Sydnonimines represent a class of chemical compounds promising for the development of new drugs [1]. These substances are known to possess anti-inflammatory, antipyretic [2], and antitumor [3, 4] activities, act as monoamine oxidase inhibitors [5, 6], and exhibit spasmolytic effects [1]. However, sydnonimines are best known as nitric oxide donors with vasodilatory activity [7]. The most well-known drug in this group is molsidomine, currently used as an antianginal agent [8]. Another representative of this compound group, the drug candidate pirsidomine (and its hydrolyzed form, darsidomine) [9], which exhibited vasodilatory activity, did not pass phase II clinical trials and is not currently used [2]. The use of sydnonimine drugs as psychostimulants is also known. Representatives of this pharmacological group include mesocarb and its active enantiomer, armezocarb, as well as the drug feprosidnine [10].

The established vasodilatory activity of sydnonimines allows these substances to be considered as lead compounds in the early stages of developing agents for correcting cerebral circulation disorders. The selection of compounds from this chemical group is also justified by the presence of a neuroprotective effect, associated with increased glutathione content in brain tissues [11] or stimulation of soluble guanylate cyclase followed by cGMP accumulation [12]. To achieve cerebral vasodilatory activity combined with the absence of a pronounced effect on systemic hemodynamics, optimization of lead compounds is required, either through interaction with specific targets in central nervous system (CNS) structures or by improving the pharmacokinetic properties of molecules to achieve higher concentrations in CNS tissues [13, 14]. Considering the mechanism of vasodilatory activity of sydnonimines, associated with spontaneous nitric oxide release at physiological blood plasma pH values and not requiring interaction with a specific target, the pharmacokinetic approach to optimizing lead compounds in this chemical group is the most suitable. Taking into account the fact that CNS tissues are highly lipophilic, increasing the hydrophobicity of pharmacologically active molecules will promote their more pronounced distribution into brain structures and slow down elimination.

Optimization of sydnonimine lead compounds to increase their lipophilicity can be achieved by introducing various hydrophobic radicals into the molecular structure. Subsequent experimental pharmacokinetic studies, including the assessment of the distribution of lead compounds between blood plasma and brain tissues, will allow the identification of an optimal lead compound, which can then be considered a promising drug candidate for the pharmacological correction of cerebral circulation disorders.

The aim of the present study is to optimize sydnonimine lead compounds to obtain a drug candidate with predominant cerebral vasodilatory activity.

MATERIALS AND METHODS

The synthesis of sydnonimine lead compounds was carried out at the Research Laboratory of Medicinal Substance Chemistry, Research Institute of Translational Medicine, Pirogov Russian National Research Medical University (RNRMU) of the Ministry of Health of Russia. The pharmacological and bioanalytical parts of the experiment were performed at the Research and Production Center for Pharmaceutical and Bioanalytical Studies, Tver State Medical University of the Ministry of Health of Russia.

The objects of the study were compounds that are N-(ethoxycarbonyl)sydnonimine derivatives with various substituents at the 3-position of the 1,2,3-oxadiazole ring (Table 1).

To study the accumulation of lead compounds in central nervous system structures, a pharmacological experiment was conducted on rats. The aim was a comparative assessment of the concentration of each substance in blood plasma and brain tissues after a single intragastric administration, followed by calculation of tissue distribution coefficients.

The studies were performed on 65 male Wistar rats weighing approximately 300 g (bred by LLC “SMK STEZAR,” Russia). Animal cages were kept under controlled environmental conditions (20–26 °C and 30–70% relative humidity). The animal housing rooms maintained a 12-hour light cycle and an air change rate of 8–10 volumes per hour. Rats were fed complete PK-120 compound feed (LLC “Laboratorkorm”) and had ad libitum access to filtered tap water. Cage cleaning, room wet cleaning, and replacement of water bottles with fresh ones were performed daily. On the evening before the experiment, animals were deprived of food.

