N-isobutyl-1-octyl-5-phenyl-1H-imidazol-2-amine (8a). Obtained from the general procedure

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1 NMR spectr N-isoutyl-1-octyl-5-phenyl-1H-imidzol-2-mine (8). Otined from the generl procedure s n ornge oil, yield 57%. 1 H NMR (300 MHz, Chloroform-d) δ (m, 5H), 6.71 (s, 1H), 3.71 (t, J = 7.5 Hz, 3H), 3.23 (t, J = 6.4 Hz, 2H), 1.96 (dp, J = 13.4, 6.7 Hz, 1H), (m, 2H), 1.18 (s, 10H), 1.00 (d, J = 6.6 Hz, 6H), 0.86 (t, J = 6.8 Hz, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ , , , , , , , , , 51.56, 42.64, 31.67, 29.42, 29.02, 28.98, 28.94, 28.42, 26.51, 22.58, 20.32, (4-chlorophenyl)-N-isoutyl-1-octyl-1H-imidzol-2-mine (8). Otined from the generl procedure s n ornge oil, yield 46%. 1 H NMR (300 MHz, Chloroform-d) δ 7.37 (d, J = 8.1 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 6.69 (s, 1H), 3.79 (t, J = 7.5 Hz, 2H), 3.16 (d, J = 6.9 Hz, 2H), (m, 1H), 1.48 (t, J = 7.5 Hz, 2H), (m, 10H), 0.95 (d, J = 6.6 Hz, 6H), 0.85 (t, J = 6.9 Hz, 3H). 13 C NMR (75 MHz, CDCl3) δ , , , , , , , 51.07, 43.09, 31.61, 28.93, 28.84, 28.80, 28.50, 28.27, 26.15, 22.54, 22.52, 19.99, (4-romophenyl)-N-isoutyl-1-octyl-1H-imidzol-2-mine (8c). Otined from the generl procedure s yellow oil, yield 52%. 1 H NMR (300 MHz, Chloroform-d) δ (m, 2H), (m, 2H), 6.72 (s, 1H), (m, 3H), 3.23 (t, J = 6.3 Hz, 2H), 1.95 (dt, J = 13.4, 6.7 Hz, 1H), 1.57 (t, J = 7.5 Hz, 2H), (m, 10H), 1.00 (d, J = 6.6 Hz, 6H), 0.87 (t, J = 6.8 Hz, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ , , , , , , , 51.50, 42.68, 31.68, 29.44, 29.03, 28.95, 28.41, 26.50, 22.60, 20.30, (3,4-dichlorophenyl)-N-isoutyl-1-octyl-1H-imidzol-2-mine (8d). Otined from the generl procedure s n ornge oil, yield 41%. 1 H NMR (300 MHz, Chloroform-d) δ (m, 2H), 7.06 (dt, J = 8.3, 1.9 Hz, 1H), 6.56 (d, J = 29.1 Hz, 1H), (m, 3H),

