DOI:
10.1039/C4MD00236A
(Concise Article)
Med. Chem. Commun., 2015,
6, 230-238
Received 4th June 2014 , Accepted 2nd October 2014
First published on 13th October 2014
Introduction
COMPOUND LINKS
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Explore further on Open PHACTSBetulinic acid [3β-hydroxy-lup-20(29)-en-28-oic acid] (BA, Fig. 1), a naturally occurring pentacyclic COMPOUND LINKS
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Explore further on Open PHACTSlupane-type triterpenoid, represents an attractive scaffold for the development of new biologically active compounds, since it exhibits a variety of biological and medicinal properties, such as antiviral, anticancer, anti-bacterial, antimalarial as well as anti-inflammatory activities.1 However, a low water solubility of this compound limits its applications in medicine. A number of structural modifications of BA, mostly at the C-3, C-20 and C-28 positions, were performed in order to increase the water solubility of this triterpenoid, as well as to investigate structure–activity relationships and obtain derivatives with improved pharmacological properties.2 For example, the introduction of sugar moieties at both C-3 and C-28 positions of BA led to a dramatic increase in the anti-cancer activity compared to BA.3 The introduction of amino acids at the C-3 or C-28 position of BA resulted in improved COMPOUND LINKS
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Explore further on Open PHACTSwater solubility as well as selective cytotoxicity of the obtained conjugates.4
The copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC, “click” reaction)5 has been recognized as a powerful tool for the conjugation and decoration of biologically active molecules.6 1,2,3-Triazole moieties are ideal linkers, because they are extremely stable under typical physiological conditions and can form hydrogen bonds.7 Moreover, the 1,2,3-triazole fragment can be considered as a bioisostere of the amide bond.8
Recently, we have reported the synthesis of biohybrids of betulonic acid via CuAAC.9 It was demonstrated that the amides of betulonic acid modified with a 1,2,3-triazole moiety showed anti-inflammatory properties, as well as high antioxidant activity, which exceeded the activity of the reference compound dihydroquercetin by a factor of 1.4.9a In continuation of our studies on the modification of plant-derived pentacyclic triterpenoids, we decided to explore biohybrids of COMPOUND LINKS
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Explore further on Open PHACTSbetulinic acid as potential anti-inflammatory agents with improved solubility in biological media. Herein we report the synthesis and investigation of pharmacological properties of new betulinic acid derivatives containing COMPOUND LINKS
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Explore further on Open PHACTS1,2,3-triazole peptide fragments at the C-3 position.
The starting BA derivatives 2a,b bearing an alkyne group at the C-3 position were obtained via the reaction of ethynylmagnesium bromide with betulonic acid 1 (Scheme 1).10 The reaction furnished the major diastereoisomer 2a with the axially oriented alkynyl group in a 82% yield along with the minor isomer 2b having the equatorially oriented ethynyl group (11% yield). The absolute stereochemistry for 2a,b was assigned using 2D NMR experiments.11 The observed stereochemistry of the nucleophilic addition to betulonic acid 1 can be explained by sterical reasons; in this case, axial methyl groups at 4 and 10 positions control the addition of acetylenic Grignard reagent to the carbonyl group of 1. As a result, the major isomer of the prepared 3-ethynyl substituted COMPOUND LINKS
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Explore further on Open PHACTSbetulinic acid has the same orientation of the hydroxyl group as a natural betulinic acid.
The starting azidopeptides 3 (ref. 12) are accessible by the Ugi reaction13 of chiral isocyanoazides14 with carbonyl compounds (COMPOUND LINKS
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Explore further on Open PHACTSacetone), 2,4-dimethoxybenzyl (DMB) amine, and Boc-protected amino acids. These compounds bearing the azide function can be conjugated with other biologically active molecules via CuAAC.
Having in hands the set of betulinic acid derivatives 2a,b and azidopeptides 3, we investigated the conjugation process. To find the optimal reaction conditions, various reaction conditions have been studied (sodium ascorbate – CuSO4 in COMPOUND LINKS
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Explore further on Open PHACTSH2O; and COMPOUND LINKS
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Explore further on Open PHACTSCuCl in COMPOUND LINKS
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Explore further on Open PHACTSbutan-1-ol) (Table 1). First, betulinic acid derivative 2a bearing the axially oriented ethynyl group at the C-3 position was reacted with azidopeptides 3 in the presence of the corresponding Cu(I) source to afford conjugates 4a–f in 60–88% yields (Scheme 2).11 As an example, a minor product of ethynylation 2b was also reacted with peptide 3e to give compound 5 in a 73% yield (Scheme 2).
