Synthesis and Biological Evaluation of Some Novel 1,8-Naphthyridine Derivatives

A series of substituted 1,8-naphthyridine derivatives was synthesized to be used as cytotoxic and antioxidant agents by applying 1,4-dihydro-4-oxo-1,8-naphthyridine-3-carbohydrazide (1) as the starting material. Compound 1 was reacted with different reagents to afford the corresponding 3-heterarylcarbonyl-1,8-naphthyridine derivatives 3–19 which were tested for their in vitro cytotoxicity against Ehrlich Ascites Carcinoma, and antioxidant activity. Compound 15 showed the best cytotoxicity and antioxidant activity.


Introduction
2][3] Nalidixic acid (1-ethyl-3-carboxy-7methyl-1,8-naphthyridine-4-one) has been found to be effective particularly against gram negative bacteria found in chronic urinary tract infections. 41,8-Naphthyridine derivatives were found to display moderate cytotoxic activity against murine p388 leukemia, when changes were carried out at N-1 and N-7 positions. 5,6It has been reported that C-3 carboxamide derivatives with a spacer have shown good cytotoxicity along with anti-inflammatory activity. 7harmacologically, pyrazole and its derivatives represent one of the most important classes of organic heterocyclic compounds, possessing anti-bacterial, anti-fungal, 8 herbicidal 9 and anti-viral activities. 10oreover, the chemistry of carbohydrazoles has gained increased interest in both synthetic organic chemistry and biological fields and has considerable value in many useful applications, such as the assessment process of the three dimensional ultra structure examination techniques of interphase nuclei and tissues, besides their therapeutic importance. 11

1. 1. Chemicals and Reagents
All the chemicals and solvents used in this study were obtained from Merck (Germany) and Sigma-Aldrich chemical company (Germany).

1. Instruments
All melting points were recorded on Gallenkamp electric melting point apparatus and are uncorrected.The IR spectra ν cm -1 (KBr) were recorded on Perkin-Elmer Infrared Spectrophotometer Model 157, Grating.The 1 H and 13 C NMR spectra were run on Varian Spectrophotometer at 400 MHz and 100 MHz using TMS as the internal reference and DMSO-d 6 as the solvent.Chemical shifts (δ) are given in ppm.The mass spectra (EI) were recorded on 70 eV with Kratos MS equipment and/or Varian MAT 311 A Spectrometer at Cairo University, Giza, Egypt, and at Assuit University Central Laboratory.Elemental analyses (C, H, and N) were carried out at the Microanalytical Center of Cairo University, Giza, Egypt (automatic analyzer CHNS, Vario ELIII-elementar, Ger-Abu-Melha : Synthesis and Biological Evaluation ... many).The results were found to be in good agreement with the calculated values.

Pathway 2
A mixture of 1 (2.04 g, 10 mmol) in ethanol (25 mL) containing a few drops of glacial acetic acid and malononitrile (0.66 g, 10 mmol) were refluxed for 3 h, then left to stand at room temperature overnight.The separated solid product was filtered off, dried and recrystallized from ethanol to give 19.Yield 71%; gray powder; m.p.

3. Antitumor Activity (or Cytotoxicity) Using In Vitro Ehrlich Ascites Assay
The isolated compounds were screened for their antitumor activity.The viability of the cells used in control experiments exceeded 95%.Different concentrations of the tested compounds were prepared (100, 50 and 25 µL from 1 mg/mL in DMSO (< 00.05%, v/v) and complete to 1 mL using RPMI-1640 medium).5-Fluorouracil (25 µg/ mL) was prepared in 100 µL DMSO and complete to 1 mL using RPMI-1640 medium.Ehrlich ascites Carcinoma (EAC) were derived from ascetic fluid from diseased mouse (purchased from National Cancer Institute, by National Medical Research Ethics Committee).Ascites fluid from the peritoneal cavity of the diseased mouse (containing Ehrlich cells) was aseptically aspirated.The cells were grown partly floating and partly attached in a suspension culture in RPMI 1640 medium, supplemented with 10% fetal bovine serum.They were maintained at 37 °C in a humidified atmosphere with 5% CO 2 for 2 h.The viability of the cells was determined by the microscopical examination using a hemocytometer and using trypan blue stain (that stains only the dead cells). 14

