Patent Grant
10730891
The present invention relates to a compound of general formula (L) for selective determination of free cysteine and a process for the preparation thereof.
wherein R1 is selected from phenyl, toluene, naphthalene and pyrene.
Biological thiols such as Cysteine (Cys), Homocysteine (Hcy) and Glutathione (GSH) play crucial roles in maintaining cellular antioxidant defence system. Among them cysteine plays many important roles in living systems. Cysteine is one of the three main precursors required for GSH synthesis. The deficiency of this compound causes many diseases such as slowed growth in children, depigmentation of hair, edema, liver damage, skin lesions, and weakness. An elevated level of Hcy is a risk factor for cardio-vascular disease, dementia and Alzheimer's disease. Abnormal levels of GSH is connected to many diseases such as HIV, cell death and aging. Thus, detection and discrimination of these thiol containing molecules are of great importance. Because of the similar structure and reactivity, distinction among biothiols is a challenging task. The respective concentration level of Cys and His in human plasma is typically 240-360 μM and 15-75 μM. Estimation of these amino thiols in human blood plasma is essential for understanding the role of these in the pathogenesis of vascular diseases.
The past two decade has seen significant effort being devoted to the development of optical probes for the selective recognition of thiol containing amino acids. There are many strategies for sensing biothiols, based on Micheal addition, cyclization with aldehydes, disulfide cleavage and others.
Das et al. (Chem. commun., 2014, 50, 9899-9902) discloses chemo dosimetric reagents for biothiol. A Cu (II)-complex based probe for detection of Cys and Histidine was reported.
P Das et al. (Org. Biomol. Chem., 2013, 11, 6604-6614) reports a new and simple chemodosimetric probe L11 is utilized for the selective detection of biothiols in the presence of other relevant amino acids under physiological conditions (pH=7.4). Furthermore, the studies with human blood plasma demonstrated the possibility of using this reagent for the quantitative optical detection of total biothiols in biological fluid.
X Yang et al. (Angewandte Chemie International Edition, Nov. 4, 2011, Volume 50, Issue 45, pages 10690-10693) reports a benzothiazole derivative used to detect cysteine (Cys) and homocysteine (Hcy) simultaneously in neutral media. The method involves thioether formation followed by cyclization to release 2-(2′-hydroxy-3′-methoxyphenyl) benzothiazole (HMBT) and a lactam.
P Das et al. (Chemistry—A European Journal, Nov. 26, 2012, Volume 18, Issue 48, pages 15382-15393) reports rationally designed and synthesized two new reagents L1 {(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzaldehyde)} and L2 {(4-(6,11-dioxo-6,11-dihydro-3H-anthra[1,2-d]imidazol-2-yl)benzaldehyde)}, each bearing a pendant aldehyde functionality. This aldehyde group can take part in cyclization reactions with 13- or γ-amino thiols to yield the corresponding thiazolidine and thiazinane derivatives, respectively. These two chemodosimetric reagents could be used for the quantitative detection of cysteine present in blood plasma by using a pre-column HPLC technique.
However, the development of probes for specific discrimination of biothiols is an unmet need in the art. Probes that are selective to any one of these amino biothiols are very rare in literature. Probes that give specific response with colour change as well as emission change are much needed. Especially those probes which allows real time monitoring without the aid of any instrumental techniques are highly recommended as for as the practical utility is concerned. There are no reports on detection of thiols using simple test strips. Particularly, a further challenge that is unaddressed in the art is that of a simple process for the determination of free cysteine.
Therefore, there is need in the art to develop a reagent for selective detection of free cysteine, especially with a high degree of selectivity towards free cysteine. Accordingly, the inventors of present invention developed a novel ligand for the selective detection of free cysteine.
The main objective of present invention is to provide a novel ligand of formula (L) useful for the selective detection of free cysteine and process for the preparation thereof.
