Introduction

Ketoprofen (Ket) is a non-steroidal anti-inflammatory drug (NSAID) with pronounced analgesic and antipyretic properties. Numerous ketoprofen derivatives have been synthesized in order to minimize side-effects, prolong plasma half-life and increase solubility (Bonina et al., 2002a, b, 2003). Some amides have proved to be useful prodrugs, while the others possess anti-inflammatory activity independent of the parent compound. It has been demonstrated that amidation of NSAIDs improves selectivity towards COX-2 (Kalgutkar et al., 2000), while modification of the carboxylic group to hydroxamic acid leads to inhibition of both cyclooxygenase and 5-lipoxygenase, two enzymes crucial in inflammatory processes (Flynn et al., 1990; Muri et al., 2002). Glycine amides of ketoprofen and several other well-known NSAIDs are significantly less irritating to gastric mucosa, while their anti-inflammatory activities are comparable to their parent drugs (Shanbhag et al., 1992; Singh et al., 1990). Ketoprofen glycinate methyl ester has higher anti-inflammatory and analgesic activity than the parent drug (Dhaneshwar and Chaturvedi, 1994). Ketoprofenamides with heterocyclic residues (2-thiazolinyl, 4-methylpyridyl, 3-hydroxypyridyl, pyridyl, 1,5-dimethyl-2-phenylpyrazolonyl or thiazolyl) also possess significant analgesic and anti-inflammatory activities (Spickett et al., 1976), while ketoprofen 2-hydroxyethylamide and ketoprofen esters with bis-(hydroxyalkylthio)-alkanes are useful in the treatment and prevention of atherosclerosis (Lafon, 1977). Numerous studies suggest that NSAIDs are promising anticancer drugs as well and may be associated with reduced risk of colon, lung, liver and other types of cancers (Thun et al., 2002; Sivak-Sears et al., 2004).

In our previous research potency of amides and hydroxamic acid derivatives of ketoprofen and related NSAIDs as cytostatic and antioxidant agents was screened (Zovko et al., 2003; Marjanović et al., 2007; Wittine et al., 2009). Our articles and the extensive literature data describe the effect of carboxylic group derivatization. To our knowledge, a modification of both carboxylic and carbonyl functionalities in ketoprofen molecule has not been studied. In this article, a series of novel derivatives of ketoprofen bearing amide and carbamate moieties were prepared, characterized and screened for their antioxidative, cytostatic and antiviral activities.

Results and discussion

Chemistry

Benzotriazolides 3a,b were prepared from the reduced ketoprofen derivative 2 and 1-benzotriazole carboxylic acid chloride (1), following our previously developed procedure (Scheme 1) (Butula and Jadrijević Mladar Takač, 2000; Zorc et al., 1993). If the reaction was performed with one equivalent of chloride 1, product 3a with free hydroxy group was obtained in 75% yield. When the reaction was carried out with two equivalents of chloride 1, the main product was benzotriazolide 3b, in which both carboxylic and hydroxy groups were acylated. Minor amount of product 3a was detected as well, even if the reaction was performed with the excess of chloride 1. The reaction was run at room temperature in order to avoid benzotriazolide 3a polycondensation.

Scheme 1
scheme 1

Synthesis of compounds 4a–j

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Compounds 4a–j were prepared by the reaction of benzotriazolide 3b with two equivalents of an appropriate amine, in the presence of five equivalents of triethylamine (Scheme 1). All reactions were performed in toluene, at room temperature, for 0.5–48 h. Triethylamine formed a water soluble salt with benzotriazole, a by-product of the reaction, which was readily extracted with water.

Structures of compounds 3a,b and 4a–j were deduced from the analysis of their IR, 1H- and 13C-NMR spectra and confirmed by the elemental analysis. The chemical shifts were consistent with the proposed structures of the novel compounds (Fig. 1, Table 1).

Fig. 1
figure 1

Atom enumeration of compounds 3a and 3b

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Table 1 1H- and 13C-NMR data for compounds 4a–j
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Biological studies

Antioxidant activity

The interaction of the examined compounds with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) was studied. Interaction with DPPH indicates radical scavenging ability in an iron-free system. Interactions were monitored after 20 and 60 min at two concentrations of DPPH (0.05 and 0.1 mM). Ketoprofen, the prototype compound, benzotriazolide 3b as well as all the tested compounds presented very low interaction values. The results are shown in Table 2.

