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Design, Synthesis and Potency of Pyruvate Dehydrogenase Complex E1 Inhibitors against Cyanobacteria Yuan Zhou, Jiangtao Feng, Hongwu He, Leifeng Hou, wen jiang, Dan Xie, Lingling Feng, Meng Cai, and Hao Peng Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00636 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Biochemistry

Design, Synthesis and Potency of Pyruvate Dehydrogenase Complex E1 Inhibitors against Cyanobacteria Yuan Zhou1, Jiangtao Feng1, Hongwu He*, Leifeng Hou, Wen Jiang, Dan Xie, Lingling Feng, Meng Cai, Hao Peng*

College of Chemistry, Central China Normal University; Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, 152 Luoyu Road, Wuhan, 430079, China.

*

Corresponding author: (Tel: +86-27-67867958; Fax: +86-27-67867960; E-mail:

[email protected]; [email protected])

1

These authors equally contributed to this work.

1 ACS Paragon Plus Environment

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ABSTRACT: Safe and effective algaecides are needed to control agriculturally and environmentally significant algae species. Four series (6, 10, 17, and 21) of 29 novel 4-aminopyrimidine derivatives were rationally designed and synthesized. A part of 10, 17, and 21 displayed potent inhibition against Escherichia coli pyruvate dehydrogenase complex E1 (E. coli PDHc-E1) (IC50 = 2.12-18.06 µM) and good inhibition against Synechocystis sp. PCC 6803 (EC50 = 0.7-7.1 µM) and Microcystis sp. FACH 905 (EC50 = 3.7-7.6 µM). The algaecidal activity of these compounds positively correlated with their inhibnitio against E. coli PDHc-E1. In particular, 21l and 10b exhibited potent algaecidal activity against PCC 6803 (EC50 = 0.7 and 0.8 µM, respectively), which were 2-fold increase on the potency if compared to copper sulfate (EC50 = 1.8 µM), also showed the best inhibition against cyanobacteria PDHc-E1 (IC50 = 5.10 and 6.06 µM, respectively). 17h and 21e with the best inhibition against E. coli PDHc-E1 were studied by molecular docking, site-directed mutagenesis and enzymatic assays. These results revealed that the improved inhibition of novel inhibitors compared with the lead compound I was due to forming new hydrogen bond with Leu264 at the active site of E. coli PDHc-E1. The results proved a high potential to obtain effective algaecides by rational design of PDHc-E1 inhibitors.


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Biochemistry

Cyanobacterial harmful algal blooms (Cyano-HABs) are one of the serious consequences of eutrophication in many parts of the world.1-3 Such blooms pose a serious water pollution problem that impacts human health, living resources, and water safety.4,5 Various approaches have been developed to deal with HABs, which can be classified as mechanical, physical, chemical, and biological controls.6 Chemical control is regarded as one of the most effective methods against Cyano-HABs. Chemical control involves use of herbicides, copper-based compounds, and chemical oxidants.7 At present, copper sulfate, copper oxychloride, and copper citrate are the most commonly used algaecides. It is noteworthy that the application of these compounds containing copper can lead to the accumulation of cupric ion in water, which would result in ecological risks.8 In addition, several herbicides, such as diquat, paraquat, atrazine, and simazine have been used as effective algaecides.9,10 However, these herbicides were non-specific and could increase cyanotoxins in the water.11 Moreover, some chemical oxidants, such as hydrogen peroxide, chlorine dioxide, calcium peroxide, and potassium permanganate are also used as potential algaecides due to their fast effect and good sustainability.12,13 However the effect of some oxidants as specific algaecides need to be further evaluated.12,14 Although these chemicals could act as effective algaecides, none of them was specifically designed targeting the enzymes of cyanobacteria or developed as an exclusive algaecide. Thus, it is an interesting and valuable research topic to design specially effective and safe algaecide. Recently, the computer aided virtual screening


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for bioactive pharmacophores has been proved to be successful for some protein targets.15-17 Therefore, the computer aided design of new inhibitors toward critical enzyme of bacteria or cyanobacteria may provide useful solution for the development of potential algaecides. The pyruvate dehydrogenase complex (PDHc) in microorganisms plays a vital role in cellular metabolism and catalyzes the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A (CoA) to acetyl-CoA.18-20 The overall reaction of oxidative decarboxylation was simply shown in Figure 1. PDHc contains three

enzymatic

components

including

pyruvate

dehydrogenase

(E1),

dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and a number of cofactors.21,22 The pyruvate dehydrogenase complex E1 component (PDHc-E1) catalyzes the first step of the multistep processes, using thiamine diphosphate (ThDP) and Mg2+ as cofactors.23 Particularly, this PDHc-E1 catalyzed process is a rate limiting step among multistep processes. PDHc-E1 is, therefore, a viable target to discover novel algaecides.

