Comparative Analysis of the Structure and Pharmacological Properties of Some Piperidines and Host–Guest Complexes of β-Cyclodextrin

Pain and anesthesia are a problem for all physicians. Scientists from different countries are constantly searching for new anesthetic agents and methods of general anesthesia. In anesthesiology, the role and importance of local anesthesia always remain topical. In the present work, a comparative analysis of the results of pharmacological studies on models of the conduction and terminal anesthesia, as well as acute toxicity studies of the inclusion complex of 1-methyl-4-ethynyl-4-hydroxypiperidine (MEP) with β-cyclodextrin, was carried out. A virtual screening and comparative analysis of pharmacological activity were also performed on a number of the prepared piperidine derivatives and their host–guest complexes of β-cyclodextrin to identify the structure–activity relationship. Various programs were used to study biological activity in silico. For comparative analysis of chemical and pharmacological properties, data from previous works were used. For some piperidine derivatives, new dosage forms were prepared as beta-cyclodextrin host–guest complexes. Some compounds were recognized as promising local anesthetics. Pharmacological studies have shown that KFCD-7 is more active than reference drugs in terms of local anesthetic activity and acute toxicity but is less active than host–guest complexes, based on other piperidines. This fact is in good agreement with the predicted results of biological activity.


Introduction
Local anesthetics are currently used in almost all areas of practical medicine [1].The interest in local anesthetics is due to the negative side effects of general anesthesia on the cardiovascular system, central nervous system, gastrointestinal tract, and individual organs.Although a large number of local anesthetic drugs are known, a rather limited number of drugs are used in practice [2].This is due to the fact that most local anesthetics do not correspond to modern standards and requirements [3].Thus, they must have a short latent period, a long period of action and high activity, and be non-irritating and low-toxic.
One of the most rational drug design approaches towards pharmacologically active molecules is based on the structural modification of compounds with reported high activity.As we can see from the papers [4,5], some 1-alkoxyalkyl-4-hydroxypiperidine hydrochlorides and previously reported 1-ethoxyethyl analogs have revealed local anesthetic effects [6].At the same time, as reported previously, the corresponding benzoates were found to be the strongest local anesthetics [7].
It is a well-known fact that there is no clear correlation between the chemical structure of a drug and its biological effects [1].Thus, minor changes in the structure of a molecule may lead to a complete disappearance or a strong change in the biological activity (e.g., methyl and ethyl alcohol).Modern pharmaceutical research and development is a high-risk investment that typically faces setbacks at various stages of drug development [8].Because of that, a molecular design based on the use of prediction software has attracted so much attention in recent years [9].The structure-activity relationship analysis of the known drugs can help predict the chemical structure of new molecules with the desired properties [8].
One of the main reasons for failure in drug research and development is the lack of efficacy and safety, which are substantially correlated with absorption, distribution, metabolism, and excretion (ADME), as well as with toxicity (T) [10].Therefore, a rapid evaluation of the ADMET parameters is necessary to minimize failures in the drug discovery process.The ADME parameters [11,12] cover pharmacokinetics, which determine whether the intended drug molecule will reach the target protein in the body and how long it will remain in the bloodstream.
Cyclodextrins are widely used in the pharmaceutical industry for transporting and modification of an active substance [13,14].The formation of inclusion complexes makes it possible to change the properties of the biologically active component in the desired direction, i.e., to increase the bioavailability and resistance to hydrolysis, solubility, and biodegradability of many active substances [15,16].In order to improve the anesthetic effects and reduce the toxicity of the water-soluble salt forms of piperidine derivatives, we synthesized and studied [7,[17][18][19][20][21] the host-guest complexes of some of these compounds with β-cyclodextrin.
In this work, a pharmacological study of terminal anesthesia was conducted, and the acute toxicity of the 1-methyl-4-ethynyl-4-hydroxypiperidin (MEP) inclusion complex with β-cyclodextrin was analyzed.Virtual screening of the pharmacological activity for a number of piperidine derivatives was carried out in order to identify the structureactivity relationship.The results of the virtual screening were compared with their actual pharmacological effects.

