A COMPOUND FOR THE DETECTION OF HNO IN BIOLOGICAL SYSTEMS

Abstract

The present invention disclosed a compound of formula (I), process for preparation thereof and use of compound of formula (I) for detecting HNO in biological systems.

##STR00001##

Claims

1. A compound of formula (I); ##STR00005##

2. The compound as claimed in claim 1, wherein said compound of formula (I) is used for the detection of HNO in biological systems.

3. The compound as claimed in claim 1, wherein said compound of formula (I) is used as an imaging reagent for HNO detection in live HCT116 cells.

4. The compound as claimed in claim 1, wherein said compound of formula (I) is used as an imaging reagent for HNO detection in live RAW 264.7 cells.

5. A process for preparation of compound of formula (I) as claimed in claim 1, comprising the steps of: a) adding N,N′-Dicyclohexylcarbodiimide (DCC) to a solution of 2-(diphenylphosphino) benzoic acid in solvent followed by stirring at a temperature of 0° C. for a period in the range of 1 to 2 hrs to afford a reaction mixture; and b) adding (E)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-5l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)vinyl)phenol [ER-S] and 4-Dimethylaminopyridine (DMAP) to the reaction mixture of step (a) at a temperature in the range of 25 to 30° C. for a period in the range of 6 to 7 hrs.

6. The process as claimed in claim 5, wherein said solvent is dichloromethane (CH2Cl2).

7. The process as claimed in claim 5, wherein said process is carried out under nitrogen atmosphere.

8. A kit for the detection of HNO comprising at least said compound of formula (I) as claimed in claim 1.

9. A method of detection of HNO in biological systems using said compound of formula (I) as claimed in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1: (A) Change in the emission spectrum of ER-HNO (10 μM) in the absence and presence of different analytes (0.2 mM); (B) emission titration profile of ER-HNO (10 μM) with varying [HNO] (0-100 μM).

[0031] FIG. 2: (A) Change in emission intensity of ER-HNO in presence and absence of different interfering analytes; (B) effect of pH on probe ER-HNO.

[0032] FIG. 3: (A) Hct116 cells stained with 1 μM of ER-HNO in the presence of ER tracker green; (i) intensity profile of ROIs across cells: red line represents the intensity of ER-HNO and green line indicates the intensity for ER tracker green; (B) co-localization experiment: cells were co-stained with ER-HNO, ER tracker green and DAPI; λ.sub.Ext/λ.sub.Em: 530/573 nm.

[0033] FIG. 4: (A) protein SERCA2 monitoring experiment using western blot assay; (b) Flow cytometry experiment of ER-HNO in presence and absence HNO.

[0034] FIG. 5: (A-C) Confocal laser scanning microscopic (CLSM) images of RAW 264.7 cells incubated with ER-HNO (1 μM) as the control: (A) bright field; (B) dark field laser; and (C) 3D intensity plot of image (B); (D-F) CLSM images of cells incubated with ER-HNO (1 μM) for 15 min and then further exposed to AS (20 μM) for 20 min at 37° C.: (D) bright field; (E) dark field laser; (F) 3D intensity plot of image (E). λ.sub.Ext/λ.sub.Em=530/573 nm.

[0035] FIG. 6: Changes in emission intensity of ER-HNO (10 μM) induced by AS (80 μM) in the presence of a large excess (0.2 mM) of other analytes like HOCl, H.sub.2O.sub.2, .OH, TBHP, NO.sub.3—, NO2-, Benzoyl peroxide, S.sub.2—, NO, ONOO— and ascorbate. Gray bar and black bar represent emission response of ER-HNO (10 μM) in the presence and absence of HNO (80 μM), respectively. Studies were performed in aq. PBS buffer-CH.sub.3CN (9:1, v/v; pH 7.2) medium; λ.sub.Ext/λ.sub.Em: 530/586 nm.

[0036] FIG. 7: Fluorescence intensity of ER-HNO at 583 nm upon addition of HNO (0-22 μM) in acetonitrile: Aq-PBS buffer (1:9, v/v) medium at pH 7.2, λ.sub.Ext=530 nm.

[0037] FIG. 8: Time dependent Emission studies of ER-HNO (10 μM) in absence and presence of 100 μM HNO in aq-PBS-acetonitrile medium (9:1, v/v) of pH 7.2 at 37° C. λ.sub.Ext=530 nm. λ.sub.Mon=586 nm.

