COMPOUNDS FOR THE DETECTION OF HOMOCYSTEINE AND ITS METHOD OF PREPARATION THEREOF

Abstract

The present invention discloses compounds of Formula-A and its copper complex of general Formula-B. The compound of general Formula-B is used in the selective detection of homocysteine in aqueous medium at physiological pH without any interference from other challenging amino acids and thiols. Using compounds of general Formula-B, specific detection of homocysteine under UV light in aqueous solution is also possible by the naked eyes.

##STR00001##

Claims

1. A compound of Formula B ##STR00022## wherein R.sub.1CH.sub.3 or H and R.sub.2CH.sub.3 or C.sub.2H.sub.5.

2. The compound as claimed in claim 1, wherein compound of B is selected from the group consisting of: (i) [Diaqua-8-(2-dimethylamino)ethyl)minomethyl-7-phenoxo-4-methyl-2H-chromen-2-onecopper(II) nitrate] ##STR00023## (ii) [Diaqua-8-(2-(diethylamino)ethyliminomethyl-7-phenoxo-4-methyl-2H-chromen-2-onecopper(II) nitrate] ##STR00024## (iii) [Diaqua-8-(2-(dimethylamino)ethyliminomethyl-7-phenoxo-2H-chromen-2-onecopper(II) nitrate] ##STR00025## and (iv) [Diaqua-8-(2-(diethylamino)ethyliminomethyl-7-phenoxo-4-2H-chromen-2-onecopper(II) nitrate] ##STR00026##

3. A process for preparation of the compound of Formula B ##STR00027## wherein R.sub.1CH.sub.3 or H and R.sub.2CH.sub.3 or C.sub.2H.sub.5, comprising the steps of: (i) refluxing a mixture of 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde or 7-Hydroxy-2-oxo-2H-chromene-8-carbaldehyde and N,N-dimethylethylenediammine or N,N-diethylethylenediammine in a stoichiometry ratio 1:1 in a solvent for a period in the range of 3-4 h followed by evaporating the solvent to obtain a residue; (ii) triturating the residue as obtained in step (i) with diethyl ether to obtain a yellow solid; (iii) recrystallizing the solid as obtained in step (ii) to obtain crystalline Compound of Formula-A; ##STR00028## (iv) refluxing a mixture of copper salt and compound of Formula A in 1:1 stoichiometry in a solvent for a period in the range of 3-4 h followed by evaporating the solvent to obtain green coloured solid; (v) filtering the green coloured solid as obtained in step (i) followed by drying to obtain a solid; and (vi) recrystallizing the solid as obtained in step (ii) to obtain compound of Formula-B in pure form.

4. The process as claimed in claim 3, wherein compound of formula A is selected from the group consisting of: (i) [8-(2-(Dim ethyl amino)ethyl)minomethyl)-7-hydroxy-4-methyl-2H-chromen-2-one] ##STR00029## (ii) [8-(2-(Diethylamino)ethyliminomethyl-7-hydroxy-4-methyl-2H-chromen-2-one ##STR00030## (iii) [8-(2-(Dimethylamino)ethyliminomethyl-7-hydroxy-2H-chromen-2-one] ##STR00031## and (iv) [8-(2-(Diethylamino)ethyliminomethyl-7-hydroxy-2H-chromen-2-one] ##STR00032##

5. The process as claimed in claim 3, wherein solvent is selected from methanol or acetonitrile.

6. The process as claimed in claim 3, wherein the copper salt is selected from the group consisting of copper nitrate trihydrate, copper halide, copper perchlorate and copper acetate.

7. The compound as claimed in claim 1, wherein said compound is used for the fluorimetric measurement of Hcy in aqueous solution at physiological pH (7 to 7.5), even in presence of other competing species selected from the group consisting of alanine, methionine, threonine, proline, leucine, isoleucine, lysine, phenylalanine, hydroxyproline, asparagine, argenine, serine, valine, cysteine, glutathione (reduced), glycine, histidine, and glutamine or combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1. .sup.1H NMR spectrum of Compound 1.

[0046] FIG. 2. .sup.1H NMR spectrum of Compound 2.

