The document describes a facile synthesis of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFAs) via the condensation reaction of 4-aminofurazan-3-carbohydroximoyl chloride and substituted o-phenylenediamines. This synthesis proceeds under mild conditions in good yields without requiring purification of intermediates. The resulting BIFA derivatives were evaluated for their ability to destabilize microtubules in sea urchin embryos and inhibit proliferation of human cancer cell lines. Several BIFAs showed low micromolar anti-proliferative activity through both in vivo and in vitro assays. The most potent compound
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
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) 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.
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
Cellulase (Types, Sources, Mode of Action & Applications)Zohaib HUSSAIN
Cellulase is an enzyme system consisting of endo- and exo-glucanases and cellobiase that catalyzes the hydrolysis of cellulose. There are three major types of cellulases - endoglucanase, exoglucanase, and beta-glucosidase. Cellulase-producing microbes employ one of three mechanisms: free cellulase systems using individual enzymes, cellulosome complexes, or endoglucanases without other domains. The synergistic action of endo- and exoglucanases supplemented by beta-glucosidase completely degrades cellulose to glucose. Cellulases find applications in food, animal feed, textiles, biofu
The arrangement of the large (70,000 Mr) and small (30,000 Mr) subunits of succinate dehydrogenase (SDH) in the mitochondrial inner membrane was investigated using limited proteolysis and immunoblotting. Both subunits were resistant to proteinase treatment when the inner membrane integrity was preserved, suggesting neither subunit is exposed at the cytoplasmic surface. The small subunit appears to protrude into the matrix compartment, as it is extensively degraded but no membrane-associated fragment is observed. The large subunit interacts with the matrix side via two distinct domains that are detected as stable membrane-associated fragments after proteinase treatment, though one domain can be further degraded. This suggests the large subunit membrane interaction occurs via two regions,
- 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
The study aimed to detect and analyze novel diterpenoid dioxygenase genes (ditA1) involved in the degradation of resin acids, which are naturally produced by trees and released during wood pulping processes. Using newly designed PCR primers, ditA1 homolog genes were amplified from various Pseudomonas, Burkholderia, and Cupriavidus strains. All isolates containing a ditA1 homolog could grow on dehydroabietic acid and expressed ditA1 constitutively or in response to dehydroabietic acid, demonstrating their role in degradation. Evolutionary analyses indicate ditA1 and gyrB genes have coevolved from ancestral variants in Pseudomonas, Burkholderia, and Cupriavid
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
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) 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.
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
Cellulase (Types, Sources, Mode of Action & Applications)Zohaib HUSSAIN
Cellulase is an enzyme system consisting of endo- and exo-glucanases and cellobiase that catalyzes the hydrolysis of cellulose. There are three major types of cellulases - endoglucanase, exoglucanase, and beta-glucosidase. Cellulase-producing microbes employ one of three mechanisms: free cellulase systems using individual enzymes, cellulosome complexes, or endoglucanases without other domains. The synergistic action of endo- and exoglucanases supplemented by beta-glucosidase completely degrades cellulose to glucose. Cellulases find applications in food, animal feed, textiles, biofu
The arrangement of the large (70,000 Mr) and small (30,000 Mr) subunits of succinate dehydrogenase (SDH) in the mitochondrial inner membrane was investigated using limited proteolysis and immunoblotting. Both subunits were resistant to proteinase treatment when the inner membrane integrity was preserved, suggesting neither subunit is exposed at the cytoplasmic surface. The small subunit appears to protrude into the matrix compartment, as it is extensively degraded but no membrane-associated fragment is observed. The large subunit interacts with the matrix side via two distinct domains that are detected as stable membrane-associated fragments after proteinase treatment, though one domain can be further degraded. This suggests the large subunit membrane interaction occurs via two regions,
- 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
The study aimed to detect and analyze novel diterpenoid dioxygenase genes (ditA1) involved in the degradation of resin acids, which are naturally produced by trees and released during wood pulping processes. Using newly designed PCR primers, ditA1 homolog genes were amplified from various Pseudomonas, Burkholderia, and Cupriavidus strains. All isolates containing a ditA1 homolog could grow on dehydroabietic acid and expressed ditA1 constitutively or in response to dehydroabietic acid, demonstrating their role in degradation. Evolutionary analyses indicate ditA1 and gyrB genes have coevolved from ancestral variants in Pseudomonas, Burkholderia, and Cupriavid
This study aimed to isolate thermostable cellulases from woodland soil samples using metagenomic approaches. Genomic DNA was extracted from soil samples and used to amplify fragments of endo-β-1,4-glucanases. Twenty-five different cellulase sequences were identified, with nine cloned and tested for cellulolytic activity at 60°C. One sequence, CelMS6, displayed the highest activity at 60°C, making it a good candidate for applications requiring cellulose utilization at high temperatures.
This study synthesized eight new fluorinated quinazolinone-sulphonamide hybrid compounds and evaluated their anticancer activity. One compound showed significant anticancer activity with low toxicity compared to the reference drug mitoxantrone. Biological assays also demonstrated moderate anticancer activity for the compounds compared to reference drugs. The compound with the best activity profile was identified for further evaluation as an anticancer agent.
An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis ...Wadud Bhuiya
- An artificial monolignol 4-O-methyltransferase was created using iterative saturation mutagenesis to modify the structure and function of an isoeugenol O-methyltransferase (IEMT).
- Expressing this engineered enzyme in plants etherified the para-hydroxyls of lignin monomers, preventing them from participating in lignin polymerization and reducing lignin content. Transgenic plants also accumulated novel 4-O-methylated phenolic compounds.
- Lower lignin levels resulted in higher saccharification yields from transgenic plant biomass, demonstrating this protein engineering approach can modulate phenylpropanoid biosynthesis and improve cell wall digestibility.
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
Research from a bacterium bacillus subtilis b 3157 by fabAlexander Decker
This document discusses research into the biosynthetic pathways that produce 2H-labeled inosine in the bacterium Bacillus subtilis B-3157. The bacterium was grown in heavy water medium containing a hydrolysate of deuterated biomass as a source of 2H-labeled substrates. Isolation and analysis of the produced 2H-labeled inosine found incorporation of 5 deuterium atoms, with 3 in the ribose residue and 2 in the hypoxanthine residue. The non-exchangeable deuterium atoms in ribose originated from HMP shunt reactions, while the atoms in hypoxanthine came from [2H]amino acids in the growth medium.
Enhanced endoglucanase production by Bacillus aerius on mixed lignocellulosic...Mushafau Adebayo Oke
Oke, M. A., Annuar, M. S. M., and Simarani, K. (2016). "Enhanced endoglucanase production by Bacillus aerius on mixed lignocellulosic substrates." BioResources, 11(3), 5854-5869.
The document discusses biotransformation, which is the biological process by which organic compounds are modified by enzymes in microbial, plant, and animal cells. Microbial transformation is preferred over plant or animal cell transformation due to microbes having a higher surface-to-volume ratio, growth rate, and metabolism rate, as well as being easier to maintain sterility. Microbial transformations can occur under mild conditions and achieve high yields, regioselectivity, stereoselectivity, and multi-step conversions using different microorganisms.
Bioactive compounds and antioxidant capacities of fresh and canned fruit,of p...GC University Faisalabad
This document summarizes a study on the bioactive compounds and antioxidant capacities of fresh and canned pineapple fruit. The study found that fresh pineapple extracts had higher levels of total phenolics, total flavonoids, and stronger antioxidant activities compared to canned pineapple extracts based on DPPH radical scavenging, inhibition of linoleic acid peroxidation, and reducing power assays. Fresh pineapple is a richer source of natural antioxidants than canned pineapple.
This document describes a study on isolating cellulase-producing fungi from termite soil. Eight fungi - Aspergillus sp, Trichoderma sp, Penicillium sp, Acremonium sp, Cladosporium sp, Neurospora sp, and Phoma sp - were isolated from termite soil samples using spread plating and identified microscopically. The fungi were screened for cellulolytic activity using various pretreated lignocellulosic substrates. Aspergillus niger and Trichoderma viridae showed the highest enzymatic activity on sugarcane bagasse.
This document summarizes research on the production of chitosan by various fungi. Key findings include:
- Absidia coerulea and Mucor rouxii were among the best chitosan-producing fungi strains examined.
- Optimum chitosan yields were observed at temperatures of 21-26°C in medium containing yeast extract, glucose and peptone.
- Continuous fermentation in a stirred tank reactor produced the highest amount of chitosan from A. coerulea, resulting in approximately a three-fold increase compared to batch culture.
Production and isolation of chitosan by submerged and solid state fermentationNgcon35
This document summarizes a study that developed a method for producing and isolating chitosan through liquid and solid-state fermentation of Lentinus edodes. Under liquid fermentation, chitosan yields of 120 mg/L were achieved. Solid-state fermentation produced significantly higher yields of 6.18 g/kg, up to 50 times greater than other fungal methods. Solid-state fermentation provides an economical process for large-scale chitosan production with characteristics suitable for industrial applications.
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.
The document describes the design, synthesis, and testing of novel lipopeptide conjugates as potential antimicrobial agents. A library of conjugates was designed based on a template consisting of a hydrophobic moiety, dipeptide, and spermidine. Conjugates containing linoleic acid exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Structure-activity relationships revealed that optimal activity required a hydrophobicity of 50-70% and a minimum charge of +2. Active conjugates were found to have different modes of action, damaging bacterial cell membranes and DNA. Two conjugates showed selective membrane disruption of pathogenic MRSA and were identified as promising leads for further optimization and development as antimicrobial agents.
1) The document examines the effects of antimycin A and monofluoroacetate treatments on reactive oxygen species (ROS) production and citrate levels in Arabidopsis thaliana leaves.
2) The treatments were found to increase ROS production, as measured by dichloroflourscein diacetate and 3,3-diaminobenzidine. Citrate levels decreased over time with the treatments.
3) Antimycin A treatment led to the highest increase in ROS production and greatest decrease in citrate levels, while monofluoroacetate treatment resulted in lower ROS production and higher citrate levels compared to the control.
This document reviews drug discovery efforts to develop new antimicrobials by targeting the diaminopimelic acid (DAP) biosynthesis pathway in bacteria. It discusses how DAP is an essential component of bacterial cell walls but not mammalian cells, making enzymes in the DAP pathway potential drug targets. The review focuses on substrate-based inhibitors of DAP pathway enzymes and how understanding the enzymology could aid inhibitor design. Developing inhibitors that selectively block DAP biosynthesis in resistant bacteria could lead to new, less toxic antimicrobial therapies.
The student synthesized demethylated curcumin to create a derivative that is more soluble in aqueous solutions than curcumin. Demethylation was achieved through a reaction involving aluminum trichloride and hydrochloric acid. While NMR analysis indicated partial demethylation occurred, results were unclear due to overlapping peaks from impurities. In the future, the student aims to test the synthesized compound's ability to disaggregate beta amyloid plaques compared to curcumin.
This document discusses optimizing the production of exopolysaccharide (EPS) by Bacillus subtilis using various carbon and nitrogen sources. Sucrose at 2% concentration produced the highest yield of EPS. Cane molasses and rice bran were also tested as carbon sources, with cane molasses at 2% giving the highest yield. Different solvents were tested for their ability to precipitate EPS, with ethanol, diethyl ether, and methanol being effective. Fourier transform infrared spectroscopy analysis revealed the extracted polymer was composed of sucrose units. In conclusion, agro-wastes like cane molasses could be used as alternative carbon sources for the economical production of EPS.
- 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.
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
This document reviews the role of bacterial extracellular polysaccharides in biofilm formation. It discusses how extracellular polymeric substances (EPS) produced by microorganisms form the matrix of microbial aggregates and biofilms. EPS are involved in the initial attachment of cells to surfaces and provide protection from environmental stresses. The production of EPS is regulated by quorum sensing and helps mediate processes like bioremediation and bioleaching that are important in industrial applications.
This document discusses personal information about Mohammad Wahid Abdullah Khan and contains inappropriate, unverified claims. It describes a person taking money from Khan near Eid and then spending it inappropriately. It also contains rude language calling someone a "rapist" and making unfounded claims about their character and actions. Overall, the document seems to be spreading misinformation about individuals in an unethical manner.
The NSCS General Body Meeting discussed upcoming officer positions, end of year social plans, and distributing membership pins. Officer applications were due by April 18th and included positions like Vice President, Secretary, and Vice presidents of PACE and Public Relations. The end of year social would be held on May 12 at 6:30pm in Trabant 209 to celebrate NSCS's 20th anniversary and the end of semester, with food from Capriotti's. Members still needing membership pins from the induction could collect them after the meeting.
This study aimed to isolate thermostable cellulases from woodland soil samples using metagenomic approaches. Genomic DNA was extracted from soil samples and used to amplify fragments of endo-β-1,4-glucanases. Twenty-five different cellulase sequences were identified, with nine cloned and tested for cellulolytic activity at 60°C. One sequence, CelMS6, displayed the highest activity at 60°C, making it a good candidate for applications requiring cellulose utilization at high temperatures.
This study synthesized eight new fluorinated quinazolinone-sulphonamide hybrid compounds and evaluated their anticancer activity. One compound showed significant anticancer activity with low toxicity compared to the reference drug mitoxantrone. Biological assays also demonstrated moderate anticancer activity for the compounds compared to reference drugs. The compound with the best activity profile was identified for further evaluation as an anticancer agent.
An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis ...Wadud Bhuiya
- An artificial monolignol 4-O-methyltransferase was created using iterative saturation mutagenesis to modify the structure and function of an isoeugenol O-methyltransferase (IEMT).
- Expressing this engineered enzyme in plants etherified the para-hydroxyls of lignin monomers, preventing them from participating in lignin polymerization and reducing lignin content. Transgenic plants also accumulated novel 4-O-methylated phenolic compounds.
- Lower lignin levels resulted in higher saccharification yields from transgenic plant biomass, demonstrating this protein engineering approach can modulate phenylpropanoid biosynthesis and improve cell wall digestibility.
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
Research from a bacterium bacillus subtilis b 3157 by fabAlexander Decker
This document discusses research into the biosynthetic pathways that produce 2H-labeled inosine in the bacterium Bacillus subtilis B-3157. The bacterium was grown in heavy water medium containing a hydrolysate of deuterated biomass as a source of 2H-labeled substrates. Isolation and analysis of the produced 2H-labeled inosine found incorporation of 5 deuterium atoms, with 3 in the ribose residue and 2 in the hypoxanthine residue. The non-exchangeable deuterium atoms in ribose originated from HMP shunt reactions, while the atoms in hypoxanthine came from [2H]amino acids in the growth medium.
Enhanced endoglucanase production by Bacillus aerius on mixed lignocellulosic...Mushafau Adebayo Oke
Oke, M. A., Annuar, M. S. M., and Simarani, K. (2016). "Enhanced endoglucanase production by Bacillus aerius on mixed lignocellulosic substrates." BioResources, 11(3), 5854-5869.
The document discusses biotransformation, which is the biological process by which organic compounds are modified by enzymes in microbial, plant, and animal cells. Microbial transformation is preferred over plant or animal cell transformation due to microbes having a higher surface-to-volume ratio, growth rate, and metabolism rate, as well as being easier to maintain sterility. Microbial transformations can occur under mild conditions and achieve high yields, regioselectivity, stereoselectivity, and multi-step conversions using different microorganisms.
Bioactive compounds and antioxidant capacities of fresh and canned fruit,of p...GC University Faisalabad
This document summarizes a study on the bioactive compounds and antioxidant capacities of fresh and canned pineapple fruit. The study found that fresh pineapple extracts had higher levels of total phenolics, total flavonoids, and stronger antioxidant activities compared to canned pineapple extracts based on DPPH radical scavenging, inhibition of linoleic acid peroxidation, and reducing power assays. Fresh pineapple is a richer source of natural antioxidants than canned pineapple.
This document describes a study on isolating cellulase-producing fungi from termite soil. Eight fungi - Aspergillus sp, Trichoderma sp, Penicillium sp, Acremonium sp, Cladosporium sp, Neurospora sp, and Phoma sp - were isolated from termite soil samples using spread plating and identified microscopically. The fungi were screened for cellulolytic activity using various pretreated lignocellulosic substrates. Aspergillus niger and Trichoderma viridae showed the highest enzymatic activity on sugarcane bagasse.
This document summarizes research on the production of chitosan by various fungi. Key findings include:
- Absidia coerulea and Mucor rouxii were among the best chitosan-producing fungi strains examined.
- Optimum chitosan yields were observed at temperatures of 21-26°C in medium containing yeast extract, glucose and peptone.
- Continuous fermentation in a stirred tank reactor produced the highest amount of chitosan from A. coerulea, resulting in approximately a three-fold increase compared to batch culture.
Production and isolation of chitosan by submerged and solid state fermentationNgcon35
This document summarizes a study that developed a method for producing and isolating chitosan through liquid and solid-state fermentation of Lentinus edodes. Under liquid fermentation, chitosan yields of 120 mg/L were achieved. Solid-state fermentation produced significantly higher yields of 6.18 g/kg, up to 50 times greater than other fungal methods. Solid-state fermentation provides an economical process for large-scale chitosan production with characteristics suitable for industrial applications.
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.
The document describes the design, synthesis, and testing of novel lipopeptide conjugates as potential antimicrobial agents. A library of conjugates was designed based on a template consisting of a hydrophobic moiety, dipeptide, and spermidine. Conjugates containing linoleic acid exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Structure-activity relationships revealed that optimal activity required a hydrophobicity of 50-70% and a minimum charge of +2. Active conjugates were found to have different modes of action, damaging bacterial cell membranes and DNA. Two conjugates showed selective membrane disruption of pathogenic MRSA and were identified as promising leads for further optimization and development as antimicrobial agents.
1) The document examines the effects of antimycin A and monofluoroacetate treatments on reactive oxygen species (ROS) production and citrate levels in Arabidopsis thaliana leaves.
2) The treatments were found to increase ROS production, as measured by dichloroflourscein diacetate and 3,3-diaminobenzidine. Citrate levels decreased over time with the treatments.
3) Antimycin A treatment led to the highest increase in ROS production and greatest decrease in citrate levels, while monofluoroacetate treatment resulted in lower ROS production and higher citrate levels compared to the control.
This document reviews drug discovery efforts to develop new antimicrobials by targeting the diaminopimelic acid (DAP) biosynthesis pathway in bacteria. It discusses how DAP is an essential component of bacterial cell walls but not mammalian cells, making enzymes in the DAP pathway potential drug targets. The review focuses on substrate-based inhibitors of DAP pathway enzymes and how understanding the enzymology could aid inhibitor design. Developing inhibitors that selectively block DAP biosynthesis in resistant bacteria could lead to new, less toxic antimicrobial therapies.
The student synthesized demethylated curcumin to create a derivative that is more soluble in aqueous solutions than curcumin. Demethylation was achieved through a reaction involving aluminum trichloride and hydrochloric acid. While NMR analysis indicated partial demethylation occurred, results were unclear due to overlapping peaks from impurities. In the future, the student aims to test the synthesized compound's ability to disaggregate beta amyloid plaques compared to curcumin.
This document discusses optimizing the production of exopolysaccharide (EPS) by Bacillus subtilis using various carbon and nitrogen sources. Sucrose at 2% concentration produced the highest yield of EPS. Cane molasses and rice bran were also tested as carbon sources, with cane molasses at 2% giving the highest yield. Different solvents were tested for their ability to precipitate EPS, with ethanol, diethyl ether, and methanol being effective. Fourier transform infrared spectroscopy analysis revealed the extracted polymer was composed of sucrose units. In conclusion, agro-wastes like cane molasses could be used as alternative carbon sources for the economical production of EPS.
- 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.
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
This document reviews the role of bacterial extracellular polysaccharides in biofilm formation. It discusses how extracellular polymeric substances (EPS) produced by microorganisms form the matrix of microbial aggregates and biofilms. EPS are involved in the initial attachment of cells to surfaces and provide protection from environmental stresses. The production of EPS is regulated by quorum sensing and helps mediate processes like bioremediation and bioleaching that are important in industrial applications.
This document discusses personal information about Mohammad Wahid Abdullah Khan and contains inappropriate, unverified claims. It describes a person taking money from Khan near Eid and then spending it inappropriately. It also contains rude language calling someone a "rapist" and making unfounded claims about their character and actions. Overall, the document seems to be spreading misinformation about individuals in an unethical manner.
