This document describes the synthesis and evaluation of a series of 4-azapodophyllotoxin derivatives as potential anticancer agents. Key findings include:
1) A series of 4-azapodophyllotoxin derivatives were synthesized using allylpolyalkoxybenzenes extracted from parsley seeds.
2) Compounds were evaluated in a sea urchin embryo assay and those inducing cleavage alteration, arrest, and embryo spinning were identified as having potential tubulin destabilizing activity.
3) The most active compounds in the sea urchin assay featured a myristicin-derived ring E substitution.
4) Selected compounds were further evaluated in cancer cell lines, demonstrating cytotoxic effects including microtubule disruption
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
The current study investigated the immunomodulatory
potential of ethyl acetate soluble supernatant of
Lactobacillus casei (LC-EAS) in vitro. The effect of
LC-EAS on nitric oxide release was analyzed in RAW
264.7 cells, wherein, an inhibition in nitric oxide production
through suppression of inducible nitric oxide synthase
mRNA expression was observed. Evaluation of LC-EAS
on LPS-induced peripheral blood mononuclear cells
showed a down-regulation in TNF-a and IL-6 genes and an
upregulation of IL-10. An inhibition in the protein
expression of NF-kB, ERK1/2 and STAT3 phosphorylation
confirms the immunomodulatory potential of LC-EAS. The
effect of LC-EAS on in vitro intestinal epithelial cells was
investigated using HT-29 human colon adenocarcinoma
cancer cells. LC-EAS exhibited an inhibition of NF-jB and
ERK1/2 phosphorylation, whereas STAT3 phosphorylation
was unregulated. To evaluate the downstream target of
STAT3 upregulation, expression of the intestinal trefoil
factor TFF3 which is a NF-jB regulator and STAT3
downstream target was studied. LC-EAS was observed to
elevate TFF3 mRNA expression. Overall the study shows
that the anti-inflammatory potential of LC-EAS is through
inhibition of NF-kB in different cell types.
Methods and Approaches for Defining Mechanism Signatures from Human Primary C...BioMAP® Systems
The document describes methods for using human primary cell-based disease models called BioMAP systems to define mechanism signatures of compounds. BioMAP systems analyze compound profiles across multiple readouts in various cell types to classify compounds according to their mechanisms of action or toxicity pathways. High reproducibility is shown for compound profiles even across different experiments and cell donors.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
This study investigated the structural, thermodynamic and unfolding properties of the kappa class glutathione transferase (CdGSTK1-1) from Camelus dromedarius. The key findings were:
1) CdGSTK1-1 was expressed in E. coli and purified. Analytical gel filtration showed it has a unique trimeric structure.
2) CdGSTK1-1 exhibited low thermal stability and unfolded through three states with intermediate species formation. The first transition melting point was 40.3°C and the second was 49.1°C.
3) Intrinsic fluorescence and near-UV CD studies indicated that substrate binding did not cause major conformational changes in
CYP2A6_HPLC_PK_2015 New Simple Method for Coumarin in Liver Cytochrome of RatsWael Ebied
This document describes the development and validation of a new liquid chromatography method for determining coumarin and its 7-hydroxy metabolite as a marker of cytochrome P450 2A6 activity in rats. The method uses a C18 column and isocratic mobile phase to separate coumarin, 7-hydroxycoumarin, and an internal standard. The method was validated and showed good linearity, precision, and accuracy. This validated method was then applied to study the effect of resveratrol, sulforaphane, and thymoquinone on hepatic CYP2A6 activity in spontaneously hypertensive rats.
The crystal structure of the sucrose synthase from the bacterium Nitrosomonas europaea was determined, revealing an open conformation. Comparative analysis showed a hinge-latch mechanism is critical for transition between open and closed forms. Unlike eukaryotic sucrose synthases, the bacterial enzyme prefers ADP-glucose over UDP-glucose as a substrate, indicating differences in sucrose metabolism between prokaryotes and plants.
This document summarizes research evaluating the trypanocidal activity of plant extracts and identifying active constituents from Persea americana seeds. Crude extracts from 65 Mexican plant species were screened, with 39 showing activity against trypomastigotes of Trypanosoma cruzi. The avocado seed methanol extract showed moderate activity, and fractionation yielded 8 trihydroxyheptadecane/nonadecane derivatives as the active compounds. These displayed similar activity against epimastigotes and trypomastigotes, in contrast to other compounds that are more active against trypomastigotes.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
The current study investigated the immunomodulatory
potential of ethyl acetate soluble supernatant of
Lactobacillus casei (LC-EAS) in vitro. The effect of
LC-EAS on nitric oxide release was analyzed in RAW
264.7 cells, wherein, an inhibition in nitric oxide production
through suppression of inducible nitric oxide synthase
mRNA expression was observed. Evaluation of LC-EAS
on LPS-induced peripheral blood mononuclear cells
showed a down-regulation in TNF-a and IL-6 genes and an
upregulation of IL-10. An inhibition in the protein
expression of NF-kB, ERK1/2 and STAT3 phosphorylation
confirms the immunomodulatory potential of LC-EAS. The
effect of LC-EAS on in vitro intestinal epithelial cells was
investigated using HT-29 human colon adenocarcinoma
cancer cells. LC-EAS exhibited an inhibition of NF-jB and
ERK1/2 phosphorylation, whereas STAT3 phosphorylation
was unregulated. To evaluate the downstream target of
STAT3 upregulation, expression of the intestinal trefoil
factor TFF3 which is a NF-jB regulator and STAT3
downstream target was studied. LC-EAS was observed to
elevate TFF3 mRNA expression. Overall the study shows
that the anti-inflammatory potential of LC-EAS is through
inhibition of NF-kB in different cell types.
Methods and Approaches for Defining Mechanism Signatures from Human Primary C...BioMAP® Systems
The document describes methods for using human primary cell-based disease models called BioMAP systems to define mechanism signatures of compounds. BioMAP systems analyze compound profiles across multiple readouts in various cell types to classify compounds according to their mechanisms of action or toxicity pathways. High reproducibility is shown for compound profiles even across different experiments and cell donors.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
This study investigated the structural, thermodynamic and unfolding properties of the kappa class glutathione transferase (CdGSTK1-1) from Camelus dromedarius. The key findings were:
1) CdGSTK1-1 was expressed in E. coli and purified. Analytical gel filtration showed it has a unique trimeric structure.
2) CdGSTK1-1 exhibited low thermal stability and unfolded through three states with intermediate species formation. The first transition melting point was 40.3°C and the second was 49.1°C.
3) Intrinsic fluorescence and near-UV CD studies indicated that substrate binding did not cause major conformational changes in
CYP2A6_HPLC_PK_2015 New Simple Method for Coumarin in Liver Cytochrome of RatsWael Ebied
This document describes the development and validation of a new liquid chromatography method for determining coumarin and its 7-hydroxy metabolite as a marker of cytochrome P450 2A6 activity in rats. The method uses a C18 column and isocratic mobile phase to separate coumarin, 7-hydroxycoumarin, and an internal standard. The method was validated and showed good linearity, precision, and accuracy. This validated method was then applied to study the effect of resveratrol, sulforaphane, and thymoquinone on hepatic CYP2A6 activity in spontaneously hypertensive rats.
The crystal structure of the sucrose synthase from the bacterium Nitrosomonas europaea was determined, revealing an open conformation. Comparative analysis showed a hinge-latch mechanism is critical for transition between open and closed forms. Unlike eukaryotic sucrose synthases, the bacterial enzyme prefers ADP-glucose over UDP-glucose as a substrate, indicating differences in sucrose metabolism between prokaryotes and plants.
This document summarizes research evaluating the trypanocidal activity of plant extracts and identifying active constituents from Persea americana seeds. Crude extracts from 65 Mexican plant species were screened, with 39 showing activity against trypomastigotes of Trypanosoma cruzi. The avocado seed methanol extract showed moderate activity, and fractionation yielded 8 trihydroxyheptadecane/nonadecane derivatives as the active compounds. These displayed similar activity against epimastigotes and trypomastigotes, in contrast to other compounds that are more active against trypomastigotes.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
1) The study examined the effects of various cytokinins, cytokinin ribosides, and their analogs on the viability of normal and cancerous human cells.
2) Only a few compounds, including isopentenyladenosine (1a) and N6-benzyladenosine (3a), significantly impaired viability in most cell lines tested, including normal endothelial cells and various cancer cell lines.
3) Colon carcinoma LoVo cells were uniquely insensitive to all compounds tested and may serve as a model for studying cytokinin resistance.
Comparative Cytotoxic Activities of the Flavonoid-Rich Ethyl Acetate Fruit Ex...inventionjournals
The fruit of Pouteriacampechianahas been previously reported to contain flavonoids and polyphenolic substances with high in vitro anti-oxidative effects. Its anti-tumorigenic activities have not been previously demonstrated. This study seeks to demonstrate the cytotoxic effects of the flavonoid-rich ethyl acetate fraction of the fruit extract of P. campechiana(EAFFPC) against K562 leukemic cell lines and healthy human whole blood cells. The standardized MTT-dye cell viability assay was carried out to measure the cytotoxicity of EAFFPC against the aforementioned cell lines cultured in RPMI. The assay showed that at a 60 µg well concentration, EAFFPC exhibited a 55.4% cell viability which is significantly lower than the cell viability obtained with 10 µg of vincristine. In contrast, EAFFPC demonstrated concentration-dependent cytoprotective properties in healthy human whole blood cells (HHWBC). This study confirms specific nonconcentration-dependent cytotoxic effects on K562 leukemic cell lines while exhibiting a concentrationdependent cytoprotective effects in HHWBC
Here you will know , how to write a critical review & what sections are suppose to analyse why reading a paper. Here is a critical review of a paper titled ' Repeated dose multi-drug testing using a microfluidic chip-based
coculture of human liver and kidney proximal tubules equivalents' published recently in 2020 in reputed journal nature.
The document describes the synthesis and evaluation of 6,7-dihydro-4H-isothiazolo[4,5-b]pyridin-5-ones (DIP) as potential antimitotic agents. A series of DIP derivatives were synthesized via a multicomponent reaction. Selected DIP derivatives were found to alter sea urchin egg cleavage at low nanomolar concentrations and showed cytotoxicity against cancer cells, including chemoresistant lines, at submicromolar to low nanomolar levels. Both the sea urchin embryo assay and cancer cell assays confirmed the DIP derivatives act by destabilizing microtubules. Structure-activity relationship studies showed the importance of a p
1. A series of 3-amino-thieno[2,3-b]pyridines (AThPs) were synthesized and evaluated for their ability to affect microtubule dynamics and inhibit cancer cell growth.
