Heterocyclic o-Aminonitriles: Preparation of Pyrazolo[3,4-d]-pyrimidines with Modification of the Substituents at the 1-Position

Novel 1-[6-(p-tolyl) pyridazin-3-yl]pyrazole-o-aminonitriles (3a-c) were formed using 3-hydrazino-6-(p-tolyl)pyridazine (2) and ketene S,S-acetals (1a), S,N-acetals (1b) or tetracyanoethylene (1c). The pyrazole-o-aminonitriles (3a-c) were in turn used as precursors for the preparation of previously unreported 1-[6-(p-tolyl)-pyridazin-3-yl]pyrazolo[3,4-d]pyrimidines (8, 9, 13-20) and 7-[6-( p-tolyl) pyridazin-3-yl]2-arylpyrazolo[3,4-d]1,2,4-triazolo[5,1-f]pyrimidines (10-12) which are expected to possess considerable chemical and pharmacological activities.


Introduction
Pyrazolo [3,4-d]pyrimidines are of considerable chemical and pharmacological importance as purine analogs [1][2][3]. Various compounds with related structures also possess anti-tumor and antileukemia activities [4,5]. On the other hand, substituted pyridazines are often used in medicine thanks to their pronounced bactericidal and fungicidal effects [6]. As a continuation of our work on azoloazines [7], we aimed to incorporate the pyridazine moiety into the 1-position of the pyrazolo [3,4-d]pyrimidine ring system to thus obtain a new heterocyclic system which is expected to possess notable chemical and pharmacological activities.

Scheme 1
The reaction sequence shown in Scheme 1 yielded only one product (3) instead of a mixture of the two possible isomeric pyrazole-o-aminonitriles 3 and 4. This fact was confirmed by the several strategies. First, by using the technique of Hecht et al. [10], who found it was possible to differentiate between the isomeric pyrazoles 5 and 6 based on their different reactivity; thus, treatment of 6 with acetic anhydride in pyridine at room temperature afforded the corresponding Nacetyl derivative, but pyrazole 5 did not form the corresponding derivative under the same conditions, although it did form at 50-60 o C (Scheme 2).

Scheme
By analogy with the results of Hecht et al, when our product was treated with acetic anhydride, it formed the corresponding acetyl derivative only at higher temperatures and not at room temperature, due to the electronic and steric factors of the substituents (Ar and CN). Secondly, only one spot was obtained in TLC. Third, the structure of the product was identified as that of 3 on the basis of the X-ray crystal structure analysis, which confirmed the substituted nitrogen of the pyrazole ring is adjacent to the amino group ( Figure 1). Finally, the structural assignment of 3 was also based on spectral evidence (IR, 1 H-NMR, 13 C-NMR and Mass spectrometry, while COSY-2D experiments indicated the (C-H) and (H-H) correlations).

Fig. 1 Single-crystal X-ray analysis. ORTEP view of 3a
showing the atom numbering scheme, The above mentioned pyrazoles were used as intermediates for the synthesis of new pyrazolo [3,4-d]pyrimidines because they contain the ß-enaminonitrile moiety which is well known to be highly reactive. Thus, condensation of 3a-c with triethylorthoformate in refluxing acetic anhydride afforded the intermediate ethoxymethyleneamino derivatives (7a-c) (Scheme 3), which were isolated and used without purification in the next step. Treatment of 7a-c with cold aqueous alcoholic ammonia yielded 4-aminopyrazolo [3,4-d]pyrimidines (8a-c). The IR spectrum of 8a,b displayed no absorption for the cyano group. We also attempted a direct synthesis of compounds 8a-c by treating pyrazoles 3a-c with formamide. When cooled compounds 7a-c were stirred with hydrazine hydrate in ethanol and then warmed to room temperature for 6h, they yielded 5-amino-4iminopyrazolo [3,4-d]-pyrimidines (9a-c) in good yield. The proposed structures for the products 9a-c were supported by their elemental analysis and spectral data (see Experimental).
The imidates 7a-c gave 2-arylpyrazolo [3,4-d]-1,2,4-triazolo[5,1-f] pyrimidine systems (10-12) when reacted with 2-furancarboxylic acid hydrazide, 2-thiophenecarboxylic acid hydrazide and 4pyridinecarboxylic acid hydrazide, respectively. The structures of the reaction products were assigned based on correct elemental analysis and spectral data (for example, the IR spectrum shows the absence of nitrile and amino bands, while the 1 H-NMR spectrum shows the signals of hydrogen atoms respectively characteristic of the aryl moiety in 2-position).