The NSCS General Body Meeting discussed upcoming officer positions, end of year social plans, and distributing membership pins. Officer applications were due by April 18th and included positions like Vice President, Secretary, and Vice presidents of PACE and Public Relations. The end of year social would be held on May 12 at 6:30pm in Trabant 209 to celebrate NSCS's 20th anniversary and the end of semester, with food from Capriotti's. Members still needing membership pins from the induction could collect them after the meeting.
Thuraya is a mobile satellite services company headquartered in Dubai that provides coverage to over 140 countries and 2/3 of the world's population using two satellites. It supports journalists, aid organizations, and NGOs through satellite connectivity solutions like the Thuraya SatSleeve and IP+ modem. During disasters like Typhoon Haiyan, Thuraya donates equipment and airtime to relief efforts.
The document contains information from surveys of TUD TUE TUT alumni on average annual income by years since graduation, most inspiring managers as nominated in a December poll, most admired employers, best secondary employment benefits, preferred years to start a new job, and a note about career resources on talentstorm.nl for Dutch technical university professionals.
This document contains a research proposal submitted by Edy Wijaya. The proposal examines the hypothesis that a husband who only does housework and does not have a paid job is considered lazy. It outlines a qualitative analysis and survey structure to gather data on public perceptions of husbands who are homemakers. The proposal provides details on developing survey questions, distributing the survey online, collecting responses, analyzing the data, and ensuring an ethical research process that is confidential, original, relevant and fair.
TALENTSTORM is a program at TU Eindhoven that aims to support 5000 students and alumni in their career mobility. Participation of women in TALENTSTORM has increased from 24% to 32%. People can join TALENTSTORM through their website at www.TALENTSTORM.nl.
This document presents the crystal structure of mature apo-caspase-6, an enzyme involved in neurodegenerative diseases. The structure reveals the canonical caspase conformation, contrasting with a previous structure of apo-caspase-6 that showed a noncanonical conformation. The authors believe the previous structure represented an inactive pH-dependent form. Comparison to other caspase structures allows visualization of loop rearrangements upon ligand binding. This new structure provides insight into the conformational dynamics of caspase-6.
This document discusses Hyper IgD Syndrome (HIDS), a rare inherited autoinflammatory syndrome characterized by recurrent fever episodes beginning in infancy accompanied by skin rash, abdominal pain, headaches and enlarged lymph nodes. It is caused by mutations in the mevalonate kinase gene resulting in reduced cholesterol synthesis and increased inflammation. Diagnosis is based on clinical criteria including onset before age 5 and fever episodes lasting less than 14 days, along with elevated IgD levels and urine mevalonic acid during attacks. Treatment aims to reduce inflammation and frequency of attacks, but with variable success. Prognosis is generally good with improvement over time, but amyloidosis can rarely occur.
This short story is about a teenage boy who imagines a rubber band named Stretchy coming to life as his friend. The boy receives a school project where he can bring his imaginations to reality. He imagines what it would be like to have a rubber band friend, how that friend would live, and what powers or abilities the rubber band would have.
The document announces an upcoming Udance event on March 23rd from 9am to 9pm at the BOB where members of the NSCS can earn 1 hour of service for every hour spent there. It provides instructions to check in with NSCS officers wearing "talk nerdy to me" t-shirts or email the appropriate contact if officers cannot be found. It also notes that t-shirts and pins from last semester can be picked up and applications are being accepted for the executive board by April 1st.
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.
This document contains photos from various photographers including widdowquinn, Earthworm, Joel Haas, hetan_hunt13, foodiesathome.com, and Dave77459. It encourages the reader to get started creating their own Haiku Deck presentation on SlideShare by including photos from different photographers.
This document discusses internet safety and provides definitions and steps related to cyber security, cyber safety, and cyber ethics. It defines cyber security as protecting networks, computers, programs and data from threats through technologies and practices. For cyber security, it recommends placing computers in high traffic areas, installing effective security software with antivirus, anti-spyware and firewalls, and enabling parental controls. Cyber safety is defined as the responsible and safe use of technology. Steps include establishing family computer rules and not sharing personal information online. Cyber ethics examines user behavior and its effects, and its steps include reporting illegal activities, understanding online dangers, applying common sense, and avoiding unsafe websites.
Integrating EQuIP Into Your State’s CCSS Implementation Strategy Achieve, Inc.
On April 29, 2014 Achieve hosted a webinar on integrating EQuIP into Common Core State Standards implementation plans. It provided an overview of the available tools and resources developed through Achieve’s EQuIP (Educators Evaluating the Quality of Instructional Products) initiative, designed to identify high-quality materials aligned to the CCSS. We then heard directly from leaders at the state and district level who have put the EQuIP resources into use to support their efforts to identify quality and aligned instructional materials to advance implementation of the CCSS, including Merri Ann Drake, Idaho Core Coach, Idaho State Department of Education; Elissa Farmer, Curriculum Specialist, Seattle Public Schools; Terri King-Hunt, Gifted Support Specialist, Atlanta Public Schools; Linda Schoenbrodt, Elementary Mathematics Program Specialist, Maryland Department of Education; and Amy Youngblood, Founder, Eduoptimus. For more and to hear the recording, go to http://www.achieve.org/meetings-webinars
JosDeVries is a retail company established in 1986 with offices in multiple countries. They have over 50 retail specialists and designers with expertise in retail trends, strategy, concept development, design, and digital solutions. Their services include retail format development, branding, interior design, graphic design, and omni-channel concept creation for clients in food retail, non-food retail, shopping centers, and other industries.
2014. Pietkiewicz, Wahyudi. Synthesis of macrocycles that inhibit protein syn...Adrian Pietkiewicz
This document describes the synthesis of seven new analogues of the macrocyclic peptide sanguinamide B (SanB) and testing of their ability to inhibit protein synthesis in cancer cells. The analogues were designed by altering the amino acids at positions I and III of the SanB backbone, inverting stereochemistry at position III, and changing the protecting group on lysine. All analogues were tested for cytotoxicity against colon cancer cell lines and ability to inhibit protein synthesis. The lead compound with an IC50 of 15.9 μM against colon cancer cells contained an N6-carboxybenzyl-lysine at position I. This establishes the importance of this moiety for biological activity.
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
This document provides details of a thesis submitted for a Doctor of Philosophy degree in Chemistry. It describes the objectives of the thesis which are to synthesize and characterize N-phenyl succinimides, glutarimides, chalcones, pyrazoles, amino-pyrimidines and malononitriles and test their biological activities. It outlines the materials and methods used which involve synthesizing cyclic imides, chalcone derivatives from the imides, and then pyrazoles, amino-pyrimidines and malononitriles from the chalcones. It also provides an abstract that summarizes the key points of the thesis and introduces the topics to be covered.
Synthesis, characterization, amdet and docking studies of novel diclofenac de...Alexander Decker
This document discusses the synthesis and characterization of novel diclofenac derivatives containing phenylalanine moieties as selective inhibitors of cyclooxygenase-2 (COX-2). Sixteen novel diclofenac derivatives were synthesized and their structures were confirmed through various analytical techniques. Molecular docking studies predicted that compounds 7, 12 and 16 showed stronger binding with COX-2 than the reference drug diclofenac, making them potential selective COX-2 inhibitors. The binding scores of these compounds were higher than diclofenac when docked into the active site of COX-2.
Synthesis, Characterization and Biological Evaluation of Oxazolone Derivativesijceronline
A series of six 4-aryl Benzelidene-2-phenyl-5- oxazolone derivatives were synthesized by condensation of aromatic aldehydes with N-benzoyl glycine (Hippuric acid) in the presence of sodium acetate and acetic anhydride at room temperature in ethanol. Six of the compounds are new derivatives. The structures of the compounds were evaluated based on 1H-NMR , IR and FTIR methods and by elemental analysis. .All the derivative compounds prepared were tested for their antimicrobial activity by disk diffusion technique. Test organisms: Bacteria like Staphylococcus aureusMTCC 7443 and Salmonella typhimuriumMTCC 733 Fungi like C.albicans and A.flavus The results were compared with those of the standard 0.5% Ciprofloxacin. The derivatives with Salicylaldehyde and cinnamaldehyde were showed excellent activities against E. coli. and Staphylococcus aureusMTCC 7443 : than Salmonella typhimuriumMTCC 733 bacteria. It also showed reasonable activity withFungi like C.albicans than A.flavus
This document analyzes the secondary metabolite compounds produced by Enterobacter aerogenes and tests its anti-fungal and anti-bacterial activity. GC-MS analysis identified 27 bioactive compounds in the methanolic extract of E. aerogenes including acids, alcohols, amines, and esters. Testing showed the extract was highly effective at suppressing the growth of Candida albicans. Coriandrum sativum plant extract also showed strong anti-bacterial activity against E. aerogenes, with a zone of inhibition of 6.75±0.22 mm. The results suggest secondary metabolites of E. aerogenes have potential for development as anti-fungal and anti-bacterial drugs.
Targeted Intermediates of Eudesmic Acid: Synthesis and X-ray Investigationsijtsrd
it was carried out synthesis of esters and their dinitro derivatives of 3,4,5-trimethoxybenzoic (eudesmic) acid. Esterification of eudesmic acid carried out n absolute methanol or ethanol and corresponding methyl and ethyl 3,4,5-trimethoxybenzoates (2, 3) have been synthesized in good yields. It was revealed that nitration of these esters gives only dinitro products. The structure of the synthesized compounds of the methyl and ethyl 2,6-dinitro-3,4,5-trimethoxybenzoates (4, 5) was determined by X-ray diffraction analysis (XRD). In the asymmetric part of the crystal structures of 4, 5 one and two molecules are observed, respectively. In crystalline structures a flat nitro groups and carboxylic groups do not participate in the conjugation with aromatic rings. In the crystal structure of 4, an intermolecular C8-H...O9 hydrogen bond is observed, these H bonds link the molecules along the [010] axis. In the crystal structure of 5, intermolecular C9B-H...O4A and C10B-H...O8A hydrogen bonds form chains along the [011] axis. The formed chains are cross linked by the intermolecular C9B-H...O5A hydrogen bonds. Olimova M.I. | Okmanov R. Ya. | Elmuradov B. Zh."Targeted Intermediates of Eudesmic Acid: Synthesis and X-ray Investigations" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-1 | Issue-6 , October 2017, URL: http://www.ijtsrd.com/papers/ijtsrd2417.pdf http://www.ijtsrd.com/chemistry/other/2417/targeted-intermediates-of-eudesmic-acid--synthesis-and-x-ray-investigations/olimova-mi
A STUDY TO EVALUATE THE IN VITRO ANTIMICROBIAL ACTIVITY AND ANTIANDROGENIC E...Dr. Pradeep mitharwal
The present paper deals with synthesis and characterization
of some new chromium (III) Schiff base complexes using microwave irradiation
technique as well as conventional heating. The S∩N donor benzothiazolines, 1-
(2-furanyl) ethanone benzothiazoline (Bzt1N
∩
SH), 1-(2-thienyl) ethanone
benzothiazoline (Bzt2N
∩
SH) and 1-(2-pyridyl) ethanone benzothiazoline
(Bzt3N
∩
SH) were prepared by the condensation of ortho-aminothiophenol with
respective ketones in ethanol.
Novel Hybrid Molecules of Isoxazole Chalcone Derivatives: Synthesis and Study...Ratnakaram Venkata Nadh
medicine due to their significant role in the treatment of different health problems.
Methods: We have synthesized new series of isoxazole-chalcone conjugates (14a-m) by the
Claisen-Schmidt condensation of suitable substituted acetophenones with isoxazole aldehydes (12a-d).
In vitro cytotoxic activity of the synthesized compounds was studied against four different selected
human cancer cell lines by using sulforhodamine B (SRB) method.
Results: The adopted scheme resulted in good yields of new series of isoxazole-chalcone
conjugates (14a-m). Potent cytotoxic activity was observed for compounds -14a, 14b, 14e, 14i, 14j
and 14k against prostate DU-145 cancer cell line.
Conclusion: The observed potent cytotoxic activities were due to the presence of 3,4,5-
trimethoxyphenyl group.
International Journal of Engineering and Science Invention (IJESI)inventionjournals
International Journal of Engineering and Science Invention (IJESI) is an international journal intended for professionals and researchers in all fields of computer science and electronics. IJESI publishes research articles and reviews within the whole field Engineering Science and Technology, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
This document describes a study that synthesized a new class of tyrosinase inhibitors called azachalcones. Azachalcone derivatives were tested for their ability to inhibit the enzyme tyrosinase, which is involved in melanin biosynthesis. Two compounds that were reduction products of pyridinyl azachalcones strongly inhibited tyrosinase activity and were more potent inhibitors than the positive control kojic acid. Kinetic studies showed that these two compounds act as competitive inhibitors of tyrosinase by binding to the enzyme's active site. This new class of azachalcone inhibitors could potentially be used as depigmenting agents or to prevent browning in foods.
This document describes the synthesis of 1,3-Di(substituted-phenyl)-thiourea intermediates. The synthesis involves two steps: 1) reaction of substituted anilines with hydrochloric acid to form substituted-phenylammonium chlorides; and 2) reaction of the chlorides with ammonium thiocyanate to form the 1,3-Di(substituted-phenyl)-thiourea products. The products were characterized using techniques like melting point determination, UV-Vis spectroscopy, thin layer chromatography and Fourier-transform infrared spectroscopy. The final products have potential for use as intermediates in synthesizing the heterocyclic compound 2-aminobenzothiazole.
Studies on Aminobenzothiazole and Derivatives: Part-1. Synthesis of Intermedi...BRNSS Publication Hub
1,3-Di(substituted-phenyl)-thiourea is used as intermediate in different reactions because they play an important role in synthesizing the different heterocyclic compounds. These reactions involve the synthesis of an intermediate, substituted-phenylammonium chloride which is converted to 1,3-Di(substituted-phenyl)-thiourea using ammonium thiocyanate. The final product formed, 1,3-Di(substituted-phenyl)-thiourea has potential to use as an intermediate in the synthesis of a building block for the heterocyclic compound, 2-aminobenzothiazole.
This document describes the synthesis and evaluation of novel 2-phenyl-3-sulphonamido quinazolin-4(3H)-one derivatives for anti-HIV activity and cytotoxicity. A series of derivatives were synthesized by condensing 2-phenyl-1,3-benzoxazine-4-one with primary aromatic amines. The compounds were screened for anti-HIV activity against HIV-1 and HIV-2 in MT-4 cells and for cytotoxicity in uninfected MT-4 cells. Most compounds showed cytotoxicity between 1.93-87 μg/ml. Compound SPB-III displayed the highest cytotoxic activity with a CC50 of 1.93 μg/ml. While most
This document describes the synthesis and evaluation of novel 2-phenyl-3-sulphonamido quinazolin-4(3H)-one derivatives as potential anti-HIV agents. A series of derivatives were synthesized by condensing 2-phenyl-1,3-benzoxazine-4-one with primary aromatic amines containing sulphonamide groups. The compounds were evaluated for anti-HIV activity against HIV-1 and HIV-2 in MT-4 cells and for cytotoxicity in uninfected MT-4 cells. Most compounds showed cytotoxicity between 1.93-87 μg/ml. Compound SPB-III displayed the highest activity with a CC50 of 1.93 μg/ml.
Green synthesis of well dispersed nanoparticles using leaf extract of medicin...tshankar20134
Green synthesis of gold nanoparticles was achieved using an extract of the medicinal plant Adhatoda vasica.
The nanoparticles formed were predominantly spherical and monodisperse, with sizes ranging from 22 to 47 nm as determined through transmission electron microscopy analysis. Ultraviolet-visible spectroscopy and X-ray diffraction data confirmed the formation and crystalline nature of the gold nanoparticles. Functional groups present in the plant extract, such as hydroxyl and carboxyl groups, were found to play a role in both the reduction of gold ions and stabilization of the resulting nanoparticles. This green synthesis method using A. vasica extract could provide a means of producing biocompatible gold nanoparticles for applications such as drug delivery.
This document describes a study that assessed the antimicrobial properties of selenium nanoparticles (SeNPs) using cyclic voltammetry. SeNPs were synthesized using a microwave-assisted method with controllable size distributions. Cyclic voltammetry experiments showed that adding SeNPs to E. coli cells caused a continuous decrease in characteristic peak intensities over time, demonstrating a reduction in viable bacteria. This indicates that SeNPs interact with and disrupt the integrity of bacterial cell membranes, likely by forming reactive oxygen species, and can be used as potential antimicrobial agents.
Al rawi 2018-j._phys.__conf._ser._1003_012012Muna AL-rawi
new Schiff base [I] was prepared by refluxing Amoxicillin trihydrate and 4-Hydroxy-
3,5-dimethoxybenzaldehyde in aqueous methanol solution using glacial acetic acid as a catalyst. The
new 1,3-oxazepine derivative [II] was obtained by Diels- Alder reaction of Schiff base [I] with
phthalic anhydride in dry benzene. The reaction of Schiff base [I] with thioglycolic acid in dry
benzene led to the formation of thiazolidin-4-one derivative [III]. While the imidazolidin-4-one [IV]
derivative was produced by reacting the mentioned Schiff base [I] with glycine and triethylamine in
ethanol for 9 hrs. Tetrazole derivative [V] was synthesized by refluxing Schiff base [I] with sodium
azide in dimethylformamid DMF. The structure of synthesized compounds[I-V] was characterized
by their melting points, elemental analysis CHN-S and by their spectral data; FTIR and 1H NMR
spectroscopy. Two cancer cell lines include: (RD) human pelvic rhabdomyosarcoma
and (L20B) the mice intestines carcinoma cell line
This document summarizes key signaling pathways in muscle-invasive bladder carcinoma. It discusses molecular markers that indicate basal or luminal subtypes of bladder cancer, which differ in response to treatment. Basal cancers often overexpress EGFR and respond to chemotherapy, while luminal cancers involve alterations in genes like FGFR3, ERBB2/3 and are generally less aggressive. The document also reviews markers for cancer stem cells, receptor tyrosine kinase signaling pathways, cytoskeleton proteins, the PI3K-Akt-mTOR pathway, and VEGF/VEGFR pathways that are clinically significant for modeling and optimizing treatment of muscle-invasive bladder cancer.
This document discusses biological markers for predicting response to Bacillus Calmette–Guerin (BCG) treatment in patients with non-muscle invasive bladder cancer (NMIBC). It identifies several promising markers based on a literature review, including tumor associated macrophages (TAMs), human leukocyte antigen (HLA) class I, a combination of Ki-67/CK20 proliferation markers, and certain cytokine levels like IL-2, IL-8, and the IL-6/IL-10 ratio. The document advocates using mathematical models along with molecular/cellular biology and clinical data to better understand these complex markers and their relationship to individual patient responses to BCG therapy for NMIBC.
1) Researchers used virtual and biomolecular screening to identify the first selective agonist for GPR30, a G protein-coupled receptor that binds estrogen. They identified a compound called G-1 that selectively binds and activates GPR30 with high affinity and specificity over estrogen receptors ERα and ERβ.
2) Experiments showed that G-1 competes for binding of a fluorescent estrogen to GPR30 with high affinity but does not bind to ERα and ERβ. G-1 also selectively activated GPR30 signaling pathways like calcium mobilization and PI3K activation, but did not activate these pathways through ERα and ERβ.
3) G-1 was able to selectively target and bind GPR30 over ERα
The document describes the discovery of leukotriene A4 hydrolase (LTA4H) inhibitors using a fragment-based drug discovery approach. A novel fragment library called "fragments of life" (FOL) was designed, which includes natural small molecules, derivatives, and molecules that mimic protein structures. Screening the FOL library against LTA4H by X-ray crystallography identified several fragment hits, including derivatives of resveratrol and nicotinamide. These fragments were elaborated into potent LTA4H inhibitors representing novel chemotypes.
1. Researchers determined the crystal structure of human caspase-6 bound to the irreversible inhibitor Z-VAD-FMK at acidic pH.
2. The structure adopted a non-canonical conformation not seen previously, with helix extensions blocking the peptide binding site and the inhibitor binding in a unique mode.
3. This suggests the non-canonical conformation observed for caspase-6 at acidic pH is capable of peptide binding, though the physiological relevance is uncertain given the non-physiological conditions.
Wityak 2015 JMC Lead optimization towards PoC tools for HD with a 4-pyrazol-...Alex Kiselyov
1. Researchers identified compound 9t as a promising pan-JNK inhibitor for testing in Huntington's disease models based on its sub-micromolar activity in cellular assays, favorable permeability properties, and low potential for efflux.
2. Compound 9t showed brain penetration in mice after oral dosing but its exposure was limited by rapid plasma clearance. Co-administering 9t with a cytochrome P450 inhibitor increased its brain exposure levels.
