Design, Synthesis and Preliminary Evaluation of the Cytotoxicity and Antibacterial Activity of Novel Triphenylphosphonium Derivatives of Betulin

For several decades, natural products have been widely researched and their native scaffolds are the basis for the design and synthesis of new potential therapeutic agents. Betulin is an interesting biologically attractive natural parent molecule with a high safety profile and can easily undergo a variety of structural modifications. Herein, we describe the synthesis of new molecular hybrids of betulin via covalent linkage with an alkyltriphenylphosphonium moiety. The proposed strategy enables the preparation of semi-synthetic derivatives (28-TPP⊕ BN and 3,28-bisTPP⊕ BN) from betulin through simple transformations in high yields. The obtained results showed that the presence of a lipophilic cation improved the solubility of the tested analogs compared to betulin, and increased their cytotoxicity. Among the triphenylphosphonium derivatives tested, analogs 7a (IC50 of 5.56 µM) and 7b (IC50 of 5.77 µM) demonstrated the highest cytotoxicity against the colorectal carcinoma cell line (HCT 116). TPP⊕-conjugates with betulin showed antimicrobial properties against Gram-positive reference Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228 bacteria, at a 200 µM concentration in water. Hence, the conjugation of betulin’s parent backbone with a triphenylphosphonium moiety promotes transport through the hydrophobic barriers of the mitochondrial membrane, making it a promising strategy to improve the bioavailability of natural substances.


Introduction
Advancements in medical science have allowed for the treatment of a wide range of diseases, however, many disorders lack necessary pharmaceuticals. The high systemic toxicity of medicinal preparations and increasing resistance of tumor cells to a significant number of drugs often limits anticancer therapy success. According to World Health Organization (WHO) reports, cancerous diseases are one of the biggest problems of modern medicine and are one of the main causes of death in the world in the 21st century [1]. Therefore, drug design is an important issue in modern medicinal chemistry. Despite many innovative tools that allow for the development of extremely advanced methods of treatment, many therapies are still based on active substances of natural origin. These substances act as basic structures that can be subjected to various chemical modifications in order to improve their physicochemical and pharmacokinetic properties.
For several decades, natural products (NPs) have been widely researched in terms of searching for new drugs. It is NPs that are an invaluable source of native scaffolds, which are the basis for the design and development of new potential therapeutic agents. Naturally occurring pentacyclic lupane-type triterpenoids have attracted a lot of attention including betulin (BN, 3-lup-20(29)-ene-3,28-diol), which is one of the most available terpenoids in the plant kingdom. BN is a cheap, easily accessible natural active substance that can be readily extracted from the bark of several species of trees, especially white birch (Betula pubescens) [2,3]. Due to the presence of simply transformable functional groups in its skeleton (including C 3 -OH, C 28 -OH), BN has high synthetic potential for numerous semisynthetic derivatives. BN is an interesting example of a biologically attractive natural parent molecule with a high safety profile and the possibility of making a variety of structural modifications (Figure 1) [3][4][5].
Molecules 2022, 27, x FOR PEER REVIEW 2 of 21 treatment, many therapies are still based on active substances of natural origin. These substances act as basic structures that can be subjected to various chemical modifications in order to improve their physicochemical and pharmacokinetic properties. For several decades, natural products (NPs) have been widely researched in terms of searching for new drugs. It is NPs that are an invaluable source of native scaffolds, which are the basis for the design and development of new potential therapeutic agents. Naturally occurring pentacyclic lupane-type triterpenoids have attracted a lot of attention including betulin (BN, 3-lup-20(29)-ene-3,28-diol), which is one of the most available terpenoids in the plant kingdom. BN is a cheap, easily accessible natural active substance that can be readily extracted from the bark of several species of trees, especially white birch (Betula pubescens) [2,3]. Due to the presence of simply transformable functional groups in its skeleton (including C 3 -OH, C 28 -OH), BN has high synthetic potential for numerous semi-synthetic derivatives. BN is an interesting example of a biologically attractive natural parent molecule with a high safety profile and the possibility of making a variety of structural modifications (Figure 1) [3][4][5]. The multidirectional biological activity of natural BN has been confirmed by numerous research articles. Additionally, the reported derivatives of BN have shown a broad spectrum of bio-activity in terms of anticancer [4,[6][7][8][9][10], antimalarial activity [11], antibacterial [12][13][14], antiviral [4,[15][16][17], anti-inflammatory [18,19], or hepatoprotective properties [3]. Moreover, BN has a positive effect on the treatment of atopic dermatitis [20]. However, despite the abundance of BN, well-developed isolation methods from plant material as well as many studies confirming its very good biological properties, its use as a potential therapeutic agent is limited due to its low bioavailability, high hydrophobicity, and insufficient intracellular accumulation. The pentacyclic molecule and hydrophobic nature of the skeleton of BN hinders its ability to reach the target in vivo and obtain the desired therapeutic effect in acceptable therapeutic doses [21].
