The Role of Pyrazolopyridine Derivatives on Different Steps of Herpes Simplex Virus Type-1 In Vitro Replicative Cycle.

Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.

Deciphering the robustness of pyrazolo-pyridine carboxylate core structure-based compounds for inhibiting α-synuclein in transgenic C. elegans model of Synucleinopathy.

Parkinson's disease (PD), a calamitous neurodegenerative disorder with no cure till date, is closely allied with the misfolding and aggregation of α-Synuclein (α -Syn). Inhibition of α-Syn aggregation is one of the optimistic approaches for the treatment for PD. Here, we carried out hypothesis-driven studies towards synthesising a series of pyrazolo-pyridine carboxylate containing compounds (7a-7m) targeted at reducing deleterious α-Syn aggregation. The target compounds were synthesized through multi-step organic synthesis reactions. From docking studies, compounds 7b, 7g and 7i displayed better interaction with the key residues of α-Syn with values: -6.8, -8.9 and -7.2 Kcal/mol, respectively. In vivo transgenic C. elegans model of Synucleinopathy was used to evaluate the ability of the designed and synthesized compounds to inhibit α-Syn aggregation. These lead compounds 7b, 7g and 7i displayed 1.7, 2.4 and 1.5-fold inhibition of α-Syn with respect to the control. Further, the strategy of employing pyrazolo-pyridine-based compounds worked with success and these scaffolds could be further modified and validated for betterment of endpoints associated with PD.

A four-component reaction: regio- and chemoselective formation of 7-amino-2-(tert-butyl)-5-aryl-4,5-dihydropyrazolo[1,5-a]pyrimidine-6-carbonitrile.

A novel one-pot four-component reaction of an aldehyde, malononitrile, hydrazine and 4,4-dimethyl-3-oxopentanenitrile is described. As regio- and chemoselective products, 7-amino-2-(tert-butyl)-5-aryl-4,5-dihydropyrazolo[1,5-a]pyrimidine-6-carbonitriles are formed during the course of the reaction.

Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists.

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-ß gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.

Identification of 1H-pyrazolo[3,4-b]pyridine derivatives as potent ALK-L1196M inhibitors.

Anaplastic lymphoma kinase (ALK) has been recognised as a promising molecular target of targeted therapy for NSCLC. We performed SAR study of pyrazolo[3,4-b]pyridines to override crizotinib resistance caused by ALK-L1196M mutation and identified a novel and potent L1196M inhibitor, 10g. 10g displayed exceptional enzymatic activities (<0.5 nM of IC50) against ALK-L1196M as well as against ALK-wt. In addition, 10g is an extremely potent inhibitor of ROS1 (<0.5 nM of IC50) and displays excellent selectivity over c-Met. Moreover, 10g strongly suppresses proliferation of ALK-L1196M-Ba/F3 and H2228 cells harbouring EML4-ALK via apoptosis and the ALK signalling blockade. The results of molecular docking studies reveal that, in contrast to crizotinib, 10g engages in a favourable interaction with M1196 in the kinase domain of ALK-L1196M and hydrogen bonding with K1150 and E1210. This SAR study has provided a useful insight into the design of novel and potent inhibitors against ALK gatekeeper mutant.

Cytotoxic Effects of Newly Synthesized Heterocyclic Candidates Containing Nicotinonitrile and Pyrazole Moieties on Hepatocellular and Cervical Carcinomas.

In this study, a series of newly synthesized substituted pyridine 9, 11-18, naphthpyridine derivative 10 and substituted pyrazolopyridines 19-23 by using cycnopyridone 8 as a starting material. Some of the synthesized candidates are evaluated as anticancer agents against different cancer cell lines. In vitro cytotoxic activities against hepatocellular and cervical carcinoma cell lines were evaluated using standard MTT assay. Different synthesized compounds exhibited potential in vitro cytotoxic activities against both HepG2 and HeLa cell lines. Furthermore, compared to standard positive control drugs, compounds 13 and 19 showed the most potent cytotoxic effect with IC50 values of 8.78 ± 0.7, 5.16 ± 0.4 µg/mL, and 15.32 ± 1.2 and 4.26 ± 0.3 µg/mL for HepG2 and HeLa cells, respectively.