A mixture containing 270 μmol of each studied lead compound active pharmaceutical ingredient (API) was prepared in an evaporation flask. An equivalent amount of molsidomine API was additionally added to the combination. The resulting mixture was quantitatively transferred to a 10 ml volumetric flask and dissolved in dimethyl sulfoxide. An aliquot (926 μl) of the obtained combined solution (individual concentration of each substance 27 μmol/ml) was transferred to a 50 ml volumetric flask and dissolved in corn oil. The combined oil solution (0.5 μmol/ml) was used to prepare a 10% corn oil-in-water emulsion, with an individual concentration of each compound of 0.05 μmol/ml. The resulting emulsion was administered to the experimental rats as a single intragastric dose using a disposable syringe equipped with a gavage needle, at a rate of 5 ml per 300 g body weight. Individual doses of the combined preparation were determined after preliminary weighing of the rats and were ensured by adjusting the administered volume of the emulsion. Thus, the individual dose of each compound for rats was 0.82 μmol/kg, which is equivalent to a single standard human dose of molsidomine (2 mg).

Animals were euthanized by decapitation using a guillotine (Open Science) at the following time points after emulsion administration: 7.5, 15, 30, 45, 60, 90 minutes; 2, 4, 6, 8, 12, and 24 hours. Additionally, a group of intact rats (0-hour time point) was used. There were 5 rats per time point. During decapitation, blood was collected in a volume of 2 ml into polypropylene tubes containing K2EDTA, vigorously mixed by inversion, and plasma was immediately separated by centrifugation at 3500 rpm for 10 minutes using an LMC-4200R laboratory centrifuge (Biosan, Latvia). A 100 μl aliquot of plasma was combined with 400 μl of an internal standard (IS) solution (N-(ethoxycarbonyl)-3-(4-methylpentan-2-yl)sydnonimine, 100 ng/ml) in methanol containing 0.1% formic acid, mixed on a Microspin FV-2400 vortex mixer (Biosan, Latvia) for 30 seconds, and placed in a freezer.

After blood collection, the brain was immediately extracted, rinsed in physiological saline, blotted dry with filter paper, and divided into two halves along the longitudinal fissure. One half of the brain was placed into a pre-weighed 2 ml Eppendorf tube on an analytical balance. The mass of the fragment was determined by re-weighing the filled tube. A 0.1% aqueous formic acid solution was immediately added at a ratio of 400 μl per 100 mg of tissue. A quartz glass bead was added to the tube, and homogenization was performed using a Minilys homogenizer (Bertin Technologies, France) in two cycles of 2 minutes each. Sample preparation of the obtained homogenates was carried out similarly to that for blood plasma.

Brain homogenates, blood plasma, and prepared extracts were stored at –40 °C in an MDF-136 freezer (Sanyo, Japan) until analysis. In the bioanalytical laboratory, the supernatant was separated using an EBA 400R centrifuge (EAC, Russia) at 21,000 g and –10 °C for 20 minutes. A 50 μl aliquot of the supernatant was transferred into disposable polyethylene inserts placed in chromatographic vials and loaded into the autosampler of the chromatograph.

A bioanalytical HPLC-MS/MS method was developed and validated for the quantitative determination of sydnonimine lead compounds in blood plasma and brain homogenates. Separation of the studied substances was performed on an Agilent InfinityLab Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 μm) with a Phenomenex C18 guard column (4.0 × 3 mm). Elution was carried out using a gradient mixture of deionized water and acetonitrile with the addition of 0.1% formic acid. Chromatography was performed using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, Germany), and detection was carried out using a Sciex QTrap 3200 MD quadrupole mass spectrometer (AB Sciex, Singapore) in multiple reaction monitoring (MRM) mode with positive ionization. The concentrations of analytes were calculated using Analyst 1.6.3 software (AB Sciex) based on calibration curves depicting the relationship between the chromatographic peak area of the analyte normalized to the IS area and the nominal concentration of the analyte.

STUDY RESULTS

At the first stage of bioanalytical method development, two fragment ions were selected for all analytes and the internal standard. Optimization of parameters ensuring the best analysis sensitivity was performed for these ions (Table 2). The product ion mass spectra of the determined substances are presented in Figure 1.

Chromatographic separation was performed on an Agilent InfinityLab Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 μm) coupled with a Phenomenex C18 guard column (4.0 × 3 mm) using gradient elution with 0.1% formic acid in acetonitrile and deionized water at a flow rate of 0.6 mL/min (Table 3). Figure 2 shows a total ion chromatogram of a rat plasma standard sample with an individual concentration of 0.2 nmol/ mL for each studied compound.

