Meridianin D analogues display antibiofilm activity against MRSA, increase colistin efficacy in Gram-negative bacteria

Transcription

1 Supporting Information Meridianin D analogues display antibiofilm activity against MRSA, increase colistin efficacy in Gram-negative bacteria William M. Huggins, William T. Barker, James T. Baker, Nicholas A. Hahn, Roberta J. Melander, and Christian Melander Table of Contents MIC, Biofilm assay, Repotentiation assay, Growth Curve Protocols... S2 Growth curves for select compounds against MRSA and A. baumannii S4 MICs of Compounds 9b, 17a-b against Gram-positive Pathogens... S5 Complete Library of Meridianin Derivative MRSA Biofilm Inhibition Results... S6 Dose Response Curves for MRSA Biofilm Inhibition... S7 Dose Response Curves for MRSA Biofilm Dispersion... S23 Dose Response Curves for MRSA Biofilm Dispersion With 19µg/mL of Vancomycin... S28 Full Colistin Repotentiation Results... S31 General Synthetic Procedures... S33 Previously Reported Compounds... S35 Novel Compound Characterization... S44 LC Traces of Compounds 9b and 9d... S58 1 H and 13 C NMR Spectra of Novel Compounds... S59 References... S99 S1

2 Biology Experimental Broth Microdilution Method for MIC Determination (MRSA and E. faecium): Overnight cultures of bacterial strain were subcultured to 5 x 10 5 CFU/mL in Mueller-Hinton medium. The resulting bacterial suspension was aliquoted (1.0 ml) into culture tubes. Samples were prepared from these culture tubes containing either 200 µmol of specified antibiotic or no test compound as a control. Samples were then aliquoted (200 µl) into the first row of wells of a 96-well microtiter plate in which subsequent wells were prefilled with 100 µl of Mueller-Hinton medium based 5 x10 5 CFU/mL bacterial subculture. Using the multichannel pipettor set at 100 µl, row one wells were mixed 8-10 times. Then, 100 µl were withdrawn and transferred to row two. Row two wells were mixed 8-10 times followed by a 100 µl transfer from row two to row three. This procedure was used to serial dilute the rest of the rows of the microtier plate. The microtiter plate sample was then covered with a microtiter plate lid and then placed in a covered plastic container. The chamber was incubated under stationary conditions at 37 C. After 16 hours, the lid was removed and MIC values were recorded. Broth Microdilution Method for MIC Determination (A. baumannii, K. pneumoniae, E. coli): Overnight cultures of bacterial strain were subcultured to 5 x 10 5 CFU/mL in cation adjusted Mueller- Hinton medium. The resulting bacterial suspension was aliquoted (1.0 ml) into culture tubes. Samples were prepared from these culture tubes containing either 200 µmol of specified antibiotic or no test compound as a control. Samples were then aliquoted (200 µl) into the first row of wells of a 96-well microtiter plate in which subsequent wells were prefilled with 100 µl of Mueller-Hinton medium based 5 x10 5 CFU/mL bacterial subculture. Using the multichannel pipettor set at 100 µl, row one wells were mixed 8-10 times. Then, 100 µl were withdrawn and transferred to row two. Row two wells were mixed 8-10 times followed by a 100 µl transfer from row two to row three. This procedure was used to serial dilute the rest of the rows of the microtier plate. The microtiter plate sample was then covered with a microtiter plate lid and then placed in a covered plastic container. The chamber was incubated under stationary conditions at 37 C. After 16 hours, the lid was removed and MIC values were recorded. Procedure to Determine the Inhibitory Effect of Test Compounds on MRSA ATCC# Biofilm Formation: Inhibition assays were performed by taking an overnight culture of bacterial strain and subculturing it at an OD 600 of 0.04 into tryptic soy broth (Sigma-Aldrich) with a 2.0% glucose supplement (TSBG). Stock solutions of predetermined concentrations of the test compound were then made in TSBG. These stock solutions were aliquoted (100 µl) into the wells of the 96-well PVC microtiter plate. Sample plates were then wrapped in GLAD Press n Seal followed by an incubation under stationary conditions for 24 h at 37 C After incubation, the medium was discarded from the wells and the plates were washed thoroughly with water. Plates were then stained with 100 µl of 0.1% solution of crystal violet (CV) and then incubated at ambient temperature for 30 min. Plates were washed with water again and the remaining stain was solubilized with 200 µl of 95% ethanol. A sample of 125 µl of solubilized CV stain from each well was transferred to the corresponding wells of a polystyrene microtiter dish. Biofilm inhibition was quantitated by measuring the OD 540 of each well in which a negative control lane wherein no biofilm was formed served as a background and was subtracted out. Procedure to Determine the Dispersal Effect of Test Compounds on MRSA ATCC# Preformed Biofilms: Dispersion assays were performed by taking an overnight culture of MRSA ATCC in tryptic soy broth (Sigma-Aldrich) with a 2.0% glucose supplement (TSBG) and subculturing it at an OD 600 of 0.04 into TSBG. The resulting bacterial suspension was aliquoted (100 µl) into the wells of a 96-well PVC microtiter plate. Plates were then wrapped in GLAD Press n Seal followed by an S2

3 incubation under stationary conditions at 37 C to establish the biofilms. After 24 h, the medium was discarded from the wells and the plates were washed thoroughly with water. Stock solutions of predetermined concentrations of the test compound were then made in the necessary medium. These stock solutions were aliquoted (100 µl) into the wells of the 96-well PVC microtiter plate with the established biofilms. Medium alone was added to a subset of the wells to serve as a control. Sample plates were then incubated for 24 h at 37 C. After incubation, the medium was discarded from the wells and the plates were washed thoroughly with water. Plates were then stained with 110 µl of 0.1% solution of crystal violet (CV) and then incubated at ambient temperature for 30 min. Plates were washed with water again and the remaining stain was solubilized with 200 µl of 95% ethanol. A sample of 125 µl of solubilized CV stain from each well was transferred to the corresponding wells of a polystyrene microtiter dish. Biofilm dispersion was quantitated by measuring the OD 540 of each well in which a negative control lane wherein no biofilm was formed served as a background and was subtracted out. Broth Microdilution Method for Antibiotic Resensitization: Overnight cultures of bacteria in cationadjusted Mueller-Hinton Broth (CAMHB) were subcultured to CFU/mL in CAMHB. Aliquots (5 ml) were placed in culture tubes and dosed with compound from stock solutions to give concentrations of 30% of the MIC of the compound against the particular bacterial strain. One milliliter of the resulting solution was placed in a separate culture and dosed with antibiotic at the highest concentration to be tested. Bacteria treated with antibiotic alone were used as the control. Row 1 of a 96-well plate was filled with 200 µl of the antibiotic/adjuvant, and rows 2 8 were filled with 100 µl each of the remaining 4 ml of bacterial subculture. Row 1 was then mixed five times, and 100 µl was transferred to row 2, which was then mixed five times before being transferred to row 3. This process was repeated until all rows had been mixed, except for row eight, which would have only compound, to serve as a control. The 96-well plate was then covered in Glad Press n Seal and incubated under stationary conditions at 37 C for 16 h. MIC values were determined as the lowest concentration at which no bacterial growth was observed; fold reductions were determined by comparison to control lane. Growth curves for compounds in biofilm conditions: MRSA ATCC# was grown overnight in TSBG, and this culture was used to inoculate fresh TSBG (OD 600 =0.04). Inoculated medium was aliquoted (3 ml) into culture tubes, and compound was added, with untreated inoculated medium serving as the control. Tubes were incubated at 37 C with shaking. Samples were taken at 2, 4, 6, 8, and 24 h time points, serially diluted in fresh TSBG, and plated on nutrient agar. Plates were incubated at 37 C overnight in stationary conditions, and the number of colonies was enumerated. Growth curves for compounds in resensitization conditions: Cultures were grown overnight in CAMHB, and this culture was used to inoculate fresh CAMHB ( CFU/mL). Inoculated medium was aliquoted (3 ml) into culture tubes, and compound was added, with untreated inoculated medium serving as the control. Tubes were incubated at 37 C with shaking. Samples were taken at 2, 4, 6, 8, and 24 h time points, serially diluted in fresh CAMHB, and plated on nutrient agar. Plates were incubated at 37 C overnight in stationary conditions, and the number of colonies was enumerated. S3

