Synthesis of 1 , 8-Naphthyridine Derivatives under Ultrasound Irradiation and Cytotoxic Activity against HepG 2 Cell Lines

Novel pyrazole derivatives 3a,b, 5, 1,3,4-oxadiazole 6, 1,3,4-thiadiazole 8, and 1,2,4-triazole 9a–c incorporated into 1,8naphthyridine have been synthesized using the versatile synthon 2-(2,7-dimethyl-1,8-naphthyridin-4-yloxy) acetohydrazide 1. An improvement in rates and yields was observed when the reactions were carried out under ultrasonic irradiation compared with the classical synthesis. The newly synthesized compounds were evaluated for HepG2 cell growth inhibition. The results obtained revealed that the tested compounds possess inhibitory effect on the growth of HepG2 liver cancer cells. The results were compared to doxorubicin (DOX) as a reference drug (IC 50 : 0.04 μM). Compounds 9b showed the highest inhibition activity against HepG2 cell line (IC 50 : 0.048 μM) among all tested compounds.

On the other hand, an important class of heterocyclic compounds such as pyrazoles [14,15], 1,2,4-triazole derivative [16], 1,3,4-thiadiazoles [17], and 1,3,4-oxadiazoles [18] showed a remarkable anticancer effect [14][15][16][17][18]. Based on these observations it was of interest to incorporate the 1,8-naphthyridine ring system into the abovementioned nitrogen, sulphur, and oxygen heterocyclic systems in one molecule in a trial to obtain a new target and product of dual mode of biological function.The application of ultrasound in synthetic organic chemistry became more and more interesting."Sonochemistry" is a new trend in organic chemistry, offering a versatile and pathway for a large variety of syntheses.Therefore, a large number of organic reactions can be carried out under ultrasonic irradiation in high yields, short time, and mild conditions [19][20][21][22][23][24].
As an extension of our efforts directed towards development of convenient synthetic approaches for the construction of biologically active heterocycles and as a part of growing interest in sonochemistry [25][26][27], our strategy is to develop a facile sonochemical synthesis and high yield procedure to prepare some novel 1,3,4-oxadiazole, 1,3,4-thiadiazole, 1,2,4triazole, and pyrazole incorporated into 1,8-naphthyridine and the findings of their biological activities in suppressing the growth of HepG2 liver cancer cells.The structure of the new pyrazolone 5 was confirmed on the basis of its elemental analysis and IR, 1 HNMR, 13 CNMR, and mass spectral data, and its 1 HNMR revealed the disappearance of the two singlet signals from  3.99 and 9.28 corresponding to NH 2 and NH protons and new two singlet To find the specific effect of ultrasound on this reaction, all previously mentioned reactions were carried out under the same conditions in absence of ultrasound irradiations (Table 1).The data cited in Table 1 showed that the reaction time increased and the yields of the products decreased in absence of ultrasonic irradiation.Thus, the ultrasound irradiation was found to have beneficial effect on the synthesis of the pyrazole derivatives.
Upon subjecting the reaction mixture to ultrasonic irradiation at 65 ∘ C, the elemental analysis and spectral data of the reaction product were compatible only with the corresponding oxadiazole derivative 6. IR spectra revealed that weak absorption band at 3200 cm −1 correspond to NH, and at 1293 cm −1 band due to C=S group.On the other hand the acid hydrazide 1 with excess CS 2 in ethanolic KOH was subjected to ultrasound irradiation at room temperature; it gave the dithiocarbazate potassium salt 7 (Scheme 2).The IR spectra for 7 showed band due to NH at 3132 cm −1 , C=O amidic at 1686 cm −1 , and C=S at 1291 cm −1 .The dithiocarbazate potassium salt 7 was added to concentrated H 2 SO 4 at 0 ∘ C and then subjected to ultrasound irradiation (Scheme 2); it afforded 5-((2,7-dimethyl-1,8naphthyridin-4-yl)oxy)-methyl)1,3,4-thiadiazole-2-thiol 8 methyl.The IR spectra showed the disappearance of NH band at 3200 cm −1 and new band at 2352 cm −1 for SH was observed.The 1 HNMR spectrum showed only one D 2 O exchangeable signal due to SH at  10.5.Its mass spectrum revealed a peak corresponding to the molecular ion m/z 304.Furthermore, reaction of the potassium salt 7 with hydrazinehydrate in ethanol under ultrasound irradiation at 60-65 ∘ C afforded only one isolable product (as examined by TLC) identified as 4-amino-5-((2,7-dimethyl-1,8-naphthyridin-4-yloxy)methyl)-4H-1,2,4-triazole-3-thiol 9a (Scheme 2); under the same reaction conditions the pot salt 7 allowed to react with phenyl hydrazine and methyl hydrazine; it gave the corresponding aminophenyl and aminomethyltriazole derivative 9b and 9c (Scheme 2).All structures were established according elemental analysis and spectral data.The IR spectra of 9a showed the disappearance of carbonyl group of potassium salt at 1660 cm −1 and two Generally, ultrasound showed beneficial effect on the synthesis of some novel pyrazoles, 1,3,4-oxadiazole, 1,3,4-thiadiazole, and 1,2,4-triazole incorporated into 1,8-naphthyridine which decreases the time of the above reactions from several hours in conventional procedure to few minutes with high yield under ultrasound irradiation.The improvement induced by ultrasound in the abovementioned reaction can be attributed to the well-established theory for the cavitation.These reactions according to sonochemical classification of Cabello et al. [28] and Ikawa et al. [29] are considered as false sonochemistry type in which cavitation effect provides the mechanical energy for all subsequent chemical reactions, including bond scission induced by viscous frictional forces.

