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Compound 7 (2 g, 6.34 mmol, 1.0 equiv) was dissolved in 15 mL of EtOH and hydrazine hydrate (7.5 mL, 154 mmol, 23 equiv) was added. The mixture was.
SUPPORTING INFORMATION

Synthetic lethality triggered by combining Olaparib with BRCA2-Rad51 disruptors

Federico Falchi,1 Elisa Giacomini,1 Tiziana Masini,1 Nicolas Boutard,1 Lorenza Di Ianni,2 Marcella Manerba,2 Fulvia Farabegoli,3 Lara Rossini,4 Janet Robertson,4 Saverio Minucci,5,6 Isabella Pallavicini,5 Giuseppina Di Stefano,2 Marinella Roberti,3 Roberto Pellicciari,4 and Andrea Cavalli*1,3

1

CompuNet, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova, Italy.

2

Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via S.

Giacomo 14, 40126 Bologna, Italy. 3

Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126

Bologna, Italy. 4

TES Pharma S.r.l., Via Palmiro Togliatti 22bis, I-06073 Loc. Terrioli, Corciano, Perugia, Italy.

5

Department of Experimental Oncology at the European Institute of Oncology, IFOM-IEO Campus,

Via Adamello 16, 20100 Milan, Italy. 6

Department of Biosciences, University of Milan, Via Celoria 26, 20100 Milan, Italy.

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Table of Contents: Figure S1. Combination index between Olaparib and Compound 2, measured in BxPC-3 and Capan-1 cells. ................................................................................................................................. 3 Figure S2. Stable Rad51 Silencing. ................................................................................................. 3 Figure S3. Transient silencing. Expression of RAD51 assessed by RT-PCR in BxPC-3 cells treated with scalar doses of siRNA. ............................................................................................................ 4 Figure S4. Blotted PVDF membranes showing γH2AX and Actin signals in treated cultures. ......... 5 Virtual Screening ............................................................................................................................ 6 Protein Preparation .................................................................................................................. 6 Database Preparation ............................................................................................................... 6 High Throughput Docking ....................................................................................................... 6 Synthetic Procedures ....................................................................................................................... 7 Synthetic plan.......................................................................................................................... 7 General experimental details ................................................................................................... 8 Synthetic procedures to obtain compounds 1-14 ...................................................................... 9 References .................................................................................................................................... 16

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Figure S1. Combination index between Olaparib and compound 2, measured in BxPC-3 and Capan-1 cells.

Figure S1. Combination index was calculated according to the procedure reported in ref.1. For each combination dose, the reported values are means ± SE of the results measured from day 3 to day 6. As explained in Materials and Methods, a result ranging from 0.8 to 1.2 denotes an additive effect. Synergism is indicated by a result < 0.8; antagonism by a result > 1.2.

Figure S2. Stable Rad51 Silencing.

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Figure S2. (a) Immunoblot analysis of the protein levels of RAD51 (or vinculin as a loading control) after transfection of BxPC3 cells transduced with the control (pLKO scr) or sh-RNA targeting Rad51 (shRNA 77). (b) densitometric analysis of the western blot shown in panel (a). Figure S3. Transient silencing. Expression of RAD51 assessed by RT-PCR in BxPC-3 cells

RAD51 Expression

treated with scalar doses of siRNA.

100 p < 0.01, vs CTR 75 50 25

NA si 50 R nM N A 10 0n M

25 nM si R

NA

10 nM si R

SC R si R

N A

C

TR

0

Figure S3. Experimental details are reported in Materials and Methods; the statistical evaluation was performed by ANOVA followed by Dunnet’s post-test.

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Figure S4. Blotted PVDF membranes showing γH2AX and Actin signals in treated cultures.

Figure S4. Treatments of cell cultures and Western Blot procedure are described in Materials and Methods. 1: Control; 2: Cisplatin, 24h; 3: Cisplatin, 48h; 4: 2 20 µM, 48h; 5: 2 30 µM, 48h; 6: Cisplatin + 2 20 µM, 48h; 7: Cisplatin + 2 30 µM, 48h.

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Virtual Screening

Protein Preparation The crystal structure of a Rad51-BRCA2 BRC repeat complex was downloaded from the Protein Data Bank (PDB code 1N0W)2. The structure was then treated with the Schrödinger Suite 2014-4 Protein Preparation Wizard tool3. All the selenomethionines were mutated to methionine, water molecules and ions were removed, and an exhaustive sampling of the orientations of groups, whose hydrogen bonding network needs to be optimized, was performed. Finally, the protein structure was refined to relieve steric clashes with a restrained minimization with the OPLS2005 force field until a final RMSD of 0.30 Å with respect to the input protein coordinates.

