Synthesis, Thermal Stability and Antifungal Evaluation of Two New Pyrrole Esters

To develop new chemicals that are stable at high temperatures with biological activity, a pyrrole intermediate was firstly synthesized using glucosamine hydrochloride as raw materials through cyclization and oxidation. Further, two novel pyrrole ester derivatives were prepared via Steglich esterification from pyrrole intermediate with vanillin and ethyl maltol, respectively. Nuclear magnetic resonance (1H‐NMR, 13C NMR), infrared spectroscopy (IR) and high resolution mass spectrometry (HRMS) were used to confirm the target compounds. Thermal behavior of the compounds was investigated by thermogravimetry (TG), differential scanning calorimeter (DSC) and the pyrolysis‐gas chromatography/mass spectrometry (Py‐GC/MS) methods. The plausible pyrolytic mechanism was proposed. Additionally, their biological activities against the pathogens Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Phytophthora nicotianae, and Rhizoctonia solani were assessed. These target compounds showed outstanding antifungal activities and the highest inhibitor rates of 62.50 % and 68.75 % against R. solani with EC50 values of 0.0296 and 0.0200 mg mL−1, respectively. SDHI protein sequence was molecularly docked to identify the binding mechanisms in the active pocket and examine the interactions between both the molecules and the SDHI protein.


Introduction
Pyrrole derivatives are adaptable essential intermediates that are often employed in foods, [1,2] dyes, [3] materials, [4][5][6][7][8] pharmaceuticals and physiologically active chemicals. [9,10]For example, pyrrole subunit has diverse applications in therapeutically active compounds including fungicides, antibiotics, antiinflammatory, [11] cholesterol reducing drugs, [12] antitumor agents [13] and many more.In addition, pyrroles are extremely important compounds in scent that may be used as food flavoring additives because they have distinct aromas or organoleptic qualities including bitter, roasted, peanut, buttery, and meaty. [14]Pyrroles are also highly valuable compounds in perfume and biological chemistry, and substantial attention has been paid to develop mild and efficient methods for synthesis pyrroles in recent past.[20] Nevertheless, often such processes suffer from (i) generation of stoichiometric waste, (ii) harsh reaction conditions, (iii) and increase of cost.In our continuous effort to the construction of N-heteroaryl compounds, here, we present a green, effective method for creating novel pyrrole esters by cyclizing, oxidizing, and esterifying using glucosamine hydrochloride as the starting material.The two synthesized novel pyrrole ester derivatives were confirmed by nuclear magnetic resonance ( 1 H-NMR, 13 C NMR), infrared spectroscopy (IR) and high resolution mass spectrometry (HRMS).
As is well known, the Maillard process or nonenzymatic browning reaction results in the formation of pyrrole chemicals possessed drawbacks including high fragrance volatility and low threshold, which could limit their application in the area of food flavorings. [21]Based on some literature reports, the synthesized new precursor could solve this problem by the production of volatile monomer scents during high-temperature pyrolysis operations, [22] including glycosides, [23][24][25] nitrogen heterocyclic esters, [26] Amadori analogues, [27] and others.Thermogravimetry, derivative thermogravimetry and differential scanning calorimeter (TG-DTG-DSC) were used to studying thermal behavior.Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) are useful tools for the synthesized compounds.
Moreover, it was reported that pyrrole derivatives isolated from medicinal fungi could protect nerves and fight neuroinflammation. [28][31][32] In particular, pyrrole esters have been found application in the bioactive molecules, such as novel medications and therapeutic targets. [33]However, less attention has been paid on study antifungal activity of pyrrole ester derivatives, especially for F. graminearum, F. oxysporum, F. moniliforme, P. nicotianae, and R. solani. [34,35]38][39][40][41] In view of the above issues, we designed two new pyrrole ester analogues using pyrrole-2-carboxylic acid and perfume alcohols.Then the thermal behavior of synthesized compounds was evaluated by TG-DTG-DSC and Py-GC/MS.Their plausible pyrolytic mechanism was proposed.Meanwhile, the antifungal activities of these target compounds were evaluated.The results showed that pyrrole esters possess good thermal stability with release of safe and volatile flavor products upon thermal pyrolysis, and excellent antifungal activities against F. graminearum, F. oxysporum, F. moniliforme, P. nicotianae, and R. solani.Moreover, the potential antifungal mechanism of the produced target compounds was investigated using molecular docking.The results can promote and accelerate the discovery of new and safe pesticides.

