INTRODUCTION:

azo dyes contain chemical groups that bind to metal ions. Among the many metal salts used with these dyes, chromium and copper are the most common; Oftentimes, the metal ion also combines with the fibers, improving the dye's resistance to washing. There are a few anthraquinone dyes and some disperse dyes which also belong to azo compounds, the latter are not soluble in water but can be suspended in soapy water in which case they are absorbed from the suspension by cellulose acetate fibres, Azo compounds has grabbed the attention of chemists in the field of organic chemistry since its bright colors including yellow, red, green, blue or orange depending on the molecular structure and the compounds are very important as pigments^1,2. In fact, the structural properties in colored compounds that give color are:–N = O, > C = C <, –N=N–,> C = O, aromatic rings, and –NO₂^3,4.

The moiety that gives the color always is Azo(–N=N–) or nitroso(–N=O) groups. In addition, azo dyes have been widely studied because of their excellent thermal and optical properties, and most of them have been prepared from diazonium salts⁵. Also the Azo compounds show a variety of biological activities, including anti-bacteria, fungi, viruses, infections and insecticides^6,7,8. In this strategy the key step of the synthesis of Azo compounds (F1 to F3) is the Schiff base reaction that contain (-HC=N-) group. Hugo Schiff (1864) has been synthesized many Schiff bases compounds by intensification of primary amine with a compound containing carbonyl group under mild conditions. Moreover, Schiff bases ligands are used widely because of their high stability and good solubility⁹. Furthermore, the structure of some schiff bases that carry aryl groups or heterogeneous residues shows an excellent biological, antimicrobial activities, antiviral and anticancer¹⁰.

Various dyes have been documented utilizing Isatin as an intermediate compound to reach our target. Isatin (1H-indole-2,3-dione) is classify as a heterocyclic organic compound with a bicyclical structure consisting of a six-membered benzene ring fused to a five-membered nitrogen containing ring. The structure is based on the indoline compound but where two carbonyl groups are situated at 2- and 3-position of the five membered ring. Isatin is one of the few compounds has been synthesized before it was discovered in nature¹¹. The unique structure of Isatin and its derivatives which led to wide range use of these compounds as key intermediates in the field of organic synthesis. Moreover, Isatin is a basic compound of many alkaloids and many drugs as well as dyes, pesticides and analytical reagents and they have different biological function as antibacterial, antifungal, antiviral, anti-HIV¹².

MATERIALS AND METHODS:

1-Chemical part:

Materials: All the chemicals were supplied by BHD and Fluka and used without further purification.

Measurement: The melting points(m.p) were recorded and expressed in degree (0^oC) by using the electro thermal 9300 melting point LTD, UK. Thin layer chromotography T.L.C was performed on aluminum and glass plates coated with 0.25mm layer of silica-gel (Fluka). FT-IR spectra, Fourier transform infrared (SHIMADZU, 8400) spectrophotometer, Japan the prang 4000-600cm^-1. The samples were run in KBr disc. ¹³C,¹H-NMR spectra in (ppm) unit were operating in DMSO -d6 as solvent using (Bruker-Ultra Shield 300 MHz Switzerland), Iran. Elemental analyses were carried out by means of micro analytical unit of 1180 C. H. N elemental analyzer.

Synthesis of Basic compound(Azo) (F1)[2-amino-4-nitro-6-(pyrimidin-2-yldiazenyl)phenol]¹³.

Successful coupling reaction of diazonium salt has been carried out to produce Azo comp. (F1),a mixture of 2-amino pyrimidine with appropriate amount of 2-amino-4-nitrophenol as coupling component. A is prepared by dissolving To prepare diazonium solution, 2-amino pyrimidine (0.01mol, 0.95gm) was dissolved in 4ml of concentrated hydrochloric acid and 30ml of distilled water. To this reaction mixture was added drop wise (0.69gm, 0.01`mole) of sodium nitrite in 10ml of distilled water at (0-5)^oC and left to stand 30 min. This diazonium solution was added drop wise into (1.54gm, 0.01mole) 2-amino-4-nitrophenolwhichdissolved in 50 ml of ethanol at (0-5)^oC. The mixture was allowed to stand overnight. The precipitate was filtered off, washed for many times with distilled water and re-crystallized twice from hot ethanol and then dried in oven at 50^oC for 12hours to give Azo compound (F1) as a green solid, yield (75%), m.p (230^oC decomp.) scheme 1.

