Synthesis, Characterization, and Antifungal Activity of Phenylpyrrole-Substituted Tetramic Acids Bearing Carbonates

For the aim of discovering new fungicide, a series of phenylpyrrole-substituted tetramic acid derivatives bearing carbonates 6a–q were designed and synthesized via 4-(2,4-dioxopyrrolidin-3-ylidene)-4-(phenylamino)butanoic acids 4a–k and the cyclized products 1′,3,4,5′-tetrahydro-[2,3′-bipyrrolylidene]-2′,4′,5(1H)-triones 5a–k. The compounds were characterized using IR, 1H- and 13C-NMR spectroscopy, mass spectrometry (EI-MS), and elemental analysis. The structure of 6b was confirmed by X-ray diffraction crystallography. The title compounds 6a–q were bioassayed in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea and Rhizoctonia solani at a concentration of 100 μg/mL, respectively. Most compounds displayed good inhibitory activity.


Introduction
Tetramic acid derivatives, which represent an important class of nitrogen-containing heterocycles, have received considerable attention due to their significant biological activities [1], such as antioxidant [2], herbicidal [3,4], phytocytotoxic [5][6][7], anti-HIV [8,9], and antitumor properties [10,11]. Among the abundant bioactivity research of tetramic acid derivatives, 3-heterocycle substituted tetramic acids were proved to be more interesting. Some literatures reported that these compounds showed a wide variety of bioactivity. Fischerellin A, the most active allelochemical compound of Fischerella muscicola, exhibited a MIC (Minimal Inhibition Concentration) of 14 nM against Synechococcus PPC 6911 and had interesting herbicidal activity [12]. Benzothiadiazine-substituted tetramic acids are potent inhibitors of the hepatitis C virus RNA polymerase [13]. Vermelhotin was obtained from an unidentified fungus CRI247-01, which was found to display cytotoxic activity and antiplasmodial activity with the IC 50 values of 1-10 µM [14]. Another tetramic acid derivative produced by a plant type-III polyketide synthase showed moderate antiproliferative activity against murine leukemia P388 cells [15]. However, almost no literatures have reported the antifungal activity of 3-heterocycle substituded tetramic acids.
It was reported [22,23] that 3-(aryl or heterocyclic) tetramic acid derivatives were usually generated via Dieckmann cyclization of N-(aryl or heterocyclic-acetyl) amino acid esters (Scheme 2). In this paper, pyrroles were formed in the esterification with chloroformates after generation of the pyrrolidine-2-ones, this method was efficient and convenient, which might be useful to synthesize other 3-heterocyclic tetramic acid derivatives.  In the crystal structure of compound 6b (Figure 1), the ethyl group connected to the atom O6 appears in a disordered state. In pyrrole system, the bonds C1-N1 and C4-N1 are significantly shorter than the typical single C-N bond and longer than the typical C=N bond, which indicates a significant electron delocalization exists in the pyrrole system. The three rings pyrrole, pyrroline, and benzene are not coplanar, their dihedral angles between pyrrole and pyrrolidone, pyrrole and benzene are 36.379(89)˝and 48.522(93)˝, respectively. There are three intramolecular hydrogen bonds C9-H9¨¨¨O1, C19-H19A¨¨¨O5 and C16-H16B¨¨¨O5 (Figure 1), which ulteriorly stabilize the molecule. Moreover, other two intermolecular hydrogen bonds N2-H2A¨¨¨O7 and C16-H16A¨¨¨O2 between adjacent molecules form a two dimensional chain structure ( Figure 2). As shown in Figure 3, C-H¨¨¨π interaction exists between benzene and pyrrole. The distance between the hydrogen of benzene and the centroid of pyrrole is 3.004 Å. The C-H¨¨¨π interaction connects the two dimensional chains to form a three-dimensional supramolecular framework.

Antifungal Activity
The inhibition effects of compounds 4-6 were tested in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani using mycelium growth rate method at the concentration of 100 μg/mL [27]. Compounds 4 and 5 were almost inactive against all tested fungus, such as 4h and 5h, their inhibition rates were less than 20%, while the title compounds of 6 exihibited obvious antifungal activities.
As shown in Table 4, B. cinerea and R. solani were more sensitive than F. graminearaum to most members of the compounds. Compound 6h showed the highest activity with an inhibitory rate of 82.2% against B. cinerea. It can be noticed that compounds 6d and 6f carrying two i-propyl or i-butyl pyrrole is 3.004 Å. The C-H···π interaction connects the two dimensional chains to form a threedimensional supramolecular framework.

Antifungal Activity
The inhibition effects of compounds 4-6 were tested in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani using mycelium growth rate method at the concentration of 100 μg/mL [27]. Compounds 4 and 5 were almost inactive against all tested fungus, such as 4h and 5h, their inhibition rates were less than 20%, while the title compounds of 6 exihibited obvious antifungal activities.
As shown in Table 4, B. cinerea and R. solani were more sensitive than F. graminearaum to most members of the compounds. Compound 6h showed the highest activity with an inhibitory rate of 82.2% against B. cinerea. It can be noticed that compounds 6d and 6f carrying two i-propyl or i-butyl pyrrole is 3.004 Å. The C-H···π interaction connects the two dimensional chains to form a threedimensional supramolecular framework.

