Click chemistry inspired facile synthesis and bioevaluation of novel triazolyl analogs of D-(+)-pinitol

2.2.1 In vitro screening of the novel analogs against human cancer cell lines

As part of our ongoing research program on bio-prospection of natural products (Rehman et al., 2015; Lone et al., 2013a,b), all the newly synthesized analogs (1/1a–25/25a) prepared through click reaction were screened for anti-proliferative activity against promyelocytic leukemia (HL-60), colorectal carcinoma (HCT-116) and pancreatic carcinoma (Mia-Paca-2) cell lines using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay.

Preliminary cytotoxicity screening of the analogs was carried out at 100 μM concentration and cytotoxicity was determined. Some of the synthesized analogs displayed broad spectrum cytotoxic effect in a dose dependent manner. The analogs that exhibited greater than 50% anti-proliferative activity at 100 μM concentration were further assayed at different concentrations (10–100 μM) to determine the IC₅₀ values (Table 1). The values are the average of triplicate analysis. BEZ-235 (Dactolisib), an imidazoquinoline derivative, used as a potent antineoplastic agent was used as positive control in this assay.

Of the synthesized triazoles, only seven derivatives exhibited appreciable anti-proliferative activity against the tested cancer cell lines. Compounds 2, 3 and 7 showed comparatively better activity with IC₅₀ values of 19.2, 17.5 and 16.4 μM against leukemia (HL-60), colorectal carcinoma (HCT-116) and pancreatic carcinoma (Mia-Paca-2) cell lines respectively. Compound 22 displayed selective cytotoxicity against colorectal carcinoma (HCT-116) cell line with IC₅₀ of 24.8 μM. Among all the synthesized derivatives a few showed moderate activity, while as, others were weakly active toward the tested cancer cell lines. In addition to this, oxygen deprotected analogs (1a–25a) were unable to sensitize any of the tested cancer cell line.

From the activity profile (Table 1), a structure activity relationship (SAR) was drawn which reflects that the compounds with bromo substitution in R moiety (2 and 4) are comparatively effective cytotoxic agents. Compound 3 with a 2,5-dimethylphenyl R moiety displayed significant cytotoxicity against all the tested cancer cells, while as, compound 17 with a 5-iodo-2-methylphenyl R moiety was ineffective to sensitize any of the cancer cell line in the preliminary cytotoxicity testing, highlighting the beneficial impact of an extra methyl group at 5 position in SR-3 toward the cytotoxic activity. However, it was also observed that a nitro (NO₂) functionality at ortho and meta position in the R moiety (8 and 24) of pinitol triazoles plays a significant role in achieving better and enhanced activity against all the tested cancer cell lines, while as, compound 5 having para nitro functionality in the R moiety is inactive toward the tested cancer cell lines. Compound 22 bearing an orthocyanophenyl R moiety exhibited selective cytotoxic activity against HCT-116 cell line, while as, paracyanophenyl analog (6) did not pass the preliminary cytotoxicity testing.

2.2.2 Glucosidase inhibition studies

Glycosidases are carbohydrate processing enzymes which are implicated in an array of vital biological processes that include cell–cell and cell-virus recognition, synthesis of complex carbohydrates, N-linked glycoprotein processing (Gloster and Vocadlo, 2012; Horne et al., 2011). Inhibitors of such enzymes, both natural and synthetic, have been the subject of extensive studies in the past few decades. Such inhibitors have been found to have tremendous potential as therapeutic agents in the treatment of a number of diseases such as diabetes, obesity, high blood pressure, viral infection, lysosomal storage disorder, and tumor metastasis (Dedola et al., 2010; Sánchez-Medina et al., 2001; Mehta et al., 1998; Yoon et al., 1978; Simoes-Pires et al., 2009; Verma et al., 2012). There is no doubt that many more glycosidase inhibitors of practical use remain to be discovered. In view of the early reports of β-glucosidase inhibition by pinitol derivatives (Falshaw et al., 2000) and certain carbohydrate derived triazoles (Kumar et al., 2011) and the necessity to develop new glycosidase inhibitors, the present work was aimed to evaluate the possible β-glucosidase inhibitory activity of the triazolyl derivatives of D-(+)-pinitol.

Preliminary screening of the derivatives was carried out at 166.7 μM concentration and percent glucosidase inhibition was determined. The inhibitory effects of all the synthesized derivatives were compared with those of castanospermine, a well-known and potent β-glycosidase inhibitor (Saul et al., 1983). The analogs that exhibited significant β-glucosidase inhibition (>50%) at the preliminary screening concentration were further assayed at different concentrations (16.6, 41.5, 83.3, 166.7 and 250.0 μM) to generate the IC₅₀ values (Table 2). The values are the average of triplicate analysis.

