A novel class of inhibitors of peptide deformylase discovered through high-throughput screening and virtual ligand screening

Article abstract of DOI:10.1021/jm049222o

Peptide deformylase (PDF) has been identified as a promising antibacterial and herbicide target. A structurally novel class of inhibitors containing a 2-thioxo-thiazolidin-4-one heterocycle substituted by an arylidene group at the 5-position and a hexanoi

Full text of DOI:10.1021/jm049222o

JM049222O - Supporting Information
A Novel Class of Inhibitors of Peptide Deformylase
Discovered Through High Throughput Screening and
Virtual Ligand Screening
Michael H. Howard,*,† Teodorica Cenizal,† Steven Gutteridge,† Wayne S. Hanna,† Yong
Tao,† Maxim Totrov,‡ Vernon A. Wittenbach† and Ya-jun Zheng†
† DuPont Crop Protection, Stine-Haskell Research Center, 1094 Elkton Road, Newark,
Delaware 19711 and ‡ Molsoft LLC, 3366 North Torrey Pines Court, Suite 300, La Jolla,
California 92037
michael.h.howard-1@usa.dupont.com
JM049222O
SUPPORTING INFORMATION
Experimental procedures for new compounds…………………………………..…….S2
Analytical data for compounds from corporate collection and vendors...………..……S10
Biological assay protocols. ………………………………………………………..…..S14
Virtual Ligand Screening: additional details…………………………………………..S15
References for Supporting Information………………………………………………..S16
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JM049222O - Supporting Information
General Procedures. Common reagents were purchased from commercial suppliers. 4-
oxo-2-thioxo-3-thiazolidineacetic acid was purchased from Aldrich (Milwaukee, WI) and
4-oxo-2-thioxo-3-thiazolidinepropanoic acid was purchased from TCI-America
(Portland, OR). All reactions were carried out under an inert nitrogen atmosphere with
dry solvents, under anhydrous conditions, unless otherwise stated. Organic solutions
were dried over anhydrous Na2SO4 before being concentrated. TLC was performed on
silica gel 60 F254 (Merck) with detection by UV light. Column chromatography was
performed on silica gel (60-120 micron). Analytical reversed-phase HPLC was
performed on a BDS C-18 column (250mm x 4.6 mm) eluted with a gradient of MeCN in
H2O containing 0.1% TFA (flow rate of 0.50 mL/min, with a detection range of 200-400
1
nm using maximum chromatogram). H and 13C spectra were recorded at 300 or at 400
MHz and 75 or at 100 MHz, respectively, for solutions in CDCl3 [residual CHCl3 (δH
7.26 ppm) or CDCl3 (δC 77.0 ppm) as internal standard.
4-Oxo-2-thioxo-3-thiazolidinehexanoic acid (17e) was prepared according to the
literature procedure.1 The corresponding butanoic (17c), pentanoic (17d), heptanoic
(17f) and octanoic acids (17g) were prepared in the same manner from the appropriate
amino acids and their characterization data is provided, below.
4-Oxo-2-thioxo-3-thiazolidinebutanoic acid (17c): 4-aminobutanoic acid (10.31 g, 100
mmol), 22% aq. KOH (50 mL), carbon disulfide (9.7 mL, 100 mmol), chloroacetic acid
(9.45 g, 100 mmol), and conc. H2SO4 (32 mL, 180 mmol) afforded the corresponding
1
butanoic acid (14.78 g, 67 %) as an off-white solid: mp 124-127 °C; H NMR (300
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JM049222O - Supporting Information
MHz, CDCl3) δ 1.99-2.06 (m, 2H), 2.45 (t, 2H, J = 7.2), 3.98 (s, 2H), 4.09 (t, 2H, J =
7.2); MS AP(−) m/z 218.01 (M-H)−. Anal. Calcd for C7H9NO3S2: C, 38.34; H, 4.14;
N, 6.39. Found: C, 38.64; H, 3.98; N, 6.32.
