Synthesis of Pyrazolo[3,4-b]pyridine Derivatives and Their In-Vitro and In-Silico Antidiabetic Activities.

In the current study, new pyrazolo[3,4-b]pyridine esters, hydrazides, and Schiff bases have been synthesized starting from 3-methyl-1-phenyl-1H-pyrazol-5-amine. The first step involved solvent-free synthesis of pyrazolo[3,4-b]pyridine-6-carboxylate derivatives (2a-d) with 55%-70% yield in the minimum time frame compared with the conventional refluxing method, which was followed by the synthesis of corresponding hydrazides (3a-d) and hydrazones (4a-e). The structures of the synthesized derivatives were confirmed using element analysis, FT-IR, 1H NMR, 13C NMR, and LC-MS techniques. Synthesized hydrazides (3a-d) and hydrazones (4a-e) were also tested for their in-vitro antidiabetic activity and found that all the compounds exhibited significant antidiabetic activity, while 3c (IC50 = 9.6 ± 0.5 µM) among the hydrazides and 4c (IC50 = 13.9 ± 0.7 µM) among the hydrazones were found to be more active in comparison to other synthesized derivatives. These in-vitro results were further validated via docking studies against the α-amylase enzyme using the reference drug acarbose (200.1 ± 10.0 µM). The results were greatly in agreement with their in-vitro studies and these derivatives can be encouraging candidates for further in-vivo studies in mice models.

Differentiation of regioisomeric N-alkylation of some indazoles and pyrazolopyridines by advanced NMR techniques.

Indazole scaffold have two interconvertible tautomeric forms. Regioselectivities were determined for N-benzylations and alkylation of some non-substituted and substituted indazoles, under basic conditions (K2CO3) in DMF. The ratio of regioisomers occurrence between N1:N2 is almost equal. Their structures were established through a combination of NOESY and 1H-13C/15N HMBC NMR methods. Additionally, pyrazolo[3,4-b]pyridines have also three possible tautomeric forms; primarily 1H and 2H, with 7H isomers being rare. Pyrazolo[4,3-b]pyridines have only known two possible tautomeric forms so far; 1H and 2H.

Identification of 1H-pyrazolo[3,4-b]pyridine derivatives as novel and potent TBK1 inhibitors: design, synthesis, biological evaluation, and molecular docking study.

sABSTRACTTANK-binding kinase 1 (TBK1), a noncanonical member of the inhibitor-kappaB kinases (IKKs) family, plays a vital role in coordinating the signalling pathways of innate immunity, involving in the process of neuroinflammation, autophagy, and oncogenesis. In current study, based on rational drug design strategy, we discovered a series of 1H-pyrazolo[3,4-b]pyridine derivatives as potent TBK1 inhibitors and dissected the structure-activity relationships (SARs). Through the several rounds of optimisation, compound 15y stood out as a potent inhibitor on TBK1 with an IC50 value of 0.2 nM and also displayed good selectivity. The mRNA detection of TBK1 downstream genes showed that compound 15y effectively inhibited TBK1 downstream IFN signalling in stimulated THP-1 and RAW264.7 cells. Meanwhile, compound 15y exhibited a micromolar antiproliferation effect on A172, U87MG, A375, A2058, and Panc0504 cell lines. Together, current results provided a promising TBK1 inhibitor 15y as lead compound for immune- and cancer-related drug discovery.

3-Aminopyrazolo[3,4-b]pyridine: Effective Precursor for Barium Hydroxide-Mediated Three Components Synthesis of New Mono- and Bis(pyrimidines) with Potential Cytotoxic Activity.