One of the most promising strategies for the design and synthesis of effective therapeutic agents is the conjugation of a native skeleton (e.g., BN) with triphenylphosphonium cation (TPP ⊕ ) of low molecular weight, which promotes accumulation inside the cell's mitochondria. Furthermore, the presence of the TPP ⊕ group in a molecular hybrid improves the pharmacokinetic properties including solubility, bioavailability, and intermembrane transport as well as selectivity in targeting drugs for a specific purpose. The high lipophilicity and large ionic radius of TPP ⊕ effectively reduce the activation energy required for membrane passage. The presence of a delocalized lipophilic cation can accelerate the transport of biologically active molecules across the mitochondrial membrane [22]. Studies have shown that, in contrast to other cellular organelles, mitochondria have a high negative transmembrane potential (Δψm). This potential is much higher for tumor cells, providing an opportunity for the selective delivery and accumulation of anticancer agents in mitochondria-targeted therapies [21][22][23][24]. The multidirectional biological activity of natural BN has been confirmed by numerous research articles. Additionally, the reported derivatives of BN have shown a broad spectrum of bio-activity in terms of anticancer [4,[6][7][8][9][10], antimalarial activity [11], antibacterial [12][13][14], antiviral [4,[15][16][17], anti-inflammatory [18,19], or hepatoprotective properties [3]. Moreover, BN has a positive effect on the treatment of atopic dermatitis [20]. However, despite the abundance of BN, well-developed isolation methods from plant material as well as many studies confirming its very good biological properties, its use as a potential therapeutic agent is limited due to its low bioavailability, high hydrophobicity, and insufficient intracellular accumulation. The pentacyclic molecule and hydrophobic nature of the skeleton of BN hinders its ability to reach the target in vivo and obtain the desired therapeutic effect in acceptable therapeutic doses [21].
One of the most promising strategies for the design and synthesis of effective therapeutic agents is the conjugation of a native skeleton (e.g., BN) with triphenylphosphonium cation (TPP ⊕ ) of low molecular weight, which promotes accumulation inside the cell's mitochondria. Furthermore, the presence of the TPP ⊕ group in a molecular hybrid improves the pharmacokinetic properties including solubility, bioavailability, and intermembrane transport as well as selectivity in targeting drugs for a specific purpose. The high lipophilicity and large ionic radius of TPP ⊕ effectively reduce the activation energy required for membrane passage. The presence of a delocalized lipophilic cation can accelerate the transport of biologically active molecules across the mitochondrial membrane [22]. Studies have shown that, in contrast to other cellular organelles, mitochondria have a high negative transmembrane potential (∆ψm). This potential is much higher for tumor cells, providing an opportunity for the selective delivery and accumulation of anticancer agents in mitochondria-targeted therapies [21][22][23][24].
