Introduction

1,2,4-Triazines and their derivatives have been widely studied in terms of their synthetic methodologies and reactivity, since some of these derivatives were reported to have promising biological activities and used as a drugs in medicinal chemistry (Nyffenegger et al., 2007), including antifungal (Sztanke et al., 2005; Holla et al., 2001), anti-HIV (El-Barbary et al., 2005; Makk et al., 2014), anticancer (Abdel-Monem, 2004; El-Gendy et al., 2003), anti-inflammatory (Kumar et al., 2014; Mullick et al., 2009), analgesic (Makhlouf and Maklad, 2004), antiamebic activity (Singh et al., 2005), antimicrobial activity (El-Barbary et al.; 2014; Hegde et al., 2008; Bishnoi et al., 2014), antiasthma agents (Paul et al., 1985) and diuretic and antihypertensive activities (Abdel-Monem, 2004; Paul et al., 1985; Heilman et al., 1979; Ibrahim et al., 2008; Abd-El-All et al., 2013).

In addition, furochromones are very interesting heterocycles, having a wide range of biological activities (Magd-El-Din et al. 2012). They have been reported as antimicrobial (Ragab et al., 1993; Kim et al., 2006; Galal et al., 2009; Hayakawa et al., 2005; Hishmat et al., 1999; Hishmat et al., 1983) and bacteriostatic, bactericidal, fungistatic and fungicidal agents (Hishmat et al., 1999).

On the other hand, chalcone is a biosynthetic product of the shikimate pathway and is a separate class of compounds, having a wide range of biological properties. Chalcones are useful synthons in the synthesis of a large number of bioactive molecules such as pyrazolines that are well-known nitrogen-containing heterocyclic compounds. Considerable interest has been focused on the pyrazoline structure, which possesses a broad spectrum of biological activities such as antiamebic, antimicrobial, monoamine oxidase inhibitors, antimycobacterial, antidepressant, anticonvulsant and anti-inflammatory. Moreover, the pyridine nucleus is prevalent in numerous natural products and is extremely important in the chemistry of biological systems. Pyridine derivatives have been used as bactericides, fungicides and anticancer agents (Bondock et al., 2013).

Based on these findings, our aim is to design and synthesize novel pyrazolines 8, 9, N-phenyl pyrazolines 10, 11, oxazole 12, 13 and pyridine 14, 15 compounds from chalcone derivatives 6, 7 containing the 1,2,4-triazine moiety and to study their anticancer, cytotoxicity, antimicrobial and antifungal activities of the newly synthesized compounds 6, 8, 10, 12 and 14 which have union between 1,2,4-triazine and furochromone moieties.

Results and discussion

Chemistry

Mannich base 2-((4-acetylphenyl amino)methyl)-6-methyl-3-thioxo-1,2,4-triazin-5(2H)-one 2 was prepared as previously reported by condensation of 3,4-dihydro-6-methyl-3-thioxo-1,2,4-triazin-5(2H)-one 1 with formaldehyde and 4-amino acetophenone in the presence of absolute ethyl alcohol as solvent (Abd-El-All et al., 2013) (Fig. 1).

Fig. 1
figure 1

1,2,4-Triazine derivate and its Mannich with 4-amino acetophenone

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Also, the naturally occurring furochromone “khellin” 3 yields khellinone 4 upon hydrolysis with potassium hydroxide. When 4 is subjected to Vilsmeier–Haack reaction, the corresponding 4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-6-carboxaldehyde 5 “formyl khellin” is obtained (Abdel-Aziz et al., 1990; Eidin and Schünemann 1983) (Fig. 2).

Fig. 2
figure 2

Furochromone structure khellin 3, khellinone 4, Khellin aldehyde 5

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It has been found that chalcone derivatives 6, 7 were prepared via addition of Mannich base 2 and formyl khellin 5 or vanillin in a glacial acetic acid and fused sodium acetate upon refluxing for 4 h (Scheme 1). Structure of compounds 6, 7 was confirmed on the basis of their analytical and spectral data. IR spectra of compounds 6, 7 found a new band of (C=O) at 1655 cm−1 and 1665 cm−1 indicating the formation of chalcone, while IR of compound 7 showed a band at 3567 cm−1 of (OH) indicating the attachment of vanillin. Its 1H-NMR spectrum (DMSO-d6, δ ppm) showed doublet at 8.70–8. 76 due to CH=CH aliphatic chalcone, and two singlet peaks appeared at 3.79 and 10.58 are assigned to methoxy and hydroxyl protons of vanillin. 13C NMR spectrum of compound 6 was revealed signal at δ 163.1(C=O), 176.9(C=O) and 178.5(C=S). The other bands of chalcone derivatives 6, 7 appeared in IR, 1H-NMR and 13C NMR spectra in the expected regions.