Table 1 Yields and conditions for the synthesis of conjugates 4
Unfortunately, all attempts to remove the 2,4-dimethoxybenzyl protecting groups from 4 and 5 under various conditions were not successful. Nevertheless, we evaluated the influence of amino acid fragments on the pharmacological activity of the synthesized conjugates.
The anti-inflammatory activity was chosen as a testing characteristic, since it is an intrinsic property of triterpenoids. It has been studied using the standard model of inflammation in vivo. In addition, we were intrigued to study the influence of amino groups at the C3 position of conjugates on the molecule affinity and its thermodynamic and conformational characteristics in relation to a known molecular target. The Kelch domain of the Keap1 protein was selected as a molecular target of triterpenoids.15,16 It was assumed that the data of docking could explain possible differences in the properties of agents, and thus, would be amplified with the data obtained in vivo. This statement is based on the relationship between the antioxidant and anti-inflammatory activity of tritepenoids observed upon Nrf2/Keap1/ARE activation. It has been recently demonstrated that the activation of this signaling pathway is accompanied not only by the induction of antioxidant factors, but also the suppression of pro-inflammatory enzymes, such as iNOS and iCOX.15,17 The testing of compounds 2, 4 and 5in vivo was carried out using a histamine-induced paw edema model.
It was found that intraperitoneally administered compounds 4b, 4c, 4d and 4a containing alanine, serine, tryptophan and histidine residues, respectively, exhibited significant anti-inflammatory effect, decreasing COMPOUND LINKS
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Explore further on Open PHACTShistamine-induced edema in mice by 31.5, 23.0, 38.8, and 33.4%, respectively, relative to the control (Table 2).
Table 2 Anti-inflammatory activity of compounds 2, 4 and 5 in the histamine-induced paw edema modela
Compound 4e having isoleucine residue has reduced inflammation in the paw of mice by 31.2%, in contrast to its epimer 5 having an opposite arrangement of the hydroxyl group and a triazole fragment at the C-3 position, which demonstrated no significant anti-inflammatory effect (14.5%). The conjugate 4f showed no anti-inflammatory activity at all. Compound 4d is comparable with COMPOUND LINKS
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Explore further on Open PHACTSindomethacin by the intensity of the effect, while compounds 4a,b,e are insignificantly (1.2–1.3 times) inferior to COMPOUND LINKS
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Explore further on Open PHACTSindomethacin given intraperitoneally at the same dose. In addition, the activities of the latter are comparable with the activity of COMPOUND LINKS
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Explore further on Open PHACTSindomethacin, when administered orally at a mean effective dose for mice (20 mg kg−1). The activity of compound 4c is 1.5 times lower than that of the reference drug, in the case of intragastric administration.
It should be noted that the starting 3-ethynyl betulinic acid 2a exhibits significant anti-inflammatory effect (25.2%). Based on the obtained data, the contribution of each amino acid residue to the anti-inflammatory activity of this compound could be conventionally evaluated. Taking into account that the activity of the derivative 2a is 40% less than the activity of COMPOUND LINKS
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Explore further on Open PHACTSindomethacin, when administered intraperitoneally, the introduction of tryptophan, histidine, alanine and isoleucine fragments enhanced its activity by 32.4, 19.5, 15.0 and 14.3%, respectively, while the introduction of a serine fragment resulted in almost the same level of activity. In the case of compound 5, the introduction of isoleucine amino acid fragment led to a decrease in the activity of the starting compound by 25.5%.
Besides anti-inflammatory activity, triterpenoids also exhibit significant antioxidant activity, which is associated with the induction of the cytoprotective signaling pathway Nrf2/Keap1/ARE.15 The activation of this pathway leads to the ARE-controlled gene expression of enzymes of the second phase of biotransformation and other antioxidant molecules. Cysteine residues in Kelch-domain of repressor protein Keap1 represent a well-known molecular target of triterpenoids, binding with which leads to disintegration of the Keap1-Nrf2 complex and activation of ARE genes.16 In order to study the conformational features and the nature of the relationships that occur during the interaction of the initial triterpenoid 2a and its peptide conjugates with the molecular target of Keap1, their docking at the binding site of the Keap1 Kelch-domain model (PDB ID 4IQK) with the ranking of values of the minimum binding energy in comparison with the native ligand Cpd16 (N,N′-naphthalene-1,4-diylbis (4-methoxybenzenesulfon-amide)) has been performed.
