INTRODUCTION:

The author proposed 1,3-diphenyl-1H-pyrazol-4-yl-methylbenzenamine synthesis methodologies were reported in few journals^1-4, but none of the synthetic procedure having adequate commercial or scalable process information with respect to the cost of production, process safety and environmental requirements.

Heterocyclic compounds have attracted considerable attention in the design of biologically active molecules and advanced organic materials^5.Hence a practical method for the preparation of such compounds is of great interest in synthetic organic chemistry.

Pyrazoles and their derivatives, a class of well-known nitrogen containing heterocyclic compounds, occupy an important position in medicinal and pesticide chemistry having a wide range of bioactivities such as anti-microbial,⁶ anti-cancer,⁷ anti-inflammatories,⁸ antidepressants,⁹ anti-convulsant¹⁰, ¹¹ anti-hyperglycemic,¹² anti-pyretic,¹³ anti-bacterial,¹⁴ antifungal activities,¹⁵ CNS regulants,¹⁶ and selective enzyme inhibitory activities¹⁷.

It has been found that pyrazoles are also known as inhibitors and deactivators of liver alcohol dehydrogenase and oxidoreductases. It has been shown in vivo that some of the pyrazole derivatives have appreciable anti-hypertensive activity. These compounds also exhibit properties such as cannabinoid hCB1 and hCB2 receptor, inhibitors of p38 Kinase, and CB1 receptor antagonists ^{18, 19, 20}. As early as in 1884, Knorr discovered the anti-pyretic action of a pyrazole derivative in man, he named the compound antipyrine. This stimulated the interest in pyrazole chemistry^{21,
22, 23}.

Scheme 1: Proposed route of synthesis

Scheme 2: retro synthetic approach of proposed route

RESULTS AND DISCUSSION:

MATERIALS AND METHODS:

Synthesized materials melting points were determined using a manual POLMON electro thermal apparatus (Range 0–300°C) in glass capillary tubes and are uncorrected. IR spectra were recorded on a Perkin- Elmer FT IR 1600. 1H & 13CNMR spectra were recorded on a Bruker-400 MHz spectrometer and are expressed in a using TMS as internal reference. All the reactions were monitored by thin layer chromatography over pre-coated silica gel plates, using UV lamp, iodine vapors or KMnO4 spray as developing agents. The purification was carried out by using 60-120 mesh silica gel by column chromatography (obtained from Merck) with a suitable eluting system with various polarity mobile phase ratio’s such as n-Hexane, Ethyl acetate, Chloroform, Chloroform: Methanol.

The biological activity of chemically synthesized molecules was studied by Inhibition zone diameter calculation method against Candida Albicam, Aspergillus Niger cell cultures with reference drug standard Fluconazole.

Inoculum Preparation:

Potato Dextrose Broth PDB (commercially available).

Procedure for Antifungal activity:

Weigh 80mg of Potato Dextrose Broth powder. Add to a 1 Liter Erlenmeyer flask containing 800mL of water, stir to dissolve into solution. Transfer the solution to a 1 Liter volumetric flask and dilute to 1 Liter. Transfer the 1 Liter media solution to a 1 Liter media storage bottle and label with appropriate information, Autoclave the solution at 121°C for 15 mines to ensure that the LB is sterilized of all foreign matter and contaminants. After cooling of the broth, using a sterile pipette tip, select a single spore from a pure culture plate. Drop the tip into the liquid PDB and swirl. Incubate fungal culture at 25°C for 5-7 days in shaking incubator at 80rpm.

Preparation of Compound Stock Solution:

1mg/ml stock solution of each extract was prepared in a suitable solvent.

Preparation of Potato Dextrose Agar PDA: (Agar Well Diffusion):

Weigh PD Agar as per requirement and dissolve in distilled water as per the guidelines given by manufacturer. Autoclave the media at 121°C for 15 minutes, after autoclaving, pour the media in sterile Petri plates and keep for solidification.

