View of Synthesis and Characterization of Some New Formazan- Cefixime and Study of Against Breast Cancer Cells

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Synthesis and Characterization of Some New Formazan- Cefixime and Study of Against Breast Cancer Cells

Dr. Nagham Mahmood Aljamali

^1*

, Hussein Mejbel Azeez

²

1*Professor, Organo-Synthetic Field, Iraq.E-mail: [email protected]

2M.Sc-Student, (First Part of Thesis), Department of Chemistry, College of Education, Iraq.

ABSTRACT

In this study, the starting of synthesis from the esterification of Cefixime of the two carboxylic groups, then its reaction with hydrazine to produce hydrazo cefixime, flowed by reaction with some different aldehydes compounds (R-CHO) to yield imine containing a group (-C=N-) known as azomethine,the last step via more than three reactions fromCoupling and linking with diazo compounds fromgroup (-N=N-) to produce formazan- Cefiximederivatives fromdrugs. All the synthesized compounds studied with spectroscopy techniques (FT.IR, H¹NMR, C¹³HNMR) and were examined to demonstrate chemical bonds, active groups and different environments for their components, and some other chemical properties such as melting point measurement and thin layer chromatography (TLC), in addition to the biomechanical test likebreast cancer (MTT assay).

KEYWORDS

Cefixime,Azo,Imine,Breast Cancer, Schiff Base, Hydrazine, MTTAssay,Formazan, Bioactive.

Introduction

Cefixime is a semi-synthetic antibiotic that belongs to the third generation of the Cephalosporin group⁽¹⁾and its molecular weight is (453.45 g.mol^-1) and it has the structural formula below⁽²⁾:

Photo1.CefiximeStructure

Chemically, 7-(2-(2-aminothiazol-4-yl)-2-(carboxymethoxy)imino)acetamido)-8-oxo-3-vinyl-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid is a white powder with very little moisture. As for its solubility in some materials, it is slightly soluble in water, has a high solubility in methanol and ethanol, influenced by air, so it is stored in tightly closed containers, away from light ⁽³⁾. Cefixime is given orally, and among the cases in which it is prescribed is its use to treat bacterial infections, including infections that occur in the pharynx and stomatitis, as well as otitis and bronchitis, as well as urinary tract infections. In 1989 the medical use of cefixime was approved^(2,4).

Hydrazine, Chemically(N₂H₄) is a versatile inorganic bi-amine compound⁽³⁾. It is a very toxic substance and a very dangerous that causes cancer as well as many symptoms such as burns of the eyes and nose,short-term vision loss, fainting, vomiting, and loss of consciousnessand others^(4,5). Hydrazine is involved in many different chemical reactions as a catalyst or in the synthesis of many chemical compounds ^(6,7).

Formazan: Imine compounds comprise (-HC=N-) the azomethine group⁽⁶⁾, when they bond directly to the group (- N=N-) contained in the Azo dyes^(3-7), resulting in compounds containing a distinct sequence of atom-bonding (-N=N- C=N-NH), characterized by pigments ranging in color from red to orange or blue depending on their structure⁽⁸⁾,If the ring was closed, it was tetrazolium, but the open structure was called formazan⁽⁹⁾,bTetrazolium / formazan is a special biological system represented by oxidation and regeneration (the process of oxidation of formazan

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compounds into conversion to tetrazolium salts, and when given to a living organism and by the action of enzymes in its cells, the opposite occurs)^(10-15) and from applying this property to various branches of science such as medicine, pharmacology and immunology and botany, but especially in biochemistry and histochemistry, as it is a distinct marker that enables the identification of cancer cell activity, as well as of great benefit in determining the quality and selection of drugs and anti-cancer drugs^(15-20).The most common synthetic pathway for formazan compounds involves coupling of aryl diazonium salts with imines in the presence of certain catalysts, for example (coupling) of (phenylhydrazine-imine) with diazonium salts to formulate derivatives of formazan^(21-28).The anti-viral and antimicrobial properties of formazan industrial and biological applications are attributed to the presence of the amine group on the one hand and the azo group on the other hand on the same compound^(29-35) and its applications as an antimicrobial⁽³⁶⁾, pain reliever⁽³⁷⁾, anti-fungal, anti-cancer, anti-HIV, and many applications as an effective inhibitor^{(38, 43)}, Corrosion, and other applications of formazan compounds in polymers and in other fields.

