WO2016005992A1

WO2016005992A1 - SUBSTITUTED NAPHTHO[2,1-b][1,10]PHENANTHROLINE BASED FLUORESCENT DYES AND APPLICATION THEREOF

Info

Publication number: WO2016005992A1
Application number: PCT/IN2015/000076
Authority: WO
Inventors: Atul Goel; Shahida Umar; Pankaj Nag; Aamir Nazir; Lalit Kumar; Shamsuzzama; Jiaur Rahaman GAYEN; Zakir Hossain
Original assignee: Council Of Scientific And Industrial Research
Priority date: 2014-07-11
Filing date: 2015-02-09
Publication date: 2016-01-14

Abstract

The present invention relates to novel dihydronaphtho[2,l-b][l,10]phenanthrolines and naphtho[2,l-b][l,10]phenanthrolines of the general formula I which can be used potentially as fluorescent probes in chemical and biological sciences such as cell imaging applications, diagnostics, fluorescent tags, ion sensor, pharmaceuticals and other useful applications, and a process of preparing said novel compounds and quantification of reactive oxygen species. The compounds are prepared by reacting lactones in isolated or rigid conformations with methylene carbonyl moiety in the presence of a base in an organic solvent.

Description

SUBSTITUTED NAPHTHO[2,1-6][1,10]PHENANTHROLINE BASED

FLUORESCENT DYES AND APPLICATION THEREOF

FIELD OF THE INVENTION

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.

Particularly, the present invention relates to a compound of formula I their ion chelators and salts thereof:

I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of . hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, C_\-Cg alkyl, C₂-C₈ alkenyl, C_\-C» alkoxyl, C₆-C₂₀ aryl, C₅-C₈ cyclo, C₆-C₂₀ aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C_\-C& alkyl esters, C₆- C₂o aryl esters, C]-C₈ alkylthio, Cj-C₈ alkyl amino, C₆-C₂₀ aryl amino, Q-Cg cycloalkyl amino, C6-C₂o cycloaryl amino, Q-Cg acylamino, Q-Cg acylthio, d-Cg acyl, C6-C₂₀ aroyl, C Cg acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C3-C20 arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂o heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C -C₂o arylthioamido, C₃-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃- C₂o heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S). More particularly, 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.

More particularly, 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.

BACKGROUND OF THE INVENTION 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. Only a fraction of iron is free and exists as cytosolic nonprotein bound labile iron pool (LIP). Transient changes in the LIP status can either be caused locally due to ischaemia or systematically as in case of genetic haemochromatosis or transfusion-induced iron overload. Prooxidant-mediated intracellular reductive stress can also result in reductive iron release from intracellular stores, including ferritin. If the cellular homeostasis of iron gets disturbed its catalytically active labile form triggers Fenton reaction leading to various diseases, such as 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 (Kell, D.B. BMC Medical Genomics, 2009, 2, 1). Fenton reaction involves the generation of hydroxyl radicals from hydrogen peroxide and the formation of aldehydes and lipid peroxy radicals from lipid hydroperoxides. These reactive oxygen species (ROS) interacts with various biological macromolecules such as lipids, proteins, nucleic acids etc causing peroxidative tissue damage (Dixon, S. J.; Stockwell, B. R. Nat. Chem. Biol. 2014, 10, 9).

Commercially available 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. 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). Further, apart from quenching issues, calcein also suffers from selectivity problems not only between the two ionic forms of Fe but also shows significant interference from other divalent metal ions. Similarly other iron probes, Phen green SK (excitation maximum, 507 nm; emission maximum, 532 nm) and Phen green FL (excitation maximum, 492 nm; emission maximum, 517 nm) also respond to both major spin states of iron. Once again, selectivity is a major issue associated with these probes as other metal ions can exhibit a nonspecific turn-off quenching response. To obtain a better selectivity, naturally occurring 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. 1,10-Phenanthroline is characterized by a weak fluorescence quantum yield due to close lying π- π * and n- π* singlet excited states. Substantial efforts have been made to improve its emission efficiency as novel chelating ligands by introducing aromatic substituents at various positions. We aimed to introduce donor-acceptor and chromophore groups to develop fluorescent dihydronaphtho[2,l-i>][l,10]phenanthrolines and naphtho[2,l- 6][l,10]phenanthrolines operating on the classical push-pull system.

In the present invention 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. OBJECT OF THE INVENTION

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.

SUMMARY OF THE INVENTION 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 ¾nd processes of preparing said novel compounds;

The present invention more particularly relates to a compound of formula I:

I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Ci-C₈ alkyl, C₂-C₈ alkenyl, d-Cg alkoxyl, C₆-C₂o aryl, C₅-C₈ cyclo C₆-C₂o aryl, C3-C8 cycloalkyl, C3-C20 heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C3-C₂o cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Q-Cg alkyl esters, C₆-C₂₀ aryl esters, Ci-C₈ alkylthio, Ci-Cg alkyl amino, C6-C20 aryl amino, Ci-Cg cycloalkyl amino, C₆- C₂₀ cycloaryl amino, Ci-C acylamino, Ct-Cg acylthio, Ci-Cg acyl, C₆-C₂o aroyl, Ci-C₈ acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C3-C2₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C3-C20 heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₅-C₂₀ arylthioamido, C3-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S),

More particularly, 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. 11 -(naphthalen-2-yl)-9-(piperidin- 1 -yl)- 12, 13-dihydronaphtho[2, 1 - b][ 1 , 10]phenanthroline-8-carbonitrile (3). iv. 1 1 -(biphenyl-4-yl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [ 1 , 10]phenanthroline-8-carbonitrile (4). v. 9-(piperidin- 1 -yl)- 1 1 -(pyren- 1 -yl)- 12,13 -dihydronaphtho[2, 1 -b] [ 1 , 1 Ojphenanthroline- 8-carbonitrile (5). vi. 1 1 -(4-bromophenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [ 1 , 10]phenanthroline-8-carbonitrile (6). vii. 11 -(4-chlorophenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [1 , 10]phenanthroline-8-carbonitrile (7). viii. 1 l-(4-methoxyphenyl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [1 , 10]phenanthroline-8-carbonitrile (8).

11 -(4-hydroxyphenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho[2, 1 - b] [ 1 , 10]phenanthroline-8-carbonitrile (9).

11 -(biphenyl-4-yl)-9-(piperidin- 1 -yl)naphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (10).