Thus, the TPP ⊕ moiety not only affects the physical properties, but also the mechanism of action of a potential drug. In addition, it increases the selectivity, which often reduces the drug dose, and in turn diminishes the harmful side effects [24,25]. Therefore, research into mitochondria-targeted anticancer drugs is an attractive prospect [24][25][26][27][28]. The strategy of modification of the native skeleton via conjugating with the TPP ⊕ group has been used successfully for anticancer drugs such as doxorubicin [29], cisplatin [26], chlorambucil [30], metformin [31], and tamoxifen [32] because it facilitates their transport and selective accumulation in cancer cells ( Figure 2). Thus, the TPP ⊕ moiety not only affects the physical properties, but also the mechanism of action of a potential drug. In addition, it increases the selectivity, which often reduces the drug dose, and in turn diminishes the harmful side effects [24,25]. Therefore, research into mitochondria-targeted anticancer drugs is an attractive prospect [24][25][26][27][28]. The strategy of modification of the native skeleton via conjugating with the TPP ⊕ group has been used successfully for anticancer drugs such as doxorubicin [29], cisplatin [26], chlorambucil [30], metformin [31], and tamoxifen [32] because it facilitates their transport and selective accumulation in cancer cells ( Figure 2).
Mitochondria play a vital role in a wide range of physiological and pathological processes. They are the main source of reactive oxygen species, and at the same time, are particularly susceptible to oxidative damage, contributing to the development of many diseases. Due to their functions, mitochondria may be an important molecular target for anticancer drugs as well as in the treatment of cardiovascular diseases or neurodegenerative diseases (e.g., Alzheimer's disease or Parkinson's disease) [36]. Non-targeted antioxidant therapeutics show low effectiveness, therefore, attempts have been made to modify them to increase the drug accumulation in the mitochondrial matrix. For example, Mito-Q10, (coenzyme Q10) has been described as a potential agent for the treatment of sepsis or Parkinson's disease ( Figure 2) [24,36].
A relatively novel group of potential mitocans (acronym derived from the terms mitochondria and cancer) is conjugates of pentacyclic lupane-type triterpenoids including BN or betulinic acid (BA) with the lipophilic cation TPP ⊕ [28]. Spivak et al. [21,28,[37][38][39], Tsepaeva et al. [23,40,41], Ye et al. [42], and Xu et al. [43] reported the preparation of BA Mitochondria play a vital role in a wide range of physiological and pathological processes. They are the main source of reactive oxygen species, and at the same time, are particularly susceptible to oxidative damage, contributing to the development of many diseases. Due to their functions, mitochondria may be an important molecular target for anticancer drugs as well as in the treatment of cardiovascular diseases or neurodegenerative diseases (e.g., Alzheimer's disease or Parkinson's disease) [36]. Non-targeted antioxidant therapeutics show low effectiveness, therefore, attempts have been made to modify them to increase the drug accumulation in the mitochondrial matrix. For example, Mito-Q10, (coenzyme Q10) has been described as a potential agent for the treatment of sepsis or Parkinson's disease ( Figure 2) [24,36].
A relatively novel group of potential mitocans (acronym derived from the terms mitochondria and cancer) is conjugates of pentacyclic lupane-type triterpenoids including BN or betulinic acid (BA) with the lipophilic cation TPP ⊕ [28]. Spivak et al. [21,28,[37][38][39], Tsepaeva et al. [23,40,41], Ye et al. [42], and Xu et al. [43] reported the preparation of BA or BN conjugates, in which one or two TPP ⊕ moieties were linked to the triterpenoid skeleton at positions C-2, C-3, C-28, or C-30 by the carbon-carbon or ester bonds, as shown in or BN conjugates, in which one or two TPP ⊕ moieties were linked to the triterpenoid skeleton at positions С-2, С-3, С-28, or С-30 by the carbon-carbon or ester bonds, as shown in Figure 3 and Figure 4. The cytotoxic effect of these TPP ⊕ -analogs against various types of tumor cells toward Schistosoma Mansoni and antibacterial activity was analyzed [21,23,28,[37][38][39][40][41][42][43][44]. In the library of known triphenylphosphonium derivatives of pentacyclic lupanetype triterpenoids, betulinic acid derivatives definitely dominate (TPP ⊕ -conjugated with BA, about 30 compounds, Figure 3). Both BN and BA are common in the plant kingdom, especially in the outer layer of the birch bark (Betulaceae, Betula, Betula pendula). However, BN is considerably more available (BN content was up to 34%, and BA was only 0.3% of dry weight [6]), which may be an advantage in terms of the economic analysis of methods in obtaining potential therapeutic agents. In the library of known triphenylphosphonium derivatives of pentacyclic lupane-type triterpenoids, betulinic acid derivatives definitely dominate (TPP ⊕ -conjugated with BA, about 30 compounds, Figure 3). Both BN and BA are common in the plant kingdom, especially in the outer layer of the birch bark (Betulaceae, Betula, Betula pendula). However, BN is considerably more available (BN content was up to 34%, and BA was only 0.3% of dry weight [6]), which may be an advantage in terms of the economic analysis of methods in obtaining potential therapeutic agents.  