Molecular Sciences Synthesis and Bioactivity of 5-substituted-2-furoyl Diacylhydazide Derivatives with Aliphatic Chain

A series of 5-substituted-2-furoyl diacylhydazide derivatives with aliphatic chain were designed and synthesized. Their structures were characterized by IR, 1 H NMR, elemental analysis, and X-ray single crystal diffraction. The anti-tumor bioassay revealed that some title compounds exhibited promising activity against the selected cancer cell lines, especially against the human promyelocytic leukemic cells (HL-60). Their fungicidal tests indicated that most of the title compounds showed significant anti-fungal activity. The preliminary structure-activity relationship showed that the aliphatic chain length and differences in the R 2 group had obvious effects on the anti-tumor and anti-fungal activities. The bioassay results demonstrated that the title compounds hold great promise as novel lead compounds for further drug discovery.

In our previous study, we found that 5-phenyl-2-furoyl diacylhydrazides containing aromatic rings (IV) [16][17][18][19][20][21][22] not only exhibited anti-tumor activity, but also fungicidal and insecticidal activity. In order to discover compounds with better bioactivities, we replaced the rigid aromatic rings (IV) with the flexible aliphatic chains (III), as shown in Scheme 1. A series of 5-phenyl-2-furoyl diacylhydrazides bearing different aliphatic chains were designed and synthesized by the route shown in Scheme 2. Their anti-tumor, anti-fungal, and insecticidal activities were evaluated. The structure-activity relationships were elucidated.

Synthesis and Structure Elucidation
By utilizing the method of Meerwein arylation with copper(II)-catalyzed decomposition of diazonium salts, a series of 5-substituted phenyl-2-furoic acid I were prepared in good yields, based on substituted anilines and furoic acid as starting reagents. The nucleophilic reaction with 2-furoic acid exhibited a high region-selectivity at the 5-position of the furan ring. Then the 5-substituted phenyl-2-furoyl hydrazides II were prepared as previously described [16][17][18][19][20][21][22], and subsequently reacted with different aliphatic acids and thionyl chloride to obtain the title compounds. The synthesis route of title compounds III is shown in Scheme 2.
All the structures of the title compounds were confirmed by IR, 1 H NMR, and elemental analyses. In the IR spectra, the compounds showed absorption bands around 3300 cm −1 , originating from the N-H stretching vibration. The strong bands around 1680 cm −1 could be assigned to the C=O stretching vibration. The bands around 1620 cm −1 were attributed to the secondary amide. Absorption bands around 1510 and 1480 cm −1 were attributed to the frame vibration of the phenyl and furan rings.
In the 1 H NMR spectra, one or two sharp peaks in the range from 10.30 to 10.80 ppm were due to the presence of hydrazine. Mostly, the signals for protons on the phenyl rings appeared as multiplets in the range from 7.10 to 8.20 ppm and the signals for protons on the furan ring were split into two doublets in the range from 6.90 to 7.05 ppm. All the proton signals of the aliphatic chain appeared in the high field around 0.80 to 2.20 ppm.
The crystal data and structure are presented in Table 1 and Figure 1, and allowed a perspective view of compound III-3-2. Some important bond lengths, angels, and torsion angles of compound III-3-2 are given in Table 2. It can be seen from the X-ray single crystal analysis of III-3-2 that the distance of single bonds C11-N1 and C12-N2 (1.355(6) and 1.343(6) Å) were equal to the C-N double bond (1.35 Å), the single bonds C4-C7 and C10-C11 (1.455(6) and 1.465(6) Å) were shorter than the standard C-C single bond (1.54 Å), but longer than the C-C double bond (1.34 Å). N1-N2 (1.396(5) Å) single bonds was shorter than the standard N-N single bond (1.45 Å), but longer than N-N double bond (1.25 Å). These results clearly indicated that the p orbital of the N atoms conjugated with the π molecular orbital and formed the delocalized π-bonds with the conjoint furan and benzene ring. However, unexpectedly, the p orbitals of N1 and N2 seemed not to be conjugated with the π molecular orbital of the C11-O2 and C12-O3 double bonds, which was explained by the bond length of C11-O2 and C12-O3 (1.238(5) and 1.218(5) Å) that followed the normal range for C-O double bond lengths (1.19-1.23 Å).   Table 2. Selected bond lengths, angels, and torsion angles of compound III-3-2. In the crystal structure, C(1), C(2), C(3), C(4), C(5), and C(6) formed a plane with a mean deviation of 0.0153 Å, defined as plane I; C(7), C(8), C(9), C(10), and O(1) formed a plane with a mean deviation of 0.0150 Å ,defined as plane II; O(2), C (11), N(1), and N(2) formed a plane with a mean deviation of 0.0047 Å, defined as plane III; O(3), C(12), C(13), and C (14) were not coplanar, which were defined factitiously as plane IV with a mean deviation of 0.3708 Å ( Figure 1 and Table 3). Planes II, III, IV formed a dihedral angle with plane I of 10.0°, 12.4°, and 56.9°. Planes III and IV formed a dihedral angle with plane II of 6.8° and 47.7°, and the dihedral angle between planes III and IV was 50.6°. The related data is summarized in Table 3.

