Synthesis, Identification, Theoretical Study and effect of 1,3,4-Oxadiazole Compounds Substituted on Creatinine ring on the activity of some Transfers Enzymes

 

Zahraa T. Khudhair, Entesar O. Al-Tamimi

Department of Chemistry, College of Science, University of Baghdad, Jadiriya, Baghdad, Iraq

 

ABSTRACT:

The present report describes the synthesis of 1,3,4-oxadiazole compounds on shiff base substituted on creatinine ring, the Synthetic route started from reaction shiff base derivatives with α-chloroethylacetate to give compounds(1e-2e). Hydrazide derivatives were synthesized by the reaction compounds (1e-2e) with hydrazine hydrate to give compounds (3e-4e). The compounds (3e-4e) reacts with phenylisothiocyanate to give compounds (5e-6e). The synthesized compounds characterized by FT-IR and¹HNMRspectroscopy.Beside the experimental work, we worked theoretical study involving calculated the spectra, total energy, dipole moment etc. Also this study was designed to show the effects of creatinine derivatives on the activities of some transferase enzymes such as: GOT, and GPT enzymes in sera. This compounds demonstrated activation effects on GOT and GPT activities. These effects increased with increasing the concentration of the compounds. The causes of the increases in the enzymes activities are discussed.

 

 

 

1.    INTRODUCTION:

It is well known in the literature that nitrogen and oxygen-containing compounds are mainly used in medicine to treat various types of fungal and bacterial infections, gastric ulcer, cancer, etc. [1]. The organic moiety with nitrogen atoms results in higher efficiency against different diseases [2]. Five heterocyclic compounds have different types of biological activity. 2,5-Disustituted 1,3,4-oxadiazole also exhibits a wide range of activities such as antibacterial [3], antimalarial [4], anti-inflammatory [5], antifungal [6] and anticonvulsant [7]. Replaced 1,3,4-oxadiazoles are of considerable pharmaceutical and material interest, as evidenced by a steadily increasing number of patents and publications. For example, 2-amino-1,3,4-oxadiazoles actas muscle relaxants [8] and 5-aryl-2-hydroxymethyl-1,3,4-oxadiazole derivatives have antimitotic activity analgesic, anti-inflammatory, diuretic and antiemetic properties [9].

 

Some material applications of derivatives of 1,3,4-oxadiazole are in the field of photosensitizers and liquid crystals [10-11]. Common synthetic oxadiazoles approaches [12] involve the diacylhydrazine cycling. A variety of conditions of reaction affect the cyclization reaction. In general, heat and anhydrous reagents, including thionyl chloride [13], promote the reaction. Phosphorous oxychloride [14], pentoxide phosphorus [15], triphenylphosphine [16], Lawesson’s reagent [17] and triflic anhydride [18]. Alternative synthetic methods include carboxylic hydrazide reaction with keteneylidene triphenylphosphorane [19] or trichloroacetic acid hydrazone cyclization reaction [20].

 

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 compounds (1e-2e) [21]:

Shiff base derivatives (0.01mole) were dissolved in absolute ethanol (20 mL), then NaOH (1M, 10 mL) were 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 (1e-2e) are given in Table1.

 

2.2.2. Synthesis of compounds (3e-4e)[22]:

Generally, a solution of compounds (1e-4e) (0.01mole), hydrazine hydrate(0.01 mole,85%)in absolute ethanol (50mL) were prepared. The reaction mixture was refluxed for 24hr. The obtained product was filtered, and recrystallized from ethanol. The physical properties of synthesized compounds (3e-4e) are given in Table 1.

 

2.2.3. Synthesis of compounds (5e-6e) [23]:

A mixture of (5e-8e) compounds (6.39, 0.01mol) and phenylisothiocynate (0.04 mol, 4.3 ml) in DMF (50 ml) were refluxed for 10 hours on steam bath. the reaction mixture was recrystallized from ethanol. The physical properties of synthesized compounds (5e-6e) are given in Table 1.

 

Table1: The physical properties of synthesized compounds (1e- 6e).

