Triazolopyrimidines as a New Herbicidal Lead for Combating Weed

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Triazolopyrimidines as New Herbicidal Lead for Combating Weed Resistance Associated with Acetohydroxyacid Synthase Mutation Yu-Chao Liu, Ren-Yu Qu, Qiong Chen, Jing-Fang Yang, Congwei Niu, Zhen Xi, and Guang-Fu Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00720 • Publication Date (Web): 05 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016

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Journal of Agricultural and Food Chemistry

Triazolopyrimidines as New Herbicidal Lead for Combating Weed Resistance Associated with Acetohydroxyacid Synthase Mutation

Yu-Chao Liu,1 Ren-Yu Qu,1 Qiong Chen,1 Jing-Fang Yang,1 Niu Cong-wei2, Xi Zhen2,3 and Guang-Fu Yang1,2,3* 1

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P.R.China;

2

State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, P. R. China; 3

Collaborative Innovation Center of Chemical Science and Engineering, Tianjing 30071, P.R.China

*corresponding authors: E-mail: [email protected] (G.-F. Yang), Tel: +86-27-67867800, Fax: +86-27-67867141.

1

ACS Paragon Plus Environment


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ABSTRACT: Acetohydroxyacid synthase (AHAS; also known as acetolactate synthase;

2

EC 2.2.1.6; formerly EC 4.1.3.18) is the first common enzyme in the biosynthetic pathway

3

leading to the branched-chain amino acids in plants and a wide range of microorganisms.

4

Weed resistance to AHAS-inhibiting herbicides, increasing at an exponential rate, is

5

becoming a global problem and leading to an urgent demand of developing novel

6

compounds against both resistant and wild AHAS. In the present work, a series of (total 55)

7

novel 2-aroxyl-1,2,4-triazolopyrimidine derivatives were designed and synthesized with the

8

aim to discover anti-resistant lead compound. Fortunately, the screening results indicated

9

that many of newly synthesized compounds showed better even excellent inhibition effect

10

against both the wild-type A. thaliana AHAS and P197L mutants. Among them, compounds

11

5-3 to 5-17, compounds 5-19 to 5-26, compounds 5-28 to 5-45 and compound 5-48 have

12

the lower values of resistance factor (RF) and display a potential power to overcome

13

resistance associated with the P197L mutation in the enzyme levels. Further green house in

14

vivo assay showed that compounds 5-15 and 5-20 displayed “moderate” to “good”

15

herbicidal activity against both the wild-type and resistant (P197L mutation) Descurainia

16

sophia even at a rate as low as 0.9375 g.ai/ha. The above results indicated that these two

17

compounds could be used as new leads for the future development of anti-resistance

18

herbicides.

19 20

KEYWORDS: molecular design, herbicide, weed resistance, acetohydroxyacid synthase,

21

triazolopyrimidines.

22 2


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23


INTRODUCTION

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As the first common enzyme in the biosynthetic pathway leading to the branched-chain

25

amino acids in plants and a wide range of microorganisms, acetohydroxyacid synthase

26

(AHAS; also known as acetolactate synthase; EC 2.2.1.6; formerly EC 4.1.3.18) has been

27

identified as a promising target for structurally diverse commercial herbicides such as

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chlorsulfuron (CS), flumetsulam (FS) and bispyribac (BP) (Figure 1).1-7 AHAS-inhibiting

29

herbicides have been widely and rapidly adopted because they combined the advantages of

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low use rates, low mammalian toxicity, broad-spectrum weed control and flexible

31

application timing in a wide variety of crops. However, despite the great success of

32

commercial AHAS-inhibiting herbicide in last decades, weed resistance has become one of

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the most serious problems to overcome. To date, resistance to AHAS-inhibiting herbicides

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has been proved to be caused by an altered AHAS enzyme in most cases.6, 7 Some single

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point mutations conferring resistance to AHAS inhibitors such as A122T, P197A, P197S,

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P197E, P197L, A205V, D376E, W574L and S653T (numbered according to the

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Arabidopsis thaliana AHAS) have been identified.6 Among these mutations, P197L is one

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of the most comprehensively characterized mutation, which results in at least 10-fold

39

resistance to all types of AHAS inhibitors. Moreover, this mutation has been reported

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around the world, and no effective way to solve this problem are available up to now.6, 8-11

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Thus, it is of the active and practical significance for design of novel compounds to

42

overcome weed resistance associated with the P197L mutation.

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Previously, we have designed and synthesized a series of novel triazolopyrimidine-2-

44

sulfonamides12-14, and with the further structural optimization, we have proposed that 3



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conformational flexible AHAS inhibitors might be used as herbicidal leads for combating

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weed resistance.15 X-ray diffraction analysis16-19 indicated that the pyrimidine ring of

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sulfonylureas formed strong π-π interactions with the side chain, which plays an important

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role in defining the shape of the active-site channel and serves to anchor both classes of

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herbicide to the enzyme. Our computational simulations results15 showed that the

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triazolopyrimidine ring of triazolopyrimidine-2-sulfonamide also formed strong π-π

51

interactions with the side chain. Therefore, we proposed that the triazolopyrimidine ring20-22

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of triazolopyrimidine-2-sulfonamide should be regarded as the bioisoster of the pyrimidine

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moiety of sulfonylureas. In addition, comparison of the binding models of structurally

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diverse

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pyrimidinylbenzoates) indicated that oxygen atom should be acted as a more flexible bridge

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than the SO2NHCONH and SO2NH groups in the view of anti-resistance.15 Hence, based on

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the above considerations, also as a continuing work, we designed and synthesized a series of

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2-aroxyl-1,2,4-triazolopyrimidine derivatives with the aim to discover new lead compounds

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showing higher potency towards both the wild-type AHAS and P197L mutant (Figure 2).

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The synthetic chemistry, structural characterization, in vitro and in vivo assay of these

61

compounds are described in this work.

AHAS

inhibitors

(sulfonylureas,

triazolopyrimidine-2-sulfonamide

and

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MATERIALS AND METHODS

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Chemicals. 5-methyl-2-(methylthio)-[1,2,4]triazolo[1,5-a]pyrimidine; 5,7-dimethyl-2-

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(methylthio)-[1,2,4]triazolo[1,5-a]pyrimidine; 2-(methylthio)-5-phenyl-7-(trifluoromethyl)-

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[1,2,4]triazolo[1,5-a]pyrimidine;

5-(furan-2-yl)-2-(methylthio)-7-(trifluoromethyl)4


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2-(methylthio)-5-(thiophen-2-yl)-7-(trifluoromethyl)-

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5,7-dimethyl-2-(methylthio)-[1,2,4]triazolo[1,5-

69

c]pyrimidine; hydrogen peroxide; acetic acid; sodium hydride; toluene; the functionalized

70

salicylates; sulfuric acid; sodium tungstate dihydrate; sodium sulfite; sodium sulfate; water.

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Unless otherwise noted, reagents were purchased from commercial suppliers and used

72

without further purification, as all solvents were redistilled before use. 1H NMR spectra

73

were recorded on a Mercury-Plus 400 or 600 spectrometer in CDCl3 or DMSO-d6 with

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TMS as the internal reference. 13C NMR spectra were recorded on a Mercury-Plus 500 (125

75

MHz) spectrometer in DMSO-d6 with TMS as the internal reference. MS spectra were

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determined using a Trace MS 2000 organic mass spectrometer. Elemental analyses were

77

performed on a Vario EL III elemental analysis instrument. Melting points were measured

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on a Buchi B-545 melting point apparatus and are uncorrected. Intermediates 1 were

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prepared according to the reported methods.23 Intermediates 3 were prepared according to

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the reported methods.24-29

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Preparation of Compounds 2a~2f. To a stirred mixture of compounds 1 (0.01 mol)

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and 8 mL of acetic acid, 0.1g of NaWO4.H2O was added at room temperature. To the

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vigorously stirred solution, 0.02 mol of hydrogen peroxide as a 30% aqueous solution was

84

added slowly at 40℃. Stirring was continued at 50℃ for an addition 3 hours. The excess of

85

hydrogen peroxide was destroyed by the addition of an aqueous solution of sodium sulfite,

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and the solid filtered off and recrystallized from ethanol or purified by flash

87

chromatography to give the pure products 2a~2f.

5



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General Procedure for the Synthesis of Compounds 4-1~4-55. A mixture of 0.002

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mol of the functionalized 6-arylsalicylates (3) and 0.003 mol of sodium hydride (60%) in 30

90

mL of anhydrous toluene was stirred at 55~60℃ for 1h, then 0.002 mol of compounds 2

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was added and the mixture was refluxed until the reaction was complete monitored by TLC.

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The reaction mixture was cooled to room temperature, after diatomite filtration, the solvent

93

was removed under reduced pressure to obtain crude product. The crude product was

94

purified by flash chromatography to afford the desired product or reacted on the next step

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directly.

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General Procedure for the Synthesis of Compounds 5-1~5-55. 0.001 mol of

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compounds 4 was dissolved in the mixture solution of H2SO4 (4 mL), HOAc (7 mL) and

98

H2O (1 mL). Then it was heated to reflux until the reaction was completed according to

99

TLC detection. The reaction medium was cooled to room temperature, poured into ice-cold

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water (50 mL). Then the aqueous layer was extracted with CH2Cl2 (15 mL) for three times,

101

dried by anhydrous Na2SO4, and then concentrated by rotary evaporation. The residue was

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purified via flash chromatography to give title compounds 5 in yields of 15-65%.

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Enzyme inhibition activities against wild-type AHAS and P197L mutant. The Ki

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values of compounds 5 against wild-type AtAHAS and P197L mutant were determined

105

respectively according to the methods reported previously15, 30-32 and the results were shown

106

in Table 1.

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Greenhouse herbicidal activities against resistant weeds. The post-emergent

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herbicidal activities of compounds 5, against the wild-type Descurainia Sophia (DS) and

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the resistant D. Sophia (RDS) (Pro197Leu mutation took place in AHAS). The seed of D. 6


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Sophia with 0.05% GA3 for 24 hours and break dormancy, then seeding them after washed

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by clean water. Earth up the seeds equably and keep the temperature at 15 °C (night time) /

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25 °C (day time) in phytotron. Move the seedings to greenhouse to harden after cotyledons

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spread and process them in the 4 leaf stage, then moving them to phytotron after processing.

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Herbicidal activity was evaluated visually after 35 days from post treatment. The results of

115

herbicidal activities are shown in Table 2, with three replicates per treatment and

116

averaging figures.

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Computational Methods: The molecular structure of wild type AHAS in Arabidopsis

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thaliana was obtained from RCSB Protein Databank (PDB) (PDB accession code: 1YHZ)

119

which is monomer. Because the pocket of ligand between the binding surface of two

120

monomers. So we build up dimer based on the structure of AHAS in yeast (PDB accession

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code: 1T9B). The mutation P197L was introduced into the wild type ligand-protein

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complexes by the use of biopolymer mutation modeling tool in SYBYL7.0. The

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conformations of mutant complexes were further minimized using the Tripos force field and

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the Powell method for 300 steps for all residues within 6 Å of the mutated residue. The

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gradient was lower than 0.005 kcal·mol-1Å-1. Computational modeling studies were

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completed by using AutoDock Tools and AutoDock4. The structure was prepared as

127

follows: 1) All waters and ligand except for FAD and P22 were removed from the structure,

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2). All hydrogens were added as ‘Polar Only’, and 3) A grid box for the 1CS binding site

129

was created (center x = 77.679, center y = 75.624, center z = -80.86/ size x = 15, size y = 15,

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size z = 15). The atomic Gasteiger-Huckel charges were assigned to the ligand and receptor.

131

Compounds to be computationally modeled were assigned appropriate rotatable bonds using 7



132

AutoDock Tools. A total of 40 runs were launched for each compound. Most of the

133

parameters for the docking calculation were set to the default values recommended by the

134

software. Each docked structure was scored by the built-in scoring function and was

135

clustered by 2 Å of RMSD criterions. After modeling, the results were visualized and

136

analyzed with PyMOL.

