Background

Nowadays, the synthesis of nitrogen containing heterocycles is an important direction in the fields of pesticidal chemistry [1–3], medicinal chemistry [4], polymer chemistry [5], coordination chemistry [6] and industrial chemistry [7]. 1,2,4-Triazole derivatives and pyridine derivatives often display broad and diverse biological activities [8–10]. Some reports found that fused heterocycles generally displayed mixed properties of the corresponding single heterocycles. Many references proved that the fusing of triazole and pyridine rings was a good way to produce highly active compounds, such as herbicidal [11, 12], antifungal [13, 14], anticonvulsant [15], antibacterial activity [16]. Furthermore, the acylhydrazone structure is considered an important pharmacophore in drug discovery [17]. In the past years, there have been many reports in the literature for the synthesis and biological activities of hydrazone derivatives [18–20], such as acaricidal, anti-cancer, insecticidal, antifungal, antibacterial, antimicrobial and antileishmanial activity. In addition, hydrazones also are very useful starting materials in bioactive heterocycles, such as β-lactams, pyrazoles, and pyrazines.

In our previous work, many 1,2,4-triazolo[4,3-a]pyridine derivatives were designed and synthesized, which exhibit excellent and diverse activity [11–14]. In line with our continuous efforts to synthesize bioactive lead compounds for crop protection [21–27], the title 1,2,4-triazolo[4,3-a]pyridine derivatives were designed and synthesized by introducing acylhydrazone pharmacophore into the lead compound (Scheme 1).

Scheme 1
scheme 1

Design strategy of compounds 4a–4q

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Results and discussion

Synthesis

The key intermediate 8-chloro-[1, 2, 4]triazolo[4,3-a]pyridine-3-carbohydrazide was synthesized according to the Ref. [28]. Microwave technology was applied to the synthetic reaction to shorten the reaction time and increase the yield. First, the one pot synthesis of intermediate 1 under microwave irradiation was applied, but the result was not better than that of conventional condition. Then intermediate 1 was cyclized with diethyl oxalate lead to the intermediate 2 by a condensation reaction. At last, the 8-chloro-[1, 2, 4]triazolo[4,3-a]pyridine-3-carbohydrazide reacted with different aldehyde in ethanol was synthesized under microwave irradiation conditions. This reaction was completed with higher yields compared with the conventional mode of heating. The synthetic route is showed in Scheme 2.

Scheme 2
scheme 2

The synthetic route of compounds 4a–4q

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The reaction parameters were optimized for the synthesis of title compounds. The title compounds were prepared from 8-chloro-[1, 2, 4]triazolo[4,3-a]pyridine-3-carbohydrazide and substituted aldehyde under microwave irradiation condition, leading to the desired compounds in 82–94 % yields. The compound 4d was chosen as a model reaction under different conditions. Several key reaction conditions were studied, such as reaction temperature, reaction times, reaction mode (conventional or microwave irradiation). The results are illustrated in Table 1. From Table 1, it is indicated that the microwave irradiation method allowed a shorter reaction time, compared with the room temperature or reflux condition. Also we can see that the yield of compound 4d is higher under microwave irradiation condition than that of room temperature or reflux condition. Under the microwave irradiation condition, the yield increase, when the reaction time is prolonged (10 min). Meanwhile, the longer reaction time held higher yield. From the Table 1, the best reaction condition is 78 °C and 10 min.

Table 1 Comparison of yields of 4d through methods with or without microwave irradiation
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All the compounds were identified and characterized by FTIR, 1H NMR, 13C NMR, 19F NMR, MS and elemental analysis. In the 1H NMR spectra of target compounds, all the –NH proton signals of the title compounds can be found around 10–13 ppm. The appearance of signals at ~7.0, ~7.5 and ~9.3 ppm are assigned to pyridine ring. The infrared spectrum of acyl hydrozone derivatives 4 showed absorption bands at 3139–3500 cm−1 for N–H stretching. The characteristic stretching vibrations ν (C=O) and ν (C=N) appears at 1657–1724, 1618–1686 cm−1 respectively. Meanwhile, most of the title compounds exhibited the M + H+ peak in the ESI–MS results.