4-Oxo-2-thioxo-3-thiazolidinepentanoic acid (17d): 5-Aminopentanoic acid (15.36 g,
100 mmol), 22% aq. KOH (50 mL), carbon disulfide (9.7 mL, 100 mmol), chloroacetic
acid (9.45 g, 100 mmol) and conc. H2SO4 (32 mL, 180 mmol) afforded the corresponding
1
pentanoic acid (9.44 g, 41 %) as an off white solid: mp 117-119 °C; H NMR (300
MHz, CDCl3) δ 1.62-1.81 (m, 4H), 2.41 (t, 2H, J = 6.9), 3.93-4.08 (m, 4H); MS AP(−)
m/z 232.02 (M-H)−. Anal. Calcd for C8H11NO3S2: C, 41.19; H, 4.75; N, 6.00; S, 27.49.
Found: C, 40.98; H, 4.65; N, 5.90; S, 27.70.
4-Oxo-2-thioxo-3-thiazolidineheptanoic acid (17f): 7-aminoheptanoic acid (1.0 g, 6.9
mmol), aq. KOH (0.78 g, 13.7 mmol), carbon disulfide (0.53 g, 6.9 mmol), chloroacetic
acid (0.65 g, 6.9 mmol) and conc. H2SO4 (1.22 g, 12.4 mmol) afforded the corresponding
1
heptanoic acid (0.55 g, 30%) as an off-white solid: mp 74-76 °C; H NMR (300 MHz,
CDCl3) δ 1.27-1.46 (m, 4H), 1.56-1.72 (m, 4H), 2.35 (t, 2H, J = 7.4), 3.91-4.03 (m, 4H);
13C NMR (75 MHz, CDCl3) δ 24.4, 26.3, 26.5, 28.5, 33.8, 35.3, 44.6, 173.9, 179.7, 201.2;
MS AP(−) m/z 260.11 (M-H)−.
4-Oxo-2-thioxo-3-thiazolidineoctanoic acid (17g): 8-Aminooctanoic acid (1.0 g, 6.3
mmol), aq. KOH (0.71g, 12.5 mmol), carbon disulfide (0.48 g, 6.3 mmol), chloroacetic
acid (0.59 g, 6.3 mmol) and conc. H2SO4 (1.2 g, 11.3 mmol) afforded the corresponding
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1
octanoic acid (0.50 g, 29%) as a yellow solid: mp 82-84 °C; H NMR (300 MHz,
CDCl3) δ 1.29-139 (m, 6H), 1.56-1.70 (m, 4H), 2.35 (t, 2H, J = 7.3), 3.93-4.00 (m, 4H);
13C NMR (75 MHz, CDCl3) δ 24.5, 26.5, 26.6, 28.7, 28.8, 33.9, 35.3, 44.7, 173.9, 179.7,
201.2; MS AP(−) m/z 274.11 (M-H)−. Anal. Calcd for C11H17NO3S2: C, 47.98; H, 6.22;
N, 5.09; S, 23.29. Found: C, 48.17; H, 6.06; N, 4.93; S, 22.94.
5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidinehexanoic acid (2). Ethylenediamine diacetate (2.18 g, 12.0 mmol) and 2,5-
dimethyl-1-phenylpyrrole-3-carboxaldehyde (2.41 g, 12.0 mmol), was added to stirring
solution of 4-oxo-2-thioxo-3-thiazolidinehexanoic acid (17e) (3.00 g, 12.0 mmol) in
methanol (45 mL) at rt. After 1 h, a yellow precipitate formed. Stirring was continued
overnight at room temperature. The precipitate was filtered, washed with water and dried
1
under high vacuum to give 2 (4.12 g, 79%) as a bright yellow solid: mp 184-186 °C; H
NMR (300 MHz, CDCl3) δ 1.35-1.52 (m, 2H), 1.64-1.83 (m, 4H), 2.03 (s, 3H), 2.18 (s,
3H), 2.38 (t, 2H, J = 7.5), 4.12 (t, 2H, J = 7.6), 6.20 (s, 1H), 7.18-7.21 (m, 2H), 7.45-7.56
(m, 3H), 7.75 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 11.1, 12.8, 24.2, 26.2, 26.6, 33.8,
44.1, 105.8, 114.8, 116.4, 127.8, 127.8, 128.9, 129.6, 132.6, 137.2, 137.6, 168.0, 179.3,
193.5; MS AP(−) m/z 427.12 (M-H)−. Anal. Calcd for C22H24N2O3S2: C, 61.66; H,
5.64; N, 6.54; S, 14.96. Found: C, 61.30; H, 5.43; N, 6.48; S, 15.13.