2. The most active AThPs in a sea urchin embryo assay, which indicates microtubule destabilization effects, had a tricyclic core with a fused cycloalkyl substituent and an unsubstituted or alkyl-substituted phenyl group.
3. Molecular modeling suggested these compounds inhibit the colchicine binding site on tubulin, supporting a microtubule destabilizing mechanism.
4. Several compounds strongly inhibited the growth of multidrug-resistant cancer
Proteome-wide covalent ligand discovery in native biological systemsMegha Majumder
The document describes a new method for finding drug candidates that bind to specific proteins called isoTOP-ABPP. It involves applying small molecule fragments that covalently bind to cysteine amino acids on proteins. This allows researchers to detect which proteins the fragments bind to. They tested a library of these fragments on cancer cell proteins and found they bound to over 750 cysteines on more than 600 proteins, including some previously considered undruggable. Further experiments showed some fragments could selectively target and inhibit certain proteins like IDH1/2 and label inactive pro-forms of proteins like caspase-8, but not the active forms.
This summary provides the key points from the document in 3 sentences:
This study explored how sulforaphane affects calcium levels and cell viability in renal tubular cells. The results showed that sulforaphane increased calcium levels in the cells in a concentration-dependent manner by promoting calcium entry from outside the cell rather than calcium release from internal stores. Sulforaphane also decreased cell viability in a concentration-dependent manner, which was independent of changes in calcium levels and may have involved the induction of apoptosis.
- The document examines the role of membrane potential in the biogenesis of cytochrome c oxidase subunit II, a mitochondrial gene product.
- Using an in vitro mitochondrial translation system, the authors find that accumulation of unprocessed subunit II precursor (pre-II) occurs when mitochondrial gene products are synthesized under conditions that prevent formation of a normal membrane potential.
- The majority of pre-II generated in this way is inserted into the lipid bilayer, as judged by resistance to sodium carbonate extraction, indicating that membrane potential is required for a step in subunit II biogenesis other than precursor insertion into the membrane.
Yang_et_al-2016-ChemMedChem-Retro-1 Analogues Differentially Affect Oligonucl...Bing Yang
1) Retro-1 is a small molecule that both blocks certain toxins by altering their intracellular trafficking and enhances oligonucleotide activity by releasing them from endosomes.
2) The authors tested several Retro-1 analogues to determine if the two effects involve the same or different molecular targets.
3) They found analogues that affected toxins but not oligonucleotides, and vice versa, indicating the targets are distinct. Retro-1 was the only compound that affected both.
This document summarizes research on sortase enzymes. Sortases are bacterial transpeptidase enzymes that covalently attach secreted proteins to the bacterial cell wall. They play important roles in virulence and pathogenesis. The document discusses various classes of sortases, their mechanisms of action, roles in antibiotic resistance and biofilm formation. It also outlines applications of sortases in areas like vaccine development, drug targeting, and protein immobilization. Overall, the document provides an overview of the sortase enzyme family and their significance in microbial physiology and disease.
1) The study examined the role of Rho kinase in T cell activation and immune responses.
2) Inhibition of Rho kinase attenuated T cell proliferation, cytokine gene expression, actin polymerization, and aggregation of T cell receptors.
3) Treatment with a Rho kinase inhibitor prolonged survival of allogeneic heart transplants in mice and diminished cytokine mRNA expression in the transplants.
4) Rho kinase promotes structural rearrangements in T cells that are critical for T cell signaling and activation during cellular immune responses.
This document describes the synthesis and anticancer activity of novel 1,2,3-triazole derivatives tethered to a 1,2-benzisoxazole scaffold. Specifically:
- Compounds were synthesized via copper-catalyzed azide-alkyne cycloaddition between benzisoxazole-3-azide and various alkynes.
- The most potent compound, PTB, showed low micromolar anticancer activity against acute myeloid leukemia cell lines via apoptosis induction and cell cycle arrest.
- PTB was found to inhibit histone deacetylases, leading to increased acetylation of histone H3 and tubulin, as well as upregulation of p21
1) Adamantyl-tethered-biphenylic compounds were synthesized and found to induce apoptosis in cancer cells.
2) Compound 30-(adamantan-1-yl)-40-methoxy-[1,10-biphenyl]-3-ol (AMB) showed cytotoxic activity against hepatocellular carcinoma cell lines without harming normal cells.
3) AMB was found to target and downregulate anti-apoptotic Bcl-2 family proteins like Bcl-2 and Bcl-xL, leading to cell cycle arrest and induction of apoptosis in cancer cells.
The complement system is a series of more than 30 proteins that interact in a precise order to enhance host defense. It can be activated through three pathways: the classical pathway involves antibody binding and nine proteins; the alternative pathway is antibody-independent; and the lectin pathway involves mannose-binding lectin binding to pathogens. Complement activation results in cytolysis of target cells, opsonization to enhance phagocytosis, chemotaxis to recruit immune cells, and anaphylaxis.
This document summarizes research identifying a small molecule inhibitor of Holliday junctions (HJs). Researchers screened over nine million compounds and identified an N-methyl aminocyclic thiourea that accumulates HJs during site-specific DNA recombination in vitro and inhibits the HJ-processing enzyme RecG helicase. This small molecule binds specifically to protein-free HJs, like peptide inhibitors previously identified, but showed only modest antibacterial activity, likely due to poor membrane permeability. Nonetheless, this small molecule represents a potential functional analog of peptide HJ inhibitors.
This document describes the efficient synthesis of glaziovianin A (GVA) and related isoflavones starting from readily available plant metabolites. A six-step reaction sequence involving bromination, alkylation, oxidation, condensation, epoxidation, and cyclization produced GVA and various alkoxyphenyl derivatives. Both an in vivo sea urchin embryo assay and screening of human cancer cell lines showed that GVA and some derivatives have antimitotic effects by destabilizing microtubules. Structure-activity relationship studies found that certain substituents, such as a methylenedioxy or trimethoxy group, influenced the compounds' potency. GVA was generally the most active compound, inhibiting cancer cell
- Ostreococcus tauri is a species of marine microalgae that is able to grow in minimal media lacking cobalt and cobalamin, even though it lacks the gene for the cobalamin-independent methionine synthase METE. This suggests it can synthesize methionine without cobalamin.
- The author proposes that the sole methionine synthase in O. tauri, METH, may be able to function similar to METE by facilitating direct methyl group transfer from methyltetrahydrofolate to homocysteine in the absence of cobalamin.
- Experiments transforming O. tauri to disrupt or replace METH could help determine if it is able to function as both a
1) Researchers used fragment-based crystallography screening and medicinal chemistry to develop inhibitors of leukotriene A4 hydrolase (LTA4H), identifying compound 20 (DG-051) as a potent inhibitor.
2) They identified initial fragment hits that bound to LTA4H's active site through crystallography screening and optimized fragments through iterative chemistry and structural analysis, guided by ligand efficiency.
3) Compound 20 (DG-051) was identified as a potent, orally bioavailable inhibitor of LTA4H currently in clinical trials for myocardial infarction and stroke.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive function. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
1) The study examined the effects of various cytokinins, cytokinin ribosides, and their analogs on the viability of normal and cancerous human cells.
2) Only a few compounds, including isopentenyladenosine (1a) and N6-benzyladenosine (3a), significantly impaired viability in most cell lines tested, including normal endothelial cells and various cancer cell lines.
3) Colon carcinoma LoVo cells were uniquely insensitive to all compounds tested and may serve as a model for studying cytokinin resistance.
Comparative Cytotoxic Activities of the Flavonoid-Rich Ethyl Acetate Fruit Ex...inventionjournals
The fruit of Pouteriacampechianahas been previously reported to contain flavonoids and polyphenolic substances with high in vitro anti-oxidative effects. Its anti-tumorigenic activities have not been previously demonstrated. This study seeks to demonstrate the cytotoxic effects of the flavonoid-rich ethyl acetate fraction of the fruit extract of P. campechiana(EAFFPC) against K562 leukemic cell lines and healthy human whole blood cells. The standardized MTT-dye cell viability assay was carried out to measure the cytotoxicity of EAFFPC against the aforementioned cell lines cultured in RPMI. The assay showed that at a 60 µg well concentration, EAFFPC exhibited a 55.4% cell viability which is significantly lower than the cell viability obtained with 10 µg of vincristine. In contrast, EAFFPC demonstrated concentration-dependent cytoprotective properties in healthy human whole blood cells (HHWBC). This study confirms specific nonconcentration-dependent cytotoxic effects on K562 leukemic cell lines while exhibiting a concentrationdependent cytoprotective effects in HHWBC
Here you will know , how to write a critical review & what sections are suppose to analyse why reading a paper. Here is a critical review of a paper titled ' Repeated dose multi-drug testing using a microfluidic chip-based
coculture of human liver and kidney proximal tubules equivalents' published recently in 2020 in reputed journal nature.
The document describes the synthesis and evaluation of 6,7-dihydro-4H-isothiazolo[4,5-b]pyridin-5-ones (DIP) as potential antimitotic agents. A series of DIP derivatives were synthesized via a multicomponent reaction. Selected DIP derivatives were found to alter sea urchin egg cleavage at low nanomolar concentrations and showed cytotoxicity against cancer cells, including chemoresistant lines, at submicromolar to low nanomolar levels. Both the sea urchin embryo assay and cancer cell assays confirmed the DIP derivatives act by destabilizing microtubules. Structure-activity relationship studies showed the importance of a p
1. A series of 3-amino-thieno[2,3-b]pyridines (AThPs) were synthesized and evaluated for their ability to affect microtubule dynamics and inhibit cancer cell growth.
2. The most active AThPs in a sea urchin embryo assay, which indicates microtubule destabilization effects, had a tricyclic core with a fused cycloalkyl substituent and an unsubstituted or alkyl-substituted phenyl group.
3. Molecular modeling suggested these compounds inhibit the colchicine binding site on tubulin, supporting a microtubule destabilizing mechanism.
4. Several compounds strongly inhibited the growth of multidrug-resistant cancer
Proteome-wide covalent ligand discovery in native biological systemsMegha Majumder
The document describes a new method for finding drug candidates that bind to specific proteins called isoTOP-ABPP. It involves applying small molecule fragments that covalently bind to cysteine amino acids on proteins. This allows researchers to detect which proteins the fragments bind to. They tested a library of these fragments on cancer cell proteins and found they bound to over 750 cysteines on more than 600 proteins, including some previously considered undruggable. Further experiments showed some fragments could selectively target and inhibit certain proteins like IDH1/2 and label inactive pro-forms of proteins like caspase-8, but not the active forms.
