Synthesis and Identification of some new β-Lactam derivatives from 6-amino-1,3-dimethyluracil and study their antioxidant activity

 

Huda Jamal Al-Adhami*, Suaad M. H. Al-Majidi, Thikra Hasan Mathkor

¹Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq.

 

ABSTRACT:

In this work the synthesis of a 6-( amino acetyl chloride)-1,3-dimethylpyrimidine-2,4-dione-6-yl [1] by reaction of 6-amino-1,3-dimethyluracil with chloroacetyl chloride, triethylamine in DMF as a solvent, the product [1] then, reacted with a hydrazine hydrate in absolute ethanol to produce hydrazide derivative [2]. A series of Schiff bases (3-8) were synthesized in moderate yields via a reaction of 6-(hydrazinoacetamide)-1,3-dimethyl pyrimidine-2,4-dione-6-yl [2], substituted aryl aldehydes and glacial acetic acid under reflux conditions. The resulting [3-8] could be useful for the synthesis of [9-26] β-Lactams derivatives. They have been achieved by cyclization of Schiff bases compounds with different reagents (chloroacetyl chloride, phenyl isocyanate and phenyl isothiocyanate) in THF solvent. All newly synthesized compounds conformation were characterized in detail by FT-IR and NMR spectroscopy including 1D NMR experiments (¹H and ¹³C). The study antioxidant of some new compounds.

 

 

 

INTRODUCTION:

Uracil as a specific base in RNA plays an important role in gene coding and various physiological processes of organisms. It's derivatives are anti-tumor agent used for the treatment of prostate cancer^(1,2). Many uracil derivatives have been reported to have antioxidative and anti-inflammatory activities^(3-5). Oxidative stress is defined as the alteration produced by the imbalance between oxidants and an­tioxidants species, It can be caused by an excess of oxidants, antioxidant deficiency, or both factors simultaneously. Currently, there is a growing interest in oxidative stress because it can lead to cell death and contribute to the development of multiple pathologies⁽⁶⁾. and it is reported that uracils show anti-viral and antitumour, Anti-HIV characteristics in addition to bactericidal, herbicidal and insecticidal features^(7-9). The heterocyclic ring can be bound to a uracil ring using different strategies.⁽¹⁰⁾

 

Uracil and its modified derivatives belong to a class of compounds important in drug discovery with a wide range of biological activities and as pharmaceutical intermediates and/or prodrugs, play an important role in drug development, and as chemotherapeutic agents^(11,12). β-lactam antibiotics are a class of antibiotic consisting of all antibiotic agents, it plays an essential role in medicine and consequently in pharmaceutical industry. The discovery of penicillin and cephalosporin were undoubtedly a milestone in the history of modern medicine, antifungal, anticancer, antiviral, inhibition of HIV and anti-hyperglycemic activity^(13-16). In various diseases such as infectious diseases, oxidative stress occurs secondary to the initial disease but plays an important role in immune or vascular complications, the relative antioxidant effect of some β-lactam antibiotics such as ampicillin on oxygen-reactive species has been reported and a possible therapeutic role for β-lactam agents in protecting host tissues from oxidative damage has been proposed^(17-19)

 

EXPERIMENT SECTION:

Material and Instruments:

Chemicals used in this work are supplied from BDH, Fluka, Merck and Sigma Aldrich companies and used without further purification. Melting points were uncorrected and registered via digital Stuart scientific SMP3 melting point device. Thin layer chromatography (TLC) used to check purity and homogeneity of synthesis compounds. FTIR spectra of the compounds in the (4000-600) cm^-1 spectral range were recorded on SHIMAZU FTIR-8400 Fourier transform Infrared spectrophotometer using KBr discs. ¹H-NMR and ¹³C-NMR spectra were recorded on VARIAN 500MHz in Iran, instrument using TMS as internal reference and DMSO-d₆ as a solvent. UV-VIS spectra were recorded by Shimadzu-spectrophotometer and Apel PD-303- spectrophotometer, Japan.