5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidinebutanoic acid (18). Following the same procedure as for 2, ethylenediamine
diacetate (0.252 g, 1.14 mmol), 2,5-dimethyl-1-phenylpyrrole-3-carboxaldehyde (0.227
g, 1.14 mmol) and 4-oxo-2-thioxo-3-thiazolidinebutanoic acid (0.250 g, 1.14 mmol)
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1
afforded 18 (290 mg, 63%) as a yellow solid: mp 87-89 °C; H NMR (300 MHz,
CDCl3) δ 2.03 (s, 3H), 2.04-2.15 (m, 2H), 2.18 (s, 3H), 2.46 (t, 2H, J = 7.5), 4.22 (t, 2H, J
= 6.8), 6.20 (s, 1H), 7.16-7.23 (m, 2H), 7.45-7.57 (m, 3H), 7.76 (s, 1H); MS AP(+) m/z
401.03 (M+H)+ . Anal. Calcd for C20H20N2O3S2 : C, 59.98; H, 5.03; N, 6.99; S, 16.01.
Found: C, 60.11; H, 5.02; N, 6.83; S, 15.85.
5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidinepentanoic acid (19). Following the same procedure as for 2,
ethylenediamine diacetate (2.31 g, 12.8 mmol), 2,5-dimethyl-1-phenylpyrrole-3-
carboxaldehyde (2.56 g, 12.8 mmol) and 4-oxo-2-thioxo-3-thiazolidinepentanoic acid
1
(3.00 g, 12.8 mmol) afforded 19 (3.55 g, 67%) as an orange solid: mp 88-90 °C; H
NMR (300 MHz, CDCl3) δ 1.67-1.87 (m, 4H), 2.03 (s, 3H), 2.18 (s, 3H), 2.44 (t, 2H, J =
7.2), 4.15 (t, 2H, J = 7.2), 6.20 (s, 1H), 7.18-7.23 (m, 2H), 7.47-7.53 (m, 3H), 7.75 (s,
1H); 13C NMR δ 24.2, 25.2 (2C), 25.6, 26.2, 26.6, 31.2(2C), 33.8, 41.6, 44.2, 125.0,
142.9, 166.6, 179.7, 194.0; MS AP(+) m/z 415.15 (M+H)+ . Anal. Calcd for
C21H22N2O3S2: C, 60.85; H, 5.35; N, 6.76; S, 15.47. Found: C, 60.90; H, 5.31; N,
6.76; S, 15.50.
5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidineheptanoic acid (20): Following the same procedure as for 2,
ethylenediamine diacetate (1.09 g, 6.7 mmol), 2,5-dimethyl-1-phenylpyrrole-3-
carboxaldehyde (1.33 g, 6.7 mmol) and 4-oxo-2-thioxo-3-thiazolidineheptanoic acid
1
(1.75 g, 6.7 mmol) afforded 20 (2.1 g, 71%) as an orange solid: mp 162-163 °C; H
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NMR (300 MHz, CDCl3) δ 1.35-1.47 (m, 4H), 1.64-1.74 (m, 4H), 2.03 (s, 3H), 2.18 (s,
3H), 2.36 (t, 2H, J = 7.4), 4.12 (t, 2H, J = 7.6), 6.21 (s, 1H), 7.20 (m, 2H), 7.51 (m, 3H)
and 7.75 (s, 1H); MS (+ ve mode): m/z 443.2 (M+H)+; HPLC purity 91% (BDS).