[0047] FIG. 3. .sup.1H NMR spectrum of Compound 3.

[0048] FIG. 4. .sup.1H NMR spectrum of Compound 4.

[0049] FIG. 5. .sup.13C NMR spectrum of Compound 1.

[0050] FIG. 6. .sup.13C NMR spectrum of Compound 2.

[0051] FIG. 7. .sup.13C NMR spectrum of Compound 3.

[0052] FIG. 8. .sup.13C NMR spectrum of Compound 4.

[0053] FIG. 9. Fluorimetric responses of 20 M Compounds 5 to 8 toward 25 equivalents of different amino acids, upon excitation at 370 nm (slit width, 2 nm) at physiological pH (7.4).

[0054] FIG. 10. Bar diagram showing selective fluorescence turn on responses of Compound 5 (20 M in HEPES buffer) with Hcy (25 equivalent) in presence of equivalent amount of other competing amino acids.

[0055] FIG. 11. Bar diagram showing selective fluorescence turn on responses of Compound 6 (20 M in HEPES buffer) with Hcy (25 equivalent) in presence of equivalent amount of other competing amino acids.

[0056] FIG. 12. Bar diagram showing selective fluorescence turn on responses of Compound 7 (20 M in HEPES buffer) with Hcy (25 equivalent) in presence of equivalent amount of other competing amino acids.

[0057] FIG. 13. Bar diagram showing selective fluorescence turn on responses of Compound 8 (20 M in HEPES buffer) with Hcy (25 equivalent) in presence of equivalent amount of other competing amino acids.

[0058] FIG. 14. Fluorimetric titration profiles for Compound 5 with increasing concentrations of Hcy at physiological pH 7.4.

[0059] FIG. 15. Fluorimetric titration profiles for Compound 6 with increasing concentrations of Hcy at physiological pH 7.4.

[0060] FIG. 16. Fluorimetric titration profiles for Compound 7 with increasing concentrations of Hcy at physiological pH 7.4.

[0061] FIG. 17. Fluorimetric titration profiles for Compound 8 with increasing concentrations of Hcy at physiological pH 7.4.

[0062] FIG. 18. Limit of detection (LOD) determination plots from fluorescence titration data of Compounds 5 and 6 in presence of different concentrations of Hcy.

[0063] FIG. 19. Limit of detection (LOD) determination plots from fluorescence titration data of Compounds 7 and 8 in presence of different concentrations of Hcy.

[0064] FIG. 20. Nonlinear fittings of the data points obtained from fluorescence titration of Compounds 5 to 8 in presence of varying concentrations of Hcy.

[0065] FIG. 21. Crystal structure of Compound 1.

[0066] FIG. 22. Crystal structure of Compound 2.

[0067] FIG. 23. Crystal structure of Compound 4.

[0068] FIG. 24. Crystal structure of Compound 5.

DETAIL DESCRIPTION OF THE INVENTION

[0069] Present invention provides compound of Formula A and its copper complex of Formula B. The present invention provides a general process for the preparation of compound of Formula-A by reacting 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde or 7-Hydroxy-2-oxo-2H-chromene-8-carbaldehyde with stoichiometric (1:1) amount of N,N-dimethylethylenediammine or N,N-diethylethylenediammine in methanol.

[0070] The present invention provides a process for the preparation of compound of Formula B by reacting stoichiometric amount (1:1) of compound of Formula A with different copper salts such as copper nitrate/copper halide/copper perchlorate/copper acetate in methanol or acetonitrile.

[0071] The present invention provides a process for the synthesis of Compound 1 comprising the steps of: [0072] (i) 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (204 mg, 1 mmol) and N,N-dimethylethylenediamine (88 mg, 1 mmol) in 1:1 stoichiometry were dissolved in 15 mL methanol and refluxed for ca. 3-4 h; [0073] (ii) residue as obtained in step (i) was triturated with diethyl ether after evaporating the solvent using rotary evaporator to get a yellow solid; [0074] (iii) recrystallization of the yellow solid from diethyl ether resulted crystalline

[0075] Compound 1.

