Synthesis and Molecular docking studies of some new Pyrazoline derivatives for Antimicrobial properties

 

Moqbel Ali Moqbel Redhwan, Gitima Deka, Melvin Mariyam Varghese

Department of Pharmacology, Karnataka College of Pharmacy, Bangalore-560064, India

 

ABSTRACT:

Molecular docking is a well-established computational technique which predicts the interaction energy between two molecules and used for understanding drug-receptor interaction. In the present study eight Pyrazoline derivatives containing substituted Pyrazole moiety (1a-8a) were synthesized. Structures of the newly synthesized compounds were characterized by spectral studies (UV, IR, and NMR). Compounds were screened for their antibacterial activity. 4a Compound was found to be potent antimicrobial activity against Pseudomonas aeruginosa compared to the standard drug Ciprofloxacin and other test compounds. All the compounds were subjected to molecular docking studies using PATCHDOCK software for the inhibition the activity of serval types of bacterial (Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli, Pseudomonas putida). The in silico molecular docking study results showed that, all the synthesized compounds having minimum binding energy and have good affinity toward the active pocket, thus, they may be considered as good inhibitor of bacterial activity.

 

 

 

1. INTRODUCTION:

Molecular docking is a well-established computational technique which predicts the interaction energy between two molecules. This technique mainly incorporates algorithms like molecular dynamics, Monte Carlo stimulation, fragment-based search methods. Molecular docking studies are used to determine the interaction of two molecules and to find the best orientation of ligand which would form a complex with overall minimum energy. The small molecule, known as ligand usually fits within protein’s cavity which is predicted by the search algorithm. These protein cavities become active when they come in contact with any external compounds and are thus called as active sites.^[1] Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to predict the affinity and activity of the small molecule. Hence docking plays an important in the rational drug design.

 

Given the biological and pharmaceutical significance of molecular docking, considerable efforts have been directed towards improving the methods used to predict docking. The results are analyzed by a statistical scoring function which converts interacting energy into numerical values called as the docking score; and also the interaction energy is calculated.^[2] The 3D pose of the bound Ligand can be visualized using different visualizing tools like Pymol, Rasmol etc which could help in inference of the best fit of ligand. Predicting the mode of protein ligand interaction can assume the active site of the protein molecule and further help in protein annotation. Moreover molecular docking has major application in drug designing and discovery. The recent expansion of antimicrobial drug research has occurred because there is a critical need for new antimicrobial agents to treat these life threatening invasive infections. The development of antimicrobial resistance has increased in this century and there is a need for developing new antimicrobial agents which will be more selective, potent and less toxic compared to the existing drugs in clinical treatment. Heterocyclic compounds have been extensively explored for developing Pharmaceutically important molecules like Pyrazolines, Isoxazolines, Imidazoles, Benzimidazoles, Furans, Benzofurans, Triazoles and Tetrazoles etc. these molecules exhibited important medicinal properties.^[3-6] Nitrogen containing heterocyclic compounds eg.alkaloids, amides, nucleosides/nucleotides etc are widely distributed in nature and possess a wide range of biological activities.^[7] Among the medicinally important heterocyclic compounds, the pyrazoline function is quite stable and inspired chemists to incorporate this stable fragment on bioactive moieties in an attempt to synthesis new compounds possessing biological activities.^[8-9] Variously substituted pyrazolines and their derivatives are important biological agents and a significant amount of research activity has been directed towards this class. In particular, they are used as antitumor, antiviral, antitubercular, cardiovascular activities, antimicrobial and insecticidal agents. Some of these compounds also possess anti-inflammatory, antidiabetics and analgesic properties. Moreover pyrazolines have also been used extensively as useful synthons in organic synthesis.^[10-12] A classical synthesis of these compounds involves the base catalyzed aldol condensation reaction of aromatic ketones and aldehydes to give alpha beta unsaturated ketones(chalcones), which undergo a subsequent cyclization reaction with hydrazine affording 2-pyrazolines in the presence of a suitable cyclizing reagent like acetic acid.^[13] Several new quinazolinonyl chalcones, quinazolinonyl pyrazolines and quinazolinonyl isoxazoles possess anticonvulsant activity. In view of the diverse biological activities of the heterocyclic compounds, it is planned to synthesize substituted Pyrazolines and to evaluate them for various biological activities.

