Influence of the Type of Amino Acid on the Permeability and Properties of Ibuprofenates of Isopropyl Amino Acid Esters

Modifications of (RS)-2-[4-(2-methylpropyl)phenyl] propanoic acid with amino acid isopropyl esters were synthesised using different methods via a common intermediate. The main reaction was the esterification of the carboxyl group of amino acids with isopropanol and chlorination of the amino group of the amino acid, followed by an exchange or neutralisation reaction and protonation. All of the proposed methods were very efficient, and the compounds obtained have great potential to be more effective drugs with increased skin permeability compared with ibuprofen. In addition, it was shown how the introduction of a modification in the form of an ion pair affects the properties of the obtained compound.


Introduction
Currently, the most commonly used drugs for combating pain of various origin, fever, and inflammation are included in the nonsteroidal anti-inflammatory drugs (NSAIDs) group. It is estimated that 30-50 million people daily use pharmacological agents containing medicinal substances belonging to NSAIDs. This is attributed to their broad spectrum of activity, but above all, their availability. Most of the known preparations are available on the market without a prescription. NSAIDs are a large and diverse group of compounds in terms of chemical structure, showing an acidic nature. They include compounds that are medium-strength acids (pKa [3][4][5]. Most of this group of drugs are derivatives of carboxylic and enol acids with free functional groups of an acidic nature. Analgesic, antipyretic, and anti-inflammatory effects characterise NSAIDs. In connection with their mechanism of action and acidic character, they can cause a number of side effects, especially concerning the gastrointestinal tract, such as gastrointestinal bleeding, reduction in the volume of gastric juices, and the associated risk of ulceration, nausea, vomiting, diarrhoea, or side effects concerning the circulatory system, such blood clots and severe bronchospasm and attacks of asthmatic cough [1][2][3][4][5]. In order to avoid side effects, e.g., ulcerogenic effects related to the inhibition of PGI 2 biosynthesis, many preparations have been introduced in which the active group's acidic ones are obtained after enzymatic hydrolysis. After oral administration, drugs of this type pass through the stomach practically unchanged. These are prodrugs, esters (e.g., bonorylate), or other structures that allow the formation of an acidic moiety in the process of biotransformation (e.g., oxidation). There are new prodrugs with improved pharmacological activity and a lower risk of gastrointestinal side effects, such as ibuprofen amides formed by the reaction of the ibuprofen carboxyl group with heteroaromatic amines [6], ibuprofen ester derivatives obtained by the reaction of the ibuprofen carboxyl group with menthol, thymol and eugenol [7], or ibuprofen esters obtained as a result of the reaction of the ibuprofen carboxyl group with compounds such as quercetin, salicylic alcohol, or gallic acid [8].
Ibuprofen ((RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid) is one of the most widely used NSAIDs that is commonly used in the treatment of rheumatoid arthritis and osteoarthritis, as well as for relieving pain and fever [9]. Today, it is one of the most widely used over-the-counter (OTC) medications. The most common route of administration of this drug is the oral route (it is available as tablets, capsules, and paediatric suspensions). In the treatment of pain and fever, the drug should be effective as quickly as possible, which is directly related to achieving its appropriate concentration in the blood (therapeutic dose) [10]. The rate of ibuprofen uptake into the blood depends on the dissolution rate in the body fluids. Ibuprofen is a relatively high lipophilic carboxylic acid (determined log P in the range of 2.41-4.00), with low water solubility (0.076 g dm −3 at 25 • C) and low permeation through the skin [11]. The solubility of ibuprofen depends on the pH and is particularly low in the stomach's acidic environment, which requires high doses. This results in serious risk of side effects, i.e., damage to the gastric mucosa due to the acidic nature of the drug. In the pH range 1-4, the changes in ibuprofen solubility are small [12][13][14]. The increase in the solubility of ibuprofen starts from the moment of reaching the pKa. After it is reached, the solubility increases significantly, which is caused by the molecule's ionisation under these conditions [14,15]. For example, the solubility of ibuprofen at 37 • C is 0.038 mg·cm −3 (pH 1), 0.084 mg·cm −3 (pH 4.5), 0.685 mg·cm −3 (pH 5.5), and 3.37 at pH 6.8 it is mg·cm −3 [13].
The most straightforward modification of ibuprofen is the formation of its salt. In polar solvents, including water, the solubility increases significantly by replacing an acid proton with a sodium cation [16]. Potassium, calcium, magnesium, copper, and zinc salts are also known. This has a measurable impact on faster action, including more rapid pain relief [17][18][19][20].