[0076] In the present invention, both .sup.1H (FIG. 1) and 13 C NMR (FIG. 5) confirms the preparation of Compound 1.

[0077] The invention provides a process for the synthesis of Compound 2 comprising the steps of: [0078] (i) 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (204 mg, 1 mmol) and N,N-diethylethylenediamine (116 mg, 1 mmol) in 1:1 stoichiometry was refluxed in 15 mL methanol for ca. 3-4 h; [0079] (ii) residue as obtained in step (i) was triturated with diethyl ether after evaporating the solvent to get a brownish yellow solid; [0080] (iii) recrystallization of the brownish yellow solid from diethyl ether resulted crystalline Compound 2.

[0081] In the present invention, both .sup.1H (FIG. 2) and 13 C NMR (FIG. 6) confirms the preparation of Compound 2.

[0082] The invention provides a process for the synthesis of Compound 3 comprising the steps of: [0083] (i) 7-Hydroxy-2-oxo-2H-chromene-8-carbaldehyde (190 mg, 1 mmol) and N,N-dimethylethylenediamine (88 mg, 1 mmol) in 1:1 stoichiometry was refluxed in 15 mL methanol for ca. 3-4 h; [0084] (ii) residue as obtained in step (i) was triturated with diethyl ether after evaporating the solvent to get a yellow solid; [0085] (iii) recrystallization of the yellow solid from diethyl ether resulted crystalline Compound 3.

[0086] In the present invention, both .sup.1H (FIG. 3) and 13 C NMR (FIG. 7) confirms the preparation of Compound 3.

[0087] The invention provides a process for the synthesis of Compound 4 comprising the steps of: [0088] (i) 7-Hydroxy-2-oxo-2H-chromene-8-carbaldehyde (190 mg, 1 mmol) and N,N-diethylethylenediamine (116 mg, 1 mmol) in 1:1 stoichiometry was refluxed in 15 mL methanol for ca. 3-4 h; [0089] (ii) residue as obtained in step (i) was triturated with diethyl ether after evaporating the solvent to get a brownish residue; [0090] (iii) recrystallization of the brownish solid from diethyl ether resulted crystalline Compound 4.

[0091] In the present invention, both .sup.1H (FIG. 4) and 13 C NMR (FIG. 8) confirms the preparation of Compound 4.

[0092] The invention provides a process for the synthesis of Compound 5 comprising the steps of: [0093] (i) a mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 1 (274 mg, 1 mmol) in 1:1 stoichiometry was refluxed in methanol solution (30 mL) for ca. 3-4 h; [0094] (ii) green solid precipitated at the bottom of the round bottom flask, was obtained by evaporating half of the solvent, which was filtered and the residue was dried in a desiccator; [0095] (iii) recrystallization from methanol solution produced crystalline Compound 5.

[0096] The invention provides a process for the synthesis of Compound 6 comprising the steps of: [0097] (i) A mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 2 (302 mg, 1 mmol) in 1:1 stoichiometry was refluxed in methanol solution (30 mL) for ca. 3-4 h; [0098] (ii) green at the bottom of the round bottom flask, was obtained by evaporating half of the solvent, which was filtered and the residue was collected and dried in a desiccator; [0099] (iii) recrystallization from methanol solution resulted deep green Compound 6.

[0100] The invention provides a process for the synthesis of Compound 7 comprising the steps of: [0101] (ii) A mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 3 (260 mg, 1 mmol) in 1:1 stoichiometry was refluxed in methanol solution (30 mL) for ca. 3-4 h; [0102] (iii) deep green solid precipitated at the bottom of the round bottom flask, was obtained by evaporating half of the solvent, which was filtered and the residue was dried in a desiccator; [0103] (iv) recrystallization from methanol solution resulted green powder of Compund 7.

[0104] The invention provides a process for the synthesis of Compound 8 comprising the steps of: [0105] (i) A mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 4 (288 mg, 1 mmol) in 1:1 stoichiometry was refluxed in methanol solution (30 mL) for ca. 3-4 h; [0106] (ii) deep green solid at the bottom of the round bottom flask was obtained by evaporating half of the solvent, which was filtered and dried in a desiccator; [0107] (iii) recrystallization from methanol solution produced green powder of Compound 8.

