Pyridine Carboxamides Based on Sulfobetaines: Design, Reactivity, and Biological Activity

The synthesis of the products of the 1,3-propanesultone ring opening during its interaction with amides of pyridinecarboxylic acids has been carried out. The dependence of the yield of the reaction products on the position (ortho-, meta-, para-) of the substituent in the heteroaromatic fragment and temperature condition was revealed. In contrast to the meta- and para-substituted substrates, the reaction involving ortho-derivatives at the boiling point of methanol unexpectedly led to the formation of a salt. On the basis of spectroscopic, X-Ray, and quantum-chemical calculation data, a model of the transition-state, as well as a mechanism for this alkylation reaction of pyridine carboxamides with sultone were proposed in order to explain the higher yields obtained with the nicotinamide and its N-methyl analog compared to ortho or meta parents. Based on the analysis of ESP maps, the positions of the binding sites of reagents with a potential complexing agent in space were determined. The in silico evaluation of possible biological activity showed that the synthetized compounds revealed some promising pharmacological effects and low acute toxicity.


Introduction
For many years, our group has focused on discovering and developing new heterocyclic compounds that have nootropic, anti-inflammatory, and antidepressant activities. Our main goal is to develop effective medications for treating cognitive impairments, including ischemic stroke and its complications [1][2][3][4]. The recent COVID-19 pandemic highlighted the emerging need for these kinds of therapeutic agents. Moreover, the greatest danger to the patient's mental health is not the disease itself, but the consequences it causes. Currently available medications have not been able to deal with emerging post-COVID-19 disorders alone. One of the reasons is insufficient bioavailability and multiple side effects. Previously, we managed to increase the bioavailability of Phenotropil by introducing chemical modification, which led to the production of a water-soluble form based on taurine [1]. The same approach was shown for homotaurine [3].
In continuation, in this work we studied the synthetic possibilities of the propansultone ring opening reaction, leading to the formation of sulfobetaines as end products.
The reaction comes from the opening of the sultone ring, as shown in Scheme 1.

Scheme 1. Representative synthetic routes for compounds 8-14.
(The decoding of substituents R 1,2 is presented in Table 1; see below). In contrast to the sultone ring formation (which could be considered as well described [24]), the chemistry of the sultone ring opening remains unclear. In the current study, we aim to study the subtle features of the reaction presented in Scheme 1, including the influence of the structure of the substrate and temperature on its mechanism. We further aim to evaluate the possible anti-inflammatory activity of the synthesized compounds.
The aim of this study was to study and characterize the compounds presented in Table 1.

Results and Discussion
The reaction yields of sultone alkylation of pyridine carboxamides are presented below (Table 1).
Yield analysis revealed that, when an amide group (substrates 3-7) is in a metaor para-position, it increases reaction yield, while in ortho positions (substrates 1, 2) reaction yield decreases.
To further investigate this phenomenon, we combined spectroscopic, X-Ray, and quantum-chemical calculation data.

Ortho-Pyridine Carboxamides
We have collected IR-spectra of the starting compounds ( Figure 1). Figure 1. IR-spectra of compounds 1 (ortho), 2 (ortho) and 3 (meta), and 6 (para) pyridine carboxamides. The inset shows the zoomed 1800-1400 region. Note that the amide-II peak of ortho derivative 1 and para derivative 6 are overlaid with two peaks of C=C bending.
There is a notable difference in the position of the amide-II peak (NH bending) of orthoand para-/meta-derivatives. The first is being right-shifted. Moreover, the shift of the amide-II peak of compound 2 is bigger. The shift of the NH bending peak is due to the formation of the hydrogen bond. The last is stronger in the case of compound 2 than in the case of compound 1.
To understand the structure and the dynamic behavior, we investigated compounds 1 and 2 using 2D NMR approaches. Implementation of HSQMBC techniques allowed measurement of the vicinal 3 J HC couplings. Namely, in compound 1 the amide proton at 7.61 ppm is in the trans-position to the carbon of the pyridine ring (149.4 ppm), with 3 J HC equal to 8 Hz, while the 3 J HC of the second amide proton at 8.10 ppm is too small to give rise to a detectable cross-peak in HSQMBC. The amide proton in compound 2 (8.07 ppm) is in cis-position to the carbon of the aromatic ring, because no cross-peak is observed in HSQMBC, while the 2 J-coupling to the carbonyl (~7 Hz) is measurable ( Figure 2). The NH 2 -group protons of compound 1 experience the slow flips-their signals become broader upon heating and exchange cross-peaks are observed in both NOESY and ROESY spectra, which follows from the identical sign of cross-and diagonal peaks. We assessed the rate of such flips using the equations of ZZ-exchange spectroscopy and found that k = 0.21 ± 0.01 s −1 .
For the case of compound 2, only the single NH and N-methyl group signals are observed, which implies that amide bond flips either do not take place or the population of the cis-conformation is lower than the detection limit.
Estimated as a result of quantum-chemical calculations using the M052X-D3/TZVP (methanol PCM, implicit) method, the N-H hydrogen bond breaking energy was 15.3 kJ mol −1 .
We conclude that steric hindrance of the pyridine nitrogen atom with the amide hydrogen atom of the carbamoyl group is the second point (along with low ESP) that decreases the yield of the reaction.