No. of compd.	Structure and name of compounds	Chemical formula	Color	Molecular weight	M. P. °C Dec.	Yield%
1e	ethyl 2-(1-methyl-4-oxo-2-(2-(1-phenylethylideneamino) oxazol-5-ylamino)-4,5-dihydro-1H-imidazol-5-yl) acetate	C15H15N5O2	Yellow	383.40	152-155	79
2e	ethyl 2-(2-(2-( benzylideneamino) thiazol-5-ylamino)-1-methyl-4-oxo-4,5-dihydro-1H-imidazol-5-yl)acetate	C18H19N5O3S	Yellow	385.44	168-170	98
3e	2-(1-methyl-4-oxo-2-(2-(1-phenylethylideneamino)oxazol-5-ylamino)-4,5-dihydro-1H-imidazol-5-yl)acetohydrazide	C16H16N8O5	White	383.40	209-210	79
4e	2-(2-(2-(benzylideneamino)thiazol-5-ylamino)-1-methyl-4-oxo-4,5-dihydro-1H-imidazol-5-yl)acetohydrazide	C16H17N7O2S	White	371.42	185-187	70
5e	1-methyl-5-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)methyl)-2-(2-(1-phenylethylideneamino)oxazol-5-ylamino)-1H-imidazol-4(5H)-one	C24H22N8O2S	Off white	486.55	210-212	68
6e	2-(2-(benzylideneamino)oxazol-5-ylamino)-1-methyl-5-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)methyl)-1H-imidazol-4(5H)-one	C23H20N8OS2	Off white	488.59	227-230	76

 

3. BIOLOGICAL ACTIVITY:

3.1. Effect of compounds (5e-6e) 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 compound (5e-6e):

A stock solution (0.01 M) of compounds (5e-6e) 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 (5e-6e). The activation percentage was calculated by comparing the activity with and without compounds (5e-6e) 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

 

The activation constant (K_i) was calculated according to the following equation:

 

V_max+A= V_max–A/ (1+ [A]/ K_i)

Where A is activation constant.

+A is with activator

-A is without activator

[A] is activator concentration

 

3.3. A constant concentration of compound (5e-6e) (10^-2 M):

A constant concentration of compounds (5e-6e) (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, SGPT (phosphate buffer pH=7.40, 100 mmol/L). The enzymes velocity were determined with and without compounds (5e-6e), by using the Lin weaver and Burke equation and plotting 1/v against 1/[s] were evaluated values; Ki, apparent V_max (V_mapp), apparent Km (K_mapp), type of inhibition or activation [24].

 

 

 

4. RESULT AND DISCUSSION:

4.1. Synthesis:

Scheme 1 included synthesiscreatininederivatives. Thecharacterization data of all compounds 1e–6e 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.

 

Scheme1: The chemical steps for the synthesis of compounds (1e-6e).

 

4.2. FT-IR spectra:

4.2.1.The FTIR spectra of compounds (1e-2e) have important characteristic stretching vibration bands that corresponds to (C=O) ester band The FT-IR spectrum of compounds (1e-2e) arelisted in Table 2[25].

 

Table 2: FT-IR Spectral data of synthesized compounds (1e-2e) in cm^-1.

Comp. No.	n C-H Aromatic	n C=O Ester	n C=C Aromatic	n C=O Cycl. amide	n N-H
1e	3072	1749	1670	1699	3269
2e	3041	1747	1643	1697	3249

 

4.2.2.The FTIR spectra of compounds (2e-3e) have important characteristic stretching vibration bands that corresponds to (C=O) ester band which are disappeared and stretching vibration bands that corresponds to (C=O) amide band which are appeared[25], show Table 3.

 

Table 3: FT-IR Spectral data of synthesized compounds (3e-4e) in cm^-1.

Comp. No.	n C-H Aromatic	n C-H Aliphatic	n C=O Amide	n NH2	n N-H
18	3002	2964	1668	Asy.= 3454 Sy.=3413	3250
19	3020	2902	1666	Asy.= 3353 Sy.=3413	3303

 

 

4.2.3. The FTIR spectra of compounds (5e-6e) have important characteristic stretching vibration bands that corresponds to (-C-O) of oxadiazole ring band which are appeared, also stretching vibration bands that corresponds to (NH₂) and (C=O) amide band which are disappeared, Show Table 4.

 

Table 4: FT-IR Spectral data of synthesized compounds (5e-6e) in cm^-1.

Comp. No.	n C-O	n C=N oxadiazole ring	n C=O Amide	n NH2	n C-H Aromatic
5e	1049	1604	------	------	3002
6e	1081	1600	-----	-----	3002

 

4.3. ¹HNMR Spectra:

The ¹HNMR spectra of compounds (5eand 6e) are listed in Table 5[26].

