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1 School of Chemical science, Swami Ramanand Treeth Marathwada University, Nanded- 431606, India

2 Gramin (Art's, commerce and science) Mahavidyalaya, Vasant Nagar (Kotgyal) TQ.Mukhed, Dist.Nanded- 431715, India

3 Department of Chemistry, Dr.D. Y. Patil ACS Womens College Pimpri, Pune-411018; Affiliated to Savitribai Phule Pune University, India

4 Department of Biochemistry, Gramin Science (Vocational) College, Vishnupuri, Nanded-431 606 (MS), India

5 Department of Chemistry, Dr. D. Y. Patil ACS College Pimpri, Pune - 411018; Affiliated to Savitribai Phule Pune University, India


A new series of (E)-4-((4-substitutedphenyl)diazenyl)-1H-pyrazole-3,5-diamine derivatives were synthesized by using greener poly ethylene glycol-400 as a reaction solvent. The drug resistance capacity and growing incidence of Mycobacterium tuberculosis infections have been increasing day by day. There is high demand for the synthesis of multidrug resistance anti-tubercular drugs. Herein, all the newly prepared ligand was subjected to the Vitro anti-tubercular activity against Mycobacterium tuberculosis against H37Rv strains with, pyrazinamide, isoniazid, ethambutol, and streptomycin as the standard drugs. The compounds 4a, 4b and 5a showed excellent activity while 5b exhibit moderate activity against Mycobacterium tuberculosis. Further, the molecular docking was done with an enzyme PDB:1G3U. The ligand thymidine monophosphate was completed in the crystal structure of mycobacterium tuberculosis, while Mtb Pks13 Thioesterase domain in complex with inhibitor TAM16 with PDB:5V3Y Crystal Structure showed excellent docking results. These newly synthesized derivatives of pyrazole may be useful in the development of new anti-tubercular agents.

Graphical Abstract

Design, Synthesis, Docking and Biological Study of Pyrazole-3,5-diamine Derivatives with Potent Antitubercular Activity


Main Subjects


Multidrug resistance tuberculosis (TB) is one of the major problems in the world, accounting for morbidity and mortality [1, 2]. The literature report and per World Health Organization (WHO) report indicates that there is one person infected out of three-person and the world representing 2–3 billion people are infected with Mycobacterium tuberculosis (TB), which is a bacterial, communicable disease transmitted from person to person due to droplets of people suffering from active respiratory disease. TB as one of the leading causes of death has become one of the most dangerous health problems all over the world [3]. The infection of TB is caused by the bacteria bacillus Mycobacterium tuberculosis. The main symptoms of the disease are coughing, sneezing, and spitting. TB infects the lungs (pulmonary TB) as well as other organs of the body. As per a medical survey report, about 90% of infections occur in males than women. About 1/4 of the world’s population is infected with M. tuberculosis [4]. TB is curable, preventable, and treatable. The number of people acquiring infection is on the rise due to poverty, malnutrition, HIV infection, smoking, and diabetes. WHO has given a global report on TB every year since 1997, providing an up-to-date report on the TB epidemic and its progress in the research at national, regional, and global levels. The death caused by TB was 1.3 million in 2020, nearly similar to death toll caused by HIV/AIDS (0.68 million). TB is one of the 13th ranks in the death-caused element worldwide. As per a new WHO report, TB is one of the second leading causes of death after the COVID-19 outbreak [5]. As per the WHO report in the year 2020, about 10 million people have died from the infection caused by TB. The mutation occurs in some strains of TB bacteria, which are not responding to the available treatment worldwide. The new challenging task in front the of the pharmacist and chemist is to discover a new drug for the treatment of Multidrug resistance (MRD) bacterial strains. The medicine will be good and safe, has a no-side effect, is non-toxic, and eco-friendly. The conformation for the bacteriological TB is to detect the drug-resistant strain with the rapid molecular test or culture methods or sequencing technologies. Globally total of 5.8 million people is newly diagnosed with TB. Out of these 4.8 million, 59% were bacteriologically confirmed [6]. In the world, the present situation is most people suffer from TB, the main infection caused in low and middle-income countries such as Sub-Saharan Africa and Southeast Asia [7]. The pyrazole and their derivatives have a broad range of biological activities such as glycation, antibacterial, antifungal, anti-Alzheimer's, anticancer, anti-diabetic activity, antidepressant, anti-inflammatory, anti-tuberculosis, antioxidant as well as antiviral agents [8-13]. Nowadays, pyrazole systems, as biomolecules, have attracted more attention due to their interesting pharmacological properties (Scheme 1) [14].