Figure 1. Oxidative decarboxylation catalyzed by PDHc. To the best of our knowledge, the crystal structure of PDHc-E1 from cyanobacteria has not been reported. At the moment it is difficult to perform the structure-based rational design of novel inhibitors against PDHc-E1 from cyanobacteria directly. However, the crystal structure of E. coli PDHc-E1 has been


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Biochemistry

determined.24 On that basis, we built an optimal binding model for inhibitors into active site of PDHc-E1 from E. coli using molecular simulation.25 In recent years, a systematic study for the design and synthesis of inhibitors against E. coli PDHc-E1 was carried out in our laboratory. A series of 2-methylpyrimidine-4-ylamine derivatives as ThDP analogs, such as I, II, and III (Figure 2) had been chemically synthesized and demonstrated to be effective inhibitors against E.coli PDHc-E1.26-32 Some of the compounds, by the modification of I, exhibited moderate to good inhibitory activity against both E. coli PDHc-E1 and cyanobacteria.30,31 The results showed that the inhibitory potency of some compounds against cyanobacteria was related to their inhibition against E.coli PDHc-E1. On the other hand, the cyanobacteria, which is similar to bacteria, belong to prokaryotes. They all have cytoplasm, ribosome and nucleoid. We thought that it is possible to obtain an effective algaecide by designing potent PDHc-E1 inhibitors with the binding model of active site of E. coli PDHc-E1. Therefore, we tried to design more potent PDHc-E1 inhibitors by the modification of I with the aid of E. coli PDHc-E1 structure-based molecular docking methods. Our previous studies have shown that the 2-methylpyrimidine-4-ylamine and 1,2,3-triazole ring is a pharmacophore acting on E. coli PDHc-E1. Therefore, the moieties of 2-methylpyrimidine-4-ylamine and 1,2,3-triazole ring were kept in structure I, and further optimization was focused on the modification of substituted phenoxy moiety. The analysis of molecular docking indicated no interaction between oxygen atom of phenoxy moiety and residues in the


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active site of PDHc-E1. It is expected that the inhibition would be enhanced by introducing a heterocyclic ring to replace phenoxy moiety in structure I.

Figure 2. Structures of ThDP and known PDHc-E1 inhibitors. Therefore, the phenoxy moiety in I was replaced with tetrahydrophthalimide moiety, which is a structural unit of fungicide captan (Figure 3), to give the new compound 6. As a comparison, compound 10a was designed by introducing a phthalimide moiety into I to replace the phenoxy moiety. To explore the effect of different heterocycles on E. coli PDHc-E1 inhibition, compound 17 was designed by replacing the phthalimide moiety in 10a with the benzotriazinone moiety. Furthermore, in order to improve the inhibition against both E. coli PDHc-E1 and cyanobacteria, a better hydrophobic group quinazolinone moiety (LogP: 0.98) was introduced by replacing the benzotriazinone moiety (LogP: 0.84) of 17 to give the new compound 21. Meanwhile, the study of structure-activity relationship (SAR) of I indicated that both inhibitory activity against E. coli PDHc-E1 and antifungal activity could be further increased by introduction of iodine at the 5-position of 1,2,3-triazole.26 Therefore, iodine atom was also introduced in a series of compounds 6 ACS Paragon Plus Environment

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Biochemistry

10, 17, and 21.

Figure 3. Design of title compounds. Herein, four series (6, 10, 17, and 21) which included 29 4-aminopyrimidine derivatives were rationally designed and synthesized. The interaction between inhibitors and E. coli PDHc-E1 was studied by molecular docking and site-directed mutagenesis. The inhibitory activities against E. coli PDHc-E1 and two common algae species (Synechocystis sp. PCC 6803 and Microcystis sp. FACH 905) were tested. Some representative compounds were further examined their inhibitory activity against cyanobacteria PDHc-E1. To find safe inhibitors for mammals, the enzyme-selectivity of representative compounds between porcine and E. coli PDHc-E1 was also examined. MATERIALS AND EXPERIMENTAL DETAILS Chemicals Characterization and Reagents. Melting points (m.p.) were measured on an electrothermal melting point apparatus and were uncorrected. 1H NMR and 19F NMR spectra were recorded on a Bruker spectrometer at 400 and 376 MHz, respectively, with deuterated dimethyl sulfoxide (DMSO-d6) as the solvent and TMS as the internal standard. Chemical shifts are reported in δ parts per million


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(ppm). High-resolution electron impact mass spectra (HR-EIMS) were recorded under electron impact (70 eV) conditions using a MicroMass GCT CA 055 spectrometer. Elemental analysis (EA) was measured on a Vario ELIII CHNSO elemental analyzer. Crystal structure was recorded by Bruker AEPEX DUO CCD diffractometer. Unless otherwise noted, reagents were purchased from commercial suppliers and used without further purification. Column chromatography purification was carried out using silica gel. ThDP, pyruvate, and porcine PDHc-E1 were purchased from Sigma-Aldrich. Synthesis Procedures General

