (0.192 ml, 3.2 mmol). The crude compound was purified on preparative HPLC using a reversed-phase C18 column (RP C18, flow 20.0 ml/min, solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in acetonitrile, from 10% B to 100% B). The solvent was freeze dried to afford the pure product as a white solid (TFA salt). 1 H NMR (700 MHz, CDCl 3 ) δ 8.04 (s, 1H), 3.00 (m, 2H), 2.76 (m, 2H), 2.28 (s, 3H), 1.72 (m, 2H), 1.67 1.54 (m, 5H, including CH, Cy), 1.10 (m, 4H), 0.89 (m, 2H). 13 C NMR (176 MHz, CDCl 3 ) δ 166.0 (C 5 ), 150.6 (C 3 H), 109.2 (C 4 ), 54.2 (CH 2 ), 48.3 (CH 2 ), 35.0 (CH, Cy), 30.4 (2 CH 2 ), 25.7 (2 CH 2 ), 25.3 (CH 2 ), 19.3 (CH 2 ), 10.3 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 13 H 23 N 2 O: 223.1732, found: 223.1794. N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1-cyclopentyl-N4-(2-(5- methylisoxazol-4-yl)ethyl)benzene-1,4-disulfonamide, 20 A solution of N-(cyclohexylmethyl)-2-(5-methylisoxazol-4-yl)ethan-1- amine (99 mg, 0.45 mmol) and triethylamine (74 ul, 0.54 mmol) in dichloromethane (15 ml/mmol) was treated with a solution of 4-(N-(4- chlorobenzyl)-n-cyclopentylsulfamoyl)benzenesulfonyl chloride (200 mg, 0.45 mmol) in dichloromethane (10 ml/mmol) and stirred at 0 C for

20 minutes. Water was then added and the product was extracted into DCM. The combined extracts were dried over MgSO 4 and filtered. The solvent was evaporated and purification of the resulting mixture was carried out by flash column chromatography using silica gel (9:1 cyclohexane/ethyl acetate) to afford the titled compound (215 mg, 76% yield) as a brown solid. 1 H NMR (700 MHz, CDCl 3 ) δ 7.98 (s, 1H, CH 3 ), 7.83 (dd, J = 24, 8 Hz, 4H, 4 CH, Ar), 7.21 (q, J = 9 Hz, 4H, 4 CH, Ar), 4.27 (s, 2H, CH 2 -Ar), 4.20 (m, 1H, CH, Cp), 3.11 (m, 2H), 2.88 (d, J = 7 Hz, 2H), 2.63 (m, 2H), 2.30 (s, 3H, CH3), 1.64 (m, 2H), 1.55 (m, 6H), 1.44 (m, 2H), 1.38 (m, 3H, including CH, Cy), 1.19 (m, 2H), 1.09 (m, 2H), 0.80 (m, 2H). 13 C NMR (176 MHz, CDCl 3 ) δ 164.5 (C5, isoxazole), 149.8 (C 3 H, isoxazole), 143.5 (C), 142.1 (C), 135.8 (C), 132.2 (C), 127.6 (2 CH, Ar), 127.5 (2 CH, Ar), 126.9 (2 CH, Ar), 126.8 (2 CH, Ar), 109.5 (C4, isoxazole), 58.7 (CH, Cp), 54.8 (CH 2 ), 48.4 (CH 2 ), 46.0 (CH 2 -Ar), 35.6 (CH, Cy), 29.7 (2 CH 2 ), 28.4 (2 CH 2 ), 25.2 (CH 2 ), 24.7 (2 CH 2 ), 22.2 (2 CH 2 ), 22.2 (CH 2 ), 9.7 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 31 H 41 ClN 3 O 5 S 2 : 634.2098, found: 634.2025. N1-(4-chlorobenzyl)-N1-cyclopentyl-N4-(2-(5-methylisoxazol-4-yl)ethyl)-N4- (piperidin-4-ylmethyl)benzene-1,4-disulfonamide, 23 First, a mixture of the aldehyde (500 mg, 4 mmol) and the amine (942 mg, 4.4 mmol) in methanol was stirred at room temperature overnight. The mixture was cooled down and sodium borohydride (151 mg, 4 mmol) was added. The mixture was stirred at room temperature for 1h. After addition of water, methanol was removed under reduced pressure and the resulting aqueous phase was extracted with DCM, washed with