General procedures of Schiff bases:[2-(4-(dimethylamino)benzylideneamino)-4-nitro-6-(pyrimidin-2-yldiazenyl)phenol ](F2), [(3E)-3-(2-hydroxy-5-nitro-3-(pyrimidin-2-yldiazenyl)phenylimino)indolin-2-one](F3)^14-17:

A mixture of Azo compound (F1) (0.052gm, 0.0002mol) was added to a stirred solution of (p-N,N-dimethyl benzaldehyde (0.03gm, 0.0002mole) to produce (F2) and Isatin to produce (F3), in (25ml) absolute ethanol and (3) drops of glacial acetic acid. This mixture was refluxed for (30) hrs at (80⁰C). The progress of the reaction was followed by TLC. The reaction mixture was cool down to room temperature then the solid re-crystallized from absolute ethanol to give F2 as dark brown solid; R_f = 0.90 in (EtOH: Benzene) (3: 2), 60%, m.p.(73-75)⁰C and F3: black solid; R_f =0.83 in (EtOH: Benzene) (2:3), 50%, m.p.(122-124)⁰C scheme 1..

Scheme-1: Preparation of Azo comp.F1and Azo-Schiff bases F2,F3

2-Biological part:

All Chemicals and biological materials were supplied from Sigma, Difco, USA, Santacruze biotechnology Inc, Europe, BDH, Flow laboratories, GCC, UK, Merk, Germany were supplied from Arnold Sons, Genex, Beckman Model J2-21, Lab-TeK and Nunc, USA, Memmert, Hermle, Leica, Sartorius, Leitz Germany, Marubeni, Ogawa seiki, Japan, Gallen-kamp, UK, Eppendroff Oxford, LKB Sweden, Nunc Denmark.

Cell culture media:

In this assay two types of cell culture media were used: Growth media (GM) and maintenance media (MM), The PH has been checked and adjusted to about (6.8-7.1). Additionally, the antibiotics have been added to culture medium at final concentration of 100 IU/ml and 100 µg/ml of penicillin G and streptomycin, respectively (1ml of antibiotic solution to 100ml of culture medium), then Nystatin was also added to give the final concentration of 25 IU/ml. Finally, filtration of media was carried out in biohazard safety cabinet using 0.22 µm Millipore filter. Another step is to prepare 100ml GM and MM, the components were mixed up to prepare media necessary for Hep G2 (human liver cancer) cell line.

Cytotoxicity assay^15,16:

Cell line has been seeded on 96 well plates with a concentration of1.0 x l05 cell/ml. After incubation at 37°C for 24 - 48 hr, when the confluent monolayer of Hep G2 cells were complete 80- 100% concentrations (6.5, 12.5, 25, 50, 100, 200,, 400) µg/ml of micro tittered composites were added to cultured wells at a final volume of 100 µl in each well except cells control in triplicate. After 24 hrs incubation at 37°C in 5% CO2, the micro titer 96 wells plates have been marched out and transferred to biohazard safety cabinet by sterilized environments to avoid any contamination, all used wells media were discarded, The Hep G2 cell mono layers have been washed by PBS (Phosphate buffered saline) solution to remove any residual amount of composites or standard anticancer drugs used that may be interacts with MTT (Methyl thiazolyl tetrazolium) reagents. Then 100 µl of maintenance media has been added to all wells included drugs treated cells, drugs untreated cells and blank wells, then, MTT reagent 20µl has been added to each well. After 4 hrs of incubation at 37°C, 5% CO2, the formazan particles were formed as a mitochondrial enzymatic process of the non-effected viable Hep G2 cells, the dead or viral effected cells were didn’t form formozan particles because it is mitochondria organelles were disrupted. The formazan was solubilized by adding diluted DMSO (dimethylsulfoxide) (1:1) in isopropanol on each wells included blank wells, the absorbance was read at 490nm with a reference wavelength of 630 nm by an ELISA reader, this protocol of MTT assay measurement was mentioned by many reports ^18-21.