Materials and Instruments

Chemicals used in the present study were supplied from different companies like (C.D.H., B.D.H,and othercompanies), The purity ofdrugsfrom Cefiximeandthe synthesized Imine and formazan derivatives was checked by thin layer chromatography (TLC) stains were appear by iodine vapor.The infrared (FT.IR) spectra all compounds was recorded on Shimadzu FT-IR using (KBr) disk technology and Proof of all structures mediated by measurement (FT-IR, H¹-NMR, C¹³-NMR) withDMSO-d6as a solvent. The melting points was determined in Capillary tubes using Electrothermal device.

Synthesis of Cefixime- Imine Derivative (1):2-(2-aminothiazol-4-yl)-N-(2-(hydrazinecarbonyl)-8-oxo-3-vinyl-5- thia-1-azabicyclo[4.2.0]oct-2-en-7-yl)-2-((2-hydrazinyl-2-oxoethoxy)imino)acetamide: In a round flask capacity (50 ml), (0.01mol, 4.53gm) of cefixime reacts in (40 ml) of absolute ethanol as a solvent with continuous stirring using a glass stirrer and gradually add a few dropsof concentrated sulfuric acid. The process of reflux for (3 hrs) at (75C^o) to giveester of cefixime, thenreacted with hydrazine 40 % by refluxing for (4 hrs) according to studies^(3-7),the reaction was followed by the thin layer chromatography (TLC) technique using a mixture of solvents (benzene, ethyl alcohol) in a ratio of (6: 4), followed by cooling and filtration of the solution, then left for drying to yield compound(1).

Synthesis of Cefixime- Imine Derivative (2):2-(2-aminothiazol-4-yl)-N-(2-((Z)-2-(4-hydroxy-3- methoxybenzylidene)hydrazinecarbonyl)-8-oxo-3-vinyl-5-thia-1-azabicyclo[4.2.0]oct-2-en-7-yl)-2-((2-((Z)-2-(4- hydroxy-3-methoxybenzylidene)hydrazinyl)-2-oxoethoxy)imino)acetamide:By reacting (0.001mol, 0.48gm) of hydrazo of cefixime with (0.003mol, 0.47gm) of Vaniline in (40ml) of ethanol, then added (3) drops from glacial acetic acid to the mixture, the process of refluxing for (6hrs) at (75C^o), and the reaction was followed by TLCaccording to studies^(3-7), after that the product was cooled,filtered, dried, and recrystallized.

Synthesis of Cefixime- Imine Derivative (3) :2-(2-((E)-(4-nitrobenzylidene)amino)thiazol-4-yl)-N-(2-(2-(4- nitrobenzylidene)hydrazinecarbonyl)-8-oxo-3-vinyl-5-thia-1-azabicyclo[4.2.0]oct-2-en-7-yl)-2-((2-(2-(4-

nitrobenzylidene)hydrazinyl)-2-oxoethoxy)imino)acetamide:By reacting (0.001mol, 0.48gm) from hydrazo of cefixime with (0.003mol, 0.45gm) of (4-nitrobenzaldehyde) in (40ml) ethanol. Then add (3) drops from glacial acetic acid to the mixture, The process of refluxing for (8 hrs) at (75C^o), and the reaction was followed by the TLCaccording to studies^(3-7),after that the product was cooled, filtered, dried, and recrystallized.

Synthesis of Cefixime- Imine Derivative (4) : 2-(2-((Z)-(2-hydroxybenzylidene)amino)thiazol-4-yl)-N-(2-(2-(2- hydroxybenzylidene)hydrazinecarbonyl)-8-oxo-3-vinyl-5-thia-1-azabicyclo [4.2.0]oct-2-en-7-yl)-2-((2-(2-(2- hydroxybenzylidene)hydrazinyl)-2-oxoethoxy)imino) acetamide:By reacting (0.001mol, 0.48gm) from hydrazo of cefixime with (0.003mol, 0.36gm) of (2-hydroxybenzaldehyde) in (40ml) of ethanol, Then add (3) drops from glacial acetic acid to the mixture, The process of reflux for (8 hrs) at (75C^o) according to studies^(3-7), and the reaction was followed by the TLC technique, after that the product was cooled, filtered, dried, and recrystallized to give compounds in scheme (1).Follow of all reaction via (TlC).

