One-pot synthesis and in vitro bioactivity of novel 4-aminopyrazolo[3,4-b]pyridine derivatives as potential antimicrobial compounds

doi:10.21203/rs.3.rs-3211765/v1

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One-pot synthesis and in vitro bioactivity of novel 4-aminopyrazolo[3,4-b]pyridine derivatives as potential antimicrobial compounds

https://doi.org/10.21203/rs.3.rs-3211765/v1

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published 23 Nov, 2023

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The present paper describes an efficient synthesis of a series of 4-aminopyrazolo[3,4-b]pyridine derivatives 11a-i in 69-81% yields by one-pot three-component domino reaction of phenylhydrazine and two different β-ketonitriles in DMSO. All the synthesized derivatives were screened in vitro for their antimicrobial potential against seven Gram-positive and Gram-negative clinical bacterial strains and one fungal strain. Most of the tested derivatives displayed promising antimicrobial potency and activity was found to be comparable to standard drugs (amoxicillin and fluconazole). Compounds 11a and 11d with the MIC value of 3.12 μg/mL were identified as broad spectrum antibacterial agents. Compounds 11b, f, h and 11i exhibited equivalent antifungal activity (MIC 6.25 μg/mL) against Candida albicans to fluconazole. Interestingly, all the compounds were found to be non-toxic to normal mammalian and plant cells at very high concentration of 1 mg/mL.

4-Aminopyrazolo[3

4-b]pyridines

Domino reaction

Antimicrobial activity

Drug resistance

Minimum inhibitory concentration

Increased antimicrobial resistance to several marketed antibiotic drugs due to overuse of antibiotics is making them less effective day by day from the last few decades [1]. According to WHO priority list 2018 on antibiotic-resistant bacteria, common bacteria in the community like Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhi and Shigella spp. should be included in long term plans to develop new antibiotics to combat their emerging antibiotic resistance [2]. Moreover, the fungal pathogens such as Candida sp., one of the leading causes of nosocomial infections, are becoming resistant to available antifungal drugs. Due to these reasons once easily curable diseases are becoming difficult to cure which is increasing the burden of healthcare costs and facilities ultimately affecting the society in terms of economy and individual’s life. Therefore, development and exploration of novel antimicrobial agents with high activity and good selectivity is urgently needed to address this global challenge of antibiotic resistance in bacteria and fungi [3, 4].

Pyrazolo[3,4-b]pyridine scaffold is a privileged fused heterocycle in medicinal chemistry [5] and its derivatives are reported to possess wide spectrum of pharmacological activities, viz. anticancer agents [6], PIM-1 kinase inhibitors [7], antimicrobial agents [8, 9, 10], antimalarial agents [11], anti-inflammatory agents [12], combretastatin A-4 analogues [13] and adenosine antagonists [14]. In particular, 4-amino derivatives of pyrazolo[3,4-b]pyridine are prevalently present in a variety of clinically anxiolytic drugs such as cartazolate (1) [15], tracazolate (2) [16], and etazolate (3) [17] and compound 4 is reported as potent glycogen synthase kinase-3 (GSK-3) inhibitor [18] (Fig. 1). Compound 5 in which tacrine is linked with pyrazolo[3,4-b]pyridine moiety by a six-carbon spacer, was found to be potent acetylcholinesterase (AChE) and phosphodiesterase 4 (PDE4D) inhibitior, with IC₅₀ values of 0.125 µM and 0.271 µM, respectively [19]. 3'-Diethylaminomethyl-substituted 4-anilino-1H-pyrazolo[3,4-b]pyridine-5-carboxylic ester 6 can exert an antileishmanial effect [20]. 4-Aminopyrazolo[3,4-b]pyridine-5-carboxylic acid ester 7 was reported as selective inhibitor of A1 adenosine receptors [14].

The foregoing observations prompted us to synthesize more bioactive molecules containing 4-aminopyrazolo[3,4-b]pyridine in the field of antimicrobials. Typically, pyrazolo[3,4-b]pyridines can be synthesized by (i) the pyrazole ring construction on an existing pyridine ring [21, 22] and (ii) annulation of the pyridine ring on a pyrazole structure [6, 12]. The latter approach provides more structural diversity and flexibility in terms of substitution at pyrazole and pyridine rings. One-pot syntheses including multicomponent reactions and domino reactions offer several advantages in terms of high efficiency, operational simplicity and sustainability [23]. In view of these observations and in continuation to our ongoing research on development of novel pyrazolo[3,4-b]pyridines [24, 25, 26], we report herein the one-pot synthesis of a series of pyarazolo[3,4-b]pyridines 11a-i for their evaluation as antimicrobial agents. Cytotoxicity study of the synthesized derivatives towards normal cells was also evaluated.

Chemistry

Initially we have carried out the reaction of phenylhydrazine (8) with 2 eq. of 3-(4-bromophenyl)-3-oxopropanenitrile (9a) in catalytic amount of PTSA (p-toluenesulphonic acid) using ethanol as solvent under reflux. PTSA being solid, non-oxidizing, non-volatile, recyclable, non-explosive and thermally robust acid catalyst [27] was used in initial studies. The idea for the choice of this catalyst was that highly efficient synthesis of pyrazolo[3,4-b]pyridines was previously reported by us using catalytic amount of PTSA [24]. Unfortunately, no fruitful result was obtained and the reaction generated 5-aminopyrazole (10a) instead of the desired 4-aminopyrazolo[3,4-b]pyridine (11a) even on prolonged heating for 24 h (Scheme 1).

