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Article

Towards Antibacterial Agents: Synthesis and Biological Activity of Multivalent Amide Derivatives of Thiacalix[4]arene with Hydroxyl and Amine Groups

by
Igor Shiabiev
1,
Dmitry Pysin
1,
Alan Akhmedov
1,
Olga Babaeva
2,
Vasily Babaev
2,
Anna Lyubina
2,
Alexandra Voloshina
2,
Konstantin Petrov
2,
Pavel Padnya
1,* and
Ivan Stoikov
1,*
1
A.M. Butlerov Chemical Institute, Kazan Federal University, Kremlevskaya, 18, Kazan 420008, Russia
2
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences, 8 Arbuzov Street, Kazan 420088, Russia
*
Authors to whom correspondence should be addressed.
Pharmaceutics 2023, 15(12), 2731; https://doi.org/10.3390/pharmaceutics15122731
Submission received: 14 November 2023 / Revised: 28 November 2023 / Accepted: 4 December 2023 / Published: 5 December 2023
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Abstract

:
Antimicrobial resistance to modern antibiotics stimulates the search for new ways to synthesize and modify antimicrobial drugs. The development of synthetic approaches that can easily change different fragments of the molecule is a promising solution to this problem. In this work, a synthetic approach was developed to obtain multivalent thiacalix[4]arene derivatives containing different number of amine and hydroxyl groups. A series of macrocyclic compounds in cone, partial cone, and 1,3-alternate stereoisomeric forms containing -NHCH2CH2R (R = NH2, N(CH3)2, and OH) and -N(CH2CH2OH)2 terminal fragments, and their model non-macrocyclic analogues were obtained. The antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains and cytotoxicity of the obtained compounds were studied. Structure–activity relationships were established: (1) the macrocyclic compounds had high antibacterial activity, while the monomeric compounds had low activity; (2) the compounds in cone and partial cone conformations had better antibacterial activity compared to the compounds in 1,3-alternate stereoisomeric form; (3) the macrocyclic compounds containing -NHCH2CH2N(CH3)2 terminal fragments had the highest antibacterial activity; (4) introduction of additional terminal hydroxyl groups led to a significant decrease in antibacterial activity; (5) the compounds in partial cone conformation had significant bactericidal activity against all studied cell strains; the best selectivity was observed for the compounds in cone conformation. The mechanism of antibacterial activity of lead compounds with terminal fragments -NHCH2CH2N(CH3)2 was proved using model negatively charged POPG vesicles, i.e., the addition of these compounds led to an increase in the size and zeta potential of the vesicles. The obtained results open up the possibility of using the synthesized macrocyclic compounds as promising antibacterial agents.

1. Introduction

According to the World Health Organization [1], antimicrobial resistance to modern antibiotics is one of the most important problems of the 21st century. Bacteria have several mechanisms to combat antibiotics, such as the formation of biofilms, membrane changes, the production of enzymes capable of modifying antibiotics, and chromosomal mutations [2,3,4,5]. The presence of such a large number of mechanisms leads to difficulties in the synthesis of new effective antibacterial agents [6]. One of the possible solutions to this problem is the use of multivalent compounds, which, due to different functional groups, are able to interact effectively with the bacterial cell membrane, leading to its destabilization and destruction [7,8,9,10,11,12,13,14]. Linear [15,16,17,18] and hyperbranched polymeric macromolecules [19,20,21,22,23,24,25,26,27,28,29] with low symmetry, as well as more symmetric dendrimers with a large number of positively charged fragments [30,31,32,33,34,35,36,37,38,39,40,41], are often used as such multivalent structures.
A problem in the design and synthesis of hyperbranched structures is usually the difficulty in controlling their hydrophilic–lipophilic balance, which is an important factor in “tuning” the macromolecule. Introduction of a lipophilic macrocyclic core, such as (thia)calix[n]arenes, into the structure of the target compounds leads to changes in the physical and biological properties of the resulting compounds [42,43,44,45,46,47,48,49,50,51]. There are a number of examples in the literature of antimicrobial agents based on these macrocyclic compounds [52,53,54,55,56,57,58]. A recent review by Prof. Duval’s group [59] detailed the benefits of the (thia)calixarene platforms, e.g., the use of a multivalent approach to obtain the target compounds, the possibility of easy functionalization with positively charged fragments (e.g., ammonium, guanidinium, and imidazolium), and the spatial organization of these fragments by obtaining different stereoisomeric forms, i.e., cone, partial cone, and 1,3-altenate. Previously, our research group has shown that thiacalix[4]arene derivatives containing eight primary terminal amino groups and their complexes with lysozyme have antibacterial properties [60]. However, the synthetic approaches proposed earlier in the literature do not allow for the possibility of “fine-tuning” the structure of the target compounds by the introduction of different functional groups.
In this work, a synthetic approach to obtaining multivalent thiacalix[4]arene derivatives containing different amounts of amine and hydroxyl groups at the lower rim was proposed and developed. The antibacterial properties of the synthesized compounds were also evaluated, and structure–activity relationships were established. The obtained results open up the possibility of using these compounds as antibacterial agents.

2. Materials and Methods

2.1. General Experimental Information

More details on the equipment, methods of confirmation, and establishment of the compounds structure (NMR, IR, and mass spectra of the synthesized compounds, antimicrobial and cytotoxic assay, dynamic light scattering data) are described in the Supplementary Materials.
Most chemicals (acryloyl chloride, triethylamine, ethylenediamine, N,N-dimethylethylenediamine, ethanolamine, and diethanolamine) were purchased from Sigma-Aldrich, Dortmund, Germany. TRIS buffer (pH = 7.4, 150 mM NaCl) was purchased from Fisher Scientific. Organic solvents were purified in accordance with standard procedures. Deionized water with resistivity >18.0 MΩ cm (Millipore-Q, Simplicity® water purification system, Merck-Millipore, Molsheim, France) was used for the solutions preparation. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (POPG) was purchased from Avanti Polar Lipids, Birmingham, AL, USA.
Thiacalix[4]arene derivatives 1–3 (cone, partial cone, and 1,3-alternate conformations, respectively) and monomer analogue (p-tert-butylphenol derivative) 19 were synthesized by the previously described procedure [61].

2.2. General Procedure for the Synthesis of Compounds 4–6

The solution of acryloyl chloride (0.27 mL, 3.27 mmol) in 10 mL of corresponding solvent was added dropwise to an ice-cooled (0 °C) mixture of compounds 1–3 (1.00 g, 0.74 mmol) and triethylamine (0.93 mL, 6.89 mmol) in 20 mL of CH2Cl2 (for compounds 1 or 3) or CHCl3 (for compound 2). The reaction mixture was stirred for 12 h at room temperature. Afterward, the solvent was removed on a rotary evaporator, and the residue was washed with water (3 × 20 mL). Then, the wet residue was dissolved in a minimal amount of hot ethanol and poured into 25 mL of water. The resulting suspension was centrifuged, and the precipitate was dried under reduced pressure.

2.2.1. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(acrylamido)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 4 in cone conformation. Yield: 0.98 g (85%). White Powder, mp 88 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.06 (s, 36H, (CH3)3C), 1.24 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.40 (m, 8H, CH2CH2CH2NH), 1.45 (m, 8H, C(O)NHCH2CH2CH2), 3.05–3.13 (m, 8H, CH2NHC(O)CH=CH2), 3.13–3.19 (m, 8H, OCH2C(O)NHCH2), 4.76 (s, 8H, OCH2C(O)), 5.54 (dd, 4H, part of ABX system, CH=CH2, 2JHH = 2.5, 3JHH = 10.0), 6.05 (dd, 4H, part of ABX system, CH=CH2, 2JHH = 2.5, 3JHH = 17.1), 6.19 (dd, 4H, part of ABX system, CH=CH2, 3JHH = 10.0, 3JHH = 17.1), 7.38 (s, 8H, ArH), 8.05 (br.t, 4H, NHC(O)CH=CH2), 8.34 (br.t, 4H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.22, 26.25, 29.05, 29.11, 30.73, 33.89, 38.43, 38.49, 73.81, 124.73, 128.00, 131.88, 134.37, 146.48, 157.70, 164.41, 167.67.
FTIR ATR (ν, cm−1): 3287 (N-H), 3075 (N-H), 1653 (C(O)NH, amide I), 1624 (C=C), 1540 (C(O)NH, amide II), 1093 (CPhOCH2).
ESI-HRMS, Calculated [M + H + NEt3]+ m/z = 1663.9219, [M + Na]+ m/z = 1583.7801, [M + 2H + NEt3]2+ m/z = 831.9629, [M + 2H]2+ m/z = 781.4027. Found [M + H + NEt3]+ m/z = 1663.9155, [M + Na]+ m/z = 1583.7760, [M + 2H + NEt3]2+ m/z = 831.9606, [M + 2H]2+ m/z = 781.3992.

2.2.2. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(acrylamido)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 5 in partial cone conformation. Yield: 1.11 g (96%). White Powder, mp 84 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.00 (s, 18H, (CH3)3C), 1.12–1.54 (m, 32H, C(O)NHCH2CH2CH2CH2, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 1.25 (s, 9H, (CH3)3C), 1.27 (s, 9H, (CH3)3C), 2.95–3.25 (m, 16H, CH2NHC(O)CH=CH2, OCH2C(O)NHCH2), 4.10 (d, 2H, OCH2C(O), 2JHH = 13.5), 4.47 (s, 2H, OCH2C(O)), 4.57 (s, 2H, OCH2C(O)), 4.87 (d, 2H, OCH2C(O), 2JHH = 13.5), 5.54 (dd, 4H, part of ABX system, CH=CH2, 2JHH = 2.3, 3JHH = 10.0), 6.04 (dd, 4H, part of ABX system, CH=CH2, 2JHH = 2.3, 3JHH = 17.1), 6.13–6.24 (m, 4H, part of ABX system), 6.99 (d, 2H, ArH, 2JHH = 2.5), 7.58 (s, 2H, ArH), 7.62 (d, 2H, ArH, 2JHH = 2.5), 7.69 (s, 2H, ArH), 8.05 (br.t, 4H, NHC(O)CH=CH2), 8.18–8.24 (m, 3H, OCH2CONH), 8.18–8.24 (br.t, 1H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.15, 26.26, 28.99, 29.08, 29.14, 30.75, 31.04, 33.81, 33.85, 33.90, 38.18, 38.51, 38.56, 68.90, 72.66, 72.89, 124.79, 126.45, 126.87, 127.38, 128.01, 131.90, 133.62, 133.96, 134.30, 135.49, 144.60, 145.44, 146.61, 156.52, 157.59, 159.45, 164.47, 166.79, 167.44, 168.08.
FTIR ATR (ν, cm−1): 3285 (N-H), 3074 (N-H), 1652 (C(O)NH, amide I), 1625 (C=C), 1538 (C(O)NH, amide II), 1088 (CPhOCH2).
ESI-HRMS, Calculated [M + H]+ m/z = 1562.8015, [M + Na]+ m/z = 1583.7801, [M + 2H]2+ m/z = 781.4027. Found [M + H]+ m/z = 1562.7946, [M + Na]+ m/z = 1583.7714, [M + 2H]2+ m/z = 781.4010.

2.2.3. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(acrylamido)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 6 in 1,3-alternate conformation. Yield: 1.07 g (92%). White Powder, mp 91 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.18 (s, 36H, (CH3)3C), 1.25 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.36–1.46 (m, 16H, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 3.02–3.14 (m, 16H, CH2NHC(O)CH=CH2, OCH2C(O)NHCH2), 3.93 (s, 8H, OCH2C(O)), 5.55 (dd, 4H, part of ABX system, CH=CH2, 2JHH = 2.5, 3JHH = 10.0), 6.05 (dd, 4H, part of ABX system, CH=CH2, 2JHH = 2.5, 3JHH = 17.1), 6.19 (dd, 4H, part of ABX system, CH=CH2, 3JHH = 10.0, 3JHH = 17.1), 7.51 (s, 8H, ArH), 7.69 (br.t, 4H, OCH2CONH), 8.05 (br.t, 4H, NHC(O)CH=CH2).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.20, 26.30, 29.04, 29.10, 30.74, 33.86, 38.45, 38.72, 70.40, 124.75, 127.47, 131.89, 132.07, 146.23, 156.52, 164.41, 166.87.
FTIR ATR (ν, cm−1): 3297 (N-H), 3072 (N-H), 1653 (C(O)NH, amide I), 1626 (C=C), 1533 (C(O)NH, amide II), 1086 (CPhOCH2).
ESI-HRMS, Calculated [M + H]+ m/z = 1562.8015, [M + 2H]2+ m/z = 781.4027. Found [M + H]+ m/z = 1562.7938, [M + 2H]2+ m/z = 781.4122.

2.3. General Procedure for the Synthesis of Compounds 7–12

The corresponding diamine (1.92 mmol) (ethylenediamine for 7–9 and N,N-dimethylethylenediamine for 10–12) was added to the solution of 4–6 (0.10 g, 0.064 mmol) in 7 mL of methanol. The reaction mixture was refluxed for 60 h (for 7–9) or 90 h (for 10–12). Then the solvent was evaporated under reduced pressure, and the remaining diamine was removed by azeotropic distillation (toluene:methanol mixture, 9:1). Afterward, the remaining toluene was removed by azeotropic distillation with methanol. The residue was dried under reduced pressure.

