Synthesis and Antibacterial Activities of Novel 2,5-Diphenylindolo[2,3-e] Pyrazolo[1',5':3",4"]pyrimido[2",1"-c][1,2,4]triazines

The formation of (E)-3-{2-(2,5-diphenylpyrazolo[1,5-c]pyrimidin-7-yl)hydrazono}indolin-2-ones 3 has been achieved by condensation of equimolar amounts of 7-hydrazino-2,5-diphenylpyrazolo[1,5-c]pyrimidine (1) and isatin (or isatin derivatives) 2at room temperature. The (E)-products could be isomerized into corresponding the (Z)-3 isomers. Reactions of the latter fused heterocyclic hydrazones towards different electro-philic reagents yielded the corresponding 3-substituted derivatives 4–7. Dehydrative cyclisation of the hydrazones 3 using phosphorus oxychloride afforded the 2,5-diphenyl- indolo[2,3-e]pyrazolo[1',5':3",4"]pyrimido[2",1"-c][1,2,4] triazines 13. The polyfused heterocyclic ring system 13 underwent electrophilic substitution reactions at position 4 rather than at position 3. The 3-bromo isomer of 17 was prepared by a sequence of reactions starting from 2,5-diphenylpyrazolo[1,5-c]pyrimidine-7(6H)-thione (11). The orientation of the electrophilic attack was supported by spectroscopic and chemical evidence. Some of the synthesized compounds were found to possess slight to moderate activity against the microorganisms Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

Isatin is known to be a colorimetric reagent for the amino acid proline, forming blue derivatives [19]. This property has been exploited for the determination of the level of this amino acid in pollens [20] or for the detection of polymer bound compounds possessing proline residues [21]. It has also been used in a colorimetric screening test for human serum hyperprolinaemia [22], in a colorimetric assay of HIV-1 proteinase [23] and for the estimation of the age of bones in crime investigation [24]. In a similar manner, isatin-3-hydrazone has been studied for the colorimetric determination of steroids [25,26].

