Supporting Information

Fragment splicing-based Design, Synthesis and Safener Activity

of Novel Substituted Phenyl Oxazole Derivatives

Ying Fua, Dong Zhanga, Tao Kanga, You-Yuan Guoa, Wen-Geng Chena, Shuang Gaoa, Fei Yea,b

a
Department of Applied Chemistry, College of Science, Northeast Agricultural University, Harbin
150030, China;
[email protected]; [email protected]; [email protected]; [email protected];
[email protected]; [email protected]
b
Correspondence: [email protected]; Tel.: +86-451-5519-1507

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1. Materials and Methods

1.1. Reagents and Analysis

All solvents and reagents were purchased from commercial sources. They were analytically pure
and used as received. Yields of all of the target compounds were not optimized. Melting points were
obtained on a X-4 binocular microscope melting point apparatus and were uncorrected. The IR spectra
were recorded on a Bruker ALPHA-T spectrometer as KBr pellets with absorption given in cm -1. 1H
and 13C nuclear magnetic resonance (NMR) spectra were taken on a Bruker AV400 spectrometer with
CDCl3 as the solvent and tetramethylsilane (TMS) as the internal reference. Chemical shifts (δ) were
given in parts per million (ppm). Reaction progress was monitored by thin-layer chromatography
(TLC) on silica gel GF254 with ultraviolet (UV) detection. Column chromatographic purification was
obtained with silica gel. High-resolution mass spectrometry (HRMS) data were performed with a
Fourier transformion cyclotron resonance mass spectrometry (FTICR-MS) spectrometer.
Crystallographic data of the compound was mounted on a Rigaku RAXIS-RAPID area detector
diffractometer (Japan). Molecular docking was performed using Tripos SYBYL-X 2.0 program.

1.2. General Synthetic Procedure for the Target Compounds I-a~I-h

The key intermediate 1-a and 1-b were synthesized following the reported procedure. 1,2 A
mixture of ethyl (2-methyl-1,3-dioxolan-2-yl) acetate and NH3·H2O were mixed and stirred for 4 days
at room temperature, then the substituted (2-methyl-1,3-dioxolan-2-yl) acetamide 1-a were achieved.
The target compounds I were obtained by cyclization with the substituted α-bromoacetophenone 1-b
(3.5 mmol, prepared via the bromination of ketone) and (2-methyl-1,3-dioxolan-2-yl) acetamide 1-a
(4.2 mmol) in 30 mL DMF, under N2 for 6h at room temperature. The solvent was removed under
vacuum. Water (30 mL) was added and extracted by CH 2Cl2 (30 mL×3), the organic extracts were
combined and dried over anhydrous MgSO 4. The solvent was removed under reduced pressure, and the
crude product was purified by column chromatography on 80-100 mesh silica gel using a mixture of
petroleum ether and EtOAc (v/v, 9:1) as eluent.

1.2.1. 2-((2-Methyl-1,3-dioxolan-2-yl)methyl)-4-phenyloxazole (I-a)

White solid; yield, 25%; mp, 99-100 ℃; IR (KBr, νmax, cm-1): 3121-2901 (C-H), 1567-1451 (C=C);
1
H-NMR (400 MHz, CDCl3): δ (ppm) 7.89 (s, 1H, O-CH=C), 7.74-7.77 (d, J=9.6 Hz, 2H, Ar-H), 7.32-
7.43 (m, 3H, Ar-H), 3.98-4.00 (m, 4H, O-CH2-CH2-O), 3.21 (s, 2H, C-CH2-C), 1.52 (s, 3H, C-CH3);
13
C-NMR (100 MHz, CDCl3): δ (ppm) 160.91, 140.86, 133.75, 131.03, 128.68, 128.68, 127.97, 125.60,
125.60, 108.21, 64.98, 64.98, 38.41, 24.42; HRMS (ESI): m/z calcd for C14H15NO3: 246.1125, found:
246.1127 [M + H+]+.

1.2.2. 2-((2-Methyl-1,3-dioxolan-2-yl)methyl)-4-(p-tolyl)oxazole (I-b)

Yellow solid; yield, 27%; mp, 77-78 ℃; IR (KBr, νmax, cm-1): 3111-2879 (C-H), 1578-1505 (C=C);
1
H-NMR (400 MHz, CDCl3): δ (ppm)7.86 (s, 1H, O-CH=C), 7.63-7.65 (d, J=8 Hz, 2H, Ar-H), 7.21-
7.23 (d, J=8 Hz, 2H, Ar-H), 3.94-4.03 (m, 4H, O-CH2-CH2-O), 3.21 (s, 2H, C-CH2-C), 2.39 (s, 3H, C-
CH3), 1.52(s, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3): δ (ppm) δ 160.74, 140.91, 137.79, 133.35,
129.38, 129.38, 128.26, 125.49, 125.49, 108.24, 65.00, 65.00, 38.43, 24.43, 21.31; HRMS (ESI): m/z
calcd for C15H17NO3: 260.1281, found: 260.1284 [M + H+]+.

1.2.3. 5-Methyl-2-((2-methyl-1,3-dioxolan-2-yl)methyl)-4-phenyloxazole (I-c)

Yellow liquid; yield, 23%; IR (KBr, νmax, cm -1): 3057-2887 (C-H), 1601-1447 (C=C); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.65-7.67 (d, J=8 Hz, 2H, Ar-H), 7.40-7.44 (t, J=7.6 Hz, 2H, Ar-H), 7.30-7.32

2 / 41
(d, J=7.2 Hz, 1H, Ar-H), 3.99-4.03 (m, 4H, O-CH 2-CH2-O), 3.15 (s, 2H, C-CH2-C), 2.54 (s, 3H, CH3-
C=C), 1.53 (s, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3): δ (ppm) 158.26, 144.05, 134.64, 132.33,
128.52, 128.52, 127.14, 126.74, 126.74, 108.25, 64.97, 64.97, 38.24, 24.39, 11.91; HRMS (ESI): m/z
calcd for C15H17NO3: 260.1281, found: 260.1279 [M + H+]+.

1.2.4. 4-(4-Fluorophenyl)-2-((2-methyl-1,3-dioxolan-2-yl)methyl)oxazole (I-d)

Yellow solid; yield, 21%; mp, 74-75 ℃; IR (KBr, νmax, cm-1): 3106-2889 (C-H), 1571-1503 (C=C);
1
H-NMR (400 MHz, CDCl3): δ (ppm) 7.84 (s, 1H, O-CH=C), 7.70-7.74 (m, 2H, Ar-H), 7.07-7.12 (t,
J=11.8 Hz, 2H, Ar-H), 3.93-4.04 (m, 4H, O-CH2-CH2-O), 3.20 (s, 2H, C-CH2-C), 1.51 (s, 3H, C-CH3);
13
C-NMR (100 MHz, CDCl3): δ (ppm) 164.17, 161.00, 140.03, 133.38, 127.37, 127.26, 127.21, 115.80,
115.51, 108.17, 64.99, 64.99, 38.37, 24.41; HRMS (ESI): m/z calcd for C14H14FNO3: 264.1030, found:
264.1033 [M + H+]+.

