New Gemcitabine Derivatives as Potent in vitro α-Glucosidase Inhibitors
Farked Wahoodi Salman 1, Ahmed Jasim Twayej 2, Hayder Ahmed Shaheed 3, Ali Jabbar Radhi 3*
1 Department of Chemistry, Faculty of Science, Kufa University, Najaf-Iraq.
2 Faculty of Health and Medical Techniques, Al-Kafeel University, Najaf, Iraq.
3 Faculty of Pharmacy, Al-Kafeel University, Najaf, Iraq.
* Corresponding author. E-mail: Alijebar56@gmail.com
Received: Jun. 28, 2018; Accepted: Feb. 13, 2019; Published: Mar. 20, 2019
Citation: Farked Wahoodi Salman, Ahmed Jasim Twayej, Hayder Ahmed Shaheed, and Ali Jabbar Radhi, New Gemcitabine Derivatives as Potent in vitro α-Glucosidase Inhibitors. Nano Biomed. Eng., 2019, 11(1): 84-90.
In this work, new heterocyclic compounds 1,2,3-triazoline derivatives starting from gemcitabine were synthesized. At first, gemcitabine was converted to 2-azido gemcitabine (G) through the reaction of gemcitabine with sodium azide. 1,2,3-Triazolines were prepared from the reaction of 2-azido gemcitabine with some unsaturated compounds such as malic anhydride, cinamic acid and acryl amide by click reaction. The products were identified by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) technique. The α-glucosidase inhibitory activities of all the synthesized compounds were determined in vitro. All the tested compounds showed α-glucosidase inhibitory activity of IC50 = 144.8 ± 1.74, 212.9 ± 3.4 and 345 ± 4.5 µM against the α-glucosidase enzyme when compared to the standard drug acarbose IC50 = 824 ± 1.73 µM.
Keywords: Gemcitabine; 1,2,3-triazolines; α-glucosidase inhibitors
Alpha-glucosidase is an enzyme that catalyzes the hydrolysis of polysaccharides into monosaccharide, leading to post-parandial hyperglycemia in diabetic patients . This high blood glucose level (hyperglycemia) causes damages to vital organs, and leads to complications, such as cataract, retinopathy, neuropathy, atherosclerosis, nephropathy and impaired wound healing [2-5]. α- Glucosidase inhibition has been reported mainly to overcome the risk of hyperglycemia after eating in diabetics, which in turn is associated with the above-mentioned health disorders [6, 7]. Acarbose, 1-dioxinogermesin and miglitol were developed as α-glucosidase inhibitors. However, many of them have adverse effects and low patient tolerance. Gemcitabine (Scheme 1), a pyrimidine fluorinated nucleoside analog, is chemically known as1-(2' deoxy-2', 2'-difluoro-D-ribofuranosyl)-4-aminopyrimidin-2-one hydrochloride and 2’,2’-difluorodeoxycytidine. Originally developed by Lilly, it is an anticancer drug marketed as the HCl salt under the trade name of Gemzar (Lilly) . Gemcitabine structure is similar to cytarabine; it has a wider spectrum of solid antitumor activity because of its difference in pharmacology and the mechanism of action . Gemcitabine is a type of chemotherapy to treat many types of cancer including pancreatic cancer, lung cancer, ovarian cancer and breast cancer [10-12]. In pancreatic cancer, gemcitabine is administered as a single agent, but in lung cancer non-cell lung cancer and bladder cancer, it is given in conjunction with cisplatin . Gemcitabine can interfere speedily with the growing cells such as cancer cells and kill this cell. Gemcitabine is a prodrug  which undergoes phosphorylation intracellular into the form of active triphosphate and diphosphate, with the effect of DNA synthesis leading to apoptosis . In this study, a series compounds of pharmacologically active 1,2,3-triazolines derivatives based on gemcitabine were successfully prepared and their in-vitro α-glucosidase inhibitory activity was investigated.
Scheme 1 Structural formula of gemcitabine.