Scheme 1
scheme 1

Preparation of chalcones 6, 7

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On the other hand, cyclization of chalcone derivatives 6, 7 with hydrazine hydrate in ethanol and a few drops of hydrochloric acid formed the corresponding new pyrazoline hydrazine derivatives 8, 9 (Heilman et al., 1979; Shawali and Gomha, 2002; Ismail, 2001). Under the same reaction conditions, chalcone derivatives 6, 7 react with phenyl hydrazine or hydroxylamine hydrochloride to afford the corresponding new N-phenyl pyrazoline 10, 11 and oxazole derivatives 12, 13, while aminocyanopyridine derivatives 14, 15 were synthesized by refluxing chalcone derivatives 6, 7 with malononitrile in the presence of ammonium acetate in ethanol solution (Scheme 2). Structures of 8– 15 were established on their analytical and spectral data. IR of compounds 8, 9 found the disappearance of carbonyl group which was assigned to chalcone and 1,2,4-triazine ring at 1655–1665 and 1672 cm−1 and the appearance of a new band at ν 3120–2894 cm−1 which was assigned to (3NH, NH2) which indicated the presence of hydrazinyl group. The 1H-NMR spectrum (DMSO-d6, δ ppm) of compound 9 displayed a doublet peak at 2.51–2.56 which is characteristic to diastereotopic (CH2), triplet peak at 2.85–3.12 due to pyrazole ring and two singlet peaks present at 7.01 and 8.41 that are corresponding to protons of the NH and NH2 of hydrazinyl group which were D2O exchangeable. IR spectra of the compounds 10– 15 showed the disappearance carbonyl group which was assigned to chalcone at ν 1655–1665 cm−1, while compounds 14, 15 showed appearance of cyano group in the expected regions at ν 2218, 2221 cm−1. The 1H-NMR of compounds 10–13 found the disappearance of signals for (CH=CH) aliphatic and appearance of signals of CH and CH2 refers to pyrazole and oxazole rings in the expected regions. The 1H-NMR(DMSO-d6, δ ppm) spectrum of compound 15, taken as example, revealed six singlet peaks at 1.04–1.08, 2.04, 3.88, 4.47, 5.26 and 10.72 due to (NH2, CH3, OCH3, 2NH and OH), doublet peak at 5.48–5.60 related to methane proton and multiplet peak appearing at 7.63–7.69 for seven aromatic protons.

Scheme 2
scheme 2

Derivatives preparation of pyrazole 8–11, oxazole 12, 13 and pyridine 14, 15

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The formation of pyrazoline derivatives 8, 9 is, therefore, assumed to proceed via cyclization, and initial attack of the amine nucleophilic on the carbonyl carbon of the chalcone by nitrogen atom of hydrazine hydrate and the carbonyl oxygen gets hydroxylation. Another end of the nitrogen atom of hydrazine hydrate bonded with β-carbon of chalcone, and the unsaturation was shifted between carbonyl and α-carbon of the chalcones. The hydroxylated group was followed by intramolecular cyclization via elimination of water molecule. Migration of proton of cyclic N-2 to C-4 of azole ring and the π-bond was shifted to N-2 and C-3 of the azole ring (Scheme 3).

Scheme 3
scheme 3

The suggested mechanism of preparation pyrazoline derivatives 8, 9

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Biological assay

In vitro cytotoxicity activities

Mosimann’s method was used to evaluate the cytotoxic activity of synthesized compounds 1, 5, 6, 8, 10, 12 and 14 against different human cancer cell lines (Table 1) including: cervical carcinoma (KB), ovarian carcinoma (SKOV-3), CNS cancer (SF-268), non-small lung cancer (NCl H460), colon adenocarcinoma (RKOP 27), antileukemia (HL60, U937, K561), melanoma (G361,SK-MEL-28) and neuroblastoma (GOTO, NB-1). The cytotoxic effect of the tested compounds over cell lines of HeLa (cervical), MCF-7(breast), HT1080 (fibrosarcoma) and HEPG2 (liver) was also tested. The results were expressed as the IC50, and the results are given in Table 1, which induces a 50 % inhibition of cell growth of treated cells when compared to the growth of control cells. There was a good reproducibility between replicate wells with standard errors below 10 %.

Table 1 Cytotoxicity activities of compounds
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In vitro inhibition of histone deacetylase

Yoshida’s method was used to evaluate the cytotoxic activity of synthesized compounds 1, 5, 6, 8, 10, 12 and 14 against antileukemia cell line (K562). The tested compounds showed a high potent activity against antileukemia cell line (K562) (Table 1).

