Fungi of the genus Aspergillus are common contaminants of food and feed stuffs. Many species are saprobes and are found in a variety of habitats and are ubiquitous agents of decay. Several Aspergillus species such as Aspergillus niger have been found associated with production of mycotoxins of public health importance [1]. As such, the occurrence of Aspergillus secondary metabolites in food stuffs is becoming an increasing environmental concern. So, in order to effectively reduce the infection of food stuffs by fungi especially, toxigenic Aspergillus (Aspergillus niger ), it is important to identify new compounds, as rapidly as possible, able to inhibit fungal activity and consequently protect our environmental [2].

In our interest in the development of synthetic strategies to obtain functionalized heterocycles, [3-8] we have concentrated much of our recent efforts in the preparation of such bioactive nitrogen-containing heterocycles, and have already reported simple and efficient procedures to reach interesting molecules such as pyridin-2-ones [9], isoxazolo[5,4-b] pyridines [10], pyrido[2,3-d] pyrimidines [11], 2- amino-5-arylpyridine-3-carbonitrile [12] and pyrazolo[3,4-b]pyridin-3-ones [13] using vinamidinium salts, with biological properties as shown in Figure 1 [14-19]. Then, the simple fact that the vast majority of currently used drugs are heterocyclic compounds has encouraged a lot of researchers to synthesize analogous pyrazolopyridines derivatives in order to find new molecules with higher activities and consequently optimize their pharmacological profile. The biological properties of pyrazolopyridines heterocycles are very promising and many of them have been studied, especially as antitumor agents. This review is mainly focused on the synthesis of various types of nitrogen containing heterocyclic pyrazolopyridines for their potential use as future drugs in medicine and in addition discusses their overall biological activities. Recently, just one team reported an antibacterial activities study of diarilpyrazolo [3,4-b] piridines. Eleven derivatives was tested against six different bacterial strains; Staphylococcus aureus (gram +), Pseudomonas (gram–), Escherichia coli (gram–) and Klebsiella (gram–).

Figure 1: Synthesis of substituted pyrazolo [3,4-b]pyridines derivatives PYR_1-5

Only four compounds showed moderate activity against S. aureus, Klebsiella, Pseudomonas and E. coli [20]. The global threat of antimicrobial resistance is a serious matter under review by the WHO and many countries throughout the world [21-24]. Currently, the widespread selective pressure and the efficient dissemination channels for multidrug-resistant organisms are major factors that may have contributed to the rapid emergence of the resistant organisms [21-25].

Fused heterocyclic containing pyrazolopyridine systems have been described associated with several biological and medicinal activities including anxiolytic [26], antiviral [27,28], antileishmanial [29] and anti-inflammatory [30] profiles [31]. However, pyrazolo[3,4-b] pyridine derivatives have been studied as antiviral [32], potential antimalarial agents [33], compounds inhibiting cholesterol formation [34], for the treatment of Alzheimer’s disease, gastrointestinal diseases, anorexia nervosa, drug and alcohol withdrawal symptoms, drug addiction and infertility [35]. They have also been reported as potent and selective inhibitors of A1 adenosine receptors [36] and phosphodiesterase 4 (PDE4) inhibitors in immune and inflammatory cells [37]. This review is the first of its kind to investigate the antimicrobial activity of various pyrazolo [3,4- b]pyridines reported until now. This review is a concerted effort to highlight design, synthetic strategies as well as biological activities of this class of condensed heterocycles. Structure-activity relationship studies and in silico approaches wherever reported have also been discussed.

General experimental procedure for the preparation of PYR_1-5

To a flame dried one-necked round-bottomed flask equipped with magnetic stirring, reflux condenser and nitrogen atmosphere was added vinamidinium salt VIN_1-5 (0.48 mmol), 3-amino-1-phenyl-2- pyrazolin-5-one 1 (0.48 mmol), sodium methoxide (0.96 mmol) and anhydrous methanol (6 mL). The mixture was allowed to reflux overnight under a nitrogen atmosphere in an oil bath. The flask was cooled to room temperature and the obtained precipitate was filtered off to give substituted pyrazolo [3,4-b] pyridines derivatives PYR_1-5, neither Work-up nor purification. For pyrazolo [3,4-b] pyridine PYR₃, after evaporation of the solvent, THF (6 mL) and 1 N HCl (6 mL) were added. The mixture was allowed to stir at room temperature for 2 h and filtration as above gave directly PYR₃.