Antifungal activities and SAR

The antifungal activities of compound 4a–4q were evaluated in vivo at 100 μg/mL against Stemphylium lycopersici (Enjoji) Yamamoto (SL), Fusarium oxysporum sp. Cucumebrium (FO) and Botrytis cinerea (BC) and the bioassay results were listed in Table 2. From Table 2, compound 4i(82.74 %) and 4k(83.53 %) possessed good activity against SL, much better than that of control zhongshengmycin (59.58 %). Among the other, compound 4h(63.99 %), 4l(62.30 %), 4p(58.63 %), 4q(61.61 %) exhibited good effect against SL, they displayed a comparable level of activity as the control zhongshengmycin. For FO, compound 4k exhibited excellent effect (88.89 %), better than that of thiophanate-methyl(81.69 %). Meanwhile, compounds 4a, 4b, 4e, 4l, 4m, 4n, 4o, 4p and 4q showed moderate effect against FO with the inhibitory values of 53.89, 75.56, 64.44, 77.22, 70.00, 66.67, 50.00, 69.81, 65.56 % respectively. Unfortunately, most of the compounds had low antifungal activities against Botrytis cinerea.

Table 2 The antifungal activity of title compounds in vivo at 100 μg/mL
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From Table 2, the preliminary structure and activity relationship (SAR) analysis indicated that compound with electron donating group at para position of benzene ring exhibited significant antifungal activity against SL. For example, compound 4h(p-F), 4l(p-N(CH3)2), 4p(p-CF3) and 4q(p-CH3) displayed >50 % inhibitory activities. Also we found that the five-membered ring (Furan ring) held better activity against SL and FO than that of alkyl or aryl group. For the substituted salicylaldehydes, only compound 4i exhibited excellent antifungal activity against SL. On the other hand, single or poly substituted compounds on the benzene ring both showed good activity against FO.

DFT calculation and SAR

In order to study their structure-active relationship, we choose a highly active compound 4k and low activity compound 4c as model compounds; the frontier orbitals and LogP were calculated. The LogP, energy of HOMO and LUMO, total energy and energy gap are listed in Table 3.

Table 3 LogP, total energy, energy gap and frontier orbital energy
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According to the frontier molecular orbital theory, HOMO has the priority to provide electrons, while LUMO can accept electrons firstly [29, 30]. As we can see from Fig. 1, the LUMO and HOMO are different between the high active compound 4k and low active compound 4c, especially in the orient of electron transition and energy gap. For the HOMO, the electron of compound 4k is mainly concentrated on the fused 1,2,4-triazolo[4,3-a]pyridine ring and a little on the acyl hydrazine bridge and furan ring, while for the compound 4c, the electron is mainly concentrated on the acyl hydrazine bridge and phenyl ring, but the fused 1,2,4-triazolo[4,3-a]pyridine ring had no electrons. As for the LUMO, The electron of compound 4k is evenly distributed among the 1,2,4-triazolo[4,3-a]pyridine ring, acyl hydrazone group and furan ring. But the electron of compound 4c is located on the 1,2,4-triazolo[4,3-a]pyridine ring. The possible reasons of different antifungal activity between the compound 4c and 4k is electron transition direction and energy gap. From Fig. 1, we assumed that the compound with higher energy gap exhibited higher antifungal activity. Also the 1,2,4-triazolo[4,3-a]pyridine ring is important for the higher active compound. The other impact fact is LogP. From Table 1, the LogP is different between the two compounds.

Fig. 1
figure 1

Frontier molecular orbitals of compound 4c and 4k

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The optimized structures of the compound 4c and 4k are presented in Fig. 2. From Fig. 2, we can find that the orientations of amide groups are different. As we known, the conformation of compound is important for the biological activity due to the bind mode between the receptor and acceptor. So we speculate that the conformation of highly active compound is perpendicular between the 1,2,4-triazolo[4,3-a]pyridine ring and the aromatic ring. Otherwise, when the conformation of low active compound, the aromatic ring is parallel with the 1,2,4-triazolo[4,3-a]pyridine ring. These important clues will be helpful in the design of more potent compounds in the future.

Fig. 2
figure 2

Overlay of energy-minimized structures of 4c and 4k

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Methods

Instruments

All the chemical reagents are analytical grade or prepared by our lab. Melting points were measured using an X-4 apparatus and were uncorrected. 1H NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer using DMSO-d6 as solvent. 13C NMR and 19F NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer using DMSO-d6 as solvent. Mass spectra were determined on a Thermo Finnigan LCQ Advantage LC/mass detector instrument. Elemental analysis data of title compounds were collected by a Perkin-Elmer 240C analyzer. CEM Discover Focused Synthesizer was used to carry out the microwave reaction (600 W, 2450 MHz).