11 -(4-bromophenyl)-9-(piperidin- 1 -yl)naphtho [2, 1 -b] [ 1 , 10]phenanthroline-8 carbonitrile (11). xii. 11 -(4-chlorophenyl)-9-(piperidin- 1 -yl)naphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (12).

11 -(4-methoxyphenyl)-9-(piperidin- 1 -yl)naphtho [2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (13)

11 -(4-hydroxyphenyl)-9-(piperidin- 1 -yl)naphtho [2, 1 -b] [ 1 , 10]phenanthroline-8 carbonitrile (14)

9-(4-bromophenyl)- 11 -(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [ 1 , 10]phenanthroline- 10-carbonitrile (15)

In one embodiment of the invention, 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;

In another embodiment of the invention, the compounds are having excitation maxima in the range of 330-700 nm and emission maxima in the range of 450-900 nm; In yet another embodiment, 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;

In a further embodiment 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:

Process 1

a. reacting a compound having general formula S-1 with a compound having general formula S-2 to furnish a compound having the general formula I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Cj-Cg alkyl, C₂-C₈ alkenyl, Ci-C₈ alkoxyl, C₆-C₂₀ aryl, C₅-C cyclo C₆-C₂o aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃- C₂o cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Ct-Cg alkyl esters, C6-C₂₀ aryl esters, Ci-C₈ alkylthio,

alkyl amino, C₆-C₂₀ aryl amino, d-C₈ cycloalkyl amino, C6-C₂₀ cycloaryl amino, C Cg acylamino, Cj-Cg acylthio, Ci-C₈ acyl, C₆-C₂₀ aroyl, Ct-Cg acyloxy, amido, thioamido, C₅-C2₀ arylamido, C₃-C₂₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₅-C₂₀ arylthioamido, C3-C20 arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C3-C20 heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), wherein, the reaction may proceed in a common organic solvent particularly DMF, THF, DMSO, DCM, isopropanol in the presence of a base particularly KOH, NaOH, NaH, KH, K₂C0₃, Cs₂C0₃ at a temperature ranging between -78°C to 150°C for a period ranging between 1 minute to 3 days, followed by oxidation in presence of an oxidant in organic solvent; b. oxidizing the isolated compound of general formula I in the presence of DDQ or different palladium catalysts without any external ligand in a common organic solvent particularly DMF, THF, DMSO, 1,4-dioxane, toluene at a temperature ranging between -78°C to 150°C for a period ranging between 1 minute to 3 days; c. isolating the compound of general formula I from the reaction mixture and purifying by chromatographic techniques;

Process 2

a. reacting a compound having general formula S-4 with a compound having general formula S-3 to furnish a compound having the general formula I wherein ¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, C C₈ alkyl, C₂-C₈ alkenyl, Ci-Cs alkoxyl, C₆-C₂₀ aryl, C₅-C₈ cyclo C -C₂₀ aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, 0 and S), C₃- C₂₀ cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Ci-Cs alkyl esters, C₆-C₂o aryl esters, Ci-Cg alkylthio, Ci-Cg alkyl amino, C -C₂₀ aryl amino, C_\-C_& cycloalkyl amino, Q-C₂o cycloaryl amino, C]-C₈ acylamino, Cj-Cg acylthio, Ci-Cg acyl, C₆-C₂₀ aroyl, Q-Cg acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C₃-C₂₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C3-C20 heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C5-C20 arylthioamido, C3-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C3-C20 heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), wherein, the reaction may proceed in a common organic solvent particularly DMF, THF, DMSO, DCM, isopropanol in the presence of a base particularly KOH, NaOH, NaH, KH, K2CO3, Cs₂C0₃ at a temperature ranging between -78°C to 150°C for a period ranging between 1 minute to 3 days, followed by oxidation in presence of an oxidant in organic solvent; b. oxidizing the isolated compound of general formula I in the presence of DDQ or different palladium catalysts without any external ligand in a common organic solvent particularly DMF, THF, DMSO, 1,4-dioxane, toluene at a temperature ranging between -78oC to 150oC for a period ranging between 1 minute to 3 days; c. isolating the compound of general formula I from the reaction mixture and purifying by chromatographic techniques; wherein process 1 is independent of process 2;

In a further embodiment, 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;

In another embodiment 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;

In a further embodiment, 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;

BRIEF DESCRIPTION OF THE DRAWINGS The present invention may be more clearly understood by reference to the following Table/Figures:

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 3 Emission spectra of compound 11 in solvents of varying polarity (solvatochromism).

Figure 4 Absorbance spectra of 10 towards increasing concentration of Fe³⁺ ions (0-6.25 x lO^ M).

Figure 5 Fluorescence spectra of 10 towards increasing concentration of Fe³⁺ (0-6.25 ^χ 10^"4 M). Figure 6 Absorption spectra of 11 towards increasing concentration of Fe³⁺ions (0-6.8 ^χ 10 M).

Figure 7 Fluorescence spectra of 11 towards increasing concentration of Fe ions (0-6.8 ^χ lO^ M).

Figure 8 Absorption spectra of 12 towards increasing concentration of Fe 3+ ions (0-6.25 x 10^"4 M).

Figure 9 Fluorescence spectra of 12 towards increasing concentration of Fe³⁺ ions (0-6.25 10^"4 M).

Figure 10 Absorption spectra of 13 towards increasing concentration of Fe 3+ ions (0-6.25 x \0 M).

Figure 11 Fluorescence spectra of 13 towards increasing concentration of Fe³⁺ ions (0-6.25 lO^ M).

Figure 12 Selective colorimetric response of 11 towards Fe 3+ in visible light.

Figure 13 Selective fluorescence response of 11 towards Fe in UV light (254nm).

Figure 14 Probable complex structure of the salt of chelator 11 and Fe³⁺.

Figure 15 Excitation and emission spectra (at 561 nm excitation) of ll'Fe³⁺ complex.

Figure 16 Increase in the fluorescence intensity ratio (Ι₆₀₅/Ι₅₄₄) of 11 upon addition of different metal ions.

Figure 17 Fluorescence response of calcein (cal, 2.5 10^" M) towards Fe (l.leq).

Figure 18 Fluorescence response of 11 (2.5 10^"5 M) towards Fe³⁺ (5 10^"4 M). Figure 19 Ratiometric fluorescence response of 11 in the presence of a) H2O2 and b) 'BuOOH with increasing Fe²⁺ (0-5.8 ^χ 10^"4 M).

Figure 20 Quantification of Fe generated in situ from Fenton reagent (Η₂0₂ and Fe ) with a Fe³⁺ standard (5 10"* M).

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 H₂0₂.

