Rapid Synthesis of Indazole derivatives using Microwave Technology, its Characterisation and Anti-Inflammatory effects Observed in Laboratory Tests

 

Rupesh Pingale¹, Kirtan Shah², Pritam Khandave¹*, Vrushali Neve³

¹Principal, Department of Pharmacognosy, NCRD’s Sterling Institute of Pharmacy, Nerul (E), Navi Mumbai, Maharashtra, India.

¹Associate Professor, Department of Pharmaceutical Chemistry, NCRD’s Sterling Institute of Pharmacy,

Nerul (E), Navi Mumbai, Maharashtra, India.

²Research Scholar, NCRD’s Sterling Institute of Pharmacy, Nerul (E), Navi Mumbai, Maharashtra, India.

³Assistant Professor, D.Y. Patil Vidyapeeth, Pimpri-Chinchwad, Pune, Maharashtra, India.

*Corresponding Author E-mail: pritamk@ncrdsip.com

 

 

INTRODUCTION:

1.1 Indazole and its derivatives:

Indazole, first described by Emil Fischer as a "fusion of pyrazole ring with benzene ring," has captivated researchers because of its intriguing various chemical as well as biological characteristics. This heterocyclic compound, part of the azole family, consists of carbon, hydrogen, and nitrogen atoms. Known as benzpyrazole or isoindazolone, indazole features two nitrogen atoms in its structure. With a ten π-electron aromatic system similar to pyrazole, it also shares structural resemblances to pyridine and pyrrole, making it a versatile and compelling molecule for scientific exploration.^1,3

 

Indazole derivatives are key to several therapeutic drugs, including Granisetron, an anti-emetic, and Benzydamine as an anti-inflammatory drug. Indazole ring, with dual nitrogen atoms, could be selectively modified at various positions, enabling the creation of numerous derivatives with potential biological and therapeutic benefits.^1,2,3,4

The indazole scaffold plays a crucial role in modern medicinal chemistry due to its adaptability and broad therapeutic potential. Various indazole derivatives have been found to demonstrate notable activities biologically, including to cure inflammation, bacterial and fungal infections and anticancer properties. The remarkable versatility has contributed to the development of several indazole-based drugs. Because of these properties, indazole continues to be an important structure in pharmaceutical research, driving the discovery of new and effective therapeutic agents.⁵

 

Indazole is not commonly found in nature, but it has been discovered in some alkaloids derived from plants like Nigella sativa (black cumin). These naturally occurring compounds exhibit distinct structural features and may offer valuable therapeutic benefits.⁶

 

1.2 Microwave technology:

Microwave heating has emerged as a powerful tool for accelerating chemical reactions. Under solvent-free conditions, it provides several benefits, including lower environmental impact, cost savings, and ease of use. The development of single-mode technology has further improved safety and consistency, making microwave synthesis increasingly popular among chemists. As a result, research in this area has grown significantly, with numerous studies exploring its application in various reaction types, such as additions, cycloadditions, substitutions, eliminations, and fragmentations.^7,8,11

 

Over the past 30 years, microwave irradiation has transformed organic synthesis by drastically cutting reaction times and improving yields. This technique, which gained traction through the work of Gedye and Giguere, uses dielectric heating to rapidly energize polar molecules, speeding up reactions compared to traditional methods. Its benefits include precise thermal control, reduced waste, fewer side reactions, and higher efficiency, making it a sustainable and eco-friendly choice in modern chemistry.^9,11

 

1.3 Mechanism of microwaves:

Microwave heating works through dielectric heating, where electromagnetic waves interact with dipole molecules like water, causing them to rotate rapidly and generate heat through molecular friction. This process also accelerates ions, further increasing the temperature. Unlike traditional heating, which relies on conduction and can create uneven heat distribution, microwave heating is more uniform and efficient. As the electromagnetic field continuously realigns dipole molecules, they release energy when returning to their natural state, resulting in localized "superheating" that enhances the overall heating process.^10,11

