Peptidomimetics as Protein Arginine Deiminase 4 (PAD4) Inhibitors

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1 Peptidomimetics as Protein Arginine Deiminase 4 (PAD4) Inhibitors Andrea Trabocchi a, icolino Pala b, Ilga Krimmelbein c, Gloria Menchi a, Antonio Guarna a, Mario Sechi b, Tobias Dreker c, Andrea Scozzafava d, Claudiu T. Supuran e* and abrizio Carta d* a University of lorence, Department of Chemistry Ugo Schiff, via della Lastruccia 13, I Sesto iorentino, lorence, Italy. b Università degli Studi di Sassari, Dipartimento di Chimica e armacia, Via Vienna 2, Sassari, Italy. c 4SC Discovery Gmb, Am Klopferspitz 19a, Planegg-Martinsried, Germany. d Università degli Studi di irenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, Sesto iorentino, lorence, Italy. e Università degli Studi di irenze, EURARBA Department, Sezione di Scienze armaceutiche e utraceutiche, Via Ugo Schiff 6, Sesto iorentino, lorence, Italy.

2 Synthesis of, -Di-Boc- -(pent-4-ynyl)-guanidine (17). ollowing the general procedure for the synthesis of alkynes, compound 17 was obtained as a white solid in 68% yield. R f 0.63 (EtAc/pet. ether, 1:2). 1 MR (CDCl 3, 200 Mz) δ 9.30 (br, 1), 4.00 (t, J = 7.2 z, 2), 2.23 (td, J 1 = 7.2 z J 2 = 2.2 z, 2), 1.93 (t, J = 2.2 z, 1), 1.83 (m, 2), 1.52 (s, 9), 1.48 (s, 9); 13 C-MR (CDCl 3, 50 Mz) δ (s), (s), (s), 83.8 (s), 83.7 (s), 78.7 (s), 68.5 (d), 43.9 (t), 28.3 (q), 28.0 (q), 27.5 (t), 16.0 (t). Synthesis of, -di-boc- -(but-3-ynyl)-guanidine (18). ollowing the general procedure for the synthesis of alkynes, compound 18 was obtained as a white solid in 72% yield. R f 0.80 (EtAc/pet. ether, 1:2). 1 MR (CDCl 3, 200 Mz) δ 9.20 (br, 1), 4.08 (t, J = 7.0 z, 2), 2.52 (td, J 1 = 7.0 z J 2 = 2.6 z, 2), 1.95 (t, J = 2.6 z, 1), 1.53 (s, 9), 1.48 (s, 9); 13 C-MR (CDCl 3, 50 Mz) δ (s), (s), (s), 83.8 (s), 81.1 (s), 78.5 (s), 69.7 (d), 42.8 (t), 28.2 (q), 27.9 (q), 18.5 (t). 3

3 Synthesis of 3(R)-(2-azido-acetylamino)-3-(4-fluoro-phenyl)-propionic acid methyl ester (19). To a solution of (R)-β-(4-fluoro)-phenylalanine methyl ester (1.00 g, 5.08 mmol) and TEA (0.71 ml, 5.10 mmol) in anhydrous DCM (5 ml) at -10 C was added drop-wise the bromoacetyl bromide (445 μl, 5.10 mmol). After stirring 15 min at -10 C, the cooling bath was removed and the mixture stirred for 30 min more. Then, water was added, and the phases were separated. The organic phase was washed by Cl, brine and dried over a 2 S 4 and evaporated to dryness to obtain the corresponding 3(R)-(2-bromo-acetylamino)-3-(4-fluoro-phenyl)-propionic acid methyl ester as yellow oil (1.40 g, 4.42 mmol) in 87% yield. 1 MR (CDCl 3, 200 Mz) δ 7.74 (br, 1), (m, 2), (m, 2), 5.36 (dt, J = 8.0, 5.6 z, 1), 3.90 (s, 2), 3.64 (s, 3), 2.88 (dd, J = 5.6, 3.8 z, 2). 13 C-MR (CDCl 3, 50 Mz) δ (s), (s), (d, J C- = 245 z), (s), (d, J C- = 8.2 z), (d, J C- = 20 z), 52.1 (q), 49.6 (d), 39.6 (t), 29.1 (t). This intermediate compound was dissolved in DM, and a 3 (0.84 g, 13.0 mmol) was added at room temperature. After 5 min, the mixture was warmed up at 80 C and stirred for 16h. Water was added to the solution, and extracted with diethyl ether. The crude mixture was then purified through CC (AcEt/petroleum ether 1:1, R f 0.53) to obtain the title compound 19 as a yellowish solid (0.932 g, 3.33 mmol) in yield 78%. [α] 23 D (c 0.5, CCl 3 ). 1 MR (CDCl 3, 200 Mz) δ 7.46 (d, J = 8 z 1), (m, 2), 7.00 (td, J - = 8.8 z, J = 1.4 z, 2), 5.36 (dt, J = 8.0, 5.6 z, 1), 3.97 (s, 2), 3.62 (s, 3), 2.88 (pseudo-t, J = 5.6 z, 2). 13 C-MR (CDCl 3, 50 Mz) δ (s), (s), (d, J C- = 245 z), (s), (d, J C- = 8.2 z), (d, J C- = 20 z), 52.5 (t), 51.9 (q), 48.9 (d), 39.7 (t). 3 Synthesis of 3(S)-(2-azido-acetylamino)-3-(4-fluoro-phenyl)-propionic acid methyl ester (20) Compound 20 was prepared as described for 19 starting from (S)-β-(4-fluoro)-phenylalanine methyl ester (1.00 g, 5.08 mmol). The intermediate 3(S)-(2-bromo-acetylamino)-3-(4-fluoro-phenyl)- propionic acid methyl ester 20 was obtained as yellow oil (1.40 g, 4.42 mmol) in 87% yield with similar MR data as reported for the bromo-intermediate of 19. The crude product was purified by