Figure 23 Confocal fluorescence images of HepG2 cells incubated with dye 11 only (a, ) and cells preincubated with Fe³⁺ then with dye 11 (c, d). (a) and (c) Green channel images, λβ_Χ = 405 nm, e_m = 505-550 nm. (b) and (d) Red channel, λε_Χ = 561 nm, λβ,η = 575 nm Long Pass.

Figure 24 shows cell viability assessment of dye 11 in Human HepG2 cells.

Figure 25 (a-b) Wild type (N2) C. elegans treated with dye 11 (3 μΜ). (c-d) ftn-1 silenced C. elegans treated with 3 μΜ dye 11. (a) and (c) Green channel images, «_x = 405 nm, e_m = 505-550 nm. (b) and (d) Red channel, λβ_Χ = 561 nm, X«_m = 575 nm Long Pass. Scale bar 10

Figure 26 The expression of C. elegans gene ftn-1 when treated with dye 11.

ABBREVIATIONS

DMEM Dulbecco's Modified Eagle's Medium

DIC Differential Interference Contrast

TDW Triple Distilled Water

'BuOOH tert-butyl hydrogen peroxide

DETAILED DESCRIPTION OF THE INVENTION In one embodiment, 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:

I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Ct-C₈ alkyl, C₂-C8 alkenyl, Cj-Cs alkoxyl, C6-C₂₀ aryl, C₅-C₈ cyclo C₆-C₂₀ aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Ci-C₈ alkyl esters, C₆-C₂₀ aryl esters, Ci-C₈ alkylthio, Ci-C₈ alkyl amino, C₆-C₂o aryl amino, Ci-C₈ cycloalkyl amino, C₆- C₂o cycloaryl amino,

acylthio, acyl, C₆-C2₀ aroyl, Q-Q acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C₃-C₂₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C5-C20 arylthioamido, C₃-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S),

More particularly, 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).

1 1 -(naphthalen-1 -yl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2, 1 - b] [1 , 10]phenanthroline-8-carbonitrile (2).

11 -(naphthalen-2-yl)-9-(piperidin- 1 -yl)- 12, 13 -dihydronaphtho [2, 1 - b] [ 1 , 10]phenanthroline-8-carbonitrile (3).

11 -(biphenyl-4-yl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [1 , 10]phenanthroline-8-carbonitrile (4).

9-(piperidin- 1 -yl)- 11 -(pyren- 1 -yl)- 12,13-dihydronaphtho[2, 1 -b] [1 , 10]phenanthroline- 8-carbonitrile (5).

11 -(4-bromophenyl)-9-(piperidin- 1 -yl)- 12,13-dihydronaphtho[2, 1 - b] [1 , 10]phenanthroline-8-carbonitrile (6).

11 -(4-chlorophenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [ 1 , 10]phenanthroline-8-carbonitrile (7).

11 -(4-methoxyphenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [1 , 10]phenanthroline-8-carbonitrile (8).

1 1 -(4-hydroxyphenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b] [ 1 , 10]phenanthroline-8-carbonitrile (9).

11 -(biphenyl-4-yl)-9-(piperidin- 1 -yl)naphtho [2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (10).

1 1 -(4-bromophenyl)-9-(piperidin- 1 -yl)naphtho [2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (11).

1 1 -(4-chlorophenyl)-9-(piperidin- 1 -yl)naphtho[2, 1 -b] [1 , 10]phenanthroline-8- carbonitrile (12).

1 1 -(4-methoxyphenyl)-9-(piperidin- 1 -yl)naphtho [2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (13). xiv. 11 -(4-hydroxyphenyl)-9-(piperidin-l -yl)naphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (14). xv. 9-(4-bromophenyl)-l l-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [ 1 , 1 OJphenanthroline- 10-carbonitrile (15)

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;

More particularly, the compounds of the present invention, their ion chelators and salts thereof 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;

In another embodiment of the present invention, 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:

Process 1

a. reacting a compound having general formula S-1 with a compound having general formula S-2 to furnish a compound having the general formula I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1}, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Ci-C₈ alkyl, C₂-C₈ alkenyl, C C₈ alkoxyl, C₆-C₂₀ aryl, C₅-C₈ cyclo C₆-C₂₀ aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃- C₂o cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Q-Cs alkyl esters, C₆-C₂₀ aryl esters, Ci-Cg alkylthio, CrC₈ alkyl amino, C6-C₂₀ aryl amino, Q-Cg cycloalkyl amino, C₆-C₂o cycloaryl amino,

acylamino, Ci-C₈ acylthio, C C₈ acyl, C₆-C₂₀ aroyl, Q-Cs acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C₃-C₂₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C5-C20 arylthioamido, C3-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), wherein, the reaction may proceed in a common organic solvent particularly DMF, THF, DMSO, DCM, isopropanol in the presence of a base particularly KOH, NaOH, NaH, KH, K₂C0₃, Cs₂C0₃ at a temperature ranging between -78°C to 150°C for a period ranging between 1 minute to 3 days; b. oxidizing the isolated compound of general formula I in the presence of DDQ or different palladium catalysts without any external ligand in a common organic solvent particularly DMF, DMSO, 1,4-dioxane, toluene at a temperature ranging between - 78°C to 150°C for a period ranging between 1 minute to 3 days; c. isolating the compound of general formula I from the reaction mixture and purifying by chromatographic techniques;

Process 2

a. reacting a compound having general formula S-4 with a compound having general formula S-3 to furnish a compound having the general formula I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, C_\-C& alkyl, C₂-C₈ alkenyl, d-C₈ alkoxyl, C₆-C₂₀ aryl, C₅-C₈ cyclo C6-C₂₀ aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃- C₂o cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C C₈ alkyl esters, C₆-C₂₀ aryl esters, Q-C₈ alkylthio, Ci-C₈ alkyl amino, C₆-C₂₀ aryl amino, Cj-Cg cycloalkyl amino, C6-C₂₀ cycloaryl amino, Q-Cg acylamino, Ci-Q acylthio, C!-C₈ acyl, C₆-C₂o aroyl, Ci-C₈ acyloxy, amido, thioamido, C₅-C₂o arylamido, C₃-C₂₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C5-C₂0 arylthioamido, C3-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, 0 and S), C₃-C₂₀ heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), wherein, the reaction may proceed in a common organic solvent particularly DMF, THF, DMSO, DCM, isopropanol in the presence of a base particularly KOH, NaOH,

NaH, KH, K2CO3, Cs₂C0₃ at a temperature ranging between -78°C to 150°C for a period ranging between 1 minute to 3 days; b. oxidizing the isolated compound of general formula I in the presence of DDQ or different palladium catalysts without any external ligand in a common organic solvent particularly DMF, DMSO, 1,4-dioxane, toluene at a temperature ranging between - 78°C to 150°C for a period ranging between 1 minute to 3 days; c. isolating the compound of general formula I from the reaction mixture and purifying by chromatographic techniques; wherein process 1 is independent of process 2; In a further embodiment, 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).