3. The goal was to optimize a 4-(1H-pyrazol-4-yl)pyrimidine class of compounds into a pan-JNK inhibitor with properties suitable for proof-of-concept studies in Huntington's disease, including
The document discusses crystal structures of phosphodiesterase 4 (PDE4) regulatory domains bound to small molecule inhibitors. It presents seven crystal structures that show the regulatory domain closed across the active site, revealing how PDE4 activity is regulated by controlling access to the active site. This structural insight allowed the authors to design PDE4 allosteric modulators that only partially inhibit cAMP hydrolysis, rather than completely inhibiting activity like existing drugs. The allosteric modulators showed reduced potential for side effects like emesis in cellular and animal studies while maintaining therapeutic effects.
This document describes the development of novel antiplatelet agents that target the EP3 receptor for prostaglandin E2 (PGE2) on platelets. Current antiplatelet drugs like clopidogrel that target the P2Y12 receptor reduce heart attacks and strokes but increase the risk of severe bleeding. The document reports:
1) PGE2 produced in inflamed atherosclerotic plaques activates platelets through the EP3 receptor, similarly to how ADP activates platelets through P2Y12.
2) A selective EP3 antagonist called DG-041 was developed that inhibits PGE2 facilitation of platelet aggregation in vitro and ex vivo without affecting bleeding time in rats, unlike
This document describes structure-activity relationship studies that identified compound DG-041 as a potent and selective antagonist of the prostaglandin EP3 receptor on platelets. A pharmacophore model was developed based on known EP3 agonists and antagonists. Various heterocyclic scaffolds were explored through rational design and synthesis. Optimization of 1,7-disubstituted indole analogues led to compounds with nanomolar activity against the human EP3 receptor. Further modifications to the indole core and substituents yielded additional potent analogs. Compound 50 (DG-041) was selected as a clinical candidate based on favorable in vitro and functional activities as an inhibitor of human platelet aggregation without prolonging bleeding.
Loss of Cilia ACS ChemBio 2008 cb700163qAlex Kiselyov
This document describes research examining the effects of synthetic derivatives of plant polyalkoxybenzenes on sea urchin embryos. The researchers synthesized isoxazoline derivatives of apiol and dillapiol, which are plant compounds with various biological activities. They found that one derivative, a p-methoxy-phenyl isoxazoline, caused sea urchin embryo immobilization by selectively removing motile cilia while leaving long sensory cilia intact. This effect was reversible through washing. The compound did not alter cell division or larval development. The researchers believe this derivative could serve as a tool for studying ciliary function and morphogenesis in sea urchin embryos.
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.
1. Accepted Manuscript
A facile synthesis and microtubule-destabilizing properties of 4-(1H-
benzo[d]imidazol-2-yl)-furazan-3-amines
Andrei I. Stepanov, Alexander A. Astrat’ev, Aleksei B. Sheremetev, Nataliya K.
Lagutina, Nadezhda V. Palysaeva, Aleksei Yu. Tyurin, Nataly S. Aleksandrova,
Nataliya P. Sadchikova, Kyrill Yu. Suponitsky, Olga P. Atamanenko, Leonid D.
Konyushkin, Roman V. Semenov, Sergei I. Firgang, Alex S. Kiselyov, Marina N.
Semenova, Victor V. Semenov
PII: S0223-5234(15)00147-6
DOI: 10.1016/j.ejmech.2015.02.051
Reference: EJMECH 7733
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2014
Revised Date: 18 January 2015
Accepted Date: 27 February 2015
Please cite this article as: A.I. Stepanov, A.A Astrat’ev, A.B Sheremetev, N.K. Lagutina, N.V. Palysaeva,
A.Y. Tyurin, N.S. Aleksandrova, N.P. Sadchikova, K.Y. Suponitsky, O.P. Atamanenko, L.D. Konyushkin,
R.V. Semenov, S.I. Firgang, A.S. Kiselyov M.N. Semenova, V.V. Semenov, A facile synthesis and
microtubule-destabilizing properties of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.051.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
2. M
ANUSCRIPT
ACCEPTED
ACCEPTED MANUSCRIPT
1
A facile synthesis and microtubule-destabilizing properties of 4-(1H-
benzo[d]imidazol-2-yl)-furazan-3-amines
Andrei I. Stepanov,a
Alexander A. Astrat’ev,a
Aleksei B. Sheremetev,b
Nataliya K. Lagutina,c
Nadezhda V. Palysaeva,b
Aleksei Yu. Tyurin,b
Nataly S. Aleksandrova,b
Nataliya P. Sadchikova,c
Kyrill Yu. Suponitsky,d
Olga P. Atamanenko,b
Leonid D. Konyushkin,b
Roman V. Semenov,b
Sergei I. Firgang,b
Alex S. Kiselyov,e
Marina N. Semenova,f
Victor V. Semenovb,*
a
Special Design and Construction Bureau SDCB “Technolog”, 33-A Sovetskii Ave., Saint
Petersburg, 192076, Russian Federation
b
N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow,
Russian Federation
c
I. M. Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, 119991 Moscow,
Russian Federation
d
A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28
Vavilov Str., 119991 Moscow, Russian Federation
e
Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology,
Institutsky Per. 9, Dolgoprudny, Moscow Region, 141700, Russian Federation
f
N. K. Kol’tsov Institute of Developmental Biology, RAS, Vavilov Str., 26, 119334 Moscow,
Russian Federation
Corresponding author: Victor V. Semenov
Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991,
Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966.
E-mail: vs@zelinsky.ru
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E-mail addresses:
Andrei I. Stepanov stepanoff@pisem.net
Alexander A. Astrat’ev astrchim@yandex.ru
Aleksei B. Sheremetev sab@ioc.ac.ru
Nataliya K. Lagutina mpcpr@yandex.ru
Nadezhda V. Palysaeva naduasha.85@mail.ru
Aleksei Yu. Tyurin tyurin@ioc.ac.ru
Nataly S. Aleksandrova natali.aleksandrova.50@mail.ru
Nataliya P. Sadchikova cska76@gmail.com
Kyrill Yu. Suponitsky kirshik@yahoo.com
Olga P. Atamanenko info@chemblock.com
Leonid D. Konyushkin LeonidK@chemical-block.com
Roman V. Semenov rs@chemical-block.com
Sergei I. Firgang sfirgang@yandex.ru
Alex S. Kiselyov akiselyov@chemdiv.com
Marina N. Semenova ms@chemical-block.com
Victor V. Semenov vs@zelinsky.ru
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ABSTRACT
A series of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFAs) were prepared in good yields
(60–90% for each reaction step) via a novel procedure from aminofurazanyl hydroximoyl chlorides
and o-diaminobenzenes. The synthetic sequence was run under mild reaction conditions, it was
robust and did not require extensive purification of intermediates or final products. Furthermore,
there was no need for protection of reactive moieties allowing for the parallel synthesis of diverse
BIFA derivatives. Subsequent biological evaluation of the resulting compounds revealed their anti-
proliferative effects in the sea urchin embryo model and in cultured human cancer cell lines. The
most active compounds showed 0.2–2 µM activities in both assay systems. The unsubstituted
benzene ring of the benzoimidazole template as well as the unsubstituted amino group in the
furazan ring were essential prerequisites for the antimitotic activity of BIFAs. Compound 57
bearing the 2-chlorophenyl acetamide substituent at the nitrogen atom of the imidazole ring was the
most active molecule in the examined set.
Keywords:
Benzoimidazolfurazanamines
Inhibitors of tubulin polymerization
Sea urchin embryo
Cytotoxicity
Abbreviations:
BIFA, 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amine;
SAR, structure-activity relationship.
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1. Introduction
Molecules exhibiting 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amine (BIFA) scaffold have
attracted considerable attention of medicinal chemists in the past decade. This core is found in
multiple inhibitors of protein kinases, enzymes that represent an important class of cellular drug
targets in the treatment of hypertension, neoplastic, autoimmune, neurodegenerative, and
inflammatory diseases [1]. BIFA derivatives blocking glycogen synthase kinase GSK-3 signaling
were introduced as agents for the treatment of diabetes, Alzheimer’s disease, and as
immunomodulators [2]. BIFA-based inhibitors of p60 ribosomal S6 kinase 1 (RSK1) involved in
the cell cycle regulation [3] were suggested as promising antitumor agents [4]. BIFAs were reported
to work as potent selective modulators of ribosomal p70S6 kinase that control cell growth [5]. They
were also shown to suppress the activity of mitogen and stress-activated rho-kinase (MSK-1,
ROCK 1 and 2) [6] involved in apoptosis, cell proliferation and migration. Basilea team completed
synthesis and screening of a series of BIFAs (Fig. 1, I) [7]. Based on their cytotoxicity and
proapoptotic properties, BIFAs were proposed as agents for the treatment of various malignancies
and autoimmune disorders [7–9]. BAL27862 (Fig. 1) exhibiting low nM cytotoxicity across
multiple cancer cell lines [7,10–12] was selected for further optimization to yield a water-soluble
prodrug BAL101553 (Fig. 1) [8]. This compound is currently undergoing phase II clinical trials as
both an antimitotic and vascular targeting agent [13].
Insert Fig. 1.
N
N
N
O
N
NH
O
R1
R2
N
N
N
O
N
NH
O
NH2
CN
N
N
N
O
N
NH
O
NH
CN
NH2
O
NH2
NH
N
OH
N
H3CO
O
H3CO N
OH
O
O
O OCH3
H
Vinblastine
H3CO
H3CO
H3CO
OCH3
O
NH
O
Colchicine
I
BAL27862
BAL101553
Fig. 1. Structures of reported BIFAs and reference compounds colchicine and vinblastine.
Mechanism of BIFAs anti-proliferative activity has been tied to microtubule impairment.
For example, BAL27862 caused unique alterations of interphase and mitotic spindle microtubules
in cultured cancer cells [10,15]. The compound was shown to inhibit purified tubulin
polymerization and to bind tubulin dimers at the colchicine site [16]. Despite of this molecular
interaction, the specific effect of BAL27862 on microtubule dynamics differed from those of
colchicine and vinblastine suggesting its novel microtubule destabilizing mode of action [16]. It
should be noted that of the reported BIFAs the anti-tubulin mechanism was proved only for
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BAL27862. The indirect anti-tubulin effect of BIFAs could be also mediated by GSK-3 known to
affect microtubule stability, mitotic spindle formation and orientation [14].
Due to the diverse biological activity of BIFAs, we developed a robust protocol yielding a
library of respective derivatives and evaluated their microtubule destabilizing activity using the in
vivo sea urchin embryo model. Selected compounds were also studied in vitro using tubulin
polymerization assay, cell cycle distribution analysis, and further screened against a panel of human
cancer cell lines to assess their cytotoxicity.
BIFA scaffold 9 was first reported by Tselinskii et al. in 2001 [17]. To date, there are three
main routes towards 9 described in the literature (Scheme 1). Route A [17] involves treatment of
(un)substituted o-phenylenediamines 3 with carbimidate 2 [18] easily accessible from the
amidoxime 1. Route B [6] is based on recyclization of 5 upon heating with o-phenylenediamines 3
in acetic acid. Route C [2,8] employs condensation of 3 with ethylcyanoacetate at high temperature
to yield 6 [19] followed by its sequential conversion to cyano oxime 7, amidoxime 8 and finally, the
targeted aminofurazan derivative 9 [20–22].
Insert Scheme 1.
CH2(CN)2
NC CN
NOH NN
O
NH2
NH2
NOH
NN
O
NH2 CN
NN
O
NH2
MeO
NH
N
NMe
OH
NH2
N
NMe
OH
NH2
NO
N
N N
O
N
Me
OH
NH2
NHR
e
g
1
3 8
2
5
3
4
6 7
9
Route A
Route B
Route C
h
a b c d
N
N CN
R2
N
N CN
R2
NOH N
N
R2
NOH
NOH
NH2
3
NH2
NHR
R1
N
N
R2
N
O
N
NH2
fa
a b
e
R1
R1R1
R1R1
Scheme 1. Reported syntheses of BIFA scaffold. Reagents and conditions: (a) NaNO2, H+
; (b)
NaOH, NH2OH⋅HCl, H2O, reflux [18]; (c) Pb2O3, AcOH; (d) MeOH, HCl or MeOH, MeONa; (e)
o-phenylenediamine 3, EtOH, reflux, 20 h; (f) Pb(OAc)4, AcOH, r.t.; (g) NCCH2CO2Et, 100–190
°C; (h) NaOH, H2O, reflux.
All three strategies summarized in Scheme 1 afford poor yields of the targeted compounds
9. They require elevated temperatures, extensive purification of intermediates, convoluted synthetic
manipulations and toxic or costly reagents. In this study, we developed a robust and rapid access to
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a library of diverse BIFAs. The described protocol does not employ anhydrous solvents, inert
atmosphere, toxic chemicals and chromatographic purification of the targeted molecules.
2. Results and discussion
2.1. Chemistry
Benzimidazoles are an important class of biologically active compounds [23–25]. A plethora
of methods exist for their syntheses, including condensation of hydroximoyl chloride and
unsubstituted o-diaminobenzene. This procedure was originally described by Sasaki et al. for aryl
hydroximoyl chloride [26] and further expanded by Paton et al. onto carbohydrate-derived
hydroximoyl chloride [27,28]. Considering these results, we turned our attention to optimize the
reaction of o-diaminobenzenes 3 and 4-aminofurazan-3-carbohydroximoyl chloride 10 [29,30]
(Scheme 2), easily available from amidoxime 1 and sodium nitrite in HCl. We found that the
reaction of 10 with o-diaminobenzene (1:2 molar ratio) in ethanol at 60 °C for 0.5 h afforded the
benzoimidazole 9a in 81% yield after work-up and recrystallization. Using these conditions, we
evaluated the reaction of hydroximoyl chloride 10 with a variety of o-diaminobenzenes 3a–n
(Scheme 2). The desired BIFA derivatives 9a–n were obtained in moderate to good yields for both
electron-rich and electron-deficient o-diaminobenzenes 3a–n. Condensation of 10 with o-Me-
substituted o-diaminobenzenes 3c,d furnished 9c,d as a mixture of two isomers exhibiting a defined
proton position(s) at different N-atoms in the imidazole ring. The existence of isomers was further
confirmed by a doubling of signals in 1
H and 13
C NMR spectra.
N-substituted o-diamines 3o,p were also effective reagents in condensation with
hydroximoyl chloride 10 (Scheme 2) to afford 26 and 29. BIFAs 9a,h,i were condensed with 2,5-
dimethoxytetrahydrofuran to afford pyrrole derivatives 11a,h,i (Scheme 2).
Insert Scheme 2.
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R1 R2 R4
63: H Et pMeO-C6
H4
-
64: Cl H C6H5-CH2-
c
d e
NN
O
NH2
Cl
NOH
10
NN
O
NH2
NH2
NOH
1
NN
O
NHR4
Cl
HON
62v,w
NN
O
NH2
R4HN
NOH
60v-x
NN
O
NHR4
NH2
HON
61v-x
59v-x
R4NH2 59-70%
78-82% 60-87%
N
H
N
N
O
N
NH2
9a-n
NH2
NHR2
3o,p
12'-57'
R2Hal
NH2
NH2
3a-n
54-92%
a
70-90%
b
a
R3COCl or (R3CO)2O
58r-u
N
N
R2
N
O
N
NH2
12-57
g
70-80%
N
N
R2
N
O
N
NH
R3
O
14r,16s,19r,20r,24r,
25r,26t,28r,32r,34s,
38r,39r,u,40r,51t
R1
N
N
R2
N
O
N
N
11a,h,i
O OMeMeO
g
R1
R1
R1
R1
R1
3f,q
NH2
NHR2
R1
N
N
R2
N
O
N
R4HN
R1
f
59-70%
o-Phenylenediamines (3a-q)
NH2
NH2
NH2
NH2
F
a e
NH2
NH2
Cl
f
NH2
NH2
MeONH2
NH2
Cl
hg
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
MeO
MeOb c d i
NH2
NH2
NC NH2
NH
F3C
OMe
NH2
NH2
NH2
NH2
NO
N
NH2
NH2
MeOOC
k l m n
NH2
NHMe
NH2
NHEt
p qo
NH2
NH2
HOOC
j
Acid anhydrates or chloroanhydrates R3COCl, (R3CO)2O (58r-u)
u
N
Cl
O
F
Cl
O
ts
O
EtOEt
O
r
Cl
O
Me
O
MeOMe
O
w
Amines R4NH2 (59v-x)
NH2
MeO MeO
NH2
x
NH2
v
Cl
O
Et
Scheme 2. Facile synthesis of BIFAs. Reagents and conditions: (a) hydroximoyl chloride 10, o-
diaminobenzene 3, EtOH, 60 °C, 0.5 h; 5 h for 9c; (b) R2Hal, K2CO3, DMF, 50−90 °C, 4–12 h; (c)
R4NH2, NEt3, EtOH, i-PrOH, r.t., 3 h; (d) KOH, (CH2OH)2, reflux, 4 h; (e) NaNO2, HCl, AcOH,
≤10 °C, 3 h, r.t., 1 h; (f) o-phenylenediamine 3f,q, EtOH, reflux, 0.5 h, r.t., 1 h; (g) R3COCl,
toluene, reflux, 8–20 h; (R3CO)2O, AlkCOONa, reflux, 3 h.
Alkylation of scaffold 9 proceeded regioselectively at a nitrogen atom of the benzimidazole
ring, whereas the amino group in the furazan ring was not affected. N-substituted benzimidazoles
12–57 (Table 1) were prepared from the unsubstituted precursors 9a–n and respective alkyl halides
12′′′′–57′′′′ (Scheme 2). BIFAs 12–57 exhibiting free amino group in the furazan ring were coupled
with the appropriate acid chlorides or anhydrides 58r–u to form amides 14r, 16s, 19r, 20r, 24r,
25r, 26t, 28r, 32r, 34s, 38r, 39r,u, 40r, and 51t (Scheme 2; Table.1).
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Aminofurazans 63 and 64 were synthesized via the recyclization route (Scheme 2).
Treatment of hydroximoyl chloride 10 with amines 59v–x in the presence of NEt3 yielded the
corresponding amidoximes 60v–x. These were converted to the isomeric amidoximes 61v–x via a
recyclization reaction [31–33] in refluxing ethylene glycol with KOH. The corresponding
hydroximoyl chlorides 62v,w were synthesized via deazotization of 61v,w with sodium nitrite in
HCl. Condensation of the hydroximoyl chloride 62v,w with 1,2-diaminobenzenes in ethanol at 60
°C for 1 h afforded the desired benzoimidazoles 63 and 64 in moderate yields. Cyclization
conditions were similar for substituted (3o–q) and unsubstituted (3a–n) o-phenylenediamines
(Scheme 2, a and f).
Treatment of compound 9a with chloroacetonitrile gave cyanomethyl derivative 40. It was
reacted with sodium azide in hot DMF to give tetrazole 65. Alternatively, the nitrile group of
compound 40 was coupled with thiosemicarbazide at reflux in trifluoroacetic acid to yield of 2-
amino-1,3,4-thiadiazole derivative 66 (Scheme 3).
Insert Scheme 3.
N
N
N
O
N
NH2
CN
N
N
N
O
N
NH2
N N
NH
N
N
N
N
O
N
NH2
N N
S
NH2
a b
63% 71%
4065 66
Scheme 3. Synthesis of N-(hetarylmethyl) benzoimidazol derivatives 65 and 66. Reagents and
conditions: (a) NaN3, NH4Cl, DMF, 100 °C, 8 h; (b) H2NC(S)NHNH2, CF3COOH, reflux, 8 h.
The structures of all synthesized products were confirmed by spectroscopy. Both 1
H and 13
C
NMR data were consistent with the presence of furazan and benzimidazole moieties. Specifically,
for the N-unsubstituted benzimidazole group, there were two distinct signals at ca. 138 ppm and ca.
124–130 ppm for C-2 and C-5/6 respectively, notably signals corresponding to other carbon atoms
were broadened and overlapped. This phenomenon has been described for benzimidazoles earlier
[34]. It was explained by the rapid proton exchange between N-1 and N-3 atoms. Signals at ca. 140
(C-C-NH2) and ca. 155 ppm (C-NH2) were attributed to carbons of the furazan ring [31,35].
Structures of benzimidazoles 27 and 29 were unequivocally established by X-ray crystallography
(Fig. 2). Experimental details are given in Supplementary data.
Insert Figure 2.