Results and Discussion
To determine the structure-activity relationship, we used piperidines of the general formula, shown in Figure 1.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 24 It is a well-known fact that there is no clear correlation between the chemical structure of a drug and its biological effects [1].Thus, minor changes in the structure of a molecule may lead to a complete disappearance or a strong change in the biological activity (e.g., methyl and ethyl alcohol).Modern pharmaceutical research and development is a high-risk investment that typically faces setbacks at various stages of drug development [8].Because of that, a molecular design based on the use of prediction software has attracted so much attention in recent years [9].The structure-activity relationship analysis of the known drugs can help predict the chemical structure of new molecules with the desired properties [8].
One of the main reasons for failure in drug research and development is the lack of efficacy and safety, which are substantially correlated with absorption, distribution, metabolism, and excretion (ADME), as well as with toxicity (T) [10].Therefore, a rapid evaluation of the ADMET parameters is necessary to minimize failures in the drug discovery process.The ADME parameters [11,12] cover pharmacokinetics, which determine whether the intended drug molecule will reach the target protein in the body and how long it will remain in the bloodstream.
Cyclodextrins are widely used in the pharmaceutical industry for transporting and modification of an active substance [13,14].The formation of inclusion complexes makes it possible to change the properties of the biologically active component in the desired direction, i.e., to increase the bioavailability and resistance to hydrolysis, solubility, and biodegradability of many active substances [15,16].In order to improve the anesthetic effects and reduce the toxicity of the water-soluble salt forms of piperidine derivatives, we synthesized and studied [7,[17][18][19][20][21] the host-guest complexes of some of these compounds with β-cyclodextrin.
In this work, a pharmacological study of terminal anesthesia was conducted, and the acute toxicity of the 1-methyl-4-ethynyl-4-hydroxypiperidin (MEP) inclusion complex with β-cyclodextrin was analyzed.Virtual screening of the pharmacological activity for a number of piperidine derivatives was carried out in order to identify the structure-activity relationship.The results of the virtual screening were compared with their actual pharmacological effects.

Results and Discussion
To determine the structure-activity relationship, we used piperidines of the general formula, shown in Figure 1.