Metaand Para-Pyridine Carboxamides
All para-derivatives (compounds 6, 7) and meta-diethylamide pyridine (compound 5) show moderate yields in sultone alkylation (products 13, 14), while meta-amide (compound 3) and meta-methylamide (compound 4) demonstrate sufficiently higher yields (products 10, 11) (Table 1). Because metaand para-derivatives in contrast to ortho-derivatives have no geometrical constraints, it could be suggested that there is some additional activation during the path of the reaction. To find out what it is, we have performed continuous monitoring of the reaction using IR spectroscopy. It appears that, in the most high-yield reaction (namely reaction of compounds 3, 4, Table 1), the carbonyl group is involved. The C=O stretching band of compound 4 during the reaction shifts from 1640 to 1670 cm −1 (Figure 3). In the case of compound 3, along with the shifting of C=O stretching from 1675 cm −1 to 1690 cm −1 , an additional band of 1645 cm −1 arises ( Figure 3). The last may point to two different stable conformers of the product. In the case of compound 5, the product formation does not affect the C=O stretching band (Figure 3). The reason for this is the absence of the proton in the amide group in this derivative.
The hydrogen bonding of the amide group could be of intermolecular or intramolecular origin. From X-Ray data (see above) it follows that, in the crystal state, the sulfo group forms an intermolecular hydrogen bond with the amide group. To check if the observable band shift is caused by the internal or external hydrogen bonding, we have collected IR spectra of the two products in different dilutions ( Figure 4). One idea is that, upon dilution, intermolecular hydrogen bonds should disappear while intramolecular hydrogen bonds should remain intact. It appears that the IR spectra of compounds 10 and 11 remain intact upon dilution. Amid bands do not shift. Thus, we conclude that hydrogen bonds between the sulfo and amide groups are internal (intramolecular) and propose the following mechanism of reaction activation in the case of meta-pyridine carboxamides with the NH group (Scheme 2). The reaction starts from the attack of sultone on the N atom. Once the intermediate is formed, the hydrogen bond appears between the sultone in-ring oxygen atom and an amide hydrogen atom. The hydrogen bond is assumed to either decrease the energy barrier of the sultone ring opening or to stabilize the transition state. This is the reason for the increased reaction yield in the case of meta-pyridine carboxamides.