In this study, an efficient one-pot procedure for preparing a new series of pyrazolo[3,4-b]pyridine-fused pyrimidines was described. The target hybrids were developed through a three-component reaction of 3-amino-1H-pyrazolo[3,4-b]pyridine, benzaldehydes, and acetophenones (molar ratio 1 : 1 : 1). The best conditions for the previous reaction were 2.5 equivalents of barium hydroxide in DMF at 150 °C for 6 h. New bis(pyrimidines) were synthesized in high yields using a similar one-pot reaction protocol with some modifications. Thus, two equivalents of each of the appropriate acetophenones and 3-aminopyrazolopyridine were reacted with one equivalent of the appropriate bis(aldehydes). The reaction was carried out at 150 °C for 8 h using 4.5 equivalents of barium hydroxide in DMF. Repeating the previous reaction with the appropriate bis(acetyl) derivatives and benzaldehydes resulted in good yields of the target bis(pyrimidines). The in vitro cytotoxic activity of new pyrimidines against the MCF-7, HEPG2, and Caco2 cell lines was evaluated using the reference doxorubicin (IC50 values of 4.34-6.97 µM). Hybrid 6h had the best activity against Caco2 and MCF-7 cell lines, IC50 values of 12.62 and 14.50 µM, respectively. The IC50 values for hybrids 6c, 6e, and 6f against MCF-7 and Caco2 cell lines were 23.99-41.69 and 33.14-43.33 µM, respectively. Furthermore, hybrid 6e displayed IC50 value of 20.06 µM HEPG2 cell lines, while the hybrids 6c, 6f and 6h exhibited IC50 values ranging between 26.29-50.51 µM. Furthermore, hybrid 6e had an IC50 value of 20.06 µM for the HEPG2 cell lines, whereas hybrids 6c, 6f, and 6h had IC50 values ranging from 26.29 to 50.51 µM.

Novel pyrazolo[3,4-b]pyridine derivatives: Synthesis, structure-activity relationship studies, and regulation of the AMPK/70S6K pathway.

A series of novel pyrazolo[3,4-b]pyridine derivatives were designed, synthesized, and biologically evaluated for anti-lung cancer activity. Structure-activity relationship and AutoGPA models were constructed based on the in vitro antiproliferative potency of the compounds against a human lung adenocarcinoma cell line (A549). Compound 9d exhibits improved potency for A549 cell growth inhibition (3.06 ± 0.05 µM) compared with A-769662 (45.29 ± 2.14 µM). Compound 9d can elevate the phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and its substrate acetyl-CoA carboxylase and reduce the level of phosphorylated ribosomal S6 kinase (p-70S6K) at 1 µM, which is comparable to the activity of A-769662 at 20 µM. 9d induced G2/M cell cycle arrest, which was rescued when co-incubated with "Compound C," a potent AMPK inhibitor. Taken together, compound 9d showed potential anti-lung cancer activity via inducing cell cycle arrest by regulation of the AMPK/70S6K pathway in A549 cells, which could provide a new lead for the discovery of anti-lung cancer agents.

Synthesis of Diversified Pyrazolo[3,4-b]pyridine Frameworks from 5-Aminopyrazoles and Alkynyl Aldehydes via Switchable C≡C Bond Activation Approaches.

A cascade 6-endo-dig cyclization reaction was developed for the switchable synthesis of halogen and non-halogen-functionalized pyrazolo[3,4-b]pyridines from 5-aminopyrazoles and alkynyl aldehydes via C≡C bond activation with silver, iodine, or NBS. In addition to its wide substrate scope, the reaction showed good functional group tolerance as well as excellent regional selectivity. This new protocol manipulated three natural products, and the arylation, alkynylation, alkenylation, and selenization of iodine-functionalized products. These reactions demonstrated the potential applications of this new method.

Molecular Hybrids of Pyazolo[3,4-b]pyridine and Triazole: Design, Synthesis and In Vitro Antibacterial Studies.

Antimicrobial resistance is on the rise, and there aren't enough new treatments to combat it. This might send the modern world back to the pre-antibiotic age. The molecular hybrids of pyrazolo[3,4-b]pyridine and triazole have been designed, synthesized, and analyzed for their drug-like molecule nature and in vitro analyses for their inhibition potentials against S. aureus and K. pneumoniae. The compounds 24 and 27 have been identified as the high potential molecules in this series based on in vitro experiments. Compound 24 has zone of inhibition values of 15 ± 0.82 mm and 14 ± 0.7 mm, whilst compound 27 has zone of inhibition values of 18 ± 0.95 mm and 16 ± 0.82 mm against S. aureus and K. pneumoniae, respectively. MIC and MIB values for compounds 24 and 27 against S. aureus and K. pneumoniae are 0.25 and 0.5, respectively.

