Patent ID: 12187696

Further, the invention shall be explained in more detail by the following Examples.

1) Materials and Methods

1.1) Reagents

Reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich Co. LLC, Thermo Fisher Scientific Inc., Merck KGaA, TCI Europe GmbH, Fluorochem Ltd. and Alfa Aesar GmbH) and used without further purification, unless otherwise indicated. HPLC-grade solvents or anhydrous solvents (max. 0.01% water content, stored over molecular sieve under an argon atmosphere) were used for all reactions. All experiments were monitored by analytical thin layer chromatography (TLC). TLC was performed on precoated silica gel plates (60 F-254, 0.25 mm, Merck KGaA) with detection by UV (λ=254 and/or 366 nm) and/or by coloration using a phosphomolybdate (PMA), and/or potassium permanganate (KMnO4) stain and subsequent heat treatment. Flash chromatography was performed on silica gel 60 (0.035-0.070 mm, mesh 60 Å, Merck KGaA) with the indicated eluent. Preparative thin layer chromatography (prep TLC) was performed on pre-coated silica layer plates (SIL G-100 UV254, 1.00 mm, Macherey-Nagel GmbH & Co. KG) with the indicated eluent. Common solvents for chromatography [n-hexane (Hex), ethyl acetate (EtOAc), dichloromethane (CH2Cl2) and methanol (MeOH)] were distilled prior to use.

1.2) NMR

1H and proton-decoupled13C NMR spectra were recorded on a Bruker Avance III HD 300 (300 MHz), a Bruker Avance I 360 (360 MHz), a Bruker Avance III HD (500 MHz) or a Bruker Avance III HD (500 MHz, equipped with a Bruker CryoProbe platform) at 298 K. Chemical shifts are reported in delta (δ) units in parts per million (ppm) relative to distinguished solvent signals [deuterated chloroform (CDCl3) δH=7.26 ppm and δC=77.16 ppm; deuterated DMSO (DMSO-d6), δH=2.50 ppm]. The following abbreviations were used for the assignment of the signals: s—singlet, d—doublet, t—triplet, q—quartet, m—multiplet. Coupling constants J are given in Hertz [Hz]. HR-MS spectra were recorded in the ESI or APCI mode on a Thermo Scientific LTQ-FT Ultra (FT-ICR-MS) coupled with an UltiMate 3000 HPLC system (Thermo Fisher Scientific Inc.).

1.3) Cell Culture

Cell culture media and supplements were obtained from Sigma Life Science and Life Technologies. A549 and Hela cells were cultured in Dulbecco's Modified Eagle Medium (DMEM high glucose, 4.5 g/L) supplemented with 10% fetal bovine serum (Sigma Life Science) and 2 mM L-glutamine (PAA). NIH/3T3 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM high glucose) supplemented with 10% fetal bovine serum (Sigma Life Science) and 4 mM L-glutamine (PAA). HepG2 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Sigma Life Science) and 2 mM L-glutamine (PAA). All cells were maintained in a humidified 37° C. incubator with 5% CO2. Cells were detached with trypsin-EDTA.

1.4) Bacterial Strains and Media

Commercially available strains were obtained from the following suppliers: Institute Pasteur, France (Staphylococcus aureusNCTC 8325,S. aureusMu 50,Listeria monocytogenesEGD-e), American Type Culture Collection, USA (USA 300 FPR3757), (Mycobacterium smegmatismc2155,Mycobacterium tuberculosisH37Rv), (Bacillus subtilis168). DSMZ (Acinetobacter baumanniiDSM-30007, Eneterococcus faeciumDSM-20477,Pseudomonas aeruginosaDSM-19882,Enterobacter cloacaesubsp.CloacaeDSM-30054,Enterobacter aerogenesDSM-30053). ClinicalS. aureusisolates (BK95395, BK97296, IS050678, IS050611, VA417350, VA418879, VA402923, VA412350, VA409044, VA402525) were a kind gift from Prof. Markus Gerhard at the Institute of Medical Microbiology and Immunology, Technische Universität München.Escherichia coliCFT073 was a kind gift from Dr. Guiseppe Magistro (Klinikum d. Universität München Urologische Klinik).

Bacterial growth media: LB-medium (1% peptone, 0.5% NaCl, 0.5% yeast extract, pH 7.5), B-medium (1% peptone, 0.5% NaCl, 0.5% yeast extract, 0.1% K2HPO4, pH 7.5); BHB-medium (Brain Heart Infusion, 0.75% brain infusion, 1% heart infusion, 1% peptone, 0.5% NaCl, 0.25% Na2HPO4, 0.2% glucose, pH 7.4); 7H99 medium (4.7 g/L 7H9 powder, 2 mL/L glycerol, 2.5 mL/L 20% Tween 80, 5 g/L BSA (fraction V), 2 g/L dextrose, 850 mg/L NaCl, 3 mg/L catalase).

2) Compounds

Probe Compound

N-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido) phenoxy)picolinamide (PK/X17-1-058)

To a solution of 4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinic acid (23.1 mg, 0.0511, 1.0 eq.) in dry DMF (0.5 mL) was added HOBt (8.28 mg, 0.0613 mmol, 1.2 eq.), EDC (11.8 mg, 0.0613, 1.2 eq.) and DIEA (17.8 μL, 13.2 mg, 0.102 mmol, 2.0 eq.). After the addition of a solution of 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-amine (Li, Z. et al. Design and synthesis of minimalist terminal alkyne-containing diazirine photo-crosslinkers and their incorporation into kinase inhibitors for cell- and tissue-based proteome profiling.Angew. Chem. Int. Ed. Engl.52, 8551-6 (2013)) (7.71 mg, 0.0562 mmol, 1.1 eq.) in dry DMF (0.5 mL) the mixture was stirred at room temperature for 24 h. The solvent was removed and the residue was purified flash column chromatography on silica (Hex/EtOAc=2/3) to yield the desired product.

Yield: 60% (17.6 mg, 0.0308 mmol); workup A (CH2Cl2/MeOH=99/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.25 (s, 1H), 9.03 (s, 1H), 8.85 (t, J=6.1 Hz, 1H), 8.52 (d, J=5.6 Hz, 1H), 8.13 (d, J=2.4 Hz, 1H), 7.68-7.58 (m, 4H), 7.37 (d, J=2.6 Hz, 1H), 7.20-7.16 (m, 3H), 3.17 (q, J=7.0 Hz, 2H), 2.83 (t, J=2.7 Hz, 1H), 2.00 (td, J=7.4, 2.7 Hz, 2H), 1.63 (t, J=7.2 Hz, 2H), 1.59 (t, J=7.4 Hz, 2H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=166.1, 163.3, 152.5, 152.2, 150.4, 147.8, 139.4, 137.1, 132.1, 126.7 (q, J=30.3 Hz), 123.2, 123.2 (m), 121.6, 122.9 (q, J=273.3 Hz), 120.5, 116.9 (q, J=5.3 Hz), 114.2, 108.7, 83.2, 71.9, 34.1, 32.0, 31.3, 27.3, 12.7 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 571.1467 calcd. for C27H23ClF3N6O3+; found, 571.1472.

2.2) General Procedure for the Synthesis of Urea and Thiourea Containing Compounds

A solution of the corresponding commercially available isocyanate or thioisocyanate (1.1 eq.) in dry dichloromethane (3 mL) was cooled to 0° C. After the addition of the corresponding amine (1.0 eq.) the reaction mixture was allowed to warm to room temperature and the reaction was stirred at room temperature for 20 h. Individual work up and purification yielded the desired urea or thiourea containing compounds. In brief, the solvent was removed followed either by purification by flash column chromatography on silica (Hex/EtOAc or CH2Cl2/MeOH; workup A) or by the precipitation from DMF through the addition of water (10 fold excess) and collection of the product by centrifugation (17000 g, 10 min) (workup B).

Example 1

Methyl 3-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)benzoate (PK/X17-1-052)

Yield: 91% (1.40 g, 3.01 mmol); workup A (CH2Cl2/MeOH=99/1).

1H NMR (250 MHz, CDCl3): δ [ppm]=7.78-7.72 (m, 1H), 7.69 (br s, 1H), 7.60-7.55 (m, 2H), 7.45 (br s, 1H), 7.41-7.27 (m, 3H), 7.20-7.09 (m, 3H), 6.92-6.84 (m, 2H), 3.89 (s, 3H).

13C NMR (63 MHz, CDCl3): δ [ppm]=167.1, 157.6, 153.9, 153.6, 137.2, 133.1, 132.1, 131.9, 130.1, 128.9 (q, J=31.6 Hz), 126.4 (m), 124.6, 124.1, 123.6, 123.4, 122.6 (q, J=273.3 Hz), 120.0, 119.2, 119.1 (m), 52.5 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 465.0824 calcd. for C22H17ClF3N2O4+; found, 465.0825.

