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1.
J Exp Bot ; 2024 May 22.
Article in English | MEDLINE | ID: mdl-38776394

ABSTRACT

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure-activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The best compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize (Zea mays) and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans, and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia, Drosera, and Plectranthus, as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (namely 82), activated a cytokinin primary response gene ARR5:GUS and cytokinin sensor TCSv2:GUS, without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the upregulation of cytokinin signalling. Overall, this work presents highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology.

2.
J Exp Bot ; 2024 Mar 25.
Article in English | MEDLINE | ID: mdl-38526483

ABSTRACT

We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms and exploits an efficient combination of hydrophilic interaction chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry. This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labelled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to the field of plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.

3.
Biomed Pharmacother ; 161: 114492, 2023 May.
Article in English | MEDLINE | ID: mdl-36931035

ABSTRACT

Targeting cyclin-dependent kinase 7 (CDK7) provides an interesting therapeutic option in cancer therapy because this kinase participates in regulating the cell cycle and transcription. Here, we describe a new trisubstituted pyrazolo[4,3-d]pyrimidine derivative, LGR6768, that inhibits CDK7 in the nanomolar range and displays favourable selectivity across the CDK family. We determined the structure of fully active CDK2/cyclin A2 in complex with LGR6768 at 2.6 Å resolution using X-ray crystallography, revealing conserved interactions within the active site. Structural analysis and comparison with LGR6768 docked to CDK7 provides an explanation of the observed biochemical selectivity, which is linked to a conformational difference in the biphenyl moiety. In cellular experiments, LGR6768 affected regulation of the cell cycle and transcription by inhibiting the phosphorylation of cell cycle CDKs and the carboxy-terminal domain of RNA polymerase II, respectively. LGR6768 limited the proliferation of several leukaemia cell lines, triggered significant changes in protein and mRNA levels related to CDK7 inhibition and induced apoptosis in dose- and time-dependent experiments. Our work supports previous findings and provides further information for the development of selective CDK7 inhibitors.


Subject(s)
Cyclin-Dependent Kinase-Activating Kinase , Cyclin-Dependent Kinases , Cyclin-Dependent Kinases/genetics , Phosphorylation , Cell Cycle , Pyrimidines/pharmacology , Pyrimidines/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry
4.
Int J Mol Sci ; 23(23)2022 Dec 02.
Article in English | MEDLINE | ID: mdl-36499496

ABSTRACT

Phenolic acids are known flavonoid metabolites, which typically undergo bioconjugation during phase II of biotransformation, forming sulfates, along with other conjugates. Sulfated derivatives of phenolic acids can be synthesized by two approaches: chemoenzymatically by 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferases or PAPS-independent aryl sulfotransferases such as those from Desulfitobacterium hafniense, or chemically using SO3 complexes. Both approaches were tested with six selected phenolic acids (2-hydroxyphenylacetic acid (2-HPA), 3-hydroxyphenylacetic acid (3-HPA), 4-hydroxyphenylacetic acid (4-HPA), 3,4-dihydroxyphenylacetic acid (DHPA), 3-(4-hydroxyphenyl)propionic acid (4-HPP), and 3,4-dihydroxyphenylpropionic acid (DHPP)) to create a library of sulfated metabolites of phenolic acids. The sulfates of 3-HPA, 4-HPA, 4-HPP, DHPA, and DHPP were all obtained by the methods of chemical synthesis. In contrast, the enzymatic sulfation of monohydroxyphenolic acids failed probably due to enzyme inhibition, whereas the same reaction was successful for dihydroxyphenolic acids (DHPA and DHPP). Special attention was also paid to the counterions of the sulfates, a topic often poorly reported in synthetic works. The products obtained will serve as authentic analytical standards in metabolic studies and to determine their biological activity.


Subject(s)
Phosphoadenosine Phosphosulfate , Sulfotransferases , Phosphoadenosine Phosphosulfate/chemistry , Phosphoadenosine Phosphosulfate/metabolism , Sulfotransferases/metabolism , Sulfates/metabolism , Hydroxybenzoates
5.
J Med Chem ; 65(13): 8881-8896, 2022 07 14.
Article in English | MEDLINE | ID: mdl-35749742