 

2. EXPERIMENTAL:

2.1. Materials and methods:

Melting point were determined in open capillaries on a Tempo melting point apparatus and were uncorrected. The UV spectra were recorded on Analytical UV-VIS Spectrometer. The IR spectra (in KBr pellets) were recorded on FT-IR spectrophotometer (Bruker Alpha). NMR spectra were recorded on a Bruker (400 MHz) using TMS as the internal standard. The completion of the reaction was checked by thin layer chromatography (TLC) on silica gel coated aluminum sheets. Commercial grade solvents and reagents were used without further purification (see Scheme 1).

 

2.2 General procedure for the synthesis of new derivatives of 1-Acetyl-5-(Substituted phenyl)-(amino phenyl)-2-pyrazoline:

A mixture of Acetanilide (0.02mol), absolute ethanol (10 ml), 2% NaOH, and benzaldehyde (0.02mol) was refluxed for for 8-12 h at 120℃. The reaction mixture was then concentrated, cooled and poured into ice. The solid thus obtained was filtered, washed with cold water and recrystallized. After competition the process the product was mixed with (0.03mol) methanol, hydrazine hydrate (0.05mol) and few drops of glacial acetic acid. The reaction mixture was concentrated and poured onto ice. The separated solid was filtered, washed with cold water and recrystallized using ethanol-DMF mixture. The various 1-Acetyl-5-(Substituted phenyl)-3-(amino phenyl)-2-pyrazoline were prepared in similar manner.^[14-15]

 

General Scheme For 1-Acetyl-5-(Substituted phenyl)-3-(amino phenyl)-2-pyrazoline

 

2.2.1. Characterization of synthesized compounds:

2.2.1.1. 1-Acetyle -5-phenyl-3-(amino phenyl)-2-pyrazoline (1a). IR (KBr, max cm^-1): 3267 (N-H), 1661 (C=O), 1599 (C=C of Aromatic ring), 1532 (C=N), 1490 (N-N); UV (ethanol): 239 nm (ℇ17, 760).

 

2.2.1.2. 1-Acetyl -5-(4¹-methyl phenyl-3-(amino phenyl) -2- pyrazoline (2a). IR (KBr, max cm^-1): 3500 (N-H), 1692 (C=O), 1605 (C=C of Aromatic ring), 1517 (C=N), 1409 (N-N); UV (ethanol): 306 nm (ℇ 23, 967).

 

2.2.1.3. 1-Acetyl-5-(4¹ methoxy phenyl)-3-(amino phenyl)-2-pyrazoline (3a). IR (KBr, max cm^-1): 3412 (N-H), 2890 (O-H), 1624(C=O), 1599 (C=C of Aromatic ring), 1561 (C=N) 1450 (N-N); UV (ethanol): 226 nm (ℇ 23, 966).

 

2.2.1.4. 1-Acetyl-5-(4^1-chloro phenyl)-3-(amino phenyl)-2-pyrazoline (4a). IR (KBr, max cm^-1): 3480 (N-H), 1692(C=O), 1555 (C=C of Aromatic ring), 1581 (C=N), 1459 (N-N); ¹H NMR (400 MHz, Bruker): δ 2.15 (s, 3H, -COCH3), 2.3 (t, 1H, -CH-Ar), 4.7(d, 2H, pyrazoline CH2) 6.6-8.6 (m, 9H, Ar-H and N-H); UV (ethanol):279 nm (ℇ 25, 760).                               