Example 2

1-(4-Benzoylphenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (PK/X17-1-144)

Yield: 70% (121 mg, 0.289 mmol); workup B.

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.35 (s, 1H), 9.31 (s, 1H), 8.16-8.11 (m, 1H), 7.77-7.62 (m, 9H), 7.56 (t, J=7.6 Hz, 2H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=194.5, 152.2, 143.8, 139.0, 137.7, 132.2, 132.1, 131.4, 130.4, 129.3, 128.5, 126.8 (q, J=30.7 Hz), 123.4, 122.8, 122.8 (q, J=273.0 Hz), 117.6, 117.0 (q, J=5.7 Hz) (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 419.0769 calcd. for C21H16ClF3N2O2+; found, 419.0766.

Example 3

4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-N-methylbenzamide (PK/X17-1-145)

Yield: 64% (98.2 mg, 0.264 mmol); workup B.

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.25 (s, 1H), 9.10 (s, 1H), 8.31 (q, J=4.2 Hz, 1H), 8.14-8.11 (m, 1H), 7.78 (d, J=8.8 Hz, 2H), 7.66-7.61 (m, 2H), 7.53 (d, J=8.8 Hz, 2H), 2.76 (d, J=4.5 Hz, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=166.1, 152.3, 141.8, 139.2, 132.1, 128.1, 128.0, 126.8 (q, J=30.5 Hz), 123.2, 122.8 (q, J=273.0 Hz), 122.5 (m), 117.6, 116.9 (q, J=5.8 Hz), 26.2 (observed complexity is due to the C—F splitting).

Example 4

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)urea (PK/X17-1-150)

Yield: 91% (148 mg, 0.375 mmol); workup A (CH2Cl2/MeOH=99/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.22 (s, 1H), 9.07 (s, 1H), 8.09 (d, J=2.3 Hz, 1H), 7.68-7.59 (m, 3H), 7.33 (d, J=8.7 Hz, 1H), 7.12 (dd, J=8.8, 2.2 Hz, 1H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.5, 142.8, 139.2, 137.8, 136.0, 132.0, 131.3 (t, J=252.2 Hz), 126.7 (q, J=30.5 Hz), 123.2, 122.8 (q, J=273.1 Hz), 122.5 (m), 116.9 (q, J=6.0 Hz), 114.2, 110.1, 101.7 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 395.0216 calcd. for C16H9ClF6N2O3+; found, 395.0211.

Example 5

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-methoxyphenyl)urea (PK/X17-1-155)

Yield: 40% (56.6 mg, 0.164 mmol); workup B.

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.08 (s, 1H), 8.64 (s, 1H), 8.10 (d, J=2.2 Hz, 1H), 7.65-7.57 (m, 2H), 7.36 (d, J=8.9 Hz, 2H), 6.87 (d, J=8.9 Hz, 2H), 3.72 (s, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=154.8, 152.6, 139.6, 132.1, 132.0, 126.7 (q, J=30.5 Hz), 122.9. 122.9 (q, J=273.0 Hz), 122.0, 120.6, 116.7 (q, J=5.6 Hz), 114.0, 55.2.

ESI-HR-MS (m/z) [M+H+] 345.0612 calcd. for C15H13ClF3N2O2+; found, 345.0608.

The analytical data corroborate with the literature data in Zhang, L., Darko, A. K., Johns, J. I. and McElwee-White, L. (2011), Eur. J. Org. Chem., 2011: 6261-6268. doi: 10.1002/ejoc.201100657.

Example 6

1-(Benzo[d][1,3]dioxol-5-yl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (PK/X17-1-159)

Yield: 26% (37.7 mg, 0.105 mmol); workup A (CH2Cl2/MeOH=99/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.11 (s, 1H), 8.74 (s, 1H), 8.08 (d, J=2.2 Hz, 1H), 7.64-7.58 (m, 2H), 7.18 (d, J=2.0 Hz, 1H), 6.84 (d, J=8.3 Hz, 1H), 6.79 (dd, J=8.4, 2.0 Hz, 1H), 5.98 (s, 2H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.5, 147.2, 142.4, 139.5, 133.5, 132.0, 126.7 (q, J=30.6 Hz), 123.0, 122.9 (q, J=273.1 Hz), 122.1 (m), 116.7 (m), 111.6, 108.1, 101.4, 100.9 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 359.0405 calcd. for C15H11ClF3N2O3+; found, 359.0407.

Example 7

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3,4-dimethoxyphenyl)urea (PK/X17-1-160)

Yield: 96% (147 mg, 0.392 mmol); workup A (CH2Cl2/MeOH=99/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.07 (s, 1H), 8.68 (s, 1H), 8.09 (d, J=2.3 Hz, 1H), 7.65-7.57 (m, 2H), 7.21-7.17 (m, 1H), 6.91-6.85 (m, 2H), 3.74 (s, 3H), 3.71 (s, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.5, 148.7, 144.4, 139.5, 132.7, 132.0, 126.7 (q, J=30.5 Hz), 123.0, 122.9 (q, J=273.1 Hz), 122.1 (m), 116.7 (q, J=5.7 Hz), 112.3, 110.7, 104.3, 55.8, 55.4 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 375.0718 calcd. for C16H16ClF3N2O3+; found, 375.0720.

Example 8

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3,4,5-trimethoxyphenyl)urea (PK/X17-1-162)

Yield: 98% (163 mg, 0.402 mmol); workup A (CH2Cl2/MeOH=99/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.10 (s, 1H), 8.79 (s, 1H), 8.09 (d, J=2.4 Hz, 1H), 7.67-7.58 (m, 2H), 6.80 (s, 2H), 3.75 (s, 6H), 3.61 (s, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.9, 152.4, 139.4, 135.3, 132.8, 132.0, 126.7 (q, J=30.6 Hz), 123.2, 122.9 (q, J=273.1 Hz), 122.3 (m), 116.8 (q, J=5.6 Hz), 96.4, 60.1, 55.7 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 405.0824 calcd. for C17H17ClF3N2O4+; found, 405.0828.

Example 9

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2,2-difluorobenzo[d][1,3]dioxol-4-yl)urea (PK/X17-1-164)

Yield: 38% (62 mg, 0.157 mmol); workup A (Hex/EtOAc=4/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.40 (br s, 1H), 9.04 (br s, 1H), 8.09 (s, 1H), 7.66 (dd, J=8.5, 1.1 Hz, 1H), 7.65-7.61 (m, 2H), 7.16 (t, J=8.3 Hz, 1H), 7.11-7.07 (m, 1H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=151.8, 143.1, 138.9, 133.0, 132.1, 131.0 (t, J=252.5 Hz), 126.8 (q, J=30.8 Hz), 124.5, 123.2, 123.0, 122.8, 122.8 (q, J=273.1 Hz), 116.9 (q, J=5.6 Hz), 116.5, 104.4 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 395.0216 calcd. for C16H9ClF6N2O3+; found, 395.0216.

Example 10

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)thiourea (PK/X17-1-166)

Yield: 74% (128 mg, 0.312 mmol); workup A (CH2Cl2/MeOH=99/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=10.17 (s, 1H), 10.05 (s, 1H), 8.06 (d, J=2.5 Hz, 1H), 7.78 (dd, J=8.7, 2.5 Hz, 1H), 7.67 (d, J=8.7 Hz, 1H), 7.61 (d, J=2.1 Hz, 1H), 7.40 (d, J=8.6 Hz, 1H), 7.14 (dd, J=8.6, 2.1 Hz, 1H).

13C NMR (126 MHz, DMSO-d6): 5 [ppm]=180.3, 142.5, 140.1, 139.1, 135.3, 131.6, 131.4 (t, J=252.5 Hz), 128.9, 126.1 (q, J=30.8 Hz), 125.5 (m), 122.9 (q, J=5.6 Hz), 122.7 (q, J=273.1 Hz), 120.7, 109.9, 107.8 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 410.9988 calcd. for C16H9ClF6N2O8S+; found, 410.9986.

Example 11

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(naphthalen-2-yl)urea (PK/X17-4-002)

Yield: 29% (43.0 mg, 0.118 mmol); workup A (Hex/EtOAc=4/1).

1H NMR (500 MHz, DMSO-d6): 5 [ppm]=9.25 (s, 1H), 9.07 (s, 1H), 8.18 (d, J=2.4 Hz, 1H), 8.13 (d, J=1.9 Hz, 1H), 7.87-7.79 (m, 3H), 7.68-7.61 (m, 2H), 7.50 (dd, J=8.8, 2.1 Hz, 1H), 7.48-7.44 (m, 1H), 7.39-7.35 (m, 1H).