Subsequently, to study the effect of elevated temperature and role of polar/nonpolar solvents on the reaction outcome, the reaction of 8 and 3-(4-bromophenyl)-3-oxopropanenitrile (9a) was carried in various high boiling solvents like toluene, DMF and DMSO at reflux temperature (Scheme 1). It was observed that in general, 5-aminopyrazole (10a) was obtained in most of the cases (Table 1, entries 1–4). Fusion of reactants under solvent-free conditions afforded mixture of products and the desired product 11a was obtained in trace amount (Table 1, entry 6). In order to improve the yield of solvent-free reaction, prolonged heating of reactants was done which resulted in charring of the contents (Table 1, entry 7).

DMSO is used as polar aprotic solvent in synthesizing various organic compounds of medicinal importance because of its low cost and toxicity [28]. DMSO can also be used to introduce various functional groups to organic compounds [29, 30]. However, use of DMSO in synthesizing heterocyclic compounds has been explored by very few researchers [31, 32]. 3-Aryl-3-oxopropanenitrile or β-ketonitrile demonstrated to be a poor electrophile in comparison with β-diketones, thus DMSO as a solvent had to be used in order to obtain the desired products. Moreover, DMSO being high boiling solvent could assists in the reaction by enhancing the electrophilicity of β-ketonitrile due to its polarizability [33]. Fortunately, formation of new spot was observed after 1 h along with the spot corresponding to aminopyrazole 10a (Table 1, entries 4 and 5 respectively) when DMSO was used as the refluxing solvent. Further heating of reaction mixture for almost 3 h in DMSO at reflux temperature resulted in formation of single product in 76% yield (Table 1, entry 5).

It was inferred that the reaction proceed through the intermediate formation of 5-aminopyrazole 10a which upon further condensation with another molecule of 3-(4-bromophenyl)-3-oxopropanenitrile (9a) resulted in the formation of desired product when the reaction was carried out in DMSO at reflux temperature. The product came out as solid on cooling to room temperature was filtered, recrystallized and characterized as 3,6-bis(4-bromophenyl)-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-amine (11a).

Table 1

Optimization of reaction conditions.
Entry	Solvent	Temp. °C	Catalyst	Time (h)	Isolated Yield (%)
Entry	Solvent	Temp. °C	Catalyst	Time (h)	10a	11a
1	Ethanol	78	PTSA	24	40	-
2	Toluene	110	PTSA	24	32	-
3	DMF	153	None	24	38	-
4	DMSO	80–90	None	1	42	-
5	DMSO	189	None	4	-	76
6	Solvent free	fusion	PTSA	5	30	trace
7	Solvent free	fusion	PTSA	Prolonged heating^a	-	-

^aCharring of products takes place.

Structure of 11a was established by ¹H, ¹³C NMR spectroscopy, IR spectroscopy and mass spectrometry. IR spectrum of the compound 11a exhibited characteristics absorption peaks at 3379 and 3418 cm^− 1 corresponding to the symmetric and asymmetric N-H stretches of amine group. ¹H NMR spectrum of 11a exhibited a singlet of one proton intensity at about δ 8.77 ppm due to the pyrazolopyridine 5-H besides signals corresponding to the protons of aryl groups. ¹³C NMR spectrum of 11a exhibited a signal at δ 101.7 ppm due to C₅ carbon besides signals for other carbon atoms.

From the encouraging results of above three-component reaction, it was further planned to study the substrate scope of this methodology by using two different β-ketonitriles and phenylhydrazine through sequential addition of different β-ketonitriles bearing electron-donating or electron-withdrawing substituents at aryl ring avoiding formation of mixture of products. The reaction of phenylhydrazine (8) was carried out with 3-(4-bromophenyl)-3-oxopropanenitrile (9a) (1 eq.) in DMSO at 80–90 ⁰C for 1 h. The reaction resulted in expected 5-aminopyrazole (10a) on TLC. After complete consumption of the reactants, second molecule of β-ketonitrile was added. The resulting mixture was refluxed for another 3 h and the completion of the reaction was observed by TLC examination. This one-pot three-component sequential reaction directly resulted in the formation of 3-(4-bromophenyl)-1-phenyl-6-(4-methylphenyl)-1H-pyrazolo[3,4-b]pyridin-4-amine (11b) bearing different aryl groups (Scheme 2) and structural confirmation was made by ¹H, ¹³C NMR and IR spectral studies. Thus with the optimized reaction conditions in hand, other representatives 11c-i were prepared and characterized. Structural substitution pattern and physical data of compounds 11a-i are presented in Table 2.

Table 2. Structural substitution pattern and physical data of compounds 11a-i.

Compound	Ar	Ar'	Yield^a	M.p. (°C)
11a	4-BrPh	4-BrPh	76%	211
11b	4-BrPh	4-MePh	71%	204
11c	4-ClPh	4-BrPh	69%	191
11d	4-FPh	4-BrPh	72%	185
11e	4-FPh	4-ClPh	75%	173
11f	4-FPh	4-MePh	73%	181
11g	4-MePh	4-BrPh	81%	216
11h	Ph	4-ClPh	68%	189
11i	Ph	4-BrPh	69%	173

^aIsolated yield.