Procedure:

Sterilize a cork borer by autoclaving or disinfect it by rinsing in alcohol followed by sterile water. On a PD agar plate aseptically punch 5 holes using a cork borer, using a marker, mark the underside of the Petri to label the wells. Aseptically spread 20µl of the indicator organism onto the MH agar plate. Let the plate stand for 5 minutes, Place 10µl, 25µl, 50µl, 100µl and 150µl of the extract in the appropriate wells. Incubate the plate at 25°C for 72-96 hrs. Measure the zones of inhibition in millimeters, using a ruler on the underside of the plate. Same Procedure is repeated with Fluconazole as a standard with concentrations of 10µg, 25µg, 50µg, 100 µg, 150µg

EXPERIMENTAL:

Experimental procedure:

Step-I: General Procedure for Substituted (E)-1-phenyl-2-(1-phenylethylidene) hydrazine derivatives (5):

In a 250ml round bottom flask taken a mixture of Acetohenone (1.0mmol), substituted phenyl hydrazine (1.2mmol), and 1% Sulfuric acid used as catalyst in absolute ethanol (5V) at room temperature. Stir the reaction mass for 4-5 hours, after completion of TLC, the solvent removed under reduced pressure and then add two volumes of water extract the aqueous layer with methylene dichloride (3X2 volumes) and then separate the layers, organic layer washed with brine solution, finally dried over anhydrous sodium sulphate. The obtained solid after distillation of dried organic layer was taken for purification in hexanes (3-4 volumes) to give desired substituted (E)-1-phenyl-2-(1-phenylethylidene) hydrazine derivatives (5p-5x) yield range: 70-90%

Step-II: General procedure for alpha bromination of 1,3-diphenyl-1H-pyrazole-4-carbaldehyde derivatives (4):

To a cooled solution of substituted (E)-1-phenyl-2-(1-phenylethylidene) hydrazine derivative (1.0mmol) individual compounds were taken in Phosphorous oxy chloride (POCl₃ 3.0 equivalents), added anhydrous Dimethyl Formamide (1.02mmol) portion wise to above solution at 0 to 5⁰C (it took 20 min) and then slowly rose the temperature up to 60 to 70⁰C, and Main titan the reaction mass under gentle stirring for 3-5 hours.

Remove the solvent under reduced pressure after TLC complies, the obtained residue taken in aqueous Sodium bicarbonate solution and extract with Methylene dichloride (3 X 5V). The combined organic layers was washed with brine and dried over anhydrous sodium sulphate, the solid obtained end products of substitutes 1, 3-diphenyl-1H-pyrazole-4-carbaldehyde yield range 65-85%

Table-1: Derived analogs details and their individual characterization data

S. No.

Chemical Name

Chemical structure

Mol. Wt.

Code

Spectral characterization Data

4-Chloro-N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)benzenamine

359.85

1H NMR (DMSO-D6):

δ 8.56(S, 1H), δ 7.85(d, 2H), δ 7.50(t, 3H), δ 7.45(m, 2H), δ 7.37(t, 1H), δ 7.30(t, 1H), δ 7.09(d, 2H), δ 6.68 (d, 2H), δ 6.18 (t,1H),δ 4.22(d, 2H),

¹³C NMR : 153.3, 152.8, 150.7,147.4, 139.4, 138.8, 132.8,131.8, 129.9, 127.9, 126.1, 122.6, 119.6, 118.5,113.6, 38.1

MASS: 359.85 (m+), 358.29(m-H)+

4-Bromo-N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)benzenamine

404.30

1H NMR (DMSO-D6):

δ 8.56(S, 1H), δ 7.85(d, 2H), δ 7.77(d, 2H), δ 7.50(t, 2H), δ 7.45(t, 2H), δ 7.37(t, 1H), δ 7.30(t, 1H), δ 7.22(d, 2H), δ 6.64 (d, 2H), δ 6.20 (t,1H),δ 4.22(d, 2H),

¹³C NMR :150.3, 147.7, 139.4, 132.7, 131.3, 129.5, 128.5, 127.7, 126.1, 122.4, 118.5, 114.1,106.7, 38.0

MASS: 404.30 (m+), 404.22(m-H)+

2-((1,3-diphenyl-1H-pyrazol-4-yl) methyl amino)-5-methylphenol

355.43

1H NMR (DMSO-D6):

δ 7.98 (S, 1H), δ 7.82 (d, 2H), δ 7.80 (d, 2H), δ 7.44 (t, 4H), δ 7.36 (t, 1H), δ 7.27 (d, 1H), δ 6.62 (t, 1H), δ 6.52 (s, 1H), δ 6.46 (t, 1H), δ δ 4.37 (s, 2H), 2.24 (S,3H),

MASS: 355.43 (m+), 356.03 (m-H)+

2-(3-nitrophenyl)-N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)ethanamine

398.60

1H NMR (DMSO-D6):