Synthesis

The key intermediate 1, 2, 3 are synthesized according to our previous work [28]. The title compounds 4a–4q was synthesized from the intermediate 3 and different aldehydes or ketones in the solution of ethanol at the condition of microwave (150 w, 78 °C, 200 psi, 10 min). All the other compounds are synthesized according to the procedure (Scheme 2).

8-Chloro-N′-(1-phenylethylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4a) white yellow crystal, yield 82 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3416, 3216, 3128, 1690, 1662, 1539, 1478, 1382, 1342, 1258, 1217, 1088, 948, 860, 794; 1H NMR (DMSO-d6, 500 MHz), δ: 2.51(s, 3H, CH3), 7.08(t, J = 7.0 Hz, 1H, Py), 7.46(s, 3H, Ar), 7.56(d, J = 7.5 Hz, 1H, Py), 7.94(s, 2H, Ar), 9.38(d, J = 7.0 Hz, 1H, Py), 10.35(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 15.19, 116.52, 120.83, 125.56, 125.80, 127.11, 128.95, 129.26, 129.39, 138.06, 139.91, 141.37, 149.04, 156.91, 168.78; MS (ESI), m/z: 314(M+1)+. Elemental anal. For C15H12ClN5O (%), calculated: C, 57.42; H, 3.86; N, 22.32; found: C, 57.65; H, 3.76; N, 22.51.

8-Chloro-N′-ethylidene-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4b) white yellow crystal, yield 92 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3219, 3116, 2995, 1724, 1686, 1578, 1491, 1239, 1109, 1039, 855, 794, 688, 535; 1H NMR (DMSO-d6, 500 MHz), δ: 1.95(s, 3H, CH3), 7.24(t, J = 7.2 Hz, 1H, Py), 7.82(d, J = 7.3 Hz, 1H, Py), 9.11(d, J = 7.0 Hz, 1H, Py), 10.03(s, 1H, CH), 11.02(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 21.04, 116.52, 120.82, 125.55, 129.25, 139.90, 149.03, 156.90, 168.78; MS (ESI), m/z: 238(M+1)+. Elemental anal. For C9H8ClN5O (%), calculated: C, 45.49; H, 3.39; N, 29.47; found: C, 45.55; H, 3.21; N, 29.65.

8-Chloro-N′-(2-hydroxy-4-methoxybenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4c) white yellow crystal, yield 90 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3500, 3141, 3083, 1709, 1677, 1631, 1606, 1508, 1457, 1251, 1230, 1204, 1170, 1092, 1022, 965, 832, 742; 1H NMR (DMSO-d6, 500 MHz), δ: 3.79(s, 3H, OCH3), 6.55(d, J = 9.0 Hz, 1H, Ar), 7.25(t, J = 7.2 Hz, 1H, Py), 7.43(d, J = 8.5 Hz, 1H, Py), 7.83(d, J = 7.3 Hz, 1H, Ar), 8.74(s, 1H, Ar), 9.22(d, J = 7.0 Hz, 1H, Py), 11.49(s, 1H, CH), 11.97(s, 1H, NH), 13.01(s, 1H, OH); 13C NMR (150 MHz, DMSO-d6) δ 55.84, 101.69, 107.15, 112.19, 116.42, 120.78, 125.92, 129.38, 131.79, 140.42, 149.14, 150.93, 153.82, 160.03, 162.86; MS (ESI), m/z: 346(M+1)+. Elemental anal. For C15H12ClN5O3 (%), calculated: C, 52.11; H, 3.50; N, 20.26; found: C, 51.98; H, 3.44; N, 20.43.

8-Chloro-N′-(2-hydroxybenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4d) white yellow crystal, yield 92 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3178, 3143, 3052, 1667, 1619, 1549, 1486, 1445, 1354, 1271, 1271, 1238, 1220, 1096, 1039, 954, 848, 762; 1H NMR (DMSO-d6, 500 MHz), δ: 6.92–6.96(m, 2H, Ar), 7.23–7.33(m, 2H, Ar), 7.54(t, J = 7.2 Hz, 1H, Py), 7.82(s, 1H, CH), 8.28(d, J = 7.3 Hz, 1H, Py), 9.21(d, J = 7.0 Hz, 1H, Py), 11.13(s, 1H, NH), 13.10(s, 1H, OH); 13C NMR (150 MHz, DMSO-d6) δ 116.48, 116.98, 119.15, 119.94, 120.79, 125.93, 129.43, 130.02, 132.25, 140.40, 149.17, 150.41, 154.08, 159.03; MS (ESI), m/z: 316(M+1)+. Elemental anal. For C14H10ClN5O2 (%), calculated: C, 53.26; H, 3.19; N, 22.18; found: C, 53.35; H, 3.22; N, 22.41.