1.2.5. 4-(4-Fluorophenyl)-5-methyl-2-((2-methyl-1,3-dioxolan-2-yl)methyl)oxazole (I-e)

Yellow liquid; yield, 19%. IR (KBr, νmax, cm -1): 3065-2888 (C-H), 1593-1509 (C=C); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.59-7.64 (m, 2H, Ar-H), 7.07-7.13 (t, J=11.6Hz, 2H, Ar-H), 3.97-4.02 (m, 4H,
O-CH2-CH2-O), 3.13 (s, 2H, C-CH2-C), 2.51 (s, 3H, CH3-C=C), 1.52 (s, 3H, C-CH3); 13C-NMR (100
MHz, CDCl3): δ (ppm) 163.61, 160.35, 158.25, 143.68, 133.93, 128.45, 128.35, 115.58, 115.29,
108.20, 64.96, 64.96, 38.25, 24.35, 11.80; HRMS (ESI): m/z calcd for C15H16FNO3: 278.1187, found:
278.1190 [M + H+]+.

1.2.6. 4-(4-Chlorophenyl)-2-((2-methyl-1,3-dioxolan-2-yl)methyl)oxazole (I-f)

Yellow solid; yield, 20%; mp, 92-93 ℃; IR (KBr, νmax, cm-1): 3101-2875 (C-H), 1574-1486 (C=C);
1
H-NMR (400 MHz, CDCl3): δ (ppm) 7.88 (s, 1H, O-CH=C), 7.66-7.71 (m, 2H, Ar-H), 7.35-7.40 (m,
2H, Ar-H), 3.93-4.04 (m, 4H, O-CH2-CH2-O), 3.20 (s, 2H, C-CH2-C), 1.51 (s, 3H, C-CH3); 13C-NMR
(100 MHz, CDCl3): δ (ppm) 161.10, 139.92, 133.88, 133.66, 129.55, 128.88, 128.88, 126.86, 126.86,
108.15, 64.99, 64.99, 38.37, 24.41; HRMS (ESI): m/z calcd for C14H14ClNO3: 280.0735, found:
280.0737 [M + H+]+.

1.2.7. 4-(2,4-Dichlorophenyl)-2-((2-methyl-1,3-dioxolan-2-yl)methyl)oxazole (I-g)

Brown liquid; yield, 13%; IR (KBr, νmax, cm -1): 184-2887 (C-H), 1587-1469 (C=C); 1H-NMR (400
MHz, CDCl3): δ (ppm) 8.29 (s, 1H, O-CH=C), 8.09-8.12 (d, J=11.6 Hz, 1H, Ar-H), 7.45-7.46 (d, J=2.8
Hz, 1H, Ar-H), 7.31-7.34 (m, 1H, Ar-H), 3.93-4.04 (m, 4H, O-CH 2-CH2-O), 3.20 (s, 2H, C-CH2-C),
1.51 (s, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3): δ (ppm) 161.68, 139.40, 137.99, 135.37, 133.66,
132.50, 131.68, 130.25, 129.09, 109.93, 66.79, 66.79, 39.99, 26.29; HRMS (ESI): m/z calcd for
C14H13Cl2NO3: 314.0345, found: 314.0347 [M + H+]+.

1.2.8. 2-((2-Methyl-1,3-dioxolan-2-yl)methyl)-4-(4-(trifluoromethyl)phenyl)oxazole (I-h)

Light yellow solid; yield, 10%; mp, 92-93 ℃; IR (KBr, νmax, cm-1): 3115-2902 (C-H), 1622-1564
(C=C); 1H-NMR (400 MHz, CDCl3): δ (ppm) 7.97 (s, 1H, O-CH=C), 7.85-7.88 (d, J=10.8 Hz, 2H, Ar-
H), 7.64-7.67 (d, J=10.8 Hz, 2H, Ar-H), 3.94-4.05 (m, 4H, O-CH2-CH2-O), 3.22 (s, 2H, C-CH2-C), 1.52
(s, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3): δ (ppm) 161.34, 139.79, 134.84, 134.59, 134.57, 130.04,
129.61, 125.74, 125.66, 122.35, 108.16, 65.02, 65.02, 38.40, 24.43; HRMS (ESI): m/z calcd for
C15H14F3NO3: 314.0999, found: 314.0997 [M + H+]+.

1.3. General Synthetic Procedure for the Target Compounds II-a~II-h

Substituted phenyloxazolidine 2-a were prepared by cyclization with various substituted
benzaldehyde and amino alcohols, and there was a slight modification from the literature report
procedure.3 Substituted amino alcohol (0.03 mol) and benzaldehyde (0.03 mol) were added in 35 mL
toluene and the reaction system was stirred for 1 h at 35 °C. The mixture was heated to reflux until

3 / 41
water was stripped off, and then cooled to 0 °C. Afterwards, 0.03 mol propyl chloroformate with 4-
dimethylaminopyridine (DMAP, 0.06 mol) was added slowly and dropwise. After completion of the
addition, the whole system was allowed to warm gradually to room temperature and stirred for 1 h. The
reaction progress was monitored by the TLC. The resulting mixture was added to water (30 mL) and
extracted with EtOAc (30 mL×3), and the organic layer was dried over anhydrous MgSO 4 and filtered.
The solvent was removed under reduced pressure, and the crude product was purified as compounds I.

1.3.1. Propyl 2-phenyloxazolidine-3-carboxylate (II-a)

Light yellow liquid; yield, 36%; IR (KBr, νmax, cm-1): 3041-2859 (C-H), 1695 (C=O); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.35-7.42 (m, 5H, Ar-H), 6.12 (s, 1H, N-CH-O), 3.61-4.12 (m, 6H, O-CH 2-C,
O-CH2-CH2-N), 1.51-1.74 (m, 2H, C-CH2-C), 0.76-0.97 (m, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3):
δ (ppm) 154.27, 139.23, 128.73, 128.37, 128.37, 126.58, 126.58, 89.20, 67.20, 65.60, 45.15, 22.24,
10.26; HRMS (ESI): m/z calcd for C13H17NO3: 258.1101, found: 258.1105 [M + Na+]+.

1.3.2. Propyl 5-methyl-2-phenyloxazolidine-3-carboxylate (II-b)

Light yellow liquid; yield, 33%; IR (KBr, νmax, cm-1): 3042-2858 (C-H), 1696 (C=O); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.35-7.43 (m, 5H, Ar-H), 5.94 (s, 1H, N-CH-O), 3.72-4.36 (m, 5H, N-CH 2-C,
O-CH-C, O-CH2-C), 1.62 (s, 1H, C-CH-C), 1.42-1.44 (d, J=6.0 Hz, 3H, O-C-CH3), 1.28-1.37 (m, 1H,
C-CH-C), 0.67-0.94 (m, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3): δ (ppm) 153.88, 128.89, 128.30,
128.30, 127.06, 127.06, 126.30, 89.93, 74.23, 67.05, 51.43, 22.19, 17.76, 10.23; HRMS (ESI): m/z
calcd for C14H19NO3: 272.1257, found: 272.1261 [M + Na+]+.

1.3.3. Propyl 2-(p-tolyl) oxazolidine-3-carboxylate (II-c)

Light yellow liquid; yield, 43%; IR (KBr, νmax, cm-1): 2944-2859 (C-H), 1694 (C=O); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.18-7.31 (m, 4H, Ar-H), 6.10 (s, 1H, N-CH-O), 3.56-4.11 (m, 6H, N-CH 2-CH2-
O, O-CH2-C), 2.37 (s, 3H, Ar-CH3), 1.53-1.77 (m, 2H, C-CH2-C), 0.78-0.97 (m, 3H, C-CH3); 13C-NMR
(100 MHz, CDCl3): δ (ppm) 154.25, 138.51, 136.33, 129.04, 129.04, 126.50, 126.50, 89.12, 67.14,
45.12, 22.27, 21.25, 21.25, 10.29; HRMS (ESI): m/z calcd for C14H19NO3: 272.1257, found: 272.1261
[M + Na+]+.