137

Binding Free Energy Calculation by the Method of MM/PBSA The molecular

138

mechanics Poisson-Boltzmann surface area (MM-PBSA) method in the AMBER14 package

139

was employed to perform the free energy analyses. The binding free energy was computed

140

through calculating the free energy differences of ligand, receptor and their complex as

141

follows:

142

∆Gbinding = Gcomplex − Gligand − Greceptor (1)

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In MM-PBSA, the free energy (G) of each state is estimated from molecular

144

mechanical energy EMM, solvation free energy GSOLV and vibrational, rotational, and

145

translational entropies S, respectively.

146

G = EMM + GSOLV − TS (2)

147

EMM = Eint + Evdw + Eele (3)

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GSOLV = Gpb/solv + Gnp/solv (4)

149

where T is the temperature; Eint is internal energy, i.e., the sum of bond, angle, and

150

dihedral energies; Evdw is van der Waals energy; Eele is electrostatic energy; GSOLV is the

151

sum of electrostatic solvation free energy, Gpb/solv, and the non-polar salvation free energy,

152

Gnp/solv. The entropy S is estimated by a normal mode analysis of the harmonic vibrational

153

frequencies, calculated using the Nmode module in Amber14 package. 8


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RESULTS AND DISCUSSION

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Synthetic chemistry of the title compounds 5. As shown in Figure 3, 2-methylthio-

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1,2,4-triazolopyrimidines 1a-e, prepared from acetimidamide hydrochloride according to

157

the reported method,23 were easily oxidized to the corresponding intermediates 2a-f using

158

the oxidation system of H2O2/AcOH. Then, in the presence of sodium hydride, the

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intermediates 2a-f underwent substitution reaction with various functionalized 6-

160

arylsalicylates (3) which were synthesized by a Suzuki cross-coupling reaction-based

161

synthetic strategy24-26 to afford the compounds 4 in good yields. Finally, in the mixture

162

solution of H2SO4, HOAc and H2O, the hydrolysis of compounds 4 afforded the

163

corresponding title compounds 5 in yields of 15-65%. The structures of all the target

164

compounds 5 (as shown in Figure 4) were characterized by 1H NMR, 13C NMR, EI-MS and

165

elemental analyses.

166

Data for 2-methyl-6-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)benzoic

167

acid (5-1): White solid, 15% yield, mp: 214-215℃. 1H NMR (600 MHz, DMSO-d6) δ 13.26

168

(s, 1H), 9.09 (d, J = 6.6 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.21 (dd,

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J = 11.4, 7.2 Hz, 2H), 2.59 (s, 3H), 2.35 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 169.52,

170

167.38, 165.17, 153.46, 149.91, 136.25, 135.92, 129.96, 127.61, 127.08, 118.73, 111.18,

171

24.53, 19.06. EI-MS: m/z = 284.13 (M+). Anal. calcd for (C14H12N4O3): C, 59.15; H, 4.25;

172

N, 19.71. Found: C, 59.38; H, 4.37; N, 19.92.

173

Data for 3-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-2-

174

carboxylic acid (5-2): White solid, 37% yield, mp: 210-211 ℃. 1H NMR (600 MHz,

175

DMSO-d6) δ 13.23 (s, 1H), 9.13 (d, J = 7.2 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.46 (d, J = 9



176

4.2 Hz, 5H), 7.41 (s, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H), 2.60 (s, 3H).

177

13

178

136.00, 130.09, 128.47, 128.14, 127.78, 127.27, 126.64, 120.23, 111.27, 24.55. EI-MS: m/z

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= 346.03 (M+). Anal. calcd for (C19H14N4O3): C, 65.89; H, 4.07; N, 16.18. Found: C, 65.90;

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H, 3.99; N, 16.31.

C NMR (125 MHz, DMSO-d6) δ 169.44, 167.30, 165.26, 153.47, 149.90, 140.07, 139.15,

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Data for 4'-(tert-butyl)-3-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-[1,1'-

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biphenyl]-2-carboxylic acid (5-3): White solid, 18% yield, mp: 221-222℃. 1H NMR (600

183

MHz, DMSO-d6) δ 13.20 (s, 1H), 9.12 (d, J = 7.2 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.48 (d,

184

J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 7.8 Hz, 1H),

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7.24 (d, J = 7.2 Hz, 1H), 2.60 (s, 3H), 1.32 (s, 9H).

186

169.49, 167.46, 165.26, 153.49, 150.20, 149.89, 139.97, 136.27, 135.97, 130.03, 127.87,

187

127.27, 126.61, 125.32, 120.02, 111.26, 34.28, 31.06, 24.56. EI-MS: m/z = 402.63 (M+).

188

Anal. calcd for (C23H22N4O3): C, 68.64; H, 5.51; N, 13.92. Found: C, 68.59; H, 5.69; N,

189

13.70.

13

C NMR (125 MHz, DMSO-d6) δ

190

Data for 4'-fluoro-5-methyl-3-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-

191

[1,1'-biphenyl]-2-carboxylic acid (5-4): White solid, 42% yield, mp: 215-216℃. 1H NMR

192

(600 MHz, DMSO-d6) δ 13.17 (s, 1H), 9.14 (d, J = 7.2 Hz, 1H), 7.47 (dd, J = 8.4, 5.4 Hz,

193

2H), 7.33-7.26 (m, 3H), 7.24 (d, J = 7.2 Hz, 1H), 7.17 (s, 1H), 2.60 (s, 3H), 2.38 (s, 3H).

194

13

195

140.35, 138.98, 135.99, 135.75, 135.73, 130.19, 130.12, 127.40, 124.47, 120.58, 115.37,

196

115.20, 111.23, 24.54, 20.74. EI-MS: m/z = 378.06 (M+). Anal. calcd for (C20H15FN4O3): C,

197

63.49; H, 4.00; N, 14.81. Found: C, 63.61; H, 4.21; N, 14.98.

C NMR (125 MHz, DMSO-d6) δ 169.47, 167.37, 165.23, 162.83, 160.89, 153.48, 150.05,

10


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Data for 2-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-6-methylbenzoic

199

acid (5-5): White solid, 17% yield, mp: 210-211℃. 1H NMR (600 MHz, DMSO-d6) δ 7.39

200

(t, J = 7.8 Hz, 1H), 7.26-7.10 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.16 (s, 1H),

201

4.02 (s, 1H), 2.63 (s, 3H), 2.54 (s, 3H), 2.35 (s, 3H).

202

168.83, 167.51, 164.05, 153.60, 150.04, 146.83, 136.17, 129.95, 127.49, 126.88, 118.34,

203

110.86, 24.40, 19.09, 16.46. EI-MS: m/z = 298.29 (M+). Anal. calcd for (C15H14N4O3): C,

204

60.40; H, 4.73; N, 18.78. Found: C, 60.21; H, 4.47; N, 18.74.

13

C NMR (125 MHz, DMSO-d6) δ

205

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-

206

2-carboxylic acid (5-6): White solid, 57% yield, mp: 224-225℃. 1H NMR (600 MHz,

207

DMSO-d6) δ 13.23 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 6.0 Hz, 5H), 7.41 (d, J =

208

6.0 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H), 2.65 (s, 3H), 2.55 (s, 3H).

209

(125 MHz, DMSO-d6) δ 168.74, 167.47, 164.07, 153.58, 150.06, 146.79, 140.04, 139.19,

210

130.05, 128.50, 128.18, 127.81, 127.11, 126.39, 119.79, 110.87, 24.37, 16.42. EI-MS: m/z =

211

360.10 (M+). Anal. calcd for (C20H16N4O3): C, 66.66; H, 4.48; N, 15.55. Found: C, 66.46; H,

212

4.59; N, 15.32.

13

C NMR

213

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-5-methyl-[1,1'-

214


215

MHz, DMSO-d6) δ 13.08 (s, 1H), 7.45 (d, J = 3.6 Hz, 4H), 7.40 (s, 1H), 7.25 (s, 1H), 7.18

216

(s, 1H), 7.15 (s, 1H), 2.65 (s, 3H), 2.55 (s, 3H), 2.37 (s, 3H). 13C NMR (125 MHz, DMSO-

217

d6) δ 168.91, 167.35, 161.20, 151.48, 149.89, 144.89, 140.07, 139.11, 130.08, 128.48,

218

128.15, 127.81, 127.05, 126.53, 119.73, 117.76, 23.76, 14.97. EI-MS: m/z = 374.34 (M+).

11



219

Anal. calcd for (C21H18N4O3): C, 67.37; H, 4.85; N, 14.96. Found: C, 67.65; H, 4.81; N,

220

14.75.

221

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-4'-methoxy-5-

222

methyl-[1,1'-biphenyl]-2-carboxylic acid (5-8): White solid, 18% yield, mp: 208-209℃.

223

1

224

(s, 1H), 7.12 (s, 1H), 7.01 (d, J = 8.4 Hz, 2H), 3.79 (s, 3H), 2.65 (s, 3H), 2.55 (s, 3H), 2.37

225

(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 168.76, 167.70, 163.98, 158.94, 153.56, 150.16,

226

146.77, 139.94, 139.55, 131.63, 129.29, 126.91, 124.19, 119.45, 113.89, 110.81, 55.13,

227

24.35, 20.81, 16.44. EI-MS: m/z = 404.06 (M+). Anal. calcd for (C22H20N4O4): C, 65.34; H,

228

4.98; N, 13.85. Found: C, 65.44; H, 4.88; N, 13.67.

H NMR (600 MHz, DMSO-d6) δ 13.04 (s, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.20 (s, 1H), 7.18

229

Data for 4'-(tert-butyl)-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-

230

[1,1'-biphenyl]-2-carboxylic acid (5-9): White solid, 48% yield, mp: 215-216℃. 1H NMR

231

(600 MHz, DMSO-d6) δ 13.20 (s, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H),

232

7.44 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H),

233

2.65 (s, 3H), 2.55 (s, 3H), 1.32 (s, 9H). 13C NMR (125 MHz, DMSO-d6) δ 168.71, 167.50,

234

164.05, 153.57, 150.17, 149.96, 146.83, 139.83, 136.28, 129.90, 127.87, 127.12, 126.31,

235

125.31, 119.50, 110.88, 34.28, 31.06, 24.37, 16.43. EI-MS: m/z = 416.74 (M+). Anal. calcd

236

for (C24H24N4O3): C, 69.21; H, 5.81; N, 13.45. Found: C, 69.23; H, 5.72; N, 13.39.

237

Data

for

3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-5-methyl-4'-

238

nitro-[1,1'-biphenyl]-2-carboxylic acid (5-10): White solid, 37% yield, mp: 234-235℃. 1H

239

NMR (600 MHz, DMSO-d6) δ 13.29 (s, 1H), 8.32 (d, J = 8.4 Hz, 2H), 7.80-7.67 (m, 2H),

240

7.35 (s, 1H), 7.24 (s, 1H), 7.19 (s, 1H), 2.65 (s, 3H), 2.56 (s, 3H), 2.41 (s, 3H). 13C NMR 12


Page 12 of 50

Page 13 of 50


241

(125 MHz, DMSO-d6) δ 168.67, 167.07, 164.08, 153.58, 150.44, 146.94, 146.85, 146.18,

242

140.77, 138.04, 129.55, 127.15, 124.28, 123.56, 121.32, 110.89, 24.36, 20.75, 16.43. EI-

243

MS: m/z = 419.02 (M+). Anal. calcd for (C21H17N5O5): C, 60.14; H, 4.09; N, 16.70. Found:

244

C, 60.05; H, 3.94; N, 16.87.

245

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-4'-fluoro-[1,1'-

246


247

MHz, DMSO-d6) δ 13.30 (s, 1H), 7.57 (s, 1H), 7.53-7.45 (m, 3H), 7.31 (dd, J = 14.4, 7.8 Hz,

248

3H), 7.18 (s, 1H), 2.65 (s, 3H), 2.55 (s, 3H).

249

167.32, 164.09, 162.90, 162.08, 160.96, 153.58, 150.02, 146.85, 138.92, 135.57, 135.55,

250

130.28, 130.21, 130.05, 126.38, 119.85, 115.45, 115.28, 110.90, 24.37, 16.43. EI-MS: m/z =

251

378.02 (M+). Anal. calcd for (C20H15FN4O3): C, 63.49; H, 4.00; N, 14.81. Found: C, 63.41;

252

H, 4.27; N, 14.79.