Insecticidal Activity
Considering that our previous aromatic diacylhydrazide compounds displayed good insecticidal activities against dipterous and lepidopterous insects, Plutella xylostella, Mythimna separata, and Culex pipiens pallens were chosen to evaluate whether the aliphatic compounds retained the insecticidal activity. The results in Table 4 show that compounds III had moderate to poor insecticidal properties. The best one was compound III-8-1, with activities of 60%, 50%, and 60% against P. xylostella, M. separata, and C. pipiens pallens at 200, 200, and 10 mg·L −1 , respectively. Comparison of the activity between the title compounds and the aromatic diacylhydrazide compound IV-1 from our previous study [16][17][18][19][20][21][22] revealed that the replacement of the rigid aromatic rings (IV) by the flexible aliphatic chains (III) had a negative effect on the insecticidal activity.

Fungicidal Activity
The in vivo anti-fungal activities of the title compounds against Fusarium oxysporum, Corynespora cassiicola, Botrytis cinerea, and Rhizoctonia solanii are listed in Table 5. Most of the title compounds showed moderate to good activity against B. cinerea and R. solanii at 500 μg·mL −1 in vivo. For instance, the inhibitory rates of III-11-1 and III-3-1 against B. cinerea were 92.52% ± 2.71% and 84.23% ± 2.43%, which was equal to the positive control 40% pyrimethanil SC (89.57% ± 2.15%). The inhibitory rates of III-3-1 and III-9-1 against R. solanii were 93.43% ± 1.63% and 88.62% ± 1.62%, which was the same as the control level of 3% validamycin AS (92.21% ± 2.41%). The aliphatic chain length showed significant effects on the fungicidal activities against B. cinerea and R. solanii. In general, the shorter the chain, the better the corresponding activity was. The structure and position of R 2 also affected the fungicidal activity against R. solanii. When R 2 was NO 2 and Cl at the ortho or para position, such as in III-3-1, III-1-1, III-9-1, and III-11-1, a higher fungicidal activity was observed. The results showed that meta substitution on the phenyl ring decreased the activity, and the electron-donating groups at the para position significantly decreased the activity. All the compounds showed moderate to poor activity against F. oxysporum and C. cassiicola. Compared with aromatic diacylhydrazide IV-1, the aliphatic derivatives showed improved and increased fungicidal activity.
The preliminary structure-activity relationship showed that the aliphatic chain length had a significant impact on the anti-tumor activity against HL-60. Generally, the shorter the chain, the better the corresponding activity was. The structure and position of R 2 also affected the anti-tumor activity against HL-60. When R 2 was Cl and F at the ortho or para position, such as in III-3-1, III-1-1, and III-8-1, a higher anti-tumor activity was observed. The anti-tumor activity of the title compounds was much better than that of the aromatic diacylhydrazides.

