Synthesis of New Azoles and Azolopyrimidines Incorporating Morpholine Moiety as Potent Anti-Tumor Agents

A new series of morpholinyl-chalcones 2a–d was prepared by reaction of 2-oxo-N,4-diarylbut-3-enehydrazonoyl chlorides 1a–d with morpholine. These chalcones were used as a building block for constructing pyrazoles 3a–d and 3,4-dihydropyrimidine-2(1H)-thione 6 via their reaction with phenylhydrazine and thiourea, respectively. Moreover, a new series of azolopyrimidine derivatives 11a,b, 15, 17, 19, and 21 incorporating morpholine moiety were synthesized by reaction of 1-morpholino-4-phenyl-1-(2-phenylhydrazono)but-3-en-2-one (2a) with a number of heterocyclic amines in the presence of a catalytic amount of acetic acid. The assigned structures for all the newly synthesized compounds were confirmed on the basis of elemental analyses and spectral data and the mechanisms of their formation were also discussed. All the synthesized compounds were tested for in vitro activities against two antitumor cell lines, human lung cancer (A-549) and human hepatocellular carcinoma (HepG-2) compared with the employed standard antitumor drug (cisplatin) and the results revealed that compounds 6, 8c and 17 have promising activities compared with cisplatin.


INTRODUCTION
YDRAZONOYL halides are compounds which have the characteristic functionality -C(X):NNH-, where X is a halogen (Br or Cl).An increasing flow of work has appeared on the chemistry of such a class of compounds.[36][37][38][39] In view of all these reports and in continuation of our previous work in synthesis of bioactive heterocyclic compounds, [40][41][42][43][44][45][46][47] herein, we are interested in synthesizing a new series of pyrazoles and azolopyrimidines using morpholinylchalcones as common precursor and evaluate these compounds for their anticancer activities.

H Synthesis of Pyrazoles 3a-d
A mixture of chalcones 2a-d (1 mmol) and phenylhydrazine (0.108 g, 1 mmol) in 15 mL of ethanol containing 1 mL of glacial acetic acid was refluxed for 4-10 h (monitored through TLC).The desired product precipitated from reaction mixture was filtered, washed with ethanol and recrystallized from proper solvent to give the respective pyrazoles 3a-d, respectively.

Evaluation of Cytotoxic Effects of Compounds
MAMMALIAN CELL LINES A-549 cells (human Lung cancer cell line, HepG-2 cells (human Hepatocellular carcinoma) were obtained from VACSERA Tissue Culture Unit.

CHEMICALS USED
Dimethyl sulfoxide (DMSO), crystal violet and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA).

CRYSTAL VIOLET STAIN (1 %)
It composed of 0.5 % (w / v) crystal violet and 50 % methanol then made up to volume with ddH 2O and filtered through a Whatmann No.1 filter paper.

CELL LINE PROPAGATION
The cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum, 1 % L-glutamine, HEPES buffer and 50 µg mL -1 gentamycin.All cells were maintained at 37 °C in a humidified atmosphere with 5 % CO2 and were subcultured two times a week.

CYTOTOXICITY EVALUATION USING VIABILITY ASSAY
For cytotoxicity assay, the cells were seeded in 96-well plate at a cell concentration of 1 × 10 4 cells per well in 100µl of growth medium.Fresh medium containing different concentrations of the test sample was added after 24 h of seeding.Serial two-fold dilutions of the tested chemical compound were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette.The microtiter plates were incubated at 37 °C in a humidified incubator with 5 % CO2 for a period of 48 h.Three wells were used for each concentration of the test sample.Control cells were incubated without test sample and with or without DMSO.The little percentage of DMSO present in the wells (maximal 0.1 %) was found not to affect the experiment.After incubation of the cells for at 37 °C, various concentrations of sample were added, and the incubation was continued for 24 h and viable cells yield was determined by a colorimetric method.
In brief, after the end of the incubation period, media were aspirated and the crystal violet solution (1 %) was added to each well for at least 30 minutes.The stain was removed and the plates were rinsed using tap water until all excess stain is removed.Glacial acetic acid (30 %) was then added to all wells and mixed thoroughly, and then the absorbance of the plates were measured after gently shaken on Microplate reader (TECAN, Inc.), using a test wavelength of 490 nm.All results were corrected for background absorbance detected in wells without added stain.Treated samples were compared with the cell control in the absence of the tested compounds.All experiments were carried out in triplicate.The cell cytotoxic effect of each tested compound was calculated.The optical density was measured with the microplate reader (SunRise, TECAN, Inc, USA) to determine the number of viable cells and the percentage of viability was calculated as [1-(ODt/ODc)] × 100 % where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells.The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound.The 50 % inhibitory concentration (IC50), the concentration required to cause toxic effects in 50 % of intact cells, was estimated from graphic plots of the dose response curve for each conc.using Graphpad Prism software (San Diego, CA.USA). [48]