Synthesis and bioevaluation of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amines as tubulin polymerization inhibitors with anti-angiogenic effects.

A new series of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine derivatives as tubulin polymerization inhibitors were synthesized, and evaluated for the anti-proliferative activities. A structure-activity relationship study revealed that the free amino moiety of 1H-pyrazolo[3,4-b]pyridin-3-amine played an essential role in the anti-proliferative activities. Especially, compound 15c displayed the strongest anti-proliferation against MCF-7 cells with IC50 value of 0.067 ± 0.003 µM, and high selectivity over the normal human embryonic lung WI-38 cells with IC50 value of 23.41 ± 1.53 µM. Further mechanistic studies revealed that 15c showed strong anti-tubulin polymerization activity, changed the morphology of tubulin, and arrested the cell cycle at the G2/M transition in MCF-7 cells. Molecular docking analysis suggested that 15c well occupied the colchicine-binding pocket of tubulin. Additionally, 15c demonstrated anti-angiogenic activities with blocking the migration, invasion and tube formation, disrupting the newly formed tube, and regulating both MMP-9 and TIMP-1 in HUVEC cells. In summary, our results highlight that compound 15c is a potential antitumor compound that are worthy of further development.

Trypanocidal activity of new 1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine derivatives: Synthesis, in vitro and in vivo studies.

Despite the serious public health problems caused by Chagas disease in several countries, the available therapy remains with only two drugs that are poorly active during the chronic phase of the disease in addition to having severe side effects. In search of new trypanocidal agents, herein we describe the synthesis and biological evaluation of eleven new 1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine compounds containing the carbohydrazide or the 2,3-dihydro-1,3,4-oxadiazole moieties. Two of them showed promising in vitro activity against amastigote forms of T. cruzi and were evaluated in vivo in male BALB/c mice infected with T. cruzi Y strain. Our results suggest that the substitution at the C-2 position of the phenyl group connected to the carbohydrazide or to the 2,3-dihydro-1,3,4-oxadiazole moieties plays an important role in the trypanocidal activity of this class of compounds. Moreover, the compound containing the 2,3-dihydro-1,3,4-oxadiazole moiety has demonstrated more favorable structural requirements for in vivo activity than its carbohydrazide analog.

Discovery of New Pyrazolopyridine, Furopyridine, and Pyridine Derivatives as CDK2 Inhibitors: Design, Synthesis, Docking Studies, and Anti-Proliferative Activity.

New pyridine, pyrazoloyridine, and furopyridine derivatives substituted with naphthyl and thienyl moieties were designed and synthesized starting from 6-(naphthalen-2-yl)-2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (1). The chloro, methoxy, cholroacetoxy, imidazolyl, azide, and arylamino derivatives were prepared to obtain the pyridine--C2 functionalized derivatives. The derived pyrazolpyridine-N-glycosides were synthesized via heterocyclization of the C2-thioxopyridine derivative followed by glycosylation using glucose and galactose. The furopyridine derivative 14 and the tricyclic pyrido[3',2':4,5]furo[3,2-d]pyrimidine 15 were prepared via heterocyclization of the ester derivative followed by a reaction with formamide. The newly synthesized compounds were evaluated for their ability to in vitro inhibit the CDK2 enzyme. In addition, the cytotoxicity of the compounds was tested against four different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549). The CDK2/cyclin A2 enzyme inhibitory results revealed that pyridone 1, 2-chloro-6-(naphthalen-2-yl)-4-(thiophen-2-yl)nicotinonitrile (4), 6-(naphthalen-2-yl)-4-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridin-3-amine (8), S-(3-cyano-6-(naphthaen-2-yl)-4-(thiophen-2-yl)pyridin-2-yl) 2-chloroethanethioate (11), and ethyl 3-amino-6-(naphthalen-2-yl)-4-(thiophen-2-yl)furo[2,3-b]pyridine-2-carboxylate (14) are among the most active inhibitors with IC50 values of 0.57, 0.24, 0.65, 0.50, and 0.93 µM, respectively, compared to roscovitine (IC50 0.394 µM). Most compounds showed significant inhibition on different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549) with IC50 ranges of 31.3-49.0, 19.3-55.5, 22.7-44.8, and 36.8-70.7 µM, respectively compared to doxorubicin (IC50 40.0, 64.8, 24.7 and 58.1 µM, respectively). Furthermore, a molecular docking study suggests that most of the target compounds have a similar binding mode as a reference compound in the active site of the CDK2 enzyme. The structural requirements controlling the CDK2 inhibitory activity were determined through the generation of a statistically significant 2D-QSAR model.