 

4.4. Biological activity of transferase enzymes (SGOT and SGPT).

This research addresses investigation of the effects of compounds (5e-6e) of SGOT and SGPT enzymes. The biochemical tests revealed that these compounds caused stimulation effects on SGOT and SGPT enzymes activities. Table (6) is listed below shows the effect of different concentration of compounds (5e-6e) on the activity of SGOT and GPT enzymes in human serum. This research addresses investigation of the effects of compounds (5e-6e) of SGOT and SGPT enzymes. The biochemical tests revealed that these compounds caused activatory effects on SGOT and SGPT enzymes activities. The normal value of the SGOT and SGPT enzyme activities were (14 and 16 U/L) respectively. The relationship between compounds (5e-6e) concentrations versus and the activity of enzymes were shown in Figures (5e-6e). These results observed that any increase in compound concentrations caused increase in percentage of activation of enzymes.

 

Table (5) ¹HNMR data of compounds (5e and 6e) in ppm.

Comp. No.	Compound structure	1HNMR data of (δ-H) in ppm
5e		Singlet 1H of -NH (8.35); multiplet 11H of aromatic rings (7.37-7.87); Singlet 1H of –NH group (4.64); Singlet 1H of –CO-CH group (3.70); Singlet 3H of -N-CH3 group (3.12); Singlet 2H of -CH2 group (2.91); Singlet 3H of –CH3 group (1.25).
6e		Singlet 1H of -NH (8.10); singlet 1H of –N=CH (8.08); multiplet 11H of aromatic rings (7.33-7.72); Singlet 1H of –NH group (4.37); Singlet 1H of –CO-CH group (3.56); Singlet 3H of -N-CH3 group (3.44); Singlet 2H of -CH2 group (3.13).

 

Table 6: The effect of different concentration of compounds (5e-6e) on the activity of SGOT and SGPT enzymes in human serum.

Concentration (M)	GOT activity (U/L)	Activation (%)	GPT activity (U/L)	Activation (%)
Sample
0	14	0.000	16	0.000
Compound (5e)
10-2	77	450.000	97	506.250
10-3	43	207.142	63	293.750
10-4	31	121.428	39	143.750
10-5	17	21.285	20	25.000
Compound (6e)
10-2	117	680.000	98	476.470
10-3	73	386.666	75	341.176
10-4	51	240.000	40	135.294
10-5	22	46.666	21	23.529

 

Figure.1: (a) The relationship between concentration of compounds (5e) and SGOT enzyme activity. (b) The relationship between concentration of compounds (5e) and SGPT enzyme activity.

 

Figure. 2: (a) The relationship between concentration of compounds (5e) and SGOT enzyme activity. (b) The relationship between concentration of compounds (5e) and SGPT enzyme activity.

 

 

Figure. 3: (a) The percentage of activation SGOT enzyme and compounds (6e) concentration. (b) The percentage of activation SGPT enzyme and compounds (6e) concentration.

 

Figure. 4: (a) The percentage of activation SGOT enzyme and compounds (6e) concentration. (b) The percentage of activation SGPT enzyme and compounds (6e) concentration.

 

 

Competitive, non-competitive and uncompetitive activators can easily be distinguished by using the Lineweaver–Burk plot's double reciprocal plot. Two sets of rate determination were performed in which the concentration of enzymes was kept constant. In the first experiment, the speed of the unactivated enzyme was determined, and in each enzyme test the second experimental constant amount of activator was included. Various substances are capable of reducing or eliminating the catalytic activity of a particular enzyme. [27].

 

Table 7 and Figures (5-6) showed that the type of enzyme activation using Lineweaver–Burk plot for compounds (5e-6e) on SGOT and SGPT activity. The Vmax and Km values determined with 10^-2 M of compounds (5e-6e) and without it. Vmax without compounds (5e-6e) were greater than Vmax in the presence compounds (5e-6e). A liquate 10^-2 M of compounds (5e-6e) were noncompetitive activation for enzymes activity. Noncompetitive activation changed the Vmax of the enzyme but not the Km. By using Lineweaver–Burk equation, the Ki values of enzyme for compound which was studied in different concentrations.

 

Figure.5: Lineweaver-Burk plots for compounds (5e) effects on (a) SGOT, (b) SGPT.

 

Figure.6: Lineweaver-Burk plots for compounds (6e) effects on (a) SGOT, (b) SGPT.

 

 

 

 

 

Table 7: The kinetic properties of SGOT and SGPT with compounds (5e-6e).