Investigations under High Temperature
The study of the effect of temperature on the yield of the target product (Scheme 1) was carried out in boiling methanol for 4 h [28,29]. In the case of substrates with meta-and para-orientation of the amide fragment (compounds 3, 6), the yield of the final product (compounds 10, 13) practically did not change (see Experimental part).
At the same time, for substrates with ortho-orientation of the amide fragment (compounds 1, 2), a similar reaction unexpectedly led to the formation of salt (compounds 15, 16) (Scheme 3). Compound 15 was also synthesized in methanol-d 4 . The signal (s) at 3.23 ppm, which corresponds to the CH 3 O-CH 2 group in the CH 3 O(CH 2 ) 3 SO 3 − anion, disappears in the 1 H NMR spectrum. The degree of deuteration of the OCD 3 group is > 98%. As a result of the exchange processes with the solvent, mobile acidic protons SO 3 H(D) and C(O)NH 2 (D 2 ) are partially replaced by deuterium (deuteration degree~50%) (see Supplementary Materials, Figure S2).
The data of quantum chemical calculations (M052X-D3/TZVP) confirm the proposed scheme. Indeed, for compound 1, the conformation in which the oxygen atoms of the amide group and the nitrogen atoms in the pyridine ring are located in the same plane turned out to be preferable due to stabilization by an intramolecular hydrogen bond (Scheme 3). When heated, the breaking of the hydrogen bond (structure A) is accompanied by protonation with the formation of a cation (structure B), which is characterized by a conformation in which the oxygen and nitrogen atoms are located opposite each other. For compounds 3 and 6, the position of the substituent affects the energy characteristics of the cations to a lesser extent (see SM). In addition, attention is drawn to the increase in the values of charges on the nitrogen atom in the pyridine ring in the series of compounds 1, 3, and 6 in the following sequence: −0.21; −0.15; −0.14. In other words, the nitrogen atom in the pyridine ring of ortho-derivatives more readily accepts the H + cation compared to compounds 3 and 6.

X-ray Investigation
The structures of compounds 9-14 were studied by single crystal X-ray diffraction ( Figure 5A).
The values of bond length and angles of the abovementioned compounds are very close to organic compounds with similar fragments [30]. The substituted pyridine ring and amide CONR 1 R 2 moieties in molecules 10, 11, and 13 are coplanar. In the rest of the compounds, carbonyl atoms are deviated from the plane of the pyridine ring (the angle of these moieties are equal to 45.7, 68.3, and 17.8 • for 9, 12, and 14, respectively). The presence of amino groups leads to the formation of 1D H-bonded motifs in crystal packing ( Figure 5B). At the same time, the crystal packing of 11 molecules form centrosymmetric dimers via N-H . . . O bonds.

Electrostatic Potential Analysis
The ring opening of 1,3-propanesultone during its reaction with amides could proceed under charge control [31,32]. Analysis of electrostatic potential (ESP) maps makes it possible to find the position of the binding sites of reagents with a potential complexing agent in space. Below are the ESP maps of compounds 1-7 and sultone in the gas phase ( Figure 6). The blue spheres show the ESP extrema corresponding to the regions with electron donor properties. Naturally, the largest extremum corresponds to the region located near the oxygen atom of the carbonyl group. However, the most interesting is the region near the nitrogen atom in the pyridine ring of the substrates. Changing the position of the ring substituent from the parato the metaand ortho-positions leads to a reduction in potential dropping from −60.1 kJ·mol −1 (compound 1, Figure 6) to −138.7 kJ·mol −1 for the meta-position (compound 3) and −147.0 kJ·mol −1 for para-substitution (compound 6). The introduction of a methyl group into the amide fragment of compounds 1, 3, and 6 is accompanied by a decrease in the ESP values at the nitrogen atom (compounds 2, 4, 7, Figure 6). The presence of two ethyl fragments in the amide substituent in the metaposition (compound 5) leads to a decrease in the ESP value to −176.8 kJ·mol −1 . ESP extrema corresponding to regions exhibiting pronounced electron-withdrawing properties are marked with red spheres in sultone and pyridine substrates 1-7. The regions located near the carbon atom of the sultone are capable of the nucleophilic ring opening of 1,3propanesultone by substrates 1-7. The extremum regions on the ESP maps of the reagent and substrates can be considered as reaction centers, and the carbon atom located next to the sulfur atom in 1,3-propanesultone can be considered as an electron acceptor in this reaction ( Figure 6). Compound 1 has some additional peculiarities, in contrast to other substrates; instead of one local minimum at the reaction center (nitrogen atom), two symmetric points are observed equidistant from the plane of the ring. This may indicate the presence of steric hindrances in nucleophilic addition reactions. A change in the position of substituents in the aromatic fragment of the substrates 1-7 affects the electron-donating properties of the reaction center. Indeed, a satisfactory correlation is observed between the charge on the nitrogen atom and the reaction yields. Thus, compounds 3-5 (meta-substitution), as well as 6, 7 (para-substitution) are characterized by the highest charges and yields of reaction.