Although BN derivatives have been extensively explored, to date, no structures have demonstrated the desired biological properties at a satisfactory dosage that would allow them to be used as drugs. The aim of the presented study was to evaluate the relationships between the biological effects and the structure of the new triphenylphosphonium derivatives of BN. Hence, we designed and synthesized molecular hybrids of BN by covalently linking a lipophilic alkyltriphenylphosphonium moiety (shown in dark red, Scheme 1) to the parent skeleton of BN through a linker (shown in blue, Scheme 1). In addition, in order Although BN derivatives have been extensively explored, to date, no structures have demonstrated the desired biological properties at a satisfactory dosage that would allow them to be used as drugs. The aim of the presented study was to evaluate the relationships between the biological effects and the structure of the new triphenylphosphonium derivatives of BN. Hence, we designed and synthesized molecular hybrids of BN by covalently linking a lipophilic alkyltriphenylphosphonium moiety (shown in dark red, Scheme 1) to the parent skeleton of BN through a linker (shown in blue, Scheme 1). In addition, in order to obtain a library of compounds intended for the initial assessment of biological activity (e.g., cytotoxicity, antibacterial activity), structural modifications of the BN skeleton were conducted via the introduction of one or two TPP ⊕ moieties at the C-28 or C-3 and C-28 positions. We prepared a series of the monoand bisTPP ⊕ derivatives of BN through the multi-step synthesis (3 or 5 step) and their bio-activity was analyzed. Although BN derivatives have been extensively explored, to date, no structures have demonstrated the desired biological properties at a satisfactory dosage that would allow them to be used as drugs. The aim of the presented study was to evaluate the relationships between the biological effects and the structure of the new triphenylphosphonium derivatives of BN. Hence, we designed and synthesized molecular hybrids of BN by covalently linking a lipophilic alkyltriphenylphosphonium moiety (shown in dark red, Scheme 1) to the parent skeleton of BN through a linker (shown in blue, Scheme 1). In addition, in order to obtain a library of compounds intended for the initial assessment of biological activity (e.g., cytotoxicity, antibacterial activity), structural modifications of the BN skeleton were conducted via the introduction of one or two TPP ⊕ moieties at the C-28 or C-3 and C-28 positions. We prepared a series of the mono-and bisTPP ⊕ derivatives of BN through the multi-step synthesis (3 or 5 step) and their bio-activity was analyzed. Scheme 1. The synthetic route for the preparation of 28-TPP ⊕ BN and 3,28-bisTPP ⊕ BN.  [45]. In the first step, betulin was esterified with an excess of acetic anhydride in pyridine (Py) in the presence of 4-(dimethylamino)pyridine (DMAP) to give ester 2 in 98% yield. Then, the C-28 position of the BN backbone was selectively deprotected by treating crystalline ester 2 with aluminum isopropoxide ((i-PrO) 3 Al) in i-PrOH. After 2 h at 80 • C, followed by column chromatography purification, analog 3 was obtained in 78% yield. The next step involved the introduction of a linker terminated with a carboxyl group (O(CO)CH 2 CR 2 COOH, R = H or Me) at position C-28 (mono-substituted BN analog 4) or C-3 and C-28 (disubstituted BN analog 5) of BN. The carboxyacyl moiety seemed to be an ideal linker component as it allowed for further structural modifications. In addition, numerous literature reports have suggested that the combination of the triterpene skeleton with this type of moiety improved the biological properties including anti-HIV, antibacterial, or antifungal activity [46,47]. In accordance with the published procedures [48,49] with some modifications, analog 3 was reacted with succinic anhydride (SA) or 2,2-dimethylsuccinic anhydride (DMSA) in dry pyridine in the presence of DMAP at reflux for 18-20 h. The synthesis of 3,28-O,O'-bis(3-carboxypropanoyl)betulin 5 was performed in a similar manner, but the reagents were used in a molar ratio of BN:SA (1:10) at reflux for 9 h. The crude products were purified by chloroform extraction and washed with the water and HCl merous literature reports have suggested that the combination of the triterpene skeleton with this type of moiety improved the biological properties including anti-HIV, antibacterial, or antifungal activity [46,47]. In accordance with the published procedures [48,49] with some modifications, analog 3 was reacted with succinic anhydride (SA) or 2,2-dimethylsuccinic anhydride (DMSA) in dry pyridine in the presence of DMAP at reflux for 18-20 h. The synthesis of 3,28-O,O'-bis(3-carboxypropanoyl)betulin 5 was performed in a similar manner, but the reagents were used in a molar ratio of BN:SA (1:10) at reflux for 9 h. The crude products were purified by chloroform extraction and washed with the water and HCl solution. The BN analogs were obtained in excellent yields (4a: 99%, 4b: 99%, 5: 65%, Scheme 2).