13C NMR (126 MHz, DMSO-d6): 5 [ppm]=152.5, 139.4, 136.9, 133.7, 132.1, 129.3, 128.5, 127.5, 127.1, 126.8 (q, J=30.6 Hz), 126.4, 124.2, 123.1, 122.9 (q, J=273.0 Hz), 122.4, 119.8, 116.8 (q, J=5.6 Hz), 114.0 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 365.0663 calcd. for C18H13ClF3N2O+; found, 365.0662.

Example 12

1-(4-Benzylphenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (PK/X17-4-003)

Yield: 72% (120 mg, 0.296 mmol); workup A (Hex/EtOAc=3/1).

1H NMR (300 MHz, DMSO-d6): δ [ppm]=9.13 (br s, 1H), 8.78 (br s, 1H), 8.10 (d, J=2.0 Hz, 1H), 7.66-7.56 (m, 2H), 7.37 (d, J=8.5 Hz, 2H), 7.32-7.11 (m, 7H), 3.88 (s, 2H).

13C NMR (75 MHz, DMSO-d6): δ [ppm]=152.4, 141.6, 139.4, 137.1, 135.2, 132.0, 129.0, 128.6, 128.4, 126.7 (q, J=30.5 Hz), 125.9, 122.9, 122.8 (q, J=273.0 Hz), 122.1 (m), 118.9, 116.6 (q, J=6.0 Hz), 40.5 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 405.0976 calcd. for C21H17ClF3N2O+; found, 405.0975.

Example 13

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-ethylphenyl)urea (PK/X17-4-004)

Yield: 78% (110 mg, 0.320 mmol); workup A (Hex/EtOAc=3/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.11 (br s, 1H), 8.74 (br s, 1H), 8.10 (d, J=2.4 Hz, 1H), 7.64-7.58 (m, 2H), 7.36 (d, J=8.5 Hz, 2H), 7.12 (d, J=8.5 Hz, 2H), 2.54 (q, J=7.6 Hz, 2H), 1.15 (t, J=7.6 Hz, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.4, 139.5, 137.7, 136.8, 132.0, 128.0, 126.7 (q, J=30.5 Hz), 123.0, 122.9 (q, J=273.0 Hz), 122.1 (m), 118.8, 116.7 (q, J=5.7 Hz), 27.6, 15.8 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 343.0820 calcd. for C16H15ClF3N2O+; found, 343.0819.

Example 14

1-(4-Chloro-3-methylphenyl)-3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)urea (PK/X17-4-017)

Yield: 60% (84.0 mg, 0.247 mmol); workup A (Hex/EtOAc=3/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=8.91 (br s, 1H), 8.78 (br s, 1H), 7.65 (d, J=2.1 Hz, 1H), 7.43 (br s, 1H), 7.32-7.29 (m, 3H), 7.08 (dd, J=8.8, 2.2 Hz, 1H), 2.29 (s, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.5, 142.8, 138.4, 137.5, 136.4, 135.6, 131.3 (t, J=252.1 Hz), 129.0, 125.9, 120.7, 117.5, 113.7, 110.1, 101.3, 19.9 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 341.0499 calcd. for C16H12F2N2O3+; found, 341.0498.

Example 15

1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-3-(3-(trifluoromethyl)phenyl)urea (PK/X17-4-018)

Yield: 85% (149 mg, 0.414 mmol); workup A (Hex/EtOAc=3/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.09 (br s, 1H), 9.00 (br s, 1H), 8.00 (s, 1H), 7.66 (d, J=2.1 Hz, 1H), 7.58 (d, J=8.6 Hz, 1H), 7.51 (t, J=7.9 Hz, 1H), 7.32 (d, J=8.7 Hz, 2H), 7.12 (dd, J=8.7, 2.2 Hz, 1H).

13C NMR (75 MHz, DMSO-d6): δ [ppm]=152.5, 142.8, 140.4, 137.7, 136.2, 131.3, 129.9, 129.5 (q, J=31.4 Hz), 124.2 (q, J=272.5 Hz), 122.0, 118.3 (q, J=3.9 Hz), 114.3 (q, J=4.1 Hz), 114.0, 110.0, 101.6 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 361.0606 calcd. for C16H10F6N2O3+; found, 361.0605.

Example 16

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-phenylurea (PK/X17-3-004)

Yield: 13% (44.0 mg, 0.140 mmol); A (CH2Cl2/MeOH=2/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.15 (br s, 1H), 8.83 (br s, 1H), 8.11 (d, J=2.3 Hz, 1H), 7.66-7.59 (m, 2H), 7.46 (d, J=7.7 Hz, 2H), 7.29 (t, J=7.9 Hz, 2H), 7.00 (t, J=7.4 Hz, 1H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.4, 139.4, 139.2, 132.0, 128.8, 126.7 (q, J=30.5 Hz), 123.0, 122.9 (q, J=273.1 Hz), 122.3, 122.2 (m), 118.6, 116.7 (q, J=5.5 Hz) (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 315.0507 calcd. for C14H11ClF3N2O+; found, 315.0507.

Example 17

Butyl 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benzoate (PK/X17-3-005)

Yield: 93% (200 mg, 0.482 mmol); workup B.

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.30-9.23 (m, 2H), 8.11 (d, J=2.1 Hz, 1H), 7.89 (d, J=8.7 Hz, 2H), 7.68-7.58 (m, 4H), 4.24 (t, J=6.5 Hz, 2H), 1.72-1.64 (m, 2H), 1.46-1.37 (m, 2H), 0.93 (t, J=7.4 Hz, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=165.4, 152.1, 143.9, 139.0, 132.1, 130.4, 126.8 (q, J=30.8 Hz), 123.3, 123.2, 122.8 (q, J=273.0 Hz), 122.7 (m), 117.7, 117.0 (q, J=5.5 Hz), 64.0, 30.3, 18.8, 13.7 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 415.1031 calcd. for C19H19ClF3N2O3+; found, 415.1032.

Example 18

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-phenoxyphenyl)urea (PK/X17-3-006)

Yield: 92% (203 mg, 0.499 mmol); workup B.

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.13 (s, 1H), 8.84 (s, 1H), 8.11 (d, J=2.3 Hz, 1H), 7.66-7.59 (m, 2H), 7.48 (d, J=8.9 Hz, 2H), 7.36 (t, J=8.0 Hz, 2H), 7.09 (t, J=7.4 Hz, 1H), 7.02-6.94 (m, 4H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=157.5, 152.5, 151.1, 139.4, 135.1, 132.0, 130.0, 126.7 (q, J=30.5 Hz), 123.0, 122.9, 122.9 (q, J=273.0 Hz), 122.2 (m), 120.5, 119.7, 117.7, 116.7 (q, J=5.7 Hz) (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H]+407.0769 calcd. for C20H16ClF3N2O2+; found, 407.0770.

Example 19

1,1′-(1,3-Phenylene)bis(3-(4-chloro-3-(trifluoromethyl)phenyl)urea (PK/X17-3-003)

A solution of 4-chloro-3-(trifluoromethyl)phenyl isocyanate (339 mg, 1.53 mmol, 2.2 eq.) in dry dichloromethane (10 mL) was cooled to 0° C. After the addition of m-phenylenediamine (75.0 mg, 0.694 mmol, 1.0 eq.) the reaction mixture was allowed to warm to room temperature and the reaction was stirred at room temperature for 20 h. The solvent was removed followed by the precipitation from DMF through the addition of water (10 fold excess) and collection of the product by centrifugation (17000 g, 10 min).

Yield: 50% (192 mg, 0.348 mmol).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=9.08 (s, 2H), 8.92 (s, 2H), 8.14 (s, 2H), 7.74 (t, J=2.0 Hz, 1H), 7.61 (d, J=1.4 Hz, 4H), 7.22-7.17 (m, 1H), 7.09 (dd, J=7.9, 2.0 Hz, 2H).

ESI-HR-MS (m/z) [M+H+] 551.0471 calcd. for C22H15Cl2F6N4O2+; found, 551.0486.

Reference Example 1

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-3-phenylurea (PK/X17-4-011)

Yield: 17% (38.0 mg, 0.130 mmol); workup A (Hex/EtOAc=4/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=8.88 (s, 1H), 8.72 (s, 1H), 7.66 (d, J=2.1 Hz, 1H), 7.46-7.43 (m, 2H), 7.32-7.26 (m, 3H), 7.08 (dd, J=8.7, 2.2 Hz, 1H), 7.00-6.95 (m, 1H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.6, 142.8, 139.5, 137.4, 136.6, 131.3 (t, J=252.2 Hz), 128.8, 122.1, 118.4, 113.5, 110.1, 101.2 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 293.0732 calcd. for C14H11F3N2O3+; found, 293.0732.

