The Activity of Synthesized N-heteroaryl Substituted Benzene ...

6 Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348 -7658 Impact Factor (SJIF): 2.092 March -May 2015; 3(1):06-16

Available online at http://www.jpcbs.info

Original Research Article

The Activity of Synthesized N-heteroaryl Substituted Benzene Sulphonamides on the Liver and Kidney Marker Enzymes of Plasmodium berghei Infected Mice Igwe Christiana N1*, Okoro Uchechukwu C1, Egba Simeon I2, Nwankwo Nicodemus E3 Anaduaka Emeka G3 1

Synthetic Organic Chemistry Division, Department of Pure and Industrial Chemistry, University of Nigeria, Nsukka. 2 Michael Okpara University of Agriculture, Umudike Abia State. 3 Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka. * Corresponding Author Email: : [email protected]; [email protected] Received: 22 November 2014

Revised: 15 February 201

Accepted: 14 March 2015

ABSTRACT N-heteroaryl substituted benzene sulphonamides were screened for biological activities. The compounds were administered for their antiplasmodial activity against Plasmodium berghei and effect on liver and kidney markers enzymes. The parameters; ALT (Alanine Aminotransferase) and AST (Aspartate Aminotransferase) enzymes activities were used for liver function test while serum Urea and Creatinine tests were used as markers for renal function. The results of the activities of the synthesized compounds showed appreciable inhibition of the growth of test organism. The liver and kidney function tests showed improved activity when compared with the positive control. Keyword: Alanine Aminotransferase; Aspartate Aminotransferase; creatinine; N-benzene sulphonamides; urea; Plasmodium berghei

INTRODUCTION Sulphonamides have, for many years, been widely studied for their chemotherapeutic activity1. A very large number of other derivatives are constantly being synthesized and evaluated in

order to obtain compounds with lower toxicity or augmented activity against viruses resistant to the first generation of such drugs [2]. The basic sulphonamide group –SO2NH- occurs in various

J Pharm Chem Biol Sci, March-May 2015; 3(1):06-16

Christiana et al biological active compounds widely used as antimicrobial drugs, antithyroid agents, antitumor, antibiotics and carbonic anhydrase inhibitors [3,4]. The liver is a versatile organ which is involved in metabolism and independently involved in many other biochemical functions. Regenerating power of liver cells is tremendous [5], it is endowed with various functions such as, metabolism of carbohydrates, lipids and proteins. The LFTs are used to determine if the liver has been damaged or its function impaired. The liver cells can detoxify drugs, hormones and convert them into less toxic substances for excretion [6,7]. Aspartate transaminase (AST) or aspartate aminotransferase, also known as (AspAT) or serum glutamic oxaloacetic transaminase (SGOT), is a pyridoxal phosphate (PLP)-dependent transaminase enzyme [8] found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells, and it is commonly measured clinically as a marker for liver health. AST is present in both the mitochondrial and cytosol of the hapatocytes and catalyzes the reversible transfer of α-amino group of L - aspartate to α – ketoglutarate resulting into formation of oxaloacetate and L – glutamate. Normal range of AST in human is 3 to 15 IU/L5. ALT is an enzyme that catalyzes the transfer of amino groups to form the hepatic metabolite oxaloacetate9. It is composed of 496 amino acids, which are encoded by a gene located in the long arm of chromosome [8,10,11]. ALT levels differ with gender, with higher values in men than in women12. Additional factors that affect serum ALT levels include body mass index (BMI) and triglyceride levels, regardless of gender [13-15]. Total cholesterol levels and alcohol consumption among men have a positive correlation, whereas smoking; physical activity and age have a negative correlation with ALT levels [15-17]. Alkaline phosphatase (ALP) levels