The one-pot multicomponent synthesis of pyrazolo[3,4-b]pyridin-4-amines 11a-i is supposed to follow the proposed mechanism as shown in Scheme 3. Initially, phenylhydrazine 8 condenses with one mole 3-aryl-3-oxopropanenitrile 9 to give the intermediate 5-aminopyrazole 10. Then, intermediate 10via its exocyclic amino group assists the nucleophilic attack of C-4 double bond on nitrile group of another 3-aryl-3-oxopropanenitrile 9 to afford the intermediate 12 which further undergoes intramolecular condensation involving the reaction of amino group on the carbonyl group to give intermediate 13. Finally, dehydration and aromatization of intermediate 13 produces desired product 11 through the intermediacy of 14.

Biological Activity

Antimicrobial evaluation

The antimicrobial activity of newly synthesized compounds 11a-i was evaluated in vitro against three Gram-positive (Staphylococcus aureus ATCC 6538P, Bacillus cereus ATCC 11110, Listeria monocytogenes MTCC 657) and four Gram-negative (Pseudomonas aeruginosa ATCC 15442, Escherichia coli MTCC 143, Salmonella typhi MTCC 733, Shigella flexneri ATCC 9199) bacterial strains and one fungal strain (Candida albicans MTCC 183). The antimicrobial activity of compounds (11a-i) is reported as their minimum inhibitory concentration (MIC) and depicted in Table 3. The antimicrobial potential of the compounds was compared with standard antibiotic (amoxicillin) and antifungal drug (fluconazole).

It was interesting to note that all newly synthesized compounds 11a-i displayed moderate to good antimicrobial activity (Table 3, significant activity is indicated by green and blue entries) and showed MIC values in the range of 3.12–31.25 µg/mL. Among these, compounds 11a, 11b, 11d and 11e were as potent as standard drug amoxicillin with MIC value for each 3.12 µg/mL against P. aeruginosa. The same compounds (11a, 11b, 11d and 11e) as well as 11g were also active against E. coli with two-fold higher MIC than standard drug. Compounds 11a, 11d and 11f with the same MIC value were found to inhibit S. typhi (MIC: 3.12 µg/mL). The five compounds (11a, 11c-d and 11f-g) exhibited potent activity against S. aureus. Compounds 11a and 11i have shown the same MIC against L. monocytogenes (MIC: 3.12 µg/mL). The bacterial strain B. cereus was found to be effectively inhibited by the compounds 11g and 11i. The compounds 11i and 11d inhibited the growth of S. flexneri bacterial strain. Compound 11i was found to be the least active against S. typhi and exhibited highest MIC value of 31.25 µg/mL. Compound 11h was found to be the least active against all the tested bacterial strains due to high MICs (Table 3), however, the same compound showed equipotent antifungal activity to standard fluconazole. Almost all of the compounds were found effective against tested fungal strain C. albicans but interestingly, the four compounds 11b, 11f, 11h and 11i displayed potent antifungal effect with MICs equal to standard drug fluconazole (MIC: 6.25 µg/mL) (Table 3). The results suggested that pyrazolo[3,4-b]pyridin-4-amines might be promising in new antifungal drug development.

Table 3. MIC values of compounds 11a-i against tested bacterial and fungal strains.

^aMean of three replicates. ^bAmoxicillin (Amox) was used as positive control for antibacterial activity. ^cFluconazole (Fluc) was used as positive control for antifungal activity.

Overall, most of the tested compounds 11 were more effective against Gram-negative bacteria than Gram-positive bacteria. Interestingly, compounds 11a and 11d exhibited equipotent antibacterial activity against P. aeruginosa, E. coli, S. typhi, and S. aureus with MIC value of 3.12 µg/mL which was comparable to that of amoxicillin, thus revealing their broad-spectrum antibacterial efficacy.

Structure activity relationship

In this series of compounds, variation in antimicrobial potential is related to the nature of substituents at para-position of phenyl rings present at C₃ and C₆ position of pyrazolo[3,4-b]pyridine scaffold. In general, presence of chloro substituent at any phenyl ring on pyrazolo[3,4-b]pyridine 11c, 11e and 11h resulted in a clear decrease of antimicrobial potency especially antibacterial except 11e against P. aeruginosa and E. coli (Table 3). Compounds 11b and 11f possessing p-methyl group at benzene ring at C₆ position of pyrazolo[3,4-b]pyridine scaffold also exhibited decreased antibacterial activity against various bacterial strains. However, the same compounds (11b and 11f) showed good antifungal activity. In particular, the presence of bromo substituent on both aryl rings (11a) or fluoro substituent at phenyl ring attached to C₆ position and bromo at phenyl ring attached to C₃ position of pyrazolo[3,4-b]pyridine (11d) was seen to favor the broad spectrum antibacterial activity. In addition, 11b and 11e with fluoro and bromo group at phenyl ring at C₃ position displayed antibacterial activity selectively for P. aeruginosa.

Acute toxicity studies

Compounds 11a-i were evaluated for their cytotoxic studies against one mammalian cell line (mouse fibroblast cell line) and one plant cell line (plant seed germination cell line) at very high concentration of 1 mg/mL using MTT assay. The results of the cytotoxic studies are presented in Table 4. It can be seen from cytotoxicity screening that after 24 h of exposure time, compounds 11a-i were found to be safe to normal cells with 90–100% cell viability even at much higher doses of the corresponding MICs. Therefore, compounds 11 possessed another positive point that is highly desirable in the antimicrobial agents.