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Fig. 2. Molecular structure of compounds 27 and 29 showing the atom numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
The N–O bonds in the furazan ring of both structures showed different length (N1–O1 of
1.40–1.41 Å, N2–O1 of 1.37–1.38 Å) affected by substituents [36]. Crystal packing of 27 and 29
and the details of X-ray data collection are presented in Fig. S2 and Table S1, Supplementary data
[37].
2.2. Biological effects
2.2.1. Antiproliferative activity in the sea urchin embryo model
The synthesized BIFA analogs were evaluated for their antiproliferative activity using in
vivo phenotypic sea urchin embryo assay [38]. This assay has been extensively validated in our lab
to afford a reliable insight into specific antimitotic, cytotoxic, and microtubule destabilizing effects
of tested compounds. A typical experimental protocol includes (i) fertilized egg test for antimitotic
activity displayed by cleavage alteration/arrest, and (ii) swimming pattern observation of blastulae
treated by compounds after hatching. The lack of forward movement, settlement to the bottom of
the culture vessel, and rapid spinning of embryos around the animal–vegetal axis suggests a
microtubule destabilizing activity caused by a molecule (video illustrations are available at
http://www.chemblock.com). The attainment of specific tuberculate shape of arrested eggs, which is
typical for microtubule destabilizing agents, is considered an indirect evidence of targeting
tubulin/microtubules [38–40]. The test results are listed in Table 1. Colchicine and vinblastine
sulfate served as reference microtubule destabilizing compounds.
Insert Table 1.
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Table 1.
Structures of BIFAs and their effects on sea urchin embryos and human cancer cells.
Compd R1 R2 R3
Sea urchin embryo effects, EC (µM)a
NCI60 screen
Cleavage
alteration
Cleavage
arrest
Embryo
spinning
Mean GI50,
µMb
Mean cell
growth, %c
Colchicine 50 100 TEd
50 0.132e
Vinblastine 0.1 0.2 TEd
2 0.00137f
BAL 27862 0.0065–0.017g
9a H H – 2 >4 >4 102.97
11a H H R3C(O)NH=Pyrrole >4 >4 >4 104.11
9b 5-Me H – NDh
9c 4-Me H – NDh
9d 4,5-diMe H – NDh
9e 5-F H – 2 >4 >5 97.83
9f 5-Cl H – 2 >4 >5 97.13
R3COCl or (R3CO)2O
58r-u
9-57
N
N
R2
N
O
N
NH
R3
O
14r,16s,19r,20r,24r,
25r,26t,28r,32r,34s,
38r,39r,u,40r,51t
R1N
N
R
N
O
N
H2N
2
1
R
2
4
7
6
5
1
3O
OMeOMe
N
N
R2
N
O
N
N
11a,h,i
R1
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9g 4-Cl H – 1 >4 >4 99.8
9h 5-OMe H – 0.5 >4 >5 101.01
11h 5-OMe H R3C(O)NH=Pyrrole 2 >4 >4 104.43%
9i 4,7-diOMe H – 2 >4 >4 NDh
11i 4,7-diOMe H R3C(O)NH=Pyrrole NDh
9j 5-COOH H – NDh
9k 5-COOMe H – NDh
9l 5-CN H – >4 >4 >4 107.07%
9m 6
5
H – 0.5 4 >4 NDh
9n
NO
N
6
7
H – NDh
12 H OH – NDh 102.07
13 H CH – >4 >4 >4 99.56
14 H CH2 – NDh
14r H CH2 Me >4 >4 >4 96.53
15 H
F
– 1 >4 >4 3.09
16 H
F
– 0.5 >4 >4 NDh
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16s H
F
Et 4 >4 >4 7.76
17 H
F
– 2 >4 >4 8.91
18 H
Br
– 0.5 >4 2 0.603
19 H
F3C
– 2 >4 >4 0.676
19r H
F3C
Me >4 >4 >4 82.18
20 H
MeO
– 0.2 2 TEd
>5 0.457
20r H
MeO
Me 1 >4 >4 3.98
21 H
MeO
Me
– 0.2 2 TEd
>4 0.479
22 H
EtO
EtO
– >4 >4 >4 80.97
23 H
Me
– NDh
70.38
24 H
t-Bu
– >4 >4 >4 67.38
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24r H
t-Bu
Me NDh
73.48
25 H
NC
– 4 >4 >4 79.81
25r H
NC
Me >4 >4 >4 94.08
26 H Me – NDh
26t H Me
F
NDh
99.5
27 H Et – NDh
28 H
MeO
– NDh
28r H
MeO
Me >4 >4 >4 84.96
29 5-CF3
MeO
– >4 >4 >4 81.06
30 H
Cl
– NDh
3.39
31 H
Cl
– >4 >4 >4 84.97
32 H
Cl
Cl
– NDh
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32r H
Cl
Cl
Me NDh
76.68
33 H
F
Cl
– >4 >4 >4 91.9
34 H
F
Cl
– NDh
34s H
F
Cl
Et >4 >4 >4 64.55
35 H
Cl
O
O
– >4 >4 >4 88.34
36 H
Br
O
O
– >4 >4 >4 86.07
37 H – 0.2 2 TEd
2 NDh
38 H
N
– 2 >4 >4 89.31
38r H
N
Me >4 >4 >4 103.6
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39 H N
– NDh
96.96
39r H N
Me >4 >4 >5 101.38
39u H N N
>4 >4 >4 97.07
40 H NC – >4 >4 >4 102.45
40r H NC Me NDh
65 H
NN
NH
N – >4 >4 >4 102.74
66 H
NN
SNH2
– NDh
41 H N
N
N
NH2
NH2
– >4 >4 >4 101.61
42 H N
N
N
NH2
NMe2
– 2 >4 >4 80.77
43 H
N
N
N
NH2
N
– 4 >4 >4 87.71
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44 H
NH
O
Me
Me
– >4 >4 >4 108.54
45 H
NH
O
Me
Me
– >4 >4 >4 90.2
46 H
NH
O
MeO
OMe
MeO
– 1 4 TEd
>5 9.77
47 H
NH
O
F
F
– 1 >4 >4 3.98
48 H
NH
O
F
F
– 1 4 >4 3.55
49 H
NH
O
F
Cl
– 2 >4 >4 70.49
50 H
NH
O
Cl
MeO
– >4 >4 >4 100.44
51 H
NH
O
Me
F
– NDh
51t H
NH
O
Me
F
F
NDh
71.77
52 H
NH
O
O
F3C – 4 >4 >4 104.79
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53 H
NH
O
O
O
– 2 4 >4 80.78
54 H
NH
O
Me
NHS
NH
– >4 >4 >4 NDh
55 H NH
F
O
– 4 >4 >4 101.92
56 H
NH
O
Cl
– 4 >4 >4 NDh
57 H
NH
O
Cl
– 0.05 0.5 TEd
>5 0.24
57h 5-OMe
NH
O
Cl
– 2 >4 >4 NDh
57h′′′′ 6-OMe
NH
O
Cl
– >4 >4 >4 NDh
57i 4,7-diOMe
NH
O
Cl
– Not tested due to poor solubility
a
The sea urchin embryo assay was conducted as described previously [38]. 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
GI50: concentration required for 50% cell growth inhibition.
c
Cell growth percent at 10 µM concentration.
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d
TE: tuberculate eggs typical for microtubule destabilizing agents.
e
NCI60 screen data for colchicine NSC 757. For the structure see Fig. 1.
f
NCI60 screen data for vinblastine NSC 49842. For the structure see Fig. 1.
g
Ref. [11].
h
ND: Not determined.
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As evidenced from the data, compounds 18 and 37 exhibited antimitotic activity at ca. 0.2–
0.5 µM concentrations. This effect was likely related to their microtubule destabilizing activity, as
suggested by embryo spinning. Compounds 20, 21, 46, and 57 were also considered to target
tubulin/microtubules. This conclusion was based on the observation that the arrested sea urchin
eggs acquired the tuberculate shape typical of the microtubule destabilizers [38–40]. The additional
24 molecules, including 16, 9h, and 9n, that induced cleavage abnormalities at 0.5–4 µM
concentration but failed to cause both cleavage arrest and embryo spinning, were classified as
tubulin independent antiproliferative agents.
It is worth noting that the most active BIFAs were endowed with the unsubstituted phenyl
ring in benzimidazole fragment and aminofurazan moiety (R1 = R3 = H). For the R1 substituents, the
maximal activity was observed for R1 = 5-MeO. For derivatives with R2 = R3 = H, the activity order
was as follows: 5-MeO (9h) = naphtyl (9m)> 4-Cl (9g) > H (9a) = 5-F (9e) = 5-Cl (9f). For the
analogs 57, the activity decreased in the order: R1-unsubstituted 57 >6-MeO (57h′)>5-MeO (57h),
where 57h was inactive up to 4 µM. For the R2 substituents the order of activity was as follows: R1
= R3 = H; R2 = benzyl, m-MeO (20) = m-MeO-p-Me (21) = o-naphtyl (37) > m-F (16) = m-Br (18) >
o-F (15) > p-F (17) > p-CN (25). Compounds substituted with m,p-diEtO (22), p-t-Bu (24), p-Cl
(31), o-Cl-methylenedioxy (35), o-Br-methylenedioxy (36) in R2 = benzyl were inactive in the
assay. Notably, meta-substitution in R2 with MeO or Hal group was favorable to compound activity.
In contrast, presence of bulky groups especially in para-position was deteriorating. Similarly,
compounds with R2 = alkyl showed no activity, as evidenced by 13, 14r, 40, and 63. Heterocyclic
derivatives with R1 = H; R2 = -CH2-pyridin were inactive up to 4 µM (38r, 39r, and 39u) except for
38 that exhibited antiproliferative effect at 2 µM. For arylacetamide derivatives 44–57 (R2 = -CH2-
CO-NH-Ar) compound 57 substituted with o-Cl-phenyl group was the most active microtubule
destabilizer. There was a significant reduction in activity for the related p-Cl-phenyl analog 56. In
evaluating the effect of R3 substitution (aminofurazane fragment), we found that the best activity
was exhibited by the unsubstituted compounds. Specifically, the NH2 derivatives were more active
than the respective pyrrole analogs (9a vs 11a; 9h vs 11h). It is worth noting that as opposed to
BIFA, their regioisomers 3-amino-4-[5-aryl-1H-1,2,3-triazol-1-yl]furazans I (Fig. 3) were generally
inactive in the sea urchin embryo assay. However, the respective 3-pyrrole-substituted furazans II
were reported to be potent antimitotic microtubule destabilizing agents [41].
Insert Figure 3.
N N
N
N O
N
NH2
N N
N
N O
N
N
(AlkO)n
I
(AlkO)n
II
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Fig. 3. Structures of 3-amino-4-[5-aryl-1H-1,2,3-triazol-1-yl]furazans [41].
Furthermore, unsubstituted NH2 derivatives were generally more active than the respective
N-substituted analogs including NHAc (19 vs 19r; 20 vs 20r; 25 vs 25r; 38 vs 38r). Similarly, NH2
derivative was more potent that the COEt compound (16 vs 16s). Thus, the replacement of NH2 in
furazan ring with pyrrole, acetyl- or propionamide groups resulted in a marked decrease or loss of
the antiproliferative effect. In summary, our assay data suggested that for the synthesized series of
BIFAs both the unsubstituted phenyl ring in benzimidazole and the unsubstituted amino group of
aminofurazan were essential for the antimitotic activity. In contrast, the nature of R2 was critical to
the microtubule destabilizing mode of action. For example, 9h and 9m with R2=H altered cell
division in the sea urchin embryos at submicromolar concentrations likely via a tubulin-independent
manner (Table 1).
2.2.2. In vitro cytotoxicity
The sea urchin embryo test results for the derivatives of 9 correlated well with their
cytotoxicity against a panel of human cancer cell lines (NCI60 anticancer drug screen) (Table 1).
Compound 57 was the most active in both assays. All potent molecules that exhibited GI50 less than
1 µM (18, 19, 20, 21, and 57) featured the unsubstituted phenyl ring in the benzimidazole fragment
and the unsubstituted amino group in the aminofurazan ring. The NCI60 screen mean graphs for
these compounds are presented in Supplementary data, Figures S3–S7. These SAR results are in
agreement with the cytotoxicity and apoptosis induction data reported previously for the series of
related furazanobenzimidazoles [7]. Four human cancer cell lines, namely melanoma MDA-MB-
435, CNS cancer SF 539, renal cancer RXF 393, and ovarian cancer OVCAR-3 cells were the most
sensitive to 57 (Table 2). Notably, in melanoma MDA-MB-435 cells, this compound caused total
cell growth inhibition and 50% reduction of cell number at concentrations of 0.088 µM and 0.84
µM, respectively. In addition, 57 displayed higher cytotoxicity against NCI/ADR-RES multidrug
resistant ovarian cancer cells over expressing P-glycoprotein than against the parent OVCAR-8 cell
line (Table 2).
Insert Table 2
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Table 2.
Effects of compound 57 on human cancer cells
Panel Cell line
57 Vinblastinea
GI50, µMb
TGI, µMc
LC50, µMd
GI50, µMb
TGI, µMc
LC50, µMd
Melanoma MDA-MB-435 0.028 0.088 0.84 0.00025 0.00025 0.001
Renal cancer RXF 393 0.087 0.366 19.2 0.001 0.05 2.51
CNS cancer SF 539 0.152 0.511 16.9 0.0006 0.0025 0.79
Ovarian cancer OVCAR-3 0.122 0.427 8.05 0.0003 0.0016 0.5
OVCAR-8 0.371 >100 >100 0.0016 0.32 2
NCI/ADR-RESe
0.184 16.0 >100 0.1 0.79 1.58
a
NCI60 screen data for vinblastine NSC 49842.
b
GI50: concentration required for 50% cell growth inhibition.
c
TGI: concentration required for total (100%) cell growth inhibition.
d
LC50: concentration required for 50% reduction in cell number.
e
NCI/ADR-RES: P-glycoprotein-overexpressing multi-drug resistant cell line derived from OVCAR-8.
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2.2.3. Inhibition of tubulin polymerization and cell cycle analysis
Compounds 16, 18, 19, 37, and 57 that caused pronounced cleavage alteration/arrest in the
sea urchin embryos were evaluated for their anti-tubulin properties using in vitro inhibition of
purified tubulin polymerization assay [42] (Table 3). As indicated in Table 1, compounds 18 and 37
induced sea urchin embryo spinning suggestive of their direct microtubule destabilizing activity.
These molecules also inhibited tubulin polymerization (Table 3). Compound 57 that caused
formation of tuberculate eggs typical for microtubule destabilizers showed IC50 of 7.36 µM in in
vitro tubulin polymerization assay. Derivative 16 altered sea urchin embryo cleavage at 0.5 µM
(Table 1) whereas the inhibition of tubulin polymerization was observed with the IC50 value of 7.9
µM. Compound 19 was a less potent in vitro tubulin inhibitor. Correspondingly, it exhibited only
moderate antiproliferative activity in the sea urchin embryo assay.
Insert Table 3.
Table 3. Tubulin polymerization inhibition of selected BIFAs.
Compd ITP IC50, µMa
16 7.9
18 2.04
19 13.27
37 7.03
57 7.36
Vinblastineb
0.6
a
ITP IC50: concentration required for 50% inhibition of in vitro tubulin polymerization.
b
Data from [43].
Active BIFA derivatives 16, 18, 19, 37, and 57 were further tested for their effects on cell
cycle distribution in the mouse fibroblast 3T3 cell line at 1 µM concentration. Molecules 19 and 57
showed G2/M arrest confirming their anti-tubulin mode of action. These compounds were found to
induce G2/M block in human epidermoid carcinoma A431 cell line. Namely, A431 cells were
treated with 1 µM of 19 and 57 for 24 hrs followed by flow cytometry analysis to display induction
of cell cycle arrest of ca. 55% and 60% (percent of cells in G2/M phase, average of 2 experiments
with SD < 15%). A subsequent dose-response studies for 19 and 57 yielded EC50 values (the
compound concentration that causes 50% cells to arrest, average of 3 experiments with SD < 15%)
of ca. 1 µM and 0.5 µM, respectively. Once again, these data correlated well with the sea urchin
embryo data and NCI60 GI50 values (0.767 µM and 0.24 µM, respectively, Table 1). In our hands,
BIFAs were consistently less cytotoxic than BAL 27862. The sea urchin embryo assay data
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provided insight into the structure-activity relationship for these new series and further confirmed
the micritubule-destabilizing mechanism of their activity.
3. Conclusions
In summary, 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFA) were prepared in good
yields using a novel robust procedure from aminofurazanyl hydroximoyl chlorides and o-
diaminobenzenes (Scheme 2). As opposed to the reported sequences, the developed protocol used
mild reaction conditions and accommodated a wide variety of functional groups to afford a diverse
array of targeted compounds. Furthermore, our approach to BIFAs requires neither protection of the
reactive moieties nor chromatographic purification of the respective intermediates. A subsequent
biological evaluation of the resulting library using the sea urchin embryo model and human cancer
cell lines revealed the antiproliferative effect of several derivatives. The activity of BIFAs in our
assay systems could be attributed to both direct microtubule destabilization and tubulin independent
mechanisms. The unsubstituted phenyl ring of benzoimidazole moiety as well as the unsubstituted
amino group in the furazan ring were essential prerequisites for the antimitotic activity of BIFAs.
The most active compound 57 was substituted with the 2-chlorophenyl acetamide moiety at the N
atom of the imidazole fragment. The potent synthetically feasible tubulin-targeting BIFA series will
be further evaluated as lead candidates for in vivo experiments.
4. Experimental protocols
4.1. Chemistry and chemical methods
Elemental microanalyses were obtained on an Perkin-Elmer 2400 CHN analyzer. Mass
spectra were collected on the Varian MAT-CH-6 spectrometer with direct sample injection at an
ionization voltage of 70 eV. IR spectra were recorded on IFS-113v Bruker in KBr pellets (1:200);
the frequencies were expressed in cm−1
. The 1
H NMR spectra were recorded on Bruker DRX-500
(500 MHz) and Bruker AM-300 (300 MHz) using internal standard with DMSO-D6 as the solvent;
the chemical shifts were reported in ppm (δ) and coupling constants (J) values were given in Hertz
(Hz).
The 13
C, and 15
N NMR spectra were recorded on Bruker AM-300 at 75.47 and 50.7 MHz,
respectively. Melting points were measured on a Kofler bench. Completion of the reactions and
purity of the obtained products were monitored by thin layer chromatography on the Silufol UV-
254 plates using hexane-acetone mixture (5:3) as an eluent and iodine vapor as a stain.
4.1.1. General procedure for synthesis of benzimidazoles 9a–n from hydroximoyl chloride 10
Hydroximoyl chloride 10 (0.16 g, 1 mmol) was added slowly by small portions to a solution
of the appropriate 1,2-diaminobenzene 3 (0.3 g, 1.5 mmol) in ethanol (5–10 mL) at room
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temperature. The mixture was stirred at reflux for 0.5 h followed by an additional hour at room
temperature, diluted with water (10 mL) and 0.1 M aq HCl (5 mL). The heterogeneous mixture was
stirred for 1 h. The precipitate was filtered, washed with water, and recrystallized from iPrOH/H2O.
4.1.1.1. 3-Amino-4-(1H-benzimidazol-2-yl)-furazan (9a). White solid; yield 0.16 g (80%); mp 268–
269 °C (lit. [17] 264–265 °C); 1
H NMR (DMSO-d6, 500 MHz): 6.84 (s, 2H, NH2), 7.31 (t, J = 7.3
Hz, 1H, H-5), 7.37 (t, J = 7.8 Hz, 1H, H-6), 7.59 (d, J = 7.8 Hz, 1H, H-7), 7.81 (d, J = 7.3 Hz, 1H,
H-4), 13.69 (s, 1H, NH); 13
C NMR (DMSO-d6): 112.6 (br), 120.2 (br), 123.8 (br), 135.0 (br), 139.1,
140.8, 143.2 (br), 156.1; EIMS m/z 201 [M]+
(20), 144 (100), 143 (29), 118 (42), 116 (11), 92 (18),
90 (20), 77 (6), 63 (35); Anal. Calcd for C9H7N5O: C 53.73; H 3.51; N 34.81. Found: C 53.61; H
3.47; N 34.93; IR (KBr): ν max 3406, 3303, 1635, 1621, 1604, 1561, 1495, 1459, 1423, 1322, 1278,
1125, 1012, 1000, 955, 900, 864, 748, 732, 697, 611.