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usually include two similar enzymes (isoenzymes) that mainly come from the liver and catalyze hydrolysis of phosphate esters at an alkaline pH[18]. Proteins are known that the albumin is solely synthesized in the liver and also to some extent α and β globulins5 and their levels indicate synthetic capability of the liver. Altered serum protein levels are common in liver disease but are nonspecific [19]. Many medications can affect the total protein test results. If total protein is abnormal, additional tests must be performed to identify which specific protein is low or high before a diagnosis can be made [20]. The liver is found in the body as total and conjugated bilirubin. An altered bilirubin level is indicative of liver damage when compared to plasma activities of serum enzymes in the liver [18]. Serum creatinine (a blood measurement) is an important indicator of renal health because it is an easily measured by-product of muscle metabolism that is excreted unchanged by the kidneys. Under certain condition; oxygen can seriously affect our well-being through the formation of reactive oxygen species (ROS) representing both free radical and non-free radical species which leads to the potential deleterious effects such as atherosclerosis, ischemic heart disease, ageing, inflammation, diabetes, immune suppression, neurodegenerative diseases, cancer and other diseases [21]. Creatine is then transported through blood to the other organs, muscle, and brain, where, through phosphorylation, it becomes the high-energy compound phosphocreatine [22]. During the reaction, creatine and phosphocreatine are catalyzed by creatine kinase, and a spontaneous conversion to creatinine may occur [23]. In a recent Japanese study, a lower serum creatinine level was found to be associated


Christiana et al with an increased risk for the development of type 2 diabetes in Japanese men [24]. The metabolism of protein in the body, the liver creates ammonia, which is broken down into a by-product called urea. ROS exert beneficial effects on cellular responses and immune function but at high levels, free radicals and oxidants generate oxidative stress, a deleterious process that can damage cell structures, including lipids, proteins and DNA [25]. Oxidative stress plays a major part in the development of chronic and degenerative ailments such as cancer, autoimmune disorders, rheumatoid arthritis,cataract, aging, cardiorascular and neurodegerative diseases [26]. Liver cells can detoxicate drugs, hormones and convert them into less toxic substances for excretion [27-28]. In continuation of our investigation in the research of new bioactive compounds using benzene sulphonyl chloride and aminopyridine as starting material, we describe here the biological activity of the synthesized N-heteroaryl substituted benzene sulphonamides [29]. This work describes the antimalarial activity of some N-heteroaryl substituted benzene sulphonamides against Plasmodium berghei, and their effects on organs using liver function tests; (Alanine Aminotransferase and Aspartate Aminotransferase activities) and kidney function tests; urea and creatinine concentrations.

MATERIALS AND METHODS Spectrophotometer (SPM721-2000), Digital Photo Colorimeter, Olympus Microscope, Centrifuge (model 800D), Water bath, Microscope Slides, Nheteroaryl substituted benzene sulphonamides synthesized samples. Malaria parasitaemia (Plasmodium berghei) was obtained from malaria infected mice at Veterinary Medicine Department of University of Nigeria, Nsukka.

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Methods Induction of Parasitaemia Parasitaemia was maintained in the laboratory by the method of David et al. (2004)[31]. Ten drops of the parasitized blood obtained with the aid of a capillary tube through the ocular region of the mice, was diluted with 1ml of a normal saline. Thereafter, 0.2ml of the diluted parasitized blood was used to infect the three mice that served as the host from where other experimental animals were infected. Determination of the Malaria parasite (Mp+) The inhibition pattern of parasitic growth of the synthesized samples at 10 nm was carried out according to the method of Dacie and Lewis (2000) [32]. A pair of scissors was used to cut the tail which was squeezed gently to obtain a small drop of blood that was placed on the Centre of a microscope slide. Immediately the thin film was spread using a smooth edged slide spreader. The slide was labeled with a black lead pencil and air – dried in horizontal position. Determination of the Aminotransferase (AST) Activity

Aspartate

The activity of aspartate aminotransferase was assayed by the method of Reitman and Frankel (1957) [33] as outline in Randox Kit. Aspartate aminotransferase activity was measured by monitoring the following information of oxaloacetate hydrazine with 2,4-dinitrophenylhydrazine. Measurement against Reagent Blank The AST substrate phosphate buffer of 0.5 mL each was pipetted into both the reagent blank (B) and sample test (T) test tubes respectively. The serum sample of 0.1 mL was added to the sample test (T) test tubes only and mixed thoroughly.


