Analogs of biologically active compounds IX. 1 Synthesis of several new uracil and pteridine 6 ‐ azaanalogs based on cyclization of arylhydrazones derived from mesoxalic acid

A series of 5 C and 5 N substituted 3 ‐ oxo ‐ 2 ‐ phenyl ‐ 1,2,4 ‐ triazine ‐ 6 ‐ carbonitriles and 8 ‐ imino ‐ 2 ‐ arylpyrimido[4,5 ‐ e ][1,2,4]triazine ‐ 3,6(2 H ,5 H ) ‐ diones (6 ‐ aryl ‐ 6 ‐ azapteridines) are described via a cyclocondensation reaction from the corresponding aryliminohydrazones with CDI. The required aryliminohydrazones were obtained from the starting compound – malononitrile. These prepared compounds were tested for cytotoxic activity on cancer cell lines.


Results and Discussion
In this paper we utilize a cyclization principle based on the cyclization of arylhydrazones leading to 2-arylderivatives of 1,2,4-triazines.The most usual and proven syntheses of this type include the cyclization of ethyl arylhydrazono-cyanoacetyl carbamates resulting in 1-aryl-6-azauracils [12][13][14] and the analogous cyclization of arylhydrazones of ethoxycarbonylated amidines of the mesoxalic acid providing 1-aryl-6-azacytosines. 1 On a modified principle we cyclized aryliminohydrazones of last type by direct cyclocondensation reaction with 1,1 /carbonyldiimidazole (CDI) which had been used earlier for the formation of 6-azalumazin-7(6H)ones. 15 The starting compound utilized for the synthesis of both uncondensed and condensed 1,2,4-triazines was malononitrile.In the case of the formation of 6-azapteridine derivatives 4, malononitrile was converted via reaction with diethyl malonimidate dihydrochloride 1 to malonimidamide dihydrochloride 2 as according to the previously described literature procedure. 16,17This compound possessing a reactive methylene group was coupled with various diazonium salts to give corresponding 2-arylhydrazonomalonimidamides 3a-3c which were isolated as dihydrochloride salts.The purity and yields of hydrazones were very dependent on the pH and method of preparation.The optimal method found was to add the diazonium salt to an aqueous solution of malonimidamide 2 and adjust the pH to 5-6 with sodium acetate with vigorous stirring.If the pH of the reaction mixture was higher then the formed hydrazones were less pure due to the formation of gummy products making separation of the compounds difficult.Additional to this, a lower pH was found to be insufficient for initiation of the azo-coupling reaction.Three arylhydrazones differing in substitution of the para position (3a-3c) were obtained in good yields.From the literature only phenyl derivative 3a is known and it was described as an azocompound, 17,18 however herein we have proven it exists in a hydrazono form on the basis of the NMR spectra measured in DMSO-d 6 .In fact, the molecule is an asymmetric compound including the diamidine moiety.Apart from the aromatic hydrogens, five different hydrogen atoms were present.One sharp signal at 11.85-12.34ppm with integral intensity one belongs to HN-aryl hydrogen and four signals with integral intensity two belong to the diamidine functionality.Additionally from the 13 C NMR spectroscopy, using APT method, it was evident that the methine hydrogen was missing; and so the other suggested structures are not probable.