Increasingly, the metal cation in acid-derived drugs is replaced with an organic cation due to limitations related to solubility, bioavailability, and the possibility of introducing metallic salts into finished drug forms. Lysine ibuprofenate, obtained by combining (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid and lysine in a molar ratio of 1:1.14 is also known [21]. This compound is part of the commercial preparation of Lizymax. This new ibuprofen derivative is absorbed much faster after oral administration and achieves the maximum plasma concentration 3 times faster than ibuprofen acid. It is also known from the publication by Furukawa et al. that ibuprofen salt, in which the cation is derived from L-proline, is obtained by the reaction of L-proline ethyl ester and ibuprofen [22].
There have been reports that the reduction in gastrointestinal side effects can be achieved by converting the active substance into a prodrug using amino acids that are non-toxic and that additionally have a therapeutic effect on gastric damage caused by NSAIDs. For example, amides formed in the reaction of proline and ibuprofen are known, which show antiinflammatory and neuroprotective effects while being well-tolerated by the gastric wall. In addition, L-cysteine ethyl ester ibuprofenate is known, which has a potent anti-inflammatory and antioxidant effect and reduced side effects on the gastrointestinal tract [23][24][25][26][27][28].
The topical application of drugs is one of the most important methods of delivering them to the body. A significant factor in limiting penetration is the skin barrier which reduces the penetration efficiency and limits the absorption of the compounds. This layer is the greatest obstacle to the transport of active substances and is considered the primary barrier to the permeation of molecules. It is mainly made of ceramides, cholesterol, fatty acids, cholesterol esters, and minor phospholipids. Among the available and topical medications, a significantly small group can passively cross the skin barrier in amounts sufficient to obtain a therapeutic effect. Ibuprofen is the active ingredient in many topical formulations. This route of administration is used to reduce undesirable side effects, avoid first-pass metabolism in the liver, and minimise gastrointestinal side effects [29]. However, it is difficult to obtain an effective concentration due to its poor penetration capacity through the stratum corneum (SC). It is related to the construction of the SC. SC consists mainly of lipids-namely, ceramides, cholesterol, and fatty acids [30].
In our previous work, we presented the greater permeation compared with pure IBU of the ion pairs of ibuprofen with L-valine alkyl esters [ValOR][IBU], in which the alkyl chain R was extended from C1 to C8 [11,31]. In the presented study, we decided to extend the modifications of IBU about other amino acids and then estimate the penetration of new compounds as well as the amount of their accumulation in the skin. Penetration in vitro was investigated using Franz diffusion cells using abdomen porcine skin. This work presents the synthesis, complete identification, and characterisation of new salts: amino acid isopropyl ester ibuprofenates [AAOR][IBU].

Results
Our previous work investigated the effect of the amino acid alkyl ester chain length on the properties of the obtained amino acid alkyl ester ibuprofenates. It has been shown that to obtain a compound with good skin permeability and good solubility in water and buffers solutions while having high biodegradability and medium lipophilicity, using an isopropyl chain is most advantageous [11,31]. As part of the research, 14 ibuprofenates of isopropyl esters of protein amino acids were obtained and tested. The studies used glycine, L-alanine, L-valine, L-isoleucine, L-leucine, L-serine, L-threonine, L-cysteine, L-methionine, L-aspartic acid, L-lysine, L-phenylalanine, and L-proline. Unfortunately, the remaining derivatives of protein amino acids could not be obtained using the presented methodology. Interestingly, the hydrochloride of the L-aspartic acid ester was obtained in the reaction of L-asparagine with isopropanol and a chlorinating agent.
As a result of the three-step reaction, the ibuprofenates of amino acid alkyl esters were obtained in high yields (85-99%). In the last step of the reaction, the amino acid isopropyl ester was combined with ibuprofen without using a solvent. The use of the solvent-free method is associated with the limitation of volatile organic compounds and the costs of the process itself. Furthermore, due to the last reaction step, no reaction by-products are formed, and the product is obtained with high purity.