There is an immediate need for the promotion and research on the finding of new anti-TB drugs. All over the world, the efforts have been made by respective countries to eradicate TB. The regulatory guideline, the treatment of TB involves 2 months of intensive phase treatment with isoniazid (H), rifampicin (R), pyrazinamide (Z), and ethambutol (E). The novel thiosemicarbazide and its analogues have a very high range of bio-activities. These are used to conflict with HIV-TB co-infection [15], malaria [16], tumors [17], and fungal infections [18].

Hence it is very essential to develop new, secure, and more adequate antibiotics against tuberculosis. In an effort to discover new and effective chemotherapeutic agents for the treatment of TB, we recently screened the antimicrobial activity of various pyrazole derivatives of hydrazine hydrate and thiosemicarbazide. In continuation of ongoing research on finding new lead scaffolds with potential antitubercular activity [19-22], herein, we have synthesized a series of novel pyrazole-3,5-diamine derivatives as a potential TB character.

Scheme 1: Pyrazole derivatives with anti TB activity

We performed an in-silico docking study of novel substituted pyrazole-3,5-diamine derivatives with attractive antituberculosis targets.

The solvent polyethylene glycol (PEG) has appeared as a novel greener and low expensive solvent for the various organic functional group transformation [23]. PEG is an excellent solvent and behaves as a polymeric, durable and biodegradable agent in the organic synthesis and can be reused several times without loss of their catalytic/ solvent prosperity. Polyethylene glycol is an eco-friendly solvent as compared with other halogenated, toxic solvents [24]. PEG solvent is flexible and cost-effective and has properties like biostability, surfactant, and high hydration capacity [25]. Furthermore, the solvent shows some outstanding properties low inflammability, non-volatility, easy degradability, reduced toxicity, recyclability, low cost, solubility in organic compounds, and stability even at high temperatures. For the last decades, research laboratory has always been interested in the use of PEG as a greener solvent for the organic functional group transformation [26-29].

Materials and Methods

The M.P. was determined with the open capillary tube. All the solvent was dried and purified before use. The progress of the reaction was determined with TLC, made up of silica gel and the stained with iodine vapors. The functional group was determined by FT-IR spectroscopy with FT-IR Shimadzu spectrophotometer (8400s). The 1H-NMR was recorded in a DMSO-d6 with an Avance spectrometer (Bruker, Germany) at 400 MHz frequency and TMS was used as internal standard. Mass spectra were recorded by an EI-Shimadzu QP 2010 PLUS GC-MS system (Shimadzu, Japan).

General procedure for the synthesis of derivatives

An equimolar quantity of (E)-2- derivatives of diazenylmalanonitrile with hydrazine hydrate/ thiosemicarbazide (0.001 mmol) was stirred in PEG-400 (20 mL) as reaction solvent at 60 °C temperature for 5 to 6 hours. Completion of reaction was monitored by TLC. Content was poured on crushed ice, and neutralized it with acid or base if necessary. The solid was separated by suction, washed with distilled water and crude product was recrystallized from ethanol to afford the pure product (70-90%). The synthesized compounds were identified by comparison of its melting point and spectroscopic data (FT-IR, 1H-NMR, 13C-NMR and Mass).

(E)-4-((4-chlorophenyl)diazenyl)-1H-pyrazole-3,5-diamine (4a): FT-IR (cm-1): 3405, 3302, 2925, 1617, 1563. 1H-NMR (300 MHz, DMSO-d6): δ 10.76 (s, 1H, NH), 7.70 (dd, 2H, Ar-CH), 7.4 (dd, 2H, Ar-CH), 7.05 (s, 1H, NH), 6.36 (s, 1H, NH), 5.86 (s, 1H, NH), 5.21 (s, 1H, NH). The HRMS for Formula: C9H9ClN6 [M+H+] 236.0577; observed 235.1