RESULTS:

The following section will discuss the formation of Azo compound(F1) and there derivatives that prepared by diazotization reaction between 2-amino pyrimidine with 4-nitro- 2-aminophenol as coupling component. The major pathway demonstrated that the successful reaction between sodium nitrite and concentrated acidic acid led to the formation of nitroso ion which add to the 2-amino pyrimidine would then produce diazoniumsalt⁽²²⁾ .The next step involved formation the Schiff bases derivatives F2 and F3 were prepared by condensation reaction between F1 and two carbonyl compounds, p-N,N-dimethyl benzaldehyde (F7) and Isatin (F4) respectively. The carbonyl group that far away from (-NH) group inside the ring undergo the condensation reaction and not the carbonyl close to the group mentioned due to being is amide carbonyl and tautomeric transition^(23-25) of the proton from the atom of nitrogen to the carbon carbonyl atom nearby.

Micro analysis:

The elemental analysis data for the derivatives show in Table (1) it was found that the theoretical values are in a good agreement with the found data .the purity of the Schiff bases were tested by TLC technique with using Ethanol and Benzene as Eluent and elemental analysis(C,H,N).

Table.1: elemental analysis

Comp.	Found(calculated) %
Comp.	C	H	N
F1	-46.15	-3.07	-32.3
F1	45.93	3.34	31.82
F2	-58.31	-4.35	-25.06
F2	58.83	4.01	25.82
F3	-55.5	-2.83	-25.19
F3	54.88	2.34	25.72

DISCUSSION:

The FT-IR technique provide valuable information regarding the nature of the functional group in the compound. Through which the knowledge of the disappearance of groups and the emergence of new groups. The composition of the compound can also be ascertained by observing the change in the value of the absorption frequency, the change in the form of the absorption band and the intensity of the band. The structure of (F1)was assigned by ¹H-NMR and FT-IR experiments; FT-IR showed strong sharp absorption band due to (-C=N)endocyclic for pyrimidine ring at (1624) Cm^-1, emergence of a hydroxyl group is combined with an aromatic primary amine group, where it appears in the form of a broad band at (3439) Cm ^-1.The Azo group band was absorbed at (1498) Cm^-1, in addition to the nitro group was showed two stretching bands at (1543,1332) Cm^-1, the aromatic( C = C)) at (1564) Cm^-1, and the aromatic (C-H)was appeared at (3066) Cm^-1.

Also (F2) characterized by disappearance of aromatic amine band and appearance the absorption band of Imine group(-CH=N) at (1591) cm^-1, (-C=N) endocyclic for pyrimidine ring appeared in higher frequency than that of (F1) at (1647) cm^-1,while the azo group was absorbed at (1494) cm^-1. Additionally, the band of aromatic (C=C) appeared in combination with the nitro group band at (1537) cm^-1, and two bands back to nitro group at (1537, 1334) cm^-1. Also the analysis data show aromatic (C-H) stretched at (3066-3014) cm^-1 while an aliphatic (C-H) at (2918-2951) cm^-1.

Diagnosis of the Shiff base (F3) by the infrared spectrum through disappearance of aromatic amine band and appearance the absorption band of Imine group(-CH=N) at )1595(cm^-1.Furthermore, the hydroxyl group's stretching band is combined with the stretching band of (NH-) for Isatine ring which is present at approximately (3194) cm ^-1. (-C=N) endocyclic for pyrimidine ring appeared in less Frequency than that of F1 at (1618) cm^-1.More importantly, the carbonyl amide group was absorbed in the wave number (1730) cm^-1 and the Azo group was absorbed at (1527) cm^-1 and Nitro group stretches at (1500, 1336) cm^-1, while (C= C) aromatic group at (1562 ) cm^-1, (C-H) aromatic showed at (3111)cm^-1.