Another objective of present invention is to provide a kit for the selective detection of free cysteine characterized in that the selectivity of novel ligand of formula (L) to cysteine is very high and a process for detection using the kit.
Yet another objective of present invention is to provide a visual as well as fluorescence test for detection of cysteine using novel ligand of formula (L).
Accordingly, the present invention provides a compound of general formula (L) which is useful for the selective detection of free cysteine and process for the preparation thereof.
Wherein R1 is selected from
The representative compounds of formula (L) being the ligand of formula (L) are;
In an aspect, the present invention provides a kit for the selective detection of free cysteine characterized in that the selectivity of novel ligand of formula (L) to cysteine is 100% and a process for detection using the kit.
In another aspect, the present invention provides a visual as well as fluorescence test for detection of cysteine using novel ligands of formula (L).
Abbreviations Used:
GSH: Glutathione
Hcy: Homocysteine
Cys: Cysteine
NAC: N-Acetylcysteine
AAs: Aminoacids
Probe L: Novel ligand of formula (L)
HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
BODIPY—Boron-dipyrromethene
DCM—Dicholomethane
For the purpose of this invention, the expression “probe L” and “novel ligand of formula (L)” are used interchangeably throughout the specification and both having the same meaning.
The present invention provides a novel ligand of formula (L) which is useful for the selective detection of free cysteine and process for the preparation thereof. This probe L is used for the detection of free cysteine by a simple, convenient, selective and fast visual method in aqueous medium as well as biological fluids. The present invention also provides a kit for the selective detection of free cysteine characterized in that the selectivity of novel ligand of formula (L) to cysteine is 100% and a process for detection using the kit.
Accordingly, the main embodiment of the present invention provides a compound for selective determination of free cysteine having general formula (L)
wherein R1 is selected from
Another embodiment of the present invention provides a novel ligand of formula (L) useful for the detection of free cysteine.
wherein R1 is selected from
In another embodiment of the present invention the compounds of formula (L) comprises:
Another embodiment of the present invention provides a process for the preparation of compound of formula (L), comprising the steps of:
Another embodiment of the present invention provides a process for the synthesis of ligand of formula (L) comprising the steps of:
In another embodiment of the present invention, a mixture of BODIPY (boron-dipyrromethene) (400 mg, 1.23 mmol), 4-hydroxybenzaldehyde (165 mg, 1.35 mmol), 0.9 ml piperidine and 0.6 ml glacial acetic acid was reflux in 30 ml toluene in a Dean-Stark apparatus for 3 h. Then water was added into it and crude organic layer was extracted with dichloromethane. The organic layer was collected and dried over anhydrous sodium sulphate and solvent was removed under reduced pressure. It was then subjected to column chromatography using silica gel (100-200 mesh) as stationary phase and 10% EtOAc in hexane as mobile phase to get compound R′ as red solid.
In yet another embodiment, BODIPY compound R′ (30 mg, 0.07 mmol) was dissolved in 10 ml anhydrous dichloromethane in a 100 ml round bottom two neck flask. Then 100 uL of Et3N was added to the reaction mixture and allowed to stir for 10 mins at room temperature under N2 atmosphere. 20 uL of acryloyl chloride was added to this and resulting mixture was stirred at room temperature until all the starting material was consumed monitored by TLC. Then water was added to it and organic layer was extracted using dichloromethane. The organic layer was collected and dried over anhydrous sodium sulphate and solvent was removed under reduced pressure. It was then subjected to column chromatography using silica gel (100-200 mesh) as stationary phase and 5% EtOAc in hexane as mobile phase to get probe L as solid as 70% yield.
In yet another embodiment, the present invention discloses the process for the synthesis for novel ligand of formula (L) is depicted in scheme 1 below;
The BODIPY of formula R″ is prepared by known methods comprising reacting aldehyde or acid chloride with 2, 4 dimethyl pyrrole. The aldehyde is used appropriately to obtain the BODIPY of formula R″ wherein the substituent R represents toluene, naphthalene and pyrene. The acid chloride is selected from benzoyl chloride to obtain BODIPY of formula R″ wherein the substituent R is phenyl.