Table 4

*In vitro* cytotoxicity analysis of compounds **11a-i** against normal cells at the concentration of 1 mg/mL.
Cell line	11a	11b	11c	11d	11e	11f	11g	11h	11i
	(Cell Viability %)^a
Mouse Fibroblast cell	94.5	96.6	90.2	90.5	91.8	96.6	94.5	94.5	94.5
Plant seed germination	98	98	98	100	97	96	98	98	98

^aTested in triplicates at 1 mg/mL for 24 h.

A series of new 4-aminopyrazolo[3,4-b]pyridines were synthesized by efficient domino approach, characterized and evaluated as antimicrobial agents. The in vitro biological evaluation revealed that some compounds exhibited promising antimicrobial activity which was comparable to standard drugs, amoxicillin and fluconazole. Compounds 11a and 11d were identified as broad spectrum antibacterial agent and represent a promising lead for further optimization to overcome Gram-negative resistance. Compounds 11b, f, h and 11i displayed superior antifungal activity against C. albicans (MIC 6.25 µg/mL) which was equivalent to standard drug fluconazole. Furthermore, all the compounds were found safe to normal cells.

General information

Commercial solvents and other reagents were used as purchased without any purification. Melting points were determined in open capillaries on digital melting point apparatus (MEPA), Lab India and were uncorrected. IR spectra were recorded on Buck Scientific IR M-500 spectrophotometer using KBR pellets (ν_max in cm^− 1). ¹H and ¹³C NMR spectra for analytical purpose were recorded in CDCl₃ on a Bruker instrument operating at 400 and 100 MHz, respectively at Central Instrumentation Laboratory, Guru Jambheshwar University of Science & Technology, Hisar, India. ¹H and ¹³C NMR spectra were recorded in CDCl₃ on a Bruker instrument at 400 and 100 MHz, respectively. Elemental analyses were performed at Sophisticated Analytical Instrument Facility, Panjab University, Chandigarh. All the compounds gave C, H and N analysis within ± 0.5 of the theoretical values.

Synthesis

General procedure for the synthesis of 3-(aryl)-6-substituted-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-amines (11a-i)

A solution of phenylhydrazine (8, 0.001 mol) and 3-(4-bromophenyl)-3-oxopropanonitrile (9a, 0.001 mol) in DMSO (15 mL) was stirred at 90°C for 1 h. Reaction progress was monitored on TLC and after consumption of starting materials, 3-(4-methylphenyl)-3-oxopropanonitrile (9b, 0.001 mol) was added and the reaction mixture was refluxed for another 3 h. After consumption of 3-(4-methylphenyl)-3-oxopropanonitrile (9b) on TLC, the reaction mixture was cooled to get a solid product as 3-(4-bromophenyl)-1-phenyl-6-(4-methylphenyl)pyrazolo[3,4-b]pyridin-4-amine (11b) which was recrystallized in ethanol.

All other derivatives 11c-i were prepared following the same procedure. However, compound 11a was prepared by refluxing the phenylhydrazine (8, 0.001 mol) and 3-(4-bromophenyl)-3-oxopropanonitrile (9a, 0.002 mol) in DMSO for 4 h.

11a: 3,6-Bis(4-bromophenyl)-1-phenylpyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3379, 3418 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 7.26–7.30 (d, 2H, J = 8.0 Hz, 3''',5'''-H), 7.37–7.39 (m, 1H, 4'-H), 7.53–7.57 (m, 2H, 3',5'-H), 7.70–7.73 (d, 2H, J = 8.0 Hz, 3'',5''-H), 7.91–7.93 (d, 2H, J = 8.0 Hz, 2''',6'''-H), 7.99–8.03 (m, 2H, 2', 6'-H), 8.33–8.36 (d, 2H, J = 8.0 Hz, 2'',6''-H), 8.77 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 101.7, 116.5, 116.7, 121.5, 125.5, 127.1, 129.2, 129.3, 129.4, 131.0, 132.1, 136.1, 138.1, 138.7, 158.6; MS: m/z 521.12 [M + 1]⁺, 523.11 [M + 1 + 2]⁺, 525.13 [M + 1 + 4]⁺, (1:2:1); Anal. Calc. for C₂₄H₁₆Br₂N₄: C, 55.41; H, 3.10; N, 10.77%; Found: C, 54.95; H, 2.86; N, 10.36%.

11b: 3-(4-Bromophenyl)-1-phenyl-6-(4-methylphenyl)pyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3379, 3448 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 2.46 (s, 3H, CH₃), 7.34–7.39 (m, 3H, 4',3''',5'''-H), 7.52–7.65 (m, 4H, 2',3',5',6'-H), 7.89–7.91 (d, 2H, J = 8.0 Hz, 3'',5''-H), 7.97–7.99 (d, 2H, J = 8.0 Hz, 2''',6'''-H), 8.34–8.36 (d, 2H, J = 8.0 Hz, 2'',6''-H), 8.78 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 21.5, 101.4, 113.4, 118.5, 121.4, 121.49, 126.8, 127.3, 129.1, 130.3, 138.8, 131.0, 132.1, 136.1, 138.1, 138.7, 139.98, 150.76 ,158.3; MS: m/z 455.19 [M + 1]⁺, 457.11 [M + 1 + 2]⁺, (1:1); Anal. Calc. for C₂₅H₁₉BrN₄: C, 65.94; H, 4.21; N, 12.30%; Found: C, 65.45; H, 4.06; N, 11.93%.