In Silico Pharmacology
In drug development, efficient target binding is not only important, but it also ensures oral bioavailability and drug-like properties.In this regard, the study of the physicochemical properties of compounds is crucial for drug development.
The predictive analysis and in silico studies of possible targets, ADME parameters (absorption, distribution, metabolism, and excretion), and compliance with the bioavailability criteria [11,22] were carried out for the studied compounds.
An analysis of the structures for compliance with Lipinski's rule of five (molecular weights (MW) ≤ 500, cLogP ≤ 5.0, TPSA ≤ 140 Å 2 , number of H-acceptors ≤ 10, Hdonors ≤ 5) [23,24] was performed, using the SwissADME software package [25].Compliance with Lipinski's rule makes the compounds active drug candidates.The substance is unlikely to become an active drug candidate if Lipinski's rule is violated even by one parameter.
The analysis of lipophilicity (LogP) is provided in Table 2. Optimal values for LogP (P is the partition coefficient of all forms of the molecule between n-octanol and water) are between 0 and 3. LogP < 0 corresponds to the bad permeability of the lipid bilayer; LogP > 3 indicates poor water solubility [26].Compounds with high cLogP values may have difficulty in achieving the therapeutic targets due to their lipophilicity, which potentially limits their effectiveness.The LogP value shows moderately good (0.33) absorption and permeability for the MEP.For EEHP and MEBP, the cLogP values are 1.07 and 2.42, respectively.For the other compounds, the distribution coefficient is significantly higher and ranges from 3.30 to 4.93.More positive cLogP values usually indicate a higher concentration of the compound in the lipid phase.
LogS values (logarithm of water solubility value, expressed in log mol/L) above −4 logmol/L and below 10 µg/mL indicate low solubility.In the range of 10-60 µg/mL, the compounds have moderate solubility.All LogS values higher than 60 µg/mL indicate high solubility [27].
The TPSA parameter for EEHP, MEBP, and MEP has a low value of 23.47 Å 2 and meets the criteria for oral bioavailability.The MEP compound meets the Lipinski, Egan, and Weber criteria.The Egan filter (Pharmacia filter) is based on the LogP and TPSA parameters.It anticipates drug absorption, depending on the processes involved in the membrane permeability of a small molecule, and considers the molecule drug-like if it has WLOGP ≤ 5.88 and TPSA ≤ 131.6, respectively [28].The Muegge filter (the Bayer filter) is the independent pharmacophore point filter that separates drug-like and nondrug-like molecules.The Ghose filter (Amgen) describes small molecules based on their physicochemical properties and the existence of functional groups and substructures [28].EEHP only fails to meet the Muegge criteria due to its low molecular weight.BBB•HCl has failed to meet the Ghose criteria because the calculation was carried out for a hydrochloride form.The remaining compounds correspond to all the criteria provided in Table 3.All compounds have shown favorable bioavailability values (0.55).This indicates good suitability for oral drug administration and implies achieving a therapeutic result at lower concentrations.
The radar diagrams (Figure 2) show the distribution of the physicochemical properties of the compounds: lipophilicity (LIPO), size (SIZE), polarity (POLAR), solubility (INSOLU), saturation (INSATU), elasticity (FLEX), presence of donors (nHD), and proton acceptors (nHA).The pink area represents the optimal range for each property (lipophilicity: XLOGP3 −0.7 to +5.0, size: molecular weight 150 to 500 g/mol, polarity: TPSA 20 to 130 Å 2 , solubility: log S not above 6, saturation: the fraction of carbons in sp 3 hybridization is at least 0.25 and flexibility no more than nine rotating bonds) [25].The analyses of the diagrams show that prosidol, kazcaine, and AEPP have the best distribution of parameters, though all the compounds, in principle, meet the requirements for a medicinal substance.BVBP and BBB have a slight excess in the FLEX parameter, and for MEP, the size, polarity, and flexibility indicators are at the lower limit.
To predict possible biological effects, the open software products PASS Online, AntiBac-Pred, and AntiFun Pred [29][30][31] were used.Here, and below, the score function F = Pa − Pi is used, which is the difference in the probabilities that a substance will be active (Pa) or inactive (Pi) for the corresponding biological activity.In Table 4, the results for MEP are provided (for F > 0.1).Based on these data, the most probable biological activity of MEP is the suppression of ovulation; there is also a very high probability of its influence on the hormones responsible for reproductive functions.The substance can be used as an anticonvulsant.The other activities (anesthetic, anabolic, nootropic, antidepressant, analgesic, and muscle relaxant) have a rather low probability.Comparative data on the major types of activity for all the substances under consideration are provided in Table 5.The results are provided for the substances in the form of bases since the calculation programs, in most cases, cannot work with the substances in the form of salts and complex compounds (including inclusion complexes).Possible protein targets (for Homo sapiens) were evaluated using the Swiss Target Prediction service.The results are shown in Table 6.The score for each target is called "confidence", which is the difference between probabilities of chemical compounds interacting and not interacting with a particular target.Higher confidence means a higher chance of a positive prediction being true.The first 5-6 results are listed and the rest are provided in the Supplementary Materials.The probabilities for MEP are very low, but we can conclude that the substance may affect mechanisms that occur in the central nervous system.