 

6-(amino acetylchloride)-1,3-dimethylpyrimidine-2,4-dione-6-yl [1]⁽²⁰⁾:

In a round bottom flask (50mL) equipped with magnetic stirring bar, 6-amino-1,3-dimethyluracil (2.0g, 0.0129 mol), triethylamine (1.8mL, 0.0129mol) were dissolved in DMF (15mL). The round bottom flask was cooled to 0°C for 30 min with stirring and then added drop wise chloroacetyl chloride (1.1mL, 0.0129mol). The mixture was stirred 4 h at room temperature, and afterwards refluxed for (75- 85)°C for 7 h. After the completion of reaction, the reaction mixture was poured into ice water. Then solid product was filtered off, washed with water. The obtained product was purification by ether. Some of the physical properties and yield of compound [1] are listed in Table (1).

 

6-(hydrazinoacetamide)-1,3-dimethyl pyrimidine-2,4-dione-6-yl[2]⁽²¹⁾:

6-amino acetyl chloride-(1,3-dimethyl pyrimidin-6-yl) [1] (1.0g, 0.0043 mol) and hydrazine hydrate (0.3mL, 0.0086mol) were dissolved in absolute ethanol (10mL) with continuous stirring. The stirring reaction mixture was refluxed for 4 h. Subsequently, the combined organic phases were cooled off before pouring into crushed ice. The obtained precipitate has been filtered, washed with ether, dried and purified by recrystallization from ether. Physical properties are listed in Table (1).

 

N-[6-(Substituted benzylidene hydrazone)acetamide]-1,3-dimethyl pyrimidine-2,4-dione-6-yl [3-8]⁽²²⁾ :

A mixture of compound [2] (1.0g, 0.0044mol), different substituted aromatic aldehydes (0.0044mol) in absolute ethanol (10mL) and (3-5) drops of glacial acetic acid was refluxed in water bath for about (6-7) h. The excess solvent was evaporating under reduced pressure. Then the crude product was dried, recrystallized from Dioxan. Some of the physical properties and yield of compounds [3-8] are listed in Table (2).

 

N-[6-((3-chloro-2-substituted phenyl)-4-oxoazetidin-1-yl)amino)acet amide]-1,3-dimethylpyrimidine-2,4-dione-6-yl [9-14]⁽²¹⁾ :

A solution of appropriate Schiff base derivatives [3-8] (0.0016mol) in (10mL) of dioxan as solvent; chloroacetyl chloride (0.2mL, 0.0016mol); triethylamine (Et₃N) (0.1mL, 0.0016mol) was added. The mixture was refluxed for (14-16) h. The reaction mixture was cooled at room temperature and the precipitate was filtered, washed with cool water and recrystallized from ethanol. Physical properties of compounds [9-14] are listed in Table (3).

 

N-[6-((2-oxo-3-phenyl-2-substituted phenyl)-1,3-diazetidine-1-yl) amino) acetamide]-1,3-dimethylpyrimidine-2,4-dione-6-yl [15-20]:

N-[6-((3-phenyl-2-substituted phenyl-4-thio)-1,3-diazetidine-1-yl) amino) acetamide]-1,3-dimethylpyrimidine-2,4-dione-6-yl [21-26] ⁽²³⁾:

A mixture of Schiff bases [3-8] (0.0016 mol) with THF (10mL) , phenyl isocyanate (0.2mL, 0.0016mol)  or phenyl isothiocyanate (0.2mL, 0.0016mol) was added in little amounts in round bottomed flask with continuous stirring and also the reaction mixture was gradually heated to laboratory temperature degree, then refluxed for 6 h. the solvent was removed under reduced pressure and the residue treated with a mixture of (1:1) ethyl acetate and petroleum ether. The resulted precipitate was filtered and dried. Some of the physical properties and yield of compounds [11-26] and [21-26] are listed in Table (4) and Table (5) respectively.