253

Data

for

13

C NMR (125 MHz, DMSO-d6) δ 168.66,

3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-4'-fluoro-5-

254


255

1

256

7.26 (s, 1H), 7.18 (s, 1H), 7.15 (s, 1H), 2.65 (s, 3H), 2.55 (s, 3H), 2.38 (s, 3H). 13C NMR

257

(125 MHz, DMSO-d6) δ 168.74, 167.49, 164.03, 162.86, 160.91, 153.57, 150.22, 146.79,

258

140.25, 138.91, 135.79, 135.77, 130.21, 130.15, 127.11, 124.30, 120.10, 115.37, 115.20,

259

110.84, 24.35, 20.77, 16.42. EI-MS: m/z = 392.06 (M+). Anal. calcd for (C21H17FN4O3): C,

260

64.28; H, 4.37; N, 14.28. Found: C, 64.06; H, 4.35; N, 14.32.

261 262

H NMR (600 MHz, DMSO-d6) δ 13.13 (s, 1H), 7.54-7.42 (m, 2H), 7.29 (t, J = 9.0 Hz, 2H),

Data

for


methyl-[1,1'-biphenyl]-2-carboxylic acid (5-13): White solid, 40% yield, mp: 218-219℃. 13



Page 14 of 50

263

1

264

(m, 4H), 7.19 (d, J = 8.4 Hz, 2H), 2.65 (s, 3H), 2.55 (s, 3H), 2.39 (s, 3H). 13C NMR (125

265

MHz, DMSO-d6) δ 168.73, 167.41, 164.05, 162.92, 160.98, 153.57, 150.25, 146.80, 141.75,

266

141.68, 140.43, 138.53, 130.50, 130.43, 127.09, 124.42, 124.41, 124.26, 120.55, 115.03,

267

114.85, 114.63, 114.47, 110.86, 24.35, 20.77, 16.42. EI-MS: m/z = 392.26 (M+). Anal. calcd

268

for (C21H17FN4O3): C, 64.28; H, 4.37; N, 14.28. Found: C, 64.04; H, 4.35; N, 14.12.

269

H NMR (600 MHz, DMSO-d6) δ 13.24 (s, 1H), 7.51 (dd, J = 14.4, 7.8 Hz, 1H), 7.32-7.22

Data

for


270


271

1

272

7.29 (s, 1H), 7.27 (dd, J = 8.4, 4.5 Hz, 2H), 7.18 (s, 1H), 7.13 (s, 1H), 2.65 (s, 3H), 2.55 (s,

273

3H), 2.38 (s, 3H).

274

157.92, 153.56, 150.51, 146.77, 140.53, 134.65, 131.01, 129.96, 129.90, 128.09, 127.15,

275

127.03, 124.63, 124.29, 124.27, 121.06, 115.49, 115.31, 110.80, 24.35, 20.70, 16.43. EI-

276

MS: m/z = 392.11 (M+). Anal. calcd for (C21H17FN4O3): C, 64.28; H, 4.37; N, 14.28. Found:

277

C, 64.17; H, 4.56; N, 14.56.

H NMR (600 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.47-7.41 (m, 1H), 7.39 (t, J = 7.8 Hz, 1H),

13

C NMR (125 MHz, DMSO-d6) δ 168.79, 166.65, 163.96, 159.87,

278

Data for 4'-chloro-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-[1,1'-

279


280

MHz, DMSO-d6) δ 13.31 (s, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.51-

281

7.45 (m, 3H), 7.33 (d, J = 7.2 Hz, 1H), 7.18 (s, 1H), 2.65 (s, 3H), 2.55 (s, 3H).

282

(125 MHz, DMSO-d6) δ 168.66, 167.27, 164.08, 153.56, 150.10, 146.80, 138.77, 138.00,

283

132.86, 130.22, 129.99, 128.51, 126.99, 126.32, 120.16, 110.88, 24.36, 16.42. EI-MS: m/z =

14


13

C NMR

Page 15 of 50


284

394.26 (M+). Anal. calcd for (C20H15ClN4O3): C, 60.84; H, 3.83; N, 14.19. Found: C, 60.78;

285

H, 3.92; N, 14.34.

286

Data

for

4'-chloro-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-5-

287


288

1

289

Hz, 2H), 7.27 (s, 1H), 7.18 (s, 1H), 7.16 (s, 1H), 2.65 (s, 3H), 2.55 (s, 3H), 2.38 (s, 3H). 13C

290

NMR (125 MHz, DMSO-d6) δ 168.73, 167.40, 164.02, 153.56, 150.29, 146.76, 140.41,

291

138.74, 138.24, 132.72, 129.94, 128.43, 127.02, 124.20, 120.39, 110.82, 24.35, 20.78, 16.42.

292

EI-MS: m/z = 408.80 (M+). Anal. calcd for (C21H17ClN4O3): C, 61.69; H, 4.19; N, 13.70.

293

Found: C, 61.82; H, 4.23; N, 13.54.

H NMR (600 MHz, DMSO-d6) δ 13.15 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4

294

Data for 4'-bromo-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-[1,1'-

295


296

MHz, DMSO-d6) δ 13.32 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.58 (t, J = 7.8 Hz, 1H), 7.49 (d,

297

J = 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H), 2.65 (s, 3H),

298

2.55 (s, 3H).

299

146.84, 138.77, 138.39, 131.43, 130.31, 130.19, 126.98, 126.24, 121.43, 120.15, 110.90,

300

24.37, 16.43. EI-MS: m/z = 439.03 (M+). Anal. calcd for (C20H15BrN4O3): C, 54.69; H,

301

3.44; N, 12.75. Found: C, 54.86; H, 3.64; N, 12.58.

13

C NMR (125 MHz, DMSO-d6) δ 168.64, 167.25, 164.09, 153.56, 150.08,

302

Data for 2-methyl-6-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-

303

2-yl)oxy)benzoic acid (5-18): White solid, 61% yield, mp: 211-212℃. 1H NMR (600 MHz,

304

DMSO-d6) δ 13.37 (s, 1H), 8.43 (s, 1H), 8.37 (d, J = 7.2 Hz, 2H), 7.68-7.57 (m, 3H), 7.45 (t,

305

J = 7.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 2.38 (s, 3H). 13C NMR 15



306

(125 MHz, DMSO-d6) δ 170.13, 167.34, 160.70, 154.54, 149.75, 136.36, 134.98, 134.26,

307

133.95, 133.65, 133.34, 132.07, 130.01, 129.15, 127.87, 127.51, 127.29, 122.08, 119.90,

308

118.43, 117.71, 115.53, 106.29, 19.08. EI-MS: m/z = 414.15(M+). Anal. calcd for

309

(C20H13F3N4O3): C, 57.98; H, 3.16; N, 13.52. Found: C, 57.79; H, 3.26; N, 13.30.

310

Data for 3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-

311

[1,1'-biphenyl]-2-carboxylic acid (5-19): White solid, 50% yield, mp: 236-237℃ . 1H

312

NMR (600 MHz, DMSO-d6) δ 13.37 (s, 1H), 8.47 (s, 1H), 8.39 (d, J = 7.2 Hz, 2H), 7.63 (d,

313

J = 7.2 Hz, 4H), 7.53 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 7.2 Hz, 4H), 7.42 (s, 1H), 7.39 (d, J =

314

7.8 Hz, 1H).

315

140.13, 139.10, 135.01, 134.33, 134.02, 133.72, 133.41, 132.14, 130.16, 129.20, 128.52,

316

128.17, 127.92, 127.85, 127.17, 126.82, 122.10, 119.91, 119.86, 117.73, 106.42. EI-MS:

317

m/z = 476.33 (M+). Anal. calcd for (C25H15F3N4O3): C, 63.03; H, 3.17; N, 11.76. Found: C,

318

63.28; H, 3.16; N, 11.60.

319

Data

13

C NMR (125 MHz, DMSO-d6) δ 170.01, 167.29, 160.80, 154.57, 149.76,

for

4'-(tert-butyl)-3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

320

a]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-2-carboxylic acid (5-20): White solid, 16% yield,

321

mp: 226-227℃. 1H NMR (600 MHz, DMSO-d6) δ 13.32 (s, 1H), 8.46 (s, 1H), 8.38 (d, J =

322

7.2 Hz, 2H), 7.62 (dt, J = 12.6, 7.2 Hz, 4H), 7.54-7.47 (m, 3H), 7.43 (d, J = 8.4 Hz, 2H),

323

7.38 (d, J = 7.8 Hz, 1H), 1.32 (s, 9H).

324

160.77, 154.55, 150.26, 149.72, 140.03, 136.19, 134.96, 134.01, 133.70, 132.10, 130.10,

325

129.15, 127.88, 127.11, 126.78, 125.35, 122.10, 119.91, 119.66, 117.73, 115.50, 106.40,

326

34.29, 31.05. EI-MS: m/z = 532.04 (M+). Anal. calcd for (C29H23F3N4O3): C, 65.41; H, 4.35;

327

N, 10.52. Found: C, 65.36; H, 4.36; N, 10.68.

13

C NMR (125 MHz, DMSO-d6) δ 170.05, 167.41,

16


Page 16 of 50

Page 17 of 50

328


Data

for

5-methyl-4'-nitro-3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

329


330


331

7.2 Hz, 2H), 8.33 (d, J = 9.0 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.64 (dd, J = 10.8, 7.2 Hz,

332

3H), 7.43 (s, 1H), 7.31 (s, 1H), 2.43 (s, 3H).

333

166.95, 160.80, 154.56, 150.20, 146.98, 146.06, 141.04, 138.25, 134.95, 134.32, 134.02,

334

133.72, 133.41, 132.13, 129.56, 129.17, 127.89, 127.63, 124.20, 123.58, 122.09, 121.52,

335

119.91, 117.72, 115.54, 106.43, 20.79. EI-MS: m/z = 535.36 (M+). Anal. calcd for

336


13

C NMR (125 MHz, DMSO-d6) δ 170.02,

337

Data for 4'-fluoro-3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-

338

2-yl)oxy)-[1,1'-biphenyl]-2-carboxylic acid (5-22): White solid, 38% yield, mp: 218-

339

219℃. 1H NMR (600 MHz, DMSO-d6) δ 13.38 (s, 1H), 8.46 (s, 1H), 8.38 (d, J = 7.2 Hz,

340

2H), 7.63 (dt, J = 7.8, 5.4 Hz, 4H), 7.56-7.50 (m, 3H), 7.38 (d, J = 7.8 Hz, 1H), 7.32 (t, J =

341

9.0 Hz, 2H).

342

154.56, 149.75, 139.08, 135.45, 135.43, 134.99, 134.33, 134.02, 133.72, 133.45, 132.14,

343

130.29, 130.22, 129.19, 127.91, 127.11, 126.83, 122.09, 119.98, 119.90, 117.72, 115.49,

344

115.32, 106.46, 99.47. EI-MS: m/z = 494.14 (M+). Anal. calcd for (C25H14F4N4O3): C,

345

60.73; H, 2.85; N, 11.33. Found: C, 60.45; H, 3.07; N, 11.12.

13

C NMR (125 MHz, DMSO-d6) δ 169.94, 167.20, 162.93, 160.99, 160.81,

346

Data for 4'-fluoro-5-methyl-3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

347


348


349

7.8 Hz, 2H), 7.63 (d, J = 7.2 Hz, 3H), 7.50 (dd, J = 8.4 5.4 Hz, 2H), 7.35-7.27 (m, 3H), 7.20 17



13

350

(s, 1H), 2.41 (s, 3H).

C NMR (125 MHz, DMSO-d6) δ 170.02, 168.92, 167.31, 162.86,

351

160.91, 160.75, 154.55, 149.91, 140.35, 138.98, 135.69, 135.00, 134.00, 133.67, 132.14,

352

130.21, 130.14, 129.20, 127.91, 127.53, 126.18, 120.20, 119.91, 117.70, 117.35, 115.39,

353

115.22, 115.12, 106.42, 20.79. EI-MS: m/z = 507.98 (M+). Anal. calcd for (C26H16F4N4O3):

354

C, 61.42; H, 3.17; N, 11.02. Found: C, 61.32; H, 3.35; N, 10.89.

