Synthesis, Crystal Structure and Antifungal Activity of New Furan-1,3,4-oxadiazole Carboxamide Derivatives①

SUN Yue YANG Zi-Hui GU Wen

(Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-forest Biomass,Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, Co-inovation Center for Efficient Processing and Utilization of Forest Products, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China)

ABSTRACT A series of novel furan-1,3,4-oxadiazole carboxamide derivatives (5a ～5e) were designed,synthesized and characterized by spectroscopic methods including HR-MS, 1H- and 13C-NMR. The crystal structure of compound 5a was determined by single-crystal X-ray diffraction. The compound crystallizes in the triclinic system, space group P1 with a = 4.7261(5), b = 10.4672(11), c = 14.5886(13) ?, α = 106.081(4)°, β = 91.043(3)°,γ = 99.456(4)°, Z = 2, V = 682.48(12) ?3, Mr = 348.16, Dc = 1.694 Mg/m3, S = 1.008, = 3.025 mm-1, F(000) = 348,the final R = 0.0775 and wR = 0.2080 for 2774 observed reflections (I 2σ(I)). There are two kinds of hydrogen bonds (N(3)–H(3A) N(2) and C(8)–H(8A) O(3)) present in its crystal structure. The preliminary antifungal assay showed that compounds 5b and 5c exhibited significant antifungal activities against several plant pathogenic fungi.

Keywords: furan-1,3,4-oxadiazole carboxamide, synthesis, crystal structure, antifungal activity;

1 INTRODUCTION

Antifungal agrochemicals have been playing an important role in reducing the huge losses in agricultural production caused by plant diseases. Nowadays, the application of antifungal agrochemicals meets the progressive reduction of efficiency due to the increasing resistance by pathogenic fungi[1,2]. Therefore, there is still an urgent need to develop new antifungal agents with better efficacy against plant pathogens.

Heterocyclic compounds have drawn extensive interests in the development of medicines and pesticides because of their prominent physiochemical and pharmaceutical properties[3-5].Oxadiazole derivatives received increasing attention due to their various biological activities, such as antitumor[6],antibacterial[7], antifungal[8], insecticidal[9]and herbicidal activities[10]. Furan moiety is also a key building block in many drugs or pesticides due to its biological activities including antimicrobial[11], antitumor[12,13], antioxidant[14],antimalarial[15], fungicidal[16-18]and herbicidal properties[19,20].Lots of furan compounds were developed as commercial medicines or pesticides such as fenfuram and furalaxyl. In addition, carboxamide is also a key building block in many drugs or pesticides, for example, fluopyram, penflufen,bixafen and fluxapyroxad. Especially, aromatic carboxamide derivatives have attracted great attention since a number of carboxamide derivatives, such as thifluzamide, isopyrazam and benodanil, were commercialized as fungicides for crop protection. Recently, Zhu et al designed and synthesized the novel compound containing the carboxamide group that exhibited impressive spore germination inhibitory properties[21]. Yu et al. also synthesized a series of pyrazolethiazole carboxamides which showed promisingin vivoantifungal activity againstRhizoctonia solani[22].

Based on these findings, a series of novel furan-1,3,4-oxadiazole carboxamide derivatives were synthesized as potential antifungal agents. Their structures were characterized by1H-NMR,13C-NMR and HR-MS spectra, and the crystal structure of one title compound was determined by single-crystal X-ray diffraction analysis. Thein vitroantifungal activities of the title compounds against several crop pathogenic fungi were also preliminarily evaluated.

2 EXPERIMENTAL

2. 1 Reagents and measurements

The melting points were determined by an XT-4 melting point apparatus (Taike Corp., Beijing, China) and were uncorrected.1H-NMR and13C-NMR spectra were obtained by using a Bruker AV-600 spectrometer (Bruker Co. ltd,Switzerland) with TMS as the internal standard. The crystal structure was recorded on a Bruker APEXII CCD diffractometer. High-resolution mass spectrometry (HRMS) data were obtained on a Waters G2-xs (LC/ESI) instrument(Waters, United States). SP-300B Biochemistry incubator(Nanjing Hengyu Co., Ltd., China) was used forin vitroantifungal assay. Reactions were monitored by TLC which was carried out on silica gel IB-F flexible sheets(Mallinckrodt Baker Inc., Germany) and visualized in UV light (254 and 365 nm). Silica gel (300～400 mesh) for column chromatography was purchased from Qingdao Marine Chemical Factory, China. The reagents and chemicals of AR grade were purchased from commercial suppliers and used without further purification.