The Development of Novel Compounds Against Malaria: Quinolines, Triazolpyridines, Pyrazolopyridines and Pyrazolopyrimidines.

Based on medicinal chemistry tools, new compounds for malaria treatment were designed. The scaffolds of the drugs used to treat malaria, such as chloroquine, primaquine, amodiaquine, mefloquine and sulfadoxine, were used as inspiration. We demonstrated the importance of quinoline and non-quinoline derivatives in vitro with activity against the W2 chloroquine-resistant (CQR) Plasmodium falciparum clone strain and in vivo against Plasmodium berghei-infected mouse model. Among the quinoline derivatives, new hybrids between chloroquine and sulfadoxine were designed, which gave rise to an important prototype that was more active than both chloroquine and sulfadoxine. Hybrids between chloroquine-atorvastatin and primaquine-atorvastatin were also synthesized and shown to be more potent than the parent drugs alone. Additionally, among the quinoline derivatives, new mefloquine derivatives were synthesized. Among the non-quinoline derivatives, we obtained excellent results with the triazolopyrimidine nucleus, which gave us prototype I that inspired the synthesis of new heterocycles. The pyrazolopyrimidine derivatives stood out as non-quinoline derivatives that are potent inhibitors of the P. falciparum dihydroorotate dehydrogenase (PfDHODH) enzyme. We also examined the pyrazolopyridine and pyrazolopyrimidine nuclei.

Discovery and biological evaluation of novel pyrazolopyridine derivatives as potent and orally available PI3Kδ inhibitors.

Phosphatidylinositol-3-kinase (PI3K)δ inhibition is one of the most attractive approaches to the treatment of autoimmune diseases and leukocyte malignancies. Through the exploration of pyrazolopyridine derivatives as potential PI3Kδ inhibitors, compound 12a was identified as a potent PI3Kδ inhibitor but suffered from poor oral exposure in mice. With a modified amide linkage group, compound 15a was developed as an orally available PI3Kδ inhibitor with reduced selectivity against other PI3Ks. To improve the trade-off between selectivity and PK profile, structure-activity relationship (SAR) studies of terminal substituents on the pyrolidine ring were conducted. As a result, we developed potent PI3Kδ inhibitors with good oral availability. In particular, the representative compound 15j showed excellent selectivity for PI3Kδ over other PI3Ks with good oral exposure in mice.

Optimization and in vivo evaluation of pyrazolopyridines as a potent and selective PI3Kδ inhibitor.

Chemical optimization of pyrazolopyridine 1, focused on cellular potency, isoform selectivity and microsomal stability, led to the discovery of the potent, selective and orally available PI3Kδ inhibitor 5d. On the basis of its desirable potency, selectivity and pharmacokinetic profiles, 5d was tested in the trinitrophenylated aminoethylcarboxymethyl-Ficoll (TNP-Ficoll)-induced antibody production model, and showed higher antibody inhibition than a 4-fold oral dose of the starting compound 1. These excellent results suggest that 5d is a potential candidate for further studies in the treatment of autoimmune diseases and leukocyte malignancies.

Design and synthesis of 1H-pyrazolo[3,4-c]pyridine derivatives as a novel structural class of potent GPR119 agonists.

Design and synthesis of a novel class of 1H-pyrazolo[3,4-c]pyridine GPR119 receptor agonists are described. Lead compound 4 was identified through the ligand-based drug design approach. Modification of the left-hand aryl group (R(1)) and right-hand piperidine N-capping group (R(2)) led to the identification of compound 24 as a single-digit nanomolar GPR119 agonist.

Design, synthesis and anti-P. falciparum activity of pyrazolopyridine-sulfonamide derivatives.