In one embodiment, 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.

In another embodiment, 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.

In a yet another embodiment, 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;

Synthesis:

EXAMPLES:

Following examples are given by way of illustration and should not construe the scope of the present invention.

EXAMPLE ! ll-phenyl-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l-A][1 0]phenanthroline-8- carbonitrile (1)

A mixture of 2-oxo-6-phenyl-4-(piperidin-l -yl)-2H-pyran-3-carbonitrile having general formula S-l (280 mg, 1 mmol, 1 equiv.), 10,1 l-dihydrobenzo[6][l ,10]phenanthrolin-8(9H)- one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), and NaH (60% dispersion in oil, 1.5 mmol, 1.5 equiv.) in dry DMF (5 mL) was stirred at 25°C for 15 min. On completion the reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HC1. The precipitate obtained was filtered and purified on a silica gel column with 0.5% methanol in chloroform as the eluent to afford 350 mg (75%) as a yellow solid; R_f = 0.56 (chloroform/methanol, 20: 1 , v/v); mp (chloroform/methanol) 266-268 °C; MS (ESI) 467 [M + H⁺];

EXAMPLE-2 ll-(naphthalen-l-yl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [l,10]phenanthroline-8-carbonitrile (2)

A mixture of 6-(naphthalen-l-yl)-2-oxo-4-(piperidin-l-yl)-2H-pyran-3-carbonitrile having general formula S-l (330 mg, 1 mmol, 1 equiv.), 10,1 l-dihydrobenzo[6] [1,1 OJphenanthrolin- 8(9H)-one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), and NaH (60% dispersion in oil, 1.5 mmol, 1.5 equiv) in dry DMF (5 mL) was stirred at 27°C for 20 min. On completion the reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HCl. The precipitate obtained was filtered and purified on a silica gel column with 0.5% methanol in chloroform as the eluent to afford 410 mg (79%) as a yellow solid; R_f = 0.53 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 517[M + H⁺];

EXAMPLE-3 ll-(naphthalen-2-yl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b][l,10]phcnanthroline-8-carbonitrile (3)

A mixture of 6-(naphthalen-2-yl)-2-oxo-4-(piperidin-l-yl)-2H-pyran-3-carbonitrile having general formula S-l (330 mg, 1 mmol, 1 equiv.), 10,l l-dihydrobenzo[b][l,10]phenanthrolin- 8(9H)-one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), and NaH (60% dispersion in oil, 1.5 mmol, 1.5 equiv.) in dry DMF (5 mL) was stirred at 25°C for 15 min. On completion reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HCl. The precipitate obtained was filtered and purified on a silica gel column with 0.5% methanol in chloroform as the eluent to afford 390 mg (76%) as a yellow solid; R_f = 0.50 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 517 [M + H⁺].

EXAMPLE-4 ll-(biphenyI-4-yl)-9-(piperidin-l-yl)-12 3-dihydronaphtho[2 -A][1 0]phenanthroIine- 8-carbonitrile (4)

A mixture of 6-(biphenyl-4-yl)-2-oxo-4-(piperidin-l-yl)-2H-pyran-3-carbonitrile having general formula S-l (356 mg, 1 mmol, 1 equiv.), 10,1 l-dihydrobenzo[6] [1,1 OJphenanthrolin- 8(9H)-one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), KH(60% dispersion in oil, 1.5 mmol, 1.5 equiv.) in dry DMF (5 mL) was stirred 27°C for 20 min. On completion the reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HCl. The precipitate obtained was filtered and purified on a silica gel column with 0.5% methanol in chloroform as the eluent to afford 450 mg (83%) as a yellow solid; R_f = 0.55 (chloroform/methanol, 20:1 , v/v); mp (chloroform/methanol) 176-178 °C; MS (ESI) 543 [M + H⁺].

EXAMPLE S

9-(piperidin-l-yI)-ll-(pyren-l-yl)-12,13-dihydronaphtho[2,l-6] [l,10]phenanthroline-8- carbonitrile (5)

A mixture of 2-oxo-4-(piperidin-l-yl)-6-(pyren-l-yl)-2H-pyran-3-carbonitrile having general formula S-l (404 mg, 1 mmol, 1 equiv.), 10,l l-dihydrobenzo[0][l,10]phenanthrolin-8(9H)- one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), and NaH(60% dispersion in oil, 1.5 mmol, 1.5 equiv.) in dry THF (5 mL) was stirred at 25°C for 15 min. On completion the reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HCl. The precipitate obtained was filtered and purified on a silica gel column with 0.8% methanol in chloroform as the eluent to afford 450 mg (76%) as a yellow solid; R_f = 0.52 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 591 [M + H⁺];

EXAMPLE-6 ll-(4-bromophenyI)-9-(piperidin-l-yI)-12,13-dihydronaphtho[2,l- b] [l,10]phenanthroline-8-carbonitrile (6)

A mixture of 6-(4-bromophenyl)-2-oxo-4-(piperidin-l-yl)-2H-pyran-3-carbonitrile having general formula S-l (359 mg, 1 mmol, 1 equiv.), 10,1 l -dihydrobenzo[6] [1,1 OJphenanthrolin- 8(9//)-one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), and KH(60% dispersion in oil, 1.5 mmol, 1.5 equiv.) in dry DMF (5 mL) was stirred at 25°C for 10 min. On completion the reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HCl. The precipitate obtained was filtered and purified on a silica gel column 1% methanol in chloroform as the eluent to afford 410 mg (75%) as a yellow solid; Rf = 0.45 (chloroform/methanol, 20: 1 , v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 545 [M + H⁺].