This summary provides the key points from the document in 3 sentences:
This study explored how sulforaphane affects calcium levels and cell viability in renal tubular cells. The results showed that sulforaphane increased calcium levels in the cells in a concentration-dependent manner by promoting calcium entry from outside the cell rather than calcium release from internal stores. Sulforaphane also decreased cell viability in a concentration-dependent manner, which was independent of changes in calcium levels and may have involved the induction of apoptosis.
- The document examines the role of membrane potential in the biogenesis of cytochrome c oxidase subunit II, a mitochondrial gene product.
- Using an in vitro mitochondrial translation system, the authors find that accumulation of unprocessed subunit II precursor (pre-II) occurs when mitochondrial gene products are synthesized under conditions that prevent formation of a normal membrane potential.
- The majority of pre-II generated in this way is inserted into the lipid bilayer, as judged by resistance to sodium carbonate extraction, indicating that membrane potential is required for a step in subunit II biogenesis other than precursor insertion into the membrane.
Yang_et_al-2016-ChemMedChem-Retro-1 Analogues Differentially Affect Oligonucl...Bing Yang
1) Retro-1 is a small molecule that both blocks certain toxins by altering their intracellular trafficking and enhances oligonucleotide activity by releasing them from endosomes.
2) The authors tested several Retro-1 analogues to determine if the two effects involve the same or different molecular targets.
3) They found analogues that affected toxins but not oligonucleotides, and vice versa, indicating the targets are distinct. Retro-1 was the only compound that affected both.
This document summarizes research on sortase enzymes. Sortases are bacterial transpeptidase enzymes that covalently attach secreted proteins to the bacterial cell wall. They play important roles in virulence and pathogenesis. The document discusses various classes of sortases, their mechanisms of action, roles in antibiotic resistance and biofilm formation. It also outlines applications of sortases in areas like vaccine development, drug targeting, and protein immobilization. Overall, the document provides an overview of the sortase enzyme family and their significance in microbial physiology and disease.
1) The study examined the role of Rho kinase in T cell activation and immune responses.
2) Inhibition of Rho kinase attenuated T cell proliferation, cytokine gene expression, actin polymerization, and aggregation of T cell receptors.
3) Treatment with a Rho kinase inhibitor prolonged survival of allogeneic heart transplants in mice and diminished cytokine mRNA expression in the transplants.
4) Rho kinase promotes structural rearrangements in T cells that are critical for T cell signaling and activation during cellular immune responses.
This document describes the synthesis and anticancer activity of novel 1,2,3-triazole derivatives tethered to a 1,2-benzisoxazole scaffold. Specifically:
- Compounds were synthesized via copper-catalyzed azide-alkyne cycloaddition between benzisoxazole-3-azide and various alkynes.
- The most potent compound, PTB, showed low micromolar anticancer activity against acute myeloid leukemia cell lines via apoptosis induction and cell cycle arrest.
- PTB was found to inhibit histone deacetylases, leading to increased acetylation of histone H3 and tubulin, as well as upregulation of p21
1) Adamantyl-tethered-biphenylic compounds were synthesized and found to induce apoptosis in cancer cells.
2) Compound 30-(adamantan-1-yl)-40-methoxy-[1,10-biphenyl]-3-ol (AMB) showed cytotoxic activity against hepatocellular carcinoma cell lines without harming normal cells.
3) AMB was found to target and downregulate anti-apoptotic Bcl-2 family proteins like Bcl-2 and Bcl-xL, leading to cell cycle arrest and induction of apoptosis in cancer cells.
The complement system is a series of more than 30 proteins that interact in a precise order to enhance host defense. It can be activated through three pathways: the classical pathway involves antibody binding and nine proteins; the alternative pathway is antibody-independent; and the lectin pathway involves mannose-binding lectin binding to pathogens. Complement activation results in cytolysis of target cells, opsonization to enhance phagocytosis, chemotaxis to recruit immune cells, and anaphylaxis.
This document summarizes research identifying a small molecule inhibitor of Holliday junctions (HJs). Researchers screened over nine million compounds and identified an N-methyl aminocyclic thiourea that accumulates HJs during site-specific DNA recombination in vitro and inhibits the HJ-processing enzyme RecG helicase. This small molecule binds specifically to protein-free HJs, like peptide inhibitors previously identified, but showed only modest antibacterial activity, likely due to poor membrane permeability. Nonetheless, this small molecule represents a potential functional analog of peptide HJ inhibitors.
This document describes the efficient synthesis of glaziovianin A (GVA) and related isoflavones starting from readily available plant metabolites. A six-step reaction sequence involving bromination, alkylation, oxidation, condensation, epoxidation, and cyclization produced GVA and various alkoxyphenyl derivatives. Both an in vivo sea urchin embryo assay and screening of human cancer cell lines showed that GVA and some derivatives have antimitotic effects by destabilizing microtubules. Structure-activity relationship studies found that certain substituents, such as a methylenedioxy or trimethoxy group, influenced the compounds' potency. GVA was generally the most active compound, inhibiting cancer cell
- Ostreococcus tauri is a species of marine microalgae that is able to grow in minimal media lacking cobalt and cobalamin, even though it lacks the gene for the cobalamin-independent methionine synthase METE. This suggests it can synthesize methionine without cobalamin.
- The author proposes that the sole methionine synthase in O. tauri, METH, may be able to function similar to METE by facilitating direct methyl group transfer from methyltetrahydrofolate to homocysteine in the absence of cobalamin.
- Experiments transforming O. tauri to disrupt or replace METH could help determine if it is able to function as both a
1) Researchers used fragment-based crystallography screening and medicinal chemistry to develop inhibitors of leukotriene A4 hydrolase (LTA4H), identifying compound 20 (DG-051) as a potent inhibitor.
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1. r XXXX American Chemical Society A dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
ARTICLE
pubs.acs.org/jmc
Polyalkoxybenzenes from Plants. 5. Parsley Seed Extract in Synthesis
of Azapodophyllotoxins Featuring Strong Tubulin Destabilizing
Activity in the Sea Urchin Embryo and Cell Culture Assays
Marina N. Semenova,†,‡
Alex S. Kiselyov,§
Dmitry V. Tsyganov,||
Leonid D. Konyushkin,||
Sergei I. Firgang,||
Roman V. Semenov,||
Oleg R. Malyshev,||
Mikhail M. Raihstat,||
Fabian Fuchs,^
Anne Stielow,^
Margareta Lantow,^,#
Alex A. Philchenkov,3
Michael P. Zavelevich,3
Nikolay S. Zefirov,O
Sergei A. Kuznetsov,^
and Victor V. Semenov*,‡,||
†
Institute of Developmental Biology, RAS, 26 Vavilov Street, 119334 Moscow, Russian Federation
‡
Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus
§
CHDI Foundation, 6080 Center Drive, Suite 100, Los Angeles California 90045, United States
)
N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation
^
Institute of Biological Sciences, University of Rostock, 3 Albert-Einstein-Strasse, D-18059 Rostock, Germany
#
Department of Pathology, Immunology and Laboratory Medicine, University of Florida, 1600 Archer Road, Gainesville Florida 32610,
United States
3
R. E. Kavetsky Institute of Experimental Oncology, Pathology, and Radiobiology, National Academy of Sciences of Ukraine,
45 Vasyl0
kivska Street, 03022 Kyiv, Ukraine
O
Lomonosov Moscow State University, GSP-1, Leninskie Gory, 119991 Moscow, Russian Federation
bS Supporting Information
’ INTRODUCTION
Podophyllotoxin (PT) (Figure 1) is a lignan of aryltetralin
family found in plants of Podophyllum genus. It is a strong
microtubule destabilizing agent that binds to the colchicine site
of tubulin.1,2
PT and its derivatives exhibit anticancer, antiviral,
and insecticide activities based on their ability to dissociate
microtubules.3À9
However, clinical trials of PT as an anticancer
agent were unsuccessful due to its considerable toxicity.3
Nu-
merous attempts have been made to improve PT safety profile
while maintaining its potency. For example, semisynthetic PT
derivatives, including etoposide (Figure 1), teniposide, and
etopophos, are currently in clinical use for the treatment of a
variety of malignancies. A reported mechanism of action for these
compounds is distinct from that of the parent PT and involves
strong DNA-topoisomerase II inhibition leading to late S-G2 cell
cycle arrest and subsequent apopotosis.10À13
An alternative
nontubulin and nonantitopoisomerase mode of action for some
PT derivatives has been proposed recently, associated with cell
death and sub-G1 apoptotic cell accumulation without any
significant cell cycle arrest.11,14
PT structure (Figure 1) features four chiral centers, making it a
challenge for the structureÀactivity relationship studies. Abso-
lute stereochemistry of substituents at the C1 atom (the ring C)
was reported to have major influence on compound’s antitumor
activity.3
Several synthetically feasible structural analogues of PT
Received: June 8, 2011
ABSTRACT: A series of 4-azapodophyllotoxin derivatives with
modified rings B and E have been synthesized using allylpo-
lyalkoxybenzenes from parsley seed oil. The targeted molecules
were evaluated in vivo in a phenotypic sea urchin embryo assay
for antimitotic and tubulin destabilizing activity. The most
active compounds identified by the in vivo sea urchin embryo
assay featured myristicin-derived ring E (4e, 6e, and 8e). These
molecules were determined to be more potent than podophyl-
lotoxin. Cytotoxic effects of selected molecules were further
confirmed and evaluated by conventional assays with A549 and
Jurkat human leukemic T-cell lines including cell growth
inhibition, cell cycle arrest, cellular microtubule disruption, and induction of apoptosis. The ring B modification yielded 6-OMe
substituted molecule 8e as the most active compound. Finally, in Jurkat cells, compound 8e induced caspase-dependent apoptosis
mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic
pathway.