MEP prosidol kazcaine AEPP
The PASS Targets program provides a slightly different prediction of possible molecular targets.It is advisable to consider results with a confidence value greater than 0.5.Table 7 shows the values greater than 0.5 for MEP, EEHP, and MEBP and greater than 0.25 for the remaining compounds.The full list is presented in Table S1.
According to Table 7, MEP has the largest number of possible targets with a confidence value greater than 0.5.It looks most similar to kazcaine according to the list of possible targets, though the character of the data obtained (a large number of targets and high probability values) should rather be considered an anomaly.The substances MEP, EEHP, and MEBP actively bind to protein kinases.In silico prediction of acute toxicity values (LD50) for rats for four types of administration (oral, intravenous, intraperitoneal, subcutaneous, and inhalation) was carried out using the GUSAR program [32].This program compares the structure of a substance with structures from the SYMYX MDL toxicity database.In order to assess which of these drugs best corresponds to the optimal characteristics required for an ideal drug, the acute toxicity parameter LD50 (known as the "lethal dose, 50%" or oral acute dose for rats) was calculated.High toxicity was indicated by values of 1-50 mg/kg; average toxicity was in the range of 51-500 mg/kg.Low toxicity values were 501-5000 mg/kg [33].The GUSAR program could not calculate data for BBB HCl in the form of either hydrochloride (which is expected) or a base.
The acute toxic class is provided according to the OECD.Low concentrations of the substance reduce the risk of side effects and toxicity.Analyzing the data in Table 8, it can be argued that the acute toxicity values of the compounds exceed the values of the average toxicity range for the compounds prosidol, AEPP, and BVBP.MEP showed a fairly low predicted toxicity risk for intraperitoneal, intravenous, and subcutaneous administration but higher toxicity for all routes of administration compared to the other study drugs.* IP-intraperitoneal route of administration, IV-intravenous route of administration, Oral-oral route of administration, and SC-subcutaneous route of administration.** BOLD = in AD: the compound falls within the range of applicability of the models; italic = out of AD: compound outside the range of applicability of models.
The prognosis of adverse effects (arrhythmia, heart failure, hepatotoxicity, myocardial infarction, and nephrotoxicity) was made using ADVER Pred [34].The results are shown in Table 9 and Figure 3.The prognosis of adverse effects (arrhythmia, heart failure, hepatotoxicity, myocardial infarction, and nephrotoxicity) was made using ADVER Pred [34].The results are shown in Table 9 and Figure 3.The compounds may exhibit side effects such as arrhythmia (prosidol, kazcaine, AEPP, BVBP, and BBB), hepatotoxicity (MEP, kazcaine, AEPP, EEHP, and MEBP), myocardial infarction (kazkain and MEBP), and nephrotoxicity (MEBP and EEHP).Kazcaine was predicted to cause the highest number of adverse effects compared to the other compounds.However, their probability, excluding hepatotoxicity, was low.The calculated results also indicate a high probability of hepatotoxicity for MEP.For most compounds, a high probability of arrhythmia was predicted as an adverse effect.In order to improve the bioavailability parameters and reduce the toxic side effects, it is advisable to use active compounds in the form of inclusion complexes with cyclodextrin.

Host-Guest Complexes with β-Cyclodextrin
The severity of adverse effects, such as hepatotoxicity and nephrotoxicity, can be reduced using drug inclusion complexes with β-cyclodextrin.Cyclodextrins usually improve the solubility of guest molecules in water, significantly reduce their toxicity, and increase the period of action due to the slow dissociation of the inclusion complex in the body.
Usually, the drugs are used not in a pure form but in a so-called "dosage form".For example, water-soluble drugs are used in the form of isotonic solutions containing a local anesthetic, while fat-soluble drugs are administered subcutaneously in the form of an oil solution, from which the drug slowly passes into the interstitial fluid.
Earlier, piperidines have been often used as water-soluble salt forms, such as hydrochlorides, to prepare useful dosage forms.
However, along with a high anesthetic effect, such dosage forms also have significant toxicity.Therefore, the preparation of new dosage forms with minimal adverse effects is an actual problem.
The preparation of new dosage forms based on inclusion (host-guest) complexes of cyclodextrins seems to be a promising solution to the problem.
Inclusion complexes are effective as delivery tools.With the conventional type of administration, only nearly one-tenth of the drug molecules can reach the site of application (nerves, tumors, etc.).When the drug is delivered in the form of an inclusion complex and released directly near the site of application, the effective local concentration is increased.Therefore, less amount of drug is required, which can also reduce overall toxicity.
In our previous works [7,[13][14][15][16][17][18][19][20][21], we reported the preparation of host-guest complexes of the above piperidines with β-CD and studied their structure (Table 10).All the compounds except MEP formed inclusion complexes with a guest-host ratio of 1:2.For MEP, the 1:1 complex was isolated, which is most likely due to the smaller size of the guest molecule.The structures of inclusion complexes were studied by NMR during their complex formation in the solutions as well as by X-ray diffraction in their crystalline form.Due to the flexibility of the piperidine ring, piperidines can exist in two main conformations.In inclusion complexes, they can either remain in their starting conformation (for example, BBB) or have a different conformation compared to their free form (kazcaine and prosidol).In addition, in a solution (CDCl 3 and D 2 O), BBB-HCl exists as two isomers in a 2:1 ratio with different orientations of benzoyloxy groups: 1e-(3-n-butoxypropyl)-4abenzoyloxypiperidine hydrochloride and 1e-(3-n-butoxypropyl)-4e-benzoyloxypiperidine hydrochloride.BBB-HCl forms inclusion complexes with β-CD with a stoichiometry of 2 β-CD:1 BBB-HCl.The same conformation also exists in the inclusion complex isolated in the solid form.
The structure of the inclusion complex of β-CD with MEP (KFCD-7) was studied using NMR and X-ray diffraction [21].Below (Figure 4), the expansion from the ROESY NMR spectrum in addition to the data published earlier are shown.The cross peaks between inner (3 and 5) protons of β-CD and 2 and 6 protons of the piperidine ring clearly show that the structure of the MEP:β-CD complex in the solution corresponds to the one obtained from the X-ray data in the solid state.
tween inner (3 and 5) protons of β-CD and 2 and 6 protons of the piperidine ring clearly show that the structure of the MEP:β-CD complex in the solution corresponds to the one obtained from the X-ray data in the solid state.
An analysis of the predicted biological activity shows that MEP, as well as its β-CD inclusion complex, should be significantly different in biological activity from the other piperidine derivatives and their inclusion complexes.Because of that, we conducted a pharmacological study of KFCD-7 and compared its acute toxicity, infiltration, and conduction anesthesia with the data for previously obtained piperidine derivatives and reference drugs.

