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WO2016005992A1 - Colorants fluorescents à base de naphtho[2,1-b][1,10]phénanthroline substitué et application associée - Google Patents

Colorants fluorescents à base de naphtho[2,1-b][1,10]phénanthroline substitué et application associée Download PDF

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WO2016005992A1
WO2016005992A1 PCT/IN2015/000076 IN2015000076W WO2016005992A1 WO 2016005992 A1 WO2016005992 A1 WO 2016005992A1 IN 2015000076 W IN2015000076 W IN 2015000076W WO 2016005992 A1 WO2016005992 A1 WO 2016005992A1
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heteroatoms
phenanthroline
carbonitrile
piperidin
preferably selected
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PCT/IN2015/000076
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English (en)
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Atul Goel
Shahida Umar
Pankaj Nag
Aamir Nazir
Lalit Kumar
Shamsuzzama
Jiaur Rahaman GAYEN
Zakir Hossain
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Council Of Scientific And Industrial Research
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Publication of WO2016005992A1 publication Critical patent/WO2016005992A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to novel substituted dihydronaphtho[2,l- 6][l ,10]phenanthrolines and naphtho[2,l-6][l,10]phenanthrolines their ion chelators and salts thereof as Fe(III) selective dual colorimetric and ratiometric fluorescent probes for cell imaging applications, as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction, and processes of preparing said novel compounds.
  • the present invention relates to a compound of formula I their ion chelators and salts thereof:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 are independently selected from the groups consisting of . hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, C ⁇ -Cg alkyl, C 2 -C 8 alkenyl, C ⁇ -C» alkoxyl, C 6 -C 20 aryl, C 5 -C 8 cyclo, C 6 -C 20 aryl, C 3 -C 8 cycloalkyl, C 3 -C 20 heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C 3 -C 20 cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C ⁇ -C& alkyl esters,
  • the present invention relates to 12,13 -dihydronaphtho [2,1 - Z > ][l,10]phenanthrolines and naphtho[2,l-6][l ,10]phenanthrolines, processes for preparing the said compounds and their applications as fluorescent probes in chemical and biological sciences such as cell imaging applications, diagnostics, fluorescent tags, ion sensors, pharmaceuticals.
  • the present invention relates to the compounds having the general formula I as Fe(III) selective dual colorimetric and ratiometric fluorescent probes for cell imaging applications, as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction.
  • Iron is ubiquitously found in nature, from the earth's crust to the biological systems. It is an essential trace element in all living organisms possessing indispensable physiological as well as pathophysiological functions. By virtue of its interesting oxygen affinity and facile redox chemistry it is efficiently involved in an array of biological pathways including oxygen uptake and metabolism, regulation of cell growth and differentiation, electron transfer and transcriptional regulation. Therefore highly sophisticated regulatory approaches are required to balance the demands of cells as well as to avoid its deficiency/excess accumulation (Hentze, M. W.; Muckenthaler, M. U.; Andrews, N. C. Cell 2004, 117, 285). The bulk of the iron present in biological systems is tightly bound to various enzymes, storage and regulatory proteins.
  • LIP cytosolic nonprotein bound labile iron pool
  • Fenton reaction involves the generation of hydroxyl radicals from hydrogen peroxide and the formation of aldehydes and lipid peroxy radicals from lipid hydroperoxides.
  • ROS reactive oxygen species
  • iron selective fluorescence probes are Calcein (Epsztejn, S.; Kakhlon, O.; Glickstein, H.; Breuer, W.; Cabantchik Z. I. Anal. Biochem. 1997, 248, 31), phenanthroline-fluorescein hybrids Phen green SK and Phen green FL (Petrat, F.; Rauen, U.; Groot H. D. Hepatology, 1999, 29, 1171), and siderophore-fluorescein hybrid FL-DFO (Petrat, F.; Rauen, U.; Groot H. D. Hepatology, 1999, 29, 1171).