All chemicals and solvents were obtained from Merck, BDH, Fluke, and Sigma-Aldrich and were used without further purification. Fourier-transform infrared spectroscopy (FTIR) spectra were recorded by using Fourier transformation infrared Shimadzu IR-408” at Faculty of Pharmacy, University of Kufa. Elemental analysis was performed using a Perkin-Elmer 204E Instrument. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on Bruker 300 MHz. Thin layer chromatography (TLC) was performed on a silica gel SG-40 (Merck) and developed with the solvents mentioned; spots were visualized with Iodine vapor.
General procedure for synthesis of 2-azido gemcitabine (G)
60 mmol of gemcitabine and 60 mmol of sodium azide were dissolved in 30 mL of tetrahydrofuran (THF). The resulting mixture was stirred at 60 ℃ for 4 h. Then, the TLC showed that the reaction was completed by using (ethyl acetate: petroleum ether, 1 : 4). Then, it was extracted with 40 mL of diethyl ether three times and then the organic phase was treated with 100 mL of water (three times). Subsequently, the diethyl ether phase was dried by adding anhydrous magnesium sulfate MgSO4 and then filtered. The diethyl ether was evaporated and the residue was dried and purified by recrystallization from ethanol (Scheme 2). 2-azido gemcitabine (G) was prepared as a white solid powder, Chemical formula: C9H11FN6O4; yield: 64%; m.p.: 240-242 ºC. FTIR: 3454 cm-1, 3411 cm-1 due to OH stretching, 2971 cm-1, 2870 cm-1 due to CH stretching, 2105 cm-1 due to azide group stretching, 1671 cm-1 due to carbonyl group stretching, 1580 cm-1 due to C=N stretching, 1276 cm-1, 1119 cm-1, 1075 cm-1 due to C-O stretching and 796 cm-1 due to C-H bonding. 1H-NMR (301 MHz, DMSO-d6) (Fig. 1): δ 9.79 ppm (s, 1H), 7.83-7.71 ppm (m, 2H), 7.63 ppm (d, J = 7.5 Hz, 1H), 7.18 ppm (s, 1H), 7.01-6.90 ppm (m, 2H), 6.07 ppm (d, J = 4.0 Hz, 1H), 5.72 ppm (d, J = 7.4 Hz, 1H), 4.03-3.88 ppm (m, 2H), 3.77 ppm (td, J = 5.2, 3.1 Hz, 1H), 3.70–3.53 ppm (m, 2H), 1.92 ppm (s, 1H). Elemental analysis: calc. C, 37.77; H, 3.87; N, 29.36; Found: C, C, 37.01; H, 3.81; N, 29.11.
Scheme 2 Synthesis of 2-azido gemcitabine (G).
Fig. 1 1H-NMR of compound G.
Synthesis of 1,2,3-triazoline derivatives (G1-G3)
1.1 mmol of 2-azido gemcitabine (G) and 1.1 mmol of unsaturated compounds (maleic anhydride, acrylamide and cinamic acid) were dissolved in 20 mL of ethanol. To this mixture was added sodium ascorbate (0.4 mmol) and CuCl (0.2 mmol). The solution was stirred at 60 ℃ until TLC indicated the reaction was completed and the consumption of 2-azido gemcitabine compound. The mixture was diluted with organic solvent (diethyl ether) and water. The organic phase was separated, and the water phase was extracted two times with diethyl ether. The organic phase was dried over MgSO4. The solvent was removed and recrystallized from hexane–chloroform (Scheme 3). 