Synthesis of furo[2',3':4,5]pyrrolo[1,2-d ][1,2,4]triazine derivatives and their antibacterial activity

Furo[2',3':4,5]pyrrolo[1,2-d ][1,2,4]triazin-8(7 H )-ones 5 were synthesized either by reaction of carbohydrazide 2 with triethyl orthoesters or by acetylation of methyl 4 H -furo[3,2-b ]pyrrole-5-carboxylate 1 followed by thionation of 4-acetylfuro[3,2-b ]pyrrole-5-carboxylate 3 and cyclisation of thione 4 with hydrazine. Triazine 5a afforded the corresponding thione 7 by reaction with P 2 S 5 . Upon reaction with alkyl-or acylhalogenides compounds 5 and 7 gave N (7)-substituted products 6 and 8 , respectively. Finally, triazino-triazinone derivative 9 was synthesized by cyclisation of thione 8b with hydrazine. Compounds 5 - 9 were evaluated for their antibacterial activity.


Introduction
The synthesis and the study of physical and chemical properties of pyrrolo-fused heteroaryl compounds, such as furo-, thieno-and selenopyrroles has been well documented over the last few decades.Furo [3,2-b]pyrroles are isosteres of the indole ring system in which the benzene ring is replaced by the furan ring.Efficient synthetic routes to these heterocycles are of great interest [1][2][3][4] as the furo [3,2-b]pyrrole core has been found in compounds with diverse biological activities [5][6][7] or they are used as the fluorescent dyes 8 .
In order to exploit the new route to the formation of triazine ring of 5c, we have realized N-acetylation of 1 by method of Krutošíková 20 in acetic anhydride for 4 h, when methyl N-acetylfuro [3,2-b]pyrrole-5-carboxylate 3 was obtained in 60% yield.Compound 3 was subsequently converted to the corresponding thione 4 in 60% yield by reaction with phosphorus pentasulfide in pyridine for 10 h.Finally, thione 4 cyclised into 5c in 65% yield by heating with hydrazine hydrate in ethanol (Scheme 1, path b).