355

Data for 4'-chloro-3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-

356


357


358

2H), 7.70-7.57 (m, 4H), 7.55 (d, J = 8.4 Hz, 3H), 7.50 (d, J = 8.4 Hz, 2H), 7.39 (s, 1H). 13C

359

NMR (125 MHz, DMSO-d6) δ 169.95, 167.12, 160.81, 154.55, 149.80, 138.88, 137.92,

360

134.98, 134.02, 133.71, 132.88, 132.13, 130.39, 130.33, 130.00, 129.18, 128.53, 127.91,

361

126.75, 122.09, 120.25, 119.87, 117.71, 106.45. EI-MS: m/z = 510.00 (M+). Anal. calcd for

362

(C25H14ClF3N4O3): C, 58.78; H, 2.76; N, 10.97. Found: C, 58.69; H, 2.72; N, 10.82.

363

Data

for

4'-chloro-5-methyl-3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

364


365


366

7.2 Hz, 2H), 7.63 (dd, J = 10.8, 7.2 Hz, 3H), 7.53 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz,

367

2H), 7.34 (s, 1H), 7.21 (s, 1H), 2.41 (s, 3H).

368

167.23, 160.77, 154.56, 150.01, 140.57, 138.86, 138.15, 134.99, 134.01, 133.71, 133.40,

369

132.76, 132.14, 131.52, 129.95, 129.20, 128.47, 127.91, 127.47, 124.24, 122.10, 120.50,

370

119.91, 117.73, 115.54, 106.40, 20.79. EI-MS: m/z =524.66 (M+). Anal. calcd for

371

(C26H16ClF3N4O3): C, 59.50; H, 3.07; N, 10.67. Found: C, 59.41; H, 3.18; N, 10.43.

13

C NMR (125 MHz, DMSO-d6) δ 170.02,

18


Page 18 of 50

Page 19 of 50


372

Data for 4'-bromo-3-((5-phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-

373


374


375

2H), 7.68 (d, J = 8.4 Hz, 2H), 7.63 (dt, J = 11.4, 6.6 Hz, 4H), 7.55 (d, J = 8.4 Hz, 1H), 7.43

376

(d, J = 8.4 Hz, 2H), 7.39 (d, J = 7.8 Hz, 1H).

377

169.97, 167.09, 160.81, 154.55, 149.83, 138.96, 138.28, 134.99, 134.93, 134.32, 134.02,

378

133.71, 133.42, 132.12, 131.46, 130.39, 130.30, 129.18, 127.91, 126.99, 126.71, 122.08,

379


380

(C25H14BrF3N4O3): C, 54.07; H, 2.54; N, 10.09. Found: C, 54.02; H, 2.57; N, 9.87.

13

C NMR (125 MHz, DMSO-d6) δ 170.27,

381

Data for 2-((5-(furan-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-

382

yl)oxy)-6-methylbenzoic acid (5-27): Yellow solid, 62% yield, mp: 218-219℃. 1H NMR

383

(600 MHz, DMSO-d6) δ 13.35 (s, 1H), 8.12 (d, J = 3.6 Hz, 2H), 7.78 (d, J = 3.6 Hz, 1H),

384

7.43 (t, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 6.85 (d, J = 1.8

385

Hz, 1H), 2.37 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 169.99, 167.37, 154.55, 151.48,

386

150.00, 149.74, 147.98, 136.34, 134.21, 133.90, 133.59, 133.29, 129.97, 127.48, 127.25,

387

121.92, 119.73, 118.32, 117.55, 116.36, 115.37, 113.59, 105.11, 19.07. EI-MS: m/z =

388

404.05 (M+). Anal. calcd for (C18H11F3N4O4): C, 53.47; H, 2.74; N, 13.86. Found: C, 53.56;

389

H, 2.77; N, 13.78.

390

Data

for

3-((5-(furan-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-

391

yl)oxy)-[1,1'-biphenyl]-2-carboxylic acid (5-28): White solid, 30% yield, mp: 228-229℃.

392

1

393

(t, J = 7.8 Hz, 1H), 7.50 (dd, J = 17.4, 7.8 Hz, 5H), 7.42 (s, 1H), 7.38 (d, J = 7.8 Hz, 1H),

H NMR (600 MHz, DMSO-d6) δ 13.37 (s, 1H), 8.15 (d, J = 6.0 Hz, 2H), 7.80 (s, 1H), 7.62

19



13

394

6.86 (s, 1H).

C NMR (125 MHz, DMSO-d6) δ 169.84, 167.31, 154.57, 151.54, 149.99,

395

149.74, 148.04, 140.12, 139.10, 134.24, 133.94, 133.63, 133.32, 130.11, 128.51, 128.17,

396

127.85, 127.14, 126.77, 119.73, 117.55, 116.45, 113.63, 105.22. EI-MS: m/z = 466.17 (M+).

397

Anal. calcd for (C23H13F3N4O4): C, 59.23; H, 2.81; N, 12.01. Found: C, 58.96; H, 2.92; N,

398

11.79.

399

Data for 4'-(tert-butyl)-3-((5-(furan-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

400

a]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-2-carboxylic acid (5-29): hite solid, 57% yield,

401


402

3.0 Hz, 1H), 7.59 (s, 1H), 7.49 (d, J = 7.8 Hz, 3H), 7.43 (d, J = 7.8 Hz, 2H), 7.36 (d, J = 7.8

403

Hz, 1H), 6.86 (s, 1H), 1.30 (d, J = 26.8 Hz, 9H). 13C NMR (125 MHz, DMSO-d6) δ 169.83,

404

167.38, 154.56, 151.53, 150.25, 149.99, 149.68, 148.04, 139.97, 136.18, 134.24, 133.93,

405

133.63, 133.32, 130.04, 127.87, 127.04, 126.70, 125.34, 121.91, 119.73, 119.48, 117.55,

406


407


408

Data

for

4'-fluoro-3-((5-(furan-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

409


410

mp: 216-217℃. 1H NMR (600 MHz, DMSO-d6) δ 13.39 (s, 1H), 8.13 (d, J = 3.0 Hz, 2H),

411

7.79 (s, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 3H), 7.37 (d, J = 7.8 Hz, 1H), 7.31

412

(t, J = 8.4 Hz, 2H), 6.85 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 169.77, 167.21, 162.93,

413

160.98, 154.56, 151.55, 149.98, 149.72, 148.05, 139.05, 135.44, 133.94, 133.63, 130.29,

414

130.22, 130.16, 127.11, 126.77, 121.89, 119.83, 119.72, 117.54, 116.47, 115.48, 115.31,

20


Page 20 of 50

Page 21 of 50


415

113.63, 105.24. EI-MS: m/z = 484.08 (M+). Anal. calcd for (C23H12F4N4O4): C, 57.03; H,

416

2.50; N, 11.57. Found: C, 57.26; H, 2.76; N, 11.35.

417

Data

for


418

a]pyrimidin-2-yl)oxy)-5-methyl-[1,1'-biphenyl]-2-carboxylic acid (5-31): White solid,

419

55% yield, mp: 222-223℃. 1H NMR (600 MHz, DMSO-d6) δ 13.23 (s, 1H), 8.14 (s, 2H),

420

7.80 (d, J = 3.6 Hz, 1H), 7.49 (dd, J = 8.4, 5.4 Hz, 2H), 7.31 (dd, J = 10.2, 7.8 Hz, 3H), 7.19

421

(s, 1H), 6.86 (d, J = 1.8 Hz, 1H), 2.40 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 169.88,

422

167.36, 162.89, 160.94, 154.56, 151.51, 150.00, 149.92, 148.03, 140.37, 139.03, 135.69,

423

135.67, 134.24, 133.94, 133.63, 133.33, 130.22, 130.16, 127.49, 124.29, 121.93, 120.11,

424

119.74, 117.56, 116.42, 115.40, 115.23, 113.63, 105.20, 20.80. EI-MS: m/z = 498.00 (M+).

425

Anal. calcd for (C24H14F4N4O4): C, 57.84; H, 2.83; N, 11.24. Found: C, 57.98; H, 2.67; N,

426

11.48.

427

Data

for


428


429

36% yield, mp: 215-216℃. 1H NMR (600 MHz, DMSO-d6) δ 13.33 (s, 1H), 8.15 (d, J = 6.0

430

Hz, 2H), 7.81 (d, J = 3.0 Hz, 1H), 7.56-7.48 (m, 1H), 7.34 (s, 1H), 7.33-7.22 (m, 4H), 6.86

431

(s, 1H), 2.41 (s, 3H).

432

154.56, 151.53, 149.99, 149.94, 148.04, 141.64, 141.57, 140.54, 138.64, 134.24, 133.94,

433

133.64, 133.33, 130.51, 130.45, 127.47, 124.42, 124.40, 124.24, 121.92, 120.54, 119.74,

434

117.56, 116.44, 115.38, 115.04, 114.87, 114.69, 114.52, 113.63, 105.20, 20.80. EI-MS: m/z

435

= 498.07 (M+). Anal. calcd for (C24H14F4N4O4): C, 57.84; H, 2.83; N, 11.24. Found: C,

436

57.80; H, 3.00; N, 11.34.

13

C NMR (125 MHz, DMSO-d6) δ 169.85, 167.26, 162.92, 160.99,

21



437

Data

for

Page 22 of 50


438


439

44% yield, mp: 220-221℃. 1H NMR (600 MHz, DMSO-d6) δ 13.01 (s, 1H), 8.14 (s, 2H),

440

7.79 (d, J = 3.6 Hz, 1H), 7.45 (dd, J = 13.2, 6.0 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.35 (s,

441

1H), 7.29 (dd, J = 13.2, 6.6 Hz, 2H), 7.18 (s, 1H), 6.86 (dd, J = 3.6, 1.8 Hz, 1H), 2.40 (s,

442

3H).

443

150.26, 150.00, 148.03, 140.74, 134.84, 134.21, 133.91, 133.60, 133.30, 131.03, 131.01,

444

130.03, 129.97, 128.52, 127.09, 126.97, 124.58, 124.33, 124.31, 121.92, 121.15, 119.73,

445

117.55, 116.39, 115.51, 115.33, 113.63, 105.14, 20.73. EI-MS: m/z = 498.06 (M+). Anal.

446

calcd for (C24H14F4N4O4): C, 57.84; H, 2.83; N, 11.24. Found: C, 57.70; H, 2.68; N, 11.46.

447

13

C NMR (125 MHz, DMSO-d6) δ 169.98, 166.52, 159.89, 157.94, 154.53, 151.47,

Data

for

4'-chloro-3-((5-(furan-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

448


449

mp: 220-221℃. 1H NMR (600 MHz, DMSO-d6) δ13.36 (s, 1H), 8.14 (d, J = 7.2 Hz, 2H),

450

7.80 (d, J = 3.6 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.54 (dd, J = 13.2, 8.4 Hz, 2H), 7.50 (d, J

451

= 8.4 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 8.4 Hz, 2H), 6.86 (s, 1H).

452

(125 MHz, DMSO-d6) δ 169.78, 167.11, 164.73, 154.56, 151.55, 149.99, 149.76, 148.07,

453

138.75, 137.80, 133.93, 132.85, 132.85, 130.02, 129.94, 128.52, 127.42, 126.67, 124.67,

454

122.54, 120.08, 120.03, 119.16, 116.48, 113.65, 105.29. EI-MS: m/z = 500.13 (M+). Anal.

455

calcd for (C23H12ClF3N4O4): C, 55.16; H, 2.42; N, 11.19. Found: C, 55.23; H, 2.45; N,

456

11.02.

457 458

Data

for

13

C NMR

4'-bromo-3-((5-(furan-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

a]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-2-carboxylic acid (5-35): White solid, 31% yield, 22


Page 23 of 50


459

mp: 219-220℃. 1H NMR (600 MHz, DMSO-d6) δ 13.42 (s, 1H), 8.15-8.11 (m, 2H), 7.79 (d,

460

J = 3.6 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H),

461

7.43 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 7.8 Hz, 1H), 6.86 (dd, J = 3.6, 1.8 Hz, 1H). 13C NMR

462

(125 MHz, DMSO-d6) δ 169.75, 167.09, 154.55, 151.55, 149.97, 149.80, 148.04, 138.93,

463

138.28, 134.24, 133.94, 133.63, 133.33, 131.46, 130.33, 130.30, 126.92, 126.64, 121.51,

464


465

(C23H12BrF3N4O4): C, 50.66; H, 2.22; N, 10.28. Found: C, 50.52; H, 2.30; N, 10.02.