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Scheme1.Synthesis ofCefixime- Imine Compounds[1-6]

Synthesis of Cefixime-Formazane Derivatives (7, 8, 9, 10)

(0.01mol) Variousaromaticamines was dissolved in (3 ml) of hydrochloric acid and (5ml) distilled water with stirring, The mixture was cooled in freeze bath at (0-5Cº), then adding solution of (0.67 gm) sodium nitrite (NaNO₂) dropwise with stirring at (0–5Cº) in an freeze bath via two steps,(0.001ml) fromImine compoundswere dissolvedin (20ml)inabsolute ethanol with basic medium ofsolution (10ml) a diluteof (10% Sodium hydroxide) with coolingat (0-

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5Cº) and stirring.Then followed by mixing the prepared solution ofdiazonium salt(in first step) to the solution Imine compound (of second step) gradually, while maintaining the temperature at (0-5C^o), then leaving (from 10 to 15 hrs), then filtered, dried, and the crystallized in ethanol according to studies^(3-7),to get a product of pure Cefixime- formazan derivatives (7, 8, 9, 10) in scheme (2).

Scheme2.Synthesis ofCefixime- FormazanDerivatives[7, 8, 9 10]

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Results and Discussion

Newlyprepared Cefixime- Formazan derivativesandCefixime- Imine derivatives were identifiedvia[FT.IR-spectra, H¹.NMR-spectra and some ofthem by C¹³.NMR-spectra, melting, melting points], and study of behaviorof one of drugderivativesagainst breast cancer cells:

SpectralIdentification (FT.IR, H.NMR, C.NMR)

FT-IR_spectrum data for derivative (1): it appearedband at (3387, 3315 cm^-1) for (-NH₂),bands for (-CO-NH) amine OF amide at (3200 cm^-1),(1681cm^-1) for (CO-N) Carbonyl of amide,(2950 cm^-1) for (C- H) aliphatic, (1650 cm^-1)for (C=N) endocyle, (771 cm^-1) for (C-S),(3100cm^-1) for (=CH) Alkene. H¹NMR spectrum: show Signals at (1.03 - 1.15) refer to the protons of the instance groups (-CH₂-) present in the two Cefixime rings (CH-CH-S-CH₂-), signals for the alkene (-CH=CH2) at the sites PPM (6.84-6.88-6.86), Signals at (4.64-4.63) for amine groups (NH), at the ends of compound 2 for the presence of more than one amine group with different environment, signals at (8.69- 9.76) refer to the amide protons (OC-NH), signal at (7.76) It refers to the protons of the aromatic ring of thiazole, a signal at (1.23) refers to the protons of methylene (CO-CH₂-O).In all spectra.

FT-IR_spectrum data for derivative (2): it appeared band at (3406cm^-1) for (OH) Phenol,bands for (-NH-CO) amine of amide at (3332 cm^-1), and (1618cm^-1) for (CH=N) Imide group,(1163cm^-1) for (OCH₃) ether, (2981cm^-1)for (C-H) aliphatic,(=CH) Alkene at (3100 cm^-1), (1664cm^-1) for (CO-N) Carbonyl of amide, (752 cm^-1) for (C-S). H¹NMR spectrum: showWenoted the emergence of signals at (1.06-1.91) that refer to the protons of the methylene groups in (-CH-CH-S-CH₂) in the two cefixime rings. Signals refer to the alkene (-CH=CH₂), which is PPM (6.81-6.79-6.75).

Signals at (9.93-8.99) refer to the protons of the amide group of different environments (OC-NH). Signal at (6.99- 7.93) refers to the aromatic ring protons in the compound. Signal at (3.75) refers to the protons of the methylene group (CO-CH₂-O). Signal at (8.00) PPMrefers to the proton of the amine group (HC=N). Signal at (2.23) refers to the (O-Me) methoxy group protons. A signal at (11.12) PPM refers to the proton of the hydroxy (OH) group in the phenol.C¹³NMR spectrum: a signal at (40.00ppm) belonging to the solvent DMSO-d6., Where a signal appeared at (56.00) belonging to the methoxy group (-OCH₃), a signal at (60.42) belonged to the carbon of the methylene group (CO-CH2-O), and a signal at (21.5) related to the carbon of the methylene group inside the ring (-CH2-C), multiple signals belong to the carbon atoms in the aromatic rings at (113-136), a signal appeared at (153) related to the imine carbon (HC=N) in the compound as a result of the formation of the amine complex, signals at (163,165, 169) refers to the carbon of the amide groups (OC-NH) repeated in the compound, a signal refers to the alkene carbon (CH=CH2) at the PPM (106) sites, and a signal at (12.56) refers to the carbon (-CH-CH)) in the four membered ring.