[0108] In the present invention, a fluorimetric process for the selective recognition of Homocysteine (Hcy) using the Compounds 5 to 8 in aqueous solution at physiological pH (7 to 7.5) has been developed.

[0109] Fluorescence enhancement at 439 nm of Compounds 5 to 8 (20 M) was observed on addition of Hcy solution, which was monitored via emission technique upon exciting the solution at 370 nm (slit width, 2 nm).

[0110] Fluorescence intensity enhancement of the mixtures of Compounds 5 to 8 (20 M) and equivalent each of different amino acids such as L-glycine, L-alanine, L-leucine, L-isoleucine, L-valine, L-glutamine, L-lysine, L-proline, L-hydroxyproline, L-phenylalanine, L-aspargine, L-serine, L-threonine, L-histidine, L-arginine, L-aspartic acid, L-glutamic acid, L-methionine, L-cysteine, and L-glutathione (reduced) was studied in HEPES buffer (10 mM) at physiological pH (7 to 7.5). The mixtures were excited at 370 nm (slit width, 2 nm) and the emission spectra were recorded between 380 and 580 nm in the liquid mode after 1 h incubation of the mixture. Selective detection of Hcy by Compounds 5 to 8 without any interference from the other competing amino acids was noticed.

[0111] Turn on fluorescence response of Compounds 5 to 8 (20 M) in HEPES buffer (10 mM) at physiological pH (7 to 7.5) was perceived by the naked eyes under UV lamp (360 nm) in presence of Hcy.

[0112] Spectrofluorimetric titration of Compounds 5 to 8 against Hcy was carried out upon excitation at 370 nm (slit width, 2 nm). Non-linear fitting of the data points corresponding to the maximum emission intensity at 439 nm (Compounds 5 and 6) and 445 nm (Compounds 7 and 8) yielded binding constant 1.7510.sup.3, 2.1510.sup.3, 1.2310.sup.3, and 1.6810.sup.3 M.sup.1, respectively for Compounds 5, 6, 7, and 8. Lower detection limit (LOD) was found to be 15, 2.5, 12, and 8 M for Compounds 5, 6, 7, and 8 respectively.

[0113] The present invention discloses that the Compounds 5, 6, 7, and 8 are capable of detecting Hcy fluorimetrically among other competing amino acids like alanine, methionine, threonine, proline, leucine, isoleucine, lysine, phenylalanine, hydroxyproline, asparagine, argenine, serine, valine, cysteine, glutathione (reduced), glycine, histidine, and glutamine (FIG. 9).

[0114] Fluorescence based interference studies of Compound 5 to 8 in presence of other amino acids like alanine, methionine, threonine, proline, leucine, isoleucine, lysine, phenylalanine, hydroxyproline, asparagine, argenine, serine, valine, cysteine, glutathione (reduced), glycine, histidine, and glutamine showed no interference from other amino acids (FIGS. 10 to 13).

[0115] As disclosed, the LODs for Hcy detection by the Compounds 5 and 6 are 15 and 2.5 M, respectively (FIG. 18).

[0116] As disclosed, the LODs for Hcy detection by the Compounds 7 and 8 are 12 and 8 M, respectively (FIG. 19).

[0117] Hcy concentration variable fluorescence titration data of Compounds 5, 6, 7, and 8 resulted the binding constants as 1.7510.sup.3 M.sup.1, 2.1510.sup.3 M.sup.1, 1.2310.sup.3 M.sup.1, and 1.6810.sup.3 M.sup.1, respectively (FIG. 20).