Transition States Search
The search for transition states was carried out in two steps: relaxed scanning along the reaction coordinate to find the maximum point (transition state) on the potential energy surface (PES) projection and the subsequent standard procedure for localizing the stationary maximum point. As a result, the transition states of reactions of nucleophilic ring opening of 1,3-propanesultone in the gas phase and in the presence of a solvent were obtained. Figure 7 shows the structure of the transition state of the considered reaction with the participation of substrate 1, with the formation of compound 8, the geometric parameters of which are optimized in methanol. The structures of transition states with the participation of other substrates (Table 1) have a similar structure (see Supplementary Materials, Figure S1).
As can be seen from the example of the transition state in reaction for substrates 1-8 (Figure 7), the 1,3-propanesultone ring opens at the C-O bond, followed by coordination through the carbon atom to the nitrogen atom of the pyridine fragment at a distance of slightly more than 2 Å. Note that the reaction center of the reagent (1,3-propanesultone), based on the analysis of the ESP map, has pronounced electron-withdrawing properties ( Figure 6).

Thermodynamic and Activation Parameters
Optimization of geometric parameters of reagents, products, and transition states of nucleophilic ring-opening reactions of 1,3-propanesultone with amides of pyridine derivatives made it possible to calculate the total energies of the systems under study and evaluate the thermodynamic and activation parameters of the reactions ( Table 2).
As can be seen from Table 2, in all cases the reaction under study has a pronounced exothermic character. However, the Gibb's energy of reactions 1 → 8 and 2 → 9 is, on average, four times higher than for other reactions. The highest activation barrier is also characteristic of 1,3-propanesultone ring opening reactions with substrates 1 and 2. Note that the nitrogen atom in the pyridine ring of compounds 1 and 2 is characterized by the lowest electron-donor properties compared to compounds 3-7, according to ESP maps ( Figure 6). These results are consistent with the lowest yields obtained with compounds 8 and 9 (Table 1). Table 2. Thermodynamic and activation parameters of 1,3-propanesulton cycle opening reactions by amides. Figure 2 Product Gas Phase, T = 298. 15 Methanol, T = 298.15 For the rest of the reactions, the values of the thermodynamic and activation parameters are generally comparable. The lower values of the Gibb's free energy of reactions and low activation barriers for the formation of products 10-14 in comparison with reactions 1 → 8 and 2 → 9 allow us to conclude that the rate of formation of more stable products of the reaction under consideration is higher. A lower yield of the product reaction 5 → 12 in comparison with the expected one (Tables 1 and 2) can be caused by the influence of steric factors. The results obtained by MD simulation that do not contradict this assumption are presented in SM ( Figure S3).

ESP (N), kJ/mol See
Interesting patterns were found when comparing the dependence of the product yield on the thermodynamic parameters ( Figure 8). It appeared that the reaction of sultone for meta-derivatives differs from the reaction with paraand ortho-derivatives. While increasing ESP increases reaction yield in the case of paraand ortho-derivatives, it decreases yield in the case of meta-derivatives. At the same time, Gibb's energy could not be used to assign high reaction yields to the meta-derivatives, because para-derivatives have lower Gibb's energy but their reaction yields are also lower.
Thus, the minimum reaction yield obtained for ortho-derivatives could be assigned to the lowest ESP on the nitrogen atom of the starting compound. Meta-derivatives have increased reaction yield, and the last is hard to assign to higher ESP or decreased Gibb's energy. To evaluate the question further, we have performed spectroscopic investigations.