 

2.2.1.5. 1-Acetyl-5-(4¹-hydroxy phenyl)-3-(amino phenyl)-2-pyrazoline (5a). IR (KBr, max cm^-1): 3490 (N-H), 2930 (O-H), 1610(C=O), 1500 (C=C of Aromatic ring), 1590 (C=N), 1466 (N-N); ¹H NMR (400 MHz, Bruker): δ 2.15 (s, 3H, -COCH3), 2.3 (t, 1H, -CH-Ar), 4.7(d, 2H, pyrazoline CH2) 6.6-8.6 (m, 9H, Ar-H and N-H), 7.6-7.8 (m, 7H, Ar-H), 7.9 (s, 1H, NH), 8.1(s, 1H, Ar-OH); UV (ethanol): 330 nm (ℇ 36, 166).                                

 

2.2.1.6. 1-Acetyl-5-(4 nitro phenyl)-3-(amino phenyl)-2-pyrazoline (6a). IR (KBr, max cm^-1): 3400 (N-H), 1600(C=O), 1543 (C=C of Aromatic ring), 1520 (C=N), 1430 (N-N); UV (ethanol): 310 nm (ℇ 34, 188).

 

2.2.1.7. 1-Acetyl -5-(2 nitro phenyl)-3-(amino phenyl)-2-pyrazline (7a). IR (KBr, max cm^-1): 3400 (N-H), 1600(C=O), 1543 (C=C of Aromatic ring), 1520 (C=N), 1430 (N-N); UV (ethanol): 330 nm (ℇ 36, 178).

2.2.1.8. 1-Acetyl -5-(3¹-methoxy, 4¹-hydroxy phenyl)-3-(amino phenyl)-2-pyrazoline (8a). IR (KBr, max cm^-1): 3400 (N-H), 1600(C=O), 1543 (C=C of Aromatic ring), 1520 (C=N), 1430 (N-N); ¹H NMR (400 MHz, Bruker): : δ 2.15 (s, 3H, -COCH3), 3.3 (s, 3H, Ar-OCH3), 3.7(d, 2H, pyrazoline CH2) 6.6-8.6 (m, 7H, Ar-H ), 8.6 (s, 1H, NH), 9.2 (Ar-OH); UV (ethanol): 345 nm (ℇ 82, 804). (see Table 1).

 

Table 1 Characterization data of the compounds (1a-8a)

 

2.3 Antibacterial studies:

The antibacterial activity of newly synthesized compounds 2a, 4a and 8a were determined by well plate method in Muller-Hinton Broth (Merck) medium. The in vitro antibacterial activity was carried out for 24 h at 37°C. In this work, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Pseudomonas putida used to investigate the activity. Final bacterial numbers were standardized to 1×10 ⁶ CFU/ml. Then 60 ul of bacterial suspension was poured on a plate, containing Muller-Hinton Agar (MHA, Merck). The lawn culture was prepared by sterile cotton swab and allowed to remain in contact for 1 min. Thereafter, 1mg/ml the concentration of each test compounds was prepared using Dimethyl sulfoxide (DMSO). Then with the borer 9-mm diameter were bored on the lawn culture and 100ul of the each test compounds were poured in to the wells and 24 h after incubation at 37^°C, the inhibition zone was measured in mm. The plates were incubated for 24 h at 37°C. The inhibition zone that appeared after 24 h, around the well in each plate were measured as zone of inhibition in mm. Experiments were triplicates and standard deviation was calculated.^[16-18] The antibacterial results were compared with Ciprofloxacin in Table 2.

 

Table 2 Antibacterial activity of the compounds 2a, 4a and 8a

 