ABSTRACT

3,5,7-Trisubstituted pyrazolo[4,3-d]pyrimidines have been identified as potent inhibitors of cyclin-dependent kinases (CDKs), which are established drug targets. Herein, we describe their further structural modifications leading to novel nanomolar inhibitors with strong antiproliferative activity. We determined the crystal structure of fully active CDK2/A2 with 5-(2-amino-1-ethyl)thio-3-cyclobutyl-7-[4-(pyrazol-1-yl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine (24) at 1.7 Å resolution, confirming the competitive mode of inhibition. Biochemical and cellular assays in lymphoma cell lines confirmed the expected mechanism of action through dephosphorylation of retinoblastoma protein and RNA polymerase II, leading to induction of apoptosis. Importantly, we also revealed an interesting ability of compound 24 to induce proteasome-dependent degradation of cyclin K both in vitro and in a patient-derived xenograft in vivo. We propose that 24 has a dual mechanism of action, acting as a kinase inhibitor and as a molecular glue inducing an interaction between CDK12 and DDB1 that leads to polyubiquitination of cyclin K and its subsequent degradation.


Subject(s)
Antineoplastic Agents , Cyclin-Dependent Kinases , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinase 2 , Cyclins/metabolism , Humans , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Structure-Activity Relationship
6.
Bioorg Med Chem ; 33: 115993, 2021 03 01.
Article in English | MEDLINE | ID: mdl-33497938

ABSTRACT

Kinetin (N6-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect Friedreich́s ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.


Subject(s)
Kinetin/pharmacology , Purines/pharmacology , Cell Survival/drug effects , Cells, Cultured , Cytoprotection , Dose-Response Relationship, Drug , Humans , Kinetin/chemical synthesis , Kinetin/chemistry , Molecular Structure , Oxidative Stress/drug effects , Purines/chemical synthesis , Purines/chemistry , Structure-Activity Relationship
7.
J Exp Bot ; 72(2): 355-370, 2021 02 02.
Article in English | MEDLINE | ID: mdl-32945834

ABSTRACT

Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10-8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture.


Subject(s)
Arabidopsis , Oxidoreductases , Biotechnology , Cytokinins
8.
J Med Chem ; 62(9): 4606-4623, 2019 05 09.
Article in English | MEDLINE | ID: mdl-30943029

ABSTRACT

Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2) H-pyrazolo[4,3- d]pyrimidines with various substitutions at position 5 with potent antiproliferative activity in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and the cocrystal with CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation of XIAP and MCL-1, and activation of caspases, which collectively confirmed ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a combination therapy with the BCL2-targeting venetoclax. These findings support further studies of combinatorial treatment based on CDK inhibitors.


Subject(s)
Antineoplastic Agents/therapeutic use , Cyclin-Dependent Kinases/antagonists & inhibitors , Lymphoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Female , Humans , Mice , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Structure-Activity Relationship , Sulfonamides/pharmacology , Xenograft Model Antitumor Assays
9.
R Soc Open Sci ; 5(11): 181322, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30564417

ABSTRACT

Cytokinins (CKs) and their metabolites and derivatives are essential for cell division, plant growth regulation and development. They are typically found at minute concentrations in plant tissues containing very complicated biological matrices. Therefore, defined standards labelled with stable isotopes are required for precise metabolic profiling and quantification of CKs, as well as in vivo elucidation of CK biosynthesis in various plant species. In this work, 11 [15N]-labelled C6-purine derivatives were prepared, among them 5 aromatic (4, 5, 6, 7, 8) and 3 isoprenoid (9, 10, 11) CKs. Compared to current methods, optimized syntheses of 6-amino-9H-[15N5]-purine (adenine) and 6-chloro-9H-[15N4]-purine (6-chloropurine) were performed to achieve more effective, selective and generally easier approaches. The chemical identity and purity of prepared compounds were confirmed by physico-chemical analyses (TLC; HRMS; HPLC-MS; 1H, 13C, 15N NMR). The presented approach is applicable for the synthesis of any other desired [15N4]-labelled C6-substituted purine derivatives.

10.
Steroids ; 128: 58-67, 2017 12.
Article in English | MEDLINE | ID: mdl-29100780

ABSTRACT

A series of amphiphilic derivatives of (3ß,17ß)-3-hydroxyandrost-5-ene-17-carboxylic acid (1) with the polyamine spermine and three other diamines, 1,2-diaminoethane, piperazine and cadaverine, were synthesized and their antimicrobial activity and cytotoxicity were investigated. Among the target compounds, several ones showed antimicrobial activity on Gram positive and Gram negative microorganisms. The most active compounds were 20 (Streptococcus mutans CCM 7409, 3.125 µM), 16 (Streptococcus mutans CCM 7409, 12.5 µM) and 10d (Escherichia coli CCM 3954, 12.5 µM). In addition, compounds 5d, 10d, 13 and 20 displayed cytotoxicity on CEM (12.1 ±â€¯2.1 µM, 7.6 ±â€¯1.0 µM, 19.0 ±â€¯0.4 µM and 5.9 ±â€¯0.7 µM, respectively). Two additional compounds displayed medium cytotoxicity on CEM, 5a (34.6 ±â€¯5.2 µM) and 5c (37.7 ±â€¯5.9 µM). The compound 13 and 20 displayed high toxicity also on normal fibroblasts.