All products were characterized from their ¹H NMR, ¹³C NMR and mass spectroscopic data. The chemical structures are summarized in Figure 2, in which the five synthesized compounds are illustrated.

Figure 2: Pyrazolo [3,4-b]pyridines derivatives PYR_1-5 structures

In vitro antibacterial and antifungal assay

Antimicrobial screening of the substituted pyrazolo [3,4-b]pyridines derivatives (PYR1-5) was performed by the Agar well diffusion method [38] and the dilution method (agar and liquid broth). The antimicrobial activity was evaluated using two different laboratory control strains of bacteria, i.e., Gram positive Bacillus subtilis CIP 5262, Gram negative, Escherichia coli CIP 53126. Two fungal strains were Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231. Fungal strains were procured from the culture maintained at Pasteur Institute of Tunis. All tests were triplicates in Muller Hinton broth for the bacterial strains and were swabbed on sterile Muller Hinton agar plates using sterile cotton swab followed by punching wells of 6 mm with the help of sterile cork borer. Overnight broth cultures of each strain were prepared, and the final concentration in each well was adjusted to (20, 10 and 5 μM) concentration against both bacterial and fungal strains. DMSO was used as a vehicle. Spiramycin (20, 10 and 5 μM), Streptomycin (20, 10 and 5 μM) and Fluconazole (20, 10 and 5 μM) were used as standard drugs for comparison of antibacterial and antifungal activities respectively. The zone of inhibition was measured after 24 h of incubation at 37°C for antibacterial activity and 72 h at 25°C for antifungal activity.

Statistical analysis

Data of antibacterial and antifungal activity assays were subjected to one-way analysis of variance (ANOVA) using the SPSS 21 Difference between means were tested through Student-Newman-Keuls test and values of P ≤ 0.05 were considered significantly different.

Antibacterial and antifungal activities of substituted pyrazolo [3,4-b]pyridines derivatives PYR1-5

The pyrazolopyridines compounds were tested for antibacterial and antifungal activities in vitro against respectively Bacteria (Escherichia coli CIP 53126 and Bacillus subtilis CIP 5262) and Fungi (Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404). According statistical analysis, all tested compounds showed that pyrazolopyridines significantly reduced the growth of all tested Fungi and Bacteria in dose-dependent manner (Tables 1-5). The five substituted derivatives exhibited different inhibition effects on the growth of Fungi and Bacteria. In general, there was a correlation between the antibacterial, antifungal activities and structures.

Table 1: Inhibitory activity of standard drugs against Escherichia coli, Bacillus subtilis and Candida albicans (means ± SE). Means in the same column with the same upper case letter are not significantly different according to the Student-Newman-Keuls test (P ≤ 0.05). Values are means of three replicates ± SE.

Table 2: Inhibitory activity of PYR1 against Escherichia coli, Bacillus subtilis and Candida albicans (means ± SE). Means in the same column with the same upper case letter are not significantly different according to the Student-Newman-Keuls test (P ≤ 0.05). Values are means of three replicates ± SE.

Table 3: Inhibitory activity of PYR2 against Escherichia coli, Bacillus subtilis and Candida albicans (means ± SE). Means in the same column with the same upper case letter are not significantly different according to the Student-Newman-Keuls test (P≤ 0.05). Values are means of three replicates ± SE.

Table 4: Inhibitory activity of PYR₃ against Escherichia coli, Bacillus subtilis and Candida albicans (means ± SE). Means in the same column with the same upper case letter are not significantly different according to the Student-Newman-Keuls test (P ≤ 0.05). Values are means of three replicates ± SE.