All compounds obtained were identified by 1 H and 13 C NMR, FTIR, elemental analysis, and UV-Vis. The individual results are presented in the Supplementary Materials. In addition, a comparison of the 1 H NMR spectra for ibuprofen and all obtained ibuprofenates is shown in Figure 1. Significant from the point of view of confirming the ionic structure of the compounds obtained was to prove, among other things, protonation of the amino group of the amino acid. As is shown in Figure 2, the value of the NH 3 signals shifts ranges from 4.67 ppm (for the L-methionine derivative) to 9.13 ppm (for the L-proline derivative). The integration of these signals clearly confirmed the obtained ionic structure.
Another confirmation of obtaining the ionic structure of the ibuprofenates of isopropyl esters of amino acids was signal shifts of the carbonyl carbon of ibuprofen from 0.83 ppm (for [L-ThrOiPr][IBU]) to 5.04 ppm (for [L-CysOiPr][IBU]) in comparison with the value for this carbon in unmodified ibuprofen (181.16 ppm) [11,[32][33][34]. Figure 2 presents the 13 C NMR spectra for ibuprofen and the obtained ibuprofenates; signals of the ibuprofen carbonyl group are marked in red square and the signals of the carbonyl group in green square from the amino acid part. 1      The characteristic IR absorption bands also confirmed the ionic structure. The FTIR spectra for ibuprofen and obtained ibuprofenates are compared in Figure 3. Two characteristic absorption bands are visible, establishing the ionic structure of the compounds obtained-asymmetric v (COO − ) as and symmetric stretching vibrations v (COO − ) sym. The difference above 200 cm −1 between the frequency values of these two bands confirmed the presence of the carboxylate anion COO − and the ionic structure of ibuprofenates [35]. FTIR and UV-Vis spectra are in the Supplementary Materials ( Figures S3-S57). 0.83 ppm (for [L-ThrOiPr][IBU]) to 5.04 ppm (for [L-CysOiPr][IBU]) in comparison with the value for this carbon in unmodified ibuprofen (181.16 ppm) [11,[32][33][34]. Figure 2 presents the 13 C NMR spectra for ibuprofen and the obtained ibuprofenates; signals of the ibuprofen carbonyl group are marked in red square and the signals of the carbonyl group in green square from the amino acid part. 1 H and 13 C NMR spectra are in the Supplementary Materials ( Figures S2-S29).
The characteristic IR absorption bands also confirmed the ionic structure. The FTIR spectra for ibuprofen and obtained ibuprofenates are compared in Figure 3. Two characteristic absorption bands are visible, establishing the ionic structure of the compounds obtained-asymmetric v(COO − )as and symmetric stretching vibrations v(COO − )sym. The difference above 200 cm −1 between the frequency values of these two bands confirmed the presence of the carboxylate anion COO − and the ionic structure of ibuprofenates [35]. FTIR and UV-Vis spectra are in the Supplementary Materials ( Figures  S3--S57).

4,000
3,500 3,000 2,500 2,000 1,500 1,000 500  LysOiPr][IBU] (cyan), [L-LysOiPr][IBU]2 (yellowish-green) [L-PheOiPr][IBU] (pink), [L-ProOiPr][IBU] (gray); (νsym.) (COO − ), and asymmetric (νasym.) (COO − ) stretching vibrations marked in green and red, respectively). Figure 4 shows the XRD patterns for ibuprofen and ibuprofen salts. Reflections that can be attributed to the ibuprofen phase ((RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid (PDF 96-230-0213) were identified in the ibuprofen diffraction pattern. As can be seen, the diffractograms of the derivatives obtained are different from that of the starting ibuprofen. The obtained results confirmed the crystalline nature of both unmodified ibuprofen and its derivatives. XRD patterns are in the Supplementary Materials ( Figures  S58-S71). The physicochemical properties such as melting point, specific rotation, thermal stability, morphology, and solubility were determined for all the compounds obtained. Table 1 summarises the results. The melting point was determined from the DSC curves. Table 1  Moreover, the specific and molar rotations were determined ( Table 1). As can be seen, the specific rotation depends on the type of amino acid. In most cases, the specific rotation of the resulting salt follows the specific rotation of the starting amino acid, with the exception of [L-CysOiPr][IBU] and [L-PheOiPr][IBU] in the opposite direction. The value of specific rotation of ibuprofenates is lower than that of the starting amino acids, which results from the proportion of the amino acid part in the whole molecule. A racemic mixture of ibuprofen was used for the tests. In the case of glycine salts, the obtained salt shows the ability to rotate the plane of polarised light, which may suggest a slight excess of the S(+)-ibuprofen enantiomer.