2.4 In silico molecular docking studies:

The ligands were drawn in ChemDraw Ultra 8.0 (Chem Office package) assigned with proper 2D orientation and the structure of each compound was analyzed for connection error in bond order. The molecule thus obtained were saved in PDB format. Docking software PATCHDOCK was used to dock the microbial genome with the test molecule. Fig:1 3D structure for Pseudomonas aeruginosa. Patchdock algorithm is inspired by object recognition and image segmentation techniques used in Computer Vision. Docking can be compared to assembling a jigsaw puzzle. When solving the puzzle should try to match two pieces by picking one piece and searching for the complementary one. Concentrating on the patterns that are unique for the puzzle element and look for the matching patterns in the rest of the pieces.^[19] PatchDock employs a similar technique. Given two molecules, their surfaces are divided into patches according to the surface shape. These patches correspond to patterns that visually distinguish between puzzle pieces. molecular Shape Representation - in this step should compute the molecular surface of the molecule. Next, apply a segmentation algorithm for detection of geometric patches (concave, convex and flat surface pieces). The patches are filtered, so that only patches with 'hot spot' residues are retained. surface Patch Matching - apply a hybrid of the Geometric Hashing and Pose-Clustering matching techniques to match the patches detected in the previous step. Concave patches are matched with convex and flat patches with any type of patches. Filtering and Scoring - the candidate complexes from the previous step are examined. discard all complexes with unacceptable penetrations of the atoms of the receptor to the atoms of the ligand. Finally, the remaining candidates are ranked according to a geometric shape complementarity score.^[20]

 

Fig:1 3D structure for Pseudomonas aeruginosa

 

3. RESULTS AND DISCUSSION:

3.1. Synthesis of Pyrazolines derivatives and characterization:

1-Acetyl-5-(Substituted phenyl)-3-(amino phenyl)-2-pyrazoline were synthesized by A mixture of Acetanilide (0.02mol), absolute ethanol (10ml), 2% NaOH, and benzaldehyde (0.02mol) was refluxed for for 8-12 h at 120℃. The reaction mixture was then concentrated, cooled and poured into ice. The solid thus obtained was filtered, washed with cold water and recrystallized. After competition the process the product was mixed with (0.03mol) methanol, hydrazine hydrate (0.05mol) and few drops of glacial acetic acid. The reaction mixture was concentrated and poured onto ice. The separated solid was filtered, washed with cold water and recrystallized using ethanol-DMF mixture. 1-Acetyl-5-(4¹ methoxy phenyl)-3-(amino phenyl)-2-pyrazoline (3a) and 1-Acetyl -5-(3¹-methoxy, 4¹-hydroxy phenyl)-3-(amino phenyl)-2-pyrazoline (8a) were obtained in excellent yields. The reaction pathway has been summarized in Scheme 1. Newly synthesized compounds (1a-8a) were characterized by IR, NMR, UV and Thin layer chromatography. Formation of all the newly synthesized compounds (1a-8a) were confirmed by recording their IR, NMR, UV and thin layer chromatography. All compounds were characterized after recrystallization from appropriate solvents. IR spectrum of compound 4a showed absorption at 3480 (N-H), 1692(C=O), 1555 (C=C of Aromatic ring), 1581 (C=N), 1459 (N-N).The NMR(400 MHz, Bruker): spectrum of 4a showed δ 2.15 (s, 3H, -COCH3), 2.3 (t, 1H, -CH-Ar), 4.7(d, 2H, pyrazoline CH2) 6.6-8.6 (m, 9H, Ar-H and N-H); 279 nm (ℇ 25, 760). The UV (ethanol) of compound 4a showed 279 nm (ℇ 25, 760). Similarly the spectral values for all the compounds are given in the experimental part and the characterization is provided in Table 1.

 

3.2 Antibacterial studies:

The in vitro antibacterial activity of newly synthesized compounds 2a, 4a and 8a were determined by well plate method. The antibacterial screening revealed that some of the tested compounds showed good inhibition against various tested microbial strains. The result indicated that, the compound 2a showed the significant activity in the following order: Pseudomonas aeruginosa > Klebsiella pneumoniae > Escherichia coli > Pseudomonas putida. The compound 42 showed the significant activity in the following order: Pseudomonas aeruginosa > Klebsiella pneumoniae > Pseudomonas putida > Escherichia coli. The compound 8a showed the significant activity in the following order: Pseudomonas aeruginosa > Escherichia coli > Pseudomonas putida > Klebsiella pneumoniae. 4a showed good activity against Pseudomonas aeruginosa compared to standard drug and all the other test compounds, while, the anti-bacterial activity of the other tested compounds against the tested organisms were found to be less than that of standard anti-bacterial drug ciprofloxacin at tested dose level. results of antibacterial studies have been presented in Table 2. As regards the relationships between the structure of the heterocyclic scaffold and the detected antibacterial properties, it showed varied biological activity. Compounds showed moderate antimicrobial activity. Pyrazole nucleus which is present in both the series is responsible for the biological activity. However the presence of other substituents is responsible for the varied biological activity of the compounds.