Synthesis and biological evaluation of novel pyrazolo[3,4-b]pyridines as cis-restricted combretastatin A-4 analogues.

Twenty-six novel pyrazolo[3,4-b]pyridine-bridged analogues of combretastatin A-4 possessing 3,4,5-trimethoxylphenyl groups, were synthesized and evaluated for their antiproliferative and tubulin polymerization inhibitory activities. Preliminary biological evaluation demonstrated that some of the target compounds displayed significant antiproliferative effectagainst four different cell lines including MCF-7, MDA-MB-231, HeLa and Kyse150. The most active analogue 6n was found to induce HeLa cells arrest in the G2/M phase in a dose-dependent manner. Molecular modeling studies indicated that derivative 6n most likely occupies the colchicine site of tubulin. The initial results suggest that the 3,4,5-trimethoxyphenyl substituted pyrazolo[3,4-b]pyridine could serve as a promising scaffold for development of potent tubulin inhibitors as anticancer agents.

Three-component reactions of aromatic amines, 1,3-dicarbonyl compounds, and α-bromoacetaldehyde acetal to access N-(hetero)aryl-4,5-unsubstituted pyrroles.

N-(Hetero)aryl-4,5-unsubstituted pyrroles were synthesized from (hetero)arylamines, 1,3-dicarbonyl compounds, and α-bromoacetaldehyde acetal by using aluminum(III) chloride as a Lewis acid catalyst through [1 + 2 + 2] annulation. This new versatile methodology provides a wide scope for the synthesis of different functional N-(hetero)aryl-4,5-unsubstituted pyrrole scaffolds, which can be further derived to access multisubstituted pyrrole-3-carboxamides. In the presence of 1.2 equiv of KI, a polysubstituted pyrazolo[3,4-b]pyridine derivative was also successfully synthesized.

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.

A series of tacrine-pyrazolo[3,4-b]pyridine hybrids were synthesised and evaluated as dual cholinesterase (ChE) and phosphodiesterase 4D (PDE4D) inhibitors for the treatment of Alzheimer's disease (AD). Compound 10j, which is tacrine linked with pyrazolo[3,4-b]pyridine moiety by a six-carbon spacer, was the most potent acetylcholinesterase (AChE) with IC50 value of 0.125 µM. Moreover, compound 10j provided a desired balance of AChE and butylcholinesterase (BuChE) and PDE4D inhibition activities, with IC50 value of 0.449 and 0.271 µM, respectively. The above results indicated that this hybrid was a promising dual functional agent for the treatment of AD.

Discovery of 3,6-diaryl-1H-pyrazolo[3,4-b]pyridines as potent anaplastic lymphoma kinase (ALK) inhibitors.

A new series of 3,6-diaryl-1H-pyrazolo[3,4-b]pyridine compounds have been discovered as potent anaplastic lymphoma kinase (ALK) inhibitors. The 4-hydroxyphenyl in the 6-position of 1H-pyrazolo[3,4-b]pyridine were crucial and a fluorine atom substitution could give promising inhibitory activity. The IC50 of compound 9v against ALK was up to 1.58 nM and a binding mechanism was proposed.

Structural development of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivatives as human peroxisome proliferator-activated receptor alpha (PPARα)-selective agonists.

We previously reported that 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivative 6 is an agonist of human peroxisome proliferator-activated receptor alpha (hPPARα). Here, we prepared a series of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivatives in order to examine the structure-activity relationships (SAR). SAR studies clearly indicated that the steric bulkiness of the substituent on 1H-pyrazolo-[3,4-b]pyridine ring, the position of the distal hydrophobic tail part, and the distance between the distal hydrophobic tail part and the acidic head part are critical for hPPARα agonistic activity. These SAR results are somewhat different from those reported for fibrate-class hPPARα agonists. A representative compound (10f) was as effective as fenofibrate in reducing the elevated plasma triglyceride levels in a high-fructose-fed rat model.