1.3.4. Propyl 5-methyl-2-(p-tolyl)oxazolidine-3-carboxylate (II-d)

Light yellow liquid; yield, 40%; IR (KBr, νmax, cm-1): 3032-2857 (C-H), 1695 (C=O); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.32 (m, 2H, Ar-H), 7.17-7.19 (d, J=7.6 Hz, 2H, Ar-H), 5.92 (s, 1H, N-CH-O),
4.19-4.28 (m, 1H, N-CH2-C), 3.96-4.06 (m, 3H, O-CH2-C, O-CH-C), 3.14-3.19 (t, J=9.8 Hz, 1H, N-
CH2-C), 2.36 (s, 3H, Ar-CH3), 1.29-1.65 (m, 5H, C-CH2-C, O-C-CH3), 0.71-0.95 (m, 3H, C-CH3); 13C-
NMR (100 MHz, CDCl3): δ (ppm) 153.92, 138.66, 136.79, 128.99, 128.99, 126.97, 126.97, 89.80,
74.09, 67.00, 52.15, 22.17, 21.30, 17.77, 10.26; HRMS (ESI): m/z calcd for C15H21NO3: 286.1414,
found: 286.1417 [M + Na+]+.

1.3.5. Propyl 2-(4-chlorophenyl)oxazolidine-3-carboxylate (II-e)

Light yellow liquid; yield, 31%. IR (KBr, νmax, cm-1): 3032-2859 (C-H), 1695 (C=O); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.35 (s, 4H, Ar-H), 6.07 (s, 1H, N-CH-O), 3.54-4.13 (m, 6H, O-CH 2-CH2-N, N-
CH2-C), 1.53-1.76 (m, 2H, C-CH2-C), 0.78-0.94 (m, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3): δ
(ppm) 154.20, 137.85, 134.55, 128.55, 128.55, 128.09, 128.09, 88.58, 67.28, 65.81, 45.09, 22.24,
10.27; HRMS (ESI): m/z calcd for C13H16ClNO3: 292.0711, found: 292.0712 [M + Na+]+.

1.3.6. Propyl 2-(4-chlorophenyl)-5-methyloxazolidine-3-carboxylate (II-f)

Light yellow liquid; yield, 29%; IR (KBr, νmax, cm-1): 3031-2858 (C-H), 1696 (C=O); 1H-NMR (400
MHz, CDCl3): δ (ppm) 7.33-7.35 (m, 4H, Ar-H), 5.91 (s, 1H, N-CH-O), 3.11-4.32 (m, 5H, N-CH 2-C,
O-CH-C, O-CH2-C), 1.64-1.78 (m, 1H, C-CH-C), 1.41-1.42 (d, J=6.0 Hz, 3H, O-C-CH3), 1.35-1.36 (d,

4 / 41
J=6.0 Hz, 1H, C-CH-C), 0.71-0.94 (m, 3H, C-CH3); 13C-NMR (100 MHz, CDCl3): δ (ppm) 153.79,
138.25, 134.66, 128.54, 128.54, 128.50, 128.50, 89.19, 74.36, 67.12, 51.38, 22.18, 17.73, 10.26;
HRMS (ESI): m/z calcd for C14H18ClNO3: 306.0867, found: 306.0869 [M + Na+]+.

1.3.7. Propyl 2-(4-methoxyphenyl)oxazolidine-3-carboxylate (II-g)

White solid; yield, 36%; IR (KBr, νmax, cm-1): 3015-2817 (C-H), 1693 (C=O); 1H-NMR (400 MHz,
CDCl3): δ (ppm) 7.34 (m, 2H, Ar-H), 6.89-6.91 (d, J=8.8 Hz, 2H, Ar-H), 6.06 (s, 1H, N-CH-O), 4.00-
4.13 (m, 4H, O-CH2-C, O-CH2-C), 3.88-3.91 (m, 1H, N-CH2-C), 3.82 (s, 3H, O-CH3), 3.56-3.64 (m,
1H, N-CH2-C), 1.52-1.65 (m, 2H, C-CH2-C), 0.77-0.98 (m, 3H, C-CH 3); 13C-NMR (100 MHz, CDCl3):
δ (ppm) 159.91, 154.23, 131.46, 127.91, 127.91, 113.71, 113.71, 89.00, 67.12, 65.45, 55.32, 45.16,
22.25, 10.29; HRMS (ESI): m/z calcd for C14H19NO4: 288.1206, found: 288.1203 [M + Na+]+.

1.3.8. Propyl 2-(4-methoxyphenyl)-5-methyloxazolidine-3-carboxylate (II-h)

White solid; yield, 36%; IR (KBr, νmax, cm-1): 2948-2857 (C-H), 1692 (C=O); 1H-NMR (400 MHz,
CDCl3): δ (ppm) 7.35(m, 2H, Ar-H), 6.89-6.91 (d, J=8.4 Hz, 2H, Ar-H), 5.89 (s, 1H, N-CH-O), 4.19-
4.25 (m, 1H, N-CH-C), 3.95-4.04 (m, 3H, O-CH-C, O-CH2-C), 3.82 (s, 3H, O-CH3), 3.14-3.19 (t, J=9.8
Hz, 1H, N-CH-C), 1.28-1.76 (m, 5H, C-CH2-C, O-C-CH3), 0.70-0.95 (m, 3H, C-CH3); 13C-NMR (100
MHz, CDCl3): δ (ppm) 160.02, 153.94, 131.94, 128.37, 128.37, 113.68, 113.68, 89.65, 74.00, 66.99,
55.32, 52.16, 22.21, 17.75, 10.29; HRMS (ESI): m/z calcd for C15H21NO4: 302.1363, found: 302.1361
[M + Na+]+.

1.4. X-ray Diffraction Analysis

Compound I-f was recrystallized from EtOAc/n-hexane to afford a suitable single crystal. Both
cell intensities and dimensions were measured at 293(2) K on a Rigaku RAXIS-RAPID area detector
diffractometer (Japan) with graphite monochromatic Mo Ka radiation (λ = 0.71073 Å). A total of 6491
reflections were measured, of which 2978 independent reflections (Rint = 0.0152) were obtained in the
range of 2.99° < θ < 27.48° (h, -6 to -7; k, -13 to 13; l, -15 to 15), and 2421 ascertained reflections with
I > 2σ (I) were used in the refinement on F2. The structure was performed by direct methods using the
SHELXS-97 refined by full-matrix least-squares on F2.4,5 The maximum and minimum differences of
peak and hole are 0.252 and -0.446 e/Å -3, respectively. The crystallographic data for crystal has been
deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication
number CCDC 1843486.