Table 1. Physical properties and FT-IR spectral data cm^-1 of the synthesized compounds [1 and 2]

 

Table 2. Physical properties and FT-IR spectral data cm^-1 of the synthesized Schiff bases compounds [3-8]

 

Table 3. Physical properties and FT-IR spectral data cm^-1 of the synthesized compounds [9-14]

Table 4. Physical properties and FT-IR spectral data cm^-1 of the synthesized compounds [15-20]

 

Table 5. Physical properties and FT-IR spectral data cm^-1 of the synthesized compounds [21-26]

 

Total antioxidant capacity⁽²⁴⁾:

The total antioxidant capacity of the synthesized compounds was evaluated by the phosphomolybdenum method. A different concentrations (50, 100, 150μg/mL) of an aliquot compound solutions  was combined with 1 mL of reagent solution ((0.6M) sulfuric acid, (28mM) sodium phosphate and (4 mM) ammonium molybdate). The tubes containing the reaction solution were capped and incubated at 95°C for 90 min. Then, the tubes were cooled to room temperature, the absorbance of each solution was measured at 695nm using a spectrophotometer against blank. The total antioxidant activity is expressed as the number of gram equivalent of ascorbic acid. Different concentrations (10, 20, 30, 50, 70, 90, 120, 180, 200µg/mL) of ascorbic acid with DW where used to plot the calibration curve.

 

Reducing power assay⁽²⁵⁾:

The reducing power assay of β-Lactam derivatives was determined by using various concentrations of samples (50, 100, 150μg/mL), which were mixed with (1mL) sodium phosphate buffer (0.2M, pH6.6) and (1mL) potassium ferric cyanide 1%. The solutions were incubated at 50°C for 20 min. Then (1mL) of  trichloroacetic acid 10% was added. The tubes were centrifuged at 2000 rpm for 10 min. The upper layer of solution (2.5mL) was mixed with (2.5ml) of distilled water and (0.3mL) of fresh prepared ferric chloride 0.1%. The absorbance was measured at 700nm using a spectrophotometer against blank. Ascorbic acid was used as standard.

 

Nitric Oxide Radical Scavenging Assay⁽²⁶⁾:

The nitric oxide radical scavenging assay was determined the nitric oxide radical scavenging activity of the synthesized compounds by using Griess reagent, the absorbance were measured and recorded as A₀. After that a solution of (2mL, 10mM) sodium nitroprusside dissolved in (0.5mL) phosphate buffer (0.2M, pH 7.4) were mixed with (0.5mL) of various concentrations of samples (20, 50, 100, 150, 200μg/mL). Tubes were capped and incubated at 25°C for 150min. Then (0.5mL) of incubation solution was withdrawn and mixed with (0.5mL) of Griess reagent; ((1.0mL) sulfanilic acid reagent (0.1% w/v) and (1mL) of naphthylethylenediamine dichloride (0.1% w/v)). The solution was incubated at room temperature for 30min. The absorbance was measured at 540 nm using a spectrophotometer against blank and ascorbic acid was used as standard. The inhibition percentage of nitric oxide radical was calculated using the following equation:

 

                                A₀ - A_t

% Inhibition of No. = ––––––––––––– × 100

                                   A₀

Where A₀ is the absorbance before the reaction and 𝐴_𝑡 is the absorbance after the reaction.

 

Superoxide free radical scavenging activity⁽²⁷⁾:

The superoxide free radical scavenging activity of β-Lactam derivatives were measured as described by using the NBT method. The reaction mixture containing of (6.6μM) EDTA, (3μg) NaCN, (2μM) riboflavin, (50μM) NBT and (58mM, pH 7.8) of phosphate buffer with various concentrations of the synthesized compounds and  in a final volume of (3mL). Optical density was measured at 560nm using a spectrophotometer. The test tubes were uniformly illuminated for 15 min with an incandescent lamp, after which the optical density was measured again at 560 using the same spectrophotometer against blank. Ascorbic acid was used as standard. The inhibition percentage of superoxide radical generation was measured by comparing mean absorbance values of the control and those of the test substances. IC50 values were obtained in μg versus percentage inhibition from the linear plot drawn of concentration of tested compound solution (50, 100 and 150μg/mL) and the ordinate represented the average percent of antioxidant activity from three separate tests.