4.1.1.2. 3-Amino-4-(4-methyl-1(3)H-benzimidazol-2-yl)-furazan (9c).
Ratio of isomers 3:2.
White solid; yield 0.16g (73%); mp 213–216 °С; 1
H NMR (DMSO-d6): δ 2.55, 2.59 (s/s = 3/2, 3H,
Me-4), 6.88, 6.90 (s/s = 3/2, 2H, NH2), 7.05 (t, J = 7.8 Hz, 2H, H-6,7) and 7.20 (m, 2H, H-6,7),
7.38, 7,58 (2d, J = 7.8 Hz, 1H, H-5), 13.58, 13.64 (s/s = 3/2, 1H, NH); 13
C NMR (DMSO-d6): 16.3,
17.1, 109.5, 117.0, 122.4, 122.6, 122.7, 124.4, 124.9, 129.3, 133.9, 134.0, 138.6, 139.4, 140.2,
142.3, 142.6, 155.6; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.86; H 4.18;
N 32.47; IR (KBr): ν max 3430, 3323, 3194, 1635, 1620, 1591, 1516, 1456, 1422, 1327, 1268,
1239, 1157, 1138, 1005, 949, 899, 874, 748, 671, 561.
4.1.1.3. 3-Amino-4-(4,5-dimethyl-1(3)H-benzimidazol-2-yl)-furazan (9d).
White solid; yield 0.211g (92%); mp 242–243 °C; 1
H NMR (DMSO-d6): δ 2.32, 2.46 (s/s = 3/1, 6H,
Me-4,5), 6.85, 6.88 (2s, 2H, NH2), 7.11 (m, 1H, H-6), 7.27, 7.47 (2d, J = 8.2 Hz, 1H, H-7), 13.43
(br. s, 2H, NH); 13
C NMR (DMSO-d6): 13.6, 14.3, 19.3, 19.6, 109.1, 116.9, 120.5, 125.7, 127.2,
127.5, 129.8, 132.4, 132.6, 135.1, 139.0, 139.8, 140.3, 141.5, 143.2, 156.0; Anal. Calcd for
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C11H11N5O: C 57.63; H 4.84; N 30.55. Found: C 57.69; H 4.81; N 30.48; IR (KBr): ν max 3425,
3284, 3202, 1634, 1619, 1596, 1504, 1458, 1425, 1373, 1323, 1006, 950, 903, 874, 793, 767, 741,
717, 661, 631, 561, 501.
4.1.2. General procedure for the synthesis of pyrroles 11a, 11h, and 11i
2,5-Dimethoxytetrahydrofuran (1.7 mmol) was added to a slurry of respective BIFAs (9a,
9h, 9i) (1.7 mmol) in AcOH (3 mL) at room temperature. The mixture was refluxed for 15 min,
cooled to room temperature, and the resulting suspension was stirred for 1 h. The precipitate was
filtered, washed with ice water (2×5 mL) followed by 70% iPrOH (2×5 mL) and dried.
Crystallization of the crude product from 70% iPrOH afforded pure pyrroles 11a, 11h, and 11i
(white solids, 70–90% yield).
4.1.3. General procedure for alkylation of 9a–n
4.1.3.1. Alkylation by benzylhalides 12′′′′–39′′′′ and 41′′′′–43′′′′
A mixture of 3-amino-4-(1H-benzimidazol-2-yl)-furazan 9a–n (0.01 mol), benzylhalide
12′′′′–43′′′′ (0.011 mol), and K2CO3 (1.52 g, 0.011 mol), in dry DMF (30 mL) was stirred at 80–90 °C
for 4–5 h (reflux condenser was used for volatile benzylhalides). The mixture was cooled to room
temperature and diluted with water (100 mL). The precipitate was filtered and recrystallized from
acetic acid. Yields of 12–39 and 41–43 were 70–90%.
4.1.3.2. Synthesis of [2-(4-amino-furazan-3-yl)-benzoimidazol-1-yl]-acetonitrile 40
BIFA 9a (2g, 0.01 mol) was alkylated by ClCH2CN (1.27 g, 0.02 mol) as described in the
following procedure 4.1.3.3 at ≤50 °C. The product was isolated and recrystallized from
EtOH:AcOH 3:1 v/v to yield 1.56 g (65%) of the targeted nitrile 40 as white solid.
4.1.3.3. Alkylation by N-aryl-acetamides 44′′′′–57′′′′
Alkylation by N-Aryl-acetamides 44′′′′–57′′′′ and separation of products was conducted as
alkylation by benzylhalides as described in 4.1.2.1., however at lower temperature (60 °C) and
increased reaction time (8–12 h). The precipitate was crystallized from acetic acid or mixture of
acetic acid–DMF (10–30% by volume) to give 44–57 (white solid, 70–90% yield).
4.1.3.4. Synthesis of isomeric 2-[2-(4-aminofurazan-3-yl)-5(6)-methoxy-1H-benzimidazol-1-yl]-N-
(2-chlorophenyl)acetamides 57h and 57h′′′′
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A mixture of 3-amino-4-(6-methoxy-1H-benzimidazol-2-yl)-furazan 9h (0.23 g, 0.001 mol),
2-chloro-N-(2-chlorophenyl)acetamide 57′′′′ (0.222 g, 0.0011 mol), K2CO3 (0.152 g, 0.0011 mol),
KBr (0.03 g), and glym (5 mL) was stirred at reflux for 9 h. The mixture was cooled to room
temperature and diluted with water (50 mL). The precipitate was filtered and washed with water (30
mL) to give a mixture of the respective isomers 57h and 57h′′′′. White solids, 91% yield (0.364 g);
mp 252–253 °C. Pure isomers were isolated by fractional crystallization from MeCN. Specifically,
isomer 57h′′′′ exhibited lower solubility in MeCN and was obtained in 38% yield (0.14 g). More
soluble isomer 57h was isolated in 46% yield (0.17 g).
4.1.4. Acylation of 4-(1-R-1H-Benzoimidazol-2-yl)-furazan-3-yl-amines
4.1.4.1. Acylation by anhydrates of aliphatic carbonic acids 58r,s
Corresponding BIFA (0.01 mol) was added to the neat acetic or propionic anhydrate (20
mL) and refluxed for 3 h in presence of 5 mmol of dry MeCO2Na or EtCO2Na, respectively. The
mixture was cooled to room temperature and diluted with water (100 mL). After a day at room
temperature, the precipitate was filtered and recrystallized from DMF–EtOH to yield 80% of the
desired products as white solids.
4.1.4.2. Acylation by chloroanhydrates of aromatic carbonic acids (aroylchloroanhydrates) 58t,u
i) A mixture of corresponding BIFA (26, 39 or 51) (0.01 mol) and ArCOCl 58t,u (0.015
mol) in toluene (50 mL) was refluxed for 8–20 h until the evolution of HCl stopped. Toluene was
evaporated in vacuo, and the residue was recrystallized from DMF–EtOH to yield 80% of the
desired products as white solids.
ii) A mixture of corresponding BIFA (26, 39 or 51) (0.01 mol) and ArCOCl 58t,u (0.015
mol) in freshly distilled pyridine (30 mL) was refluxed for 3 h, cooled, and diluted with water (100
mL). After 24 h the residue was filtered and recrystallized from DMF–EtOH to afford targeted
products as white solids (70% yield).
Crude alkylating products 14, 28, 32, 34, and 51 were filtered and without purification
acylated to afford 14r, 28r, 32r, 34s, and 51t.
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4.1.5. 4-Aminofurazan-3-carbox-N-(p-methoxyphenyl)amidoxime (60v). A solution of 4-
aminofurazan-3-carbohydroxymoyl chloride 10 (8.1 g, 50 mmol) in EtOH (100 mL) was added
drop wise to a solution of p-anizidine 59v (11.9 g, 97 mmol) and NEt3 (6 g, 60 mmol) in iPrOH (50
mL) at 0 °C over 10 min. The mixture was stirred at room temperature for 3 h, the solvent was
removed in vacuo and the residue was treated with 80 mL of water. The precipitate was filtered and
washed with H2O (2×50 mL) followed by benzene (30 mL). The solid residue (14 g, 86 mmol) was
further recrystallized to give 60v. Gray solid, yield 13.3 g, (62%); mp 197–198 °C (from
benzene/iPrOH); 1
H NMR (DMSO-d6) δ 3.69 (s, 3H, MeO), 6.18 (s, 2H, NH2), 6.80 (s, 4H, Ar), 8.4
(s, 1H, NH), 11.02 (s, 1H, NOH); 13
C NMR (DMSO-d6) δ 55.1, 113.6, 123.7, 133.2, 140.3, 140.7,
155.3, 155.4; Anal. Calcd for C10H11N5O3 (%): C, 48.19; H, 4.45; N, 28.10. Found (%): C, 48.23;
H, 4.51; N, 28.05. IR (KBr, cm-1
) 3472, 3372, 3272, 2964, 2840, 1652, 1612, 1568, 1536, 1516,
1440, 1400, 1304, 1252, 1156, 1108, 1044, 952.
4.1.6. 4-Aminofurazan-3-carbox-(N-benzylamid)oxime (60w). Gray solid; 7.93 g, 68% yield; mp
124–125 °C (from benzene/iPrOH); 1
H NMR (DMSO-d6) δ 4.63 (d, J = 7.3 Hz, 2H, CH2), 6.27 (s,
2H, NH2), 6.89 (t, J = 7.3 Hz, 1H, NH), 7.21 (m, 5H, Ph), 10.80 (s, 1H, NOH); 13
C NMR (DMSO-
d6) δ 46.3 (C4), 126.6 and 128.3 (C6, C7, C8), 139.7 (C5), 140.9 (C2), 144.8 (C3), 155.2 (C1);
Anal. Calcd for C10H11N5O2 (%): C, 51.50; H, 4.75; N, 30.03. Found (%): C, 51.55; H, 4.68; N,
28.75.
4.1.7. 4-Aminofurazan-3-carbox-N-(p-methoxybenzylamid)oxime (60x). Light-brown solid; 7.77 g,
59% yield; mp 141–142 °C (from benzene) (lit. [44] 140–142 °C); 1
H NMR (DMSO-d6) δ 3.69 (s,
3H, MeO), 4.54 (d, J = 7.1 Hz, 2H, CH2), 6.26 (s, 2H, NH2), 6.83 (m, 3H, NH, H-3',5'), 7.12 (d, J =
8.4 Hz, 2H, H-2',6'), 10.79 (s, 1H, NOH); 13
C NMR (DMSO-d6) δ 45.8, 55.0, 113.7, 128.1, 132.8,
139.7, 144.8, 155.3, 158.2; EIMS m/z 263 (M+), 233 [M+
–NO]; Anal. Calcd for C11H13N5O3 (%):
C, 50.19; H, 4.98; N, 26.60. Found (%): C, 50.27; H, 5.01; N, 26.54.
4.1.8. 4-(p-Methoxyphenylamino)furazan-3-carboxamidoxime (61v). A solution of 60v (13.3 g, 53
mmol) and KOH (2.92 g, 53 mmol) in ethylene glycol (50 mL) was refluxed for 4 h. The reaction
mixture was cooled, diluted with water (30 mL), and neutralized with 36% aqueous HCl. The
residue was filtered, washed with H2O (100 mL), benzene (15 mL), and recrystallized from
benzene–iPrOH to afford 61v. Light-brown solid; yield 10.84 g (82%); mp 191 °C (from
benzene/iPrOH) (lit. [44] 190–191 °C); 1
H NMR (DMSO-d6) δ 3.74 (s, 3H, MeO), 6.39 (s, 2H,
NH2), 6.97 (d, 2H, J = 8.6 Hz, 2H, ArH-3',5'), 7.38 (d, 2H, J = 8.6 Hz, 2H, ArH-2',6')), 8.79 (s, 1H,
NH), 10.71 (s, 1H, NOH); 13
C NMR (DMSO-d6) δ 55.2, 114.5, 118.4, 132.5, 139.7, 143.9, 151.0,
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154.5; EIMS m/z 249 (M+), 219 [M+
–NO]; Anal. Calcd for C10H11N5O3 (%): C, 48.19; H, 4.45; N,
28.10. Found (%): C, 48.22; H, 4.49; N, 28.03.
4.1.9. 4-(Benzylamino)furazan-3-carboxamidoxime (61w). White solid; yield 9.76 g (79%); mp 104
°C; 1
H NMR (DMSO-d6) δ 4.44 (d, J = 7.0 Hz, 2H, CH2), 6.25 (s, 2H, NH2)), 6.51 (t, J = 7.0 Hz, 1H,
NH), 7.31 (m, 5H, Ph), 10.50 (s, 1H, NOH)); 13
C NMR (DMSO-d6) δ 47.6 (C4), 127.3, 127.6,
128.5 (C6, C7, C8), 138.4 (C2), 139.7 (C5), 144.0 (C1), 154.9 (C3).
4.1.10. 4-(p-Methoxybenzylamino)furazan-3-carboxamidoxime (61x). A solution of 60x (5.2 g, 20
mmol) and KOH (1.1 g, 20 mmol) in ethylene glycol (15 mL) was refluxed for 4 h. The reaction
mixture was cooled, diluted with water (30 mL) and neutralized with 36% aqueous HCl. The
residue was filtered, washed with H2O (100 mL), benzene (15 mL), and recrystallized from
benzene–iPrOH to furnish 61x. Gray solid; yield 4 g (78%); mp 113–117 °C; 1
H NMR (DMSO-d6)
δ 3.72 (s, 3H, MeO), 4.36 (d, J = 6.0 Hz, 2H, CH2), 6.22 (s, 2H, NH2), 6.40 (br. s, 1H, NH), 6.89 (d,
J = 8.2 Hz, 2H ArH-3',5'), 7.33 (d, J = 8.2 Hz, 2H, ArH-2',6'), 10.45 (s, 1H, NOH); 13
C NMR
(DMSO-d6) δ 47.1, 55.1, 113.9, 129.2, 130.3, 139.7, 144.1, 154.9, 158.7; EIMS m/z 263 (M+);
Anal. calcd for C11H13N5O3 (%): C, 50.19; H, 4.98; N, 26.60. Found (%): C, 50.25; H, 5.00; N,
26.52
4.1.11. General procedure for synthesis of 4-(R-amino)furazan-3-carbohydroxymoyl chlorides
62v,w
A solution of NaNO2 (0.14 g, 2 mmol) in H2O (2 mL) was added drop wise to a stirred
solution of amidoxime 61v or 61w (2 mmol) in a mixture of conc. HCl (3.6 mL), AcOH (6 mL),
and H2O (3.2 mL) at < 10 °C. The reaction was allowed to stir for 3 h at 10 °C and for 1 h at room
temperature. The solid residue was filtered and washed with H2O (4×5 mL) to give 62v,w (60%–
87% yield) as white crystals.
4.1.11.1. N-hydroxy-4-((4-methoxyphenyl)amino)-1,2,5-oxadiazole-3-carbimidoyl chloride (62v).
White solid; yield 0.4 g (60%); mp 202–203 °C; 1
H NMR (DMSO-d6) δ 3.72 (s, 3H, OMe), 6.94 (d,
2H, J = 8.6, ArH-3'',5'' ), 7.38 (d, 2H, J = 8.6, Ar2H, H-2'',6'' ), 8.21 (s, 1H, NOH), 13.62 (s, 1H,
NH); 13
C NMR (DMSO-d6) δ 55.1, 114.3, 119.0, 126.1, 132.2, 141.8, 150.6, 154.7; Anal. Calcd for
C10H9N4O3: C 44.71; H 3.38; N 20.85. Found (%): C 44.75; H 3.34; N 20.24; IR (KBr): ν max
3363, 3253, 1617, 1602, 1566, 1512, 1466, 1444, 1270, 1231, 1183, 1025, 945, 876, 824, 792, 754,
701, 564, 516.
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4.1.11.2. 4-(Benzylamino)-N-hydroxy-1,2,5-oxadiazole-3-carbimidoyl chloride (62w). White solid;
yield 0.44 g (87%); mp 164–165 °C; 1
H NMR (DMSO-d6) δ 4.45 (d, J=5.8 Hz, 2H, CH2), 6.35 (s,
1H, NOH), 7.34 (m, 5H, Ph), 13.41 (s, 1H, NH); 13
C NMR (DMSO-d6) δ 47.8, 126.8, 127.4, 127.7,
128.6, 138.2, 141.6, 154.4; Anal. Calcd for C10H9ClN4O2: C 47.54; H 3.59; N 22.18. Found (%): C
47.58; H 3.52; N 22.01; IR (KBr): ν max 3392, 3263, 1619, 1601, 1562, 1440, 1354, 1233, 1031,
967, 946, 909, 856, 754, 696, 610, 557, 508.
4.1.12. 3-(1-Ethyl-1H-benzimidazol-2-yl)-4-(4-methoxyphenyl)amino-furazan (63). Hydroximoyl
chloride 62v (0.27 g, 1 mmol) was added in several portions to a vigorously stirred solution of 1,2-
diaminobenzene 3q (0.2 g, 1.5 mmol) in ethanol (5–10 mL) at room temperature. The mixture was
stirred at reflux for 0.5 h followed by stirring for 1 h at room temperature, dilution with water (10
mL) and 0.1 M aqueous HCl (5 mL). The heterogeneous mixture was stirred for 1 h, the resulting
precipitate was filtered, washed with water, and recrystallized from iPrOH–H2O to afford 63. White
solid; yield 0.22 g (65%); mp 135–136 °C; 1
H NMR (DMSO-d6): δ 1.43 (t, J = 7.1 Hz, 3H, CH3),
3.77 (s, 3H, OMe-4''), 4.76 (q, J = 7.1 Hz, 2H, CH2), 7.03 (d, J = 9.0 Hz, 2H, ArH-3'',5''), 7.43 (t, J
= 8.0 Hz, 1H, H-5), 7.49 (t, J = 8.2 Hz, 1H, H-6), 7.60 (d, J = 9.0 Hz, 2H, ArH-2'',6''), 7.86 (d, J =
8.2 Hz, 1H, H-7), 7.97 (d, J = 8.0 Hz, 1H, H-4), 9.97 (s, 1H, NH) 13
C NMR (DMSO-d6): δ 15.0,
40.6, 55.6, 110.1, 114.5 (C2), 119.0 (C2), 120.6, 123.5, 124.9, 133.2, 134.9, 137.9, 140.4, 142.1,
152.9, 155.0; 15
N NMR (DMSO-d6): δ 21.3, -20.9, -137.0, -221.7, -297.0; EIMS m/z 335 [M]+
, 295
[M+
- NO]; Anal. Calcd for C18H17N5O2: C 64.47; H 5.11; N 20.88. Found (%): C 64.54; H 5.14; N
20.77; IR (KBr): ν max 3249, 2977, 1623, 1577, 1513, 1439, 1334, 1245, 1180, 1115, 1977, 1035,
1008, 908, 872, 825, 753, 742, 717, 608, 557, 524.
4.1.13. 3-Benzylamino-4-(5-chloro-1H-benzimidazol-2-yl)-furazan (64). White solid; yield 0.19 g
(59%); mp 196 °C; 1
H NMR (DMSO-d6): δ 4.56 (d, 2H, J = 5.89 Hz, CH2), 7.34 (m, 6H, Ph, H-6),
7.68 (s, 2H, CH) 13.85 (br, s, 2H, NH); 13
C NMR (DMSO-d6): δ 47.5, 116.0 (br, s), 123.9, 127.1,
127.4, 127.5, 128.3, 128.4, 128.5, 128.4, 137.9, 138.5, 141.3, 155.7; EIMS m/z 325 [M]+
, 295 [M+
-
NO], 220, 178, 106, 91; Anal. Calcd for C16H12ClN5O: C 58.99; H 3.71; N 21.50. Found (%): C
59.02; H 3.69; N 21.42; IR (KBr): ν max 3358, 3259, 1630, 1594, 1519, 1496, 1432, 1371, 1332,
1299, 1262, 1236, 1141, 1063, 1029, 986, 955, 927, 855, 815, 741, 705, 640.
4.1.14. Synthesis of 3-amino-4-[1-((1H-tetrazol-5-yl)methyl)-1H-benzimidazol-2-yl]-furazan 65
A solution of nitrile 40 (2.4 g, 0.01 mol), NaN3 (1 g, 0.015 mol), and NH4Cl (0.8g, 0.015
mol) in dry DMF (30 mL) was stirred at 100 °C for 8 h, cooled, and diluted with water (100 mL).