2. B dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
were designed to address epimerization at positions 1À4.3
For
example, 4-aza-PT derivatives with a chiral center C1 (Figure 1)
showed antiproliferative and pro-apoptotic activity in HeLa and
Jurkat cells (4a and 18a)15,16
and inhibited growth of human liver
cancer cells HepG2 (4a and 4i).17
Two racemic analogues of
4-aza-PTs (4a and 6a) were more cytotoxic against P388 murine
leukemia cells than the parent PT.18,19
Several 4-aza-PT deriva-
tives were reported to be potent tumor inhibitors (ex., 4a)20
and
vascular-disrupting agents.21
In addition, insecticidal (larvicidal)
activity possibly mediated by microtubule alterations was re-
ported for this class of molecules.6
However, experimental
evidence for the mechanism of action and cellular targets of
4-aza-PTs has been obtained only recently. Namely, it was shown
that some of 4-aza-PTs with hetero aryl, benzo hetero aryl, or
tetrahydronaphthalene rings A and B exhibited cytotoxicity
involving microtubule depolymerization, G2/M cell cycle arrest,
and apoptotic cell death.22,23
Considering synthetic feasibility, antimitotic, antiproliferative,
and tubulin destabilizing activity of 4-aza-PT derivatives, these
compounds continue to attract a considerable interest. In the
present study, we prepared a series of 4-aza-PT derivatives using
plant polyalkoxybenzenes easily available from parsley and dill
seed extracts.24,25
It has been suggested that rotation of the axial
E-ring in the parent PT molecule is restricted as it is almost
orthogonal to the other rings.26
Therefore a modification of the
rings B and E with additional methoxy functionality could affect
the rotation of the ring E yielding PT derivatives with promising
antimitotic potencies. Moreover, tetraalkoxybenzene pharmaco-
phore is featured in several natural products with reported
antiproliferative activity including Cactaceae tetrahydroiso-
quinolines27
and flavonoids and isoflavonoids.28À30
In designing the evaluation sequence for test molecules, we
aimed at identifying potent novel aza-analogues of PT and
confirming their specific antitubulin activity via a variety of
cell-based assays. The initial in vivo sea urchin embryo assay
allowed for the identification of hits featuring desirable antimi-
totic potency and phenotypic effect (vide infra) in comparison
with the parent molecule (PT). This primary screen based on the
simple organism model has been carried out to yield active
molecules that were likely to affect tubulin dynamics as shown by us
earlier.31À34
Tubulin targeting activity of these compounds would
be further confirmed in A549 human lung epithelial carcinoma cell
line. A detailed insight into the effects of selected active compounds
on cellular proliferation, cell cycle progression, and microtubule
distribution in cultured A549 cells was intended to confirm their
specific tubulin destabilizing mode of action. Moreover, the link
between compound-induced microtubule disruption and respective
downstream intracellular mechanisms responsible for initiation of
caspase-mediated apoptotic events was studied.
All molecules were evaluated in a phenotypic sea urchin
embryo assay in order to assess their antiproliferative and
tubulin-destabilizing activities.31
The assay includes (i) fertilized
egg test for antimitotic activity displayed by cleavage alteration/
arrest and (ii) behavioral monitoring of a free-swimming blas-
tulae treated immediately after hatching. In our earlier validation
efforts, we concluded that lack of forward movement, settlement
to the bottom of the culture vessel and rapid spinning of an
embryo around the animalÀvegetal axis suggests a tubulin-
destabilizing activity caused by a molecule (video illustrations
are available at http://www.chemblock.com). An assay setup
provides the possibility to identify a molecule with antimitotic or
nonspecific cytotoxic activity and a novel mode of action.32
Data
generated by the sea urchin embryo assay have been further
confirmed using conventional cell-based and in vitro tubulin
polymerization assays.33,34
As a result, we identified several
promising candidates for the advanced in vivo evaluation.
’ CHEMISTRY
4-Aza-PTs 4À13 and intermediate quinolines 40
À150
were
synthesized as described earlier6,15À20,35À37
via the cyclization of
4-chloro-acetoacetic acid with the corresponding amines, fol-
lowed by the addition of aldehydes 2aÀk in the presence of an
Figure 1. Structures of podophyllotoxin, etoposide, and general struc-
ture of synthesized 4-aza-analogues.
Scheme 1a
a
Reagents and conditions: (a) (i) powdered KOH, (n-Bu)4N+
BrÀ
, heat,
100 °C, 40 min; (ii) O3, CHCl3ÀMeOHÀpyridine (80:20:3 v/v), À15
°C, 1À2 h;25
(b) C6H6ÀAcOH, reflux, 8 h; (c) CF3COOH, rt, 24 h;
(d) AcOHÀNaBH3CN, rt, 15 h; (e) ref 43; (f) EtOHÀEt3N, reflux, 3 h.
Compounds 4À13 (fÀk), 17i, 18e, 19e were synthesized from the
corresponding commercial aldehydes.
3. C dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
oxidizing reagent p-chloranil (Scheme 1). Aldehydes 2aÀe were
synthesized using natural polyalkoxybenzenes extracted from
parsley seed oil.25
For the SAR study, a panel of 4-aza-PTs with
the ring E derived from commercially available aldehydes 2fÀk
has been obtained. Quinolines 40
À130
were reduced with
NaBH3CN in a glacial acetic acid to yield compounds 4À13 in
12À86% yields. 4-Aza-PTs 4À13 were stable as solids, however,
they were readily oxidized to respective quinolines 40
À130
when
dissolved in DMSO or ethanol. Dihydropyridopyrazole deriva-
tives 17À19 were obtained by a three-component condensation
of respective aminopyrazole, aldehyde 2, and tetronic acid 16 as
reported in the literature.16
Notably, compounds 4g, 40
g, 4f, and
40
f are aza-analogues of the natural lignans taiwanin C and
chinensin.36,38À42
’ RESULTS AND DISCUSSION
All synthesized aza-PTs 4À19 and 40
À150
were evaluated for
their antiproliferative, antimitotic, and microtubule destabilizing
activities using in vivo sea urchin embryo assay. Cytotoxicity,
cellular microtubule depolymerization, and apoptosis-inducing
effects of selected compounds have been further assessed using
A549 human lung epithelial carcinoma and Jurkat human
leukemic T-cell lines.
Evaluation of 4-aza-PTs in the Phenotypic Sea Urchin
Embryo Assay. Effects of 4-aza-PTs on the sea urchin embryo
are summarized in Table 1 and Table S1 of the Supporting
Information. PT and etoposide were used as references.
As evidenced from Table 1, a number of 4-aza-PTs (4a,c,eÀg,
iÀk, 5e, 6a,e, 7e, 8e,h, 9e, 11e, 110
e, 12a, and 13e) displayed
significant cleavage alteration, cleavage arrest, and embryo spinning,
suggesting their antimitotic microtubule destabilizing activity. Fig-
ure 2 illustrates typical developmental changes of an embryo when
treated with a representative compound 4e. The observed pheno-
typic changes suggest direct tubulin dynamic effects of 4-aza-PT
derivatives. Interestingly, molecules 4a, 4e, and 8h were reported as
larvicidal agents against Phaedon cochleariae beetle, whereas 4j, 4k,
Table 1. Effects of 4-Aza-podophyllotoxins on Sea Urchin Embryo and Cancer Cells
sea urchin embryo effects EC (nM)a
cytotoxicity IC50 (nM)
compd cleavage alteration cleavage arrest embryo spinning A549b
P388c
A549 cell total microtubule disruption (μM)d
PT 20 50 500 7.47 ( 0.41 10.4 0.1
etoposide 2000 >68000 >68000 7140e
1180e
NDf
4a 50 500 2000 2.71 ( 0.92 4.5 0.1
40
a >4000 >4000 >4000 11928.33 ( 5368.30 >252900 >5
4b 1000 2000 >5000 NDf
NDf
NDf
4c 100 500 4000 NDf
NDf
NDf
4d 1000 4000 >10000 299.32 ( 58.35 NDf
1
4e 5 50 50 55.18 ( 7.54 NDf
1
4f 10 50 200 NDf
85.4 NDf
4g 5 100 2000 595.58 ( 46.18 130.7 1
4i 50 200 2000 >3200 385.4 NDf
4j 5 20 100 NDf
15.7 NDf
4k 5 20 100 63.00 ( 5.21 17.2 NDf
5a 2000 >2000 >2000 291.43 ( 143.53 NDf
5
5e 50 500 2000 297.63 ( 164.54 NDf
>5
5i 2000 >2000 >2000 NDf
NDf
NDf
6a 200 1000 1000 9.20 ( 1.22 4.1 1
6e 5 50 100 64.67 ( 11.34 NDf
0.5
7e 10 50 500 282.27 ( 96.99 NDf
5
8e 0.5 5 50 15.14 ( 2.98 NDf
1
8h 2 10 200 NDf
NDf
NDf
9e 20 200 2000 533.83 ( 111.47 NDf
>5
10f >4000 >4000 >4000 NDf
NDf
NDf
11e 5 50 100 159.05 ( 8.24 NDf
NDf
110
e 200 2000 5000 NDf
NDf
NDf
12a 50 200 100 56.54 ( 6.83 NDf
0.5
13e 2 20 50 264.09 ( 52.32 NDf
5
130
e 100 1000 >5000 NDf
NDf
NDf
140
e 100 >4000 >5000 NDf
NDf
NDf
a
The sea urchin embryo assay was conducted as described previously;31
fertilized eggs and hatched blastulae were exposed to 2-fold decreasing
concentrations of compounds; duplicate measurements showed no differences in effective threshold concentration (EC) values. b
A549: human lung
epithelial carcinoma cells; data are expressed as the mean ( SD from the doseÀresponse curves of three independent experiments. c
P388: murine
leukemia cells; data from ref 19. d
A549 cells were incubated with compounds at concentrations of 0.1, 0.5, 1, and 5 μM during 8 h. Data are expressed as
compound concentration that caused total microtubule disassembly from three independent experiments (see Figure 4B,DÀF as an example). e
Data
from ref 44. f
ND: not determined.
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Journal of Medicinal Chemistry ARTICLE
and 8h were toxic against Spodoptera frugiperda larvae.6
We believe
that less potent compounds 4b,d, 18e, 60
b, and 130
e were tubulin
destabilizers as well, although they did not cause embryo spinning.
As evidenced from the detailed microscopic analysis, these com-
pounds induced formation of tuberculate eggs typical of tubulin
destabilizers (Figure 2C).31
In our hands, topoisomerase inhibitor
etoposide featured cleavage alteration but failed to induce cleavage
arrest and embryo spinning. When applied to hatched blastulae,
etoposide at 4À5 μM caused larval (pluteus) malformations. At
higher concentrations (10À20 μM), marked abnormalities of
gastrulation and morphogenesis were detected, whereas at 40À68
μM, developmental abnormalities and eventual embryo death were
observed after 2.5 h of treatment.