Ten 1-phenyl-1H-pyrazolo[3,4-b]pyridine derivatives connected by a linker group to benzenesulfonamide moieties with different substituents in the 4-position were synthesized and assayed against Plasmodium falciparum. These ten compounds exhibited activity in vitro against the chloroquine-resistant clone W2 with IC50 values ranging from 3.46 to 9.30µM. The most active derivatives with substituent R2=Cl or CH3 at the benzenesulfonamide moiety exhibited the lowest IC50. Compounds with an R1=CO2Et substituent at the 5-position of the 1H-pyrazolo[3,4-b]pyridine ring presented lower activity than those with a CN substituent. The 1H-pyrazolo[3,4-b]pyridine system appears to be promising for further studies as an antimalarial for overcoming the burden of resistance in P. falciparum.

Antiplatelet pyrazolopyridines derivatives: pharmacological, biochemical and toxicological characterization.

Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N'-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3 h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE2 and TXB2 through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.

Evaluation of the protective properties and genotoxic potential of pyrazolo pyridine derivatives against neutron and gamma radiation using the Ames/Salmonella test system.

PURPOSE: Nuclear applications are being increasingly used in various fields, necessitating studies to protect from radiation hazards and their effects. In this study, five different chemical structures of pyrazolo [3,4-b] pyridine derivatives were synthesized. The gamma and neutron radiation protective abilities of these samples were determined and demonstrated their potential use as ingredients in radioprotective drugs. MATERIAL AND METHODS: Gamma radiation absorption parameters were calculated both theoretical and experimental. Important attenuation parameters for fast neutrons (4.5 MeV energy radiation) were figured out using the Monte Carlo simulation Geant4 code. Additionally, experimental dose rates were measured for each sample and compared to those of Paraffin and high-density polyethylene, an organic substance. Besides, Ames/Salmonella test system was aimed to detecting genotoxicity features of pyrazolo pyridine derivatives. RESULTS: All results demonstrated that each sample possesses both gamma and neutron radiation attenuation capabilities. It was determined that sample PPC4 (C20H14BrN5) exhibited the highest gamma radiation attenuation capacity among all samples, while sample PPC2 (C22H20N6) displayed an excellent neutron stopping capacity. The genotoxic properties of pyrazolo[3,4-b] pyridine derivatives were examined using the Ames/Salmonella test, and as a result, it was determined that these substances did not exhibit genotoxic effects at test doses up to 5 mM. CONCLUSION: All obtained results indicate that all PPC (pyrazolo[3,4-b] pyridine derivatives) samples do not possess a toxic effect, and they can be utilized as an active substance for the development of a drug or cream with protective properties against both gamma and neutron radiations.

Pyridine Derivatives as Insecticides. Part 5. New Thieno[2,3-b]pyridines and Pyrazolo[3,4-b]pyridines Containing Mainly Ethyl Nicotinate Scaffold and Their Insecticidal Activity toward Aphis gossypii (Glover,1887).

Ethyl 5-cyano-1,6-dihydro-2-methyl-4-(2'-thienyl)-6-thioxonicotinate (A) was synthesized and reacted with ethyl chloroacetate in the presence of sodium acetate or sodium carbonate to give ethyl 5-cyano-6-((2-ethoxy-2-oxoethyl)thio)-2-methyl-4-(2'-thienyl)nicotinate (1a) or its isomeric thieno[2,3-b]pyridine 2a. 3-Aminothieno[2,3-b]pyridine-2-carboxamide 2b was also synthesized by the reaction of A with 2-chloroacetamide. The reaction of 1a with hydrazine hydrate in boiling ethanol gave acethydrazide 3. Heating ester 1a with hydrazine hydrate under neat conditions afforded 3-amino-1H-pyrazolo[3,4-b]pyridine 10. Compounds 2b, 3, and 10 were used as precursors for synthesizing other new thieno[2,3-b]pyridines and pyrazolo[3,4-b]pyridines containing mainly the ethyl nicotinate scaffold. Structures of all new compounds were confirmed by elemental and spectral analyses. Most of the obtained compounds were evaluated for their insecticidal activity toward the nymphs and adults of Aphis gossypii (Glover,1887). Some compounds such as 4, 9b, and 9c showed promising results. The effect of some sublethal concentrations, less than LC50, of compounds 4, 9b, and 9c on the examined Aphis was subjected to a further study. The results demonstrated that exposure of A. gossypii nymphs to sublethal concentrations of compounds 4, 9b, and 9c had noticeable effects on their biological parameters, i.e., nymphal instar duration, generation time, and adult longevity. The highest concentration C1 of all three compounds increased the nymphal instar duration and generation time and decreased adult longevity and vice versa.