Table 2 Interaction with DPPH, in vitro inhibition of soybean lipoxygenase (LOX) and lipid peroxidation (LP)
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Soybean lipoxygenase inhibition

Compounds were further evaluated for the inhibition of soybean lipoxygenase (LOX) by the UV absorbance based enzyme assay (Pontiki and Hadjipavlou-Litina, 2007). Lipoxygenases oxidize certain fatty acids at specific positions to hydroperoxides, precursors of leukotrienes, which contain a conjugated triene structure, i.e. soybean lipoxygenase converts linoleic to 13-hydroperoxylinoleic acid. Leukotrienes play an important role as mediators of a variety of inflammatory and allergic processes (Kühn et al., 1990). Inhibitors of LOX have attracted attention initially as potential agents for the treatment of inflammatory and allergic diseases but their therapeutic potential has now been expanded to certain types of cancer and cardiovascular diseases (Pontiki and Hadjipavlou-Litina, 2005). Most of the LOX inhibitors are antioxidants or free radical scavengers, since lipoxygenation occurs via a carbon-centred radical (Muller, 1994). Perusal of IC50 values shows that compound 3b is the most active by far, followed by compounds 4g, 4d and 4f (IC50 = 21–95 μM). From Table 2 it is obvious that aromatic and cycloalkyl derivatives 4g, 4d and 4f are more potent lipoxygenase inhibitors than the other amidocarbamates.

Inhibition of linoleic acid lipid peroxidation

Azo compounds generating free radicals through spontaneous thermal decomposition are useful for free radical production studies in vitro. The water soluble azo compound 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) has been extensively used as a clean and controllable source of thermally produced alkylperoxyl free radicals. In our studies, AAPH was used as a free radical initiator to follow oxidative changes of linoleic acid to conjugated diene hydroperoxide. The results indicated that all the compounds are excellent inhibitors of lipid peroxidation (LP) (54.5–99.5%), significantly higher than ketoprofen (69.3%) at 0.1 mM concentration (Table 2). This inhibition was found to be concentration dependent.

Regression analysis of the LP inhibition at 100 μM revealed that the overall molar refractivity (CMR) is the main physicochemical parameter influencing the inhibition. The linear CMR model suggests that the compounds with high CMR value will be more active. No correlation for lipophilicity was found.

$$ \log \;{\text{LP}}\% = 0.047(0.018)\;{\text{CMR}}-1.317(0.234) $$
$$ n = 11,\;r = 0.894,\;r^{2} = 0.800,\;q^{2} = 0.680,\;s = 0.045,\;F_{1,8} = 31.555,\;\alpha = 0.01 $$

Antiviral and cytostatic evaluation

Antiviral evaluation was performed on a broad series of DNA and RNA viruses (as listed under the “Experimental” section). No antiviral effects were noted for any of the tested compounds against any of the viruses evaluated at subtoxic compound concentrations (data not shown). Only 4g showed minor antiviral activity against vesicular stomatitis virus in HeLa cell cultures (EC50: 3 μM), Sindbis virus (EC50: 11 μM) and Punta Toro virus in Vero cell cultures (EC50: 11 μM). However, the activity was found at compound concentrations close to their cytostatic activities (CC50: 2.7–14 μM) (Table 3), pointing to a toxic rather than a specific antiviral effect. The compounds have also been evaluated for their cytostatic activity against murine leukaemia L1210, murine mammary carcinoma FM3A and human T-lymphoblast CEM cell cultures. The 50% inhibitory concentrations of the test compounds ranked between 2.7 and 422 μM depending on the nature of the compound and the tumour cell line evaluated (Table 3). The majority of the compounds show IC50 values around 10–25 μM (i.e. 4c, 4d, 4e, 4f, 4g, 4h and 4j), pointing to a relatively minor role of the R-substituents on the core structure for cytostatic activity, as long as a bulky lipophilic (cyclic) entity has been present. Also, the presence of the amide groups might play an important role to eventually exert cytostatic potential.

