Hetareno[e]pyrrole-2,3-diones and their derivatives include compounds exhibiting antimicrobial, analgesic, antiinflammatory, and antihypoxic activity [1,2,3,4]. The presence and structure of the pharmacophoric fragments are known to be largely responsible for the pharmacological effects of synthetic drugs. Considering the antituberculosis, antimicrobial, and antitumor activity of thiosemicarbazide derivatives [5,6,7,8], it seemed promising to synthesize new hetareno[e]pyrrole-2,3-diones containing a thiosemicarbazone fragment.

In continuation of research on the pharmacological activity of hetareno[e]pyrrole-2,3-diones, we studied reactions of pyrrolobenzoxazinetriones with a series of aromatic and heteroaromatic aldehyde thiosemicarbazones, i.e., benzaldehyde, o-fluoro and o-nitrobenzaldehydes, o- and p-hydroxybenzaldehydes [9], and nicotinic aldehyde thiosemicarbazone [10]. The structures of the synthesized compounds were studied using x-ray crystal structure analysis (XSA).

Their antinociceptive and antimicrobial activities were also studied.

Reactions of 3-aroylpyrrolo[1,2-c][4,1]benzoxazine-1,2,4-triones I-V with aromatic aldehyde thiosemicarbazones in a 1:1 ratio in refluxing anhydrous MeCN for 1.5 – 2 h produced 9-aroyl-8-hydroxy-2-{2-[2-benzylidene(pyrid-3-ylmethylene] hydrazono}-6-(2-hydroxyphenyl)-1-thia-3,6-diaz aspiro[4.4]non-8-en-4,7-diones VI-XXII (Scheme 1) [11].

scheme 1

Scheme 1

Reactions of 3-aroylpyrrolo[1,2-c][4,1]benzoxazine-1,2,4-triones with nicotinic aldehyde thiosemicarbazone were studied first. The reactions of 3-aroylpyrrolo[1,2-c]- [4,1]benzoxazine-1,2,4-triones I-V with nicotinic aldehyde thiosemicarbazone in a 1:1 ratio with stirring in dioxane at 60°C for 6 – 8 h produced 9-aroyl-8-hydroxy-2-(pyrid-3-ylmethylene) hydrazono-6-(2-hydroxyphenyl)-1-thia-3,6-diazaspiro[4.4]non-8-ene-4,7-diones XXIII-XXVII (Scheme 1).

Compounds VI-XXVII were colorless or light-yellow high-melting crystalline compounds that melted with decomposition that were soluble in DMSO, DMF, Me2CO, 1,2-dichloroethane, and EtOAc; slightly soluble in aromatic hydrocarbons and CHCl3; and insoluble in alkanes and H2O. They gave positive tests (wine-red color) for enol and phenol hydroxyl groups with ethanolic FeCl3.

IR spectra of VI-XXVII showed bands for OH and NH stretching vibrations (3400 – 3595 cm–1), lactam carbonyls C4=O and C7=O (1696 – 1775), and aroyl ketone carbonyl (1611 – 1643).

PMR spectra of VI-XXVII showed resonances for the aromatic-ring protons and the groups bonded to them, a singlet for the amide NH proton in the region of aromatic protons or after it (6.77 – 8.88 ppm), a singlet for the methine proton (8.19 – 8.44), a singlet for the phenol OH proton (9.64 – 9.77), and a broad singlet for the enol OH proton (12.22 – 12.62). PMR spectra of VI, VII, IX, X, XIV, and XVIII also exhibited a singlet for the hydroxybenzylidene OH proton (9.94 – 10.54).

The structures of the synthesized compounds were elucidated by an XSA of IX [11].

Apparently, VI-XXVII formed via initial addition of the thiolimide SH of salicylaldehyde thiosemicarbazone to the C3a atom of pyrrolidones I-V followed by closing of the thiazole ring as a result of intramolecular attack of the thiolimide NH2 at the lactone carbonyl of the benzoxazine ring and its opening at the C4–O5 bond.

The direction of the reaction was not affected if various substituents were introduced on the thiosemicarbazone aryl fragment. Table 1 presents the characteristics of the synthesized compounds.