Reference Example 2

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-3-(p-tolyl)urea (PK/X17-4-013)

Yield: 63% (131 mg, 0.428 mmol); workup A (Hex/EtOAc=4/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=8.83 (s, 1H), 8.61 (s, 1H), 7.66 (d, J=2.1 Hz, 1H), 7.34-7.28 (m, 3H), 7.10-7.04 (m, 3H), 2.24 (s, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=152.6, 142.8, 137.4, 136.9, 136.7, 131.3 (t, J=252.2 Hz), 130.9, 129.2, 118.5, 113.4, 110.1, 101.1, 20.4 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 307.0889 calcd. for C16H13F2N2O3+; found, 307.0887.

Reference Example 3

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-3-mesitylurea (PK/X17-4-014)

Yield: 39% (74.0 mg, 0.221 mmol); workup A (Hex/EtOAc=4/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=8.93 (br s, 1H), 7.68 (br s, 1H), 7.65 (d, J=2.1 Hz, 1H), 7.27 (d, J=8.8 Hz, 1H), 7.08 (dd, J=8.8, 2.1 Hz, 1H), 6.88 (s, 2H), 2.22 (s, 3H), 2.14 (s, 6H).

ESI-HR-MS (m/z) [M+H+] 335.1202 calcd. for C17H17F2N2O3+; found, 335.1201.

Reference Example 4

1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-3-octylurea (PK/X17-4-020)

Yield: 31% (59.0 mg, 0.180 mmol); workup A (Hex/EtOAc=3/1).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=8.62 (s, 1H), 7.63 (d, J=2.1 Hz, 1H), 7.23 (d, J=8.7 Hz, 1H), 6.96 (dd, J=8.8, 2.1 Hz, 1H), 6.17 (t, J=5.6 Hz, 1H), 3.05 (q, J=6.8 Hz, 2H), 1.45-1.37 (m, 2H), 1.26 (br s, 10H), 0.84 (t, J=6.9 Hz, 3H).

13C NMR (126 MHz, DMSO-d6): δ [ppm]=155.1, 142.8, 137.6, 136.8, 131.3 (t, J=252.1 Hz), 112.6, 109.9, 100.4, 39.1, 31.3, 29.7, 28.8, 28.8, 26.4, 22.1, 14.0 (observed complexity is due to the C—F splitting).

ESI-HR-MS (m/z) [M+H+] 329.1671 calcd. for C16H23F2N2O3+; found, 329.1671.

Reference Example 5

N-Methyl-4-(4-(3-phenylureido)phenoxy)picolinamide (PK/X17-2-011)

Yield: 63% (93.9 mg, 0.259 mmol); workup A (Hex/EtOAc=3/2).

1H NMR (500 MHz, DMSO-d6): δ [ppm]=8.81 (s, 1H), 8.76 (q, J=4.6 Hz, 1H), 8.70 (s, 1H), 8.50 (d, J=5.6 Hz, 1H), 7.58 (d, J=8.9 Hz, 2H), 7.46 (d, J=7.7 Hz, 2H), 7.38 (d, J=2.5 Hz, 1H), 7.31-7.26 (m, 2H), 7.18-7.13 (m, 3H), 7.00-6.96 (m, 1H), 2.78 (d, J=4.9 Hz, 3H).

13C NMR (75 MHz, DMSO-d6): δ [ppm]=166.0, 163.8, 152.6, 152.4, 150.4, 147.4, 139.6, 137.6, 128.8, 121.9, 121.5, 119.9, 118.3, 114.0, 108.6, 26.0.

ESI-HR-MS (m/z) [M+H+] 363.4152 calcd. for C20H19N4O3+; found, 363.1450.

3) Biological and Pharmacological Tests

3.1) Cytotoxicity Assay (MTT)

The MTT assay was performed in 96 well plates. A549, HeLa and HepG2 cells were seeded with 4000 cells/well, whereas NIH/3T3 cells were seeded with 2000 cells/well. Cells were grown to 30-40% confluence at 37° C. and 5% CO2over a time span of 24 h. The medium was removed and 100 μL medium/well containing varying concentrations of the respective compound and a final DMSO concentration of 0.1% were added to the cells in triplicates and incubated at 37° C. and 5% CO2for 24 h. 20 μL Thiazolyl blue tetrazolium bromide (5 mg/mL in PBS, Sigma Aldrich) were added to the cells and incubated at 37° C. and 5% CO2for 4 h until complete consumption was observed. After removal of the medium, the resulting formazan was dissolved in 200 μL DMSO. Optical density was measured at 570 nm (562 nm) and background subtracted at 630 nm (620 nm) by a TECAN Infinite M200 Pro.

The results of MTT testing in various cell lines (FIG.4) reveal toxicity of PK/X17-1-150 (example compound 4) at concentrations higher compared to the antibacterial MICs providing a therapeutic window for efficacy studies.

3.2) Plasma Stability Assay

The in vitro stability was tested by a LC-MS based method. Mouse plasma was purchased from biowest (mouse plasma w/lithium heparin, sterile filtered S2162-010) and used as a 1:1 dilution with potassium phosphate buffer (0.1 M, pH 7.4). Final assay concentration of DMSO from compound stocks was 1%. AV1, a β-lactone with known low plasma stability, was used as positive control at a concentration of 50 μM. The compound stability test in plasma was initiated by the addition of 10 μM compound of interest (50 μM in the case of AV1) to 250 μL of diluted mouse plasma at 37° C. Directly after compound addition the reaction mixture was shortly mixed by vortexing and the first sample of 25 μL was withdrawn (time point 0 min). Every sample was quenched immediately by the addition 30 μL of pre-chilled acetonitrile. The reaction mixture was incubated at 37° C. with gentle shaking at 600 rpm. At certain time points (5, 10, 20, 30, 60, 120, 240, 360 min) additional samples (25 μL) were taken for every test compound, quenched as described and stored at −20° C. For analysis by LC-MS all samples were allowed to warm to rt and centrifuged at 17000 g for 5 min. The supernatants were filtered through modified nylon centrifugal filters (0.45 μM) and transferred to LS-MS glass vials. Quantitative LC-MS analysis was performed by LCQ-Fleet Ion Trap Mass Spectrometer equipped with an APCI ion source and a DionexHPLC system using a Waters Xbridge BEH130 C18 column (5 μM 4.6×100 mm). Data analysis was performed by Thermo Scientific Xcalibur software. Shortly, ion peaks from single ion monitoring mass detection were integrated. Peak areas at the time point 0 min were set to 100% and peak decline with time was expressed relative to 100% at t=0 min. Plasma stability was determined in three independent experiments.

Sorafenib as well as PX/X17-1-150 (example compound 4) exhibit excellent stability in plasma for several hours which represents an ideal condition for clinical studies (FIG.5).

3.3) Minimal Inhibitory Concentration (MIC)

Minimum inhibitory concentrations (MICs) represent the lowest concentration of sample that will inhibit the visible growth of a microorganism after overnight incubation, and was obtained by a 96 well plate-based assay (Thermo Scientific) with serial dilutions of the probes tested. In the case ofStaphylococcus aureus,5 ml of fresh media was inoculated with 5 μL of the corresponding bacterial overnight culture (1:100) and incubated at 37° C. with gentle shaking (200 rpm) until the cultures reached an OD600of 0.4-0.6. Bacteria were diluted in fresh medium to a concentration of 105CFU/mL. In the case of all other bacteria species tested, fresh media was inoculated 1/10000 and directly used for testing. Diluted bacterial cultures (99 μL) were added to various concentrations of probe (1 μL of the respective stock in DMSO). A growth control containing DMSO (1 μL) and cultivated medium (99 μL) and a sterile control containing fresh medium (100 μL) were run on every 96 well plate in triplicates. After incubation at 37° C. with gentle shaking (200 rpm) for 24 h, the dilution series was analysed for microbial growth, usually indicated by turbidity and/or a pellet of bacteria at the bottom of the well. The lowest concentration in the dilution series at which no growth of bacteria could be observed by eye was defined as the minimum inhibitory concentration (MIC) of the probe. MIC values were determined by three independent experiments with at least triplicate runs for each concentration.

The antibacterial activity of example compound 4 was demonstrated in an in vitro test. Sorafenib was tested as a reference. Both compounds were tested against various bacterial strains. LB medium: 1% peptone, 0.5% NaCl, 0.5% yeast extract, pH 7.5; B medium: 1% peptone, 0.5% NaCl, 0.5% yeast extract, 0.1% K2HPO4, pH 7.5; BHB medium: 0.75% brain infusion, 1% heart infusion, 1% peptone, 0.5% NaCl, 0.25% Na2HPO4, 0.2% glucose, pH 7.4.