Of the synthesized analogs, only ten exhibited appreciable enzyme inhibition against the sweet almond β-glucosidase. Compounds 6a, 18a and 25a showed the best activity with IC₅₀ of 148.5, 139.2 and 142.4 μM respectively, while as majority of the derivatives were weakly active against the tested enzyme. The activity profile (Table 2), reflects that the compounds with –NO₂ or –CN substitutions in R-moiety display strong β-glucosidase inhibitory effects. Compounds 6a, 22a and 23a with –CN functionality at p, o and m positions in R moiety displayed strong inhibitory effects with IC₅₀ of 148.5, 186.3 and 208.9 μM respectively. Similarly, 8a, 24a and 25a with –NO₂ group at o, m and p positions in R moiety also showed satisfactory β-glucosidase inhibition with IC₅₀ of 169.3, 216.8 and 142.4 μM respectively. These observations reveal that the analogs with p-CN and p-NO₂ substitutions in R moiety are more active than the corresponding ortho and meta substituted derivatives. Additionally, Compounds 12a, 13a and 15a with nitrogen in R moieties displayed significant β-glucosidase inhibition. However, 18a with n-octyl R moiety showed strong β-glucosidase inhibition with IC₅₀ of 139.2 μM highlighting the importance of aliphatic hydrocarbon chain for achieving the better and enhanced inhibitory activity. Hence, these results suggest that N-containing R moieties along with long chain aliphatic hydrocarbon R moieties seem to be essential for β-glucosidase inhibition. Based on the notable β-glucosidase inhibitory activity of triazolyl derivatives of D-(+)-pinitol, it would not be surprising that such molecules could become the center of attraction for the medicinal chemists working in this field.

It is noteworthy to mention that it is not only the effect of a particular group but also its position plays a significant role in bioactivity. The most active compounds could become a better arsenal toward the corresponding sensitive cell lines or enzymes after further polishing and fine tuning.

4.1.1 Preparation of 3-O-Methyl-1,2:5,6-bis-O-isopropylidene-D-chiro-inositol/(pinitol diacetonide) SR-A

D-(+)-pinitol (3.0 g, 15.5 mmol, 1 eq) was taken in acetone (30 mL) and p-toluene sulfonic acid monohydrate (0.94 g, 5 mmol, 0.3 eq) and 2,2-dimethoxy propane (5.7 mL, 46.5 mmol, 3 eq) were added with stirring. After completion of the reaction (2 h), triethylamine (1 mL) was added and the solvents were removed in vacuo. The residue was subjected to column chromatography using hexane:ethyl acetate (8:2) as mobile phase to give SR-A as white crystalline solid (4.2 g, 15.3 mmol). Yield: 96%; mp. 132 °C; ¹H NMR (400 MHz, CDCl₃) δ: 4.23 (4H, m), 3.59 (4H, s), 3.22 (1H, dd, J = 6.0, 5.2), 1.51 (6H, s), 1.37 (6H, s); ¹³C NMR (100 MHz, CDCl₃) δ: 110.2, 110.0, 81.5, 79, 78, 77.1, 76.8, 71.6, 59.3, 29.9, 29.4, 25.4, 25.3; HR-ESIMS m/z calcd for C₁₃H₂₂O₆ [M + H]⁺ 275.1424, found 275.1488.

4.1.2 Preparation of 3-O-Methyl-1,2:5,6-bis-O-isopropylidene-4-propargyl-D-chiro-inositol SR-B

To a solution of diacetonide (SR-A) (2.0 g, 7.3 mmol, 1.0 eq) in acetonitrile (10 mL), NaH (192 mg, 8.7 mmol, 1.2 eq) was added portionwise and stirred for 15 min at 0 °C. Solution of propargyl bromide (0.82 mL, 10.9 mmol, 1.5 eq) in acetone (3.0 mL) was then added and the suspension was stirred for 6 h. Progress of reaction was monitored using TLC in hexane:ethyl acetate (9:1) at regular intervals. After the completion of reaction, the product was extracted with ethyl acetate (30 mL × 3) which on subsequent purification over silica gel column resulted in the isolation of pure product SR-B in 90% yield. Cream colored solid; Yield: 93%; mp. 117 °C; ¹H NMR (400 MHz, CDCl₃) δ: 4.46 (m, 2H), 4.24 (s, 2H), 4.19 (m, 1H), 3.59 (s, 3H), 3.54 (m, 1H), 3.30 (m, 1H), 2.45 (s, 1H), 1.52 (s, 6H), 1.35 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 109.75, 109.65, 81.55, 79.90, 78.87, 78.72, 78.60, 76.42, 76.35, 74.46, 59.85, 58.85, 27.81, 27.74, 25.33, 25.28; HR-ESIMS m/z calcd for C₁₆H₂₄O₆ [M + H]⁺ 313.1583, found 313.1632.