In Figure 5, the thermal stability of ibuprofen and amino acid isopropyl ester ibuprofenates was determined and compared. All TG, DTG, and c-DTA curves are in the Supplementary Materials (Figures S87-S100). The decomposition onset temperature (T onset ) and the temperature of the maximum rate of mass loss (T max ) were determined. The least stable is [L-AlaOiPr][IBU] (T onset = 78.3 • C), while the most stable is [L-CysOiPr][IBU] (T onset = 190.2 • C), which shows the onset of decomposition at a temperature higher than ibuprofen. In other cases, the decomposition onset temperature is lower for the obtained salts than for ibuprofen (T onset = 189.8 • C). As can be seen, derivatives of aliphatic amino acids show lower stabilitydown to temperatures below 110 • C. least stable is [L-AlaOiPr][IBU] (Tonset = 78.3 °C), while the most stable is [L-CysOiPr][IBU] (Tonset = 190.2 °C), which shows the onset of decomposition at a temperature higher than ibuprofen. In other cases, the decomposition onset temperature is lower for the obtained salts than for ibuprofen (Tonset = 189.8 °C). As can be seen, derivatives of aliphatic amino acids show lower stability-down to temperatures below 110 °C. The maximum weight loss rate (Tmax) temperatures were similar for ibuprofen and Lamino acid isopropyl esters ibuprofenates (Table 1). [L-ValOiPr][IBU] had the lowest Tmax, while [L-PheOiPr][IBU] was the compound with the highest Tmax ( Figure 6). The maximum weight loss rate (T max ) temperatures were similar for ibuprofen and L-amino acid isopropyl esters ibuprofenates (Table 1). [L-ValOiPr][IBU] had the lowest T max , while [L-PheOiPr][IBU] was the compound with the highest T max ( Figure 6). least stable is [L-AlaOiPr][IBU] (Tonset = 78.3 °C), while the most stable is [L-CysOiPr][IBU] (Tonset = 190.2 °C), which shows the onset of decomposition at a temperature higher than ibuprofen. In other cases, the decomposition onset temperature is lower for the obtained salts than for ibuprofen (Tonset = 189.8 °C). As can be seen, derivatives of aliphatic amino acids show lower stability-down to temperatures below 110 °C. The maximum weight loss rate (Tmax) temperatures were similar for ibuprofen and Lamino acid isopropyl esters ibuprofenates (Table 1). [L-ValOiPr][IBU] had the lowest Tmax, while [L-PheOiPr][IBU] was the compound with the highest Tmax ( Figure 6). One of the most critical factors affecting solubility and bioavailability is crystal morphology (size and shape of crystals). The SEM of ibuprofen and amino acid isopropyl ester ibuprofenates crystals is given in Figure 7. The shape of unmodified ibuprofen crystals is needle-type crystals. In other cases, the formation of agglomerates is visible-all derivatives of aliphatic amino acids behave similarly and form layer agglomerates. For example, the crystals of L-methionine and L-serine derivatives are needle-shaped like ibuprofen. A layered structure can also be seen for the salt of [L-Asp(OiPr) 2 ][IBU], [L-LysOiPr][IBU], and [L-LysOiPr][IBU] 2 . In contrast, [L-CysOiPr][IBU] and [L-ProOiPr][IBU] were amorphous. [L-ThrOiPr][IBU] was liquid at the measurement temperature.
As is well known, knowledge of the solubility of new drugs is essential in the drug discovery and development process. The solubility in water and organic solvents was tested to classify the compounds obtained accordingly. Due to the great importance of dissolution testing in drug discovery and development, we determined the solubility in water and typical organic solvents. The solubility data are summarised in Table 2. All L-amino acid isopropyl ester ibuprofenates were soluble in ethanol and dimethyl sulfoxide and insoluble in n-hexane. L-lysine derivatives-both mono and bis (ibuprofenate) saltwere dissolved only in ethanol and DMSO; in the other solvents used for the tests, they were insoluble. One of the most critical factors affecting solubility and bioavailability is crystal morphology (size and shape of crystals). The SEM of ibuprofen and amino acid isopropyl ester ibuprofenates crystals is given in Figure 7. The shape of unmodified ibuprofen crystals is needle-type crystals. In other cases, the formation of agglomerates is visibleall derivatives of aliphatic amino acids behave similarly and form layer agglomerates. For example, the crystals of L-methionine and L-serine derivatives are needle-shaped like ibuprofen. A layered structure can also be seen for the salt of As is well known, knowledge of the solubility of new drugs is essential in the drug discovery and development process. The solubility in water and organic solvents was Solvents were ranked with decreasing value of empirical solvent polarity parameters, E T (30) [41] ("+": soluble >100 mg cm −3 ; "+/−": partially soluble 33-100 mg cm −3 ; "−": insoluble <33 mg cm −3 ) at the temperature 25 • C by modified Vogel's method [42].