Database Preparation A commercially available library of compounds composed of ASINEX4 and LifeChemicals5 databases collected from ZINC6 was prepared with the LigPrep7 tool of the Schrödinger Suite. The 2D (smi file) structures were converted to 3D structures and for each entry all stereoisomers were generated. The resulting molecules were submitted to Epik8 and all the tautomers and ionization states at pH 7.0 ± 2.0 were calculated. Finally, duplicates, compounds with more than 2 chiral centers, Pan-Assay Interference Compounds (PAINS), compounds with Michael acceptor groups, and frequent hitters were deleted. To enrich the database with potential Protein Protein Interaction Inhibitors, the database was filtered with the PPI-HitProfiler9 tool using the “soft” methods.

High Throughput Docking All filtered ligands (about 750K) were docked with Glide10 SP by centering the grid on the position of BRCA Phe1524, which was preferred to Phe1546 on the basis of the druggability index calculated by SiteMap11. The 10K top-scoring compounds were re-docked with Glide XP and the 6

1K top-scoring compounds were selected. Both grid generation and docking calculations were performed with the default settings. The selected compounds were visually inspected to identify compounds able to match the interactions between RAD51 and BRCA and 90 compounds were selected and purchased.

Synthetic Procedures

Synthetic plan Synthesis of final compounds 1-4 is illustrated in Scheme S1 and briefly described below. Nalkylation of benzothiazolone 5 with benzyl bromoacetate 6 gave intermediate ester 7, which was subjected to hydrazinolysis to give hydrazide 8. Cyclization to obtain triazole 10-13 was performed by reaction between hydrazide 8 with appropiate isothiocyanates 9a-d in the presence of triethylamine (TEA). Ultimately, S-alkylation of 10-13 with 14 led to final compounds 1-4.

Scheme S1

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General experimental details Solvents and reagents were obtained from commercial suppliers and were used without further purification. If required, solvents were distilled prior to use. For simplicity, solvents and reagents are indicated as follows: acetonitrile (MeCN), 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU), cyclohexane (Cy), dichloromethane (DCM), diethyl ether (Et2O), dimethylsulfoxide (DMSO), ethanol (EtOH), ethyl acetate (EtOAc), methanol (MeOH), triethylamine (TEA). Thin layer chromatography analyses were performed using pre-coated Supelco silica gel on TLC Al foils 0.2 mm and visualized by UV (254 nm), and/or KMnO4 stain. Automated column chromatography purifications were done using a Teledyne ISCO apparatus (CombiFlash® Rf) with pre-packed silica gel columns of different sizes (from 4 g until 120 g). Mixtures of increasing polarity of Cy and EtOAc or DCM and MeOH were used as eluents. Reactions involving microwave irradiation were performed using Explorer® -48 positions instrument (CEM). NMR experiments were run on a Bruker Avance III 400 system (400.13 MHz for 1H, and 100.62 MHz for 13C), equipped with a BBI probe and Z-gradients. Spectra were acquired at 300 K, using deuterated dimethylsulfoxide (DMSO-d6) or deuterated chloroform (Chloroform-d) as solvent. Chemical shifts for 1H and

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C

spectra were recorded in parts per million using the residual non-deuterated solvent as the internal standard (for DMSO-d6: 2.50 ppm, 1H; 39.52 ppm,

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C; for Chloroform-d: 7.26 ppm, 1H; 77.16

ppm, 13C). Data are reported as follows: chemical shift (ppm), multiplicity (indicated as: bs, broad signal; s, singlet; d, doublet; t, triplet; q, quartet; p, quintet; sx, sextet; m, multiplet and combinations thereof), coupling constants (J) in Hertz (Hz) and integrated intensity. UPLC/MS analyses were run on a Waters ACQUITY UPLC/MS system consisting of a SQD (Single Quadrupole Detector) Mass Spectrometer equipped with an Electrospray Ionization interface and a Photodiode Array Detector. PDA range was 210-400 nm. Analyses were performed on an ACQUITY UPLC BEH C18 column (50 x 2.1 mmID, particle size 1.7 µm) with a VanGuard BEH C18 pre-column (5 x 2.1 mmID, particle size 1.7 µm). Mobile phase was 10 mM NH4OAc in H2O 8

at pH 5 adjusted with AcOH (A) and 10 mM NH4OAc in CH3CN/H2O (95:5) at pH 5 (B). Electrospray ionization in positive and negative mode was applied. Analyses were performed with a gradient: 5 to 95% B over 3 min; flow rate 0.5 mL/min; temperature 40 °C. Compounds were named using the naming algorithm developed by CambridgeSoft Corporation and used in ChemBioDraw Ultra 15.0. All final compounds displayed ≥ 98% purity as determined by UPLC/MS analysis.