4 Growth curves for select compounds against MRSA 43300: 1.00E+10 MRSA Growth Curves CFU/mL 1.00E E E E E E E E+02 0 µm Compound 9a µm Compound 9b 9.62 µm Compound 9e 73.1 µm 1.00E Time Growth curves for select compounds against A. baumannii 4106: 1.00E E E+08 Compounds 8d and 8e v. A. baumannii4106 CFUs/mL 1.00E E E E E E E+01 0 µm 9d 60 µm 9e 60 µm 1.00E Time S4

5 MICs of Compounds 9b, 17a-b against Gram-positive Pathogens Strain MRSA ATCC 1556 MSSA ATCC 6538 VRE ATCC Compound 9b MIC (µm) Compound 9b MIC (µg/ml) Compound R 1 = MIC (µm) v. MRSA a 5-Br b 6-Br MIC (µg/ml) v. MRSA S5

6 Complete Library of Meridianin Derivative MRSA Biofilm Inhibition Results Compound R 1 R 2 R 3 MIC (µm) IC 50 (µm) 1 6-Br H H > ±4.0 9a 5-Br H H ±2.2 9b 5-Br Bn H ±1.4 9c 5-Br Me Me ±1.8 9d 5-Br Et H ±0.7 9e 5-Br Me H ±2.3 9f 6-I H H > ±8.1 9g 5-I H H ±5.1 9h 5-Cl H H ±8.8 9i 6-Cl H H ±14.1 9j 7-Br H H ±15.2 9k 4-Br H H >200 >100 9l 5-F H H >200 >100 9m 6-F H H >200 >100 9n 5-Me H H >200 >100 9o H H H >200 >100 13a 6-Br n-pr H ±6.8 13b 5-Br n-bu H ±2.8 13c 6-Br Me H ±2.3 13d 6-Br Et H ±8.8 13e 5-Br Et H ±7.6 13f 5-Br Pr H ±3.6 13g 5-Br Me H 200 >100 13h 5-Br n-pn H >200 >100 13i 6-Br Bn H >200 >100 13j 6-Br n-bu H >200 >100 13k 5-Br i-bu H >200 >100 13l 6-Br i-bu H >200 >100 13m 6-I n-pr H >200 >100 13n 6-Br n-me Et >200 >100 S6

7 Dose Response Curves for MRSA Biofilm Inhibition 100 Compound 1 Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 9a Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S7

8 Compound 9b Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 9c Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S8

9 Compound 9d Inhibition v. MRSA Inhibition (%) Compound Conentration (µm) Compound 9e Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S9

10 Compound 9f Inhibition v. MRSA Inhibiiton (%) Compound Concentration (µm) Compound 9g Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S10

11 Compound 9h Inhibition v. MRSA Inhibiiton (%) Compound Concentration (µm) Compound 9i Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S11

12 Compound 9j Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 9k Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S12

13 Compound 9l Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 9m Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S13

14 Compound 9n Inhbition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 9o Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S14

15 Compound 13a Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 13b Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S15

16 Compound 13c Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 13d Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S16

17 Compound 13e Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 13f Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S17

18 Compound 13g Inhibition v. MRSA Inhibtion (%) Compound Concentration (µm) Compound 13h Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S18

19 Comopound 13i Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 13j Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S19

20 Compound 13k Inhibition v. MRSA Inhbition (%) Compound Concentration (µm) Compound 13l Inhibition v. MRSA Inhbition (%) Compound Concentration (µm) S20

21 Compound 13m Inhibition v. MRSA Inhibition (%) Compound Conentration (µm) Compound 13n Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S21

22 Compound 17a Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) Compound 17b Inhibition v. MRSA Inhibition (%) Compound Concentration (µm) S22

23 Dose Response Curves for MRSA Biofilm Dispersion Compound 9a Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) 60 Compound 9b Dispersion v. MRSA Dispersion (%) Concentration (µm) S23

24 20 Compound 9c Dispersion v. MRSA Dispersion (%) Concentration (µm) Compound 9d Dispersion v. MRSA Dispersion (%) Concentration (µm) S24

25 Compound 9e Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) Compound 13a Dispersion v. MRSA Dispersion (%) Concentration (µm) S25

26 Compound 13b Dispersion v. MRSA Dispersion (%) Concentration (µm) Compound 17a Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) S26

27 Compound 17b dispersion v. MRSA Dispersion (%) Compound Concentration (µm) Vancomycin Dispersion v. MRSA y = 5.116x R² = Dispersion (%) Concentration (µg/ml) S27

28 Dose Response Curves for MRSA Biofilm Dispersion With 19µg/mL of Vancomycin Compound 9a+Vancomycin Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) Compound 9d+Vancomycin Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) S28

29 Compound 9e+Vancomycin Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) Compound 17a+Vancomycin Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) S29