466

Data

for

3-((5-(furan-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-

467

yl)oxy)-4'-(trifluoromethyl)-[1,1'-biphenyl]-2-carboxylic acid (5-36): White solid, 27%

468

yield, mp: 216-217℃. 1H NMR (600 MHz, DMSO-d6) δ 13.46 (s, 1H), 8.14 (d, J = 4.8 Hz,

469

2H), 7.86 (d, J = 7.8 Hz, 2H), 7.80 (d, J = 3.6 Hz, 1H), 7.70 (d, J = 7.8 Hz, 2H), 7.67 (s,

470

1H), 7.58 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 6.86 (d, J = 1.8 Hz, 1H), 2.09 (s,

471

3H).

472

149.81, 148.08, 147.70, 143.31, 143.03, 138.86, 138.70, 133.95, 133.65, 130.26, 129.11,

473

128.45, 128.20, 126.80, 125.41, 125.38, 125.30, 123.14, 120.61, 119.73, 117.55, 116.50,

474

113.66, 105.30. EI-MS: m/z = 534.11 (M+). Anal. calcd for (C24H12F6N4O4): C, 53.94; H,

475

2.26; N, 10.48. Found: C, 53.89; H, 2.35; N, 10.24.

476

13

C NMR (125 MHz, DMSO-d6) δ 169.77, 166.97, 154.57, 151.74, 151.57, 150.00,

Data

for

2-methyl-6-((5-(thiophen-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

477

a]pyrimidin-2-yl)oxy)benzoic acid (5-37): Yellow solid, 55% yield, mp: 234-235℃. 1H

478

NMR (600 MHz, DMSO-d6) δ 13.36 (s, 1H), 8.43 (s, 2H), 7.99 (d, J = 4.8 Hz, 1H), 7.43 (t,

479

J = 7.8 Hz, 1H), 7.32 (d, J = 4.2 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H),

480

2.37 (s, 3H).

13

C NMR (125 MHz, DMSO-d6) δ 169.89, 167.35, 156.11, 154.25, 149.75, 23



481

141.05, 136.32, 134.06, 133.86, 133.76, 133.45, 133.15, 132.05, 129.99, 129.31, 127.49,

482

127.25, 122.01, 119.83, 118.39, 117.65, 115.46, 105.49, 19.07. EI-MS: m/z = 420.55 (M+).

483

Anal. calcd for (C18H11F3N4O3S): C, 51.43; H, 2.64; N, 13.33. Found: C, 51.51; H, 2.53; N,

484

13.58.

485

Data for 3-((5-(thiophen-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-

486

yl)oxy)-[1,1'-biphenyl]-2-carboxylic acid (5-38): White solid, 27% yield, mp: 246-247℃.

487

1

488

(t, J = 7.8 Hz, 1H), 7.53-7.45 (m, 5H), 7.42 (t, J = 6.6 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H),

489

7.32 (t, J = 4.2 Hz, 1H). 13C NMR (125 MHz, DMSO-d6) δ 169.76, 167.28, 156.18, 154.26,

490

149.74, 141.05, 140.10, 139.09, 133.94, 133.80, 133.50, 133.19, 132.11, 130.12, 129.35,

491

128.50, 128.16, 127.84, 127.16, 126.77, 119.83, 117.64, 105.59, 99.47. EI-MS: m/z =

492

482.56 (M+). Anal. calcd for (C23H13F3N4O3S): C, 57.26; H, 2.72; N, 11.61. Found: C,

493

57.24; H, 2.65; N, 11.73.

494

H NMR (600 MHz, DMSO-d6) δ 13.33 (s, 1H), 8.43 (s, 2H), 8.00 (d, J = 4.8 Hz, 1H), 7.62

Data

for

5-methyl-3-((5-(thiophen-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

495

a]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-2-carboxylic acid (5-39): Yellow solid, 49% yield,

496


497

4.8 Hz, 1H), 7.46 (d, J = 3.6 Hz, 4H), 7.41 (s, 1H), 7.35-7.31 (m, 1H), 7.30 (s, 1H), 7.20 (s,

498

1H), 2.41 (s, 3H).

499

149.96, 141.07, 140.37, 140.14, 139.36, 134.13, 133.93, 133.83, 133.53, 133.22, 132.11,

500

129.36, 128.48, 128.16, 127.78, 127.56, 124.41, 122.07, 120.18, 119.88, 117.70, 115.53,

501

105.57, 20.86. EI-MS: m/z = 496.88 (M+). Anal. calcd for (C24H15F3N4O3S): C, 58.06; H,

502

3.05; N, 11.29. Found: C, 57.99; H, 3.03; N, 11.27.

13

C NMR (125 MHz, DMSO-d6) δ 169.93, 167.50, 156.18, 154.30,

24


Page 24 of 50

Page 25 of 50

503


Data

for

4'-fluoro-3-((5-(thiophen-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

504


505


506

3.6 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.56-7.48 (m, 3H), 7.38 (d, J = 7.8 Hz, 1H), 7.33 (d, J

507

= 8.4 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 169.75, 167.24, 162.95, 161.00, 156.22,

508

154.28, 149.77, 141.05, 139.09, 135.48, 135.46, 134.12, 133.95, 133.83, 133.52, 133.22,

509

132.13, 130.30, 130.23, 129.36, 127.14, 126.81, 122.03, 119.98, 119.84, 117.66, 115.49,

510

115.32, 105.61. EI-MS: m/z = 500.88 (M+). Anal. calcd for (C23H12F4N4O3S): C, 55.20; H,

511

2.42; N, 11.20. Found: C, 55.46; H, 2.39; N, 11.33.

512

Data

for

4'-fluoro-5-methyl-3-((5-(thiophen-2-yl)-7-(trifluoromethyl)-

513

[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-2-carboxylic

514

White solid, 65% yield, mp: 222-223℃. 1H NMR (600 MHz, DMSO-d6) δ 13.22 (s, 1H),

515

8.43 (s, 2H), 8.00 (d, J = 4.8 Hz, 1H), 7.54-7.46 (m, 2H), 7.31 (t, J = 9.0 Hz, 4H), 7.20 (s,

516

1H), 2.40 (s, 3H).

517

156.16, 154.27, 149.94, 141.05, 140.40, 139.04, 135.71, 135.69, 134.11, 133.92, 133.81,

518

133.51, 133.20, 132.10, 130.23, 130.16, 129.33, 127.53, 124.38, 122.04, 120.26, 119.85,

519

117.67, 115.40, 115.23, 105.56, 20.81. EI-MS: m/z = 514.00(M+). Anal. calcd for

520

(C24H14F4N4O3S): C, 56.03; H, 2.74; N, 10.89. Found: C, 56.00; H, 2.72; N, 11.02.

521

Data

13

for

acid

(5-41):

C NMR (125 MHz, DMSO-d6) δ 169.85, 167.38, 162.89, 160.94,


522


523

Yellow solid, 59% yield, mp: 220-221℃. 1H NMR (600 MHz, DMSO-d6) δ 13.29 (s, 1H),

524

8.43 (s, 2H), 8.00 (d, J = 4.8 Hz, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.36-7.22 (m, 6H), 2.41 (s, 25


acid

(5-42):


13

Page 26 of 50

525

3H).

526

149.96, 141.65, 141.59, 141.04, 140.58, 138.67, 134.12, 133.93, 133.82, 133.51, 133.21,

527

132.11, 130.51, 130.45, 129.33, 127.50, 124.42, 124.40, 124.32, 122.04, 120.69, 119.85,

528

117.67, 115.48, 115.04, 114.87, 114.68, 114.52, 105.61, 20.80. EI-MS: m/z = 514.23 (M+).

529

Anal. calcd for (C24H14F4N4O3S): C, 56.03; H, 2.74; N, 10.89. Found: C, 55.96; H, 2.57; N,

530

11.01.

531

C NMR (125 MHz, DMSO-d6) δ 169.82, 167.27, 162.93, 160.99, 156.18, 154.27,

Data

for


532


533

Yellow solid, 41% yield, mp: 217-218℃. 1H NMR (600 MHz, dmso) δ 13.01 (s, 1H), 8.43

534

(s, 2H), 7.99 (s, 1H), 7.42 (d, J = 27.6 Hz, 3H), 7.38-7.23 (m, 3H), 7.18 (s, 1H), 2.41 (s, 3H).

535

13

536

141.06, 140.75, 134.82, 133.93, 133.77, 133.46, 132.10, 131.02, 131.00, 130.03, 129.96,

537

129.35, 128.53, 127.08, 126.95, 124.61, 124.33, 124.31, 122.02, 121.24, 119.83, 117.65,

538


539

(C24H14F4N4O3S): C, 56.03; H, 2.74; N, 10.89. Found: C, 56.03; H, 2.58; N, 11.18.

540

acid

(5-43):

C NMR (125 MHz, DMSO-d6) δ 169.90, 166.50, 159.87, 157.92, 156.11, 154.23, 150.25,

Data

for

4'-chloro-3-((5-(thiophen-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

541


542


543

4.2 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 11.4 Hz, 3H), 7.49 (d, J = 7.2 Hz, 2H),

544

7.39 (d, J = 7.2 Hz, 1H), 7.33 (s, 1H).

545

156.23, 154.28, 149.85, 141.03, 138.94, 137.95, 134.11, 133.93, 133.84, 133.53, 133.23,

546

132.95, 132.12, 130.36, 130.02, 129.33, 128.55, 127.11, 126.77, 122.03, 120.32, 119.85,

13

C NMR (125 MHz, DMSO-d6) δ 169.80, 167.20,

26


Page 27 of 50


547

117.67, 115.48, 105.61. EI-MS: m/z = 516.04 (M+). Anal. calcd for (C23H12ClF3N4O3S): C,

548

53.44; H, 2.34; N, 10.84. Found: C, 53.28; H, 2.26; N, 11.04.

549

Data

for

4'-bromo-3-((5-(thiophen-2-yl)-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-

550


551

mp: 217-218℃. 1H NMR (600 MHz, DMSO-d6) δ 13.43 (s, 1H), 8.43 (s, 2H), 7.99 (s, 1H),

552

7.68 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8

553

Hz, 2H), 7.38 (d, J = 7.8 Hz, 1H), 7.32 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 169.73,

554

167.12, 156.21, 154.26, 149.83, 141.04, 138.93, 138.30, 134.12, 133.95, 133.82, 133.51,

555

133.21, 132.13, 131.46, 130.35, 130.31, 129.36, 127.01, 126.67, 122.00, 121.51, 120.27,

556

119.83, 117.64, 115.46, 105.62. EI-MS: m/z = 560.98 (M+). Anal. calcd for

557

(C23H12BrF3N4O3S): C, 49.21; H, 2.15; N, 9.98. Found: C, 49.02; H, 2.10; N, 9.92.

558

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-[1,1'-biphenyl]-

559

2-carboxylic acid (5-46): White solid, 45% yield, mp: 215-216℃. 1H NMR (600 MHz,

560

DMSO-d6) δ 13.26 (s, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 4.8 Hz, 6H), 7.42 (d, J =

561

3.0 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 2.77 (s, 3H), 2.48 (s, 3H).

562

DMSO-d6) δ 168.68, 167.35, 154.96, 152.10, 149.98, 149.06, 140.07, 139.13, 130.11,

563

128.48, 128.16, 127.81, 127.16, 126.58, 119.96, 105.75, 23.43, 19.25. HRMS (MALDI):

564

Calcd for C20H16N4O3 [M+Na]+ 383.1120. Found 383.1117.

13

C NMR (125 MHz,

565

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-5-methyl-[1,1'-

566


567

MHz, DMSO-d6) δ 13.05 (s, 1H), 7.51-7.42 (m, 5H), 7.40 (s, 1H), 7.24 (s, 1H), 7.16 (s, 1H),

568

3.32 (s, 3H), 2.78 (s, 3H), 2.38 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 168.78, 167.49, 27



569

154.90, 152.09, 150.16, 149.02, 140.26, 140.04, 139.38, 128.41, 128.12, 127.69, 127.35,

570

124.40, 120.22, 105.75, 23.43, 20.79, 19.26. HRMS (MALDI): Calcd for C21H18N4O3

571

[M+Na]+ 397.1277. Found 397.1277.