(E)-4-((4-nitrophenyl)diazenyl)-1H-pyrazole-3,5-diamine (4b): FT-IR (cm-1): 3453, 3380, 3351, 3257, 3157. 1H-NMR (300 MHz, DMSO-d6): δ 10.99 (s, 1H), 8.27 (dd, 2H, Ar-H), 7.87 (dd, 2H, Ar-H), 7.51 (s, 1H, NH), 6.82 (s, 1H, NH), 6.15 (s, 1H, NH), 5.47 (s, 1H, NH); 13C-NMR (DMSO-d6, 75 MHz): δ 158.6, 144.1, 124.7, 124.7, 124.7, 120.49, 117.5. The HRMS for Formula C9H9N7O2 [M+H+] 248.0818; obtained 248.0815

(E)-3,5-diamino-4-((4-chlorophenyl)diazenyl)-1H-pyrazole-1-carbothioamide (5a): FT-IR (cm-1): 3407, 3382, 3272, 1638 1584, 1089. 1HNMR (300 MHz, DMSO-d6): δ 9.44 (S, 1H, NH), 8.55 (S, 1H, NH), 7.84 (dd, 2H, Ar-H), 7.54 (dd, 1H, Ar-H), 7.43 (s, 2H, NH2); 13C-NMR (DMSO-d6, 75 MHz): δ 175.6, 151.7, 131.9, 129.3, 128.9, 122.5, 118.5, 113.9. Chemical Formula: C10H10ClN7S, found: 295.0407; obtained 295.8 (296.06).

(E)-3,5-diamino-4-((4-nitrophenyl)diazenyl)-1H-pyrazole-1-carbothioamide (5b): FT-IR (cm-1): 3424, 3301, 3175, 1612, 1101. 1HNMR (300 MHz, DMSO-d6): δ 10.04 (s, 1H, NH), 9.54 (s, 1H, NH), 9.30 (s, 1H, NH), 8.61 (s, 1H, NH), 8.31 (dd, 2H, Ar-H), 8.02 (dd, 2H, Ar-H), 6.63 (s, 1H, NH), 6.35 (s, 1H, NH). 13CNMR (DMSO-d6, 75 MHz): δ 175.6, 157.3, 145.5, 124.5, 121.7, 121.7, 121.3, 116.1. HRMS calculated for formula Chemical Formula: C10H11N8O2S, [M+H+] 307.0726; observed 307.0722.

Results and discussion

The starting material was prepared by the reaction of diazotization with appropriate aniline followed by the reaction with malononitrile in a sodium acetate in EtOH solvent at 0 °C. Next, the intermediate 3a-d was treated with the appropriate reagent in a PEG-400 solvent at 60 °C temperature for 5-6 hrs giving the desired product (Scheme 2). When the intermediate 2-(phenyldiazenyl) malononitrile 3a-d was treated with the hydrazine hydrate in the PEG-400 solvent at 60 °C temperature for 5-6 hrs giving novel 4-(phenyldiazenyl)-1H-pyrazole-3,5-diamine 4a-d derivatives with 75-88% yield. Similarly, the intermediate 3a-d was treated with thiosemicarbazide at 60 °C temperature in PEG-400 solvent for the 5-6 hrs that produced the desired 3,5-diamino-4-(phenyldiazenyl)-1H-pyrazole-1-carbothioamide 5a-c derivatives with 70 to 85% yield. All the newly synthesized compounds were confirmed by spectral analysis (Table 1).

Scheme 2: Synthesis of novel 4-(phenyldiazenyl)-1H-pyrazole-3,5-diamine and 3,5-diamino4-(phenyldiazenyl)-1H-pyrazole-1-carbothioamide derivatives

The formation of the final scaffold was confirmed by the spectral techniques, the compound ((E)-3,5-diamino-4-((4-chlorophenyl)diazenyl)-1H-pyrazole-1-carbothioamide) shows the characteristic peak for the FT-IR spectroscopy techniques; the band for the NH2 and NH appeared at the 3424 cm-1 and 3301 cm-1 respectively, while the absorption band at 1612cm-1 resembled –C=N stretching (Figure 1). The compound (E)-4-((4-chlorophenyl)diazenyl)-1H-pyrazole-3,5-diamine was confirmed with 1H-NMR spectroscopy. In 1H-NMR, the peak for the NH group was observed at δ 10.76 (s, 1H), while the peak for the aromatic proton appeared at δ 7.70-7.67 (dd, 2H), δ 7.43-7.40 (dd, 2H). The four protons belonging to the NH2 appeared separately singlets and were observed at δ 7.05 (s, 1H), δ 6.36 (s, 1H), δ 5.86 (s, 1H), δ 5.21 (s, 1H) (Figure 2). From the observed spectral data, the structure of the desired compound was confirmed by mass spectra.