General Information
Melting points were determined with a Cole-Parmer melting point apparatus (Cole-Parmer, Vernon Hills, IL, USA) (thermometer was uncorrected). IR spectra were recorded on a NEXUS-470 FTIR spectrometer (International Equipment Trading Ltd., Vernon Hills, IL, USA) with KBr pellets. 1 H NMR spectra were recorded with a Bruker DPX300 instrument (Bruker, Billerica, MA, USA) and tetramethylsilane was used as an internal standard. Analytical thin-layer chromatography (TLC) was carried out on precoated plates (silica gel 60 F254) and spots were visualized under ultraviolet light. Elemental analyses (C, H and N) were carried out with a Flash EA 1112 elemental analyzer (Thermo Finnigan, Bremen, Germany). Mass spectra were measured on a Bruker ESQUIRE-LC spectrometer (Bruker, Fallanden, Switzerland). The X-ray crystal diffraction (Bruker, Fallanden, Switzerland) data were collected with a Rigaku Saturn diffractometer at 294(2) K and the crystal structures were calculated using the SHELXL program package and refined by full-matrix least squares procedures at Nankai University (Tianjin, China).

Crystallography
Compound III-3-2 was recrystallized from methanol to give colorless crystals suitable for X-ray single crystal diffraction. Cell constants at 113(2) K were determined by a least-square fit to the setting parameters of independent reflections measured on a Bruker SMART [12] 1000 CCD area-detector diffractometer (Bruker, Fallanden, Switzerland) with graphite-monochromated Mo K α radiation (λ = 0.071070 nm) and operating in the phi and scan modes. The structure was solved by the direct method with SHELXS-97 [24][25][26][27] and refined by the full-matrix least squares method on F2 data using SHELXL-97 [27,28]. The empirical absorption corrections were applied to all intensity data. H atom of N-H was initially located in a different Fourier map and was refined with the restraint U iso (H) = 1.2 U eq (N). Other H atoms were positioned geometrically and refined using a riding model, with d (C···H) = 0.093-0.097 nm and U iso (H) = 1.2 U eq (C) or 1.5 U eq (C-methyl). The crystal data in CIF format have been deposited at the Cambridge Crystallographic Data Centre with deposition number CCDC 935116.

Insecticidal Activity
Assessments were made on a dead/live basis. Evaluations are based on a percentage scale of 0-100, which 0 equals no activity and 100 equals total kill. The bioassay was repeated three times and the result of bioactivity was the average of these replicates. Error in the experiments was 5%. The commercialized insecticide RH-5849 and an aromatic derivative (Scheme 1, VI-1: R 1 = 4-CH 3 , R 2 = 4-Cl) were tested as controls under the same conditions. EXCEL2007 was applied to analyze bioassay data.
Larvicidal activity against Culex pipiens pallens. The larvicidal activity was evaluated at the preliminary test concentration of 10 mg·L −1 against the fourth-instar Culex pipiens pallens by the water immersion method under conditions of 27 ± 2 °C, photoperiod of 10:14 (light:dark), and relative humidity 50%-70%. All the test beakers containing twenty Culex pipiens pallens were evaluated 8 days after treatment.
Larvicidal activity against the oriental armyworm (Mythimna separata). The larvicidal activity of the title compounds against the oriental armyworm was evaluated by foliar application. For the foliar armyworm tests, individual corn leaves were placed on moistened pieces of filter paper in Petri dishes. The leaves were then sprayed with the test solution at the preliminary test concentration of 200 mg·L −1 and allowed to dry. The dishes were infested with 10 fourth-instar oriental armyworm larvae. Percentage mortalities were evaluated 4 days after treatment.
Larvicidal activity against the diamondback moth (Plutella xylostella). The larvicidal activity of the title compounds against the diamondback moth was tested by the leaf-dip method. Leaf disks (1.8 cm diameter) were cut from fresh cabbage leaves and then dipped in the test solution for 15 s at the preliminary test concentration of 200 mg·L −1 . After air-drying, the treated leaf disks were placed in a Petri dish (9 cm diameter), lined with a piece of filter paper and then 10 second-instar diamondback moth larvae were transferred to the Petri dish. Percentage mortalities were evaluated 6 days after treatment.