Biological Activity 2.4.1. In Silico Evaluation of Possible Biological Activity
The study of possible pharmacological effects was made by the PASS Online web application [33][34][35]. The PASS (Prediction of Activity Spectra for Substances) score is based on structure-activity analysis of a training set that includes structures of tested compounds and data on more than 4000 known therapeutic effects and molecular mechanisms of action. Biological activity is presented in a qualitative way (active/inactive). The chemical structure is described in the form of MNA descriptors (Multilevel Neighborhoods of Atoms-multiple atomic neighborhoods) [34]. The algorithm for creating classification "structure-activity" relationship models is based on the structures of substances of the training set and Bayesian-like estimates [33][34][35]. The average prediction accuracy calculated by a leave-one-out cross-validation procedure for all compounds in the training set and all types of biological activity presented is approximately 95%. The analysis of prediction results revealed the most frequent and probable pharmacological activities for compounds 8-14. They are shown in Table 3. IDs-identifiers of compounds; Pa-probability to be active for appropriate activity; Pi-probability to be inactive for appropriate activity. Table 3 shows that the cerebral anti ischemic effect was predicted with high probability for all compounds. Antianginal activity was also predicted for all compounds with medium likelihood. The effects related to phobic disorder treatment were predicted with high probability for compound 14 and medium probability for compounds 8, 10, 11, and 13. Several other pharmacotherapeutic effects, including some types of antineoplastic activity, were predicted for some compounds with medium probability.
CLC-Pred web service (Cell Line Cytotoxicity Predictor, http://way2drug.com/Cellline, accessed on 25 September 2022) was used to evaluate the possible cytotoxicity of compounds in tumor and normal cell lines [36]. CLC-Pred is based on the technology and methods used in PASS Online and predicts cytotoxicity against 278 tumor and 27 normal cell lines, with an average accuracy of 95% calculated by a leave-one-out cross-validation procedure. The training set for CLC-Pred was created based on data from the ChEMBL database. The results of prediction showed that cytotoxicity for normal cell lines was not predicted for the synthesized compounds. The predicted safety indicates the further possibility to use these compounds in drug development. For some cell lines, the toxicity was predicted as moderate (Table 4). To determine the potential acute toxicity of the synthesized compounds, the LD 50 values for rats with different routes of administration were predicted using the GUSAR Online acute toxicity web service [37]. It uses four QSAR models for prediction, which correspond to the route of administration of the substance (oral, intravenous, intraperitoneal, and subcutaneous). The models were based on the data from the RTECS database, and their accuracy is sufficient to be used in these types of assessments. Based on LD 50 values (mg/kg), a toxicity class from 1 to 5 is determined, where class 1 is a highly toxic compound and class 5 is non-toxic. Table 4 shows that all compounds were predicted as non-toxic (class 5) or slightly toxic (class 4).

In Vitro Studies
Sulfobetaines are used as antifouling and antimicrobial materials, as a drug delivery system, and much more [38][39][40][41][42]. Synthesized compounds 8-13 were tested for the ability to influence the viability of four cell lines using the MTT assay (Table 5). Based on MTT assay, all tested compounds have low cytotoxicity against the tested cell lines. We further tested the ability of these substances to exhibit anti-inflammatory activity. The study was carried out on the RAW 264.7 macrophage cell line. Macrophages play a critical role in the development, progression, and resolution of many inflammatory responses. By secreting various cytokines, these cells can direct the immune response towards a more or less pro-inflammatory profile. In our experiment, we treated the RAW 264.7 cell line with lipopolysaccharide (LPS) and measured the levels of expression of interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) genes involved in inflammation. COX2 and iNOS were chosen as they are the main inflammatory mediators. IL-1β is one of the main pro-inflammatory cytokines. Figure 9 shows real-time PCR data on gene expression for compound 12 (Gene expression results for all compounds are presented in the Supplementary Material (SM) Inflammatory Gene Expression). According to our results of the LPS-induced RAW 264.7 cell inflammation model, the tested compounds had multidirectional effects (see Supplementary Materials Figure S5). However, compound 12 had a pronounced anti-inflammatory potential, so with respect to the COX2 gene, iNOS and IL-1b, it caused a dose-dependent inhibitory effect.

General
The purities of compounds were assessed by NMR to be ≥95%. 1 H, 2 H, 13  Determinations of the structures of the obtained compounds and assignments of 1 H, 2 H, 13 C, and 15 N signals were made with the aid of 2D COSY, NOESY, DOSY-LED, edited-HSQC, HMBC, 1 H-15 N HSQC, and 1 H-15 N HMBC spectra. Spin multiplicities were described as s (singlet), d (doublet), t (triplet), or q (quartet).
IR spectra in the solid state were recorded on a Bruker Tensor-27 (Bruker Corporation, Bremen, Germany) spectrometer with the attenuated total internal reflectance (ATR) module. Refraction parameters were measured with an IRF-454B2M refractometer (KOMZ, Kazan, Russia). Melting points were determined on a Stuart SMP10 instrument (Barloworld Scientific Ltd., Stone, UK). Elemental analyses were carried out on a ELEMENTAR vario MI-CRO cube Microanalyzer (Abacus Analytical Systems GmbH, Babenhausen, Germany). All initial reagents and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).
High-resolution mass spectra were obtained on a Bruker maXis Q-TOF instrument (Bruker Daltonik GmbH, Bremen, Germany) equipped with an electrospray ionization (ESI) ion source. The spectra were processed using Bruker Data Analysis 4.0 software.