MATERIALS AND PHYSICAL MEASUREMENTS

[0118] All chemicals were procured from Aldrich unless otherwise stated. All solvents were acquired from Finar and used without any further purification. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-c arb aldehyde and 7-Hydroxy-2-oxo-2H-chromene-8-c arb aldehyde were prepared following the procedure described by Kamoto, M. et al. published in Chem.-Eur. J. 2008, 14, 8004-8012. The UV-vis spectra were recorded with Shimadzu 3600 UV-Vis-NIR spectrophotometer and Varian Cary-500 UV-Vis spectrophotometer. Elemental analyses (C, H, and N) were performed on an Elementar Vario MICRO CUBE analyser. Fluorescence experiments were performed on a Fluorolog FL 1065 Horiba Jobin Yvon Spectrometer instrument. IR spectra were recorded using Agilent Technologies Cary 600 Series spectrometer. pH of the solutions were measured using Thermo Scientific Orion Versa-star Advanced Electrochemistry meter at 298 K. JEOL Resonance ECZR 600 MHz spectrometer was used for .sup.1H and 13 C NMR spectra. The ESI-MS was measured on Micromass Q-ToF micro and Agilent technologies 6545 QTOF LCMS/Infinity. The melting points of the ligands were measured using a Mettler-Toledo FP-62 instrument.

EXAMPLES

[0119] Following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.

Example 1: Preparation of Compound 1

[0120] [8-(2-Dimethylamino)ethyliminomethyl-7-hydroxy-4-methyl-2H-chromen-2-one]

##STR00014##

[0121] A mixture of 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (204 mg, 1 mmol) and N,N-dimethylethylenediammine (88 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 15 mL of methanol was added to this mixture. The mixture was refluxed under stirring for 3 h. TLC then monitored the progress of the reaction. After completion of the reaction, yellow solid was obtained after evaporating the solvent under pressure using rotary evaporator, Further, trituration with diethyl ether, the solid was collected and air dried further. Dried solid was then recrystallized using diethyl ether and further dried at room temperature to obtain the crystalline product.

[0122] Yield: 68% (recrystallized). MP: 157 C. .sup.1H NMR (CDCl.sub.3, 600 MHz, ppm)=14.83 (s, 1H, Phenolic OH), 8.90 (s, 1H), 7.47 (d, 1H, J=9 Hz), 6.72 (d, 1H, J=9 Hz), 6.00 (s, 1H), 3.72 (t, 2H, J=6 Hz), 2.64 (t, 2H, J=6 Hz), 2.36 (s, 3H), 2.31 (s, 6H). 13 C NMR (CDCl.sub.3, 600 MHz, ppm)=19.04, 45.63, 52.67, 59.06, 104.89, 108.08, 109.05, 118.43, 130.02, 153.90, 155.44, 160.35, 160.82, 174.05. Anal. Calcd. for C.sub.15H.sub.18N.sub.2O.sub.3: C, 65.68; H, 6.61; N, 10.21. Found: C, 64.13; H, 6.4; N, 9.9. ESI-MS (+ive, m/z): 275.14 [M+H.sup.+]. Selected IR bands (cm.sup.1): 3435, 2941, 2770, 1723, 1636, 1578, 1504, 1436, 1424, 1340, 1245, 1174, 1057, 834. UV-vis (H.sub.2O) [.sub.max, nm (, L.sub.mol.sup.1 cm.sup.1)]: 350 (36000) and 277 (17400).

Example 2: Preparation of Compound 2

[0123] [8-(2-Diethylamino)ethyliminomethyl-7-hydroxy-4-methyl-2H-chromen-2-one]

##STR00015##

[0124] A mixture of 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (204 mg, 1 mmol) and N,N-diethylethylenediammine (116 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 15 mL of methanol was added to this mixture. The mixture was refluxed under stirring for 4 h. TLC monitored the progress of the reaction. After completion of the reaction, brownish yellow solid was obtained by the evaporation of the solvent under pressure using rotary evaporator. Yellow solid was obtained upon trituration with diethyl ether, which was air dried further. Solid was then recrystallized using diethyl ether and dried further at room temperature.