Because of their marginal solubility in DMSO, ethanol, and
seawater, we were unable to test 80
e, 110
a, and 120
a or reach
higher concentrations for 4b,d, 18e, 60
b, and 130
e. 140
e showed
nontubulin antiproliferative activity, as evidenced by the lack of
embryo spinning and arrested tuberculate eggs.
StructureÀActivity Studies in the Sea Urchin Embryo
Assay. 1. The Ring C: Saturated vs Unsaturated Compounds.
A number of 1,4-aza-PT analogues (4aÀg,iÀk, 5e, 6a,e, 7e,
9e, and 13e) featuring 1,4-dihydropyridine system for the ring
C exhibited potent antimitotic activity with EC50 values of
0.5 nMÀ2 μM in the sea urchin assay. These results are in
agreement with the published data.19
Conversely, the respective
aromatic pyridine derivatives 40
aÀg,iÀk, 50
e, 60
a,e, 70
e, 90
e, and
130
e) were inactive up to 4 μM concentration or exhibited
moderate cleavage alteration effect (Table 1; Table S1, Support-
ing Information), with a notable exception of 110
e, featuring
myristicin substituent for the ring E. This molecule did exhibit a
tubulin destabilizing activity although it was significantly lower
than that for the corresponding unsaturated analogue 11e. In
addition, aromatization of the ring C yielded decrease in cyto-
toxicity and microtubule effects, presumably due to the inactive
conformation of the ring E.3
Several saturated compounds were found to lose their activity
in DMSO solution at room temperature. For example, the
antimitotic effect of 4k on sea urchin embryos decreased
significantly (by ca. 5-fold) within 2 days after storage in DMSO.
According to the literature data, aromatization of 4a to yield 40
a
required harsh experimental conditions such as refluxing acetic
acid.37
We observed this conversion to take place in DMSO at
room temperature, as confirmed by the time course NMR studies
of 4k. Specifically, in 2 and 6 days, the rate of its conversion to the
respective aromatic analogue 40
k were 25% and 45%, respec-
tively. A similar activity reduction was observed for 4i and 6a,
whereas DMSO stocks of compounds 4f, 8e, 9e, and 11e
retained their activities for 2À4 days.
2. Other Substitutions: Rings A, B, and E. Introduction of a
methoxy-group to C8 position of the ring B dramatically de-
creased antiproliferative activity in sea urchin embryo assay
(Table 1, compounds 4a . 5a, 4e . 5e, and 4i . 5i).
Replacement of methylenedioxy group (the ring A) with ethy-
lenedioxy substituent did not result in a definitive outcome. For
example, the potency of respective ethylenedioxy derivative
(Table 1, 4a vs 6a) in the sea urchin embryo cleavage stages
was reduced significantly, similar to the reported insecticidal
activity.6
However, for the pair 4e vs 6e, the activity was not
affected.19
Replacement of the ring A with two methoxy substit-
uents was reported to either result in a marked reduction of
cytotoxicity19,45,46
or was negligible.16
Similar derivatives featur-
ing a single methoxy group at C7 displayed reduced potency
compared to the parent 4-aza-PT molecule (Table 1, 4e vs 7e).
On the contrary, 6-methoxy derivative 8e exhibited the strongest
antiproliferative effect of all tested 4-aza-PTs being ca. 40 times
more potent than the parent PT. Another 6-methoxy analogue
8h displayed high activity in the sea urchin assay as well as
pronounced larvicidal effect.6
Compound 9e lacking the ring A
was less active, however it did retain the antimitotic activity.
Substitution of the ring A with cyclopentane moiety was reported
to result in a moderate decrease in cytotoxicity.19
In our assay
system, this modification slightly enhanced the in vivo effect
(Table 1, 4e and 13e), however a nontubulin antiproliferative
activity of 5,6-cyclopentyl derivative 140
e was also detected. In
contrast, replacement of the ring A with a cyclohexyl group did
not influence the antimitotic potency at the sea urchin embryo
cleavage stages (Table 1, 4e and 11e; 4a and 12a). At later
developmental stages of the embryo (ex., after hatching), 12a
displayed formidable toxicity. Similarly, cyclohexyl derivative 12a
exhibited cytotoxicity against human cervical (HeLa) and breast
(MCF-7) carcinomas cells at nanomolar concentration range.23
A replacement of the AB ring system of 4-aza-PTs with
pyrazole was reported to furnish derivatives that retained both
cytotoxic and proapoptotic activities at 5 μM concentration.15,16
However, cellular target(s) or mode of action for these molecules
have not been determined. In our hands, all tested pyrazole-
containing compounds 17a,i, 18aÀc, and 19aÀc,e failed to affect
sea urchin embryo development at any stage up to 4 μM
concentration, the only exception being myristicin-derived 18e
that exhibited weak tubulin-destabilizing properties (Table S1,
Supporting Information).
As described above, 1,4-dihydropyridine derivatives (the ring C)
showed the best activities in the sea urchin embryo assay. We further
evaluated the effect of C1 substituent (the ring E) on compounds
activity. Notably, molecules featuring m-methoxybenzene- or myr-
isticin-derived substituent (Table 1, 4e,j, 5e, 6e, 7e, 8e, 9e, 11e, and
13e) consistently exhibited the highest potency. 2,3-Dimethoxy and
Figure 2. Effect of 4e on sea urchin embryo development. Time after
fertilization (21 °C): 6 h. (A) Intact embryo at early blastula stage.
Fertilized eggs were exposed continuously to 4e at 10 nM (B) and 100
nM (C). Note abnormal cleavage of cells with different size and shape
that are irregularly positioned and formation of large multinucleated
blastomeres (B). Tuberculate shape of an arrested egg seen on (C) is
typical for tubulin destabilizing agents.
Figure 3. Structures of 4e enantiomers.
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Journal of Medicinal Chemistry ARTICLE
ethylenedioxy derivatives (ex., 4f,g) were less potent, displaying
activities slightly higher than the parent PT. (see Table 1). Respec-
tive p-methoxybenzene and 3,4,5-trimethoxybenzene analogues
showed further reduced antitubulin effects in the assay (Table 1,
4a,i, 5a,i, and 6a), whereas 4-aza-PT molecules substituted with
dillapiol-, apiol-derived or tetramethoxybenzene moieties 4bÀd
were the least active. Compound 4k with unsubstituted benzene
ring E exhibited strong antimitotic tubulin destabilizing activity,
suggesting that methoxy groups in the ring E may not directly affect
antitubulin activity. Similarly, 30
,40
,50
-demethoxy PT retained sig-
nificant cytotoxicity.47
For the molecules featuring 8-methoxy
moiety, the myristicin-derived analogue 5e displayed the highest
activity(5evs5a,i).Asimilar patternwasobservedfortherespective
ethylenedioxy derivatives 6a and 6e.
In the next round of structureÀactivity studies, the effect of
stereochemistry on compound’s activity has been studied. It was
reported previously that (+)-configuration of antitubulin isoqui-
noline derivatives was essential for their cytotoxicity and inhibi-
tion of tubulin polymerization.48
For 4-aza-PTs, only R-enantiomer
of 8h was found to be cytotoxic against cultured insect cells and
larvae, showing cellular behavior consistent with its microtubule
effect.6
Figure 4. Effects of selected compounds on the interphase microtubule networks in A549 lung carcinoma cells as analyzed by indirect
immunofluorescence microscopy. Cells were treated with 0.1% DMSO (A) and with test compounds (1 μM) for 8 h: 4a (B), 40
a (C), 4e (D), 6a
(E), and 8e (F). Bar: 25 μm. Note microtubule disappearance in B, DÀF.
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Similarly, R-enantiomer of 4-aza-PT mimetic with naphtha-
lene A,B-ring system was about 1000 times more potent in HeLa
and MCF-7 cells than S-enantiomer.23
In the present study, a
racemic 4e was separated by chiral HPLC to yield pure enantio-
mers (Figure 3 and Figure S1, Supporting Information). Both
R-isomer and a respective racemate of 4e were found to cause
cleavage alteration and arrest of the sea urchin embryo at 5 and 50
nM, respectively. The respective S-isomer was less active, ex-
hibiting cleavage abnormalities at 50 nM and cleavage arrest at
200 nM. Both enantiomers triggered embryo spinning, suggest-
ing their tubulin destabilizing properties.
In summary, the sea urchin embryo profiling of the new aza-
PT derivatives suggested that the most potent representative in
each structural group featured myristicin-derived ring E (ex., 4e,
5e, 6e, 8e, 11e, and 13e, Table 1). Notably, methylenedioxy ring
A was not required for the activity. For example, introduction of
6-methoxy or 7-methoxy groups into the ring B yielded potent
molecules (ex., 7e, 8e, and 8h).
Cell Growth Inhibitory Activity. Following outcome of the
sea urchin embryo assay, we evaluated the inhibitory effect of
selected active compounds on proliferation of A549 human lung
carcinoma cells and Jurkat leukemia T-cells using MTT49
and
trypan blue assays, respectively. Relevant A549 cellular IC50 data
for 4a,d,e,g,i,k, 5a,e, 6a,e, 7e, 8e, 9e, 11e, 12a, 13e, 40
a, and
reference compounds (PT, etoposide) are summarized in
Table 1. Reported cytotoxicity data for selected molecules
against P388 murine leukemia cells are included as well (Table 1).19
Notably, in Jurkat cells, 8e and PT exhibited IC50 values of 17.5
and 25 nM, respectively.
In general, sensitivity of sea urchin embryos to 4-aza-PTs at the
cleavage stage, when cells divide synchronously every 35À40
min, was markedly higher than that of cancer cells, probably due
to their higher frequency of mitosis. Key features of the most
active molecules identified in both assays included: (i) 1,4-
dihydropyridine template for the ring C (4a vs 40
a), (ii) a
myristicin-derived substitution in the ring E (8e), (iii) 6-methoxy
substituent replacing the ring A (4e vs 8e), and (iv) lack of
8-methoxy substitution in the ring B (4a vs 5a, 4e vs 5e). Results
of structureÀactivity relationship studies for the ring E correlated
well between both cellular (A549 and P388) and sea urchin
embryo assays. Compounds 4f and 4j exhibited high cytotoxicity
against P388 cells (Table 1).19
Molecules featuring 3,4,5-tri-
methoxybenzene substituent (4a and 6a) as well as the parent PT
were the most cytotoxic agents against both human A549 and
murine P388 cancer cell lines, 4a being more potent than PT.