 

Hydrogen Peroxide Radical Scavenging Assay⁽²⁸⁾:

Hydrogen peroxide scavenging activity (H₂O₂) of synthesized compounds was determined as follow: (40 mM) of hydrogen peroxide solution, in phosphate buffer (0.2M, pH 7.4). The synthesized compounds with concentration of (20, 30 and 40) μg/mL were added to a hydrogen peroxide solution (0.6mL, 40mM). The absorbance of H₂O₂ measured at 230nm using a UV-VIS spectroscopy and determined after 10 min. against blank and ascorbic acid. The percentage of hydrogen peroxide scavenge activity was then calculated using Equation 1

 

                                   A₀ - A_t

% Scavenging activity = ––––––––––––– × 100

                                         A₀

 

RESULT AND DISCUSSION:

The steps of the synthesis 6-( amino acetyl chloride)-1,3-dimethylpyrimidine-2,4-dione-6-yl [1], 6-(hydrazinoacetamide)-1,3-dimethyl pyrimidine-2,4-dione-6-yl [2], it's Schiff bases derivatives [3-8], chloroazetidine-2-one and β-Lactams are shown in the Scheme (1):

 

Scheme (1)

 

Compound [1] was prepared via the nucleophilic acetyl substitution of 6-amino-1,3-dimethyluracil with chloroacetyl chloride in the presence of triethylamine as a catalyst in dimethylformamide at 0°C. Scheme(1).The mechanism of this reaction involved nucleophilic attack of amino group in substituted primary aromatic amines on carbonyl group of chloroacetyl chloride, followed by elimination of HCl molecule⁽²⁹⁾,shown in Scheme(2).

 

Scheme (2): Mechanism of the prepared compound [1]

 

Compound[1] was afforded as brown crystals of (85%)  yield with a melting point of (88-90^oC), also, Silver nitrate test gave (+ve) in the presence of chlorine group ⁽³⁰⁾. FTIR spectrum data of compound[1] showed disappearance of a υ(NH₂) while υ(N-H) at (3317) and υ(C=O) of amide at (1745) cm^-1 was appeared with still appearance of υ(C=O) of amides⁽³¹⁾ at(1699)cm^-1as shown in Table (1)

 

Compound[1] was reacted with hydrazine hydrate to give hydrazine derivative [2] in absolute ethanol as a solvent. FTIR spectrum of this compound indicates that υ(C-Cl) (779) cm^-1 was disappeared from the spectrum while appearance of  the asymmetric and symmetric stretching bands of υ(NH₂) absorption band at (3421)cm^-1 Asym., (3334)cm^-1 sym, also the presence of a strong sharp bands of  υ(N-H) at (3182) cm^-1, υ(C-H) aliphatic⁽³¹⁾ at (2962, 2829)cm^-1 and υ(C=O) at (1710, 1662) cm^-1 as shown in Table (1). ¹H-NMR spectrum of compound [2] showed a singlet signal at δ=(3.03)ppm due to (-CH₃) protons, a singlet signal at δ=(3.25)ppm due to () protons, a singlet signal at δ=(3.52)ppm is due to (-CH₂-) protons, a singlet signal at δ=(4.19)ppm for (N-N-H) proton, a singlet signal at δ=(4.91)ppm due to (=CH-) proton, signals at δ=(6.85)ppm belonged to  (-NH₂) protons and a singlet signal at δ=(8.57)ppm due to (O=C-N-H) proton as listed in Table (6) and shown in Figure(1). ¹³C-NMR spectral data were listed in Table (2) and shown in Figure(2).

 

Compound[3-8] was synthesized by condensation reaction of compound [1] with different substituted aromatic aldehydes and few drops of glacial acetic acid in absolute ethanol to form Schiff's bases [3-8]. Scheme(1).FTIR spectra showed absorption bands at (3340-3303)cm^-1 for υ(N-H) and confirmed the formation of compound [3-8] from the appearance of  the bands at (1637-1623) cm^-1 due to υ(C=N) of Schiff's bases [3-8] respectively. With the disappearance of υ(NH₂) at (3421)cm^-1 Asym., (3334)cm^-1 sym. All details of FTIR Spectral data of compounds [3-8] were listed in Table (2). ¹H-NMR spectrum of compound [5] showed a singlet signal at δ= (2.87)ppm due to (N-(CH₃)₂) protons, singlet signal at δ=(3.07)ppm due to (-CH₃) protons, a singlet signal at δ=(3.08)ppm due to () protons, a singlet signal at δ=(3.31)ppm is due to (-CH₂-) protons, a singlet signal at δ=(4.19)ppm for (N-N-H) proton, a singlet signal at δ=(4.93)ppm due to (=CH-) proton, a singlet signal at δ=(6.65)ppm is due to (N=CH-) protons, signals at δ=(6.66,7.62) ppm are due to aromatic rings protons and a singlet signal at δ=(9.03) ppm belonged to (O=C-N-H) proton as shown in Table (6). ¹³C-NMR spectral data of compound[5] are listed in Table (7).