5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidineoctanoic acid (21): Following the same procedure as for 2, ethylenediamine
diacetate (590 mg, 3.6 mmol), 2,5-dimethyl-1-phenylpyrrole-3-carboxaldehyde (720 mg,
3.6 mmol) and 4-oxo-2-thioxo-3-thiazolidineoctanoic acid (1.0 g, 3.6 mmol) afforded 21
1
(600 mg, 38%) as an orange solid: mp 128-129 °C; H NMR (300 MHz, CDCl3) δ 1.38
(m, 4H), 1.64 (m, 6H), 2.03 (s, 3H), 2.18 (s, 3H), 2.36 (t, 2H, J = 7.4), 4.12 (t, 2H, J =
7.6), 6.21 (s, 1H), 7.20 (m, 2H), 7.51 (m, 3H) and 7.75 (s, 1H); 13C NMR (75 MHz,
CDCl3) δ 11.1, 12.7, 24.5, 26.5, 26.8, 28.8 (2C), 33.9, 44.4, 105.8, 115.0, 116.4, 127.8
(2C), 128.9, 129.6, 137.3, 137.5, 168.0, 179.5 and 193.5; MS (+ ve mode): m/z 457.2
(M+H)+; HPLC purity 85%. Anal. Calcd for C24H28N2O3S2: C, 63.13; H, 6.18; N, 6.13;
S, 14.04. Found: C, 62.98; H, 6.03; N, 6.21; S, 14.05.
N-Hydroxy-5-[(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidinehexamide (22). O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (160 mg, 1.4
mmol) was added to a solution of the acid 2 (500 mg, 1.2 mmol) in dry dichloromethane
(10 mL) at rt and cooled it to 0 oC. To this cold solution was added 1-
hydroxybenzotriazole hydrate (190 mg, 1.4 mmol) and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (270 mg, 1.4 mmol) sequentially. The mixture was then
allowed to warm to rt and stir overnight. The reaction mixture was diluted with
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dichloromethane (20 mL) and the dichloromethane layer was washed with water (2 x 10
mL), brine (10 mL) and dried (Na2SO4), filtered and concentrated. The crude material so
obtained was taken directly to the next step without further purification.
p-Toluenesulfonic acid (20 mg, 0.12 mmol) was added to a solution of the crude O-THP
hydroxamate in dry MeOH (10 mL) and stirred at rt for 2 h, at which point the reaction
was judged to be complete as indicated by formation of an FeCl3 active spot on TLC. The
reaction was quenched by adding water (30 mL) and extracted with ethyl acetate (3 x 15
mL). Ethyl acetate layer was washed with water (2 x 15 mL) and brine (15 mL), then
dried (Na2SO4), filtered and concentrated. The crude compound was purified by column
chromatography (silica gel, CHCl3 / MeOH) to obtain 22 in pure form as yellow solid
1
(310 mg, 60%). mp 110 – 113 °C; H NMR (400 MHz, CDCl3) δ 1.42 (m, 2H), 1.77 (m,
4H), 2.04 (s, 3H), 2.19 (s, 3H), 2.23 (m, 2H), 4.12 (t, 2H, J = 7.4), 6.21 (s, 1H), 7.20 (m,
2H), 7.52 (m, 3H) and 7.76 (s, 1H); 13C NMR (75 MHz) δ 11.1, 12.7, 24.5, 25.8, 26.2,
32.4, 43.8, 105.7, 114.6, 116.4, 127.7, 128.0, 128.8, 129.5, 132.6, 137.2, 137.8, 168.1,
170.9, 193.4; MS (+ ve mode): m/z 444 (M+H)+; HPLC purity 92% (BDS).
5-Cyclohexylmethylene-4-oxo-2-thioxo-3-thiazolidinehexanoic acid (23): Following
the same procedure as for 2, ethylenediamine diacetate (95.5 mg, 0.53 mmol) and
cyclohexanecarboxaldehyde (0.0554 mL, 0.53 mmol), afforded 23 (75 mg, 42%) as a
1
yellow solid: mp 130-133 °C; H NMR (300 MHz, CDCl3) δ 1.21-1.47 (m, 6H), 1.63-
1.87 (m, 10 H), 2.07-2.21 (m, 1H), 2.37 (t, 2H, J = 7.4), 4.06 (t, 2H, J = 7.6), 6.83 (d, 1H,
J = 9.4); MS AP(−) m/z 340.10 (M-H)−. Anal. Calcd for C16H23NO3S2: C, 56.28; H,
6.79; N, 4.10; S, 18.78. Found: C, 55.95; H, 6.56; N, 4.02; S, 18.46.