1.5. Biological Assays

1.5.1. The Growth Indexs of Maize

Maize seeds (Dongnong 253) were soaked in a solution of synthesized compounds (0.025
mmol/L) for 12 h, whereas the control was soaked in water. Afterwards, the seeds were germinated in
dishes in a growing chamber for 24 h. After, the seeds were sown in paper cups (10 cm × 15 cm), seven
seeds per cup, containing soil added with chlorsulfuron (20 mg/kg) with a depth of 13 cm, maize seeds
were incubated in a growth chamber with a 12:12 h photoperiod, 26.5 ± 1 °C, and 75% relative
humidity. Each treatment was replicated three times in a completely randomized design. The recovery
rate of growth index (plant height, root length, fresh weight of shoot, fresh weight of root) of maize
was measured 7 d after treatment.6,7
The shoots and roots tissues of maize at the optimum concentration of safener were collected for
enzyme activities (GSH, GST, ALS). The treatments were replicated three times in a completely
randomized design.

1.5.2. GSH Content

5 / 41
 The detoxification ability of safener was determined to some extent by the level of GSH
conjugation in plant. GSH content was assayed according to the procedure of Gronwald et al. 8 GSH
content in root and shoot of maize was analyzed spectrophotometrically with 5,5’-dithiobis(2-
nitrobenzoic) acid (DTNB) used as chromogenic agent. Absorbance determined at 412 nm and GSH
content was calculated by comparing with the known concentration.9

1.5.3. GST Activity

GST extraction and GST enzyme assay in vivo were carried out according to the published
literature.10 The GST activity was expressed in amount of conjugate constituted by GSH and 1-chloro-
2,4-dinitrobenezene (CDNB) catalyzed by GST per minute per milligram of enzyme (μmol·min -1·mg-1
protein). Assay of GST against chlorsulfuron in vitro was carried out by the Scarponi et al. method. 11
Chlorsulfuron solution was added to glutathione and GST extract. The reaction mixture was cultivated
2 h and stopped. The GST activity was measured by the differential value between the initial
concentration of chlorsulfuron and the chlorsulfuron residue concentration after reaction. The
concentration of chlorsulfuron lost in non-enzymatic reaction, measured by a blank sample in which
the enzymatic extract was substituted by buffer solution, was reduced from the result. The GST activity
was expressed as amount of herbicide consumed by GSH catalyzed by GST per minute per milligram
of enzyme (μmol·min-1·mg-1 protein). The protein content of the seedlings was determined
spectrophotometrically by the published literature using Coomassie blue G-250 reagent solution at 340
nm with bovine serum albumin as a standard. 12

1.5.4. ALS Activity

The treatment of maize seeds and soil was the same as that in the biological activity tests. ALS
activity determination was carried out following the procedure of Kobayashi and Sugiyama with a few
modifications.13 ALS activity was expressed as amount of acetoin composed from acetolactate
catalyzed by ALS per hour per milligram of enzyme (nmol·h-1·mg-1 protein).

1.6. Computational Methods

The three-dimension structures of compound I-f, commercial safener isoxadifen-ethyl and
chlorsulfuron were generated with the sketch module of SYBYL-X 2.0. Subsequently, the Gasteiger‐
Huckel charges were calculated, and the tested molecules were optimized. The crystal structure of ALS
was downloaded from the Protein Data Bank (PDB code 1YHY). The docking modeling used the
CDOCKER method in Accelrys Discovery Studio 3.0. 14 Before the docking, the CHARMM force field
was obtained from the protein structure, and other co-crystallized small molecules and water were
removed. After the protein preparation, the active site was defined, with a subset region of 13.0 Å
through the centre of the known ligand. During the docking process the top 10 conformations were
deposited for each ligand based on -CDOCKER_ENERGY after the energy minimization via the smart
minimize method.

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6 / 41
3. Zhu N, Ling Y, Lei X, Handratta V, Brodie AMH. Novel P450 17α inhibitors: 17-(2'-oxazolyl)- and 17-(2'-
thiazolyl)-androstene derivatives. Steroids. 2003;68:603-611. https://doi.org/10.1016/S0039-128X(03)00082-
5.
4. Yüksektepe Ç, Çalişkan N, Genç M, Servi S. Synthesis, crystal structure, HF and DFT calculations of 1-(2-
chlorobenzyl)-N-(1-(2-chlorobenzyl)-4,5-dihydro-1H-imidazol-2-yl)-1H-benzimidazol-2-amine. Crystallogr.
Rep. 2010;55:1188-1193. https://doi.org/10.1134/S106377451007014X.
5. Sheldrick GM. A short history of SHELX. Acta Crystallogr. F. 2008;64:112-122.
https://doi.org/10.1107/S0108767307043930.
6. Ercoli L, Mariotti M, Masoni A, Arduini I. Growth responses of sorghum plants to chilling temperature and
duration of exposure. Eur. J. Agron. 2004;21:93-103. https://doi.org/10.1016/S1161-0301(03)00093-5.
7. Zhao LX, Qu HT, Fu Y, Gao S, Ye F. Alleviation of injury from chlorimuron-ethyl in maize treated with
safener 3-dichloroacetyl oxazolidine. Can. J. Plant Sci. 2015;95:897-903. https://doi.org/10.4141/CJPS-2014-
437.
8. Gronwald JW, Fuerst EP, Eberlein CV, Egli MA. Effect of herbicide antidotes on glutathione content and
glutathione S-transferase activity of sorghum shoots. Pestic. Biochem. Phys. 1987;2:66-76.
https://doi.org/10.1016/0048-3575(87)90085-X.
9. Ismaiel AA, Papenbrock J. The effects of patulin from Penicillium vulpinum on seedling growth, root tip
ultrastructure and glutathione content of maize. Eur. J. Plant Pathol. 2014;139:497-509.
https://doi.org/10.1007/s10658-014-0406-9.
10. Jablonkai I, Hatzios KK. Role of glutathione and glutathione S-transferase in the selectivity of acetochlor in
maize and wheat. Pestic. Biochem. Phys. 1991;41:221-231. https://doi.org/10.1016/0048-3575(91)90076-X.
11. Scarponi L, Quagliarini E, Del Buono D. Induction of wheat and maize glutathione S-transferase by some
herbicide safeners and their effect on enzyme activity against butachlor and terbuthylazine. Pest Manag. Sci.
2006;62:927-932. https://doi.org/10.1002/ps.1258.
12. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem. 1976;72:248-254. https://doi.org/10.1016/0003-
2697(76)90527-3.
13. Kobayashi K, Sugiyama H. Selective action of pyrazosulfuron-ethyl on growth and acetolactate synthase
activity between rice and cyperus serotinus. Weed Res. 1991;36:251-256.
https://doi.org/10.3719/weed.36.251.
14. Catalyst, Version 4.10. Accelrys, Inc., San Diego, CA, USA, 2005.