Table 5: Inhibitory activity of PYR₄ against Escherichia coli, Bacillus subtilis and Candida albicans (means ± SE). Means in the same column with the same upper case letter are not significantly different according to the Student-Newman-Keuls test (P ≤ 0.05). Values are means of three replicates ± SE.

Table 6: Inhibitory activity of PYR₅ against Escherichia coli, Bacillus subtilis and Candida albicans (means ± SE). Means in the same column with the same upper case letter are not significantly different according to the Student-Newman-Keuls test (P ≤ 0.05). Values are means of three replicates ± SE.

Despite significant progresses in antimicrobial therapy, infectious diseases caused by bacteria and fungi remain a major worldwide health problem because of the rapid development of resistance to existing antimicrobial drugs. Therefore, there is a constant need for new antimicrobial agents. There are a large number of heterocyclic derivatives containing nitrogen atoms that possess a broad spectrum of biological activities including pyridine and pyrazole, which are two of the most important heterocycles in medicinal chemistry.

Refer to the Ibrahim et al. [39] works new pyridine derivatives are synthesized. Most of these compounds were tested and evaluated as antimicrobial agents and the results indicated that many of the obtained compounds exhibited high antimicrobial activity comparable to ampicillin, which was used as the reference compound. Most of the synthesized compounds were found to exhibit strong inhibitory effects on the growth of Gram-positive bacteria especially Bacillus subtilis CIP 5262. Furthermore, a large number of structurally diverse natural compounds containing azole nucleus constitute an important class of biologically active heterocycles that are gaining more attention in the field of medicinal chemistry. Among azoles, pyrazoles are rarely found in nature probably due to difficulty in the formation of N-N bond by living organisms. However, they exhibit numerous biological activities, including anti-diabetic, antiviral, anticancer, anti-inflammatory, antibacterial and antifungal activities. Therefore, Kumar et al. review, for example, [40] was an attempt to understand the chemistry along with medicinal importance of pyrazole containing natural products reported till date which would certainly help the scientific community to bring further developments in the isolation and synthetic methodologies for pyrazole based novel bioactive compounds.

In addition, refer to the literature, 3,5-dimethylpyrazole (1.29%) and pyrazole (0.16%) are two of the chemical composition of the essential oil isolated from the floral parts of Nandina domestica Thunb. Bajpai et al. [41] tested the efficacy of essential oil and various organic extracts against a panel of food-borne pathogenic and spoilage bacteria such as Bacillus subtilis ATCC6633, Listeria monocytogenes ATCC19166, Staphylococcus aureus KCTC1916, S. aureus ATCC6538, Pseudomonas aeruginosa KCTC2004, Salmonella typhimurium KCTC2515, Salmonella enteridis KCCM12021, Escherichia coli 0157- Human, E. coli ATCC8739, E. coli 057:H7 ATCC43888 and Enterobacter aerognes KCTC2190. Thus, the oil had strong detrimental effect on the viable count of the tested bacteria. The obtaned results indicate the potential efficacy of plant-based natural products such as essential oil and organic extracts of N. domestica to control food-borne pathogenic and spoilage bacteria. In this context, we were intrigued by the number of reports that derivatives of pyrazole and pyridine have a wide range of important biological activities. Consequently, we synthesized analogues that contain both pharmacophores in the framework of one molecule. In addition, the synthesized pyrazolopyridine derivatives were subjected to antibacterial activity. Herein we report the result of that study and discuss their structureactivity relationship.

PYR₁ is a pyrazolo[3,4-b]pyridine derivative with phenyl moiety substituted, so in comparison with the Chemical compositions of the essential oils having the same moiety, we can make mention of phenylalanine, phenylpropanoids and 1,2-dimethoxybenzene (veratrole) studies.

So, in the aim to determinate Chemical composition of Gongronema latifolium leaves, Eleyinmi AF [42] demonstrated that phenylalanine is dominant essential amino acids then concluded that the aqueous extracts show activity against E. coli and P. aeruginosa while methanol extracts are active against P. aeruginosa and L. monocytogenes . Thus, G. latifolium has potential food and antibacterial uses.