2. 2 Synthesis of the title compounds 5a～5e

The synthetic route for compounds 5a～5e was outlined in Scheme 1. The intermediate 2 was synthesized according to the method in the previous report[23]. To a solution of compound 2 (7.0 mmol) and Et3N (14.0 mmol) in 10 mL of anhydrous CH2Cl2was added methyl oxalyl chloride (7.7 mmol) dropwise in an ice bath. The mixture was stirred at 0 ℃ for 30 min and then stirred at room temperature for 14 h.At the end of reaction, the solvent was evaporated to give a crude product of compound 3 which could be used directly for the next step. The intermediate 3 was re-dissolved with 15 mL of POCl3, and the reaction mixture was refluxed for 15 h. Then the mixture was cooled to room temperature and poured slowly into ice water (50 mL). The mixture was stirred for 15 minutes and then the precipitate formed was filtered and dried to afford compound 4. Subsequently, to a solution of compound 4 (1.0 mmol) in DMF (10 mL) was added different substituted benzylamine (1.2 mmol). The mixture was stirred at 80 ℃ for 2～5 h. At the completion of reaction, the mixture was cooled to room temperature and poured into ice water (50 mL), and the solid was filtered and dried. Finally, the crude product was recrystallized from petroleum ether and ethanol to give the title compounds 5a～5e.

Scheme 1. Synthetic route of compounds 5a～5e

Compound 5a: Yellow solid; yield, 66%, m.p. 165～166 ℃;1H-NMR (600 MHz, DMSO-d6)δ4.45 (d,J= 6.2 Hz, 2H), 6.84 (dd,J= 3.6, 1.7 Hz, 1H), 7.32 (d,J= 8.4 Hz,2H), 7.48 (d,J= 3.5 Hz, 1H), 7.54 (d,J= 8.4 Hz, 2H), 8.12(d,J= 1.1 Hz, 1H), 9.91 (t,J= 6.1 Hz, 1H);13C-NMR (150 MHz, DMSO-d6)δ42.0, 112.9, 115.9, 120.1, 129.7, 131.2,137.8, 138.1, 147.7, 153.0, 157.5, 157.8; ESI-HRMS:m/zcalcd. for C14H11BrN3O3[M+H]+: 347.9984; found:347.9981.

Compound 5b:Yellow solid; yield, 85%, m.p. 136.5～138.5 ℃;1H-NMR (600 MHz, DMSO-d6)δ4.47 (d,J= 6.2 Hz, 2H), 6.84 (dd,J= 3.5, 1.7 Hz, 1H), 7.37 (d,J= 8.5 Hz,2H), 7.41 (d,J= 8.5 Hz, 2H), 7.48 (d,J= 3.5 Hz, 1H), 8.12(s, 1H), 9.91 (t,J= 6.1 Hz, 1H);13C-NMR (150 MHz,DMSO-d6)δ41.9, 112.9, 115.9, 128.3, 129.3, 131.6, 137.4,138.1, 147.7, 153.0, 157.6, 157.8; ESI-HRMS:m/zcalcd. for C14H11ClN3O3: 304.0489[M + H]+; found 304.0493.

Compound 5c: White solid; yield, 84%, m.p. 137.1～139.5 ℃;1H-NMR (600 MHz, DMSO-d6)δ4.46 (d,J= 6.2 Hz, 2H), 6.84 (t,J= 3.8, 1.7 Hz, 1H), 7.17 (t,J= 8.9 Hz,2H), 7.39 (dd,J= 8.4, 5.7 Hz, 2H), 7.48 (d,J= 3.5 Hz, 1H),8.12 (s, 1H), 9.90 (t,J= 6.0 Hz, 1H);13C-NMR (150 MHz,DMSO)δ42.4, 113.4, 115.5 (d,J= 21.1 Hz), 116.4, 130.0 (d,J= 8.1 Hz), 135.0 (d,J= 2.9 Hz), 138.6, 148.2, 153.4, 158.1,158.3, 161.8 (d,J= 241.0 Hz); ESI-HRMS:m/zcalcd. for C14H11FN3O3: 288.0784[M + H]+; found 288.0781.

Compound 5d: White solid; yield, 86%, m.p. 149.5～151.2 ℃;1H-NMR (600 MHz, DMSO-d6)δ2.28 (s, 3H),4.43 (d,J= 6.2 Hz, 2H), 6.84 (dd,J= 3.0, 1.2 Hz, 1H), 7.14(d,J= 7.8 Hz, 2H), 7.23 (d,J= 7.8 Hz, 2H), 7.47 (d,J= 3.5 Hz, 1H), 8.12 (s, 1H), 9.85 (t,J= 6.1 Hz, 1H);13C-NMR(150 MHz, DMSO-d6)δ20.6, 42.3, 112.9, 115.9, 127.4,128.8, 135.3, 136.1, 138.1, 147.6, 152.8, 157.6, 157.8;ESI-HRMS:m/zcalcd. for C15H14N3O3[M + H]+: 284.1035;found 284.1039.