 

MATERIALS AND METHOD:

Raw material characterization:

Table 1: Raw material characterization

 

Experimental scheme:

 

Figure 1 General scheme

 

Table 2: Reagent and Product

Sr. No.	Reagent	Product
1	o-chloro benzaldehyde	1-H indazole
2	o-nitro benzaldehyde	1-H indazole
3	2,6-Dichloro benzaldehyde	4-chloro-1-H indazole

 

Procedure for synthesis:

1.       Synthesis of 1-H indazole (derivative 2a): A reaction mixture consisting of o-chlorobenzaldehyde (1 mmol), hydrazine hydrate (2mmol), and 10% LPP in 10 mL of distilled water was subjected to ultrasonic irradiation at 425MW for 18 minutes. The reaction's progress was tracked by performing thin layer chromatography (TLC) using a mobile phase n-hexane and ethyl acetate in the ratio (1:1). After the reaction was completed, the mixture was diluted with hot ethanol and filtered. The catalyst was rinsed properly using ethanol (5ml) for three times and all the filtrates were concentrated to obtain the final product. The final product was purified by recrystallization from ethanol.¹²

 

Figure 2 Scheme for synthesis of 2a

 

2.       Synthesis of 1-H indazole (derivative 2b): Reaction mixture of o-nitro benzaldehyde (1 mmol), hydrazine hydrate (2 mmol), and 10% LPP in 10 mL of distilled water was subjected to ultrasonic irradiation at 425 MW for 18 minutes. In process thin layer chromatography (TLC) was performed with a mobile phase of n-hexane and ethyl acetate in ratio (1:1). After that, the reaction mix was diluted with hot ethanol and filtered. The catalyst was washed nicely with ethanol (5ml) thrice, and the filtrates were concentrated to get the final product. Finally, the product was purified by recrystallization from ethanol.¹²

 

Figure 3 Scheme for synthesis of 2b

 

3.       Synthesis of 4-chloro-1-H indazole (derivative 2c): A mixture consisting of 1 mmol of 2,6-dichlorobenzaldehyde, 2 mmol of hydrazine hydrate, and 10% LPP in 10 mL of distilled water was exposed to ultrasonic irradiation at 425 MW for 18 minutes. In process thin-layer chromatography (TLC) was performed using an n-hexane and ethyl acetate mobile phase in the ratio (1:1). Afterwards, the mixture was diluted with hot ethanol and filtered to remove any insoluble materials. The catalyst was washed thrice with ethanol (5 mL), and all the filtrates were concentrated to yield the end product. The purified compound was obtained through recrystallization using ethanol.¹²

 

Figure 4 Scheme for synthesis of 2c

 

RESULT AND DISCUSSION:

Identification and characterization:

To confirm that the synthesized compounds were chemically distinct from their respective parent compounds, various identification and characterization techniques were employed. These methods ensured the structural integrity and uniqueness of each prepared compound.

 

1.       Melting point determination:

Open capillary tube method was used to determine the melting point temperatures of each compound. Melting point is a key indicator of purity, as pure crystalline substances typically have a sharp and well-defined melting point. However, purity should not be solely judged by this measure and should be confirmed by observing any variations in the melting point. Following recrystallization, the synthesized compounds showed only minor changes in their melting points, providing additional evidence of their purity.