11c: 6-(4-Bromophenyl)-1-phenyl-3-(4-chlorophenyl)pyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3402, 3464 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 7.38–7.39 (d, 2H, J = 8.0 Hz, 3''',5'''-H), 7.54–7.57 (m, 3H, 3', 4',5'-H), 7.69–7.72 (d, 2H, J = 8.0 Hz, 3'',5''-H), 7.90–7.98 (m, 4H, 2'',6'',2''',6'''-H), 8.34 (m, 2H, 2',6'-H), 8.77 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 101.7, 121.5, 121.6, 126.9, 127.1, 127.3, 128.6, 129.3, 129.7, 130.1, 131.0, 137.0, 137.1, 150.8, 158.1; MS: m/z 474.99 [M + 1]⁺_, 476.98 [M + 1 + 2]⁺, 478.98 [M + 1 + 4]⁺, (3:4:1); Anal. Calc. for C₂₄H₁₆BrClN₄: C, 60.59; H, 3.39; N, 11.78%; Found: C, 60.93; H, 2.99; N, 11.31%.

11d: 6-(4-Bromophenyl)-1-phenyl-3-(4-fluorophenyl)pyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3340, 3402 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 7.30–7.34 (m, 3H, 3',4',5'-H), 7.57–7.61 (m, 4H, 3'',5'',3''',5'''-H), 8.01–8.06 (m, 4H, 2',6',2''',6'''-H), 8.37–8.39 (d, 2H, J = 8.0 Hz, 2'',6''-H), 8.80 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 101.6, 113.1, 116.4, 118.3, 121.46, 129.0, 129.2, 129.3, 130.7, 135.5, 136.9, 138.0, 138.6, 144.1, 150.7, 158.1; MS: m/z 458.98 [M + 1]⁺_, 460.99 [M + 1 + 2]⁺, (1:1); Anal. Calc. for C₂₄H₁₆BrFN₄: C, 62.76; H, 3.51; N, 12.20%; Found: C, 62.27; H, 3.01; N, 12.63%.

11e: 6-(4-Chlorophenyl)-1-phenyl-3-(4-fluorophenyl)pyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3286, 3356 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 7.51–7.66 (m, 5H, 3',4',5',3'',5''-H), 8.06–8.08 (m, 2H, 3''',5'''-H), 8.48–8.52 (m, 4H, 2',6', 2'',6''-H), 8.70–8.71 (m, 2H, 2''',6'''-H), 8.88 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 101.8, 116.2, 121.3, 127.6, 127.7, 129.8, 131.4, 131.7, 133.2, 134.6, 138.5, 144.1, 149.0, 153.3, 167.3; MS: m/z 415.09 [M + 1]⁺_, 417.08 [M + 1 + 2]⁺, (3:1); Anal. Calc. for C₂₄H₁₆ClFN₄: C, 69.48; H, 3.89; N, 13.51%; Found: C, 69.89; H, 3.41; N, 13.05%.

11f: 6-(4-Methylphenyl)-1-phenyl-3-(4-fluorophenyl)pyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3387, 3425 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 2.46 (s, 3H, CH₃), 7.34–7.39 (m, 3H, 3', 4',5'-H), 7.52–7.56 (m, 4H, 2',6',3''',5'''-H), 7.89–7.91 (d, 2H, J = 8.0 Hz, 3'',5''-H), 7.97–7.99 (d, 2H, J = 8.0 Hz, 2''',6'''-H), 8.34–8.36 (d, 2H, J = 8.0 Hz, 2'',6''-H), 8.78 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 21.5, 101.4, 113.4, 118.6, 121.41, 121.43, 121.46, 121.49, 121.51, 126.8, 127.3, 129.1, 129.3, 130.8, 138.3, 138.8, 139.9, 150.7, 158.4; MS: m/z 395.21 [M + 1]⁺_; Anal. Calc. for C₂₅H₁₉FN₄: C, 76.12; H, 4.86; N, 14.20%; Found: C, 75.92; H, 4.51; N, 13.77%.

11g: 3-(4-Methylphenyl)-1-phenyl-6-(4-bromophenyl)pyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3348, 3433 (NH₂ str.); ¹H NMR (500 MHz, CDCl₃): 2.47 (s, 3H, CH₃), 7.35–7.37 (m, 1H, 4'H), 7.38–7.40 (d, 2H, J = 8.5 Hz, 3'',5''-H), 7.53–7.57 (m, 4H, 3',5',2',6'-H), 7.91–7.93 (d, 2H, J = 8.0 Hz, 3''',5'''-H), 7.98–8.01 (d, 2H, J = 8.5 Hz, 2'',6''-H), 8.35–8.37 (d, 2H, J = 8.0 Hz, 2''',6'''-H), 8.80 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 18.9, 103.0, 113.7, 117.2, 121.7, 126.9, 128.9, 129.2, 130.8, 131.0, 131.1, 136.0, 136.7, 138.5, 145.9, 149.9, 150.8, 159.3; MS: m/z 455.03 [M + 1]⁺_, 457.02 [M + 1 + 2]⁺, (1:1); Anal. Calc. for C₂₅H₁₉BrN₄: C, 65.94; H, 4.21; N, 12.30%; Found: C, 65.45; H, 3.82; N, 11.86%.