Antifungal Activity
The inhibition effects of compounds 4-6 were tested in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani using mycelium growth rate method at the concentration of 100 μg/mL [27]. Compounds 4 and 5 were almost inactive against all tested fungus, such as 4h and 5h, their inhibition rates were less than 20%, while the title compounds of 6 exihibited obvious antifungal activities.
As shown in Table 4, B. cinerea and R. solani were more sensitive than F. graminearaum to most members of the compounds. Compound 6h showed the highest activity with an inhibitory rate of 82.2% against B. cinerea. It can be noticed that compounds 6d and 6f carrying two i-propyl or i-butyl

Antifungal Activity
The inhibition effects of compounds 4-6 were tested in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani using mycelium growth rate method at the concentration of 100 µg/mL [27]. Compounds 4 and 5 were almost inactive against all tested fungus, such as 4a and 5a, their inhibition rates were less than 20%, while the title compounds of 6 exihibited obvious antifungal activities.
As shown in Table 4, B. cinerea and R. solani were more sensitive than F. graminearaum to most members of the compounds. Compound 6h showed the highest activity with an inhibitory rate of 82.2% against B. cinerea. It can be noticed that compounds 6d and 6f carrying two i-propyl or i-butyl groups displayed relatively better antifungal activity against the three kinds of fungi than compounds 6a-c, 6e, and 6g carrying two methyl, ethyl, n-propyl, n-butyl, or benzyl groups did. Screening data of compounds 6a-6g indicated that introducing medium-sized alkyls to the carbonate moiety may elevate the antifungal activity. Meanwhile, there is no direct relationship between antifungal activities and substituents of phenyl ring compared with the antifungal activities of compounds 6f and 6h-6q. That is, neither electronegative nor electropositive substitutions at phenyl have played a crucial role in the activity.

General
All melting points of the title compounds were determined on an uncorrected WRS-1B digital melting point apparatus. IR spectra (4000-400 cm´1) were recorded on a Bruker Tensor 27 FT-IR spectrometer, using KBr disks. 1 H-NMR and 13 C-NMR spectra were measured on Bruker 400 spectrometer (DMSO-d 6 or CDCl 3 as solvent, TMS as internal standard). Mass spectra were recorded on a TRACE 2000 spectrometer. The elemental analyses were performed on an Elementar Vario EL cube analyzer. CCDC-1432180 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336033; E-mail: deposit@ccdc.cam.ac.uk). Progress of the reactions was monitored by thin layer chromatography (TLC). All reagents and solvents were obtained from commercial suppliers. Reagents were analytically or chemically pure and were not further purified. All the solvents were dried by standard methods in advance.

General Procedure for the Synthesis of Compounds 3
A mixture of DMAP (14.2 g, 116.2 mmol) in dichloromethane (50 mL) was added to a mixture of pyrrolidine-2,4-dione 1 (5.0 g, 50.5 mmol) and ethyl succinyl chloride (8.3 g, 50.5 mmol) in dichloromethane (100 mL) at 0˝C. The resulting mixture was stirred at 25˝C for 10 h. Then the mixture was washed successively with 10% aqueous HCl, saturated brine and water, dried with Na 2 SO 4 , filtered and concentrated in vacuo to yield the crude product 2 (9.5 g) as a yellow solid which was used directly in the next step.
10% aqueous NaOH (100 mL) was added to the above obtained product 2 (9.5 g). The resulting mixture was stirred at 110˝C for 2 h. Then the mixture was allowed to cool to room temperature, acidified with 10% aqueous HCl to pH = 2-3 and precipitated. The yellow solid was collected by filtration, rinsed with water, and dried in the air to afford the desired product 3 (7.

General Procedure for the Synthesis of Compounds 5
A mixture of compound 4 (4 mmol), EDCI (4.8 mmol) and DMAP (4.6 mmol) in dichloromethane (30 mL) was stirred at room temperature for 24 h. The resulting solid product was collected by filtration and recrystallized from MeOH to give the desired products 5.

Antifungal Activity Test
Compounds 4-6 were screened in vitro for antifungal activity against the phytopathogenic fungi F. graminearum, B. cinerea, and R. solani with the mycelium growth rate method according the reported procedure [27]. Drazoxolon was co-tested as positive control. Every tested compound was dissolved in 0.5 mL DMSO and mixed with PSA (potato sucrose agar) medium (45 mL). The final concentration was 100 µg/mL. Meanwhile, 0.5 mL DMSO in 45 mL PSA medium was used as the control experiment. The medium was poured into three 9 cm petri plates uniformly, cooled, and solidified. The fungi were inoculated to the center of the medium. Then the treatments were cultured at 25˘1˝C for 3-5 days in the dark. The diameters of the fungal colonies were measured to calculate the growth inhibition rate when the Petri dishes had been covered two-thirds by the fungal colonies in the control treatment.

Conclusions
In this paper, a convenient synthesis of novel bioactive heterocycle compounds, phenylpyrrol-substituted tetramic acid derivatives bearing carbonates, was reported. The structures were well supported by spectroscopic data and single crystal X-ray diffraction analysis. The antifungal activity test indicated that these compounds showed obvious antifungal activities.