 

2.       Thin Layer Chromatography (TLC):

Thin Layer Chromatography (TLC) was performed using pre-coated silica plates as the solid phase. The solvent mobile phase consisted of a 1:1 solution of n-hexane solution and ethyl acetate liquid (v/v). A small amount of sample was carefully applied to the plate using a capillary tube, ensuring accurate placement from the baseline. The plate was then placed upright in a developing chamber containing the mobile phase, allowing the solvent to rise through capillary action. Once the solvent front reached about three-quarters of the plate’s length, the plate was removed and allowed to air dry. Spots were visualized under UV chamber, and Rf values were calculated by comparing the distance each spot travelled with the distance moved by the solvent front.¹³

 

 

 

Table 3: Melting point, % yield and Rf value

Sr. No	Aldehyde	Product	Reaction time	% yield	Melting point	Rf value
1			15min	77%	145°C	0.76
2			15min	81.5%	145°C	0.69
3			15min	85.79%	153°C	0.589

 

3.       Infrared Spectroscopy (IR Spectroscopy):

Infrared (IR) spectroscopy method is quick and with no damage used to analyse the chemical characteristics of various substances. The IR spectrum has three regions: near-infrared (NIR), mid-infrared (MIR), and far-infrared. NIR spectroscopic method, in particular, is commonly used in food analysis because it is cost-effective and can penetrate samples more deeply. This technique detects overtones and combination bands of IR-active bonds, offering a rapid analysis with minimal sample preparation. Some of its main advantages include high sensitivity, low sample volume requirements, and the ability to analyse different states of matter—solids, liquids, and gases—making it a valuable tool in analytical chemistry.¹⁴

 

2a (1-H Indazole):

Figure 5: IR of 2a

 

Table 4: IR stretching of 2a

Sr. No.	Frequency		Functional group
	Range (cm-1 )	Reported (cm-1 )
1	1400-1600	1432-1587.46	C-C
2	1600-1700	1614.56-1690.15	C=C
3	3000-3100	3005.19-3069.38	C-H
4	1600-1650	1614.56	C=N
5	1300-1350	1316.47	N-N
6	3300-3500	3069.38	N-H

 

2b (1-H Indazole):

Figure 6: IR of 2b

 

Table 5: IR stretching of 2b

Sr. No.	Frequency		Functional group
	Range (cm-1 )	Reported (cm-1 )
1	1400-1600	1517.57	C-C
2	1600-1700	1607.43	C=C
3	3000-3100	3069.38-3102.18	C-H
4	1600-1650	1607.43	C=N
5	1300-1350	1313.61-1340.71	N-N
6	3300-3500	3367.47-3461.61	N-H

 

2c (4-chloro-1-H Indazole):

Figure 7: IR of 2c

 

Table 6: IR stretching of 2c

Sr. No.	Frequency		Functional group
	Range (cm-1 )	Reported (cm-1 )
1	1400-1600	1430.57-1558.93	C-C
2	1600-1700	1617.41	C=C
3	3000-3100	3052.26-3110.74	C-H
4	1600-1650	1617.41	C=N
5	1300-1350	1313.61	N-N
6	3300-3500	3373.18	N-H
7	600-800	892.86	C-Cl

 

4.       1H NMR Spectroscopic technique:

Proton Nuclear Magnetic Resonance (¹H NMR) spectroscopic technique is a highly effective method for analysing hydrogen atoms in a molecule, offering key insights into their chemical environment and bonding. It is indispensable for determining the structure of organic compounds. Renowned for its sensitivity and low sample volume requirements, ¹H NMR provides detailed structural information without altering the sample, making it a vital tool in chemical analysis and research.^{15, 16}

 

2a (1-H Indazole): 1H NMR (500 MHz, cdcl3) δ 9.08 (s, 1H), 8.21 (dd, J = 7.7, 1.2 Hz, 1H), 7.47 – 7.30 (m, 3H).

Figure 8: NMR of 2a

2b (1-H Indazole): 1H NMR (500 MHz, cdcl3) δ 8.25 (s, 2H), 8.04 (d, J = 7.7 Hz, 2H), 7.94 (t, J = 9.6 Hz, 2H), 7.60 – 7.53 (m, 2H), 7.44 – 7.37 (m, 2H), 5.95 (d, J = 38.8 Hz, 5H).