EXAMPLE-7 ll-(4-chlorophenyl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [l,10]phenanthroline-8-carbonitrile (7) A mixture of 6-(4-chlorophenyl)-2-oxo-4-(piperidin-l-yl)-2H-pyran-3-carbonitrile having general formula S-l (315 mg, lmmol, 1 equiv.), 10,1 l-dihydrobenzo[6] [1,1 Ojphenanthrolin- 8(9H)-one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), and NaH(60% dispersion in oil, 1.5 mmol, 1.5 equiv.) in dry DMF (5 mL) was stirred at 25°C for 15 min. On completion the reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HC1. The precipitate obtained was filtered and purified on a silica gel column with 1% methanol in chloroform as the eluent to afford 395 mg (79%) as a yellow solid; R_f = 0.46 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 501 [M + H⁺]. EXAMPLE-8 ll-(4-methoxyphenyl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [l,10]phenanthroline-8-carbonitrile (8) A mixture of 6-(4-methoxyphenyl)-2-oxo-4-(piperidin-l-yl)-2H-pyran-3-carbonitrile having general formula S-l (310 mg, lmmol, 1 equiv.), 10,1 l-dihydrobenzo[&] [1,10]phenanthrolin- 8(9H)-one having general formula S-2 (248 mg, 1 mmol, 1 equiv.), and ΚΟΗ (1.5 mmol, 1.5 equiv.) in dry THF (5 mL) was stirred at 25°C for 6 hr. On completion the solvent was evaporated and added ice cooled water with vigorous stirring and finally neutralized with 10% HC1. The precipitate obtained was filtered and purified on a silica gel column with 1% methanol in chloroform as the eluent to afford 400 mg (80%) as a yellow solid; R_f = 0.47 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) 180 °C; MS (ESI) 497 [M + H ; EXAMPLE-9 ll-(4-hydroxyphenyl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [1 ,1 Ojphenanthroline-8-carbonitrile (9) 11 -(4-methoxyphenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2, 1 -b] [ 1 , 10]phenanthroline- 8-carbonitrile (496mg, 1 mmol, lequiv.) was dissolved in dry CH2CI2 (10 ml) and BBr₃ ( 2 mmol, 2 equiv.) was added to the solution at -78°G. The reaction mixture was stirred for 50 min at -78°C and then 12 hours at 27°C. After completion, the reaction mixture was quenched with ice-cooled water and neutralized with 10% HC1. The reaction mixture was then extracted with CH₂C1₂ and the organic layer separated, washed with brine, dried over anhydrous Na₂S04, and concentrated. The residue was purified by silica gel column chromatography using 1.5% methanol in chloroform as the eluent to afford 390mg (81%) yield as a yellow solid; R_f = 0.53 (chloroform/methanol, 16:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 483 [M + H⁺] ;

EXAMPLE-10

11 -(bipheny l-4-yl)-9-(piperidin-l -y l)naphtho [2,1 -b] [ 1 ,10] phenanthroline-8-carbonitrile (10)

Compound 11 -(biphenyl-4-yl)-9-(piperidin- 1 -yl)- 12, 13 -dihydronaphtho [2, 1 - b][l,10]phenanthroline-8-carbonitrile (543mg, 1 mmol,leq.) was refluxed in 1,4-dioxan (5 ml) at 110°C for 1 hr. with DDQ(2 mmol, 2equiv.). On completion the reaction was cooled to 27°C and then was filtered. The filterate was collected and solvent was evaporated. Finally the residue was purified by silica gel column chromatography using 0.5% methanol in chloroform as the eluent to afford 200 mg (37%) as an orange solid; R_f — 0.58 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 541 [M + H⁺];

EXAMPLE-11 ll-(4-bromophenyl)-9-(piperidin-l-yl)naphtho[2,l-^][l_»i0]phenanthroline-8- carbonitrile (11)

11 -(4-bromophenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (545 mg, 1 mmol, 1 equiv.) was heated in DMSO (5 mL) at 100°C for 12 hr. with Pd(OAc)₂(0.5 mmol, 0.5 equiv.). After completion, ice-cooled water was added to the reaction mixture and then extracted with CH₂C1₂ and the organic layer separated, washed with brine, dried over anhydrous Na₂S0₄, and concentrated. Finally the residue was purified by silica gel column chromatography using 1% methanol in chloroform as the eluent to afford 345 mg (63%) as an orange solid. R_f = 0.52 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 543 [M + H⁺];

EXAMPLE-12 ll-(4-Qhlorophenyl)-9-(piperidin-l-yl)naphtho[2,l-6] [l,10]phenanthroline-8- carbonitrile (12)

11 -(4-chlorophenyl)-9-(piperidin- 1 -yl)- 12,13-dihydronaphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (501 mg, 1 mmol, 1 equiv.) was heated in DMSO (5 mL) at 100°C for 12 hr. with Pd(TFA)₂ (0.5 mmol, 0.5 equiv.). After completion, ice-cooled water was added to the reaction mixture and then extracted with CH₂Cl₂ and the organic layer separated, washed with brine, dried over anhydrous Na₂S0₄, and concentrated. Finally the residue was purified by silica gel column chromatography using 1% methanol in chloroform as the eluent to afford 360 mg (72%) as an orange solid; R_f = 0.50 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 499 [M + H⁺]; EXAMPLE-13 ll-(4-methoxyphenyl)-9-(piperidin-l-yl)naphtho[2,l-6] [l,10]phenanthroline-8- carbonitrile (13) 11 -(4-methoxyphenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2, 1 -b] [ 1 , 10]phenanthroline- 8-carbonitrile (497 mg, 1 mmol, 1 equiv.) was refluxed in 1,4-dioxane (5 mL) at 1 10°C for 1 hr. with DDQ (2 mmol, 2 equiv.). On completion the reaction was cooled to 27°C and then was filtered. The filterate was collected and solvent was evaporated. Finally the residue was purified by silica gel column chromatography using 1% methanol in chloroform as the eluent to afford 180 mg (36%) as an orange solid R_f = 0.53 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 495 [M + H⁺].