In another report, Silveira e Sá et al. [43] aimed the search for alternative drugs capable of disrupting the inflammatory process especially with reference to the use of natural substances (essential oils). So, they made an overview of the anti-inflammatory action exerted by phenylpropanoids from essential oils and discussed possible mechanisms of action involved in the anti-inflammatory response, assessed through specific experimental models. A previous study showed that 1,2-dimethoxybenzene (veratrole) is a potent pollinator attractant to the nocturnal moth Hadena bicruris emited by white campion (Silene latifolia: a dioecious plant). Akhtar et al. [44] explored firstly the biosynthetic route to veratrole. Then, suggested that it was derived by the methylation of guaiacol (2-methoxyphenol), which itself originates from phenylalanine via BA (benzoic acid) and SA (salicylic acid), and therefore implies a novel branch point of the general phenylpropanoid pathway.

In the other hand, PYR2 is a pyrazolo[3,4-b]pyridine derivative with methoxyphenyl moiety substituted, so in comparison with the Chemical compositions of the essential oils having the same moiety, we can make mention of anethole and methyl chavicol studies. According to Soylu et al. [45] study, major compounds found in essential oils of fennel (Foeniculum vulgare) were anethole (82.8%), it is a cyclic hydrocarbon with methoxyphenyl moiety substituted. Thus, the aim of this group was to find an alternative to synthetic fungicides currently used in the control of devastating oomycete pathogen Phytophthora infestans, causal agent of late blight disease of tomato. So, antifungal activities of essential oils obtained from aerial parts of aromatic plants such as fennel were investigated against P. infestans. These essential oil were found to inhibit the growth of P. infestans in a dose-dependent manner. Complete growth inhibition of pathogen by essential oil of fennel was, however, observed at 0.4–2.0 μg/ml air concentrations. For the determination of the contact phase effects of the tested essential oil, fennel oil at 6.4 μg/ml were found to inhibit the growth of P. infestans completely. Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. Sporangial production was also inhibited by the fennel oil.

Besides, Sarin Tadtong et al. [46] claimed that the major composition of the essential oil from rhizome of Etlingera punicea (Roxb.) is methyl chavicol, (a cyclic hydrocarbon with methoxyphenyl moiety substituted). Their study aims to determine the chemical compositions and the antimicrobial activities of the essential oil from rhizome of Etlingera punicea (Roxb.) R.M. Smith. The rhizome of plant is used as spice and ingredient in noodle and curry in Thailand. This essential oil possesses fungicidal activity against Candida albicans ATCC 10231 and shows bacteriostatic activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella albany DMST 10641, whilst no activity was found on Gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 by agar disc diffusion assay. So, they concluded that The bacteriostatic and anitifungal activities of the essential oil from the rhizome of E. punicea might be due to the presence of methyl chavicol as previously reported [47]. The possible antimicrobial action of methyl chavicol might be arrived from the complexation between the protein or other components of the cell membrane of the microbes and the phenolic components in the essential oil as previous reported [48]. Furthermore, methyl chavicol showed limited and weak antibacterial activity [49] which was similar to our report. According to its antifungal activity, the volatile oil from this plant might be used as an active ingredient in antifungal formulations.

In the case of our bacteriological study, both of PYR₁ and PYR₂ have a better activity against Bacillus subtilis Gram-positive as compared with the standard drug Spiramycin. However, the compound PYR₂ exhibited significantly higher activity than PYR₁ against Escherichia coli CIP 53126 Gram-negative. But PYR1 exhibit significantly higher activity than PYR₂ against Bacillus subtilis CIP 5262 Gram-positive as compared with standard drugs (Streptomycin and Spiramycin). It means that the substitution of methoxy moiety, an electron donating at the p-position in the phenyl ring in PYR₂ increased its activity against Escherichia coli CIP 53126 Gram-negative and decreased it against Bacillus subtilis CIP 5262 Gram- positive.