4.1.3 General procedure for synthesis of azides

To a solution of particular aromatic amine in 1,4-dioxane (at the concentration of 50 mg/mL) at −15.0 °C, 5 equivalents of 2 M Sulphuric acid was added in small instalments while stirring. After 5 min 2 equivalents of 3 M sodium nitrite was added drop wise and after 30 min 3 equivalents of 3 M sodium azide was added drop wise carefully. Reaction was brought to room temperature and extracted with diethyl ether for at least three times. Organic layers were washed with saturated sodium bicarbonate solution two times, dried over anhydrous sodium sulphate and concentrated to a minimum volume under reduced pressure on Rotary evaporator without making use of heating from water bath (Dedola et al., 2010).

4.1.4 General procedure for synthesis of triazolyl derivatives 1–25 and 1a–25a

To a solution of SR-B (50 mg, 0.16 mmol, 1 eq) in t-BuOH:H₂O (2:1, 5 mL), sodium ascorbate (4.0 mg, 0.024 mmol) and CuSO₄⋅5H₂O (4 mg, 0.015 mmol) were added at room temperature. To this mixture, freshly prepared organic azides (0.24 mmol) were added and the reaction mixture was sonicated till its completion. The crude mixture was extracted with ethyl acetate (3 × 20 mL) and the combined organic layer was dried over sodium sulphate and purified through column chromatography to give pure 1–25 in 88–96% yield. 1–25 (25 mg each) were further treated with trifluoroacetic acid:H₂O (1:1; 5 mL) for the deprotection of 1,2 and 5,6 hydroxyls and the reaction mixture was allowed to stir at room temperature for 3 h to afford corresponding analogs (1a–25a) in 44–59% yield.

4.2.1 MTT cytotoxicity assay

All the compounds were evaluated against a panel of three different human cancer cell lines viz. promyelocytic leukemia (HL-60), colorectal carcinoma (HCT 116) and pancreatic carcinoma (Mia-Paca-2) using MTT assay in a 96 well plate. Cells were routinely maintained in RPMI 1640 (Sigma Aldrich) supplemented with 10% FBS (Merck) and 1% penicillin G and streptomycin (Sigma Aldrich) at 37 °C in a humidified incubator with 5% CO₂ and were subcultured at 1:5 ratio once a week. For antiproliferative activity, compounds were dissolved in cell culture grade DMSO. Briefly, cells (10⁴ cells/well) were cultured in 96 well tissue culture plates and treated with different concentrations of compounds for 48 h. At the end of incubation, 20 μL of MTT (2.5 mg/mL) was added to the wells and incubated for 4 h. Absorbance was recorded at 570 nm using Eliza Plate Reader. Inhibition of formation of colored MTT formazan was taken as an index of cytotoxicity activity. The IC₅₀ values on the cancer cells of different tissue origin used for screening were determined by nonlinear regression analysis using graph pad software (Chashoo et al., 2011).

4.2.2 β-D-glycosidase inhibitory assay

All buffers and solutions were prepared using Millipore filtered water. Assays were performed in triplicate at 37 °C using 96 well microtitre plates with a final assay volume of 300 μl. All assays used 200 μL of 250 μM p-nitrophenyl glycoside (substrate) solution, 50 μl test inhibitor solution and 50 μL enzyme solution (0.2 U/mL), buffered to pH 5.0 in 0.12 M phosphate (Pi) buffer. The liberated p-nitrophenol (PNP) was measured at 405 nm after quenching the reactions with 100 μL borate buffer (pH 9.8, 0.2 M). Reaction mixture containing Pi buffer (pH 5.0) in place of inhibitory samples was used as negative control and castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), a commercially available β-D-glycosidase inhibitor was included in the assay as positive control. The percentage inhibition was calculated using the following formula: $$\text{Inhibition}(\%)=1-\frac{{\text{ABS}}_{\text{sample}}-{\text{ABS}}_{\text{positive control}}}{{\text{ABS}}_{\text{negative control}}}\times 100$$

For the time-scale inhibition studies, at each pre-incubation time-step enzyme (50 μl of 0.2 U/mL) was added to the test inhibitor (50 μl of 1000 μM) or Pi buffer for the negative control (50 μl). At the end of the pre-incubation time, the corresponding p-nitrophenyl glycopyranoside (200 μl of 250 μM) was added to each well and incubated for further 5 min at 37 °C. To determine the inhibitory concentrations for the various inhibitors, enzyme (50 μl of 0.2 U/mL) was added to each well containing 50 μl of various concentrations of inhibitor (0, 100, 250, 500, 1000, 1500 μM; to give final assay concentrations of 0, 16.6, 41.5, 83.3, 166.7 or 250.0 μM, respectively) and pre-incubated for 1 h. After this time, the corresponding p-nitrophenyl glycopyranoside was added (200 μL of 250 μM) and incubated for a further 5 min. The results are expressed as percentage activity relative to the control wells. The IC₅₀ values were determined using graph pad prism 5 software.