TABLE 1IC50values for inhibition of bacterial growth.MIC (μM)of ExampleMIC (μM)strainmediumcompound 4of SorafenibStaphylococcusB0.33aureusUSA300S. aureusMu50B0.33StaphylococcusB0.33aureusDSM18827StaphylococcusB0.33aureusNCTC8325StaphylococcusB0.35aureusBk95395StaphylococcusB0.35aureusBk97296StaphylococcusB0.35aureusIS050678StaphylococcusB0.33aureusIS050611StaphylococcusB0.35aureusVA417350StaphylococcusB0.35aureusVA418879StaphylococcusB0.35aureusVA402923StaphylococcusB0.35aureusVA412350StaphylococcusB0.33aureusVA409044StaphylococcusB0.35aureusVA402525MycobacteriumLB16smegmatismc2155Mycobacterium7H9225tuberculosisH37RvBacillus subtilisLB15AcinetobacterB10>100baumanniiPseudomonasB>100>100aeruginosaEnterobacter cloacaeB>100>100subsp.CloacaeEnterobacterB>100>100aerogenes

The antibacterial activity of compounds of the invention was demonstrated in an in vitro test in which the compounds were tested againstS. aureusNCTC 8325 by minimum inhibitory concentration (MIC) assays. The assay was performed as described above.

TABLE 2IC50values for inhibition of bacterialgrowth ofS. aureusNCTC 8325.Compound of example no.IC50(μM)10.52133040.35361073083091101111121131141151163170.6180.6191Reference Example 1>100Reference Example 2>100Reference Example 3>100Reference Example 4>100Reference Example 5>100Probe compound (PK/X17-1-058)10
3.4) Resistance Development Assay

For resistance development by sequential passaging, exponential growingS. aureusNCTC 8325 was diluted 1:100 in 1 mL MHB medium containing sorafenib, example compound 4 (PK/X17-1-150) or Ofloxacin as positive control as well as DMSO or 0.1 M NaOH as growth/negative controls. Bacteria were incubated at 37° C. with shaking at 200 rpm, and passaged in 24 h intervals in the presence of sorafenib, example compound 4 (PK/X17-1-150) or Ofloxacin at different concentrations (0.25×MIC, 0.5×MIC, 1×MIC, 2×MIC, 4×MIC). Cultures from the second highest concentrations that allowed growth (OD600≥3) were diluted 1:100 into fresh media containing different concentrations of the respective antimicrobial (0.25×MIC, 0.5×MIC, 1×MIC, 2×MIC, 4×MIC). If a shift in MIC levels was observed, concentrations of the respective antimicrobial were adjusted accordingly for the following passaging. This serial passaging was repeated for 27 days.

Serial passaging ofS. aureusin the presence of subinhibitory levels of example compound 4 (PK/X17-1-150) over a period of 27 days showed no resistant development, whereas serial passaging ofS. aureusin the presence sorafenib resulted in resistance development within the same time frame (FIG.6). Bacteria showed first signs of lower sensitivity against sorafenib within the first 5 days, while the minimal inhibitory concentration increased by a factor 40 within the first 10 days. Furthermore, preliminary results indicate that PK/X17-1-150 (example compound 4) is still active againstS. aureusthat developed resistance against sorafenib.

3.5) Activity Based Protein Profiling with Photoprobe X17PP1 (pABPP, Probe Compound) inS. aureusNCTC8325

The gel-free affinity-based protein profiling (AfBPP) platform (Evans, M. J.; Cravatt, B. F.Chem. Rev.2006, 106 (8), 3279-3301) was utilized to identify the protein target of sorafenib and structurally related compounds inS. aureus. A photoreactive derivative of sorafenib (PK/X17-1-058 (Probe compound)) bearing a terminal alkyne handle was incubated withS. aureuscells in vivo. After irradiation the cells were lysed and the terminal alkyne modified with a biotin-containing linker via click chemistry. Proteins, which were in this way irreversibly attached to a biotin molecule, were enriched on avidin beads, which bind biotins via affinity-based interaction. Following tryptic digest the samples were measured by LC-MS/MS and analyzed using MaxQuant and Perseus. We identified type I signal peptidase (SpsB), an essential serine-protease, as a possible protein target of this compound class (FIG.3). Further in vitro experiments will be conducted to biochemically validate SpsB as the molecular target of sorafenib and related compounds.

For overnight culture 5 mL of B medium (1% peptone, 0.5% NaCl, 0.5% yeast extract, 0.1% K2HPO4, pH 7.5) were inoculated with 50 μL of a cryostock (1:100) and incubated by shaking at 37° C. (200 rpm) for 14 h. The overnight culture was diluted 1:10 into 100 mL B medium. After 7 h growth an equivalent of OD600=20 of the culture was harvested at 6000×g and 4° C. for 10 min and washed with PBS. Cells were resuspended in 0.5 mL PBS. For competition experiments samples were incubated with 0.5 mM sorafenib in DMSO or DMSO only as control (final concentration of 1%) for 45 min at 25° C. and 700 rpm. After preincubation 50 μM photoprobe X17PP1 in DMSO or DMSO as control (final concentration of 2%) were added and incubated for another 45 min at 25° C. and 700 rpm. After compound treatment samples were diluted in 4 mL PBS, transferred to petri dishes and irradiated with UV light at 360 nm (Philips TL-D BLB UV) for 30 min on ice. The suspension was transferred to falcons and bacteria were harvested by centrifugation at 6000×g and 4° C. for 10 min and washed with PBS.

Cell pellets were resuspended in 0.5 mL PBS with 1×EDTA-free Complete mini protease inhibitors (Roche) on ice and transferred to Precellys Glass/Ceramic Kit SK38 2.0 mL tubes. Cells were lysed with a Precellys®24 Homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) (at 5500 rpm for 15 s. Lysis was performed 6 times with 2 min cooling breaks on ice after each run. 300 μL of the lysates were transferred to 1.5 mL microcentrifuge tubes and treated with 8 μg/mL lysostaphin (Sigma) for 20 min at 37° C. and 700 rpm. Membranes were separated from cytosol by centrifugation for 1 h at 4° C. and 21,000×g. Membrane fraction was then washed twice with PBS using an ultrasonic rod (Bandelin Sonopuls, Berlin, Germany) at 10% intensity for 10 s for resuspension. Protein concentrations were determined using bicinchoninic acid assay (Pierce BCA Protein assay kit, Thermo Fisher Scientific, Pierce Biotechnology, Rockford, IL, USA) and used for normalization.

For click chemistry 300 μL of membrane and cytosol fractions were treated with 60 μM Biotin-PEG3-N3(CLK-AZ104P4-100, Jena Bioscience, Jena, Germany), 1 mM TCEP, 0.1 mM TBTA ligand and 1 mM CuSO4. The samples were incubated for 1 h at RT in the dark. Subsequently proteins were precipitated using 1.2 mL of cold acetone over night at −80° C.

The precipitated proteins were centrifuged at 16900×g and 4° C. for 15 min and formed protein pellets were washed two times with 1 mL cold methanol (−80° C.). Resuspension was achieved by sonication (15 sec at 10% intensity with an ultrasonic rod). Pellets were resuspended in 0.5 mL 0.4% SDS in PBS at RT by sonication (15 sec at 10% intensity). For enrichment 50 μL avidin-agarose beads (Sigma) were prepared by washing the three times with 1 mL 0.4% (w/v) SDS in PBS. Protein solution were added to the washed avidin-agarose beads and incubated under continuous inverting at 20 rpm and RT for 1 h. Beads were washed three times with 1 mL 0.4% SDS in PBS, two times with 1 mL 6 M urea in water and three times with 1 mL PBS. All centrifugation steps were conducted at 400 g for 2 min at RT.

