Synthesis, reactions and antitumor activity of new β -aminovinyl 3-pyrazolyl ketones

A new series of enaminones 4a-e was prepared and their reaction with the hydrazonoyl chloride 2c gave 3,4'- bis (pyrazolyl)ketones 8a-d . Hydrazinolysis of 8 proved to be site selective as it yielded the respective 4-(pyrazol-3-yl)-2 H -pyrazolo[3,4-d ]pyridazines 9 . The results of screening of the antitumor activity of enaminones 4a-e against human breast cell line MCF-7 revealed that all such compounds exhibited lower activity in relation to the reference drug Doxorubicin and the activity of the enaminone having electron-accepting substituent is more than that having electron donating substituent.


Introduction
Hydrazonoyl halides, R-C(X)=NNHAr, are well known versatile precursors for the nitrilimines, R-C( + )=N-N( -)-Ar.Their reactions have been extensively studied and reviewed by one of the authors. 1-10Furthermore, it is well known that the β-enaminones of the general formula R 2 N-CH=CH-COR' combine the ambident nucleophilicity of enamines with the ambident electrophilicity of enones. 11,12Because of this, they have been utilized as versatile valuable precursors for heterocyclic synthesis. 13,14Their synthetic applications have been extensively reviewed. 12,15-27Furthermore, many enaminones were found to exhibit several biological activities as antitumor, antibacterial and anticonvulsant agents. 28,29In the light of these facts, it was thought interesting to synthesize a new series of β-dimethylaminovinyl 3-pyrazolyl ketones 4 which are novel enaminones that have been unreported hitherto and study their 1,3-dipolar cycloaddition to the nitrilimines, generated in situ by base-catalyzed dehydrohalogenation of hydrazonoyl halides R-C(X)=NNH-Ar 2. Our objectives after such a study are on one hand to screen the antitumor activity of the target enaminones and shed some light on structure activity relationship (SAR) and on the other hand to explore the regiochemistry of the reactions to be Next, the reactions of the enaminones 4a-d, as dipolarophiles, with the nitrilimine generated in situ by dehydrochlorination of N-phenyl-2-oxopropanehydrazonoyl chloride 2c were examined.Thus, in our hands, reaction of 4a-d each with 2c in refluxing dioxane in the presence of triethylamine yielded, in each case, a single product that was identified, on the basis of its elemental analysis and spectral (IR, 1 H NMR and MS) data as 3,4'-bis(pyrazolyl)ketone 8 (Scheme 2).The other possible isomeric structure namely 3,5'-bis(pyrazolyl)ketone 7 was discarded.For example, the 1 H NMR spectra of the products isolated showed in each case a singlet signal in the region δ 8.6 -9.22, which corresponds to H-5 of the pyrazole ring residue in the products 8.This assignment is based on the fact that in the pyrazole ring system, the C-4 is the most electron rich carbon and so H-4 is expected to be more shielded than H-5 linked to C-5 which is bonded to nitrogen atom.Typically the signal of H-5 usually appears at δ 8.66-8.69 33 whereas that of H-4 appears at δ 5.81-5.89. 34,35Thus, the formation of 8 rather than 7 indicates that the studied reaction of 4 with 2 is regiospecific.To account for the formation of 8, it is suggested that the reaction of 4 with 2 proceeds via 1,3-dipolar cycloaddition of the nitrilimine, derived from 2c, to the activated double bond in the enaminone 4 to give the respective cycloadduct 6 which in turn undergoes in situ elimination of dimethylamine to afford 8 as the end product.

Scheme 2
Reaction of hydrazine with the products 8 was next examined to explore its site selectivity as such a reaction can lead to the production of fused pyrazoles 9 and/or 10 (Scheme 3).In our hands, treatment of compound 8a with hydrazine hydrate in refluxing ethanol yielded a product whose spectroscopic (IR, MS, 1 H NMR) and elemental analysis data were consistent with structure 9.For example, its mass spectrum showed its molecular ion peak at m/z 530 and its IR spectrum revealed one carbonyl absorption band at ν 1712 cm -1 assignable to the ester carbonyl group.Its 1 H NMR spectrum showed, in addition to the aromatic proton signals, the following signals : δ 1.06, 4.16, 2,26 and 3.78 assignable to CH 3 CH 2 OCO, CH 3 and CH 3 O groups.
To provide further evidence for structure of the product 9, it was compared with an authentic sample of compound 10 prepared by alternate unambiguous route (Scheme 3).Thus, hydrazinolysis of the pyrazole 3a in refluxing ethanol gave product that was identified as 11 (Scheme 3) on the basis of its spectra (IR, 1 H NMR and MS) and elemental analysis (see Experimental).Reaction of 11 with DMF-DMA gave the enamine 12 which upon treatment with 2c in dry dioxane in the presence of triethylamine under reflux yielded the product 10 that was found different from compound 9 isolated from hydrazinolysis of 8a (Scheme 2).The structures of compounds 10, 11 and 12 were elucidated on the basis of their spectra and elemental analyses (see Experimental).For example, in addition to the aromatic proton signals, their 1

Antitumor screening and SAR
The cytotoxic effects of the newly synthesized enaminones 4a-e against human breast cell line MCF-7 were evaluated at the National Institute of Cancer, Cairo, Egypt.Doxorubicin was used as a reference to evaluate the potency of the tested compounds.Five different concentrations of each compound and the reference were used in such screening tests and determination of IC 50 values.The results are given in Table 1.As shown in this table, compounds have lower antitumor activity against MCF-7 cell line as their IC 50 values are much higher (10.1-11.9)than that of the reference doxotrubicin (IC 50 = 0.70).However, the data show that the antitumor activity of the studied enaminones 4 having electron withdrawing substituents is little bit more than that of those having electron donating ones.

Conclusion
In conclusion, a series of new enaminones 4a-e was prepared and their reaction with hydrazonoyl chloride 2c gave 3,4'-bis(pyrazolyl)ketones 8a-d.Reaction of 8 with hydrazine proved to be site selective as it yielded 9.The structures of the newly synthesized compounds

Synthesis of compounds 9 and 11
A mixture of compound 8a (5mmole) and hydrazine hydrate (5mL) in absolute ethanol was refluxed for 10 h and the reaction mixture was cold.The solid that precipitated was filtered off and crystallized from ethanol to give compound 9.When the above procedure was repeated using compound 3a in place of 8a, it yielded the respective pyrazolo [3,4-d]

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H NMR spectra revealed the absence of both the triplet and quartet signals of the protons of the CH 3 CH 2 OCO group.Compound 10 exhibits four singlet signals at δ 2.54, 3.82, 8.05 and 12.0 assignable to the protons of the CH 3 CO, CH 3 O, pyrazole-H and NHCO, respectively.Compound

Table 1 .
In vitro cytotoxic activity of the new compounds 4a-e against MCF-7 cell line a Doxotrobicin, an antitumor reference.