Accordingly, the process for preparation of BODIPY of formula R″, wherein R represents toluene, naphthalene and pyrene comprises reacting the corresponding aldehyde with 2, 4 dimethyl pyrrole in presence of DDQ, Et3N:BF3 in presence of trifluroacetic acid and DCM.
The process for preparation of BODIPY of formula R″, wherein R is phenyl comprises reacting benzoyl chloride and 2, 4 dimethyl pyrrole in presence of triethylamine, BF3.OEt2 in dichloromethane under nitrogen atmosphere.
Another embodiment of the present invention provides for a diagnostic kit for selective detection of free cysteine.
In another embodiment of the present invention, the diagnostic kit comprises:
(a) Ligand of Formula (L)
(b) HEPES aqueous buffer medium
(c) TLC Test strips
(d) HEPES:acetonitrile medium
(e) Instruction manual
Another embodiment of the present invention provides a method for selective detection of free cysteine using the diagnostic kit comprising;
In another embodiment of the present invention the emission response of ligand L with various amino acids (AAs) as well as anionic analytes in aqueous buffer-acetonitrile (9:1, v/v) at pH 7.2 solution (
In another embodiment of the present invention time dependent fluorescence assay of probe L (10 uM) is carried out in presence of 5 eqv. of Cys, Hcy and GSH by monitoring emission changes at λem at 583 nm in aq. HEPES buffer-acetonitrile (9:1, v/v) at pH 7.2 medium (
In another embodiment of the present invention the fluorescence intensity of probe L is determined to be linearly proportional with Cys concentration and the detection limit of free cysteine by the fluorescence spectroscopy method is found to be 15 nM. The effect of pH towards probe L indicated ligand L is stable up to pH 9.
In another embodiment, probe L is used for the detection of free cysteine by a simple, convenient, selective and fast visual method in aqueous medium as well as biological fluids. Accordingly, a TLC plate is immersed into acetonitrile solution of probe L (5 μL) and dried. This TLC plate coated with probe L is exposed to aqueous solution of different analytes under identical condition. Upon addition of Cysteine solution a prominent change in fluorescence (green to reddish) using UV lamp with excitation at 365 nm and little colour (light pink to dark pink) are observed in TLC plate. In case of other analytes including Hcy, GSH, NAC, and BSA protein (contain terminal Cys residue) no detectable changes are induced.
In another embodiment, the probe L is tested for biological properties. In a preferred embodiment, the probe L is tested for cytotoxicity and at micro molar concentration of probe L used for cellular studies 85% of cell servility. The in vitro cytotoxicity of L on Hct116 cells (Colon cancer cell) are determined by conventional MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow tetrazole) assay. Hct116 colon cancer cells (7×103) are seeded in each well of a 96 well plate and cultured in a 37° C. incubator supplied with 5% CO2. Cells are maintained in DMEM medium, supplemented with 10% Foetal Bovine Serum and 100 Units of Penicillin Streptomycin antibiotics. After 24 hours the cells are treated with different concentrations of the L in triplicates for 12 hours. After treatment cells are added with 0.5 μg/ml of MTT reagent. The plate is then incubated for 4 hours at 37° C. 100 μl of Isopropyl Alcohol is added to each well.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention:
A mixture of BODIPY (boron-dipyrromethene) (400 mg, 1.23 mmol), 4-hydroxybenzaldehyde (165 mg, 1.35 mmol), 0.9 ml piperidine and 0.6 ml glacial acetic acid was reflux in 30 ml toluene in a Dean-Stark apparatus for 3 h. Then water was added into it and crude organic layer was extracted with dichloromethane. The organic layer was collected and dried over anhydrous sodium sulphate and solvent was removed under reduced pressure. It was then subjected to column chromatography using silica gel (100-200 mesh) as stationary phase and 10% EtOAc in hexane as mobile phase to get compound R′ as red solid. Yield: 56%; 1H NMR (500 MHz, CDCl3, J in Hz, δ ppm): 7.52 (1H, d, J=16.5), 7.47 (5H, m), 7.3 (2H, d, J=8), 7.18 (1H, d, J=16), 6.83 (2H, d, J=8.5), 6.58 (1H, s), 5.99 (1H, s), 5.4 (1H, s), 2.59 (3H, s), 1.42 (3H, s), 1.38 (3H, s); 13C NMR (125 MHz, CDCl3, δ ppm): 156.80, 154.68, 153.47, 142.74, 142.40, 140.08, 136.24, 135.13, 132.84, 131.68, 129.38, 129.22, 129.09, 128.93, 128.25, 121.14, 117.55, 116.90, 115.82, 14.69, 14.59, 14.32. HRMS (ESI): m/z calculated for C26H23BF2N2O [M+H]: 429.1872, found 429.1943.