Pharmacology.
Preliminary screening showed that all selected compounds exhibited a moderate to strong growth inhibition activity on the tested cell line (IC 50 : 0.048-0.091M) concentrations in comparison to the traditional anticancer drug doxorubicin (DOX).It can be deduced from the results cited in Table 3 that compound 9b showed a growth inhibition activity quite similar to that observed to DOX.The pronounced activity might be due to the presence of N-aminophenyltriazole attached to (2,7-dimethyl-1,8naphthyridine-4-yloxy) methyl moiety [30].

Conclusion
We have synthesized a class of novel substituted pyrazoles, 1,3,4-oxadiazole, 1,3,4-thiadiazole, and 1,2,4-triazole incorporated into 1,8-naphthyridine nucleus under both sonication and classical conditions.In general, improvements in rates and yield of the reactions are observed when reactions were carried out under sonication compared with classical condition.The cytotoxicity screening of the new compounds revealed that the selected compounds showed reasonable  (
Method B: Sonicated Reaction.A mixture of acid hydrazide 1 (16 mmol) and ethylacetoacetate (65 mmol) in 50 mL Erlenmeyer flask was subjected to ultrasound irradiation for suitable time (cf.Table 1) until the starting material was no longer detectable by TLC.All the reactions were kept at temperature 60-65 ∘ C (the temperature inside reaction vessel was 60 ∘ C).The precipitate formed was filtered off and washed with pet.ether and finally recrystallized from ethanol to afford the corresponding ethyl-
Method B: Sonicated Reaction.A mixture of acid hydrazide 1 (2 mmol) was dissolved in hot ethanolic potassium hydroxide solution (0.11 gm KOH in 5.5 mL absolute ethanol) and carbon disulfide (23mmol) in 50mL Erlenmeyer flask.The mixture was subjected to ultrasound irradiation for suitable time (cf.Table 2) until the evolution of H 2 S ceased and the starting material was no longer detectable by TLC.The reaction was kept at temperature 60-65 ∘ C. The reaction mixture was cooled and acidified with acetic acid.The formed solid precipitate was filtered off, washed with water, and recrystallized from methanol to afford the corresponding 5-((2,7-dimethyl-1,8-naphthyridin-4-yl)oxy)methyl)-1,3,4-oxadiazole-2(3H)-thione 6, m.p. 261 (7) Method A: Silent Reaction.Carbon disulphide (0.6 mL, 10 mmol) was added dropwise to an ice cold solution of potassium hydroxide (0.56 gm, 10 mmol) in absolute ethanol (20 mL) containing the respective hydrazide 1 (2.46 gm, 10 mmol).The mixture was stirred at room temperature for 8 h.The separated solid was filtered off and washed several times with ether.The product obtained in quantitative yield was employed in the next reactions without further purification.
Method B: Sonicated Reaction.Carbon disulphide (0.6 mL, 10 mmol) was added dropwise to an ice cold solution of potassium hydroxide (0.56 gm, 10 mmol) in absolute ethanol (20 mL) containing the respective hydrazide 1 (2.46 gm, 10 mmol).The mixture subjected to ultrasound irradiation at room temperature for a suitable time (cf.(8) Method A: Silent Reaction.The potassium salt 7 (0.36 gm, 1 mmol) was added in small portions to H 2 SO 4 (d = 1.836, 1.75 mL) under gentle stirring at 0 ∘ C.After complete addition of the salt the stirring was continued for further 2 h.; then the solution was poured into ice.The precipitate formed was filtered off, washed with water, dried, and recrystallized from ethanol to give the title compound as chestnut crystals.