30 Compound 17b+Vancomycin Dispersion v. MRSA Dispersion (%) Compound Concentration (µm) S30

31 Full Colistin Repotentiation Results Colist in MIC 1 (19.5 µg/m L) E. coli ATCC m cr-1 8 µg/ml E. coli ATCC par ent 0.5 µg/ml A. bauman nii mcr µg/ml A. bauman nii pare nt 1 µg/ml A. bauman nii µg/ml A. bauman nii µg/ml K. pneumon iae C3 512 µg/ml K. pneumon iae B9 512 µg/ml A. bauman nii ATCC A. bauman nii 5075 K. pneumon iae ATCC 2146 NDM-1 1 µg/ml 1 µg/ml 1 µg/ml 4 (2) 0.5 (0) 0.5 (32) 0.5 (2) 128 (4) 16 (64) 2 (256) 16 (32) 0.5 (2) 0.25 (4) 0.5 (2) 9a (19.5 µg/m L) 4 (2) 0.5 (0) 1 (16) 0.5 (2) 256 (2) 256 (4) 4 (128) 16 (32) 0.5 (2) 0.5 (2) 0.5 (2) 9b (24.9 µg/m L) 2 (4) (8) 0.25 (64) (16) 4 (128) 4 (256) 1 (512) 2 (256) (16) (8) 0.25 (4) 9c (21.2 µg/m L) 4 (2) 0.25 (2) 2 (8) 0.25 (2) 128 (4) 32 (32) 64 (8) 512 (0) 0.25 (4) 0.25 (4) 1 (0) 9d (21.2 µg/m L) 0.5 (16) (8) (128) (16) 4 (128) 2 (512) 1 (512) 1 (512) (16) (16) (16) 9e (20.4 µg/m L) 1 (8) (4) 0.5 (32) 0.25 (2) 16 (32) 4 (256) 1 (512) 1 (512) (16) (8) 1 (0) 9f (22.4 µg/m L) 9g (22.4 µg/m L) 9h (16.9 µg/m L) 9i (17.0 µg/m L) 9j (19.5 µg/m L) 9k (19.5 µg/m L) 2 (4) 0.5 (0) 1 (16) 0.5 (2) 128 (4) 16 (64) 32 (16) 32 (16) 0.5 (2) 0.25 (4) 0.24 (4) 2 (4) 0.25 (2) 2 (8) 0.5 (2) 32 (16) 32 (32) 64 (8) 4 (128) 0.5 (2) 0.5 (2) 1 (0) 4 (2) 0.5 (0) 16 (0) 0.5 (2) 256 (2) 128 (8) 32 (16) 128 (4) 0.5 (2) 0.5 (2) 0.5 (2) 4 (2) 0.5 (0) 0.5 (32) 0.5 (2) 128 (4) 32 (32) 4 (128) 16 (32) 0.5 (2) 0.5 (2) 0.5 (2) 4 (2) 0.5 (0) 16 (0) 0.5 (2) 512 (0) 128 (8) 256 (2) 256 (2) 0.5 (2) 0.5 (2) 0.5 (2) 4 (2) 0.5 (0) 16 (0) 0.5 (2) 512 (0) 512 (2) 128 (4) 512 (0) 0.5 (2) 1 (0) 0.5 (2) S31

32 9l (15.9 µg/m L) 9m (15.9 µg/m L) 9n (15.6 µg/m L) 9o (14.8 µg/m L) 4 (2) 0.5 (0) 16 (0) 0.5 (2) 512 (0) 256 (4) 64 (8) 512 (0) 0.5 (2) 0.5 (2) 0.5 (2) 4 (2) 0.5 (0) 16 (0) 0.5 (2) 512 (0) 128 (8) 64 (8) 512 (0) 0.5 (2) 0.5 (2) 0.5 (2) 8 (0) 0.5 (0) 16 (0) 0.5 (2) 512 (0) 256 (4) 64 (8) 512 (0) 0.5 (2) 0.5 (2) 0.5 (2) 8 (0) 0.5 (0) 16 (0) 0.5 (2) 512 (0) 512 (2) 256 (2) 512 (0) 0.5 (2) 1 (0) 1 (0) 13a (22.1 µg/m L) 13b (22.9 µg/m L) 4 (2) 1 (8) 0.5 (0)** (16) 2 (8) 1 (0)** 32 (16) 32 (32) 8 (64)* 16 (32) 1 (16) (16) 32 (16) 32 (32) 8 (64) 8 (64) (16) (16) (8)* (16) 1 (0)* 1 (0) 13c (20.4 µg/m L) 1 (8) (8) 0.25 (64) 0.5 (2) 16 (32) 16 (64) 32 (16) 2 (256) (8) 0.25 (4) 0.25 (4) 13d (21.2 µg/m L) 4 (2) (4) 2 (8) (8) 32 (16) 32 (32) 4 (128) 4 (128) (8) (8) 1 (0) 13e (21.2 µg/m L) 2 (4) (4) 4 (4) (8) 32 (16) 32 (32) 8 (64) 8 (64) 0.25 (4) (8) 0.25 (4) 13f (22.1 µg/m L) 2 (4) (16) 4 (4) (16) 8 (64) 16 (64) 2 (256) 1 (512) (8) (128) (16) 13g (20.4 µg/m L) 2 (4) 0.25 (2) 8 (2) 0.25 (2) 128 (4) 64 (16) 32 (16) 64 (8) 0.25 (4) 0.5 (2) 0.25 (4) 13h (23.7 µg/m L) 4 (2) (4) 4 (4) 0.25 (2) 128 (4) 128 (4) 64 (8) 128 (4) 0.5 (2) 0.5 (2) 1 (0) 13i (24.9 µg/m L) 13j (22.9 µg/m L) 4 (2) 0.5 (0) 4 (4) 0.5 (2) 256 (2) 256 (4) 32 (16) 256 (2) 0.25 (4) 0.5 (2) 0.5 (2) 8 (0) 0.5 (0) 16 (0) 1 (0) 512 (0) 1024 (0) 256 (2) 512 (0) 1 (0) 0.5 (2) 1 (0) S32

33 13k (22.9 µg/m L) 1 (8) (16) 0.5 (32) (16) 32 (16) 32 (32) 32 (16) 8 (64) (16) (16) 1 (0) 13l (22.9 µg/m L) 4 (2) (8) 2 (8) 0.25 (2) 64 (8) 32 (32) 64 (8) 128 (4) 0.5 (2) 0.5 (2) 1 (0) 13m (24.9 µg/m L) 4 (2) (8) 2 (8) (4) 64 (8) 64 (16) 32 (16) 64 (8) (8) 0.25 (4) 1 (0) 13n (22.1 µg/m L) 17a (18.8 µg/m L) N/A N/A N/A N/A N/A N/A N/A 512 (0) N/A N/A N/A 2 (4)* 0.5 (0)* 16 (0) 1 (0)** 128 (4)* 32 (32)* 256 (2) 512 (0)** 2 (0)** 0.5 (2)* 1 (0)* 17b (18.8 µg/m L) 1 (8)* 0.5 (0)* 16 (0) 1 (0)** 512 (0)* 128 (8)* 256 (2)* 512 (0)* 2 (0)** 0.5 (2)* 1 (0)* * compound s MIC = 200 µm, ** compound s MIC = 100 µm, tested at 30 µm, compound s MIC = 50 µm, tested at 15 µm General Synthetic Procedures General synthetic procedure for compounds 6a-l: Compounds were synthesized according the procedure by MacKay et al. 1 General synthetic procedure for compounds 7a-l: Compounds were synthesized according the procedure by MacKay et al. 1 General synthetic procedure for compounds 8a-l: Compounds were synthesized according to the procedure by Fresnada et al. 2 General synthetic procedure for compounds 1, 9a, 9f-9o: Compounds were synthesized according to the procedure by Fresnada et al. 2 General synthetic procedure for Compounds 9b-9e: Compounds were synthesized according to the procedure by Fresnada et al. 2 with minor modifications. The crude solid was then purified with flash chromatography ( % MeOH-NH 3 /DCM). The solid was then washed with cold hexanes (3 x 3 ml) to yield the product as a yellow solid. General synthetic procedure for compounds 10a-k: To a solution of compound 5a-l (1.68 mmol) and tetrabutylammonium bromide ( mmol) in DCM (4 ml) and 50% sodium hydroxide (2 ml) at room temperature, the desired alkyl halide (3.36 mmol) was added dropwise. The reaction was allowed to stir for 24 hours at room temperature. After completion, the solution was washed with water (1 x 10 ml), and brine (1 x 10 ml), dried (MgSO 4 ) and concentrated under reduced pressure. The residue was purified using flash column chromatography (15-50% EtOAc/Hex) to yield the product as a white solid. General synthetic procedure for compounds 11a-b: To a stirring solution of compound 5a or 5l (1.68 mmol) and potassium carbonate (8.40 mmol) in acetone at reflux, 1-bromo-2-methylpropane (2.52 mmol) was added dropwise. The reaction was allowed to stir overnight for 16 hours. After completion, the S33