  • Calcein (excitation maximum, 494 nm; emission maximum, 517nm), most commonly used for quantification of LIP undergoes intracellular fluorescence quenching on binding to iron in a manner proportional with the concentration of labile iron present. Since the experimental systems reach steady state levels of fluorescence within minutes, it is assumed that the LIP determination per se does not perturb short-term intracellular iron distribution. Determination of the cell-associated CAL-Fe is based on dequenching of the fluorescence signal by addition of a highly permeant chelator (mostly 2,2'-Dipyridyl) that displays a high binding affinity for the metal.
  • a highly permeant chelator mostly 2,2'-Dipyridyl
  • Calcein undergoes fluorescence quenching on binding to both the ionic forms of iron present in the LIP (Tenopoulou, M.; KURZ, T.; Doulias, P.T.; Galaris, D.; Brunk, U.T. Biochem. J. 2007, 403, 261). It is suggested in many reports that it binds (quenches) more effectively to Fe(III) rather than Fe(II) ( Thomas, F.; Serratrice, G.; Be ' guin, C; Aman, E. S.; Pierre, J. L.; Fontecave, M.; Laulhe're, J. P. J. Biol. Chem. 1999, 274, 13375).
  • iron chelators derived/inspired from siderophores are explored by tagging them with different fluorophores like fluorescein (as FL-DFO; excitation maximum, 493 nm; emission maximum, 515 nm;), coumarin and nitrobenzoxadiazole.
  • fluorescein as FL-DFO; excitation maximum, 493 nm; emission maximum, 515 nm;
  • 1,10-Phenanthroline is characterized by a weak fluorescence quantum yield due to close lying ⁇ - ⁇ * and n- ⁇ * singlet excited states.
  • Substantial efforts have been made to improve its emission efficiency as novel chelating ligands by introducing aromatic substituents at various positions.
  • novel Dihydronaphtho[2,l-b][l,10]phenanthrolines and Naphtho[2,l- 6][l,10]phenanthrolines have been synthesized and investigated as Fe (III) selective dual (colorimetric and ratiometric) fluorescent probes for cell imaging applications and quantification of ROS in Fenton reaction.
  • the present invention also relates to a highly rapid synthesis of novel 12,13-dihydronaphtho[2,l-6][l,10]phenanthrolines and naphtho[2,l- b] [ 1 , 10]phenanthrolines.
  • Main object of the present invention is to provide novel dihydronaphtho[2,l- b][l,10]phenanthroline and Naphtho[2,l-6][l,10]phenanthrolines, having the general formula I their ion chelators and salts thereof;
  • Another object of the invention is to provide processes for the preparation of the novel dihydronaphtho [2, 1 -b] [ 1 , 1 OJphenanthroline and naphtho[2, 1 -b] [ 1 , 10]-phenanthrolines, having the general formula I;
  • a further object of the invention is to provide the compounds having the general formula I their ion chelators and salts thereof as Fe(III) selective dual colorimetric and ratiometric fluorescent probes for cell imaging applications, as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction;
  • Another object of the present invention is to provide a method for fluorescence-based imaging or analysis of cells and/or cellular components using the compound of general formula I;
  • a yet another object of the present invention is to provide a method of detecting and estimating peroxides using the compound of general formula I their ion chelators and salts thereof in the chemical mixture, biological fluid, or in the cellular system;
  • a further object of the present invention is to provide a method of detecting and estimating reactive oxygen species using the compound of general formula I their ion chelators and salts thereof through measuring changes in fluorescence in the chemical mixture, biological fluid, or in the cellular system;
  • Another object of the present invention is to provide the compounds of general formula I their ion chelators and salts thereof for the treatment of diabetes, osteoporosis, cancer, central nervous system disorders, cardiovascular disorders, tuberculosis, reproductive health disorders, Alzheimer, Parkinson's, reactive oxygen species (ROS)-induced disorders and disorders associated with iron-imbalance.