Infiltration Anesthesia
The test was performed using the Bulbring-Wade method.All the compounds were tested as 0.5% aqua solutions.The results are summarized in Table 11.An analysis of the predicted biological activity shows that MEP, as well as its β-CD inclusion complex, should be significantly different in biological activity from the other piperidine derivatives and their inclusion complexes.Because of that, we conducted a pharmacological study of KFCD-7 and compared its acute toxicity, infiltration, and conduction anesthesia with the data for previously obtained piperidine derivatives and reference drugs.

Pharmacological Study 2.3.1. Infiltration Anesthesia
The test was performed using the Bulbring-Wade method.All the compounds were tested as 0.5% aqua solutions.The results are summarized in Table 11.
As we can see from Table 11, all the drugs have an anesthetic effect that exceeds both novocaine and lidocaine.KFCD-7 shows a slightly longer duration of complete anesthesia than lidocaine, higher than trimecaine in terms of the anesthesia index (35.4± 1.3) and duration of complete anesthesia, but less in total duration of anesthesia.The other piperidine derivatives revealed the best values for all parameters of the infiltration anesthesia.
The only exception is kazcaine which has a duration of complete anesthesia comparable to lidocaine but a higher total duration of anesthesia.The formation of an inclusion complex significantly (two times) increases the duration of complete anesthesia up to the KFCD-6 value.
Overall (Figure 5), the kazcaine:β-CD inclusion complex is comparable to BVBP, while KFCD-6 has a slightly shorter duration of complete anesthesia.KFCD-6 and KFCD-4 are better than procaine by 5.9 and 4.8 times, lidocaine by 2.3 and 1.9 times, and trimecaine by 2.0 and 1.6 times, respectively.They have a longer total duration of effect than trimecaine by approximately 2 and 1.7 times, lidocaine by 2.3 and 1.3 times, and procaine by 4.0 and 3.3 times, respectively (statistically significant at p < 0.05).
Molecules 2024, 29, x FOR PEER REVIEW 17 of 24 Figure 5.The comparison of local anesthetic activity of the compounds and reference drugs for the infiltration anesthesia, using the Bulbring-Wade method (concentration 0.5%).

Conduction Anesthesia
A modified "tail flick" method was used in the study of conduction anesthesia [36].It was developed at the Department of Pharmacology of the St. Petersburg Medical University, named after Academician I.P. Pavlov.The principle of the method is to determine the latent period of tail withdrawal during the thermal exposure of its middle part with a The duration of complete anesthesia (min.), (М±m) Total duration of the effect (min.), (М±m) . The comparison of local anesthetic activity of the compounds and reference drugs for the infiltration anesthesia, using the Bulbring-Wade method (concentration 0.5%).