Subject(s)
Androstenes/pharmacology , Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Polyamines/pharmacology , Androstenes/chemical synthesis , Androstenes/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Carboxylic Acids/chemical synthesis , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , Escherichia coli/pathogenicity , Microbial Sensitivity Tests , Polyamines/chemical synthesis , Polyamines/chemistry
11.
Eur J Med Chem ; 110: 291-301, 2016 Mar 03.
Article in English | MEDLINE | ID: mdl-26851505

ABSTRACT

A series of 5-substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine derivatives was synthesized and evaluated for their cyclin-dependent kinase (CDK) inhibition activity. The most potent compounds contained various hydroxyalkylamines at the 5 position and possessed low nanomolar IC50 values for CDK2 and CDK5. Preliminary profiling of one of the most active compounds on a panel of 50 protein kinases revealed its high selectivity for CDKs. The compounds arrested cells in S and G2/M phases, and induced apoptosis in various cancer cell lines. Significant dephosphorylation of the C-terminus of RNA polymerase II and focal adhesion kinase (FAK), well-established substrates of CDKs, has been found in treated cells. Cleavage of PARP-1, down-regulation of Mcl-1 and activation of caspases correlated well with CDK inhibition and confirmed apoptosis as the primary type of cell death induced in cancer cells treated with the compounds in vitro. A comparison of known purine-based CDK inhibitor CR8 with its pyrazolo[4,3-d]pyrimidine bioisosteres confirmed that the novel compounds are more potent in cellular assays than purines. Therefore, pyrazolo[4,3-d]pyrimidine may emerge as a novel scaffold in medicinal chemistry and as a source of potent CDK inhibitors.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinases/metabolism , G2 Phase/drug effects , Humans , Neoplasms/drug therapy , Neoplasms/enzymology , Pyrazoles/chemistry , Pyrazoles/pharmacology
12.
Chem Biol Drug Des ; 86(6): 1528-40, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26198005

ABSTRACT

Selective inhibitors of kinases that regulate the cell cycle, such as cyclin-dependent kinases (CDKs) and aurora kinases, could potentially become powerful tools for the treatment of cancer. We prepared and studied a series of 3,5,7-trisubstituted pyrazolo[4,3-d]pyrimidines, a new CDK inhibitor scaffold, to assess their CDK2 inhibitory and antiproliferative activities. A new compound, 2i, which preferentially inhibits CDK2, CDK5, and aurora A was identified. Both biochemical and cellular assays indicated that treatment with compound 2i caused the downregulation of cyclins A and B, the dephosphorylation of histone H3 at Ser10, and the induction of mitochondrial apoptosis in the HCT-116 colon cancer cell line. It also reduced migration as well as tube and lamellipodia formation in human endothelial cells. The kinase inhibitory profile of compound 2i suggests that its anti-angiogenic activity is linked to CDK5 inhibition. This dual mode of action involving apoptosis induction in cancer cells and the blocking of angiogenesis-like activity in endothelial cells offers possible therapeutic potential.


Subject(s)
Aurora Kinase A/antagonists & inhibitors , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cyclin-Dependent Kinase Inhibitor Proteins/chemical synthesis , Cyclin-Dependent Kinase Inhibitor Proteins/chemistry , Cyclin-Dependent Kinase Inhibitor Proteins/pharmacology , Drug Design , Drug Screening Assays, Antitumor , HCT116 Cells , Human Umbilical Vein Endothelial Cells , Humans , Molecular Docking Simulation , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity Relationship
13.
Bioorg Med Chem Lett ; 25(11): 2298-301, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-25937014

ABSTRACT

Here we describe the leishmanicidal activities of a library of 2,6,9-trisubstituted purines that were screened for interaction with Cdc2-related protein kinase 3 (CRK3) and subsequently for activity against parasitic Leishmania species. The most active compound inhibited recombinant CRK3 with an IC50 value of 162 nM and was active against Leishmania major and Leishmania donovani at low micromolar concentrations in vitro. Its mode of binding to CRK3 was investigated by molecular docking using a homology model.