572

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-4',5-dimethyl-

573

[1,1'-biphenyl]-2-carboxylic acid (5-48): White solid, 33% yield, mp: 214-215℃. 1H

574

NMR (600M Hz, DMSO-d6) δ 13.04 (s, 1H), 7.46 (s, 1H), 7.34 (d, J = 7.2 Hz, 2H), 7.25 (d,

575

J = 7.2 Hz, 2H), 7.21 (s, 1H), 7.13 (s, 1H), 2.77 (s, 3H), 2.47 (s, 3H), 2.37 (s, 3H), 2.34 (s,

576

3H).

577

140.15, 139.98, 137.05, 136.49, 129.01, 128.01, 127.23, 124.38, 119.99, 105.74, 23.43,

578

20.81, 20.68, 19.25. HRMS (MALDI): Calcd for C22H20N4O3 [M+H]+ 389.1614. Found

579

389.1610.

13

C NMR (125 MHz, DMSO-d6) δ 168.82, 167.57, 154.90, 152.09, 150.16, 149.01,

580

Data for 3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-4'-methoxy-5-

581


582

1

583

(s, 1H), 7.13 (s, 1H), 7.01 (d, J = 9.0 Hz, 2H), 3.79 (s, 3H), 2.77 (s, 3H), 2.48 (s, 3H), 2.37

584

(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 168.79, 167.65, 158.96, 154.88, 152.09, 150.13,

585

149.02, 140.07, 139.66, 131.61, 129.31, 127.17, 124.30, 119.71, 113.89, 105.74, 55.13,

586

23.42, 20.80, 19.25. HRMS (MALDI): Calcd for C22H20N4O4 [M+H]+ 405.1563. Found

587

405.1562.

588

H NMR (600M Hz, DMSO-d6) δ 13.07 (s, 1H), 7.47 (s, 1H), 7.38 (d, J = 9.0 Hz, 2H), 7.20

Data

for

3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-5-methyl-4'-

589

nitro-[1,1'-biphenyl]-2-carboxylic acid (5-50): White solid, 28% yield, mp: 215-216℃. 1H

590

NMR (400M Hz, DMSO-d6) δ 13.31 (s, 1H), 8.32 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 28


Page 28 of 50

Page 29 of 50


13

591

2H), 7.47 (s, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 2.77 (s, 3H), 2.48 (s, 3H), 2.41 (s, 3H).

592

NMR (125 MHz, DMSO-d6) δ 168.71, 167.01, 154.97, 152.11, 150.42, 149.05, 146.93,

593

146.14, 140.97, 138.18, 129.55, 127.43, 124.28, 123.55, 121.62, 105.78, 23.43, 20.76, 19.24.

594

HRMS (MALDI): Calcd for C21H17N5O5 [M+H]+ 420.1308. Found 420.1309.

595

Data

for

C

3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-4'-fluoro-5-

596


597

1

598

16.0, 7.2 Hz, 3H), 7.15 (s, 1H), 2.77 (s, 3H), 2.48 (s, 3H), 2.38 (s, 3H). 13C NMR (126 MHz,

599

DMSO-d6) δ 168.72, 167.38, 162.85, 160.90, 154.91, 152.10, 150.15, 149.04, 140.36,

600

138.98, 135.74, 135.72, 130.20, 130.14, 127.36, 124.37, 120.33, 115.37, 115.20, 105.76,

601

23.42, 20.77, 19.25. HRMS (MALDI): Calcd for C21H17FN4O3 [M+Na]+ 415.1182. Found

602

415.1181.

H NMR (400M Hz, DMSO-d6) δ 13.12 (s, 1H), 7.47 (d, J = 12.0 Hz, 3H), 7.28 (dd, J =

603

Data

for


604


605

1

606

= 7.2 Hz, 3H), 7.24 (d, J = 9.0 Hz, 1H), 7.20 (s, 1H), 2.77 (s, 3H), 2.48 (s, 3H), 2.39 (s, 3H).

607

13

608

149.05, 141.67, 141.61, 140.51, 138.57, 130.48, 130.41, 127.32, 124.40, 124.31, 120.74,

609

115.01, 114.83, 114.64, 114.47, 105.78, 23.42, 20.75, 19.25. HRMS (MALDI): Calcd for

610

C21H17FN4O3 [M+H]+ 393.1363. Found 393.1366.

611 612

H NMR (600M Hz, DMSO-d6) δ 13.20 (s, 1H), 7.53-7.48 (m, 1H), 7.47 (s, 1H), 7.28 (d, J

C NMR (125 MHz, DMSO-d6) δ 168.68, 167.27, 162.89, 160.95, 154.92, 152.10, 150.15,

Data

for


methyl-[1,1'-biphenyl]-2-carboxylic acid (5-53): White solid, 20% yield, mp: 211-212℃. 29



613

1

614

7.29 (s, 1H), 7.27 (s, 1H), 7.25 (s, 1H), 7.14 (s, 1H), 2.77 (s, 3H), 2.48 (s, 3H), 2.38 (s, 3H).

615

13

616

149.02, 140.72, 134.79, 131.05, 129.99, 129.93, 128.37, 127.15, 127.03, 124.69, 124.32,

617

124.30, 121.33, 115.50, 115.32, 105.74, 23.43, 20.71, 19.25. HRMS (MALDI): Calcd for

618

C21H17FN4O3 [M+H]+ 393.1363. Found 393.1360.

H NMR (400M Hz, DMSO-d6) δ 12.90 (s, 1H), 7.48-7.39 (m, 2H), 7.37 (d, J = 8.0 Hz, 1H),

C NMR (125 MHz, DMSO-d6) δ 168.82, 166.62, 159.89, 157.94, 154.91, 152.10, 150.52,

619

Data for 4'-chloro-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-[1,1'-

620

biphenyl]-2-carboxylic acid (5-54): White solid, 29% yield, mp: 224-225℃. 1H NMR

621

(400M Hz, DMSO-d6) δ 13.34 (s, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.53 (s, 1H),

622

7.49 (d, J = 2.4 Hz, 2H), 7.46 (s, 2H), 7.35 (d, J = 7.6 Hz, 1H), 2.77 (s, 3H), 2.48 (s, 3H).

623

13

624

137.95, 132.85, 130.29, 129.99, 128.51, 127.08, 126.52, 120.35, 105.76, 23.43, 19.24.

625

HRMS (MALDI): Calcd for C20H15ClN4O3 [M+H]+ 395.0911. Found 395.0912.

C NMR (125 MHz, DMSO-d6) δ 168.62, 167.16, 154.97, 152.10, 150.02, 149.07, 138.81,

626

Data

for

4'-chloro-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)oxy)-5-

627


628

1

629

Hz, 3H), 7.27 (s, 1H), 7.17 (s, 1H), 2.77 (s, 3H), 2.48 (s, 3H), 2.38 (s, 3H). 13C NMR (125

630

MHz, DMSO-d6) δ 168.73, 167.29, 154.92, 152.09, 150.22, 149.02, 140.51, 138.81, 138.20,

631

132.72, 129.94, 128.43, 127.27, 124.29, 120.62, 105.76, 23.42, 20.77, 19.25. HRMS

632

(MALDI): Calcd for C21H17ClN4O3 [M+H]+ 409.1067. Found 409.1071.

H NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4

633

Enzyme inhibition activities against wild-type AHAS and P197L mutant. In order

634

to evaluate the potency of the newly synthesized compounds, the Ki values of compounds 5 30


Page 30 of 50

Page 31 of 50


635

against wild-type AtAHAS and P197L mutant were determined respectively according to

636

the methods reported previously.15, 30-32 In the meantime, we also investigated the binding

637

modes of the new compounds by using seven representative compounds 5 (Figure 5). And

638

then, we defined resistant factor (RF) as the ratio between the Ki values against the mutant

639

to that against the wild-type enzyme for evaluating the effects of the compounds 5 on

640

resistance. So the larger the value of RF, the worse resistance the compounds 5.

641

Flumetsulam (FS) was used as a positive control and the results were listed in Table 1.

642

Although FS exhibited strong inhibition activity against wild-type AtAHAS, it showed

643

about 68.07 folds lower potency towards P197L mutant. However, as expected, many of

644

newly synthesized compounds 5 showed better even excellent inhibition effect against both

645

the wild-type A. thaliana AHAS and P197L mutants simultaneously. Among them, the Ki

646

values of compound 5-1 (R1 = H, R2 = CH3) against wide-type and P197L AHAS were

647

determined to be (2.00±0.09)×10-4 M and >1.00×10-3 M, respectively. These results indicate

648

that the P197L mutation confers more than 1000-fold greater resistance toward compound

649

5-1, and its values of RF is >1000. And then, when we placed a benzene ring at R3,

650

compound 5-2 (R1 = H, R2 = CH3) displayed greater inhibition activities against both the

651

wild-type AHAS and P197L mutants, and its values of RF is reduced to 1491. The reason

652

for the high level of resistances could be explained according to the binding modes of the

653

new compounds with the tested enzymes. As shown in Figure 5A, 5B and 5C, we can know

654

that after mutation there exists steric hindrance between L197 and compound 5-2 (the

655

distance of these only 2.3 Å) from the binding model. Further, a hydrophobic interaction

656

between the benzene ring of R3 and residue P197 improved the activity of compound 5-2 31



657

against the wild-type AHAS. Moreover, this interaction didn’t exist on compound 5-1. For

658

the further optimization, a series of substituents on the benzene ring of R3 were introduced.

659

As shown in Table 1, compound 5-3 and 5-4 (R1 = H, R2 = CH3) displayed higher

660

inhibition activities against both the wild-type AHAS and P197L mutants than compound 5-

661

2, and their values of RF is only 463.7 and 26.72 respectively. Moreover, when a methyl

662

group was introduced at the position of R1, the inhibition activities of compounds 5-5~5-17

663

(R1 = CH3, R2 = CH3) against the wild-type AHAS and P197L mutants usually displayed

664

an enhanced effects, and the values of RF were further reduced. Such as compound 5-6, the

665

Ki values against wide-type and P197L AHAS were determined to be (4.81±0.62)×10-8 M

666

and (7.22±0.21)×10-6 M, respectively, and its values of RF was only 150.1. The interaction

667

analysis shown in Figure 5D and 5E indicates that compound 5-6 seems to be more fitness

668

to pocket of AHAS when a methyl was replaced at the R1 position. Moreover, comparing

669

compound 5-6 with compound 5-7 or compound 5-11 with compound 5-12, it could be

670

discovered that a methyl group at the position of R4 was beneficial to reduce the values of

671

RF (5-6 > 5-7, 5-11 > 5-12). So the structure-drug resistance relationship at these position

672

can be summarized as follows: R1: CH3 > H; R3: 4-tert-butylPh > Ph > 2-FPh > 4-

673

OCH3Ph > 4-FPh > 3-FPh > 4-NO2Ph > CH3; R4: CH3 > H.