8-Chloro-N′-(3-nitrobenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4e) white yellow crystal, yield 94 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3322, 3158, 1683, 1620, 1533, 1486, 1451, 1353, 1275, 1214, 1146, 1078, 959, 897, 853, 788, 736; 1H NMR (DMSO-d6, 500 MHz), δ: 7.27(t, J = 7.0 Hz, 1H, Py), 7.73(t, J = 7.8 Hz, 1H, Ar), 7.84–7.87(m, 2H, Py & Ar), 8.10 ~ 8.17(m, 2H, Ar), 9.08(s, 1H, CH), 9.21(d, J = 6.8 Hz, 1H, Py), 13.21(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 116.58, 120.83, 121.57, 125.08, 125.87, 129.48, 131.09, 134.06, 136.39, 140.48, 147.71, 148.75, 154.48, 162.17; MS (ESI), m/z: 345(M+1)+. Elemental anal. For C14H9ClN6O3 (%), calculated: C, 48.78; H, 2.63; N, 24.38; found: C, 48.86; H, 2.76; N, 24.99.

8-Chloro-N′-(5-chloro-2-hydroxybenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4f) white yellow crystal, yield 88 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3246, 3135, 1676, 1618, 1521, 1477, 1458, 1342, 1267, 1211, 1184, 1145, 1084, 846, 724; 1H NMR (DMSO-d6, 500 MHz), δ: 6.98(d, J = 8.7 Hz, 1H, Ar), 7.28(t, J = 7.2 Hz, 1H, Py), 7.35(d, J = 8.5 Hz, 1H, Py), 7.68(s, 1H, Ar), 7.93(d, J = 7.4 Hz, 1H, Ar), 8.82(s, 1H, CH), 9.21(d, J = 6.9 Hz, 1H, Py), 11.05(s, 1H, NH), 13.16(s, 1H, OH); 13C NMR (150 MHz, DMSO-d6) δ116.51, 118.80, 120.80, 121.29, 123.54, 125.92, 127.86, 129.45, 131.62, 140.41, 147.80, 149.18, 154.25, 156.59; MS (ESI), m/z: 350(M+1)+. Elemental anal. For C14H9Cl2N5O2 (%), calculated: C, 48.02; H, 2.59; N, 20.00; found: C, 47.80; H, 2.75; N, 20.21.

N′-(5-bromo-2-hydroxybenzylidene)-8-chloro-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4g) white yellow crystal, yield 90 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3249, 3134, 1674, 1619, 1613, 1519, 1473, 1455, 1341, 1266, 1210, 1182, 1077, 958, 846, 742; 1H NMR (DMSO-d6, 500 MHz), δ: 6.92(d, J = 8.7 Hz, 1H, Ar), 7.27(t, J = 7.2 Hz, 1H, Py), 7.45(d, J = 8.5 Hz, 1H, Py), 7.80(s, 1H, Ar), 7.84(d, J = 7.4 Hz, 1H, Ar), 8.82(s, 1H, CH), 9.21(d, J = 6.9 Hz, 1H, Py), 11.10(s, 1H, NH), 13.15(s, 1H, OH); 13C NMR (150 MHz, DMSO-d6) δ 116.58, 120.83, 121.57, 125.08, 125.87, 129.48, 131.09, 134.06, 136.39, 140.48, 147.71, 148.75, 149.21, 154.48; MS (ESI), m/z: 395(M+1)+. Elemental anal. For C14H9BrClN5O2 (%), calculated: C, 42.61; H, 2.30; N, 17.75; found: C, 42.45; H, 2.25; N, 17.71.