Enzymes	Km (mmole/L)	Vmax (mmole/ L). min.	Ki (mmole/L)	Type of effect
SGOT
Without compound	200	1.649	------	------
Compound (5e)	200	0.023	0.00014	Noncompetitive
SGPT
Without compound	100	0.061	------	-------
Compound (6e)	100	0.031	0.0103	Noncompetitive

 

The enzymes play important role in amino acid metabolism and in urea and tricarboxylic acid cycles. We suggested that compounds (5e-6e) have (N– and O=) groups by which, it activities the active sides of amino acids of GOT and GPT enzymes by increasing affinity of active sides of enzymes to react with the substrates.

 

5. Theoretical details:

5.1. Optimize geometry of compound (6e).

The Optimize geometry of atoms forthe compound (6e) calculating byusing semi-empirical (PM3) method was depicted in figure 7

 

Figure 7. a) The Optimize geometry, b) The numbering Optimize geometry for compound (6e).

 

The result of the calculated optimized structural parameters for compound (6e) such as bonds length and bonds angle (A^˚) were calculating by using semi-empirical (PM3) method. The Table 8 and 9 respectively revealed that the results of this work were in good agreement with experimental data.

 

Table 8. Some calculated bonds length for the compound (6e).

 

Table 9. Some calculated bonds angle for thecompound (6e).

 

Table 10. Some energies and physical properties for compound (6e).

Property	PM3 method
Point group	C1
Symmetry	A
Etot(kcal/mole)	-115127.5667
Eb (kcal/mole)	-5889.6391
ΔHof (kcal/mole)	179.2298
EHOMO ( a.u.)	-0.3295
ELOMO ( a.u.)	-0.0514
∆E HOMO-LUMO( a.u.)	0.2781
µ (debye)	7.9605

 

Figure 8. The calculated a- HOMO, b- LUMO for the compound (6e).

 

The following figure 9 electron distribution governs the electrostatic potential of the molecules. The electrostatic potential (E.P) describes the interaction of energy of the molecular system with a positive point charge. 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 [28,29].

 

 

Figure 9. The calculated electrostatic potential a- 2D, b- 3D for the compound (6e).

 

5.2- Partial atomic charge of compound (6e)

The calculated partial atomic charge using the PM3 method for individual atoms were illustrated in figure 9. The PM3 method give more reasonable value, showing that the O, N atoms have negative partial charge and positive in C atoms, figure 9Since the O, N atom have more electronegativity than C atom.

 

Figure 10. The partial atomic charges for the compound (6e).

 

5.3. The vibrational spectra of compound (6e)

compound (6e) belongs to (C₁) point group and symmetry (A),The Table are shown below revealed that the theoretical data of this work were in good agreement with experimental data, calculating by using semi-empirical method (PM3).

 

Table 11. PM3 vibration frequencies and IR absorption intensities for compound (6e).

Description	Theoretical		Experimental
	Frequency cm-1	Intensity Km/mole	Frequency
C-H str.(aromat.)	3005	14.56	3002
C=N str.	1623	13.86	1625
C=C str.	1674	32.92	1672
C-H sr. (aliphat. )	2927	16.05	2929

 

Figure 11. Some Modes of vibration frequencies for compound (6e).

5.4. The ¹HNMR spectra of compound (6e)

The Tables are shown below revealed that the theoretical data were in good agreement with experimental data, calculating by using DFT and B3LYP methods (3-21G).

 

Table 12. DFT and B3LYP (3-21 G)¹HNMR for compound (6e).

Description	Chemical shift ppm
	Experimental	Theoretical
1H of –C=N group	3.56	3.55
1H of –CH-CO	3.44	3.40
1H of -CH=	4.68	4.66
1H of -NH	3.81	3.79

 

6. CONCLUSION:

Shiff base derivatives substituted on creatinine ring were synthesized and structurally characterized by using spectroscopic techniques. The Synthetic route started fromreaction shiff base derivatives with α-chloroethylacetate to give compounds(1e-2e). Hydrazide derivatives were synthesized by the reaction compounds (1e-2e) with hydrazine hydrate to give compounds (3e-4e). The compounds (3e-4e) reacts with phenylisothiocyanate to give compounds (5e-6e).The biochemical studies revealed that the creatinine derivatives caused activator effects on GOT and GPT 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.

 

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Received on 11.02.2019           Modified on 02.03.2019

Accepted on 02.04.2019         © RJPT All right reserved