Likewise, PYR₃ is an aldehyde, however aldehydes are oxygenated compounds chemical constituent of essential oils, such as Citral and Citronellal. So, according to Grace O. Onawunmi work [50], citral showed appreciable antimicrobial activity against Gram-positive and Gram-negative bacteria as well as fungi. Media composition and inoculum size had no observable effect on activity but alkaline pH increased citral activity. The growth rates of Escherichia coli cultures were reduced at concentrations of citral ≥ 0.01% v/v while concentrations ≥ 0.03% v/v produced rapid reduction in viable cells followed by limited regrowth. In a non-growth medium, 0.08% and 0.1% v/v showed rapid bactericidal effects. Citral may therefore be of preservative use in addition to its other uses in the food, soap and cosmetic industries.

Moreover, Manosroi et al. [51] reported that the essential oil of kaffir lime peels was the mixture of many compounds such as β-pinene (30.6 %), limonene (29.2%), sabinene (22.6 %) and citronellal (4.2 %), which was correlated to the study of Chanthaphon1 et al. who investigayed Ethyl acetate extracts and hydrodistillated-essential oils (from peels of Citrus spp.) for their antimicrobial activities against food related microorganisms by broth microdilution assay. Overall, ethyl acetate extracts from all citrus peels showed stronger antimicrobial activities than their essential oils obtained from hydrodistillation. The ethyl acetate extract of kaffir lime (Citrus hystrix DC.) peel showed broad spectrum of inhibition against all Gram-positive bacteria, yeast and molds including Staphylococcus aureus, Bacillus cereus , Listeria monocytogenes, Saccharomyces cerevisiae var. sake and Aspergillus fumigatus TISTR 3180. They concluded that the higher antibacterial activity against Gram-positive bacteria of the ethyl acetate extract than the hydrodistillated-essential oil from kaffir lime peel was related to citronellal content which was also higher in the ethyl acetate extract. Therefore citronellal could be a potential component, which contributed to the antibacterial activity.

In this study, compound PYR₃ displayed excellent activity against Bacillus subtilis CIP 5262 Gram-positive and Candida albicans ATCC 10231 as compared with standard drugs (Spiramycin and Fluconazole) respectively.

Then, PYR₃ exhibited moderate activity against Escherichia coli CIP 53126 Gram-negative.

Therefore, the presence of aldehyde function in the pyrazolopyridines compounds increased its activity against Bacillus subtilis CIP 5262 Gram-positive and Candida albicans ATCC 10231.

Furthermore, PYR₄ exhibited excellent activity against Escherichia coli CIP 53126 Gram-negative and Bacillus subtilis CIP 5262 Gram-positive as compared with standard drugs (Streptomycin and Spiramycin respectively).

In the other hand, and recording to the literature [52], antibacterial and antifungal activity of N-phthaloylamino acid hydroxamates 1–3 C₆H₄(CO)₂N-X-CONHOH, X = amino acid residues of glycine, alanine or D-phenylglycine was examined against 44 strains of Gram-positive and Gram-negative bacteria, and 10 species of yeasts. N- phthaloyl-D-phenylglycine-hydroxamic acid (3), the substance with the highest lipophilicity (log P), showed the best antibacterial activity, especially against Gram-negative bacteria.

Therefore, coordination compounds derived from N-phthaloylglycine and N-phthaloyltyrosine were synthesized [53]. The ligands were formed by 1:2 molar condensation of phthalic anhydride with tyrosine and glycine respectively. The complexes were formulated as [Zn(PHG)₂ (H₂O)₂] (OAc)₂, [Ni(PHG)₂ (H₂O)₂] (OAc)₂ and [Cu(PHT)₂ ] (OAc)₂. The antimicrobial studies using four test organisms (P. aerugenosa, E. Coli , S. aureus and C. albicans) revealed that the metal complexes exhibit higher activity than their respective ligands.

According to Begum et al. [54] study, two series of Nphthaloylglycine derivatives were synthesized under SchottenBaumann conditions. The first series consists of Nphthaloylglycine amides (4ah), and the second one consists of benzimidazole derivatives of Nphthaloylglycine. All the synthesized analogues were evaluated for their in vitro antimicrobial activity. All the synthesized compounds exhibited a wide range of antibacterial activity against all of the Staphylococcus aureus resistant strains tested.