8-Chloro-N′-(1-(4-fluorophenyl)ethylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4h) white yellow crystal, yield 93 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3345, 3223, 3137, 1698, 1662, 1538, 1504, 1497, 1382, 1342, 1217, 1158, 1089, 949, 858, 794, 742; 1H NMR (DMSO-d6, 500 MHz), δ: 2.47(s, 3H, CH3), 7.05-7.13(m, 3H, Ar and Py), 7.54(t, J = 7.2 Hz, 1H, Py), 7.90–7.93(m, 2H, Ar), 9.35(d, J = 6.9 Hz, 1H, Py), 10.31(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 15.23, 115.80, 115.93, 116.51, 120.83, 125.55, 125.78, 129.25, 129.40, 139.91, 149.04, 156.91, 168.78; 19F NMR (564 MHz, DMSO-d6) δ -111.38; MS (ESI), m/z: 332(M+1)+. Elemental anal. For C15H11ClFN5O (%), calculated: C, 54.31; H, 3.34; N, 21.11; found: C, 54.18; H, 3.52; N, 21.31.

8-Chloro-N′-(4-(diethylamino)-2-hydroxybenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4i) white yellow crystal, yield 92 %, m.p. >300 °C; FT-IR (KBr, cm−1): ν 3212, 2970, 2931, 1676, 1629, 1596, 1518, 1488, 1350, 1247, 1132, 1078, 1040, 850, 758, 738; 1H NMR (DMSO-d6, 500 MHz), δ: 1.18–1.25(m, 6H, 2CH3), 3.36-3.42(m, 4H, 2CH2), 6.62(m, 2H, Ar), 7.03–7.10(m, 2H, Py and Ar), 7.52(d, J = 7.2 Hz, 1H, Py), 8.25(s, 1H, Ar), 8.45(s, 1H, CH), 9.34(d, J = 7.1 Hz, 1H, Py), 10.22(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 13.02, 44.30, 97.93, 104.31, 106.31, 116.30, 120.75, 125.92, 129.25, 132.37, 140.55, 149.06, 150.92, 152.06, 153.35, 160.35; MS (ESI), m/z: 387(M+1)+. Elemental anal. For C18H19ClN6O2 (%), calculated: C, 55.89; H, 4.95; N, 21.73; found: C, 55.99; H, 4.76; N, 21.69.

8-Chloro-N′-(4-(dimethylamino)benzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4j) white yellow crystal, yield 90 %, m.p. 280–281 °C; FT-IR (KBr, cm−1): ν 3487, 1674, 1596, 1525, 1466, 1367, 1255, 1189, 1087, 809, 740; 1H NMR (DMSO-d6, 500 MHz), δ: 2.99(s, 6H, 2CH3), 6.78(d, J = 8.8 Hz, 2H, Ar), 7.24(t, J = 7.2 Hz, 1H, Py), 7.54(d, J = 8.7 Hz, 1H, Ar), 7.81(d, J = 7.2 Hz, 1H, Py), 8.49(s, 1H, CH), 9.21(d, J = 6.9 Hz, 1H, Py), 12.50(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 40.22, 112,25, 116.26, 120.75, 121.72, 125.86, 129.18, 138.76, 140.77, 149.03, 150.89, 152.21, 153.69; MS (ESI), m/z: 343(M+1)+. Elemental anal. For C16H15ClN6O (%), calculated: C, 56.06; H, 4.41; N, 24.52; found: C, 55.89; H, 4.47; N, 24.46.

8-Chloro-N′-(furan-2-ylmethylene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4k) white yellow crystal, yield 91 %, m.p. 278–279 °C; FT-IR (KBr, cm−1): ν 3269, 3160, 3065, 1666, 1626, 1541, 1479, 1349, 1298, 1220, 1156, 1081, 1012, 935, 851, 803, 732; 1H NMR (DMSO-d6, 500 MHz), δ: 6.93(d, 2H, Furan), 7.07(t, J = 6.9 Hz, 1H, Py), 7.54–7.57(m, 2H, Py and Furan), 8.32(s, 1H, CH), 9.33(d, J = 6.9 Hz, 1H, Py), 11.20(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 112.84, 114.79, 116.44, 120.77, 125.91, 129.36, 139.57, 140.54, 146.06, 149.13, 149.74, 154.12; MS (ESI), m/z: 290(M+1)+. Elemental anal. For C12H8ClN5O2 (%), calculated: C, 49.75; H, 2.78; N, 24.18; found: C, 49.68; H, 2.76; N, 24.44.