[0038] FIG. 9: MTT assay to determine the cell viability percentage in presence of ER-HNO in RAW 264.7 cells. IC.sub.50 was found to be 90 μM.

[0039] FIG. 10: Emission titration of ER-HNO with different concentrations of AS.

[0040] FIG. 11: Bright field & fluorescence images of Artemia after 24 h hatching: Artemia exposed to 1 μM ER-HNO; 20 μM AS (Upper row indicate bright field images; lower row represent the corresponding fluorescence images).

DETAILED DESCRIPTION OF THE INVENTION

[0041] The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

[0042] The terms “compound of formula (I)”, “ER-HNO”, “ER-HNO” and “Probe ER-HNO” are used interchangeably in the specification; however they have the same meaning.

[0043] In the view of above, the present invention provides a compound of formula (I);

##STR00003##

[0044] The IUPAC name of compound of formula (I) is (E)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-5l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)vinyl)phenyl 2-(diphenylphosphanyl)benzoate.

[0045] In another embodiment, the present invention provides a process for preparation of compound of formula (I) comprising the steps of: [0046] a) adding N,N′-Dicyclohexylcarbodiimide (DCC) to a solution of 2-(diphenylphosphino) benzoic acid in solvent followed by stirring the mixture at temperature 0° C. for the period in the range of 1 to 2 hrs to afford reaction mixture; [0047] b) adding (E)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-5l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)vinyl)phenol [ER-S] and 4-Dimethylaminopyridine (DMAP) to the reaction mixture of step (a) at a temperature in the range of 25 to 30° C. for the period in the range of 6 to 7 hrs.

[0048] In preferred embodiment, said solvent is dichloromethane (CH.sub.2Cl.sub.2).

[0049] In another preferred embodiment, said process is carried out under nitrogen atmosphere.

[0050] The process for the preparation of probe ER-HNO is as shown scheme 1;

##STR00004##

[0051] In yet another embodiment, the present invention provides use of compound of formula (I) for the detection of HNO in biological systems.

[0052] In preferred embodiment, the present invention provides a fluorescent OFF-ON based molecular probe ER-HNO for the specific detection of HNO under physiological condition with excellent sensitivity.

[0053] In another preferred embodiment, the present invention provides use of compound of formula (I) as imaging reagent for HNO detection in live HCT116 cells or RAW 264.7 cells.

[0054] The compound of formula (I) exhibits high selectivity, good sensitivity and low cytotoxicity in the detection of HNO. Besides, the compound of formula (I) shows perfect endoplasmic localization in living cells. It is observed that the compound of formula (I) can detect HNO without interference from other analytes.

[0055] In one embodiment, the present invention provides a method of detection of HNO in biological systems using compound of formula (I).

[0056] The spectroscopic properties of ER-HNO in aq. PBS buffer medium under physiological pH medium. No significant difference is observed in the electronic spectra of ER-HNO and ER-S. In order to check the response of ER-HNO probe (10 uM) with in presence of 20 mole equivalent of various ROS and RNS (HNO, HOCl, H.sub.2O.sub.2, —OH, TBHP, NO.sub.3.sup.−, NO.sub.2.sup.−, benzoyl peroxide, S.sup.2− and ascorbate) emission spectra are recorded in aq. PBS buffer medium are shown in FIG. 1. From FIG. 1 it is observed that there is no change in emission intensity in presence of HOCl, H.sub.2O.sub.2, —OH, TBHP, NO.sub.3—, NO.sub.2—, benzoyl peroxide, S.sub.2—, Ascorbate, except HNO. A turn ON emission response is observed for probe ER-HNO, only in presence of HNO with λ.sub.Ems.sup.Max=580 nm (λ.sub.Ext=530 nm). Moreover, no significant changes are observed in presence of different anionic, cationic as well as biothiols under identical experimental condition. The effects of pH on emission spectrum of probe ER-HNO is also investigated. The emission intensity of the probe remains unaltered within the pH range 4-10. Therefore, to explore the bio analytical application, all the studies are carried out at physiological pH medium.