The beads with bound proteins were resuspended in 200 μl denaturation buffer (7 M urea, 2 M thiourea in 20 mM pH 7.5 HEPES buffer). Proteins were reduced on-bead with 5 mM TCEP at 37° C. and 1200 rpm for 1 h. Subsequent alkylation was performed with 10 mM Iodoacetamide at 25° C. and 1200 rpm for 30 min in the dark. Alkylation was quenched by the addition of 10 mM dithiothreitol for 30 min at RT. For digestion 1 μL LysC (0.5 μg/μL) (Wako Pure Chemical Industries, Richmond, VA, USA) was added to each sample and incubated at RT and 1200 rpm for 2 h. Afterwards samples were diluted 1:4 with 50 mM TEAB and digested with 1.5 μL trypsin (0.5 μg/μL) (Promega Sequencing Grade Modified, Promega, Madison, WI, USA) over night at 37° C. The reaction was stopped by adding formic acid (FA) to a final concentration of 0.5% (final pH of 2-3). Peptides were desalted and labelled by stable isotope dimethyl labeling (Boersema P. J. et al.,Nat protoc2009, 4 (4), 484-94)) on-column using 50 mg SepPak C18 columns (Waters). For this SepPak C18 columns were equilibrated with 1 mL acetonitrile, 1 mL elution buffer (80% ACN, 0.5% FA) and 3×1 mL aqueous 0.5% FA solution. Subsequently the samples were loaded by gravity flow, washed with 5×1 mL aqueous 0.5% FA solution and labeled with 5 mL of the respective dimethyl labeling solution. The following solutions were used: 30 mM NaBH3CN, 0.2% CH2O, 10 mM NaH2PO4, 35 mM Na2HPO4, pH 7.5 (light (L)), 30 mM NaBH3CN, 0.2% CD2O, 10 mM NaH2PO4, 35 mM Na2HPO4, pH 7.5 5 (light (M)) and 30 mM NaBHD3CN, 0.2%13CD2O, 10 mM NaH2PO4, 35 mM Na2HPO4, pH 7.5 5 (heavy (H)). For technical replicates the labels were permuted. Labeled peptides were eluted with 500 μL of elution buffer, mixed for quantification and lyophilized using a vacuum centrifuge.

Prior to mass spectrometry samples were dissolved in 0.5% FA and filtered using 0.45 μm centrifugal filter units (VWR). Samples were analyzed via HPLC-MS/MS using an UltiMate 3000 nano HPLC system (Dionex, Sunnyvale, California., USA) equipped with Acclaim C18 PepMap100 75 μm ID×2 cm trap and Acclaim C18 PepMap RSLC, 75 μM ID×15 cm separation columns coupled to an Orbitrap Fusion (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Peptides were loaded on the trap and washed for 10 min with 0.1% formic acid, then transferred to the analytical column and separated using a 120 min gradient from 3% to 25% acetonitrile (Orbitrap Fusion) in 0.1% formic acid and 5% dimethyl sulfoxide (at 200 nL/min flow rate). LTQ Orbitrap Fusion was operated in a 3 second top speed data dependent mode. Full scan acquisition was performed in the orbitrap at a resolution of 120000 and an ion target of 4e5 in a scan range of 300-1700 m/z. Monoisotopic precursor selection as well as dynamic exclusion for 60 s were enabled. Precursors with charge states of 2-7 and intensities greater than 5e3 were selected for fragmentation. Isolation was performed in the quadrupole using a window of 1.6 m/z. Precursors were collected to a target of 1e2 for a maximum injection time of 250 with “inject ions for all available parallelizable time” enabled (“Universal” method, Eliuk et al, Thermo Scientific Poster Note PN40914). Fragments were generated using higher-energy collisional dissociation (HCD) and detected in the ion trap at a rapid scan rate. Internal calibration was performed using the ion signal of fluoranthene cations (EASY-ETD/IC source).

Peptide and protein identifications were performed using MaxQuant 1.5.1.2 software with Andromeda as search engine using following parameters: Carbamidomethylation of cysteines as fixed and oxidation of methionine as well as acetylation of N-termini as dynamic modifications, trypsin/P as the proteolytic enzyme, 4.5 ppm for precursor mass tolerance (main search ppm) and 0.5 Da for fragment mass tolerance (ITMS MS/MS tolerance). Searches were performed against the Uniprot database forS. aureusNCTC 8325 (taxon identifier: 93061, downloaded on 8.5.2014). Quantification was performed using dimethyl labeling with the following settings: light: DimethLys0, DimethNter0; medium: DimethLys4, DimethNter4 and heavy: DimethLys8, DimethNter8 with a maximum of 4 labeled amino acids. Variable modifications were included for quantification. The “I=L”, “requantify” and “match between runs” (default settings) options were used. Identification was done with at least 2 unique peptides and quantification only with unique peptides.

For statistics with Perseus 1.5.1.6 three biological replicates consisting of three technical replicates each were analysed. Putative contaminants, reverse hits and proteins, identified by side only, were removed. Dimethyl labeling ratios were log 2(x) transformed and filtered to contain at least two valid values within technical replicates. Ratios were z-score normalized and average values of technical replicates were calculated. P-values were obtained by a two sided one sample t-test over the three biological replicates.

3.6 SpsB FRET with Membrane Fraction

Cells were grown according to stationary phase, harvested (12.000×g, 10 min, 4° C.), digested with lysostaphin (end conc: 20 U/mL, 37° C., 1 h) and sonicated (30 s, 20%, Bandelin Sonoplus, Berlin, Germany). Intact cells and debris were removed by centrifugation: 12.000×g, 10 min, 4° and membranes collected: 39.000×g, 75 min, 4° C. Membranes were resuspended in 2 mL cold 50 mM sodium phosphate buffer pH 7.5 and protein concentration determined by BCA (Roti®-Quant universal, Carl Roth GmbH+Co. KG, Karlsruhe, Germany) assay.

0.1 mg/mL membranes in 50 mM sodium phosphate buffer pH 7.5 were used for the FRET (Förster resonance energy transfer) assay and incubated with 1 μL compound (in DMSO) and 10 μM SPase I FRET substrate (Sequence of SceD peptide): DABCYL-AGHDAHASET-EDANS (Protein AGHDAHASET has SEQ ID NO. 1, DABCYL: 4-(4-dimethylaminophenylazo)benzoic acid; EDANS: 5-((2-aminoethyl)amino)-1-naphthalenesulfonic acid, Anaspec Inc., Fremont, CA, USA). Fluorescence turnover was determined on a TECAN plate reader (Tecan infinite 200Pro, Tecan Group Ltd., Zurich, Switzerland) at 37° C. using 340 nm as excitation and 510 nm as emission wavelengths in fluorescence top reading mode.

Addition of sorafenib and PK/X17-1-150 increased SpsB peptidase activity (FIG.7A-C) demonstrating that binding to the enzyme stimulates substrate turnover.

3.7) Analysis ofS. aureusNCTC8325 Secretome after Treatment with Sorafenib

The following protocol is based on the publication from Schallenberger et al. (Schallenberger, M. A.; Niessen, S.; Shao, C.; Fowler B. J.; Romesberg, F. E.; J Bacteriol 2012, 194 (10), 2677-2686). For overnight cultures 50 mL of B medium (1% peptone, 0.5% NaCl, 0.5% yeast extract, 0.1% K2HPO4, pH 7.5) were inoculated with 50 μL of a cryostock (1:100) and incubated by shaking at 37° C. (200 rpm) for 16 h. The overnight culture was diluted to OD600of 0.1 into 40 mL B medium per biological replicate. After 5 h growth at 37° C. OD600were measured, cells harvested by centrifugation at 3000×g and 4° C. for 15 min and washed with PBS. Cells were resuspended in fresh B medium to a cell density of ca. 1.5×109CFU/mL. 10 mL of the cells were incubated with 0.5×MIC of PK/X17-1-150 (0.15 μM) or sorafenib (1.5 μM) or DMSO as control in 50 mL tubes for 1.5 h at 37° C. (200 rpm). After treatment OD600were measured and serial dilutions plated for cell number determination. Cells were pelleted by centrifugation at 3000×g for 15 min and 6000×g for 5 min. The supernatants were collected and filtered (0.22 μM filter). Subsequently proteins were precipitated using 20% (wt/vol) trichloroacetic acid and an overnight incubation @ 4° C. Proteins were harvested by centrifugation at 9000×g, and washed two times with 90% acetone. Protein pellets were air dried and dissolved in 8 M urea in 50 mM Tris pH 8.0. Protein concentrations were measured using BCA assay (Pierce BCA Protein assay kit, Thermo Fisher Scientific, Pierce Biotechnology, Rockford, IL, USA). Protein concentrations were normalized according to protein concentrations (as determined by BCA assay), as no change in cell numbers at 0.5×MIC could be observed.