8-Chloro-N′-(3,4,5-trimethoxybenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4l) white yellow crystal, yield 88 %, m.p. > 300 °C; FT-IR (KBr, cm−1): ν 3139, 2994, 1657, 1629, 1569, 1519, 1346, 1246, 1226, 1066, 913, 850, 739; 1H NMR (DMSO-d6, 500 MHz), δ: 3.96(s, 9H, 3OCH3), 7.04(s, 2H, Ar), 7.28(t, J = 7.0 Hz, 1H, Py), 7.84(d, J = 6.9 Hz, 1H, Py), 8.55(s, 1H, CH), 9.21(d, J = 7.2 Hz, 1H, Py), 12.83(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 56.46, 60.62, 104.95, 116.44, 120.80, 125.85, 129.34, 130.05, 139.98, 139.98, 140.60, 149.13, 153.70, 154.16; MS (ESI), m/z: 391(M+1)+. Elemental anal. For C17H16ClN5O4 (%), calculated: C, 52.38; H, 4.14; N, 17.97; found: C, 52.51; H, 4.28; N, 8.21.

8-Chloro-N′-(2-nitrobenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4m) white yellow crystal, yield 90 %, m.p. >300 °C; FT-IR (KBr, cm−1): ν 3290, 1679, 1628, 1584, 1536, 1470, 1452, 1361, 1226, 1211, 1153, 1094, 1049, 914, 843, 743; 1H NMR (DMSO-d6, 500 MHz), δ: 7.27(t, J = 7.0 Hz, 1H, Py), 7.72(t, J = 7.8 Hz, 1H, Py), 7.84–7.87(m, 2H, Ar), 8.10-8.17(m, 2H, Ar), 9.08(s, 1H, CH), 9.21(d, J = 6.8 Hz, 1H, Py), 12.21(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 116.52, 120.81, 125.19, 125.89, 128.63, 128.98, 129.46, 131.50, 134.29, 140.51, 145.52, 148.85, 149.19, 154.54; MS (ESI), m/z: 345(M+1)+. Elemental anal. For C14H9ClN6O3 (%), calculated: C, 48.78; H, 2.63; N, 24.38; found: C, 48.95; H, 2.45; N, 24.43.

8-Chloro-N′-(2,4-dichlorobenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4n) white yellow crystal, yield 91 %, m.p. >300 °C; FT-IR (KBr, cm−1): ν 3323, 3158, 3082, 1684, 1619, 1532, 1487, 1451, 1353, 1214, 1146, 1078, 1048, 958, 897, 853, 788, 735, 694; 1H NMR (DMSO-d6, 500 MHz), δ: 7.27(t, J = 7.0 Hz, 1H, Py), 7.53(d, J = 8.3 Hz, 1H, Ar), 7.74(s, 1H, Ar), 7.85(d, J = 7.2 Hz, 1H, Py), 8.03(d, J = 8.5 Hz, 1H, Ar), 9.05(s, 1H, CH), 9.21(d, J = 6.9 Hz, 1H, Py), 12.13(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 116.55, 120.83, 125.88, 128.58, 128.64, 129.46, 129.97, 131.09, 134.72, 135.92, 140.49, 145.14, 149.20, 154.40; MS (ESI), m/z: 369(M+1)+. Elemental anal. For C14H8Cl3N5O (%), calculated: C, 45.62; H, 2.19; N, 19.00; found: C, 45.65; H, 2.33; N, 19.21.

8-Chloro-N′-(2-methylbenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4o) white yellow crystal, yield 86 %, m.p. >300 °C; FT-IR (KBr, cm−1): ν 3222, 3060, 1680, 1598, 1548, 1523, 1488, 1454, 1359, 1234, 1222, 1112, 1085, 1039, 949, 849, 784, 742, 693; 1H NMR (DMSO-d6, 500 MHz), δ: 2.37(s, 3H, CH3), 7.24–7.28(m, 1H, Ar), 7.37(t, J = 7.5 Hz, 1H, Py), 7.53(d, J = 7.5 Hz, 1H, Ar), 7.57(s, 1H, Ar), 7.73(d, J = 7.2 Hz, 1H, Py), 7.84(d, J = 7.2 Hz, 1H, Py), 8.51(s, 1H, CH), 9.21(d, J = 6.9 Hz, 1H, Py), 12.91(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 21.35, 116.43, 120.79, 125.15, 125.88, 128.06, 129.29, 129.33, 131.62, 134.52, 138.66, 140.59, 149.13, 150.21, 154.21; MS (ESI), m/z: 315(M+1)+. Elemental anal. For C15H12ClN5O (%), calculated: C, 57.42; H, 3.86; N, 22.32; found: C, 57.38; H, 4.01; N, 22.11.