Optimization of pyrazolopyridine 4-carboxamides with potent antimalarial activity for which resistance is associated with the P. falciparum transporter ABCI3.

Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class.

Discovery of New 1,4,6-Trisubstituted-1H-pyrazolo[3,4-b]pyridines with Anti-Tumor Efficacy in Mouse Model of Breast Cancer.

Purine analogues are important therapeutic tools due to their affinity to enzymes or receptors that are involved in critical biological processes. In this study, new 1,4,6-trisubstituted pyrazolo[3,4-b]pyridines were designed and synthesized, and their cytotoxic potential was been studied. The new derivatives were prepared through suitable arylhydrazines, and upon successive conversion first to aminopyrazoles, they were converted then to 1,6-disubstituted pyrazolo[3,4-b]pyridine-4-ones; this served as the starting point for the synthesis of the target compounds. The cytotoxic activity of the derivatives was evaluated against several human and murine cancer cell lines. Substantial structure activity relationships (SARs) could be extracted, mainly concerning the 4-alkylaminoethyl ethers, which showed potent in vitro antiproliferative activity in the low µM level (0.75-4.15 µΜ) without affecting the proliferation of normal cells. The most potent analogues underwent in vivo evaluation and were found to inhibit tumor growth in vivo in an orthotopic breast cancer mouse model. The novel compounds exhibited no systemic toxicity; they affected only the implanted tumors and did not interfere with the immune system of the animals. Our results revealed a very potent novel compound which could be an ideal lead for the discovery of promising anti-tumor agents, and could also be further explored for combination treatments with immunotherapeutic drugs.

Multi-targeted anti-Alzheimer's agents: Synthesis, biological evaluation, and molecular modeling study of some pyrazolopyridine hybrids.

A new series of compounds bearing a pyrazolopyridine scaffold was synthesized as integrated anti-Alzheimer's disease (AD) multi-targeted ligands. Compounds 49 and 51 showed remarkable activity as hAChE inhibitors with IC50 values of 0.17 and 0.16 µM, respectively; and proved to be active hBuChE inhibitors with IC50 values 0.17 and 0.69 µM, eight and two-fold more active than the reference compound rivastigmine, respectively. Compounds 49 and 51 showed potent GSK3ß inhibition with IC50 values of 0.21 and 0.26 µM, respectively compared to L807mts. Also, 49 and 51 showed 66.0 and 60.0% as tau protein aggregation inhibitors; and Aß1-42 self-aggregation inhibitors with 79.0 and 75.0% respectively. Furthermore, 49 and 51 could bind virtually with the PAS affecting Aß aggregation, thus preventing Aß-dependent neurotoxicity. They proved to have the ability to chelate bio-metals such as Fe2+, Cu2+, and Zn2+ preventing their oxidative damage in the brain of AD patients, in addition to their safety upon WI-38 cell line. Both compounds could virtually penetrate BBB and obeyed Lipinski's rule of five. Compounds 49 and 51 could be considered as MTDLs for AD patients and the obtained model and pattern of substitution could be used for further development of new multi-targeted anti-Alzheimer's agents.

In silico and in vitro evaluation of newly synthesized pyrazolo-pyridine fused tetrazolo-pyrimidines derivatives as potential anticancer and antimicrobial agents.