δ 8.45 (S, 1H), δ 7.82 (d, 2H), δ 7.94 (d, 1H), δ 7.49 (t, 3H), δ 7.25 (m, 5H), δ 3.77 (s, 2H), δ 2.92 ( t, 2H), 2.83 (t, 2H),

MASS: 398.46 (m+), 397.2(m-H)+

2-(4-nitrophenyl)-N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)ethanamine

398.60

1H NMR (DMSO-D6):

δ 8.61 (S, 1H), δ 7.90(d, 2H), δ 7.80(d, 2H), δ 7.53(m, 6H), δ 7.39(t, 2H), δ 7.31(t, 2H), ,δ 4.22(d, 2H), δ 3.3 (t,4H)

MASS: 398.46 (m+), 397.3(m-H)+

2-(2-nitrophenyl)-N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)ethanamine

398.60

1H NMR (DMSO-D6):

δ 8.66(S, 1H), δ 7.87(d, 2H), δ 7.81(d, 2H), δ 7.51(t, 2H), δ 7.35(m, 6H), δ 7.30(t, 1H), δ 4.63 (s, 2H),

MASS: 398.46 (m+), 397.1 (m-H)+

N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)pyridin-2-amine

419.32

1H NMR (DMSO-D6):

δ 9.22(S, 1H), δ 8.66(s, 1H), δ 7.88(dd, 2H), δ 7.81(d, 2H), δ 7.66 (d, 1H), δ 7.51(m, 5H), δ 7.37 (m, 4H), δ 4.63(d, 2H), δ 2.27 (s, 1H), δ 6.20 (t,1H),δ 4.22(d, 2H),

MASS: 326.39 (m+), 327.2 (m-H)+

2,6-dimethyl-N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)benzenamine

353.46

1H NMR (DMSO-D6):

δ 8.65 (S, 1H), δ 7.44 (d, 1H), δ 7.84 (m, 1H), δ 7.66 (d, 1H), δ 7.53(m, 3H), δ 7.37(m, 1H), δ 6.78(d, 4H), δ 6.40 (t, 2H), δ 4.45 (s, 2H), ,δ 2.05(S, 6H),

MASS: 353.19 (m+), 352.3 (m-H)+

Table-2: In-vitro biological activity test results of synthesized new chemical entities against selected antifungal cultures

S. No	Compound Code	*Candida Albicam*					*Aspergillus Niger*
		Zone Inhibition diameter in μg/mm					Zone Inhibition diameter in μg/mm
		10	25	50	100	150	10	25	50	100	150
1.	Sp	-	2	-	-	-	-	-	-	-	2
2.	Sq	-	-	-	-	-	-	-	1	-	-
3.	Sr	-	-	1	-	-	-	-	-	-	-
4.	Ss	3	-	-	-	-	-	5	-	-	-
5.	St	-	-	-	-	-
6.	Su	-	-	-	2	-	-	-	-	-	-
7.	Sv	-	-	6	-	-	-	-	-
8.	Sw	-	-	-	-	-	-	-	-	3	-
9.	Sx	4	8	10*	12*	12*	-	-	-	-	-
10.	Fluconazole(Std)*	-	-	12	18	18	-	-	6	10	15

CONCLUSION:

The synthetic methodology which is described in this research and biology (in-vitro) of derived substituted biphenyl aminothaizoles analogues such as Sp, Sq, Sr, Ss, St, Su, Sv, Sw and Sx were successful. The Anti-fungal activity of these compounds has been studied against cell cultures like Candida Albicam, Aspergillus Niger. Among all synthesized new molecular entities S_X is said to be promising molecule which exhibits more potentiality towards anti-fungal activity and equivalent to the standard drug substance Fluconazole. Hence, it is believed that these molecules having more scope to study further stage of biology studies to understand the routing to a new drug moiety for future medical applications.

ACKNOLEDGEMENT:

We are very thankful to Department of chemistry, Banasthali University and to Prof. Dr. Dharma Kishore, Dr. Bhavani Singh, and Dr. Jaya Dwivedi for giving this opportunity to do this research and for their continuous support in this regards. Additionally, we are grateful to have valuable support for biology study Synteny life sciences Pvt. Ltd. and SMS Pharmaceuticals Ltd for giving this opportunity to explore all this research work.

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Received on 24.08.2020 Modified on 29.11.2020

Research J. Pharm.and Tech 2021; 14(12):6442-6446.

DOI: 10.52711/0974-360X.2021.01114