Table 1. Physicochemical and Spectral Characteristics of VI–XXVII
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Experimental Chemical Part

PMR spectra were recorded with HMDS internal standard on Bruker Avance III HD 400 spectrometers (400 and 100 MHz, respectively). IR spectra were recorded from mineral-oil mulls on a PerkinElmer Spectrum Two spectrophotometer. Elemental analyses were obtained on a vario Micro cube analyzer. The purity of the synthesized compounds were confirmed by TLC on Sorbfil and Merck silica gel 60 F254 plates using MeOH–EtOAc (1:1) + 1% formic acid and toluene–EtOAc (5:1) eluents with detection by I2 vapor and UV light at 254 nm. Starting pyrrolidones I-V were synthesized from the corresponding enamines and oxalyl chloride using the literature method [12].

8-Hydroxy-6-(2-hydroxyphenyl)-2-[2-(4-methoxybenzylidene) hydrazono]-9-(4-methylbenzo)-1-thia-3,6-diazaspiro[4.4]non-8-ene-4,7-dione (XI). A solution of II (0.5 mmol) and anisaldehyde thiosemicarbazone (0.5 mmol) in anhydrous MeCN (10 mL) was stirred and refluxed for 2 h and cooled. The resulting precipitate was filtered off. Yield 64%, mp 212 – 214°C (MeCN). IR spectrum, ν, cm– 1: 3500 br (OH, NH), 3180 br (OHenol), 1740 (C7=O), 1711 (C4=O), 1625 (COC6H4Me-4). PMR spectrum, δ, ppm: 2.43 (s, 3H, OMe), 6.85 – 7.73 (gr.s, 13H, HAr, 3C6H4 + NHamid), 8.26 (s, 1H, CH), 9.73 (s, 1H, OHPhOH), 12.38 (br.s, 1H, OHenol). C28H22N4O6S.

Compounds XV and XVII-XXII were synthesized analogously. Compounds VI-X, XII-XIV, and XVI were previously reported [11].

9-Benzoyl-8-hydroxy-6-(2-hydroxyphenyl)-2-(pyrid-3-ylmethylene)hydrazono-1-thia-3,6-diazaspiro[4.4]non-8-ene-4,7-dione (XXIII). A solution of I (0.5 mmol) and nicotinic aldehyde thiosemicarbazone (0.5 mmol) in anhydrous 1,4-dioxane (10 mL) was stirred, refluxed for 7 – 8 h, and cooled. The resulting precipitate was filtered off. Yield 79%, mp 208 – 210°C (dioxane). IR spectrum, ν, cm– 1: 3340 br (OH, NH), 3188 (OHenol), 1746 (C7=O), 1707 (C4=O), 1637 (COPh). PMR spectrum, δ, ppm: 6.86 – 8.62 (gr.s, 13H, Ph + 2C6H4), 8.42 (s, 1H, CH), 8.82 (s, 1H, NHamid), 9.75 (s, 1H, OHPhOH), 12.49 (br.s, 1H, OHenol). C25H17N5O5S.

Compounds XXIV-XXVII were synthesized analogously.

Experimental Biological Part

Antinociceptive activity of the synthesized compounds was determined using laboratory female white mice (18 – 22 g) and thermal irritation of paws (hot plate test of Eddy and Leimbach) [13] (Table 2). Animals with an initial onset time of a defensive reflex of ≤15 sec were used in the test. The nociceptive parameter was the residence time in seconds of the animal on the hot plate until licking of hind paws, shaking them, or attempts to jump when placed on a metal plate heated to 55°C. The effect was assessed 0.5, 1, and 2 h after administration of the compounds before onset of the nociceptive response (latent period). Each compound was tested in six animals. Results were assessed from the increase of onset time of a defensive reflex as compared to the initial data.

Table 2. Antinociceptive Activity of VI-XXVII in the Hot Plate Test
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The tested compounds were injected i.p. at a dose of 50 mg/kg according to methodical recommendations for experimental (preclinical) studies of new drugs [14] as suspensions in starch paste (2%) 0.5 h before placing the animal on the instrument. The reference drugs were commercially available metamizole sodium (analgin) drug substance (Farmkhimkomplekt OOO) at a dose of 93 mg/kg, which corresponded to ED50 for i.p. injection [15, 16], and ibuprofen (Pharmaceutical Secondary Standard, Supelco) at a dose of 50 mg/kg.