In a 100 ml round bottom two neck flask BODIPY compound R′ (30 mg, 0.07 mmol) was dissolved in 10 ml anhydrous dichloromethane. Then 100 uL of Et3N was added to the reaction mixture and allowed to stir for 10 mins at room temperature under N2 atmosphere. 20 uL of acryloyl chloride was added to this and resulting mixture was stirred at room temperature until all the starting material was consumed monitored by TLC. Then water was added to it and organic layer was extracted using dichloromethane. The organic layer was collected and dried over anhydrous sodium sulphate and solvent was removed under reduced pressure. It was then subjected to column chromatography using silica gel (100-200 mesh) as stationary phase and 5% EtOAc in hexane as mobile phase to get probe L as solid as 70% yield. 1H NMR (500 MHz, CDCl3, J in Hz, δ ppm): 7.57 (1H, s), 7.53 (3H, d, J=8.2), 7.41 (3H, d, J=5.8), 7.25-7.21 (2H, m), 7.13 (1H, d, J=16.3), 7.07 (2H, d, J=8.4), 6.56 (1H, s), 6.52 (1H, s), 6.25 (1H, dd, J=17.3, 10.5), 5.95 (2H, d, J=11.1), 2.52 (3H, s), 1.35 (3H, s), 1.31 (3H, s); 13C NMR (125 MHz, CDCl3, δ ppm): 164.35, 155.92, 152.21, 150.90, 143.27, 142.34, 140.65, 135.03, 134.69, 134.39, 132.78, 132.05, 129.15, 129.01, 128.44, 128.14, 127.86, 121.88, 121.55, 119.41, 117.48, 14.79, 14.57, 14.42. HRMS (ESI): m/z calculated for C29H26BF2N2O2 [M+H]: 483.1977 found 483.2057.
Stock solution of probe L (1×10−4 M) was prepared in HPLC grade acetonitrile. All the analytes stock solution (1×10−2 M) was prepared in aqueous HEPES buffer medium at pH 7.2. 500 μL of this stock solution of probe L was added to 4.5 ml of HEPES (10 mM) aqueous buffer medium having solution pH 7.2 to make the effective ligand concentration of 1×10−5 M. This solution was used for all the photophysical studies. All the photo physical studies were performed in HEPES:acetonitrile medium (9:1, v/v) at pH 7.2. The detection limit was calculated based on the fluorescence titration. To determine the S/N ratio, the emission intensity of L without Cys was measured 10 times and the standard deviation of blank measurements was determined. The detection limit (DL) of L for Cys was determined from the following equation:
DL=K*Sb1/S,
Where K=2 or 3 (we took 3 in this case);
Sb1 is the standard deviation of the blank solution;
S is the slope of the calibration curve.
From the graph we get slope=4.99×108, and Sb1 value is 2.49
Thus using the formula we get the Detection Limit=15×10−9M.