 

Figure 9: NMR of 2b

 

2c (4-chloro-1-H Indazole): 1H NMR (500 MHz, cdcl3) δ 7.92 (s, 2H), 7.32 (d, J = 8.1 Hz, 4H), 7.14 (t, J = 8.1 Hz, 2H), 5.99 – 5.71 (m, 5H).

 

Figure 10: NMR of 2c

 

5.       13C NMR Spectroscopic technique:

6.       13C NMR (carbon-13 nuclear magnetic resonance) is a method that employs radio waves to study organic compounds. It serves as a valuable tool for detecting carbon atoms within molecules and analysing their structural layout and how they are connected.

 

2a: ¹³C NMR (100 MHz, CDCl₃) δ 139.90 (s), 133.40 (s), 125.80 (s), 122.80 (s), 120.40 (s), 120.10 (s), 110.00 (s).

Figure 11: 13C NMR of 2a

 

2b: ¹³C NMR (126 MHz, cdcl₃) δ 136.44 (s), 130.88 (s), 123.33 (s), 120.34 (s), 117.93 (s), 117.71 (s), 107.71 (s).

Figure 12: 13C NMR of 2b

 

2c: ¹³C NMR (100 MHz, CDCl₃) δ 139.50 (s), 131.70 (s), 126.10 (s), 122.60 (s), 119.60 (s), 117.90 (s), 115.60 (s).

 

Figure 13: 13C NMR of 2c

 

7.       It’s a method which integrates TLC with mass spectrometry (MS) to detect and identify compounds. This technique is most widely used in many industries, such as food and beverage, pharmaceuticals, & cosmetics.

 

Figure 14: TLC MS of 2a

 

Figure 15: TLC MS of 2b

 

Figure 16: TLC MS of 2c

 

Anti-inflammatory activity:

Egg protein denaturation method:

Principle:

Protein denaturation is often associated with inflammatory processes, and a substance's ability to prevent this denaturation can suggest its anti-inflammatory potential. One approach to evaluate this is the denaturation test, which involves using fresh egg whites. These contain various proteins such as ovalbumin, ovoinhibitor and prostaglandin D2 synthase. In which, extent to which albumin denaturation is inhibited is compared to the effects of Diclofenac sodium, a standard anti-inflammatory drug. Plant extracts can also be assessed similarly to determine their anti-inflammatory properties, particularly if they behave like nonsteroidal anti-inflammatory drugs (NSAIDs). The test measures the reduction in prostaglandin activity by observing how proteins respond to heat, with the amount of inhibition of albumin denaturation reflecting the extent of anti-inflammatory property. A higher level of inhibition indicates a stronger anti-inflammatory effect.^18,19

 

METHODOLOGY:

Dilutions were prepared with concentrations of 50, 100, 150, 200, 250µg/ml. Inuprofen was used as a control drug and its concentration series was prepared as above.

 

For the test samples, 2.8mL of PBS, 0.2mL of egg albumin, and 0.2mL of drug dilutions were combined. For the control (Ibuprofen), the same procedure was followed, but 0.2mL of Ibuprofen solution was used instead of the test sample. A control sample was also prepared by mixing 2.8mL of PBS, 2mL of distilled water, and 0.2mL of egg albumin.

 

All the prepared samples were subjected to incubation in a water bath at 37°C for 15-20 minutes and then heated at 70°C for 5 minutes. After cooling at room temperature for 10 minutes, absorbance readings were taken at 660 nm using a double-beam UV-visible spectrophotometer. The entire process was repeated three times. The percentage inhibition of denaturation of egg albumin was calculated by the following formula:

 

% Inhibition of denaturation = [(Absorbance _control– Absorbance _test) / Absorbance _control] × 100.