2.3.1. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-aminoethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 7 in cone conformation. Yield: 0.11 g (97%). White Solid Foam, mp 90 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.07 (s, 36H, (CH3)3C), 1.23 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.36 (m, 8H, CH2CH2CH2NH), 1.45 (m, 8H, C(O)NHCH2CH2CH2), 2.18 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.47 (m, 8H, NH2CH2CH2NH), 2.57 (m, 8H, NH2CH2CH2NH), 2.66 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.96–3.05 (m, 8H, CH2NHC(O)CH2CH2), 3.10–3.22 (m, 8H, OCH2C(O)NHCH2), 4.75 (s, 8H, OCH2C(O)), 7.38 (s, 8H, ArH), 7.92 (br.t, 4H, NHC(O)CH2CH2), 8.34 (br.t, 4H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.19, 29.10, 30.70, 33.84, 35.95, 38.28, 38.42, 41.13, 45.51, 51.69, 73.79, 127.98, 134.34, 146.45, 157.68, 167.62, 171.15.
FTIR ATR (ν, cm−1): 3287 (N-H), 1649 (C(O)NH, amide I), 1545 (C(O)NH, amide II), 1094 (CPhOCH2).
ESI-HRMS, Calculated [M + Na]+ m/z = 1825.0584, [M + 2H]2+ m/z = 902.0419, [M + 3H]3+ m/z = 601.6970. Found [M + Na]+ m/z = 1825.0580, [M + 2H]2+ m/z = 902.0439, [M + 3H]3+ m/z = 601.6988.

2.3.2. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-aminoethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 8 in partial cone conformation. Yield: 0.11 g (92%). White Solid Foam, mp 88 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.00 (s, 18H, (CH3)3C), 1.13–1.53 (m, 32H, C(O)NHCH2CH2CH2CH2, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 1.26 (s, 9H, (CH3)3C), 1.29 (s, 9H, (CH3)3C), 2.18 (br.t, 8H, NHCH2CH2CO), 2.48 (m, 8H, NH2CH2CH2NH), 2.58 (m, 8H, NH2CH2CH2NH), 2.66 (br.t, 8H, NHCH2CH2C(O)), 2.94–3.13 (m, 16H, CH2NHC(O)CH2CH2, OCH2C(O)NHCH2), 4.11 (d, 2H, OCH2C(O), 2JHH = 13.4), 4.47 (s, 2H, OCH2C(O)), 4.58 (s, 2H, OCH2C(O)), 4.86 (d, 2H, OCH2C(O), 2JHH = 13.4), 7.00 (br.d, 2H, ArH), 7.59 (s, 2H, ArH), 7.62 (br.d, 2H, ArH), 7.70 (s, 2H, ArH), 7.92 (br.t, 4H, NHC(O)CH2CH2), 8.21 (m, 3H, OCH2CONH), 8.18–8.24 (br.t, 1H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.20, 26.26, 28.96, 29.06, 29.12, 30.72, 30.77, 31.01, 33.76, 33.86, 35.74, 38.18, 38.33, 38.53, 45.24, 49.40, 68.92, 72.64, 72.84, 126.39, 126.82, 127.38, 127.96, 133.57, 133.91, 134.28, 135.46, 144.60, 145.36, 146.56, 156.50, 157.56, 159.41, 166.74, 167.38, 167.99, 171.11.
FTIR ATR (ν, cm−1): 3279 (N-H), 3064 (N-H), 1646 (C(O)NH, amide I), 1545 (C(O)NH, amide II), 1089 (CPhOCH2).
ESI-HRMS, Calculated [M + 2H]2+ m/z = 902.0419, [M + 3H]3+ m/z = 601.6970. Found [M + 2H]2+ m/z = 902.0443, [M + 3H]3+ m/z = 601.6992.

2.3.3. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-aminoethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 9 in 1,3-alternate conformation. Yield: 0.10 g (90%). White Solid Foam, mp 88 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.19 (s, 36H, (CH3)3C), 1.24 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.37 (m, 8H, CH2CH2CH2NH), 1.43 (m, 8H, C(O)NHCH2CH2CH2), 2.18 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.47 (m, 8H, NH2CH2CH2NH), 2.56 (m, 8H, NH2CH2CH2NH), 2.66 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.96–3.03 (m, 8H, CH2NHC(O)CH2CH2), 3.04–3.11 (m, 8H, OCH2C(O)NHCH2), 3.93 (s, 8H, OCH2C(O)), 7.52 (s, 8H, ArH), 7.70 (br.t, 4H, OCH2CONH), 7.92 (br.t, 4H, NHC(O)CH2CH2).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.18, 26.30, 29.13, 30.75, 33.86, 36.04, 38.27, 38.72, 41.31, 45.62, 52.01, 70.45, 127.47, 132.11, 146.22, 156.54, 166.87, 171.17.
FTIR ATR (ν, cm−1): 3299 (N-H), 3064 (N-H), 1646 (C(O)NH, amide I), 1534 (C(O)NH, amide II), 1085 (CPhOCH2).
ESI-HRMS, Calculated [M + Na]+ m/z = 1825.0584, [M + 2H]2+ m/z = 902.0419, [M + 3H]3+ m/z = 601.6970, [M + 4H]4+ m/z = 451.5246. Found [M + Na]+ m/z = 1825.0567, [M + 2H]2+ m/z = 902.0445, [M + 3H]3+ m/z = 601.6989, [M + 4H]4+ m/z = 451.5266.

2.3.4. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-(N,N-dimethylamino)ethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 10 in cone conformation. Yield: 0.12 g (98%). White Solid Foam, mp 60 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.06 (s, 36H, (CH3)3C), 1.23 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.35 (m, 8H, CH2CH2CH2NH), 1.45 (m, 8H, C(O)NHCH2CH2CH2), 2.08 (s, 24H, N(CH3)2), 2.18 (t, 8H, NHCH2CH2C(O), 3JHH = 6.7), 2.25 (br.t, 8H, (CH3)2NCH2CH2NH), 2.53 (br.t, 8H, (CH3)2NCH2CH2NH), 2.66 (t, 8H, NHCH2CH2C(O), 3JHH = 6.7), 2.95–3.04 (m, 8H, CH2NHC(O)CH2CH2), 3.10–3.20 (m, 8H, OCH2C(O)NHCH2), 4.75 (s, 8H, OCH2C(O)), 7.37 (s, 8H, ArH), 7.94 (br.t, 4H, NHC(O)CH2CH2), 8.34 (br.t, 4H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.26, 29.17, 30.75, 33.90, 35.80, 38.31, 38.45, 45.27, 45.75, 46.65, 58.79, 73.84, 128.02, 134.38, 146.48, 157.74, 167.66, 171.14.
FTIR ATR (ν, cm−1): 3300 (N-H), 3073 (N-H), 1648 (C(O)NH, amide I), 1542 (C(O)NH, amide II), 1095 (CPhOCH2).
ESI-HRMS, Calculated [M + Na]+ m/z = 1937.1836, [M + 2H]2+ m/z = 958.1045, [M + 3H]3+ m/z = 639.0721. Found [M + Na]+ m/z = 1937.1793, [M + 2H]2+ m/z = 958.1057, [M + 3H]3+ m/z = 639.0736.

2.3.5. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-(N,N-dimethylamino)ethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 11 in partial cone conformation. Yield: 0.11 g (94%). White Solid Foam, mp 66 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.00 (s, 18H, (CH3)3C), 1.13–1.54 (m, 32H, C(O)NHCH2CH2CH2CH2, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 1.26 (s, 9H, (CH3)3C), 1.28 (s, 9H, (CH3)3C), 2.09 (s, 24H, N(CH3)2), 2.19 (br.t, 8H, NHCH2CH2C(O)), 2.27 (br.t, 8H, (CH3)2NCH2CH2NH), 2.55 (br.t, 8H, (CH3)2NCH2CH2NH), 2.70 (br.t, 8H, NHCH2CH2C(O)), 2.96–3.22 (m, 16H, CH2NHC(O)CH2CH2, OCH2C(O)NHCH2), 4.11 (d, 2H, OCH2C(O), 2JHH = 13.4), 4.46 (s, 2H, OCH2C(O)), 4.58 (s, 2H, OCH2C(O)), 4.86 (d, 2H, OCH2C(O), 2JHH = 13.4), 6.99 (br.d, 2H, ArH), 7.59 (s, 2H, ArH), 7.62 (br.d, 2H, ArH), 7.70 (s, 2H, ArH), 7.93 (br.t, 4H, NHC(O)CH2CH2), 8.20 (m, 3H, OCH2CONH), 8.29 (br.t, 1H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.21, 26.29, 28.98, 29.14, 29.22, 30.74, 30.79, 31.03, 33.78, 35.17, 38.17, 38.31, 38.38, 38.54, 45.20, 45.43, 46.29, 58.21, 68.91, 72.64, 72.85, 126.42, 126.84, 127.37, 127.97, 133.60, 133.92, 134.28, 135.48, 144.56, 145.36, 146.56, 156.51, 157.57, 159.44, 166.73, 167.37, 168.00, 170.90.
FTIR ATR (ν, cm−1): 3290 (N-H), 3072 (N-H), 1648 (C(O)NH, amide I), 1542 (C(O)NH, amide II), 1091 (CPhOCH2).
ESI-HRMS, Calculated [M + Na]+ m/z = 1937.1836, [M + 2H]2+ m/z = 958.1045, [M + 3H]3+ m/z = 639.0721, [M + 4H]4+ m/z = 479.5559. Found [M + Na]+ m/z = 1937.1832, [M + 2H]2+ m/z = 958.1070, [M + 3H]3+ m/z = 639.0745, [M + 4H]4+ m/z = 479.5578.

2.3.6. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-(N,N-dimethylamino)ethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 12 in 1,3-alternate conformation. Yield: 0.12 g (97%). White Solid Foam, mp 62 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.19 (s, 36H, (CH3)3C), 1.20–1.26 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.36 (m, 8H, CH2CH2CH2NH), 1.43 (m, 8H, C(O)NHCH2CH2CH2), 2.09 (s, 24H, N(CH3)2), 2.18 (br.t, 8H, NHCH2CH2C(O)), 2.25 (br.t, 8H, (CH3)2NCH2CH2NH), 2.54 (br.t, 8H, (CH3)2NCH2CH2NH), 2.69 (br.t, 8H, NHCH2CH2C(O)), 2.96–3.02 (m, 8H, CH2NHC(O)CH2CH2), 3.03–3.12 (m, 8H, OCH2C(O)NHCH2), 3.92 (s, 8H, OCH2C(O)), 7.51 (s, 8H, ArH), 7.70 (br.t, 4H, OCH2CONH), 7.95 (br.t, 4H, NHC(O)CH2CH2).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.20, 26.34, 29.15, 30.77, 33.87, 35.82, 38.28, 38.74, 45.29, 45.76, 46.65, 58.80, 70.42, 127.49, 132.11, 146.23, 156.54, 166.87, 171.13.
FTIR ATR (ν, cm−1): 3299 (N-H), 3072 (N-H), 1647 (C(O)NH, amide I), 1536 (C(O)NH, amide II), 1086 (CPhOCH2).
ESI-HRMS, Calculated [M + Na]+ m/z = 1937.1836, [M + 2H]2+ m/z = 958.1045, [M + 3H]3+ m/z = 639.0721, [M + 4H]4+ m/z = 479.5559. Found [M + Na]+ m/z = 1937.1816, [M + 2H]2+ m/z = 958.1060, [M + 3H]3+ m/z = 639.0745, [M + 4H]4+ m/z = 479.5575.

2.4. General Procedure for the Synthesis of Compounds 13–15

Ethanolamine (0.23 mL, 3.84 mmol) was added to the solution of 4–6 (0.20 g, 0.128 mmol) in methanol (10 mL). The reaction mixture was refluxed for 90 h. Afterward, the solvent was evaporated under reduced pressure. Then 15 mL of water was added to the residue, and the resulting suspension was centrifuged. The precipitate was dried under reduced pressure.

2.4.1. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-hydroxyethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 13 in cone conformation. Yield: 0.19 g (84%). White Powder, mp 72 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.07 (s, 36H, (CH3)3C), 1.23 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.36 (m, 8H, CH2CH2CH2NH), 1.44 (m, 8H, C(O)NHCH2CH2CH2), 2.18 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.53 (t, 8H, NHCH2CH2OH, 3JHH = 5.8), 2.68 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.95–3.03 (m, 8H, CH2NHC(O)CH2CH2), 3.11–3.19 (m, 8H, OCH2C(O)NHCH2), 3.41 (t, 8H, NHCH2CH2OH, 3JHH = 5.8), 4.75 (s, 8H, OCH2C(O)), 7.38 (s, 8H, ArH), 7.90 (br.t, 4H, NHC(O)CH2CH2), 8.33 (br.t, 4H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.25, 29.15, 30.74, 33.90, 35.91, 38.33, 38.45, 45.67, 51.47, 60.30, 73.82, 128.02, 134.38, 146.48, 157.73, 167.67, 171.14.
FTIR ATR (ν, cm−1): 3299 (N-H), 3076 (N-H), 1646 (C(O)NH, amide I), 1542 (C(O)NH, amide II), 1094 (CPhOCH2).
ESI-HRMS, Calculated [M + 2H]2+ m/z = 904.0099, [M + 3H]3+ m/z = 603.0090. Found [M + 2H]2+ m/z = 904.0121, [M + 3H]3+ m/z = 603.0109.