procedure

for

preparation

of

5-azidomethyl-2-methyl

pyrimidine-4-ylamine (1). Compound 1 was prepared according to the procedure previously reported.33 To a solution of Vitamin B1 (thiamine chloride) (10.3 g, 30.6 mmol) and sodium azide (4.9 g, 75.4 mmol) in water (100 mL) was added sodium sulfite (0.38 g, 3.0 mmol) and the mixture was stirred at 65 oC for 5 h. Citric acid (21.0 g, 100 mmol, to pH ≈ 4) was added and the aqueous solution was washed with dichloromethane. Potassium carbonate (to pH ≈ 8) was added, upon which some precipitation of the product occurred. The suspension was filtered and the filtrate was extracted with ethyl acetate and the combined organic layers were washed with brine, dried with MgSO4 and evaporated under reduced pressure. The solid residue was pooled with the precipitate and recrystallized from ethyl acetate-hexane to give the azide (1) as fine needles in 60%


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Biochemistry

yield. General procedure for preparation of N-iodomorpholine (3). Compound 3 was prepared according to the procedure previously reported.34 To a stirred solution of iodine (12.7 g, 0.05 mol) in 200 mL of methanol was added dropwise morpholine 2 (4.35 g, 0.05 mol). The reaction mixture was stirred at 20-25 o

C for 1 h. The mixture was then filtered and washed with methanol to obtain orange

crystals in 75-80% yield. General procedure for preparation of alkynes (5 and 8). Compound 5 was prepared according to the procedure previously reported.35 To a solution of tetrahydrophthalimide 4 (1.51 g, 10 mmol) in acetone (20 mL), potassium carbonate (2.76 g, 20 mmol) and propargyl bromide (1.43 g, 12 mmol) were added sequentially. The resulting mixture was refluxed for 8 h. After cooling to room temperature, water (50 mL) was added, which was extracted with EtOAc (100 mL) for 3 times. The organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated to give compounds (5). Under this condition, 8 was also prepared. General procedure for preparation of iodoalkynes (9). Compound 9 was prepared according to the procedure previously reported.34 To a solution of terminal alkyne 8 (2 mmol) in THF (10 mL) was added N-iodomorpholine (3) (1.36 g, 4 mmol), followed by CuI (0.04g, 0.2 mmol). The reaction mixture was stirred at 20-25 oC for 4-6 h. The reaction mixture was filtered.


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The filtrate was evaporated under reduced pressure to afford yellow solid 9, which were used directly without further purification. General procedure for synthesis of the 2-methylpyrimidine-4-ylamine derivatives 6 and 10. CuI

(0.04

g,

0.2

mmol)

was

added

to

a

stirred

solution

of

5-azidomethyl-2-methylpyrimidine-4-ylamine (1) (0.33 g, 2 mmol) and alkynes 5 (2 mmol) in THF (10 mL) followed by Et3N (0.24 g, 2.4 mmol). After overnight stirring at room temperature, the reaction mixture was poured into water (30 mL). The precipitate was collected by filtration and dried under atmospheric pressure. The crude product was further purified by recrystallization (methanol/dichloromethane) to give the title compound 6. Under this condition, 10 were also prepared. General procedure for preparation of isatoic anhydride (12). Compound 12 was prepared according to the procedure previously reported.36 A mixture of anthranilic acid 11 (50 mmol) and tetrahydrofuran (THF, 100 mL) was stirred at -10 oC for 30 min. Then, a solution of triphosgene (BTC, 50 mmol) in THF (30 mL) was added dropwise to the above mixture. After that, the mixture was stirred for 1 h at -10 oC, followed by 18-24 h at 20-25 oC. The solvent was removed under reduced pressure, and anhydrous ether (150 mL) was added to the obtained residue with vigorous stirring. The precipitate was collected by filtration, washed with anhydrous ether, and dried to give compound 12 in yields of 80%-90%. General procedure for preparation of anthranilamide (13).


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Biochemistry

Compound 13 was prepared according to the procedure previously reported.37 A suspension of isatoic anhydride 12 (40 mmol), ammonium carbonate (160 mmol), and 1,4-dioxane (150 mL) was heated at 60 oC. After stirring for 5-8 h, the reaction mixture was cooled to room temperature and evaporated under reduced pressure, and then water (200 mL) was added to the residue, which was extracted with EtOAc (300 mL ) for three times. The organic layer was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated to afford compound 13 in yields of 80-85%. General procedure for preparation of 1,2,3-benzotriazin-4-ones (14). Compound 14 was prepared according to the procedure previously reported.38 A solution of sodium nitrite (4.14 g, 60 mmol) in 0.5 N HCl (240 mL) was stirred at 0 o

C for 20 min. Anthranilamide 13 (30 mmol) dissolved in N,N-dimethylformamide

(DMF, 15 mL) was then added dropwise to the above solution for 40 min. After another 1 h of stirring at 0 oC, 30% aqueous ammonia was added slowly to adjust the pH to 10.0. The reaction mixture was allowed to stir for 15 min and then reacidified to pH 2.0. After stirring for 30 min, the precipitated product was filtered off with suction, washed with water (200 mL), and dried to afford compound 14 in yields of 75-83%. General procedure for preparation of alkynes (15) and iodoalkynes (16). Compounds 15 and 16 were prepared according to the procedures described for 5 and 9, respectively. General procedure for synthesis of the 2-methylpyrimidine-4-ylamine derivatives