4. Antioxidant Activity Screening Assay 2,2'-Azino-bis-3-ethylbenzthiazoline-6sulfonic Acid Method
For each of the investigated compounds, 2 mL of ABTS solution (60 µM) was added to 3 mL MnO 2 solution (25 mhg/mL), all prepared in 5 mL aqueous phosphate buffer solution (pH 7; 0.1 M).The mixture was shaken, centrifuged, filtered, and the absorbance of the resulting green-blue solution (ABTS radical solution) at λ 734 nm was adjusted to approximately 0.5.Then, 50 µL of 2 Mm solution of the tested compound in spectroscopic grade MeOH/phosphate buffer (1:1) was added.The absorbance was measured and the reduction in color intensity was expressed as inhibition percentage.L-Ascorbic acid was used as the standard antioxidant (positive control).Blank sample was run without ABTS and using MeOH/phosphate buffer (1:1) instead of the tested compounds.Negative control was run with ABTS and MeOH/phosphate buffer (1: 1) only. 15,16The inhibition ratio (%) was calculated using the following formula: (1)
The IR spectrum showed absorption bands at 3230-3238, 1668, 1671, 1614, and 1569 cm -1 corresponding to stretching vibrations of two NH, amidic carbonyl groups, α,β-unsaturated ketone, and C=N groups. 1 H NMR spectrum revealed singlet signal at δ 1.93 ppm due to methyl protons, in addition to the classical pattern of 1,8-naphthyridine protons.The mass spectrum provided more evidence for the correct structure, which showed the molecular ion peak at m/z 347 (M + ).
Compound 1 when reacted with ethyl acetoacetate in refluxing ethanol containing a catalytic amount of glacial acetic acid afforded the acyclic intermediate ethyl Structures 4-7 were proved by elemental and spectral analyses.The IR spectra of compounds 4 and 6 in general showed absorption frequencies at 1567-1569, 1724, and 1689 cm -1 corresponding to C=N and two C=O due to ketonic (ester), and amidic carbonyl functional groups, while the 1 H NMR spectrum of compound 4 showed a characteristic signal at δ 0.91 ppm as a singlet signal for CH 3 protons, δ 1.38 ppm as a triplet signal for CH 3 protons, δ 2.35 ppm as a singlet signal for CH 2 protons and at δ 4.25 ppm as a quartet signal for CH 2 protons besides the aromatic protons of pyridine ring at δ 7.60-8.24ppm, and a singlet signal at 8.61 ppm due to C 2 -H of naphthyridine ring.On the other hand, the 1 H NMR spectrum of compound 6 showed three singlet signals at δ 1.20, 2.11, 2.54 ppm corresponding to two methyl protons and CH 2 protons, respectively.In addition, the mass spectrometry measurement gave m/z 316 (M + ) and 286 (M + ) corresponding to the molecular ion peaks of compounds 4 and 6, respectively.For the pyrazole derivative 5 the IR spectrum showed a new absorption band at 1669 cm -1 corresponding to a new amidic carbonyl and absorption frequency at 1606 cm -1 corresponding to C=N stretching frequency while the IR spectrum of compound 7 showed stretching frequency at 1567 cm -1 due to C=N functional group. 1 H NMR of compound 5 showed two new singlet signals at δ 1.22 and 2.07 ppm corresponding to methyl and methylene protons of pyrazolone moiety.On the other hand, the 1 H NMR spectrum of compound 7 revealed three singlet signals at δ 1.74, 1.79, and 6.08 ppm attribut- able to two methyl groups and methine protons of pyrazole moiety.The mass spectra of compounds 5 and 7 showed their molecular ion peak at m/z 270 (M + ) and 270 (M + +2), respectively.