Subject(s)
Leishmania donovani/drug effects , Leishmania major/drug effects , Proto-Oncogene Proteins c-crk/antagonists & inhibitors , Purines/chemistry , Purines/pharmacology , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Binding Sites , Models, Molecular , Molecular Structure , Protein Binding , Protein Conformation , Structure-Activity Relationship
14.
Steroids ; 100: 27-35, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25963549

ABSTRACT

ß-Sitosterol and betulinic acid were used in designing their conjugates with selected polyamines bearing either an amide bond, or an ester and an amide bond simultaneously in the target molecule. The synthesized compounds were subjected to basic cytotoxic and antimicrobial tests. The synthetic protocol is described separately for each of the three series of the target amides, because each series of compounds required a different synthetic approach. The cytotoxicity was tested on cells derived from human T-lymphoblastic leukemia, breast adenocarcinoma and cervical cancer, and compared with the tests on normal human fibroblasts. Most of the target compounds (5a-5c, 11a-11c and 16a-16c) showed medium to high cytotoxicity (0.7-7.8 µM), however, in some cases the compounds showed high cytotoxicity even toward normal human fibroblasts (11a-11c). Two compounds of this series (11c and 16c) also displayed antimicrobial activity with high and selective microbe specificity. The compound 11c was potent against Escherichia coli (minimal inhibition concentration (MIC) 6.25 µg mL(-1), i.e. 9.75 nM mL(-1)) and Staphylococcus aureus (MIC 12.5 µg mL(-1), i.e. 19.5 nM mL(-1)), and showed medium activity against Pseudomonas aeruginosa. The compound 16c was highly active against Enterococcus faecalis and S. aureus (both, MIC 3.125 µg mL(-1), i.e. 4.22 nM mL(-1)), both Gram-positive bacteria, however showed only weak activity against E. coli and no activity against P. aeruginosa, both Gram-negative bacteria, which indicates possible microbe specificity of 16c. Comparing ß-sitosterol-based series (5a-5c) and betulinic acid series (11a-11c and 16a-16c) of the target compounds, the latter one gave more promising structures. The compounds 11c and 16c showed effects which may be described as multifarious activity (pleiotropic effects).


Subject(s)
Anti-Bacterial Agents/chemistry , Polyamines/chemistry , Sitosterols/chemistry , Triterpenes/chemistry , Anti-Bacterial Agents/pharmacology , Cell Survival/drug effects , Escherichia coli/drug effects , HeLa Cells , Humans , MCF-7 Cells , Microbial Sensitivity Tests , Pentacyclic Triterpenes , Polyamines/toxicity , Pseudomonas aeruginosa/drug effects , Sitosterols/pharmacology , Staphylococcus aureus/drug effects , Triterpenes/pharmacology , Betulinic Acid
15.
Steroids ; 78(14): 1347-52, 2013 Dec 20.
Article in English | MEDLINE | ID: mdl-24145008

ABSTRACT

The current interest of the team has been focused on investigation of novel amides with potential cytotoxicity. The presented series of compounds was synthesized from selected steryl hemiesters and heteroaromatic amines. The synthetic protocol was designed in a simple and economic way, and divided into several general methodologies applicable to the compounds synthesized. The cytotoxicity was tested on cells derived from human T-lymphoblastic leukemia, breast adenocarcinoma and cervical cancer, and compared with tests on normal human fibroblasts. Most of the lanosterol-based compounds (3-5 and 7-10) showed medium to good cytotoxicity, while only two derivatives of cholesterol (18 and 19) showed medium cytotoxicity on human T-lymphoblastic leukemia cell line. The compounds 8 and 9 displayed the reasonable cytotoxicity among this series of amides, tested on the cell lines of T-lymphoblastic leukemia [14.5±0.4 µM (8) and 18.5±3.9 µM (9)], breast adenocarcinoma [19.5±2.1 µM (8) and 23.1±4.0 µM (9)] and cervical cancer [24.8±5.3 µM (8) and 29.1±4.7 µM (9)]. Only the compound 8 was adequately less active on normal human fibroblasts (40.4±11.1 µM).