¹HNMR Spectra:

The derivatives (F1), (F2) and (F3) were synthesized gave a satisfactory spectral data and the molecular structure was assigned on the basic of ¹H NMR chemical shift. ¹H NMR spectra were determined in solution of DMSO with tetra methyl saline as an internal reference. The ¹H NMR spectrum of F1 showed clear signals involved singlet at δ (2.5) ppm belong to the proton of solvent (DMSO) and multiples signals at δ (7.041-8.391) ppm which were assigned to aromatic protons of phenyl and pyrimidine rings. Also the spectrum showed the proton signal at δ (6.937-6.967) ppm which belong to aromatic amine group. Singlet signal at δ (11.01) ppm for the proton of hydroxyl group(OH), and broad signal at 3.41 ppm due to water dissolved in DMSO-d6²⁶.

Fig.1: ¹H- NMR of (F1)

The ¹H NMR spectrum of F2 showed clear signal singlet at δ (2.5) ppm belong to the proton of solvent (DMSO) and singlet signal at δ (3.06) ppm which was assigned to six protons of (-N(CH₃)₂. It also shows multiples signals at δ (6. 78 -8.15) ppm were assigned to aromatic protons of phenyl and pyrimidine rings, singlet signal at δ (9.68) duo to proton Imine group (1H, -CH=N)^(27-29). Additionally, the broad signal at 3.38 ppm due to water dissolved in DMSO-d6,Finally, singlet signal at δ (11.06) ppm belong to the proton of hydroxyl group(-OH).

The¹H NMR spectrum of (F3) revealed the presence of singlet signal at δ (2.5) ppm belong to the proton of solvent (DMSO), multiples signals at δ (6. 84 -8.10) ppm were assigned to aromatic protons of phenyl and pyrimidine rings. In addition, singlet signal at δ (8.13) duo to proton of Imine group (1H, -CH=N),and singlet signal at δ(10.50) ppm due to (-NH) group of Isatine, and singlet signal at δ (11.06) ppm belong to the proton of hydroxyl group(OH)as in the derivative F2.

Fig.2: ¹H- NMR of (F3)

¹³C -NMR Spectra:

"Nuclear magnetic resonance spectroscopy" of carbon, which is of big interest for Organic Chemistry, is limited to the realization of ¹³C. The magnetic moment of the ¹³C is smaller^30-33 than that of (H) by a factor of 4., The signal at 40ppm due to the solvent (DMSO)

Fig.3: C¹³- NMR of F3

2D NMR HSQC ¹H-¹³C experiments:

The 2D NMR HSQC ¹H-¹³C spectrum of the F3 showed a correlation of the Imine proton signal of F3 at δ 8.13 ppm with carbon at δ 164.39 ppm, which to the assignment of Imine group carbon. The HSQC spectrum showed a correlation between proton signal at 11 ppm with carbon signal at 159 ppm which to the assignment of Carbon Bearing hydroxyl group. The aromatic protons due to benzene and pyrimidine rings signals from 6. 84 -8.10ppm have been correlation with carbon aromatic signals at112.0-159 ppm.

Fig. 4: 2D- NMR HSQC of (F3)

Table 2: Physical properties for Azo compound and its derivatives

Comp.	Molecular	M. Wt	m. p C°	Color	*R_f*	Yield
	Formula				Solvent	%
	(M.F)
F1	C₁₀H₈ O₃N₆	260	230 decomp.	Green	-----	75
F2	C₁₉H₁₇ O₃N₇	391	73-75	Dark Brown	0.9	60
					EtOH: Benzne
					3ml:2ml
F3	C₁₈H₁₁ O₄N₇	389	122-124	Black	0.83	50
					EtOH: Hexane
					2ml:3ml

Cytotoxicity Activity:

Chemotherapy is the use of anti-cancer drugs designed to slow or stop the growth of rapidly dividing cancer cells in the body. The concentrations used to inhibit cell growth ranged from (6.25- 400) μg/ml for Hep G2 ( human liver cancer )carcinoma cells and thus to healthy cells (WRL). The number of living cells left after reactivation with F3 ranged from 17.86% to 84. 57% for Hep G2 and 65.70-96.95% for cell line cells for (WRL). The best rate of inhibition of cell growth of the cancer line of the remaining living cells and healthy cells after F3 reactivation was at concentration 400μg/ml. The results showed that the concentration of the derivative used was an important factor in determining the rate of inhibition of cells, as detailed in Table (23). It was showed that the highest rate of inhibition of F3 for the human liver cancer cell (Hep G2) at 400μg/ml after treatment with F3 was 82.14% while the highest WRL was observed with the same concentration above after reacting with F3 is 34.3%, which is an excellent result. Concentration of 100μg/ml of the above-mentioned derivative will kill more than half of the infected cells Hep G2 and have less effect Healthy cells WRL, and this in turn indicates the possibility of using this derivative as a treatment for the human liver cancer^34-36. The half inhibitory concentration (IC50)that have lowest concentration which kills 50% of cells. The(IC50) for F3 derivative on infected cells Hep G2 is equal 25.46μg/ ml while on the WRL cell was observed 163μg/ml, This means that the derivative is likely to be a killer of the cancer human liver cell without affecting the normal cells when using its lethal concentration for half cells that which 25.46μg/ml .

Table 3: Effect of the derivative (F3) on human liver cellular cancerous cells Hep G2 viability and comparison with natural cells line (WRL) for same concentration using 24-hour MTT test at 37 °c

Conc. μg / ml	Mean percentage (%) for each cell line
	Cancerous line cells of Hep G2	Normal line cells of WRL
	Cell Cell viability inhibition	Cell Cell viability inhibition
6.25	84.57 15.43	96.10 3.9
12.5	76.12 23.88	94.91 5.09
25	60.07 39.93	96.95 3.05
50	44.29 55.71	96.18 3.82
100	35.19 64.81	92.13 7.87
200	28.97 71.03	74.46 25.54
400	17.86 82.14	65.70 34.3

Table.4 :Calculate the best concentration for the highest inhibition

Conc. μg / ml	Cell inhibition for Hep G2 -- Cell inhibition for WRL	Result
6.25	15.43-3.9	11.53
12.5	23.88-5.09	18.79
25	39.93-3.05	36.88
50	55.71-3.82	51.89
100	64.81-7.87	56.94
200	71.03-25.54	45.49
400	82.14-34.3	47.84

Fig.5: Showing anticancer activity data (F3) against human Liver cancer cell line

Antibacterial Activity:

Photolysis is not considered a degradation pathway for azo dyes. In order to extend the life of products dyed with azo dyes, it is necessary to ensure stability against microbial attack, and tests have shown that azo dyes degrade significantly in short-term tests under aerobic conditions. However, under anaerobic conditions, color change can be observed as a result of biodegradation Photolysis is not considered a degradation pathway for azo dyes. In order to extend the life of products dyed with azo dyes, it is necessary to ensure stability against microbial attack, and tests have shown that azo dyes degrade significantly in short-term tests under aerobic conditions. However, under anaerobic conditions, color change can be observed as a result of biodegradation³⁷., The synthesized derivatives exhibited activity against both "Gram- negative" and "Gram- positive" bacteria. The biological activity was examined in this work against two types of bacteria Granutic telladiacens as (Gram +),and Enterobacter as (Gram--). Two concentrations of each derivative were prepared (0.5, 0.1) μg/L. the results were shown in the Table (5).

Table 5: Zone inhibition (mm) of all derivatives against two types of microorganisms

S. No.	Concentration μg / L	Inhibition Zone
S. No.	Concentration μg / L	Enterobacter *(-ve)G*	*Granutic telladiacens* *(+ve)G*
F1	0. 5 0.1	26 16	32 20
F2	0.5 0.1	20 21	20 18
F3	0.5 0.1	18 12	11 14

CONCLUSION:

In this paper, a new Azo compounds have been prepared and fully characterized by spectral and analytical data. These compounds was showed biological activity, which exhibit antimicrobial properties. The cytotoxicity of (F3) on human Liver Cancer (Hep G2) and normal cells (WRL) were studied using MTT assay. It is appeared higher selective cytotoxicity versus cancer cell line, whereas it was very secure on normal cells line. The results signalize undoubtedly the possibility of utilize it as anti-cancer drug in the domain of medicine.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank staff of bio-center for measurements and bio-studies.

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Received on 27.06.2022 Modified on 18.07.2022

Research J. Pharm. and Tech 2023; 16(3):1289-1295.

DOI: 10.52711/0974-360X.2023.00212