Assay of in vivo acute toxicity

HEpG2 cells

Compounds 1, 5, 6, 8, 10, 12 and 14 were tested tumor cell growth inhibition against HEpG2 cells (1 × 105 cells in 0.1 ml PBS) (Table 3). Tested compounds were injected subcutaneously (10 μg/50 μl) daily for four doses. Monoclonal antiplatelet antibody (PY-13) and sterile PBS were used as isotype and negative controls, respectively. Tumor growth was observed every 3 days by measuring its diameter with vernier calipers. Tumor weight (TW) was calculated by TW (g) = tumor volume (cm3) = d2 × D/2, where d is the shortest and D is the longest diameter, respectively. Mice were killed when the tumor size reached 2.0 cm in diameter, and samples were collected.

The results are presented in Table 2.

Table 2 In vivo acute toxicity of prepared compounds against HEpG2 cells of liver cancer
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All tested compounds 1, 5, 6, 8, 10, 12 and 14 were indicating a significance activity tumor cell growth inhibition against HEpG2 compared with control drug doxorubicin. Concerning the new pyridine derivative combining with 1,2,4-triazine and furochromone moieties 14 was the most active compared with the other synthesized chalcone 6, pyrazoles 8, 10 and oxazole 12 combining with 1,2,4-triazine and furochromone moieties, and it is obvious that the least active compound in this tested compounds is start material 1,2,4-triazine 1.

MCF-7 xenograft model

Tested compounds 1, 5, 6, 8, 10, 12 and 14 were evaluated against MCF-7 mouse xenograft model of breast cancer (Table 3).

Table 3 Evaluated compounds 1, 5, 6, 8, 10, 12 and 14 in an MCF-7 mouse xenograft model of breast cancer
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The results are presented in Table 3.

Seven compounds 1, 5, 6, 8, 10, 12 and 14 showed marked acute activity against MCF-7 mouse xenograft model of breast cancer. It is obvious that new pyridine derivative union with 1,2,4-triazine and furochromone moieties 14 was the most active, while the least active compound was the start material 1,2,4-triazine 1.

Antimicrobial activity

In vitro antimicrobial screening

The results given in Table 4 revealed that compound 5 exhibited a wide range of antimicrobial activities against both gram-positive and gram-negative bacteria and fungi, and its inhibitory activity against fungi (especially A. alternata and F. oxysporium) was higher than of the bacteria, while compound 5 exhibited a moderate antimicrobial activity against all the tested pathological strains except A. niger and A. t enuissima, in comparison with the standard drugs. Compounds 6, 8, 10, 12 and 14 exhibited low inhibitory effect against gram-negative bacteria E. c oli and P. aeruginosa (zones of inhibition range from 7 to 9 mm). Results indicated that compound 5 was the most effective against all tested microorganisms with zones of inhibition ranging from 11 to 30 mm and minimum inhibitory concentrations (MIC) ranging between 6.25 and 25 μg. Compared with the standard antifungal drug (triflucan), compound 5 exhibited excellent antifungal activity against all tested fungi, with minimum inhibitory concentration better than that of the standard drug (Table 5).

Table 4 Antimicrobial activity of compounds (1, 5, 6, 8, 10, 12, 14)
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Table 5 Minimal inhibitory concentration (μg/disk) of compound 5 against the pathological strains
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Experimental

Chemistry

All melting points were uncorrected. Elemental analysis made at the Micro-analytic Center, Cairo University. The IR spectra (4000–400 cm−1) were recorded using KBr pellets in a Jasco FT/IR 300 E Fourier. The 1H-NMR spectra were measured on 1H-NMR spectrophotometers and recorded using Joel EX-200, 500 MHz. The mass spectra were performed using Finnigan MAT SSQ 7000 (Thermo. Inst. Sys. Inc., USA) spectrophotometer at 70 and 20 eV.

Preparation of chalcone (6, 7)

General procedure

Mannich base 2 (10 mmol) and (10 mmol) of furochromone-6-carboxaldehyde 5 or vanillin in 30 ml of glacial acetic acid and 10 mmol of fused sodium acetate was heated under reflux for 4 h and then poured onto ice water, and the resulting solid was collected by filtration and crystallized from ethanol.