Structures 8 and 9 were proved based on the correct analytical and spectral data.The IR of compound 8 showed bands at 3450 and 1574 cm -1 corresponding to the hydroxyl group and stretching vibration of C=N function.Compound 9 was confirmed by analytical and spectral data, beside it was confirmed chemically by an alternative synthesis.Thus, when hydrazide 1 reacted with 2-hydroxy-1-naphthaldehyde in refluxing ethanol containing a catalytic amount of pipridine or triethylamine or sodium ethoxide afforded directly the corresponding pyrazole derivative 9.The IR spectrum of compound 9 showed bands at 1579 cm -1 corresponding to C=N function, and the disappearance of the band around 3450 cm -1 corresponding to hydroxyl group which indicates that the hydroxyl group was involved in the cyclization process.The 1 H NMR spec-trum showed a characteristic singlet signal at δ 8.20 ppm due to the pyrazole CH proton plus the classical 1 H NMR pattern of the rest of 1,8-naphthyridine protons.The mass spectroscopic measurement gives an additional confirmation for compound 9 which showed the molecular ion peak at m/z 340 (M + ).
The IR spectrum of pyrazole derivative 10 showed a characteristic absorption band at 1569 cm -1 corresponding to C=N function and the disappearance of any band around the region 3400 cm -1 corresponding to hydroxyl group. 1 H NMR spectrum showed singlet signal at δ 8.70 ppm attributable to the pyrazole proton.The mass spectroscopic measurement gives an additional confirmation for compound 10 which showed the molecular ion peak at m/z 290 (M + ).
An interesting reaction was observed when hydrazide 1 was heated with malononitrile dimmer (11)  Abu-Melha : Synthesis and Biological Evaluation ... tion of NHNH 2 moiety to the two cyano groups with the formation of a pyrazole ring.The reaction followed by the attack of the lone pair of electrons of NH 2 of pyrazole ring to the cyano group as shown in the following mechanism affording the pyrazolopyridine 13 and not the other possibility 12a because the exocyclic double bond will be more stable in the Z-form due to less steric hindrance.This fact confirms that nucleophilic addition occurred from NH 2 group and not imino group.(Scheme 5).
The IR spectrum of compound 13 showed absorption bands at 3411, 3382 cm -1 due to NH 2 functions, besides a broad band at 3228 cm -1 for NH groups, 1671 and 1608 cm -1 corresponding to amidic C=O, C=N and the disappearance of any band due to cyano functions at 2220 cm -1 which indicate that the cyano group was involved in the cyclization reaction.The 1 H NMR showed four singlet exchangeable signals at δ 4.18, 9.81, and 11.60 ppm attributable to one amino group and three NH protons, respectively.The mass spectrum showed the molecular ion peak at m/z 336 (M + ).
The hydrazide 1 when heated with dimethylthiomethylene malonate (14) in the presence of ethanol containing a few drops of glacial acetic acid afforded the pyrazole derivative 15 (Scheme 6).The reaction proceeds according to the proposed following mechanism.
The IR spectrum of compound 15 showed absorption bands at 3413, 3382 cm -1 corresponding to NH 2 group, 3226 cm -1 due to NH function, 1670 and 1608 cm -1 corresponding to amidic C=O and C=N and a characteristic absorption band at 2220 cm -1 corresponding to CN group.The 1 H NMR revealed a characteristic singlet signal of thiomethyl group at δ 1.96 ppm and three exchangeable Structures 17 and 18 were proved based on the analytical and spectral data.The IR spectrum of both compounds showed, in general, characteristic absorption bands at 1582 and 1550 cm -1 corresponding to stretching frequencies of C=N and C=C functions, respectively.The 1 H NMR of both compounds showed a characteristic sin-glet signal at δ 5.29 and 5.69 ppm attributable to methyne protons.The mass spectra of compounds 17 and 18 showed the molecular ion peak at m/z 403 (M + ) and 392 (M + -1), respectively.
Structure 19 was established based on both analytical and spectral data.The IR spectrum showed the classical pattern for carbonyl groups which appear at 1668 and 1634 cm -1 , and showed the stretching vibration of C=N and C=C functions at 1612 and 1570 cm -1 , respectively.On the other hand, the amino groups appeared as a tautomeric equilibrium with amino-imino groups and appeared at 3441 and 3232 cm -1 , respectively.Abu-Melha : Synthesis and Biological Evaluation ... additional evidence for structure 19 which showed its molecular ion peak at m/z 270 (M + ).