Synthesis and Analysis
As shown in Scheme 1, two pyrrole esters (4a and 4b) were synthesized through oxidative (condition a) and esterification (condition b) using compound 2 as raw material as shown in Scheme 1.The compound 2 was reacted with an excess of KMnO 4 (2.0 eq) to finish the corresponding pyrrole derivatives (compound 3) in 55 % yield.Compounds 4a and 4b then were obtained by esterification from compound 3 with vanillin and ethyl maltol (1.2 eq) in the presence of EDC (1.0 eq) and DMAP (0.02 eq) resulting in 60 and 62 % yields, respectively.Compared with the dehydrating agent of DCC, by-product from EDC was easier to remove after the reaction completed.Meanwhile, the vanillin and ethyl maltol in this esterification were mostly safe and showed excellent organoleptic properties, such as milky, fruit like and burnt-sweet smelling. [42,43]Finally, the two novel compounds were obtained using the optimized conditions in the presence of compound 3.The new compounds' structures have been confirmed via 1 H-NMR, 13 C NMR, IR and HRMS.

TG-DTG-DSC Analysis of Target Compounds
The TG-DTG curves of compounds 4a and 4b undergoing degradation at a heating rate of 10 °C min À 1 are shown in Figure 1a.It was discovered that the primary mass loss stage of 4a occurred between 233.6 and 400 °C.and mass loss (TG) dropped sharply by 83.77 %.The greatest decomposition rate was obtained at a peak temperature of 293.7 °C, with a mass loss of 37.10 % from the start.The mass of 4b was lost 90.54 % from 155.4 to 450.4 °C.In this stage, the largest rate of mass loss appeared at the peak temperature of 325.7 °C and mass decreased by 58.2 %.The final stage of 4b was high heat carbonization of the residual, with a mass loss of approximately 9.46 % of the original mass.
Figure 1b depicts the DSC curves of 4a and 4b.The installation measured the change in enthalpy of a samples as well as the peak temperatures of the DSC curves.It displayed temperatures of endothermic peak of 4a and 4b were 347.4 °C and 360.4 °C, respectively, and melting points of 4a and 4b were 85.8 °C and 89.8 °C, respectively.The endothermic breakdown was revealed by DSC analysis of both samples.The results depicted in the TG-DTG-DSC curves were equivalent.Table 1 summarizes data on decomposition temperatures.
As shown in Table 3, there was no pyrolysis product of 4b under the pyrolysis temperature of 300 °C.However, pyrolysis products of ethyl maltol (66.00 %), methyl 2-methyl-1-propyl-1H-pyrrole-3-carboxylate (28.92 %) and compound 3 (5.06%) were formed from 4b at 350 °C.While the pyrolysis temperature was at 400 °C, the relative content of them were 50.13 %, 42.80 % and 1.02 %, respectively.Among them, ethyl maltol and 2-methyl-1H-pyrrole were the main products.The relative content of compound 3 from 4a and 4b decreased with the increase of pyrolysis temperatures, indicating that it might be converted into small molecular pyrrole substance through decarboxylation.
Among the pyrolysis products of 4a and 4b, vanillin shows a strong milky aroma, and ethyl maltol possess malt and caramel aroma, and 2-methyl-1H-pyrrole has sweet aroma. [14,42]hese flavor compounds have been detected in various foods, including chocolate, biscuits, puddings, cakes, beverages, soya bean, and milk powder. [42,43]Furthermore, recent research revealed that these compounds also have appetite-enhancing effects. [46,47]he possible pyrolytic mechanism of 4a and 4b were presented in Figure 2. Interestingly, the main pyrolytic products of 4a and 4b were raw materials which were utilized to  synthesize them.Originally, compound 3 was observed when the ester bond O=C-O of target compounds were broken, meanwhile vanillin and ethyl maltol from 4a and 4b were released, respectively.Depropyl reaction at 1-position and decarbonylative reaction of compound 3 happened under pyrolytic conditions, culminating in the synthesis of 2-methyl-1H-pyrrole. [47] These findings corroborated our original concepts for constructing the target compounds' synthetic routes.It was discovered that pyrrole ester derivatives have good thermal stability and have the effect of taste slow-release at high temperatures.