Following sample preparation, rat blood plasma and brain homogenate samples were analyzed in separate analytical runs. Each run included a blank sample (containing neither the studied compounds nor the internal standard), a zero sample (containing only the internal standard), calibration samples, quality control samples at four concentration levels, and the study samples. Concentrations of the sydnonimine group compounds were calculated based on the calibration curve depicting the relationship between the chromatographic peak area of the analyte normalized to the internal standard area and the nominal analyte concentration. Linear regression was used to construct the calibration curves. Accuracy and precision (CV%) control of the analysis in each analytical run was performed using quality control samples. Deviations from the nominal analyte concentrations and the CV% between replicate injections of calibration and quality control samples were within 15% at all concentration levels, demonstrating the acceptability of the analytical run.

Based on the averaged results of the quantitative determination of the sydnonimine group compounds in biological material, pharmacokinetic curves were plotted for blood plasma (Fig. 3) and rat brain tissues (Fig. 4).

For a comparative assessment of the penetration of the studied compounds into rat brain structures, tissue distribution coefficients (Kd) were calculated as the ratio of the individual concentration of each substance in brain tissues to its concentration in blood plasma. This parameter was calculated for all animals at each time point. Based on the individual Kd values, the mean values of the parameter were calculated for each time point after excluding outliers detected using the Grubbs’ statistical test (Fig. 5).

In addition, mean brain/plasma distribution coefficient (Kd) values for each studied sydnonimine group compound, averaged over all time points (Fig. 6).

DISCUSSION OF RESULTS

Based on the results of pharmacokinetic studies, it was established that for all studied sydnonimine group compounds, the time to reach maximum concentration (Tmax) in rat blood plasma is 15 minutes. The absence of significant differences in this parameter between the studied compounds is likely due to the employed dosage form (oil emulsion), which ensures rapid entry into the systemic circulation from the gastrointestinal tract for both lipophilic and hydrophilic substances. Furthermore, all substances exhibit a second concentration peak in plasma corresponding to 90 minutes, which is explained by enterohepatic circulation, as established by previously conducted studies [15]. The highest maximum concentration (Cmax) values in blood plasma were found for compounds containing the following radicals: 4-morpholinyl (molsidomine), ethyl (925), and 3-methoxy-propyl (935). The lowest Cmax values were observed for sydnonimines with structures containing the radicals: decyl (933), octyl (934), and 4-methylhexan-2-yl (BBP2023).

Comparative analysis of the pharmacokinetic curves of the studied sydnonimine group compounds in rat brain tissues reveals pronounced differences. For substances BBP2023, 933, and 934, the Tmax value averages 45 minutes, while for the remaining compounds it is 15 minutes. Additionally, for BBP2023, 933, and 934, a more gradual increase and decrease in concentration is observed in brain tissues.

Comparative assessment of the mean brain/plasma tissue distribution coefficients shows significant differences in this parameter for the aforementioned compounds compared to molsidomine (Fig. 6). This fact indicates the relative predominance of compounds BBP2023, 933, and 934 in central nervous system structures relative to blood plasma throughout the observation period.

Furthermore, a direct correlation is observed between the increase in the mean brain/plasma Kd value and the number of hydrocarbon groups in the radical at the 3-position of the 1,2,3-oxadiazole ring in the structure of the studied substances. This finding correlates with the behavior of the compounds during reversed-phase HPLC analysis (Fig. 2). Thus, increasing the hydrophobicity of sydnonimine compounds by introducing a hydrocarbon radical into their structure promotes more pronounced distribution of these substances into brain structures, which is important in the development of agents for the pharmacological correction of cerebral circulation disorders.

CONCLUSIONS

Among the studied sydnonimine group compounds, the lead compound with the laboratory code BBP2023 is of greatest interest. Additional biotransformation studies revealed that one of the metabolites of BBP2023 is 1,3-dimethylamylamine (geranamine), a compound with established psychostimulant activity [16]. This fact may be beneficial in the treatment of cerebrovascular disorders accompanied by central nervous system depression.

The selected lead compound possesses experimentally confirmed cerebral vasodilatory activity, exhibits an optimal pharmacokinetic profile in brain tissues, and is characterized by the presence of pleiotropic effects (procognitive, psychostimulant), making it a promising drug candidate recommended for further expanded preclinical studies of efficacy and safety.