Annellation of Triazole and Tetrazole Systems onto Pyrrolo[2,3-d]pyrimidines: Synthesis of Tetrazolo[1,5-c]-pyrrolo[3,2-e]-pyrimidines and Triazolo[1,5-c]pyrrolo-[3,2-e]pyrimidines as Potential Antibacterial Agents

Syntheses of several novel 4-chloropyrrolo[2,3-d]pyrimidines (1), 4- hydrazinopyrrolo[2,3-d]pyrimidines (2) and 3-amino-4-iminopyrrolo[2,3-d]pyrimidines (7) and their use in the synthesis of tetrazolo[1,5-c]pyrrolo[3,2-e]pyrimidines (3) and triazolo[1,5-c]pyrrolo[3,2-e]pyrimidines (4) required for biological screening are reported.


Results and Discussion
Reaction between 1 and sodium azide in presence of ammonium chloride and DMSO as a solvent afforded the target tetrazolopyrrolopyrimidines 3. Ammonium chloride was used for in situ generation of ammonium azide and the reaction was found to be incomplete in the absence of ammonium chloride. The same 7,9-disubstituted 7H-tetrazolo [1,5-c]pyrrolo [3,2-e]pyrimidines (3) can also be synthesized by the diazotization of 5,7-disubstituted 4-hydrazino-7H-pyrrolo [2,3-d]pyrimidines (2) [17][18][19], obtained from 1 on treatment with hydrazine hydrate in hot alcohol. Thus obtained compounds 2 were reacted with sodium nitrite in acetic acid under cooling (0-5 °C) to give the same tetrazolopyrrolopyrimidines 3. The complete reaction sequences leading to compounds 3 is depicted in Scheme The synthesis of novel 7,9-disubstituted 7H-triazolo [1,5-c]pyrrolo [3,2-e]pyrimidines (4) has also been carried out from 2 upon reaction with hot formic acid. The same triazolopyrrolopyrimidines 4 have been prepared by another method, starting from 2-amino-3-cyanopyrroles (5) [17][18][19][20][21], whereby compounds 5 were treated with triethyl orthoformate giving 1,4-disubstituted N-ethoxymethylene 2amino-3-cyanopyrroles (6), which on condensation with hydrazine hydrate afforded 5,7-disubstituted 3-amino-4-imino-7H-pyrrolo[2,3-d]pyrimidines (7). Cyclocondensation of 7 with hot formic acid or triethyl orthoformate resulted in the formation of the same 7,9-disubstituted 7H-triazolo [1,5c]pyrrolo [3,2- The identity of compounds 3 and 4 obtained by both methods was proven on the basis of melting points (mp), thin layer chromatography (TLC) and spectral data. The IR spectra of compounds 3 and 4 exhibited C=C and C=N stretching vibrations near 1624-1488 cm -1 . The absence of absorptions near 2100 cm -1 excluded the possibility of azide formation [22] in case of compounds 3. The formation of 3 from 2, and 4 from 2 as well as of 4 from 7 was also supported by absence in their IR spectra of bands at 3400-3150 and at 1648-1640 cm -1 for the respective stretching and bending vibrations of NH in imino, amino as well hydrazino functionalities, which were found to be present in case of compounds 2 [17,18] and 7. In the IR spectra of compounds 6 the cyano and ester carbonyl groups exhibited bands in the 2210-2200 and 1720-1710 cm -1 regions, respectively. The 1 H-NMR spectra of the compounds 3 and 4 displayed resonances due to aromatic protons near δ 8.3-6.95, and, in case of 4, the C 2 proton resonated at δ 9.15−9.1. In the 1 H-NMR spectra of the compounds 6 and 7 the aromatic protons appeared as a multiplet near δ 6.9-7.95. Amino protons appeared as a broad peak in the δ 5.46-5.43 region, integrating for 2H, while the imino proton appeared at δ 8.1-8.0 in the spectra of compounds 6. Both imino and amino protons were found to be D 2 O exchangeable. The CH proton of the NCH=OEt group in 6 also appeared downfield at δ 8.5-8.45, whereas a triplet (3H) at δ 1.42-1.2 and quartet (2H) at δ 4.3-4.2 were assigned to the methyl and methylene protons of the ethyl group in the same functionality. The mass spectra of tetrazolopyrrolopyrimidine 3e was found to show a regular fragmentation pattern [22] giving a molecular ion peak at m/e 342 along with the fragments at 314, 287 and 286, resulting from successive elimination of nitrogen and hydrogen cyanide or subsequent elimination of two nitrogen molecules (Scheme 3). The mass fragmentation pattern of triazolopyrrolopyrimidine 4b was also in agreement with that reported for fused triazolopyrimidines [23][24], showing a M + peak at 341 together with signals at m/e 313, 314 and 286 resulting from subsequent removal of nitrogen and hydrogen cyanide or hydrogen cyanide and nitrogen molecules (Scheme 4).

Scheme 4 Antibacterial activity
The antibacterial activity of all the newly synthesized compounds 3 and 4 was investigated. Assays against different bacterial cultures comprising E. coli, E. entericus, P. aeruginosa, S. aureous and B. subtilis were carried out by the agar plate diffusion method [25][26]. The bacterial cultures were streaked across grooves filled with 0.6 mL of a 1 % solution in DMSO of each compound 3 and 4, which were then allowed to diffuse in the agar nutrient plate. The antibiotic ampicillin and the DMSO solvent were used as positive and negative controls, respectively. The results are recorded in Table 1 as the relative extent of inhibition observed around the grooves compared to the negative control. Against E. coli all the compounds tested displayed comparable activities except for 3e and 3j. Compounds 4 were found to be moderately active. Compounds 3c and 3j exhibited better activity than ampicillin against E. entericus while the remaining compounds were found to be either less active or inactive. Activity against P. aeruginosa was similar for all active compounds, while 3g, 3k and 4d failed to give .
-N 2 (28) any inhibition. Against S. typhi better to good activity was shown by most of the derivatives 3 and 4, except 3l and 4a, which were inactive. 3h and 3k gave better inhibition than the positive control against S. aureous species while 4c and 4d showed remarkable activity against B. subtilis.

Experimental Section
General Melting points were determined on an Electrothermal apparatus in an open capillary tube and are uncorrected. The IR spectra were recorded in cm -1 for KBr pellets on a Buck Scientific spectrophotometer. 1 H-NMR spectra were recorded on a Varian 300MHz spectrometer using DMSO-d 6 or CDCl 3 as the solvent and TMS as the internal reference standard. Chemical shifts are expressed in δ ppm. Mass spectra were taken on a LKB 9000 mass spectrometer. The purity of the compounds was routinely checked by TLC using Silica G and the spots were exposed in iodine vapour for visualization. Synthesis of 7,9-disubstituted 7H-tetrazolo [1,5-c]pyrrolo [3,2-e]pyrimidines 3a-l: General Procedure.
Method A: A mixture of sodium azide (0.011 mole, 0.072 g) and ammonium chloride (0.011 mole, 0.059 g) in DMSO (20 mL) was stirred for five min. at 90°C and the 5,7-disubstituted 4-chloro-7Hpyrrolo[2,3-d]pyrimidine (1, 0.01 mole) was added in portions with stirring. After the addition the reaction mixture was further stirred for 2.0-2.5 hr at the same temperature and for 1 hr at room temperature. Then it was decomposed on ice. The solid obtained was filtered, washed with water, dried and crystallized from toluene or a mixture of ethanol-chloroform (8:2) to give the title compounds 3.