brine, dried over MgSO 4, filtered and concentrated in vacuo to give corresponding crude compound that was directly subjected to the next reaction. A solution of the previous mixture and triethylamine (1.1 ml, 8 mmol) in dichloromethane (15 ml/mmol) was treated with a solution of 4-(N-(4-chlorobenzyl)-Ncyclopentylsulfamoyl)benzenesulfonyl chloride (1.8 g, 4 mmol) in dichloromethane (10 ml/mmol) and stirred at 0 C for 20 minutes. Water was then added and the product was extracted into DCM. The combined extracts were dried over MgSO 4 and filtered. The solvent was reduced and Boc deprotection was then done in situ by slowly adding to the mixture TFA (~10 eq), and this reaction was then stirred for 2 h at room temperature. The solvent was evaporated and purification of the resulting mixture was carried out on preparative HPLC using a reversed-phase C18 column (RP C18, flow 20.0 ml/min, solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in acetonitrile, from 10% B to 100% B). The solvent was freeze dried to afford the pure product ((1.42 g, 48% yield, two steps) as a white solid (TFA salt). 1 H NMR (700 MHz, DMSO) δ 8.40 (s, 1H), 8.06 (dd, J = 28, 8 Hz, 4H, 4 CH, Ar), 7.41 (m, 4H, 4 CH, Ar), 4.40 (s, 2H, CH2-Ar), 4.26 (m, 1H, CH, Cp), 3.29 (m, 2H), 3.23 (m, 2H), 3.05 (m, 2H), 2.82 (m, 2H), 2.56 (m, 2H), 2.32 (m, 3H), 1.82 (m, 1H, CH, Cy), 1.75 (m, 2H), 1.45 (m, 4H), 1.38 1.22 (m, 4H), 1.19 (m, 2H). 13 C NMR (176 MHz, DMSO) δ 165.6 (C 5 ), 152.0 (C 3 H), 144.1 (C), 142.8 (C), 138.8 (C), 132.0 (C), 129.1 (2 CH, Ar), 128.7 (2 CH, Ar), 128.5 (2 CH, Ar), 128.5 (2 CH, Ar), 111.2 (C 4 ), 59.6 (CH, Cp), 53.7 (CH 2 ), 49.0 (CH 2 ), 46.6 (CH 2 -Ar), 43.3 (CH 2 ), 32.3 (CH, Cy), 29.0 (2 CH 2 ), 28.6 (CH 2 ), 26.5 (2 CH 2 ), 23.3 (2 CH 2 ), 22.2 (CH 2 ), 10.7 (CH 3 ). HRMS (ESI): calc. for [M+H] + C 30 H 40 ClN 4 O 5 S 2 : 635.2123, found: 635.2110.

4-(2-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N- (cyclohexylmethyl)phenyl)sulfonamido)ethyl)-2,5-dimethylisoxazol-2-ium, 14 A solution of N1-(4-chlorobenzyl)-N4-(cyclohexylmethyl)-N1- cyclopentyl-n4-(2-(5-methylisoxazol-4-yl)ethyl)benzene-1,4- disulfonamide in DCM was treated with methyl trifluoromethanesulfonate (1.5 eq), and the reaction was stirred until TLC indicated completion (4 h). The volatiles were evaporated under vacuum to afford pure compound 14 (quant.). 1 H NMR (500 MHz, DMSO) δ 9.47 (s, 1H, CH 3 ), 7.96 (dt, J = 24, 12 Hz, 4H, 4 CH, Ar), 7.33 (q, J = 9 Hz, 4H, 4 CH, Ar), 4.31 (s, 2H, CH 2 -Ar), 4.26 (s, 3H, N-CH 3 ), 4.18 (m, 1H, CH, Cp), 3.26 (m, 2H), 2.94 (m, 2H), 2.70 (m, 2H), 2.54 (s, 3H, Me), 1.52 (m, 6H), 1.31 (m, 5H, including CH, Cy), 1.25 (m, 2H), 1.07 (m, 4H), 0.72 (m, 2H). 13 C NMR (126 MHz, DMSO) δ 172.3 (C 5 ), 150.3 (C 3 H), 144.1 (C), 143.2 (C), 138.9 (C), 132.0 (C), 129.1 (2 CH, Ar), 128.7 (2 CH, Ar), 128.7 (2 CH, Ar), 128.5 (2 CH, Ar), 117.1 (C 4 ), 59.5 (CH, Cp), 54.6 (CH 2 ), 47.7 (CH 2 ), 46.5 (CH 2 -Ar), 41.2 (N-CH 3 ), 35.9 (CH, Cy), 30.5 (2 CH 2 ), 29.0 (2 CH 2 ), 26.4 (CH 2 ), 25.6 (2 CH 2 ), 23.3 (2 CH 2 ), 21.8 (CH 2 ), 11.3 (CH 3 ). HRMS (ESI): calc. for [M] C 32 H 43 ClN 3 O 5 S + 2 : 648.2327, found: 648.2326.