Effect of Drugs on the Viability of Ehrlich Ascites Cells In Vitro
The synthesis, antitumor evaluation and QSAR studies of novel pyrazol derivatives employed against Ehrlich Ascites Carcinoma (EAC, in vitro) cells are described.These novel analogues were molecularly designed with the goal of having significant potent cytotoxic effect against EAC cells.
Pyrazoles and related analogues were tested for cytotoxicity against EAC in vitro.EAC cells were used because they have a very well known established model of activity. 20Results for the ED25 value of the active compounds are summarized in Table 1.The data showed clearly that compound 15 showed moderate activity (~45%) compar-ing with the drug reference (5-FU, 98% activity).The rest of compounds showed weak activity.Thus, it would appear that introducing thiomethyl tautomeric moiety enhanced the cytotoxic properties.
Comparing the obtained cytotoxic activity of tested compounds in this study, the following structure-activity relationships (SAR) were postulated: 1) Compound 15 showed a mild cytotoxic activity (~45%), this may be due to the presence of thiomethyl and cyano groups which have toxic activity in nature.
2) All pyrazole derivatives showed weak activity at ED25.Thus the position and nature of substituents in the structure of pyrazole derivatives seems to modulate cytotoxic activity.

2. 2. ABTS Antioxidant Activity Screening
The antioxidant activity assay employed here is one of the several assays that depends on measuring the consumption of stable free radicals, i.e. evaluates the free rad- Where ED25 is the effective dose at 25 µL of the compounds used.The dead % refers to the % of the dead tumor cells and 5-Flu is 5-fluorouracil as a well known cytotoxic agent.
ical scavenging activity of the investigated component.The methodology assumes that the consumption of the stable free radical (X') will be determined by the reaction as follows: (2) The rate and/or the extent of the process measured in terms of the decrease in X' concentration, would be related to the ability of the added compounds to trap free radicals.The decrease in color intensity of the free radical solution due to scavenging of the free radical by the antioxidant material is measured colorimetrically at a specific wavelength.The assay employs the radical cation derived from 2,2'-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) (ABTS) as a stable free radical to assess antioxidant potential of the isolated compounds and extracts.The advantage of ABTS-derived free radical method over other methods is that the produced color remains stable for more than one hour and the reaction is stoichiometric.The inhibition ratio (%) was calculated using the following formula: (3) The antioxidant activity of some newly synthesized compounds was evaluated by ABTS method. 21The data in Table 2 showed clearly that compound 15 demonstrated moderate antioxidant activities.Thus, it would appear that introducing sulfur atoms and the presence of thiomethyl tautomeric equilibrium enhances the antioxidant properties of 1,8-naphthyridine derivatives.By comparing the results obtained of antioxidant of the compound reported in this study to their structures, the following SAR was postulated: compound 15 was nearly inpotent to vitamin C which may be attributed to the presence of amino and imino groups which trap the free radical X.

Conclusion
This work aimed to synthesize a new series of pyrazole derivatives containing 1,8-naphthyridine ring via carboxamide linkage.All the structures of the synthesized compounds were confirmed by different spectroscopic data and screened for their in vitro cytotoxicity against EAC and antioxidant activity.Results obtained show that compound 15 displayed the best cytotoxicity and antioxidant activity.

Scheme 9 Table 1 .
In vitro cytotoxicity of pyrazol against Ehrlich Ascites Carcinoma

Table 2 .
Antioxidant assay for some prepared new compounds.