11h: 6-(4-Chlorophenyl)-1,3-diphenylpyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3387, 3448 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 7.34–7.36 (m, 1H, 4'-H), 7.52–7.61 (m, 7H, 2',3',6',3''',5''',3'',5''-H), 7.98–8.01 (m, 4H,, 3',5',2''',6'''-H), 8.35–8.37 (d, 2H, J = 8.0 Hz, 2'',6''-H), 8.82 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 101.6, 113.4, 118.5,8, 145.2 121.2, 121.5, 127.5, 129.1, 129.3, 129.4, 130.8, 135.5, 138.3, 138.7, 145.2; MS: m/z 397.01 [M + 1]⁺_; 399.03 [M + 1 + 2]⁺, (3:1); Anal. Calc. for C₂₄H₁₇ClN₄: C, 72.63; H, 4.32; N, 14.12%; Found: C, 72.18; H, 3.85; N, 13.79%.

11i: 6-(4-Bromophenyl)-1,3-diphenylpyrazolo[3,4- b ]pyridin-4-amine

IR (KBr, cm^− 1): 3379, 3448 (NH₂ str.); ¹H NMR (400 MHz, CDCl₃): 7.37–7.39 (m, 1H, 4'-H), 7.53–7.59 (m, 5H, 2',3',5',6',4''-H), 7.70–7.72 (d, 2H, J = 8.0 Hz, 3''',5'''-H), 7.91–7.93 (d, 2H, J = 8.0 Hz, 3'',5''-H), 8.01–8.03 (d, 2H, J = 8.0 Hz, 2''',6'''-H), 8.35–8.37 (d, 2H, J = 8.0 Hz, 2'',6''-H) 8.81 (s, 1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ 101.5, 118.4, 121.5, 125.4, 126.9, 127.5, 129.2, 129.3, 129.4, 129.8, 131.0, 131.4, 132.0, 136.1, 138.3, 138.7; MS: m/z 441.05 [M + 1]⁺_; 443.04 [M + 1 + 2]⁺, (1:1); Anal. Calc. for C₂₄H₁₇BrN₄: C, 65.32; H, 3.88; N, 12.70%; Found: C, 65.66; H, 3.29; N, 13.19%.

Antimicrobial activity

Antimicrobial activities of the tested compounds are reported as the MICs which were evaluated using the broth microdilution method in 96-well microtiter plates, following the CLSI guidelines [34]. The microorganisms like Pseudomonas aeruginosa (ATCC 15442), Bacillus cereus (ATCC 11770), Escherichia coli (MTCC 143), Listeria monocytogenes (MTCC 657), Salmonella typhi (MTCC 733), Staphylococcus aureus (ATCC 6538P), and Shigella flexneri (ATCC 9199) and one fungal strain Candida albicans (MTCC 183) were used for investigating the antimicrobial potential of compounds. The standard drugs used for the antibacterial and antifungal activities were amoxicillin and fluconazole, respectively. The tested compounds were dissolved in DMSO to prepare stock solution (2 mg/mL). The stock solutions were further diluted as required in PBS (pH 7.4). Colonies of microorganisms were picked off freshly from isolation plate and were inoculated in corresponding tubes containing 5 ml of Brain Heart Infusion (BHI) broth. This broth was incubated for 6 h at 37°C. The MC Farland No. 5 standard was prepared by adding 0.05 mL of 1% w/v BaCl₂.2H₂O in Phosphate Buffered Saline (PBS) to 9.95 ml of 1% v/v H₂SO₄ in PBS. The growth of all the cultures was adjusted to Mc Farland No. 5 turbidity standard using sterile PBS to achieve 10⁸ CFU/mL suspension. The working inoculums of aforementioned different microorganisms containing 10⁵ CFU/mL suspension was prepared by diluting the 10⁸ CFU/mL suspension, 10³ times in Brain Heart Infusion broth. The MIC is the lowest concentration of a compound inhibiting visible growth of the microbe after 24 h of incubation.

Toxicity studies

Cell culture

Mouse fibroblast cells were cultured in Dulbecco's Modified Eagle medium (DMEM). The cells were supplemented with 10% fetal serum albumin along with 50 µg per mL of penicillin and streptomycin each. However, plant seed germination cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. These cell lines were also supplemented with 10% fetal serum albumin and 1% of gentamycin. All the cell lines were kept in an incubator containing 5% CO₂ at 37°C for 5–10 minutes.

Cell viability assay

The cells, under consideration, were seeded in a 96-well plate at a density of 10⁵ per ml and were grown throughout the night. The cells were treated with test compounds at a concentration of 1 mg/mL and were incubated for 24 h in humidified atmosphere of 5% CO₂ and 95% air at 37°C. The cell viability assay was performed with MTT cell proliferation kit from American Type Culture Collection (ATCC) (#30-1010K). At the end, 10 µL of MTT reagent was added to the well followed by placing the cells back in incubator for another 4 h. Subsequently, the absorbance was measured at 570 nm using a UV–visible spectrophotometer. MTT assays were repeated in three separate experiments. Data was processed for calculation of % cell survival with help of following formula

% Cell survival = (100/A_t X A_s)

A_t- Absorbance of wells treated with test compounds.

A_s-Absorbance of wells treated with solvent control.

Author Information

ORCID Ranjana Aggarwal http://orcid.org/0000-0001-8848-9602

Acknowledgements

We thank the University Grants Commission, New Delhi for financial assistance in the form of Rajiv Gandhi National Fellowship to Suresh Kumar.

Conflicts of interest: The author(s) confirm that this article's content has no conflict of interest.

Supplementary material

Supplementary data to this article can be found attached.