7 / 41
 IR
Transmittance [%]
65 70 75 80 85 90 95 100

3500
compounds I and II
3121.96

2981.69

3000
2901.47

2500

8 / 41
Compound I-a

2000
Wavenumber cm-1
1567.74

1500
1451.36
1354.89 1388.01
1271.97 1298.31
1214.17
1128.79
1047.91 1065.73

1000
945.12
863.96
793.03
750.32
693.24

502.09

500
2. Copies of detailed analytical IR, 1H NMR, 13C NMR, and HRMS spectra of
1
H NMR
7.89
7.77
7.74
7.43
7.41
7.38
7.34
7.32
7.28

4.00
3.98

3.21

1.52
1000

900

800

700

600

Intensity
500

400

300

200

100

0
0.89
1.98

2.94

4.06

2.03

3.00

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR

9 / 41
 420000

160.91

140.86
133.74
131.03
128.68
127.97
125.59

108.21

77.44
77.02
76.60

64.98

38.41

24.42
400000

380000

360000

340000

320000

300000

280000

260000

240000

220000

Intensity
200000

180000

160000

140000

120000

100000

80000

60000

40000

20000

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS
F:\Users\...\fuying-36_160331172302 3/31/2016 8:29:19 PM 36#
Error=0.8 ppm

NL:
246.1127 7.57E5
100
fuying-
90 36_160331172302#1
5 RT: 0.12 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50
268.0946
40

30

20
247.1160
10 209.1075 245.0787 338.3420
269.0979 284.0685 301.1412
311.1293
195.0878 202.0865 210.1109 221.0950 235.0980 248.1197 266.1183 299.1103 315.9830 330.3366
0
NL:
246.1125 8.49E5
100
C14 H15 N1 O3 +H:
90 C14 H16 N1 O3
paChrg1
80

70

60

50

40

30

20 247.1158
10
248.1192
0
190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340
m/z

Compound I-b
IR

10 / 41
 1
Transmittance [%]
60 65 70 75 80 85 90 95 100

H NMR
3500
3111.06
3091.96
2987.13

3000
2924.06
2879.49

2500

11 / 41
2000
Wavenumber cm-1
1731.27

1560.85 1577.53
1505.19
1500

1379.86

1213.77

1042.66
1000

946.37
837.75

694.83
604.72
550.44
508.48
500
 7.86
7.65
7.63
7.28
7.23
7.21

4.03
4.02
4.01
4.01
4.00
4.00
4.00
3.98
3.98
3.97
3.97
3.97
3.96
3.94
3.21

2.39

1.52
13000

12000

11000

10000

9000

8000

Intensity
7000

6000

5000

4000

3000

2000

1000

0
0.94
1.89

1.92

3.80

1.88

3.08

3.00
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5
f1 (ppm)

13
C NMR
160.74

140.91
137.79
133.35
129.38
128.26
125.49

108.24

77.37
77.05
76.73

65.00

38.43

24.43
21.31
8500

8000

7500

7000

6500

6000

5500

5000

4500
Intensity

4000

3500

3000

2500

2000

1500

1000

500

-500

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS

12 / 41
F:\Users\...\fuying-54_161109161909 11/9/2016 9:22:06 PM 54#
Error=1.2 ppm

NL:
260.1284 1.34E6
100
fuying-
90 54_161109161909#6
RT: 0.05 AV: 1 T:
80 FTMS + p ESI Full
ms [100.00-2000.00]
Relative Abundance

70

60
282.1104
50

40

30

20 261.1315 298.0843
10 262.1351 283.1136
279.1595 301.1413 360.3242 376.2983 383.2045
205.0862 216.1022 228.1961 238.0843 254.0583 284.1172 317.1155 330.3371 338.3420
0
NL:
260.1281 8.40E5
100
C15 H17 N1 O3 +H:
90 C15 H18 N1 O3
paChrg1
80

70

60

50

40

30

20 261.1315

10
262.1348
0
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390
m/z

Compound I-c
IR
100
90
Transmittance [%]

545.30
914.14
80

3465.51

856.90
3057.40

804.34
70

968.11
1495.07
1600.91
1716.55

950.44
1381.03
2887.06

1447.34
60

770.43
1129.34
1256.40
2985.78

1571.39
1683.24

1193.49
1357.64
2934.34

698.44
1047.31

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
13 / 41
 11000

7.67
7.65
7.44
7.42
7.40
7.32
7.30
7.28

4.03
4.03
4.02
4.01
4.00
3.99

3.15

2.54

1.53

0.02
10000

9000

8000

7000

6000

Intensity
5000

4000

3000

2000

1000

0
1.80
1.98
0.81

4.14

1.71

3.00

2.69
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
158.26

144.05

134.64
132.33
128.52
127.14
126.74

108.25

77.38
77.06
76.74

64.97

38.24

24.39

11.91
11000

10000

9000

8000

7000

6000
Intensity

5000

4000

3000

2000

1000

-1000
160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
f1 (ppm)

MS
14 / 41
F:\Users\...\fuying-55_161109161909 11/9/2016 9:17:39 PM 55#
Error=0.8 ppm

NL:
260.1279 1.96E6
100
fuying-
90 55_161109161909#1
4 RT: 0.10 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60 282.1099

50

40

30

20 261.1312
298.0838
262.1347 283.1131
10 254.0487 301.1407
200.1068 218.1174 231.0474 246.1276 279.1590 295.1944 321.1309 330.3364 338.3413 353.0641 365.1570 383.2040
0
NL:
260.1281 8.40E5
100
C15 H17 N1 O3 +H:
90 C15 H18 N1 O3
paChrg1
80

70

60

50

40

30

20 261.1315

10
262.1348
0
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380
m/z

Compound I-d
IR
100

1889.34
80
Transmittance [%]

2991.82
60

1310.15
3105.67

602.18
1289.86
1381.98
2888.93
40

1571.24

947.02
20

1503.29

1213.19

1042.88

836.15

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
15 / 41
 1700

7.84
7.74
7.73
7.71
7.70
7.28
7.12
7.09
7.07

4.04
4.02
4.01
4.01
4.00
4.00
3.98
3.98
3.97
3.97
3.96
3.93
3.20

1.51
1600

1500

1400

1300

1200

1100

1000

900

Intensity
800

700

600

500

400

300

200

100

0
0.94
2.04

2.07

4.23

2.04

3.00
-100
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
164.17
161.00

140.03

133.38
127.37
127.26
127.21

108.17

360000
115.80
115.51

77.46
77.03
76.61

64.99

38.37

24.41
340000

320000

300000

280000

260000

240000

220000

200000
Intensity

180000

160000

140000

120000

100000

80000

60000

40000

20000

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20

f1 (ppm)

MS
16 / 41
F:\Users\...\fuying-37_160331203559 3/31/2016 8:36:00 PM 37#
Error=1.1 ppm

NL:
264.1033 1.34E6
100
fuying-
90 37_160331203559#5
RT: 0.04 AV: 1 T:
80 FTMS + p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50

40
286.0852
30
246.1127
20 265.1065
258.0242
10 268.0945 302.0591
209.1075 247.1161 287.0885
220.0771 230.2482 239.5813 284.0686 299.1103 311.1294 318.3004 330.3369 346.3318 358.3681
0
NL:
264.1030 8.49E5
100
C14 H14 F1 N1 O3 +H:
90 C14 H15 F1 N1 O3
paChrg1
80

70

60

50

40

30

20 265.1064
10
266.1098
0
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360
m/z

Compound I-e
IR
100
80
Transmittance [%]
60

3065.35
40

732.09
1725.10
20

540.35
2986.49

1381.15

600.49
1593.30

840.71
1047.55
1223.84
1508.73

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
17 / 41
 1200

7.64
7.62
7.61
7.59
7.28
7.13
7.10
7.07

4.02
4.01
4.01
4.00
4.00
3.99
3.98
3.98
3.97
3.97
3.13

2.51

1.52
1100

1000

900

800

700

Intensity
600

500

400

300

200

100

0
2.04

2.05

4.14

2.07

2.82

3.00
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
163.61
160.35
158.25

143.68

133.93
128.45
128.35

108.20
115.58
115.29

77.47
77.04
76.62

64.96

38.25

24.35

11.80
65000

60000

55000

50000

45000

40000

35000
Intensity

30000

25000

20000

15000

10000

5000

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS
18 / 41
F:\Users\...\fuying-38_160331203559 3/31/2016 8:41:13 PM 38#
Error=1.1 ppm