Diversely functionalized pyrazolo-pyridine fused tetrazolo-pyrimidines 10aa-am and 10ba-bn were successfully synthesized via a catalyst-free synthetic protocol with moderate to very good yields. The compounds were evaluated for cytotoxicity against MCF-7 and HEK-293 cells using MTT assay. Among the tested compounds, 10ab (IC50- 23.83 µM) and 10ah (IC50- 23.30 µM) demonstrated the highest potency against MCF-7 cells, while 10bc (IC50- 14.46 µM) and 10bh (IC50- 2.53 µM) exhibited excellent cytotoxicity against HEK-293 cells. Additionally, antibacterial screening was performed against three Gram-negative bacteria (E. coli, P. aeruginosa, and S. enterica) and three Gram-positive bacteria (S. aureus, B. megaterium, and B. subtilis) using broth dilution method, while antifungal activity was assessed against three fungal strains (A. niger, Penicillium, and S. cerevisiae) using agar well diffusion method. In antimicrobial screening, the majority of the compounds demonstrated significant antibacterial efficacy compared to antifungal activity. We also conducted comprehensive computational studies, including DFT calculations, molecular docking and dynamics, and drug-likeness assessments. In the DFT study, compounds 10ac and 10bc displayed stable conformations, indicating their potential for higher therapeutic activity. Molecular docking analyses revealed compelling interactions, with compound 10ah demonstrating docking score -7.42 kcal/mol against catalytical domain PARP1 (PDB ID: 7KK4) and 10bh exhibiting a best docking score -10.77 kcal/mol against human corticotropin-releasing factor receptor 1 (PDB ID: 4Z9G). A 100 ns molecular dynamics (MD) simulation study of compounds 10ah and 10bh revealed the stable conformation and binding energy in a stimulating environment. In drug-likeness assessments, both the compounds 10ah and 10bh adhere all the established guidelines.Communicated by Ramaswamy H. Sarma.

"Walking the nitrogen around the ring": Chemical synthesis and spectroscopic characterization of novel 4-, 5-, 6-, and 7-azaindazole analogs of the synthetic cannabinoid receptor agonist MDMB-PINACA.

Over the past decade, synthetic cannabinoid receptor agonists (SCRAs) have rapidly evolved to encompass a wide range of structurally diverse new psychoactive substances (NPS), including derivatives that incorporate indole, indazole, 7-azaindole, γ-carbolinone, or carbazole heterocyclic scaffolds. The introduction of legislative measures seeking to control the availability of NPS on the recreational drug scene has likely contributed to the continued emergence of novel SCRA analogs, which often evade regulatory control. However, the detection and/or identification of azaindazole-type SCRAs in seized material has not yet been reported (September, 2021). It is plausible that SCRAs bearing a 1,3-disubstituted azaindazole scaffold may possess cannabimimetic activity, given their structural similarity with known indole, indazole, and azaindole SCRAs. In view of these antecedents, a set of four novel isomeric 4-, 5-, 6-, and 7-azaindazole analogs of the known potent indazole SCRA, MDMB-PINACA, were synthesized using a Pd-catalyzed aminocarbonylation strategy. The complementary use of ultraviolet (UV) and infrared (IR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), high resolution mass spectrometry (HRMS), 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy, and high performance liquid chromatography (HPLC) has permitted the spectroscopic differentiation, unambiguous structural assignment, and rapid separation of novel isomeric 4-, 5-, 6-, and 7-azaindazole analogs of the indazole SCRA, MDMB-PINACA.

Pyrazolopyridine: An efficient pharmacophore in recent drug design and development.

Among the various heterocyclic molecules employed for drug design and discovery, pyrazolopyridine is one of the promising pharmacophores. Pyrazolopyridine is a result of fusion of pyrazole and pyridine rings. The potent pharmacology of pyrazolopyridine may be the synergistic effect of pyrazole and pyridine moieties in a single framework. It has been used in drug design of a wide range of diseases such as anticancer, antimicrobial, anti-inflammatory, and neuroprotection. Cancer has become a common disease among elderly people now a days that might be because of genetic inheritance to some extent, carcinogens, pollution, and some infectious diseases. Whatever may be the reason, cancer is one of the major causes of deaths worldwide. In addition, over-usage and improper usage of antibiotics have led to drug resistance of microbes. Further, inflammation is a cause of various diseases such as arthritis, and other diseases. Thus, proinflammatory kinases are considered as primary target for inhibition of inflammation. In view of this, a work that compiles potent pharmacology of recently reported pyrazolopyridine analogs has been planned. The review is aimed to discuss pharmacology in brief along with structure-activity relationship (SAR). The review would emphasize importance of pyrazolopyridines in future drug design and discovery and may help in design of potent pharmacological agents.