Table 3 Cytostatic activity of the test compounds in cell cultures
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Conclusions

A series of novel ketoprofen amidocarbamate derivatives 4a–j were prepared and screened for antioxidative, antiviral and cytostatic activities. Antioxidative screenings revealed that the prepared compounds possess excellent LP inhibition. Compounds 3b and 4g also showed high soybean lipoxygenase inhibition activity. No selective antiviral effects were noted for the tested compounds against a broad variety of DNA and RNA viruses. Most compounds were endowed with a moderate cytostatic activity.

Experimental

Melting points were determined on a Stuart Melting Point Apparatus SMP3 and were uncorrected. IR spectra were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer. 1H- and 13C-NMR spectra were recorded on a Varian Gemini 300 spectrometer, operating at 300 and 75.5 MHz for the 1H and 13C nuclei, respectively. Samples were measured in DMSO-d 6 solutions at 20°C in 5-mm NMR tubes. Chemical shifts (δ) were referred to TMS. Coupling constants (J) are given in Hz. Elemental analysis was determined on CHN-LECO-932. For thin-layer chromatography, precoated Merck silica gel 60 F254 and solvent system cyclohexane/ethyl acetate/methanol (3:1:0.5) were used. Spots were visualized by short-wave UV light and iodine vapour. Column chromatography was performed on Merck silica gel 0.063–0.200 mm with cyclohexane/ethyl acetate (1:2, 2:1 → 1:1, 1:1) as eluents. O-ethylhydroxylamine hydrochloride was obtained from Fluka and ketoprofen from PLIVA. All chemicals were purchased from Sigma-Aldrich. All solvents were of analytical grade purity and were dried prior to use.

1-Benzotriazole carboxylic acid chloride (BtCOCl, 1)

Solution of benzotriazole (1.191 g, 10 mmol) and triphosgene (2.523 g, 8.5 mmol) in dry toluene was refluxed for 3 h. The reaction mixture was evaporated under reduced pressure. The crude product was used in the following reactions without further purification (Butula and Jadrijević Mladar Takač, 2000).

2-(3-(Hydroxy(phenyl)methyl)phenyl)propanoic acid (2)

2-(3-(Hydroxy(phenyl)methyl)phenyl)propanoic acid (2) was prepared by the catalytic hydrogenation of ketoprofen using H2/Pd/C(en)/tetrahydrofurane (Hattori et al., 2001), according to the modified published procedure (Allegretti et al., 2003; 2005).

2-(3-(Hydroxy(phenyl)methyl)phenyl)propanoic acid benzotriazolide (3a)

To a solution of 2 (2.561 g, 10 mmol) and triethylamine (1.4 ml, 10 mmol) in dry toluene (20 ml), a solution of chloride 1 (1.696 g, 10 mmol) in dry toluene (20 ml) was added dropwise (0.25 h). The reaction mixture was stirred at room temperature for 1 h and washed four times with water. The organic layer was dried over anhydrous sodium sulphate, filtrated and evaporated. Thus, obtained crude product was purified by the trituration with ether. Yield: 2.681 g (75%); mp 96–99°C; IR (KBr): ν max 3342, 3072, 3027, 3003, 2942, 2874, 1738, 1060, 1597, 1486, 1452, 1376, 959, 771, 751, 746, 710 cm−1; 1H-NMR (DMSO-d 6) δ 8.26–8.21 (m, 2H, arom.), 7.80–7.75 (m, 1H, arom.), 7.62–7.57 (m, 1H, arom.), 7.52–7.50 (m, 1H, arom.), 7.33–7.14 (m, 8H, arom.), 5.88 (d, 1H, 11, J = 4.00 Hz), 5.65 (d, 1H, 10, J = 3.83 Hz), 5.32 (q, 1H, 2, J = 6.89 Hz), 1.63 (d, 3H, 3, J = 6.94 Hz); 13C-NMR (DMSO-d 6) δ 173.50 (1), 146.82, 145.88, 145.83, 140.01, 131.18 (4, 8, 12, 1′, 6′), 131.39, 129.08, 128.45, 127.14, 127.02, 126.66, 126.63, 126.58, 125.84, 120.53, 114.44 (5–7, 9, 13–17, 2′–5′), 74.49 (10), 45.12 (2), 19.07 (3). Atom enumeration is given in Fig. 1. Anal. Calcd. for C22H19N3O2: C, 73.93; H, 5.36; N, 11.76. Found: C, 73.64; H 5.39; N, 11.39.