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containing 1,2,3-benzotriazin-4-ones moiety 17. CuI

(0.04

g,

0.2

mmol)

was

added

to

a

stirred

solution

of

5-azidomethyl-2-methylpyrimidine-4-ylamine (1) (0.33 g, 2 mmol) and alkynes 15 or iodoalkynes 16 (2 mmol) in THF (10 mL) followed by Et3N (0.24 g, 2.4 mmol). After overnight stirring at room temperature, the reaction mixture was poured into water (30 mL). The precipitate was collected by filtration and dried under atmospheric pressure. The

crude

product

was

further

purified

by

recrystallization

(methanol/dichloromethane) to give the title compound 17. General procedure for preparation of 4(3H)-quinazolinones (18). Compound 18 was prepared according to the procedure previously reported.39 A mixture of anthranilic acid 11 (0.1 mol) and formamide (18 g, 0.4 mol) was heated at 130-135 oC. After stirring for 4 h, water (40 mL) was added to the mixture. The reaction mixture was cooled to 60 oC, water (20 mL) was added to the mixture. After stirring for 30 min, the precipitated product was filtered off with suction. The crude products were recrystallized with ethanol to give compound 18 in yields of 80-95%. General procedure for preparation of alkynes (19) and iodoalkynes (20). Compounds 19 and 20 were prepared according to the procedures described for 5 and 9, respectively. General procedure for synthesis of the 2-methylpyrimidine-4-ylamine derivatives containing quinazolinone moiety 21. CuI

(0.04

g,

0.2

mmol)

was

added

to

a

stirred

solution

of


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Biochemistry

5-azidomethyl-2-methylpyrimidine-4-ylamine 1 (0.33 g, 2 mmol) and 19 or 20 (2 mmol) in THF (10 mL) followed by Et3N (0.24 g, 2.4 mmol). After overnight stirring at room temperature, the reaction mixture was poured into water (30 mL). The precipitate was collected by filtration and dried under atmospheric pressure. The crude product was further purified by recrystallization (methanol/dichloromethane) to give title compounds 21. Crystallographic Study The crystal of the title compound 21f was cultured from the mixture of methanol and dichloromethane. X-ray single-crystal diffraction data for 21f were collected on a Bruker AEPEX DUO CCD diffractometer at 298(2) K using Mo Kα radiation (λ = 0.71073 Å) by π and ω scan mode. The program SAINT+ was used for integration of the diffraction profiles. The structure was solved by the direct method using the SHELXS program of the SHELXTL package and refined by full-matrix least-squares methods with SHELXL.40 All non-hydrogen atoms of 21f were refined with anisotropic thermal parameters. All hydrogen atoms were placed in geometrically idealized position and constrained to ride on their parent atoms. Enzymatic Inhibition Activity and Half Maximum Inhibition Concentrations (IC50) To evaluate the inhibition of title compounds against E. coli PDHc-E1, IC50 were determined. The cloning, expression, purification and activity of E. coli PDHc-E1 were carried out according to the reported method.25 Enzymatic activities were


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measured according to the published method.25 For IC50 determination, a standard reaction

mixture

containing

50

mM

K3PO4

(pH

6.4),

0.4

mM

2,6-dichlorophenolindophenol (DCIP), 50 µM sodium pyruvate as the substrate, 5 µg PDHc-E1 purified enzyme and 50 µM ThDP. Title compounds in concentrations ranging from 0 to 200 µM were incubated for 5 min with E. coli PDHc-E1 at 37 oC prior to addition of the substrate pyruvate. The IC50 values were determined by nonlinear least-squares fitting of the data using the Hill kinetic equations in the Growth/sigmoidal model from origin 7.0 software as described previously.41 Inhibitory Assays (in vivo) on Cyanobacteria Synechocystis sp. PCC 6803 and Microcystis sp. FACH 905 were cultured photoautotrophically in BG11 medium42 at 28 oC for 7 days at a 12:12 h of light/dark cycle at 50-55% relative humidity.43 The inhibition of title compounds against cyanobacteria growth was calculated according to the equation: I = {1-[∆, − ∆ ,]/∆, }× 100%, where, I is the relative inhibition (%); ∆, and ∆, are the average data of optical densities at 680 nm for the cyanobacteria in the blank control and treatment, respectively; and ∆ , is the average data optical density at 680 nm for the test compound in each assay. When cyanobacteria growth was significantly inhibited, the maximum half effective concentrations (EC50) were estimated with logistic fitting. The widely used algaecide copper sulfate was the positive control, while DMSO was the solvent control. Each test was measured in triplicate.


