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CHAPTER 4. EXPERIMENTAL PART
All solvents, organic and inorganic reagents, unless otherwise noted, were used without further purification.
Mass spectra were obtained on a Bruker micrOTOF spectrometer with electrospray ionisation. The solvent is a MeOH/CH2Cl2 mixture, the detection range is m / z = 50–3000. The m / z values are given for the signals with the highest intensity.
Elemental analysis (C, H, N) was carried out on a Euro EA 3028 HT CHNSO analyser. IR spectra were recorded on a Shimadzu IRAffinity-1S FT-IR spectrometer (4000–400
cm–1) in pellets with KBr for solid samples.
1H (400 MHz), 13C{1H} NMR spectra were measured on a Bruker Avance spectrometer. Chemical shifts (in ppm) were determined relative to the solvent signal, the waveforms were c
— singlet, d — doublet, t — triplet, dd — doublet of doublets, td — triplet of doublets, m — multiplet.
13C solid-state NMR spectra were recorded on a Bruker Avance III 400 WB spectrometer; operating frequency 100.64 MHz.
X-ray diffraction experiments were carried out on SuperNova, Dual, (CuKα, λ = 1.54184) and Xcalibur, Eos diffractometer (MoKα, λ = 0.71073) diffractometers at 100 K.
Crystal structures were solved by direct methods and refined using the SHELX program, built into the OLEX2 complex. Absorption corrections were introduced empirically in the CrysAlisPro software package using spherical harmonics implemented in the SCALE3 ABSPACK scaling algorithm. Hydrogen atoms were included in the refinement with fixed positional and temperature parameters.
X-ray diffraction analysis of the GO-based sample was performed using a Rigaku MiniFlex II diffractometer.
Raman spectra were recorded on a Horiba Jobin-Yvon LabRam HR800 spectrometer; IR spectra were recorded on a Nicolet 8700 IR Fourier spectrometer.
The morphology of the resulting GO-based nanoparticles was determined using a JSM7001F scanning electron microscope. The thermal stability of the samples was studied on a NETZSCH TG 209 F1 Libra thermogravimetric analyser in the temperature range 30–900 °C in a nitrogen atmosphere at a heating rate of 5 °C min–1.
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Qualitative and quantitative analysis of GO samples was performed by X-ray photoelectron spectroscopy (XPS) on a Fisher Scientific ESCAlab 250Xi thermospectrometer using monochromatic AlKα radiation (photon energy 1486.6 eV).
The temperature and concentration dependences of the physicochemical properties of aqueous solutions were studied using the following instruments:
1)Anton Paar DSA 5000 unit for density measurement;
2)microviscometer Lovis 2000 M Anton Paar (Austria) for measuring viscosity;
3)automatic multiwave refractometer Abbemat WR/MW (Austria) for measuring the refractive index.
Description of experimental techniques, as well as metrological characteristics are presented in the works [118-129].
The study of solubility in water at atmospheric pressure in the temperature range T = 293.15–318.15 K was carried out by the method of isothermal saturation in a shaker-thermostat LAUDA ET 20 with a shaking frequency of 80 Hz. Aliquots of the liquid phase of the saturated solution, obtained using vacuum filtration, were analysed on an SF-2000 spectrophotometer (Russia). Fig. 4.1 shows the UV spectrum of an aqueous solution of compound 1.57 (C = 0.055 g dm−3) at λ = 200–400 nm and the validity of the Bouguer–Lambert–Beer law (R2 = 0.989) at
λ = 220 nm.
A / a. u.
1.0221 nm
0.8
0.6
0.4
0.2
0.0 |
|
|
|
|
200 |
250 |
300 |
350 |
400 |
l / nm
Fig. 4.1. UV spectrum of an aqueous solution of compound 1.57.
To determine the concentration of compound 1.57 in a saturated solution, Eq. 4.1 was used (the length of the optical path is 1 cm):
C = 0.049 Ч A220 , |
(4.1) |
where C is the volume concentration of compound 1.57 (g∙dm−3), A220 is the optical density of the solution at λ = 220 nm.
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The composition of the equilibrium crystalline hydrate of compound 1.57 was determined by the thermogravimetric method on a NETZSCH TG 209 F1 Libra thermogravimetric analyser in the temperature range 40–600 °C in a nitrogen atmosphere and a heating rate of 5 °C·min−1.
The hydrolysis reaction of compound 1.57 in D2O was studied at 25 °C by recording the
1H NMR spectra of the reaction mixture on a Bruker Avance III 400 instrument (400.13 MHz) for 24 h in automatic mode, while the spectra were recorded every 30 min.
To study the stability of compound 1.57 in an acidic medium (pH 3 and 4), 20 mg of compound 1.57 was dissolved in 40 ml of aqueous hydrochloric acid solutions of various concentrations (C = 0.001 and 0.0001 M). After completion of the reaction and removal of the solvent, the product was dried in air. To study the stability of compound 1.57 in an alkaline medium (pH 10), 20 mg of compound 1.57 was dissolved in 40 ml of an aqueous solution of NaOH (C = 0.0001 M). After completion of the reaction and removal of the solvent, the product was dried in air.
The distribution of compound 1.57 in the octan-1-ol–H2O system was studied using a LAUDA ET 20 thermostatic shaker (shaking frequency 80 Hz). The temperature was maintained with an accuracy of 0.1 K, the experiment time was 5 h. For the experiment, a solution of compound 1.57 (C = 1 g∙dm−3) was prepared in deionised water (electrical conductivity 5.5∙10−6 Sm∙m−1), to which was added an equivalent volume of octan-1-ol (25.0 ml). After reaching equilibrium, an aliquot of the aqueous phase was taken. The concentration of the compound 1.57 in the aqueous phase was determined by the spectrophotometric method on an SF-2000 instrument according to Eq. 3.1.
4.1 Synthesis of 1,3,5-triazine derivatives
Synthesis of (5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)-2,2-dimethyl-1,3-dioxan-5-
yl)methanol (3.1)
Cyanuric chloride (1.5 mol) was dissolved in 50 ml of acetone.
Tris(hydroxymethyl)aminomethane (1.4 mol) dissolved in 15 ml of acetone was added dropwise to the resulting solution. With vigorous stirring, p-toluenesulphonic acid was introduced into the
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reaction mixture in a catalytic amount. The completeness of the reaction was monitored using TLC (mobile phase, chloroform/methanol 9.5/0.5). Next, the reaction mixture was separated
from the precipitate by filtration and dried.
The yield of the final product was 2.07 g (96 wt.%), beige crystals, melt. temp. 193–195 °C. 1H NMR spectrum (DMSO-d6),
δ, ppm: 1.49 (s, 9H, 3CH3), 3.93 (s, 4H, 2CH2), 3.78 (d, J = 5.9 Hz,
2H, 2CH). 13C NMR spectrum (DMSO-d6), δ, ppm: 172.4 (Ctriazine), 
166.9 (Ctriazine), 99.8 (C), 65.8 (CH2), 63.9 (CH2), 59.2 (CH2), 19.9 (CH3). Elemental analysis, exp. (%): C, 37.22; H, 5.10; N, 18.91 (C6H11N5O2). Calc. (%):
C 38.85; H 4.56; N 18.12. Mass spectrum (ESI-), m / z: 307.0366 [M]-, [C10H13Cl2N4O]-.
Calc. [M]-: 307.0443. |
|
|
X-ray diffraction data |
C10H14Cl2N4O3; |
M =309.02 g/mol, a =11.77100 |
(10) Å, b =12.72050 (10) Å, c = |
18.5584 (2) Å, |
β =93.5730 (10)°, V =2773.40(4) Å3, |
monoclinic crystal system, space group P21/n, temperature 100 K, 24051 reflections, Rsigma = 0.0319, R1 = 0.0355, wR2 = 0.09764.
Synthesis of (5-((4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl)amino)-2,2-dimethyl-1,3-
dioxan-5-yl) methanol (1.57)
Aminobromoethanol (0.016 mol), Na2CO3 (0.032 mol), NaOH (0.032 mol) were dissolved in 10 ml of water and stirred for 1 h at 50°C. Then the reaction mixture was cooled and added dropwise to (5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)-2,2-dimethyl-1,3-dioxane-5- yl)methanol (0.008 mol). The resulting reaction mass was stirred for 3 h at room temperature, monitoring the course of the reaction by TLC (mobile phase, chloroform/methanol 9.5/0.5). After completion of the reaction, the solvent was removed under reduced pressure, and the precipitate was dried in a stream of air.
The yield of the final product was 1.36 g (56 wt. %), white crystals. 1H NMR spectrum (400 MHz, CDCl3), δ: 6,14 (t, J =
6.4 Hz, 1H, CH), 6,05 (s, 1H, CH), 3.92 (s, 4H, 2CH2), 3.75 (d,
J = 5.9 Hz, 2H, 2CH), 2.36 (s, 8H, 4CH2), 1.47 (d, J = 6.0 Hz, 6H, 2CH3). 13C NMR spectrum (101 MHz, CDCl3), δ: 176.2 (Ctriazine), 175.5 (Ctriazine), 166.2 (Ctriazine), 98.9 (C), 64.9 (CH2), 64.3 (CH2), 54.7 (CH2), 27.7
(CH2aziridine), 26.9 (CH2aziridine), 19.4 (CH3). IR (cm−1): 3344,71, 2978,22, 1537,33, 1448.60,
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1417.74, 1225.82, 1072.47, 822.68. Mass spectrum (ESI+), m / z: 345.1646 [M+Na]+, [C14H22N6O3Na]+. Calc. [M+Na]+: 345.1641.
X-ray diffraction data C14H22N6O3; M = 322.37 g/mol, a =9.077 (2) Å, b = 9.0823
(2) Å, c = 9.8721 (2) Å, α = 79.883(2)°, β = 83.960(2)°, γ = 72.365(2)°, V = 762.38(3) Å3, triclinic crystal system, space group P-1, temperature 99.97, 20067 reflections of which 3083 are unique, Rsigma = 0.0231, R1 = 0.0382, wR2 = 0.1013.
4.2 Synthesis of tetrazole derivatives
Synthesis of 5-phenyltetrazole (3.2)
3.71 g (0.057 mol) of sodium azide and 4.57 g (0.056 mol) of dimethylammonium chloride were dissolved in 50 ml of dimethylformamide. Then 5.95 g (0.050 mol) of benzonitrile and 25 ml of dimethylformamide were added. The resulting reaction mixture was stirred for 8 h at 110–115 °C. Then the mixture was cooled and separated by filtration from the precipitated NaCl precipitate. After that, the filtrate was evaporated on a rotary evaporator. A small amount of water was added to the remaining mass, and the mixture
was acidified with a solution of hydrochloric acid (ω = 10%) to pH 2–
3. The resulting precipitate of 5-R-2H-tetrazole was filtered off, washed with water, and allowed to dry in air. The yield of the final product was
5.19 g (64 wt. %).
1H NMR spectrum (400 MHz, DMSO-d6), δ: 6.94–7.89 (m, 4H, Ar). 13C NMR spectrum (101 MHz, DMSO-d6), δ: 156.2 (Ctetrazole), 127.35 (C6H5), 128.97 (C6H5), 130.59 (C6H5), 135.66 (C6H5).
Synthesis of N-substituted 5-R-tetrazoles
Synthesis of 5-phenyl-2H-tetrazol-2-ylacetic acid ethyl ester (3.3)
To a suspension of 10.0 g (0.068 mol) of 5-phenyltetrazole in 100 ml of acetonitrile was added 9.5 ml (0.068 mol) of triethylamine with stirring. Then, 8.4 g (0.069 mol) of chloroacetic acid ethyl ester was added dropwise to the solution. The reaction mass was heated to 50 °C and kept for 4 h. After that, the reaction mixture was cooled and the precipitation of triethylamine hydrochloride was filtered off. The filtrate was evaporated in vacuo and the residue was dissolved in chloroform. The solution in chloroform was
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washed with a 5 % aqueous solution of sodium bicarbonate (3 × 50 ml) and distilled water
(2 × 50 ml). After drying over Na2SO4, chloroform was removed in vacuo. After drying in a stream of air, the final product was obtained with Rf = 0.81 (mobile phase was chloroform/methanol 9.5/0.5).
The yield of the final product was 10.39 g (65 wt. %). Colourless crystals, m. p. 85–86 °C. 1,22 (t, 3H, CH3), 4.20 (q, 2H, CH2), 5.9 (s, 2H, N2-CH2), 7.55– 7.60 (m, 3H, C6H5), 8.09 (m, 2H, C6H5). 13C NMR spectrum (101 MHz, DMSO-d6), δ:166.4 (C=O), 164.7 (Ctetrazole), 130.8 (C6H5), 129.6 (C6H5), 126.9 (C6H5), 126.6 (C6H5), 62.2 (CH2), 53.7 (CH2), 14.1 (CH3). IR, ν, cm−1: 1754 (C=O), 1H NMR spectrum (400 MHz, DMSO-d6), δ: 2973, 2941 (C–H), 1452, 1281, 1154, 1076, 1049, 1021, 932 (CN4), 1220 (C-O-C), 732, 695 (C6H5).
Synthesis of 5-phenyl-2H-tetrazol-2-ylacetic acid (3.4)
To a solution containing 10.0 g (0.043 mol) of 5-phenyltetrazol-2-ylacetic acid ethyl ester was added 125 ml of a solution of sodium hydroxide in methanol (C = 3 M). After incubation for 1 h at room temperature, the solvent was removed in vacuo, the residue was dissolved in a minimum amount of water. The aqueous solution was acidified to pH 2–3 with hydrochloric acid. Then the precipitate formed was filtered off and dried on the filter in a stream of air.
The yield of the final product was 6.24 g (71 wt. %). White crystals, m. p. 182–184 °C. 1H NMR spectrum (400 MHz, DMSO-d6), δ: 5.75 (s, 2H, N2-CH2), 7,66– 7.50 (m, 3H, C6H5), 8.15–8.05 (m, 2H, C6H5), 13.8 (s 1H, OH). 13C NMR spectrum (101 MHz, DMSO-d6), δ: 168.3 (C=O), 165.1 (Ctetrazole), 131.5 (C6H5), 130.2 (C6H5), 127.6 (C6H5), 127.2 (C6H5), 54.5 (CH2). IR, ν, cm−1: 1718 (C=O), 3434, 2675 (OH bonds), 2999, 2952 (aliphatic C–H bonds), 1451, 1254, 1161, 1074, 1045, 1025, 920 (CH4), 726, 687 (C6H5). Exp., %: C 52.72, H 4.02, N 27.46. C9H8N4O2. Calc., %: C 52.94, H 3.95, N 27.44.
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Synthesis of 5-phenyl-2H-tetrazol-2-ylacetic acid chloride (3.5)
The 5.00 g (0.024 mol) of crushed 5-phenyl-2H-tetrazol-2-ylacetic acid was heated in a flat-bottomed flask with 5.20 g (0.025 mol) of phosphorus pentachloride with constant stirring in a water bath at 85 °C until gas evolution ceased. After the completion of the reaction, the resulting mixture was poured into boiling hexane. The precipitate formed was filtered off and dried under vacuum.
The yield of the final product was 3.61 g (68 wt. %). White crystals. 1H NMR spectrum (400 MHz, DMSO-d6), δ: 5.73 (s, 2H, N2-CH2), 7.68–7.52 (m, 3H, C6H5), 8.13–8.07 (m, 2H, C6H5). 13C NMR spectrum (101 MHz, DMSO-d6), δ: 168.9 (C=O), 165.4 (Ctetrazole), 131.5 (C6H5), 130.1 (C6H5), 126.9 (C6H5), 127.1 (C6H5), 56.2 (CH2).
4.3 Synthesis of hybrid triazinyltetrazoles
Synthesis of (5-((4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl)amino)-2,2-dimethyl-1,3-
dioxan-5-yl)methyl 2-(5-phenyl-2H-tetrazol-2-yl)acetate (3.6)
To a solution of 1.24 mmol [5-[[4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl]-amino]- 2,2-dimethyl-1,3-dioxan-5-yl]-methanol (1.57) in 40 ml of acetonitrile was added 1.48 mmol of 5-phenyltetrazol-2-ylacetic acid chloride and 1.48 mmol of triethylamine. The reaction mass was stirred for 1 h at room temperature. After completion of the reaction, the solvent was distilled off under reduced pressure. The product was purified by silica gel column chromatography, eluent chloroform/methanol (9.5:0.5). The purified product was dried in air.
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The yield of the final product was 0.34 g (54 wt. %), beige powder, m. p. 226–228 °C. 1H NMR spectrum (400 MHz, CDCl3), δ: 8.17–8.12 (m, 2H, 2CHAr), 7.48
(dd, J = 5.1, 1.9 Hz, 3H, 3CHAr), 5.48 (s, 2H, CH2), 5.37
(s, 1H, NH), 4.77 (s, 2H, CH2), 4.11 (d, J = 11.9 Hz, 2H, CH2), 3.88 (d, J = 11.9 Hz, 2H, CH2), 2.31 (s, 8H, 4CH2), 1.45 (s, 3H, CH3), 1.36 (s, 3H, CH3). 13C NMR spectrum (101 MHz, CDCl3), δ: 176.2
(C=O), 166.9 (Ctriazine), 165.8 (Ctriazine), 164.8 (CN4), 130.8 (C6H5), 129.1 (C6H5), 127/1 (C6H5), 127.0 (C6H5), 99.0 (C), 65.1 (CH2), 63.1 (CH2), 53.4 (CH2), 52.6 (CH2), 27.1 (CH2), 23.8 (CH3), 23.3 (CH3). ESI+-MS, m / z: 509.2373 [M + H]+ (calc. for C23H28N10O4: 509.2368). IR (KBr): ν (cm−1) 2994.10, 1759.37, 1552.42, 1409.76, 1199.78, 1043.59, 830.44, 694.45.
4.4 Study of the stability of compounds 1.57 and 3.6
Compounds 1.57 and 3.6 (30 mg) were dissolved in 5 ml of 1 M HCl and stirred at room temperature for 72 h. The solvent was removed in a stream of air at room temperature.
2-((4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl)amino)-2-(hydroxymethyl)propan-1,3-diol
(3.7)
The yield of the final product was 0.025 g (95 wt %), white crystals, m. p. 189–191 °C. 1H NMR spectrum (400 MHz, DMSO-d6),
δ: 4.95 (br. s, 3H), 3.69 (s, 6H), 3.67 (s, 6H), 1.63 (s, 8H).
2-((4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl)amino)-3-hydroxy-2-
(hydroxymethyl)propyl-2-(5-phenyl-2H-tetrazol-2-yl)acetate (3.9)
The yield of the final product was 0.027 g (96 wt. %), white powder, m. p. 211–213 °C. 1H NMR spectrum (400 MHz, DMSO-d6), δ: 8.08–7.97 (m, 2H), 7.55 (td, J = 4.9, 2.2 Hz, 3H), 5.53 (s, 2H), 4.26-3.89 (m, 6H), 3.87–3.72 (m, 5H), 3.67 (s, 6H), 1.81 (s, 8H).
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4.5 GO synthesis
GO was synthesised from graphite via a modified oxidation reaction by Hummers and Offeman. Graphite powder (3 g) was dispersed in concentrated sulphuric acid (200 ml) in an ice bath with stirring for 30 min. Then solid KMnO4 (8 g) was slowly added with continuous stirring for 45 min, after which NaNO3 (2 g) was added with stirring for 2 h at
5°C. The reaction mixture was then stirred for 90 min at 96°C, with deionised water (200 ml) gradually added. Additional portions of deionised water (350 ml) and 30% H2O2 solution (10 ml) were added to complete the oxidation and remove excess KMnO4. The GO precipitate was separated by filtration and washed many times in a 5% HCl solution and deionised water until a neutral pH was reached. The precipitate was dried at 65 °C for
5 h, then redispersed in deionised water under sonication for 1 h, and then centrifuged (20 min at 4000 rpm). As a result, a brown precipitate of GO was obtained with a yield of 90 wt. % [130,131].
4.6 Synthesis of GO conjugate with 2,4,6-trisubstituted-1,3,5-triazine 1.57
GO powder (5 g) was dispersed in 200 ml alkalised deionised water (pH 9) under sonication for 15 min. Then, a solution of compound 1.57 in water (5 g, C = 0.0155 M) was added to the resulting mixture, and the reaction mixture was kept in an ultrasonic bath for 45 min. The GO-1.57 precipitate isolated from the reaction mixture was washed with methylene chloride and then with deionised water. The resulting GO-1.57 was dried at 37 °C for 10 h.
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4.7 Biocompatibility study
Haemocompatibility study
Spontaneous haemolysis of erythrocytes
Erythrocyte haemolysis was studied by measuring the optical density of supernatants at a wavelength of λ = 540 nm using a Hitachi U2900 spectrophotometer (Hitachi, Ltd., Tokyo, Japan) in accordance with the procedure described in [108]. The test mixture with a volume of 1.5 ml was prepared from 0.75 ml of solutions of compounds
1.57, 3.6, and GO-1.57 dispersion (in terms of loading 1.57) with various concentrations and 0.75 ml of erythrocyte suspension in physiological saline. As a result, solutions of compounds 1.57, 3.6 and dispersions of GO-1.57 with final concentrations of 1, 5, 10, 25,
50, 75, 100, and 200 μM were obtained. After preparing the mixture, tubes containing a suspension of erythrocytes and the test substance at various concentrations were incubated at 37.0 ± 0.2 °C for 1 and 3 h, then centrifuged for 10 min at 2500 rpm. Erythrocyte suspensions supplemented with equivalent volumes of distilled water and saline were used as positive and negative controls.
Platelet aggregation
After obtaining informed consent, blood for the study was taken from donors (n = 8), persons of both sexes who did not receive drugs that affect platelet function for 7–10 days. To prevent platelet activation, blood was taken into vacutainers containing 3.8 % sodium citrate as a stabiliser in the sodium citrate : blood ratio of 1:9.
Stabilised blood was centrifuged for 7 min at room temperature and 1000 rpm. Part of the platelet rich plasma (PRP) was taken into a plastic tube in the amount necessary to perform the analysis. Platelet-poor plasma was obtained from the remaining blood by centrifugation for 30 min at 3600 rpm. Platelet-poor plasma was used to calibrate the optical density scale of the aggregometer. The number of platelets in the test plasma affects the measurement result, therefore, plasma was standardised to obtain a platelet concentration of 200–250∙109 cells/l, taking into account the addition of the test substance. Platelet concentration was determined in the platelet counting mode on an aggregometer. If the platelet concentration in the plasma was above the standard level, then it was diluted by adding platelet-free plasma.
Platelet aggregation in PRP was studied using a platelet aggregation analyser model
Solar AP 2110 at 37 and a magnetic stirrer speed of 1200 rpm [109]. In the test of ADP-
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