Scheme 1. Synthesis of triazine derivatives 5
Although the yields of 5c by the path b (65%) and the path a (71%), were comparable, path b requires long reaction time (168h) and this fact limits the applicability of this approach.
Compound 3 displays in its 1 H NMR spectrum doublet due to H-2 proton at 7.79 ppm (J = 2.4 Hz), doublet of doublets of the pyrrole H-6 proton at 6.76 ppm (J = 2.4, 0.9 Hz) and doublet of the H-3 proton at 6.61 ppm (J = 2.4, 0.9 Hz).The most distinct signals of 3 in the 13 C NMR spectrum were the carbonyl carbons at 168.9 and 162.1 ppm.
The structures of compounds 6a-6e were established by 1 H NMR, 13 C NMR and IR spectroscopy.They display in their 1 H NMR spectra, in addition to other signals, singlets of H-5 protons at 8.81-8.83ppm, doublets of the pyrrole H-2 protons in the region of 7.99-8.09ppm (J = 2.4 Hz), H-3 protons resonate as doublets of doublets at 7.09-7.11ppm (J = 2.1, 0.9 Hz).The H-9 protons appear as singlets at 7.14-7.20 ppm. 13C NMR spectra show the signals of C-8 carbonyl carbons at 153.9-154.6 ppm, region, signals of C-5 carbon at 149.7-151.6 ppm region.In the spectrum of 6e there is the signal of the carbonyl carbon of acetyl group at 170.6 ppm and carbonyl carbon of methoxycarbonyl group of 6d appears at 168.2 ppm.The characteristic bands observed at 1641-1652 cm -1 in IR spectra correspond to the C=O groups at C-8. Scheme 2. Synthesis of triazines 6-8 and triazino-triazinone 9.
The structures of compounds 7 and 8 were established by 1 H NMR, 13 C NMR and IR spectroscopy. 1 H NMR spectra show doublets of H-5 protons at 10.98-9.16ppm region (J = 0.6 Hz), doublets of the pyrrole H-2 protons in the region of 8.09-8.31ppm (J = 2.1 Hz), H-3 protons resonate as doublets of doublets at 7.15-7.34ppm (J = 2.1, 0.9 Hz).The H-9 protons appear as singlets at 7.76-7.29 ppm. 13C NMR spectra show the signals of C-8 thione carbons at 160.3-173.3ppm and signals of C-5 carbon at 144.8-150.9ppm.The characteristic band observed at 1257 cm -1 in IR spectrum of 7 corresponds to the C=S group at C-8.
Antibacterial activity.Compounds 2, 3, 5a, 5c, 6a, 6b, 6c, 6d, 7, 8a, 8b and 9 were screened on their antibacterial activity on G -bacterial species Escherichia coli CCM 7929, Pseudomonas syringae CCM 2114 and G + bacterial species Micrococcus luteus CCM 732, Bacillus pumilus CCM 2218.Antibacterial activity of all tested structures has been compared to standard 6-aminopenicillanic acid (6-APA) as known building block of successful antibiotics -penicillins.The result of antibacterial activity is presented in Table 1.As it is shown in Table 1, only compounds 5a, 5c and 6b exhibit higher antibacterial activity than standard 6-APA on all bacterial strains with MIC values in the range 0.016 -2.56 mM.Moderate antibacterial activity against Micrococcus luteus has been observed for compounds 5a and 6a (MIC values 0.016 and 0.024 mM, respectively) and compounds 3 and 6d showed poor antibacterial activity against Micrococcus with MIC value 0.64 mM.
From structural point of view, compounds with lower molecular weight expressed a higher measure of antibacterial activity and the derivatization of the N-7 nitrogen atom of triazine ring has been concluded as counter-productive in relation to antibacterial activity.According to Lipinski's rule of five 21,22 the trend of increasing antibacterial activity indicates the predominance of the hydrogen bond acceptors over the hydrogen bond donors, comparing the results between compounds 2 and 3.As it is shown in Table 1, all tested compounds were more effective on two G + bacterial species (M.luteus and B. pumilus) than on G ─ bacterial targets (E. coli and P. syringae CCM 2114), what is typical for inhibitors of bacterial cell wall peptidoglycan synthesis like -lactam antibiotics.

Experimental Section
General.Melting points of products were determined on a Kofler hot plate apparatus and are uncorrected. 1H NMR / 13 C NMR spectra were obtained on a 300 MHz/75 MHz spectrometer VARIAN GEMINI 2000 in DMSO-d6 with tetramethylsilane as the internal standard.The infrared spectra were taken on a FTIR IRAffinity-1 spectrophotometer using KBr technique.Elemental analyses were performed on FlashEA 2000 CHNS/O-OEA analyser.MS spectra were measured at Agilent Technologies 1200 Series apparatus.All solvents were distilled and dried appropriately prior to use.The course of reactions was monitored by TLC in ethyl acetate -hexane.

Determination of Minimal Inhibition Concentration (MIC) parameters.
On determination of MIC parameters there were used sterile microplates (type P), where the suspension of bacterial species in nutrient broth medium with dissolved tested compound has been achieved by convenient dilution method using automatic multichannel pipets.The concentration from the column 1 to column 12 was in the decreasing order: 20.48 mM; 10.24 mM; 5.12 mM; 2.56 mM; 1.28 mM; 0.64 mM; 0.32 mM; 0.16 mM; 0.08 mM; 0.04 mM; 0.02 mM and 0.01 mM.The inoculum concentration of bacterial species suspension in nutrient broth medium was before filling set by McFarland Densitometer DEN-1 (UK) on the value 0.1.The first two rows A and B were occupied by the standard 6-aminopenicilanic acid (6-APA) on each microplate and the tested compounds were in the rows C-G.After 24h of cultivation at 37 °C in the bacteriological thermostat 30 µl of 0.03% solution of Thiazolyl Blue (MTT) in water was added to each well and incubated again for 1h under the same conditions.Bacterial proliferation led to the production of bacterial mitochondrial dehydrogenase, which turned yellow colored solution of MTT to intensively blue colored formazan product.MIC parameter was identified visually as the last not colored well in the row.All experiments were carried out as triplicate.