  • ROS reactive oxygen species
  • the present invention relates to novel substituted dihydronaphtho[2,l- b][l,10]phenanthrolines and naphtho[2,l-b][l,10]phenanthrolines of the general formula I their ion chelators and salts thereof as fluorescent probes, and processes of preparing said novel compounds;
  • the present invention more particularly relates to novel 12,13 -dmydronaphtho [2,1 - 6][l,10]phenanthroline and naphtho[2,l-b][l,10]phenanthrolines, their ion chelators and salts thereof as Fe(III) selective dual colorimetric and ratiometnc fluorescent probes for cell imaging applications, as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction 3 ⁇ 4nd processes of preparing said novel compounds;
  • the present invention more particularly relates to a compound of formula I:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Ci-C 8 alkyl, C 2 -C 8 alkenyl, d-Cg alkoxyl, C 6 -C 2 o aryl, C 5 -C 8 cyclo C 6 -C 2 o aryl, C3-C8 cycloalkyl, C3-C20 heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C3-C 2 o cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Q-Cg alkyl esters, C 6 -C 20 ary
  • the representative compounds of the general formula I are selected from the group comprising; i. 11 -phenyl-9-(piperidin- 1 -yl)- 12, 13 -dihydronaphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (1). ii. 11 -(naphthalen- 1 -yl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [1 , 10]phenanthroline-8-carbonitrile (2). iii.
  • the compounds are useful for Fe(III) selective dual colorimetric and ratiometnc fluorescent sensing for cell imaging applications/analysis and quantification of ROS in Fenton reaction;
  • the compounds are having excitation maxima in the range of 330-700 nm and emission maxima in the range of 450-900 nm;
  • the present invention provides compounds having the general formula I their ion chelators and salts thereof as Fe(III) selective dual colorimetric and ratiometric fluorescent probes for cell imaging applications, and useful biological applications as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction;
  • the present invention provides processes for the preparation of novel 12,13-dihydronaphtho[2,l-b][l,10]phenanthroline-8-carbonitrile and naphtho[2,l- 6][l,10]phenanthrolines, having the general formula I, as shown in drawing accompanying the specification wherein the said processes comprise:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Cj-Cg alkyl, C 2 -C 8 alkenyl, Ci-C 8 alkoxyl, C 6 -C 20 aryl, C 5 -C cyclo C 6 -C 2 o aryl, C 3 -C 8 cycloalkyl, C 3 -C 20 heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C 3 - C 2 o cycloheteroaryl containing 1 to 3
  • the present invention provides a method for fluorescence-based imaging or analysis of cells and/or cellular components with the compound of general formula I, their ion chelators and salts thereof;
  • the present invention provides a method of detecting and estimating peroxides using the compound of general formula I, their ion chelators and salts thereof in the chemical mixture, biological fluid, or in the cellular system; In a yet another embodiment, the present invention provides a method of detecting and estimating reactive oxygen species using the compound of general formula I, their ion chelators and salts thereof through measuring changes in fluorescence in the chemical mixture, biological fluid, or in the cellular system;
  • the present invention is to provide the compounds of general formula I their ion chelators and salts thereof for the treatment of diabetes, osteoporosis, cancer, central nervous system disorders, cardiovascular disorders, tuberculosis, reproductive health disorders, Alzheimer, Parkinson's, reactive oxygen species (ROS)-induced disorders and disorders associated with iron-imbalance;
  • ROS reactive oxygen species
  • Table 1 represents the photophysical properties of the compounds of the invention.
  • Figure 1 illustrates the reaction sequences resulting in the preparation of various phenanthroline derivatives.
  • Figure 2 illustrates absorption and emission spectra of compounds 1-9 in TDW.DMSO, 9:1 (v/v).
  • Figure 4 Absorbance spectra of 10 towards increasing concentration of Fe 3+ ions (0-6.25 x lO ⁇ M).
  • Figure 14 Probable complex structure of the salt of chelator 11 and Fe 3+ .
  • Figure 16 Increase in the fluorescence intensity ratio ( ⁇ 605 / ⁇ 544 ) of 11 upon addition of different metal ions.
  • Figure 21 Changes in emission intensity of 11 (2.5x 10 " M) in the presence of Fe and hydroxylamine hydrochloride (5X 10 " M, sodium acetate (6.0 lO “4 M)).
  • Figure 22 Ratiometric fluorescence response of 11 in the presence of Fe with increasing H 2 0 2 .