Synthesis of new pyrazoles and pyrozolo [3,4-b] pyridines as anti-inflammatory agents by inhibition of COX-2 enzyme.

New pyrazoles and pyrazolo[3,4-b] pyridines were synthesized and their structure was confirmed by elemental analyses as well as IR, 1H NMR, 13C NMR, and mass spectral data. All the newly synthesized derivatives were evaluated in vitro for inhibitory activity against COX-1 and COX-2 enzymes and their IC50 values were calculated, most of the derivatives showed good inhibitory activity with derivatives IVb, IVh and IVJ showing inhibitory activity better than celecoxib. Moreover, the eight most potent derivatives IVa, IVb, IVc, IVd, IVe, IVh, IVJ, and IVL were selected for in vivo assay to measure their effect on paw edema in rates and their ulcerogenic effect. Compounds IVa, IVb and IVc were found to be the most active and selective as COX-2 inhibitors and most effective in protection from edema, they were also found to have lowest ulcerogenic effect among all derivatives.

Synthesis and structure-activity relationships of pyrazolo-[3,4-b]pyridine derivatives as adenosine 5'-monophosphate-activated protein kinase activators.

A series of pyrazolo[3,4-b]pyridine derivatives were designed, synthesized, and evaluated for their activation activity toward adenosine 5'-monophosphate-activated protein kinase (AMPK). According to the enzyme activity, the pyrazole N-H exposure and para substitution on the diphenyl group were proved to be essential for the activation potency. Compound 17f showed equal activation compared with A-769662. In the molecular modeling study, compound 17f exhibited important hydrogen bond interaction with Lys29, Asp88, and Arg83. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays on the NRK-49F cell line showed that potent enzyme activators could effectively inhibit cell proliferation, especially for 17f (EC50 [AMPKα1γ1ß1] = 0.42 µM, efficacy = 79%; IC50 [NRK-49F cell line] = 0.78 µM). These results might provide new insights to explore novel AMPK activators.

Alcohol Participates in the Synthesis of Functionalized Coumarin-Fused Pyrazolo[3,4-b]Pyridine from a One-Pot Three-Component Reaction.

A concise and efficient approach to synthesizing coumarin-fused pyrazolo[3,4-b]pyridine via silica sulfuric acid (SSA) catalyzed three-component domino reaction under microwave irradiation has been demonstrated. Participation of various alcohols in construction of coumarin derivatives has been described for the first time. Short reaction time, high yields, one-pot procedure, usage of eco-friendly catalyst, and solvent are the key features of this method.

Doebner-type pyrazolopyridine carboxylic acids in an Ugi four-component reaction.

Substituted 1H-pyrazolo[3,4-b]pyridine-4- and 1H-pyrazolo[3,4-b]pyridine-6-carboxamides have been synthetized through a Doebner-Ugi multicomponent reaction sequence in a convergent and versatile manner using diversity generation strategies: combination of two multicomponent reactions and conditions-based divergence strategy. The target products contain as pharmacophores pyrazolopyridine and peptidomimetic moieties with four points of diversity introduced from readily available starting materials including scaffold diversity. A small focused compound library of 23 Ugi products was created and screened for antibacterial activity.

Discovery of novel CDK inhibitors via scaffold hopping from CAN508.

Cyclin-dependent kinases (CDKs) are promising drug targets for various human diseases, especially for cancers. Scaffold hopping strategy was applied on CAN508, a known selective CDK9 inhibitor, and a series of pyrazolo[3,4-b]pyridine compounds were synthesized and evaluated in vitro as CDK2 and CDK9 inhibitors. Most compounds exhibited moderate to potent inhibitory activities against both CDK2/cyclin A and CDK9/cyclin T1 systems. Among them, compound 2e showed IC50 values of 0.36 µM for CDK2 and 1.8 µM for CDK9, respectively. Notably, the scaffold alteration seems to cause a shift in the selectivity profile of the inhibitors. In contrast to CAN508, compound 2k demonstrated remarkable selectivity toward CDK2 (265-fold over CDK9). Docking studies on compound 2k provided hints for further design of more potent and selective CDK2/CDK9 inhibitors.