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Then, 0.1 mL of distilled water was added to the reagent blank (B). Then, the entire reaction medium was well mixed and incubated for 30 minutes in a water bath at 37°C.Immediately after incubation, 0.5 mL of 2,4dinitrophenylhydrazine was added to the reagent blank (B) and the sample test tubes. It was mixed thoroughly and allowed to stand for exactly 20 minutes at 25°C. Finally, 5.0 mL of sodium hydroxide solution was added to both the blank and the reagent and the reagent test tubes respectively and mixed thoroughly, the absorbance of sample A was read at a wavelength of 550 nm against the reagent blank after 5 minutes. Measurement against Sample Blank The AST substrate phosphate buffer of 0.5 mL each was pipetted into both the reagent blank (B) and sample test (T) test tubes respectively. The serum sample of 0.1 mL was added to the sample test (T) test tubes only and mixed thoroughly. Then, 0.1 mL of distilled water was added to the reagent blank (B). Then, the entire reaction medium was well mixed and incubated at 37°C for 30 minutes in a water bath. A volume of 0.5 mL of 2,4-dinitrophenylhydrazine was added to the reagent blank (B) and the sample test tubes immediately after incubation. Also, 0.1 mL of the sample was added to blank (B) only. The medium was mixed and allowed to stand for exactly 20 minutes at 25°C. Finally, 5.0 mL of sodium hydroxide (NaOH) solution was added to both the blank (B) and sample test (T), test tubes and mixed thoroughly. The absorbance of sample A was read at a wavelength of 550 nm against the reagent blank after 5 minutes. The activity of AST in mice serum was obtained from the already calibrated table below (Randox Company).

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Determination of the Alanine Aminotransferase (ALT) Activity The activity of aspartate aminotransferase was assayed by the method of Reitman and Frankel (1957) [33] as outline in Randox Kit. The ALT substrate phosphate buffer of 0.5 mL each was pipetted into both the reagent blank (B) and sample test (T) test tubes respectively. The serum sample of 0.1 mL was added to the sample test (T) test tubes only and mixed thoroughly. Then, 0.1mL of distilled water was added to the reagent blank (B). Then, the entire reaction medium was well mixed and incubated for 30 minutes in a water bath at 37°C. Immediately after incubation, 0.5 mL of 2,4dinitrophenylhydrazine was added to the reagent blank (B) and the sample test tubes. It was mixed thoroughly and allowed to stand for exactly 20 minutes at 250C. Finally, 5.0 mL of sodium hydroxide solution (NaOH) was added to both the blank and the reagent and the reagent test tubes respectively and mixed thoroughly, the absorbance of sample A was read at a wavelength of 550 nm against the reagent blank after 5 minutes. Determination of the Urea Concentration The concentration of serum urea was determined using the method of Tietz (1994) [34] as outlined in Randox Kit, UK. A known volume, 10 μl of the sample was pipetted into the sample tube; 10 μl of the standard was also pipette into the standard tube followed by addition of 10 μl of distilled water to the blank tube. A volume of 10 μl sodium nitroprusside and urease were added to each of the three tubes. The tube were mixed and incubated at 37% for 15 minutes. Then, 2.50 mL of phenol was added to each of the three tubes followed by addition of 2.50 mL of sodium hypochlorite also. These were mixed and


Christiana et al incubated for 15 minutes 370C and the absorbance of the sample was read against the reagent blank at 546 nm. Determination of the Creatinine Concentration The concentration of serum urea was determined using the method of Tietz (1994) [34] as outlined in Randox Kit, UK. Creatinine in alkaline solution reacts with picric acid to form a coloured complex. The amount of the complex formed is directly proportional to the creatinine concentration. A known volume, 100 μl, of distilled water was pipette into the blank tube,

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also 100 μl 0f the standard was added to the standard tube while 100 μl of the sample was pipetted into the sample tube. Then, 100 μl of the working reagents were pipetted into the three tubes and mixed. The absorbance of the sample was read against the blank at 492 nm. Experimental Design Wistar albino mice of either sex weighing 20–34g were housed in separate cages, acclimatized for one week and then divided into eleven groups of five mice each. The route of administration was via oral route with aid of an incubation tube.