2.4.2. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-hydroxyethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 14 in partial cone conformation. Yield: 0.19 g (82%). White Powder, mp 65 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.00 (s, 18H, (CH3)3C), 1.12–1.53 (m, 32H, C(O)NHCH2CH2CH2CH2, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 1.26 (s, 9H, (CH3)3C), 1.28 (s, 9H, (CH3)3C), 2.19 (br.t, 8H, NHCH2CH2C(O)), 2.54 (br.t, 8H, NHCH2CH2OH), 2.68 (br.t, 8H, NHCH2CH2C(O)), 2.94–3.03 (m, 8H, CH2NHC(O)CH2CH2), 3.04–3.12 (m, 8H, OCH2C(O)NHCH2), 3.41 (br.t, 8H, NHCH2CH2OH), 2.95–3.03 (m, 8H, CH2NHC(O)CH2CH2), 3.05–3.23 (m, 8H, OCH2C(O)NHCH2), 4.11 (d, 2H, OCH2C(O), 2JHH = 13.4), 4.47 (s, 2H, OCH2C(O)), 4.57 (s, 2H, OCH2C(O)), 4.86 (d, 2H, OCH2C(O), 2JHH = 13.4), 6.99 (br.d, 2H, ArH), 7.59 (s, 2H, ArH), 7.62 (br.d, 2H, ArH), 7.70 (s, 2H, ArH), 7.90 (br.t, 4H, NHC(O)CH2CH2), 8.20 (m, 3H, OCH2CONH), 8.29 (br.t, 1H, OCH2CONH).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.22, 26.27, 28.97, 29.07, 29.14, 30.73, 30.78, 31.02, 33.78, 33.82, 33.89, 35.92, 38.18, 38.32, 38.37, 38.55, 45.68, 51.47, 60.30, 68.94, 72.66, 72.87, 126.40, 126.84, 127.38, 127.98, 133.59, 133.93, 134.30, 135.48, 144.61, 145.39, 146.57, 156.51, 157.57, 159.43, 166.75, 167.39, 168.00, 171.14.
FTIR ATR (ν, cm−1): 3290 (N-H), 3076 (N-H), 1648 (C(O)NH, amide I), 1540 (C(O)NH, amide II), 1088 (CPhOCH2).
ESI-HRMS, Calculated [M + Na]+ m/z = 1828.9945, [M + 2H]2+ m/z = 904.0099, [M + 3H]3+ m/z = 603.0090. Found [M + Na]+ m/z = 1828.9949, [M + 2H]2+ m/z = 904.0124, [M + 3H]3+ m/z = 603.0111.

2.4.3. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(2-hydroxyethyl)aminopropanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 15 in 1,3-alternate conformation. Yield: 0.18 g (79%). White Powder, mp 80 °C

1H NMR (DMSO-d6, δ, ppm, J/Hz): 1.19 (s, 36H, (CH3)3C), 1.22 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.37 (m, 8H, CH2CH2CH2NH), 1.43 (m, 8H, C(O)NHCH2CH2CH2), 2.19 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.54 (t, 8H, NHCH2CH2OH, 3JHH = 5.7), 2.68 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.94–3.03 (m, 8H, CH2NHC(O)CH2CH2), 3.04–3.12 (m, 8H, OCH2C(O)NHCH2), 3.41 (t, 8H, NHCH2CH2OH, 3JHH = 5.7), 3.94 (s, 8H, OCH2C(O)), 7.52 (s, 8H, ArH), 7.69 (br.t, 4H, OCH2CONH), 7.90 (br.t, 4H, NHC(O)CH2CH2).
13C{1H} NMR (DMSO-d6, δ, ppm): 26.19, 26.32, 29.13, 30.76, 33.87, 35.95, 38.30, 38.74, 45.70, 51.48, 60.31, 70.43, 127.48, 132.11, 146.23, 156.54, 166.89, 171.13.
FTIR ATR (ν, cm−1): 3300 (N-H), 1647 (C(O)NH, amide I), 1536 (C(O)NH, amide II), 1085 (CPhOCH2).
ESI-HRMS, Calculated [M + 2H]2+ m/z = 904.0099, [M + 3H]3+ m/z = 603.0090. Found [M + 2H]2+ m/z = 904.0129, [M + 3H]3+ m/z = 603.0113.

2.5. General Procedure for the Synthesis of Compounds 16–18

Diethanolamine (0.38 mL, 3.84 mmol) was added to the solution of 4–6 (0.20 g, 0.128 mmol) in methanol (10 mL) in glass cylindrical pressure vessel equipped with magnetic stirrer. The reaction mixture was stirred at 100 °C for 40 h. Afterward, the solvent was evaporated under reduced pressure. Then 15 mL of water was added to the residue, and the resulting suspension was centrifuged. The precipitate was dried under reduced pressure.

2.5.1. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(N,N-di(2-hydroxyethyl)amino)propanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 16 in cone conformation. Yield: 0.20 g (78%). White Powder, mp 85 °C

1H NMR (CD3OD, δ, ppm, J/Hz): 1.14 (s, 36H, (CH3)3C), 1.37 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.52 (m, 8H, CH2CH2CH2NH), 1.60 (m, 8H, C(O)NHCH2CH2CH2), 2.35 (t, 8H, NHCH2CH2C(O), 3JHH = 6.7), 2.65 (t, 16H, N(CH2CH2OH)2, 3JHH = 5.7), 2.83 (t, 8H, NHCH2CH2C(O), 3JHH = 6.7), 3.17 (m, 8H, CH2NHC(O)CH2CH2), 3.33 (m, 8H, OCH2C(O)NHCH2), 3.60 (t, 16H, N(CH2CH2OH)2, 3JHH = 5.7), 4.89 (s, 8H, OCH2C(O)), 7.44 (s, 8H, ArH).
13C{1H} NMR (CD3OD, δ, ppm): 27.76, 27.78, 30.34, 30.54, 31.63, 31.66, 34.70, 35.20, 40.29, 52.11, 57.24, 60.70, 75.23, 129.77, 136.00, 148.79, 159.20, 170.57, 174.97.
FTIR ATR (ν, cm−1): 3300 (N-H), 1648 (C(O)NH, amide I), 1546 (C(O)NH, amide II), 1094 (CPhOCH2).
ESI-HRMS, Calculated [M + 2H]2+ m/z = 992.0623, [M + 3H]3+ m/z = 661.7107, [M + 4H]4+ m/z = 496.5348. Found [M + 2H]2+ m/z = 992.0648, [M + 3H]3+ m/z = 661.7132, [M + 4H]4+ m/z = 496.5368.

2.5.2. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(N,N-di(2-hydroxyethyl)amino)propanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 17 in partial cone conformation. Yield: 0.20 g (79%). White Powder, mp 81 °C

1H NMR (CD3OD, δ, ppm, J/Hz): 1.08 (s, 18H, (CH3)3C), 1.20–1.72 (m, 32H, C(O)NHCH2CH2CH2CH2, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 1.08 (s, 18H, (CH3)3C), 2.34 (br.t, 8H, NHCH2CH2C(O)), 2.65 (br.t, 16H, N(CH2CH2OH)2), 2.83 (t, 8H, NHCH2CH2C(O)), 3.10–3.25 (m, 16H, CH2NHC(O)CH2CH2, OCH2C(O)NHCH2), 3.60 (br.t, 16H, N(CH2CH2OH)2), 4.21 (d, 2H, OCH2C(O), 2JHH = 13.8), 4.60 (s, 2H, OCH2C(O)), 4.86 (s, 2H, OCH2C(O)), 5.02 (d, 2H, OCH2C(O), 2JHH = 13.8), 7.11 (br.d, 2H, ArH), 7.60 (br.d, 2H, ArH), 7.65 (s, 2H, ArH), 7.83 (s, 2H, ArH).
13C{1H} NMR (CD3OD, δ, ppm): 27.71, 27.78, 27.84, 30.37, 30.47, 30.59, 31.68, 31.76, 31.85, 34.71, 35.20, 35.29, 39.94, 40.30, 40.37, 52.13, 57.26, 60.72, 70.59, 74.00, 74.53, 128.03, 129.45, 130.07, 134.67, 135.45, 135.94, 137.50, 147.11, 147.63, 148.87, 157.81, 159.38, 160.83, 170.20, 170.91, 175.06.
FTIR ATR (ν, cm−1): 3290 (N-H), 3083 (N-H), 1646 (C(O)NH, amide I), 1542 (C(O)NH, amide II), 1085 (CPhOCH2).
ESI-HRMS, Calculated [M + 2H]2+ m/z = 992.0623, [M + 3H]3+ m/z = 661.7107, [M + 4H]4+ m/z = 496.5348. Found [M + 2H]2+ m/z = 992.0648, [M + 3H]3+ m/z = 661.7134, [M + 4H]4+ m/z = 496.5369.

2.5.3. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis[N-(6-(N-(3-(N,N-di(2-hydroxyethyl)amino)propanoyl)amino)hexyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene 18 in 1,3-alternate conformation. Yield: 0.20 g (79%). White Powder, mp 92 °C

1H NMR (CD3OD, δ, ppm, J/Hz): 1.28 (s, 36H, (CH3)3C), 1.34 (m, 16H, C(O)NHCH2CH2CH2CH2), 1.47–1.60 (m, 16H, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 2.35 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 2.65 (t, 16H, N(CH2CH2OH)2, 3JHH = 5.7), 2.84 (t, 8H, NHCH2CH2C(O), 3JHH = 6.8), 3.12–3.23 (m, 16H, CH2NHC(O)CH2CH2, OCH2C(O)NHCH2), 3.61 (t, 16H, N(CH2CH2OH)2, 3JHH = 5.7), 4.16 (s, 8H, OCH2C(O)), 7.59 (s, 8H, ArH).
13C{1H} NMR (CD3OD, δ, ppm): 26.65, 26.79, 29.55, 31.21, 33.92, 34.33, 38.80, 39.21, 51.30, 56.64, 59.65, 70.88, 127.93, 132.59, 146.70, 157.00, 167.36, 171.77.
FTIR ATR (ν, cm−1): 3300 (N-H), 3074 (N-H), 1646 (C(O)NH, amide I), 1539 (C(O)NH, amide II), 1265 (C(O)NH, amide III), 1027 (CPhOCH2).
ESI-HRMS, Calculated [M + 3H]3+ m/z = 661.7107, [M + 4H]4+ m/z = 496.5348. Found [M + 3H]3+ m/z = 661.7132, [M + 4H]4+ m/z = 496.5368.

2.6. Procedure for the Synthesis of Compound 20

The solution of acryloyl chloride (0.16 mL, 1.94 mmol) in 3 mL of CH2Cl2 was added dropwise to an ice-cooled (0 °C) mixture of 19 (0.54 g, 1.76 mmol) and triethylamine (0.49 mL, 3.52 mmol) in 5 mL of CH2Cl2. The reaction mixture was stirred for 3 h at room temperature. Afterward, the reaction mixture was washed with water (5 × 10 mL). Then the organic layer was separated and dried under the anhydrous Na2SO4. The solvent was removed on a rotary evaporator, and the residue was dried under reduced pressure.

N-(6-(2-(4-(tert-butyl)phenoxy)acetamido)hexyl)acrylamide 20. Yield: 0.58 g (91%). White Powder, mp 77 °C

1H NMR (CDCl3, δ, ppm, J/Hz): 1.29 (s, 9H, (CH3)3C), 1.35 (m, 4H, C(O)NHCH2CH2CH2CH2), 1.54 (m, 4H, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 3.33 (m, 4H, OCH2C(O)NHCH2, CH2NHC(O)CH=CH2), 4.46 (s, 2H, OCH2C(O)), 5.62 (dd, 1H, part of ABX system, CH=CH2, 2JHH = 1.6, 3JHH = 10.2), 5.82 (br.t, 1H, NHC(O)CH=CH2), 6.10 (dd, 1H, part of ABX system, CH=CH2, 3JHH = 10.2, 3JHH = 17.0), 6.28 (dd, 1H, part of ABX system, CH=CH2, 2JHH = 1.6, 3JHH = 17.0), 6.65 (br.t, 1H, OCH2CONH), 6.85 (m, 2H, ArH), 7.33 (m, 2H, ArH).
13C{1H} NMR (CDCl3, δ, ppm): 26.18, 26.23, 29.44, 29.56, 31.58, 34.30, 38.72, 39.29, 67.60, 114.27, 126.35, 126.70, 131.07, 145.06, 155.10, 165.69, 168.60.
FTIR ATR (ν, cm−1): 3277 (N-H), 3094 (N-H), 1651 (C(O)NH, amide I), 1624 (C=C), 1513 (C(O)NH, amide II), 1094 (CPhOCH2).