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Biochemistry

Inhibition against Porcine PDHc-E1 To test the inhibitory activity against porcine PDHc-E1, the relative inhibition (%) of title compounds were assayed at 100 µM. The total volume of 100 µL reaction mixture

contained

1.0

mM

MgCl2,

0.2

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

mM bromide

ThDP, (MTT),

0.5 6.5

mM mM

phenazine methosulfate (PMS), 50 mM K3PO4, pH 7.0, 5 µL enzyme and 100 µM compound. The reaction mixture was incubated for 3 min at 37 oC, then added sodium pyruvate (5 µL, 40 mM) to initiate reaction. The optical density at 566 nm (OD566) was recorded. The reaction mixture was then incubated for 5 min at 37 oC, and the OD566 was recorded. The relative inhibition was calculated according to the equation: I= [(∆, − ∆, )/∆, ] × 100%, where, I is the relative inhibition (%); ∆, is the average data of optical density at 566 nm in the blank control; and ∆, is the average data of optical density at 566 nm in the presence of compound. Each test was measured in triplicate. Molecular Docking The binding modes of selected compounds were investigated using an AutoDock 4.2 program.44 The three-dimensional (3D) structure of the PDHc-E1 was obtained from PDB database (PDB code: 1L8A). Hydrogens were added to the protein structure by pdb2pqr server.45 The structure of co-crystal ligand ThDP and compounds (Ic, 10a, 17h and 21e) were respectively prepared with SYBYL7.0 and Gasteiger charges were used for these inhibitors. The original ligand ThDP was first


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docked into the binding site. In the docking process, the Lamarckian genetic algorithm (LGA) was applied for the conformational search of the binding complex of ThDP with PDHc-E1. The grid size was set to be 60×60×60 and the used grid space was the default value of 0.375 Å, other parameters were set to default. Among a set of 100 conformational candidates of the docked complex structures, the top-ranked one was selected according to the interaction energy. The predicted binding mode of ThDP was compared with the conformation of ThDP extracted from the crystal structure. The same docking parameters were subsequently adopted to perform molecular docking of Ic, 10a, 17h and 21e, respectively. Site-Directed Mutagenesis of PDHc-E1 Site-directed mutagenesis of PDHc-E1 was accomplished by the introduction of specific base changes into a double-standard DNA plasmid according to reported method.41

DNA

encoding

of

the

wild-type

PDHc-E1

cloned

into

the

pMAL-C2x-PDHc-E1 was used as a template for mutagenesis. The standard PCR mixture contained 50-100 ng of template DNA and 100-200 ng of each mutagenizing primer. The methylated plasmid was digested with DpnI, and 4 µL of each reaction was used to transform the DH5α competent cells. All mutations were confirmed by DNA sequencing. Verified plasmids containing the desired mutations were transformed into the E.coli TB1 strain. The mutant PDHc-E1 proteins were purified by the same manner as the purification of wild-type PDHc-E1. RESULTS AND DISCUSSION


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Biochemistry

Synthesis A total of 29 new compounds in the series of 6, 10, 17, and 21 was designed and synthesized (Scheme 1, 2, and 3). The synthetic route is simple and convenient. In this synthetic route, 5-azidomethyl-2-methylpyrimidine-4-ylamine (1) that was prepared starting from thiamine hydrochloride (Vitamin B1) is a key intermediate in the synthesis of 6, 10, 17, and 21. The skeleton of all title compounds 6, 10, 17, and 21 contain 1,2,3-triazole ring. The 1,2,3-triazole ring in the skeleton of title compounds could be readily constructed by applying ‘click chemistry’. In the present work, 6 or 10 was synthesized via 1,3-dipolar cycloaddition reaction employing copper(I) iodide and triethyl amine from 1 by reacting with terminal alkynes 5, 8 or iodoalkynes 9, which was prepared by the reaction of propargyl bromide and tetrahydrophthalimide 4 or phthalimide 7 (Scheme 1).


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Scheme 1. Synthetic route for the title compounds 6 and 10. The title compound 17 was further synthesized by this Cu-catalyzed 1,3-dipolar cycloaddition reaction of 1 with 15 or 16, which was prepared by the reaction of 1,2,3-benzotriazin-4-ones 14 with propargyl bromide (Scheme 2). 14 was prepared from anthranilamides 13 through the combination of diazotization, nucleophilic addition, and cyclization in one pot. 13 was prepared starting from anthranilic acids 11, which was converted to the desired isatoic anhydride 12 via the annulation reaction with BTC. Then, 12 reacted with ammonium to give the anthranilamide 13.


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Biochemistry

Scheme 2. Synthetic route for the title compound 17. The title compound 21 was also synthesized by 1 reacting with 19 or 20 via 1,3-dipolar cycloaddition in the presence of CuI and Et3N (Scheme 3). 19 was prepared from the key intermediate 4(3H)-quinazolinone 18, which was prepared by the reaction of 11 with formamide.