[0057] The probe ER-HNO itself shows a weak emission spectrum (Φ=0.002, for λ.sub.Ext=530 nm) in aq-PBS buffer medium. An enhancement in emission signal are observed at λ.sub.Max=586 nm (Φ=0.105, for λ.sub.Ext=530 nm) with gradual increasing [HNO] (FIG. 1B). A good linearity is observed between emission intensity and concentration of HNO in the range of 2-15 uM, with a lower detection limit of 2 μM. All these result demonstrate that probe ER-HNO detect trace amount of HNO quantitatively with high sensitivity under physiological pH in aq. PBS buffer medium.

[0058] The interferences studies also confirmed the emission response of ER-HNO in presence of HNO, remain unchanged even in presence of excess amount of other analytes, used for the studies (FIG. 2). This conformed that the reagent ER-HNO could be used as a switch ON fluorescence probe for the specific detection of HNO in aq. PBS medium under physiological pH.

[0059] An MTT assay confirmed the non-toxic nature of probe ER-HNO towards live Hct116. Therefore, ER-HNO is tested as an imaging reagent for the detection of HNO in Hct116 colon cancer. The cells are incubated with probe ER-HNO (1 μM) for 15 min at 37° C. in acetonitrile-PBS buffer (0.2:99.8, v/v) at pH 7.2 and no intracellular fluorescence is observed. CLSM images for cells that are further incubated with HNO (20 μM) showed a strong intracellular fluorescence (FIG. 3). The results shown in FIG. 3 clearly show that the reagent ER-HNO could be used as an imaging reagent for the detection of HNO uptake in live CT116 cells.

[0060] From CLSM images as shown in FIG. 3 it is observed that this reagent ER-HNO as well as the product (ER-S) are preferentially localized in the lipid dense region, Endoplasmic Reticulum (ER) of Hct116 cells, the results of the co-localization studies with ER-tracker green (ER-specific reagent) and DAPI (nuclei specific reagent, with little or no cytoplasmic labelling) as well as the high Pearson's co-localization coefficient of 0.975 for this co-localization study confirmed this (FIG. 3). Thus, this reagent offered an option of mapping HNO in the ER region of Hct116 cells.

[0061] The SERCA2 is monitored using ER-HNO using Western blot experiment. The FIG. 4 results confirmed that with using ER-HNO it is possible to monitor the protein activity. Moreover, the reagent ER-HNO could also successful to counteracts the stabilized expression of SERCA2 to at least 50%. DCFDA staining of HCT116 cells too proved that RNS induced by Angeli's Salt is reduced when cells were treated with compound “ER-HNO”. This indicate that's the reagent is capable of regulating the RNS activity in the living cells (FIG. 4B).

[0062] The toxicity of the probe molecule toward live RAW 264.7 macrophages cells and cytotoxicity assays confirmed the low toxicity of the probe. The results encouraged to explore the use of the probe as an imaging reagent for the intracellular detection of HNO in these macrophage cells. Cells in DMEM culture media are incubated with ER-HNO (1.0 μM) for 15 min at 37° C. and subsequently viewed under confocal laser scanning microscope (CLSM) using a 530 nm laser as an excitation source. No intracellular fluorescence is observed until these pretreated cells are also exposed to AS, then a strong intracellular fluorescence was observed under CLSM (FIG. 5).

[0063] As for the CLSM experiments, RAW macrophage cells incubated with ER-HNO (1 μM) for 15 min at 37° C. in acetonitrile-PBS buffer (0.2:99.8, v/v) at pH 7.2 displayed no intracellular fluorescence. In contrast, cells incubated with ER-HNO (1 μM) and then treated with Angeli's salt (Na.sub.2N.sub.2O.sub.3) for 30 min, showed strong intracellular fluorescence (FIG. 6). The feasibility of the reagent for biological in vivo imaging is confirmed by imaging the digestive system of the small brine shrimp, Artemia nauplii, a small marine invertebrate as the model.

[0064] In still another embodiment, the present invention provides a kit for the detection of HNO comprising at least compound of formula (I).