Holmes AH, Moore LSP, Sundsfjord A, Steinbakk M, Regmi S, Karkey A et al (2016) Understanding the mechanisms and drivers of antimicrobial resistance. Lancet 387(10014):176–187. https://doi.org/10.1016/S0140-6736(15)00473-0
Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL et al (2018) Discovery, research, and development of new antibiotics: The WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 18(3):318–327. https://doi.org/10.1016/S1473-3099(17)30753-3
Miethke M, Pieroni M, Weber T, Brönstrup M, Hammann P, Halby L et al (2021) Towards the sustainable discovery and development of new antibiotics. Nat Rev Chem 5(10):726–749. https://doi.org/10.1038/s41570-021-00313-1
Terreni M, Taccani M, Pregnolato M (2021) New antibiotics for multidrug-resistant bacterial strains: Latest research developments and future perspectives. Molecules 26(9):2671. https://doi.org/10.3390/molecules26092671
Zhao B, Li Y, Xu P, Dai Y, Luo C, Sun Y et al (2016) Discovery of substituted 1H-pyrazolo[3,4- b]pyridine derivatives as potent and selective FGFR kinase inhibitors. ACS Med Chem Lett 7(6):629–634. https://doi.org/10.1021/acsmedchemlett.6b00066
Barghash RF, Eldehna WM, Kovalová M, Vojáčková V, Kryštof V, Abdel-Aziz HA (2022) One-pot three-component synthesis of novel pyrazolo[3,4-b]pyridines as potent antileukemic agents. Eur J Med Chem 227:113952. https://doi.org/10.1016/j.ejmech.2021.113952
Nafie MS, Amer AM, Mohamed AK, Tantawy ES (2020) Discovery of novel pyrazolo[3,4-b]pyridine scaffold-based derivatives as potential PIM-1 kinase inhibitors in breast cancer MCF-7 cells. Bioorg Med Chem 28(24):115828. https://doi.org/10.1016/j.bmc.2020.115828
El-Gohary NS, Gabr MT, Shaaban MI (2019) Synthesis, molecular modeling and biological evaluation of new pyrazolo[3,4-b]pyridine analogs as potential antimicrobial, antiquorum-sensing and anticancer agents. Bioorg Chem 89:102976. https://doi.org/10.1016/j.bioorg.2019.102976
Hawas SS, El-Gohary NS, Gabr MT, Shaaban MI, El-Ashmawy MB (2019) Synthesis, molecular docking, antimicrobial, antiquorum-sensing and antiproliferative activities of new series of pyrazolo[3,4-b]pyridine analogs. Synth Commun 49(19):2466–2487. https://doi.org/10.1080/00397911.2019.1618873
Salem MS, Ali MAM (2016) Novel pyrazolo[3,4-b]pyridine derivatives: Synthesis, characterization, antimicrobial and antiproliferative profile. Biol Pharm Bull 39(4):473–483. https://doi.org/10.1248/bpb.b15-00586
Al-Zharani M, Al-Eissa MS, Rudayni HA, Ali D, Alkahtani S, Surendrakumar R et al (2022) Pyrazolo[3,4-b]pyridin-3(2H)-one derivatives: Synthesis and their investigation of mosquito larvicidal activity. J King Saud Univ-Sci 34(2):101767. https://doi.org/10.1016/j.jksus.2021.101767
Mohamed LW, Shaaban MA, Zaher AF, Alhamaky SM, Elsahar AM (2019) Synthesis of new pyrazoles and pyrozolo[3,4-b]pyridines as anti-inflammatory agents by inhibition of COX-2 enzyme. Bioorg Chem 83:47–54. https://doi.org/10.1016/j.bioorg.2018.10.014
Jian X-E, Yang F, Jiang C-S, You W-W, Zhao P-L (2020) Synthesis and biological evaluation of novel pyrazolo[3,4-b]pyridines as cis-restricted combretastatin A-4 analogues. Bioorg Med Chem Lett 30(8):127025. https://doi.org/10.1016/j.bmcl.2020.127025
Tuccinardi T, Zizzari AT, Brullo C, Daniele S, Musumeci F, Schenone S et al (2011) Substituted pyrazolo[3,4-b]pyridines as human A1 adenosine antagonists: Developments in understanding the receptor stereoselectivity. Org Biomol Chem 9(12):4448. https://doi.org/10.1039/c0ob01064b
Müller CE, Jacobson KA (2011) Recent developments in adenosine receptor ligands and their potential as novel drugs. Biochim Biophys Acta - Biomembr 1808(5):1290–1308. https://doi.org/10.1016/j.bbamem.2010.12.017
Thompson S-A, Wingrove PB, Connelly L, Whiting PJ, Wafford KA (2002) Tracazolate reveals a novel type of allosteric interaction with recombinant γ-aminobutyric acid A receptors. Mol Pharmacol 61(4):861–869. https://doi.org/10.1124/mol.61.4.861
Whiting PJ, McAllister G, Vassilatis D, Bonnert TP, Heavens RP, Smith DW et al (1997) Neuronally restricted RNA splicing regulates the expression of a novel GABA A receptor subunit conferring atypical functional properties. J Neurosci 17(13):5027–5037. https://doi.org/10.1523/JNEUROSCI.17-13-05027.1997
Witherington J, Bordas V, Gaiba A, Garton NS, Naylor A, Rawlings AD et al (2003) 6-Aryl-pyrazolo[3,4-b]pyridines: potent inhibitors of glycogen synthase kinase-3 (GSK-3). Bioorg Med Chem Lett 13(18):3055–3057. https://doi.org/10.1016/S0960-894X(03)00645-0