Notably, derivatives endowed with the myristicin moiety showed
the greatest antimitotic activities in sea urchin embryo assay.
Similarly, in Jurkat cells a myristicin derivative 8e was more
potent than PT. Replacement of the ring A with cyclopentyl or
cyclohexyl moieties markedly decreased cytotoxicity against
A549 cells (4e vs 13e; 4a vs 12a), but this effect was almost
negligible in the sea urchin embryo test. The observed discre-
pancy could be attributed to the differences between mitotic
spindle microtubules in frequently dividing sea urchin blasto-
meres vs predominantly interphase microtubules in cultured
cancer cells targeted by tubulin destabilizing agents.
The Effect on Microtubule Distribution in Cells. Next, we
evaluated the effect of selected active compounds on microtubule
stability and distribution in cultured A549 cells. Specifically, cells
were incubated with test articles (0.1À5 μM) or PT (0.1 and
5 μM) as a positive control and 0.5% DMSO as a negative control.
Microtubules were subsequently labeled by the indirect immu-
nofluorescence method and analyzed by fluorescence micro-
scopy. Figure 4 exemplifies the microtubule destabilization effect
of selected 4-aza-PTs in A549 cells.
As shown in Table 1, compounds 4a,d,e,g, 6a, 6e, 7e, 8e, 12a,
and 13e identified as tubulin destabilizers in the sea urchin
embryo assay also affected cellular microtubule structure
(Figure 4B,DÀF). It should be noted that 5a and 5e precipitated
from 10 mM stock solutions in DMSO, therefore the data
pertinent to these molecules could not be accurately interpreted.
Compound 40
a (an aromatic analogue of 4a) featuring low cyto-
toxicity failed to induce sea urchin embryo development alterations
and to depolymerize cellular microtubules (Figure 4C). Surprisingly,
compound 9e that exhibited modest cytotoxicity without the
disruption of interphase microtubules in A549 cells did show a
considerable antimitotic tubulin destabilizing activity in the sea
urchin assay. The effect could be attributed to a somewhat selective
interaction of this molecule with spindle microtubules in the sea
urchin blastomeres vs interphase microtubules in cultured cancer
cells. Treatment with PT at 0.1 and 5 μM resulted in complete
disassembly of cellular microtubules (data not shown).
Cell Cycle Analysis. The ability of selected 4-aza-PTs to
interfere with A549 and Jurkat cell cycle progression was
estimated by propidium iodide staining followed by flow cyto-
metry analysis. In A549 cells, PT as well as its aza-analogues 4a,g
and 6e caused G2/M cell cycle arrest, exhibiting EC50 values of
16.5 ( 0.35, 16.2 ( 0.71, 230 ( 7.07, and 119 ( 1.41 nM,
respectively. Compound 40
a failed to affect the cell cycle
progression even at 50 μM concentration. Notably, all three
cell-based assays ranked the tested molecules similarly. Cyto-
toxicity and microtubule dynamics effects of 4-aza-PTs decreased
in the following order: 4a > 6e > 4g . 40
a. In Jurkat cells, both 8e
and PT were potent G2/M blockers as well. In contrast, etopo-
side caused a pronounced cell cycle arrest in G0/G1 phase
(Figure 5).
Apoptosis-Inducing Activity. It has been well established that
tubulinbindingcompounds interferewiththe dynamics andstructure
of both mitotic spindle and interphase microtubules, eventually
leading to cell death by apoptosis. However the link between
microtubule alteration and respective intracellular mechanisms
responsible for initiation of apoptotic events remains unclear.50À52
Caspases (intracellular proteases) play a key role in the apoptotic
response. Their activation by specific signals triggers proteolysis of
cellularsubstratestherebyexecutingapoptoticevents.53
Therearetwo
Figure 5. Cell cycle distribution of Jurkat cells treated with 8e (20 nM),
PT (25 nM), and etoposide (2 μM) for 24 h. The cells were stained with
propidium iodide to analyze DNA content by flow cytometry.
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Journal of Medicinal Chemistry ARTICLE
main apoptotic pathways, namely the extrinsic pathway that
involves membrane-bound death receptors and leads to activa-
tion of caspase-8 and the mitochondria-related caspase-9-depen-
dent intrinsic pathway. Both pathways converge onto the effector
caspase-3 activated by the apical (initiator) caspases-8 and -9.53,54
Caspase-2 combines both initiator and effector features.53
Its
processing requires caspase-9 and caspase-3.55
Generally, microtubule interfering agents induce caspase-
dependent apoptotic cell death through intrinsic caspase-9-
dependent pathway.50À52,56
For instance, tubulin destabilizers
combretastatin A-4 and its analogues, as well as nocodazole,
trigger apoptosis through the intrinsic pathway accompanied by
an activation of caspase-9 and caspase-3.57À59
However, other
tubulin-targeting compounds were found to induce apoptosis in
human cancer cells via the activation of caspase-8 and -2 rather
than caspase-9, suggesting the extrinsic pathway as a primary
apoptotic mechanism.60À63
Whether this difference is cell-spe-
cific or dependent on the compound remains unclear. Because
numerous malignancies are characterized by molecular defects in
the apoptotic pathways,54
the investigation of apoptotic mechan-
isms is of particular importance.
There are a few data concerning the participation of effector
caspases-3 and -7 in apoptosis caused by PT derivatives.64À66
For
4-aza-PTs, caspase-3 activation in Jurkat and A549 cells has been
described.16,22,23
Up to now there is no evidence on the involve-
ment of apical caspases-9 and -2 in cell death caused by PT
derivatives. In the present study, the details of apoptosis induc-
tion by compound 4a found to be the most cytotoxic against
Figure 6. Jurkat cells treated with PT or 8e: flow cytometry assessment of hypodyploid cells (A), cells with active form of caspase-3 (B), and with
cleaved PARP (C). (a) Control (untreated cells); (b) PT, 50 nM, 48 h; (c) 8e, 50 nM, 48 d. In (d), cells were pretreated with caspase inhibitor z-VAD-
FMK (50 μM) for 2 h, followed by the incubation with 8e as in (c). M1 comprises the hypodiploid cells (A), cells with active form of caspase-3 (B), and
cleaved PARP-positive cells (C) with corresponding percentages given in each plot.
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A549 cells, and 8e, the most potent in the sea urchin embryo
assay, were studied in A549 and Jurkat cells, respectively.
The proapoptotic activity of compound 8e was examined in
Jurkat cells. Apoptosis induction, cell cycle distribution, expres-
sion of active effector caspase-3 in cells, and cleavage of PARP
were assessed quantitatively by flow cytometry. Procaspase-8 and
the active form of apical caspase-2 and -9 were analyzed by
Western blot. As evidenced by the flow cytometric assay, after 48
h, both 8e and PT increased the hypodiploid subpopulation peak
(sub-G1) indicative of apoptosis from 12% (control) to 55% of
the total cell population (Figure 6A). To confirm the involve-
ment of caspases in the 8e-induced Jurkat cell death, an effect of
pan-caspase inhibitor Z-VAD-fmk67
has been examined. Jurkat
cells were preincubated with Z-VAD-fmk at 50 μM for 2 h,
followed by incubation with 50 nM of 8e for additional 48 h.
Pretreatment with Z-VAD-fmk decreased the content of apop-
totic cells from 55% to 28% (Figure 6A), suggesting that cell
death induced by 8e was related to caspase activation. Approxi-
mately 30% of cells treated with 8e or PT possessed active form
of caspase-3, as compared to 7% in untreated cells (Figure 6B).
Cleaved PARP, the product resulting from caspase-3 activation,
has been identified in ca. 40% of the cells treated by 8e or PT
(Figure 6C).
Activation of caspases-9, -2, and -8 in cells treated with 8e was
further analyzed by Western blot. The exposure of Jurkat cells to
8e resulted in the processing of procaspase-9 and -2, as evidenced
by the appearance of their active forms (Figure 7A,B). The
intensities of bands corresponding to active forms of caspase-9
and -2 upon 24 h of treatment with 8e or PT were higher than
those at 48 h, while the intensity of procaspase-8 55/57 kDa
traces was unaltered (Figure 7C), suggesting lack of active
caspase-8. As expected, etoposide caused the reduction of
procaspase-8 level in Jurkat cells (Figure 7D).68
Finally, the ability of compound 4a to trigger apoptotic and
necrotic events in A549 cells was analyzed using double staining with
Annexin VÀFITC and 7-AAD (7-amino actinomycin D) dyes. The
procedure allows for the differentiation between living (intact)
(Annexin VÀFITCÀ
/7-ADDÀ
), early apoptotic (Annexin VÀ
FITC+
/7-ADDÀ
), late apoptotic (Annexin VÀFITC+
/7-ADD+
),
and necrotic cells (Annexin VÀFITCÀ
/7-ADD+
).69,70
Compound
4a as well as nocodazole (positive control) induced a significant
increase of early apoptosis and to a lesser degree necrosis without
affecting late apoptosis (Figure S2, Supporting Information). The
effects of 4a were concentration-independent, suggesting that this
compound might trigger apoptosis and necrosis even at lower
concentrations. 4a was more potent apoptotis- and necrosis-indu-
cing agent than nocodazole.
On the basis of these data, we concluded that compounds 4a and
8e caused G2/M cell cycle arrest and apoptotic response in A549
and Jurkat human cancer cells, notably, that in Jurkat cells 8e as well
as PT induced caspase-dependent apoptosis mediated by the apical
caspases-2 and -9 and not caspase-8, implying the involvement of
the intrinsic caspase-9-dependent apoptotic pathway.