FT-IR_spectrum data for derivative (3):band at (3412cm^-1) for (NH-CO),bands for (OH) Phenol at (3441cm^-1),(1618cm^-

1) for (CH=N) Imine group, (1662cm^-1) for (CO-N) Carbonyl of amide, (2991cm^-1)for (C-H) aliphatic, (752cm^-1) for (C-S),(3115cm^-1) for (=CH₂) Alkene.H¹NMR spectrum: showSignals appear at (1.06-1.65) that refer to the protons of the two methylene groups present in (-CH-CH-S-CH₂) in the two cefixime double rings. Signals refer to the alkene (CH=CH₂) at (6.50-6.45-6.40) σ. Signals at (9.30-9.00) refer to the proton amide (OC-NH) in different environments.

Signal at (7.64-6.99) refers to the aromatic ring protons. Signal at (3.19) refers to the protons of the methylene group (CO-CH₂-O). A signal at (8.50) PPM refers to the proton of the amine group (HC=N) in the compound as a result of the disappearance of the amine group and the formation of the amine group in the formed compound.

FT-IR_spectrum data for derivative (4):band at (3383cm^-1) for (NH-CO),bands for (OH) Phenol at (3441cm^-1),(1618cm^-

1) for (CH=N) Imine group, (1662cm^-1) for (CO-N) Caronyl of amide, (748cm^-1) for (C-S), (3100cm^-1) for (=CH2) Alkene, (2993cm^-1)for (C-H) aliphatic. H¹NMR spectrum: signals at (0.83 - 1.65) σ due to the protons of the methylation groups in (-CH-CH-S-CH₂) in the two cefixime double Rings. Signals related to the alkene (CH=CH₂) appeared at the sites (6.62-6.11-6.06) PPM. Signals appeared at (9.05,9.00) going to the proton amide (OC-NH).

Signal at (7.96-6.96) refers to the protons of the aromatic ring. Signal at (3.17) refers to the methylene group (CO- CH₂-O) proton. A signal at (8.36) PPM refers to the amine group proton (HC=N). A signal at (11.45) PPM refers to the proton of the hydroxy (OH) group in the phenol.

FT-IR_spectrum data for derivative (5): show band at (3354cm^-1) for (OH) Phenol,bands for (-NH) amide at (3169cm^-1), (1654cm^-1) for (CO-N) Carbonyl of amide, (2995cm^-1) for (C-H) aliphatic, (759cm^-1) for (C-S),(=CH) Alkene at (3024 cm^-1), (1616cm^-1) for (CH=N) Imine group. H¹NMR spectrum: showSignals appear at (1.10-2.28) related to

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the protons of the two methylene groups present in (-CH-CH-S-CH₂) in the two cefixime double rings. Signals of alkene protons (-CH=CH₂) appeared at the PPM sites (6.55,6.20,6.00). Signals appeared at (9.33,9.00) due to the protons of the two amide groups (OC-NH). Signal at (7.40-6.93) refers to the aromatic ring protons. Signal at (3.46) refers to the methylene group (CO-CH₂-O) proton. A signal at (8.30) PPMrefers to the proton of the amine group (HC=N) repeating in the synthesis. A signal at (10.20) PPM refers to the proton of the hydroxy (OH) group in the phenol.