Fungicidal Activity
Using pot culture tests according to references [12,26], the in vivo fungicidal activities of the title compounds against B. cinerea, C. cassiicola, F. oxysporum, and R. solanii were evaluated in a greenhouse with four commercial fungicides, 40% pyrimethanil SC, 75% chlorothalonil WP, 40% flusilazole EC, and 3% validamycin AS and an aromatic derivative (Scheme 1, VI-1: R 1 = 4-CH 3 , R 2 = 4-Cl) as controls. All the strains were conserved at the Institute of Plant Protection, Chinese Academy of Agricultural Science, Beijing, China. B. cinerea, C. cassiicola, F. oxysporum, and R. solanii were maintained on potato dextrose agar (PDA) medium at 4 °C. The culture plates were cultivated at 24 ± 1 °C. Germination was conducted by soaking cucumber seeds in water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °C in an incubator. When the radicles were 0.5 cm, the seeds were grown in plastic pots containing a 1:1 (v/v) mixture of vermiculite and peat. Cucumber plants used for inoculations were at the stage of two seed leaves.
Tested compounds and commercial fungicides were sprayed with a hand spray onto the surface of the seed leaves and were used for B. cinerea, C. cassiicola, and R. solanii at the standard concentration of 500 μg·mL −1 . Tested compounds and commercial fungicide were applied to the cucumber plants by the irrigating method and used for F. oxysporum. Three replicates were used per treatment.
After drying, inoculation of C. cassiicola was carried out by spraying a conidial suspension, F. oxysporum was innoculated by embryo root inoculation, while inoculations of B. cinerea and R. solanii were carried out by spraying mycelial suspensions. Three replicates were performed. After inoculation, the plants were maintained at 24 ± 1 °C and above 80% relative humidity.
The fungicidal activity was evaluated when the untreated cucumber plant (blank control) fully developed symptoms. The area of inoculated treated leaves covered by disease symptoms was assessed and compared to that of untreated ones to determine the average disease index. The relative control efficacy of compounds compared to the blank assay was calculated via Equation (1): where I is relative control efficacy, CK is the average disease index during the blank assay, and PT is the average disease index after treatment during testing.

Anti-Tumor Activity
All the title compounds were dissolved in DMSO and screened for preliminary anti-tumor activity against four different cell lines: a human promyelocytic leukemic cell line (HL-60), a human hepatocellular carcinoma cell line (Bel-7402), a human gastric carcinoma cell line (BGC-823), and a human nasopharyngeal carcinoma cell line (KB) at a concentration gradient of 0.1, 1.0, 10, 50, and 100 μM. The commercial drug doxorubicin and an aromatic derivative (Scheme 1, VI-1: R 1 = 4-CH 3 , R 2 = 4-Cl) were used as controls in the bioassay. Three replicates were performed.

Conclusions
In summary, a series of novel 5-substituted-2-furoyl diacylhydazide derivatives with aliphatic chain III was designed and synthesized. Their anti-tumor and anti-fungal tests indicated that most of the title compounds showed significant bioactivity against the tested tumor cell lines and fungi. The bioactivity was improved and better than that of the aromatic diacylhydrazides. The aliphatic chain length and the structure and position of the R 2 group had a significant impact on the bioactivities. Generally, the shorter the chain, the better the corresponding activity was. When R 2 was an electron-withdrawing group, such as NO 2 and Cl at the ortho or para position, the compounds exhibited higher activity. While the electron-donating groups at the para or meta position on the phenyl ring decreased the activity. The title compounds with aliphatic chains exhibited moderate or marginal insecticidal activity, which was neither improved nor decreased compared to that of the aromatic diacylhydrazides.