PT-Related Compounds: Mode of Action. In the sea urchin
embryoassay, deathofarrested eggs exposedtotubulindestabilizing
agents was observed after 6À8 h of treatment with a test article. It
has been suggested that apoptosis never occurs during sea urchin
embryo cleavage, first appearing only at blastula stage just before
hatching.71
This explains the absence ofquick cell divisionarrest and
death at cleavage after treatment by various DNA-targeting agents
known to trigger apoptosis such as etoposide, aloe-emodin, apigen-
in, 5-fluorouracil, and 4-phenylbutyric acid.31
Correspondingly, the
formation of multinucleated cells (Figure 1B) typical of tubulin
destabilizers is an inherent morphological characteristic of mitotic
catastrophe, a cell death different from apoptosis.72
Tubulin destabilizers including colchicine, nocodazole, combre-
tastatins, phenstatin, indibulin, dolastatin 15, vinblastin, and others
interacting with different binding sites on tubulin caused embryo
spinning when applied at the swimming blastula stage. Spinning
continued for several hours, followed by slow movements near the
bottom of the vessel suggesting embryo viability.31,33,34,73
In con-
trast, PT andsomeof4-aza-PTs (4a,e,g,j, 6e, 7e, 8e,h, 12a, and13e)
induced sea urchin embryo disintegration and death after spinning
independently of compound concentration, probably owing to yet
another microtubule-unrelated effect. In addition, compound 140
e
caused cleavage alteration at 100 nM, whereas it failed to produce
cleavage arrest and embryo spinning, suggesting nontubulin antu-
proliferative activity. Similarly, 9e exerted cytotoxic effect against
A549 cells without microtubule disruption (Table 1). These results
are correlated well with the reported ability of PT derivatives to
induce cell death and sub-G1 apoptotic cell accumulation without
cell cycle arrest and interphase microtubule damage.10,14
Moreover,
in a series of 4-aza-PTs with hetero aryl or benzo hetero aryl system
instead of the A and B rings, chemical modifications of the ring E
resulted in a significant decrease of antitubulin activity and retention
of cytotoxicity.22
A combination of our results and literature data
suggests an alternative tubulin-independent mechanism of 4-aza-
PTs embryotoxicity that could be evident at postcleavage stages of
the sea urchin embryo development.
’ CONCLUSIONS
In conclusion, we synthesized a series of 4-aza-PT analogues
from the easily accessible plant-derived polyalkoxybenzenes.
A developed reaction sequence allows for an expedited synthesis
Figure 7. Western blot analysis of procaspase-8, cleaved caspase-2, and
caspase-9 in total lysates of Jurkat cells. The lysates treated with PT or 8e
were prepared in SDSÀLaemmli sample buffer. Immunoblotting was
performed using monoclonal anticaspase-9 (A), anticaspase-2 (B), and
antiprocaspase-8 (C, D) antibodies. β-Actin served as the loading
control. Lanes: (1, 6) control (untreated cells); (2) PT, 50 nM, 24 h;
(3) PT, 50 nM, 48 h; (4) 8e, 50 nM, 24 h; (5) 8e, 50 nM, 48 h; (7)
etoposide, 2 μM, 24 h.
9. I dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
of the targeted molecules in 12À86% overall yields from naturally
occurring building blocks. The resulting compounds were ex-
tensively evaluated in both phenotypic sea urchin embryo and
cellular assays linking their observed cytotoxicity to a potent
antimitotic tubulin destabilizing effect. Notably, the phenotypic
sea urchin embryo assay allowed for rapid and reproducible
identification of antitubulin molecules. These compounds were
further reconfirmed as potent antimitotic agents by a panel of
conventional cell-based assays. The most potent representative
in each structural group featured myristicin-derived ring E (4e,
6e, 8e, 11e, and 13e), as evidenced by the in vivo sea urchin
embryo assay. The activity of these new compounds compares
favorably to the reported antimitotic agents containing myristi-
cin-derived pharmacophore including corresponding phenstatin
analogue and combretastatin A-2.25,73
The ring B modification
yielded 6-methoxy substituted molecule 8e as the most active
compound. Compound 4a featuring 3,4,5- trimethoxybenzene
substituent was the most cytotoxic against A549 human lung
epithelial carcinoma cells, being more potent than the parent PT.
The activity of 4-aza-PT derivatives correlated well between the
sea urchin phenotypic and conventional cell-based assays. In
Jurkat cells, both 8e and PT induced caspase-dependent apop-
tosis mediated by the apical caspases-2 and -9 and not caspase-8,
implying the involvement of the intrinsic caspase-9-dependent
apoptotic pathway. The structureÀactivity data reported in this
paper warrant further synthetic studies and biological evaluation
of novel heterocyclic myristicine derivatives. Four specific mol-
ecules (4e, 7e, 8e, and 8h) have been selected for advanced
NCI60 human tumor cell line anticancer drug screen. Their
antiproliferative activity will be reported separately.
’ EXPERIMENTAL SECTION
Chemistry. Materials and Methods. NMR spectra were col-
lected on a Bruker DR-500 instrument [working frequencies of 500.13
MHz (1
H) and 75.47 (13
C)]. Mass spectra were obtained on a Finnigan
MAT/INCOS 50 instrument (70 eV) using direct probe injection.
Elemental analysis was accomplished with the automated Perkin-Elmer
2400 CHN microanalyzer. HPLC was performed using a Laboratorni
Pristroje (Praha, Czech Republic) chromatograph. Ozonolysis was
conducted using a custom-designed apparatus (Science and Technology
Park, St. Petersburg State Polytechnic University, Russia) equipped with
an IR detector of O3 concentration (Japan) and an automated shut-
down circuit. The device allowed for the controlled generation of ozone,
with a maximal capacity of 10 g of O3 per h from O2. The compound
purity has been determined by NMR, HPLC, and elemental analyses.
Purity of compounds 40
À150
, 4À13, and 17À19 was determined to be
g95%.
Isolation of Plant Allylpolyalkoxybenzenes24
. Liquid CO2
extraction of parsley and dill seeds was carried out earlier by Company
Karavan Ltd. (Krasnodar, Russia). Allylpolymethoxybenzenes 1bÀe
with 98À99% purity were obtained by high-efficiency distillation using
a pilot plant device at N. D. Zelinsky Institute of Organic Chemistry,
RAS (Moscow, Russia). The seed essential oils of parsley varieties
cultivated in Russia contained 70À75% of 1b (var. Sakharnaya), 21% of
1d (var. Slavyanovskaya), and 40À46% of 1e (var. Astra). Indian dill
seeds were purchased from Vremya & Co. (St. Petersburg, Russia). The
dill seed essential oil contained 30À33% of 1c.
General Procedure A for the Synthesis of Quinolines
40
À15036
. A solution of aromatic amine (50 mmol), ethyl 4-chloroa-
cetoacetate (50 mmol) and acetic acid (3 mL) in 100 mL of benzene was
refluxed for 8 h. The reaction mixture was cooled to room temperature,
and the precipitated product was collected by vacuum filtration and
washed with EtOH (30 mL). In most cases, the resulting products 3
were obtained with >85% purity as judged by NMR analysis. A solution
of corresponding anilinolactone 3 (2 mmol), appropriate aromatic
aldehyde (2aÀk) (2.2 mmol), and p-chloranil (2 mmol) in TFA
(3 mL) was stirred at room temperature for 24 h. The reaction mixture
was quenched with water (50 mL) and extracted with CH2Cl2 (3 Â
20 mL). The organic layers were combined, washed with 5% NaHCO3
(2 Â 20 mL), water (2 Â 20 mL), dried over anhydrous sodium sulfate,
and concentrated in vacuum to obtain the crude product. Flash
chromatography purification (petroleum ether/EtOAc, 4:1À1:1) af-
forded targeted quinolines 40
À150
as a white solid.
General Procedure B for the Synthesis of 4-Aza-podo-
phyllotoxin Derivatives 4À13. A suspension of quinoline 40
À130
(0.26 mmol) in a glacial AcOH (3.96 mmol, 3 mL) was treated with
NaBH3CN (0.78 mmol), and the resulting mixture was stirred for 3 h at
room temperature and treated with the ice water (15 mL). The
precipitate was filtered, washed with ethanol (2 Â 1 mL), and the
product was purified by column chromatography (petroleum ether/
EtOAc, 2:1) to afford the targeted 4-aza-podophyllotoxins 4À13 as a
white solid. NMR analysis confirmed the absence of intermediates
40
À130
.
General Procedure C for the Synthesis of Dihydropyrido-
pyrazole Derivatives 17À19. A mixture of of corresponding
pyrazole (1 mmol), tetronic acid 16 (1 mmol), triethylamine
(0.05 mL), and appropriate aldehyde (2aÀk) (1 mmol) in EtOH
(5 mL) was refluxed for 3 h. The reaction mixture was cooled to
+4 °C, and the precipitated product was collected by vacuum filtration
and washed with EtOH (2 mL) at 0 °C. Purification by flash chroma-
tography (petroleum ether/EtOAc, 4:1À2:1) afforded the targeted
4-aza-dihydropodophylltoxins 17À19 as a white solid.
Synthetic and Analytical Data for Selected 4-Aza-podo-
phyllotoxins. Data for the other compounds are presented in
Supporting Information.
9-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)[1,3]dioxolo[4,5-
g]furo[3,4-b]quinolin-8(6H)-one (40
c). The title compound was
obtained according to the general procedure A. Yield 27%, mp
241À244 °C. 1
H NMR (DMSO-d6): δ 3.35 (s, 3H, OCH3-60
), 4.02
(s, 3H, OCH3-70
), 5.42 (d, J = 15.4 Hz, 1H, OCH2-6), 5.49 (d, J = 15.4
Hz, 1H, OCH2-6), 6.11 and 6.13 (s, 2H, OCH2O-20
), 6.28 (s, 2H,
OCH2O-2), 6.51 (s, 1H, H-40
), 6.89 (s, 1H, H-10), 7.52 (s, 1H, H-4).
EIMS m/z 409 [M]+
(25), 394 (1), 351 (15), 350 (68), 224 (23), 167
(100). Anal. Calcd for C21H15NO8: C, 61.62; H, 3.69; N, 3.42. Found:
C, 61.69; H, 3.72; N, 3.36.
9-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)-6,9-dihydro-
[1,3]dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (4c). The ti-
tle compound was obtained according to the general procedure B. Yield
26%, mp 295À298 °C. 1
H NMR (DMSO-d6): δ 3.73 (s, 3H, OCH3-60
),
3.94 (s, 3H, OCH3-70
), 4.83 (d, J = 15.6 Hz, 1H, OCH2-6), 4.95 (d, J =
15.7 Hz, 1H, OCH2-6), 5.13 (s, 1H, H-9), 5.88 and 5.94 (s, 2H,
OCH2O-2), 5.89 and 5.91 (s, 2H, OCH2O-20
), 6.24 (s, 2H, H-40
),
6.42 (s, 1H, H-10), 6.49 (s, 1H, H-4), 9.81 (s, 1H, NH-5). EIMS m/z
411 [M]+
(20), 380 (100), 366 (17), 230 (39), 172 (7). Anal. Calcd for
C21H17NO8: C, 61.32; H, 4.17; N, 3.40. Found: C, 61.29; H, 4.14;
N, 3.44.