Antifungal Properties of Target Compounds
The antifungal properties of raw materials (compound 3, Ethyl maltol and Vanllin) and target compounds (4a and 4b) were evaluated using the mycelial growth rate technique against F. graminearum, F. oxysporum, F. moniliforme, P. nicotianae, and R. solani at the concentration of 0.2 mg/mL.For comparison, control and normal triadimefon were also evaluated under comparable settings.These fungi are the well-known causative agents of wheat, maize, and tobacco early blight diseases.They infiltrate diverse plant tissues, particularly stems, leaves, and fruits, and then extract nutrition from host cells, resulting in severe crop defoliation and yield decline. [48] As a result, research into antifungal agents against these diseases is critical.The fungistatic activities of samples against various diseases were studied and compared to triazolone solutions, and the findings are presented in Figure 3.The samples were shown to have variable degrees of antifungal activity against five fungal infections investigated.The antifungal efficiency of target compounds (4a, 4b) and raw materials against the five pathogens calculated by the inhibitory index formula (Section 3.7) was shown as follows.
The inhibitory indexes of the 4a against the five fungi were 23.53, 56.25, 31.25,55.00, and 62.50 %, respectively.As for 4b, they were 29.41, 52.50, 61.25, 60.00 and 68.75 %, respectively.However, the antifungal rate of the triazolone against these five fungi was not ideal, they were 5.88, 25.00, 18.75, 25.00, and 6.25 %.While the inhibitory indexes of the raw materials (compound 3, Ethyl maltol and Vanllin) against the five fungi were less than target compounds.It can be assumed that the newly synthesized target molecules showed higher antifungal activity compared to the raw materials.According to the literature report inhibitory indexes of Boscalid against R. solani was 92.00 % at the concentration of 0.05 mg/mL. [49]Moreover, the inhibitory indexes of 4a and 4b against R. solani were less than Boscalid in spite of ten times than that of triazolone.It indicated that the target compounds (4a and 4b) synthesized exhibited potent antifungal properties against a wide range of fungus infections.Further, the target compounds could be used to control plant diseases caused by R. solani.Then we performed the following tests to estimate the EC 50 values of 4a and 4b.