TLC test strips were prepared by coating 5 μM of probe solution in acetonitrile on silica TLC plates. 5 μl of Cys (1×10−1M) in 10 mM aq. HEPES buffer (pH 7) was added on it, dried and the visual as well as fluorescence colour changes were observed after 5 min. The same was repeated for Hcy, GSH and other analytes as well.
Cipla made effervescent tablets of N-Acetyl-Cysteine (mucinac 600) were purchased from commercially available sources. Based on the quantity of NAC present in the tablet, 1×10−1M tablet solution was prepared in 10 mM aq. HEPES buffer solution (pH7). Enzyme solution was prepared according to the requirement by dissolving 1 mg/ml in 10 mM aq. HEPES buffer solution (pH7). A fixed concentration of NAC (200 equiv.) was added to the 10 μM probe in HEPES: ACN (9:1). Since 1 mg of solid enzyme contains 3301 units of protein and 1 unit can hydrolyse 1 μM of substrate, accordingly enzyme concentration was varied with respect to the substrate concentration.
Hct116 cells (3×105) (ATCC® CCL247™) were seeded on cover slips placed in 6 well plates. After 24 hours, cells were treated with L (10 μM) for 30 minutes. Cells were then washed thrice with Phosphate Buffer Saline (1×PBS) and fixed with 4% PFA for 20 minutes and washed again with 1×PBS. Nail paint was used to seal the cover slips mounted on the glass slides. Images were acquired in Olympus Fluoview Microscope.
The in vitro cytotoxicity of L on Hct116 cells (Colon cancer cell) were determined by conventional MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow tetrazole) assay. Hct116 colon cancer cells (7×103) were seeded in each well of a 96 well plate and cultured in a 37° C. incubator supplied with 5% CO2. Cells were maintained in DMEM medium, supplemented with 10% Foetal Bovine Serum and 100 Units of Penicillin Streptomycin antibiotics. After 24 hours the cells were treated with different concentrations of the L in triplicates for 12 hours. After treatment cells were added with 0.5 μg/ml of MTT reagent. The plate was then incubated for 4 hours at 37° C. 100 μl of Isopropyl Alcohol was added to each well. Optical density was measured at 570 nm using Multiskan Go (Thermo Scientific) to find the concentration of the cell inhibition. IC50 value has been calculated to be 100 μM.
The formula used for the calculation of the MTT assay for evaluation of the cell viability was as follows:
Cell viability (%)=(means of Absorbance value of treated group/means of Absorbance value of untreated control)×100.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2354/DEL/2015 | Jul 2015 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2016/050254 | 7/29/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/021980 | 2/9/2017 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040076547 | Carney | Apr 2004 | A1 |
| 20120070382 | Liu | Mar 2012 | A1 |
| Entry |
|---|
| Reedy et al., “A Novel Fluorescence Probe for Estimation of Cysteine/Histidine in Human Blood Plasma and Recoginition of Endogenous Cysteine in Live Hct116 Cells”, Chem. Commun., 2014, vol. 50, pp. 9899-9902. |
| Das et al., “Desigining a Thiol Specific Fluorescent Probe for Possible Use as a Reagent for Intercellular Detection and Estimation in Blood Serum: Kinetic Analysis to Probe the Role of Intramolecular Hydrogen Bonding”, Org. Biomol. Chem., 2013, vol. 11, pp. 6604-6614. |
| Yang et al., “Conjugate Addition/Cyclization Sequence Enables Selective and Simultaneous Fluorescence Detection of Cysteine and Homocysteine”, Angew. Chem. Int. Ed., 2011, vol. 50, pp. 10690-10693. |
| Das et al., “New Chemodosimetric Reagents as Ratiometric Probes for Cysteine and Homocysteine and Possible Detection in Living Cells and in Blood Plasma”, Chem. Eur. J., 2012, vol. 18, pp. 15382-15393. |
| Number | Date | Country | |
|---|---|---|---|
| 20180354975 A1 | Dec 2018 | US |