  • FIG. 23 Confocal fluorescence images of HepG2 cells incubated with dye 11 only (a, ) and cells preincubated with Fe 3+ then with dye 11 (c, d).
  • Figure 24 shows cell viability assessment of dye 11 in Human HepG2 cells.
  • Figure 26 The expression of C. elegans gene ftn-1 when treated with dye 11.
  • the present invention relates to novel dihydronaphtho[2,l- 6][l,10]phenanthrolines and naphtho[2,l-b][l,10]phenanthrolines of the general formula I, their ion chelators and salts thereof as Fe(III) selective dual colorimetric and ratiometric fluorescent probes for cell imaging applications, and useful biological applications as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction and processes of preparing said novel compounds;
  • the term 'fluorescent probe' refers to a fluorophore, suitable to localize within a specific region of a biological specimen or to respond to a specific analyte/substance;
  • the present invention more particularly relates to a compound of formula I:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Ct-C 8 alkyl, C 2 -C8 alkenyl, Cj-Cs alkoxyl, C6-C 20 aryl, C 5 -C 8 cyclo C 6 -C 20 aryl, C 3 -C 8 cycloalkyl, C 3 -C 20 heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C 3 -C 20 cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Ci-C 8 alkyl esters, C 6 -C 20 aryl esters, C
  • the representative compounds of the general formula I are selected from the group comprising; 11 -phenyl-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (1).
  • the compounds of the present invention having the general formula I, their ion chelators and salts thereof as Fe(III) selective dual colorimetric and ratiometric fluorescent probes for cell imaging applications, and useful biological applications as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction;
  • the compounds of the present invention are as fluorescent probes, tags, markers, diagnostics, ion sensor, pharmaceuticals for detecting/trapping iron III in fluorescence-based imaging and/or analysis of cells, biological fluids, chemical mixture and/or as cellular components in fixed or live cell imaging applications;
  • the present invention relates to the processes for the preparation of novel 12,13-Dihydronaphtho[2,l-&][l,10]phenanthroline-8-carbonitrile and Naphtho[2,l-6][l,10]phenanthrolines, having the general formula I as shown in drawing accompanying the specification wherein the said processes comprises:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 1 1 , R 12 are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Ci-C 8 alkyl, C 2 -C 8 alkenyl, C C 8 alkoxyl, C 6 -C 20 aryl, C 5 -C 8 cyclo C 6 -C 20 aryl, C 3 -C 8 cycloalkyl, C 3 -C 20 heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C 3 - C 2 o cycloheteroaryl containing 1 to 3 heteroatom
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, C ⁇ -C& alkyl, C 2 -C 8 alkenyl, d-C 8 alkoxyl, C 6 -C 20 aryl, C 5 -C 8 cyclo C6-C 20 aryl, C 3 -C 8 cycloalkyl, C 3 -C 20 heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C 3 - C 2 o cycloheteroaryl containing 1 to
  • the present invention provides a method for fluorescence-based imaging or analysis of cells and/or cellular components using the compound of general formula I, their ion chelators and salts thereof, wherein the said method comprises the following steps: a) trapping cation in bound or free state in cellular components with a dye/compound of general formula I; ⁇ b) exciting the dye-cation complex of step a) with laser light in the wave length range c) detecting the light emitted by the dyes or complex used in step a) and step b); d) optionally generating images with the emission data obtained in step c); e) optionally performing an analysis with the data obtained in step c) or the images obtained in step d).
  • the present invention relates to a method for fluorescence-based imaging or analysis of cells and/or cellular components, the cellular matrix/components are stained with the dye/compound of the general formula I.
  • the present invention relates to a method of detecting and estimating peroxides using the compound of general formula I, their ion chelators and salts thereof in the chemical mixture, biological fluid, or in the cellular system.
  • the present invention relates to a method of detecting and estimating reactive oxygen species using the compound of general formula I, their ion chelators and salts thereof through measuring changes in fluorescence in the chemical mixture, biological fluid, or in the cellular system.