The reaction mixture was stirred with activated charcoal (100 mg) for 15 min, filtered, and the pH
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was adjusted to ca. 1 with 36% aqueous HCl. The resulting precipitate of 65 was recrystallized from
EtOH to furnish the pure product (white solid, 1.78 g, 63% yield).
4.1.15. Synthesis of 3-Amino-4-[1-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-1H-benzimidazol-2-yl]-
furazan 66
A solution of nitrile 40 (2.4 g, 0.01 mol) and thiosemicarbazide (1.4 g, 0.015 mol) in neat
CF3COOH (30 mL) was refluxed for 8 h at stirring. The reaction mixture was cooled, diluted with
water (100 mL), and stirred for an additional 15 min with activated charcoal (100 mg). The mixture
was filtered and neutralized with aqueous NH4OH (25%) to afford the residue of crude 66. It was
further recrystallized from EtOH to afford the targeted pure thiadiazol (white solid, 2.23 g, 71%
yield).
Synthetic and analytical data for the other compounds are presented in Supplementary data.
4.2. Biology. Materials and methods
4.2.1. Phenotypic sea urchin embryo assay [38]
Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the
Mediterranean Sea on 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 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 Biolam light microscope (LOMO, St. Petersburg, Russia). For treatment with the
test compounds, 5 mL aliquots of embryo suspension were transferred to six-well plates and
incubated as a monolayer at a concentration up to 2000 embryos/mL. Stock solutions of compounds
were prepared in DMSO at 10 mM concentration followed by a 10-fold dilution with 96% EtOH.
This procedure enhanced the solubility of the test compounds in the salt-containing medium
(seawater), as evidenced by microscopic examination 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 (≥0.1%) or EtOH (>1%) caused nonspecific
alteration and retardation of the sea urchin embryo development independent of the treatment stage.
The compound solubility in the seawater was estimated by microscopic examination of sample
wells. Colchicine and vinblastine sulfate (Sigma-Aldrich) were applied as reference compounds,
using 20 mM and 5 mM stock solutions in distilled water, respectively.
The antiproliferative activity was assessed by exposing fertilized eggs (8–20 min after
fertilization, 45–55 min before the first mitotic cycle completion) to 2-fold decreasing
concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5–5.5 h
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after fertilization. The effects were estimated quantitatively as an effective threshold concentration,
resulting in cleavage alteration and embryo death before hatching or full mitotic arrest. At these
concentrations all tested microtubule destabilizers caused 100% cleavage alteration and embryo
death before hatching, whereas at 2-fold lower concentrations the compounds failed to produce any
effect. For microtubule-destabilizing activity, the compounds were tested on free-swimming
blastulae just after hatching (8–10 h after fertilization), which originated from the same embryo
culture. Embryo spinning was observed after 15 min to 20 h of treatment, depending on the
structure and concentration of the compound. Both spinning and lack of forward movement were
interpreted to be the result of the microtubule-destabilizing activity of a molecule. Video
illustrations are available at http://www.chemblock.com. Both sea urchin embryo assay and DTP
NCI60 cell line activity data are available free of charge via the Internet at http://www.zelinsky.ru.
4.2.2. In vitro tubulin polymerization assay [42]
In vitro tubulin polymerization was determined using modified turbidity assay, developed
by Cytoskeleton Inc. (Cytodynamix 12), for maximized throughput and maintained sensitivity.
Lyophilized bovine tubulin (HTS02, Cytoskeleton Inc.) was re-suspended in G-PEM buffer (80
mM PIPES pH 7, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, 5% glycerol) to a final concentration of
3 mg/mL and kept at 4 °C. Compounds in 100× stock solutions in DMSO were dotted into pre-
warmed 96-well plates (Corning Costar 3696), with the plates immediately transferred to a 37 °C
plate reader (SPECTRAmax Plus, Molecular Devices). Cold tubulin was added to the wells, plates
were mixed by shaking, and absorbance at 340 nm was read every minute for 30 min. Kinetic
curves with 30 points each were collected for tested compound, with a dynamic range between 0
and 0.4 OD units. Percentage inhibition values were calculated using the 30 minute data point,
based on control samples (1% DMSO). IC50 values were determined by sigmoidal curve fitting
using Excel-based software.
4.2.3. Cell cycle analysis [42]
Cell cycle analysis was assessed by flow cytometry. 3T3 mouse fibroblasts were cultured in
DMEM supplemented with 10% fetal bovine serum, 1 mg/mL L-glutamate, 100 units/mL penicillin
G, and 0.2 mg/mL streptomycin sulfate. Cells were plated onto 6-well plates at a final density of
500,000 cells/well at the time of treatment, treated with compounds at a final concentration of 1 µM
(0.1% final concentration of DMSO) for 24 h, then trypsinized, collected, rinsed in phosphate
buffer saline (PBS), and fixed in 70% cold ethanol overnight at 4 °C. Cells were rinsed in PBS, re-
suspended in PBS with 0.2% Tween, incubated with RNAse (final concentration of 1 µg/mL) at 37
°C for 15 min, followed by addition of propidium iodide (final concentration of 50 µg/mL) and 30
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min incubation at room temperature. Cell cycle distribution was determined by flow cytometry
using cell sorter Guava PCA-96. A compound was reported as a mitotic arrest inducer when the
amount of cells in G2/M phase exposed to 1 µM concentration of an agent was twice or more than
in control (DMSO).
Acknowledgments
The authors acknowledge the compounds screening at the National Cancer Institute (NCI)
(Bethesda, MD, USA) by the Developmental Therapeutics Program NCI/NIH
(http://dtp.cancer.gov). KYS is thankful to the Russian Scientific Foundation for financial support
(project no. 14-13-00884).
Supplementary data
Supplementary data associated with this article can be found in the online version, at
........................ These data include MOL files, experimental details regarding syntheses, analytical
data, and X-ray analysis.
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References
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Supplementary data
A facile synthesis and antiproliferative properties of 4-(1H-benzo[d]imidazol-2-yl)-
furazan-3-amines
Andrei I. Stepanov,a
Alexander A. Astrat’ev,a
Aleksei B. Sheremetev,b
Nataliya K. Lagutina,c
Nadezhda
V. Palysaeva,b
Aleksei Yu. Tyurin,b
Nataly S. Aleksandrova,b
Nataliya P. Sadchikova,c
Kyrill Yu.
Suponitsky,d
Olga P. Atamanenko,b
Leonid D. Konyushkin,b
Roman V. Semenov,b
Sergei I. Firgang,b
Alex S. Kiselyov,e
Marina N. Semenova,f
Victor V. Semenovb,*
a
Special Design and Construction Bureau SDCB “Technolog”, 33-A Sovetskii Ave., Saint Petersburg,
192076, Russian Federation
b
N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow, Russian
Federation
c
I. M. Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, 119991 Moscow,
Russian Federation
d
A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov
Str., 119991 Moscow, Russian Federation
e
Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology,
Institutsky Per. 9, Dolgoprudny, Moscow Region, 141700, Russian Federation
f
N. K. Kol’tsov Institute of Developmental Biology, RAS, Vavilov Str., 26, 119334 Moscow, Russian
Federation
Corresponding author: Victor V. Semenov
Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991,
Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966.
E-mail: vs@zelinsky.ru
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Table of contents
1) Single crystal X-ray crystallography of benzimidazoles 27 and 29
and Fig. S1. Molecular structure of compounds 27 and 29 showing the atom
numbering scheme. Page S3
2) Table S1. Crystallographic data for 27 and 29. Page S5
3) Figure S2. Fragment of the crystal packing of 27 and 29. Page S6
4) Figure S3. NCI60 5 dose screen. GI50 Mean Graph for 18. Page S7
5) Figure S4. NCI60 5 dose screen. GI50 Mean Graph for 19. Page S8
6) Figure S5. NCI60 5 dose screen. GI50 Mean Graph for 20. Page S9
7) Figure S6. NCI60 5 dose screen. GI50 Mean Graph for 21. Page S10
8) Figure S7. NCI60 5 dose screen. GI50 Mean Graph for 57. Page S11
9) The Mean Graphs interpretation. Page S12
9) Chemistry. General experimental procedures and synthetic and analytical
data for 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines. Pages S13–S45
10) References. Page S46
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Single crystal X-ray crystallography of benzimidazoles 27 and 29.
X-ray experiments were carried out using SMART APEX2 CCD (λ(Mo-Kα)=0.71073 Å,
graphite monochromator, ω-scans) at 120K. Collected data were analyzed by the SAINT and SADABS
software incorporated into APEX2 package (APEX2 and SAINT; Bruker AXS Inc., Madison,
Wisconsin, USA, 2009). All structures were solved by the direct methods and refined by the full-matrix
least-squares procedure against F2
in anisotropic approximation. The hydrogen atoms of the NH2 groups
were found in the difference Fourier synthesis. The H(C) positions were calculated. All the hydrogen
atoms were included in the refinement within isotropic approximation by the riding model with the
Uiso(H) = 1.5Ueq(Ci) for methyl groups and 1.2Ueq(Ci) for other carbon atoms, where Ueq(C) are
equivalent thermal parameters of the parent atoms. The refinement was carried out with the SHELXTL
software [i]. The details of data collection and crystal structures refinement are summarized in Table S1.
Fig. S1. Molecular structure of compounds 27 and 29 showing the atom numbering scheme.
In the molecular structure of 27 (Fig. S1), the aminofurazan moiety is nearly coplanar to the
benzimidazole ring (torsion angle C1–C2–C3–N4 is -1.0(7)°) suggesting conjugation between these
functionalities and stabilization by the intramolecular hydrogen bond N3–H3A…N4 (H…N 2.35Å,
N…N 2.883(5)Å, <NHN 118°). The N–O bonds in the furazan ring showed different length (N1–O1 of
1.402(5)Å, N2–O1 of 1.376(5)Å) affected by substituents [ii].
In the structure of 29 featuring anisole and CF3 substituents, the furazan and benzimidazole rings
are also coplanar (torsion angle C1–C2–C3–N4 is -1.0(4)°). For this groups, the conjugation is more
pronounced presumably due to the influence of the electron-withdrawing CF3 group as evidenced by the
greater difference in the N–O bonds of the furazan ring (N1–O1 of 1.413(3)Å, N2–O1 of 1.368(3)Å).
Further stabilization of the planar structure is likely attained by the intramolecular hydrogen bond N3–
H3A…N4 (H…N 2.23Å, N…N 2.859(4)Å, <NHN 127°), which is stronger than that in 27. It was
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further determined that the torsion angle C9–N5–C11–C12 of 92.2(3)° is defined by the intramolecular
steric effects, while the rotation of the anisole moiety about the C11–C12 bond is most probably
induced by crystal packing.
In addition to intramolecular H-bond, in the crystal structure of 27 the second hydrogen of the
amino group formed H-bond with the nitrogen atom of the furazan ring [N3–H3B…N1(1-x, -0.5+y, 1-z)
(H…N 2.14Å, N…N 3.025(5)Å, <NHN 169°)]. However, instead of formation of H-bonded dimers,
this resulted in a formation of H-bonded chains along the axis b. In these chains molecules were related
by the two-fold axis (Fig. S2).
In the crystal structure of 29 the second hydrogen atom of the amino group was bonded to the
oxygen atom of the methoxy group [N3-H3B…O2(x, 1+y, z) (H…O 2.23Å, N…O 3.056(3)Å, <NHO
152°)] causing a formation of chains along axis b (Fig. S2). Obviously this H-bond was weaker than the
N–H…N bond observed in 27 suggesting better nucleophilic properties of the furazan ring N3 atom as
compared to the O-atom of the methoxygroup [ii]. However the N–H…N bond was not observed in 29
that could be a consequence of the cumulative effect of the numerous weak intermolecular interactions
contributing to a stabilization of the 3-D crystal structure. Probably, for the same reason the H-bonded
chains, other than the H-bonded dimers, were formed in the crystal structure of 27.
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Table S1.
Crystallographic data for 27 and 29.
Parameter 27 29
Empirical formula
Fw
Crystal system
Space group
a, Å
b, Å
c, Å
α, deg
β, deg
γ, deg
V, Å3
Z
dcalc, g·cm-3
µ, mm-1
F(000)
θ range, deg.
Reflections collected
Independent reflections
Rint
Refined parameters
Completeness to theta θ, %
GOF (F2
)
Reflections with I>2σ(I)
R1(F) (I>2σ(I))a
wR2(F2
) (all data)b
Largest diff. peak/hole, e⋅Å-3
C11H11N5O
229.25
Monoclinic
P21
11.180(2)
4.1948(8)
11.823(2)
90.00
107.621(3)
90.00
528.44(17)
2
1.441
0.100
240
1.81 – 29.03
6268
1591
0.0403
155
99.6
1.021
1304
0.0667
0.1792
0.337 / -0.383
C18H18F3N5O2
393.37
Triclinic
P-1
5.0273(6)
11.0378(13)
16.3137(19)
107.435(3)
91.072(3)
97.262(3)
855.20(17)
2
1.528
0.125
408
1.95 – 27.00
9133
3715
0.0531
263
99.2
1.020
2229
0.0609
0.1648
0.735 / -0.443
a
R1 = ∑|Fo – |Fc||/∑(Fo).
b
wR2 = (∑[w(Fo
2
– Fc
2
)2
]/∑[w(Fo
2
)2
]1/2
.
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The Mean Graphs interpretation.
From: Methodology of the in vitro cancer screen (http://dtp.nci.nih.gov/branches/btb/ivclsp.html
Mean graphs facilitate visual scanning of data for potential patterns of selectivity for particular
cell lines or for particular subpanels with respect to a selected response parameter. Bars extending to the
right represent sensitivity of cell line to the test agent in excess of the average sensitivity of all tested
cell lines. Since the bar scale is logarithmic a bar 2 units to the right implies the compound achieved the
response parameter (e.g. GI50) for the cell line at a concentration one-hundredth the mean concentration
required over all cell lines, and thus the cell line is usually sensitive to that compound. Bars extending
to the left correspondingly imply sensitivity less than the mean.
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Alternative representative scale-up procedure for 3-amino-4-(1H-benzimidazol-2-yl)-furazan
9a. Step 1. Synthesis of 1H-benzimidazol-2-yl(hydroxyimino)acetonitrile 7.
7
N
H
N CN
NOH
6
N
H
N CN
A solution of NaNO2 (69 g, 1 mol) in H2O (100 mL) was added dropwise to a vigorously stirred
solution of 2-cyanomethylbenzimidazol 6 [iii] (152g, 1 mol) in AcOH (200 mL) at 5–10 °C. The
reaction mixture was allowed to stir for additional 0.5 h at 10–15 °C and diluted with water (200 mL).
The residue was filtered, washed with cold water, and dried to give crude acetonitrile 7 (150–160 g, 80–
86% yield), which was further used without purification.
Step 2. Synthesis of 2-(1H-benzimidazol-2-yl)-N'-hydroxy-2-(hydroxyimino)-ethanimidamide
and 2-(1H-benzoimidazol-2-yl)-N-hydroxy-2-hydroxyimino-acetamidine (mixture of isomers of 8).
87
N
H
N CN
NOH N
H
N
NOH
NOH
NH2
N
H
N
NOH
NH
NH
OH
+
A solution of NH2OH•HCl (77 g, 1.2 mol) in water (150 mL) was added to a solution of
1H-benzimidazol-2-yl(hydroxyimino)acetonitrile 7 (150 g, 0.8 mol) in EtOH or iPrOH (500 mL)
followed by pouring of K2CO3 (60 g, 0.6 mol) portionwise of 5–10 g. The reaction was stirred for 1 h at
room temperature, 1 h at 30 °C, 1 h at 40 °C, and 2 h at 50 °C, and cooled to room temperature. Most of
EtOH was evaporated in vacuo from the reaction mixture; the residue was diluted with hot water (70–80
°C) and cooled to room temperature. The precipitate was filtered, washed with cold water, and dried in
air to give crude glyoxime 8 (120–125 g, 68–71% yield), which was further used without purification.
Step 3. Synthesis of N,N'-bis(acetyloxy)-2-[(acetyloxy)imino]-2-(1H-benzimidazol-2-yl)-
ethanimidamide and 2-(1H-Benzoimidazol-2-yl)-N-acetoxy-2-acetoxyimino-N'-acetylacetamidine
(mixture of isomers of 8-triacetate).
8
isomers
N
H
N
NOH
NOH
NH2
N
H
N
NOAc
NOAc
AcNH
8-triacetate
isomers
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Acetic anhydrate (181 mL, 196 g, 1.92 mol) was added dropwise to a solution of glyoxime 8 (120 g,
0.55 mol) and dry AcONa (10 g 0.12 mol) in acetic acid at 25–30 °C. The reaction mixture was heated
to 90–95 °C during 1 h and stirred for 1 h. Acetic acid (ca. 250 mL) was evaporated in vacuo, the
residue was diluted with H2O (300 mL) and cooled to room temperature. The precipitate was filtered,
washed with cold water, and dried in air to afford crude 8-triacetate (155–165 g, 82–87% yield), which
was further used without purification.
Step 4. Synthesis of 3-amino-4-(1H-benzimidazol-2-yl)-furazan 9a.
N
H
N
NOAc
NOAc
AcNH
8-triacetate 9{1,1,1}
N
H
N
N
O
N
NH2
8-triacetate (150 g, 0.43 mol) was added to a solution of NaOH (86 g, 2.15 mol) in hot H2O (250 mL,
50 °С). The reaction mixture was refluxed for 1 h and cooled to room temperature. The insoluble
impurities were removed by filtration, and the pH was adjusted to ca. 6 with acetic acid. The resulting
precipitate was filtered, washed with water, and recrystallized from acetic acid to furnish the pure
product 9a. White solid; 58–60 g; 67–70% yield; mp 269 °С (lit. [iv] 264–265 °С).
3-Amino-4-(5-methyl-1H-benzimidazol-2-yl)-furazan (9b).
N
H
N
N O
N
NH2
Me
White solid; yield 183 mg (85%); mp 268–269 °С; 1
H NMR (DMSO-d6): δ 2.41 (s, 3H, Me-5), 6.84 (s,
2H, NH2), 7.12 (s, 1H, (H-4), 7.50 (m, 2H, ArH-6,7, 13.53 (br. s, 1H, NH); 13
C NMR (DMSO-d6): δ
21.3, 111.6, 119.1, 124.4, 126.0, 134.1, 138.6, 139.8, 140.9, 155.5; Anal. Calcd for C10H9N5O: C 55.81;
H 4.22; N 32.54. Found: C 55.86; H 4.18; N 32.47; IR (KBr): ν max 3430, 3332, 3171, 1635, 1597,
1457, 1422, 1317, 1274, 1237, 1134, 1005, 952, 897, 877, 798, 761, 732, 668, 597, 562.
3-Amino-4-(4-methyl-1(3)H-benzimidazol-2-yl)-furazan (9c).
N
N
H
N
O
N
H2NMe
N
N
H
N
O
N
H2N
Me Ratio of isomers 3:2
White solid; yield 157 mg (73%); mp 213–216 °С; 1
H NMR (DMSO-d6): δ 2.55, 2.59 (s/s = 3/2, 3H,
Me-4), 6.88, 6.90 (s/s = 3/2, 2H, NH2), 7.05 (t, J = 7.8 Hz, 2H, H-6,7) and 7.20 (m, 2H, H-6,7), 7.38,
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7,58 (2d, J = 7.8 Hz, 1H, H-5), 13.58, 13.64 (s/s = 3/2, 1H, NH); 13
C NMR (DMSO-d6): δ 16.3, 17.1,
109.5, 117.0, 122.4, 122.6, 122.7, 124.4, 124.9, 129.3, 133.9, 134.0, 138.6, 139.4, 140.2, 142.3, 142.6,
155.6; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.86; H 4.18; N 32.47; IR
(KBr): ν max 3430, 3323, 3194, 1635, 1620, 1591, 1516, 1456, 1422, 1327, 1268, 1239, 1157, 1138,
1005, 949, 899, 874, 748, 671, 561.
3-Amino-4-(4,5-dimethyl-1(3)H-benzimidazol-2-yl)-furazan (9d).
