4 Synthesis and Fabrication of Nanoparticles for
Scheme 2.1 Schematic presentation for the synthesis of 5-Fu-FF dipeptide conjugate
2.4 Experimental Section
2.4.1 Synthetic procedures for compound 2a-2e.
A. Synthesis of prodrug (4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) methyl)-3-nitrobenzoic acid) (2a): The prodrug was synthesized according to the literature.11 Initially 5-Fu was silylated by using N,O-bis(trimethylsilyl) acetamide to form the intermediate 5-fluoro-2,4-bis((trimethylsilyl)oxy)-1,2,3,4-tetrahydropyrimidine.
This intermediate (1.07 g, 3.8 mmole) was reacted in reflux condition for 14 hours with 4-bromomethyl-3-nitrobenzoic acid (1.17 g, 4.5 mmole) in the dark under nitrogen atmosphere using dry acetonitrile (5 mL) as solvent. After completion of the reaction, the mixture was cooled down to room temperature and treated with 7 mL methanol. The solvent was evaporated under vacuum and the obtained crude residue was purified by using (1:3:0.004 v/v methanol:ethyl acetate:acetic acid) solvent system to obtain a pale yellow powder (508 mg, 42%). Characterization of (4-((5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl) methyl)-3-nitrobenzoic acid) 2a: 1H NMR (600MHz, DMSO-d6) δppm 8.53 (s, 1H), 8.18 (m, 2H), 7.46 (d, 1H, J = 6Hz), 5.23(s, 2H); 13C NMR (150 MHz, DMSO-d6) δppm 166.3, 158.2 (d, C-4, JC-F = 25 Hz), 150.3, 147.8, 141.3 (s, C- 5, JC-F = 228 Hz), 135.9, 134.8, 130.5 (d, C-6, JC-F = 34 Hz), 128.9, 125.9, 49.0: IR (KBR): ṽ=3436, 3134, 1703, 1647, 1543, 1400, 1233 cm-1; HRMS (ESI) m/z [M + H]+calcd. for C12H8FN3O6: 310.0470, found: 310.0488.
B. Synthesis of ethyl L-phenylalanyl-L-phenylalaninate (2b and 2c): The peptide was synthesized according to the solution phase protocol. Boc protected L-phenylalanine (1
g, 3.8 mmole) was coupled with C-terminal ester protected L-phenylalanine (867 mg. 3.8 mmole) utilizing N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 1.57 g, 4.1 mmole) and N, N-Diisopropylethylamine (DIPEA, 637 mg, 4.9 mmole) in room temperature for 12 h. After the completion of the reaction, the product was extracted with brine solution using ethyl acetate as the organic solvent. The organic layer was dried over sodium sulphate and further purified by column chromatography using 1:4 ethyl acetate: hexane as the mobile phase. The dipeptide (compound 2b) was obtained as white powder (1.19 g, 72%). Characterization of ethyl L-phenylalanyl-L-phenylalaninate: 1H NMR (600 MHz, CDCl3) δ 7.30 (dd, J = 12.9, 5.4 Hz, 3H), 7.25 (dt, J = 10.0, 5.0 Hz, 4H), 7.21 (d, J = 7.8 Hz, 2H), 7.04 – 7.00 (m, 2H), 6.39 (s, 1H), 5.01 (s, 1H), 4.77 (d, J = 5.2 Hz, 1H), 4.15 (ddd, J = 17.2, 10.6, 7.3 Hz, 2H), 3.06 (dd, J = 10.9, 5.5 Hz, 4H), 1.42 (s, 9H), 1.21 (t, J = 7.1 Hz,3H).13C NMR (150 MHz, CDCl3) δppm 170.9, 170.8, 155.3, 136.5, 135.7, 129.5, 129.3, 128.7, 128.5, 128.3, 127.1, 126.9, 80.2, 77.3, 77.1, 76.9, 61.4, 55.6, 53.3, 38.0, 28.3, 27.9, 14.1;
IR (KBR): ṽ=3412, 3277, 2982, 1742, 1694, 1527, 1170, 1010, 704 cm-1; HRMS (ESI) m/z [M + H]+ calcd. for C25H32N2O5: 441.2384, found: 441.2397. Thus synthesized peptide was deprotected at the N- terminal by using trifluoroacetic acid (TFA) giving the desirable compound 2c (92%).
