Role Of Novel Carbazole Linked 1, 2, 3- Triazole Analogs in Alleviating Methylglyoxal-Mediated Late Diabetic Vascular Complications

Abstract

The present intervention identifies the antiglycating inhibitors from a series of carbazole-linked 1,2,3 triazole derivatives through in vitro MGO-mediated glycating BSA model. Derivatives 12, and 13 established a remarkable antiglycation activity at the receptor level in human monocytes. These compounds were found non-toxic, and possess the potential to halt AGE-RAGE/ROS- mediated NF- kB-dependent COX-2, and its proinflammatory product, PGE.sub.2, production in monocytes. Hence, carbazole-linked 1,2,3 triazole derivatives provide treatment modalities to delay or prevent the onset of late diabetic micro- and macro-vascular complications in diabetic patients.

Claims

1. A method for reducing the development of diabetes-associated late vascular impairment diabetic atherogenesis progression comprising administration to humans and animals an effective dose of a carbazole-linked 1,2,3-triazole compounds, in combination with inert pharmaceutical ingredients.

2. The method of claim 1, wherein the carbazole-linked 1,2,3-triazole compounds are 2-(4-((9H-Carbazole-9-yl)methyl)-1H-1,2,3-triazole-1-yl)-1-(4-nitrophenyl)ethan-1-one and 9-((1-(2-(2-Methyl-5-nitro-1 H-imidazol-1 -yl)ethyl)-1 H1 ,2,3-triazol-4-yl)methyl)-9H-carbazole.

3. The method of claim 1, wherein the diabetes-associated late vascular impairment and diabetic atherogenesis progression comprise AGEs formation, pro-inflammatory biomarkers, further comprising COX-2 and its associated PGE2 formation.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0010] FIG. 1 depicts the chemical structure of carbazole-linked 1,2,3 triazole derivatives 2-16, studied for their antiglycation activity at various levels of the Maillard reaction.

[0011] FIG. 2 depicts an illustration of antiglycation activity of derivatives 2-16 in MGO-mediated BSA glycation model.

[0012] FIG. 3 depicts the antioxidant activity of lead antiglycation derivatives in human monocyte.

[0013] FIGS. 4a and 4b depict the inhibition of AGE-RAGE/ROS mediated NF- kB translocation by selected carbazole-linked 1,2,3 triazole derivatives 12-13 in human monocytes.

[0014] FIGS. 5a, and 5b depict the levels of COX-2 inhibition in human monocytes-cotreated with MGO-AGEs (50 .Math.g/mL) and carbazole- triazole derivatives 12-13 by employing immunoblotting.

[0015] FIG. 6 depicts the inhibition of COX-2 proinflammatory product, PGE.sub.2, induced by MGO-mediated BSA glycation via 12-13, representing the potential of compounds to inhibit inflammatory pathway leads to vascular dysfunction.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention elucidates the potent glycation inhibitors from a series of synthetic carbazole-linked 1,2,3 triazole derivatives to inhibit AGEs formation, and alleviate AGEs activated proinflammatory signaling cascade in human monocytes.

Example 1

Materials

[0017] Methylglyoxal (MGO), bovine serum albumin (BSA), standard antiglycation agents (quercetin, and rutin hydrate), and DCFH-DA were purchased from Sigma-Aldrich Chemical Corporation (St. Louis, Missouri, United States). Dimethyl sulfoxide (DMSO) was purchased from Amersco L.L.C (Ohio, United States). 96-Well flat-bottom non-sterile and sterile polystyrene black fluorescence plate was procured from Corning Inc. (New York, USA). Sodium azide was obtained from Merck (Darmstadt, Germany). Spectrofluorimeter, NF-.sub.KB (p.sup.65), COX-2 antibody, and DAPI were obtained from Thermo Fisher Scientific (Waltham, MA, USA). β-Actin (Cloud-Clone Corp., Wuhan, China). Sodium dihydrogen peroxide (NaH.sub.2PO.sub.4) and disodium hydrogen phosphate (Na.sub.2HPO.sub.4) were purchased from Duskan Pure Chemicals Co Ltd (Gyeoniggi-do, South Korea). While, ECL, and β-actin antibody were obtained from Sangon Biotech (Shanghai, China), and Cloud-Clone Corp (Wuhan, China), respectively. The Nikon 90i microscope (Tokyo, Japan) was used for the imaging of NF-.sub.KB (p.sup.65) translocation. The power blotter XL System Invitrogen, Thermo Fisher Scientific (Waltham, MA, USA) was used to transfer the blot. All chemicals were prepared in deionized water under an aseptic environment at 37° C.