Conclusions
We have presented various methods for synthesis of novel pyrazolo [3,4-d]pyrimidines with a pyridazine moiety at the1-position.

Acknowledgments
The authors thank Dr. M. Fettouhi, King Fahad University of Petroleum and Minerals, for his efforts in facilitating the analysis of the X-ray analysis and the laboratory of Dr. Lahcene Ouahab, LCISM, UMR 6511, Rennes l University, France, for providing the X-ray structure analysis. The author extend their gratitude to the General Presidency for Girls Education for their partial financial support of this study.

General
Melting points were determined on a Reichert hot stage microscope and are uncorrected. IR spectra were measured with a Nicolet Magna 520 instrument, using potassium bromide and results are given in cm -1 . 1 H-, 13 C NMR and 2D H-H, C-H, Cosy NMR spectra were recorded in DMSOd 6 on either a JEOL JNM-GX270, ( 1 H-NMR at 270 MHz), ( 13 C-NMR at 52.89MHz) or a Varian Gemini ( 1 H-NMR at 200 MHz), ( 13 C-NMR at 90.56 MHz ) spectrometer. The chemical shifts are reported in part per million (ppm) downfield from internal tetramethylsilane. Electron impact MS spectra were obtained on a JEOL JMS-HX 100 at 70 eV. Elemental microanalysis were done on a CARLO Erba analyzer model 110. The progress of all reactions was monitored by TLC on 2.0 cm x 6.0 cm aluminium sheets precoated with silica gel 60 containing a fluorescent indicator (Alugram SIL G/UV254 fur die DC Macherey-Nagel/ Germany), to a thickness of 0.25 mm. The developed chromatograms were viewed under ultraviolet light. Column chromatography was performed on [Merck] silica gel (70-230 mesh) (elution with 1:1 cyclohexane-ethyl acetate). The X-ray structure was determined using a NONIUS Kappa Diffractomer. Suitable crystals were grown by slow crystallization from ethanol.
General Procedure for the Preparation of To a cold solution of hydrazine 2 (2.0g , 10 mmol) in methanol (100 mL) was added 1 (10 mmol). The reaction mixtures were then stirred at room temperature for 3-6h, left overnight, then the solvent was evaporated and the residue recrystallized from a suitable solvent. -3-methylthio-1-[6-(p-tolyl)

General Procedure for Preparation of 5-Methoxymethyleneamino
A mixture of pyrazole-o-aminonitriles 3a-c (10 mmol), triethylorthoformate (3mL) and acetic anhydride (3 mL) was refluxed for 6h. The solvent was removed under reduced pressure and the resulting solid was recrystalized from ethanol.

General procedure for the Preparation of 3-Substituted-1-[6-(p-tolyl)pyridazin-3-yl]-pyrazolo
Methanimidates 7a-c (3 mmol) were added to methanol (20 mL) saturated with ammonia at 0 o C for 1h, warmed to room temperature and the reaction mixture stirred for 6h. The solid which precipitated was collected and recrystallized from an appropriate solvent.

4-Imino
Compound 3a-c (5 mmol) and urea (10 mmol) or thiourea (10 mmol) or guanidine carbonate (5 mmol) were mixed in a mortar, The mixture was then heated at 180 o C in an oil bath for 20 minutes, heating was continued 2h, at the melting point of the pyrazole derivatives using reduced pressure. The molten product was boiled 10 minutes with water, cooled and filtered. The product was finally recrystallized from a suitable solvent. [3,4-d]pyrimidine-6(7H)-one (18a).  H 4.14,N 26.83,S 8.77;Found: C 55.87,H 4.08,N 26.90,S 8.75.