34 reaction mixture was concentrated under reduced pressure, and diluted in dichloromethane (20 ml). The insoluble impurities were removed by vacuum filtration, and the filtrate was dried (MgSO 4 ) and concentrated under reduced pressure. The residue was purified using flash column chromatography (15-25% EtOAc/Hex) to the product as a white solid. General synthetic procedure for compounds 12a-m: Compounds were synthesized according to the procedure of Simon et al. 3 General synthetic procedure for compounds 13a-m: Compounds were synthesized according to the procedure by Fresnada et al. 2 with minor modifications. Compounds were purified by recrystallization in chloroform after which the solid residue was isolated using vacuum filtration to yield the product as a yellow solid. General synthetic procedure for compounds 14a-b: Compounds were synthesized according to the procedure of Roy et al. 4 General synthetic procedure for compounds 15a-b: A solution of compound 13a-b (3.00 mmol), boc-anhydride (4.51 mmol), and 4-dimethylaminopyridine (0.3 mmol) in THF (10 ml) was allowed to stir at rt for 4 hours. After completion, the solvent was removed under reduced pressure and the reaction mixture was dissolved in DCM (10 ml). The organic layer was washed with water (1 x 20 ml) and brine (1 x 20 ml), dried with MgSO 4, and concentrated under reduced pressure. The crude reside was then purified using flash column chromatography (10-30% EtOAc/ Hex) to yield the product as a white solid. General Synthetic Procedure for the N-boc-3-(N-boc-2-aminoimidazoles) (Compounds 17ab): A solution of compound 15a-b (2.07 mmol), sodium iodide (8.27 mmol), and N-boc guanidine (12.40 mmol) in DMF (25 ml) was allowed to stir at rt for 48 hours. After completion, the reaction mixture was washed with water (75 ml) and the aqueous layer was extracted with ethyl acetate (3 x 25 ml). The organic layer was then washed with brine (1 x 30 ml), dried with MgSO 4, and concentrated under reduced pressure. The crude residue purified using flash chromatography (10-30% EtOAc/Hex) to yield the product as a white solid. The product was dried under high vacuum for two hours. The cyclized protected product was then placed under an inert atmosphere (0.274 mmol) and a solution in DCM (3.2 ml) was made at 0 C. Trifluoracetic acid (0.8 ml) was then added dropwise, and the solution was allowed to stir for 16 hours overnight warming to rt. After completion, the precipitate was isolated by vacuum filtration and washed with cold DCM (3 x 3 ml) to yield the product as a brown solid (80%). S34

35 Previously Reported Compounds 4-(6-Bromo-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (1): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 1-(5-Bromo-1H-indol-3-yl)ethan-1-one (6a): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 1-(6-Iodo-1H-indol-3-yl)ethan-1-one (6b): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 6 1-(5-Chloro-1H-indol-3-yl)ethan-1-one (6d): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 7 1-(6-Chloro-1H-indol-3-yl)ethan-1-one (6e): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 8 S35

36 1-(7-Bromo-1H-indol-3-yl)ethan-1-one (6f): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 5 1-(4-Bromo-1H-indol-3-yl)ethan-1-one (6g): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 1 1-(6-Fluoro-1H-indol-3-yl)ethan-1-one (6i): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 9 1-(1H-Indol-3-yl)ethan-1-one (6k): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 5 1-(6-Bromo-1H-indol-3-yl)ethan-1-one (6l): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 S36

37 1-(5-Bromo-1-tosyl-1H-indol-3-yl)ethan-1-one (7a): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 1-(5-Chloro-1-tosyl-1H-indol-3-yl)ethan-1-one (7d): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 1-(6-Chloro-1-tosyl-1H-indol-3-yl)ethan-1-one (7e): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 1-(7-Bromo-1-tosyl-1H-indol-3-yl)ethan-1-one (7f): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 5 1-(4-Bromo-1-tosyl-1H-indol-3-yl)ethan-1-one (7g): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 1 S37

38 1-(6-Fluoro-1-tosyl-1H-indol-3-yl)ethan-1-one (7i): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 1-(1-Tosyl-1H-indol-3-yl)ethan-1-one (7k): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports (6-Bromo-1-tosyl-1H-indol-3-yl)ethan-1-one (7l): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 (E)-1-(5-Bromo-1-tosyl-1H-indol-3-yl)-3-(dimethylamino)prop-2-en-1-one (8a): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 (E)-1-(5-Chloro-1-tosyl-1H-indol-3-yl)-3-(dimethylamino)prop-2-en-1-one (8d): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 S38

39 (E)-1-(6-Chloro-1-tosyl-1H-indol-3-yl)-3-(dimethylamino)prop-2-en-1-one (8e): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 (E)-1-(7-Bromo-1-tosyl-1H-indol-3-yl)-3-(dimethylamino)prop-2-en-1-one (8f): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 5 (E)-1-(4-Bromo-1-tosyl-1H-indol-3-yl)-3-(dimethylamino)prop-2-en-1-one (8g): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 5 (E)- 3-(Dimethylamino)-1-(6-fluoro-1-tosyl-1H-indol-3-yl)prop-2-en-1-one (8i): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 (E)-3-(Dimethylamino)-1-(1-tosyl-1H-indol-3-yl)prop-2-en-1-one (8k): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 S39

40 (E)-1-(6-Bromo-1-tosyl-1H-indol-3-yl)-3-(dimethylamino)prop-2-en-1-one (8l): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 4-(5-Bromo-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9a): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 2 4-(5-Chloro-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9h): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 4-(6-Chloro-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9i): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 S40

41 4-(7-Bromo-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9j): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 5 4-(4-Bromo-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9k): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 5 4-(5-Fluoro-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9l): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 4-(6-Fluoro-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9m): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 S41