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5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidinehexanoic acid methyl ester (24). Iodomethane ( 0.168 mL, 0.52 mmol) was
added to stirring mixture of 2 (200 mg, 0.47 mmol) and potassium carbonate (193 mg,
1.4 mmol) in acetonitrile (15 mL) and allowed to react at rt overnight. The reaction
mixture was filtered and the filtrate was diluted with ethyl acetate (20 mL). The organic
phase was washed with 1 N NaOH (3 x 20 mL) and brine (1 x 10mL), dried over MgSO4,
filtered and concentrated to an orange oil. This oil was then purified by silica gel
chromatography (10-40% EtOAc /hexane gradient over 20 min.) and dried under high
vacuum to afford the methyl ester 24 (120 mg, 58%) as a yellow solid: mp 124-126 °C;
1H NMR (300 MHz, CDCl3) δ 1.33-1.49 (m, 2H), 1.61-1.85 (m, 4H), 2.03 (s, 3H), 2.18
(s, 3H), 2.33 (t, 2H, J = 7.8), 4.12 (t, 2H, J = 7.6), 6.21 (s, 1H), 7.17-7.21 (m, 2H), 7.44-
7.56 (m, 3H), 7.47 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 11.0, 12.7, 24.3, 26.1, 26.4,
33.7, 44.0, 51.3, 76.6, 76.9, 77.4, 105.6, 114.7, 116.3, 127.6 (2C), 128.8, 129.5, 132.5,
137.1, 137.4, 167.8, 173.8, 193.3; MS AP(+) m/z 443.04 (M+H)+. Anal. Calcd for
C23H26N2O3S2: C, 62.42; H, 5.92; N, 6.38; S, 14.49. Found: C, 62.25; H, 5.70; N,
6.20; S, 14.40.
5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidineacetic acid (25). 2,5-dimethyl-1-phenylpyrrole-3-carboxaldehyde (1.99 g,
10.0 mmol) and 4-oxo-2-thioxo-3-thiazolidineacetic acid (1.91 g, 10.0 mmol) was
allowed to stir in 15 mL ethanol under reflux for 18 h. The resulting precipitate was then
filtered, washed with water and dried under high vacuum to afford 25 (2.49 g, 67%) as an
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orange solid: mp 248-250 °C; 1H NMR (300 MHz, CDCl3) δ 2.03 (s, 3H), 2.17 (s, 3H),
4.83 (s, 2H), 6.21 (s, 1H), 7.20 (d, 2H, J = 8.1), 7.50-7.53 (m, 3H), 7.8 (s, 1H); MS AP
(+) m/z 373.02 (M+H)+ , MS AP(−) m/z 371.04 (M-H)−.
5-[(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidinepropionic acid (26). Following the same procedure as for 25, 2,5-dimethyl-
1-phenylpyrrole-3-carboxaldehyde (1.99 g, 10.0 mmol) and 4-oxo-2-thioxo-3-
thiazolidinepropanoic acid (2.05 g, 10.0 mmol) afforded 26 (2.21 g, 57%) as an orange
solid: mp 242-244 °C; 1H NMR (300 MHz, CDCl3) δ 2.02 (s, 3H), 2.18 (s, 3H), 2.84 (t,
2H, J = 7.8), 4.44 (t, 2H, J = 7.8), 6.20 (s, 1H), 7.20 (d, 2H, J = 8.1), 7.50-7.53 (m, 3H),
7.78 (s, 1H); MS AP(+) m/z 387.02 (M+H)+.
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The other active compounds presented in Tables 1 and 2, from our corporate compound
collection and from commercial sources (for the virtual ligand screening experiment),
were characterized as follows:
5-[(1,3-Diphenyl-1H-pyrazol-4-yl)methylene]-4-oxo-2-thioxo-3-thiazolidinehexanoic
1
acid (1). mp 174-176 °C; H NMR (300 MHz, DMSO) δ 1.25-1.38 (m, 2H), 1.47-1.70
(m, 4H), 2.19 (t, 2H, J = 7.3), 4.03 (t, 2H, J = 7.3), 7.42 (t, 1H, J = 7.8), 7.54-7.61 (m,
6H), 7.64-7.68 (m, 2H), 8.06 (d, 2H, J = 7.7), 8.81 (s, 1H); MS AP(−) m/z 476.01 (M-
H)−.