4 CC (AcEt/petroleum ether 3/2, R f 0.50) to give a yellowish oil (0.99 g, 3.54 mmol) in yield 80% with similar MR data as reported for 19. [α] 23 D 22.0 (c 0.6, CCl 3 ). 3 Sinthesis of 3-(2-azido-acetylamino)-propionic acid methyl ester (21). Compound 21 was prepared as described for 19 starting from β-alanine methyl ester (1.12 g, 8 mmol). The intermediate 3-(2-bromo-acetylamino)-propionic acid methyl ester was obtained as yellow oil (1.02 g, 4.57 mmol) in 57% yield. 1 -MR (CDCl 3, 200 Mz) δ 7.10 (br, 1), 3.83 (s, 2), 3.75 (s, 3), 3.57 (q, 2), 2.57 (t, 2). 13 C-MR (CDCl 3, 50 Mz) δ (s), (s), 51.9 (t), 35.5 (t), 33.4 (t), 29.1 (q). The crude product was purified by CC (AcEt/petroleum ether 2:1, R f 0.4) to give a yellowish oil (170 mg, 0.91 mmol) in 20% yield. 1 -MR (CDCl 3, 200 Mz) δ 6.91 (br, 1), 3.94 (s, 3), 3.68 (s, 3), 3.53 (q, 2), 2.55 (t, 2). 13 C-MR (CDCl 3, 50 Mz) δ (s), (s), (t), 51,98 (t), (t), (q). 2 Cl Synthesis of 3(R)-(4-fluoro-phenyl)-3-{2-[4-(3-guanidino-propyl)-[1,2,3]triazol-1-yl]- acetylamino}-propionic acid hydrochloride salt (4). ollowing the CuAAC general procedure previously reported, alkyne 17 (0.146 g, 0.45 mmol) and azide 19 (0.125 g, 0.45 mmol) in 2 /t-bu 1:1 (2.0 ml) gave, after work-up and purification by CC (AcEt/ Petroleum ether 4:1, R f = 0.6) the corresponding protected derivative 4a (0.096 g, 37%) as a yellow oil. 1 MR (CDCl 3, 200 Mz) δ 9.34 (br, 1), 7.86 (br, 1), (m, 3), (m, 2), 5.33 (m, 1), 5.03 (s, 2), 3.88 (m, 2), 3.55 (s, 3), 2.77 (t, 4), 1.63 (m, 2), 1.48 (s, 9), 1.46 (s, 9).