Conduction Anesthesia
A modified "tail flick" method was used in the study of conduction anesthesia [36].It was developed at the Department of Pharmacology of the St. Petersburg Medical University, named after Academician I.P. Pavlov.The principle of the method is to determine the latent period of tail withdrawal during the thermal exposure of its middle part with a focused beam of light from an optoelectronic analgesimeter TF-003 before and after anesthesia.The intensity of the thermal nociceptive stimulus is adjusted so that initial tail flick responses occur with a latency ranging from 3 to 6 s.
The activity of compounds and reference drugs for the conduction anesthesia wasstudied in 1% solutions.The following parameters were determined: the rate of onset of anesthesia, the duration of the complete anesthesia, and the total duration of effect.
The results are shown in Table 12.A comparison of the duration of the complete anesthesia and the total duration of effect is shown in Figure 6a,b.a Deviations in relation to reference preparations are statistically authentic at p < 0.001.b Deviations in relation to kazcaine are statistically authentic at p < 0.01.c Deviations in relation to reference preparations are statistically authentic at: lidocaine-p i < 0.05, trimecaine-p i < 0.001, procaine-p i < 0.02.d rate of anesthesia induction-3 min.e Local anesthetic activity for the conduction anesthesia, using the method of electrical stimulation of a rabbit inferior dental nerve.f Local anesthetic activity for the conduction anesthesia, using a modified "tail flick" method.
As can be seen from Table 12, all the complexes have an apparent local anesthetic effect, and the rate of anesthesia induction is comparable in all cases.
As for the other drugs under consideration, the best result of conduction anesthesia at a 1% solution was exhibited by BVBP, almost three times longer than its complex with CD (KFCD-4); that is, the same picture was observed as for infiltration anesthesia.
Kazcaine initially had a good activity (three times better than procaine), and its complex with CD improved the duration of complete anesthesia and the total duration of a local anesthetic effect (but not so dramatically, approximately 30%).
The results for BBB-HCl look interesting.Similar to the infiltration anesthesia, the formation of the complex did not provide an increase in the activity for a 0.5% concentration.However, what is unexpected is that for the 1% concentration, the duration of complete anesthesia was shorter, but the total duration of anesthesia was longer than for the 0.5% concentration.This may probably be due to the different measurement methods used for the 1% solution.

Acute Toxicity
Behavior changes, reflector breath excitability, rate of development and mitigation of external poisoning symptoms, and mortality (LD50) were registered (Figure 7).
The toxicity of kazcaine (which is slightly less than procaine) remained virtually unchanged upon the formation of the inclusion complex, becoming comparable to KFCD-7.* Deviations for this complex compared to the reference preparations are statistically authentic at pi < 0.001.** Deviations for the KFCD-6 and KFCD-7 compared to the reference preparations are statistically authentic at p1 < 0.01; p2 and p3 < 0.005.

Terminal Anesthesia
The comparison of the activity of the tested compounds with the reference anesthetic, dicaine, was carried out using the Rainier indices, duration of the complete anesthesia, and total duration of effect.
All the studied compounds were tested in 1% and 3% solutions.The experimental results showed that the compounds KFCD-4, KFCD-6, and KFCD-7 in all tested concentrations were significantly inferior both in strength (the Ragnier index) and in the duration of the local anesthetic effect to dicaine and in all concentrations they did not show irritating effects.
At the same time, the formation of inclusion complexes does not always lead to higher activity and depends both on the characteristics of the "guest" and on the type of anesthesia.The most effective in this sense was the inclusion complex of cyclodextrin with The toxic reactions were of the same character for KFCD-4, KFCD-6, and KFCD-7.The higher the dose, the faster poisoning was evident.The phenomena of intoxication began to develop after 20-30 min.The initial stage started with general oppression and resulted in a deferred response, absence of reflex to exogenous irritants, and dyspnea, which later developed into a short period of motional excitation, followed by muscular twitching and clonic-tonic spasms.Mice assumed a lateral position and their breathing became slower and irregular.Death was caused by primary respiratory standstill 30-90 min after injection.The surviving mice recovered from stagnation in 2-2.5 h and were as active as the untreated mice by the end of the first day.
An analysis of the data obtained for the entire group of the drugs under consideration (Table 13) showed that the formation of the inclusion complexes significantly decreases the acute toxicity of substances.The resulting inclusion complexes of piperidine derivatives with β-CD were significantly less toxic than the guests themselves.
The KFCD-6 compound turned out to be the most active and less toxic than procaine by 1.7 times, lidocaine by 3.3 times, and trimecaine by 2.2 times in all experiments (Table 13).KFCD-7 was less toxic than the reference anesthetics.
The most toxic was BBB-HCl, but in the form of an inclusion complex, its toxicity dropped by more than three times and became comparable to procaine.The formation of an inclusion complex reduced the toxicity of AEPP by 2.4 times and BVBP by 2.2 times.The toxicity of kazcaine (which is slightly less than procaine) remained virtually unchanged upon the formation of the inclusion complex, becoming comparable to KFCD-7.* Deviations for this complex compared to the reference preparations are statistically authentic at p i < 0.001.** Deviations for the KFCD-6 and KFCD-7 compared to the reference preparations are statistically authentic at p 1 < 0.01; p 2 and p 3 < 0.005.