2 (m, 1H), (m, 2H), (m, 12H), (m, 4H), (m, 4H). 13 C NMR (75 MHz, CDCl 3 ) δ , , , , , , , , 41.74, 39.10, 30.63, 28.52, 28.12, 26.49, 25.61, 25.50, 22.86, 22.66, 21.56, 19.94, 16.61, N-cyclopentyl-1-octyl-5-phenyl-1H-imidzol-2-mine (8e). Otined from the generl procedure s n ornge oil, yield 67%. 1 H NMR (300 MHz, Chloroform-d) δ 7.32 (tt, J = 12.4, 7.3 Hz, 5H), 6.73 (s, 1H), 4.20 (q, J = 6.3 Hz, 1H), 3.69 (t, J = 7.6 Hz, 2H), 3.53 (d, J = 6.1 Hz, 1H), 2.10 (tt, J = 12.2, 5.0 Hz, 2H), (m, 3H), 1.53 (dq, J = 12.1, 6.3, 5.7 Hz, 4H), (m, 10H), 0.86 (t, J = 6.8 Hz, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ , , , , , , , , , 55.21, 42.51, 35.46, 33.71, 33.69, 31.63, 29.32, 28.96, 28.86, 26.37, 26.35, 23.73, 23.32, (4-chlorophenyl)-N-cyclopentyl-1-octyl-1H-imidzol-2-mine (8f). Otined from the generl procedure s n ornge oil, yield 41%. 1 H NMR (300 MHz, Chloroform-d) δ (m, 2H), 7.26 (d, J = 3.9 Hz, 2H), 6.73 (s, 1H), 4.19 (q, J = 6.2 Hz, 1H), 3.66 (t, J = 7.6 Hz, 2H), 3.44 (d, J = 6.2 Hz, 1H), (m, 2H), (m, 2H), 1.52 (ddd, J = 12.6, 7.9, 5.4 Hz, 4H), (m, 12H), 0.87 (t, J = 6.8 Hz, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ , , , 55.40, 42.80, 33.72, 31.68, 29.25, 29.02, 28.90, 26.40, 23.76, 22.60, (4-romoophenyl)-N-cyclopentyl-1-octyl-1H-imidzol-2-mine (8g). Otined from the generl procedure s n ornge oil, yield 52%. 1 H NMR (300 MHz, Chloroform-d) δ 7.52 (dd, J = 8.5, 2.0 Hz, 2H), 7.20 (dd, J = 8.4, 2.0 Hz, 2H), 6.75 (d, J = 2.1 Hz, 1H), 4.20 (q, J = 6.3 Hz, 1H), 3.67 (t, J = 7.5 Hz, 2H), 3.44 (d, J = 6.2 Hz, 1H), (m, 2H), (m, 4H), 1.53 (dq, J = 11.6, 5.7 Hz, 4H), 1.19 (s, 10H), 0.88 (t, J = 6.5 Hz, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ , , , , , , 55.29, 42.66, 33.81, 31.69, 29.42, 29.03, 28.92, 26.46, 23.75, 22.61,

3 5-(3,4-dichlorophenyl)-N-cyclopentyl-1-octyl-1H-imidzol-2-mine (8h). Otined from the generl procedure s yellow oil, yield 49%. 1 H NMR (300 MHz, Chloroform-d) δ (m, 2H), 7.14 (dd, J = 8.3, 2.1 Hz, 1H), 6.76 (s, 1H), 4.19 (q, J = 6.3 Hz, 1H), (m, 2H), 3.50 (d, J = 6.2 Hz, 1H), (m, 2H), (m, 4H), (m, 4H), 1.19 (d, J = 3.1 Hz, 10H), 0.87 (t, J = 6.8 Hz, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ , , , , , , , , , 55.27, 42.74, 33.79, 31.68, 29.42, 29.04, 28.91, 26.43, 23.74, 22.60,

4 TABLE S1 Effect of literture compounds on pnel of monospecies iofilms S. ureus ATCC6538 S. ureus SH1000 S. epidermidis E. coli TG1 P. eruginos PA14 37 C 37 C 37 C 37 C 25 C compd iclein > > > > > ~ 23.5 > > nifuroxzide > > ~ 43.4 c > > > tnnic cid ~ > > > > ~ > > compd S. Typhimurium ATCC14028 S. liquefciens MG44 B. cepci LMG1222T C. licns SC C 25 C 25 C 37 C iclein > > > > > nifuroxzide > > > > > > > tnnic cid > > > > > : concentrtion of compound needed to inhiit iofilm formtion y 50%. : concentrtion of compound needed to inhiit plnktonic growth y 50%. c ~: The B(IC) 50 vlues could not e ccurtely clculted due to the steepness of the curve. Compounds tht hve iofilmspecific ctivity (2 x < ) re mrked in grey.

5 TABLE S2 Effect of 5-Ar-2AIs on pnel of monospecies cteril nd fungl iofilms compd S. ureus ATCC6538 S. ureus SH1000 S. epidermidis P. gingivlis ATCC33277 E. coli TG1 37 C 37 C 37 C 37 C 37 C > ~ d ~ ~ ~ 5.6 ~ ~ ~ 96.1 ~ ~ > ~ > > ~ > > > > > ~ compd P. eruginos PA14 S. Typhimurium ATCC14028 S. liquefciens MG44 B. cepci LMG1222T C. licns SC C c 37 C c 25 C 25 C 25 C 37 C ~ > ~ > > > > > > > > > > > > > > > > > : concentrtion of compound needed to inhiit iofilm formtion y 50%. : concentrtion of compound needed to inhiit plnktonic growth y 50%. c Biofilm formtion ws studied t 25 C nd 37 C to simulte environmentl nd in vivo conditions, respectively. d ~: The B(IC) 50 vlues could not e ccurtely clculted due to the steepness of the curve. The effect on the plnktonic growth hs previously een determined y growth curve nlysis (1-3). Compounds tht hve iofilm-specific ctivity (2 x < ) re mrked in grey.