NL:
278.1190 5.53E5
100
fuying-
90 38_160331203559#3
RT: 0.02 AV: 1 T:
80 FTMS + p ESI Full ms
[100.00-2000.00]
Relative Abundance

70
300.1009
60

50

40

30

20 279.1223
301.1043 316.0749
10 282.1091
234.0928 245.1152 253.0664 264.1034 274.2746 290.0803 302.1074 317.0781 338.3422 350.0804 360.3243 376.0871
0
NL:
278.1187 8.40E5
100
C15 H16 F1 N1 O3 +H:
90 C15 H17 F1 N1 O3
paChrg1
80

70

60

50

40

30

20 279.1221

10
280.1254
0
230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380
m/z

Compound I-f
IR
100
90
80
Transmittance [%]
70

2990.59

1344.56
3100.51
60

506.88
1406.41

1015.26
2875.03
50

785.72
1573.59

559.03
1382.29

945.88 972.07
1486.42

1162.06
40

1066.90
1117.59

835.63
30

1188.54 1211.89

1043.61
1096.23

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
19 / 41
 7.88
7.71
7.70
7.69
7.68
7.67
7.66
7.40
7.39
7.38
7.37
7.36
7.35
7.28

4.04
4.02
4.01
4.01
4.00
3.98
3.97
3.97
3.96
3.93
3.20

1.51
1600

1500

1400

1300

1200

1100

1000

900

Intensity
800

700

600

500

400

300

200

100

0
0.90
1.92
1.90

3.96

1.97

3.00
-100
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
161.10

139.92
133.88
133.66
129.55
128.88
126.86

108.15

77.45
77.03
76.61

64.99

38.37

24.41
140000

130000

120000

110000

100000

90000

80000
Intensity

70000

60000

50000

40000

30000

20000

10000

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20

f1 (ppm)

MS
20 / 41
F:\Users\...\fuying-39_160331203559 3/31/2016 8:45:47 PM 39#
Error=0.7 ppm

NL:
280.0737 1.63E6
100
fuying-
90 39_160331203559#9
RT: 0.06 AV: 1 T:
80 FTMS + p ESI Full ms
[100.00-2000.00]
Relative Abundance

70
302.0556
60

50

40
282.0706
30
278.1190 304.0526
20 300.1009 383.2043
274.2743 384.2077
10 318.0295 360.3239 374.3629
283.0739 305.0559 330.3369 399.1781
209.1075 220.0525 236.0475 246.2428 261.1310 338.3419 361.3271
0
NL:
280.0735 6.44E5
100
C14 H14 Cl1 n1 O3 +H:
90 C14 H15 Cl1 N1 O3
paChrg1
80

70

60

50

40
282.0705
30

20

10
283.0739
0
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410
m/z

Compound I-g
IR
100
80
Transmittance [%]

3073.67
60

3183.94
40

530.00
20

1292.89
2887.12
2986.03

942.81
1586.63

1191.54
1380.14
1469.30
1737.41

1048.02

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
21 / 41
 13
171.28

MS
8.5

170
0.72 8.29
8.12
161.68 0.75 8.09

C NMR
8.0

160
7.46
7.45
7.34

7.5
0.73

150
0.77 7.34
139.40 7.31
137.99 7.31

7.0

140
135.37 7.28
133.66
132.50

6.5
131.68

130
130.25
129.09

6.0

120
109.93

5.5

110
4.04

5.0
4.02

100
4.01
4.00
3.99

90
4.5
3.97

f1 (ppm)
3.97

f1 (ppm)
79.26
4.11 3.97

80
4.0
78.84
3.96

22 / 41
78.41
3.96
3.95

70
3.5
66.79 3.93
66.59 1.55 3.20
62.35

60
3.0
2.68

50
2.5
46.07

39.99

40
2.0

3.10 1.51

30
1.5

26.29
26.22

20
1.0

15.99

10
0.5

0
0.0

0
0
100
200
300
400
500
600
700
800
900
1100

1000
1200

20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000

Intensity Intensity
F:\Users\...\fuying-57_161109161909 11/9/2016 9:07:23 PM 57#
Error=0.6 ppm

NL:
336.0167 1.66E6
100
fuying-
90 57_161109161909#17
RT: 0.12 AV: 1 T: FTMS
80 314.0347 + p ESI Full ms
[100.00-2000.00]
Relative Abundance

70
338.0137
60
316.0317
50

40

30 342.0085

20 330.3369
301.1411
355.9855
318.0289 322.0008 351.9906 357.9827
10 300.0189 302.3055 313.3500 331.3403 343.0118 363.0033
296.2198 307.9800 325.9958 349.1833 370.0221
0
NL:
314.0345 4.88E5
100
C14 H13 Cl 2 N1 O3 +H:
90 C14 H14 Cl 2 N1 O3
paChrg1
80

70
316.0316
60

50

40

30

20
318.0286
10
319.0320 323.0429
0
295 300 305 310 315 320 325 330 335 340 345 350 355 360 365 370
m/z

Compound I-h
IR
90
80

3114.81
3093.52
2994.39
70
Transmittance [%]

2902.42

1563.78
60

1415.21
50

1622.14

948.05
40

842.90
30
20

1130.19
1328.88

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
23 / 41
 7.97
7.88
7.85
7.67
7.64
7.28

4.05
4.03
4.02
4.01
4.00
3.98
3.97
3.97
3.96
3.94
3.22

1.52

0.01
750

700

650

600

550

500

450

400

Intensity
350

300

250

200

150

100

50

0
0.96
1.92
1.94

3.95

1.86

3.00
-50

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
161.34

139.79
134.84
134.59
134.57
130.04
129.61
125.74
125.66
122.35

108.16

220000
77.47
77.04
76.62

65.02

38.40

24.43
23.73
210000
200000

190000
180000

170000
160000
150000
140000
130000

120000
Intensity

110000

100000
90000
80000

70000

60000
50000

40000

30000
20000

10000
0

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20

f1 (ppm)

MS
24 / 41
F:\Users\...\fuying-56_161109161909 11/9/2016 9:12:30 PM 56#
Error=0.6 ppm

NL:
314.0997 1.15E6
100
fuying-
90 56_161109161909#19
RT: 0.15 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-2000.00]
Relative Abundance

70 336.0817

60

50

40

30

20 315.1030
337.0851
10 301.1409
279.1590 360.3235 372.2433
239.1617 255.1355 317.1147 396.1140 413.2662 429.2401 442.0781 468.1605 484.1917 498.2069
0
NL:
314.0999 8.40E5
100
C15 H14 F3 N1 O3 +H:
90 C15 H15 F3 N1 O3
paChrg1
80

70

60

50

40

30

20 315.1032

10
316.1066
0
240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z

Compound II-a

IR
100
90 80
Transmittance [%]
50 60 4070

3040.89
3011.78
2944.15
2858.97

1694.77

1447.62
1408.65
1353.14

1119.53
1057.07

935.26

755.30
693.05

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
25 / 41
 7.42
7.41
7.40
7.38
7.37
7.35