  • the present invention provides compounds of general formula I, their ion chelators and salts thereof for the treatment of diabetes, osteoporosis, cancer, central nervous system disorders, cardiovascular disorders, tuberculosis, reproductive health disorders, Alzheimer, Parkinson's, reactive oxygen species (ROS)-induced disorders and disorders associated with iron- imbalance;
  • ROS reactive oxygen species
  • EXAMPLE-7 ll-(4-chlorophenyl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [l,10]phenanthroline-8-carbonitrile (7)
  • EXAMPLE-8 ll-(4-methoxyphenyl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [l,10]phenanthroline-8-carbonitrile (8)
  • the ll'Fe 3+ complex ( ⁇ / ⁇ 561/605 nm) was prepared using ferric perchlorate (10 eq) and 11 (2.5x l0 "J M) in TDW-DMSO. The solvent was removed and analyzed by mass spectrometry suggesting a 2:1 complex formation as perchlorate salt between 11 and Fe 3+ ( Figure 14). The excitation and emisssion spectra of the complex is given in figure 15.
  • Haber-Weiss Cycle The rapid interconversion between the two spin states of iron (Fe and Fe ) associated with Fenton reaction results in the generation of ROS via a catalytic cycle called as Haber Weiss cycle.
  • the high selectivity of 11 for trapping Fe in situ generated during Fenton reaction causes the removal of the catalytic iron thereby attenuating the hazardous ROS cycle.
  • Human HepG2 cells (Origin- derived from the liver tissue of a 15 -year-old Caucasian American male with a well-differentiated hepatocellular carcinoma, Source- ATCC, USA) were cultured in Phenol Red free Low Glucose Dulbecco's Modified Eagle Medium (LG DMEM, Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 ug/ml streptomycin, 2mM 1-glutamine (Sigma,USA), and incubated at 37°C in a humidified atmosphere of 5% C0 2 in air. For experiments, cells were seeded and treated with compounds after 24 hours or when 70% confluence is reached. Confocal microscopic analysis
  • the cells were seeded in a 24-well plate at 1 *10 5 cells/ml and incubated under 5% C0 2 and 95% humidity at 37°C. When 70% confluence was reached, it was treated with Ferric citrate dissolved in Phenol Red free LG DMEM media. After 24 h, 3 ⁇ / 1 ⁇ of dye was added to the wells for 24h and the images of the phase contrast and fluorescence were obtained under a laser scanning microscope LSM 510 META (Carl Zeiss, Jena, Germany). Images were acquired with a 63x Plan Apochromat Oil Phase II 1.4 objective. Lasers used were Diode 405 nm and DPSS 561 nm.
  • control group containing dye was excited at 405 nm and emissions collected within band pass 505-550 nm.
  • the iron loaded group containing dye + Iron were excited at 561 nm and emissions collected with a long pass of 575 nm. Further, florescence intensity was quantified using image J software (Image J, National Institute of Health, Bethesda, MD). Analysis
  • Human HepG2 cells were seeded into 96-well plates at a density of 2> ⁇ 10 cells/well and cultured for 24h. After incubation of the cells with compounds at different concentrations for 48h, the cytotoxicity of the isolated compounds was determined by the MTT assay. Percent cell viability was calculated based on the absorbance measured relative to the absorbance of control cells exposed to the vehicle alone.
  • the wild type strain of nematode C. elegans N2 (var. Bristol), obtained from Caenorhabditis Genetics Center (University of Minnesota, USA), was employed for the studies.
  • the nematodes were maintained in the laboratory on Nutrient Growth Medium (NGM) Agar plates seeded with bacteria E. coli strain OP50 as the food for nematodes. Plates were maintained at 22 °C and each experiment was carried out in synchronized nematode populations for nullifying any effects caused as a result of difference in age/ developmental stages of the nematodes.
  • the synchronization was achieved by isolating embryos from gravid nematodes employing standard axenisation method.
  • NGM plates seeded with E. coli strain OP50 served as culture plates for the control group. The seeded plates were incubated overnight at 22 ° C for culturing of bacteria into a fine lawn. Isolated embryos were added onto these plates and the nematodes were raised on these plates for 48 h in order to obtain an age synchronized population of early-adult worms for further analysis.