FT-IR_spectrum data for derivative (7): bands for (-NH) amine group at (3194cm^-1), band at (3313cm^-1) for (OH) Phenol, (1683cm^-1) for (CO-N) Caronyl of amide, (1192cm^-1) for (O-CH₃) ether, (1635cm^-1) for (C=N) Formazan, (1359,1435,1462cm^-1) for (N=N) of formazan, (771 cm^-1) for (C-Cl), (698cm^-1) for (C-S), (2902cm^-1) for (C-H) aliphatic, (3035cm^-1)for (C-H) aromatic.H¹NMR spectrum: several signals due to the protons of the methylene groups represented by (-CH-CH-S-CH₂) in the cefixime double at (0.80-1.91). Signals of alkene protons (CH=CH₂) appeared at the sites (6.60-6.59-6.09) PPM. The appearance of signals at (9.50,9.00) belonging to the proton of the amide groups (OC-NH). Signal at (7.89-6.98) σ refers to the protons of the aromatic rings. Signal at (3.58) refers to the methylene group proton (CO-CH2-O). Signal at (3.63) refers to the (O-Me) methoxy group protons. A signal at (10.40) PPM refers to the proton of the hydroxy (OH) group in the phenol. In this compound that represents formazan, we notice the disappearance of the Burton signal of the imine group, due to the interaction of the iso group with it, and the formation of formazan represented by (N=N-C=N-). C¹³NMR spectrum: A signal at (12.49) refers to the carbon of (-CH-CH-) in the quadrupole ring, another signal appeared at (55.0) refers to carbon of the methoxy group (-OCH₃), a signal at (60.0) refers to the carbon of the methylene group (CO-CH₂-O), we notice the emergence of signals at (166.0-170.0) related to the carbon of the amide groups (CO-N) repeated in the compound, a signal at (18.00) belongs to the carbon group (-CH₂-C) inside the six memberedring, the appearance of several Signals refer to the carbon atoms in the aromatic rings at (113.0-135.0) δ, the appearance of the formazan carbon signal at (95.7) δ after the disappearance of the Imine group signal, the appearance of a signal at (100.0) refers to the alkene carbon (H₂C=C).

FT-IRspectrum data for derivative (8):bands for (-NH) amine group at (3331cm^-1), (1683cm^-1) for (CO-N) Caronyl of amide, (1670cm^-1) for (C=N) Formazan, (1365,1380,1460cm^-1) for (N=N) of formazan, (756cm^-1) for (C-S), (2929cm^-1) for (C-H) aliphatic, (=CH) Alkene at (3151 cm^-1), (3059cm^-1)for (C-H) aromatic.H¹NMR spectrum:

show the rise of several signals related to the protons of the two methylene groups represented by (-CH-CH-S-CH₂) in the cefixime rings at (1.09-2.09) σ. Signals appear at (6.54-6.40-6.20) PPM related to alkene protons (CH=CH₂).

Signals appear at (9.00) that refer to the proton of the amide groups (CO-NH). Signal at (7.69-6.94) refers to the aromatic ring protons. Signal at (3.61) refers to the methylene group (CO-CH₂-O) proton. Note the disappearance of the protons signal of the imine group and the Synthesis of formazans.

FT-IR_spectrum data for derivative (9):bands for (-NH) amine group at (3319cm^-1),band at (3441cm^-1) for (OH) Phenol, (1691cm^-1) for (CO-N) Caronyl of amide, (1653cm^-1) for (C=N) Formazan, (1381,1408,1438cm^-1) for (N=N) of formazan, (721cm^-1) for (C-S), (2988cm^-1) for (C-H) aliphatic, (=CH) Alkene at (3030cm^-1).H¹NMR spectrum:

show the rise of several signals related to the protons of methylene groups in (-CH-CH-S-CH₂) in the cefixime rings at (1.05-1.91). Signals appeared at (6.56-6.12-6.09) PPM due to alkene protons (-CH=CH₂). Signal at (7.90-6.97) refers to the aromatic ring protons. The appearance of two signals at (9.50-9.00) related to the proton of repeating amide groups (OC-NH). Signal at (3.59) refers to the methylene group (CO-CH₂-O) proton. A signal at (10.36) PPM refers to the proton of (OH) phenol.

FT-IR_spectrum data for derivative (10): show bands for (-NH) amine group at (3292cm^-1),band at (3373cm^-1) for (OH) Phenol, (1680cm^-1) for (CO-N) Caronyl of amide, (1631cm^-1) for (C=N) Formazan, (1355,1429,1517cm^-1) for (N=N) of formazan,(=CH) Alkene at (3207cm^-1), (2900cm^-1) for (C-H) aliphatic, (711cm^-1) for (C-S), (3006cm^-1)for (C-H) aromatic.H¹NMR spectrum: show the emergence of several signals related to the protons of the methylene groups in (-CH-CH-S-CH₂) in the cefixime rings at (1.06-1.90) σ. Signals appeared at (6.48-6.37-6.35) PPM related to alkene protons (-CH = CH₂). The emergence of a signal at (9.30) belonging to the proton of the amide groups (CO-NH).