Dynamic NMR
Samples 1 and 2 were dissolved in CDCl 3 and placed into the 5 mm NMR tube. To narrow down the signals of amide protons, the temperature was set at 0 • C for 1 and 10 • C for 2. The structure of the compounds was studied by heteronuclear J-couplings and NOESY. J-couplings were measured using the pure in-phase HSQMBC pulse sequences, optimized for the 10 Hz couplings [43]. Two spectra-in-phase (IP) and anti-phase (AP) were recorded in the interleaved manner, and the J-coupling was measured as the peak distance in the sum (IP+AP) and difference (IP-AP) spectra. The rate of the NH 2 flips was assessed at 30 • C from the exchange cross-peak intensities in 300 ms NOESY spectra, assuming that the equilibrium constant equals unity and equal on-and off-rate constants. Under these conditions, the ratio of cross and diagonal peak intensities would have the following form: where k ex = k 1 +k −1 = 2k, and t equals the mixing time in the NOESY experiment [44].

Synthesis
The initial amides of pyridine carboxylic acids 1-7 were used as commercial (Sigma-Aldrich, Saint Louis, MO, USA) reagents without additional purification.
To 8.5 mmol of 1,3-propanesultone, a solution of 8.5 mmol of amide 1-7 was added with stirring (Scheme 1). The mixture was stirred at room temperature or was heated under reflux for 4 h. The next day, the solvent was evaporated, and the methanol or ethanol was added. The formed crystals were filtered off. Spin multiplicities were described as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet).

Calculation Details
All quantum-chemical calculations were carried out using GAUSSIAN 09 RevC software [45]. The popular hybrid functional (M052X [46]) with empirical dispersion [47] combined with the basis set TZVP [48] was used throughout the work for geometry optimization and frequency calculations. This calculation method was chosen by the results of comparable experimental and computational chemical shifts in the NMR spectra. In this case, the correlation index is more than 99% (see details in SM, 1 H and 13 C NMR chemical shifts).
Geometrical parameters of possible reagents and products were carried out. Estimation of the value of the activation barrier was carried out by searching for transition states. Hessian matrix was positively denoted as the total energy, then the stationary point attained a local minimum of the PES. If the Hessian had the only negative eigenvalue (imaginary frequency), then the stationary point corresponded to a saddle point (transition state, TS). The search for transient states was carried out in two stages: a relaxed scan by the reaction coordinate to find the maximum point (transition state) on the PES, followed by the standard procedure for localizing the stationary maximum point. The reaction products and the pre-reaction complex are predicted by the IRC procedure, for which 30 points on the PES in both directions were selected. Thermodynamic parameters were calculated in the gas-phase approximation at 298 K and also by simulating the experimental conditions: methanol as the solvent (polarized continuum model [49]) at 298 K and a pressure of 1 atm. Pre-reaction complexes of disclosure reactions sulton cycle were created based on analysis of ESP electrostatic potential maps that were calculated in the MultiWFN program version 3.8 [50]. ESP maps were rendered using VMD [51]. Chemical shifts were calculated using the Continuous Set of Gauge Transformations (CSGT) method [52][53][54].

X-ray Diffraction Studies
Single crystal X-ray studies of products 9, 12, and 14 were carried out in the Center for Molecule Composition Studies of A.N. Nesmyanov Institute of Elementoorganic Chemistry, Russian Academy of Sciences (Moscow; Russia) with Bruker APEX II diffractometer. X-ray datasets for products 10, 11, and 13 were collected in the Kurchatov Centre for Synchrotron Radiation and Nanotechnology using the 'Belok' beamline. All structures were solved with the ShelXT version 2014/4 [55] program and refined with the ShelXL [56] program. Molecular graphics were drawn using the OLEX2 version 1.3 [57] program.
CCDC 2125076-2125081 contains the supplementary crystallographic data for 9-14 (see Supplementary Materials, Table S2). These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures, accessed on 25 September 2022.