  • RNAi induced silencing of gene ftn-l Silencing of ftn-l was achieved by employing RNAi induced gene silencing approach via standard feeding protocol as described previously.
  • the freshly cultured clones were seeded onto NGM plates containing ImM isopropyl ⁇ -D- 1- thiogalactopyranoside (IPTG; Sigma, Cat. No. 16758) and 25mg/L carbenicillin (Sigma; Cat. No. CI 389) and incubated overnight at 22°C for successful induction.
  • IPTG ImM isopropyl ⁇ -D- 1- thiogalactopyranoside
  • carbenicillin Sigma; Cat. No. CI 389
  • Age synchronized worms under study were washed 2 to 3 times using M9 buffer, to remove adhering bacteria and transferred onto 2% agarose padded slides carrying a drop of mounting medium and sealed with a cover slip.
  • the mounting medium was pre-mixed with 100 mM sodium azide (Sigma, Cat no. 71289) which aids in immobilization of the worms without killing them.
  • Imaging of these live immobilized worms was performed using confocal microscope LSM 510 META (Carl Zeiss, Jena, Germany). Images were acquired with a 63x Plan Apochromat Oil Phase II 1 .4 objective.
  • the analysis of unbound/ free dye 11 was carried out with an excitation of 405 ran and emissions collected within band pass 505-550 nm.
  • RNAzol® RT method Sigma, Cat. No. R4533
  • NanoDrop Thermo, Quawell, UV-Vis Spectrophotometer, Q5000.
  • About 5ug of total RNA was used for the synthesis of cDNA using RevertAid First Strand cDNA synthesis kit (Thermo Scientific, Cat. No. #K1622). Quantification of mRNA levels was carried out using SYBR Green (Thermo Scientific Cat. No. #K0251) chemistry.
  • cDNA equivalent to 125ng was amplified in 25ul maxima using Stratagene MX3005P detection system (Agilent Technologies).
  • the program for amplification was, 50°C for 2 minutes 95°C for 10 minutes (1 cycle), followed by 95°C for 30 seconds, 55°C for 30 seconds and 60°C for 30 seconds (40 cycle) and melting curve detection (95°C for 5 sec, 65°C for 1 min).
  • Example of each sample was carried out in duplicate sets. Fold change of all samples were analyzed using comparative 2 " ⁇ 0 ⁇ .
  • Integrated DNA Technologies (IDT) software was used for designing of primers of desired genes, act-1 mRNA was used as endogenous control for normalization.
  • Dye 11 binds to Fe 3+ present in LIP in both wild type and ftn-1 silenced C. elegans
  • dye 11 responded to the elevated levels of endogenous iron after silencing of iron regulatory gene ftn-1 providing a tool to fathom the secrets of LIP.
  • C. elegans gene ftn-1 plays an important role in handling of iron within the system. We, hence, endeavored to study the effect of compound 11 on the expression of ftn-1.

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Abstract

La présente invention concerne de nouvelles dihydronaphtho[2,l-b][l,10]phénanthrolines et naphtho[2,l-b][l,10]phénanthrolines de formule générale I qui peuvent être utilisées potentiellement en tant que sondes fluorescentes en sciences chimiques et biologiques telles que dans des applications d'imagerie cellulaire, des diagnostics, des marqueurs fluorescents, des capteurs d'ions, des produits pharmaceutiques et autres applications utiles, ainsi qu'un procédé de préparation desdits nouveaux composés et de quantification d'espèces d'oxygène réactives. Les composés sont préparés en faisant réagir des lactones, dans des conformations isolées ou rigides, avec une fraction méthylènecarbonyle en présence d'une base dans un solvant organique.
PCT/IN2015/000076 2014-07-11 2015-02-09 Colorants fluorescents à base de naphtho[2,1-b][1,10]phénanthroline substitué et application associée WO2016005992A1 (fr)

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CN105647520A (zh) * 2016-03-23 2016-06-08 中节能万润股份有限公司 一种新型电致发光材料及其应用
CN109312387A (zh) * 2016-06-14 2019-02-05 剑桥显示技术有限公司 用于分析物检测的方法、组合物和传感器

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