The bioavailability and effectiveness of a drug are assessed, inter alia, by assessing the solubility of the substance in water. Therefore, the solubility parameter in water and in a buffered saline solution was determined. The results, expressed in g of compound per dm 3 and the amount of active compound in g per dm 3 , are presented in Table 3. As can be seen, the solubility of amino acid isopropyl ester ibuprofenates was markedly higher than ibuprofen, exceeding even 66 times higher (based on the concentration of active substance). Converting the drug into a salt form is a well-used technique for increasing the water solubility of a drug. Furthermore, the highest solubility in water was obtained for [L-LysOiPr][IBU] (5.047 ± 0.060 g IBU dm −3 ), while [L-PheOiPr][IBU] had the lowest (0.297 ± 0.024 g IBU dm −3 ). A typical relationship between solubility and alkyl chain length was observed: solubility decreased with increasing chain length in the case of derivatives of aliphatic amino acids. A similar dependence was observed for the solubility in PBS (pH 7.4) buffer solution. For all the tested compounds, the solubility in PBS (pH 7.4) buffer was markedly higher than in water, but these tests were conducted at a higher temperature (32 • C for PBS (pH 7.4), 25 • C for water). In this case, the highest solubility in PBS was also obtained for [L-LysOiPr] The partition coefficient is the key parameter used to predict drug hydrophobicity and partitioning in biological systems. Table 4 summarises the results of the n-octanol-water partition coefficient determined by the shake flask method. All obtained ibuprofen and amino acid isopropyl ester ibuprofenates showed a positive log P, but they were lower compared with ibuprofen. All the salts obtained are therefore more hydrophilic. The lipophilicity of the obtained salts was ranked in ascending order: Individual permeation profiles for ibuprofen and amino acid isopropyl ester ibuprofenates are plotted in Figure 8. The content of IBU and its derivatives in the acceptor fluid collected during 24 h permeation is summarised in  Figure 8). The formation of structural modifications of ibuprofen in the form of charged compounds with lower lipophilicity has an influence on better permeability compared with the more lipophilic ibuprofen. The cluster analysis graph shows the cumulative mass of the IBU and its derivatives measured over the entire 24 h permeation period ( Figure S101). In this diagram, three distinct groups characterised by similar permeation can be distinguished (circles A, B, C). Generally, the cumulative mass of all ibuprofen derivatives in the acceptor phase after 24 h of permeation was significantly higher than free ibuprofen ( Figure S102). The similarity between derivatives was found using Tukey's test, which showed a statistically significant difference between all derivatives and pure ibuprofen (p < 0.05) (  Figure 8). The formation of structural modifications of ibuprofen in the form of charged compounds with lower lipophilicity has an influence on better permeability compared with the more lipophilic ibuprofen. The cluster analysis graph shows the cumulative mass of the IBU and its derivatives measured over the entire 24 h permeation period ( Figure S101). In this diagram, three distinct groups characterised by similar permeation can be distinguished (circles A, B, C). Generally, the cumulative mass of all ibuprofen derivatives in the acceptor phase after 24 h of permeation was significantly higher than free ibuprofen ( Figure S102). The similarity between derivatives was found using Tukey's test, which showed a statistically significant difference between all derivatives and pure ibuprofen (p < 0.05) ( Table S1 in Supplementary Materials).   For NSAIDs, including ibuprofen, faster permeation through the skin is preferable to achieve a rapid therapeutic effect. Fast and increased permeation causes a quicker decrease in inflammation in the underlying tissues [38]. The permeation rate determined at each time interval is presented in Figure 9. The highest permeation rate to the acceptor fluid generally was observed in samples collected between 3 and 5 h. For NSAIDs, including ibuprofen, faster permeation through the skin is preferable to achieve a rapid therapeutic effect. Fast and increased permeation causes a quicker decrease in inflammation in the underlying tissues [38]. The permeation rate determined at each time