Cell Culture
RAW 264.7, a murine macrophage cell line, was kindly provided by laboratory of the mechanism of chemical carcinogenesis, N.N. Blokhina Russian Cancer Research Center, was grown in high-glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) in a humidified incubator flushed continuously at 37 • C with 5% CO 2 . The culture medium was refreshed every 3 days.

Cell Viability Assay
The cell viability was measured according to the cell ability of tetrazolium dye reduction. The Thiazolyl Blue Tetrazolium Bromide solution was added to the culture medium at a final concentration of 0.5 mg/mL, and the cells were left in darkness for 4 h at 37 • C. After that, the solution was removed, and formazan crystals were dissolved in dimethyl sulfoxide. The formazan absorption at 492 nm was measured using a Zenyth 1100 (Anthos Labtec Instruments, GmbH, Salzburg, Austria) microplate reader. The experiments were carried out in triplicate and repeated three times.

Quantitative Real-Time PCR Analysis
After overnight culture in a 6-well plate (25 × 104 cells/well, 2000 µL medium/well), the cells were treated with LPS (Escherichia coli L4130 0111:B4, Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 10 µg/mL and were incubated with testing compounds (0.001, 0.01, 0.1 and 0.5 mg/mL) for an additional 6 h. The anti-inflammatory effect of compounds was compared with the effect caused by LPS within 24 h. After 24 h of total incubation of cells with LPS and 6 h of incubation with compounds, the cells were scraped to analyze the expression of target genes.
Analysis of the level of gene expression was carried out using real-time reverse transcription-polymerase chain reaction (PCR).
Total RNA was isolated from cells using the RNA-Extran kit (Synthol, Moscow, Russia, Ex-515-50) following the manufacturer's protocol. The corresponding cDNA (cDNA) was obtained during a reverse transcription reaction using the OT-1 reagent kit (Synthol, Moscow, Russia) and a PTC (100)  The primers were selected using the Primer-BLAST (NCBI) datebase and are presented in Table 6. To determine the change in the expression level, the threshold cycle calculation method was used. Samples were normalized to the amount of mRNA of the Rpl27 gene. Table 6. Primer sequences used for gene amplification in the LPS-induced inflammation model.

Conclusions
The position of the substituent in the aromatic ring of pyridine carboxamides based on sulfobetaines affects not only the yield of the target product, but also the reaction mechanism. Following the results of quantum-chemical calculations, the interaction of the studied substrates with 1,3-propanesultone is subject to charge control. According to IR and NMR spectroscopy data, in substrates 1 and 2 an intramolecular hydrogen bond is formed between the hydrogen atom of the amide group in the ortho-position and the endocyclic nitrogen atom of the heteroaromatic fragment. As a result, the yield of the reaction product in the pre-reaction complex decreases to 20-40% as compared to the metaand para-substituted analogs (62-97%).
In addition to the charge factor, the high yield (89-97%) of meta-substituted derivatives 10 and 11 can be influenced by additional activation of the "substrate-1,3-propanesultone" transition state due to the formation of a hydrogen bond with the participation of the endocyclic oxygen atom of 1,3-propanesultone and a hydrogen atom of the amide group.
Steric factors or lack of internal hydrogen bond formation also influence the yield of the targeted sulfobetaines because, with the tertiary carboxamide analog of nicotinamide (substrate 5), global yield decreases from 10 to 24% compared to nicotinamide 3 or N-methyl analog 4, respectively.
The combination of experimental data, the thermodynamic parameters of the cations, and charge control explain the difference in the reactivity of ortho-substituted 1 and 2 at high temperature compared to compounds 3 and 6.
The results of in silico analysis and preliminary in vitro analysis showed low toxicity of these compounds and possible anti-inflammatory activity. Therefore, we may consider these compounds as potential therapeutic candidates for further research.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27217542/s1, Figure S1: The structures of transition states systems under study; Figure S2  Data Availability Statement: All spectra and XRD data are available from the authors.