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The treatment started after 3 days of malaria inoculation and lasted for 5 days after which analyses were performed on day 7 of post treatment.

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view that serum levels of transaminase return to negative control with healing of malaria.

O

O

O

RESULT AND DISCUSSION

O S

S

NH

The N-heteroaryl substituted benzene sulphonamide derivatives (Fig 1)3a-3h synthesized samples was assessed for their antiplasmodial activity and their effects on liver and kidney using enzyme markers. From the results presented in tables 1 and 2, sample3c, 3e and 3g, the compounds showed better and more promising antiplasmodial activity than samples 3a, 3b, 3d, 3f and 3h. The antiplasmodial activity of the analogues was compared with standard drug Artesunate and was observed that the standard drug produced better activity. Among the synthesized compounds, 3d produced highest AST (Aspartate Aminotransferase) activity (40 IU/L) while ALT (Alanine Aminotransferase) activity was found to be highest in compounds 3c and 3e (30 IU/L). Urea and creatinine concentrations in the serum were used for kidney functions test. With respect to the synthesized compounds, urea and creatinine concentrations was found to be at the similar levels to that of animals without parasitaemia indicating that the compounds have no adverse effects on renal function [35-37]. This effect is in agreement with the commonly accepted

NH N

N H3C

N-(4-methylpyridin-2-yl)benzenesulfonamide

N-(pyridin-2-yl)benzenesulfonamide

O

O

O

O

S

S

NH

NH N

O 2N

N

NO 2 N-(5-nitropyridin-2-yl)benzenesulfonamide

O

O

O

N-(3-nitropyridin-2-yl)benzenesulfonamide

O S

S

NH

NH

N

HO

N Cl

N-(3-hydroxypyridin-2-yl)benzenesulfonamide

O

O

O

O

N-(5-chloropyridin-2-yl)benzenesulfonamide

S

S

NH

NH Cl

N

N Cl

Cl Cl

N-(4,6-dichloropyridin-2-yl)benzenesulfonamide

N-(3,5-dichloropyridin-2-yl)benzenesulfonamide

Fig 1: Structures of N-heteroaryl substituted benzene sulphonamides.


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Table 1. Sulphonamides of some aminopyridine and benzene sulphonyl chloride. Substrate

Sulphonylation Agent

NH2

O

O S

N

Solvent/ Temperature Acetone and pyridine/ r.t

Time (h) 24

Yield (%) 85.8

Acetone and pyridine/ r.t

24

79.3


24

65.3


24

70.8


24

70.9


24

70.8


24

72.1


24

73.1

Cl

NH2

O

O S Cl

N H3C NH2

O

O S

O 2N

N

Cl

NH2

O

O S

N

Cl NO 2

NH2

O

O S

HO N

Cl

NH2

O

O S

N

Cl

Cl

NH2

O

O S

N Cl

Cl Cl

NH2

O

O S

Cl N

Cl

Cl

Table 2: Result of Percentage Parasitaemia before Treatment

Animal

3a

3b

3c

1st animal

6+

7+

7+

Synthesized samples 3d 3e 3f 6+

8+

2nd animal 5+ 5+ 9+ 7+ 8+ 3rd animal 7+ 6+ 6+ 6+ 6+ 4th animal 6+ 5+ 6+ 7+ 7+ 5th animal 6+ 6+ 8+ 5+ 9+ PP 6% 5.8% 7.2% 6.2% 7.6% NC = Negative Control, PC = Positive Control and SC = Standard Control (Artesunate) UC =Untreated Control PP = Percentage parasitaemia