42 4-(1H-Indol-3-yl)pyrimidin-2-amine hydrochloride (9o): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 1-(6-Bromo-1-methyl-1H-indol-3-yl)ethan-1-one (10a): Compound was synthesized using the general procedure for N-alkylation of 3-acetylindoles. Spectral data was consistent with previous reports (5-Bromo-1-methyl-1H-indol-3-yl)ethan-1-one (10f): Compound was synthesized using the general procedure for N-alkylation of 3-acetylindoles. Spectral data was consistent with previous reports (5-Bromo-1-ethyl-1H-indol-3-yl)ethan-1-one (10g): Compound was synthesized using the general procedure for N-alkylation of 3-acetylindoles. Spectral data was consistent with previous reports (5-Bromo-1-methyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13g): Compound was synthesized using previously reported methods. 5 Spectral data was consistent with previous reports. 3 S42

43 1-(5-Bromo-1H-indol-3-yl)-2-chloroethan-1-one (14a): Compound was synthesized using previously reported methods. 4 1 H NMR spectral data confirmed the structure of 3-chloroacetylindole intermediate 14a and were in accordance with literature (6-Bromo-1H-indol-3-yl)-2-chloroethan-1-one (14b): Compound was synthesized using previously reported methods. 4 1 H NMR spectral data confirmed the structure of 3-chloroacetylindole intermediate 14b and were in accordance with literature. 14 S43

44 Novel Compound Characterization 1-(5-Iodo-1H-indol-3-yl)ethan-1-one (6c): The title compound was synthesized from 5c following the general procedure to afford 6c as a white solid (0.600g, 72%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.53 (s, 1H), 8.30 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 2.44 (s, 3H) ppm; 13 C NMR (100 MHz, DMSO-d 6 ) δ 192.8, 135.8, 135.2, 130.8,129.7, 127.8, 115.9, 114.6, 86.1, 27.2 ppm; UV (λ max nm) 300; IR v max (cm -1 ) 1733, 1662, 1522, 1425, 1286, 876, 424; HRMS (ESI) calcd for C 10 H 8 INO [M+H] , found (6-Iodo-1-tosyl-1H-indol-3-yl)ethan-1-one (7b): The title compound was synthesized from 6b following the general procedure to afford 7b as a white solid (0.350g, 98%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.29 (s, 1H), 8.11 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 11.4 Hz, 2H), 7.60 (dd, J = 5.4, 1.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 193.3, 146.4, 135.8, 134.3, 133.9, 132,3, 130.5, 127.2, 127.0, 124.7, 122.0, 90.4, 27.9, 21.8 ppm; UV (λ max nm) 290; IR v max (cm -1 ) 3105, 1659, 1533, 1379, 743; HRMS (ESI) calcd for C 17 H 14 INO 3 S [M+H] , found (5-Iodo-1-tosyl-1H-indol-3-yl)ethan-1-one (7c): The title compound was synthesized from 6c following the general procedure to afford 7c as a white solid (0.370g, 77%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.66 (s, 1H), 8.15 (s, 1H), 7.80 (d, J = 9.0 Hz, 2H), 7.66 (m, 2H), 7.26 (d, J = 11.4, 2H), 2.54 (s, 3H), 2.32 (s, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 193.1, 146.3, 134.3, 134.2, 134.1, 132.6, 131.9, 130.4, 129.5, , 114.8, 89.6, 27.8, 21.7 ppm; UV (λ max nm) 300; IR v max (cm -1 ) 1664, 1535, 1294, 1158, 746; HRMS (ESI) calcd for C 17 H 14 INO 3 S [M+H] , found (E)-3-(Dimethylamino)-1-(6-iodo-1-tosyl-1H-indol-3-yl)prop-2-en-1-one (8b): The title compound was synthesized from 7b following the general procedure to afford 8b as a yellow solid (0.406g, 46%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.24 (s, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.93 (s, 1H), 7.73 (d, J = 8.7 Hz, 2H), S44

45 7.66 (s, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 5.53 (d, J = 12.3 Hz, 1H), 3.02 (brs, 3H), 2.84 (m, 3H), 2.23 (s, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 183.2, 153.1, 145.7, 135.8, 134.3, 133.0, 130.1, 128.3, 128.2, 126.8, 124.8, 123.3, 121.7, 92.9, 89.5, 21.5 ppm; UV (λ max nm) 350; IR v max (cm -1 ) 1635, 1524, 1283, 798; HRMS (ESI) calcd for C 20 H 19 IN 2 O 3 S [M+H] , found (E)-3-(Dimethylamino)-1-(5-iodo-1-tosyl-1H-indol-3-yl)prop-2-en-1-one (8c): The title compound was synthesized from 7c following the general procedure to afford 8c as a yellow solid (0.375g, 46%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.72 (s, 1H), 8.00 (s, 1H), 7.76 (d, J = 9.9 Hz, 3H), 7.67 (d, J = 8.7, 1H), 7.57 (d, J = 7.2, 1H), 7.22 (d, J = 8.1 Hz, 2H), 5.55 (d, J = 12.3 Hz, 1H), 3.10 (brs, 3H), 2.92 (m, 3H), 2.31 (s, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) 183.2, 153.3, 145.7, 134.4, 134.2, 133.5, 132.1, 131.0, 130.1, 128.7, 126.9, 122.7, 114.7, 92.9, 88.8, 29.2, 21.5 ppm; UV (λ max nm) 346; IR v max (cm -1 ) 1635, 1527, 1366, 774; HRMS (ESI) calcd for C 20 H 19 IN 2 O 3 S [M+H] , found (E)-3-(Dimethylamino)-1-(5-methyl-1-tosyl-1H-indol-3-yl)prop-2-en-1-one (8j): The title compound was synthesized from 7j following the general procedure to afford 8j as a yellow solid (0.388g, 51%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.13 (s, 1H), 8.05 (s, 1H), 7.76 (m, 4H), 7.14 (m, 3H), 5.59 (d, J = 12.6, 1H), 3.02 (brs, 6H), 2.38 (s, 3H), 2.20 (s, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 184.1, 153.0, 145.3, 134.8, 133.3, 130.0, 129.1, 128.5, 126.9, 126.5, 123.6, 123.1, 112.7, 93.4, 21.5 ppm; UV (λ max nm) 348; IR v max (cm -1 ) 1635, 1525, 1309, 726; HRMS (ESI) calcd for C 21 H 22 N 2 O 3 S [M+H] , found N-Benzyl-4-(5-bromo-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9b): The title compound was synthesized from 8a following the general procedure to afford 9b as a white Solid (0.080g, 39%). 1 H NMR (400 MHz, CD 3 OD) δ 8.63 (s, 1H), 8.17 (s, 1H), 8.14 (s, 1H), 8.02 (d, J = 6.0 Hz, 1H), 7.47 (d, J = 7.2 Hz, 1H), 7.33 (m, 4H), 7.24 (d, J = 7.2 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 4.75 (s, 2H) ppm; 13 C NMR (100 MHz, CD 3 OD) δ 166.5, 165.6, 152.7, 140.5, 137.6, 132.3, 129.6, 128.4, 128.2, 128.0, 126.7, 126.0, S45