Synthetic procedures to obtain compounds 1-14 Benzyl 2-(2-oxobenzo[d]thiazol-3(2H)-yl)acetate (7) A mixture of benzothiazolone 5 (2.00 g, 13.23 mmol, 1.00 equiv), K2CO3 (1.92 g, 13.89 mmol, 1.05 equiv), a catalytic amount of NaI and 20 mL of acetone was stirred at room temperature. Benzyl bromoacetate 6 (2.3 mL, 14.55 mmol, 1.1 equiv) was added dropwise to the mixture which was then refluxed for 2 h. The reaction mixture was next cooled and poured into ice-water (300 mL). The yellow granulous precipitate formed was filtered and recrystallized from ethanol (80 mL) to give 7 (3.74 g, 94% yield) as white needles. 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.27 (m, 6H), 7.25 (t, J = 7.8 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 5.18 (s, 2H), 4.69 (s, 2H);

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C NMR (101 MHz, CDCl3) δ 170.1, 166.9, 136.6,

135.1, 128.7 (2C), 128.6, 128.3 (2C), 126.6, 123.7, 122.8, 122.2, 110.5, 67.5, 43.5; UPLC-MS (ESI, m/z) Rt = 2.55 min – 300 (M+H)+.

2-(2-oxobenzo[d]thiazol-3(2H)-yl)acetohydrazide (8) Compound 7 (2 g, 6.34 mmol, 1.0 equiv) was dissolved in 15 mL of EtOH and hydrazine hydrate (7.5 mL, 154 mmol, 23 equiv) was added. The mixture was refluxed for 90 min, cooled down and after 5 min stirred was stopped, and the reaction flask is placed in an ice bath for 20 min then filtered, washed with EtOH and dried under vacuum to give 8 9

(1.37 g, 49% yield) as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 7.65 (d, J = 7.4 Hz, 1H), 7.42 – 7.27 (m, 1H), 7.26 – 7.13 (m, 2H), 4.56 (s, 2H), 4.32 (s, 2H); 13C NMR (101 MHz, DMSO-d6) δ 169.6, 165.8, 137.7, 126.9, 123.7, 123.2, 121.7, 111.8, 43.8; UPLC-MS (ESI, m/z) Rt = 1.30 min – 224 (M+H)+.

General procedures for compounds 10-13 In distinct reactors, a mixture of hydrazide 8 (1.0 equiv) and commercially available isothiocyanate 9a-d (1.1 equiv) in 50 mL of EtOH containing TEA (2.1 equiv) was refluxed for 12 h. When the UPLC/MS analysis revealed about 1/1 open/cyclized product, the reaction mixtures were evaporated, suspended in dioxane (20 mL), 0.5 mL of DBU added and heated at 150 ⁰C for 20 min under microwave.

3-((5-mercapto-4-phenethyl-4H-1,2,4-triazol-3-yl)methyl)benzo[d]thiazol-2(3H)-one (10) A mixture of hydrazide 8 (1.00 g, 4.48 mmol) and isothiocyanate 9a (735 µL, 4.93 mmol) in 50 mL of EtOH containing TEA (1.31 mL, 9.41 mmol) were allowed to react according to general procedure. After microwave irradiation, the reaction mixture was cooled down, the solvent was removed under reduced pressure, and the solid obtained was triturated in water for 1 h, filtered and dried. The off-white solid was dissolved in the minimum amount of hot chloroform and filtered over a pad of silica gel, eluting with EtOAc/Cy 1/1. The filtrate was evaporated to give a white/light yellow solid which was triturated in 30 mL of Cy/DCM 9/1, filtered and dried to give 10 (1.12 g, 68 % yield) of white solid. 1

H NMR (400 MHz, DMSO-d6) δ 13.82 (s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.49 – 7.17 (m, 7H), 7.11

(d, J = 7.9 Hz, 1H), 5.09 (s, 2H), 4.24 (t, J = 7.0 Hz, 2H), 2.99 (t, J = 6.8 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ 169.6, 167.8, 147.3, 138.0, 136.7, 129.4, 129.2, 127.3, 127.1, 124.1, 123.5, 121.6, 112.1, 45.5, 37.5, 33.4; UPLC-MS (ESI, m/z) Rt = 2.41 min – 369 (M+H)+.