2-(3-(N-benzotriazolcarbonyloxy)(phenyl)methyl)phenyl)propanoic acid benzotriazolide (3b)

To a solution of 2 (2.561 g, 10 mmol) and triethylamine (3.9 ml, 28 mmol) in dry toluene (50 ml), a solution of chloride 1 (5.080 g, 28 mmol) in dry toluene (50 ml) was added dropwise (0.25 h). The reaction mixture was stirred at room temperature for 1 h and washed four times with water. The organic layer was dried over anhydrous sodium sulphate, filtrated and evaporated. Thus, obtained crude products was purified by the trituration with ether. Yield: 3.52 g (70%); mp 124–127°C; IR (KBr): ν max 3091, 3032, 2980, 2937, 1764, 1732, 597, 1486, 1451, 1398, 1250, 1036, 951, 781, 760, 748, 708, 583 cm−1; 1H-NMR (DMSO-d 6) δ 8.28–7.98 (m, 3H, arom.), 7.78–7.28 (m, 15H, arom.), 7.24 (s, 1H, 10), 5.38 (q, 1H, 2, J = 6.84 Hz), 1.66 (d, 3H, 3, J = 6.94 Hz); 13C-NMR (DMSO-d 6) δ 173.28 (1), 147.91 (1′′), 145.80, 145.77, 140.72, 140.02, 139.17, 131.73 (4, 8, 11, 1′, 6′, 2′′, 7′′), 131.36, 131.11, 129.83, 129.20, 128.29, 127.36, 127.23, 126.97, 126.79, 126.55, 126.38, 120.70, 120.45, 114.39, 113.67 (5–7, 9, 12–16, 2′–5′, 3′′–6′′), 81.48 (10), 45.04 (2), 18.98 (3). Atom enumeration is given in Fig. 1. Anal. Calcd. for C29H22N6O3: C, 69.31; H, 4.41; N, 16.72. Found: C, 69.39; H 4.63; N, 16.99.

(3-(1-(Methoxycarbamoyl)ethyl)phenyl)(phenyl)methyl methoxycarbamate (4a)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), O-methylhydroxylamine hydrochloride (0.092 g, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 10 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. Thus, obtained the crude product was purified by the column chromatography (eluent cyclohexane/ethyl acetate 1:2). Yield: 0.143 g (80%); oil; IR (film): ν max 3455, 3217, 3065, 2978, 2938, 1725, 1668, 1606, 1489, 1454, 1254, 1115, 1043, 705 cm−1. Anal. Calcd. for C19H22N2O5: C, 63.67; H, 6.19; N, 7.82. Found: C, 63.75; H 6.03; N, 7.48.

(3-(1-(Ethoxycarbamoyl)ethyl)phenyl)(phenyl)methyl ethoxycarbamate (4b)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), O-ethylhydroxylamine hydrochloride (0.107 g, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 25 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. Thus, obtained the crude product was purified by the column chromatography (eluent cyclohexane/ethyl acetate 2:1 → 1:1). Yield: 0.140 g (73%); oil; IR (film): ν max 3454, 3219, 3064, 2981, 2937, 2891, 1724, 1715, 1668, 1606, 1494, 1454, 1384, 1253, 1113, 1041, 704 cm−1. Anal. Calcd. for C21H26N2O5: C, 65.27; H, 6.78; N, 7.25. Found: C, 65.34; H 6.43; N, 7.29.

(3-(1-(Benzyloxycarbamoyl)ethyl)phenyl)(phenyl)methyl benzyloxycarbamate (4c)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), O-benzylhydroxylamine hydrochloride (0.175 g, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 48 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude product was purified by the column chromatography (eluent cyclohexane/ethyl acetate 1:1). Yield: 0.115 g (45%); mp 42–45°C; IR (KBr): ν max 3215, 3064, 3032, 2974, 2936, 1722, 1665, 1605, 1495, 1454, 1249, 1107, 1027, 749, 699 cm−1. Anal. Calcd. for C31H30N2O5: C, 72.92; H, 5.92; N, 5.49. Found: C, 73.00; H 6.04; N, 5.77.