Synthesis of 28-TPP ⊕ -Conjugates Derivatives of BN
The synthesis method of new molecular BN hybrids (28-TPP ⊕ BN) with one TPP moiety attached via the linker C 28
were isolated by extraction with ethyl acetate and subsequent purification by column chromatography to produce the products in satisfactory yields (42-86%). The final step of the 28-TPP ⊕ BN synthesis was the substitution of the bromide anion of analog 6 with the TPP group by heating a homogenous mixture of 28-(bromoalkoxycarbonyl)propanoyl)betulin 6 and triphenylphosphine without a solvent under an Ar atmosphere. Optimization of the procedure consisted of examining the proportions of reagents, the temperature and reaction time, where at a molar ratio of analog 6/triphenylphosphine (1:2) at 120 °C for 6-12 h, the highest yields were obtained. Additionally, column chromatography was not necessary for all analogs, but all required extraction with diethyl ether and then crystallization from the diethyl ether/ethyl acetate (1:4, v/v), resulting in a high yield (72-99%, Scheme 3).

Synthesis of 3,28-bisTPP ⊕ -Conjugates Derivatives of BN
We also investigated the influence of two TPP ⊕ groups in the molecular hybrids of BN toward their pharmacokinetic properties. The synthetic route of 3,28-bisTPP ⊕ BN is depicted in Scheme 4.
The final step of the 28-TPP ⊕ BN synthesis was the substitution of the bromide anion of analog 6 with the TPP group by heating a homogenous mixture of 28-(bromoalkoxycarbonyl) propanoyl)betulin 6 and triphenylphosphine without a solvent under an Ar atmosphere. Optimization of the procedure consisted of examining the proportions of reagents, the temperature and reaction time, where at a molar ratio of analog 6/triphenylphosphine (1:2) at 120 • C for 6-12 h, the highest yields were obtained. Additionally, column chromatography was not necessary for all analogs, but all required extraction with diethyl ether and then crystallization from the diethyl ether/ethyl acetate (1:4, v/v), resulting in a high yield (72-99%, Scheme 3).

Synthesis of 3,28-bisTPP ⊕ -Conjugates Derivatives of BN
We also investigated the influence of two TPP ⊕ groups in the molecular hybrids of BN toward their pharmacokinetic properties. The synthetic route of 3,28-bisTPP ⊕ BN is depicted in Scheme 4.
The structures of all of the synthesized compounds (2-9) were confirmed by spectroscopic methods ( 1 H, 13 C, 31 P NMR, FTIR, and HRMS, Supplementary Materials). The 31 P NMR spectra of analogs 7 and 9 showed signals confirming the presence of TPP ⊕ in the range of 19.4-24.8 ppm. A characteristic feature in the 13 C NMR spectra of the organophosphorus compounds was the splitting of specific signals into doublets caused by coupling the phosphorus atom with selected carbon atoms. Chemical shifts and J C-P coupling constants of great diagnostic value observed for the TPP ⊕ group are summarized in Table 1.