6.12

4.12
4.07
3.90
3.61

1.73
1.51

0.97
0.76
6500

6000

5500

5000

4500

4000

3500

Intensity
3000

2500

2000

1500

1000

500

0
4.54

0.87

4.87

0.94

2.33

3.00
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
154.27

139.23

128.73
128.37
126.58

89.20

77.39
77.07
76.75

67.20
65.60

45.15

22.24

10.26
12000

11000

10000

9000

8000

7000
Intensity

6000

5000

4000

3000

2000

1000

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS
26 / 41
F:\Users\...\fuying-15_170107120924 1/7/2017 12:20:05 PM 15#
Error=1.6 ppm

NL:
258.1105 1.49E6
100
fuying-
90 15_170107120924#7
RT: 0.05 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50

40

30
236.1286 274.0845
20
259.1138 301.1415
10 255.1021
175.1096 185.1152 237.1318 279.1595 295.1950 304.2616 317.1155
205.0862 225.1768 260.1172 334.0969 353.2668 364.1075 372.6627 381.2981
0
NL:
258.1101 8.58E5
100
C13 H17 N1 O3 +Na:
90 C13 H17 N1 O3 Na1
paChrg 1
80

70

60

50

40

30

20
259.1134
10
260.1168
0
180 200 220 240 260 280 300 320 340 360 380
m/z

Compound II-b
IR
100
90
80
Transmittance [%]
40 50 30
2060 70

3041.73
3012.58
2948.83
2858.40

1695.69

1447.65
1407.94
1350.66

1200.73

1063.86

940.74

755.97
692.73
618.00

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

F:\Sample description.6 Sample description Instrument type and / or accessory 06/03/2017

Page 1/1

1
H NMR
27 / 41
 13
8.0

MS
160
7.43
153.88 7.38

7.5
4.91 7.36

C NMR

150
7.35
7.28

7.0

140
6.5
128.89

130
128.30
127.06
126.30

6.0
0.80 5.94

120
5.5

110
4.37

5.0
4.36

100
4.34
4.28
4.27

4.5
89.93

90
4.26
4.24
4.23 4.23

4.0
77.37 4.05

80
77.05 3.97

f1 (ppm)
f1 (ppm)
76.73 3.71
74.23

3.5
3.37

70

28 / 41
67.05 1.10 3.20
3.18
3.15

3.0

60
51.43

2.5

50
2.0

40
1.62
1.44
0.81
1.42
1.5

3.22

30
1.37
0.97
1.36
22.19 1.28
1.0

20
0.94
17.76 3.11 0.75
0.67
10.23
0.5

10
0
0.0

0
0
500

-500

1000
2000
3000
4000
5000
6000
7000
8000
9000
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000

10000

Intensity Intensity
F:\Users\...\fuying-16_170107120924 1/7/2017 12:36:24 PM 16#
Error=2.2 ppm

NL:
272.1261 3.07E6
100
fuying-
90 16_170107120924#1
1 RT: 0.08 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50

40

30 250.1441
288.1000
20 273.1294 295.1949
10 269.1177 301.1414
251.1474 279.1595 309.2041 321.2104 330.3372 338.3430 348.1123 353.2666
206.1543 215.0594 225.1767 239.1323 245.0787 267.1633
0
NL:
272.1257 8.49E5
100
C14 H19 N1 O3 +Na:
90 C14 H19 N1 O3 Na1
paChrg1
80

70

60

50

40

30

20 273.1291
10
274.1324
0
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360
m/z

Compound II-c
IR
100
90
Transmittance [%]
70 60
50 80

2944.25
2858.81

1694.37

1502.06
1447.25
1408.54
1353.34

1119.71
1058.73

932.51

805.47
767.65

569.40

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
29 / 41
 22000

7.31
7.28
7.20
7.18

6.10

4.05
4.04
4.02
3.89
3.62
3.60
3.60
3.59
3.58
3.56

2.37

1.77
1.67
1.53

0.97
0.78
4.11
21000
20000
19000
18000
17000
16000
15000
14000
13000
12000

Intensity
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
4.10

0.98

4.21
0.96

1.02

3.16

2.23

3.00
-1000

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
154.25

138.51
136.33

129.04
126.50

89.12

77.42
77.10
76.78

67.14

57.51

45.12

22.27
21.25

10.29
7000

6500

6000

5500

5000

4500

4000
Intensity

3500

3000

2500

2000

1500

1000

500

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS
30 / 41
F:\Users\...\fuying-17_170107120924 1/7/2017 12:39:05 PM 17#
Error=1.5 ppm

NL:
272.1261 3.35E6
100
fuying-
90 17_170107120924#1
6 RT: 0.12 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50

40

30
250.1441
288.1000
20 273.1294 289.1034
10 269.1178 301.1414
251.1474 261.0529 309.2041
202.5911 213.1388 223.0968 239.1322 245.0788 279.1595 321.2105 330.3372 348.1122 358.3688
0
NL:
272.1257 8.49E5
100
C14 H19 N1 O3 +Na:
90 C14 H19 N1 O3 Na1
paChrg1
80

70

60

50

40

30

20 273.1291
10
274.1324
0
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360
m/z

Compound II-d
IR
100
90 80
Transmittance [%]
60 70
50
40

3031.52
2949.49
2912.83
2856.90

1695.21

1503.81
1445.96
1412.85
1349.74

1200.12
1109.65
1069.07

942.26

805.82
768.15

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
31 / 41
 7.32
7.29
7.19
7.17

5.92

4.28
4.26
4.25
4.24
4.23
4.22
4.21
4.19
4.06
3.96
3.19
3.17
3.14

2.36

1.65

0.95
0.71
20000

19000

18000

17000

16000

15000

14000

13000

12000

11000

Intensity
10000

9000

8000

7000

6000

5000

4000

3000

2000

1000

0
1.75
1.97

0.95

1.02
3.03

0.99

3.00

5.28

3.02
-1000

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
153.92

138.66
136.79

128.99
126.97

89.80

77.45
77.13
76.81
74.09
67.00

52.15

22.17
21.30
17.77

10.24
10000

9000

8000

7000

6000
Intensity

5000

4000

3000

2000

1000

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS
32 / 41
F:\Users\...\fuying-18_170107120924 1/7/2017 1:29:14 PM 18#
Error=1.0 ppm

NL:
286.1417 1.59E7
100
fuying-
90 18_170107120924#9
RT: 0.06 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50

40

30
264.1598 302.1156
20 287.1449
283.1335
10
265.1631 301.1413 303.1190 309.2177
240.1147 250.1443 261.1022 281.1865 288.1485 320.1173 330.3372 344.0839 348.1122
0
NL:
286.1414 8.40E5
100
C15 H21 N1 O3 +Na:
90 C15 H21 N1 O3 Na1
paChrg 1
80

70

60

50

40

30

20 287.1447

10
288.1481
0
240 250 260 270 280 290 300 310 320 330 340 350
m/z

Compound II-e
IR
100
90 80
Transmittance [%]
60 7050
40

3032.38
2945.03
2859.36

2342.54

1694.79

1479.48
1404.34
1352.79
1321.16

1169.12
1120.71
1080.93
1060.22
1007.26
933.05
801.74
764.01

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
33 / 41
 7.35
7.29

6.07

4.13

4.10
4.06
4.04
3.88
3.60
3.58
3.56
3.54

1.76
1.65
1.53

0.94
0.93
0.77
4.11
10000

9000

8000

7000

6000

Intensity
5000

4000

3000

2000

1000

0
3.55

0.87

4.93

1.02

2.00

3.12
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
154.20

137.85
134.55
128.55
128.09

88.58

77.41
77.09
76.77

67.28
65.81

45.09

22.24

10.27
3800

3600

3400

3200

3000

2800

2600

2400

2200
Intensity

2000

1800

1600

1400

1200

1000

800

600

400

200

-200

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS
34 / 41
F:\Users\...\fuying-19_170107120924 1/7/2017 1:34:14 PM 19#
Error=0.3 ppm