Similarly, the compounds 5a-c were synthesized via a similar protocol. The intermediate moiety was treated with thiosemicarbazide giving the corresponding 5a, and the formed final scaffolds were confirmed with the spectral study. The 1H-NMR shows the characteristic peak at δ 9.44 (s, 1H) and 8.55 (S, 1H), NH2 belonging to NH2 proton and the aromatic proton would appear at δ 7.84 (d, 2H), δ 7.54 (d, 2H) ppm for four hydrogen's. The FT-IR peak shows the NH2 peak at 3407.19 and 3382.25 cm-1 for the characteristic functional group NH2 proton. Then, all the synthesized scaffolds were tested for the Mycobacterium strain M. tuberculosis (MTCC 300).

Biological investigation

Anti-mycobacterial activity

The M. tuberculosis (MTCC 300) strain was used in the present study for the assessment of anti-mycobacterial activity. The strain used for the study was procured from Microbial Type Culture and Gene Bank, Institute of Microbial Technology, Chandigarh (PB), India. The mentioned strain was sub-cultured and persevered as per the earlier depicted method on the Lowenstein Jensen medium [30]. The Agar diffusion method was used to assess the sensitivity of M. tuberculosis strain against the synthesized compounds [31]. Different stock solutions such as 0.1, 0.5, and 1.0 mg/mL of all compounds were prepared in dimethyl sulfoxide (DMSO). A sterile cork borer of 9-mm diameter was further employed to prepare holes into the Middlebrook 7H9 agar, already inoculum seeded and solidified. Firstly, the wells were labelled appropriately according to the compounds, and afterward a volume of 40 µL of each compound was added by using a sterile pipette. The test was executed in triplicates. To achieve the sample pre-diffusion, the plates were stored in the refrigerator and further incubated at room temperature for 48 h. After the incubation, the growth of the mentioned strain was detected and the diameter of the inhibition zone was measured. Rifampicin was used as a positive control for the mentioned experiment.

Resazurin microtiter assay (REMA) plate method for MIC determination

The MIC of synthesized compounds against MTCC 300 strain of M. tuberculosis was determined by using REMA assay.

It is a colorimetric method that is mostly used for the determination of the Susceptibility of multidrug-resistant M. tuberculosis strain. The experiment was performed as per earlier reported protocol with minor alterations [32-34].

The results of the antimycobacterial activity of the synthesized compounds are summarized in Table 2, which clearly shows the differential sensitivity of Mycobacterial strain MTCC 300 toward the test compounds. The compounds 4b (MIC-12.5±0.61µg/ml) and 5c (MIC-12.5±0.89µg/ml) were found to be most effective growth inhibitors of this strain. The compounds 5b (MIC25±0.58µg/ml), and 4c (MIC- 50±0.67µg/ml) were also found to be effective and significant growth inhibitors of Mycobacterial strains, whereas the remaining compounds 4a, and 5a showed moderate activity against M. tuberculosis.

Docking study of synthesized compounds

The molecular docking was performed in order to understand the types of interaction of the designed ligand with the mycobacterium tuberculosis enzyme. The molecular docking was performed with Auto dock software [35]. Based on the structure of the designed ligand and literature search, we performed the molecular docking with the enzyme PDB:1G3U; the crystal structure of mycobacterium tuberculosis thymidylate kinase was complexed with thymidine monophosphate (TMP) [36] and the enzyme PDB:5V3Y Crystal Structure of Mtb Pks13 Thioesterase domain in complex with inhibitor TAM16 [37-39].

The docking of synthesized ligand (4a-d and 5a-c) shows the binding interaction in the active sites of the enzyme PDB:1G3U and displays acceptable H-bonding interaction. Figures 3a,b show that compound 4a displays an exactly similar orientation in the binding pocket of the enzyme PDB:1G3U with bonding energy -8.9 kcal/mol. The molecule inhibitor displays excellent hydrogen bonding with the GLU-166, ASP163, and Van der Waals interaction with LEU:52, GLN:172, TYR;165, ARG;74, TYR;39, SER;99, ARG;74, ASN;100 amino acids. The compound 4a also exhibits Pi-cation and Pi-anion interaction with the ASP;9, TYR;103, ARG;95 amino acids. The 4a exhibits the hydrogen bonding with the GLU-166 amino acid via the COOH------HN bond having a bond distance of 2.2Ǻ, while the residues ASP-163 show the hydrogen bond with the COOH-------HN with a bond distance of 2.5Ǻ (Figure 3).

