Synthesis and biological evaluation of thienopyrrolizines, a new family of CDK/GSK-3 inhibitors

Fifteen new thieno[2,3-b ]- and thieno[3,4-b]pyrrolizines were synthesized and tested against two protein kinases, CDK1/cyclin B and GSK-3. Among these compounds, 3-(3-hydroxy-4-methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-8-one 4g was identified as a moderate inhibitor of these kinases. Its molecular modeling study brought to the fore the pivotal role of the 2-methoxyphenol grouping and the interest in replacing it by bioisosteric moieties in future pharmacomodulations.     

Synthesis and evaluation of pyrazolo[3,4-b]pyridine CDK1 inhibitors as anti-tumor agents

A series of 3,5-disubstituted pyrazolo[3,4-b]pyridine cyclin-dependent kinase (CDK) inhibitors was synthesized. These compounds showed potent and selective CDK inhibitory activities and inhibited in vitro cellular proliferation in cultured human tumor cells. Selected compounds were evaluated in an in vivo tumor xenograft model. The synthesis and biological evaluation of these pyrazolo[3,4-b]pyridines and related compounds are reported.     

5-aryl-pyrazolo[3,4-b]pyridines: potent inhibitors of glycogen synthase kinase-3 (GSK-3)

A novel series of pyrazolo[3,4-b]pyridines has been identified that are potent inhibitors of glycogen synthase kinase-3 (GSK-3).     

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.     

Synthesis and Biological Evaluation of Thienopyrrolizines,a New Family of CDK/GSK-3 Inhibitors

Fifteen new thieno[2,3-b]- and thieno[3,4-b]pyrrolizines were synthesized and tested against two protein kinases, CDK1/cyclin B and GSK-3. Among these compounds, 3-(3-hydroxy-4-methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-8-one 4g was identified as a moderate inhibitor of these kinases. Its molecular modeling study brought to the fore the pivotal role of the 2-methoxyphenol grouping and the interest in replacing it by bioisosteric moieties in future pharmacomodulations.     

Thiazolo[5,4-f]quinazolin-9-ones, inhibitors of glycogen synthase kinase-3

In an effort to identify new protein kinase inhibitors with increased potency and selectivity, we have developed the microwave-assisted synthesis of thiazolo[5,4-f]quinazolin-9-ones. The effects of eighteen derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3 were investigated. Several turned out to inhibit GSK-3 in the micromolar range. Molecular modeling studies suggest that the most selective GSK-3 inhibitors 7a-d bind into the ATP-binding site through a key hydrogen bond interaction with Val135 and target the specific hydrophobic backpocket of the enzyme.     

Synthesis of novel 5-substituted indirubins as protein kinases inhibitors

In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we synthesized and evaluated new 5-substituted indirubins. The effects of 34 indirubin derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3, as well as on SH-SY5Y human neuroblastoma cell survival, were investigated.     

Novel,potent and selective cyclin D1/CDK4 inhibitors: indolo[6,7-a]pyrrolo[3,4-c]carbazoles

The synthesis and CDK inhibitory properties of a series of indolo[6,7-a]pyrrolo[3,4-c]carbazoles is reported. In addition to their potent CDK activity, the compounds display antiproliferative activity against two human cancer cell lines. These inhibitors also effect strong G1 arrest in these cell lines and inhibit Rb phosphorylation at Ser780 consistent with inhibition of cyclin D1/CDK4.     

Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 beta, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described.     