3g

3h

NC

UC

SC

6+

6+

5+

0

8+

13+

8+ 5+ 5+ 7+ 6.2%

8+ 5+ 6+ 9+ 6.8%

6+ 7+ 8+ 6+ 6.4%

0 0 0 0 0%

5+ 6+ 7+ 10+ 7.2%

9+ 13+ 10+ 7+ 10.4%


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12 10 8 6 4 2 0 3a 3b 3c 3d 3e 3f 3g 3h NC PC SC Parasitaemia before Treatment

Fig. 1: Result of Percentage Parasitaemia before Treatment

Fig. 2: Result of Percentage Parasitaemia after Treatment

Table3: Result of Percentage Parasitaemia after Treatment

Animal

Synthesized samples 3a 3b 3c

3d

3e

3f

3g

3h

NC

UC

SC

1st animal 2nd animal 3rd animal 4th animal 5th animal PP

2+ 1+ 3+ 2+ 1+ 1.8%

2+ 3+ 2+ 3+ 3+ 2.6%

2+ 1+ 0+ 2+ 2+ 1.4%

1+ 2+ 3+ 1+ 2+ 1.8%

2+ 1+ 2+ 0+ 3+ 1.6%

0+ 2+ 1+ 2+ 2+ 1.4%

0 0 0 0 0 0%

11+ 8+ 9+ 10+ 13+ 10.2%

1+ 2+ 0+ 1+ 2+ 1.2%

2+ 3+ 1+ 2+ 3+ 2.2%

1+ 2+ 2+ 2+ 3+ 2%

Table 4: Result of the Liver Function Test Compounds

Urea (mg/dL)

3a 3b 3c 3d 3e 3f 3g 3h Normal control Positive control Standard control

68 68 68 67 67 68 68 68 68

Creatinine (mg/dL) 2.2 2.2 2.2 2.2 2.2 2.1 2.2 2.2 2.1

68

2.2

68

2.2

Table 5: Result of the Kidney Function Test Compound

AST (IU/L)

ALT (IU/L)

3a

37

29

3b

39

26

3c

37

30

3d

40

28

3e

38

30

3f

36

27

3g

37

29

3h

39

28

Normal control

40

30

Positive control

42

34

Standard control

39

29


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ALT (IU/L)

AST (IU/L) 35 30 25 20 15 10 5 0

42 40 38 36 34 32 3a 3b 3c 3d 3e 3f 3g 3h NC PC SC

3a 3b 3c 3d 3e 3f 3g 3h NC PC SC

Fig. 3: Result of the AST (IU/L)

Fig. 4 Result of the ALT (IU/L)

Urea (mg/dL)

Creatinine (mg/dL)

68

2.2

67.5

2.15

67

2.1

66.5

2.05 3a 3b 3c 3d 3e 3f 3g 3h NC PC SC

3a 3b 3c 3d 3e 3f 3g 3h NC PC SC

Fig.5: Result of Urea (mg/dL)

Fig.5: Result of Creatinine (mg/dL)

the search for development of new antimalarial drug

CONCLUSION The sulphonamide derivatives were screened for their antiplasmodial activity against Plasmodium berghei and it was observed that some of the compounds showed very promising activity against the susceptible organism. The liver enzymes AST and ALT activities were not affected adversely showing that these compounds have little or no liver damaging effect. In the same vein, the serum levels of creatinine and urea were moderate showing that the compounds have no serious damaging effect on the kidneys. All these findings suggest that these compounds should be explored in

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Cite this article as: Igwe Christiana N, Okoro Uchechukwu C, Egba Simeon I, Nwankwo Nicodemus E, Anaduaka Emeka G. The Activity of Synthesized N-heteroaryl Substituted Benzene Sulphonamides on the Liver and Kidney Marker Enzymes of Plasmodium berghei Infected Mice. J Pharm Chem Biol Sci 2015; 3(1): 6-16.