6 TABLE S3 Effect of 5-Ar-2AIs on pnel of mixed E. coli/p. eruginos nd S. ureus/s. epidermidis iofilms E. coli TG1 + P. eruginos PA14 S. ureus ATCC S. epidermidis 37 C 37 C compd ~ c ~ 7.2 ~ > ~ 26.3 ~ > ~ > ~ 66.6 ~ > ~ : concentrtion of inhiitor needed to inhiit iofilm formtion y 50%. : concentrtion of inhiitor needed to inhiit plnktonic growth y 50%. c ~: The B(IC) 50 vlues could not e ccurtely clculted due to the steepness of the curve. Compounds tht hve iofilm-specific ctivity (2 x < ) re mrked in grey.

7 TABLE S4 Effect of novel 5-Ar-2AIs on pnel of monospecies iofilms of cteri nd fungi. S. ureus ATCC6538 P. eruginos PA14 E. coli TG1 C. licns SC C 25 C 37 C 25 C 37 C 37 C compd for for for for for for for for for for for 8 ~ 22.9 c > > ~ > > c > > > > > > d > > > > > > > > e > > ~ ~ ~ f ~ 24.1 > > > > > g > > > > > > h > > > > > > > > > : concentrtion of inhiitor needed to inhiit iofilm formtion y 50%. : concentrtion of inhiitor needed to inhiit plnktonic growth y 50%. c ~: The B(IC) 50 vlues could not e ccurtely clculted due to the steepness of the curve. Compounds tht hve iofilmspecific ctivity (2 x < ) re mrked in grey.

8 TABLE S5 Effect of novel 5-Ar-2AIs on pnel of mixed species iofilms C. licns SC S. epidermidis S. ureus ATCC S. epidermidis E. coli TG1 + P. eruginos PA14 CFUs % survivl 37 C 37 C 25 µm 100 µm compd C.. S.e. C.. S.e. for ~ 26.1c 6.6 for > ~ 22.2 > > c > > > d > > > > e ~ 3.0 ~ > f ~ > g ~ > > h ~ 25.1 > > for C.. : Cndid licns; S.e. : Stphylococcus epidermidis. : concentrtion of inhiitor needed to inhiit iofilm formtion y 50%. : concentrtion of inhiitor needed to inhiit plnktonic growth y 50%. c ~: The B(IC) 50 vlues could not e ccurtely clculted due to the steepness of the curve. Compounds tht hve iofilm-specific ctivity (2 x < ) re mrked in grey nd compounds with < 75 % CFU survivl re mrked in drk grey.

9 REFERENCES 1. Steenckers HP, Ermolt'ev DS, Svliy B, De Weerdt A, De Coster D, Shh A, Vn der Eycken EV, De Vos DE, Vnderleyden J, De Keersmecker SC Structure-ctivity reltionship of 4(5)-ryl-2-mino-1H-imidzoles, N1-sustituted 2-minoimidzoles nd imidzo[1,2-]pyrimidinium slts s inhiitors of iofilm formtion y Slmonell Typhimurium nd Pseudomons eruginos. J Med Chem 54: Steenckers HP, Ermolt'ev DS, Svliy B, Weerdt AD, Coster DD, Shh A, Vn der Eycken EV, De Vos DE, Vnderleyden J, De Keersmecker SC Structure-ctivity reltionship of 2-hydroxy-2-ryl-2,3-dihydro-imidzo[1,2-]pyrimidinium slts nd 2N-sustituted 4(5)-ryl- 2-mino-1H-imidzoles s inhiitors of iofilm formtion y Slmonell Typhimurium nd Pseudomons eruginos. Bioorg Med Chem 19: Steenckers H, Ermolt'ev D, Trng TT, Svli B, Shrm UK, De Weerdt A, Shh A, Vnderleyden J, Vn der Eycken EV Microwve-ssisted one-pot synthesis nd ntiiofilm ctivity of 2-mino-1H-imidzole/trizole conjugtes. Org Biomol Chem 12: