Vol. 20 No. 1(77) (2022)
Aim. To synthesize hexahydrospiro[cyclopropane-1,10’-pyrido[1,2-c]quinazoline] and 2-λ5-benzo[f][1,4,2]diazaphosphepine derivatives – new N-P containing heterocyclic compounds with the 6-azaspiro[2.5]octane fragment.
Results and discussion. A new analog of the powerful electrophilic reagent – “Alder dimer” ‒ was obtained from the interaction of triflic anhydride and spiro(4-cyclopropane) piperidinyl formamide, and further used to synthesize new Nʹ-PV- and PIII-substituted Νʹ-phenyl, Νʹʹ-hexahydro(azaspiro)octylformamidinium salts – precursors of acyclic N-phosphorylated diamino carbenes with a spiroamine group. It has been shown that acyclic N-phosphorylated diaminocarbenes are transient species affording various products. The structure of the final product is primarily determined by nature of the phosphorus-bearing substituent, namely a phosphoryl or phosphino-group. N-PV-substituted carbene undergoes a 1,2-phosphorus shift with the formation of (selenophosphoryl)formamidine in a high yield. For N-PIII-substituted carbene a compatible 1,3-H shift also occurs with the formation of an intermediate azomethine ylide converted into a new heterocyclic system – hexahydrospirocyclopropane -1,10’-pyrido[1,2-c]quinazoline. Under the action of acid an unexpected further expansion of the 6-member ring occurs with the formation of a diazepine derivative.
Experimental part. The reaction of Alder reagent with N-PV-seleno phosphoryl arylamides afforded N-phosphorus substituted formamidinium salts, which are easily reduced to PIII analogues. In addition to the corresponding formamidines, the new N-phosphorylated spiroamine-containing polycyclic system was isolated from the reaction mixture formed by the deprotonation of such salts with a strong non-nucleophilic base.
Conclusions. The Alder reagent approach allows synthesizing precursors of acyclic formamidine carbenes with the spiroamine group. Such carbenes are unstable. By converting these compounds N-PIII-substituted tetrahydropyrimidine and diazaphosphepine derivatives with the 6-azaspiro[2.5]octane fragment have been obtained for the first time.
The synthesis and the anticonvulsant activity screening of new 5-substituted 2-imino-4-thiazolidinone derivatives
Aim. To synthesize 5-ene-4-thiazolidinones containing heterocyclic rings in the molecule as potential anticonvulsants, and screen their anticonvulsant activity on a model of pentylenetetrazole (PTZ) seizures.
Results and discussion. A straightforward and convenient synthesis of novel 5-ene-derivatives of thiazol/oxazole-bearing 4-thiazolidinones as possible anticonvulsant agents was performed. Compounds were characterized using methods of spectral analysis (1H NMR and LC-MS). 5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde underwent the aminolysis on a chlorine atom by a molecule of monoethanolamine (MEA) in the Knoevenagel reaction with thiazole/oxazole-bearing 4-thiazolidinones. The preliminary screening of the anticonvulsant activity was performed for the compounds synthesized on the model of PTZ-induced seizures, and active derivatives were identified.
Experimental part. Commercially available 2-aminothiazole and 5-methylisoxazol-3-amine were used as starting compounds for the synthesis of 2-chloro-N-(hetaryl)acetamides. The latter were transformed into thiazole/oxazole-bearing 4-thiazolidinones by the treatment with ammonium isothiocyanate. Modification at C5 position of the heterocycles synthesized was performed by the Knoevenagel reaction with aromatic/heteroaromatic aldehydes and MEA as a catalyst (either equimolar or 0.1 mol% amount) in the ethanol medium. The structure of novel derivatives was confirmed by 1H NMR and LC-MS spectra. The anticonvulsant activity of all derivatives synthesized was studied in vivo on the model of PTZ-induced seizures. Latency of the seizures, the number of clonic-tonic seizures in one mouse, the percent of animals with clonic and tonic seizures, the duration of the seizure period, and the lifetime of the animals before death were evaluated and calculated.
Conclusions. The results obtained are promising for further design of potential anticonvulsants among oxazole-bearing 4-thiazolidones with the possible mechanism of the anticonvulsant action through the GABA-ergic impact and inhibition of the prostaglandin and leukotriene synthesis.
Application of the enzymatic method for the quantitative determination of dequalinium chloride in lozenges
Aim. To develop a new kinetic spectrophotometric enzymatic method suitable for the quantitative determination of dequalinium chloride in lozenges.
Materials and methods. An enzymatic kinetic spectrophotometric method for the quantitative determination of dequalinium chloride in lozenges has been proposed. It is based on the ability of dequalinium chloride to inhibit the enzymatic hydrolysis reaction of acetylcholine by cholinesterase in the presence of the acetylcholine (AСh) excess and H2O2. The degree of inhibition was determined by the kinetic method using two conjugated reactions: ACh perhydrolysis (interaction with an excess of hydrogen peroxide) followed by oxidation with the peroxyacid formed. Peracetic acid formed in situ by the reaction between unreacted ACh and H2O2 interacts with p-phenetidine forming a product, which absorbs at λmax = 354 nm, in the phosphate buffer solution with pH 8.3 at room temperature.
Results and discussion. The linear dependence of the calibration graph for the quantitative determination of dequalinium chloride was in the concentration range of 0.2 – 0.8 μg mL–1 (r = 0.999). LOD and LOQ were 0.01 × 10–6 and 0.03 × 10–6 mol L–1, respectively. For the quantitative determination of dequalinium chloride in lozenges, RSD ≤ 2.65 % (accuracy, δ = -1.10…+1.78 %).
Conclusions. A new enzymatic kinetic spectrophotometric method has been developed, and its applicability to the quantitative determination of dequalinium chloride in lozenges has been shown. It does not require a complicated treatment of the analyte and a tedious extraction procedure. The method proposed is sensitive enough to determine a small amount of the active pharmaceutical ingredient. These advantages encourage the application of the method proposed in routine quality control of the drugs studied in analytical laboratories.
Aim. To determine the optimal extraction frequency of green tea leaves with 60 % ethanol by the antioxidant method.
Materials and methods. Chun Myn green tea leaves were the object of the study, the raw material was collected in Anhui province (China) from March to April. Dry green tea leaves were standardized according to the European Pharmacopeia 9.0. Spectrophotometry was used to quantify biologically active substances. The antioxidant activity was determined by the potentiometric method. Potentiometric measurements were performed on a HANNA 2550 pH meter (Germany) with a combined platinum EZDO 50 PO electrode (Taiwan). A UV-1000 spectrophotometer (China) was used to measure the optical density.
Results and discussion. The total content of phenolic compounds was 9.60 ± 0.17, 1.30 ± 0.03 and 0.12 ± 0.002 %, catechins – 9.20 ± 0.18, 1.20 ± 0.02 and 0.07 ± 0.002 %, flavonoids – 0.27 ± 0.005, 0.04 ± 0.001, 0.005 ± 0.001, hydroxycinnamic acids – 0.49 ± 0.01, 0.07 ± 0.002 and 0.007 ± 0.001 %, dry residue – 10.75 ± 0.11, 1.59 ± 0.02 and 0.15 ± 0.002 %, the antioxidant activity was 474.08 ± 9.48, 67.70 ± 1.35 and 7.01 ± 0.14 mmol-equiv mdry res-1 for the first, second and third extraction, respectively. According to the results obtained, the optimal number of extractions of the raw material with 60 % ethanol was found to be two.
Conclusions. The dynamic of extractions of biologically active substances of green tea leaves has been studied by triple extraction of the raw material to find the optimal extraction frequency; for the first time, a method for determining the extraction frequency based on the antioxidant activity of the extracts has been developed and proposed. It has been found that the optimal extraction rate is 2 times. The results obtained will be used in the further production of herbal medicines, dietary supplements, and cosmetic products with a green tea extract.
A comparative study of the effect of phenothiazine derivatives and their S-oxides on cholinesterase investigated by a new kinetic spectrophotometric method
Aim. To develop a new kinetic spectrophotometric method for determining acetylcholinesterase (AChE) inhibitors – phenothiazine antipsychotic drugs (PhT) and their sulfoxide metabolites (S-oxides) without adding an exogenous catalyst to obtain a chromogenic agent.
Materials and methods. The bases of S-oxides of promethazine (PMZ), chlorpromazine (CPM) and thioridazine (THZ) were obtained by oxidizing the corresponding PhT hydrochlorides with diperoxyadipic acid. The structure of the S-oxides of the corresponding PhT synthesized was proven by melting points, spectral characteristics (1H NMR and IR methods) and oscillopolarography results. 1H NMR spectra were recorded on a Varian XL-200 spectrometer. IR spectra were recorded within the range of 4000-400 cm–1 on a SPECORD M-80 spectrometer (Zeiss, Jena, Germany). To register polarograms, a “PO 03 CLA” oscillopolarograph with a three-electrode cell was used. The purity of S-oxides was determined by the high-performance liquid chromatography method on a Zorbax SB, C-18 (250 × 4.6) mm column. Measurements of absorbance of solutions were performed in a 1 cm cuvette on an Evolution 60S UV-Visible Thermo-Scientific Spectrophotometer (USA) (λ = 358 nm).
Results and discussion. Acetylcholine (ACh) was found to mimic the activity of peroxidase; based on it, a spectrophotometric system containing ACh–H2O2–p-Ph for a sensitive and selective assessment of the AChE activity and determination of its inhibitors was developed. According to the plots of inhibition efficiency vs inhibitors concentration, the inhibiting ability of chlorpromazine, promethazine and thioridazine and their S-oxides was determined. The IC50 values of CPM, PMZ and THZ and their metabolites in relation to the AChE activity were estimated as 11 ng mL−1 (CPM) and 1.8 ng mL−1 (CPM S-oxide), 17 ng mL−1 (PMZ) and 2.5 ng mL−1 (PMZ S-oxide) and 27 ng mL−1 (THZ 2S,5S-dioxide). The results obtained indicate that S-oxides of the corresponding PhT are selective and potent inhibitors of AChE. The values of the inhibition efficiency obtained for S-oxides of PhT derivatives were an order of magnitude lower than those of the corresponding PhT derivatives.
Conclusions. The spectrophotometric method proposed without the addition of other exogenous catalysts holds promise for the on-site determination of PhT antipsychotics and can be additionally used for sensory applications in areas related to environmental protection and food safety, as well as in the chemical-toxicological analysis.
The allelopathic activity of water-soluble biologically active substances from Hyssopus officinalis L. of Marquis varieties and their effect on the root growth of Lepidium sativum L.
Aim. To study the effect of secretions of bioactive water-soluble compounds from leaves, stems, flowers, and the root system of Hyssopus officinalis L. on the root growth of the test sample (Lepidium sativum L.).
Materials and methods. To determine the effect of biologically active water-soluble compounds released by hyssop plants, a series of biotests was conducted; the allelopathic activity of leaves, stems, and flowers of H. officinalis L. and its root system were determined. The preparation of extracts of physiologically active substances was carried out according to the method of A. M. Grodzinsky. The allelopathic effect of H. officinalis L. was assessed by the effect of the water extracts of various concentrations (100 %, 50 % and 10 %) on the root length of seedlings of L. sativum L. test objects. The inhibition index was determined by Williamson method.
Results and discussion. According to the results of the research, it was found that the allelopathic activity of water-soluble compounds of common hyssop affected the germination and length of seed roots of L. sativum L. as it changed with the age of the plant : in the first year of vegetation, the stimulating effect of aqueous solutions of hyssop on seed germination was observed, in the second year, the stimulating effect decreased, and in the third year of vegetation, the inhibitory effect on seed germination of the test object was observed. Thus, the index of the allelopathic activity also changed.
Conclusions. It was found that the highest allelopathic activity of biologically active water-soluble compounds of root secretions, leaves, stems and flowers of H. officinalis L. was during the flowering phase in concentrations of 100 %, 50 % and 10 %, affecting the length of the roots of seedlings of L. sativum L. test objects. At the same time, the maximum stimulating effect on the length of the roots of the test object was in the variant using an aqueous extract of plant flowers of the second year of vegetation in a 10 % concentration. The use of aqueous solutions of plants of the third year of vegetation had an inhibitory effect on the root length of L. sativum L seedlings.
The quantitative determination of glutathione by the effect of the chemiluminescence inhibition in the catalytic oxidation reaction of luminol with hydrogen peroxide in the presence of hemoglobin
Aim. To develop a method for the quantitative determination of reduced glutathione in a lyophilized powder for the preparation of “Hepaval®” solution for intravenous and intramuscular administration by the effect of inhibiting chemiluminescence in the luminol (H2L)–Н2О2–hemoglobin (Hb) system.
Materials and methods. The study object was the reduced glutathione substance and lyophilized powder for the preparation of “Hepaval®” solution for injection, ampoules of 4 mL No. 10 manufactured by “Valartin pharma” (Italy). The glutathione content in powder was determined using the chemiluminescence method by the effect of inhibiting the luminol oxidation reaction with hydrogen peroxide in the presence of Hb as a catalyst. The analysis was performed by the standard addition method.
Results and discussion. As a result of the studies, it has been found that under optimal conditions glutathione shows a noticeable inhibitory effect on chemiluminescence in the H2L–H2O2–Hb system. This phenomenon was used to develop a new procedure for the quantitative determination of glutathione in the substance and lyophilized powder for the preparation of “Hepaval®” aqueous solution for injection. The linear dependence of the integral chemiluminescence intencity (S) on the molar concentration of glutathione was maintained in the concentration range of (2 – 20) × 10–7 mol L-1. The graph equation was S = (–1.6 ± 0.2) × 107 × c + (198.9 ± 2.0), (r = 0.999). The relative standard deviation (RSD) was ±1.82 % (n = 7, P = 0.95).
Conclusions. The method has been developed, and the possibility of the quantitative determination of glutathione in powder for the preparation of “Hepaval®” solution for injection by the method of the chemiluminescence inhibition of the H2L–Н2О2–Нb system has been shown. The content of glutathione in powder calculated with reference to dried substance was 91.49 % (against 90.8 % by the certificate). The accuracy was +0.76 %.
The quantitative content determination of main groups of biologically active substances in batches of Viburnum opulus fruits
Aim. To determine the quantitative content of the main groups of biologically active substances (BAS) in batches of Viburnum opulus fruits using pharmacopeial methods for their subsequent use in the standardization of the plant raw material.
Materials and methods. For the study, 6 batches of air-dried Viburnum opulus fruits crushed to a particle size of 1 – 2 mm and harvested in different regions of Ukraine in the mass fruiting phase in October-November 2020 were used. The quantitative determination of polyphenols and tannins was performed using the spectrophotometric method at a wavelength of 760 nm calculated with reference to pyrogallol and dried substance. The total amount of hydroxycinnamic acids was determined according to the method of the SPhU (State Pharmacopoeia of Ukraine) 2.0 described in the monograph “Kidney TeaN” calculated with reference to rosemary acid. The total amount of organic acids was determined by the titrimetric method according to the method of the monograph of the SPhU 2.1 “Rose hipsN” calculated with reference to malic acid.
Results and discussion. The quantitative content of polyphenols (calculated with reference to pyrogallol and dried substance) in batches of Viburnum opulus fruits varied by about 1.4 times from 1.74 ± 0.01 % to 2.36 ± 0.01 %. According to the results obtained, the quantitative content of tannins (calculated with reference to pyrogallol and dried substance) in batches of Viburnum opulus fruits varied by 1.7 times from 0.73 ± 0.01 % to 1.23 ± 0.01 %. The quantitative content of the total amount of hydroxycinnamic acids calculated with reference to rosemary acid in batches of Viburnum opulus fruits ranged by 1.2 times from 3.96 ± 0.01 % to 4.73 ± 0.01 %. The quantitative content of the total amount of organic acids calculated with reference to malic acid in batches of Viburnum opulus fruits fluctuated more than 1.3 times from 6.80 ± 0.01 % to 9.08 ± 0.01 %.
Conclusions. The quantitative content of the main groups of biologically active substances has been determined in 6 batches of Viburnum opulus fruits harvested in different regions of Ukraine using pharmacopoeial methods: polyphenols (varied by 1.4 times), tannins (varied by 1.7 times), the total amount of hydroxycinnamic (varied by 1.2 times), and the total amount of organic acids (varied by 1.3 times) calculated with reference to dried substance. The content of the groups of biologically active substances in batches of Viburnum opulus fruits slightly correlates with the place of the raw material harvesting. Thus, the selected methods of the SPhU 2.0 are quite suitable for determining the quantitative content of polyphenols, tannins and of the total amount of hydroxycinnamic and organic acids in batches of Viburnum opulus fruits; they can be used in further studies to standardize the raw material.