46 115.8, 114.9, 114.5, 106.5, 45.9 ppm; UV (λ max nm) 360; IR v max (cm -1 ) 1630, 1523, 1288, 799; HRMS (ESI) calcd for C 19 H 15 BrN 4 [M+H] , found (5-Bromo-1H-indol-3-yl)-N,N-dimethylpyrimidin-2-amine hydrochloride (9c): The title compound was synthesized from 8a following the general procedure to afford 9c as a yellow solid (0.102g, 52%). 1 H NMR (300 MHz, CD 3 OD) δ 8.68 (d, J = 1.5 Hz, 1H), 8.07 (d, J = 5.1 Hz, 1H), 7.99 (s, 1H), 7.33 (d, 8.7 Hz, 1H), 7.27 (dd, J = 8.7, 1.5 Hz, 1H), 6.87 (d, J = 5.7 Hz, 1H), 3.21 (s, 6H) ppm; 13 C NMR (175 MHz, Acetone-d 6 ) δ 162.7, 161.9, 156.9, 136.1, 128.8, 127.6, 124.9, 124.8, 114.7, 113.6, 104.1, 36.4 ppm; UV (λ max nm) 360; IR v max (cm -1 ) 2822, 1609, 1521, 1290, 796; HRMS (ESI) calcd for C 14 H 13 BrN 4 [M+H] , found (5-Bromo-1H-indol-3-yl)-N-ethylpyrimidin-2-amine hydrochloride (9d): The title compound was synthesized from 8a following the general procedure to afford 9d as a white Solid (0.120 g, 26%). 1 H NMR (400 MHz, CD 3 OD) δ 8.73 (s, 1H), 8.05 (s, 1H), 8.00 (s, 1H), 7.33 (d, J = 6.0, 1H), 7.29 (d, J = 6.0 Hz, 1H) 6.94 (d, J = 5.7 Hz, 1H), 3.52 (q, J = 6.8 Hz, 2H), 1.31 (t, J = 6.8 Hz, 3H) ppm; 13 C NMR (175 MHz, CD 3 OD) δ 165.3, 162.6, 155.7, 137.5, 130.8, 128.6, 126.3, 126.0, 115.3, 115.1, 114.3, 106.0, 37.2, 15.2 ppm; UV (λ max nm) 358; IR v max (cm -1 ) 2915, 1629, 1520, 1282, 792; HRMS (ESI) calcd for C 14 H 13 BrN 4 [M+H] , found (5-Bromo-1H-indol-3-yl)-N-methylpyrimidin-2-amine hydrochloride (9e): The title compound was synthesized from 8a following the general procedure to afford 9e as a white Solid (0.088 g, 36%). 1 H NMR (300 MHz, CD 3 OD) δ 8.72 (s, 1H), 8.03 (d, J = 5.7 Hz, 1H), 7.99 (s, 1H), 7.30 (m, 2H), 6.89 (d, J = 5.1 Hz, 1H), 3.01 (s, 3H) ppm; 13 C NMR (100 MHz, CD 3 OD) δ 164.9, 163.9, 156.7, 137.4, 130.3, 128.6, S46

47 126.2, 126.0, 115.1, 114.2, 106.1, 28.6 ppm; UV (λ max nm) 358; IR v max (cm -1 ) 2917, 1633, 1523, 1282, 795; HRMS (ESI) calcd for C 13 H 11 BrN 4 [M+H] , found (6-Iodo-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9f): The title compound was synthesized from 8b following the general procedure to afford 9f as a yellow solid (0.200 g, 41%). 1 H NMR (400 MHz, DMSO-d 6 ) δ (s, 1H), 8.70 (s, 1H), 8.46 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 4.8, 1H), 7.91 (s, 1H), 7.49 (d, J = 8.4, 1H), 7.42 (d, J = 5.2, 1H) ppm; 13 C NMR (100 MHz, DMSO-d 6 ) δ 168.0, 155.5, 143.4, 138.9, 135.2, 130.3, 124.8, 124.5, 121.2, 112.5, 105.2, 87.8 ppm; UV (λ max nm) 360; IR v max (cm -1 ) 1633, 1526, 1179, 801; HRMS (ESI) calcd for C 12 H 9 IN 4 [M+H] , found (5-Iodo-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9g): The title compound was synthesized from 8c following the general procedure to afford 9g as a tan solid (0.158 g, 53%). 1 H NMR (400 MHz, DMSO-d 6 ) δ (brs, 1H), 8.92 (s, 1H), 8.71 (s, 1H), 8.16 (s, 1H), 7.53 (d, J = 5.1 Hz, 1H), 7.38 (d, J = 6.3 Hz, 2H) ppm; 13 C NMR (100 MHz, DMSO-d 6 ) δ 167.9, 155.4, 143.1, 136.7, 135.4, 131.6, 130.6, 127.4, 114.9, 111.7, 105.2, 87.1 ppm; UV (λ max nm) 358; IR v max (cm -1 ) 1632, 1529, 1289, 785; HRMS (ESI) calcd for C 12 H 9 IN 4 [M+H] , found (5-Methyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (9n): The title compound was synthesized from 8j following the general procedure to afford 9n as an orange solid (0.177 g, 53%). 1 H NMR (300 MHz, CD 3 OD) δ 8.37 (s, 1H), (s, 1H), 7.82 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.20 (s, 1H), 7.09 (s, J = 7.8 Hz, 1H), 2.46 (s, 3H) ppm; 13 C NMR (100 MHz, CD 3 OD) δ 170.3, 156.5, 143.2, 137.3, 135.9, 133.1, 126.7, 126.2, 123.5, 113.9, 112.8, 107.0, 21.8 ppm; UV (λ max nm) 362; IR v max (cm -1 ) 2917, 1632, 1207, 788; HRMS (ESI) calcd for C 13 H 12 N 4 [M+H] , found S47

48 1-(6-Bromo-1-ethyl-1H-indol-3-yl)ethan-1-one (10b): The title compound was synthesized from 6l following the general procedure to afford 10b as a white solid (0.420 g, 84%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.20 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H), 7.43 (s, 1H), 7.34 (dd, J = 9.2, 0.8 Hz, 1H), 4.08 (q, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.46 (t, J = 7.6, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 192.7, 137.3, 134.4, 125.6, 125.2, 123.9, 117.0, 116.8, 112.8, 41.8, 27.5, 15.1 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 2910, 1615, 1560, 1250, 862; HRMS (ESI) calcd for C 12 H 12 BrNO [M+H] , found (6-Bromo-1-propyl-1H-indol-3-yl)ethan-1-one (10c): The title compound was synthesized from 6l following the general procedure to afford 10c as a white solid (0.450 g, 79%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.22 (d, J = 8.4 Hz, 1H) 7.63 (s, 1H), 7.44 (d, J = 1.6, 1H), 7.34 (dt, J = 8.4, 0.8 Hz, 1H), 3.99 (t, J = 7.2 Hz, 2H), 2.47 (s, 3H), 1.86 (q, J = 7.2 Hz, 2H), 0.92 (t, J = 7.2 Hz, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 192.8, 137.6, 135.2, 125.5, 125.2, 123.9, 116.8, 112.9, 48.8, 27.5, 23.1, 11.3 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 2908, 1640, 1572, 1275, 870; HRMS (ESI) calcd for C 13 H 14 BrNO [M+H] , found (6-Bromo-1-butyl-1H-indol-3-yl)ethan-1-one (10d): The title compound was synthesized from 6l following the general procedure to afford 10d as a white solid (0.467 g, 85%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.21 (d, J = 8.0, 1H), 7.62 (s, 1H), 7.42 (d, J = 1.6 Hz, 1H), 7.33 (dd, J = 8.4, 1.6 Hz, 1H), 3.99 (t, J = 7.6 Hz, 2H), 2.44 (s, 3H), 1.77 (m, 2H), 1.32 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 192.7, 137.5, 135.1, 125.4, 125.1, 123.8, 116.8, 116.7, 112.8, 46.8, 31.8, 27.4, 20.0, 13.6 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 2924, 1629, 1530, 1466, 1204, 736; HRMS (ESI) calcd for C 14 H 16 BrNO [M+H] , found S48