Pan T, Xie S, Zhou Y, Hu J, Luo H, Li X et al (2019) Dual functional cholinesterase and PDE4D inhibitors for the treatment of alzheimer’s disease: Design, synthesis and evaluation of tacrine-pyrazolo[3,4-b]pyridine hybrids. Bioorg Med Chem Lett 29(16):2150–2152. https://doi.org/10.1016/j.bmcl.2019.06.056
de Mello H, Echevarria A, Bernardino AM, Canto-Cavalheiro M, Leon LL (2004) Antileishmanial pyrazolopyridine derivatives: Synthesis and structure-activity relationship analysis. J Med Chem 47(22):5427–5432. https://doi.org/10.1021/jm0401006
Sizova EE, Arshinov EV, Kotsareva YA, Glizdinskaya LV, Sagitullina GP (2017) A simple synthesis of 1H-pyrazolo[3,4-b]pyridines. Chem Heterocycl Compd 53(9):1026–1032. https://doi.org/10.1007/s10593-017-2165-y
Bhukya B, Korra R, Guguloth H (2023) Synthesis of novel amide/amino acid functionalized pyrazolo[3,4-b]pyridine derivatives; their anticancer activity and docking studies. J Heterocycl Chem 60(5):872–878. https://doi.org/10.1002/jhet.4636
Aggarwal R, Mamta, Sumran G (2019) An expeditious one-pot multicomponent synthesis of sterically hindered bis-1,2,4-triazolopyridazines under solvent-free conditions. Arkivoc 2019(6):190–199. https://doi.org/10.24820/ark.5550190.p011.009
Aggarwal R, Kumar V, Bansal A, Sanz D, Claramunt RM (2012) Multi-component solvent-free versus stepwise solvent mediated reactions: regiospecific formation of 6-trifluoromethyl and 4-trifluoromethyl-1H-pyrazolo[3,4-b]pyridines. J Fluor Chem 140:31–37. https://doi.org/10.1016/j.jfluchem.2012.04.007
Aggarwal R, Singh G, Kumar S, McCabe T, Rozas I (2016) Preparation of 4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-nitriles in a multicomponent domino process. Chem Select 1(18):5990–5994. https://doi.org/10.1002/slct.201601201
Aggarwal R, Kumar S, Singh G (2019) Multi-component reaction to access a library of polyfunctionally substituted 4,7-dihydropyrazolo[3,4-b]pyridines. Synth Commun 49(7):973–985. https://doi.org/10.1080/00397911.2019.1582064
Baghernejad B (2011) Application of p-toluenesulfonic acid (PTSA) in organic synthesis. Curr Org Chem 15(17):3091–3097. https://doi.org/10.2174/138527211798357074
Xie C, Zhang Z, Li D, Gong J, Han X, Liu X et al (2017) Dimethyl sulfoxide involved one-pot synthesis of quinoxaline derivatives. J Org Chem 82(7):3491–3499. https://doi.org/10.1021/acs.joc.6b02977
Zhang Z, Tian Q, Qian J, Liu Q, Liu T, Shi L et al (2014) Dimethyl sulfoxide participant iron-mediated cascade oxidation/α-formylation reaction of substituted 2,3-dihydropyrroles under air and protonic acid free condition. J Org Chem 79(17):8182–8188. https://doi.org/10.1021/jo501385w
Ren X, Chen J, Chen F, Cheng J (2011) The palladium-catalyzed cyanation of indole C-H bonds with the combination of NH₄HCO₃ and DMSO as a safe cyanide source. Chem Commun 47(23):6725. https://doi.org/10.1039/c1cc11603g
Lv Y, Li Y, Xiong T, Pu W, Zhang H, Sun K et al (2013) Copper-catalyzed annulation of amidines for quinazoline synthesis. Chem Commun 49(57):6439. https://doi.org/10.1039/c3cc43129k
Wu X, Zhang J, Liu S, Gao Q, Wu A (2016) An efficient synthesis of polysubstituted pyridines via C_sp3-H oxidation and C-S cleavage of dimethyl sulfoxide. Adv Synth Catal 358(2):218–225. https://doi.org/10.1002/adsc.201500683
Um I-H, Han J-Y, Buncel E (2009) Effects on the reactivity by changing the electrophilic center from C = O to C = S: Contrasting reactivity of hydroxide, p-chlorophenoxide, and butan-2,3-dione monoximate in DMSO/H₂O mixtures. Chem-A Eur J 15(4):1011–1017. https://doi.org/10.1002/chem.200801534
Wikler MA (2009) Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically, Approved Standard M7-A8;

Schemes 1 and 3 are available in the Supplementary Files section

GA.png
Scheme1.png
Scheme 1. Strategy for synthesis of 11a via multicomponent approach.
Scheme2.png
Scheme 2. Synthetic route of 4-aminopyrazolo[3,4-b]pyridine derivatives 11b-i.
Scheme3.png
Scheme 3. Plausible mechanistic route for the formation of compounds 11.
RevisedSupplementaryfiles.docx

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One-pot synthesis and in vitro bioactivity of novel 4-aminopyrazolo[3,4-b]pyridine derivatives as potential antimicrobial compounds

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Synthesis

General procedure for the synthesis of 3-(aryl)-6-substituted-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-amines (11a-i)

Antimicrobial activity

Toxicity studies

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