2.7. General Procedure for the Synthesis of Compounds 21 and 22

The corresponding diamine (3.10 mmol) (ethylenediamine for 21 and N,N-dimethylethylenediamine for 22) was added to the solution of 20 (0.14 g, 0.39 mmol) in 6 mL of methanol. The reaction mixture was refluxed for 15 h. Then the solvent was evaporated under reduced pressure, and the remaining diamine was removed via azeotropic distillation (toluene:methanol mixture, 9:1). Afterward, the remaining toluene was removed via azeotropic distillation with methanol. The residue was dried under reduced pressure.

2.7.1. 3-((2-aminoethyl)amino)-N-(6-(2-(4-(tert-butyl)phenoxy)acetamido)hexyl)propanamide 21. Yield: 0.16 g (98%). Viscous Oil

1H NMR (CDCl3, δ, ppm, J/Hz): 1.29 (s, 9H, (CH3)3C), 1.33 (m, 4H, C(O)NHCH2CH2CH2CH2), 1.48 (m, 2H, CH2CH2CH2NH), 1.54 (m, 2H, C(O)NHCH2CH2CH2), 2.37 (t, 2H, NHCH2CH2C(O), 3JHH = 5.9), 2.69 (t, 2H, NH2CH2CH2NH, 3JHH = 5.8), 2.83 (br.t, 2H, NH2CH2CH2NH), 2.89 (t, 2H, NHCH2CH2C(O), 3JHH = 5.9), 3.22 (m, 2H, CH2NHC(O)CH2CH2), 3.32 (m, 2H, OCH2C(O)NHCH2), 4.46 (s, 2H, OCH2C(O)), 6.66 (br.t, 1H, OCH2CONH), 6.85 (m, 2H, ArH), 7.32 (m, 2H, ArH), 7.48 (br.t, 1H, NHC(O)CH2CH2).
13C{1H} NMR (CDCl3, δ, ppm): 26.33, 26.45, 29.42, 29.54, 31.58, 34.29, 35.22, 38.86, 39.15, 40.49, 45.28, 50.25, 67.58, 114.28, 126.69, 145.04, 155.09, 168.63, 172.43.
FTIR ATR (ν, cm−1): 3289 (N-H), 3065 (N-H), 1647 (C(O)NH, amide I), 1512 (C(O)NH, amide II), 1060 (CPhOCH2).

2.7.2. N-(6-(2-(4-(tert-butyl)phenoxy)acetamido)hexyl)-3-((2-(dimethylamino)ethyl)amino)propanamide 22. Yield: 0.13 g (75%). Viscous Oil

1H NMR (CDCl3, δ, ppm, J/Hz): 1.29 (s, 9H, (CH3)3C), 1.33 (m, 4H, C(O)NHCH2CH2CH2CH2), 1.48 (m, 2H, CH2CH2CH2NH), 1.54 (m, 2H, C(O)NHCH2CH2CH2), 2.22 (s, 6H, N(CH3)2), 2.36 (t, 2H, NHCH2CH2C(O), 3JHH = 5.9), 2.41 (t, 2H, (CH3)2NCH2CH2NH, 3JHH = 6.0), 2.70 (br.t, 2H, (CH3)2CH2CH2NH), 2.88 (br.t, 2H, NHCH2CH2C(O)), 3.20 (m, 2H, CH2NHC(O)CH2CH2), 3.32 (m, 2H, OCH2C(O)NHCH2), 4.46 (s, 2H, OCH2C(O)), 6.63 (br.t, 1H, OCH2CONH), 6.85 (m, 2H, ArH), 7.32 (m, 2H, ArH), 7.75 (br.t, 1H, NHC(O)CH2CH2).
13C{1H} NMR (CDCl3, δ, ppm): 26.45, 26.56, 29.51, 29.58, 31.57, 34.28, 35.35, 38.90, 39.01, 45.55, 45.69, 46.66, 58.53, 67.59, 114.26, 126.67, 145.02, 155.09, 168.50, 172.62.
FTIR ATR (ν, cm−1): 3300 (N-H), 3065 (N-H), 1649 (C(O)NH, amide I), 1512 (C(O)NH, amide II), 1056 (CPhOCH2).

2.8. General Procedure for the Synthesis of Compounds 23 and 24

The corresponding alkanolamine (3.10 mmol) (ethanolamine for 23 and diethanolamine for 24) was added to the solution of 20 (0.14 g, 0.39 mmol) in 6 mL of methanol. The reaction mixture was refluxed for 15 h. Then the solvent was evaporated under reduced pressure. Then, 5 mL of water was added to the residue, and the resulting emulsion was centrifuged. The precipitate was dried under reduced pressure.

2.8.1. N-(6-(2-(4-(tert-butyl)phenoxy)acetamido)hexyl)-3-((2-hydroxyethyl)amino)propanamide 23. Yield: 0.08 g (49%). Viscous Oil

1H NMR (CDCl3, δ, ppm, J/Hz): 1.29 (s, 9H, (CH3)3C), 1.34 (m, 4H, C(O)NHCH2CH2CH2CH2), 1.49 (m, 2H, CH2CH2CH2NH), 1.55 (m, 2H, C(O)NHCH2CH2CH2), 2.39 (t, 2H, NHCH2CH2C(O), 3JHH = 5.9), 2.80 (t, 2H, NHCH2CH2OH, 3JHH = 5.1), 2.92 (t, 2H, NHCH2CH2C(O), 3JHH = 5.9), 3.23 (m, 2H, CH2NHC(O)CH2CH2), 3.33 (m, 2H, OCH2C(O)NHCH2), 3.70 (t, 2H, NHCH2CH2OH, 3JHH = 5.1), 4.46 (s, 8H, OCH2C(O)), 6.68 (br.t, 1H, OCH2CONH), 6.85 (m, 2H, ArH), 7.29 (br.t, 1H, NHC(O)CH2CH2), 7.32 (m, 2H, ArH).
13C{1H} NMR (CDCl3, δ, ppm): 26.22, 26.33, 29.34, 29.46, 31.58, 34.30, 35.43, 38.78, 39.07, 45.32, 50.94, 60.76, 67.55, 114.27, 126.71, 145.09, 155.06, 168.76, 172.39.
FTIR ATR (ν, cm−1): 3307 (N-H), 3065 (N-H), 1633 (C(O)NH, amide I), 1540 (C(O)NH, amide II), 1066 (CPhOCH2).

2.8.2. 3-(Bis(2-hydroxyethyl)amino)-N-(6-(2-(4-(tert-butyl)phenoxy)acetamido)hexyl)propanamide 24. Yield: 0.10 g (56%). Viscous Oil

1H NMR (CDCl3, δ, ppm, J/Hz): 1.28 (s, 9H, (CH3)3C), 1.32 (m, 4H, C(O)NHCH2CH2CH2CH2), 1.45–1.59 (m, 4H, CH2CH2CH2NH, C(O)NHCH2CH2CH2), 2.43 (t, 2H, NHCH2CH2C(O), 3JHH = 6.0), 2.75 (t, 4H, N(CH2CH2OH)2, 3JHH = 5.0), 2.94 (t, 2H, NHCH2CH2C(O), 3JHH = 6.0), 3.22 (m, 2H, CH2NHC(O)CH2CH2), 3.31 (m, 2H, OCH2C(O)NHCH2), 3.68 (t, 4H, N(CH2CH2OH)2, 3JHH = 5.0), 4.45 (s, 8H, OCH2C(O)), 6.79 (br.t, 1H, OCH2CONH), 6.84 (m, 2H, ArH), 7.04 (br.t, 1H, NHC(O)CH2CH2), 7.31 (m, 2H, ArH).
13C{1H} NMR (CDCl3, δ, ppm): 25.96, 26.09, 29.16, 29.32, 31.59, 33.86, 34.31, 38.71, 39.35, 51.24, 56.58, 59.03, 67.53, 114.29, 126.72, 145.09, 155.06, 168.88, 172.21.
FTIR ATR (ν, cm−1): 3289 (N-H), 3096 (N-H), 1645 (C(O)NH, amide I), 1512 (C(O)NH, amide II), 1056 (CPhOCH2).