EXAMPLE-14 11 -(4-hy droxy pheny l)-9-(piperidin-l-y l)nap htho [2, 1 -b] [ 1 , 10] phenanthrolinc-8- carbonitrile (14)

1 1 -(4-methoxyphenyl)-9-(piperidin- 1 -yl)naphtho [2, 1 -b] [ 1 , 10]phenanthroline-8-carbonitrile (494mg, 1 mmol, lequiv.) was dissolved in dry DCM (10 ml) and BBr3 ( 2 mmol, 2 equiv.) was added to the solution at -78°C. The reaction mixture was stirred for 50 min at -78°C and then 12 hours at 25°C. After completion, the reaction mixture was quenched with ice-cooled water and neutralized with 10% HC1. The reaction mixture was then extracted with CH₂C1₂ and the organic layer separated, washed with brine, dried over anhydrous Na₂S0₄, and concentrated. The residue was purified by silica gel column chromatography using 2% methanol in chloroform as the eluent to afford 380mg (79%) yield as yellow solid R_f = 0.56 (chloroform/methanol, 16:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 481 [M + H ; EXAMPLE-15

9-(4-bromophenyl)-ll-(piperidin-l-yl)naphtho[2,l-^][ljl0]phenanthroline-10- carbonitrile (15) A mixture of 2-oxo-4-(piperidin- 1 -yl)-5,6-dihydro-2H-chromeno[7,8-0] [1,10]phenanthroline- 3-carbonitrile having general formula S-4 (408mg, lmmol, lequiv.), l-(4- bromophenyl)ethanone having general formula S-3 (199mg, lmmol, lequiv.) and KH(60% dispersion in oil, 1.5 mmol, 1.5 equiv.) in dry DMF (5 mL) was stirred at 25°C for 15 min. On completion the reaction mixture was poured onto crushed ice with vigorous stirring and finally neutralized with 10% HC1. The precipitate obtained was filtered and purified on a silica gel column with 1% methanol in chloroform as the eluent to afford 109 mg (20%) as an orange solid; Rf = 0.52 (chloroform/methanol, 20:1, v/v); mp (chloroform/methanol) >290 °C; MS (ESI) 543 [M + H⁺]; Preparation of perchlorate salt of chelator ll'Fe³⁺ complex

The ll'Fe³⁺ complex (λβχ/λεη 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 Fe³⁺(Figure 14). The excitation and emisssion spectra of the complex is given in figure 15.

Photophysical studies of the compounds of general formula I

The photophysical properties of all the synthesized compounds 1-15 were examined by UV- vis and fluorescence techniques. Table 1 showed quantum yields in TDW:DMSO, 9:1 (v/v) and DMSO. These compounds produced different color emissions depending upon the nature and position of electron donor-acceptor substituents and chromophores attached on phenanthroline scaffolds (Figure 2).

Table 1. Photophysical properties of phenanthrolines (Examples 1-15) entry

Water³ DMSO"

1 0.053 0.30

2 0.0528 0.39

3 0.0456 0.42

4 0.09 0.66

5 0.024 0.38

6 0.0274 0.43

7 0.026 0.50

8 0.08 0.48

9 0.036 0.49

10 0.029 0.146

11 0.014 0.11

12 0.026 0.18

13 0.025 0.12

14 0.01 0.15

15 - -

Fluorescence quatum yield (cp_f) in

aTDW:DMSO, 9:1 (v/v), ^bDMSO

relative to harmine in 0.1 M H2S04 as a

standard (φ = 0.45). Colo rim etric and ratiometric fluorescence response of dyes towards Fe The iron-chelating studies of all the representative compounds having general formula I in the presence of perchlorate salts of Fe²⁺ and Fe³⁺ revealed that compounds 10, 11, 12, 13 exhibited a visible color change selectively on binding with Fe³⁺. On gradual addition of Fe³⁺ ions a new absorption peak emerged at 580 run in the absorption spectra of the dyes (Figure 4, 6, 8, 10). Such a large red shift in the absorption behavior of the iron chelators (2.5xl0^"5 M, λ«χ =365 nm) changed the solution from colourless to pink, suggesting colorimetric detection of Fe³⁺ with the "naked eye" (Figure 12). In agreement to the absorption spectra the fluorescence emission exhibited a ratiometric shift to 605 nm (Figure 5, 7, 9, 11) suggesting the ratiometric fluorescence detection of Fe by the iron chelators 10, 11, 12, and 13 (Figure 13).

Selectivity of the dye towards different metal ions To investigate the ratiometric sensing selectivity of the dye l l(2.5xl0^"5 M, λβ_Χ =365 nm), other metal ions such as Li⁺, Na⁺, K⁺, Cs⁺, Mg²⁺, Co²⁺, Ca²⁺, Ba²⁺, Fe²⁺, Ni ⁺, Cu²⁺, Mn²⁺, Al³⁺, Cr³⁺, Zn²⁺, Hg²⁺, Pb²⁺, Cd²⁺ were also screened under the same conditions(5xl0^"4 M each). Only Fe³⁺ induced a dramatic increment of emission ratio I₆o₅ I 44, while others were either silent or triggered minor changes. None of them interfered with the ratiometric fluorescence response towards Fe 3+ (Figure 16).

Comparative study of naphtho-phenanthroline 11 and commercial probe calcein

Comparative study of naphtho-phenanthroline 11 and commercial probe calcein revealed that calcein exhibits a major turn off /quenching response upon addition of Fe³⁺ (Figure 17) while a dual colorimetric as well as ratiometric fluorescence response was observed in case of 11 selectively for Fe³⁺ (Figure 18).

Application of phenanthroline dye in Fenton Chemistry and method for detection/quantification of ROS

Fascinated by the remarkable ratiometric selectivity of dyes towards Fe³⁺ in the presence of Fe²⁺, we endeavored to explore the selectivity and sensitivity of dye to detect the Fe³⁺ expected to be generated in situ via Fenton reaction between Fe²⁺ and H2O2. Despite its obvious importance, the mechanism of Fenton's reaction is not fully understood. Further considering the strong ratiometric selectivity of dye 11 for Fe³⁺ over Fe²⁺ it was tested for in situ binding of Fe³⁺ during Fenton reaction. Gradual addition of Fe²⁺ (10^"5-10^-4 M) ions in the solution containing dye (2.5xl0^"5 M) and Η₂0₂ ΐ¼00Η (lO^-lO^-4 M) showed the desired ratiometric fluorescence response with a visible colour change at 365nm excitation maxima (Figure 19) thereby proving its sensitivity and application as fluorescent tool in Fenton chemistry and associated ROS generation. Furthermore, based on the ratiometric response of

3+ 3+

dye 11 , a ratiometric curve (I605 I544 vs [Fe ]) is generated using Fe standards of known

3Ί^~

concentration to quantify the unknown levels of Fe and free radicals (ROS) generated during Fenton reaction (Figure 20). Considering the sensitivity of Fe and Fe in reducing

3 2

environment, the dye was tested to detect the conversion of Fe into Fe in the presence of hydroxylamine hydrochloride. No response was observed in this case as hydroxylamine hydrochloride reduced Fe to Fe (Figure 21). Method for detecting and estimating peroxides using the ion chelator 11 having general formula I using Fenton reaction

Considering the biological oxidative stress conditions, we varied the levels of H₂0₂ been

2+

added to the solution of 1 1 and Fe and increasing ratiometric response was observed (Figure 22). In order to detect and estimate the peroxides, a ratiometric curve (I o5 I₅44 vs [H₂0₂]) of chelator 11 in the presence of a known concentration of Fe²⁺is generated using standards of H₂0₂ of known concentration in order to quantify the unknown levels of H₂0₂/other organic peroxides. Treatment of iron/ROS associated disorders

R

ROO^* ROOH

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. The potential of 11 to capture Fe³⁺ generated in situ during Fenton cycle and the biological compatibility of the phenanthroline dye (in both in vitro HepG2 cells and in vivo model C.elegans) thereby proves the wide therapeutic potential of this dye in diseases associated to iron imbalance/ Fenton associated ROS generation such as haemochromatosis, thalessemia, diabetes, osteoporosis, cancer, central nervous system disorders, cardiovascular disorders, tuberculosis, reproductive health disorders, Alzheimer, Parkinson's, and other ROS-induced disorders and disorders associated with iron-imbalance.