Subject(s)
Amides/chemistry , Amines/chemistry , Antineoplastic Agents/chemical synthesis , Cholesterol/analogs & derivatives , Cytotoxins/chemical synthesis , Lanosterol/analogs & derivatives , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cholesterol/chemical synthesis , Cholesterol/pharmacology , Cytotoxins/pharmacology , Drug Screening Assays, Antitumor , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Lanosterol/chemical synthesis , Lanosterol/pharmacology , Magnetic Resonance Spectroscopy
16.
PLoS One ; 8(1): e54607, 2013.
Article in English | MEDLINE | ID: mdl-23336010

ABSTRACT

Current inhibitors of angiogenesis comprise either therapeutic antibodies (e.g. bevacicumab binding to VEGF-A) or small molecular inhibitors of receptor tyrosin kinases like e.g. sunitinib, which inhibits PDGFR and VEGFR. We have recently identified cyclin-dependent kinase 5 (Cdk5) as novel alternative and pharmacologically accessible target in the context of angiogenesis. In the present work we demonstrate that trisubstituted pyrazolo[4,3-d]pyrimidines constitute a novel class of compounds which potently inhibit angiogenesis. All seven tested compounds inhibited endothelial cell proliferation with IC(50) values between 1 and 18 µM. Interestingly, this seems not to be due to cytotoxicity, since none of them showed acute cytotoxic effects on endothelial cells at a concentration of 10 µM,. The three most potent compounds (LGR1404, LGR1406 and LGR1407) also inhibited cell migration (by 27, 51 and 31%, resp.), chemotaxis (by 50, 70 and 60% in accumulative distance, resp.), and tube formation (by 25, 60 and 30% of total tube length, resp.) at the non-toxic concentration of 10 µM. Furthermore, angiogenesis was reduced in vivo in the CAM assay by these three compounds. A kinase selectivity profiling revealed that the compounds prevalently inhibit Cdk2, Cdk5 and Cdk9. The phenotype of the migrating cells (reduced formation of lamellipodia, loss of Rac-1 translocation to the membrane) resembles the previously described effects of silencing of Cdk5 in endothelial cells. We conclude that especially LGR1406 and LGR1407 are highly attractive anti-angiogenic compounds, whose effects seem to largely depend on their Cdk5 inhibiting properties.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/toxicity , Cell Death/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Cyclin-Dependent Kinase 5/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Protein Transport/drug effects , Pseudopodia/drug effects , Pseudopodia/metabolism , Pyrazoles/chemistry , Pyrazoles/toxicity , Pyrimidines/chemistry , Pyrimidines/toxicity , rac1 GTP-Binding Protein/metabolism
17.
Bioorg Med Chem Lett ; 21(14): 4233-7, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21683592

ABSTRACT

We report here results of screening directed to finding new anti-leishmanial drugs among 2,6-disubstituted purines and corresponding 3,7-disubstituted pyrazolo[4,3-d]pyrimidines. These compounds have previously been shown to moderately inhibit human cyclin-dependent kinases. Since some compounds reduced viability of axenic amastigotes of Leishmania donovani, we screened them for interaction with recombinant leishmanial cdc-2 related protein kinase (CRK3/CYC6), an important cell cycle regulator of the parasitic protozoan. Eighteen pairs of corresponding isomers were tested for viability of amastigotes and for inhibition of CRK3/CYC6 kinase activity. Some compounds (9A, 12A and 13A) show activity against amastigotes with EC(50) in a range 1.5-12.4µM. Structure-activity relationships for the tested compounds are discussed and related to the lipophilicity of the compounds.


Subject(s)
Leishmania donovani/drug effects , Purines/chemistry , Pyrazoles/chemistry , Pyrimidines/chemistry , Trypanocidal Agents/chemistry , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Purines/chemical synthesis , Purines/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/pharmacology
18.
Angiogenesis ; 14(3): 281-91, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21487803