Synthesis and kinase inhibitory activity of novel substituted indigoids

The bis-indole indigoids are a promising protein kinase inhibitor scaffold to be further evaluated against the numerous human diseases that imply abnormal regulation of kinases including neurodegenerative disorders. In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we designed, synthesized new 5,7-disubstituted or 6-substituted bis-indole derivatives. On the basis of our previous synthetic work, 22 selected compounds were tested on CDK1/cyclin B, CDK5/p25, DYRK1A, CK1, and GSK-3α/β kinases, five kinases involved in Alzheimer’s disease. Some of them were also evaluated for their cytotoxic and antiproliferative activities. 6-Nitro-3′-N-oxime-indirubin and 5-amino-3′-N-oxime-indirubin derivatives exhibited inhibitory activity in a submicromolar range against CDK1/cyclin B (0.18 and 0.1 μM, respectively), CK1 (0.6 μM and 0.13 μM) and GSK3 (0.04 μM and 0.36 μM).     

Synthesis and evaluation of pyrazolo[1,5-b]pyridazines as selective cyclin dependent kinase inhibitors

A novel series of pyrazolo[1,5-b]pyridazines have been synthesized and identified as cyclin dependant kinase inhibitors potentially useful for the treatment of solid tumors. Modification of the hinge-binding amine or the C(2)- and C(6)-substitutions on the pyrazolopyridazine core provided potent inhibitors of CDK4 and demonstrated enzyme selectivity against VEGFR-2 and GSK3beta.     

Structure-guided design of pyrazolo[1,5-a]pyrimidines as inhibitors of human cyclin-dependent kinase 2

The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3beta and other kinases.     

Solvent free microwave synthesis and evaluation of antimicrobial activity of pyrimido[4,5-b]- and pyrazolo[3,4-b]quinolines

A series of 2-oxopyrimido[4,5-b]-, 2-thio[4,5-b]-, 1-(p-tosyl)pyrazolo[3,4-b]- and 1-(2',4'-dinitrophenyl)pyrazolo[3,4-b]-quinolines have been synthesized in good to excellent yields by environmentally benign solvent free microwave-induced techniques involving the condensation of 2-chloro-3-formylquinolines with urea, thiourea, p-toluenesulfonylhydrazide and 2,4-dinitrophenylhydrazine, respectively, using PTSA as a catalyst. All the synthesized compounds were evaluated for their antibacterial and antifungal activities. Most of the compounds showed the best activity against Escherichia coli and Pseudomonas aeruginosa.     

Pyrazolo[4,3-d]pyrimidines as new generation of cyclin-dependent kinase inhibitors

A search among analogues of anti-CDK purines led to the identification of substituted pyrazolo[4,3-d]pyrimidines as novel inhibitors of CDK1/cyclin B. Some of these derivatives also show antiproliferative activity on cancer cell line K-562, thus may find an application as anticancer agents.     

4-Acylamino-6-arylfuro[2,3-d]pyrimidines: potent and selective glycogen synthase kinase-3 inhibitors

Modeling studies of a furo[2,3-d]pyrimidine GSK-3 hit compound 1 superimposed onto the X-ray crystal structure of a legacy pyrazolo[3,4-c]pyridazine GSK-3 inhibitor 2 led to the identification of 4-acylamino-6-arylfuro[2,3-d]pyrimidine template 3. Synthesis of analogues based on template 3 has resulted in a number of potent and selective GSK-3beta inhibitors. The most potent and selective compound was the m-pyridyl analogue 24.     

Pyrazoloheteroaryls: novel p38alpha MAP kinase inhibiting scaffolds with oral activity

A test library with three novel p38alpha inhibitory scaffolds and a narrow set of substituents was prepared. Appropriate combination of substituent and scaffold generated potent p38alpha inhibitors, for example, pyrazolo[3,4-b]pyridine 9, pyrazolo[3,4-d]pyrimidine 18a and pyrazolo[3,4-b]pyrazine 23b with potent in vivo activity upon oral administration in animal models of rheumatoid arthritis.     