3. Results and Discussion

3.1. Development of an Approach to the Synthesis of Multivalent Derivatives of Thiacalix[4]arene

According to the literature [43,59], most of the previously reported macrocyclic antibacterial compounds based on calix[n]arenes are in cone conformation, in which four lower rim substituents are located on one side of the macrocyclic core (Scheme 1). It was noted that the macrocycle conformation (and consequently the spatial orientation of the substituents) has a significant influence on its antibacterial activity. Therefore, the synthesis of new (thia)calixarenes in different conformations is a promising task. Its solving will allow us to establish new structure–activity relationships for macrocycle conformations and its antibacterial activity and to find the most effective derivatives of (thia)calixarene with improved antibacterial properties. Therefore, we specifically chose thiacalix[4]arene as a macrocyclic platform because its three conformations (cone, partial cone, and 1,3-alternate) can be synthesized in high yields (Figure 1). As starting compounds, we chose thiacalix[4]arene derivatives 1–3, previously obtained in our research group [61], containing four amidoamine fragments at the lower rim of the macrocycle. These macrocycles can be easily obtained in high yields in two steps from the starting p-tert-butylthiacalix[4]arene in three conformations, i.e., cone, partial cone, and 1,3-alternate. The presence of terminal primary amino groups in these compounds opens up wide possibilities for further modification of the macrocycle. At the same time, the hexylidene spacer provides sufficient distance between the reaction centers to reduce the possibility of side reactions.
In order to introduce additional amino groups of different nature (i.e., primary, secondary, and tertiary) into the structure of the starting thiacalixarenes, one of the priority tasks was to design of macrocyclic precursors capable of easily forming new C-N bonds without changing the amine nature of the reagent. For this purpose, we have selected an acrylamide fragment that is a Michael acceptor and can be readily involved in the addition reaction of N-nucleophiles to electron-deficient alkenes (aza-Michael reaction) [62,63], hence ideally suited to the above requirements (Figure 1).
The interaction of such macrocyclic precursors with a series of diamines and alkanolamines (ethylenediamine, N,N-dimethylethylenediamine, ethanolamine, and diethanolamine) will lead to polyfunctional derivatives of thiacalixarene in various conformations (cone, partial cone, and 1,3-alternate), containing various amine fragments (primary, secondary, and tertiary) and hydroxyl groups, potentially possessing antimicrobial activity (Figure 1). In addition, once the optimal conditions for these reactions are established, it will be possible in the future to introduce the macrocyclic acrylamide precursors obtained into the reaction with more complex reagents containing an N-nucleophilic site.
The first stage of the synthetic work involved the synthesis of precursors of target multivalent thiacalixarene derivatives, i.e., macrocycles containing acrylamide groups. For this purpose, starting macrocycles 1–3 were introduced into the acylation reaction with acryloyl chloride. For compounds 1 (cone) and 3 (1,3-alternate), the synthesis was carried out in dichloromethane. In the case of compound 2 in partial cone conformation, chloroform was chosen as the solvent due to its poor solubility in dichloromethane. After addition of the acryloyl chloride at low temperature, the reaction mixtures were stirred for 12 h at room temperature. A number of methods, e.g., extraction, recrystallization, and column chromatography, were successively tried for the purification of acrylamide precursors 4–6, but these methods were found to be inefficient. It was found that the most optimal method for purification of these compounds was water re-precipitation from saturated ethanol solution. As a result, macrocyclic precursors 4–6 in cone, partial cone, and 1,3-alternate conformations were obtained in high yields (85–96%).
In the second synthetic step, obtained acrylamide precursors 4–6 as Michael acceptors were reacted with a series of amines and alkanolamines (ethylenediamine, N,N-dimethylethylenediamine, ethanolamine, and diethanolamine). Initially, the reaction of compounds 4–6 with ethylenediamine was investigated. To avoid side reactions (e.g., intermolecular and intramolecular cross-linking), a 30-fold excess of the corresponding amine per macrocycle molecule was used. The reaction mixture in methanol was refluxed for 60 h. To remove excess amine from the reaction mixture, a convenient and practical method of azeotropic distillation with toluene:methanol mixture (9:1), which we had previously used in the synthesis of PAMAM-calix-dendrimers, was used [64,65]. As a result, macrocycles 7–9 in cone, partial cone, and 1,3-alternate conformations were isolated with 97%, 92%, and 90% yields, respectively.
In the next step, compounds 4–6 were reacted with N,N-dimethylethylenediamine and ethanolamine containing only one primary amino group. The reactions were carried out under similar conditions, with excess reagent in methanol under reflux. After 60 h, according to the 1H NMR spectroscopy data, the reaction was not complete, as indicated by residual signals of acrylamide fragments in the range of 5.55–6.18 ppm in the 1H NMR spectra of the reaction mixtures. We therefore decided to extend the reaction time to 90 h. As a result, the target polyfunctional thiacalix[4]arene derivatives 10–12 and 13–15 were isolated in 94–98% and 79–84% yields, respectively.
The reactions of compounds 4–6 with diethanolamine were initially carried out analogously to the previous syntheses. However, even after 170 h of the reflux, the conversion of the reagent into the product was not complete. Apparently, this is due to the increased steric hindrance of the diethanolamine amino group compared to the primary amines used. A glass autoclave was used to increase the conversion and speed up the reaction. This allowed us to significantly reduce the synthesis time and achieve complete conversion (Scheme 1). The reaction was carried out at 100 °C for 40 h in methanol. Upon completion of the reaction, macrocycles 16–18 were isolated from the reaction mixtures in yields of 78–79%.
In order to evaluate the influence of the macrocyclic platform on the biological activity of the obtained compounds, we have additionally synthesized monomeric analogues of thiacalix[4]arenes 7–18, i.e., p-tert-butylphenol derivatives containing identical substituents. For this purpose, acylation of amine 19 with acryloyl chloride was carried out. This resulted in compound 20, which was involved in the next step in the aza-Michael addition reaction with the above-described series of amines and alkanols (ethylenediamine, N,N-dimethylethylenediamine, ethanolamine, and diethanolamine). Thus, monomeric compounds, p-tert-butylphenol derivatives 21–24, were obtained in 49–98% yields.
All the obtained compounds were fully characterized by a number of physical methods such as 1H, 13C{1H} NMR, IR spectroscopy, and ESI high resolution mass spectrometry (ESI-HRMS) (Figures S1–S75).
Thus, in all the 1H NMR (DMSO-d6) spectra of obtained acrylamide derivatives of thiacalix[4]arene 4–6, the protons at the double bond of the acrylamide fragment were presented as ABX-system expressed as doublets of doublets at 5.55, 6.06, and 6.18 ppm. The protons at the nitrogen atoms of the acrylamide fragments appeared as a broadened triplet at 8.05 ppm. The proton signals of the acrylamide fragment were observed in the same regions of the 1H NMR spectra regardless of the macrocycle conformation due to their sufficient distance from the macrocyclic platform. In the 1H NMR spectra of synthesized macrocycles 7–18, the signals of the acrylic fragments in the above-mentioned regions were completely absent. Together with the ratio of the signal intensities and the multiplicity of peaks, this confirmed the completion of the reaction. In all 1H NMR spectra of compounds 7–15, triplets of two methylene groups of C(O)–CH2–CH2–R fragments were observed at 2.18 and 2.68 ppm. The signals of the methylene group protons of the terminal ethylidene fragment closest to the secondary amino group appeared as a broadened triplet at 2.53–2.57 ppm. The proton signals of the end methylene group of the terminal ethylidene fragment appeared as broadened triplets at 2.47, 2.25, and 3.40 ppm for macrocycles with terminal primary, tertiary amino or hydroxyl groups, respectively. The thiacalixarene conformation only affected the chemical shifts of the protons closest to the macrocyclic platform. Thus, in the 1H NMR spectra (DMSO-d6) of all the compounds obtained in cone conformation, the proton signals of the tert-butyl, oxymethylene, and aromatic fragments appeared as singlets at 1.06, 4.75, and 7.38 ppm, respectively. At the same time, the signals of these protons for the compounds in 1,3-alternate conformation were located at 1.19, 3.94, and 7.52 ppm, due to the shielding of these protons by the aromatic rings of the macrocycle in this conformation. All of the above trends are also fully presented in the 1H NMR spectra of compounds 16–18, which were recorded in CD3OD to simplify the interpretation.
In the IR spectra of acrylamide compounds 4–6, a narrow intense absorption band related to the C=C bond vibrations was observed at 1625 cm−1. This characteristic absorption band was completely absent in the IR spectra of compounds 7–18, which further confirmed the structure of the obtained compounds. Along with this, the IR spectra of compounds 4–18 contained broad bands at the 3000 and 3075 cm−1 corresponding to N-H vibrations, bands at 1650 and 1540 cm−1 (amide I and amide II), as well as a band at the 1080–1095 cm−1 characteristic for arylalkyl ethers (CPhOCH2 fragment).
The obtained compounds were additionally characterized by mass spectrometry (ESI-HRMS). Peaks of mono- or diprotonated molecules [M + 1H]1+ and [M + 2H]2+ were registered in all mass spectra of acrylamide thiacalixarene derivatives 4–6. In the case of compounds 7–18, the peaks of di- and triprotonated molecules [M + 2H]2+ and [M + 3H]3+ were observed in all mass spectra. Additionally, peaks of tetraprotonated ions [M + 4H]4+ were found in mass spectra of compounds 10–12 and 15–18 containing the tertiary amino groups.
Thus, a convenient synthetic approach for multivalent thiacalix[4]arene derivatives containing amide, hydroxyl, and amino groups, consisting of a stepwise modification of the macrocyclic platform with acrylic fragments and the subsequent reaction with diamines and alkanolamines has been developed.

3.2. Antibacterial Properties and Cytotoxicity of the Obtained Multivalent Derivatives of Thiacalix[4]arene

According to the literature [66,67,68], the mechanism of antibacterial action of most compounds containing amine and ammonium groups is based on interaction with the negatively charged cell membrane of bacteria. Sufficient examples of ammonium and amine derivatives of (thia)calixarenes with antibacterial activity have been reported in the literature [44,69]. However, structure–activity relationship data establishing the antibacterial activity of the macrocycle with its conformation are currently lacking. Moreover, the partial cone conformation data are limited to individual examples [52]. The establishment of such structure–activity relationships will allow for the design of macrocyclic antibiotics for specific applications by exploiting the features of each conformation.
It is well known that (thia)calixarene derivatives in 1,3-alternate stereoisomeric form containing long substituents with polar terminal fragments are able to incorporate into lipid bilayers via “bouquet”-type (Figure 2), forming ion channel-type structures [70,71]. In this case, the lipophilic macrocyclic part of the molecule is incorporated in the middle of the phospholipid membrane, and substituents with polar terminal fragments are built at the boundaries of the membrane bilayer next to the polar groups. Such incorporation causes loosening of the lipid bilayer, leading to the destruction of bacterial cell membranes. This “bouquet”-type incorporation into membranes can also be expected in the case of macrocycles in partial cone conformation. However, prior to this work, there are no literature data describing the antibacterial activity of (thia)calixarene derivatives in partial cone conformation relative to 1,3-alternate.
To establish the regularities between the structure of the synthesized compounds and their biological activity, the antibacterial activity of obtained macrocyclic compounds 7–18 and monomeric analogues 21–24 against Gram-positive (Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains was further studied. The well-known antibiotics Ciprofloxacin and Norfloxacin were chosen as the standards. The dilutions of the compounds were prepared immediately in nutrient media; 5% DMSO was added for better solubility and the test strains were not inhibited at this concentration. The investigated macrocyclic compounds were found to have high antibacterial activity against the studied bacterial strains, while the monomeric compounds were characterized by low activity (Table 1). These results clearly indicate the advantage of the multivalent approach for the development of antibacterial agents. It should be noted that thiacalixarene derivatives in partial cone conformation had better antibacterial activity compared to the compounds in cone and 1,3-alternate stereoisomeric forms. In our opinion, this is due to the fact that partial cone is the most dissymmetric in cone-partial cone-1,3-alternate conformer series. Therefore, compounds in partial cone conformation tend to loosen membrane bilayers most strongly upon incorporation (Figure 2). This seems to be the reason for the highest activity of compounds in partial cone conformation. In turn, compounds in 1,3-alternate conformation are symmetric and capable of tighter packing when incorporated into lipid bilayers, resulting in less membrane loosening in bacteria. Therefore, the antibacterial activity of macrocycle compounds in 1,3-alternate conformation was lower. At the same time, compounds in cone conformation are not able to incorporate into membranes in the “bouquet”-type manner described above due to their amphiphilic structure in which the lipophilic tert-butyl groups and the hydrophilic substituents of the lower rim are on opposite sides of the macrocyclic platform. For incorporation into a membrane, a pair of macrocycles in cone conformation must be oriented toward each other with lipophilic parts (Figure 2). Thus, two molecules of the cone macrocycle are required to overlap the membrane bilayer, corresponding to the “slot” type of structures [40]. This is an important difference from the macrocycles obtained in 1,3-alternate and partial cone conformations, in the case of which a single macrocycle molecule is required to overlap the bilayer.
Among macrocycles with different substituents, compounds 10–12 containing -NHCH2CH2N(CH3)2 fragments had the highest antibacterial activity against all studied bacterial strains. These results are explained by their greater lipophilicity—the calculated miLogP values for these compounds were the highest (Table 1, miLogP values were calculated using an online platform http://www.molinspiration.com/cgi-bin/properties (accessed on 10 October 2023). The calculated values of MW (molecular weight), miLogP (logarithm of the octanol–water partition coefficient), HBA (hydrogen bond acceptor atoms), HBD (hydrogen bond donor atoms), TPSA (topological polar surface area), and solubility data are also presented in Table S3. The introduction of additional terminal hydroxyl groups (when comparing compounds 13–15 with -NHCH2CH2OH fragments and compounds 16–18 with -N(CH2CH2CH2OH)2 fragments) resulted in a dramatic decrease in antibacterial activity against all bacterial strains studied. This can be explained by the reduction of surface positive charge in thiacalixarene derivatives 16–18 upon introduction of additional terminal hydroxyl groups.
An important characteristic in the development of new drugs is their cytotoxic effect on mammalian cells. Therefore, the next step of this work was to study the cytotoxicity of lead compounds 10–12 against normal Chang liver cell line (Human liver cells) (Table 2). The advantage of in vitro models is the ability to work directly on human cell cultures, which makes the data obtained more adequate when applied to the human body. In addition, the use of cell cultures makes it possible to establish the nature of the biological activity of the studied compounds directly at the cellular level and take into account the complex synergistic or multidirectional effects of mixtures of chemical compounds [72]. The lowest cytotoxicity value was observed for macrocycle 11 in partial cone conformation (IC50 = 3.6 ± 0.3 M). This fact also confirmed the previously proposed mechanism of incorporation of the obtained compounds into the membrane bilayer. The cytotoxicity values for macrocycles 10 (IC50 = 52.0 ± 4.2 M) in cone conformation and 12 in 1,3-alternate conformation (IC50 = 25.3 ± 1.8 M) were concentrationally consistent (2-fold difference) with the mechanism proposed above, according to which two macrocycle molecules are involved in bilayer overlapping in the case of compounds in cone conformation. Compound 11 (partial cone) with the most significant antibacterial activity was predominantly non-selective against cells (highest selective index (SI) = 4.0 for S. aureus). Compound 10 (cone) showed the highest selectivity against S. aureus (SI = 3.3) and E. faecalis (SI = 13.3).
Thus, the compounds in partial cone conformation had significant bactericidal activity against all studied cell strains. This can be used to create drugs with universal action against all microorganisms. At the same time, the best selectivity was observed for the compounds in cone conformation, which can be used to design antibacterial agents with low toxicity and activity against a specific type of bacteria. Certainly, this work is a proof of concept, and further optimization of the structure–activity relationship is required. However, we already believe that the proposed universal approach to the synthesis of multivalent antimicrobial agents has great potential for the future.

3.3. Study of the Mechanism of Antibacterial Activity of the Obtained Compounds

The bacterial membrane mimetic systems were further used to prove the proposed mechanism of interaction of the compounds obtained with bacteria. Lead compounds 10–12 in cone, partial cone, and 1,3-altenate conformations, were chosen for this experiment. Initially, the self-association of compounds 10–12 was investigated by dynamic light scattering (DLS) in TRIS buffer (pH = 7.4, 150 mM NaCl) at concentration 1 × 10−5 M and 1 × 10−4 M. Typically, supramolecular systems with low polydispersity index (PDI < 0.25) values are considered stable and monodisperse, but no formation of stable supramolecular systems based on compounds 10–12 (PDI = 0.32–0.70) was found (Table S2). Consequently, the antibacterial activity of the obtained compounds was due to the action of the macrocycles themselves rather than their self-associates.
Model vesicles based on 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (POPG) were used as Gram-positive negatively charged membrane mimetic systems [73,74,75]. POPG vesicles were prepared in TRIS buffer (pH = 7.4, 150 mM NaCl). The resulting vesicles were studied by DLS and Doppler microelectrophoresis to evaluate the effect of macrocycles addition (Table 3). The values of the hydrodynamic diameter and the electrokinetic potential (zeta potential) of the vesicles were measured in presence and absence of compounds 10–12. Measurements were carried out in molar ratios [POPG:macrocycle] = 1:0.1 and [POPG:macrocycle] = 1:1. As the concentration of macrocycles 10–12 increased, the hydrodynamic diameter of the resulting vesicles increased. The most noticeable changes occurred in the case of 12 (1,3-altertane) (up to 290 nm) and 11 (partial cone) (up to 571 nm). Moreover, in the case of 11 (partial cone), the monodispersity of the POPG vesicles was disrupted (PDI 0.56) and the hydrodynamic diameter was very different from the initial one. This can be explained by partial destruction of the POPG vesicles. The zeta potential values also increased with increasing macrocycle concentration, which clearly indicated the adsorption of the compounds on the surface of lipid vesicles and further incorporation into the membrane structure.
The obtained data additionally confirmed the previously proposed mechanism of interaction of the obtained compounds with the membrane bilayer. The largest changes in the size of model vesicles (1:1 ratio) were observed in the case of macrocycle 11 (partial cone) since the compounds in this conformation loosen the bilayer the most during incorporation. Smaller changes were found for compound 12 (1,3-alternate). In the case of macrocycle 10 (cone), POPG vesicle size changes were the smallest. This is due to the fact that twice as many macrocycle molecules in cone conformation, as opposed to partial cone and 1,3-alternate, are required to overlap the bilayer upon incorporation. Thus, the concentration of compound 10 used is not sufficient to induce significant changes in POPG vesicle size.
Thus, the mechanism of antibacterial activity of lead compounds 10–12 was proved using model negatively charged POPG vesicles. The addition of 12 (partial cone), which had the best biological activity, was also found to result in partial destruction of the vesicles.