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3-((5-mercapto-4-(3-phenylpropyl)-4H-1,2,4-triazol-3-yl)methyl)benzo[d]thiazol-2(3H)-one (11) A mixture of hydrazide 8 (100 mg, 0.45 mmol) and isothiocyanate 9b (78 µL, 0.47 mmol) in 5 mL of ethanol containing TEA (130 µL, 0.95 mmol) were allowed to react according to general procedure. After refluxing overnight, it was observed the formation of cyclization product, so the microwave step was not necessary. The reaction is cooled, solvent evaporated, and dried under vacuum for few hours to give 11 (195 mg, crude product) that was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ 13.73 (s, 1H), 7.76 – 7.66 (m, 1H), 7.43 – 7.33 (m, 2H), 7.33 – 7.16 (m, 6H), 5.36 (s, 2H), 4.09 – 4.01 (m, 2H), 2.68 – 2.55 (m, 2H), 1.95 – 1.84 (m, 2H); 13C NMR (101 MHz, DMSO-

d6) δ 169.1, 167.3, 147.0, 140.7, 136.4, 128.3 (2C), 128.1 (2C), 126.7, 125.9, 123.7, 123.1, 121.1, 111.8, 43.3, 37.2, 32.1, 29.1; UPLC-MS (ESI, m/z) Rt = 2.32 min – 383 (M+H)+.

3-((4-benzyl-5-mercapto-4H-1,2,4-triazol-3-yl)methyl)benzo[d]thiazol-2(3H)-one (12) A mixture of hydrazide 8 (100 mg, 0.45 mmol) and isothiocyanate 9c (62 µL, 0.47 mmol) in 5 mL of ethanol containing TEA (130 µL, 0.95 mmol) were allowed to react according to general procedure. After microwave irradiation, the reaction mixture was cooled down, the solvent was removed under reduced pressure. The slurry was dissolved in EtOAc, washed with water x 2, dried over MgSO4, filtered and concentrated. The solid was triturated with petroleum ether to give 12 (133 mg, 83% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.78 (s, 1H), 7.65 – 7.50 (m, 1H), 7.41 – 6.97 (m, 8H), 5.35 (s, 2H), 5.19 (s, 2H);

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C NMR (101 MHz, DMSO-d6) δ 169.0, 168.3, 147.3, 136.2, 134.9, 128.6 (2C), 127.7,

126.5, 126.4 (2C), 123.5, 122.9, 121.3, 111.4, 45.9, 37.3; UPLC-MS (ESI, m/z) Rt = 2.09 min –355 (M+H)+.

3-((5-mercapto-4-(4-phenylbutyl)-4H-1,2,4-triazol-3-yl)methyl)benzo[d]thiazol-2(3H)-one (13) 11

A mixture of hydrazide 8 (100 mg, 0.45 mmol) and isothiocyanate 9d (92 µL, 0.47 mmol) in 5 mL of ethanol containing TEA (130 µL, 0.95 mmol) were allowed to react according to general procedure. After refluxing overnight, it was observed the formation of cyclization product, so the microwave step was not necessary. The reaction is cooled, solvent evaporated, and the crude was placed over a pad of silica gel, eluting with EtOAc 20-50 % in Cy, to obtain 13 (178 mg, quantitative yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.74 (s, 1H), 7.73 – 7.67 (m, 1H), 7.39 – 7.30 (m, 2H), 7.30 – 7.20 (m, 3H), 7.20 – 7.13 (m, 3H), 5.34 (s, 2H), 4.06 – 4.01 (m, 2H), 2.61 – 2.53 (m, 2H), 1.63 – 1.48 (m, 4H);

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C NMR (101 MHz, DMSO-d6) δ 169.1, 167.4, 147.0, 136.4, 128.3 (2C),

128.2 (2C), 126.7, 125.8, 123.7, 123.1, 121.2, 111.7, 43.3, 37.3, 34.7, 28.0, 27.5; UPLC-MS (ESI, m/z) Rt = 2.38 min – 397 (M+H)+.