Compound 5e: White solid; yield, 43%, m.p. 148 ～149.5 ℃;1H-NMR (600 MHz, DMSO-d6)δ4.48 (d,J= 6.2 Hz, 2H), 6.84 (dd,J= 3.4, 1.6 Hz, 1H), 7.26 (m, 1H), 7.33～7.35 (m, 4H), 7.48 (d,J= 3.5 Hz, 1H), 8.12 (s, 1H), 9.89 (t,J= 6.1 Hz, 1H);13C-NMR (150 MHz, DMSO-d6)δ42.5,112.9, 115.9, 127.0, 127.4, 128.3, 138.1, 138.3, 147.7, 152.9,157.6,157.8; ESI-HRMS:m/zcalcd. for C14H12N3O3[M+H]+: 270.0879; found 270.0880.

2. 3 X-ray structure determination

The crystals suitable for X-ray diffraction were obtained by slow evaporation of the solutions of the title compound 5a in petroleum ether/ethanol at room temperature. X-ray diffraction data were collected on a Bruker APEXII CCD diffractometer equipped with graphite-monochromated MoKαradiation (λ= 0.71073 ?) by usingφandωscan modes in the range of 2.06≤θ≤28.24 (–6≤h≤6, –13≤k≤13, –19≤l≤19) at 296(2) K. A total of 6166 reflections were collected, of which 3302 were independent (Rint=0.0579) and 2774 were observed withI> 2σ(I). The structure was solved by direct methods using SHELXS-2014/7 and refined by full-matrix least-squares procedure onF2with SHELXL-2014/7. All non-hydrogen atoms were refined anisotropically using all reflections withI> 2σ(I).All H atoms were generated geometrically and refined in terms of the riding model. The final refinement gaveR=0.0775,wR= 0.2080 (w= 1/[σ2(Fo2) + (0.0580P)2], whereP= (Fo2+ 2Fc2)/3,S= 1.008, (Δ/σ)max= 0.000, (Δρ)max= 1.506 and (Δρ)min= –1.537 e/?3. The selected bond distances and bond angles are listed in Table 1.

Table 1. Selected Bond Lengths (?) and Bond Angles ( ) of Compound 5a

2. 4 Antifungal activity

Antifungal activities of compounds 5a～5e were testedin vitroagainst seven plant pathogenic fungi includingRhizoctonia solani(R.solani),Botrytis cinerea(B. cinerea),Fusarium oxysporum(F. oxysporum),Sclerotinia sclerotiorum(S. sclerotiorum),Colletotrichum capsici(C.capsici),Alternaria solani(A. solani)andGibberella zeae(G. zeae),which were provided by the Agricultural Culture Collection of China (ACCC). These tests were carried out by an mycelia growth inhibition method, and the procedures are consistent with the literature[24,25]. The stock solution was added into PDA medium, and the concentration of target compounds was 50 mg/L. Pure DMSO without the target compounds was added into PDA medium as blank control,and boscalid was co-assayed as positive control. Fresh dishes with a diameter of 5 mm were taken from the edge of the PDA-cultured fungal colonies, and inoculated on the above three PDA media. Each treatment was processed for three replicates, and the fungicidal effect was averaged.Their relative inhibitory rateI(%) was calculated according to the following equation:I(%) = [(C–T)/(C–5)] × 100,whereIis the inhibitory rate,Cthe colony diameter of the control (mm), andTthe colony diameter of treatment (mm).

3 RESULTS AND DISCUSSION

In this work, 2-furan carboxylic acid (1) was used as the starting material, which was converted to the corresponding hydrazide (2) by successive methyl esterification and hydrazidation reactions. Compound 2 reacted with methyl oxalyl chloride to afford intermediate 3, which was subjected to an intramolecular cyclization to give the furan-1,3,4-oxadiazole derivative 4 with the yield of 90%. Subsequently,the title compounds 5a～5e were obtained by reacting compound 4 with different substituted benzylamine in 43～86% yield.