Terminal Anesthesia
The comparison of the activity of the tested compounds with the reference anesthetic, dicaine, was carried out using the Rainier indices, duration of the complete anesthesia, and total duration of effect.
All the studied compounds were tested in 1% and 3% solutions.The experimental results showed that the compounds KFCD-4, KFCD-6, and KFCD-7 in all tested concentrations were significantly inferior both in strength (the Ragnier index) and in the duration of the local anesthetic effect to dicaine and in all concentrations they did not show irritating effects.
At the same time, the formation of inclusion complexes does not always lead to higher activity and depends both on the characteristics of the "guest" and on the type of anesthesia.The most effective in this sense was the inclusion complex of cyclodextrin with 1-(2-ethoxyethyl)-4-ethynyl-4-benzoyloxypiperidine, which is two times better for infiltration anesthesia and 30% better for conduction anesthesia than its salt form (1-(hydrochloride 2-ethoxyethyl)-4-ethynyl-4-benzoyloxypiperidine).
According to the literature, the extension of the alkyl chain at the N atom of the piperidine derivative to the ethoxyethyl substituent leads to the anesthesia index exceeding trimecaine by 1.5 times, lidocaine by 5.1, procaine by 5.3 times, including piperidine derivatives with butoxypropyl substituent.The EC50 value for conduction anesthesia of 1-(3-n-butoxypropyl)-4-benzoyloxypiperidine hydrochloride exceeds the ethoxyethyl homologue by 140 times, and the reference drugs pyromecaine, trimecaine, and procaine by 270, 446, and 670 times, respectively [38].
This pattern is also confirmed by good results for BVBP and BBB-HCl.Elongation of the radical at the nitrogen atom of the piperidine ring from ethoxyethyl to butoxypropyl led to a significant increase in activity during infiltration, especially during the conduction of anesthesia.However, these same drugs have the highest toxicity among those considered.The formation of inclusion complexes leads to a significant reduction in toxicity (comparable to trimecaine) but, at the same time, to a significant reduction in the anesthesia time.
The infiltration anesthesia test was performed with the Bulbring-Wade method [41].The studies were conducted on male guinea pigs with average masses of 200-250 g.The samples of isotonic solutions of the studied compounds and reference drugs were injected intradermally (0.2 mL) in the back of each animal at four points (vertices of the square with a side of 3 cm) after hair removal.The local anesthetic activity was evaluated six to eight times for each of the selected concentrations.Sensitivity at the injection site was determined by the touch of a blunt injection needle for a series of six touches every 5 min until full recovery.
The depth of anesthesia, expressed as the "anesthesia index" (average of 6 experiments, maximum index-36), the duration of complete anesthesia, and the total duration of the anesthetic effect were determined.The activity of the compounds was compared with the reference drugs, trimecaine, lidocaine, and novocaine, in corresponding concentrations.
The study of conduction anesthesia was carried out using a modified "tail flick" method in rats [36].It allows one to determine the speed of onset of anesthesia, its depth, the duration of the complete anesthesia, and the total duration of the anesthetic effect of the drug.The study was carried out on outbred white male rats weighing 200-250 g.To study the conduction anesthesia, a solution of a compound or drug (0.5 mL) was injected under the skin of the tail into the area where the thermal effect was applied.The animals in the control group were injected with a saline solution in the same way and same volume.Irritation was applied 1 cm distal from the injection.The first test was carried out 5 min after injection; subsequent tests were carried out every 10 min until the threshold values were completely restored.Doubling of the latent period was taken as complete anesthesia.
Acute toxicity was determined after a single subcutaneous injection of the studied compound and reference drugs in mice (6-8 outbreed albino mice weighing 17.0-22.0g).
The symptoms of poisoning, speed of onset, severity of regression, and mortality rate were recorded.The animals that survived the first 24 h were monitored in terms of their behavior and full recovery of appetite.The lethal dose (LD50) was calculated using the Miller and Tainter method [42].
All the data obtained were statistically treated.