[0125] Yield: 62% (recrystallized). MP: 107 C. .sup.1H NMR (CDCl.sub.3, 600 MHz, ppm)=8.85 (s, 1H), 7.46 (d, 1H, J=9.6 Hz), 6.70 (d, 1H, J=9 Hz), 5.99 (s, 1H), 3.66 (t, 2H, J=6 Hz), 2.76 (t, 2H, J=6 Hz), 2.59 (q, 4H, J=7.2 Hz), 2.36 (s, 3H), 1.03 (t, 6H, J=7.2 Hz). 13 C NMR (CDCl.sub.3, 600 MHz, ppm)=12.03, 18.99, 47.31, 52.42, 52.70, 104.49, 107.46, 108.61, 119.08, 130.18, 153.94, 155.70, 160.25, 160.87, 175.42. Anal. Calcd. for C.sub.17H.sub.22N.sub.2O.sub.3: C, 67.53; H, 7.33; N, 9.26. Found: C, 66.47; H, 7.52; N, 9.17. ESI-MS (+ive, m/z): 303.17 [M+H.sup.+]. Selected IR bands (cm.sup.1): 3431, 2967, 2931, 2864, 2798, 1735, 1720, 1644, 1585, 1507, 1433, 1380, 1184, 1066, 829. UV-vis (H.sub.2O) [.sub.max, nm (, Lmol.sup.1 cm.sup.1)]: 350 (40235) and 279 (20660).

Example 3: Preparation of Compound 3

[0126] [8-(2-Dimethylamino)ethyliminomethyl-7-hydroxy-2H-chromen-2-one]

##STR00016##

[0127] A mixture of 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde (204 mg, 1 mmol) and N,N-dimethylethylenediammine (88 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 15 mL of methanol was added to this mixture. The mixture was refluxed under stirring for 3.5 h. TLC then monitored the progress of the reaction. After completion of the reaction, yellow solid was obtained after evaporating the solvent under pressure using rotary evaporator. So-obtained solid was further triturated with diethyl ether and then was collected and air-dried further. Solid was then recrystallized using diethyl ether to obtain crystalline product.

[0128] Yield: 68% (recrystallized). MP: 108 C. .sup.1H NMR (CDCl.sub.3, 600 MHz, ppm)=14.74 (s, 1H, Phenolic OH), 8.86 (s, 1H), 7.56 (d, 1H, J=9. Hz), 7.31 (d, 1H, J=9 Hz), 6.69 (d, 1H, J=9 Hz), 6.11 (d, 1H, J=9.6 Hz), 3.72 (t, 2H, J=6 Hz), 2.65 (t, 2H, J=6 Hz), 2.31 (s, 6H). 13 C NMR (CDCl.sub.3, 600 MHz, ppm)=45.62, 52.28, 58.97, 104.79, 107.05, 109.59, 119.21, 133.43, 144.54, 156.40, 159.89, 160.90, 174.99. Anal. Calcd. for C.sub.14H.sub.16N.sub.2O.sub.3: C, 64.60; H, 6.20; N, 10.76. Found: C, 64.13; H, 6.21; N, 10.59. ESI-MS (+ive, m/z): 261.12 [M+H.sup.+]. Selected JR bands (cm.sup.1): 3435, 2944, 2817, 2766, 1722, 1640, 1581, 1514, 1465, 1430, 1348, 1232, 1186, 1103, 994, 824. UV-vis (H.sub.2O) [max, nm (, Lmol.sup.1 cm.sup.1)]: 353 (27250) and 277 (14200).

Example 4: Preparation of Compound 4 [C.SUB.16.H.SUB.20.N.SUB.2.O.SUB.3.]

[0129] [8-(2-(Diethylamino)ethyliminomethyl-7-hydroxy-2H-chromen-2-one]

##STR00017##

[0130] A mixture of 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde (190 mg, 1 mmol) and N,N-diethylethylenediammine (116 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 15 mL of methanol was added to this mixture. The mixture was refluxed under stirring for 4 h. TLC then monitored the reaction mixture. After completion of the reaction, brown solid was obtained after evaporating the solvent under pressure using rotary evaporator, which was further triturated using diethyl ether. The solid was collected and air-dried further. Dried solid was then recrystallized using diethyl ether to obtain the crystalline product.