(E)-2-(((4-(3-(4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl)acryloyl)phenyl)amino) methyl)-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one 6

A beige powder was crystallized from ethanol, yield 70 %, m.p. 220–222 °C. 1H-NMR (500 MHz, DMSO-d6) δ 2.14 (s,3H, CH3); 3.91 (s, 3H, OCH3); 4.25 (s, 3H, OCH3); 4.90 (s, 1H, NH exchangeable with D2O); 5.32–5.52 (d, 2H, CH2); 6.42 (d, 1H, J = 2 Hz, furan C-2); 6.85 (d, 1H, J = 2 Hz, furan C-3); 7.03–7.96 (m, 5H, aromatic and 4-pyranone moiety); 8.70–8.76 (2d, 2H, CH=CH aliphatic, J = 12.6 Hz); 8.99 (s, 1H, NH exchangeable with D2O). 13C NMR (DMSO-d6) δ 15.8 (CH3), 61.5 (OCH3), 62.4(OCH3), 65.0 (CH2), 128.2, 149.0 (C=C aliphatic), 101.8, 110.5, 112.5, 119.5, 120.3, 125.1, 129.0, 130.3, 132.6, 140.9, 145.7, 147.6, 150.3, 150.8, 153.2. 154.7, 163.1 (C=O), 176.9 (C=O), 178.5 (C=S). IR (KBr, ν/cm−1): (NH) 3320 br, (NH) 3216 s, (C=O triazine ring) 1672 s, (C=O chalcone) 1665, (C=O pyrane ring) 1641 s, (C=N triazine ring) 1564 s, (C=S) 1245 s. Anal. Calc. for C27H22N4O7S: C, 59.33; H, 4.06; N, 10.25; S, 5.87. Found: C, 59.57; H, 3.85; N, 10.07.; S, 6.02.

(E)-2-(((4-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)amino)methyl)-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one 7

A yellow powder was crystallized from ethanol, yield 75 %, m.p. 137–140 °C. 1H-NMR (500 MHz, DMSO-d6) δ 2.01 (s,3H, CH3); 3.79 (s, 3H, OCH3); 4.72 (s, 1H, NH exchangeable with D2O); 5.82–5.86 (d, 2H, CH2, J = 8.4 Hz); 6.05 (s, 1H, NH exchangeable with D2O); 7.67–7.90 (m, 7H, aromatic); 8.70–8.76(2d, 2H, CH=CH aliphatic, J = 12.6 Hz); 10.58 (s, 1H, OH exchangeable with D2O). IR (KBr, ν/cm−1): ν (OH) 3567, ν (NH) 3257 br, ν (NH) 3120 s, ν (C=O triazine ring) 1672 s, ν (C=O chalcone) 1655 s, ν (C=N triazine ring) 1563 s, ν (C=S) 1239 s. Anal. Calc. for C21H20N4O4S: C, 59.42; H, 4.75; N, 13.20; S, 7.55. Found: C, 59.76; H, 4.49; N, 13.01.; S, 7.84.

Preparation of compounds 8– 15

General procedure

A mixture contains compound 6 or 7 (10 mmol) with (10 mmol) each of (hydrazine hydrate, phenyl hydrazine, hydroxylamine hydrochloride or malononitrile in case of malononitrile, ammonium acetate is used) dissolve in 50 ml ethanol as solvent and a few drops of HCl. Then, the reaction mixture was heated under reflux and poured onto ice water, the resulting solid was collected by filtration and recrystallized from proper solvent.

(E)-6-(-(4-(((5-Hydrazono-6-methyl-3-thioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)methyl)amino)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-4,9-dimethoxy-5H-furo[3,2-g]chromen-5-one 8

Brown powder was crystallized from ethanol, yield 77 %, m.p. >300 °C. 1H-NMR (500 MHz, DMSO-d6) δ 2.08 (s, 3H, CH3); 2.56, 2.63 (dd, 2H, diastereotopic CH2, J = 2.4 Hz); 3.02–3.30 (t, 1H, CH, J = 12.5 Hz); 3.93 (s, 3H, OCH3); 4.04 (s, 1H, NH exchangeable with D2O); 4.42 (s, 3H, OCH3); 5.38 (s, H, NH triazine ring exchangeable with D2O); 5.97–6.01 (d, 2H, CH2 methane proton, J = 8.2 Hz); 6.8(d, 1H, J = 2.2 Hz, C-2 furan); 7.21 (s, 2H, NH2 pyrazoline ring exchangeable with D2O); 7.41–7.69 (m, 5H, aromatic and 4-pyranone moiety); 8.3 (d, 1H, J = 2.6 Hz, C-2 furan); 8.48 (s, 1H, NH pyrazoline ring exchangeable with D2O);. IR (KBr, ν/cm−1): ν (NH2) 3262, 3294 s, ν (NH) 3222 s, ν (C=O pyrane ring) 1640 s, ν (C=N ring) 1590 s, ν (C=S) 1296 s. Anal. Calc. for C27H26N8O5S: C, 56.44; H, 4.56; N, 19.50; S, 5. 58. Found: C, 56.66; H, 4.25; N, 19.27; S, 5.80.