Methodology

[0018] In vitro antiglycation activity: The MGO- BSA assay was performed as per optimized protocol of Jahan, H. et al with slight modifications [49]. Initially, the antiglycation activity of parent compound carbazole (1), and carbazole-linked 1,2,3 triazoles 2-16 were identified at 1 mM using MGO-BSA model. Compounds that showed antiglycation activity at 1 mM were diluted serially. BSA (10 mg/mL) incubated with MGO (0.1 M) for 24 h at 37° C. using phosphate- azide buffer (0.1 mM), served as a positive control. The wells with BSA only were taken as negative control. Whereas, rutin and quercetin were used as standard antiglycation agents. After 24 h of incubation, the fluorescence of each well was measured by using microplate reader (Varioskan lux, Thermo Fisher Scientific, 319 Scientific, USA) at 355 nm excitation, and 460 nm emission.

[0019] The % inhibition of MGO-BSA-mediated by carbazole-triazole derivatives was quantified by using the following formula:

[00001]$\text{\%}\text{}\text{Inhibiton}\text{of}\text{fluorescence =}\frac{1-\text{Fluorescence of test compounds}\times \text{100}}{\text{Fluorescence of glycated BSA}}$

[0020] The IC.sub.50 of active antiglycation compounds were identified by using EZ-FIT Enzyme Kinetics Program (Perrella Scientific Inc., Amherst, USA).

[0021] Results: The antiglycation activity of carbazole-linked 1,2,3 triazoles 2-16 and parent compound carbazole (1) was measured in MGO-BSA glycating model. The structures of carbazole-linked 1,2,3 triazole derivatives 2-16, and carbazole (1) are presented in FIG. 1. All these compounds exhibited greater activity, as compared to precursor carbazole (36.7% inhibition). Among all the compounds, compound 12 (83.7% inhibition, IC.sub.50: 76.1±6 .Math.M) exhibited an excellent antiglycation activity, as compared to standard compounds, i.e., rutin (67% inhibition, IC.sub.50:104±2 .Math.M), and quercetin (58% inhibition, IC.sub.50: 138±4 .Math.M). Compounds 7 (72% inhibition, IC.sub.50: 275 ±5.0 .Math.M), 8 (79% inhibition, IC.sub.50: 130 ±10.0 .Math.M), 11 (75.7% inhibition, IC.sub.50: 147 ±10.0.Math.M), 13 (77.8% inhibition, IC.sub.50: 135 ±5.0 .Math.M), and 15 (71% inhibition, IC.sub.50: 333 ±10.0.Math.M) had shown good antiglycation activity, while compound 2 (58% inhibition, IC.sub.50: 797 ±7.0 .Math.M) had exhibited a weak activity. On the other hand, compounds 3-6, 9-10, 14, and 16 (18.2, 15.6, 22, 39, 18,1, 12.1, 43.9, and 41.5% inhibition, respectively) were found to be inactive in MGO-BSA glycation model, as depicted in FIG. 2.

Example 2

[0022] Cytotoxicity analysis: The cytotoxic profile of the compounds was determined by using the MTT assay in hepatocytes (HepG2 cell line), and WST-1 assay in human monocytes (THP-1 cell line) as per the manufacturer’s protocols. The 20 × 10.sup.4 THP-1/mL, and 7 × 10.sup.4 Hep-G2/mL were plated in sterile 96-well flat bottom plates. The cells were exposed to a series of concentrations (10, 30, 50, 100, 250, and 500 .Math.M) of carbazole-linked triazole derivatives for 24 h. Doxorubicin treated cells were used as a positive control, while cells with culture medium served as negative control. Wells with culture medium served as a blank. Followed by the incubation, 20 .Math.L of MTT were added in each well. Later, 100 .Math.L of DMSO were added into wells containing Hep-G2 cells to dissolve formazan crystals. The absorbance was measured by using 540, and 450 nm, respectively (Varioskanmicroplate reader, Thermo Fisher, 319 Scientific, USA).

[0023] Results: The compounds that exhibited antiglycation activity (compounds 2, 7-8 and 11-13 and 15) were selected to quantify their cytotoxicity at different concentrations (10-500 .Math.M) using HepG2, and THP-1 cell lines. The data revealed that compounds 2, 11-13, and 15 were non-toxic till 100 .Math.M, while compounds 7-8, had shown different toxicity profiles, as presented in Table-1.

[0024] Viability Assay: The viability of cells treated with various concentrations (10, 30, 50, 100, 200, and 500 .Math.g/mL) of MGO-AGEs was measured by WST-1 metabolic assay. The data revealed that monocytes treated with 50 .Math.g/mL of AGEs were found viable, and hence selected this concentration as a reference to stimulate monocytes for studying the inhibition of intracellular mechanism by potential candidates.