4. Conclusions

In this work, a synthetic approach to obtain multivalent thiacalix[4]arene derivatives containing different amounts of amine and hydroxyl groups was developed for the first time. A series of macrocyclic compounds in cone, partial cone, and 1,3-alternate stereoisomeric forms containing -NHCH2CH2R (R = NH2, N(CH3)2, and OH) and -N(CH2CH2OH)2 fragments and their model non-macrocyclic analogs were prepared. The antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains and cytotoxicity of the obtained compounds were studied, and structure–activity relationships were established. The synthesized macrocyclic compounds were found to have better biological activity compared to monomeric analogs. The mechanism of antibacterial action of the obtained compounds lies in the interaction with negatively charged cell membrane of bacteria, which was proved on the example of model vesicles POPG. The compounds in partial cone conformation had significant bactericidal activity against all studied cell strains. The best selectivity was observed for the compounds in cone conformation.
Certainly, this work is a proof of concept, and further optimization of the structure–activity relationship is required. However, we already believe that the proposed universal approach to the synthesis of multivalent antimicrobial agents has great potential for the future. The obtained results open up the possibility of using the synthesized macrocyclic compounds as promising antibacterial agents.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/pharmaceutics15122731/s1: Figures S1–S75. 1H and 13C{1H} NMR, IR, and mass spectra of the synthesized compounds 4–18; Tables S1 and S2. Aggregation data of systems of POPG and individual macrocyclic compounds. Table S3. Values (MW, miLogP, HBA, HBD, TPSA) and solubility data for compounds 7–18 and 21–24. References [76,77] are cited in the Supplementary Materials.

Author Contributions

Conceptualization, writing—review and editing, project administration, supervision, and funding acquisition, I.S. (Ivan Stoikov); supervision, formal analysis, and writing—original draft preparation, P.P.; investigation, writing—original draft preparation, and visualization, I.S. (Igor Shiabiev), A.A., A.V. and K.P.; investigation, methodology, and formal analysis, D.P.; investigation, validation, methodology and data curation O.B., V.B. and A.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by Russian Science Foundation, Russian Federation (grant 21-73-20067, https://rscf.ru/en/project/21-73-20067/ (accessed on 1 November 2023).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the Supplementary Material.