Biological Evaluation

Cell culture of HepG2 cells

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% C0₂ 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 *10⁵ cells/ml and incubated under 5% C0₂ 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 μΜ / 1 μΜ 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. The 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

The images saved from the microscope were analyzed with the LSM Image Examiner software (Carl Zeiss). Cell viability assay

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.

Results When HepG2 cells were incubated with only dye intense fluorescence was seen in the green channel and no fluorescence was observed in the red channel. However, the cells pretreated with Fe ions when incubated with dye, intense fluorescence in red channel and very weak fluorescence in the green channel was observed. Therefore, the live cell imaging studies indicated that dye is cell permeable and suitable for selective ratiometric imaging of Fe³⁺ in living systems (Figure 23). In summary, a new class of live cell permeant, solvatochromic (Figure 3) dye was identified, which exhibited strong and selective Fe sensing in in vitro live Human HepG2 cells. The live cell imaging studies clearly demonstrated that dye is a specific and non-toxic Fe³⁺ molecular bioprobe. Cell viability experiments revealed that Compound 11 is non toxic under the studied conditions (Figure 24).

C. elegans strains and maintenance

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.

Treatment of nematodes with dye and/or exogenous Fe³⁺ Dye 11 or Ferric citrate (FC) was mixed with OP50 strain of E.coli to obtain a final concentration of 3 μΜ and 25 mM respectively, followed by seeding onto NGM plates. 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. We used bacterial clone, specific for ftn-l, from the Ahringer RNAi library that was purchased from SA Biosciences. These clones expressing ftnl specific dsRNA were cultured overnight at 37°C, in Luria Bertani Broth (Himedia, Cat. No. Ml 245) containing 50ug/ml ampicillin(Sigma, Cat. No. AO 166). The freshly cultured clones were seeded onto NGM plates containing ImM isopropyl β-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.

Confocal Microscopy Analysis

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. Further the analysis of compound bound/complexed to iron was carried out with an excitation of 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). Studies on expression of iron response gene (ftnl) using Real-time PCR (qPCR)

Age synchronized N2 worms of various groups were washed twice with 0.2% DEPC (Sigma, Cat. No.-D5758) treated water to remove adhering bacteria, following which total RNA was isolated using RNAzol® RT method (Sigma, Cat. No. R4533), quantified through 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. In brief 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).Experiment of each sample was carried out in duplicate sets. Fold change of all samples were analyzed using comparative 2^"ΛΔ0Τ. 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 Fe³⁺ present in LIP in both wild type and ftn-1 silenced C. elegans

In an attempt for the first time to detect LIP 'directly' in the organism, we endeavored to examine in vivo iron binding specificity of dye 1 1 in wild type (N2) C. elegans by confocal fluorescence imaging experiments (Figure 25). We observed that treatment of worms with 3 μΜ of 11 for 24 h exhibited staining green channel (Ex = 405 nm, Em = 490-550 nm), while staining was weak in the red channel (Ex = 561, Em = 575 LP). The weak staining in the red channel indicated that dye 11 has access to Fe³⁺ present in LIP of wild type C. elegans. Further, Fe sensing behavior of 11 in ftn-1 silenced C. elegans was also studied following the same protocol. Interestingly at this time enhanced signals in the red channel were observed as compared to wild type strain. Henceforth, 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.

The expression of C. elegans gene ftn-1 when treated with dye 11

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. We carried out quantitative real time PCR assays to quantify the levels of ftn-1 mRNA in worms from control, dye treated and Fe³⁺ treated groups. We observed 2.8 fold (p < 0.01) downregulation and 3.7 fold (p < 0.01) upregulation of ftn-1 mRNA levels in worms from dye treated and Fe³⁺ treated groups respectively (Figure 26). We further observed that upon treatment of worms with Fe³⁺ and dye exhibited a reduced level of ftn-1 as compared to the one treated with only Fe³⁺.

Claims

1. Compounds having the general formula I, and their ion chelators, salts thereof

I wherein R^l, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Q-C₈ alkyl, C₂-C₈ alkenyl, C]-C₈ alkoxyl, C₆-C₂₀ aryl, C₅-C₈ cyclo, C₆-C₂₀ aryl, C3-C8 cycloalkyl, C₃-C2₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃- C₂o cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Cj-Cs alkyl esters, C₆-C₂o aryl esters, Ci-C₈ alkylthio, C]-C₈ alkyl amino, C6-C₂₀ aryl amino, Q-Cs cycloalkyl amino, C₆-C₂₀ cycloaryl amino, C[-C₈ acylamino, Ci-C₈ acylthio, C\-C_& acyl, C₆-C₂o aroyl, Q-Q acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C₃-C₂₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₅-C₂₀ arylthioamido, C₃-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S),

The compounds as claimed in claim 1 wherein the representative compounds 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][\ ,10]phenanthroline-8-carbonitrile