General procedures for compounds 1-4 In distinct reactors, triazoles 10-13 (1 equiv), 2-chloro-N-cyclopentylacetamide 14 (1.1 equiv), Cs2CO3 (1.1 equiv) and NaI (0.05 equiv) were stirred in dry ACN at 50 ⁰C, and reactions progress was monitored by UPLC/MS. Then water was added, and the final compounds 1-4 precipitated, filtered and washed with solvents or purified by flash chromatography.

N-cyclopentyl-2-((5-((2-oxobenzo[d]thiazol-3(2H)-yl)methyl)-4-phenethyl-4H-1,2,4-triazol-3yl)thio)acetamide (1) Triazole 10 (100 mg, 0.27 mmol), 14 (48 mg, 0.30 mmol), Cs2CO3 (97

S O N

N

mg, 0.30 mmol) and a catalytic amount of NaI in 5 mL of dry ACN were

N

N

H N

S

allowed to react for 12 h according to general procedure. The desired

O

product precipitated from water, and was washed with water, Et2O and dried overnight under vacuum at 60 ⁰C to give 1 (122 mg, 92 % yield) as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 7.2 Hz, 1H), 7.75 – 7.61 (m, 1H), 7.39 – 7.14 (m, 8H), 5.13 (s, 12

2H), 4.29 (t, J = 7.5 Hz, 2H), 3.98 – 3.84 (m, 1H), 3.79 (s, 2H), 2.93 (t, J = 7.5 Hz, 2H), 1.80 – 1.63 (m, 2H), 1.63 – 1.49 (m, 2H), 1.49 – 1.37 (m, 2H), 1.37 – 1.17 (m, 2H);

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C NMR (101 MHz,

DMSO-d6) δ 169.6, 166.4, 150.8, 150.7, 137.5, 136.9, 129.4 (2C), 129.1 (2C), 127.4, 127.0, 124.0, 123.4, 121.7, 112.5, 51.1, 45.6, 37.7, 37.2, 35.7, 32.5 (2C), 23.8 (2C); UPLC-MS (ESI, m/z) Rt = 2.47 min – 494 (M+H)+; UPLC-MS purity (UV 215 nm): 98%.

N-cyclopentyl-2-((5-((2-oxobenzo[d]thiazol-3(2H)-yl)methyl)-4-(3-phenylpropyl)-4H-1,2,4triazol-3-yl)thio)acetamide (2) Triazole 11 (172 mg, 0.45 mmol), 14 (81 mg, 0.50 mmol), Cs2CO3 (163 mg, 0.5 mmol) and a catalytic amount of NaI in 5 mL of dry ACN were allowed to react for 5 hours according to general procedure. Over time, a white precipitate has formed. Water was added to facilitate precipitation and the white solid was filtered under vacuum and washed with Et2O. The solid was dried overnight under vacuum at 60 ⁰C to give 2 (128 mg, 56 % yield) as a white powder. 1H-NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 7.2 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.39 – 7.32 (m, 1H), 7.30 – 7.16 (m, 6H), 5.39 (s, 2H), 4.06 (t, J = 7.9 Hz, 2H), 3.95 – 3.85 (m, 1H), 3.82 (s, 2H), 2.59 (t, J= 7.9, 2H), 1.87-1.79 (m, 2H), 1.74-1.66 (m, 2H), 1.60-1.38 (m, 4H), 1.31-1.22 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 169.1, 165.9, 150.3, 150.2, 140.5, 136.5, 128.4 (2C), 128.1 (2C), 126.6, 126.0, 123.6, 122.9, 121.2, 112.1, 50.7, 43.6, 36.9, 36.8, 32.1 (2C), 32.0, 31.0, 23.3 (2C); UPLC-MS (ESI, m/z) Rt = 2.38 min – 508 (M+H)+; UPLC-MS purity (UV 215 nm): ≥ 99.5%.