Signal at (7.99-6.91) refers to the aromatic ring protons. Signal at (3.59) refers to the methylene group (CO-CH₂-O) proton. The emergence of two repeated signals for both protons (OH) and various positions in the compound.

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Fig.1.H¹.NMR of Compound [2]

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Fig.4. H¹.NMR of Compound [5]

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Fig.10.C¹³.NMR of Compound [3]

Fig.11. C¹³.NMR of Compound [10]

Fig. 12. FT.IR of Compound [2]

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Fig. 15.FT.IR of Compound [5]

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TestAgainst Breast Cancer

Initialization ofCancer Cell Line^(15-19)

Line processing and implantation of breast cancer cells andlive cell line were carried out at Biotechnology Center - the Nahrain (MCF-7 cell line) and (WRL cell line grew in95% of RPMI–1640) supplemented with (10% FBS), cell suspension and incubation^(15-19) at (37 °C) in incubator {(CO₂) % 5}. The suspended cells were centrifuged at (250 g) for (10 minutes) and the supernatant was removed, the cells were re-suspended in a freezing medium, then placed at (-70 °C) inbeaker for (1-3) days, the beaker was transferred from the standard freezer boxes to the liquid (N₂) container.

Procedure ofBreastCancer -Test

MTT was used to determine cell viability by chromatic examination^(15-19) of two (MCF-7 and WRL cell lines)forCefixime- FormazanDerivative [10]figure (21)andtable (1):

1. Cell suspension (100 µL) was added to the wells of a small flat plate bottom.

2. The solution was prepared by dissolving the crystals of 5 mg MTT in 1 ml of PBS solution (phosphate buffer

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solution).

3. The concentrations of each innovative derivative of the prepared derivatives were used in this research (6.15,12.5,25, 50, 100,200, 400(µg/ml of methanol, which were added to each well (three replicates per concentration).

4. A 10 ml MTT solution was added to each well of a plate containing 96 wells and then incubated for 4 hours with a test sample at 37 °C (the solution became yellow).

5. DMSO was added (200 µL( to each hole and stirred for 5 minutes (to become a purple DMSO solution).

6. After the complete dissolution of the dye, the absorption of the colored solution from the living cells was read at (575 nm) using the ELISA reader.

7. The mean absorption was calculated for each group of iterations and the validity ratio of the cells exposed to different treatments was obtained as follows^(15-19).

Cell Vitality% = [(Absorption from the treated sample /Absorption from the untreated sample) X 100

Fig.21.EffectofCefixime-Formazan [10] on Breast Cancer Cells

Table 1.Mean Percentage (%) for each cell line (Respond to Treatment) forDerivative{10}

Concentration ofCompound (10) Cefixime- Formazan Derivative (µ g/Ml^-1)

MCF-7 WRL

Mean SD Mean SD

400 43.79 2.45 72.69 1.71

200 48.92 1.20 86.81 3.75

100 63.12 2.95 94.29 1.83

50 74.46 3.99 95.18 0.96

25 86.81 5.22 95.06 1.24

12.5 96.33 0.41 93.56 0.64

6.25 94.41 1.87 94.91 1.71

Conclusions

The results indicatedto formation of these drug derivatives by appearance of new bands and disappearance of bands in starting compounds., besides to conclusions from our paper that gave good data for inhibition efficiency for these drug derivativesagainst cancer cells.Ourresults appearedgoodinhibition for carcinomacells line for allinventedderivatives, andgave highresponse forderivative {10} forinhibition andkillingof cancer cells duetoFormazane^(15-19) group(-N-C=N=N- Drug ) that linked withdrug which gaveitmore responseagainst cancer cells also due toformazan group with thiazole core and otheractive groupsin this derivative{10}.

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Acknowledgments

We would like to express our heartfelt thanks toBiotechnology Center-the Nahrainforproviding assistancesamples of cells andbioinformatics analysis.

Conflict of Interest

The authors declare that there is no conflict of interest.