The minimum binding energy is inversely proportional to the affinity of the ligand to the binding site of the receptor. According to the docking results, all compounds have lower affinity to a binding site of the 4IQK model than the native ligand Cpd16 (Table 3). However, the values of minimum binding energy are quite low for all studied ligands, which is indicative of a high probability of their interaction with Kelch-domain of Keap1. The analysis of the spatial conformation of the compounds in the binding site of 4IQK revealed two types of ligand configuration. Compound 2a, which is embedded in the binding site with a triterpenoid skeleton, is related to the first type of ligand configuration (Fig. 2). Its peptide conjugates, which are embedded in the binding site with the amino acid groups at the C3 position of the triterpenoid skeleton, as it has been shown for example on compound 4a, are related to the second type of ligand configuration (Fig. 3).
Ligand | Minimum binding energy, kcal mol−1 | Hydrophobic contacts | Non-covalent bonds | Total number of bonds |
Cpd16 | −10.6 | 4 | 5 | 9 |
2a | −9.1 | 2 | 1 | 3 |
4a | −8.3 | 5 | 3 | 8 |
5 | −8.2 | 3 | 1 | 4 |
4b | −7.6 | 5 | 3 | 8 |
4f | −7.5 | 1 | 4 | 5 |
4c | −7.2 | 5 | 1 | 6 |
4d | −7.0 | 6 | 2 | 8 |
4e | −6.8 | 3 | 3 | 6 |
|
| Fig. 2 Docking visualization of 2a in Kelch-domain. | |
|
| Fig. 3 Docking visualization of 4a in Kelch-domain. | |
The calculation of two-dimensional models of ligand–receptor complexes in the PoseView program made it possible to study in detail the nature of bonds of synthesized compounds with amino acid residues in the binding site of the 4IQK model. Non-covalent interactions between the ligands and amino acid residues at the binding site are represented by the hydrogen bonding and π-interactions with regard to the hydrophobic contacts (Table 3). Stereochemistry of the 4IQK binding site is defined by the presence of the amino acid residues arginine 415, alanine 556, tyrosine 334, 572 and 525, as well as serine 508, 602 and 555. Moreover, alanine 556 and tyrosine 572 residues form only the hydrophobic surfaces at the binding site. The hydroxyl group of serine residue 508 forms a hydrogen bond with the ligands, and the remaining amino acid residues are involved both in the hydrophobic contacts and in the formation of hydrogen and π-bonds.
In general, in the peptide conjugates under investigation, the π-system of the dimethoxyphenyl group typically participates in the formation of π-interaction with the amino acid residues in the binding site. In compound 4b, this group interacts with the phenyl groups of tyrosine 334 and phenylalanine 577 residues, and in compound 4a – with the guanidine group of arginine 415. The hydroxyl group at the C3 position forms a hydrogen bond with a hydroxyl group of serine 508 in compound 2a. In the case of compound 4a, this group reacts with a hydroxyl group of tyrosine residue 525. Oxygen atoms with a shifted electron density in the structure of the peptide fragment at the C3 position of COMPOUND LINKS
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Explore further on Open PHACTSbetulinic acid play a significant role in the formation of non-covalent interactions between the conjugates and amino acid residues in the binding site of 4IQK.