5-[(1-(4-Bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene]-4-oxo-2-thioxo-3-
1
thiazolidinehexanoic acid (3). mp 163-165 °C; H NMR (300 MHz, CDCl3) δ 1.38-
1.50 (m, 2H), 1.63-1.80 (m, 4H), 2.03 (s, 3H), 2.18 (s, 3H), 2.30 (t, 2H, J = 7.4), 4.11 (t,
2H, J = 7.8), 6.19 (s, 1H), 7.09 (d, 2H, J = 8.6), 7.64 (d, 2H, J = 8.3), 7.72 (s, 1H); MS
AP(+) m/z 508.88 (M+H)+ .
5-[(5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-oxo-2-thioxo-3-
1
thiazolidinehexanoic acid (4). mp 115-116 °C; H NMR (300 MHz, CDCl3) δ 1.39-
1.50 (m, 2H), 1.64-1.83 (m, 4H), 2.39 (t, 2H, J = 7.5), 2.44 (s, 3H), 4.12 (t, 2H, J = 7.5),
7.45-7.58 (m, 5H), 7.61 (s, 1H); MS AP(+) m/z 451.94 (M+H)+ .
5-[(3-(5-Methylfuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene]-4-oxo-2-thioxo-3-
thiazolidinehexanoic acid (5). mp 154-156°C; 1H NMR (300 MHz, CDCl3) δ 1.39-
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1.54 (m, 2H), 1.83-1.66 (m, 4H), 2.38 (t, 2H, J = 7.5), 2.45 (s, 3H), 4.13 (t, 2H, J = 7.5),
6.16 (d, 1H, J = 3.6), 6.82 (d, 1H, J = 3.3), 7.35-7.42 (m, 1H), 7.48-7.55 (m, 2H), 7.77 (d,
2H, J = 8.7), 8.07 (s, 1H), 8.18 (s, 1H); MS AP(+) m/z 482.00 (M+H)+ , MS AP(−) m/z
480.02 (M-H)−.
5-[(1-(2-Cyanoethyl)-3-methyl-1H-pyrazol-4-y)lmethylene]-4-oxo-2-thioxo-3-
1
thiazolidinehexanoic acid (6). mp 171-173 °C; H NMR (300 MHz, CDCl3) δ 1.33-
1.52 (m, 2H), 1.59-1.83 (m, 4H), 2.33 (t, 2H, J = 7.7), 2.39 (s, 3H), 3.00 (t, 2H, J = 7.1),
4.10 (t, 2H, J = 7.6), 4.41 (t, 2H, J = 6.5), 7.53 (s, 1H), 7.72 (s, 1H); MS AP(−) m/z
391.04 (M-H)−.
4-Oxo-5-[[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]methylene]-2-thioxo-3-
1
thiazolidinehexanoic acid (7). mp 163-164 °C; H NMR (300 MHz, CDCl3) δ 1.37-
1.50 (m, 2H), 1.63-1.82 (m, 4H), 2.31 (t, 2H, J = 7.4), 4.12 (t, 2H, J = 7.3), 7.19 (dd, 1H,
J = 3.6, 5.1), 7.37-7.56 (m, 5H), 7.81 (d, 2H, J = 7.5), 7.90 (s, 1H), 8.16 (s, 1H); MS
AP(−) m/z 481.96 (M-H)−.
5-[(1,3-Diphenyl-1H-pyrazol-4-yl)methylene]-4-oxo-2-thioxo-3-
1
thiazolidinepropanoic acid (8). mp 232-234 °C; H NMR (300 MHz, CDCl3) δ 2.49
(t, 2H, J = 8.1), 4.17 (t, 2H, J = 7.8), 7.16-7.22 (m, 1H), 7.26-7.36 (m, 5H), 7.44 (d, 2H, J
= 7.5), 7.51 (s, 1H), 7.52 (d, 2H, J = 1.2), 8.06 (s, 1H); MS AP(−) m/z 434.00 (M-H)−.