The prepared arylhydrazones 3 were converted into corresponding 8-imino-2-arylpyrimido[4,5e] [1,2,4]triazine-3,6(2H,5H)-diones 4 by a direct double cyclocondensation reaction with an excess of CDI in DMF and in the presence of DIPEA (Scheme 1).Interestingly, we did not succeed in preparing of compounds where only one ring would be formed by cyclization with one equivalent of CDI.A mixture of intermediates, starting hydrazone 3 and corresponding 6-azapteridine derivative 4 were always present in the reaction mixture.Other possible cyclization reagents such as ethyl chloroformate and phosgene used for similar formation of six-membered ring from amidines 1 failed to cyclize smoothly.Using CDI, the most readily formed compounds were derivatives 4a and 4b.In case of nitro derivative 4c the yield was half as expected.These low soluble derivatives exist in their 8-imino form indicative by the three different hydrogen atoms are present in 1 H NMR spectrum, which is typical for these type of compounds.Malononitrile was also used for the preparation of uncondensed 1,2,4-triazines.Both 5C and 5N substituted 3-oxo-2-phenyl-1,2,4-triazine-6-carbonitrile derivatives were prepared from common precursor -2-phenylhydrazonomalononitrile (5) which is easily accessible by azo coupling reaction of the corresponding benzenediazonium salt with malononitrile. 19n case of the preparation of 5N substituted 3-oxo-2-phenyl-1,2,4-triazine-6-carbonitrile derivatives 7 the phenylhydrazone 5 was converted to N,N-disubstituted 3-amino-2-(phenyldiazenyl)acrylonitriles 6d-6h using a described literature procedure. 20These compounds can exist in two tautomeric forms 6A-6B.Although some literature articles have described similar derivatives as hydrazones, 21,22 our results, supported by NMR spectra, are in accordance with the published literature where the azo form was reported. 20We also observed no NH proton signal in 1 H NMR spectra of compounds 6 which would correspond to the hydrazono group.Only a two-proton singlet at 7.3-7.7 ppm is apparent from the spectra of the newly prepared compounds.The azoamino form 6B was observed in both the 1 H NMR spectra in DMSO-d 6 and CDCl 3 .No mixture of the possible geometric isomers of 6B has been observed.
The cyclization of 2-(phenyldiazenyl)acrylonitriles 6 was performed with CDI in a solution of DMF at 50°C (Scheme 2).We have found that the temperature influences the purity of cyclized 1,2,4-triazines 7. If the temperature was higher (around 90 °C) more by-products are formed and the yields are lowered.At 50°C the reaction proceeds slowly but the yields are much better, exceeding 75 %.However, this method is not suitable for the cyclization of N-monosubstituted 2-(phenyldiazenyl)acrylonitriles 6 (R 1 = methyl R 2 = H 23 ; R 1 = butyl R 2 = H 23 ) in the cases that we have tried.Several by-products were presented in the reaction mixture making their separation difficult.The preparation of 5N-monosubstituted 1,2,4-triazine-6-carbonitriles 7 is possible using another method wherein substitution of chlorine atom in position 5 is carried out. 24or the preparation of 5C substituted 1,2,4-triazine derivatives 10, the required 2-(2phenylhydrazono)propanenitriles 8i-8p were prepared in good yields by addition of Grignard reagents to phenylhydrazone 5 according to the literature procedure. 25In this reference only phenyl derivative 8o was prepared and referred to as azo compound 8B.On basis of NMR spectroscopy we found that the newly prepared compounds 8i-8p in either DMSO-d 6 or CDCl 3 solutions exist in several tautomeric forms 8Ax-y and 8Bx-y in an equilibrium.In the case of DMSO-d 6 solution there is a molar ratio of approximately 5:2 while in CDCl 3 the ratio is 6:1 (results from 1 H NMR). It is difficult to say exactly which forms predominate, however it is apparent that azo-amino forms 8Bx-y predominates in the CDCl 3 solution, where broad signals at 5.5-5.9ppm indicates an amino group.On the other hand, in DMSO-d 6 , four N-H proton signals were present at 8-9 ppm.These results show that the hydrazono form predominates in their two geometric isomers 8Ax-y.Additionally the 13 C NMR spectra indicated that compound 8 exists as an equilibrium mixture of more than one of the geometric isomeric forms in the aforementioned solvents.