Cytotoxicity Studies
The obtained new molecular hybrids of BN were screened in order to initially investigate their cytotoxicity as well as examine the relationships between the structure and biological effect. The common element of all of the tested compounds was the presence of a lipophilic moiety (CH 2 ) n TPP ⊕ (n = 3, 4, 5). The research was conducted on two groups of compounds: 28-TPP ⊕ BN and 3,28-bisTPP ⊕ BN. Their cytotoxicity was investigated on two cancer cell lines: HCT 116 (colorectal carcinoma cell line) and MCF-7 (human breast adenocarcinoma cell line). The proliferation of tumor cells treated with the tested compounds (7 and 9) at 12.5-3.125 µM concentrations were determined after 24 h of incubation. Additionally, all compounds were tested against the NHDF cell line (Normal Human Dermal Fibroblast cells) to assess their safety. The effect of these compounds was compared with that of BN doses, as shown in Figure 5. Half-maximal inhibitory concentrations (IC 50 ) of triphenylphosphonium analogs of BN were determined using a CCK-8 assay and are summarized in Table 2. As expected, triphenylphosphonium derivatives of BN showed greater cytotoxicity than the parent BN toward all of the cell lines tested. The level of inhibition of cell viability depended on the concentration of the tested substances and cell type. The tested analogs of BN had the greatest effect on the viability of the HCT 116 cells (Figure 5a) and the lowest on the viability of the MCF-7 cells (Figure 5b).
When comparing the biological effect of the mono-TPP ⊕ BN derivatives (7a−7c, linker without an additional Me group), it seemed that the length of the linker did not influence their activity in the in vitro tests. The 28-TPP ⊕ BN conjugates (7a−c) with a variable length,  As expected, triphenylphosphonium derivatives of BN showed greater cytotoxicity than the parent BN toward all of the cell lines tested. The level of inhibition of cell viability depended on the concentration of the tested substances and cell type. The tested analogs of BN had the greatest effect on the viability of the HCT 116 cells (Figure 5a) and the lowest on the viability of the MCF-7 cells (Figure 5b).
When comparing the biological effect of the mono-TPP ⊕ BN derivatives (7a-7c, linker without an additional Me group), it seemed that the length of the linker did not influence their activity in the in vitro tests. The 28-TPP ⊕ BN conjugates (7a-7c) with a variable length, and an alkyl linker (n = 3, 4, 5) similarly inhibited the viability in both tumor cells (HCT 116: IC 50 = 5.56-6.48, MCF-7: IC 50 = 13.71-15.52). The exception was analog 9c, with two TPP ⊕ cations and a pentyl chain, which, compared to compounds with a shorter linker (propyl or butyl chain), showed lower cytotoxicity against the HCT 116 cells (IC 50 : 9a < 9b < 9c; Table 2). Importantly, compounds 7a-7c were almost twice less toxic toward the healthy cells (NHDF), with IC 50 values ranging from 9.68 to 11.29 µM, which demonstrated their selectivity.
Unfortunately, in the course of further studies, it was revealed that the introduction of an additional Me group into the linker 7f reduced this bio-activity compared to compound 7c against the HCT 116 and MCF-7 tumor cells whereas no significant effect of the Me groups attached to the linker was observed on the bio-activity of compound 7f in normal NHDF cells.
We observed that the presence of both one and two lipophilic cations improved the solubility of the tested analogs compared to BN, which slightly increased their cytotoxicity, especially against the colorectal carcinoma cell line (HCT 116). Among the triphenylphosphonium derivatives of BN tested, analossg 7a (IC 50 of 5.56 µM) and 7b (IC 50 of 5.77 µM) demonstrated the highest cytotoxicity against this cell line at low micromolar concentrations. This supported the hypothesis that the conjugation of the BN native backbone with the TPP ⊕ moiety allowed for its transport through the hydrophobic barriers of the mitochondrial membrane, making it a promising strategy to improve the bioavailability of natural substances.