 

¹H-NMR spectrum of compound [6] showed a singlet signal at δ=(3.03)ppm due to (-CH₃) protons, a singlet signal at δ=(3.14)ppm due to () protons, a singlet signal at δ=(3.58)ppm is due to (-CH₂-) protons, a singlet signal at δ=(3.99)ppm for (N-N-H) proton, a singlet signal at δ=(4.91)ppm due to (=CH-) proton, a singlet signal at δ=(6.50)ppm is due to (N=CH-) protons, signals at δ=(7.01-7.48)ppm are due to aromatic rings protons, a singlet signal at δ=(9.01)ppm belonged to (O=C-N-H) proton and a singlet signal at δ=(10.80)ppm is due to (O-H) proton as shown in Table (6). ¹³C-NMR spectral data of compound[6] are listed in Table (7).

¹H-NMR spectrum of compound [7] showed a singlet signal at δ=(3.18)ppm due to (-CH₃) protons, a singlet signal at δ=(3.21)ppm due to () protons, a singlet signal at δ=(3.36)ppm is due to (-CH₂-) protons, a singlet signal at δ=(3.81)ppm is due to (O-CH₃) proton,a singlet signal at δ=(4.90)ppm for (N-N-H) proton, a singlet signal at δ=(4.99)ppm due to (=CH-) proton, a singlet signal at δ=(6.52)ppm is due to (N=CH-) protons, signals at δ=(7.02-7.78)ppm are due to aromatic rings protons and a singlet signal at δ=(8.90)ppm belonged to (O=C-N-H) proton as shown in Table (6). ¹³C-NMR spectral data of compound[7] are listed in Table (7).

 

On other hand, The Schiff's bases [3-8] were reacted with different reagents by various methods to give different cyclic compounds. First method; Schiff bases[3-8] were reacted with chloroacetyl chloride followed by the addition of triethylamine producing compounds of chloroazetidine-2-one compounds [9-14] linked to N-[6-(Substituted benzylidene hydrazone)acetamide]-1,3-dimethyl pyrimidine-2,4-dione-6-yl [3-8]. The structure of azetidine-2-one has been confirmed by FTIR spectroscopy. FTIR spectrum for Compound [9-14] showed the appearance of the absorption band at (1064-1010) cm^-1 due to υ(C-Cl azetidine ring), (3461-3377) cm^-1 for υ(N-H) and bands of υ(C=O) amid appearance at (1735-1649)cm^-1 in addition to the disappearance bands of  υ(C=N) at (1637-1623) cm^-1. All details of FTIR spectral data of compounds [9-14] are listed in Table (3). ¹H-NMR spectrum of compound [13] showed a singlet signal at δ=(3.07)ppm due to (-CH₃) protons, a singlet signal at δ=(3.17)ppm due to () protons, a singlet signal at δ=(3.55)ppm is due to (-CH₂-) protons, a singlet signal at δ=(3.81)ppm is due to (O-CH₃) protons,a singlet signal at δ=(4.18)ppm for (N-N-H) proton, a singlet signal at δ=(4.91)ppm due to (=CH-) proton, a singlet signal at δ=(5.40)ppm due to (N-CH- ring) protons, a singlet signal at δ=(5.41)ppm due to (CH azetidine ring) protons, signals at δ=(7.03-7.80)ppm are due to aromatic rings protons and a singlet signal at δ=(9.51)ppm belonged to (O=C-N-H) proton as shown in Table (6). ¹³C-NMR spectral data of compound[13] are listed in Table (7).