Proteins were reduced with 10 mM TCEP at 37° C. and 1200 rpm for 1 h. Subsequent alkylation was performed with 12.5 mM Iodoacetamide at 25° C. and 1200 rpm for 30 min in the dark. Alkylation was quenched by the addition of 12.5 mM dithiothreitol for 30 min at RT. For digestion 2 μL LysC (0.5 μg/μL) was added to each sample and incubated at RT and 700 rpm for 2 h. Afterwards samples were diluted 1:5 with 50 mM TEAB and digested with 2 μL trypsin (0.5 μg/μL) over night at 37° C. The reaction was stopped by adding formic acid (FA) to a final concentration of 0.5% (final pH of 2-3). Peptides were desalted on-column using 50 mg SepPak C18 columns (Waters). For this SepPak C18 columns were equilibrated with 1 mL acetonitrile, 1 mL elution buffer (80% ACN, 0.5% FA) and 3×1 mL aqueous 0.5% FA solution. Subsequently the samples were loaded by gravity flow, washed with 3×1 mL aqueous 0.5% FA solution, eluted with 500 μL of elution buffer and lyophilized using a vacuum centrifuge.

Prior to mass spectrometry samples were dissolved in 0.5% FA and filtered using 0.45 μm centrifugal filter units (VWR). Samples were analyzed via HPLC-MS/MS using an UltiMate 3000 nano HPLC system (Dionex, Sunnyvale, California, USA) equipped with Acclaim C18 PepMap100 75 μm ID×2 cm trap and Acclaim C18 PepMap RSLC, 75 μM ID×15 cm separation columns coupled to an Orbitrap Fusion (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Peptides were loaded on the trap and washed for 10 min with 0.1% formic acid, then transferred to the analytical column and separated using a 120 min gradient from 3% to 25% acetonitrile (Orbitrap Fusion) in 0.1% formic acid (at 200 nL/min flow rate). LTQ Orbitrap Fusion was operated in a 3 second top speed data dependent mode. Full scan acquisition was performed in the orbitrap at a resolution of 120000 and an ion target of 4e5 in a scan range of 300-1700 m/z. Monoisotopic precursor selection as well as dynamic exclusion for 60 s were enabled. Precursors with charge states of 2-7 and intensities greater than 5e3 were selected for fragmentation. Isolation was performed in the quadrupole using a window of 1.6 m/z. Precursors were collected to a target of 1e2 for a maximum injection time of 250 with “inject ions for all available parallelizable time” enabled (“Universal” method, Eliuk et al, Thermo Scientific Poster Note PN40914). Fragments were generated using higher-energy collisional dissociation (HCD) and detected in the ion trap at a rapid scan rate. Internal calibration was performed using the ion signal of fluoranthene cations (EASY-ETD/IC source).

Peptide and protein identifications were performed using MaxQuant 1.5.1.2 software with Andromeda as search engine using following parameters: Carbamidomethylation of cysteines as fixed and oxidation of methionine as well as acetylation of N-termini as dynamic modifications, trypsin/P as the proteolytic enzyme, 4.5 ppm for precursor mass tolerance (main search ppm) and 0.5 Da for fragment mass tolerance (ITMS MS/MS tolerance). Searches were performed against the Uniprot database forS. aureusNCTC 8325 (taxon identifier: 93061, downloaded on 8.5.2014). Quantification was performed using MaxQuant's LFQ algorithm. The “I=L”, “requantify” and “match between runs” (default settings) options were used. Identification was done with at least 2 unique peptides and quantification only with unique peptides.

For statistics with Perseus 1.5.1.6 three biological were analysed. Putative contaminants, reverse hits and proteins, identified by side only, were removed. LFQ intensities were log 2(x) transformed and filtered to contain at least one valid value. Data was filtered to contain at least two “MS/MS count” in all three replicates of either DMSO or compound treated samples or both. Protein ratios (0.5×MIC sorafenib/DMSO and 8×MIC sorafenib/DMSO) were calculated and z-score normalized. P-values were obtained by a two sided one sample t-test over the three biological replicates.

In agreement with results of the FRET peptidase assay (FIG.7) stimulation of protein secretion was also obtained in whole cells upon incubation with 0.5×MIC of PK/X17-1-150 or sorafenib (FIG.8). Analysis of the secretome (sum of all secreted proteins) revealed a strong increase of extracellular proteins which are predicted SpsB substrates.

3.8) Minimum Biofilm Eradication Concentration (MBEC)

To each well of a flat-bottomed 96-well plate (BD Biosciences, BD 351172) 200 μL of overnight culture of bacteria diluted 1:100 in media is added. Plates are incubated for 24 hours at 37° C. to establish biofilms. After 24 hours, the wells are carefully emptied by inverting the plate and gently shaking. A pre-mixed solution of media and compound stock solution is added to each well and plates are incubated at 37° C. At 16 hours after pre-established biofilms are treated with compound, the media from each well is removed, biofilms are washed three times with 200 μL PBS to remove planktonic cells, and biofilms are regrown overnight at 37° C. in 200 μL of fresh media. 100 μL of supernatant from each well are transferred to a fresh 96-well flat bottomed plate and the OD at 595 nm is measured using a plate reader (POLARstar Omega, BMG Labtech). Concentrations of compound yielding a regrown OD of less than 0.1 correspond to the MBEC. Six replicates are completed for each concentration of compound as well as positive and negative controls.

The results are shown inFIG.10A, which shows the concentration dependent biofilm eradication effect of PK/X17-1-150 onS. aureusDSM 4910 after 20 h of compound treatment. DMSO was used as negative control compound, Oxacillin was used as positive control compound. Levels of crystal violet retained were measured spectrophotometrically at an OD of 595 nm. Concentrations of compound yielding a regrown OD of less than 0.1 correspond to the MBEC. Six replicates are completed for each concentration of compound as well as controls. PK/X17-1-150 revealed the strongest effect.

Similar results are shown inFIG.10B, which show concentration dependent biofilm eradication effect of PK/X17-1-150 onS. aureusDSM 4910 after 70 h of compound treatment. DMSO was used as negative control compound, Oxacillin was used as positive control compound. Levels of crystal violet retained were measured spectrophotometrically at an OD of 595 nm. Concentrations of compound yielding a regrown OD of less than 0.1 correspond to the MBEC. Six replicates are completed for each concentration of compound as well as controls. PK/X17-1-150 and the combination Ox+PK revealed most potent effects.

3.9) Animal Model Data

FIG.11Ashows the efficacy of PK/X17-1-150 againstS. aureusin a murine bloodstream infection model. Bacterial loads in the heart (left) and liver (right) ofS. aureus-infected mice treated with 20 mg/kg of PK/X17-1-150 (squares) or vehicle alone (circles). Each symbol represents an individual mouse. Compilation data from three independent experiments are presented. N=14 for vehicle and PK/X17-1-150. Horizontal lines represent the mean values. **, p<0.01. Bacterial loads in heart (left) and liver (right) were both significantly reduced by 2 log cfu compared to the vehicle control.

FIG.11Bshows the efficacy of PK/X17-1-150 and levofloxacin against MRSA ATCC 33591 in the neutropenic murine thigh model. PK/X17-1-150 (20 mg/kg) and the corresponding vehicle were administered orally after 30 min, 4 and 8 h after bacterial inoculation, whereas levofloxacin (5 mg/kg) and the corresponding vehicle were administered intraperitoneally after 2, 6 and 10 h after bacterial inoculation. N=6 for vehicle i.p., levofloxacin i.p. and for PK/X17-1-150; n=5 for vehicle p.o. Data are expressed as mean values±SD. **, p<0.01; ***, p<0.001. A 1-log10cfu/g thigh reduction was observed in PK/X17-1-150-treated mice in comparison with sham-treated mice. The same range of reduction was determined for mice treated with the positive control levofloxacin upon i. v. administration.

3.10) Data Obtained with Persister Cells

As the generation and treatment of persister cells is highly dependent on the conditions and there is no consistency in the scientific community, two assays with different conditions were performed to corroborate effects resulting from PK/X17-1-150 treatment.

Persister cell assay I.S. aureusNCTC 8325 cells were inoculated from an exponentially growing culture at 00600=0.4-0.5 1:1000 into tryptic soy broth (TSB, 17 g/L casein peptone, pancreas hydrolysate, 3 g/L soy peptone (papain hydrolysate), 2.5 g/L di-Potassium hydrogen phosphate, 5 g/L sodium chloride, 2.5 g/L glucose monohydrate, pH 7.3±0.2; CASO Broth, Carl Roth GmbH+Co. KG) and grown for exactly 15 h at 37° C. and 200 rpm. Cells were serially diluted and plated to determine cell numbers before any treatment. Persisters were prepared by treating the culture with 20 μg/mL gentamicin (40×MIC in NCTC 8325) for 4 h at 37° C. and 200 rpm. An H2O-treated control culture was incubated in the same way. Persisters (and control cells) were washed three times with PBS (5000×g, 5 min) and diluted to OD600=4 in PBS. Serial dilutions were prepared for plating and determination of CFU/mL. 8×MIC concentrations of PK/X17-1-150 (2.4 μM) and sorafenib (24 μM) and 5 μg/mL ciprofloxacin (20×MIC) as negative control were added 1:1000 to 10 mL aliquots of the diluted persisters in 100 mL flasks and incubated at 37° C. and 200 rpm for 70 h. At indicated times cells from 1 mL samples were harvested (10000×g, 3 min), washed with PBS to remove the compound and resuspended in 1 or 0.1 mL PBS for the determination of CFU/mL by plating serial dilutions on agar plates. Three biological replicates were prepared and means, standard deviations and p-values (unpaired parametric t-test) were determined with Prism (GraphPadPrism v6.05, GraphPad Software). After 70 h there is a significant reduction of viable cells for PK/X17-1-150 and Sorafenib treated cells compared to the DMSO control, whereas there is no change for the ciprofloxacin-treated control.