Funding Source

None

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[15] HasaneenKudhairAbdullabass, Aseel Mahmood Jawad,Nagham Mahmood Aljamali. Synthesis of drugs derivatives as inhibitors of cancerous cells., Biochem. Cell. Arch, 20 (2) – October 2020.,

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DocID:https://connectjournals.com/03896.2020.20.5315.

[16] Nagham Mahmood Aljamali, Imad Kareem AlwanAlsabri., Development of Trimethoprim Drug and Innovation of Sulfazane-Trimethoprim Derivatives as Anticancer Agents., Biomedical & Pharmacology Journal, March 2020., Vol. 13, (2), p. 613-625., http://dx.doi.org/10.13005/bpj/1925.

[17] Aseel Mahmood Jawad, Nagham Mahmood Aljamali.,"Innovation, Preparation of Cephalexin Drug Derivatives and Studying of (Toxicity & Resistance of Infection)"., International Journal of Psychosocial Rehabilitation, Vol. 24, Issue 04, 2020, 3754-3767.

[18] Imad Kareem AlwanAlsabri, HasaneenKudhairAbdullabass,Nagham Mahmood Aljamali.,Invention of (Gluta.Sulfazane-Cefixime) Compounds as Inhibitors of Cancerous Tumors., Journal of Cardiovascular Disease Research, 2020,11, 2., 44-55., DOI: 10.31838/jcdr.2020.11.02.09.

[19] Aseel Mahmood Jawad, Nagham Mahmood Aljamali, Saher Mahmood Jwad, Aseel M J, Saher M J., Development and Preparation of ciprofloxacin Drug Derivatives for Treatment of Microbial Contamination in Hospitals and Environment, Indian Journal of Forensic Medicine & Toxicology, 2020,14, 2, p:1115- 1122.

[20] Hussniya A. Aldifar, Mohammad F. Ali, Abdulrahim M. Khlafulla. Preparation and Characterization of Some of Formazan Derivatives. Research & Reviews: A Journal of Pharmacology. 2017.

[21] Moussa, Shaaban H. "Tetrazolium/formazan test as an efficient method to determine fungal chitosan antimicrobial activity." Journal of Mycology 2013 (2013).

[22] Tezcan, Habibe, Elifuzluk, and Mehmet Levent Aksu. "Electrochemical and spectroscopic properties of 1: 2 Ni complexes of 1, 3-substitued (CH3, OCH3) phenyl-5-phenylformazans." Electrochimica acta 53.18 (2008): 5597-5607.

[23] Nagham Mahmood Aljamali, Intisar Obaid Alfatlawi. "Synthesis of Sulfur Heterocyclic Compounds and Study of Expected Biological Activity", Research J. Pharm. and Tech., 2015, 8,9,1225-1242, DOI:

10.5958/0974-360X.2015.00224.3.

[24] Nagham Mahmood Aljamali.; Saher Mahmood Jawd.; Zainab M J.; Intisar, Obaid. Alfatlawi.; 2017,

"Inhibition activity of (Azo–acetyl acetone) on bacteria of mouth"., Research Journal of Pharmacy and Technology10(6):1683-1686, DOI: 10.5958/0974-360X.2017.00297.9

[25] Nagham Mahmood Aljamali., “(Synthesis, Investigation, Chromatography, Thermal)- Behavior of (Five, Seven)- Membered Ring with Azo and Anil Compounds”, Pak. J. Biotechnol.; 15(1): 219-239 (2018).

[26] Shireen R. Rasool, Nagham Mahmood Aljamali, Ali Jassim Al-Zuhairi., Guanine substituted heterocyclic derivatives as bioactive compounds., Biochem. Cell. Arch. Vol. 20, Supplement 2, pp. 3651-3655, 2020., DocID: https://connectjournals.com/03896.2020.20.3651.

[27] Nagham Mahmood Aljamali.,"Synthesis and Chemical Identification of Macro Compounds of (Thiazol and Imidazol)".,Research J. Pharm. and Tech., 2015, 8,1,78-84., DOI: 10.5958/0974-360X.2015.00016.5.

[28] Maulucci G, Labate V, Mele M, Panieri E, Arcovito G, Galeotti T, Østergaard H, Winther JR, De Spirito M, Pani G (October 2008). "High-resolution imaging of redox signaling in live cells through an oxidation- sensitive yellow fluorescent protein". ScienceSignaling, 1 (43):pl3. doi:10.1126/scisignal.143pl3.