Results and Discussion
The theoretical existence of geometric isomers of 3-{2-(2,5-diphenylpyrazolo [1,5-c]pyrimidin-7yl)hydrazono}indolin-2-ones (E and Z)-3 had been predicted for the condensation of 7-hydrazino-2,5diphenylpyrazolo [1,5-c]pyrimidine (1), which was readily obtained by sequence of reactions starting from ethyl phenylpropiolate [33,34], with isatin (or isatin derivatives) 2 (Scheme 1). But by stirring equimolar amounts of 1 with 2 at room temperature the reaction yielded only the kinetically more stable geometrical isomer (E)-3a-c, which upon heating in dioxane or stirring with conc. H 2 SO 4 at room temperature underwent isomerisation to give the thermodynamically more stable isomer (Z)-3a-c showing a possibility of hydrogen bond formation. The structure and configuration of the pyrazolopyrimidinoindolinonehydrazones (E and Z)-3 were fully differentiated by studying their spectra, which included IR, 1 H-NMR and MS. The IR spectra showed characteristic five membered ring amide carbonyl absorption bands at 1684-1710 and 1684-1692 cm −1 , in addition to the NH absorption band in the range 3459-3479 and 3451-3467 cm −1 , respectively.
The 1 H-NMR spectra of (E)-3a-c revealed, besides the aromatic protons as a multiplet at δ H 7.37-8.04, two doublets at δ H 8.07-8.11 and at δ H 8. 16-8.23, as well as other characteristic singlets at δ H 6.85-7. 23 for the H-3 pyrazole ring proton and at δ H 7.54-7.92 for the H-4 pyrimidine ring proton. The assignment of the higher field signal for the H-3 pyrazole ring proton and the lower field signal for H-4 pyrimidine ring protons is supported by the data reported for 2,5-diarylpyrazolo [1,5-c]pyrimidine-7(6H)-thiones [20]. Moreover, the spectra of (E)-3a-c exhibited exchangeable singlets at δ H 10.43-10.80 and at δ H 14. 19-14.22 which are attributed to the NH of hydrazone conformer 3 and NH of pyrimidine conformers A or B. The intensity of both singlets is equivalent to one proton. The spectra also showed an exchangeable proton as two singlets equivalent to one proton at δ H 11.03-11.36 and at δ H 11. 15-12.17 which were ascribed to the NH conformer 3 and OH conformer B of indole ring [35]. Furthermore, the spectrum of (E)-3b showed a singlet at δ H 2.33 for the CH 3 group. The previous data indicates that pyrazolopyrimidinoindolinonehydrazones (E)-3a-c exist as a mixture of the toutomers 3, A and B (Figure 1). The 1 H-NMR spectra of (Z)-3a-c showed, besides the aromatic protons as a multiplet at δ H 7.37-7.71, two doublets at δ H 8. 11-8.13 and at δ H 8. 22-8.27, as well as other characteristic singlets at δ H 7. 18-7.22 for the H-3 pyrazole ring proton and at δ H 7.88-7.95 for the H-4 pyrimidine ring proton. The spectra of (Z)-3a-c also exhibited an exchangeable NH proton at δ H 11.14-11.37 which was ascribed to the indole ring [35] and at δ H 14.21-14.23 for the chelated NH hydrazone residue. On the other hand, the spectra of (Z)-3b showed a singlet at δ H 2.33 for the CH 3 group. The above 1 H-NMR spectral data showed only a single conformer for the structure of the hydrazone (Z)-3.
Further conformation for the structure of both (E and Z)-pyrazolopyrimidinoindolinonehydrazones was obtained from their mass spectral data, where both isomers showed similar molecular ion peaks at m/z 430, 444 and 464, in addition to base peaks at m/z 77, 339 and 359 for derivatives a-c, respectively, in addition to the same fragments with similar or almost similar intensities.
In the present investigation the electrophilic substitution reactions of the geometrical isomers pyrazolopyrimidinoindolinonehydrazones (E or Z)-3a-c were studied in the hope that introduction of such substituents might enhance their biological properties, as well as, to study the more reactive position for the electrophilic attack on such fused heterocyclic rings (Scheme 2). Thus, bromination of (E or Z)-3a-c with bromine in glacial acetic acid, as well as, iodination with iodine monochloride in the same solvent yielded the respective monosubstituted (Z)-isomers 4 and 5, since the (E)-3a-c isomers were proved to convert into the respective (Z)-conformers in acidic medium.
Moreover, reaction of (E or Z)-3a-c with nitric and sulfuric acids in glacial acetic acid and with benzenediazonium chloride in the presence of sodium hydroxide afforded the (Z)-3-nitro and 3-phenyldiazenyl derivatives 6 and 7, respectively.
The structures of the 3-substituted derivatives 4-7 were confirmed by their spectral data. The 1 H-NMR spectra of 4a-c and 5a-c showed the absence of the H-3 pyrazole ring proton signals and the presence of the H-4 pyrimidine ring proton as singlet at δ H 7.61-7.93 ppm.
The 1 H-NMR spectrum of (E)-9a showed, besides the aromatic protons as a multiplet at δ H 7.87-8.05, two doublets at δ H 8.08 and at δ H 8.25, as well as another characteristic singlet at δ H 7.74 for the H-4 pyrimidine ring proton. Moreover, the spectrum of 9a exhibited exchangeable singlets at δ H 10.92 and at δ H 14.16 which are attributed to the NH of the hydrazone conformer 3 and the NH of the pyrimidine conformers A or B, respectively. The intensity of both singlets is equivalent to one proton. The spectrum also revealed an exchangeable proton as two singlets equivalent to one proton at δ H 11.21 and at δ H 11.29 which ascribed to the NH conformer 3 and OH conformer B of the indole ring [46] ( Figure 1).
The novel fused indolopyrazolopyrimidotriazines 13a-c appeared to be attractive intermediates for the synthesis of a number of substituted derivatives via reaction with some representative electrophilic reagents, and to the best of our knowledge, no reports on the electrophilic substitution reactions of the indolopyrazolopyrimidotriazine ring system have been published. We are interested in investigating the reactivity at either the C-3 or C-4 position in such heterocyclic rings. Thus, bromination of 13a-c with bromine, as well as, iodination with iodine monochloride gave the respective 4-bromo 14a-c and 4-iodo 15a-c derivatives, respectively. Moreover, nitration of 13a-c with nitric and sulfuric acids in glacial acetic acid afforded the respective 4-nitro derivatives 16a-c. The structures of the 4-substituted derivatives 14-16 were confirmed by studying their 1 H-NMR spectra, which showed the disappearance of the H-4 pyrimidine ring proton signals and the appearance of the H-3 pyrazole ring proton signals at δ H 7.28-7.47.
Furthermore, the structures of 14-16 were confirmed chemically by synthesizing the 3-substituted isomeric derivatives 17. Thus, refluxing of (Z)-4a-c with phosphorus oxychloride led to the formation of the respective isomeric 3-bromo-derivatives of the fused triazines 17a-c. The two isomeric bromo derivatives 14 and 17 were found to be completely different (TLC, mp and mixed mp, IR, 1 H-NMR and MS spectra). The 1 H-NMR spectra of 17a,b showed the absence of the H-3 pyrazole ring proton signals and the presence of the H-4 pyrimidine ring proton signals at δ H 7.69, 7.94, respectively.

General
Melting points were determined on a Kofler block and are uncorrected. Elemental analyses were carried out in the Microanalytical Laboratory of the Faculty of Science, Cairo University. The IR spectra of compounds were recorded on a Fourier Transform infrared 8400 spectrophotometer [Bruker Tensor 37] using potassium bromide pellets and frequencies are reported in cm −1 . The 1 H-NMR spectra were recorded on a JEOL JNM ECA 500 MHZ instrument and chemical shifts δ H are in ppm relative to tetramethylsilane used as internal standard. Mass spectra were recorded at 70 ev with a GCMS-QP 1000 EX spectrometer. Reactions were routinely followed by thin layer chromatography (TLC) Merck Kiesel gel; 60-F254 precoated plastic plates. The spots were detected by iodine. 5-Aryl-7-hydrazino-2-phenylpyrazolo[1,5-c]pyrimidines 1 and 10 were prepared from the respective acetylenic β-diketones as described earlier [30,33,34]. [30,33] (1, 0.30 g, 0.0010 mol) in dioxane (10 mL) was stirred with isatin (or isatin derivatives) (2, 0.0015 mol) for 24 hours at room temperature. The products that separated out as orange needles were filtered off, washed with methanol and dried.

Synthesis of Compounds
in dioxane, xylene, pyridine, acetic acid or acetic anhydride (50 mL) was heated under reflux for twenty four hours. The products that separated out were filtered off, washed with ethanol, dried and crystallized from dioxane.
The products from method A and method B showed completely similar TLC, mp, mixed mp, IR, 1 H-NMR and MS spectra.