(2). iii. 11 -(naphthalen-2-yl)-9-(piperidin- 1 -yl)- 12, 13-dihydronaphtho[2, 1 - b] [ 1 , 10]phenanthroline-8-carbonitrile (3). i v. 11 -(biphenyl-4-yl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b][l,10]phenanthroline-8-carbonitrile (4). v. 9-(piperidin-l-yl)-l l-(pyren-l-yl)-12,13-dihydronaphtho[2,l- b] [ 1 , 10]phenanthroline-8-carbonitrile (5). vi. 11 -(4-bromophenyl)-9-(piperidin-l -yl)- 12,13 -dihydronaphtho [2, 1 - b [ 1 , 10]phenanthroline-8-carbonitrile (6). vii. 11 -(4-chlorophenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho [2,1- b][l,10]phenanthroline-8-carbonitrile (7). viii. 1 l-(4-methoxyphenyl)-9-(piperidin-l-yl)-12,13-dihydronaphtho[2,l- b] [ 1 j 10]phenanthroline-8-carbonitrile (8). ix. 11 -(4-hydroxyphenyl)-9-(piperidin- 1 -yl)- 12,13 -dihydronaphtho[2, 1 - b] [ 1 , 10]phenanthroline-8-carbonitrile (9). x. 1 l-(biphenyl-4-yl)-9-(piperidin-l-yl)naphtho[2,l-&][l,10]phenanthroline-8- carbonitrile (10). xi. 11 -(4-bromophenyl)-9-(piperidin- 1 -yl)naphtho[2, 1 -b [ 1 , 10]phenanthroline-8- carbonitrile (11). xii. 11 -(4-chlorophenyl)-9-(piperidin- 1 -yl)naphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (12). xiii. 11 -(4-methoxyphenyl)-9-(piperidin- 1 -yl)naphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (13). xiv. 11 -(4-hydroxyphenyl)-9-(piperidin- 1 -yl)naphtho[2, 1 -b] [ 1 , 10]phenanthroline-8- carbonitrile (14). xv. 9-(4-bromophenyl)- 11 -(piperidin- 1 -yl)- 12,13-dihydronaphtho[2, 1 - b] [ 1 , 10]phenanthroline- 10-carbonitrile (15).

3. The compounds as claimed in claim 1, wherein the compounds are useful for imaging and/or analysis of cells, biological fluids, chemical mixture and/or as cellular components in fixed or live cell imaging applications and useful biological applications as diagnostic kits, fluorescent probe/marker, quantification of ROS in Fenton reaction;

4. The compounds as claimed in claim 1, wherein the compounds are useful as therapeutic agent in mammals with iron-imbalance induced disorders;

5. The processes for the preparation of compounds claimed in claim 1 wherein the said processes comprise;

Process 1

formula S-2 to furnish a compound having the general formula I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Ci-C₈ alkyl, C₂-C₈ alkenyl, Ci-Cs alkoxyl, C₆-C₂₀ aryl, C₅-C₈ cyclo C₆-C₂₀ aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃- C₂₀ cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), d-Cs alkyl esters, C₆-C₂₀ aryl esters, Ci-C₈ alkylthio, Ci-Cs alkyl amino, C₆-C₂o aryl amino, Ci-C₈ cycloalkyl amino, C₆-C₂₀ cycloaryl amino, Ci-C₈ acylamino, Ci-C₈ acylthio, d-C₈ acyl, C₆-C₂₀ aroyl, d-C₈ acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C₃-C₂₀ arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, 0 and S), C3-C20 heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₅-C₂o arylthioamido, C₃-C2o arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂o heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), wherein, the reaction may proceed in a common organic solvent particularly DMF, THF, DMSO, DCM, isopropanol in the presence of a base particularly KOH, NaOH, NaH, KH, K₂C0₃, Cs₂C0₃ at a temperature ranging between -78°C to 150°C for a period ranging between 1 minute to 3 days, followed by oxidation in presence of an oxidant in organic solvent; b. oxidizing the isolated compound of general formula I in the presence of DDQ or palladium catalysts without any external ligand in a common organic solvent particularly DMF, DMSO, 1,4-dioxane, toluene at a temperature ranging between - 78°C to 150°C for a period ranging between 1 minute to 3 days; c. isolating the compound of general formula I from the reaction mixture and purifying by chromatographic techniques;

Process 2

reacting a compound having general formula S-4 with a compound having general formula S-3 to furnish a compound having the general formula I wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} , R¹² are independently selected from the groups consisting of hydrogen, amine, halogens, nitriles, hydroxy, mercapto, carbontrifluoride, nitro, formyl, azido, carboxylic acid, Q-Cs alkyl, C₂-C₈ alkenyl, Ci-Cg alkoxyl, C₆-C₂₀ aryl, Cs-Cs cyclo C₆-C₂₀ aryl, C₃-C₈ cycloalkyl, C₃-C₂₀ heteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), C₃- C₂₀ cycloheteroaryl containing 1 to 3 heteroatoms (preferably selected from N, O and S), Ci-C₈ alkyl esters, C₆-C2₀ aryl esters, Ci-C₈ alkylthio, Ci-C₈ alkyl amino, C6-C₂₀ aryl amino, Q-Q cycloalkyl amino, C6-C₂₀ cycloaryl amino, Q-Q acylamino, Ci-C₈ acylthio, Ci-C& acyl, C₆-C₂₀ aroyl, C!-C₈ acyloxy, amido, thioamido, C₅-C₂₀ arylamido, C₃-C₂o arylheteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heteroamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₅-C₂₀ arylthioamido, C₃-C₂₀ arylheterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), C₃-C₂₀ heterothioamido containing 1 to 6 heteroatoms (preferably selected from N, O and S), wherein, the reaction may proceed in a common organic solvent particularly DMF, THF, DMSO, DCM, isopropanol in the presence of a base particularly KOH, NaOH, NaH, KH, K₂C0₃, Cs₂C0₃ at a temperature ranging between -78°C to 150°C for a period ranging between 1 minute to 3 days, followed by oxidation in presence of an oxidant in organic solvent; b. oxidizing the isolated compound of general formula I in the presence of DDQ or palladium catalysts without any external ligand in a common organic solvent particularly DMF, DMSO, 1,4-dioxane, toluene at a temperature ranging between - 78°C to 150°C for a period ranging between 1 minute to 3 days; c. isolating the compound of general formula I from the reaction mixture and purifying by chromatographic techniques; wherein process 1 is independent of process 2.

6. A method for fluorescence-based imaging or analysis of cells and/or cellular components, comprising the following steps: a) trapping cation in bound or free state in cellular components with a dye/compound of formula I as defined in any one of claims 1-4; b) exciting the dye-cation complex of step a) with laser light in the wave length range from 330 nm to 700 nm; 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).

7. The method as claimed in claim 6 wherein the cellular matrix components are stained with the dye/compound of the formula I.

8. A method of detecting and estimating peroxides using the dye/compound in the chemical mixture, biological fluid, or in the cellular system.

9. A method of detecting and estimating reactive oxygen species using the dye/compound through measuring changes in fluorescence in the chemical mixture, biological fluid, or in the cellular system.

10. The compounds of formula I as claimed in any one of claims 1-4 or the method according to any one of claims 5-9 for use in 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.