674

For the further optimization, a series of substituents with diversity were introduced at

675

the 5 and 7 positions of 1,2,4-triazolo[1,5-a]pyrimidine ring. As shown in Table 1, although

676

the values of RF of compounds 5-18 to 5-26 (R1 = CF3, R2 = phenyl) were further reduced

677

to < 0.001, they displayed the lower inhibitory activity against the wild-type AHAS. A

678

possible reason for this phenomenon may be that their too big or sterically bulky 32


Page 32 of 50

Page 33 of 50


679

substituents limiting its ability to form the interactions in the binding pocket. And then,

680

when we placed a furan ring (5-27 to 5-36, R1 = CF3, R2 = furan-2-yl) or a thiophene ring

681

(5-37 to 5-45, R1 = CF3, R2 = thiophen-2-yl) at R2, the inhibition activities against the

682

wild-type AHAS and P197L mutants usually displayed an enhanced effect. For example,

683

compounds 5-30, 5-31, 5-32, 5-38, 5-39 and 5-40 have shown “good” to “the excellent”

684

inhibitory activities against both the wild-type AHAS and P197L mutants, and their values

685

of RF can still keep less than 10. It also can be explained from Figure 5F, 5G and 5H. The

686

aromatic ring at R2 can form t-π stacking interaction with the FAD, however, the benzene

687

ring is too big, which nearly can’t bind in the pocket. The residues around the R2 are nearly

688

all the hydrophobic amino acid, so the activity of compounds 5-30 and 5-40 seems to be

689

better than compound 5-22. For the further optimization, we changed the 1,2,4-triazolo[1,5-

690

a]pyrimidine ring to the 1,2,4-triazolo[1,5-c]pyrimidine ring. As shown in Table 1,

691

compounds 5-46 to 5-55 (X = N, Y = CH, R1 = CH3, R2 = CH3) didn’t display the higher

692

inhibitory activity against both two tested enzymes, and their RF values rise to more than

693

1000. It seems that the 1,2,4-triazolo[1,5-c]pyrimidine ring was detrimental to improve the

694

enzyme inhibition activities. This point can be explained from Figure 5I. If we exchange the

695

position of N atom and CH at X and Y, compounds 5-46 will lose an important hydrogen

696

bond and doesn’t have high inhibitory activity against P197L mutants. So the structure-

697

resistance relationship at these position can be summarized as follows: R2: furan-2-yl,

698

thiophen-2-yl > Ph > CH3; 1,2,4-triazolo[1,5-a]pyrimidine > 1,2,4-triazolo[1,5-c]pyrimidine.

699

Herbicidal activity of compounds 5. The herbicidal activities of all the target

700

compounds against the wild-type and resistant D. sophia (P197L) were evaluated according 33



701

to a previously reported procedure.23, 32-33 Flumetsulam was used as the positive control, and

702

the results are shown in Table 2. Among them, compounds 5-15, 5-18, 5-19, 5-20, 5-21 and

703

5-22 showed moderate to good herbicidal activity against the wild-type D. sophia.

704

Moreover, compounds 5-15 and 5-20 also displayed good to excellent herbicidal activity

705

against the resistant D. sophia (P197L) at the dosage of 0.9375 g.ai/ha. It seems that they

706

displayed better anti-resistance than flumetsulam. In addition, the inhibition of weed growth

707

is weakened after adding some branched-chain amino acids (valine, leucine, and isoleucine)

708

to the in vivo assay (data not shown), which further confirmed that the herbicidal activity of

709

the target compound is due to AHAS inhibition.

710

In conclusion, based on the rule of bioisosterism and conformational flexibility

711

analysis, a series of 2-aroxyl-1,2,4-triazolopyrimidine derivatives were designed and

712

synthesized with the aim to discover anti-resistant lead compound. The in vitro assay

713

indicated that almost all the compounds 5 showed better even excellent inhibition effect

714

against both the wild-type A. thaliana AHAS and P197L mutants simultaneously. Among

715

them, compounds 5-3 to 5-17, compounds 5-19 to 5-26, compounds 5-28 to 5-45 and

716

compound 5-48 have the lower values of RF and display a potential power to overcome

717

drug resistance associated with the P197L mutation in the enzyme levels. Further green

718

house in vivo assay exhibited that compounds 5-15 and 5-20 displayed moderate to good

719

herbicidal activity against both the wild-type and resistant D. sophia (P197L) even at a rate

720

as low as 0.9375 g.ai/ha. The above results indicated that these two compounds could be

721

used as new leads for the future development of anti-resistance herbicides.

722 34


Page 34 of 50

Page 35 of 50


723

SUPPORTING INFORMATION

724

Detailed information for the preparation and the analytical data of compounds 5. This

725

material is available free of charge via the Internet at http://pubs.acs.org.

726 727

ACKNOWLEDGMENTS

728

This research was supported by the Special Fund for Agro-scientific Research in the Public

729

Interest (No. 201203022) and the National Natural science Foundation of China (No.

730

21332004).

731 732

AUTHOR INFORMATION

733

Corresponding Author

734

*E-mail: [email protected]

735 736 737

NOTES The authors declare no competing financial interest.

738 739

REFERENCES

740

1. Duggleby, R. G.; Pang, S. S. Acetohydroxyacid synthase. J. Biochem. Mol. Biol. 2000,

741

33, 1-36.

742

2. Choi, K. J.; Yu, Y. G.; Hahn, H. G.; Choi, J. D.; Yoon, M. Y. Characterization of

743

acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of

744

its new inhibitor from the screening of a chemical library. FEBS Letters 2005, 579,

745

4903-4910.

35



746 747 748 749

3. Geier, P. W.; Stahlman, P. W.; Hargett, J. G. Dose responses of weeds and winter wheat to MKH 6561. Weed Sci. 2001, 49, 788–791. 4. Gerwick, B. C.; Subermanian, V. I.; Loney-Gallant, V. I.; Chander, D. P. Mechanism of Action of the 1,2,4-Triazolo[1,5-a]pyrimidines. Pestic. Sci. 1990, 29, 357–364.

750

5. Shimizu, T.; Nakayama, I.; Nakao, T.; Nezu, Y.; Abe, H. Action Mechanism of

751

Herbicides, Pyrimidinylsalicylic Acids 1. Inhibition of Plant Acetolactate Synthase by

752

Herbicides, Pyrimidinylsalicylic Acids. J. Pestic. Sci. 1994, 19, 59–67.

753 754 755 756 757 758 759 760

6. Tranel, P. J.; Wright, T. R.; Heap, I. M. The international survey of herbicide-resistant weeds. http://www.weedscience.org (accessed Dec. 29, 2015). 7. Beckie, H. J.; Tardif, F. J., Herbicide cross resistance in weeds. Crop Protection 2012, 35, 15-28. 8. Duggleby, R. G.; Pang, S. S.; Yu, H.; Guddat, L. W. Systematic characterization of mutations in yeast acetohydroxyacid synthase. Eur. J. Biochem. 2003, 270, 2895-2904. 9. Tranel, P. J.; Wright, T. R. Resistance of weeds to ALS-inhibiting herbicides: what have we learned? 2009.

761

10. Cui, H. L.; Zhang, C. X.; Wei, S. H.; Zhang, H. J.; Li, X. J.; Zhang, Y. Q.; Wang, G. Q.

762

Acetolactate synthase gene proline (197) mutations confer tribenuron-methyl resistance

763

in flixweed (Descurainia sophia) populations from China. Weed sci. 2011, 59, 376-379.

764

11. Yu, Q.; Han, H.; Powles, S. B., Mutations of the ALS gene endowing resistance to

765

ALS-inhibiting herbicides in Lolium rigidum populations. Pest Manag. Sci. 2008, 64,

766

1229-1236.

36


Page 36 of 50

Page 37 of 50


767

12. Chen, C. N.; Lv, L. L.; Ji, F. Q.; Chen, Q.; Xu, H.; Niu, C. W.; Xi, Z.; Yang, G. F.

768

Design and synthesis of N-2, 6-difluorophenyl-5-methoxyl-1, 2, 4-triazolo [1, 5-a]-

769

pyrimidine-2-sulfonamide as acetohydroxyacid synthase inhibitor. Bioorg. Med. Chem.

770

2009, 17, 3011-3017.

771

13. Chen, C. N.; Chen, Q.; Liu, Y. C.; Zhu, X. L.; Niu, C. W.; Xi, Z.; Yang, G. F.

772

Syntheses and herbicidal activity of new triazolopyrimidine-2-sulfonamides as

773

acetohydroxyacid synthase inhibitor. Bioorg. Med. Chem. 2010, 18, 4897-4904.

774

14. Xi, Z.; Yu, Z, H.; Niu, C, W.; Ban, S, R.; Yang, G, F. Development of a general

775

quantum-chemical descriptor for steric effects: Density functional theory based QSAR

776

study of herbicidal sulfonylurea analogues. J. Comput. Chem. 2006, 27, 1571-1576.

777

15. Ji, F. Q.; Niu, C. W.; Chen, C. N.; Chen, Q.; Yang, G. F.; Xi, Z.; Zhan, C. G.

778

Computational Design and Discovery of Conformationally Flexible Inhibitors of

779

Acetohydroxyacid Synthase to Overcome Drug Resistance Associated with W586L

780

Mutation. ChemMedChem 2008, 3, 1203-1206.

781

16. Pang, S. S.; Duggleby, R. G.; Guddat, L. W. Crystal structure of yeast

782

acetohydroxyacid synthase: a target for herbicidal inhibitors. J. Mol. Biol. 2002, 317,

783

249-262.

784 785

17. Pang, S. S.; Guddat, L. W.; Duggleby, R. G. Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase. J. Biol. Chem. 2003, 278, 7639-7644.

786

18. McCourt, J. A.; Pang, S. S.; Guddat, L. W.; Duggleby, R. G. Elucidating the specificity

787

of binding of sulfonylurea herbicides to acetohydroxyacid synthase. Biochemistry 2005,

788

44, 2330-2338. 37



789

19. McCourt, J. A.; Pang, S. S.; King-Scot, J.; Guddat, L. W.; Duggleby, R. G. Herbicide-

790

binding sites revealed in the structure of plant acetohydroxyacid synthase. Proc. Natl.

791

Acad. Sci. U.S.A. 2006, 103, 569-573.

792

20. Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Afragola, J.; Hernandez, R.; Lucas, J.;

793

Gibbons, J.; Beyer, C. Synthesis and SAR of [1, 2, 4] triazolo [1, 5-a] pyrimidines, a

794

class of anticancer agents with a unique mechanism of tubulin inhibition. J. Med. Chem.

795

2007, 50, 319-327.

796

21. Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.; Moro, S.; Klotz, K.-N.;

797

Leung, E.; Varani, K.; Gessi, S.; Merighi, S. Pyrazolo [4, 3-e] 1, 2, 4-triazolo [1, 5-c]

798

pyrimidine derivatives as highly potent and selective human A3 adenosine receptor

799

antagonists: influence of the chain at the N8 pyrazole nitrogen. J. Med. Chem. 2000, 43,

800

4768-4780.

801

22. Baraldi, P. G.; Cacciari, B.; Moro, S.; Spalluto, G.; Pastorin, G.; Da Ros, T.; Klotz, K.-

802

N.; Varani, K.; Gessi, S.; Borea, P. A., Synthesis, biological activity, and molecular

803

modeling investigation of new pyrazolo [4, 3-e]-1, 2, 4-triazolo [1, 5-c] pyrimidine

804

derivatives as human A3 adenosine receptor antagonists. J. Med. Chem. 2002, 45, 770-

805

780.

806

23. Yang, G. F.; Lu, R. J.; Fei, X. N.; Yang, H. Z. Syntheses and Properties of New

807

Herbicidal 2-Arylthio-1,2,4- Triazolo[1,5-a]- pyrimidine Derivatives. Chinese J. Chem.

808

2000, 18, 435-440.

38


Page 38 of 50

Page 39 of 50


809

24. Liu, Y. C.; Huang, Z. Y.; Chen, Q.; Yang, G. F. Efficient synthesis of functionalized 6-

810

arylsalicylates via microwave-promoted Suzuki cross-coupling reaction. Tetrahedron

811

2013, 69, 9025-9032.

812

25. Liu, Y. C.; Qu, R. Y.; Chen, Q.; Wu, Q. Y.; Yang, G. F., Efficient synthesis of

813

functionalized 6-substituted-thiosalicylates via microwave-promoted Suzuki cross-

814

coupling reaction. Tetrahedron 2014, 70, 2746-2752.

815

26. Qu, R. Y.; Liu, Y. C.; Wu, Q. Y.; Chen, Q.; Yang, G. F. An efficient method for

816

syntheses of functionalized 6-bulkysubstituted salicylates under microwave irradiation.

817

Tetrahedron 2015, 71, 8123-8130.

818

27. Yawer, M. A.; Hussain, I.; Iqbal, I.; Spannenberg, A.; Langer, P., Synthesis of

819

functionalized 6 (5H)-phenanthridinones based on a [3+3]-cyclocondensation/

820

lactamization strategy. Tetrahedron Lett. 2008, 49, 4467-4469.