Example 3

[0025] Anti-oxidation activity: The measurment of effect of derivatives on AGEs-mediated ROS was based on the previously reported protocol of Soumyarani and colleagues with slight amendments. Initially, 1 × 10.sup.6 monocytes/mL were loaded in sterile 96-well flat bottom black fluorescent plate, and pre-incubated with DCFH-DA (10 .Math.M; Sigma-Aldrich Chemical Corporation, St. Louis, Missouri, USA) for 45 min at 37° C. Followed by the incubation, antiglycating, non-toxic carbazole-linked triazole derivatives (10, 30, 50, and 100 .Math.M) were added to the wells for 1 h before co-treatment of cells with MGO-AGEs (50 .Math.g/mL) for next 24 h. Next, the fluorometric measurement of converted DCFH into oxidized DCF product was carried out via spectrofluorimeter (Varioskanmicroplate reader, Thermo Fisher Scientific, USA) using 485 nm excitation and 520 nm emission wavelengths. Various controls were plated in each experiment. Wells containing culture RPMI with MGO-AGEs (50 .Math.g/mL) and H.sub.2O.sub.2 (10 .Math.M) served as positive controls, while cells containing BSA were taken as negative control.

[0026] Results: The non-toxic, antiglycating carbazole-linked 1,2,3-triazole derivatives 2, 11-13 and 15 were used to identify its anti-oxidative activity against MGO-BSA treated human monocytes. The data revealed that 50 .Math.g/mL of MGO-BSA-treated THP-1 monocytes exhibit significantly increased ROS formation than the control (untreated- and BSA-treated monocytes), with P-value <0.05. All the wells treated with selected compounds have shown maximum inhibition at 100 .Math.M. Among all, compounds 12 (6.32 RFU), and 13 (8.75 RFU) had shown an excellent antioxidant activity, comparable to standard, rutin (7.44 RFU). Compounds 11 (9.06 RFU), and 15 (10.6 RFU) had shown a moderate antioxidant activity, as compared to standard compounds rutin, PDTC (7.84 RFU) and quercetin (8.39 RFU). While, compound 2 was found to be inactive, as shown in FIG. 3.

Example 4

[0027] NF-.sub.KB (p.sup.65) translocation analysis: The monocytes at the density of 1 × 10.sup.6 cells/mL were pre-incubated with compounds (100 .Math.M) for an h, and then co-incubated with AGEs (50 .Math.g/mL) for another 1 h in a 24-well plate at 37° C. Following treatment, cells were fixed with paraformaldehyde (4% PFA; 10 min), and permeabilized with Triton X-100 (0.2%; 10 min). Next, the cells were rinsed with chilled PBS three times, and incubated with bovine serum albumin (1% BSA; 1 h) to block the non-specific binding sites. Cells were exposed to primary antibody against NF-.sub.KB (p.sup.65) (1:300 dilution, Thermo Fisher Scientific, Waltham, MA, USA) for overnight at 4° C. Subsequently followed by washing the cells with chilled PBS three times, secondary antibody (polyclonal fluorescein isothiocyanate (FITC)-conjugated antibody) to rabbit IgG (1: 1000 dilution, Abeam, Cambridge, UK) was used. Nuclei were counterstained by using DAPI (Thermo Fisher Scientific, Waltham, MA, USA). The BSA-treated monocytes (50 .Math.g/mL) had served as a negative control, while AGEs-treated monocytes (50 .Math.g/mL) as positive control. All the images were taken by using Nikon 90i microscope (Nikon, Tokyo, Japan). The quantification of images was carried out via ImageJ (Image processing and analysis in Java - NIH).

[0028] Results: Based on antiglycation, and antioxidant activities, as well as non-toxic profile of compounds 12 and 13, they were selected for the intracellular mechanistic study. Initially, the elevated kβ translocation in MGO-BSA-treated human monocytes (MGO-AGEs: 75 RFU) was compared to BSA-treated human monocytes (11 RFU). The pretreatment of monocytes with compounds 12 (33 RFU), and 13 (54 RFU) significantly reduced the NF-kβ translocation by showing P-value <0.05. Both derivatives were found comparatively more effective than standards PDTC (45 RFU), and rutin (43 RFU). While compound 12 exhibited higher activity, and compound 13 exhibited similar level of suppression of NF-kβ translocation than standard quercetin (55 RFU) in MGO-treated human monocytes (FIGS. 4a, and 4b).

Example 5

[0029] COX-2 protein levels analysis: The 50 .Math.g/mL of protein was loaded onto 10% sodium dodecyl sulfate - polymerized acrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes using power blotter XL System (Invitrogen; Thermo Fisher Scientific). Subsequently, the membrane was incubated for 1 h in 1 % bovine serum albumin in PBS with 0.1% Tween-20 to block unspecified binding. The protein bands of the membrane were probed with primary monoclonal antibody COX-2 (Thermo Fisher Scientific, Waltham, MA, USA), and β-actin (Cloud-Clone Corp., Wuhan, China) for overnight at 4° C. Following incubation, the membrane was rinsed with tris buffer saline tween (TBST) three times, and then exposed to secondary antibody (HRP mouse mono anti-rabbit IgG; Abeam, Cambridge, UK) at room temperature for an hour. The washing was again carried three times with TBST, and then the membrane was exposed to ECL (Sangon biotech, Shanghai, China) to visualize the bands in Western Blot Detection System (Thermo Fisher Scientific, Waltham, MA, USA). The densitometry of immuno-positive COX-2, and β-actin bands was used by Image J.