RESULTS AND DISCUSSION
As previously described, [49] compounds 1a-d reacted with morpholine in ethanol under reflux for 3-6 hrs to give the morpholinohydrazonobutenone derivatives 2a-d (Scheme 1), which were used as a precursors for synthesis of pyrazoles, pyrimidines, and different fused pyrimidines, such as triazolopyrimidine, tetrazolopyrimidine, thiazolopyrimidine, benzothiazolo-pyrimidine benzoimidazopyrimidine, and pyridopyrazolopyrimidine derivatives.
Compounds 2a-d were refluxed in ethanol containing catalytic amount of acetic acid with phenylhydrazine for 4-10 hrs affording the corresponding pyrazole derivatives 3a-d, rather than the compounds 4a-d (Scheme 1), what was confirmed based on the mass spectrum and IR spectrum which showed a peak at 1715 cm -1 that revealed the presence of amidic carbonyl group.All the spectral data and elemental analyses of these compounds were in agreement with the proposed structures (see Experiment).
The suggested mechanism for the formation of compounds 11a,b was outlined in Scheme 3. It was postulated that the reaction of chalcone 2 with the appropriate heterocyclic amine starts with Michael addition (route a) of the amino group to the double bond of the enone residue of 2a to give intermediate 10 which undergoes in situ dehydrative cyclization followed by autooxidation to give the final azolopyrimidine 11a,b.The spectral data and elemental analyses data are consistent with the regioisomeric structures 11 and 13.The isomeric compounds 13 produced by route b were discarded due to difficultness in condensation reaction than Michael addition (route a). [50,51]owever, an equimolar amount of compound 2a and 2-aminobenzoimidazole 14 were heated under reflux in ethanol containing a catalytic amount of acetic acid to give benzo [4,5]imidazo[1,2-a]pyrimidine derivative 15 (Scheme 4).

Cytotoxic Activity
The in vitro growth inhibitory activity of the newly synthesized compounds 3a, 6, 8a-d, 11a,b, 15, 17, 19 and 21 was investigated against two carcinoma cell lines, human lung cancer cell line (A-549) and human hepatocellular carcinoma cell line (HepG-2), in comparison with the well-known anticancer standard drug (cisplatin) under the same conditions using colorimetric MTT assay.Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50 % of cell population (IC50) was determined.The results are depicted in Table 1 and revealed that the descending order of activity of the newly synthesized compounds towards the lung carcinoma cell line (A549) were as follow: The descending order of activity of the newly synthesized compounds towards the human Hepatocellular carcinoma cell line (HepG-2) were as follow:

Structure Activity Relationship (SAR)
Examination of the SAR leads to the following conclusions.
The results revealed that all the tested compounds showed inhibitory activity to the tumor cell lines in a concentration dependent manner.
The activities of the synthesized compounds depend on the structural skeleton and electronic environment of the molecules.
For triazolopyrimidine derivatives 8a-d: compound 8c (substituted with COOEt group at position 3) has in vitro inhibitory activity more than 8a (substituted with COCH3 group at position 3).Table 1.The in vitro inhibitory activity of tested compounds against tumor cell lines expressed as IC50 values (μg mL -1 ) ± standard deviation from three replicates.

Figure 1 .
Figure 1.The most active compounds compared to cisplatin.