(3-(1-(Cyclopentylcarbamoyl)ethyl)phenyl)(phenyl)methyl cyclopentylcarbamate (4d)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), cyclopentylamine (0.109 ml, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 1 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude product was triturated with ether several times. Yield: 0.167 g (77%); mp 132–135°C; IR (KBr): ν max 3305, 3269, 3065, 2962, 2870, 1700, 1651, 1606, 1545, 1452, 1249, 1040, 1017, 702 cm−1. Anal. Calcd. for C27H34N2O3: C, 74.62; H, 7.89; N, 6.45. Found: C, 74.39; H 7.76; N, 6.28.

(3-(1-(Cyclohexylcarbamoyl)ethyl)phenyl)(phenyl)methyl cyclohexylcarbamate (4e)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), cyclohexylamine (0.126 ml, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 5 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude product was triturated with ether several times. Yield: 0.173 g (75%); mp 139–142°C; IR (KBr): ν max 3303, 3265, 3066, 2933, 2854, 1695, 1650, 1603, 1547, 1450, 1235, 1042, 702 cm−1. Anal. Calcd. for C29H38N2O3: C, 75.29; H, 8.28; N, 6.06. Found: C, 75.55; H 8.01; N, 6.16.

(3-(1-(Cyclohexanemethylcarbamoyl)ethyl)phenyl)(phenyl)methyl cyclohexanemethylcarbamate (4f)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), cyclohexanemethylamine (0.143 ml, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 0.5 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude product was triturated with ether several times. Yield: 0.184 g (75%); mp 128–129°C; IR (KBr): ν max 3340, 3278, 3064, 2922, 2851, 1707, 1654, 1604, 1551, 1449, 1249, 702 cm−1. Anal. Calcd. for C31H42N2O3: C, 75.88; H, 8.63; N, 5.71. Found: C, 75.58; H 8.22; N, 5.99.

(3-(1-(Benzylcarbamoyl)ethyl)phenyl)(phenyl)methyl benzylcarbamate (4g)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), benzylamine (0.120 ml, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 0.5 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude product was triturated with ether several times. Yield: 0.127 g (53%); mp 107–109°C; IR (KBr): ν max 3316, 3267, 3087, 3063, 3032, 2932, 1684, 1641, 1606, 1551, 1519, 1454, 1248, 699 cm−1. Anal. Calcd. for C31H30N2O3: C, 77.80; H, 6.32; N, 5.85. Found: C, 77.66; H 6.48; N, 6.01.

(3-(1-(Phenylethylcarbamoyl)ethyl)phenyl)(phenyl)methyl phenylethylcarbamate (4h)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), phenylethylamine (0.139 ml, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 0.6 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The pure product was obtained after trituration with ether. Yield: 0.190 g (75%); oil; IR (KBr): ν max 3417, 3313, 3063, 3028, 2972, 2932, 2872, 1713, 1699, 1660, 1650, 1604, 1538, 1517, 1496, 1454, 1248, 1030, 749, 700 cm−1. Anal. Calcd. for C33H34N2O3: C, 78.23; H, 6.76; N, 5.53. Found: C, 78.47; H 6.44; N, 5.70.

(3-(1-Oxo-1-(pyrrolidin-1-yl)propan-2-yl)phenyl)(phenyl)methyl pyrrolidine-1-carboxylate (4i)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), pyrrolidine (0.092 ml, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 0.5 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude product was purified by the column chromatography (eluent cyclohexane/ethyl acetate 1:1). Yield: 0.152 g (75%); oil; IR (film): ν max 3061, 3030, 2973, 2875, 1700, 1643, 1634, 1588, 1454, 1416, 1126, 1096, 764, 709 cm−1. Anal. Calcd. for C25H30N2O3: C, 73.86; H, 7.44; N, 6.89. Found: C, 73.57; H 7.67; N, 6.80.

(3-(1-Oxo-1-(piperidin-1-yl)propan-2-yl)phenyl)(phenyl)methyl piperidine-1-carboxylate (4j)

A solution of benzotriazolide 3b (0.251 g, 0.5 mmol), piperidine (0.109 ml, 1.1 mmol) and triethylamine (0.35 ml, 2.5 mmol) in toluene (5 ml) was stirred at room temperature for 0.75 h. The reaction mixture was extracted with brine (5 × 10 ml), 1% hydrochloric acid (1 × 5 ml) and washed with water till pH 7. The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. After trituration with ether afforded the pure product. Yield: 0.179 g (80%); mp 106–109°C; IR (KBr): ν max 3050, 3028, 2971, 2940, 2852, 1694, 1628, 1587, 1469, 1426, 1258, 1236, 1148, 1083, 1026, 707, 699 cm−1. Anal. Calcd. for C27H34N2O3: C, 74.62; H, 7.89; N, 6.45. Found: C, 74.36; H 7.63; N, 6.55.