5 The hydrolysis of 4a gave the title compound 4 as a yellow solid. [α] 24 D (c 0.3, 2 ). 1 MR (D 2, 400 Mz) δ 7.81 (s, 1), 7.42 (dd, J = 8.8, 5.2 z, 2), 7.17 (t, J = 8.8 z, 2), 5.32 (m, 1), 5.26 (d, J = 2.8 z, 2), 3.22 (t, J = 6.4 z, 2), 3.00 (m, 2), 2.82 (t, J = 7.4 z, 2), 1.98 (m, 2). 13 C-MR (D 2, 50 Mz) δ 171,68 (s), 164,95 (s), 161,50 (s, J C- = 242 z), 157,17 (s), 145,88 (s), 138,36 (s), 128,91 (d, J C- = 8,8, 2C), 124,05 (d), 115,49 (d, J C- = 21 z, 2C), 51,82 (d), 49,62 (t), 42,90 (t), 42,50 (t), 28,65 (t), 22,33 (t). ESI-MSMS m/z 392 [(M-) +, 25.3], 375 (100), 333 (91.3). El. An. for C1723Cl73: Calcd. C, 47.72;, 5.42;, ound: C, 47.84;, 5.63;, Cl 2 Synthesis of 3(R)-(4-luoro-phenyl)-3-{2-[4-(2-guanidino-ethyl)-[1,2,3]triazol-1-yl]- acetylamino}-propionic acid (8). ollowing the CuAAC general procedure, alkyne 18 (0.140 g, 0.45 mmol) and azide 19 (0.125 g, 0.45 mmol) in 2 /t-bu 1:1 (2.0 ml) gave, after work-up and purification by CC (AcEt/ Petroleum ether 4/1, R f = 0.43), the corresponding protected derivative 8a (0.159 g, 60%) as a yellow oil. 1 MR (CDCl 3, 200 Mz) δ 9.24 (br, 1), 7.69 (s, 1), (m, 2), (m, 2), 5.35 (m, 1), 5.01 (s, 2), 4.18 (m, 2), 3.57 (s, 3), 3.05 (m, 2), 2.80 (m, 2), 1.49 (s, 9), 1.47 (s, 9). 13 C MR (CDCl 3, 50 Mz) δ (s), (s), (d, J C = 163 z), (s), (s), (s), (s), (dd, J C = 8.3 z, 2C), (s), (dd, J C =22 z, 2C), 84.0 (s), 78.8 (s), 52.8 (t), 51.9 (q), 49.5 (d), 44.1 (t), 39.8 (t), 28.3 (q), 28.0 (q), 25.3 (t). The hydrolysis of 8a gave the title compound 8 as a yellowish solid. [α] 24 D (c 0.5, 2 ). 1 MR (D 2, 400 Mz) δ 7.73 (s, 1), 7.28 (dd, J = 8.8, 5.2 z, 2), 7.03 (t, J = 8.8 z, 2), 5.18 (m, 1), 5.13 (d, J = 4.4 z, 2), 3.39 (t, J = 6.8 z, 2), (m, 4). 13 C-MR (D 2, 50 Mz) δ (s), (s), (d, 1 J C- = 240 z), (s), (s), (s), (d, 3 J C- = 5 z, 2C), (d), (d, 2 J C- = 14 z, 2C), 52.2 (t), 50.2 (d), 40.5 (t), 39.9 (t), 24.3 (t). ESI- MSMS m/z 378 [(M-) +, 12.3], 361 (51), 333 (100). El. An. for C1621Cl73: Calcd. C, 46.44;, 5.11;, ound: C, 46.88;, 5.35;,

6 2 Cl Synthesis of 3(S)-(4-fluoro-phenyl)-3-{2-[4-(3-guanidino-propyl)-[1,2,3]triazol-1-yl]- acetylamino}-propionic acid (5). ollowing the CuAAC general procedure, alkyne 17 (0.361 g, 0.95 mmol) and azide 20 (0.250 g, 0.95 mmol) in 2 /t-bu 1/1 (4 ml) gave, after work-up and purification by CC (C 2 Cl 2 /Me 12:1, R f = 0.46), the corresponding protected derivative 5a (0.400 g, 65%). The hydrolysis of 5a gave the title 5 as a yellow solid with similar MR data as reported for compound 4. [α] 23 D (c 0.5, 2 ). El. An. for C1723Cl73: Calcd. C, 47.72;, 5.42;, ound: C, 47.94;, 5.76;, Cl Synthesis of 3-{2-[4-(3-guanidino-propyl)-[1,2,3]triazol-1-yl]-acetylamino}-propionic acid hydrochloride salt (10). ollowing the CuAAC general procedure, alkyne 17 (0.200 g, 0.62 mmol) and azide 21 (0.170 g, (0.62 mmol) in 2 /t-bu 1/1 (2.0 ml) gave, after work-up and purification by CC (C 2 Cl 2 /methanol 30:1, R f = 0.7), the corresponding protected derivative 10a in 35% yield (0.100 g). 1 MR (CDCl 3, 200 Mz) δ 9.34 (br, 2), 7,77 (s, 1), 6.72 (br, 1), 4.95 (s, 2), 3.91 (q, 2), 3.62 (s, 3), 3.49 (q, 2), 2.76 (t, 2), 2.50 (t, 2), (m, 2), 1.47 (s, 9), 1.46 (s,9). The hydrolysis of 10a gave the title product 10 as a white solid (59 mg, 95% yield). 1 - MR (D 2, 200 Mz) δ 7.84 (s, 1), 5.10 (s, 2), 3.36 (t, 2), 3.08 (t, 2), (m, 2), 2.47 (t, 2), 1.83 (t, 2). 13 C-MR (D 2, 50 Mz) δ (s), (s), (s), (s), (d), 53.0 (t), 42.2 (t), 38.9 (t), 31.0 (t), 27.0 (t), 21.0 (t). ESI-MSMS m/z [(M+) +, 5],

7 (100), (14), (8), (52). El. An. for C1120Cl73: Calcd. C, 39.58;, 6.04;, ound: C, 39.67;, 6.13;,