49 1-(1-Benzyl-6-bromo-1H-indol-3-yl)ethan-1-one (10e): The title compound was synthesized from 6l following the general procedure to afford 10e as a light orange solid (0.492 g, 75%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.28 (d, J = 8.1, 1H), 7.68 (s, 1H), 7.43 (s, 1H), 7.36 (m, 5H), 7.14 (m, 1H), 5.27 (s, 2H), 2.48 (s, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 193.0, 138.1, 135.4, 129.3, 128.6, 127.1, 126.1, 125.4, 124.2, 117.6, 117.3, 113.2, 50.8, 27.7 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 1632, 1525, 1470, 1230, 744; HRMS (ESI) calcd for C 17 H 14 BrNO [M+H] , found (5-Bromo-1-propyl-1H-indol-3-yl)ethan-1-one (10h): The title compound was synthesized from 6a following the general procedure to afford 10h as a white solid (0.490 g, 42%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.49 (s, 1H), 7.65 (s, 1H), 7.31 (dd, J = 7.8, 1.2, 1H), 7.15 (d, J = 8.4 Hz, 1H), 4.03 (t, J = 6.9 Hz, 2H), 2.44 (s, 3H), 1.89 (sept, J = 6.9 Hz, 2H), 0.91 (t, J = 7.2 Hz, 3H) ppm; 13 C NMR (100 MHz, CDCl 3 ) δ 192.6, 135.5, 135.5, 127.9, 126.1, 125.1, 116.3, 116.0, 111.3, 48.9, 27.5, 23.2, 11.4 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 2926, 1637, 1520, 1208, 794; HRMS (ESI) calcd for C 13 H 14 BrNO [M+H] , found (5-Bromo-1-butyl-1H-indol-3-yl)ethan-1-one (10i): The title compound was synthesized from 6a following the general procedure to afford 10i as a white solid (0.398 g, 80%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.49 (s, 1H), 7.65 (s, 1H), 7.31 (d, J = 9.2 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 4.06 (t, J = 6.8, 2H), 2.45 (s, 3H), 1.82 (m, 2H), 1.33 (m, 2H), 0.93 (t, J = 6.8 Hz, 3H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 192.6, 135.5, 127.8, 126.0, 125.1, 116.2, 116.0, 111.3, 47.0, 31.9, 27.4, 20.1, 13.6 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 3330, 2870, 1655, 1564, 1285, 872; HRMS (ESI) calcd for C 14 H 16 BrNO [M+H] , found S49

50 1-(5-Bromo-1-pentyl-1H-indol-3-yl)ethan-1-one (10j): The title compound was synthesized from 6a following the general procedure to afford 10h as a yellow oil (0.430 g, 85%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.45 (s, 1H), 7.62 (s, 1H), 7.25 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 3.99 (t, J = 6.3 Hz, 2H), 1.79 (m, 2H), 1.29 (m, 4H), 0.82 (t, J = 6.3 Hz, 3H) ppm; 13 C NMR (75 MHz,CDCl 3 ) δ 192.5, 135.5, 127.7, 125.8, 124.8, 116.0, 115.8, 111.2, 47.1, 29.4, 28.8, 27.3, 22.1, 13.8 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 2918, 1640, 1520, 1208, 792; HRMS (ESI) calcd for C 15 H 18 BrNO [M+H] , found (6-Iodo-1-propyl-1H-indol-3-yl)ethan-1-one (10k): The title compound was synthesized from 6b following the general procedure to afford 10k as a white solid (0.325 g, 81%). 1 H NMR (300 MHz, CDCl 3 ) δ (d, J = 8.4 Hz, 1H), 7.63 (s, 1H), 7.58 (s, 1H), 7.51 (dd, J = 8.4 Hz, 1.2 Hz, 1H), 3.98 (t, J = 6.9 Hz, 2H), 2.45 (s, 3H), 1.85 (m, 2H), 0.91 (t, J = 7.5 Hz, 3H) ppm; 13 C NMR (75 MHz,CDCl 3 ) δ 192.8, 137.9, 135.0, 131.1, 125.6, 124.2, 118.9, 116.8, 87.3, 48.7, 27.6, 23.1, 11.4 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 2910, 1632, 1524, 1208, 800; HRMS (ESI) calcd for C 13 H 14 INO [M+H] , found (5-Bromo-1-isobutyl-1H-indol-3-yl)ethan-1-one (11a): The title compound was synthesized from 6a following the general procedure to afford 11a as a white solid (0.300 g, 52%). 1 H NMR (300 MHz, CDCl 3 ) δ 8.77 (d, J = 1.5 Hz, 1H), 7.91 (s, 1H), 7.57 (dd, J = 8.7 Hz, 1.5 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 4.13 (d, J = 7.5 Hz, 2H), 2.71 (s, 3H), 2.41 (m, 1H), 1.17 (d, J = 6.6 Hz, 6H) ppm; 13 C NMR (75 MHz,CDCl 3 ) δ 192.7, 169.4, 136.0, 135.6, 127.7, 126.0, 125.0, 116.0, 111.5, 54.7, 29.2, 27.4, 20.1 ppm; S50