Conclusions
The analysis of the data obtained for the entire group of drugs under consideration shows that the formation of inclusion complexes significantly decreases the acute toxicity of substances.
Based on the results obtained, we can conclude that the inclusion complexes of piperidine derivatives under study are low-toxic local anesthetics, for which further research and development as pharmaceuticals are advisable.Of these, the inclusion complexes of kazcaine and AEPP can be considered the most promising.Moreover, recently obtained fluorine derivatives of kazcaine have shown unexpected antimicrobial activity [43,44].
The pharmacological study results determined that, in terms of local anesthetic activity and acute toxicity, KFCD-7 exceeded all the drugs in comparison but is inferior to all other considered inclusion complexes of piperidine derivatives.The predicted biological activity confirmed the results of the pharmacological study and has shown that both MEP and its complex KFCD-7 are promising molecules for further studies of anticonvulsant effects and effects on reproductive functions.

Figure 2 .
Figure 2. The bioavailability radar of the studied compounds based on the physicochemical indices ideal for oral bioavailability.Figure 2. The bioavailability radar of the studied compounds based on the physicochemical indices ideal for oral bioavailability.

Figure 2 .
Figure 2. The bioavailability radar of the studied compounds based on the physicochemical indices ideal for oral bioavailability.Figure 2. The bioavailability radar of the studied compounds based on the physicochemical indices ideal for oral bioavailability.

Figure 3 .
Figure 3.The probability of adverse effects for the compounds under study.

Figure 3 .
Figure 3.The probability of adverse effects for the compounds under study.

Figure 6 .
Figure 6.(a) The comparison of the duration of the complete anesthesia and the total duration of effect for the conduction anesthesia (0.5%).(b) The comparison of the duration of the complete anesthesia and the total duration of effect for the conduction anesthesia (1.0%).

Figure 6 .
Figure 6.(a) The comparison of the duration of the complete anesthesia and the total duration of effect for the conduction anesthesia (0.5%).(b) The comparison of the duration of the complete anesthesia and the total duration of effect for the conduction anesthesia (1.0%).

Figure 7 .
Figure 7.The acute toxicity of the compounds under study and the reference drugs.

Figure 7 .
Figure 7.The acute toxicity of the compounds under study and the reference drugs.

Table 1 .
The compounds used in the present work. 3

Table 2 .
Physical and chemical parameters of the studied compounds.

Table 3 .
The bioavailability criteria for the compounds under study.

Table 5 .
A summary of the predicted biological effects for the studied compounds.

Table 6 .
The summary of the most probable macromolecular targets for the studied compounds study (SwissTargetPrediction).

Table 7 .
The summary of the most probable macromolecular targets for the compounds under study (PASS Targets).

Table 8 .
The results of the predicted acute toxicity for the studied compounds.

Table 9 .
The prognosis of adverse effects for the compounds under study.
* Pa-probability of activity; Pi-probability of inactivity.**only as a base.*IP-intraperitoneal route of administration, IV-intravenous route of administration, Oral-oral route of administration, and SC-subcutaneous route of administration.** BOLD = in AD: the compound falls within the range of applicability of the models; italic = out of AD: compound outside the range of applicability of models.

Table 9 .
The prognosis of adverse effects for the compounds under study.

Table 10 .
The studied compounds and their host-guest complexes with β-CD.

Table 12 .
The local anesthetic activity of compounds and reference drugs for the conduction anesthesia.

Table 13 .
The acute toxicity of the compounds under study and the reference drugs.

Table 13 .
The acute toxicity of the compounds under study and the reference drugs.