1.
INTRODUCTION:
The
kidneys are one of the major chemical factories in the body that regulate the
blood flow. Remove metabolism waste products and maintain the balance of water
and make up electrolytes in the blood stream [1]. The performance of the
kidneys is generally tested by observing blood urea nitrogen levels, creatinine
clearance and also glomerular filtration rate,
the flow rate of purified fluid via the kidney [2]. Research
on plunge products such as urea and creatinine (2-amino-1-methyl-5H-
imidazol-4-one) can explore investigate the biological results and functions of
the kidneys [3]. Concentrations of the above types can also show the muscle and
thyroid functions [4]. The end product of creatine metabolism produced by
creatinine [3] is produced by the body and filtered by the kidneys from the
blood stream in relatively small quantities daily [3], normal physiological
concentration in blood is 0.90 to 1.
30 mg/dL in men and 0, 60 to 1,10 mg/dL in women, but in certain pathological
conditions, it may exceed these levels [5]. In
contrast to urea, creatinine concentrations in body fluids are not influenced
by protein consumption, so creatinine levels serve as a more reliable
indication of renal function [6]. Therefore, creatinine quantification is an
important research area in science, in particular in the areas of clinical
biochemistry, biology and medicine [7,8].
2. EXPERIMENTAL:
2.1. Materials and physical
measurements:
All starting materials and solvents were purchased
from Sigma-Aldrich and Fluka and used without further purification. Melting points
were measured on Gallen Kamp capillary melting point apparatus and were
uncorrected, FT-IR measurements were recorded on Shimadzu model FTIR-8400S. 1HNMR
spectra were obtained with Bruker spectrophotometer model ultra-shield at 400
MHz in D2O solution with the TMS as internal standard.
2.2. Synthesis of the organic compounds:
2.2.1. Synthesis of compound (1a) [9]
Literature procedure was used with modifications. In 100 mL Round-bottom flask (R.B.F) the creatinine
(0.02 mole) was dissolved in DMF (20mL) and then cooled at (0-5˚C) and 2-3
drops of trimethylamine (TEA) were added. Chloroacetyl chloride (0.02 mole) in
DMF (20 mL) was slowly added to R.B.F with vigorous stirring for 3 hours at
room temperature. The obtained product was filtered, washed with ether and
recrystallized from ethanol. The physical properties of synthesized compound
(1a) is given in Table 1.
2.2.2. Synthesis of compounds(2a-3a) [10]
Literature procedure was used with modifications. In 100 mL R.B.F (0.02 mole) of compound (1a) and
(0.02 mole) of urea/thiourea were dissolved in 1,4-dioxane (20mL) and the blend
were refluxed for 18 hours. The obtained product was filtered, and
recrystallized from ethanol. The physical properties of synthesized compounds (2a-3a)
is given in Table 1.
2.2.3. Synthesis of compounds(4a-5a) [11]
To 20 mL of hot ethanol, (0.005 mole) of benzaldehyde/m-nitro
benzaldehyde and (0.0025 mole) of compounds
(2a-3a) were dissolved. To this mixture
1.0 mL of glacial acetic acid was added. The reaction mixture was then refluxed
on a water bath in a 250 mL R.B.F for 12 hours. Completion of the reaction was
monitored by TLC. The mixture was allowed to stand for 24 hours at room
temperature. The product was collected and recrystallized with ethanol. The
Physical properties of synthesized compounds (4a-5a) are given in Table1
2.2.4. Synthesis of compounds (6a-7a) [12]
Compounds (4a-5a) respectively (0.01mole)
were dissolved in absolute ethanol (20 mL), then NaOH (1M, 10 mL) was added at
(0°C). α-chloroethylacetate (0.01) was added to the mixture. This mixture was
stirred at room temperature overnight. The precipitate was filtered and then
dried. The product was
collected and recrystallized with ethanol.
The Physical properties of synthesized compounds (6a-7a) are given in Table1.