 

3.3 Molecular docking studies:

Considering the well obtained in vitro results, it was thought worthy to perform molecular docking studies, hence screening the compounds, inculcating both in silico and in vitro results. Considering Pseudomonas aeruginosa as the target, comparative and automated docking studies with newly synthesized candidate lead compounds was performed to determine the best in silico conformation. The software inculcated in the docking program PATCHDOCK, was employed to satisfy the purpose.^[20] Fig. 1 shows the native 3D structure of Pseudomonas aeruginosa was obtained from MBGP (Microbial Genome Database for comparative analysis) (http://mbgd.genome.ad.jp/) The 3D structure of Pseudomonas aeruginosa is given in (Fig.1).The docking of Pseudomonas aeruginosa with newly synthesized ligands exhibited well established bonds in the receptor active pocket. The active pocket was considered to be the site where the inhibition of the bacterial activity occur. All the eight synthesized molecules were docked. (Fig.2) shows the docked images of selected ligands. Table 3 shows the Binding Energy of eight compounds. In silico studies revealed all the synthesized molecules showed good binding energy toward the target.

 

Table 3: Docking results of Pseudomonas aeruginosa against substituted Pyrazoline derivatives.

 

 

Ligand 2a with Pseudomonas aeruginosa

 

 

Ligand 1a with Pseudomonas aeruginosa

 

Ligand 4a with Pseudomonas aeruginosa

 

 

Ligand 3a with Pseudomonas aeruginosa

 

 

Ligand 5a with Pseudomonas aeruginosa

 

 

Ligand 6a with Pseudomonas aeruginosa

 

Ligand 7a with Pseudomonas aeruginosa

 

 

Ligand 8a with Pseudomonas aeruginosa

 

(Figs.2) shows the docked images of selected ligands with Pseudomonas aeruginosa

 

4. CONCLUSION:

Eight new Pyrazolines derivatives were synthesized in reasonably good yields. They were characterized by ¹H NMR, UV, IR studies and thin layer chromatography. All the newly synthesized compounds were tested for antimicrobial activity by Well diffusion method. Among the screened samples, compound 4a has emerged as most active against all tested microorganisms compared to the standard drug. Finally the molecular docking studies of the synthesized compounds were carried out and the results of such studies were reported. In silico studies revealed that all the synthesized compounds 1a-8a have relatively good binding energy which may be considered as a good inhibitor of bacterial activity. Hence this study has widened the scope of developing these Pyrazoline derivatives as promising antibacterial agents.

 

5. ACKNOWLEDGEMENT:

I acknowledge the support of the department of Pharmacology, Karnataka College of Pharmacy, Bangalore-560064, India

 

6. CONFLICT OF INTEREST:

The author declared no conflict of interest.

 

7. REFERENCES:

1.      Lengauer T, Rarey M, Computational methods for biomolecular docking. Curr Opin Struct Biol. 1996, 6(3): 402-6.

2.      Kitchen B, Decornez H, Furr R, Bajorath J, Docking and scoring in virtual screening for drug discovery: methods and applications. Drug Discov. 2004: 3(11): 935-49.

3.      Thomas L, Gilcrist, ”synthesis of aromatic heterocycles” J chem soc, 2001:1:2491-515.

4.      Kumar A, Archanavats, Himanshu, Sanjoy, Sing K., Sharma K, synthetic and structural studies on novel N-phenylpyrazoly coumarins” Ind.J.chem, 2002:41B:360-7.