(E)-6-(5-Hydrazono-2-(((4-(5-(4-hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)methyl)-6-methyl-3,4-dihydro-1,2,4-triazine-3(2H)-thione 9

Deep red powder was washed with DMF, yield 73 %, m.p. >300 °C. 1H-NMR (270 MHz, DMSO-d6) δ 2.08 (s, 3H, CH3); 2.51, 2.56 (dd, 2H, diastereotopic CH2, J = 2.4 Hz); 2.85–3.12 (t, 1H, CH, J = 12.5 Hz); 3.84 (s, 1H, NH exchangeable with D2O); 3.90–4.02 (m, 3H, OCH3); 5.34 (s, H, NH triazine ring exchangeable with D2O); 5.97–6.01 (d, 2H, CH2 methane proton, J = 8.4 Hz); 7.01 (s, 2H, NH2 pyrazoline ring exchangeable with D2O); 7.41–7.69 (m, 7H, aromatic); 8.41 (s, 1H, NH pyrazoline ring exchangeable with D2O); 11.4 (s, 1H, OH exchangeable with D2O). MS: m/z (M− 451.55, 0.47 %). IR (KBr, ν/cm−1): ν (OH) 3626 s, ν (NH2) 3257, 3289 s, ν (NH) 3120 s, ν (C=N rings) 1590 s, ν (C=S) 1296 s. Anal. Calc. for C21H28N8O2S: C, 55.24; H, 6.18; N, 24.54; S, 7. 02. Found: C, 55.56; H, 5.97; N, 24.15; S, 7.36.

(E)-(((4-(5-(4,9-Dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)methyl)-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one 10

Brown powder was crystallized from ethanol, yield 75 %, m.p. 110–112 oC. 1H-NMR (500 MHz, DMSO-d6) δ 2. 03 (s, 3H, CH3); 2.55, 2.63 (dd, 2H, diastereotopic CH2, J = 2.4 Hz); 2.89–3.11 (t, 1H, CH, J = 12.5 Hz); 3.65 (s, 3H, OCH3); 3.84 (s, 1H, NH exchangeable with D2O); 3.92 (s, 3H, OCH3); 5.24 (s, 1H, NH triazine ring exchangeable with D2O); 5.55–5.63 (d, 2H, CH2 methane proton, J = 6.4 Hz); 6.42 (d, 1H, J = 2 Hz, furan C-2); 7.07–7.78 (m, 10H aromatic); 8.94 (d, 1H, J = 2 Hz, furan C-2). IR (KBr, ν/cm−1): ν (NH) 3235, ν (NH) 3056 s, ν (C=O triazine ring) 1656 s, ν (C=O pyrane ring) 1641 s ν (C=N rings) 1597 and 1494 s, ν (C=S) 1285 s. MS: m/z (M− 637, 17.96 %). Anal. Calc. for C33H28N6O6S: C, 62.25; H, 4. 43; N, 13.20; S, 5.04. Found: C, 62.45; H, 4.19; N, 13.00; S, 529.

2-(((4-(5-(4-Hydroxy-3-methoxyphenyl)-1phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino) methyl) 6-methyl--3-thioxo-3,4dihydro-1,2,4-triazin-5(2H)-one 11

Yellowish powder was crystallized from ethanol, yield 70 %, m.p. 155–158 oC. 1H-NMR (500 MHz, DMSO-d6) δ 2. 05 (s, 3H, CH3); 2.56–2.60 (dd, 2H, diastereotopic CH2, J = 2.6 Hz); 2.71–3.03(t, 1H, CH, J = 12.5 Hz), 3.35 (s, 3H, OCH3); 3.88 (s, 1H, NH exchangeable with D2O); 5.25 (s, 1H, NH triazine ring exchangeable with D2O); 5.95–6.13 (d,2H, CH2 methane proton, J = 6.00 Hz); 7.17–7.71 (m, 12H aromatic); D2O); 11.4 (s, 1H, OH exchangeable with D2O). IR (KBr, ν/cm−1): ν (OH) 3403 br, ν (NH) 3235, ν (NH) 3056 s, ν (C=O triazine ring) 1656 s, ν (C=N rings) 1597 and 1494 s, ν (C=S) 1285 s.MS: m/z (M + 513, 0, 17 %). Anal. Calc. for C27H28N6O3S: C, 62.77; H, 5. 46; N, 16.27; S, 6.21. Found: C, 62.89; H, 5.19; N, 16.10; S, 6.56.