4-(2-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N-(piperidin-4- ylmethyl)phenyl)sulfonamido)ethyl)-2,5-dimethylisoxazol-2-ium, 16 A solution of 4-(3-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N- (cyclohexylmethyl)phenyl)sulfonamido)propyl)-2,5-dimethylisoxazol-2- ium in DCM was treated with methyl trifluoromethanesulfonate (1.5 eq), and the reaction was stirred until TLC indicated completion (4 h). The volatiles were evaporated under vacuum to afford pure compound 16 (quant.). 1 H NMR (500 MHz, DMSO) δ 9.42 (s, 1H, CH 3 ), 8.35 (bs, J = 10 Hz, 1H, NH), 8.03 (m, 1H, NH), 7.99 (dd, J = 27, 8 Hz, 4H, 4 CH, Ar), 7.32 (q, J = 9 Hz, 4H, 4H, 4 CH, Ar), 4.30 (s, 2H, CH 2 -Ar), 4.26 (s, 3H, N-CH 3 ), 4.18 (m, 1H, CH, Cp), 3.24 (m, 4H), 3.05 (m, 2H), 2.77 (m, 2H), 2.68 (m, 2H), 2.53 (s, 3H, Me), 1.85 (bs, 1H, CH, Cy), 1.70 (d, J = 13 Hz, 2H), 1.38 (m, 4H), 1.24 (m, 2H), 1.18 (m, 2H), 1.08 (m, 2H). 13 C NMR (126 MHz, DMSO) δ 172.3 (C 5 ), 150.2 (C 3 H), 144.2 (C), 142.8 (C), 138.8 (C), 132.0 (C), 129.1 (2 CH, Ar), 128.7 (2 CH, Ar), 128.5 (CH, Ar), 128.4 (2 CH, Ar), 117.0 (C 4 ), 59.6 (CH, Cp), 53.6 (CH 2 ), 47.9 (CH 2 ), 46.5 (CH 2 -Ar), 43.3 (CH 2 ), 41.2 (N- CH 3 ), 32.2 (CH, Cy), 29.0 (2 CH 2 ), 26.5 (2 CH 2 ), 23.3 (2 CH 2 ), 21.7 (2 CH 2 ), 11.3 (CH 3 ). HRMS (ESI): calc. for [M] C 31 H 42 ClN 4 O 5 S + 2 : 649.2280, found: 649.2279.

Synthesis of 24 (Z)-2-(2-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N-(piperidin-4- ylmethyl)phenyl)sulfonamido)ethyl)-3-hydroxy-n-methylbut-2-enamide, 24 A solution of 4-(2-((4-(N-(4-chlorobenzyl)-N-cyclopentylsulfamoyl)-N- (piperidin-4-ylmethyl)phenyl)sulfonamido)ethyl)-2,5-dimethylisoxazol- 2-ium (20 mg) in HEPES (10 ml) was stirred at 90 C for 24 h. Water was freeze-dried and the compound was purified on preparative HPLC using a reversed-phase C18 column (RP C18, flow 20.0 ml/min, solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in acetonitrile, from 10% B to 100% B). The solvent was freeze dried to afford the pure product as a white solid (TFA salt). 1 H NMR (700 MHz, DMSO) δ 8.68 (d, J = 9 Hz, 1H, NH), 8.35 (d, J = 9 Hz, 1H, NH), 8.24 (bs, 1H, NH), 8.03 (dd, J = 65, 9 Hz, 4H, 4 CH, Ar), 7.40 (dd, J = 20, 9 Hz, 4H, 4 CH, Ar), 4.39 (s, 2H, CH 2 -Ar), 4.26 (m, 1H, CH, Cp), 3.38 (m, 1H), 3.30 (d, J = 12 Hz, 2H), 3.09 2.96 (m, 4H), 2.85 (dd, J = 22, 10 Hz, 2H), 2.61 (d, J = 5 Hz, 3H, NH-Me), 2.07 (s, 3H, Me), 1.90 (m, 1H, CH, piperidine), 1.88 (m, 2H), 1.83 (m, 2H), 1.46 (m, 4H), 1.39 1.22 (m, 4H), 1.17 (m, 2H). 13 C NMR (176 MHz, DMSO) δ 203.7

(CO), 168.8 (CO), 144.1 (C), 143.1 (C), 138.8 (C), 132.0 (C), 129.1 (2 CH, Ar), 128.7 (2 CH, Ar), 128.7 (2 CH, Ar), 128.4 (2 CH, Ar), 59.6 (CH, Cp), 57.5 (CH), 53.4 (CH 2 ), 47.0 (CH 2 ), 46.6 (CH 2 -Ar), 43.3 (2 CH 2 ), 32.5 (CH, piperidine), 29.1 (2 CH 2 ), 28.8 (CH 3 ), 26.6 (CH 2 ), 26.6 (CH 2 ), 26.5 (CH 2 ), 26.4 (NH-CH 3 ), 23.3 (2 CH 2 ). HRMS (ESI): calc. for [M+H] + C 31 H 44 ClN 4 O 6 S 2 : 667.2385, found: 667.2381.

NMR spectra