2-(1,2,3-triazoline-4,6-dione-1-yl) gemcitabine (G1) was prepared as a white solid. Chemical formula: C13H13FN6O7; reaction yield: 86%; m.p.: 182 - 184 ºC. FTIR: 3476 cm-1, 3441 cm-1 due to OH stretching, 3128 cm-1 due to CH aromatic stretching, 2938 cm-1, 2823 cm-1 due to CH aliphatic stretching, 1662 cm-1 due to carbonyl group stretching, 1648 cm-1 due to benzene ring stretching, 1598 cm-1, 1579 cm-1, 1479 cm-1 due to C=N and C =C stretching, 1278 cm-1, 1111 cm-1, 1072 cm-1 due to C-O stretching, and 798 cm-1 due to C-H bonding. 1H-NMR (301 MHz, DMSO-d6): δ 9.68 ppm (s, 3H), 7.76-7.57 ppm (m, 8H), 7.10 ppm (d, J = 18.5 Hz, 3H), 6.85-6.74 ppm (m, 6H), 6.07 ppm (d, J = 4.0 Hz, 2H), 5.70 ppm (d, J = 7.4 Hz, 2H), 5.43 ppm (s, 2H), 4.01-3.88 ppm (m, 4H), 3.77 ppm (td, J = 5.2, 3.0 Hz, 2H), 3.66-3.55 ppm (m, 4H), 3.05 ppm (s, 18H), 1.93 ppm (s, 1H). Elemental analysis: calc.: C, 40.63; H, 3.4; N, 21.87; found: C, 40.01; H, 3.35; N, 21.77. 2-(1,2,3-triazoline-5-carboxamide-1-yl) gemcitabine (G2) was prepared as a white solid. Chemical formula: C12H16N7O5; reaction yield: 81%; m.p.: 202 - 204 ºC. FTIR: 3471 cm-1, 3440 cm-1 due to OH stretching, 3108 cm-1 due to CH aromatic stretching, 2944 cm-1, 2834 cm-1 due to CH stretching, 1666 cm-1due to carbonyl group stretching, 1622 cm-1 due to benzene ring stretching, 1583 cm-1, 1537 cm-1, 1473 cm-1 due to C=N and C =C stretching, 1287 cm-1, 1113 cm-1, 1070 cm-1 due to C-O stretching and 797 cm-1 due to C-H bonding. 1H-NMR (301 MHz, chloroform-d): δ 8.24 ppm (s, 1H), 7.77-7.59 ppm (m, 7H), 7.35-7.25 ppm (m, 3H), 7.17-7.06 ppm (m, 4H), 6.93 ppm (dt, J = 8.9, 2.4 Hz, 2H), 6.87-6.77 ppm (m, 2H), 6.59-6.46 ppm (m, 2H), 3.59 ppm (d, J = 18.7 Hz, 3H), 3.06 ppm (q, J = 7.3 Hz, 13H), 2.83 ppm (s, 5H), 2.16 ppm (d, J = 18.0 Hz, 8H), 1.32 ppm (t, J = 7.3 Hz, 20H). Elemental analysis: calc.: C, 40.34; H, 4.51; N, 27.44; found: C, 40.31; H, 4.48; N, 27.36. 2-(-4-phenyl-4,5-dihydro-1,2,3-triazole-5-carboxylic acid-1-yl)gemcitabine (G3) was prepared as a light white solid. Chemical Formula: C18H19FN6O6; reaction yield: 80%; m.p.: 212 - 214 ºC. FTIR: 3469 cm-1, 3417 cm-1 due to OH stretching, 3130 cm-1 due to CH aromatic stretching, 2960 cm-1, 2374 cm-1 due to CH stretching, 1671 cm-1 due to carbonyl group stretching, 1638 cm-1 due to benzene ring stretching, 1580 cm-1, 1539 cm-1, 1477 cm-1 due to C=N and C =C stretching, 1276 cm-1, 1119 cm-1, 1070 cm-1 due to C-O stretching and 798 cm-1 due to C-H bonding. 1H-NMR (301 MHz, chloroform-d): δ 9.77ppm (s, 1H), 7.76-7.57 ppm (m, 5H), 7.41-7.31 ppm (m, 2H), 7.16-7.01 ppm (m, 3H), 6.88 ppm (dd, J = 9.0, 2.5 Hz, 1H), 6.50 ppm (td, J = 8.8, 2.4 Hz, 1H), 4.06 ppm (d, J = 7.8 Hz, 1H), 3.90 ppm (s, 2H), 3.54 ppm (s, 2H), 3.08 ppm (q, J = 7.3 Hz, 7H), 2.81 ppm (s, 3H), 2.17 ppm (s, 3H), 1.32 ppm (t, J = 7.3 Hz, 11H). Elemental analysis: calc.: C, 49.77; H, 4.41; N, 19.35; found: C, 49.61; H, 4.38; N, 19.29.