[0065] The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it is being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

[0066] Examples Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1

A. Synthesis of (E)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-5l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)vinyl)phenyl 2-(diphenylphosphanyl)benzoate (ER-HNO)

[0067] Under N.sub.2 atmosphere, to a solution of 2-(diphenylphosphino) benzoic acid (100 mg, 0.32 mmol) in dry CH.sub.2Cl.sub.2, DCC (80 mg, 0.39 mmol) was added and stirred at 0° C. for 2 hr. To this, Compound ER-S (150 mg, 0.35 mmol) and DMAP (20 mg, 0.16 mmol) were added and it was stirred at 27° C. for 6 hr. Reaction was monitored by TLC. Then the mixture was concentrated under vacuum, and the crude product was purified by silica gel column chromatography by using 5% ethyl acetate in PET ether medium to give the compound ER-HNO. Yield 38%. .sup.1H NMR (CDCl.sub.3, 400 MHz, 5 ppm): 7.73 (2H, d, J 6.7), 7.68 (5H, dd, J 12.4, 7.4), 7.63 (2H, d, J 5.4), 7.56 (3H, d, J 8.5), 7.51 (5H, t, J 8.6), 7.47 (5H, d, J 5.6), 7.32 (2H, J 4.9), 7.19 (1H, d, J 16.3), 6.92 (1H, d, J 8.3), 6.60 (1H, s), 6.03 (1H, s), 2.62 (3H, s), 1.44 (3H, s), 1.41 (3H, s). .sup.13C NMR (CDCl.sub.3, 125 MHz): 190.99, 164.54, 155.27, 155.06, 153.99, 153.80, 142.51, 141.53, 137.30, 134.76, 134.53, 134.15, 133.94, 133.68, 133.48, 133.16, 132.88, 132.35, 131.94, 131.85, 131.75, 131.44, 131.11, 130.98, 128.91, 128.67, 128.60, 128.48, 128.41, 122.42, 116.15, 25.98.

B. General Experimental Procedure for UV-Vis and Fluorescence Studies

[0068] Stock solution of probe ER-HNO (1×10.sup.−4M) was prepared in HPLC grade acetonitrile. All the analytes stock solution (1×10.sup.−2M) was prepared in aqueous HEPES buffer (10 mM) medium at pH 7.2. 500 μL of this stock solution of probe ER-HNO was added to 4.5 ml of PBS (10 mM) aqueous buffer medium having solution pH 7.2 to make the effective ER-HNO concentration of (1×10.sup.−5) M. This solution was used for all the photophysical studies. All the photophysical studies were performed in aq. PBS: CH3CN medium (9:1, v/v) at pH7.2. All emission studies were done using λ.sub.Ext=530 nm with an emission slit width of 2/2 nm, unless and otherwise mentioned.

C. General Procedure for Confocal Imaging Studies

[0069] For confocal studies, Hct116 cells (3×10.sup.5) were seeded on cover slips placed in 6 well plates. After 24 hours, Hct116 cells were treated with ER-HNO (1 μM) for 30 minutes at 37° C. in a 5% CO.sub.2 air environment. Cells were then washed thrice with Phosphate Buffer (1×PBS) and fixed with 4% PFA for 20 minutes and washed again with IX PBS. Nail paint was used to seal the cover slips mounted on the glass slides for each well plates. Again Hct116 cells were pre-treated with ER-HNO (1 μM) for 15 minutes, then Hct116 cells were further expose to Angeli's salt (HNO donor), then cells were washed thrice with media and followed by incubation with ER-HNO (1 μM) for another 15 minutes under same conditions. Cells were again washed with 1×PBS buffer and fixed with 4% PFA for 20 minutes and washed again with 1×PBS. Confocal laser scanning microscopic (CLSM) images were acquired in Olympus Fluoview Microscope with λ.sub.Ext/λ.sub.Mon=530/573 nm.

D. Confocal Microscopy Experiments

[0070] RAW 264.7 cells were seeded on cover slips placed in 6 well plates. After 24 hours cells washed with DMEM medium then cells were treated with ER-HNO (1 μM) for 25 minutes. Cells were then washed thrice with culture medium and further treated with Angeli's salt (HNO donor) of different concentration for 30 min. After that cells were washed again with Phosphate Buffer Saline (3×PBS). Then cells were fixed with 4% PFA for 10 min. Again cells were washed thrice and then cover slips mounted using mounting medium. Nail paints was used to seal the coverslips mounted on the glass slides. Images were acquired by using Olympus FluoView FV-1000 Confocal Microscope.