8-Chloro-N′-(4-(trifluoromethyl)benzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4p) white yellow crystal, yield 92 %, m.p. >300 °C; FT-IR (KBr, cm−1): ν 3321, 3153, 1673, 1620, 1545, 1523, 1487, 1449, 1331, 1297, 1216, 1153, 1116, 1069, 1018, 952, 838, 794, 743; 1H NMR (DMSO-d6, 500 MHz), δ: 7.28(t, J = 7.2 Hz, 1H, Py), 7.82–7.85(m, 3H, Ar and Py), 7.97(d, J = 8.0 Hz, 2H, Ar) 8.72(s, 1H, CH), 9.21(d, J = 6.9 Hz, 1H, Py), 13.02(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 116.55, 120.82, 125.89, 126.21, 126.31, 128.36, 129.46, 138.53, 140.51, 148.32, 148.36, 149.20, 154.44; 19F NMR (564 MHz, DMSO-d6) δ -61.19; MS (ESI), m/z: 368(M+1)+. Elemental anal. For C15H9ClF3N5O (%), calculated: C, 48.99; H, 2.47; N, 19.05; found: C, 49.21; H, 2.72; N, 18.88.

8-Chloro-N′-(4-methylbenzylidene)-[1,2,4]triazolo[4,3-a]pyridine-3-carbohydrazide (4q) white yellow crystal, yield 88 %, m.p. >300 °C; FT-IR (KBr, cm−1): ν 3294, 3143, 1694, 1671, 1605, 1541, 1509, 1488, 1459, 1357, 1233, 1220, 1152, 1074, 1043, 953, 910, 842, 813, 743; 1H NMR (DMSO-d6, 500 MHz), δ: 2.36(s, 3H, CH3), 6.68(t, J = 7.5 Hz, 1H, Py), 7.65(d, J = 7.5 Hz, 2H, Ar), 7.85(d, J = 7.4 Hz, 2H, Ar), 8.07(d, J = 4.4 Hz, 1H, Py), 8.51(s, 1H, CH), 9.21(d, J = 6.9 Hz, 1H, Py), 12.78(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6) δ 21.55, 116.41, 120.78, 125.88, 127.77, 129.32, 130.01, 131.06, 140.62, 140.81, 149.12, 150.17, 154.15; MS (ESI), m/z: 314(M+1)+. Elemental anal. For C15H12ClN5O (%), calculated: C, 57.42; H, 3.86; N, 22.32; found: C, 57.65; H, 3.03; N, 22.55.

Antifungal activity

Antifungal activities of compounds 4a–4q against Stemphylium lycopersici (Enjoji) Yamamoto, Fusarium oxysporum. sp. Cucumebrium and Botrytis cinerea were determined according to our previous work [31]. The potted plants cucumber and tomato were used. The determine concentration of control zhongshengmycin, thiophanate-methyl, cyprodinil and the title compounds is 100 μg/mL. The three fungals Stemphylium lycopersici (Enjoji) Yamamoto, Fusarium oxysporum. sp. Cucumebrium and Botrytis cinerea were inoculated when the cucumber or tomato is at the stage of two seed leaves. The relative control efficacy of compounds comparing to the blank assay was calculated via the following equation:

$$ {\text{Relative control efficacy }}\left( \% \right) = \left( {CK - PT} \right)/CK \times 100\,\% $$

where CK is the average disease index during the blank assay and PT is the average disease index after treatment during testing. All experiments were replicated three times.

Therotical calculations

The theoretical calculation was carried out using DFT methods. The geometry optimization of compound 4c and 4k was carried out at the B3LYP/6-31G level. The energies of HOMO, LUMO and total energy, energy gap are calculated. All these are carried out using the Gaussian 03 package [32] on the dell computer. The LogP was calculated by Chemdraw 7.0.

Conclusions

In conclusion, a series of novel 1,2,4-triazolo[4,3-a]pyridine derivatives containing hydrazone moiety have been designed by bio-rational method based on the former lead compound by us. Many compounds were found to show good antifungal activity. The further comprehensive structure-active relationship was described by using theoretical calculation method. Among them, compound 4k possessed excellent antifungal activities against Stemphylium lycopersici (Enjoji) Yamamoto and Fusarium oxysporum. sp. Cucumebrium.