EC 50 Value of 4a and 4b
Compounds 4a and 4b with improved anti-R.solani activity were developed at various doses.Compounds 4a and 4b are effective antifungal agents against R. solani at concentrations of 0.01, 0.02, 0.05, 0.1, and 0.2 mg mL À 1 , as shown in Figure 4.When the concentration was reduced, the fungicidal activity against R. solani was reduced, but it was still significantly higher than that of blank control.
Using the SPSS 16.0 software, the half-effective inhibitory concentration (EC 50 ) of each chemical was computed in accordance with the observed inhibitory rates.A summary of the findings can be seen in Table 4, which summarized the results.The antifungal action of 4b was minimal against EC 50 = 0.0200 mg ml À 1 considering a y = 0.1719x + 0.7918 activity against the regression equation.As for 4a, EC 50 = 0.0296 mg ml À 1 considering a y = 0.1719x + 0.7918 activity against the regression equation.It showed that 4b had the best antifungal effectiveness.The next stage of the molecular docking test was completed in order to investigate the antifungal mechanism of compound 4b.Previous literature has reported the use of molecular docking between nitrogen-containing heterocyclic compounds and SDH proteins to preliminarily explore their possible antifungal mechanisms. [50]As shown in Figure 5, the binding energy between small molecules 3, 4b and ethyl maltol, as well as the reference Boscalid and the complex protein was À 5.8 kcal/mol, À 6.8 kcal/mol, À 5.5 kcal/mol, and À 7.8 kcal/mol, respectively.Generally, if the binding energy between the ligand and the target protein is less than À 5, the ligand and the receptor protein can bind stably.In the docking diagram, green dashed lines represent hydrogen bonds, light green dashed lines represent hydrocarbon bonds, and pink, purple, and brown dashed lines represent hydrophobic forces.
In Figure 5a, the ligand small molecule ethyl maltol can bind to the 207 ASN amino acid residue, 111 ASN amino acid residue, 116 ASN amino acid residue, and 247 ARG amino acid residue of the receptor complex protein through four hydrogen bonds of 2.73 Å, 5.44 Å, 2.60 Å, and 2.68 Å, respectively; Binding to the 115 ILE amino acid residue of the receptor complex protein through a hydrophobic force of 4.13 Å.
The ligand small molecule 4b can bind to the TRP amino acid residue 54 of the receptor complex protein through two hydrophobic forces of 4.86 Å and 3.50 Å, respectively as shown in Figure 5b.By binding to the 142 VAL amino acid residue of the receptor complex protein through two hydrophobic forces of 4.20 Å and 4.56 Å, respectively; By binding to the 63 PRO amino acid residues and 206 TRP amino acid residues of the receptor complex protein through two hydrophobic forces of 4.71 Å and 5.82 Å, respectively; Bind to TRP amino acid residues 205 and ILE amino acid residues 251 of the receptor complex protein through two hydrophobic forces of 4.98 Å and 4.56 Å, respectively; By binding to the 141 TYR amino acid residue of the receptor complex protein through two hydrophobic forces of 4.58 Å and 5.43 Å, respectively; By binding to the 253 ASN amino acid residues and 251 ILE amino acid residues of the receptor complex protein through two hydrogen bonds of 2.58 Å and a hydrophobic force of 4.56 Å, respectively.
As shown in Figure 5c, from the three-dimensional diagram, it can be seen that ligand small molecule 3 can bind to the 253 ASN amino acid residue and 139 HIS amino acid residue of the receptor complex protein through two hydrogen bonds of 2.09 Å and 2.75 Å, respectively; By binding to the 141 TYR amino acid residue of the receptor complex protein through two hydrophobic forces of 4.34 Å and 3.54 Å, respectively; Bind to the 202 PRO amino acid residues and 205 TRP amino acid residues of the receptor complex protein through two hydrophobic forces of 4.30 Å and 4.89 Å, respectively; By binding to the 142 VAL amino acid residues and 206 TRP amino acid residues of the receptor complex protein through two hydrophobic forces of 4.63 Å and 5.85 Å, respectively.
In Figure 5d, the ligand small molecule Boscalid can bind to the 142 VAL amino acid residues and 54 TRP amino acid residues of the receptor complex protein through two hydrophobic forces of 4.64 Å and 6.46 Å, respectively; By binding to the 202 PRO amino acid residue of the receptor complex protein through two hydrophobic forces of 4.38 Å and 4.18 Å, respectively; Bind to the 139 HIS amino acid residues and 251 ILE amino acid residues of the receptor complex protein through two hydrophobic forces of 5.14 Å and 4.87 Å, respectively; By binding to the 141 TYR amino acid residue of the receptor complex protein through four hydrophobic forces of 4.18 Å, 5.14 Å, 5.4 Å 1, and 4.82 Å, respectively.It is precisely due to the presence of the above forces that ligand small molecules 3, 4b, and ethyl maltol can stably bind to the receptor complex protein.