Biosynthesis of new indigoid inhibitors of protein kinases using recombinant cytochrome P450 2A6

Glycogen synthase kinase-3 (GSK-3) is a potential drug target for a number of human diseases. Some indigoids have been found to be potent inhibitors of GSK-3, and individual compounds with better activity, specificity, and solubility are desired. In this work, a new disubstituted indigoid generation system was developed with a tryptophanase-deficient Escherichia coli strain as a host to express the human cytochrome P450 2A6 mutant L240C/N297Q, which catalyzes the oxidation of indole to isatin and indoxyl, which in turn react to generate indigoids. Forty-five substituted 1H-indoles from commercial sources were used as substrates in the system, and indigoid mixtures were tested as potential inhibitors of GSK-3. After preliminary screening, cell extracts with high inhibitory activity towards GSK-3alpha/beta were fractionated, and the IC50 values of twelve individual indigoids were measured for GSK-3alpha/beta as well as the protein kinases CDK1/cyclinB and CDK5/p25. Several indigoids, including an indigo, showed stronger inhibition than found in previous work. The most potent towards GSK-3alpha/beta, dimethyl indirubin 5,5'-dicarboxylate (IC50 of 51 nM), was modified by chemical reactions. One product, indirubin 5,5'-dicarboxylic acid 5-methyl ester, inhibited GSK-3alpha/beta with an IC50 of 14 nM and selectivity nearly 40-fold over CDK1 and CDK5. Indirubin-5-5'-dicarbonitrile was also modified to the corresponding 3'-oxime, which had low specificity but showed very high inhibition of all three kinases with IC50 values of 5, 13, and 10 nM towards GSK-3alpha/beta, CDK1, and CDK5, respectively. Thus, this system has the potential to generate new indigoids with therapeutic potential.     

Synthesis and antiviral evaluation of some novel tricyclic pyrazolo[3,4-b]indole nucleosides

Novel pyrazolo[3,4-b]indole nucleoside analogs were synthesized from the corresponding 3-formyl-2-chloroindole and 3-cyano-2-chloroindole nucleosides by treatment with hydrazine. Very few examples of pyrazolo[3,4-b]indole heterocycles have been published in the literature and this is the first synthesis of nucleoside analogs containing this heterocycle. These new pyrazolo[3,4-b]indole nucleosides were active against human cytomegalovirus and herpes simplex virus type 1, but this activity was not well separated from cytotoxicity.     

Antitumor activity of new pyrazolo[3,4-d]pyrimidine SRC kinase inhibitors in Burkitt lymphoma cell lines and its enhancement by WEE1 inhibition

Burkitt lymphoma (BL) is a highly aggressive B cell neoplasm. Although intensive polychemotherapy regimens have proven very effective, they are associated with significant toxicities. Therefore, more rational therapies that selectively target the molecular abnormalities of BL are needed. Recent data suggest that the tyrosine kinase SRC could represent a therapeutic target for BL. We found that new pyrazolo[3,4-d]pyrimidine SRC inhibitors exerted a significant cytotoxic effect and induced apoptosis on two BL cell lines, as determined by MTS assays, cytofluorimetric analyses and caspase 3 assay. Notably, our SRC inhibitors proved to be more effective than the well-known SRC inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine] in BL cells. Moreover, our small molecules induced a G₂/M arrest in BL cells through a possible new mechanism, whereby SRC inhibition hinders an AKT-WEE1-cyclin-dependent kinase 1 (CDK1) axis, leading to inhibition of CDK1, the main trigger of entry into mitosis. By using a small-molecule inhibitor of WEE1, a crucial CDK1 negative regulator, we were able to shift the balance toward apoptosis rather than growth arrest and enhance the efficacy of the SRC inhibitors, suggesting a possible use of these selective drugs in combination for a safe and efficient treatment of BL.     

5-aryl-pyrazolo[3,4-b]pyridazines: potent inhibitors of glycogen synthase kinase-3 (GSK-3)

Introduction of a nitrogen atom into the 6-position of a series of pyrazolo[3,4-b]pyridines led to a dramatic improvement in the potency of GSK-3 inhibition. Rationalisation of the binding mode suggested participation of a putative structural water molecule, which was subsequently confirmed by X-ray crystallography.