 

While in the second method; reaction of Schiff bases [3-8] with phenylisocyanate and phenylthioisocyanate in THF under reflux to produce 𝛽-lactams [15-20] and [21-26]. The structure of 𝛽-lactam derivatives has been confirmed by FTIR spectroscopy. FTIR spectrum of the synthesized compounds [15-20] showed υ(N-H) at (3193-3406)cm^-1 and bands of υ(C=O)  at (1722-1649)cm^-1, while showed disappearance of  υ(C=N) bands at (1637-1623) cm^-1. All details of FTIR spectral data of compounds [15-20] are listed in Table (4). ¹H-NMR spectrum of compound [18] showed a singlet signal at δ=(3.08)ppm due to (-CH₃) protons, a singlet signal at δ=(3.14)ppm due to () protons, a singlet signal at δ=(3.45)ppm is due to (-CH₂-) protons,a singlet signal at δ=(4.09)ppm for (N-N-H) proton, a singlet signal at δ=(4.89)ppm due to (=CH-) proton, a singlet signal at δ=(6.84)ppm due to (N-CH- ring) protons, signals at δ=(6.91-7.98)ppm are due to aromatic rings protons, a singlet signal at δ=(8.77)ppm belonged to (O=C-N-H) proton and and a singlet signal at δ=(9.22)ppm belonged to (O-H) proton as shown in Table (6). ¹³C-NMR spectral data of compound[18] are listed in Table (7).

 

FTIR spectrum for Compound [21-26] showed υ(N-H) at (3203-3458)cm^-1 and bands of υ(C=O)  at (1689-1668)cm^-1 in addition to the disappearance bands of υ(C=N) at (1637-1623) cm^-1 with appearance of υ(C=S) at (1355- 1398)cm^-1. All details of FTIR spectral data of compounds [21-26] are listed in Table (5). ¹H-NMR spectrum of compound [23] showed a singlet signal at δ=(2.88)ppm due to (N(CH₃)₂) protons, a singlet signal at δ=(3.04)ppm due to (-CH₃) protons, a singlet signal at δ=(3.13)ppm due to () protons, a singlet signal at δ=(3.53)ppm is due to (-CH₂-) protons,a singlet signal at δ=(4.21)ppm for (N-N-H) proton, a singlet signal at δ=(4.87)ppm due to (=CH-) proton, a singlet signal at δ=(5.42)ppm due to (N-CH- ring) protons, signals at δ=(6.69-7.93)ppm are due to aromatic rings protons and a singlet signal at δ=(9.90)ppm belonged to (O=C-N-H) proton as shown in Table (6). ¹³C-NMR spectral data of compound[23] are listed in Table (7).

 

Table 6. ¹H-NMR spectral data ᵟppm for selected compounds

 

Table 7. ¹³C-NMR spectral data ᵟppm for selected compounds

 

Antioxidant activity:

Generally, five different mechanisms are used to study the antioxidant activity of the compounds, namely: total antioxidant capacity (TAOC), reducing power assay (Iron reducing Activity), nitric oxide radical scavenging assay, superoxide radical scavenging (NBT) method and hydrogen peroxide radical scavenging. All the methods were generated in vitro by non-enzymatic system and determined spectrophotometrically by photo reduction method and the compounds were tested in (3-5) different concentrations.

 

In general, the phenolic compounds have been known to have antioxidant activities by their radical scavenging activities in addition of inducing antioxidant enzyme levels. The phenolic compounds are one of the most well-studied antioxidants in vitro and in vivo in terrestrial plants. Also as a marine sources marine algae are rich in various antioxidant compounds.⁽³²⁾ Thus, the antioxidant properties of phenolic compounds were evaluated as scavengers. For example, the studied of o-, m-, and p-cresol evinced that all three cresols acted as H₂O₂ scavengers. Thus, the effect of 50% inhibitory concentration (IC₅₀) decrease as follows: o-cresol > p-cresol > m-cresol. Besides these, free phenolic hydroxyl groups are essentially reported responsible of antioxidant activity. Although, aliphatic hydroxyl groups have an inverse effect on the radical scavenging activity⁽³³⁾.