Almost, in this research we found that compound PYR₅ exhibited the best activity with regard to the others compounds against Escherichia coli CIP 53126 Gram-negative and displayed excellent activity as compared with standard drug (Spiramycin).

Fungal infection has dramatically increased in immunosuppressed patients such as those with AIDS or who have had organ or marrow transplants over the past decades [55-57]. Currently, some antifungal agents of major drug classes: azoles, polyenes, and candins, have been launched and are demonstrating remarkable success for the treatment of fungal systemic infections [56-61]. However, these usages are often limited mainly due to their unsatisfactory antifungal activity, narrow spectrum, and side effects, causing rapid development of drug resistance and a high rate of mortality [61-64]. In consideration of these facts, alternative agents for the treatment and prevention of fungal infections are urgently required, preferably with a novel mode of action [65]. In this way, we screened for Fungi against respectively (Candida albicans ATCC 10231) by the paper disc diffusion method and (Aspergillus niger ATCC 16404) by the dilution method (agar and liquid broth). So, in the present study we reported, firstly that compound PYR₃ exhibited the best activity with regard to the others compounds against Candida albicans ATCC 10231 and displayed excellent activity as compared with standard drugs (Fluconazole). Then after 72 h of incubation, at 25°C for antifungal activity, we measured a total inhibition of all substituted pyrazolo[3,4-b]pyridines derivatives PYR1-5 against (Aspergillus niger ATCC 16404). Then, we concluded that these compounds have a fungistatic action, as the mold begins to develop resistance against these derivatives, once the period of incubation is prolonged.

So, these compounds could be useful as templates for further development through modification or derivatization to design more potent antifungal agents.

The antimicrobial activity of the substituted pyrazolo[3,4- b]pyridines derivatives (PYR_1-5) synthesized was examined and it was found that some of these compounds have similar or higher activity compared with commercial antibiotic drugs (Streptomycin, Spiramycin and Fluconazole), which make them suitable for diverse applications like the manufacturing of drugs, pesticides, emulsifiers, cosmetics, etc.

Data from this study (total inhibition against (Aspergillus niger ATCC 16404) provide evidence that these pyrazole formulations could be an alternative source for the treatment of fungal infections caused by Aspergillus niger ATCC 16404. However, a specific study on the safety and efficacy of these in vivo and clinical trials is still needed, in order to evaluate the practical relevance of the in vitro results and consequently protect our environmental.

5-Phenoxy-2-phényl-1,6-dihydropyrazolo[3,4-b]pyridine-3- one (PYR₄)

Yield: 86%; yellow solid; mp=120°C; 1H NMR (300 MHz, CDCl3):

δ=8.2 (d, 1H, J=4.2), 7.9 (d, 1H, J=4.2), 7.45-7.88 (m, 10H), 6.65 (s, 1H); 13C NMR (75 MHz, CDCl3) d=164.2, 157.2, 155.09, 148.3, 135.8, 130.2, 130.3, 126.37, 125.3, 123.4, 120.4, 118.65, 115.9, 112.77. Anal. Calcd for C18H12N3ClO: C, 71.23; H, 4.29; N, 13.85. Found: C, 71.20; H, 4.27; N, 13.84%. MS (EI, 30 eV): m/z=303 (M+).

2-Phényl-5-(N-phthaloyl)-1,6-dihydropyrazolo[3,4- b]pyridine-3-one (PYR₅)

Yield: 86%; yellow solid; mp=120°C; 1H NMR (300 MHz, CDCl3):

δ=8.16 (d, 1H), 8.25 (d, 1H), 7.9-8.1 (m, 7H), 7.4-7.6 (m, 2H), 7.3 (s, 1H); 13C NMR (75 MHz, CDCl3) d=195.0, 160.2, 158.2, 144.9, 140.8, 135.2, 130.2, 130.5, 127.7, 126.3, 125.8, 115.4, 110, 108.5, 50.9. Anal. Calcd for C18H12N3ClO: C, 71.18; H, 3.41; N, 11.85. Found: C, 71.16; H, 3.40; N, 11.82%. MS (EI, 30 eV): m/z=354 (M+).