Interaction with DPPH

To a solution of DPPH (0.05 mM) in absolute ethanol an equal volume of 0.1 or 0.05 mM ethanolic solution of the tested compound was added (Pontiki and Hadjipavlou-Litina, 2007). After 20 and 60 min the absorbance was recorded at 517 nm and compared to the appropriate standard NDGA (Table 2). Ethanol was used as a control. Each in vitro experiment was performed at least in triplicate and the standard deviation of absorbance was <10% of the mean.

Soybean lipoxygenase inhibition

DMSO solution of the tested compound was incubated with sodium linoleate (0.1 mM) and 0.2 ml of soybean lipoxygenase solution (1/9 × 10−4 w/v in saline) at room temperature (Pontiki and Hadjipavlou-Litina, 2007). The conversion of sodium linoleate to 13-hydroperoxylinoleic acid was recorded at 234 nm and compared to the standard inhibitor caffeic acid, according to the procedure previously reported.

Inhibition of linoleic acid lipid peroxidation

Oxidation of linoleic acid to conjugated diene hydroperoxide in an aqueous dispersion is monitored at 234 nm (Re et al., 1999). AAPH was used as a free radical initiator. Ten microliters of the 16 mM linoleic acid dispersion was added to the UV cuvette containing 0.93 ml of 0.05 M phosphate buffer, pH 7.4 prethermostated at 37°C. The oxidation reaction was initiated at 37°C under air by the addition of 50 μl of 40 mM AAPH solution. Oxidation was carried out in the presence of compounds (10 μl, final concentration 0.1 mM). In the assay with no antioxidant lipid oxidation was measured in the presence of the same level of DMSO. The rate of oxidation was monitored at 37°C by recording the increase of absorption at 234 nm caused by conjugated diene hydroperoxides. The results were compared to the standard inhibitor trolox.

Antiviral and cytostatic activity assays

Murine leukaemia L1210, murine mammary carcinoma FM3A and human T-lymphocyte CEM cells were suspended at 300,000–500,000 cells/ml of culture medium, and 100 μl of a cell suspension was added to 100 μl of an appropriate dilution of the test compounds in wells of 96-well microtiter plates. After incubation at 37°C for 2 (L1210, FM3A) or 3 (CEM) days, the cell number was determined using a Coulter counter. The IC50 was defined as the compound concentration required to inhibit cell proliferation by 50%.

The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-induced cytopathicity in HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus and vesicular stomatitis virus], Vero (parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus), feline kidney Crandell cells [feline coronavirus (FeCoV)] (FIPV strain) and feline herpes virus (FeHV) or MDCK [influenza A (H1N1, H3N2) and influenza B] cell cultures. Most viruses have been obtained from ATCC (Rockville, MD). HIV-1(IIIB) was provided by R. C. Gallo (at that time at NIH, Bethesda, MD) and HIV-2(ROD) was provided by L. Montagnier (at that time at the Pasteur Institute, Paris, France). FCoV (FIPV) and FeHV were kindly provided by H. Egberink, Utrecht, The Netherlands. Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (CCID50 being the virus dose to infect 50% of the cell cultures). After a 1 h virus adsorption period, residual virus was removed, and the cell cultures were incubated in the presence of varying concentrations (200, 40, 8, … μM) of the test compounds. Viral cytopathicity was recorded as soon as it reached completion in the control virus infected cell cultures that were not treated with the test compounds. The methodology of the anti-HIV assays was as follows: human CEM (~3 × 105 cells/ml) cells were infected with 100 CCID50 of HIV-1(IIIB) or HIV-2(ROD)/ml and seeded in 200 μl wells of a microtiter plate containing appropriate dilutions of the test compounds. After 4 days of incubation at 37°C, HIV-induced CEM giant cell formation was examined microscopically.