2-(((4-(5-(4,9-Dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl)-4,5-dihydroisoxazol-3-yl)phenyl)amino)methyl)-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one 12

Brown powder was crystallized from ethanol, yield 78 %, m.p. 160–162 oC. 1H-NMR (270 MHz, DMSO-d6) δ 2.05 (s, 3H, CH3); 2.62,2.72 (dd, 2H, diastereotopic CH2, J = 4.2 Hz); 3.07–3.29 (t, 1H, CH, J = 12.5 Hz); 3.47 (s, 3H, OCH3); 3.80 (s, 1H, NH exchangeable with D2O); 3.90 (s, 3H, OCH3); 5.28 (s, 1H, NH triazine ring exchangeable with D2O); 5.59–5.75 (d, 2H, CH2 methane proton J = 6.6 Hz); 6.42 (d, 1H, J = 2 Hz, furan C-2); 7.07–7.78 (m, 5H aromatic); 8.94 (d, 1H, J = 2 Hz, furan C-2). IR (KBr, ν/cm−1): ν (NH) 3220 s, ν (NH) 3106 s, ν (C=O triazine ring) 1656 s, (C=O pyrane ring) 1645 s, ν (C=N rings). 1597 s and 1495 s, ν (C=S) 1226 s. Anal. Calc. for C27H23N5O7S: C, 57.75; H, 4.13; N, 12.47; S, 5.71. Found: C, 57.99; H, 4.01; N, 12.22; S, 5.82.

2-(((4-(5-(4-Hydroxy-3-methoxyphenyl)-4,5-(4-dihydroisoxazol-3-yl)phenyl)amin) methyl) 6-methyl-3-thioxo-3, 4dihydro-1,2,4-triazin-5(2H)-one 13

Brown powder was crystallized from ethanol, yield 69 %, m.p. 210–212 oC. 1H-NMR (270 MHz, DMSO-d6) δ 2.03 (s, 3H, CH3); 2.54, 2.61 (dd, 2H, CH2 diastereotopic CH2, J = 2.1 Hz); 3.09–3.30 (t, 1H, CH, J = 12.5 Hz); 3.47 (s, 3H, OCH3); 3.81 (s, 1H, NH exchangeable with D2O); 5.23 (s, 1H, NH triazine ring exchangeable with D2O); 5.38–5.52 (d, 2H, CH2 methane proton, J = 4.6 Hz); 7.63–7.82 (m, 7H aromatic); 11.4 (s, 1H, OH exchangeable with D2O). IR (KBr, ν/cm−1): ν (OH) 3366 br., ν (NH) 3220 s, ν (NH) 3106 s, ν (C=O triazine ring) 1656 s, ν (C=N rings). 1597 s and 1495 s, ν (C=S) 1226 s. MS: m/z (M+ 513, 0.17 %). Anal. Calc. for C21H23N5O4S: C, 57.13; H, 5. 25; N, 15.86; S, 7.26. Found: C, 57.49; H, 5.05; N, 15.39; S, 7.46.

2-Amino-4-(4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromen-6-yl)-6-(4-(((6-methyl-5-oxo-3-thioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)methyl)amino)phenyl)nicotinonitrile 14

A brown powder was crystallized from ethanol, yield 72 %, m.p. 255–257 °C. 1H-NMR (270 MHz, DMSO-d6) δ 1.12–1.26 (s, 2H, NH2 exchangeable with D2O); 2.43 (s, 3H, CH3); 3.43 (s, 3H, OCH3); 3.92 (s, 3H, OCH3); 4.92 (s, 1H, NH exchangeable with D2O); 5.26 (s, 1H, NH triazine ring exchangeable with D2O); 5.37–5.50 (d, 2H, CH2 methane proton, J = 6.4 Hz); 5.99–6.20 (d, 1H, J = 1 Hz, C-5 pyridine); 6.80 (d, 1H, J = 2 Hz, C-2 furan); 7.23(d, 1H, J = 2 Hz, C-3 furan); 7.63–7.69 (m, 5H aromatic); IR (KBr, ν/cm−1): ν (NH2) 3325, 3290, ν (NH triazine ring) 3265, ν (NH olefinic) 3222, ν (C ≡ N) 2218 s, ν (C=O) 1672 s, ν (C=O pyrane ring) 1640 s, ν (C=N rings) 1563 and 1496 s, ν (C=S) 1245 s. Anal. Calc. for C30H23N7O6S: C, 59.11; H, 3.80; N, 16.08; S, 5.26. Found: C, 59.33; H, 3.62; N, 16.32; S, 5.48.