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5-[[3-(4-Methylphenyl)-1-phenyl-1H-pyrazol-4-yl]methylene]-4-oxo-2-thioxo-3-
thiazolidineacetic acid (9). mp 271-272 °C; 1H NMR (300 MHz, DMSO) δ 2.41 (s,
3H), 4.51 (s, 2H), 7.34-7.47 (m, 3H), 7.64-7.49 (m, 5H), 8.07 (d, 2H, J = 8.4), 8.81 (s,
1H); MS AP(−) m/z 435.01 (M-H)−.
5-[[5-(3,4-Dichlorophenyl)-2-furanyl]methylene]-4-oxo-2-thioxo-3-
1
thiazolidinehexanoic acid (10). mp 182-188 °C; H NMR (300 MHz, CDCl3) δ 0.94-
1.07 (m, 2H), 1.19-1.36 (m, 4H), 1.85 (t, 2H, J = 7.6), 3.68 (t, 2H, J = 7.5), 6.63-6.67 (m ,
2H), 7.10 (s, 1H), 7.16-7.28 (m, 2H,), 7.45 (d, 1H, J = 2.2); MS AP(−) m/z 468.70 (M-
H)−.
5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxo-3-thiazolidinehexanoic acid (11). mp
1
203-205 °C; H NMR (300 MHz, DMSO) δ 1.26-1.38 (m, 2H), 1.48-1.59 (m, 2H), 1.60-
1.67 (m, 2H), 2.22 (t, 2H, J = 7.3), 4.02 (t, 2H, J = 7.4), 7.20-7.31 (m, 2H), 7.52 (d, 1H, J
= 7.1), 7.89 (s, 1H), 7.96 (d, 1H, J = 7.3), 8.08 (s, 1H); MS AP(+) m/z 374.91 (M+H)+ ,
MS AP(−) m/z 373.97 (M-H)−.
5-[(3-Ethoxy-4-hydroxyphenyl)methylene]-4-oxo-2-thioxo-3-thiazolidinehexanoic
1
acid (12). mp 140-143 °C; H NMR (300 MHz, CDCl3) δ 1.11-1.82 (m, 2H), 1.2 (t, 3H,
J = 7.2), 1.34-1.45 (m, 4H), 1.98 (t, 2H, J = 7.5), 3.78-3.89 (m, 4H), 6.67-6.74 (m, 3H),
7.35 (s, 1H); MS AP(−) m/z 394.02 (M-H)−.
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JM049222O - Supporting Information
5-[(2-Methoxyphenyl)methylene]-4-oxo-2-thioxo-3-thiazolidinehexanoic acid (13).
1
mp 114-116 °C; H NMR (300 MHz, CDCl3) δ 1.36-1.52 (m, 2H), 1.64-1.82 (m, 4H),
2.37 (t, 2H, J = 7.3), 3.92 (s, 3H), 4.12 (t, 2H, J = 7.3), 6.95 (d, 1H, J = 7.8), 7.00-7.08
(m, 1H), 7.37-7.47 (m, 2H), 8.09 (s, 1H); MS AP(+) m/z 366.03 (M+H)+.
5-[(4-Fluorophenyl)methylene]-4-oxo-2-thioxo-3-thiazolidinehexanoic acid (14). mp
1
160-161 °C; H NMR (300 MHz, DMSO) δ 1.23-1.34 (m, 2H), 1.43-1.72 (m, 4H), 2.18
(t, 2H, J = 7.2), 3.98 (t, 2H, J = 7.1), 7.33-7.41 (m, 2H), 7.64-7.72 (m, 2H), 7.79 (s, 1H);
MS AP(−) m/z 352.98 (M-H)−.
5-[(3-Bromo-4-methoxyphenyl)methylene]-4-oxo-2-thioxo-3-thiazolidinehexanoic
1
acid (15). mp 179-181 °C; H NMR (300 MHz, CDCl3) δ 1.36-1.48 (m, 2H), 1.62-1.79
(m, 4H), 2.30 (t, 2H, J = 8.1), 3.97 (s, 3H), 4.11 (t, 2H, J = 7.7), 7.02 (d, 1H, J = 8.6),
7.44-7.48 (m, 1H), 7.60 (s, 1H), 7.70 (s, 1H); MS AP(−) m/z 444.52 (M-H)−.