Scheme 3. Synthetic route for the title compound 21. In this synthetic method, iodine atom was introduced in title compounds by using iodoalkynes 9, 16 or 20, which was prepared via 8, 15 or 19 reacting with N-iodomorpholine 3, respectively, under the catalysis of copper(I) iodine. The title compounds and CuI were both hard to dissolve in water and most organic solvent. It was difficult to separate them by using a column chromatographic method. However,


Biochemistry

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it was found that the mixed solvent (methanol/dichloromethane) could increase the solubility of the title compounds, but CuI could not dissolve in the mixed solvent. Therefore, the crude product was dissolved in the hot mixed solvent and immediately filtered to remove CuI. The filtrate was cooled and filtered to give the desired title compounds. The abovementioned synthetic method has the advantage of being simple, mild, easy, and high yield. Those compounds were characterized with 1H NMR, 19F NMR, HRMS spectra and confirmed by elementary analysis. The characterization data were listed in the Supporting information. The Crystal Structure of Compound 21f The crystal data of 21f were presented in Table 1, and Figure 4 gives a perspective view of 21f with the atomic labeling system.46 This result indicates that 21f has a “U” shape conformation, which is close to its binding pose.

Figure 4. Crystal structure of compound 21f by X-ray diffraction determination. 20 ACS Paragon Plus Environment

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Biochemistry

Table 1. Crystallographic data of compound 21f. Parameters chemical formula formula weight crystal system space group crystal color a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å) Z Dcalc (Mg⋅m-3) θ range (deg) hkl range F (0 0 0) no. collected refl. no. ind. Refl. (Rint) data/ restraints/ parameters Absorption coefficient (mm-1) R1; wR2 [I > 2σ(I)] R1; wR2 (all data) GOOF

Data C17H15BrN8O 427.26 monoclinic C2/c colorless 15.235(4) 16.300(4) 17.449(5) 90 108.188(4) 90 4116.5(19) 8 1.482 1.99-25.50 -18 ≤ h ≤ 18; -19 ≤ k ≤ 19; -21 ≤ l ≤ 21 1872 14767 3812 (0.1054) 3812 / 2 / 286 2.029 0.0745; 0.1828 0.1751; 0.2412 0.995

In vitro Inhibition against E. coli PDHc-E1 Compounds 6 and 10 were first synthesized and evaluated for their inhibition against E.coli PDHc-E1. The IC50 values, including Ia-b,26 were summarized in Table 2. It was very interesting to find that 10a (IC50 = 9.46 ± 0.52 µM) and 10b (IC50 = 5.96 ± 0.06 µM) with a phthalimide moiety showed promising inhibitory activity, which had 6-fold and 9-fold activity higher than that of Ia, respectively. However, 6 with a tetrahydrophthalimide moiety showed very weak inhibitory activity. The reason for this is that the phthalimide moiety of 10 could form π-π interaction with His142 21 ACS Paragon Plus Environment

Biochemistry

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according to the analysis of molecular docking (Figure 5B). This result indicated that phthalimide moiety is a very important pharmacophore. Meanwhile, 10b with iodine as R1 exhibited better enzyme inhibitory activity than that of corresponding compound 10a with H as R1. Table 2. Structures and IC50 values against E.coli PDHc-E1 of compounds I, 6, and 10.

Compd. R1 IC50a (µM) H 55.15±4.65 Ia I 19.56±0.00 Ib H NTb 6 H 9.46±0.52 10a I 5.96±0.06 10b a IC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against PDHc-E1 from E. coli in vitro. b NT: not tested. The primary inhibitory potency was less than 100% at 100 µM. For exploring the effect of different heterocycles on E.coli PDHc-E1 inhibition, 17 was designed and synthesized by replacing the phthalimide group with benzotriazinone moiety. The IC50 values of 17 were summarized in Table 3. The series 17 showed moderate to good inhibition with IC50 values in the range of 18.06-3.72 µM. Most of 17 with iodine as R1 showed better enzyme inhibition than that of corresponding compounds 17 with H as R1, such as 17b (R1 = H, R2 = 6-Cl, IC50 = 18.06 ± 0.91 µM) < 17h (R1 = I, R2 = 6-Cl, IC50 = 3.72 ± 0.20 µM), 17f (R1 = H, R2 = 8-CH3, IC50 = 11.25 ± 0.44 µM) < 17l (R1 = I, R2 = 8-CH3, IC50 = 5.19 ± 0.26


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Biochemistry

µM). Among these compounds, 17h showed the best inhibition (IC50 = 3.72 ± 0.20 µM). This result proved that the inhibitory activity against E. coli PDHc-E1 could be further increased by introducing iodine at the 5-position of 1,2,3-triazole. Table 3. Structures and IC50 values against E.coli PDHc-E1 of compounds I, 17 and 21.

Compd.

R1

R2

IC50a (µM)

Compd.