interval is presented in Figure 9. The highest permeation rate to the acceptor fluid generally was observed in samples collected between 3 and 5 h. The results of the in vitro efficiency permeation experiments related to ibuprofen and its derivatives are summarised in Table 6. The permeation parameters, including flux (JSS, µg IBU cm −2 h −1 ), apparent permeability coefficient (KP·10 3 , cm h −1 ), lag time (LT, h), diffusion coefficient in the skin (D·10 4 , cm 2 h −1 ), skin partition coefficient (Km), percentage drug permeated after 24 h (Q%24 h), and enhancement factor (EF) were designated. A significant difference in the flux for ibuprofen and its derivatives was visible. The The results of the in vitro efficiency permeation experiments related to ibuprofen and its derivatives are summarised in Table 6. The permeation parameters, including flux (J SS , µg IBU cm −2 h −1 ), apparent permeability coefficient (K P ·10 3 , cm h −1 ), lag time (L T , h), diffusion coefficient in the skin (D·10 4 , cm 2 h −1 ), skin partition coefficient (K m ), percentage drug permeated after 24 h (Q %24 h) , and enhancement factor (EF) were designated. A significant difference in the flux for ibuprofen and its derivatives was visible. , for which the value was lower and equalled 0.455. The enhancement factor was also determined, as seen from the data presented in Table 6. The skin permeation enhancement value is the highest for [L-PheOiPr][IBU]. The obtained results suggest that the tested compounds may promote skin permeability. The selection of the appropriate structural modification of the active compound should result from many factors, such as molar mass, solubility, or lipophilicity.
The drugs may both permeate and accumulate in the skin. In the case of antiinflammatory drugs applied topically, faster and greater permeation is most often preferred, with less accumulation in the skin [9,29]. Figure Figure 10. Accumulation in the skin of ibuprofen and amino acid isopropyl ester ibuprofenates during the 24 h penetration; different letters indicate significant differences between the ibuprofenates and control [IBU], p < 0.0001, mean ± SD, n = 3; (mean ± SD, n = 3). The statistically significant difference was estimated by ANOVA using Tukey's test.

Materials and Methods
The materials used for the tests and the methodologies used to assess the identity and determine the properties are presented in the Supplementary Materials.

Synthesis of Amino Acids Isopropyl Ester Ibuprofenates
The general scheme of the reaction is presented in Figure 11. In the first step, the hydrochlorides of the amino acid isopropyl esters were obtained in the presence of a chlorinating agent in the reaction of amino acid with isopropanol. An excess of isopropanol was used as the reaction medium in the reaction. Trimethylsilane chloride was used as the chlorinating agent, and the reaction can be successfully carried out in the Figure 10. Accumulation in the skin of ibuprofen and amino acid isopropyl ester ibuprofenates during the 24 h penetration; different letters indicate significant differences between the ibuprofenates and control [IBU], p < 0.0001, mean ± SD, n = 3; (mean ± SD, n = 3). The statistically significant difference was estimated by ANOVA using Tukey's test.

Materials and Methods
The materials used for the tests and the methodologies used to assess the identity and determine the properties are presented in the Supplementary Materials.

Synthesis of Amino Acids Isopropyl Ester Ibuprofenates
The general scheme of the reaction is presented in Figure 11. In the first step, the hydrochlorides of the amino acid isopropyl esters were obtained in the presence of a chlorinating agent in the reaction of amino acid with isopropanol. An excess of isopropanol was used as the reaction medium in the reaction. Trimethylsilane chloride was used as the chlorinating agent, and the reaction can be successfully carried out in the presence of thionyl chloride. Due to safety, TMSCl was used. A molar excess of the chlorinating agent was used (1:2, AA/TMSCl).

[GlyOiPr][IBU]-glycine isopropyl ester ibuprofenate
Glycine isopropyl ester ibuprofenate was obtained as a white solid in 98% yield.    The next step was the neutralisation reaction. Again, NaOH, KOH or 25% ammonia solution can be successfully used for the reaction. The procedures for preparing hydrochlorides and esters are described in detail in the Supplementary Materials.