2-((4-benzyl-5-((2-oxobenzo[d]thiazol-3(2H)-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)-Ncyclopentylacetamide (3) Triazole 12 (133 mg, 0.37 mmol), 14 (66 mg, 0.41 mmol), Cs2CO3 (133 mg, 0.41 mmol) and a catalytic amount of NaI in 5 mL of dry ACN were

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allowed to react overnight according to general procedure. Over time, a white precipitate has formed. Water was added to facilitate precipitation and the white solid was filtered under vacuum and washed with Et2O. The solid was dried overnight under vacuum at 60 ⁰C to give 3 (59 mg, 33 % yield) as a white powder. 1H-NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 7.2 Hz, 1H), 7.60, (d, J = 7.8 Hz, 1H), 7.34-7.32 (m, 2H), 7.27-7.17 (m, 4H) 6.88 (dd, J = 7.7, 1.8 Hz, 2H), 5.33 (s, 4H), 3.96-3.98 (m, 1H), 3.82 (s, 2H), 1.75-1.60 (m, 2H), 1.59-1.43 (m, 4H), 1.31-1.26 (m, 2H);

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C

NMR (101 MHz, DMSO-d6) δ 169.0, 165.8, 151.1, 150.8, 136.3, 134.8, 128.7 (2C), 127.7, 126.5, 125.8 (2C), 123.5, 122.7, 121.4, 111.8, 50.7, 46.6, 36.9, 36.7, 32.1 (2C), 23.3 (2C); UPLC-MS (ESI, m/z) Rt = 2.17 min – 480 (M+H)+; UPLC-MS purity (UV 215 nm): 99%.

N-cyclopentyl-2-((5-((2-oxobenzo[d]thiazol-3(2H)-yl)methyl)-4-(4-phenylbutyl)-4H-1,2,4triazol-3-yl)thio)acetamide (4) Triazole 13 (175 mg, 0.44 mmol), 14 (78 mg, 0.48 mmol), Cs2CO3 (156 mg, 0.48 mmol) and NaI in 5 mL of dry ACN were allowed to react for 3 h according to general procedure. Afterwards, reaction was cooled down, water added and aq layers extracted with EtOAc x3. The collected organic phases were washed with brine, dried over MgSO4, filtered and evaporated in

vacuo. Crude is purified by column chromatography, eluting with EtOAc 60-100 % in heptane, thus giving 4 (167 mg, 70 % yield) as a white solid. 1H-NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 7.2 Hz, 1H), 7.69 (dd, J = 7.8, 1.2 Hz, 1H), 7.46 – 7.40 (m, 1H), 7.34 (td, J = 7.8, 1.3 Hz, 1H), 7.28 – 7.15 (m, 6H), 5.36 (s, 2H), 4.05-4.01 (m, 2H), 3.97 – 3.86 (m, 1H), 3.81 (s, 2H), 2.56 (t, J = 6.7 Hz, 2H), 1.76-1.68 (m, 2H), 1.59 – 1.42 (m, 8H), 1.32-1.23 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 169.1 , 165.9 , 150.3 , 141.5, 136.5, 128.3 (2C), 128.2 (2C) , 126.6 , 125.8 , 123.6, 122.9, 121.2, 112.0, 50.7, 43.6, 36.9, 36.8, 34.6, 32.1 (2C) , 29.2, 27.8, 23.3 (2C); UPLC-MS (ESI, m/z) Rt = 2.44 min – 522 (M+H)+; UPLC-MS purity (UV 215 nm): ≥ 99.5%.

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2-chloro-N-cyclopentylacetamide (14)

To a solution of cyclopentylamine (786 µL, 7.97 mmol, 1 equiv) in 15 mL of DCM was added chloroacetyl chloride (705 µL, 8.85 mmol, 1.1 equiv) followed by the addition of TEA (3.08 mL, 22.14 mmol, 2.5 equiv) at 0 ⁰C. The reaction was continued at the same temperature for 1 h. The reaction mixture was washed with saturated NaHCO3 solution, 2N HCl and washed with brine solution. The excess organic solvent was removed under reduced pressure and the crude compound was purified by column chromatography, eluting with EtOAc/Cy 35/65, to give 14 (880 mg, 68 % yield) as a white solid. 1H NMR (400 MHz, Chloroform-d) δ 6.47 (s, 1H), 4.35 – 4.05 (m, 1H), 4.02 (s, 2H), 2.08 – 1.94 (m, 2H), 1.79 – 1.56 (m, 5H), 1.56 – 1.32 (m, 2H);

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C NMR (101 MHz,

Chloroform-d) δ 165.4, 51.6, 42.8, 33.0 (2C), 23.8 (2C); UPLC-MS (ESI, m/z) Rt = 1.44 min – 162, 164 (M+H)+.

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References

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