R1

R2

IC50a (µM)

H 4-Cl 26.44±1.68 H 7-NO2 3.44±0.20 Ic 21b H 7-Cl 6.88±0.38 H 7-Cl 6.87±0.17 17a 21c H 6-Cl 18.06±0.91 H 6-Cl 6.54±0.20 17b 21d H 5-Cl 7.05±0.08 H 5-Cl 2.12±0.05 17c 21e H 6-Br 6.33±0.43 H 6-Br 4.29±0.17 17d 21f H 5-F 7.18±0.31 H 5-F 19.97±0.59 17e 21g H 8-CH3 11.25±0.44 H 6,7-OCH3 23.92±0.75 17f 21h I 7-Cl 4.08±0.20 I 7-Cl 4.55±0.20 17g 21i I 6-Cl 3.72±0.20 I 6-Cl 10.36±0.30 17h 21j I 5-Cl 13.35±0.54 I 5-Cl 4.71±0.14 17i 21k I 6-Br 6.70±0.34 I 6-Br 8.21±0.26 17j 21l I 5-F 8.29±0.48 I 8-CH3 8.67±0.31 17k 21m I 8-CH3 5.19±0.26 I 6,7-OCH3 9.81±0.17 17l 21n H 8-CH3 6.69±0.10 21a a IC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against PDHc-E1 from E. coli in vitro. The improved inhibition of 17 from the lead structure I motivated further optimization by replacing the benzotriazinone moiety in structure 17 with a quinazolinone moiety. The inhibitory potency of 21 was slightly improved (Table 3), compared with 17, such as 21e (R1 = H, R2 = 5-Cl, IC50 = 2.12 ± 0.05 µM) > 17c (R1 = H, R2 = 5-Cl, IC50 = 7.05 ± 0.08 µM), 21d (R1 = H, R2 = 6-Cl, IC50 = 6.54 ± 0.20 23 ACS Paragon Plus Environment

Biochemistry

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µM) > 17b (R1 = H, R2 = 6-Cl, IC50 = 18.06 ± 0.91 µM). 21e (IC50 = 2.12 ± 0.05 µM) showed the best inhibition in series 21. It was noticed that compounds with electron-withdrawing groups on the benzene ring as R2 seem to be more effective than these compounds with electron-donating alkyl counterparts as R2.

In vivo Inhibition against Cyanobacteria In this test, copper sulfate was used as a positive control. Copper sulfate is a commonly used effective algaecide against cyanobacteria. However, it is also against non-target organisms, and harmful to ecosystems due to its heavy metal ions. Most of the compounds exhibited excellent activity against the cyanobacteria PCC 6803 and FACH 905 (Table 4). The inhibitory potency against cyanobacteria is related to the inhibition against E. coli PDHc-E1 (Tables 2, 3, and 4). Generally, compounds with higher E. coli PDHc-E1 inhibition displayed potent inhibition against cyanobacteria. For instance, 10, 17, 21a-f, and 21i-n had higher E. coli PDHc-E1 inhibition (IC50 = 2.12-18.06 µM) and also exhibited potent inhibitory potency against PCC 6803 (EC50 = 0.7-7.1 µM) and FACH 905 (EC50 = 3.7-7.6 µM). 21l and 10b, particularly, exhibited excellent algaecide activity against PCC 6803, which was 2-fold better than copper sulfate (EC50 = 1.8 µM). In comparison, I, 6, and 21g-h with lower inhibition (IC50 > 19 µM) against E. coli PDHc-E1 also displayed significantly lower algaecide activities (EC50 > 50 µM).


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Biochemistry

Table 4. Structures and in vivo EC50 values against cyanobacteria of compounds I, 6, 10, 17, and 21.

EC50a (µM) inhibitory Compd.

R1

R2

against cyanobacteria PCC6803

FACH905

EC50a (µM) inhibitory Compd.