5-(2-Methyl-3-phenyl-2-propenylidene)-4-oxo-2-thioxo-3-thiazolidinehexanoic acid
1
(16). mp 155-157 °C; H NMR (300 MHz, DMSO) δ 1.23-1.37 (m, 2H), 1.46-1.70 (m,
4H), 2.18-2.29 (m, 5H), 4.00 (t, 2H, J = 7.5), 7.30-7.48 (m, 6H), 7.55 (s, 1H); MS
AP(−) m/z 375.01 (M-H)−.
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JM049222O - Supporting Information
Soybean PDF2 cloning, expression, and purification. Based on the sequence similarity
to Arabidopsis PDF2 gene, we identified soybean expressed-sequence-tagged (EST)
clones from the corporate DNA database. Soybean PDF2 gene (matured form) was
amplified from an EST cDNA clone with two PCR primers: 5’-
CCGTAAACCCTCCTCGAACCG-3’ the sequence just preceding a NcoI site in the
soybean PDF2 gene and a primer corresponding to the vector sequence with a BamHI
site. The PCR product was inserted into the bacterial expression vector pBX3 (a modified
form of pET vector) using NcoI and BamHI sites. The PDF2 gene sequence was
confirmed by the corporate DNA sequencing facility. Soybean PDF2 genes were
expressed and purified as described previously.2
PDF Inhibition Assay. Enzyme activity was measured in half-area 96-well microtiter
plates using the PDF/FDH coupled spectrophotometric assay.3 The reaction mixture
contained 50 mM Hepes buffer (pH 7.2), 0.5 unit/ml FDH, 4 mM NAD+, and PDF. Test
compounds in DMSO (final concentration of 4%) were pre-incubated with the enzyme in
reaction mix for 10 min prior to adding the substrate, for-Met-Ala-Ser (final
concentration of 2 mM). The reaction was monitored by following the conversion of
NAD+ to NADH at 340 nm. Inhibition was determined based on the reaction rates
obtained over a 10 min period.
The effect of varying substrate and inhibitor concentration on enzyme kinetics
was monitored to determine the type of inhibition. The results were analyzed using the
GraFit software package (Erithacus Software Limited). The lines in figure 2 were
generated using a Km value of 6.4 mM.
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JM049222O - Supporting Information
Virtual Ligand Screening: additional details. The database used in our virtual
screening was compiled from the “off the shelf” compounds from various vendors.
Structures of those compounds were cleaned and tautomer state checked. A set of
structure exclusion queries were applied to remove reactive and potentially toxic
compounds and well investigated areas of chemistry. The remaining structures were
combined into a single database.
Two independent ICM VLS runs were performed and lowest score of the two
runs assigned to each compound. To explore the feasibility of discovering non-chelating
inhibitors, an additional VLS was carried out with two crystallographic water molecules
present (a metal bound water and a nearby water) in the active site.
To remove some of the artifacts of VLS, two simple filters were applied: (1) only
compounds with at least one heavy atom within 3.5Å from the metal ion were retained,
since an interaction of the ligand with the active site metal was expected; and (2)
compounds which had heavy atom clashes with receptor shorter than 0.8 of the sum of
the Van der Waals radii were removed. After filtering, a list of 4526 candidate
compounds was selected.
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JM049222O - Supporting Information
References for Supporting Information
(1) Bogolyubskaya, L. T.; Bogolyubskii, V. A.; Sytnik, Z. P. 3-(ε-
Carboxyamyl)rhodanine and some of its derivatives. J. Org. Chem. USSR
(Engl.Transl.) 1966, 2, 1311-1313.
(2) Zheng, Y.J., Tao, Y., Zhang, W., and Jordan D. B. Inhibition of Fungal Aldose
Reductase. Protein and Peptide Lett. 2001, 8, 407-412.
(3) Lazennec, C. and Meinnel, T. Formate Dehydrogenase-Coupled Spectrophotometric
Assay of Peptide Deformylase. Anal. Biochem. 1997, 244, 180-182.
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