[0030] Results: The wells containing MGO-BSA-treated human monocyte models has shown elevated COX-2 enzyme levels, as compared to BSA-treated human monocytes. Followed by the treatment of compounds 12, and 13, a significant reduction in COX-2 levels (3 fold, and 3.5 fold, respectively) was observed, as compared to MGO-BSA-treated cells, by showing P value <0.05, as depicted in FIGS. 5a, and 5b. Furthermore, we found that compounds 12, and 13 were more potent in decreasing COX-2 levels, as compared to standards PDTC (1.60 fold), and rutin (1.65 fold). Whereas, compound 12 exhibited a higher inhibitory effect than quercetin (1.31 fold), and compound 13 showed almost equivalent activity as quercetin.

Example 6

[0031] PGE.sub.2levels analysis: ELISA-based method was employed to identify the concentration of PGE.sub.2 in culture supernatants of THP-1 cells that was initially exposed with compounds for 1 h, and then stimulated with AGEs (50 .Math.g/mL). All the steps were followed according to the manufacturer’s protocols.

[0032] Results: The data revealed that compounds 12, and 13 reduced PGE.sub.2 levels significantly by showing P value <0.05, as compared to control wells (FIG. 6). Consistent with the finding of COX-2 levels, standard PDTC, and rutin exhibited relatively more suppression than quercetin.

TABLE-US-00001 In vitro cytotoxic profile of selected carbazole-triazole derivatives on human monocytes and hepatocytes. Compounds IUPAC Concentrations (.Math.M) % Inhibition±SD THP-1 Hep-G2 2 2-(4-((9H-Carbazole-9-yl)methyl)-1H-1,2,3-triazole-1-yl)-1-phenylethan-1-one 1 N.sub.A N.sub.A 10 30 50 100 250 500 7 2-(4-((9H-Carbazole-9-yl)methyl)-1H-1,2,3-triazole-1-yl)-1-(o-tolyl)ethan-1-one 1 21±8.8 12.3±3.7 10 27±4.2 21.8±4.7 30 34.6±6.5 21.8±1.5 50 32.1±0.4 41.1±3.5 100 50.7±5.3 50.3±3.9 250 80.8±5.1 59.8±15.5 500 79.5±5.8 78.4±2.2 8 2-(4-((9H-Carbazole-9-yl)methyl)-1H-1,2,3-triazole-1-yl)-1-(m-tolyl)ethan-1-one 1 35.5±1.1 6.4±2.2 10 42.1±0.4 8.7±4.8 30 43±4.3 47.3±2.2 50 43.9±0.7 54.5±0.7 100 46.7±2.0 66.9±1.9 250 73.6±1.9 74.1±1.9 500 99.5±0.3 86.7±5.8 11 2-(4-((9H-Carbazole-9-yl)methyl)-1H-1,2,3-triazole-1-yl)-1-(3-nitrophenyl)ethan-1-one 1 N.sub.A N.sub.A 10 30 50 100 250 500 12 2-(4-((9H-Carbazole-9-yl)methyl)-1H-1,2,3 -triazole-1-yl)-1-(4-nitrophenyl)ethan-1-one 1 N.sub.A N.sub.A 10 30 50 100 250 500 13 9-((1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-1H1,2,3-triazol-4-yl)methyl)-9H-carbazole 1 N.sub.A N.sub.A 10 30 50 100 250 500 15 1,4-Bis ((4-((9H-carbazol-9-yl) methyl)-1H-1,2,3-triazol-lyl)methyl) benzene 1 N.sub.A N.sub.A 10 30 50 100 250 500 Rutin 2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside 1 N.sub.A N.sub.A 10 30 50 100 250 500 Quercetin 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one 1 N.sub.A N.sub.A 10 30 50 100 250 500 Standard (Doxorubicin) (7S,9S)-7-[(4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione;hydrochlorid 1 34.2±0.0 30.4±5.3 10 60.2±0.6 28.3±4.8 30 63.2±0.4 64.0±0.4 50 64.5±2.4 58.0±2.4 100 67.3±0.2 62.6±0.2 250 76.4±10 69.8±10 500 94.3±2.3 87.1±2.2 S.D. Standard deviation of mean of three independent experiments N.sub.A = Not active