R1

R2

against cyanobacteria PCC6803

FACH905

Ia

H

H

>50

>50

17l

I

8-CH3

5.6±0.8

6.5±0.5

Ib

I

H

>50

>50

21a

H

8-CH3

4.6±0.5

6.6±0.6

Ic

H

4-Cl

>50

>50

21b

H

7-NO2

5.0±0.1

7.4±0.4

>50

>50

21c

H

7-Cl

4.7±0.2

7.1±0.5

6 10a

H

2.6±0.1

4.4±0.2

21d

H

6-Cl

5.3±0.4

7.6±0.7

10b

I

0.8±0.1

4.1±0.1

21e

H

5-Cl

3.9±0.1

5.7±0.6

17a

H

7-Cl

5.5±0.8

5.7±0.3

21f

H

6-Br

4.4±0.3

5.7±0.4

17b

H

6-Cl

6.2±0.9

5.7±0.3

21g

H

5-F

>50

>50

17c

H

5-Cl

7.1±0.7

6.8±0.4

21h

H

6,7-OCH3

>50

>50

17d

H

6-Br

5.9±0.8

5.8±0.4

21i

I

7-Cl

2.5±0.2

4.1±0.1

17e

H

5-F

6.9±0.2

7.3±0.6

21j

I

6-Cl

2.8±0.1

4.4±0.2

17f

H

8-CH3

5.8±0.8

5.8±0.2

21k

I

5-Cl

2.5±0.1

4.0±0.1

17g

I

7-Cl

6.2±0.6

4.6±0.4

21l

I

6-Br

0.7±0.1

4.4±0.2

17h

I

6-Cl

4.8±0.4

3.7±0.3

21m

I

8-CH3

2.6±0.1

4.3±0.1

I

6,7-OCH3

2.8±0.1

4.4±0.1

1.8±0.1

1.5±0.1

17i

I

5-Cl

5.1±0.3

4.9±0.3

21n

17j

I

6-Br

5.5±0.6

6.6±0.5

CuSO4

17k

I

5-F

4.6±0.5

4.3±0.3

a

The inhibitory activity against synechocystis sp. PCC 6803 and Microcystis sp. FACH 905. 6 with a tetrahydrophthalimide moiety exhibited much weak inhibition against both E. coli PDHc-E1 and cyanobacteria (Tables 2 and 4). However, 10a with a phthalimide moiety exhibited prominent activities against both E. coli PDHc-E1 and cyanobacteria due to the π-π interaction between His142 and phthalimide moiety based on molecular docking (Figure 5B). When iodine as R1 was introduced into the


Biochemistry

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structure of 10a to give 10b, it was found that 10b showed more powerful inhibitory activity (EC50 = 0.8 µM) against PCC 6803 than that of 10a and control. It was further testified that inhibition against cyanobacteria could be remarkably enhanced by introducing iodine at the 5-position of 1,2,3-triazole of structure 21 (Table 4). All 21 with iodine as R1 showed higher inhibitory activity against cyanobacteria than that of these corresponding compounds with H as R1. When R2 kept same, 21n (IC50 = 9.81 ± 0.17 µM) and 21i (IC50 = 4.55 ± 0.20 µM) with iodine as R1 exhibited higher inhibition against E. coli PDHc-E1 than that of 21h (IC50 = 23.92 ± 0.75 µM) and 21c (IC50 = 6.87 ± 0.17 µM) with H as R1, respectively (Tables 3 and 4). Meanwhile, 21n (EC50 = 4.4-2.8 µM) and 21i (EC50 = 4.1-2.5 µM) with iodine as R1 also exhibited higher inhibitory activity against both PCC 6803 and FACH 905 than that 21h (EC50 > 50 µM) and 21c (EC50 = 7.1-4.7 µM) with H as R1, respectively. The results showed that it is possible to obtain an effective algaecide by designing a potent E.coli PDHc-E1 inhibitor. Enzyme-Selective Inhibition Although some reported analogs of ThDP exhibited highly inhibitory activity against E. coli PDHc-E1, they had poor enzyme-selective inhibition between mammals and microorganisms. For example, reported compound ThTTDP (Figure 2) exhibited potent inhibition against E. coli PDHc-E1 (Ki = 64 nM), however, it also displayed powerful binding to human PDHc-E1 (Ki = 74 nM).24,47 Therefore, it is important to find selective PDHc-E1 inhibitors safe to mammals. In this work,


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Biochemistry

ThTTDP was used as a positive control. 17f, 21a, and 21m with good inhibition against both E. coli PDHc-E1 (IC50 = 5.19-8.67 µM) and cyanobacteria were chosen to test their inhibition activity against porcine PDHc-E1. These compounds showed 100% inhibition against E. coli PDHc-E1 at 100 µM, but negligible inhibition (50 >50 6 2.6±0.1 10.9±0.72 10a 0.8±0.1 6.06±0.37 10b 7.1±0.7 20.8±0.90 17c 4.8±0.4 16.3±1.30 17h 5.1±0.3 19.4±1.00 17i 3.9±0.1 13.8±0.90 21e 4.4±0.3 15.4±0.70 21f 2.5±0.2 10.7±0.48 21i 2.8±0.1 11.4±0.81 21j 2.5±0.1 10.3±0.6 21k 0.7±0.1 5.10±0.05 21l a IC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against PDHc-E1 from cyanobacteria in vitro. Compd.

In conclusion, four series of 29 novel compounds containing different heterocycle as potential inhibitor targeting E. coli PDHc-E1 were designed and synthesized. Both algaecide activity and inhibitory potency against E. coli PDHc-E1 could be increased greatly by introducing phthalimide, benzotriazinone or quinazolinone moiety to substitute phenoxy moiety in lead compound, respectively. Algaecide activity was also increased by introduction of iodine at 5-position of 1,2,3-triazole in 10, 17, and 21. The algaecide activity of title compounds positively correlated with their inhibition against E. coli PDHc-E1. I, 6, and 21g-h with lower inhibition against E. coli PDHc-E1 showed almost no activity against cyanobacteria. 31 ACS Paragon Plus Environment

Biochemistry

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10, 17, 21a-f, and 21i-n displayed good inhibition against E. coli PDHc-E1 also showed potent inhibition against cyanobacteria. The study of enzyme-selective inhibition for 17f, 21a, and 21m showed that these title compounds showed 100% inhibition against E. coli PDHc-E1, but negligible inhibition (

Design, Synthesis, and Potency of Pyruvate ... - ACS Publications

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