Method B: Sonicated Reaction.The potassium salt 7 (0.36 gm, 1 mmol) was added in small portions to H 2 SO 4 (d = 1.836, 1.75 mL) in 50 mL Erlenmeyer flask under gentle stirring at 0 ∘ C.After complete addition of the salt, the mixture was subjected to ultrasound irradiation at room temperature for suitable time (cf.2) until the starting material was no longer detectable by TLC.All the reactions were kept at temperature 60-65 ∘ C (the temperature inside reaction vessel was 60 ∘ C).The reaction mixture was cooled and the solid precipitate was filtered off, dried, and finally recrystallized from glacial acetic acid to afford the corresponding 4-N-substitueted-5-((2,7-dimethyl-1,8naphthyridin-4-yloxy)methyl)-4H-1,2,4-triazole-3-thiol 9a-c.
The synthesized compounds (9a-c) with their physical data are listed below.

Cytotoxicity
Measurement of Potential Cytotoxicity by SRB Assay.The selected 1,8-naphthyridine derivatives, compounds (3a, 3b, 5, 6, 8, 9a, 9b, and 9c), were subjected to a screening system for evaluation of their antitumor activity against liver HepG2 cancer cell line in comparison to the known anticancer drugs, doxorubicin (DOX).Potential cytotoxicity of the selected 1,8-naphthyridine derivatives was tested using the method of Skehan et al. [31] as follows: cells were plated in 96multiwell plate (10 4 cells/well) for 24 h before treatment with compounds to allow attachment of cell to the wall of the plate.Different concentrations of the compound under test (5, 12.5, 25, and 50 g/mL) were added to the cell monolayer.Triplicate wells were prepared for each individual dose.Monolayer cells were incubated with the compounds for 48 h at 37 ∘ C and in atmosphere of 5% CO 2 .Cultures were fixed with trichloroacetic acid and stained for 30 min with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid.Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 M unbuffered Tris base [tris(hydroxymethyl) aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader.The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence.The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well.The relation between surviving fraction and drug concentration is plotted to get the survival curve of both cancer cell lines after the specified compound.

Table 1 : Synthesis of pyrazole derivatives 3a-b, 4, and 5 under
both ultrasonic irradiation and conventional method.

Table 2 :
Synthesis of 1,3,4-oxadiazole 6, 1,3,4-thiadiazole 8, and 1,2,4-triazole 9a-c derivatives under ultrasonic irradiation and conventional method.bands at 3151 and 3244 cm −1 corresponding to NH 2 group. 1 HNMR for 9a revealed two D 2 O exchangeable singlet signals due to NH 2 and SH protons at  2.91 and 13.55, respectively.Time of reactions and yield are shown in Table 2.It is shown from this table that the ultrasound technique reduced the time of the reactions from several hours to minutes and improved the yield from 69-83% (under conventional conditions) to 88-96%.