A 4b show hydrogen bonding with the amino acid residues GLU;166, TYR;39, ASN;100, ARG;74 amino acids, the molecules interact with the GLU;166 residues showing the C=O---HN hydrogen bond having distance 2.5Ǻ, the residues TYR;39 display the hydrogen bonding with NH2 of ligand 4A having bond distance 2.5Ǻ, and the residues ASN;100, form the hydrogen bond via NO2 of residues and N-H of the ligand with 2.8Ǻ hydrogen bond distance, while the residues form the hydrogen bond with inhibitor via N-O------H-N with 1.5Ǻ hydrogen bond distance. The inhibitor 5B displays strong hydrogen bonding with TYR; 39, ASP;9, ASP;163 and ASN;100 amino acid residues and also van der Waals force of interaction. SER;104, ALA;48, ALA;49, LEU;52, ARG;160, GLU;166, ARG; 95, TYR;165, ARG;74 amino acids residues.

The active sites of enzymePDB:1G3U form the hydrogen bonding with the designed ligand with TYR;39 residues with O-----H--O hydrogen bond with 2.5Ǻ bond distance.

The residues ASP;163 display hydrogen bonding via C-O-----H--N with 1.8 Ǻ bond distance while the amino acids residues ASP;9 display hydrogen bonding via C=O---HN with 2.2Ǻ hydrogen bond distance and the residues ASN;100 also form the hydrogen bond N-O----NH with 3.0Ǻ bond distance. The docking of synthesized ligand (4a-d and 5a-b) shows the binding interaction in the active sites of the enzyme PDB:3V3Y Crystal Structure of Mtb Pks13 Thioesterase domain in complex with inhibitor TAM16and displays acceptable H-bonding interaction.            

The compounds 4a, 4b, 5a, and 5b display potent anti-TB activity. All the compounds of 1H-pyrazole-3,5-diamine and 1H-pyrazole-1-carbothioamide series were highly potent.

Figure 6a,b represent the ligand well accommodated in the active sites of the enzyme and the molecules show hydrogen bonding interaction with ASN;1640, ALA;1667, ASP; 1644 amino acid residues, while the compound 4a displays van der Waals interaction with GLN;1638, ARG;1641, TYR;164, GLU;1671, ALA;1667, ASP;1666, ASN;1640, TYR;1637, SER;1636, GLN;1638 amino acids residues (Figure 6).


To conclude, we have prepared a series of pyrazole-3,5-diamine and 1H-pyrazole-1-carbothioamide derivatives. All the synthesized compounds were characterized by FT-IR, 1H-NMR, 13C-NMR, and mass spectroscopic techniques. The compounds were screened for the antimycobacterial activity. 12.5±0.61, 25±0.58, and 12.5±0.89 with standard Rifampicin 0.8±0.70 μM) when tested against the M.tb H37Rv strain M. tuberculosis (MTCC 300). The molecular docking study of the compounds 4a, 4b, 5a, and 5b display excellent docking score and shows excellent hydrogen bonding interaction in the active sites of enzyme PDB:1G3UB and PDB: 5V3Y. The synthesized compound could be the potential candidate for finding the M. tuberculosis inhibitors.


We highly acknowledge Karmaveer Kishanrao Rathod (Founder President, VJSS) and H.B. Rathod (Principal, Gramin ACS Mahavidyalaya, Vasantnagar, Mukhed) for their continuous support. We are thankful to the School of Chemical Sciences, S.R.T.M University, Nanded for providing a research facility.


This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


Authors' contributions

All authors contributed to data analysis, drafting and revising the paper and agreed to be responsible for all the aspects of this work.


Conflict of Interest

We have no conflicts of interest to disclose.


Devidas C. Pawar

Sunil V. Gaikwad

Sonali S. Kamble

Priya D. Gavhane

Milind V. Gaikwad

Bhaskar S. Dawane


Devidas C. Pawar, Sunil V. Gaikwad, Sonali S. Kamble, Priya D. Gavhane, Milind V. Gaikwad, Bhaskar S. Dawane. Design, Synthesis, Docking and Biological Study of Pyrazole-3,5-diamine Derivatives with Potent Antitubercular Activity. Chem. Methodol., 2022, 6(9) 677-691


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