51 UV (λ max nm) 298; IR v max (cm -1 ) 2928, 1630, 1525, 1212, 798; HRMS (ESI) calcd for C 14 H 16 BrNO [M+H] , found (6-Bromo-1-isobutyl-1H-indol-3-yl)ethan-1-one (11b): The title compound was synthesized from 6l following the general procedure to afford 11b as a white solid (0.323 g, 73%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.19 (d, J = 8.8 Hz, 1H), 7.57 (s, 1H), 7.37 (s, 1H), 7.28 (d, J = 8.4 Hz, 1H), 3.75 (d, J = 7.2 Hz, 2H), 2.46 (s, 3H), 2.09 (sept, J = 7.2 Hz, 1H), 0.83 (d, J = 9.6 Hz, 6H) ppm; 13 C NMR (100 MHz,CDCl 3 ) δ 192.6, 137.6, 135.7, 125.3, 124.9, 123.7, 116.6, 116.5, 112.9, 54.4, 28.9, 27.3, 19.9 ppm; UV (λ max nm) 298; IR v max (cm -1 ) 2928, 1630, 1525, 1212, 798; HRMS (ESI) calcd for C 14 H 16 BrNO [M+H] , found (6-Bromo-1-propyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13a): The title compound was synthesized from 10c following the general procedure to afford 13a as a brown solid (0.130 g, 61%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.82 (s, 1H), 8.62 (d, J = 8.8 Hz, 1H), 8.20 (d, J = 6.8 Hz, 1H), 7.98 (s, 1H), 7.40 (dd, J = 8.8 Hz, 1.6 Hz, 1H), 7.35 (d, J = 6.8 Hz, 1H), 4.25 (t, J = 6.8 Hz, 2H), 1.84 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H) ppm; 13 C NMR (175 MHz, DMSO-d 6 ) δ 168.3, 156.1, 144.1, 139.2, 125.9, 125.3, 117.1, 114.9, 112.4, 105.7, 48.8, 23.5, 11.7 ppm; UV (λ max nm) 364; IR v max (cm -1 ) 2910, 1618, 1530, 1487, 1212, 800; HRMS (ESI) calcd for C 15 H 15 BrN 4 [M+H] , found (5-Bromo-1-butyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13b): The title compound was synthesized from 10i following the general procedure to afford 13b as a tan solid (0.056 g, 20%). 1 H S51

52 NMR (400 MHz, DMSO-d 6 ) δ 8.86 (s, 1H), 8.81 (d, J = 2.0 Hz, 1H), 8.21 (d, J = 7.2 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.46 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.36 (d, J = 7.2 Hz, 1H), 4.27 (t, J = 7.2 Hz, 2H), 1.80 (m, 2H), 1.28 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H) ppm; 13 C NMR (175 MHz, DMSO-d 6 ) δ 168.2, 156.1, 144.0, 139.4, 137.0, 127.9, 126.8, 126.0, 116.4, 114.0, 111.8, 105.7, 47.2, 32.1, 20.1, 14.2 ppm; UV (λ max nm) 364; IR v max (cm -1 ) 2914, 1610, 1522, 1492, 1224, 788; HRMS (ESI) calcd for C 16 H 17 BrN 4 [M+H] , found (6-Bromo-1-methyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13c): The title compound was synthesized from 10f following the general procedure to afford 13c as a yellow solid (0.076 g, 38%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.75 (s, 1H), 8.59 (d, J = 5.6 Hz, 1H), 8.20 (d, J = 6.8, 1H), 7.89 (s, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 6.4 Hz, 1H), 3.90 (s, 3H) ppm; 13 C NMR (175 MHz, DMSO-d 6 ) δ 168.1, 156.3, 144.3, 139.7, 139.6, 125.8, 125.3, 117.0, 114.9, 112.2, ppm; UV (λ max nm) 364; IR v max (cm -1 ) 2903, 1625, 1530, 1494, 1217, 799; HRMS (ESI) calcd for C 13 H 11 BrN 4 [M+H] , found (6-Bromo-1-ethyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13d): The title compound was synthesized from 10b following the general procedure to afford 13d as a brown Solid (0.045 g, 20%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.81 (s, 1H), 8.60 (d, J = 7.6 Hz, 1H), 8.18 (m, 1H), 7.95 (s, 1H), 7.39 (m, 2H), 4.31 (q, J = 6.8 Hz, 2H), 1.42 (t, J = 6.8 Hz, 3H) ppm; 13 C NMR (175 MHz, DMSO-d 6 ) δ 168.2, 156.2, 144.3, 138.9, 125.9, 125.4, 117.1, 114.8, 112.5, 105.8, 42.3, 15.8 ppm; UV (λ max nm) 364; IR v max (cm -1 ) 2910, 1622, 1535, 1496, 1215, 804; HRMS (ESI) calcd for C 14 H 13 BrN 4 [M+H] , found S52

53 4-(5-Bromo-1-ethyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13e): The title compound was synthesized from 10g following the general procedure to afford 13e as a tan solid (0.072 g, 38%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.4 (s, 1H), 8.81 (s, 1H), 8.18 (d, J = 7.2 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 6.4 Hz, 1H), 4.32 (q, J = 6.8, 2H), 1.44 (t, J = 7.8 Hz, 3H) ; 13 C NMR (175 MHz, DMSO-d 6 ) δ 168.0, 155.8, 143.7, 138.2, 136.5, 127.7, 126.6, 125.5, 116.2, 113.7, 111.6, 105.4, 42.2, 15.4 ppm; UV (λ max nm) 364; IR v max (cm -1 ) ) 2907, 1600, 1535, 1500, 1210, 782; HRMS (ESI) calcd for C 14 H 13 BrN 4 [M+H] , found (5-Bromo-1-propyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13f): The title compound was synthesized from 10h following the general procedure to afford 13f as a tan solid (0.081 g, 38%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.85 (s, 1H), 8.82 (s, 1H), 8.20 (d, J = 7.2, 1H), 7.67 (d, J = 8.0, 1H), 7.46 (d, J = 8.8, 1H), 7.35 (dd, J = 6.4 Hz, 1.6 Hz, 1H), 4.25 (t, J = 6.4 Hz, 2H), 1.85 (sept, J = 6.8, 2H), 0.85 (t, J = 5.6 Hz, 3H) ppm; 13 C NMR (175 MHz, DMSO-d 6 ) δ 168.0, 155.9, 143.8, 138.8, 136.8, 127.7, 126.6, 125.5, 116.1, 113.8, 111.5, 105.4, 48.7, 23.2, 11.5 ppm; UV (λ max nm) 364; IR v max (cm -1 ) ) 2914, 1608, 1525, 1490, 1212, 780; HRMS (ESI) calcd for C 15 H 15 BrN 4 [M+H] , found (5-Bromo-1-pentyl-1H-indol-3-yl)pyrimidin-2-amine hydrochloride (13h): The title compound was synthesized from 10j following the general procedure to afford 13h as a pink solid (0.140 g, 38%). 1 H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 2H), 8.17 (d, J = 6.8 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.34 (d, J = 6.8 Hz, 1H), 4.28 (t, J = 6.4 Hz, 2H), 1.81 (m, 2H), 1.26 (m, 4H), 0.82 (dd, J = 7.2 Hz, 1.6 Hz, 3H) ppm; 13 C NMR (175 MHz, DMSO-d6) δ 168.0, 155.9, 144.0, 138.6, 136.8, 127.7, 126.6, 125.5, 116.1, 113.7, 111.5, 105.5, 47.2, 29.4, 28.7, 22.1, 14.3 ppm; UV (λ max nm) 364; IR v max (cm - 1 ) 2908, 1616, 1523, 1488, 1222, 790; HRMS (ESI) calcd for C 17 H 19 BrN 4 [M+H] , found S53