The prepared phenylhydrazones 8 were also cyclized with CDI to the corresponding 1,2,4-triazines 10 (Scheme 3).These reactions were performed in DCM as a solvent instead of DMF.Probably due to the equilibrium of tautomeric forms in a polar aprotic solvent mentioned above, we observed more by-products when using DMF instead of DCM under the same conditions.Surprisingly, on the basis on NMR spectra it was apparent that prepared 1,2,4-triazines 10 contain an alkylidene arrangement in position 5.In the case of compounds 10i, 10j, 10l and 10n only one proton at 4.88-5.87ppm was detected on the first carbon of the aliphatic chain connected to 1,2,4-triazine ring.The second proton of these compounds was observed as a broad signal corresponding to one proton of N-H group.We were not successful to cyclize phenyl derivative 8o 25 to 1,2,4-triazine 9o.No conditions (higher temperature, solvent, time) were found for the cyclization with CDI.It is possible to conclude that if starting hydrazone 8 cannot create an alkylidene arrangement on the 1,2,4-triazine ring then no cyclization can take place.The same results were observed for tert-butyl derivative 8p.The prepared compounds were tested for biological activity.Human breast adenocarcinoma cell line MCF7 and myelogenous leukemia line K-562 were used for cytotoxicity determination by the MTT assay 26 .The tested compounds showed poor cytostatic activity (IC 50 = 40-190 μmol/l), with the exception of the moderately active 6-azapteridine 4a (MCF7: IC 50 = 8.3 μmol/l; K-562: IC 50 = 12.9 μmol/l), 4b (MCF7: IC 50 = 7.2 μmol/l; K-562: IC 50 = 14.6 μmol/l) and 1,2,4-triazine 10i (MCF7: IC 50 = 8.6 μmol/l; K-562: IC 50 = 7.5 μmol/l), 10j (MCF7: IC 50 = 16.3 μmol/l; K-562: IC 50 = 11.3 μmol/l).

Experimental Section
General.All starting materials are commercially available.Commercial reagents were used without any purification.Melting points were determined with a Boetius stage apparatus and are uncorrected.Reactions were monitored by LC/MS analyses with a UHPLC-MS system consisting of a UHPLC chromatography with photodiode array detector and a triple quadrupole mass spectrometer using a C18 column at 30 °C and flow rate of 800 μL/min.The mobile phase was (A; 0.01 M ammonium acetate in water) and (B; CH 3 CN), linearly programmed from 10 to 80% B over 2.5 min, kept for 1.5 min.The column was reequilibrated with 10% B for 1 min.The APCI source operated at a discharge current of 5 mA, a vaporizer temperature of 400 °C, and a capillary temperature of 200 °C.High-resolution mass spectrometer based on the orbitrap mass analyzer was equipped with Heated Electrospray Ionization (HESI).The spectrometer was tuned to obtain a maximum response for m/z 70-700.The source parameters were set to the following values: HESI temperature 30 °C, spray voltage +3.5kV, -3kV; transfer capillary temperature 270 °C, sheath gas/aux gas (nitrogen) flow rates 35/10.The HRMS spectra of target peaks allowed evaluating their elemental composition with less than 3 ppm difference between experimental and theoretically calculated value.The 1 H and 13 C NMR spectra were measured in DMSO-d 6 , CDCl 3 at 25 °C using 400 MHz spectrometer.Chemical shift (δ) is given in ppm.

General procedure A. 2-Arylhydrazonomalonimidamide dihydrochlorides (3).
The corresponding aniline derivative (5.76 mmol) was dissolved in 35% HCl (10 mL) and diazotized with a solution of sodium nitrite (397mg, 5.76 mmol) in water (10 mL) at 0-5°C.The mixture was stirred in an ice bath for 15 min and then, in one portion, it was added to a solution of malonimidamide dihydrochloride (990.7 mg, 5.76 mmol) 2 in water (20 mL) which was pre-cooled to 0-5 °C.After 15 minutes, with efficient stirring, a saturated solution of sodium acetate was added to reach pH = 5-6 and the reaction mixture was left at 2 °C for 18 h.A small amount of precipitated solid was filtered off and the filtrate concentrated under reduced pressure to one fifth of its original volume, when hydrazone started to crystalize.The product was filtered and dried.