Acknowledgments

The investigation of the spatial structure of the compounds by NMR spectroscopy was supported by the Kazan Federal University Strategic Academic Leadership Program (‘PRIORITY–2030′). ESI-HRMS data were obtained in the CSF-SAC FRC KSC RAS with support of the State Assignment of the Federal Research Center “Kazan Scientific Center”, Russian Academy of Sciences. A.L., A.V. and K.P. are grateful for financial support from the government assignment for FRC Kazan Scientific Center of RAS in part of estimation of cytotoxicity.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. World Health Organization Fact Sheet—Antimicrobial Resistance. 2021. Available online: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (accessed on 10 November 2023).
  2. Catalano, A.; Iacopetta, D.; Ceramella, J.; Scumaci, D.; Giuzio, F.; Saturnino, C.; Aquaro, S.; Rosano, C.; Sinicropi, M.S. Multidrug Resistance (MDR): A Widespread Phenomenon in Pharmacological Therapies. Molecules 2022, 27, 616. [Google Scholar] [CrossRef] [PubMed]
  3. Khameneh, B.; Diab, R.; Ghazvini, K.; Fazly Bazzaz, B.S. Breakthroughs in Bacterial Resistance Mechanisms and the Potential Ways to Combat Them. Microb. Pathog. 2016, 95, 32–42. [Google Scholar] [CrossRef] [PubMed]
  4. Uddin, T.M.; Chakraborty, A.J.; Khusro, A.; Zidan, B.R.M.; Mitra, S.; Emran, T.B.; Dhama, K.; Ripon, M.K.H.; Gajdács, M.; Sahibzada, M.U.K.; et al. Antibiotic Resistance in Microbes: History, Mechanisms, Therapeutic Strategies and Future Prospects. J. Infect. Public Health 2021, 14, 1750–1766. [Google Scholar] [CrossRef] [PubMed]
  5. Mancuso, G.; Midiri, A.; Gerace, E.; Biondo, C. Bacterial Antibiotic Resistance: The Most Critical Pathogens. Pathogens 2021, 10, 1310. [Google Scholar] [CrossRef] [PubMed]
  6. Nwobodo, D.C.; Ugwu, M.C.; Oliseloke Anie, C.; Al-Ouqaili, M.T.S.; Chinedu Ikem, J.; Victor Chigozie, U.; Saki, M. Antibiotic Resistance: The Challenges and Some Emerging Strategies for Tackling a Global Menace. J. Clin. Lab. Anal. 2022, 36, e24655. [Google Scholar] [CrossRef]
  7. Chan, L.W.; Hern, K.E.; Ngambenjawong, C.; Lee, K.; Kwon, E.J.; Hung, D.T.; Bhatia, S.N. Selective Permeabilization of Gram-Negative Bacterial Membranes Using Multivalent Peptide Constructs for Antibiotic Sensitization. ACS Infect. Dis. 2021, 7, 721–732. [Google Scholar] [CrossRef] [PubMed]
  8. Laroque, S.; Reifarth, M.; Sperling, M.; Kersting, S.; Klöpzig, S.; Budach, P.; Storsberg, J.; Hartlieb, M. Impact of Multivalence and Self-Assembly in the Design of Polymeric Antimicrobial Peptide Mimics. ACS Appl. Mater. Interfaces 2020, 12, 30052–30065. [Google Scholar] [CrossRef]
  9. Yin, F.; Li, J.-J.; Shi, B.; Zhang, K.; Li, X.-L.; Wang, K.-R.; Guo, D.-S. Carbohydrate–Macrocycle Conjugates for Biomedical Applications. Mater. Chem. Front. 2023, 7, 5263–5287. [Google Scholar] [CrossRef]
  10. Szymura, A.; Ilyas, S.; Horn, M.; Neundorf, I.; Mathur, S. Multivalent Magnetic Nanoaggregates with Unified Antibacterial Activity and Selective Uptake of Heavy Metals and Organic Pollutants. J. Mol. Liq. 2020, 317, 114002. [Google Scholar] [CrossRef]
  11. Hoyos, P.; Perona, A.; Juanes, O.; Rumbero, Á.; Hernáiz, M.J. Synthesis of Glycodendrimers with Antiviral and Antibacterial Activity. Chem.—Eur. J. 2021, 27, 7593–7624. [Google Scholar] [CrossRef]
  12. Wei, T.; Yu, Q.; Chen, H. Responsive and Synergistic Antibacterial Coatings: Fighting against Bacteria in a Smart and Effective Way. Adv. Healthc. Mater. 2019, 8, 201801381. [Google Scholar] [CrossRef] [PubMed]
  13. Andriianova, A.N.; Latypova, L.R.; Vasilova, L.Y.; Kiseleva, S.V.; Zorin, V.V.; Abdrakhmanov, I.B.; Mustafin, A.G. Antibacterial Properties of Polyaniline Derivatives. J. Appl. Polym. Sci. 2021, 138, 51397. [Google Scholar] [CrossRef]
  14. Wang, Y.; Yang, Y.; Shi, Y.; Song, H.; Yu, C. Antibiotic-Free Antibacterial Strategies Enabled by Nanomaterials: Progress and Perspectives. Adv. Mater. 2019, 32, 201904106. [Google Scholar] [CrossRef] [PubMed]
  15. Djouhri-Bouktab, L.; Rolain, J.M.; Brunel, J.M. Mini-Review: Polyamines Metabolism, Toxicity and Potent Therapeutical Use. Anti-Infect. Agents 2014, 12, 95–103. [Google Scholar] [CrossRef]
  16. Gorbunova, M.; Lemkina, L.; Borisova, I. New Guanidine-Containing Polyelectrolytes as Advanced Antibacterial Materials. Eur. Polym. J. 2018, 105, 426–433. [Google Scholar] [CrossRef]
  17. Stelmakh, S.A.; Grigor’eva, M.N.; Garkusheva, N.M.; Lebedeva, S.N.; Ochirov, O.S.; Mognonov, D.M.; Zhamsaranova, S.D.; Batoev, V.B. Studies of New Biocidal Polyguanidines: Antibacterial Action and Toxicity. Polym. Bull. 2020, 78, 1997–2008. [Google Scholar] [CrossRef]
  18. Zhang, H.; Liu, L.; Hou, P.; Liu, J.; Fu, S. Design, Synthesis, Antibacterial, and Antitumor Activity of Linear Polyisocyanide Quaternary Ammonium Salts with Different Structures and Chain Lengths. Molecules 2021, 26, 5686. [Google Scholar] [CrossRef]
  19. Pham, P.; Oliver, S.; Boyer, C. Design of Antimicrobial Polymers. Macromol. Chem. Phys. 2022, 224, 2200226. [Google Scholar] [CrossRef]
  20. Cuneo, T.; Gao, H. Recent Advances on Synthesis and Biomaterials Applications of Hyperbranched Polymers. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2020, 12, e1640. [Google Scholar] [CrossRef]
  21. Namivandi-Zangeneh, R.; Wong, E.H.H.; Boyer, C. Synthetic Antimicrobial Polymers in Combination Therapy: Tackling Antibiotic Resistance. ACS Infect. Dis. 2021, 7, 215–253. [Google Scholar] [CrossRef]
  22. Haktaniyan, M.; Bradley, M. Polymers Showing Intrinsic Antimicrobial Activity. Chem. Soc. Rev. 2022, 51, 8584–8611. [Google Scholar] [CrossRef] [PubMed]
  23. Tavares, M.R.; Pechar, M.; Chytil, P.; Etrych, T. Polymer-Based Drug-Free Therapeutics for Anticancer, Anti-Inflammatory, and Antibacterial Treatment. Macromol. Biosci. 2021, 21, 202100135. [Google Scholar] [CrossRef] [PubMed]
  24. Chen, A.; Karanastasis, A.; Casey, K.R.; Necelis, M.; Carone, B.R.; Caputo, G.A.; Palermo, E.F. Cationic Molecular Umbrellas as Antibacterial Agents with Remarkable Cell-Type Selectivity. ACS Appl. Mater. Interfaces 2020, 12, 21270–21282. [Google Scholar] [CrossRef]
  25. Zlotnikov, I.D.; Vigovskiy, M.A.; Davydova, M.P.; Danilov, M.R.; Dyachkova, U.D.; Grigorieva, O.A.; Kudryashova, E.V. Mannosylated Systems for Targeted Delivery of Antibacterial Drugs to Activated Macrophages. Int. J. Mol. Sci. 2022, 23, 16144. [Google Scholar] [CrossRef]
  26. Pashirova, T.N.; Shaikhutdinova, Z.M.; Mironov, V.F.; Bogdanov, A.V. Ammonium Amphiphiles Based on Natural Compounds: Design, Synthesis, Properties, and Biomedical Applications. A Review. Dokl. Chem. 2023, 509, 71–88. [Google Scholar] [CrossRef]
  27. Filippova, S.S.; Deriabin, K.V.; Perevyazko, I.; Shamova, O.V.; Orlov, D.S.; Islamova, R.M. Metal- and Peroxide-Free Silicone Rubbers with Antibacterial Properties Obtained at Room Temperature. ACS Appl. Polym. Mater. 2023, 5, 5286–5296. [Google Scholar] [CrossRef]
  28. Zhao, S.; Huang, W.; Wang, C.; Wang, Y.; Zhang, Y.; Ye, Z.; Zhang, J.; Deng, L.; Dong, A. Screening and Matching Amphiphilic Cationic Polymers for Efficient Antibiosis. Biomacromolecules 2020, 21, 5269–5281. [Google Scholar] [CrossRef] [PubMed]
  29. Zhang, H.; Liu, L.; Hou, P.; Pan, H.; Fu, S. Polyisocyanide Quaternary Ammonium Salts with Exceptionally Star-Shaped Structure for Enhanced Antibacterial Properties. Polymers 2022, 14, 1737. [Google Scholar] [CrossRef]
  30. Pedziwiatr-Werbicka, E.; Milowska, K.; Dzmitruk, V.; Ionov, M.; Shcharbin, D.; Bryszewska, M. Dendrimers and Hyperbranched Structures for Biomedical Applications. Eur. Polym. J. 2019, 119, 61–73. [Google Scholar] [CrossRef]
  31. Alfei, S.; Schito, A.M. From Nanobiotechnology, Positively Charged Biomimetic Dendrimers as Novel Antibacterial Agents: A Review. Nanomaterials 2020, 10, 2022. [Google Scholar] [CrossRef]
  32. García-Gallego, S.; Franci, G.; Falanga, A.; Gómez, R.; Folliero, V.; Galdiero, S.; de la Mata, F.; Galdiero, M. Function Oriented Molecular Design: Dendrimers as Novel Antimicrobials. Molecules 2017, 22, 1581. [Google Scholar] [CrossRef] [PubMed]
  33. Quintana-Sanchez, S.; Gómez-Casanova, N.; Sánchez-Nieves, J.; Gómez, R.; Rachuna, J.; Wąsik, S.; Semaniak, J.; Maciejewska, B.; Drulis-Kawa, Z.; Ciepluch, K.; et al. The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin. Int. J. Mol. Sci. 2022, 23, 1873. [Google Scholar] [CrossRef] [PubMed]
  34. Schito, A.M.; Alfei, S. Antibacterial Activity of Non-Cytotoxic, Amino Acid-Modified Polycationic Dendrimers against Pseudomonas aeruginosa and Other Non-Fermenting Gram-Negative Bacteria. Polymers 2020, 12, 1818. [Google Scholar] [CrossRef] [PubMed]
  35. Apartsin, E.; Akhir, A.; Kaul, G.; Saxena, D.; Laurent, R.; Srivastava, K.K.; Mignani, S.; Majoral, J.-P.; Chopra, S. Low-Generation Cationic Phosphorus Dendrimers: Novel Approach to Tackle Drug-Resistant S. Aureus In Vitro and In Vivo. Biomacromolecules 2023, 24, 3215–3227. [Google Scholar] [CrossRef] [PubMed]
  36. Ramchuran, E.J.; Pérez-Guillén, I.; Bester, L.A.; Khan, R.; Albericio, F.; Viñas, M.; de la Torre, B.G. Super-Cationic Peptide Dendrimers—Synthesis and Evaluation as Antimicrobial Agents. Antibiotics 2021, 10, 695. [Google Scholar] [CrossRef] [PubMed]
  37. Staneva, D.; Manov, H.; Yordanova, S.; Vasileva-Tonkova, E.; Stoyanov, S.; Grabchev, I. Synthesis, Spectral Properties and Antimicrobial Activity of a New Cationic Water-soluble pH-dependent Poly(Propylene Imine) Dendrimer Modified with 1,8-naphthalimides. Luminescence 2020, 35, 947–954. [Google Scholar] [CrossRef]
  38. Hernando-Gozalo, M.; Aguilera-Correa, J.J.; Rescalvo-Casas, C.; Seijas-Pereda, L.; García-Bertolín, C.; de la Mata, F.J.; Sánchez-Nieves, J.; Cuadros, J.; Pérez-Tanoira, R. Study of the Antimicrobial Activity of Cationic Carbosilane Dendrimers against Clinical Strains of Multidrug-Resistant Bacteria and Their Biofilms. Front. Cell. Infect. Microbiol. 2023, 13, 1203991. [Google Scholar] [CrossRef]
  39. Dhumal, D.; Maron, B.; Malach, E.; Lyu, Z.; Ding, L.; Marson, D.; Laurini, E.; Tintaru, A.; Ralahy, B.; Giorgio, S.; et al. Dynamic Self-Assembling Supramolecular Dendrimer Nanosystems as Potent Antibacterial Candidates against Drug-Resistant Bacteria and Biofilms. Nanoscale 2022, 14, 9286–9296. [Google Scholar] [CrossRef]
  40. Mekuria, S.L.; Song, C.; Ouyang, Z.; Shen, M.; Janaszewska, A.; Klajnert-Maculewicz, B.; Shi, X. Synthesis and Shaping of Core–Shell Tecto Dendrimers for Biomedical Applications. Bioconjugate Chem. 2021, 32, 225–233. [Google Scholar] [CrossRef]
  41. Chen, S.; Huang, S.; Li, Y.; Zhou, C. Recent Advances in Epsilon-Poly-L-Lysine and L-Lysine-Based Dendrimer Synthesis, Modification, and Biomedical Applications. Front. Chem. 2021, 9, 659304. [Google Scholar] [CrossRef]
  42. Antipin, I.S.; Alfimov, M.V.; Arslanov, V.V.; Burilov, V.A.; Vatsadze, S.Z.; Voloshin, Y.Z.; Volcho, K.P.; Gorbatchuk, V.V.; Gorbunova, Y.G.; Gromov, S.P.; et al. Functional Supramolecular Systems: Design and Applications. Russ. Chem. Rev. 2021, 90, 895–1107. [Google Scholar] [CrossRef]
  43. Shurpik, D.N.; Padnya, P.L.; Stoikov, I.I.; Cragg, P.J. Antimicrobial Activity of Calixarenes and Related Macrocycles. Molecules 2020, 25, 5145. [Google Scholar] [CrossRef] [PubMed]
  44. Fang, S.; Dang, Y.-Y.; Li, H.; Li, H.; Liu, J.; Zhong, R.; Chen, Y.; Liu, S.; Lin, S. Membrane-Active Antibacterial Agents Based on Calix[4]Arene Derivatives: Synthesis and Biological Evaluation. Front. Chem. 2022, 10, 816741. [Google Scholar] [CrossRef] [PubMed]
  45. Razuvayeva, Y.; Kashapov, R.; Zakharova, L. Calixarene-Based Pure and Mixed Assemblies for Biomedical Applications. Supramol. Chem. 2020, 32, 178–206. [Google Scholar] [CrossRef]
  46. Surur, A.S.; Sun, D. Macrocycle-Antibiotic Hybrids: A Path to Clinical Candidates. Front. Chem. 2021, 9, 659845. [Google Scholar] [CrossRef] [PubMed]
  47. Ferreri, L.; Consoli, G.M.L.; Clarizia, G.; Zampino, D.C.; Nostro, A.; Granata, G.; Ginestra, G.; Giuffrida, M.L.; Zimbone, S.; Bernardo, P. A Novel Material Based on an Antibacterial Choline-Calixarene Nanoassembly Embedded in Thin Films. J. Mater. Sci. 2022, 57, 20685–20701. [Google Scholar] [CrossRef]
  48. Baldini, L.; Casnati, A.; Sansone, F. Multivalent and Multifunctional Calixarenes in Bionanotechnology. Eur. J. Org. Chem. 2020, 2020, 5056–5069. [Google Scholar] [CrossRef]
  49. Burilov, V.; Makarov, E.; Mironova, D.; Sultanova, E.; Bilyukova, I.; Akyol, K.; Evtugyn, V.; Islamov, D.; Usachev, K.; Mukhametzyanov, T.; et al. Calix[4]Arene Polyamine Triazoles: Synthesis, Aggregation and DNA Binding. Int. J. Mol. Sci. 2022, 23, 14889. [Google Scholar] [CrossRef]
  50. Lee, J.-S.; Song, I.; Shinde, P.B.; Nimse, S.B. Macrocycles and Supramolecules as Antioxidants: Excellent Scaffolds for Development of Potential Therapeutic Agents. Antioxidants 2020, 9, 859. [Google Scholar] [CrossRef]