 

Total antioxidant capacity:

The total antioxidant capacity of all synthesized compounds (1-82) was evaluated by the phosphomolybdenum assay method, which is based on the reduction of colorless Molybdenum(VI) to form blue Molybdenum(V) by using the synthesized compounds and subsequent formation of a green phosphate-Mo(V) complex in acidic pH. The antioxidant activity of the compounds were compared to standard ascorbic acid. Among the newly synthesized uracil derivatives, compounds (10, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24 and 25) possess strong antioxidant capacity and reduced Mo(VI) to Mo(V) in a better way, where that compound (18) shows possess better antioxidant activity than the standard, whereas the other compounds shows weak antioxidant capacity. The results of the total antioxidant capacity are listed in Figure 1.

 

Reducing power assay:

The reducing power assay of the synthesized compounds may serve as a significant indicator of its potential antioxidant activity at different concentrations depending on the method of reducing power of antioxidant compounds was used to determine the reduction ability of the synthesized compounds. The samples solutions changes into various shades of green and blue colors. which reduced the potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), then reacted with ferric chloride to form ferric ferrous complex. The formation of Perl’s Prussian blue can be monitored by measuring the absorbance at 700 nm. Compounds (15, 21 and 24) possesses good reducing power ability among the others where their the reducing power ability is close to the standard values of ascorbic acid. While compounds (18, 22 and 25) showed better antioxidant activity than the standard. The substitution with different substituent on the phenyl of the aldehydic group of β-Lactam moiety plays an important role in reducing potential of the synthesized compounds. Although, the presence of free NH group contributes to the reducing power assay. The results of the reducing power assay are listed in Figure 2.

 

Figure 1. Comparison of total antioxidant capacity of newly synthesized compounds

 

Figure 2.Comparison of reducing power assay of the synthesized compounds

 

Nitric Oxide Radical Scavenging Activity:

In addition to reactive oxygen species, nitric oxide is also involved in inflammation, cancer, and other pathological conditions. Sodium nitroprusside produce nitrite ions by decomposing in aqueous solution at pH 7.2 In aerobic condition. NO reacts with oxygen to produce stable products (nitrate and nitrite), that can be estimated using Griess reagent. The completion between scavengers of nitric oxide and oxygen, leading to the reduced production of nitrite ions. Suppression of the released NO may be partially attributed to direct NO scavenging. The tested of the synthesized compounds were found to be moderate to weak of nitric oxide scavenging activity. The results of the nitric oxide radical scavenging assay are listed in Figure 3.

 

 

Figure 3. Effect of the synthesized compounds toward nitric oxide

 

 

Figure 4. Effect of synthesized compounds toward superoxide

 

 

Figure 5. Effect of the synthesized compounds toward hydrogen peroxide

 

Superoxide Radical Scavenging Assay:

The superoxide anion radical is normally initially formed, and its effects can be magnified because it produces other kinds of free radicals and oxidizing agents. The generation of superoxide was estimated by the (NBT) nitroblue tetrazolium method. synthesized compounds were found to be moderate to weak of superoxide radical scavengers. It should be noted that the activity of the compounds (10, 15, 18, 21 and 22) was comparable to that of standard. The better activity of compound (10) is due to the presence of two chloro groups in the molecule and two aromatic ring and hydroxyl groups in compounds (15 and 18) respectively. while the other compounds showed less superoxide radical activity. The results of the superoxide anion radical scavenging assay are listed in Figure 4.

 

Hydrogen Peroxide Radical Scavenging Assay:

Table 5 shows hydrogen peroxide scavenging activity of β-Lactam derivatives caused a strong inhibition of hydrogen peroxide. The results show the all the synthesized compounds had potent H₂O₂ scavenging activity which may be due to the antioxidant compounds. As the antioxidant components present in the synthesized compounds are good electron donors, they may accelerate the conversion of H₂O₂ to H₂O. Figure 5 reports the H₂O₂ scavenging activity of various β-Lactam derivatives. Compounds (18, 21 and 25) were shown to scavenge the H₂O₂ radicals with a good inhibition percent of 25-62%. And all the tested compounds exerted moderate scavenging activity when compared to ascorbic acid as shown as Figure 5.

 

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Received on 12.11.2019           Modified on 27.01.2020

Accepted on 16.03.2020         © RJPT All right reserved