5.      Rajandranath, Shankar k, ”sunstituted pyrazolines and their cardiovascular activity” Ind J.chem 2002:41B:1310-3.

6.      Azarifar D, Ghasemnejad H. Microwave-Assisted Synthesis of Some 3, 5-Arylated 2-Pyrazolines. Molecules. 2003; 8(8):642-648.

7.      Archana A, Srivastava V, Chandra R, Kumar A. Synthesis of Potential Quinazolinonyl Pyrazolines and Quinazolinyl Isoxazolines as Anticonvulsant Agents. ChemInform. 2003; 34(9).

8.      Rana K, Kaur B, Kumar B. Synthesis and Antihypertensive Activity of Some Dihydropyrimidines. ChemInform. Indian J. Chem 2004; 35(45).

9.      Padmaja A, Payani T, Reddy G, Padmavathi V. Synthesis, antimicrobial and antioxidant activities of substituted pyrazoles, isoxazoles, pyrimidine and thioxopyrimidine derivatives. Eur J Med Chem. 2009; 44(11):4557-66.

10.   Farag A, Darwish E,  Atia K. Synthesis and Antimicrobial Evaluation of Some Isoxazole Based Heterocycles. heterocycles. 2014; 89(6):1393.

11.   C.S Andotra, Jatinder, Khejuria, G.B singh and surjeet singh., 1993, ”synthyesis and bkological activity of substituted pyrazolines and pyrimidines”.J.Indian.chem., soc., 70:266-267.

12.   Abunada N, Hassaneen H, Kandile N, Miqdad O. Synthesis and Antimicrobial Activity of Some New Pyrazole, Fused Pyrazolo[3, 4-d]-pyrimidine and Pyrazolo[4, 3-e][1, 2, 4]- triazolo[1, 5-c]pyrimidine Derivatives. Molecules. 2008; 13(7):1501-17.

13.   Saad H, Osman N, Moustafa A. Synthesis and Analgesic Activity of Some New Pyrazoles and Triazoles Bearing a 6, 8-Dibromo-2-methylquinazoline Moiety. Molecules. 2011; 16(12):10187-201.

14.   Bekhit A, Ashour H, Abdel Ghany Y, Bekhit A, Baraka A. Synthesis and biological evaluation of some thiazolyl and thiadiazolyl derivatives of 1H-pyrazole as anti-inflammatory antimicrobial agents. Eur J Med Chem. 2008; 43(3):456-63.

15.   Shinkai H, Onogi S, Tanaka M, Shibata T, Iwao M, Wakitani K et al. Isoxazolidine-3, 5-dione and Noncyclic 1, 3-Dicarbonyl Compounds as Hypoglycemic Agents. J Med Chem. 1998; 41(11):1927-33.

16.   Karakurt A, Özalp M, Işık Ş, Stables J, Dalkara S. Synthesis, anticonvulsant and antimicrobial activities of some new 2-acetylnaphthalene derivatives. Bioorg Med Chem. 2010; 18(8):2902-11.

17.   Galkina I, Tudriy E, Bakhtiyarova Y, Usupova L, Shulaeva M, Pozdeev O et al. Synthesis and Antimicrobial Activity of Bis-4, 6-sulfonamidated 5, 7-Dinitrobenzofuroxans. 2019.

18.   Nossier E, Fahmy H, Khalifa N, El-Eraky W, Baset M. Design and Synthesis of Novel Pyrazole-Substituted Different Nitrogenous Heterocyclic Ring Systems as Potential Anti-Inflammatory Agents. Molecules. 2017; 22(4):512.

19.   Lasker K, Phillips J, Russel D, Velázquez-Muriel J, Schneidman-Duhovny D, Tjioe E et al. Integrative Structure Modeling of Macromolecular Assemblies from Proteomics Data. Mol Cell Proteomics. 2010; 9(8):1689-702.

20.   Schneidman D, Inbar Y, Nussinov R, Wolfson H. Patch Dock and Symm Dock: servers for rigid and symmetric docking. Nucleic Acids Res. 2005; 33:363-7.

 

 

 

Received on 19.09.2019           Modified on 06.11.2019

Accepted on 21.12.2019         © RJPT All right reserved