Scheme 3 Synthesis of 1,2,3-triazoline derivatives.
Fig. 2 1H-NMR of compound G1.
Fig. 3 1H-NMR of compound G2.
Fig. 4 1H-NMR of compound G3.
Alpha-glucosidase inhibition analysis 
All synthesized 1,2,3-triazoline derivatives (G1-G3) were determined of their in-vitro α- glucosidase enzyme inhibitory activity. Acarbose was used as positive control drug and substrate (p-nitrophenyl glucopyranoside). A reaction mixture consisting of 30 µL 0.5 mM test compounds (G1-G3), 140 µL 50 mM phosphate buffer of 7.2 pH and 30 µL enzyme of 0.02 units. These contents were pre-incubated for 10 minutes at 37 ºC. The reaction was started by the addition of substrate p-nitrophenyl glucopyranoside (25 μL, 0.5 μM). After 30 minutes of incubation, the absorbance of yellow color produced due to the formation of p-nitrophenyl was measured at 405 nm. The enzymatic reaction was stopped by adding 100 µL of 200 µM Na2CO3. IC50 values were calculated and inhibition percentage for each compound was calculated by using the following formula:
% Inhibition = (Abs control - Abs sample) / Abs control × 100%.
Results and Discussion
In this article, synthesis of new 1,2,3-triazoline derivatives was achieved from gemcitabine which was converted to 2-azido gemcitabine and by the reaction of gemcitabine and sodium azide in THF as solvent. 1,2,3-triazoline derivatives (G1-G3) were synthesized by click reaction of unsaturated compounds like maleic anhydride, acrylamide and cinnamic acid with 2-azido gemcitabine in found sodium ascorbate and cuprous chloride as catalyst. The C.H.N. analysis of synthesized compounds was in agreement with the calculated percentage of elements. The FTIR spectra showed appearance of consumption of the azide of 2-azido gemcitabine and disappearance of the band at 2105 cm-1 due to the azide group. 1H-NMR spectrum was considered good evidence for the formation of our compounds (Scheme 2).
All 1,2,3-triazoline compounds (G1-G3) were evaluated for their in-vitro α-glucosidase inhibition activity. All members of the series G1-G3 exhibited a potent α-glucosidase inhibition with IC50 values. All the tested compounds were found to be more active than the standard drug, acarbose (IC50 = 824 ± 1.73 mM). Compound G3 having a phenyl ring attached to 1,2,3-triazoline skeleton was found to be the least active member of the series with an IC50 value 345 ± 4.5 mM. However, the activity increased when the 1,2,3-triazoline ring was binding with free amine group, as observed in compound G2 (IC50 = 212.9 ± 3.4 mM). Further increase in activity was observed for compound G1 (IC50 = 144.8 ± 1.74 mM) having the carbonyl groups in maleic anhydride ring. The results are summarized in Table 1.
Table 1 Results of α-glucosidase inhibition assay on compounds G1-G3
Alpha-glucosidase inhibition, IC50 (µM)
144 ± 1.74
212 ± 3.4
345 ± 4.5
824 ± 1.73
In this study, we synthesized and characterized a series of new 1,2,3-triazoline ring based on gemcitabine derivatives as α-glucosidase inhibitors. These compounds (G1-G3) were found to be more active than acarbose drug as in-vitro α-glucosidase inhibitors. The synthesized compounds could be candidates for antidiabetic drugs.
Conflict of Interests
The authors declare that no competing interest exists.
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Copyright© Farked Wahoodi Salman, Ahmed Jasim Twayej, Hayder Ahmed Shaheed, and Ali Jabbar Radhi. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.