E. Western Blot

[0071] HCT116 cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) supplemented with 100 Units of Penicillin Streptomycin antibiotics. Cells were treated with Angeli's salt 10 uM, ER-HNO 30 μM or both for 24 hrs. Prior to the treatment processes, DMEM medium was treated with N-Ethylmalemide (NEM) 1 mM and this medium was used to culture the cells. Post 24 hrs cells harvested and whole cell lysate proteins extracted. 40 ug of the proteins were resolved in SDS PAGE gel and transferred into PVDF membrane using Western Blot methods. The blot was over-night blocked with 5% Bovine Serum Albumin (BSA) at 4° C. 1:4000 dilution of SERCA2 antibody was to probe the blot for 2 hrs at room-temperature (RT). After three washes with 1×TBST, 1:4000 dilution of anti-rabbit HRP conjugated antibody was used to probe the blot for 1 hr at RT. After three washes with IX TBST, the blot was developed using auto-radiography in X-ray film.

F. Flow Cytometry

[0072] For DCFDA staining, HCT116 cells were cultured in DMEM medium containing 5 mM glucose supplemented with 10% FBS and 100 Units of antibiotics. Prior to the treatment processes, DMEM medium with 1% FBS was treated with N-Ethylmalemide (NEM) 1 mM and this medium was used to culture the cells further. Cells were treated with Angeli's salt 10 uM, L 30 uM or both for 2 hrs. After treatment cells were harvested by trypsinization and washed with complete DMEM media by centrifugation. Pellet was suspended in 1 ml of PBS buffer. 10 μM DCFDA (Sigma) was added to cells followed by incubation at 37° C. 5% CO2 incubator for 45 mins. DCFDA stained cells were analyzed in BD FACS Canto II using FITC channel. For Annexin V staining cultured HCT116 cells were harvested after trypsinization and washed with 1×PBS buffer. 500 ul of Annexin V binding buffer was used to re-suspend the cells. 5 ul of Annexin-V FITC antibody added to the re-suspended cell and incubated in dark for 5 mins. Antibody stained cells were then analyzed using BD Calibur FACs in FITC channel. The flow cytometry experiment confirmed that probe ER-HNO is capable of reducing RNS & ROS level induced by HNO [FIG. 5 (b)].

G. Cell-Culture and Microscopy Experiments

[0073] RAW 264.7 cells were seeded on Coverslips (22 mm×22 mm, 170±5 μm square Cover glasses) placed in 6-well plates in DMEM culture medium containing (10% FBS and 1% penicillin-streptomycin) for 24 h at 37° C. 5% CO2. After 24 h when 70% confluency was achieved the cells were washed with DMEM culture medium then cells were treated with ER-HNO probe (1 μM) for 30 min. Cells were then washed thrice with culture medium and further treated with different Angeli's salt (AS) for 30 min. Then cells were washed again with phosphate buffer saline (3×PBS). After carrying out the Live cell uptake of the ER-HNO probe and the small molecule, the cells were fixed with 4% PFA for 15 min and then washed thrice with PBS and two times and then the coverslips were mounted using the Mounting medium (Vectashield h-1000). The coverslips were then sealed using nail varnish, and the sample was then imaged. Because SIM relies on the cell morphology, the cells were examined with a light microscope and then imaged using SIM.

H. MTT Assay

[0074] The in vitro cytotoxicity of ER-HNO on RAW 264.7 cells were determined by conventional MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. RAW 264.7 cells (5×103) 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 ER-HNO in triplicates for 24 hours. After the 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 90 μM.

[0075] 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.

I. Artemia Culture and Imaging: Experimental Details

[0076] Imaging of Artemia nauplii by luminescence was performed using a fluorescence microscope with a 20×, 0.4 NA microscope objective (Olympus), coupled to an intensified CCD camera. The Artemia nauplii were imaged in both bright-field and epifluorescence geometries. The latter was enabled by a fluorescence filter set. The illumination intensity was about 10 W/mm.sup.2. Artemia nauplii in incubated by ER-HNO (1 μM) for 30 minutes showed no fluorescence, however on exposure to HNO, strong fluorescence was observed by fluorescence microscopy (FIG. 12).

Advantages of the Invention

[0077] 1. The reagent of the present invention specifically detects HNO over other reactive nitrogen species at neutral pH and at physiological condition. λ. The reagent of the present invention could be used to detect HNO present in the ER part of Hct116 colon cancer cells. [0078] 3. It could probe used to monitor the SERCA2a antibody which plays a critical role in initiating cardiac contraction and relaxation in our body. [0079] 4. The reagent of the present invention can be utilized to detect HNO with lower detection limit.