[29] M. N Abdmajed, Nagham Mahmood Aljamali., Preparation of Benzothiazole-Formazane Reagents and Studying of (Spectral, Thermal, Scanning Microscopy, Biological Evaluation)., International Journal of Pharmaceutical Research, 2021, 13(1), 4290-4300., DOI:https://doi.org/10.31838/ijpr/2021.13.01.641.

[30] Rabab Mahdi Ubaid Mahmood, Nagham Mahmood Aljamali., Synthesis, Spectral Investigation and Microbial Studying of Pyridine-Heterocyclic Compounds.,European Journal of Molecular & Clinical Medicine, 2020, Volume 7, Issue 11, Pages 4444-4453.

[31] Miad Mohmed,Nagham Mahmood Aljamali,Sabreen Ali Abdalrahman.,Wassan Ala Shubber., "Formation of Oxadiazole Derivatives Ligands from Condensation and Imination Reaction with References To Spectral Investigation, Thermal and Microbial Assay"., Biochem. Cell. Arch., 2018,18, 1, pp. 847-853.

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[32] Nagham Mahmood Aljamali,Rabab Mahdi Ubaid Mahmood., Synthesis, Characterization of Diazepine- Bicycles System and Study of their Bio-Behavior.,International Journal of Pharmaceutical Research, 2021, Volume 13, Issue 1, Pages 4225-4233.

[33] Vanita S.; Supriya, M. "Development of furfuraldehyde formazans as potential antitubercular agents"., Der Pharma Chemica, 2016,8,18, 144-148.

[34] Nour A., Suad S., Nagham Mahmood Aljamali., Synthesis, Characterization and Thermal Analysis for New Amoxil Ligands, Asian Journal of Chemistry; 31, 5, 1022-1026, (2019).

[35] Rajaa Abdul Ameer Ghafil, Nour AAlrazzakb, Nagham Mahmood Aljamali., Synthesis of Triazole Derivatives via Multi Components Reaction and Studying of (Organic Characterization, Chromatographic Behavior, Chem-Physical Properties)., Egypt. J. Chem. 63, No. 11, pp. 4163 - 4174 (2020). DOI:

10.21608/EJCHEM.2020.23541.2399.

[36] Ahmad, T; Kandil, F and Moustapha, M. Preparation and Characterization of Some New Azo Dyes, Azomethine Dyes and Heterocyclic-Schiff Bases Derivatives. AASCIT Journal of Chemistry,2015, 2(2): 24- 31.

[37] Shinde, A. T; Deshmukh, N. J; Kottapalle G. D and Zangade.S.B.. Synthesis Antimicrobial Evaluation of Some New FluoroFormazans. Journal of Pure and Applied Chemistry Research, 2016,5 (2) 61-66.

[38] Gurusamy M, Rejaul K, Nand M. J, Faruk A, Rajib H, Deepak K, and Tiewlasubon U. Synthesis and biological evaluation of formazan Derivatives, Journal of Advanced Pharmaceutical Technology &

Research, 2016,1(4):396-400

[39] Venkatesan, P., B. Anand, and P. Matheswaran. "Influence of formazan derivatives on corrosion inhibition of mild steel in hydrochloric acid medium." E-Journal of Chemistry 6.S1 (2009): S438-S444.

[40] Rabab Mahdi Ubaid Mahmood,Rajaa Abdul Ameer Ghafil., Synthesis and Characterization some Imidazolidine Derivatives and Study the Biological Activity., Annals of R.S.C.B., Vol. 25, Issue 3, 2021, P:

569 – 584.

[41] Nagham Mahmood Aljamali, (2015). Review in Azo Compounds and its Biological Activity. Biochem Anal Biochem,4, 169., doi:10.4172/2161-1009.1000169.

[42] Nagham Mahmood Aljamali. 2014. Synthesis and Investigation of Formazane compounds (Azo– Imine) and their complexes, Asian J. Research Chem., 7 (2), 225-231.

[43] Fei, Na; Sauter, Basilius; Gillingham, Dennis (2016). "The pK a of Brønsted acids controls their reactivity with diazo compounds". Chemical Communications, 52(47): 7501–7504.