Antibacterial Studies
Investigations were carried out using different concentrations of solutions from 25 to 250 µM. A 25 µM concentration of the tested derivatives was not enough to inhibit the growth of both Gram-positive S. aureus ATCC 25923 bacteria. When the concentration of analogs 7d-7f and 9a-9c increased to 200 µM, the growth of S. aureus ATCC 25923 and S. epidermidis ATCC 12228 was inhibited. Furthermore, the optical density of all compounds remained unchanged after 18 h of bacteria culture. In the case of analogs 7a-7c, the optical density values were between 0.9 and 2.2 when they were cultured with S. aureus ATCC 25923. The bacterial growth was slower compared to the control sample (TSB-culture medium). However, the studied TPP ⊕ -BN derivatives did not inhibit the growth of S. epidermidis ATCC 12228 bacteria. However, at a 250 µM concentration, analogs 7a and 7b greatly inhibited the growth of S. aureus ATCC 25923 and S. epidermidis ATCC 12228. In the case of analog 7c, the optical density increased up to 0.7 after 18 h of the sample culture with both kinds of Gram-positive bacteria. In contrast, the reference sample's (TSB) optical density increased to 7.5 for S. aureus ATCC 25923, and 4.9 for S. epidermidis ATCC 12228 after 18 h of bacteria culture. All of the investigated compounds did not inhibit the growth of Gram-negative Escherichia coli ATCC 25922 bacteria. All of the analog values of the measured optical density were similar to that of the reference sample (5.0-5.1, Table 3). The obtained results showed that 28-TPP ⊕ BN (7d-7f) and 3,28-bisTPP ⊕ BN (9a-9c) could be employed as agents for the inhibition of Gram-positive bacteria (S. aureus ATCC 25923 and S. epidermidis ATCC 12228) growth at a concentration of 200 µM in an aqueous solution.

General Information
NMR spectra ( 1 H and 13 C) were recorded on a Varian spectrometer at operating frequencies of 600 or 400 MHz and 150 or 100 MHz, respectively, using TMS as the resonance shift standard. CDCl 3 was used as the solvent, which was purchased from ACROS Organics (Geel, Belgium). The 31 P NMR spectra were acquired using a Varian 400 spectrometer at 161.9 MHz, where the resonance shift of H 3 PO 4 was determined as 0 ppm. All chemical shifts (δ) were reported in ppm and coupling constants (J) in Hz. The following abbreviations were used to explain the observed multiplicities: s-singlet; d-doublet; dd-double doublet; ddd-doublet of double doublet; t-triplet, dd~t-overlapping double doublet that resembles a triplet (with similar values of coupling constants); m-multiplet; brbroad. IR-spectra were measured on a Nicolet 6700 FT-IR spectrophotometer, Thermo Scientific (Waltham, MA, USA) (attenuated total reflectance method; ATR). High resolution mass spectrometry analyses were performed using a Waters Xevo G2 Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA) equipped with an ESI source operating in positive-ion mode. The accurate mass and composition for the molecular ion adducts were calculated using MassLynx software (Waters) incorporated in the instrument.
Reactions were monitored by TLC analysis on precoated plates of silica gel 60 F 254 (Merck Millipore, Burlington, MA, USA). The TLC plates were inspected under UV light (λ = 254 nm) or charring after spraying with 10% solution of sulfuric acid in ethanol.
As expected, the triphenylphosphonium derivatives of BN showed a greater cytotoxicity than natural BN toward the cell lines tested (HCT 116 and MCF-7). Importantly, analogs (7a-7c) with one triphenylphoshonium cation were almost twice less toxic against healthy cells (NHDF), which demonstrated their selectivity. TPP ⊕ -conjugates with BN showed antimicrobial properties against the Gram-positive reference S. aureus ATCC 25923 and S. epidermidis ATCC 12228 bacteria when their concentration in the water solution was 200 µM.
The obtained results show that the bioavailability of natural BN can be improved by combining its backbone via linkers with a mitochondria-targeted TPP ⊕ moiety. Additionally, our study provides important data about the properties of BN conjugates with TPP ⊕ and encourages further research on the structural modifications of the parent BN skeleton.
Supplementary Materials: The following can be downloaded at: https://www.mdpi.com/xxx/s1. Supporting information includes the 1 H, 13 C, 31 P NMR spectra of betulin and all of the synthesized compounds (1-9) as well as the gHSQC and FTIR for the selected compounds.