Control animals were injected with the corresponding volume of starch paste (2%). Experimental results were statistically processed using the Student t-criterion. An effect was considered statistically significant for p ≤ 0.05 vs. the control and reference drugs [17].

Acute toxicities of the compounds were determined in mice of both sexes (18 – 22 g) with 10 animals in a group per dose. Tested compounds were injected i.p. in starch paste (3%) in the range 100 – 2,000 mg/kg. Animals were observed for 10 d. The external appearance (fur and skin condition), color of mucous membranes, behavior, demand for feed and water, and change of body mass during this time were noted. The number of deceased animals was recorded. The toxicity parameter was the mean lethal dose (LD50) causing the death of 50% of the animals at the end of the test. Control animals were injected i.p. with starch paste (3%). Acute toxicity was calculated according to recommendations of the State Pharmacological Committee for studies of general toxicity of biologically active compounds [14].

Test animals were kept under vivarium conditions (with natural lighting at 22 – 24°C and relative humidity 40 – 50%) on a standard diet (GOST R 50258-92). Experiments were conducted according to good laboratory practice (GLP) rules for preclinical studies in the RF (GOST 3 51000.3-96 and 1000.4-96) and rules of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (1986). Animals were quarantined for 10 – 14 d before the experiments.

The results showed that all tested compounds had LD50 values >2,000 mg/kg, which corresponded to the marginally toxic class according to the Sidorov classification [18].

Experimental results for antinociceptive activity (Table 2) were analyzed and showed that all 15 tested compounds at a dose of 50 mg/kg increased the onset time of a defensive reflex in mice that exceeded that of metamizole sodium and had activities comparable to that of ibuprofen. Compounds VI, XXII, and XXVII were most active with onset times of defensive reflexes of 24.60, 24.10, and 24.60 sec, respectively.

Antimicrobial activity of the synthesized compounds was determined using double serial dilutions in growth broth [14, 19] with various concentrations of the tested compounds.

The test cultures contained conditionally pathogenic microorganisms Staphylococcus aureus No. 906 and Escherichia coli No.1257. Initial bacterial dilutions were prepared in normal saline using one-day agar cultures according to the McFarland standard and a densitometer. The final cell concentration after a series of dilutions was 2.5 × 105 microbes/mL.

Cultures (150 μL) were inoculated into the prepared series of dilutions of the tested compounds dissolved in DMSO (150 μL) in the microplate wells containing the particular concentrations of the tested compounds. The last rows contained equal volumes of growth medium and culture (controls). The maximum tested concentration of the compounds was 1,000 μg/mL.

The microplates were cultivated at 37 ± 1°C in a thermostat for 24 h and 7 d and placed into an Epoch spectrophotometer. The optical density (OD) of the culture fluid was measured at 540 nm.

The dependence of the OD on microorganism cell growth at the various concentrations of the compounds was found using Gen 5 software of the Epoch microplate spectrophotometer. The last well with growth inhibition in a series corresponded to the minimum inhibitory concentration of the compound.

Table 3 shows the analyzed results and indicates that Gram-positive S. aureus was more sensitive to the action of the studied compounds. The cultures were moderately sensitive to VIII, XVII, XXII, XXIV, and XXVII, the bacteriostatic activities of which fell in the range 125 – 500 μg/mL.

Table 3. Antimicrobial Activity of VI-XXVII
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Compounds VII, IX, X, XII, XIII, XVI, and XX had inhibitory concentrations for S. aureus of 31.2 – 62.5 μg/mL, which was comparable to that of dioxidine. However, the bactericidal activity of the compounds started at a concentration ≥500 μg/mL.

Compound XIV had the highest antimicrobial activity and inhibited growth of S. aureus culture at a concentration of 7.8 μg/mL with MBC of 500 μg/mL, which was more active than dioxidine.

Thus, the biological activities of the synthesized compounds confirmed that a search for new compounds among pyrrolobenzoxazinetrione derivatives was advisable.