2-Amino-4(4-hydroxy-3-methoxyphenyl)-6-(4-(((6-methyl-5oxo-3-thioxo-3,4-dihydro-1,2,4-triazin-2(3H)-yl) methyl)amino)phenyl)nicotinonitrile 15

A brown powder was crystallized from toluene, yield 70 %, m.p. 165–168 °C. 1H-NMR (270 MHz, DMSO-d6) δ 1.04–1.08 (s, 2H, NH2 exchangeable with D2O); 2.04 (s, 3H, CH3); 3.88 (s, 3H, OCH3); 4.47 (s, 1H, NH exchangeable with D2O); 5.26 (s, 1H, NH exchangeable with D2O); 5.48–5.60 (d, 2H, CH2 methane proton, J = 8.00 Hz); 7.63–7.69 (m, 7H aromatic); 10.72 (s, 1H, OH exchangeable with D2O);. IR (KBr, ν/cm−1): ν (OH) 3377 br, ν (NH2) 3320, 3290, ν (NH triazine ring) 3262, ν (NH olefinic) 3210, ν (C ≡ N) 2221 s, ν (C=O) 1669 s, ν (C=N rings) 1596 and 1496 s, ν (C=S) 1288 s. MS: m/z (M+468, 1.18 %). Anal. Calc. for C24H23N7O3S: C, 58.88; H, 4.74; N, 20.03; S, 6.55. Found: C, 58.99; H, 4.55; N, 19.83; S, 6.88.

Biological assays

Anticancer

The cytotoxicity of the newly synthesized compounds 1, 5, 6, 8, 10, 12 and 14 against cancer cell lines in vitro was performed with the MTT assay according to the Mosimann’s method. The MTT assay is based on the reduction of the soluble 3-(4,5-methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) into a blue-purple formazan product, mainly by mitochondrial reductase activity inside living cells. The cells used in cytotoxicity assay were cultured in RPMI 1640 medium supplemented with 10 % fetal calf serum. Cells suspended in the medium (2 × 104/ml) were plated in 96-well culture plates and incubated at 37◦C in a 5 % CO2 incubator. After 12 h, the test sample (2 μL) was added to the cells (2Ý 104) in 96-well plates and cultured at 37◦C for 3 days. The cultured cells were mixed with 20 μL of MTT solution and incubated for 4 h at 37◦C. The supernatant was carefully removed from each well, and 100 μL of DMSO was added to each well to dissolve the formazan crystals which were formed by the cellular reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well was measured by a micro-plate reader using a test wavelength of 570 nm. The results were expressed as the IC50, which induces a 50 % inhibition of cell growth of treated cells when compared to the growth of control cells. Each experiment was performed at least three times. There was a good reproducibility between replicate wells with standard errors below 10 %.

In vivo and in Vitro Screening

Inhibition of Human leukemia K562 (Histone Deacetylase) (Finney 1962; Yoshida et al., 1990).

Histone deacetylase fraction was prepared as described by Yoshida et al., and human leukemia K562 (2.5 × 108) cells were disrupted in buffer A (15 mM potassium phosphate buffer, pH 7.5, containing 5 % glycerol and 0.2 mM EDTA, 15 ml). The nuclei were collected by centrifugation (35000 g, 10 min) and resuspended with buffer A (15 ml) containing 1 M (NH4)2SO4. After sonication, the supernatant was collected by centrifugation, and ammonium sulfate was added to make the final concentration 3.5 M. After stirring for 1 h at 0 °C, the precipitate was collected by centrifugation, dissolved with buffer A (4 ml) and dialyzed against buffer A (2000 ml). The dialyzate was loaded onto a Mono Q HR5/5 column (Pharmacia) equilibrated with buffer A and eluted with a linear gradient of 0–1 M NaCl in buffer A (30 ml). A single peak of histone deacetylase activity was eluted around 0.4 M NaCl, and the fraction was stored at −80 °C until use. Inhibition histone deacetylase was estimated as described by Yoshida et al., 1990 with slight modifications. 3H-labeled histone was prepared by the method of Yoshida et al.: 3 K562 cells (108 cells) were incubated in growth medium (25 ml) containing 0.5 mg/ml [3H] sodium acetate (152.8 GBq/mmol; NEN) and 5 mM sodium butyrate at 37 °C. Histone deacetylase inhibitory activity of test compound was measured as follows: the mixture (total volume 50 μl) containing the above histone deacetylase fraction (2 μl), 3H-labeled histone (100 μg/ml) and test compound (5 μl) was incubated for 10 min at 37 °C. [3H] Acetic acid, which was liberated from 3H-labeled histone, was extracted with ethyl acetate, and radioactivity was measured by a liquid scintillation counter.