 

Table 7: Absorbances

Concentration	Absorbances
	1-H Indazole (2a)	1-H Indazole (2b)	4-chloro-1-H-indazole	Ibuprofen (standard)
50mcg	0.61	0.586	0.367	0.325
100mcg	0.559	0.54	0.304	0.317
150mcg	0.508	0.489	0.271	0.238
200mcg	0.475	0.334	0.124	0.124
250mcg	0.321	0.31	0.098	0.057

 

Table 8: % inhibition

Concentration	50 mcg	100 mcg	150 mcg	200 mcg	250 mcg
% Inhibition of Ibuprofen (Standard)	53.57	54.71	66	82.28	91.86
% Inhibition of 1-H Indazole (2a)	12.857	20.142	27.43	32.142	54.142
% Inhibition of 1-H Indazole (2b)	16.29	22.86	30.143	52.29	55.71
% Inhibition of 4-chloro-1-H Indazole (2c)	47.57	56.57	61.29	82.29	86

 

CONCLUSION:

The microwave-assisted synthesis of indazole derivatives provided a notable merit in both terms of speed as well as efficiency in contrast with conventional methods. Structural verification through various analytical techniques confirmed the successful synthesis of these compounds. Additionally, the observed anti-inflammatory activity, assessed through the egg albumin denaturation assay, indicates their potential for future development. This work highlights a promising method for synthesizing indazole derivatives, with potential applications in anti-inflammatory drug development.

 

REFERENCES:

1.      Digambar D. Gaikwad, Archana D. Chapolikar, Chandrashekhar G. Devkate, Khandu D. Warad, Amit P. Tayade, Rajendra P. Pawar, Abraham J. Domb. Synthesis of indazole motifs and their medicinal importance: An overview. European Journal of Medicinal Chemistry. 2015; 90: 707-731 https://doi.org/10.1016/j.ejmech.2014.11.029.

2.      Dilipkumar Pal, In-ho Song, Shrikant Dashrath Warkad, Keum-soo Song, Gyu Seong Yeom, Supriyo Saha, Pramod B. Shinde, Satish Balasaheb Nimse. Indazole-based microtubule-targeting agents as potential candidates for anticancer drugs discovery. Bioorganic Chemistry. 2022; 122: 105735 https://doi.org/10.1016/j.bioorg.2022.105735.

3.      Uppulapu SK, Alam MJ, Kumar S, Banerjee SK. Indazole and its Derivatives in Cardiovascular Diseases: Overview, Current Scenario, and Future Perspectives. Curr Top Med Chem. 2022; 22(14): 1177-1188. doi: 10.2174/1568026621666211214151534. 

4.      Sachin Puri, Siddhi Sawant, Kapil Juvale. A comprehensive review on the indazole based derivatives as targeted anticancer agents. Journal of Molecular Structure.  2023; 1284: 135327   https://doi.org/10.1016/j.molstruc.2023.135327.

5.      Katritzky, A. R., and Pozharskii, A. F.  Heterocycles in Life and Society: An Introduction to Heterocyclic Chemistry. Biochemistry and Applications. Wiley. 2000

6.      Khan, M. A., and Ahmad, W.  Phytochemical Constituents of Nigella sativa and Their Medicinal Properties. Phytochemical Dictionary: A Handbook of Bioactive Compounds from Plants (2nd ed.). Taylor and Francis. 2001

7.      Katritzky, Alan and Singh, Sandeep. Microwave-assisted heterocyclic synthesis. Arkivoc. 2003. 10.3998/ark.5550190. 0004.d09.

8.      Kumar, Sanjeev and Maurya, Anand. Microwave-Assisted Synthesis of Heterocyclic Scaffolds. Syn Open. 2024; 8: 138-152. 10.1055/s-0043-1775379.

9.      Meera G, Rohit KR, Saranya S, Anilkumar G. Microwave assisted synthesis of five membered nitrogen heterocycles. RSC Adv. 2020 Sep 30; 10(59): 36031-36041. doi: 10.1039/d0ra05150k. 