Skin Permeation Studies
The permeation experiments were carried out by using Franz diffusion cells (Phoenix DB-6, ABL&E-JASCO, Wien, Austria) with diffusion areas of 1 cm 2 . The acceptor chamber was 10 cm 3 and was filled with PBS solution (pH 7.4). In each diffusion unit, there was a constant temperature of 37.0 ± 0.5 • C. The acceptor chamber content was stirred with a magnetic stirring bar at the same speed for all cells. In the experiment, abdominal porcine skin coming from the local slaughterhouse was used. Porcine skin is characterised by similar permeability to human skin [43,44]. The fresh abdominal porcine skin was washed in PBS buffer pH 7.4 several times. Skin of 0.5 mm in thickness was dermatomed. The skin was then divided into 2 cm × 2 cm pieces. The skin samples were wrapped in aluminium foil and stored in a freezer at −20 • C until use, not longer than three months. This frozen storage time was safe for maintaining skin barrier properties [45]. On the day of the experiment, the skin samples were slowly thawed at room temperature for 30 min and were hydrated by PBS pH 7.4 [45][46][47]. The skin was mounted on the donor chamber. Undamaged skin pieces with an even thickness were chosen for experiments. The integrity of the skin was examined by checking its impedance, which was measured using an LCRmeter4080 (Conradelectronic, Germany), which was operated in parallel mode at an alternating frequency of 120 Hz (error at kΩ values < 0.5%). The tips of measuring probes were immersed in the donor and acceptor chamber, filled with PBS (pH 7.4) as described previously [48,49]. Only skin samples of impedance >3 kΩ were applied. These values are similar to the electrical resistance for human skin [49]. The experiment was carried out for 24 h. The samples were reported after 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 8 h, and 24 h of stirring. The donor chamber was filled with 1 cm 3 of the saturated solutions of tested compounds in PBS (pH 7.4). After this time, the acceptor fluid (0.4 cm 3 ) aliquots were withdrawn and refilled with fresh buffer at the same pH. HPLC measured the IBU and its derivative concentrations in the acceptor phase. The cumulative mass (µg IBU·cm −2 ) was calculated based on this concentration. The flux (in µg IBU·cm −2 ·h −1 ) through the pigskin into acceptor fluid was determined as the slope of the plot of cumulative mass in the acceptor fluid versus time.
The accumulation of the tested substance in the skin after penetration was determined using a modification of the methods described by Ossowicz-Rupniewska et al., Janus et al., and Haq and Michniak-Kohl [31,45,50]. After 24 h, the skin samples were removed from the Franz diffusion cell. The skin samples were carefully rinsed in PBS solution at 7.4 pH and dried at room temperature. The skin samples were weighed, cut by diffusion (1 cm 2 ), and minced using scissors. Next, skin samples were placed in 2 cm 3 methanol and were incubated for 24 h at 4 • C. After this time, skin samples were homogenized for 3 min using a homogenizer (IKA ® T18 digital ULTRA TURRAX (Staufen im Breisgau, Germany)). The homogenate was centrifuged at 3500× g rpm for 5 min. The supernatant was collected and analysed using HPLC. Accumulation of the IBU in the skin was calculated by dividing the amount of the drug remaining in the skin by a mass of skin sample and is expressed in mass of ibuprofen per mass of the skin (µg g −1 ).
The concentration of IBU and its derivatives in the acceptor fluid and accumulation in the skin was assessed with a liquid chromatography system (Knauer, Berlin, Germany). The HPLC system consisted of a model 2600 UV detector, Smartline model 1050 pump and Smartline model 3950 autosampler with ClarityChrom 2009 software (Knauer, Berlin, Germany). The detector was operated at 264 nm. A 125 × 4 mm chromatographic column filled with Hyperisil ODS (C18), particle size 5 µm, was used. The mobile phase was 0.02 M potassium dihydrogen phosphate-acetonitrile (60/40 v/v) with a flow rate of 1 cm 3 min −1 . The column temperature was set at 25 • C, and the injection volume was 20 µL.

Conclusions
Fourteen salts of amino acid isopropyl esters were prepared, described, and characterised. Twelve of these compounds have not yet been described in the literature. The obtained structural modification of ibuprofen significantly increased the solubility of this active compound in both water and PBS aqueous solution (simulating body fluids). Moreover, all the obtained derivatives showed from 2.95 to 16.52 times higher permeability of the active substance as compared with the unmodified ibuprofen. The highest permeability was obtained for L-lysine isopropyl ester monoibuprofenate, which was also characterised by higher transport rates and the greatest mass of permeating ibuprofen. It was also shown that the obtained amino acid derivatives of ibuprofen show from 3.3 to 6.7 times higher accumulation in the skin. The presented results show the possibilities of using the obtained compounds as an alternative to poorly soluble NSAIDs for application to the skin.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.