Persister cell assay II. Tryptic soy broth (50 mL in 250 mL culture flasks) was inoculated 1:1000 with overnight cultures of NCTC 8325 and grown at 37° C. and 200 rpm until an OD600of 4 was reached or overnight (ON). Serial dilution were prepared and plated to determine the cell numbers in the inoculum. The cultures were aliquoted a 1 mL and treated with 30 μg/mL oxacillin (30×MIC) combined with 8×MIC of test compounds (2.4 μM PK/X17-1-150 or PK/X17-4-011, 24 μM sorafenib or PK/X17-2-011). Additionally compounds were tested without oxacillin to exclude combinatory effects, as the majority of the cells at OD600=4 and from overnight cultures already are persisters and do not require selection by oxacillin. After 20 h (A) or 70 h (B) of treatment, cells were harvested, washed two times with PBS (10000×g, 3 min), serially diluted and plated on agar plates for determination of surviving cell numbers.

There is a significant reduction of viable cells for PK/X17-1-150 and Sorafenib treated cells compared to the DSMO control, whereas there is no change observed for ciprofloxacin-, PK/X17-4-011- or PK/X17-2-011-treated controls.

3.11) Molecular Docking

1.) Preparation of the Systems

For the preparation of the systems, the signal peptidase crystal structure with the PDB code 4wvj was used for the simulations. The bound peptide was removed and the protein was solvated in a water box using tleap module of the Amber15 (Case, D. A.; J. T. B.; Betz, R. M.; Cerutti, D. S.; Cheatham, T. E. III; Darden, T. A.; Duke, R. E.; Giese, T. J.; Gohlke, H.; Goetz, A. W.; Homeyer, N.; Izadi, S.; Janowski, P.; Kaus, J.; Kovalenko, A.; Lee, T. S.; LeGrand, S.; Li, P.; Luchko, T.; Luo, R.; Madej, B.; Merz, K. M.; Monard, G.; Needham, P.; Nguyen, H.; Nguyen, H. T.; Omelyan, I.; Onufriev, A.; Roe, D. R.; Roitberg, A.; Salomon-Ferrer, R.; Simmerling, C. L.; Smith, W.; Swails, J.; Walker, R. C.; Wang, J.; Wolf, R. M.; Wu, X.; York D. M.; Kollman, P. A.AMBER2015. In University of California, San Francisco.: 2015.) program package by applying a 12 Å buffer region around protein atoms (yielding a model consisting of ˜30,000 atoms).

2.) Molecular Dynamic Simulations

All simulations were performed using the ff03 (Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.Journal of Computational Chemistry2003, 24, 1999-2012.), GAFF (Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A.J. Comput. Chem.2004, 25, 1157-1174.) and TIP3P (Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L.The Journal of Chemical Physics1983, 79, 926-935.) force field parameters for the solute, PK/X17-1-150, and solvent, respectively. Missing bonded parameters for the probe were obtained using the antechamber package (Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A.Journal of Molecular Graphics and Modelling2006, 25, 247-260.) of Amber15, with the RESP charges calculated by the Gaussian09 software (Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; lzmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L. Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J. Raghavachari, K.; Rendell, A. P.; Burant, J. C.; lyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N. Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J. Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J. Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.Gaussian09, Gaussian, Inc.: Wallingford, CT, USA, 2009.). Prior to the minimization of the models, the density of the systems was adjusted to 1 g/cm3using an in-house python script. Hydrogens and heavy atoms were minimized consecutively using the SANDER module of Amber15. Periodic boundary conditions were applied. Long-range electrostatic interactions were calculated using the particle mesh Ewald method (Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G.J. Chem. Phys.1995, 103, 8577-8593.). A non-bonded cutoff of 12 Å and a time step of 1 fs were used. The systems were heated up to 300 K in the NVT ensemble using a stepwise fashion as performed in our previous works (Marcinowski, M.; Rosam, M.; Seitz, C.; Elferich, J.; Behnke, J.; Bello, C.; Feige, M. J.; Becker, C. F.; Antes, I.; Buchner, J.J. Mol. Biol.2013, 425, 466-474; Schneider, M.; Rosam, M.; Glaser, M.; Patronov, A.; Shah, H.; Back, K. C.; Daake, M. A.; Buchner, J.; Antes, I.Proteins: Structure, Function, and Bioinformatics2016.). The SHAKE algorithm was used to constraint all bonds involving hydrogens (Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J.Journal of Computational Physics1977, 23, 327-341.). The production runs were performed in the NPT ensemble for 150 ns and 100 ns for the PK/X17-1-150 bound complex and the apo-protein, respectively. The cuda-enabled graphics processing units (GPUS) version of the pmemd module of Amber15 was used (Götz, A. W.; Williamson, M. J.; Xu, D.; Poole, D.; Le Grand, S.; Walker, R. C.Journal of Chemical Theory and Computation2012, 8, 1542-1555; Salomon-Ferrer, R.; Gotz, A. W.; Poole, D.; Le Grand, S.; Walker, R. C.Journal of Chemical Theory and Computation2013, 9, 3878-3888.).

3.) Docking and Binding Free Energy Calculations

A stepwise and comparative protocol was followed to find the binding site of the probe. Two plausible binding sites were detected using surface based analysis and analyzing their distances to the active site. The probe was docked to these two grooves separately, using the DynaDock approach of our in-house modeling program DynaCell (Antes, I.Proteins: Structure, Function, and Bioinformatics2010, 78, 1084-1104.). The docking was performed in two steps; broad sampling and the molecular dynamic based energy refinement of the selected poses. The energetically-highest ranked five poses (total of ten poses coming from two different binding sites) were further simulated up to 5 ns using the same simulation scheme introduced above. The Molecular Mechanics-Generalized Born Surface Area approach (MMGBSA) (Srinivasan, J.; Cheatham, T. E.; Cieplak, P.; Kollman, P. A.; Case, D. A.J. Am. Chem. Soc.1998, 120, 9401-9409.) was applied to calculate the binding free energies of these 10 complexes. The pose with the lowest binding free energy was chosen for further analysis. For the MMGBSA calculations, three distinct production runs (starting with different velocities) were performed on each equilibrated structure to yield 20 ns simulation time in total (time step 1 fs, a total of 225,000 complex frames (3×75,000). The MMGBSA. py module (Miller III, B. R.; McGee Jr, T. D.; Swails, J. M.; Homeyer, N.; Gohlke, H.; Roitberg, A. E.J. Chem. Theory Comput.2012, 8, 3314-3321.) of Amber15 (Case, D.; Babin, V.; Berryman, J.; Betz, R.; Cai, Q.; Cerutti, D.; Cheatham III, T.; Darden, T.; Duke, R.; Gohlke, H. Proteins 2006, 65, 712-725.) was used to combine these frames and calculate the binding free energy. The contribution of the solvent was computed with Generalized Born Surface Area (GBSA) with a probe radius of 1.4 A and the ‘rnbondi2’ radii set (Srinivasan, J.; Trevathan, M. W.; Beroza, P.; Case, D. A.Theoretical Chemistry Accounts: Theory, Computation, and Modeling(Theoretica Chimica Acta) 1999, 101, 426-434.) using the modified GB model introduced by Case et al. (Onufriev, A.; Bashford, D.; Case, D. A.The Journal of Physical Chemistry B2000, 104, 3712-3720; Onufriev, A.; Bashford, D.; Case, D. A.Proteins: Structure, Function, and Bioinformatics2004, 55, 383-394.). The entropic contributions to the free energy of binding were not included in the calculation scheme as it has been shown that such costly computations do not significantly improve the results (Hou, T.; Wang, J.; Li, Y.; Wang, W. J. Chem.Inf. Model.2010, 51, 69-82; Genheden, S.J. Comput. Aided Mol. Des.2011, 25, 1085-1093; Genheden, S.; Ryde, U.J. Chem. Theory Comput.2011, 7, 3768-3778.).