821

28. Hussain, I.; Yawer, M. A.; Lau, M.; Pundt, T.; Fischer, C.; Görls, H.; Langer, P.,

822

Regioselective Synthesis of Fluorinated Phenols, Biaryls, 6H‐Benzo [c] chromen-6-

823

ones and Fluorenones Based on Formal [3+3] Cyclizations of 1, 3-Bis (silyl enol

824

ethers). Eur. J. Org. Chem. 2008, 2008, 503-518.

825

29. Hussain, I.; Nguyen, V. T. H.; Yawer, M. A.; Dang, T. T.; Fischer, C.; Reinke, H.;

826

Langer, P., Synthesis of Dibenzo [b, d] pyran-6-ones Based on [3+ 3] Cyclizations of 1,

827

3-Bis (silyl enol ethers) with 3-Silyloxy-2-en-1-ones. J. Org. Chem. 2007, 72, 6255-

828

6258.

829

30. Xi, Z.; Niu, C. W.; Li, Q. X.; Ouyang, Z.; Ban, S. R. Studies on Herbicide Design

830

through Mutation on Herbicidal Target--Acetohydroxyacid Synthase(I). Enzyme 39



831

kinetics of wild type and mutants of E. coli AHAS II. Chinese J. Pestic. Sci. 2005, 7,

832

215-220.

833

31. He, Y. Z.; Li, Y. X.; Zhu, X. L.; Xi, Z.; Niu, C. W.; Zhang, L.; Yang, G. F. Rational

834

Design and Interaction Mechanism of Pyrimidinylbenzoates with Acetohydroxyacid

835

Synthase Based on Bioactive Conformation Analysis by Integrating Molecular

836

Docking, CoMFA, CoMSIA and DFT Calculations. J. Chem. Inf. Model. 2007, 47,

837

2335-2344.

838

32. Li, Y. X.; Luo, Y. P.; Xi, Z.; Niu, C. W.; He, Y. Z.; Yang, G. F. Design and Syntheses

839

of Novel Phthalazin-1(2H)-one Derivatives as Acetohydroxyacid Synthase Inhibitors.

840

J. Agric. Food Chem. 2006, 54, 9135 -9139.

841

33. Luo, Y. P.; Jiang, L. L.; Wang, G. D.; Chen, Q.; Yang, G. F. Syntheses and Herbicidal

842

Activities of Novel Triazolinone Derivatives. J. Agric. Food Chem. 2008, 56, 2118-

843

2124.

844 845 846 847 848 849 850 851 852 40


Page 40 of 50

Page 41 of 50


853 854

Figure captions:

855

Figure 1 Structures of some representative commercial AHAS inhibitors.

856

Figure 2 Design of title compounds 5.

857

Figure 3 Synthetic route for the title compounds 2-5.

858

Figure 4 Chemical structures of title compounds 5.

859

Figure 5 The simulated binding models of seven representative compounds 5.

860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 41



875

Figure 1 Structures of some representative commercial AHAS inhibitors.

42


Page 42 of 50

Page 43 of 50


Figure 2 Design of title compounds 5.

43



Figure 3 Synthetic route of the title compounds 5.

44


Page 44 of 50

Page 45 of 50


R4 R1

R3 N N

X R2

O

COOH

N

Y

5

N N

N N

COOH

N

N

H3C

O

5-1

N N

O

N N

COOH

O

5-6

O

COOH

5-7

CH3

N N

O

F

N N N

H3C

O

5-9

5-10

H3C

5-11

H3C

O

COOH

Br O

N

5-16

5-17

CF3

NO2

N N

O

N N

COOH

N

N

O

N

O

N

N

O

N

N

O

O

5-29

COOH

O

O

N

N

O

5-39

5-40

COOH

O

Br

N N

COOH

N

N

N

N

CF3

Cl

N N O

COOH

N

N

S

O

S

5-42

5-43

5-44

5-45

OCH3

N N

O

N

COOH

N

N N

O

N

COOH

N

5-46

N N

O

COOH

N N

N

N

5-47

N

COOH

N N

O

COOH

5-49

5-50

F Cl

N

N N

O

N 5-51

NO2 N

COOH

N

5-48

F O

O

N

F N N

COOH

S

5-41

N

N

CF3

N N

COOH

N

N

O

N

S

S

CF3

N N

COOH

N

N

F

N N O

F

CF3

F COOH

5-35

CF3

5-38

S

N

N

N N

COOH

F

N

COOH

O

N

5-37

S

O

N

5-34

S

5-36

O

N

Br

N N

COOH

CF3

N N

COOH

N

N

N N

O

N

5-33

S

CF3

5-30

CF3

Cl

N N O

CF3

N N

COOH

N

N

N

N

O

5-32

O

COOH

O

N

N

CF3

CF3

N N

O

O

5-31

CF3

F

N N

COOH

CF3

N N

COOH

O

N

F

N

N

O

O

N

N

COOH

5-25

O

CF3

N N

COOH

O

CF3

5-28

CF3

F O

Cl

N N N

N N

COOH

F N N

CF3

COOH

5-24

O 5-27

CF3

O

N

N

N

N

O 5-26

N N

COOH

COOH

5-20

CF3

N N

COOH

O

N

N

Cl

5-23

O

N N

COOH

5-19

CF3

N N

COOH

N

O

CF3

N

5-22

N

5-15

N

N

F

N N

CF3

Br

N N

COOH

CF3

COOH N

5-21

CF3 N N

O

N

N

O

COOH

CF3

5-18

F

H3C

O

N

5-14

N

N

CF3

COOH

CF3

N N

COOH

N

O

N

N

CF3

N N H3C

N

N

H3C

Cl

N N

N N

COOH

5-13

CH3

Cl

N N

O

N

N

5-12

CH3

CH3

CH3

N N

COOH

N

N

H3C

O

COOH

F

CH3

F

N N

O

N

N

H3C

5-8

CH3

COOH

N

NO2

N N

COOH

N

N

F CH3

COOH

5-5

CH3

H3C

N

N

H3C

O

N

N

H3C

N

N

OCH3

N N

COOH

N

N

H3C

N N

COOH

5-4

CH3

N

O

5-3

CH3

N

N N

COOH H3C

5-2

CH3

H3C

O

N

N

H3C

CH3

F

N N

COOH

N

N

H3C

O

COOH

N

N N

N O

COOH

N N

O

COOH

N

5-52

5-53

Cl N

N N

O

N

N 5-54

Figure 4 Chemical structures of title compounds 5.

45


5-55

COOH


Figure 5 The simulated binding models of seven representative compounds 5.

46


Page 46 of 50

Page 47 of 50


Table 1 Inhibition activities of compounds 5 against wild-type AHAS and P197L mutants

No.

Ki, WT (M)

Ki, P197L (M)

RF*

5-1

(2.00±0.09)×10-4

>1.00×10-3

> 1000

5-2

(9.99±1.06)×10-7

(1.49±0.33)×10-3

1491

5-3

(9.08±0.97)×10-7

(4.21±0.74)×10-4

463.7

5-4

(2.65±1.18)×10-5

(7.08±0.94)×10-4

26.72

5-5

(3.61±0.38)×10-6

(3.87±0.60)×10-5

10.72

5-6

(4.81±0.62)×10-8

(7.22±0.21)×10-6

150.1

5-7

(3.21±0.98)×10-6

(1.92±0.21)×10-5

5.981

5-8

(6.32±1.12)×10-6

(8.68±0.81)×10-5

13.73

5-9

(3.94±0.28)×10

-5

-6

5-10

(6.32±0.25)×10-6

(4.63±0.41)×10-5

73.26

5-11

(1.59±0.30)×10-7

(5.19±0.29)×10-6

32.64

5-12

(2.14±0.42)×10-6

(3.43±0.27)×10-5

16.03

5-13

(9.51±0.63)×10-7

(6.16±0.34)×10-5

64.77

5-14

(1.08±0.11)×10-6

(1.18±0.15)×10-5

10.93

5-15

(2.35±0.25)×10-7

(3.11±0.35)×10-6

13.23

5-16

(8.38±0.42)×10-6

(5.65±0.34)×10-6

0.6742

5-17

(1.84±0.28)×10-7

(8.48±1.48)×10-6

46.09

5-18

>1.00×10-3

>1.00×10-3

/

5-19

(1.08±0.58)×10-3

(7.75±0.94)×10-4

0.7176

5-20

(3.48±1.89)×10-4

(1.16±0.26)×10-5

0.03333

5-21

>1×10-3

(2.48±0.38)×10-4

< 0.001

5-22

(2.07±0.58)×10-3

>1.00×10-3

/

5-23

>1.00×10-3

(4.05±0.44)×10-4

< 0.001

5-24

>1.00×10-3

(3.97±0.51)×10-4

< 0.001

5-25

>1.00×10-3

(2.69±0.24)×10-4

< 0.001

5-26

(3.82±1.04)×10-5

(6.65±0.96)×10-4

17.41

5-27

>1.00×10-3

>1.00×10-3

/

5-28

(8.14±1.19)×10-4

(4.16±0.92)×10-4

0.5111

5-29

(5.65±0.77)×10-4

(7.58±0.96)×10-4

1.342

5-30

(1.11±0.28)×10-3

(1.10±0.20)×10-3

0.9910

(3.56±0.50)×10

47


0.09036


Page 48 of 50

5-31

(1.94±0.25)×10-4

(2.07±0.21)×10-4

1.067

5-32

(3.14±0.40)×10-5

(1.34±0.14)×10-4

4.268

5-33

(5.36±0.64)×10-4

(7.92±0.85)×10-4

1.478

5-34

>1.00×10-3

(1.05±0.16)×10-4

< 0.001

5-35

(1.17±0.36)×10-5

(1.35±0.21)×10-3

115.4

5-36

(4.99±0.45)×10-4

(8.20±1.83)×10-4

1.643

5-37

(2.10±1.25)×10

-4

-3

7.762

5-38

(9.93±1.36)×10-5

(2.54±0.38)×10-4

2.558

5-39

(9.82±2.00)×10-5

(2.18±0.60)×10-4

2.220

5-40

(9.80±1.57)×10-5

(3.90±0.44)×10-4

3.980

5-41

(2.42±0.58)×10-5

(1.35±0.23)×10-4

5.579

5-42

(9.35±2.21)×10-5

(9.54±0.92)×10-5

1.020

5-43

(1.87±0.40)×10-4

(2.80±0.59)×10-4

1.497

5-44

(1.67±0.32)×10

-4

-4

2.964

5-45

(1.41±0.34)×10-4

(3.66±0.50)×10-4

2.596

5-46

(1.52±0.25)×10-6

>1.00×10-3

> 1000

5-47

(4.17±1.67)×10-4

>1.00×10-3

> 1000

5-48

(6.31±0.79)×10-5

(1.24±0.15)×10-3

19.65

5-49

(5.60±1.59)×10-4

>1.00×10-3

> 1000

5-50

(6.54±1.82)×10-4

>1.00×10-3

> 1000

5-51

(4.84±1.47)×10-4

>1.00×10-3

> 1000

5-52

(2.40±0.14)×10-4

>1.00×10-3

> 1000

5-53

(1.08±0.09)×10-4

>1.00×10-3

> 1000

5-54

(4.29±0.42)×10-6

(1.05±0.39)×10-3

244.8

5-55

(1.14±0.24)×10-3

>1.00×10-3

> 1000

FS

(3.79±0.26)×10-7

(2.58±0.32)×10-5

68.07

(1.63±0.27)×10

(4.95±0.73)×10

FS = Flumetsulam * RF = Ki, P197L/Ki, WT

48


Page 49 of 50


Table 2. Herbicidal activity against wild-type and resistant Descurainia sophia No.

g.ai/ha

DS (wild type)

RDS (P197L)

No.

g.ai/ha

DS (wild type)

RDS (P197L)

5-15

0.9375

++

++

5-20

0.9375

++

+++

5-18

0.9375

+

-

5-21

0.9375

+

-

5-19

0.9375

+

-

5-22

0.9375

++

-

FS

0.9375

+++

+

a

Abbreviations: DS (wild type) for Descurainia sophia (wild type); RDS (P197L) for the resistant

Descurainia sophia (Pro197Leu); FS = Flumetsulam b

Rating scale for the growth inhibition percentage in relation to the untreated control: +++, ≧70%; ++, ≧

50%; +, ≧20%; -,

Triazolopyrimidines as a New Herbicidal Lead for Combating Weed

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