10.   Frecentese F, Sodano F, Corvino A, Schiano ME, Magli E, Albrizio S, Sparaco R, Andreozzi G, Nieddu M, Rimoli MG. The Application of Microwaves, Ultrasounds, and Their Combination in the Synthesis of Nitrogen-Containing Bicyclic Heterocycles. International Journal of Molecular Sciences. 2023; 24(13): 10722. https://doi.org/10.3390/ijms241310722

11.   Grewal, A.S., Kumar, K., Redhu, S., Bhardwaj, S., Nayak, J., and Memorial, L. Microwave assisted synthesis: A green Chemistry approach. 2013

12.   Ghodke, Swati and Khandare, Priya and Salve, Sadhana and Kendrekar, Pravin and Rajani, Dhanji and Pawar, Rajendra. Synthesis Of 1h-Indazoles Using Lemon Peel Powder as A Natural, Green and Efficient Catalyst Under Ultrasound Irradiation. European Chemical Bulletin. 2019; 8: 405. 10.17628/ecb.2019.8.405-408.

13.   Bele, A. A., and Khale, A. An overview on thin layer chromatography. International Journal of Pharmaceutical Sciences and Research. 2011; 2(2): 256.

14.   Johnson JB, Walsh KB, Naiker M, Ameer K. The Use of Infrared Spectroscopy for the Quantification of Bioactive Compounds in Food: A Review. Molecules. 2023 Apr 4; 28(7): 3215. doi: 10.3390/molecules28073215.  

15.   Hameed, I. H., Al-Rubaye, A. F., and Kadhim, M. J. Uses of nuclear magnetic resonance spectroscopy technique in pharmaceutical analysis: A review. International Journal of Current Pharmaceutical Review and Research. 2017; 8(2): 79-84.

16.   Smith, I. C., and Blandford, D. E. Nuclear magnetic resonance spectroscopy. Analytical Chemistry. 1995; 67(12): 509-518.

17.   Samaraweera, Thummini and Samaraweera, Thumuli and Senadeera, Nimesha and Ranaweera, Chathuranaga. In vitro Anti-Inflammatory Activity of Leaves of Jeffreycia zeylanica Using the Egg Albumin Denaturation Method and Human Red Blood Cell Stabilization Method. Asian Plant Research Journal. 2023; 11: 56-64. 10.9734/aprj/2023/v11i6230.

18.   Osman NI, et al. In vitro xanthine oxidase and albumin denaturation inhibition assay of Barringtonia racemosa L. and total phenolic content analysis for potential anti-inflammatory use in gouty arthritis, Journal of Intercultural Ethnopharmacology. 2016; 5(4): 343–349. Available: https://doi.org/10.5455/jice.20160 731025522.

19.   Kashfia Nawrin et al. Protective potential of C. sinensis fruit peel aqueous extract on in vitro inflammation; 2021. Available: https://doi.org/10.13140/RG.2.2.2 5825.02408/1.

20.   Dharmadeva S, et al. In vitro anti-inflammatory activity of Ficus racemosa L. bark using albumin denaturation method. AYU (An International Quarterly Journal of Research in Ayurveda). 2018; 39(4): 239. Available: https://doi.org/10.4103/ayu.AYU_ 27_18.

21.   Berrouet C, et al. Comparison of Drug Inhibitory Effects (IC50) in Monolayer and Spheroid Cultures. Bulletin Mathematical Biology. 2020; 82(6): 68. Available: https://doi.org/10.1007/s11538 020-00746-7.

22.   Tharindu Madhuranga, Hewa Dikkumburage and Samarakoon, Nirmani. In vitro Anti-Inflammatory Egg Albumin Denaturation Assay: An Enhanced Approach. Journal of Natural and Ayurvedic Medicine. 2023; 7: 000411.

 

 

 

 

Received on 09.03.2025      Revised on 15.05.2025 Accepted on 24.06.2025      Published on 12.08.2025 Available online from August 18, 2025 Asian J. Research Chem.2025; 18(4):205-212. DOI: 10.52711/0974-4150.2025.00032 ©A and V Publications All Right Reserved
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