  51. Crowley, P.B. Protein–Calixarene Complexation: From Recognition to Assembly. Acc. Chem. Res. 2022, 55, 2019–2032. [Google Scholar] [CrossRef]
  52. Mourer, M.; Duval, R.E.; Constant, P.; Daffé, M.; Regnouf-de-Vains, J. Impact of Tetracationic Calix[4]Arene Conformation—From Conic Structure to Expanded Bolaform—On Their Antibacterial and Antimycobacterial Activities. ChemBioChem 2019, 20, 911–921. [Google Scholar] [CrossRef] [PubMed]
  53. Pan, Y.; Hu, X.; Guo, D.-S. Biomedical Applications of Calixarenes: State of the Art and Perspectives. Angew. Chem. Int. Ed. 2020, 60, 2768–2794. [Google Scholar] [CrossRef] [PubMed]
  54. Kashapov, R.R.; Razuvayeva, Y.S.; Ziganshina, A.Y.; Mukhitova, R.K.; Sapunova, A.S.; Voloshina, A.D.; Syakaev, V.V.; Latypov, S.K.; Nizameev, I.R.; Kadirov, M.K.; et al. N-Methyl-d-Glucamine–Calix[4]Resorcinarene Conjugates: Self-Assembly and Biological Properties. Molecules 2019, 24, 1939. [Google Scholar] [CrossRef] [PubMed]
  55. Gao, L.; Wang, H.; Zheng, B.; Huang, F. Combating Antibiotic Resistance: Current Strategies for the Discovery of Novel Antibacterial Materials Based on Macrocycle Supramolecular Chemistry. Giant 2021, 7, 100066. [Google Scholar] [CrossRef]
  56. Consoli, G.M.L.; Granata, G.; Ginestra, G.; Marino, A.; Toscano, G.; Nostro, A. Antibacterial Nanoassembled Calix[4]Arene Exposing Choline Units Inhibits Biofilm and Motility of Gram Negative Bacteria. ACS Med. Chem. Lett. 2022, 13, 916–922. [Google Scholar] [CrossRef]
  57. Consoli, G.M.L.; Di Bari, I.; Blanco, A.R.; Nostro, A.; D’Arrigo, M.; Pistarà, V.; Sortino, S. Design, Synthesis, and Antibacterial Activity of a Multivalent Polycationic Calix[4]Arene–NO Photodonor Conjugate. ACS Med. Chem. Lett. 2017, 8, 881–885. [Google Scholar] [CrossRef] [PubMed]
  58. Consoli, G.M.L.; Granata, G.; Picciotto, R.; Blanco, A.R.; Geraci, C.; Marino, A.; Nostro, A. Design, Synthesis and Antibacterial Evaluation of a Polycationic Calix[4]Arene Derivative Alone and in Combination with Antibiotics. Med. Chem. Commun. 2018, 9, 160–164. [Google Scholar] [CrossRef]
  59. Mourer, M.; Regnouf-de-Vains, J.-B.; Duval, R.E. Functionalized Calixarenes as Promising Antibacterial Drugs to Face Antimicrobial Resistance. Molecules 2023, 28, 6954. [Google Scholar] [CrossRef]
  60. Padnya, P.; Mostovaya, O.; Ovchinnikov, D.; Shiabiev, I.; Pysin, D.; Akhmedov, A.; Mukhametzyanov, T.; Lyubina, A.; Voloshina, A.; Petrov, K.; et al. Combined Antimicrobial Agents Based on Self-Assembled PAMAM-Calix-Dendrimers/Lysozyme Nanoparticles: Design, Antibacterial Properties and Cytotoxicity. J. Mol. Liq. 2023, 389, 122838. [Google Scholar] [CrossRef]
  61. Mostovaya, O.A.; Padnya, P.L.; Shurpik, D.N.; Shiabiev, I.E.; Stoikov, I.I. Novel Lactide Derivatives of P-Tert-Butylthiacalix[4]Arene: Directed Synthesis and Molecular Recognition of Catecholamines. J. Mol. Liq. 2021, 327, 114806. [Google Scholar] [CrossRef]
  62. Rulev, A.Y. Aza-Michael Reaction: Achievements and Prospects. Russ. Chem. Rev. 2011, 80, 197–218. [Google Scholar] [CrossRef]
  63. Rulev, A.Y. Aza-Michael Reaction: A Decade Later—Is the Research Over? Eur. J. Org. Chem. 2023, 26, e202300451. [Google Scholar] [CrossRef]
  64. Mostovaya, O.; Shiabiev, I.; Pysin, D.; Stanavaya, A.; Abashkin, V.; Shcharbin, D.; Padnya, P.; Stoikov, I. PAMAM-Calix-Dendrimers: Second Generation Synthesis, Fluorescent Properties and Catecholamines Binding. Pharmaceutics 2022, 14, 2748. [Google Scholar] [CrossRef] [PubMed]
  65. Mostovaya, O.; Padnya, P.; Shiabiev, I.; Mukhametzyanov, T.; Stoikov, I. PAMAM-Calix-Dendrimers: Synthesis and Thiacalixarene Conformation Effect on DNA Binding. Int. J. Mol. Sci. 2021, 22, 11901. [Google Scholar] [CrossRef]
  66. Zhou, Z.; Zhou, S.; Zhang, X.; Zeng, S.; Xu, Y.; Nie, W.; Zhou, Y.; Xu, T.; Chen, P. Quaternary Ammonium Salts: Insights into Synthesis and New Directions in Antibacterial Applications. Bioconjugate Chem. 2023, 34, 302–325. [Google Scholar] [CrossRef] [PubMed]
  67. Saverina, E.A.; Frolov, N.A.; Kamanina, O.A.; Arlyapov, V.A.; Vereshchagin, A.N.; Ananikov, V.P. From Antibacterial to Antibiofilm Targeting: An Emerging Paradigm Shift in the Development of Quaternary Ammonium Compounds (QACs). ACS Infect. Dis. 2023, 9, 394–422. [Google Scholar] [CrossRef] [PubMed]
  68. Seferyan, M.A.; Saverina, E.A.; Frolov, N.A.; Detusheva, E.V.; Kamanina, O.A.; Arlyapov, V.A.; Ostashevskaya, I.I.; Ananikov, V.P.; Vereshchagin, A.N. Multicationic Quaternary Ammonium Compounds: A Framework for Combating Bacterial Resistance. ACS Infect. Dis. 2023, 9, 1206–1220. [Google Scholar] [CrossRef]
  69. Padnya, P.L.; Terenteva, O.S.; Akhmedov, A.A.; Iksanova, A.G.; Shtyrlin, N.V.; Nikitina, E.V.; Krylova, E.S.; Shtyrlin, Y.G.; Stoikov, I.I. Thiacalixarene Based Quaternary Ammonium Salts as Promising Antibacterial Agents. Bioorg. Med. Chem. 2021, 29, 115905. [Google Scholar] [CrossRef]
  70. Stoikov, I.I.; Antipin, I.S.; Konovalov, A.I. Artificial Ion Channels. Russ. Chem. Rev. 2003, 72, 1055–1077. [Google Scholar] [CrossRef]
  71. Pregel, M.J.; Jullien, L.; Canceill, J.; Lacombe, L.; Lehn, J.-M. Channel-Type Molecular Structures. Part 4. Transmembrane Transport of Alkali-Metal Ions by ‘Bouquet’ Molecules. J. Chem. Soc. Perkin Trans. 2 1995, 417–426. [Google Scholar] [CrossRef]
  72. Anadón, A.; Martínez, M.A.; Castellano, V.; Martínez-Larrañaga, M.R. The Role of in Vitro Methods as Alternatives to Animals in Toxicity Testing. Expert Opin. Drug Metab. Toxicol. 2013, 10, 67–79. [Google Scholar] [CrossRef]
  73. Krok, E.; Stephan, M.; Dimova, R.; Piatkowski, L. Tunable Biomimetic Bacterial Membranes from Binary and Ternary Lipid Mixtures and Their Application in Antimicrobial Testing. Biochim. Biophys. Acta Biomembr. 2023, 1865, 184194. [Google Scholar] [CrossRef] [PubMed]
  74. Kinouchi, H.; Onishi, M.; Kamimori, H. Lipid Membrane-Binding Properties of Daptomycin Using Surface Plasmon Resonance. Anal. Sci. 2013, 29, 297–301. [Google Scholar] [CrossRef]
  75. Li, S.; Ren, R.; Lyu, L.; Song, J.; Wang, Y.; Lin, T.-W.; Brun, A.L.; Hsu, H.-Y.; Shen, H.-H. Solid and Liquid Surface-Supported Bacterial Membrane Mimetics as a Platform for the Functional and Structural Studies of Antimicrobials. Membranes 2022, 12, 906. [Google Scholar] [CrossRef] [PubMed]
  76. Voloshina, A.D.; Gumerova, S.K.; Sapunova, A.S.; Kulik, N.V.; Mirgorodskaya, A.B.; Kotenko, A.A.; Prokopyeva, T.M.; Mikhailov, V.A.; Zakharova, L.Y.; Sinyashin, O.G. The Structure—Activity Correlation in the Family of Dicationic Imidazolium Surfactants: Antimicrobial Properties and Cytotoxic Effect. Biochim. Biophys. Acta Gen. Subj. 2020, 1864, 129728. [Google Scholar] [CrossRef]
  77. Agarkov, A.S.; Nefedova, A.A.; Gabitova, E.R.; Mingazhetdinova, D.O.; Ovsyannikov, A.S.; Islamov, D.R.; Amerhanova, S.K.; Lyubina, A.P.; Voloshina, A.D.; Litvinov, I.A.; et al. (2-Hydroxy-3-Methoxybenzylidene)Thiazolo[3,2-a]Pyrimidines: Synthesis, Self-Assembly in the Crystalline Phase and Cytotoxic Activity. Int. J. Mol. Sci. 2023, 24, 2084. [Google Scholar] [CrossRef] [PubMed]
Pharmaceutics 15 02731 g001
Figure 1. The sketch image of design of multivalent thiacalixarene derivatives.
Figure 1. The sketch image of design of multivalent thiacalixarene derivatives.
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Scheme 1. Reagents and conditions: i—CH2Cl2 for compounds 4 and 6; CHCl3 for compound 5; ii—reflux, 60 h for compounds 7–9; reflux, 90 h for compounds 10–15; 100 °C, 40 h for compounds 16–18.
Scheme 1. Reagents and conditions: i—CH2Cl2 for compounds 4 and 6; CHCl3 for compound 5; ii—reflux, 60 h for compounds 7–9; reflux, 90 h for compounds 10–15; 100 °C, 40 h for compounds 16–18.
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Figure 2. The sketch image of the proposed mechanism of interaction of the obtained compounds with the membrane bilayer.
Figure 2. The sketch image of the proposed mechanism of interaction of the obtained compounds with the membrane bilayer.
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Table 1. Lipophilicity (miLogP), minimum inhibitory, and bactericidal concentrations (MIC and MBC, µM) of macrocyclic compounds 7–18, monomers 21–24, Ciprofloxacin, and Norfloxacin.
Table 1. Lipophilicity (miLogP), minimum inhibitory, and bactericidal concentrations (MIC and MBC, µM) of macrocyclic compounds 7–18, monomers 21–24, Ciprofloxacin, and Norfloxacin.
CompoundsmiLogPTerminal FragmentS. aureusB. cereusE. faecalisE. coliP. aeruginosa
MICMBCMICMBCMICMBCMICMBCMICMBC
7 (cone)8.74Pharmaceutics 15 02731 i00162.5 ± 5.362.5 ± 4.8125 ± 11250 ± 1962.5 ± 4.962.5 ± 5.262.5 ± 5.362.5 ± 4.9250 ± 19250 ± 20
8 (partial cone)31.3 ± 2.531.3 ± 2.531.3 ± 2.131.3 ± 2.315.6 ± 1.3250 ± 2115.6 ± 1.415.6 ± 1.315.6 ± 1.215.6 ± 1.2
9 (1,3-alternate)31.3 ± 2.631.3 ± 2.462.5 ± 5.4>50062.5 ± 4.862.5 ± 5.515.6 ± 1.215.6 ± 1.215.6 ± 1.315.6 ± 1.3
21 (monomer)1.97125 ± 11125 ± 9250 ± 19>500125 ± 9125 ± 10250 ± 20250 ± 21250 ± 21250 ± 19
10 (cone)9.95Pharmaceutics 15 02731 i00215.6 ± 1.315.6 ± 1.362.5 ± 4.8125 ± 103.9 ± 0.43.9 ± 0.331.3 ± 2.631.3 ± 2.4125 ± 11125 ± 9
11 (partial cone)0.90 ± 0.011.9 ± 0.115.6 ± 1.215.6 ± 1.21.9 ± 0.23.9 ± 0.37.8 ± 0.67.8 ± 0.615.6 ± 1.215.6 ± 1.3
12 (1,3-alternate)7.8 ± 0.77.8 ± 0.762.5 ± 5.362.5 ± 4.97.8 ± 0.67.8 ± 0.87.8 ± 0.77.8 ± 0.662.5 ± 5.262.5 ± 5.3
22 (monomer)3.20250 ± 19500 ± 45250 ± 21>500125 ± 11125 ± 11250 ± 19250 ± 20500 ± 44500 ± 45
13 (cone)9.47Pharmaceutics 15 02731 i0037.8 ± 0.67.8 ± 0.7125 ± 11125 ± 107.8 ± 0.6125 ± 11125 ± 10125 ± 1062.5 ± 4.862.5 ± 5.2
14 (partial cone)1.9 ± 0.13.9 ± 0.331.3 ± 2.331.3 ± 2.53.9 ± 0.362.5 ± 5.331.3 ± 2.431.3 ± 2.5125 ± 9125 ± 11
15 (1,3-alternate)3.9 ± 0.33.9 ± 0.431.3 ± 2.5>5007.8 ± 0.631.3 ± 2.831.3 ± 2.531.3 ± 2.3250 ± 20250 ± 19
23 (monomer)2.54250 ± 19250 ± 19250 ± 21>500250 ± 21250 ± 19500 ± 45500 ± 46500 ± 47>500
16 (cone)9.44Pharmaceutics 15 02731 i0047.8 ± 0.77.8 ± 0.6250 ± 21500 ± 4415.6 ± 1.2125 ± 9500 ± 42500 ± 45>500>500
17 (partial cone)7.8 ± 0.67.8 ± 0.7125 ± 10>50062.5 ± 5.462.5 ± 4.9>500>500>500>500
18 (1,3-alternate)62.5 ± 5.6250 ± 18500 ± 45>50031.3 ± 2.4>500500 ± 44500 ± 44>500>500
24 (monomer)2.50125 ± 10250 ± 21250 ± 19>500250 ± 20>500>500>500>500>500
Ciprofloxacin–0.70–0.50 ± 0.040.50 ± 0.040.50 ± 0.030.50 ± 0.033.9 ± 0.43.9 ± 0.40.25 ± 0.020.25 ± 0.020.5 ± 0.040.5 ± 0.04
Norfloxacin–0.69–3.9 ± 0.43.9 ± 0.47.8 ± 0.67.8 ± 0.67.8 ± 0.615.6 ± 1.21.5 ± 0.17.8 ± 0.63.9 ± 0.215.6 ± 1.3
Average of three values measured; ± standard deviation (SD).
Table 2. Cytotoxicity (IC50, µM) and selective indices (SI = IC50/MIC) of compounds 10–12.
Table 2. Cytotoxicity (IC50, µM) and selective indices (SI = IC50/MIC) of compounds 10–12.
CompoundsIC50Selective Index (SI = IC50/MIC)
S. aureusB. cereusE. faecalisE. coliP. aeruginosa
10 (cone)52.0 ± 4.23.30.813.31.70.4
11 (partial cone)3.6 ± 0.34.00.21.90.50.2
12 (1,3-alternate)25.3 ± 1.83.20.43.23.20.4
Average of three values measured; ± standard deviation (SD).
Table 3. Values of hydrodynamic diameters, polydispersity indexes (PDI) and zeta potentials of systems POPG (1 × 10–4 M) and POPG/Macrocycle systems.
Table 3. Values of hydrodynamic diameters, polydispersity indexes (PDI) and zeta potentials of systems POPG (1 × 10–4 M) and POPG/Macrocycle systems.
Supramolecular SystemPOPG/Macrocycle RatioD, nmPDIZeta-Potential, mV
POPG1:0106 ± 20.09–28.2 ± 4.0
POPG + 10 (cone)1:0.1145 ± 30.17–19.6 ± 2.7
1:1159 ± 40.21+26.2 ± 3.0
POPG + 11 (partial cone)1:0.1127 ± 20.13–5.8 ± 5.8
1:1571 ± 40.56+19.7 ± 2.9
POPG + 12 (1,3-altenate)1:0.1143 ± 70.17–11.2 ± 2.3
1:1290 ± 410.22+30.4 ± 2.3
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Shiabiev, I.; Pysin, D.; Akhmedov, A.; Babaeva, O.; Babaev, V.; Lyubina, A.; Voloshina, A.; Petrov, K.; Padnya, P.; Stoikov, I. Towards Antibacterial Agents: Synthesis and Biological Activity of Multivalent Amide Derivatives of Thiacalix[4]arene with Hydroxyl and Amine Groups. Pharmaceutics 2023, 15, 2731. https://doi.org/10.3390/pharmaceutics15122731

AMA Style

Shiabiev I, Pysin D, Akhmedov A, Babaeva O, Babaev V, Lyubina A, Voloshina A, Petrov K, Padnya P, Stoikov I. Towards Antibacterial Agents: Synthesis and Biological Activity of Multivalent Amide Derivatives of Thiacalix[4]arene with Hydroxyl and Amine Groups. Pharmaceutics. 2023; 15(12):2731. https://doi.org/10.3390/pharmaceutics15122731

Chicago/Turabian Style

Shiabiev, Igor, Dmitry Pysin, Alan Akhmedov, Olga Babaeva, Vasily Babaev, Anna Lyubina, Alexandra Voloshina, Konstantin Petrov, Pavel Padnya, and Ivan Stoikov. 2023. "Towards Antibacterial Agents: Synthesis and Biological Activity of Multivalent Amide Derivatives of Thiacalix[4]arene with Hydroxyl and Amine Groups" Pharmaceutics 15, no. 12: 2731. https://doi.org/10.3390/pharmaceutics15122731

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