N
N
H
N
O
N
H2N
Me
Me
N
N
H
N
O
N
H2N
Me
Me
White solid; yield 211 mg (92%); mp 242–243 °C; 1
H NMR (DMSO-d6): δ 2.32, 2.46 (s/s = 3/1, 6H,
Me-4,5), 6.85, 6.88 (2s, 2H, NH2), 7.11 (m, 1H, H-6), 7.27, 7.47 (2d, J = 8.2 Hz, 1H, H-7), 13.43 (br. s,
2H, NH); 13
C NMR (DMSO-d6): δ 13.6, 14.3, 19.3, 19.6, 109.1, 116.9, 120.5, 125.7, 127.2, 127.5,
129.8, 132.4, 132.6, 135.1, 139.0, 139.8, 140.3, 141.5, 143.2, 156.0; Anal. Calcd for C11H11N5O: C
57.63; H 4.84; N 30.55. Found: C 57.69; H 4.81; N 30.48; IR (KBr): ν max 3425, 3284, 3202, 1634,
1619, 1596, 1504, 1458, 1425, 1373, 1323, 1006, 950, 903, 874, 793, 767, 741, 717, 661, 631, 561, 501.
3-Amino-4-(5-fluoro-1H-benzimidazol-2-yl)-furazan (9e).
N
H
N
N O
N
NH2
F
White solid; yield 118 mg (54%); mp 271–272 °C; 1
H NMR (DMSO-d6): δ 6.79 (s, 2H, NH2), 7.16 (t, J
= 8.2 Hz, 1H, H-6), 7.42 (d, J = 8.2 Hz, 1H, H-7), 7.67 (s, 1H, H-4), 13.76 (s, 1H, NH); 13
C NMR
(DMSO-d6): δ 101.3 (br), 111.83, 112.2, 117.5 (br), 138.4, 141.5, 155.5, 157.8, 161.0; 19
F NMR
(DMSO-d6): -118.10, -120.20; Anal. Calcd for C9H6FN5O: C 49.32; H 2.76; N 31.95. Found: C 49.38;
H 2.80; N 31.86; IR (KBr): ν max 3453, 3418, 3298, 1624, 1601, 1562, 1505, 1491, 1456, 1432, 1415,
1321, 1265, 1224, 1142, 1112, 1005, 954, 904, 861, 808, 771, 732, 699, 633, 614, 513.
3-Amino-4-(5-chloro-1H-benzimidazol-2-yl)-furazan (9f).
N
NH2
ON
H
N
NCl
White solid; yield 148 mg (63%); mp 301–302 °C; 1
H NMR (DMSO-d6): δ 6.80 (s, 2H, NH2), 7.30 (s,
1H, H-6), 7.59 (s, 1H, H-7), 7.74 (s, 1H, H-4), 13.82 (br. s, 1H, NH); 13
C NMR (DMSO-d6): δ 112.7
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(br. d), 120.1 (br. d), 123.9 (br. d), 128.1 (br. d), 134.1 (br. d), 138.4, 141.6, 143.6, 155.7; Anal. Calcd
for C9H6ClN5O: C 45.88; H 2.57; N 29.72. Found: C 45.94; H 2.63; N 29.63; IR (KBr): ν max 3439,
3336, 3167, 3113, 632, 1623, 1597, 1454, 1414, 1309, 1271, 1228, 1134, 1057, 1005, 950, 926, 901,
875, 860, 803, 734, 676, 596, 563.
3-Amino-4-(7-chloro-1H-benzimidazol-2-yl)-furazan (9g).
N
NN
H
O
N
NH2
Cl
White solid; yield 146 mg (62%); mp 251–252 °C; 1
H NMR (DMSO-d6): δ 6.85 (s, 2H, NH2), 7.32,
7.36 (2t, J = 7.8 Hz, 1H, H-5), 7.41, 7.45 (2d, J = 7.8 Hz, 1H, H-6), 7.57, 7.79 (2d, J = 7.8 Hz, 1H, H-
4), 14.07, 14.24 (2s, 1H, NH); 13
C NMR (DMSO-d6): δ 111.2, 122.3, 123.4, 125.1, 135.4, 138.2, 139.7,
140.9, 155.5; Anal. Calcd for C9H6ClN5O: C 45.88; H 2.57; N 29.72. Found: C 45.93; H 2.62; N 29.61;
IR (KBr): ν max 3452, 3339, 3278, 1616, 1589, 1499, 1453, 1453, 1419, 1318, 1257, 1200, 1113, 1004,
975, 948, 897, 778, 739,631, 606, 569.
3-Amino-4-(5-methoxy-1H-benzimidazol-2-yl)-furazan (9h).
N
NN
H
O
N
NH2
MeO
White solid; yield 143 mg (62%); mp 210–211°C; 1
H NMR (DMSO-d6): δ 3.79 (s, 3H, OMe-5), 6.81
(s, 2H, NH2), 6.91 (d, J = 8.0, 1H, H-6), 7.04 (s, 1H, H-7), 7.60 (s, 1H, H-4), 13.49 (s, 1H, NH); 13
C
NMR (DMSO-d6): δ 56.0 (OMe), 94.7 (br), 113.5 (br), 120.7 (br), 139.0, 155.9; Anal. Calcd for
C10H9N5O2: C 51.95; H 3.92; N 30.29. Found: C 52.01; H 3.90; N 30.21; IR (KBr): ν max 3434, 3287,
1624, 1591, 1510, 1462, 1435, 1411, 1270, 1204, 1164, 1119, 1025, 1001, 953, 896, 865, 819, 627.
3-Amino-4-(4,7-dimethoxy-1H-benzimidazol-2-yl)-furazan (9i).
N
NN
H
O
N
NH2
OMe
OMe
White solid; yield 154 mg (59%); mp 269–270 °C; 1
H NMR (DMSO-d6): δ 3.91 (s, 6H, OMe-4,7, 6.70
(s, 2H, NH2), 6.72 (s, 2H, H-5,6), 13.95 (s, 1H, NH); 13
C NMR (DMSO-d6): δ 55.9 (OMe), 103.2 (br),
105.2 (br), 138.3, 139.0, 155.5; Anal. Calcd for C11H11N5O3: C 50.57; H 4.24; N 26.81. Found: C 52.63;
H 4.26; N 26.71; IR (KBr): ν max 3419, 3335, 3208, 1630,1615, 1529,1464, 1449, 1420, 1349, 1275,
1256, 1213, 1176, 1108, 1097, 1004, 983, 951, 895, 855, 778, 748, 721, 681, 567,516, 486.
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2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carboxylic acid (9j).
White solid; yield 201 mg (82%); mp 319–320 °C; 1
H NMR (DMSO-d6): δ 6.79 (s, 2H, NH2), 7.71 (s,
1H, H-7), 7.91 (d, J = 8.3, 1H, H-6), 8.26 (br. s, 1H, H-4), 12.44 (br. s, 1H, OH), 13.77 (br. s, 1H, NH);
13
C NMR (DMSO-d6): δ 20.9, 113.9 (br. s), 119.0 (br. s), 124.5, 125.8, 138.3, 142.3, 155.5, 167.5,
171.9; Anal. Calcd for C10H7N5O3: C 48.98; H 2.88; N 28.56. Found: C 49.03; H 2.85; N 28.47; IR
(KBr): ν max 3473, 3352, 3102, 2629, 2553, 1686, 1635, 1599, 1493, 1455, 1411, 1326, 1287, 1233,
1150,1137, 1088, 1013, 957, 910, 869, 837, 771, 757, 689, 670, 570, 515.
2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carboxylic acid mehtyl ester (9k).
N
N
H
N
O
N
H2N
MeOOC
White solid; yield 218 mg (84%); mp 284–285 °C; 1
H NMR (DMSO-d6): δ 3.82 (s, 3H, OMe), 6,77 (s,
2H, NH2), 7.61 (s, 1H, H-7), 7.78 (s, 1H, H-6), 8.12 (s, 1H, H-4), 13.85 (br. s, 1H, NH); Anal. Calcd for
C11H9N5O3: C 50.97; H 3.50; N 27.02. Found: C 51.01; H 3.49; N 26.85; IR (KBr): ν max 3542, 3436,
3330, 1692, 1638, 1602, 1435, 1332, 1294, 1248, 1233, 1132, 1092, 1007, 979,952, 900, 863, 826, 772,
751, 567, 501.
2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carbonitrile (9l).
N
NN
H
O
N
NH2N
White solid; yield 195 mg (86%); mp 288 °C; 1
H NMR (DMSO-d6): δ 6.75 (s, 2H, NH2), 7.62 (d, J =
8.1 Hz, 1H, H-7), 7.74 (d, J = 8.1 Hz, 1H, H-6), 8.13 (s, 1H, H-4), 14.01 (br. .s, 1H, NH); 13
C NMR
(DMSO-d6): δ 105.8, 116.1, 120.0, 122.7, 123.5, 127.3, 138.7, 139.7, 143.7, 156.0; Anal. Calcd for
C10H6N6O: C 53.10; H 2.67; N 37.15. Found: C 53.14; H 2.64; N 37.07; IR (KBr): ν max 3421, 3311,
2234, 1644, 1624, 1603, 1445, 1407, 1326, 1286, 1236, 1142, 1096, 1012, 956, 907, 872, 826, 754, 628,
571.
3-Amino-4-(1H-naphtho[2,3-d]imidazol-2-yl)-furazan (9m).
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N
N
H
N
O
N
H2N
White solid; yield 126 mg (50%); mp 276–278 °C; 1
H NMR (DMSO-d6): δ 6.94 (s, 2H, NH2), 7.42 (s,
2H, H-4,9), 8.18 (m, 4H, H-5,6,7,8), 13.70 (s, 1H, NH); 13
C NMR (DMSO-d6): δ 107.6, 116.6, 123.7,
124.5, 127.8, 127.9, 130.1, 130.9, 134.5, 138.5, 142.8, 144.4, 155.8; Anal. Calcd for C13H9N5O: C
62.15; H 3.61; N 27.87. Found: C 62.27; H 3.66; N 27.75; IR (KBr): ν max 3298, 1626, 1602, 1559,
1469, 1415, 1310, 1267, 1175, 1135, 1006, 954, 906, 874, 861, 735, 614, 477.
3-Amino-4-(6(8)H-imidazo[4',5':3,4]benzo[1,2-c][1,2,5]furazan-7-yl)-furazan (9n).
N
N
H
N
O
N
H2N
N
O N
White solid; yield 124 mg (51%); mp 319–320 °C; 1
H NMR (DMSO-d6): δ 6.74 (s, 2H, NH2), 7.84 (d, J
= 9.0 Hz, H-4,5), 14.77 (br..s, 1H, NH); 13
C NMR (DMSO-d6): δ 117.6, 127.4 (br), 140.1 (br), 143.2,
144.6, 154.1, 160.3; Anal. Calcd for C9H5N7O2: C 44.45; H 2.07; N 40.32. Found: C 44.49; H 2.01; N
40.25; IR (KBr): ν max 3450, 3354, 3148, 1614, 1627, 1605, 1562, 1525, 1486, 1443, 1414, 1376,
1331, 1261, 1195, 1121, 1081, 1005, 954, 884, 803, 782, 749, 695, 672, 606, 571, 508.
3-(1H-Benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11a).
N
N
ON
H
N
N
White solid; yield 333 mg (78%); mp 214 °С; 1
H NMR (DMSO-d6): δ 6.44 (t, J = 2.2 Hz, 2H, H-3'',4''),
7.36 (br. s, 2H, H-5,6), 7.66 (br. s, 1H, H-7), 7.79 (br. s, 1H, H-4), 7.94 (t, J = 2.2 Hz, 2H, H-2'',5''),
13.69 (s, 1H, NH); 13
C NMR (DMSO-d6): δ 111.9, 120.0 (br. s), 122.4, 134.5 (br. s), 138.1, 141.7,
143.0, 151.3; EIMS m/z 251 [M]+
; Anal. Calcd for C13H9N5O: C 62.15; H 3.61; N 27.87. Found: C
62.19; H 3.63; N 27.81; IR (KBr): ν max 2158-2586, 1565, 1504, 1480, 1435, 1399, 1390, 1374, 1324,
1281, 1232, 1195, 1149, 1116, 1065, 1034, 1003, 967, 919, 905, 886, 737, 604, 583, 484, 456.
3-(5-Methoxy-1H-benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11h).
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N
NN
H
O
N
MeO
N
White solid; yield 359 mg (75%); mp 165–166 °C; 1
H NMR (DMSO-d6): δ 3.84 (s, 3H, OMe -5), 6.44
(t, J = 2.2 Hz, 2H, H-3'',4''), 6.94, 7.02 (2dd, J = 2.4 Hz, J = 8.9 Hz, 1H, H-6), 7.04, 7.33 (2d, J = 2.2
Hz, 1H, H-4), 7.50, 7.71 (2d, J = 8.9 Hz, 1H, H-7), 7.94 (t, J = 2.2 Hz, 2H, H-2'',5''), 13.50, 13.56 (2s,
1H, NH); EIMS m/z 281 [M]+
; Anal. Calcd for C14H11N5O2: C 59.78; H 3.94; N 24.90. Found: C 59.85;
H 3.96; N 24.81; IR (KBr): ν max 3144-2672, 1631, 1594, 1566, 1483, 1461, 1430, 1388, 1334, 1276,
1202, 1159, 1113, 1065, 1032, 971, 891, 820, 732, 603.
3-(4,7-Dimethoxy-1H-benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11i).
N
NN
H
O
N
NOMe
OMe
White solid; yield 402 mg (76%); mp 188 °C; 1
H NMR (DMSO-d6): δ 3.91 (s, 6H, OMe-4,7), 6.40 (br.
s, 2H, H-3'',4''), 6.65 (d, J = 7.8 Hz, 1H, CH), 6.76 (d, J = 7.8 Hz, 1H, CH), 7.75 (m, 2H, H-2'',5''),
13.94 (s, 1H, NH). 13
C NMR (DMSO-d6): δ 55.8, 55.9, 103.4, 105.0, 112.0, 121.9, 126.4, 135.0, 136.2,
140.6, 141.4, 145.7, 151.2; Anal. Calcd for C15H13N5O3: C 57.87; H 4.21; N 22.50. Found: C 57.95; H
4.16; N 22.32; IR (KBr): ν max 3244, 3111, 1582, 1561, 1528, 1484, 1461, 1388, 1342, 1270, 1174,
1107, 1095, 1063, 1037, 1005, 986, 970, 911, 892, 858, 787, 741, 722, 666, 598.
Alkylation by benzylhalides 12′′′′–43′′′′ (yields 75–90%).
2-[2-(4-Aminofurazan-3-yl)-1H-benzimidazol-1-yl]ethanol (12).
N
N
N O
N
NH2
OH
White solid; yield 1.72 g (70%); mp 173–176 °C; 1
H NMR (DMSO-d6): δ 3.80 (q, J = 4.7 Hz, J = 5.5
Hz, 2H, CH2O), 4.74 (t, J = 5.5Hz, 2H, CH2N), 4.92 (t, J = 4.7 Hz, 1H, OH), 7.00 (s, 2H, NH2), 7.35 (t,
J = 8.1 Hz, 1H, H-5), 7.42 (t, J = 8.1 Hz, 1H, H-6), 7.77 (d, J = 8.1 Hz, 1H, H-7), 7.82 (d, J = 8.1 Hz,
1H, H-4); EIMS m/z 245 [M]+
(37), 228 (1), 215 (12), 206 (6), 188 (100), 172 (9), 157 (22), 156 (22),
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144 (96), 131 (5), 118 (12), 102 (6), 77 (10); Anal. Calcd for C11H11N5O2: C 53.87; H 4.52; N 28.56.
Found: C 53.82; H 4.50; N 28.61.
3-Amino-4-(1-propargyl-1H-benzimidazol-2-yl)-furazan (13).
N
N N
N
O
NH2
White solid; yield 1.72 g (72%); mp 210 °C; 1
H NMR (DMSO-d6): 3.33 (br. s, 1H, CH), 5.57, 5.58 (2s,
2H, CH2), 6.88 (br. s, 2H, NH2), 7.40 (t, J = 7.6 Hz, 1H, H-6), 7.49 (t, J = 7.6 Hz, 1H, H-5), 7.82 (d, J =
7.6 Hz, 1H, H-7), 7.86 (d, J = 7.6 Hz, 1H, H-4); EIMS m/z 239 [M]+
(21), 209 (31), 182 (38), 144
(100), 118 (37), 77 (8); Anal. Calcd for C12H9N5O: C 60.25; H 3.79; N 29.27. Found: C 60.29; H 3.82;
N 29.18.
N-[4-(1-Allyl-1H-benzimidazol-2-yl)-furazan-3-yl]acetamide (14r).
N
N N
N
O
NH
O
White solid; yield 2.15 g (76%); mp 160–161 °C; 1
H NMR (DMSO-d6): δ 2.31 (s, 3H, CH3CO), 5.05
(d, J = 17.0 Hz, 1H, CH2=), 5.20 (d, J = 10.0 Hz, 1H, CH2=), 5.30 (s, 2H, CH2), 6.07 (m, 1H, CH=),
7.42 (t, J = 8.0 Hz, 1H, H-6), 7.47 (t, J = 8.0 Hz, 1H, H-5), 7.75 (d, J = 8.0 Hz, 1H, H-7), 7.90 (d, J =
8.0 Hz, 1H, H-4), 10.91 (s, 1H, NH); EIMS m/z 283 [M]+
(3), 268 (5), 253 (1), 241 (1), 227 (1), 211
(10), 200 (7), 194 (4), 184 (45), 182 (5), 169 (5), 156 (5), 144 (6), 129 (2), 116 (3), 102 (5), 90 (4), 77
(9), 43 (99), 41 (100); Anal. Calcd for C14H13N5O2: C 59.36; H 4.63; N 24.72. Found: C 59.43; H 4.66;
N 24.60.
3-Amino-4-[1-(2-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan (15).
N
N
N O
N
NH2
F
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White solid; yield 2.63 g (85%); mp 184–185 °C; 1
H NMR (DMSO-d6): δ 6.03 (s, 2H, CH2), 6.81 (t, J =
7.6 Hz, 1H, H-6''), 6.95 (s, 2H, NH2), 7.06 (t, J = 7.6 Hz, 1H, H-5''), 7.23 (t, J = 8.7 Hz, 1H, H-3''), 7.32
(m, 1H, H-4''), 7.38 (t, J = 7.7 Hz, 1H, H-5), 7.41 (t, J = 7.7 Hz, 1H, H-6), 7.67 (d, J = 7.7 Hz, 1H, H-7),
7.88 (d, J = 7.7 Hz, 1H, H-4; EIMS m/z 309 [M]+
(5), 279 (4), 252 (6), 143 (8), 110 (5), 109 (100), 83
(18); Anal. Calcd for C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C 62.19; H 3.94; N 22.57.
3-Amino-4-[1-(3-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan (16).
N
N
N O
N
NH2
F
White solid; yield 2.41 g (78%); mp 158–160 °C; 1
H NMR (DMSO-d6): δ 6.00 (s, 2H, CH2), 6.89 (br. s,
2H, NH2), 6.98 (m, 3H, H-4'',5'',6''), 7.30 (t, J = 7.3 Hz, 1H, H-5), 7.36 (m, 2H, H-6,2''), 7.62 (d, J = 7.3
Hz, 1H, H-7), 7.82 (d, J = 7.3 Hz, 1H, H-4); EIMS m/z 309 [M]+
(7), 279 (6), 252 (9), 237 (6), 236 (7),
170 (1), 143 (13), 109 (100), 83 (25); Anal. Calcd for C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C
62.18; H 3.93; N 22.60.
N-(4-[1-(3-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan 3-yl)propionamide (16s).
N
N
N O
N
NH
F
O
White solid; yield 2.7 g (74%); mp 169–170 °C; 1
H NMR (DMSO-d6): 1.20 (t, J = 7.4 Hz, 3H,
CH3CH2), 2.62 (q, J = 7.4 Hz, 2H, CH2CO), 5.96 (s, 2H, CH2), 7.03 (d, J = 7.7 Hz, 1H, H-6''), 7.08 (m,
2H, H-4'',5''), 7.35 (br. k, 1H, J = 6.8 Hz, J =7.7 Hz, H-2''), 7.41 (t, J = 7.7 Hz, 1H, H-5), 7.44 (t, J =
7.7 Hz, 1H, H-6), 7.70 (d, J = 7.7 Hz, 1H, H-7), 7.90 (d, J = 7.7 Hz, 1H, H-4), 11.00 (s, 1H, NH); EIMS
m/z 365 [M]+
(6), 335 (7), 309 (11), 308 (13), 237 (8), 170 (2), 143 (15), 109 (100), 83 (23); Anal.
Calcd for C19H16FN5O2: C 62.46; H 4.41; N 19.17. Found: C 62.52; H 4.45; N 19.05.
3-Amino-4-[1-(4-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan (17).