2.2.5. Synthesis of compounds (8a-9a) [13]
(0.03 mole) of (6a-7a) compounds
in 10 ml of ethanol were added to (0.018 mole) of semithiocarbazide then
refluxed for 3 hrs. The separated precipitate was filtered and recrystallized
from ethanol. The physical properties of synthesized compounds (8a-9a) are
given in Table 1.
2.2.6. Synthesis of compounds (10a-11a) [14]
(0.003)
mole of (8a-9a) compounds were placed in a round bottomed flask equipped with
40 ml of 5% NaOH and refluxed for 5 hrs. The solution was cooled and
naturalized with dil. HCl. The separated precipitate was filtered and
recrystallized ethanol. The physical properties of synthesized compounds
(10a-11a) are given in Table 1.
3. BIOLOGICAL
ACTIVITY:
3.1. Effect of compounds (10a-11a) on SGOT, and SGPT
activities
Colorimetric determination of SGOT or SGPT activity
according
to the following reactions:
The pyruvate or oxaloacetate formed was measured in
its derived from 2,4-dinitrophenylhydrazine, which was absorbed at wave length
546 nm (SYRBIO kit).
3.2.A stock solution (0.01 M) of compounds(10a-11a)
A stock solution (0.01 M) of compounds(10a-11a) were
prepared by dissolving it in distilled water, and the following concentrations
(10-2, 10-3, 10-4, 10-5 M) were
prepared by diluting with distilled water. The enzymes SGOT, and SGPT
activities were measured in human serum by using the same methods of these
enzymes with replace 100 µl of buffer with 100 µl of compounds (10a-11a). The activation percentage was calculated by comparing
the activity with and without compounds (10a-11a) and under the same
conditions, according to the equation:
% Activation
=100× The activity in the presence of activator/The
activity in the absence of activator – 100
3.3. A constant concentration of compounds (10a-11a)
(10-2 M)
A constant concentration of compounds (10a-11a) (10-2
M) were used with different substrate concentrations of (40, 80, 120, 160, 200)
mmol/L for SGOT and SGPT to study the type of activation. Buffers were used to
prepared different substrates concentrations of these enzymes SGOT and SGPT
(phosphate buffer pH = 7.40, 100 mmol/L). The enzymes velocity was determined
with and without compounds (10a-11a), by using the Linweaver and Burke equation
and plotting 1/v against 1/[s] were evaluated values; Ki, apparent Vmax
(Vmapp), apparent Km (Kmapp), type of inhibition or
activation[15].
4. RESULT AND DISCUSSION:
4.1. Synthesis:
Scheme 1 included synthesiscreatininederivatives. Thecharacterization
data of all compounds 1a–11a are given in the experimental section. All the
newly synthesized compounds gave satisfactory analysis for the proposed
structures, which were confirmed on the basis of FTIR and1HNMR data.
The enzymes play important role in amino
acid metabolism and in urea and tricarboxylic acid cycles. We suggested that
compounds (10a-11a) have (N–, S-, O-, =S, =O, =N, and =C) groups by which, it
activities the active sides of amino acids of SGOT and SGPT enzymes by
increasing affinity of active sides of enzymes to react with the substrates.
5. THEORETICAL DETAILS:
a- HOMO
b- LUMO
Figure
4: The calculated a- HOMO, b- LUMO for the compound (11a)
The
E.P is useful for finding sites of reaction in a molecule; positively charged
species tends to attack a molecule where the electrostatic potential is
strongly negative. [19,20]
a- 2D
b- 3D
Figure
5: The calculated electrostatic potential a- 2D, b- 3D for the compound (11a).
6.
CONCLUSION:
Creatinine
derivatives were synthesized and structurally characterized by using
spectroscopic techniques..The biochemical studies revealed that the creatinine
derivatives caused activator effects on SGOT and SGPT
enzymes activities. Finally, we worked theoretical study For the purpose of
comparison with the experimental results, there are good agreement between
theoretical and experimental results.
7.
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