General Information
In this study, all reagents and solvents were commercial grade purchased from Tianjin Kermel Chemical Reagent Co., Ltd (China).Additionally, BRUKER/ AVANCE III 400 MHz, the 1 H-NMR and 13 C NMR data of new compounds were collected (400 MHz for 1 H-NMR spectroscopy and 100 MHz for 13 C NMR spectroscopy, Bruker  BioSpin GmbH), and the samples were dissolved in CDCl 3 with tetramethylsilane (TMS) as the internal reference standard.Functional groups of the target compounds were detected via Fourier transform infrared spectrophotometer (Nicolet iS50, Thermo Nicolet Co, Wal-tham, MA, USA), and the range was from 4000 to 400 cm À 1 with a resolution of 4 cm À 1 .High resolution mass spectra (HRMS) were recorded on a high-resolution mass spectrometer (Thermo Q-Exactive, USA).TLC was used to monitor the development of the reactions on glass slides precoated with silica gel (GF254, Merck, China).For compounds purification, a silica gel (0.03-0.06 mm, Qingdao Ocean Chemistry Factory, China) was used in column chromatography.

Synthesis of N-propyl Substituted Pyrrole Aldehydes (Compound 2)
Pyrrole intermediate (compound 2) was synthesized by 3 steps using glucosamine as raw material through cyclization, referring to previous report. [51]

Synthesis of 4-(Methoxycarbonyl)-5-Methyl-1-Propyl-1H-Pyrrole-2-Carboxylic Acid (Compound 3)
Compound 2, KMnO 4 (2.0 eq), acetone/water (v: v = 1 : 1), and electromagnet were introduced into a round-bottomed flask and agitated for two hours at 0 °C.TLC (petroleum ether/ethyl acetate: 2/1) was used to confirm that the reaction was complete.Acetone was then extracted using a pressure, and the aqueous phase was then acidated with aqueous HCl (4 M) to a pH of 2, then liquid 10 % NaHSO 3 was added (3 mL).Ethyl alcohol was used to extract the combination, and the content of organic extract were then washed with brine and water, dried over MgSO 4 , and condensed.By recrystallization, the anticipated compound 3 was produced. [47]

Synthesis of 4-(Methoxycarbonyl)-5-Methyl-1-Propyl-1H-Pyrrole Ester Derivatives (4a and 4b)
EDC (1.0 mmol) and compound 3 (1.0 mmol) were combined in dry DCM (10 mL), which was then agitated at room temperature for 2 hours before alcohols (1.2 mmol) and DMAP (0.02 mmol) were added.The mixture was then swirled for 10 hours.TLC (petroleum ether/ ethyl acetate: 5/ 1) was used to monitor the reaction's completion.After the mixture had evaporated, ethyl acetate (410 mL) and water (410 mL) were added, and the organic phase was isolated.Anhydrous Na 2 SO 4 was used to dry the mixture, and then condensed.In order to create pyrrole esters (4a and 4b), the crude residue underwent column chromatography purification over silica gel (100 mesh).

TG Analysis
TG-DTG and DSC curves of the target compounds were detected by a simultaneous thermal analyzer (STA 449 F3, Netzsch, Germany).Every sample was preserved at 5 mg, and spectrally pure Al 2 O 3 served as the standard.Each testing was conducted in an environment of air with a flow rate of 60 mL min À 1 and heated from 30

°C
to 450 °C with rate of 10 °C min À 1 .

Pyrolysis Analysis
The experiment was carried out in an air atmosphere.Pyrolysis was measured using Py-GC/MS, and 1 mg of each sample was placed in a 25 mm quartz tube, which broke for 10 seconds at the design temperature.The pyrolysis temperatures are set at 300, 350, and 400 °C, respectively.The injection port temperature of the capillary column DB-5MS is 300 °C, the starting oven temperature is 50 °C, and then heated to 80 °C.Finally, it is heated to 280 °C at a rate of 5 °C per minute, and helium is employed as a carrier gas at a rate of 1 mL per minute.The mass spectrometer was used to study the separated components, and the mass spectrum was acquired from 30 to 500 m/z, and the pyrolysis products were searched from the mass library linked to the GC/MS equipment (NIST 2017).