The structures of compounds 5a～5e were characterized by its HR-MS,1H- and13C-NMR spectroscopic data. As for a typical example, the molecular formula of compound 5a was determined as C14H10BrN3O3through HR-MS spectrum(m/z[M+H]+calcd. for C14H11BrN3O3: 347.9984; found:347.9981). In the1H-NMR data, the appearance of doublet atδ4.45 ppm belongs to the methylene protons of benzyl moiety. Two doublets atδ8.12 and 7.48 ppm and the double doublet atδ6.84 ppm can be attributed to three aromatic protons of furan ring at C(1), C(3) and C(2), respectively,while two doublets each containing two protons atδ7.32 and 7.54 ppm are due to the protons of the phenyl ring at C(10)/C(14) and C(11)/C(13), respectively. In addition, the triplet with one proton atδ9.91 ppm can be attributed to the carboxamide proton. The13C-NMR spectrum of compound 5a exhibits 12 well resolved resonances for 14 carbon atoms.Among them, the absorption peak atδ42.0 ppm is corresponding to the methylene carbon (C(8)), and four peaks atδ120.1, 129.7 (2C), 131.2 (2C) and 137.8 ppm are attributed to the six carbons at the benzene ring (C(9)～C(14)). On the other hand, four peaks atδ112.9, 115.9,138.1 and 147.7 ppm can be assigned to the carbons in the furan ring (C(1)～C(4)), while the three peaks atδ153.0,157.5 and 157.8 ppm are the signals of C(5), C(6) in the oxadiazole ring and the signal of amide carboxyl carbon(C(7)), respectively. The assignments of the signals in the1H- and13C-NMR spectra of compound 5a are in good accordance with its structure. Moreover, the structures of compounds 5b～5e can also be characterized by their spectral data in a similar manner.

The perspective view of compound 5a with atomic numbering scheme is given in Fig. 1, and the selected bond lengths and bond angles are listed in Table 1. As shown in Table 1, the bond lengths and bond angles within the furan and oxadiazole rings agreed well with the normal values.The dihedral angle between the furan (O(1), C(1)～C(4))and oxadiazole rings (N(1), C(5), O(2), C(6), N(2)) is 2.88(38) , indicating that they are coplanar. In addition, the torsion angles N(2)–C(6)–C(7)–N(3) and O(2)–C(6)–C(7)–O(3) are ?2.9(6) and ?4.1(6) , respectively, which indicated that the amide moiety is also coplanar with the oxadiazole ring. The bond length of N(3)–C(7) is 1.346(5) ?, shorter than the isolated N–C single bond (1.471 ?) but longer than the double bond (1.273 ?) due to the p-πconjugation effect between the nitrogen atom and the carboxyl group. Owing to the existence ofsp3-hybrid methylene carbon (C(8)), the benzene ring (C(9)～C(14)) is not coplanar with the furan-1,3,4-oxadiazole carboxamide moiety, causing a dihedral angle of 66.99(16) between the benzene and oxadiazole rings.

Fig. 1. Molecular structure of the compound 5a

The molecular packing diagram of compound 5a is displayed in Fig. 2. As can be seen from the packing diagram,there are two orientations of molecules present in the crystal structure, which are connected by two kinds of hydrogen bonds (N(3)–H(3A) N(2) and C(8)–H(8A) O(3)) (See Table 2) and aligned alternately forming a chain along thebaxis. The chains stack via Van de Waals interactions along theaandcaxes to form a three-dimensional network.

Table 2. Hydrogen Bond Lengths (?) and Bond Angles ( ) of Compound 5a

Fig. 2. Perspective view of the molecular packing of compound 5a

Compounds 5a ～5e were assayed for theirin vitroantifungal activities by the mycelia growth inhibition method against seven representative crop pathogenic fungi at 50 mg/L. As shown in Table 3, compounds 5b and 5c exhibited prominent antifungal activities againstS.sclerotiorumwith the inhibition rates of 99.3% and 95.1%,respectively, which were equipotent to that of boscalid(100%). Notably, compound 5b also showed strong inhibitory activity againstA. solani(90.6%),R. solani(80.1%),B. cinerea(86.4%) andF. oxysporum(70.7%),superior or equipotent to those of boscalid. On the other hand, compounds 5a, 5d and 5e only showed moderate activities toA. solani. From these results, it could be deduced that the introduction of electron-withdrawing substituents (-F, 5c and -Cl, 5b) could substantially increase the antifungal activities of the derivatives, while the effect of-Br substituent (5a) was not obvious compared with -H (5e).On the contrary, the introduction of the electron-donating group (-CH3, 5d) was not beneficial to the antifungal activity.These results offer a promising scaffold for the discovery of potential antifungal agents against plant pathogenic fungi.The investigations on in-depth structure-activity relationships (SAR) and the antifungal mechanisms of this class of derivatives will be carried out in the future.

Table 3. Antifungal Activities of Compounds 5a～5e