ABSTRACT

Small molecular inhibitors of Cyclin dependent kinases (Cdks) are currently being developed as anticancer therapeutics due to their antiproliferative properties. The purine Cdk specific inhibitor (R)-roscovitine (seliciclib, CYC202) represents one of the most promising of these compounds. It is currently evaluated in clinical trials concerning cancer therapy. Recently, we have shown that roscovitine exerts potent antiangiogenic effects and elucidated Cdk5 as a new player in angiogenesis. These findings introduce Cdk5 as novel target for antiangiogenic therapy, and Cdk5 inhibitors as an attractive therapeutic approach. Here, we present the antiangiogenic profile of 15 derivatives of roscovitine in vitro and in vivo and provide structure activity relationships of the roscovitine analogs. The (S)-isomer LGR561 and the respective (R)- and (S)-isomers LGR848 and LGR849 strongly inhibited proliferation and cell cycle progression, induced cell death, and reduced migration of endothelial cells in vitro. In comparison to roscovitine, these compounds showed an increased potency to inhibit Cdk2, Cdk5, Cdk7, and Cdk9. By analyzing the effects of LGR561, LGR848, and LGR849 on endothelial cell tube formation, mouse aortic ring sprouting, angiogenesis in the chick chorioallantoic membrane, and neovessel formation in the mouse cornea, we elucidate the two (S)-isomers LGR561 and LGR849 as highly potent inhibitors of angiogenesis. This study provides first information on how to modify roscovitine to develop Cdk inhibitors with increased antiangiogenic activity and suggests the application of existing and the development of new Cdk inhibitors to inhibit both, cancer cell proliferation and angiogenesis.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Aorta/metabolism , Cell Cycle/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Endothelial Cells/metabolism , Neovascularization, Physiologic/drug effects , Protein Kinase Inhibitors , Purines/pharmacology , Animals , Aorta/pathology , Cell Movement/drug effects , Cells, Cultured , Chick Embryo , Corneal Neovascularization/drug therapy , Corneal Neovascularization/metabolism , Corneal Neovascularization/pathology , Cyclin-Dependent Kinases/metabolism , Drug Evaluation, Preclinical , Endothelial Cells/pathology , Humans , Mice , Organ Culture Techniques , Protein Kinase Inhibitors/analogs & derivatives , Protein Kinase Inhibitors/pharmacology , Roscovitine , Stereoisomerism
19.
J Med Chem ; 54(8): 2980-93, 2011 Apr 28.
Article in English | MEDLINE | ID: mdl-21417417

ABSTRACT

Inhibition of cyclin-dependent kinases (CDKs) with small molecules has been suggested as a strategy for treatment of cancer, based on deregulation of CDKs commonly found in many types of human tumors. Here, a new potent CDK2 inhibitor with pyrazolo[4,3-d]pyrimidine scaffold has been synthesized, characterized, and evaluated in cellular and biochemical assays. 7-Benzylamino-5(R)-[2-(hydroxymethyl)propyl]amino-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine, compound 7, was prepared as a bioisostere of the well-known CDK inhibitor roscovitine. An X-ray crystal structure of compound 7 bound to CDK2 has been determined, revealing a binding mode similar to that of roscovitine. Protein kinase selectivity profile of compound 7 and its biological effects (cell cycle arrest, dephosphorylation of the retinoblastoma protein, accumulation of the tumor suppressor protein p53, induction of apoptosis, inhibition of homologous recombination) are consistent with CDK inhibition as a primary mode of action. Importantly, as the anticancer activities of the pyrazolo[4,3-d]pyrimidine 7 exceed those of its bioisostere roscovitine, compound 7 reported here may be preferable for cancer therapy.


Subject(s)
Cell Proliferation/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrazoles/chemistry , Pyrimidines/chemistry , Cell Line, Tumor , Crystallization , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Models, Molecular , Purines/chemistry , Recombination, Genetic , Roscovitine , Spectrophotometry, Infrared
20.
Biosci Biotechnol Biochem ; 74(3): 553-7, 2010.
Article in English | MEDLINE | ID: mdl-20208357

ABSTRACT

A number of compounds have been reported to be specific inhibitors of protein kinases mediated by structural-based selectivity, but the development of specific inhibitors has not yet been addressed in plant science. Here we tested C2, C6, and N9-trisubstituted purines to determine the basic relationship between their chemical structure and inhibitory activity versus a plant mitogen-activated protein kinase (MAPK). Modification of substitution at positions C2 and N9 caused increased inhibitory activity of 6-benzylaminopurine analogs. In the case of 6-isopentenyladenine derivatives, the addition of a methyl group at position N9 caused at least 2-fold increased inhibitory activity, as compared with the addition of an isopropyl group. The data indicate that the selectivity and potency of inhibitors can be improved by modification of the chemical structure, suggesting that trisubstituted purines are powerful tools for probing biological processes and understanding the physiological roles of MAPK signaling.


Subject(s)
Mitogen-Activated Protein Kinases/antagonists & inhibitors , Plants/enzymology , Protein Kinase Inhibitors/chemistry , Purines/chemistry , Benzyl Compounds , Cells, Cultured , Imidazoles/chemistry , Imidazoles/pharmacology , Kinetin/chemistry , Plants/drug effects , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Signal Transduction , Solanaceae/drug effects , Solanaceae/enzymology , Staurosporine/chemistry , Staurosporine/pharmacology , Structure-Activity Relationship
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