[0131] Yield: 62% (recrystallized). MP: 102 C. .sup.1H NMR (CDCl.sub.3, 600 MHz, ppm)=14.60 (s, 1H, Phenolic OH), 8.81 (s, 1H), 7.54 (d, 1H, J=9.6 Hz), 7.30 (d, 1H, J=9 Hz), 6.60 (d, 1H, J=9 Hz), 6.09 (d, 1H, J=9.6 Hz), 3.66 (t, 2H, J=6 Hz), 2.76 (t, 2H, J=6 Hz), 2.59 (q, 4H, J=7.2 Hz), 1.03 (t, 6H, J=7.2 Hz). 13 C NMR (CDCl.sub.3, 600 MHz, ppm)=12.11, 47.36, 52.16, 52.75, 104.46, 106.52, 109.15, 119.88, 133.62, 144.60, 156.74, 159.84, 160.98, 176.35. Anal. Calcd. for C.sub.16H.sub.20N.sub.2O.sub.3: C, 66.65; H, 6.99; N, 9.72. Found: C, H, 7.22; N, 9.47. ESI-MS (+ive, m/z): 289.16 [M+H.sup.+]. Selected IR bands (cm.sup.1): 3419, 2964, 2802, 1736, 1720, 1642, 1618, 1584, 1503, 1438, 1349, 1246, 1138, 1092, 981, 826. UV-vis (H.sub.2O) [.sub.max, nm (, Lmol.sup.1 cm.sup.31 1)]: 356 (21100) and 274 (8050).

Example 5: Preparation of Compound 5

[0132] [Diaqua-8-(2-(dimethylamino)ethyliminomethyl-7-phenoxo-4-methyl-2H-chromen-2-onecopper(II) nitrate]

##STR00018##

[0133] A mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 1 (274 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 30 mL methanol was added to this mixture. The mixture was refluxed under stirring for ca. 3 h and TLC monitored the progress of the reaction. Green solid was obtained as precipitates after evaporating half of the solvent using rotary evaporator, which was further filtered to obtain the solid. The dried solid was recrystallized using methanol to obtain the pure product.

[0134] Yield: 60% (recrystallized). Selected IR bands (cm.sup.1): 3432, 2926, 1720, 1628, 1583, 1526, 1469, 1406, 1381, 1280, 1184, 1101, 1057, 1012, 836 and 775. Anal. Calcd. for C.sub.15H.sub.21N.sub.3O.sub.8Cu: C, 41.43; H, 4.87; N, 9.66. Found: C, 41.11; H, 5.02; N, 9.91. UV-vis (H.sub.2O) [.sub.max, nm (c, Lmol.sup.1 cm.sup.1)]: 410 (11050), 365 (21100), 332 (33000), 289 (36200), 257 (38100), 215 (67200).

Example 6: Preparation of Compound 6

[0135] [Diaqua-8-(2-(diethylamino)ethyliminomethyl-7-phenoxo-4-methyl-2H-chromen-2-onecopper(II) nitrate]

##STR00019##

[0136] A mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 2 (302 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 30 mL methanol was added to this mixture. The mixture was refluxed under stirring for ca. 3 h and TLC monitored the progress of the reaction. Green solid was obtained as precipitates after evaporating half of the solvent using rotary evaporator, which was further filtered to obtain the solid. The dried solid was further recrystallized using methanol to obtain the pure product.

[0137] Yield: 52% (recrystallized). Selected IR bands (cm.sup.1): 3423, 2976, 2938, 1730, 1630, 1581, 1530, 1476, 1403, 1338, 1286, 1189, 1100, 1060, 1011, 840, 772 and 740. Anal. Calcd. for C.sub.17H.sub.25N.sub.3O.sub.8Cu: C, 44.11; H, 5.44; N, 9.08. Found: C, 44.73; H, 5.32; N, 9.11. UV-vis (H.sub.2O) [.sub.max, nm (c, Lmol.sup.1 cm.sup.1)]: 418 (4800), 364 (14050), 339 (18500), 281 (18900), 211 (52450).

Example 7: Preparation of Compound 7

[0138] [Diaqua-8-(2-(dimethylamino)ethyliminomethyl-7-phenoxo-2H-chromen-2-onecopper(II) nitrate]

##STR00020##

[0139] A mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 3 (260 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 30 mL of methanol was added to this mixture. The mixture was refluxed under stirring for ca. 4 h and TLC monitored the progress of the reaction. Green solid was obtained by evaporating half of the solvent using rotary evaporator, which was filtered and the residue was dried in a desiccator. The dried solid was further recrystallized using methanol to obtain the pure product.