NL:
292.0712 2.02E6
100
fuying-
90 19_170107120924#20
RT: 0.14 AV: 1 T: FTMS
80 + p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50
286.1415
40
294.0681
30
270.0893
20
301.1411
10 213.1386 308.0451
226.0994 264.1595 272.0863 309.2037 321.2401 330.3365 353.2664 365.1056
381.2976
391.2848
245.0785
0
NL:
292.0711 6.51E5
100
C13 H16 Cl1 N1 O3 +Na:
90 C13 H16 Cl1 N1 O3 Na1
paChrg1
80

70

60

50

40
294.0681
30

20

10
295.0715
0
220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390
m/z

Compound II-f
IR
100
95 90
Transmittance [%]
75 80 70
6585

3030.94
2949.63
2913.60
2858.19

1695.87

1572.43
1481.01
1415.81
1350.66

1200.49

1068.61
1007.34
941.79

811.95
765.88

524.73

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
35 / 41
 13
8.0

MS
210
7.5
7.35
4.00 7.33

200
C NMR
7.28

7.0

190
180
6.5

170
6.0
5.91

160
0.78
153.79

150
5.5
138.25
134.66 4.32

140
134.33 4.31
128.54

5.0
4.27

130
128.50 4.25
128.32 4.23
127.80

120
4.21

4.5
4.20
4.05

110
3.98

4.0
3.70
5.37 3.68

f1 (ppm)
f1 (ppm)
3.66

100 90
3.5
89.19 3.35
77.42

36 / 41
3.16
77.31

80
3.14
77.10

3.0
3.11
76.78

70
74.36
67.27

2.5

60
67.12
52.02

50
51.37 2.0
1.78
1.64

40
1.32 1.42
3.22 1.41
1.5

30
0.76 1.36
22.18 1.35
18.39 0.98

20
1.0

17.79 0.94
17.73 3.00 0.78

10
10.26 0.71
0.5

0
0.03

-10
0.0

0
0

1000
2000
3000
4000
5000
6000
7000
8000
9000
1000
2000
3000
4000
5000
6000
7000
8000
9000

11000
11000

10000
10000
12000
13000
14000

Intensity Intensity
F:\Users\...\fuying-20_170107120924 1/7/2017 1:39:55 PM 20#
Error=0.7 ppm

NL:
306.0869 1.50E6
100
fuying-
90 20_170107120924#19
RT: 0.13 AV: 1 T: FTMS
80 + p ESI Full ms
[100.00-2000.00]
Relative Abundance

70

60

50

40 284.1050
308.0838
30 286.1415
322.0608
20
301.1412
10 299.1103 324.0580
240.1152 250.1440 264.1596 279.1592 309.0872
325.0611 345.1078 353.2666 365.1057 381.2977 391.2846
0
NL:
306.0867 6.43E5
100
C14 H18 Cl1 N1 O3 +Na:
90 C14 H18 Cl1 N1 O3 Na1
paChrg1
80

70

60

50

40
308.0838
30

20

10 309.0871
0
230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
m/z

Compound II-g
IR
100
90
Transmittance [%]
70 60
50 80

3015.05
2943.27
2859.56
2816.71

1693.18
1600.79
1502.72
1406.91
1353.64

1238.48
1164.09
1119.56
1058.30
1025.59
935.31

819.16
763.62

578.04

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
37 / 41
 13

MS
159.91

7.5

160
159.89 1.84 7.34
154.23 7.28

C NMR

150
7.0
6.91
2.07
6.89

140
6.5
131.46
131.46
127.91 0.99 6.06

130
6.0
127.91
127.83

120
5.5
113.71
113.71

110
5.0
4.13
4.12

100
4.10

4.5
4.06
4.04
89.00

90
4.02
89.00 4.19 4.00

4.0
0.84 3.91
77.40 3.21

80
f1 (ppm)
f1 (ppm)
3.88
77.08 1.03 3.82

3.5
76.77 3.64

38 / 41
3.62

70
67.12
65.48 3.61

3.0
65.45 3.58
3.56

60
55.32
55.32

2.5

50
45.16
45.16
2.0

40
1.65
1.98 1.52
1.5

30
22.25 0.98
1.0

22.25 0.95

20
3.02
0.77
0.5

10.29

10
0.0

0
0
0

1000
2000
3000
4000
5000
6000
7000
8000
9000
2000
4000
6000
8000

11000

10000
12000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000

Intensity Intensity
F:\Users\...\fuying-8_170327204029 3/27/2017 9:26:48 PM 8#
Error=1.0 ppm

NL:
288.1203 3.12E7
100
fuying-
90 8_170327204029#11
RT: 0.08 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-1000.00]
Relative Abundance

70

60

50

40

30

20 289.1236
10 304.0943
256.1153 266.1385 301.1408 305.0976 320.1465
243.1013 274.2740 283.1652 290.1245 329.1382 336.1206 340.7808 351.1910 360.1418
0
NL:
288.1206 8.47E5
100
C14 H19 N1 O4 +Na:
90 C14 H19 N1 O4 Na1
paChrg 1
80

70

60

50

40

30

20 289.1240
10
290.1273
0
250 260 270 280 290 300 310 320 330 340 350 360
m/z

Compound II-h
IR
100
90
80
Transmittance [%]
40 50 60
30
20 70

2948.14
2857.36

1691.94
1601.93
1503.78
1407.99
1350.36
1292.25
1238.60
1163.38
1068.87
1026.62
941.41

820.33
769.53

573.72

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

1
H NMR
39 / 41
 22000

7.35
7.28

6.91
6.89

5.89

4.25
4.23
4.22
4.21
4.19
4.04
3.99
3.95
3.82
3.19
3.16
3.14

1.76
1.64
1.42
1.28

0.95
0.70
21000
20000
19000
18000
17000
16000
15000
14000
13000
12000

Intensity
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
1.77

1.92

0.89

0.88
2.81
3.00

0.98

5.31

3.13
-1000

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)

13
C NMR
160.02

153.94

131.94
128.37

113.68

89.65

77.42
77.10
76.78
74.00
66.99

55.32
52.16

22.21
17.75

10.29
11000

10000

9000

8000

7000

6000
Intensity

5000

4000

3000

2000

1000

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

f1 (ppm)

MS
40 / 41
F:\Users\...\fuying-9_170327204029 3/27/2017 9:31:32 PM 9#
Error=0.7 ppm

NL:
302.1361 3.69E7
100
fuying-
90 9_170327204029#12
RT: 0.08 AV: 1 T:
80 FTMS+ p ESI Full ms
[100.00-1000.00]
Relative Abundance

70

60

50

40

30

20 303.1394
10 301.1413
270.1312 280.1543 318.1101
248.1493 261.1310 288.1207 304.1424 319.1135 330.3366 340.5340 353.2436 365.1051 378.1589 384.1394 393.2976 401.1160
0
NL:
302.1363 8.38E5
100
C15 H21 N1 O4 +Na:
90 C15 H21 N1 O4 Na1
paChrg 1
80

70

60

50

40

30

20 303.1396

10
304.1430
0
250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
m/z

41 / 41