9-(7-Methoxy-1,3-benzodioxol-5-yl)-6,9-dihydro[1,3]
dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (4e). The title
compound was obtained according to the general procedure B. Yield
55%, mp 308À310 °C. 1
H NMR (DMSO-d6): δ 3.80 (s, 3H, OCH3-70
),
4.83 (s, 1H, H-9), 4.84 (d, J = 15.7 Hz, 1H, OCH2-6), 4.97 (d, J = 15.7
Hz, 1H, OCH2-6), 5.90 and 5.91 (s, 2H, OCH2O-20
), 5.90 and 5.96 (s,
2H, OCH2O-2), 6.33 (d, J = 1.4 Hz, 1H, H-40
), 6.52 (s, 1H, H-10), 6.56
(d, J = 1.4 Hz, 1H, H-60
), 6.64 (s, 1H, H-4), 9.87 (s, 1H, NH-5). EIMS
m/z 381 [M]+
(5), 231 (14), 230 (100), 152 (24), 151 (7). Anal. Calcd
10. J dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
for C20H15NO7: C, 62.99; H, 3.96; N, 3.67. Found: C, 63.06; H, 4.03;
N, 3.60.
9-Phenyl-6,9-dihydro[1,3]dioxolo[4,5-g]furo[3,4-b]quin-
olin-8(5H)-one (4k). The title compound was obtained according to
the general procedure B. Yield 73%, mp 290À293 °C (lit.6
mp
295À298 °C). 1
H NMR (DMSO-d6): δ 4.85 (d, J = 15.7 Hz, 1H,
OCH2-6), 4.91 (s, 1H, H-9), 4.94 (d, J = 15.7 Hz, 1H, OCH2-6), 5.89
and 5.95 (s, 2H, OCH2O-2), 6.53 (s, 1H, H-10), 6.56 (s, 1H, H-4), 7.15
(t, J = 7.2 Hz, 1H, H-40
), 7.19 (d, J = 7.2 Hz, 2H, H-20
,60
), 7.26 (d, J = 7.2
Hz, 2H, H-30
,50
), 9.87 (s, 1H, NH-5). EIMS m/z 307 [M]+
(20), 230
(21), 78 (12), 77 (100). Anal. Calcd for C18H13NO4: C, 70.35; H, 4.26;
N, 4.56. Found: C, 70.29; H, 4.22; N, 4.63.
10-Methoxy-9-(7-methoxy-1,3-benzodioxol-5-yl)-6,9-
dihydro[1,3]dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one
(5e). The title compound was obtained according to the general
procedure B. Yield 26%, mp 321À326 °C. 1
H NMR (DMSO-d6): δ
3.66 (s, 3H, OCH3-10), 3.77 (s, 3H, OCH3-70
), 4.78 (d, J = 15.7 Hz, 1H,
OCH2-6), 4.89 (d, J = 15.7 Hz, 1H, OCH2-6), 4.89 (s, 1H, H-9), 5.89
and 5.96 (s, 2H, OCH2O-2), 5.89 and 5.90 (s, 2H, OCH2O-20
), 6.22 (d,
J = 1.4 Hz, 1H, H-40
), 6.33 (s, 1H, H-4), 6.42 (d, J = 1.4 Hz, 1H, H-60
),
9.93 (s, 1H, NH-5). EIMS m/z 411 [M]+
(3), 384 (0.5), 260 (100), 245
(25), 152 (31), 151 (9). Anal. Calcd for C21H17NO8: C, 61.32; H, 4.17;
N, 3.40. Found: C, 61.36; H, 4.18; N, 3.38.
6-Methoxy-9-(7-methoxy-1,3-benzodioxol-5-yl)-4,9-dih-
ydrofuro[3,4-b]quinolin-1(3H)-one (8e). The title compound
was obtained according to the general procedure B. Yield 60%, mp
278À280 °C. 1
H NMR (DMSO-d6): δ 3.70 (s, 3H, OCH3-6), 3.80
(s, 3H, OCH3-70
), 4.84 (d, J = 15.5 Hz, 1H, OCH2-3), 4.86 (s, 1H, H-9),
4.97 (d, J = 15.5 Hz, 1H, OCH2-3), 5.89 and 5.90 (s, 2H, OCH2O-20
),
6.31 (d, J = 1.4 Hz, 1H, H-40
), 6.44 (d, J = 2.5 Hz, H-5), 6.52 (dd, J = 8.6,
2.5 Hz, 1H, H-7), 6.53 (d, J = 1.4 Hz, 1H, H-60
), 6.99 (d, J = 8.6 Hz, H-8),
9.94 (s, 1H, NH-4). EIMS m/z 367 [M]+
(14), 217 (15), 216 (100), 152
(16), 151 (7). Anal. Calcd for C20H17NO6: C, 65.39; H, 4.66; N, 3.81.
Found: C, 65.42; H, 4.69; N, 3.74.
9-(3,5-Dimethoxyphenyl)-6-methoxy-4,9-dihydrofuro-
[3,4-b]quinolin-1(3H)-one (8h). The title compound was obtained
according to the general procedure B. Yield 43%, mp 229À233 °C (lit.6
mp 254 °C). 1
H NMR (DMSO-d6): δ 3.68 (s, 6H, 2 Â OCH3-30
, 50
),
3.70 (s, 3H, OCH3-6), 4.85 (d, J = 15.8 Hz, 1H, OCH2-3), 4.85 (s, 1H,
H-9), 4.98 (d, J = 15.8 Hz, 1H, OCH2-3), 6.30 (t, J = 2.2 Hz, 1H, H-40
),
6.32 (d, J = 2.2 Hz, 2H, H-20
,60
), 6.45 (d, J = 2.6 Hz, H-5), 6.52 (dd, J =
8.5, 2.6 Hz, 1H, H-7), 6.99 (d, J = 8.5 Hz, H-8), 9.94 (s, 1H, NH-4).
EIMS m/z 353 [M]+
(12), 217 (15), 216 (100), 138 (16). Anal. Calcd
for C20H19NO5: C, 67.98; H, 5.42; N, 3.96. Found: C, 67.91; H, 5.38;
N, 4.04.
4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-3-methyl-1,4,7,
8-tetrahydro-5H-furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one
(18b). The title compound was obtained according to the general
procedure C. Yield 47%, mp 295À297 °C. 1
H NMR (DMSO-d6): δ 1.83
(s, 3H, CH3-3), 3.69 (s, 3H, OCH3-40
), 3.71 (s, 3H, OCH3-70
), 4.78
(d, J = 15.7 Hz, 1H, OCH2-7), 4.85 (d, J = 15.7 Hz, 1H, OCH2-7), 4.99
(s, 1H, H-4), 5.98 and 5.99 (s, 2H, OCH2O-20
), 6.23 (s, 1H, H-60
), 10.03
(s, 1H, NH-8), 11.79 (s, 1H, NH-1). EIMS m/z 371 [M]+
(31), 341
(14), 340 (74), 327 (5), 326 (21), 312 (16), 296 (14), 191 (11), 190
(100), 182 (10). Anal. Calcd for C18H17N3O6: C, 58.22; H, 4.61; N,
11.32. Found: C, 58.30; H, 4.68; N, 11.23.
Biology. Materials and Methods. Sea Urchin Embryo
Assay. Adult sea urchins Paracentrotus lividus were collected from the
Mediterranean Sea at the Cyprus coast and kept in an aerated seawater
tank. Gametes were obtained by intracoelomic injection of 0.5 M KCl.
Eggs were washed with filtered seawater and fertilized by adding drops of
a diluted sperm. Embryos were cultured at room temperature under
gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered
seawater. The embryos were observed with a light microscope Biolam
(LOMO, S.-Petersburg, Russia). For treatment with the test com-
pounds, 5 mL aliquots of embryo suspension were transferred to 6-well
plates and incubated as a monolayer at a concentration up to 2000
embryos/mL. Stock solutions of compounds were prepared in DMSO at
5À10 mM concentrations, followed by a 10-fold dilution with 95%
EtOH. This procedure enhanced solubility of the test compounds in the
salt-containing medium (seawater), as evidenced by microscopic exam-
ination of the samples. The maximal tolerated concentrations of DMSO
and EtOH in the in vivo assay were determined to be 0.05% and 1%
respectively. Higher concentrations of either DMSO (g0.1%) or EtOH
(>1%) caused nonspecific alteration and retardation of the sea urchin
embryo development independent of the treatment stage. Podophyllo-
toxin (Aldrich) and topoiomerase II inhibitor etoposide (LANS, Ver-
opharm, Russia, 20 mg/mL in 96% EtOH) served as reference com-
pounds.
The antiproliferative activity was assessed by exposing fertilized eggs
(8À20 min after fertilization, 43À55 min before the first mitotic cycle
completion) to 2-fold decreasing concentrations of the compound.
Cleavage alteration and arrest were clearly detected at 2.5À5.5 h after
fertilization. The effects were quantitatively estimated as a threshold
concentration resulting in cleavage alteration and embryo death before
hatching or full mitotic arrest. At these concentrations, all tested tubulin
destabilizers caused 100% cleavage alteration and embryo death before
hatching, whereas at 2-fold lower concentrations, the compounds failed
to produce any effect. For tubulin destabilizing activity, the compounds
were tested on free-swimming blastulae just after hatching (9À10 h after
fertilization), originated from the same embryo culture. Embryo spin-
ning was observed after 15 min to 20 h of treatment, depending on the
nature and concentration of the compound. Both spinning and lack of
forward movement were interpreted to be the result of the tubulin
destabilizing activity of a molecule according to previous studies.31
Video illustrations are available at http://www.chemblock.com). Com-
pound substructure search in sea urchin embryo screening database is
available free online at http://www.zelinsky.ru.
’ ASSOCIATED CONTENT
bS Supporting Information. Effects of compounds 40
À150
and 17À19 on the sea urchin embryo development, experimen-
tal details regarding syntheses and analytical data, biological assay
methods. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +7 (916) 620-9584. Fax: +7 (499) 137-2966. E-mail:
vs@zelinsky.ru.
’ ACKNOWLEDGMENT
This work was supported by Alexander von Humboldt
Foundation (no. 3.4-Fokoop-Rus/1015567, Germany) and a
grant from Chemical Block Ltd.
’ ABBREVIATIONS USED
PT, podophyllotoxin;SAR, structureÀactivity relationship;7-AAD,
7-amino actinomycin D;PARP, poly ADP-ribose polymerase
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