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N
N
N
N
O
NH2
F
White solid; yield 2.78 g (90%); mp 176 °C; 1
H NMR (DMSO-d6): δ 5.95 (s, 2H, CH2), 6.94 (s, 2H,
NH2), 7.12 (t, J = 8.7 Hz, 2H, H-3'',5''), 7.24 (dd, J = 5.5 Hz, J = 8.7 Hz, 2H, H-2'',6''), 7.37 (t, J = 7.6
Hz, 1H, H-5), 7.41 (t, J =7.6 Hz, 1H, H-6), 7.71 (d, J = 7.8 Hz, 1H, H-7), 7.86 (d, J = 8.1 Hz, 1H, H-4);
EIMS m/z 309 [M]+
(6), 379 (7), 263 (1), 143 (6), 110 (6), 109 (100), 83 (16), 63 (6); Anal. Calcd for
C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C 62.05; H 3.88; N 22.69.
3-Amino-4-[1-(3-bromobenzyl)-1H-benzimidazol-2-yl]-furazan (18).
N
N
N O
N
NH2
Br
White solid; yield 2.78 g (75%); mp 176–177 °C; 1
H NMR (DMSO-d6): δ 5.99 (s, 2H, CH2), 6.90 (br. s,
2H, NH2), 7.07 (d, J = 7.7 Hz, 1H, H-6''), 7.22 (t, J = 7.7 Hz, 1H, H-5''), 7.37 (m, 2H, H-5,6)), 7.40 (br.
d, J = 7.7 Hz, 1H, H-4''), 7.44 (br. s, 1H, H-2''), 7.62 (br. d, J = 7.3 Hz, 1H, H-7), 7.82 (br. d, J = 7.3 Hz,
1H, H-4); EIMS m/z 371 [M+1]+
(5), 369 [M-1]+
(5), 341 (6), 339 (6), 314 (9), 312 (9), 260 (4), 234 (5),
217 (7), 169 (100), 143 (38), 116 (10), 102 (9), 90 (84), 89 (54), 77 (12), 63 (27); Anal. Calcd for
C16H12BrN5O: C 51.91; H 3.27; N 18.92. Found: C 51.87; H 3.24; N 18.89.
3-Amino-4-[1-(3-trifluoromethylbenzyl)-1H-benzimidazol-2-yl]-furazan (19).
N
N
N O
N
NH2
CF3
White solid; yield 2.91 g (81%); mp 169–170 °C; 1
H NMR (DMSO-d6): δ 6.07 (s, 2H, CH2), 6.92 (s,
2H, NH2), 7.34 (d, J = 7.7 Hz, 1H, H-6''), 7.39 (m, 2H, H-5,6), 7.51 (t, J = 7.7 Hz, 1H, H-5''), 7.60 (d, J
= 7.7 Hz, 1H, H-4''), 7.64 (s, 1H, H-2''), 7.70 (d, J = 7.6 Hz, 1H, H-7), 7.86 (d, J = 7.7 Hz, 1H, H-4);
EIMS m/z 359 [M]+
(2), 329 (2), 302 (6), 287 (4), 286 (4), 159 (100), 143 (15), 119 (8), 109 (29), 90
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(7), 89 (6), 63 (7); Anal. Calcd for C17H12F3N5O: C 56.83; H 3.37; N 19.49. Found: C 56.77; H 3.35; N
19.55.
N-(4-[1-(3-Trifluoromethylbenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (19r).
N
N
N O
N
NH
CF3
O
White solid; yield 2.93 g (73%); mp 213–214 °C; 1
H NMR (DMSO-d6): δ 2.32 (s, 3H, CH3CO), 6.03 (s,
2H, CH2), 7.41 (m, 2H, H-5,6,6''), 7.51 (t, J = 7.7 Hz, 1H, H-5''), 7.61 (t, J = 7.7 Hz, 1H, H-4''), 7.68 (br.
s, 1H, H-2''), 7.70 (d, J = 7.7 Hz, 1H, H-7), 7.91 (d, J = 7.7 Hz, 1H, H-4), 10.95 (s, 1H, NH); EIMS m/z
401 [M]+
(0.1), 386 (1), 329 (3), 302 (5), 159 (84), 109 (21), 43 (100); Anal. Calcd for C19H14F3N5O2: C
56.36; H 3.52; N 17.45. Found: C 56.22; H 3.48; N 17.57.
3-Amino-4-[1-(3-methoxybenzyl)-1H-benzimidazol-2-yl]-furazan (20).
N
N
N O
N
NH2
OMe
White solid; yield 2.67 g (83%); mp 181–183 °C; 1
H NMR (DMSO-d6): δ 3.68 (s, 3H, OMe -3''), 5.94
(s, 2H, CH2), 6.66 (br. d, J = 7.3 Hz, 1H, H-4''), 6.76 ( br. s, 1H, H-2''), 6.82 (br. d, J = 8.2 Hz, 1H, H-
6''), 6.94 (s, 2H, NH2), 7.20 (t, J = 7.8 Hz, 1H, H-5''), 7.37 (t, J = 7.7 Hz, 1H, H-5), 7.41 (t, J = 7.3 Hz,
1H, H-6), 7.69 (d, J = 7.7 Hz, 1H, H-7), 7.86 (d, J = 7.7 Hz, 1H, H-4); EIMS m/z 321 [M]+
(6), 291
(17), 276 (3), 143 (5), 121 (100), 91 (37), 78 (19), 77 (25); Anal. Calcd for C17H15N5O2: C 63.54; H
4.71; N 21.79. Found: C 63.61; H 4.73; N 21.75.
N-(4-[1-(3-Methoxybenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (20r).
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N
N
N O
N
NH
CF3
O
White solid; yield 2.73 g (75%); mp 151 °C; 1
H NMR (DMSO-d6): δ 2.30 (s, 3H, CH3CO), 3.69 (s, 3H,
OMe-3''), 5.89 (s, 2H, CH2), 6.72 (br. d, J = 7.5 Hz, 1H, H-4''), 6.81 (br. s, 1H, H-2''), 6.83 (dd, J = 2.1
Hz, J = 8.2 Hz, 1H, H-6''), 7.21 (t, J = 7.9 Hz, 1H, H-5''), 7.40 (t, J = 7.2 Hz, 1H, H-5), 7.43 (t, J = 7.2
Hz, 1H, H-6), 7.69 (d, J = 7.7 Hz, 1H, H-7), 7.91 (d, J = 7.7 Hz, 1H, H-4), 10.94 (s, 1H, NH); EIMS m/z
363 [M]+
(32), 348 (16), 333 (2), 321 (21), 291 (59), 264 (25), 143 (10), 121 (100), 91 (25), 78 (13), 77
(14), 43 (90); Anal. Calcd for C19H17N5O3: C 62.80; H 4.72; N 19.27. Found: C 62.72; H 4.70; N 19.34.
3-Amino-4-[1-(3-methoxy-4-methylbenzyl)-1H-benzimidazol-2-yl)-furazan (21).
N
N
N O
N
NH2
MeO
Me
White solid; yield 2.72 g (81%); mp 198–200 °C; 1
H NMR (DMSO-d6): δ 2.11 (s, 3H, Me-4''), 3.84 (s,
3H, OMe-3''), 5.90 (s, 2H, CH2), 6.44 (br. s, 1H, H-2''), 6.79 (s, 2H, NH2), 6.84 (d, J = 8.3 Hz, 1H, H-
5''), 6.99 ( br. d, J = 8.3 Hz, 1H, H-6''), 7.29 (m, 2H, H-5,6), 7.40 (m, 1H, H-7), 7.78 (m, 1H, H-4');
EIMS m/z 335 [M]+
(31), 318 (3), 305 (19), 290 (1), 143 (13), 135 (100), 105 (40), 103 (9), 91 (13), 79
(10), 77 (11); Anal. Calcd for C18H17N5O2: C 64.47; H 5.11; N 20.88. Found: C 64.52; H 5.13; N 20.83.
3-Amino-4-[1-(3,4-diethoxybenzyl)-1H-benzimidazol-2-yl]-furazan (22).
N
N
N O
N
NH2
EtO
OEt
White solid; yield 3.26 g (86%); mp 168–170 °C; 1
H NMR (DMSO-d6): δ 1.34 (t, J = 7.0 Hz, 3H,
CH3CH2), 1.35 (t, J = 7.0 Hz, 3H, CH3CH2), 3.95 (q, J = 7.0 Hz, 4H, 2OCH2), 5.87 (s, 2H, CH2), 6.63
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(dd, J = 1.6 Hz, J = 8.2 Hz, 1H, H-6''), 6.73 (d, J = 8.2 Hz, 1H, H-5''), 6.81 (s, 2H, NH2), 6.86 (d, J = 1.6
Hz, 1H, H-2''), 7.33 (m, 2H, H-5',6'), 7.59 (d, J = 7.8 Hz, 1H, H-7), 7.78 (d, J = 7.6 Hz, 1H, H-4); EIMS
m/z 379 [M]+
(28), 349 (10), 179 (100), 151 (40), 144 (11), 143 (8), 133 (6), 123 (65), 105 (5), 94 (7),
77 (12); Anal. Calcd for C20H21N5O3: C 63.31; H 5.58; N 18.46. Found: C 63.40; H 5.61; N 18.56.
3-Amino-4-[1-(4-methylbenzyl)-1H-benzimidazol-2-yl]-furazan (23).
N
N
N O
N
NH2
Me
White solid; yield 2.32 g (76%); mp 184–185 °C; 1
H NMR (DMSO-d6): 2.23 (s, 3H, Me-4''), 5.93 (s,
2H, CH2), 6.95 (s, 2H, NH2), 7.05 (d, J = 8.0 Hz, 2H, H-3'',5''), 7.10 (d, J = 8.0 Hz, 2H, H-2'',6''), 7.36
(t, J = 7.8 Hz, 1H, H-6'), 7.40 (t, J = 7.8 Hz, 1H, H-5), 7.68 (d, J = 7.8 Hz, 1H, H-7), 7.86 (d, J = 7.8
Hz, 1H, H-4); EIMS m/z 305 [M]+
(18), 275 (16), 259 (1), 248 (5), 232 (7), 106 (7), 105 (100), 103 (8),
77 (10); Anal. Calcd for C17H15N5O: C 66.87; H 4.95; N 22.94. Found: C 66.94; H 4.98; N 22.80.
3-Amino-4-[1-(4-t
butylbenzyl)-1H-benzimidazol-2-yl]-furazan (24).
N
N
N O
N
NH2
t-Bu
White solid; yield 2.54 g (73%); mp 175 °C; 1
H NMR (DMSO-d6): δ 1.24 (s, 9H, C(CH3)3-4''), 5.94 (s,
2H, CH2), 6.94 (s, 2H, NH2), 7.10 (d, J = 8.1 Hz, 2H, H-3'',5''), 7.30 (d, J = 8.1 Hz, 2H, H-2'',6''), 7.35
(t, J = 7.8 Hz, 1H, H-6), 7.39 (t, J = 7.8 Hz, 1H, H-5), 7.67 (d, J = 7.8 Hz, 1H, H-7), 7.83 (d, J =7.8 Hz,
1H, H-4); EIMS m/z 347 [M]+
(15), 317 (11), 261 (11), 148 (11), 147 (100), 138 (6), 132 (18), 124 (22),
117 (23), 91 (12); Anal. Calcd for C20H21N5O: C 69.14; H 6.09; N 20.16. Found: C 69.25; H 6.14; N
20.02.
N-(4-[1-(4-t
Butylbenzyl)-1H-benzimidazol-2-yl]-furazan-3-ylacetamide (24r).
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N
N
N O
N
NH
t-Bu
O
White solid; yield 2.8 g (72%); mp 166–167 °C; 1
H NMR (DMSO-d6): 1.24 (s, 9H, C(CH3)3-4''), 2.33
(s, 3H, CH3CO), 5.91 (s, 2H, CH2), 7.14 (d, J = 8.2 Hz, 2H, H-3'',5''), 7.30 (d, J = 8.2 Hz, 2H, H-2'',6''),
7.39 (t, J = 7.6 Hz, 1H, H-6), 7.42 (t, J = 7.6 Hz, 1H, H-5), 7.70 (d, J = 7.6 Hz, 1H, H-7), 7.89 (d, J =
7.6 Hz, 1H, H-4), 10.96 (s, 1H, NH); EIMS m/z 389 [M]+
(3), 374 (1), 347 (2), 317 (14), 261 (6), 147
(100), 132 (19), 124 (6), 119 (7), 117 (21), 105 (9), 91 (11); Anal. Calcd for C22H23N5O2: C 67.85; H
5.95; N 17.98. Found: C 67.95; H 6.00; N 17.87.
3-Amino-4-[1-(4-Cyanobenzyl)-1H-benzimidazol-2-yl]-furazan (25).
N
N
N O
N
NH2
N
White solid; yield 2.66 g (84%); mp 224–225 °C; 1
H NMR (DMSO-d6): δ 6.07 (s, 2H, CH2), 6.91 (s,
2H, NH2), 7.32 (d, J = 8.2 Hz, 2H, H-2'',6''), 7.39 (m, 2H, H-5,6), 7.66 (m, 1H, H-7), 7.73 (d, J = 8.2
Hz, 2H, H-3'',5''), 7.86 (m, 1H, H-4); EIMS m/z 316 [M]+
(5), 286 (4), 259 (8), 143 (8), 117 (9), 116
(100), 89 (24); Anal. Calcd for C17H12N6O: C 64.55; H 3.82; N 26.57. Found: C 64.61; H 3.84; N 26.50.
N-(4-[1-(4-Cyanobenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (25r).
N
N
N O
N
NH
O
N
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White solid; yield 2.72 g (76%); mp 224–226 °C; 1
H NMR (DMSO-d6): δ 2.32 (s, 3H, CH3CO), 6.03 (s,
2H, CH2), 7.37 (d, J = 8.2 Hz, 2H, H-2'',6''), 7.42 (m, 2H, H-5,6), 7.66 (m, 1H, H-7), 7.73 (d, J = 8.2
Hz, 2H, H-3'',5''), 7.91 (m, 1H, H-4), 10.90 (s, 1H, NH); EIMS m/z 358 [M]+
(18), 344 (6), 343 (28),
341 (1), 316 (11), 286 (27), 259 (41), 244 (5), 117 (8), 116 (100), 89 (12), 43 (15); Anal. Calcd for
C19H14N6O2: C 63.68; H 3.94; N 23.45. Found: C 63.79; H 3.97; N 23.32.
3-Amino-4-(1-methyl-1H-benzimidazol-2-yl)-furazan (26).
N
N
Me
N
O
N
H2N
White solid; yield 1.44 g (67%); mp 206–207 °C; 1
H NMR (DMSO-d6): δ 4.12 (s, 3H, Me), 6.88 (s, 2H,
NH2), 7.32 (t, J = 7.8 Hz, 1H, H-5), 7.40 (t, J = 7.8 Hz, 1H, H-6), 7.68 (d, J = 8.2 Hz, 1H, H-7), 7.79 (d,
J = 8.2 Hz, 1H, H-4); 13
C NMR (DMSO-d6): δ 31.9, 110.6, 119.5, 122.7, 124.1, 135.6, 138.2, 140.7,
141.4, 155.9; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.83; H 4.18; N 32.46;
IR (KBr): ν max 3410, 3311, 1633, 1598, 1577, 1549, 1472, 1457, 1422, 1327, 1290, 1262, 1231, 1157,
1132, 1071, 998, 905, 866, 813, 754, 740, 717, 604, 568, 546.
4-Fluoro-N-[4-(1-methyl-1H-benzimidazol-2-yl)- furazan-3-yl]benzamide (26t).
N
N
N O
N
NH
Me
O
F
White solid; yield 2.53 g (75%); mp 251–252 °C; 1
H NMR (DMSO-d6): 4.23 (s, 3H, Me), 7.40 (t, J =
7.8 Hz, 1H, H-6), 7.48 (t, J = 7.8 Hz, 1H, H-5), 7.53 (t, J = 8.8 Hz, 2H, H-3'',5''), 7.78 (d, J = 7.8 Hz,
1H, H-7), 7.92 (d, J = 7.8 Hz, 1H, H-4), 8.15 (dd, J = 5.8 Hz, J = 8.8 Hz, 2H, H-2'',6''), 10.14 (s, 1H,
NH); EIMS m/z 337 [M]+
(11), 170 (8), 158 (100), 143 (5), 123 (32), 95 (21), 75 (6); Anal. Calcd for
C17H12FN5O2: C 60.53; H 3.59; N 20.76. Found: C 60.43; H 3.56; N 20.90.
3-Amino-4-(1-ethyl-1H-benzimidazol-2-yl)-furazan (27).
N
N
N O
N
NH2
Et
White solid; yield 1.95 g (85%); mp 168–169 °C; 1
H NMR (DMSO-d6): δ 1.35 (t, J = 7.0 Hz, 3H,
CH3CH2), 4.63 (q, J = 7.0 Hz, 2H, CH2N), 7.01 (s, 2H, NH2), 7.30 (t, J = 7.7 Hz, 1H, H-5), 7.38 (t, J =
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7.7 Hz, 1H, H-6), 7.70 (d, J = 8.0, 1H, H-7), 7.78 (d, J = 8.0, 1H, H-4); 13
C NMR (DMSO-d6): δ 14.8,
40.1, 110.8, 119.9, 123.0, 124.5, 134.7, 138.1, 140.1, 141.7, 156.2; Anal. Calcd for C11H11N5O: C
57.63; H 4.84; N 30.55. Found: C 57.67; H 4.80; N 30.48; IR (KBr): ν max 3405, 3302, 2988, 2976,
2939, 1635, 1626, 1593, 1575, 1545, 1485, 1472, 1461, 1446, 1410, 1376, 1351, 1330, 1293, 1260,
1202, 1155, 1134, 1088, 1074, 998, 955, 907, 864, 784, 757, 742, 705, 565, 466.
N-(4-[1-(4-Methoxybenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (28r).
N
N
N O
N
NH
O
MeO
White solid; yield 3.02 g (83%); mp 170–173 °C; 1
H NMR (DMSO-d6): δ 2.36 (s, 3H, CH3CO), 3.73 (s,
2H, OMe-4''), 5.89 (s, 2H, CH2), 6.80 (d, J = 8.5 Hz, 2H, H-3'',5''), 7.17 (d, J = 8.5 Hz, 2H, H-2'',6''),
7.36 (d, J = 7.6 Hz, 1H, H-6), 7.39 (t, J = 8.0 Hz, 1H, H-5), 7.64 (d, J = 7.6 Hz, 1H, H-7), 7.85 (d, J =
8.0 Hz, 1H, H-4), 10.95 (s, 1H, NH); EIMS m/z 363 [M]+
(5), 333 (1), 321 (3), 291 (15), 121 (100), 77
(10), 43 (22); Anal. Calcd for C19H17N5O3: C 62.80; H 4.72; N 19.27. Found: C 62.90; H 4.75; N 19.20.
3-Amino-4-(1-(4-methoxybenzyl)-5-(trifluoromethyl)-1H-benzimidazol-2-yl)-furazan (29).
N
N
N O
N
NH2
MeO
F3C
White solid; yield 2.3 g (59%); mp 187–188 °C; 1
H NMR (DMSO-d6): δ 3.69 (s, 3H, OMe-4''), 5.95 (s,
2H, CH2), 6.86 (d, J = 8.6 Hz, 2H, H-3'',5''), 7.02 (s, 2H, NH2), 7.16 (d, J = 8.6 Hz, 2H, H-2'',6''), 7.74
(dd, J = 1.6 Hz, J = 8.6 Hz, 1H, H-6), 7.98 (d, J = 8.6 Hz, 1H, H-7), 8.26 (d, J = 1.6 Hz, H-4); 13
C NMR
(DMSO-d6): δ 159.3, 156.7,143.3, 141.6, 138.4, 137.8, 128.7, 128.3, 126.9, 124.9, 124.4, 123.3, 121.6,
118.1, 114.6, 113.2, 55.3, 48.4; 19
F NMR (DMSO-d6): -60.24. 15
N NMR (DMSO-d6): 26.70, -18.27, -
131.45, -207.32, -332.63; EIMS m/z 389 [M]+
; Anal. Calcd for C18H14F3N5O2: C 55.53; H 3.62; N
17.99. Found: C 55.48; H 3.60; N 17.92; IR (KBr): ν max 3442, 3335, 1640, 1614, 1581, 1517, 1464,
1441, 1343, 1331, 1304, 1268, 1255, 1231, 1183, 1158, 1105, 1052, 1033, 1009, 972, 890, 867, 836,
816, 804, 780, 756, 699, 652, 631, 569.