In vivo cytotoxicity bioassay

Assay of in vivo acute toxicity

The animal study was approved by the Experimental Animal Committee of Faculty of Medicine, Chiang Mai University. All animal experiments met the animal welfare guidelines. Male BALB/c nude mice (6 weeks old) were purchased from the Institute of Experimental Animal, Mahidol University, Bangkok, Thailand. Mice were housed in laminar flow cabinets under specific pathogen-free conditions at room temperature with a 24-h night–day cycle and fed with pellets and water ad libitum. Log growth-phase HepG2 cells (1 × 105 cells in 0.1 ml PBS) were injected subcutaneously into the right flank of athymic nude mice (n = 4) to establish a model of tumor-bearing mice. On day 4 after implanting, tested compounds were injected subcutaneously (10 μg/50 μl) daily for four doses. Monoclonal antiplatelet antibody (PY-13) and sterile PBS were used as isotype and negative controls, respectively. Tumor growth was observed every 3 days by measuring its diameter with vernier calipers. Tumor weight (TW) was calculated by TW (g) = tumor volume (cm3) = d2 × D/2, where d is the shortest and D is the longest diameter, respectively. Mice were killed when the tumor size reached 2.0 cm in diameter, and samples were collected.

Human breast cancer xenograft models and animal treatment

The animal protocol was approved by the Institutional Animal Use and Care Committee of the University of Alabama at Birmingham. Female athymic pathogen-free nude mice (nu/nu, 4–6 weeks) were purchased from Frederick Cancer Research and Development Center (Frederick, MD). To establish MCF-7 human breast cancer xenografts, each of the female nude mice was first implanted with a 60-day s.c. slow-release estrogen pellet (SE-121, 1.7 mg 17β-estradiol/pellet; Innovative Research of America, Sarasota, FL). The next day, cultured MCF-7 cells were harvested from confluent monolayer cultures, washed twice with serum-free medium, resuspended and injected subcutaneously (s.c.) (5 × 106 cells, total volume 0.2 ml) into the left inguinal area of the mice. All animals were monitored for activity, physical condition, body weight and tumor growth. Tumor size was determined by caliper measurement in two perpendicular diameters of the implant every other day. Tumor weight (in g) was calculated by the formula, 1/2a × b 2 where “a” is the long diameter and “b” is the short diameter (in cm). The animals bearing human cancer xenografts were randomly divided into various treatment groups and a control group (7–10 mice/group). The untreated control group received the vehicle only. For the MCF-7 xenograft model, tested compounds were dissolved in PEG400/ethanol/saline (57.1: 14.3: 28.6, v/v/v) and were administered by intraperitoneal (i.p.) injection at dose of 1 mg/kg/day, 3 day/week for 3 weeks.

Antimicrobial assay

In vitro Antimicrobial Screening

Antimicrobial activity of compounds 1, 5, 6, 8, 10, 12 and 14 was screened in vitro against a panel of gram-positive and gram-negative bacterial pathogens and fungi, in comparison with control drugs thiophenicol (Thiamphenicol, Sanofi-Aventis, France) as an antibacterial agent and triflucan (Fluconazole, Egyptian International Pharmaceutical Industries Company (EIPICO)) as an antifungal agent, by the agar diffusion technique (Domig et al., 2007; Ajaiyeoba et al., 2003). The compounds 1, 5, 6, 8, 10, 12 and 14 were individually tested against gram-positive bacteria (Bacillus subtilis ATCC6633 and Staphylococcus aureus ATCC29213), gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC27953) and fungi (Candida albicans ATCC 10321, Aspergillus niger NRRL-363, Fusarium solani NRC15, Fusarium oxysporium NRC23, Alternaria alternata NRC43 and Alternaria tenuissima KM651985). All microorganisms used were obtained from the culture collection of the Department of Chemistry of Natural and Microbial Products, National Research Center, Cairo, Egypt. The microorganisms were passaged at least twice to ensure purity and viability. The compounds were mounted on a paper disk prepared from blotting paper (5 mm diameter) on a concentration of 100 μg/5μL DMSO/disk. Thiophenicol and triflucan were used as positive controls for antibacterial and antifungal activities in a concentration of 50 μg/disk, and DMSO showed no inhibition zone used as a negative control.

Agar plates were prepared by using 100 ml of suspension containing 1 × 108 CFU/ml of pathological tested bacteria and 1 × 106 CFU/ml of fungi spread on nutrient agar (NA) and potato dextrose agar (PDA), respectively. After the media had cooled and solidified, the disks were applied on the inoculated agar plates and incubated for 24 h at 30 ºC for bacteria and 72 h at 28 ºC for fungi. After incubation, antimicrobial activity was evaluated by measuring the zone of inhibition around the disk in millimeters (mm) and compared with that of the controls. The observed zones of inhibition against the test microorganisms are presented in Table 4.

Minimal inhibitory concentration (MIC) measurement

The active compounds [having inhibition zones (IZ) >10 mm] were then evaluated for its minimal inhibitory concentration (MIC). The final concentrations tested were: 50, 25, 12.5, 6.25 and 3.13 μg. The lowest concentration showing inhibition zone around the disk was taken as the minimum inhibitory concentration (MIC).