In Vitro Antifungal Assay
The gel well diffusion technique was used to find the target compounds' antifungal properties.For this investigation, five fungi F. graminearum, F. oxysporum, F. moniliforme, P. nicotianae, and R. solani were utilized as indicator strains.The medium was a mixture of filtered potato juice boiled, agar and glucose.The antifungal activities of the target compounds were performed based on the method of literature. [35]The materials were treated in a water solution that contains 20 % DMSO. [52]To reach the final concentration of 200 μg/mL, each solution was mixed to sterilized dextrose agar from potatoes.The fungi's mycelia were placed on the test plate once the mixture had cooled, where they were cultured for 3-7 days at 25 °C.When the fugal mycelia reached the margins of the plate containing (without the additional samples), inhibitions were calculated according to formula 1.The inhibitory index was determined by averaging the results of three separate studies.
Where Da is indeed the diameter of a test frame's growth zone and Db is the length of the control plate's growth zone.Each experiment was repeated three times, and the average value was calculated.

Molecular Docking
Molecular docking is used to verify the binding activity between active ingredients and key targets.AutoDock Vina (Vina, version 1.1.2) is a program that runs using a semi flexible docking method, with a docking accuracy of up to 78 %, and was used as the molecular docking program for this study.Download the structures of small molecules 3, 4b and ethyl wheat from the Pubchem database for docking.Obtain the SDHI protein sequence of Rhizotonia solani from the NCBI database, and use the SWISSMO-DEL server to model the SDHI protein sequence.The template protein is the SDHB protein with ID A0 A0 K6FYZ3 (Rhizotonia solani species).Download the structures of compounds FAD and Boscalid (control) from the Pubchem database.Due to the fact that SDHI proteins often exist in the form of complexes, we docked SDHI proteins (modeling proteins) with FAD small molecules and SDHB proteins (ferritin) to obtain their three complexes.The complex protein was designated as the receptor, and 3,4b and ethyl wheat, as well as the control substance Boscalid, were designated as ligands.

Conclusions
In conclusions, two novel pyrrole ester derivatives (4a and 4b) were produced and confirmed by 1 H-NMR, 13 C NMR, IR and HRMS.Then thermal behavior, pyrolytic mechanism and antifungal efficacy were evaluated.The result revealed that mass decrease of 4a was achieved 83.77 % from 233.6 to 400 °C and the peak of endothermic temperature was 347.4 °C.For 4b, between 155.4 and 450.4 °C, there was the primary mass loss stage, and overall mass decrease was 90.54 %.The endothermic peak temperature of 4b was 360.4 °C.Py-GC/MS analysis results clearly showed that the pyrolysis products formed from the broke of bond O=C-O of the target compounds and the raw materials such as vanillin, ethyl maltol and pyrrole derivatives which were all aroma ingredients and safely used in food industry.The results confirmed our early expectations that pyrrole ester derivatives possessed characteristics of thermal stability and release with safe aroma.Biological test results showed that the target compounds (4a and 4b) exhibited more excellent antifungal activity than their raw materials against five fungus.The most effective compounds against R. solani were found with inhibitor rates of 62.50 and 68.75 % with EC 50 values 0.0296 and 0.0200, respectively.To identify the binding mechanisms in the active site residues and interactions between the components and the SDHI protein, molecular dynamics simulations of 4b and raw materials with SDHI were carried out.Comparatively to its raw materials (compound 3 and ethyl maltol), the results demonstrated that compound 4b might improve protein inactivation.This study provides the pyrrole esters antifungal inhibitors with high-temperature stable and pyrolysis products safe, which were considered as promising candidates for further study.

Figure 1 .
Figure 1.TG and DTG curves (a) and DSC curves (b) of target compound 4a and 4b.

Figure 3 .
Figure 3. Antifungal activity of samples against five fungi.

Figure 4 .
Figure 4. Antifungal properties of compounds 4a and 4b at various doses.

Table 1 .
Thermal analysis data of target compounds 4a and 4b.

Table 4 .
Thermal analysis data of target compounds 4a and 4b.