C. Synthesis of ethyl (4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) methyl)-3-nitrobenzoyl)-L-phenylalanyl-L-phenylalaninate (2d): The prodrug 2a (500 mg, 1.6 mmole) was reacted with trifluoroacetate salt of diphenylalanine ethyl ester 2c (734.62 mg, 1.6 mmole) in presence of HBTU (674.59 mg, 1.8 mmole) and DIPEA (0.7 mL, 4.0 mmole) with DMF as solvent. The solution was stirred overnight under nitrogen atmosphere before being washed with cold water (50 mL). After washing, the organic layer was dried over sodium sulphate and the crude residue was purified with silica gel column chromatography to give a pale yellow solid (541 mg, 53%) yield (Mp.:
206 ºC). Characterization of ethyl (4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)methyl)-3-nitrobenzoyl)-L-phenylalanyl-L-phenylalaninate (2d): 1H NMR (600MHz, DMSO-d6) δppm 11.94 (br s, 1H), 8.99 (d, J = 6Hz, 1H), 8.61 (d, J = 6Hz 1H), 8.50 (s, 1H ), 8.11 (d, J = 6Hz, 1H), 8.06-8.04 (m, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.33-7.32 (m, 1H), 7.26-7.22 (m, 5H), 7.20-7.17 (m, 1H), 7.16-7.14 (m, 1H), 5.20 (s, 2H), 4.81-4.77
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(m, 1H), 4.51-4.47 (m, 1H), 4.04 (q, J = 7.1 Hz, 2H), 3.12-2.89 (m, 4H), 1.10 (t, J = 7.1 Hz, 3H). ; 13C NMR (150 MHz, DMSO-d6) δppm 171.3, 171.2, 163.7, 149.8, 147.2, 140.9, 139.4, 138.1, 137.0, 134.9, 134.2, 132.7, 129.7, 129.1, 128.4, 128.2, 128.0, 126.5, 126.3, 123.8, 60.5, 54.5, 53.8, 48.4, 37.0, 36.5, 13.9; IR (KBR): ṽ=3436, 3293, 3070, 2927, 2855, 1694, 1639, 1527, 1336, 1241, 704cm-1;HRMS (ESI) m/z [M + H]+calcd.
for C32H30FN5O8: 632.2151, found: 632.2166.
D. Synthesis of (4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) methyl)-3- nitrobenzoyl)-L-phenylalanyl-L-phenylalanine (2e): The above product (2d) (200 mg) was dissolved in THF (1 mL). After dissolution, 1 mL of 2(N) sodium hydroxide was added to it and the solution was stirred for 3 hours. The completion of the reaction was monitored by thin-layer chromatography (TLC). After the reaction, THF was evaporated and 4(N) HCl was added dropwise (around 1.5 mL) in cold condition to give a pale yellow precipitate. The precipitate was obtained by filtration followed by washing with cold water to get the pure compound 2e (149 mg) in 78% yield (Mp.: 216 ºC).
Characterization of (4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) methyl)-3- nitrobenzoyl)-L-phenylalanyl-L-phenylalanine (2e): 1H NMR (600MHz, DMSO-d6):
δppm 12.79 (br s, 1H), 11.94 (br s, 1H), 8.98-8.97 (m, 1H), 8.48-8.47 (m, 1H), 8.42-8.41 (m, 1H), 8.10 (d, J = 6.6 Hz, 1H),8.04-8.03 (m, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.32-7.30 (m, 1H), 7.23-7.19 (m, 6H), 7.16-7.13 (m, 2H), 5.20 (s, 2H), 4.77-4.74 (m, 1H), 4.48- 4.44 (m, 1H), 3.10-3.07 (m, 2H), 2.97-2.89 (m, 2H); 13C NMR (150 MHz, DMSO-d6):
δppm 172.8, 171.3, 163.9, 157.9 (d, C-4, JC-F = 25), 150, 147.4, 141 (s, C-5, JC-F = 237), 138.2, 137.6, 135, 134.4, 132.8, 130.2 (d, C-6, JC-F = 34), 129.3, 128.6, 128.3, 128.2, 126.6, 126.4, 124, 54.9, 53.8, 48.6, 39.9, 39.8, 39.7, 39.5, 39.4, 39.3, 39.1, 37, 36.6; IR (KBR): ṽ=3436, 3070, 2927, 1694, 1655, 1527, 1345, 1241, 701 cm-1; HRMS (ESI) m/z [M + H]+calcd. for C30H26FN5O8: 604.1838, found: 604.1848.