[0140] Yield: 51% (recrystallized). Selected IR bands (cm.sup.1): 3414, 3050, 2920, 1717, 1624, 1584, 1525, 1469, 1402, 1383, 1355, 1287, 1080, 1009, 838, 773. Anal. Calcd. for C.sub.14H.sub.19N.sub.3O.sub.8Cu: C, 39.95; H, 4.55; N, 9.98. Found: C, 40.39; H, 4.63; N, 10.11. UV-vis (H.sub.2O) [.sub.max, nm (c, Lmol.sup.1 cm.sup.1)]: 374 (18450), 350 (26800), 268 (23600), 248 (23000), 212 (62150).

Example 8: Preparation of Compound 8

[0141] [Diaqua-8-(2-(diethylamino)ethyliminomethyl-7-phenoxo-2H-chromen-2-onecopper(II) nitrate]

##STR00021##

[0142] A mixture of copper nitrate trihydrate (241.6 mg, 1 mmol) and Compound 4 (288 mg, 1 mmol) in 1:1 stoichiometry was taken in a round bottom flask and 30 mL of methanol was added to this mixture. The reaction mixture was refluxed under stirring for ca. 3 h and TLC monitored the progress of the reaction. Green solid was obtained by evaporating half of the solvent using rotary evaporator. The solid was filtered and the residue was dried in a desiccator. The dried solid was further recrystallized using methanol to obtain the pure product.

[0143] Yield: 52% (recrystallized). Selected IR bands (cm.sup.1): 3428, 3061, 2975, 2920, 1732, 1630, 1587, 1525, 1469, 1410, 1386, 1287, 1072, 1009, 832, 773. Anal. Calcd. for C.sub.16H.sub.23N.sub.3O.sub.8Cu: C, 42.81; H, 5.16; N, 9.36. Found: C, 42.57; H, 5.23; N, 9.11. UV-vis (H.sub.2O) [.sub.max, nm (c, Lmol.sup.1 cm.sup.1)]: 432 (8400), 372 (14400), 347 (20350), 273 (20150), 244 (17800), 211 (47950).

Advantages of the Invention

[0144] Elevated homocysteine levels in human plasma refers to a health condition known as hyperhomocysteinemia, which is connected to the early onset of several critical health illnesses and age related pathologies. Despite tens of thousands of publications in the literature on the relationship between the acute health disorders and homocysteine levels, we are not aware of any clinically tested optical assay for direct measurement of homocysteine in human blood. To manage various clinical pathologies, simple and rapid measurement of plasma homocysteine at the early stage is indispensable. Several analytical methods including HPLC, GCMS, CE, voltammetry, and ELISA have been developed as techniques for measuring Hcy. Even though some of these techniques are useful, most of these methods suffer from the disadvantages of the use of very expensive instruments, tedious procedures, requirement of skilled person to handle the sophisticated instruments, and complicated pre-treatment of samples to make them unsuitable for routine detection in diagnostic laboratory. There is no optical kit available in India to measure Hcy directly. The cost per test for indirect measurement of Hcy (which is enzyme based immunoassay) ranges within Rs. 1500-2500. Optical probes, which are soluble in aqueous media, are highly preferred for biological applications but most of the available or reported probes require organic solvent as co-solvent for the detection purposes. In this context, Compounds of Formula B (i.e. Compounds 5-8) are cost effective and wateraqueous soluble. Therefore, entire analysis can be performed in non-organic medium. Since homocysteine is a homologue of cysteine, differing only by an additional methylene moiety, selective detection/measurement of the former in human blood plasma is a very challenging task because of the interference from cysteine (cys) and glutathione (GSH). Compounds of Formula B can detect homocysteine directly and selectively, even in presence of cysteine and glutathione. The results obtained with the Compounds of Formula B showed excellent correlation with gold standard techniques. These promising results apparently concludes that Compounds of Formula B have the potential to be used as diagnostic kit for routine analysis and prognosis for cardiac patients in clinical laboratories with minimal resource settings.