2.4.2 Preparation and characterization of gels
10 mg (1 wt%) of compound 2d was dissolved in 1 mL of DMSO in a 5 mL glass vial and kept in room temperature. After 5 days, a white-colored gel was obtained. Formation of gel was confirmed by vial inversion test. For preparing hydrogel, 10 mg (2 wt%) of compound 2e was taken in a 5 mL glass vial. 150 µL of millipore water was added to the
vial. Then 50 µL of 1(N) NaOH was added and the pH of the solution was adjusted to nine by adding 1(N) HCl. The solution was vortexed until the compound dissolved completely. Finally, the pH of the solution was made seven by adding a total of 46 µL of 1(N) HCl gradually. The final volume of the solution was made 500 µL and kept in 37ºC for one hour to form a gel, which was assured by vial inversion test.
2.4.3 Identification of sol-gel transition temperature (Tg)
Sol-gel transition temperature was determined by dropping ball method. Briefly, a small ball was placed on the top of the hydrogel kept in a glass vial and it was heated slowly in a water bath at a rate of 1ºC/minutes. The temperature at which the ball touched the bottom of the glass vial was noted as Tg. The experiment was carried out in triplicate.
2.4.4. FESEM analysis
FESEM measurement was performed by placing a small portion of the lyophilized hydrogel sample on carbon tape. The DMSO gel was dried in a vacuum oven at 40ºC and the dried gel was used in a similar fashion like the hydrogel for FESEM analysis. All the samples were coated with gold prior to analysis.
2.4.5 Circular Dichroism
The CD spectra were recorded by using a JASCO J-600C spectropolarimeter at room temperature. The samples were measured at different concentrations of 564, 282, 141, 70, 35, 18 µM in water. A cuvette having 0.1cm path length was used. All CD profiles were an average of three scans at a scan speed of 100 nm/minutes.
2.4.6 FTIR Spectra
FTIR spectra of the dried hydrogel were recorded on Nicolet iS10 spectrometer at room temperature using KBr disk technique.
2.4.7 Rheological measurement
Rheological studies were carried out with 2 wt% hydrogel using Anton-Paar MCR 102 rheometer consisting of a measuring system of 20 mm parallel plate at 25ºC. The strain
for the Controlled Release of the Antitumor Agent
range of 0.1- 100% strain. The frequency sweep was done over a range of 0.1-100 rad/s against the constant strain of 0.1% as determined from the LVR.
2.4.8 Irradiation of 5-Fu-FF-COOH (compound 2e)
Irradiation was carried out using 365 nm LED light of 24W. 3 mL solution of compound 2e (Scheme 1) having a concentration of 85 µM was placed in a quartz cuvette and irradiated under 365 nm UV light at 25 ˚C. The experiment was monitored by UV-Vis spectrophotometer as well as by RP-HPLC. After each irradiation, the change in UV absorption was monitored at a time interval of 5, 10, 40, 60, 80 and 100 minutes by a Perkin Elmer UV-Vis spectrometer. A new broad peak gradually appeared at around 325 nm which clearly indicated the photo-cleavage of the ortho-nitro benzyl group. The release of 5-Fu was clearly observed in HPLC measurement. It was observed that the percentage of the release of 5-Fu was increased with respect to irradiation time.
The photo-induced release of 5-Fu from compound 2e was also monitored by 1H NMR.
For NMR study, 5 mg of compound 2e was dissolved in DMSO-d6. The sample was irradiated under 365 nm UV light and 1H NMR spectra were recorded after 6 hours of irradiation.
2.4.9 Cytotoxicity of compound 2e
To determine the cytotoxic effect of our peptide-drug conjugate, colorimetric assay was carried out. The cell viability assay was carried out in human cervical HeLa cell lines, obtained from National Centre for Cell Sciences (NCCS), Pune, India, by following previously demonstrated procedure.49 For this purpose, 5x103 cells were seeded into 96- well microplate in 90 µL Dulbecco’s modified Eagle’s medium (DMEM) and were incubated for 24 hours in presence of 5% CO2 at 37˚C. Cells were then treated with varying concentrations of compound 2e (33, 66, 99, 132, 165 µM) both in presence and absence of irradiation for 48 hours under similar condition. After the incubation, the cell viability assay was carried out by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT). MTT is reduced by living cells to produce blue- colored formazan and this reaction is used as a measurement of living cells. MTT was added to each plate and was incubated for 2 hours at 37˚C. The medium then was
removed and 60 µL of DMSO was added which produces formazan having absorption (A) at 550 nm. The optical density (OD) of each well-plate was measured with the background reference at 690 nm. All the tests were performed in triplicates. The control experiment was also carried out in similar way but without the addition of compound 2e.
The percentage cell viability with respect to the control was calculated by using the standard formula as given below. The cell viability percentage of the control was taken as 100%.
% 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = (𝐴550− 𝐴690) 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠
(𝐴550− 𝐴690) 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑐𝑒𝑙𝑙𝑠 × 100 %
for the Controlled Release of the Antitumor Agent