Thus, the docking data indicates that the peptide residues at the C3 position of COMPOUND LINKS
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Explore further on Open PHACTSbetulinic acid do not increase the affinity of the molecule, but play an essential role in the interaction with amino acid residues in the binding site of the Kelch-domain of Keap1. Peptide moieties actively enter into non-covalent interactions with the amino acid residues in the binding site and have a conformation, which can be embedded into the geometrical configuration of the pocket of the Keap1 C-terminal domain, due to the presence of polar oxygen atoms and the π-systems of phenyl groups. The higher affinity of triterpenoid 2a, compared with its derivatives, may be associated with the smaller size of its molecule, and, therefore, with its ability to form more strong links with complementary groups, when it is embedded into the hydrophobic cavity of the site, despite the minimal amount of non-covalent contacts. A decrease in the affinity of the peptide conjugates compared to the starting compound 2a may result in a shorter retention time of a peptide ligand on the receptor that has no significant influence on its antioxidant effect. This statement is based on the fact that the triterpenoid derivatives have no direct antioxidant activity, but act as pH-sensitive triggers by switching the activity of the signaling Nrf2/Keap1/ARE network at the change in the oxidative status of the cell.17,18
Conclusions
In summary, we described the conjugation of betulinic acid derivatives 2a,b bearing an ethynyl group at the C-3 position with azidopeptides 3via the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. The anti-inflammatory activity of obtained compounds 2, 4 and 5 was evaluated using the histamine-induced paw edema model. COMPOUND LINKS
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Explore further on Open PHACTSBetulinic acid–peptide conjugates 4a,b,d,e containing histidine, alanine, tryptophan and isoleucine amino acid fragments were found to exhibit high anti-inflammatory activity, which is comparable with that of COMPOUND LINKS
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Explore further on Open PHACTSindomethacin. The introduction of these amino acid residues in the betulinic acid core enhances its anti-inflammatory properties by 19.5, 15.0, 32.4 and 14.3%, respectively. The introduction of the serine amino acid fragment does not significantly affect the activity of the starting compound, whereas the incorporation of isoleucine (compound 5) and phenylalanine fragments reduces its anti-inflammatory activity. The molecular docking of compounds at the binding site of Kelch-domain of Keap1 revealed that the peptide conjugates form more noncovalent interactions with amino acid residues in the binding site, but have a lower affinity than their precursor triterpenoid 2a. At the same time, there are differences among compound 2a and its peptide derivatives associated with that, by which part the molecule is embedded into the binding site: triterpenoid core (2a) or an amino acid moiety (4a–f, 5). Thus, the peptide fragments modify the activity of the initial triterpenoid core not only by probably increasing the solubility, but also by presumably changing the conformational and thermodynamic features, which influence the binding of the compound with its molecular target. The absence of a direct correlation between the docking data and the anti-inflammatory activity in vivo indicates a more complex implementation of the anti-inflammatory effect of the conjugates, which may be caused by their effects on other intracellular factors. The NF-κB signaling pathway may be the most likely target. It is known that COMPOUND LINKS
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Explore further on Open PHACTSbetulinic acid and its derivatives inhibit the activity of pro-inflammatory NF-κB signaling pathways, interacting with the protein kinase inhibitor of IκB nuclear factor.15,18 Experimental
General information
Melting points were determined with a Kofler apparatus. Column chromatography was performed on SiO2 (Aldrich). Analytical TLC was performed using Merck silica gel 60 F254 plates. Visualization was accomplished by using UV light and spraying aqueous potassium permanganate (COMPOUND LINKS
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Explore further on Open PHACTSKMnO4). The IR spectra were recorded using a UR-20 spectrometer in nujol. The 1H NMR and 13C NMR spectra were recorded using a Bruker Avance 400 spectrometer (400 and 100 MHz, respectively) and a Bruker AVANCE III spectrometer (600.30 MHz and 150.96 MHz, respectively), in COMPOUND LINKS
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Explore further on Open PHACTSTMS as the internal standard. The high-resolution ESI mass spectra were recorded using a Bruker micrOTOF II spectrometer. Chemical shifts were given (δ in ppm) relative to the residual signals of COMPOUND LINKS
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Explore further on Open PHACTSCHCl3 (δH 7.24 ppm and δC 76.90 ppm). Coupling constants (J in Hz) were accurate to ±0.2 Hz. Due to the complexity of signals in the 1H NMR spectra for the betulonic acid derivatives,19 only the characteristic signals are assigned. Most protons of the triterpenoid skeleton resonate between 0.8 and 2.7 ppm. The assignment of signals in 1H and 13C NMR spectra are based on data obtained in 2D COSY and NOESY experiments, and 1H–13C-correlations HSQC and HMBC. The figures show characteristic interactions observed in the NOESY experiment (the spectrum was recorded with τm= 310 and a recovery delay of 2.0 s). 1k data points were collected for 256 increments of 16 scans, using TPPI f1 quadrature detection. Data were processed within a squared cosine-bell window in both dimensions with a single zero-fill in f1. The choice in favor of the axial location of the ethynyl group in 2a was made on the basis of NOE's lack for the axial 23-CH3 because of the largest distance between them (∼6, 4 Å). The signals of a solvent (δH = 7.26 ppm, δC = 76.9 ppm) were used as the internal standard.
General procedure for the synthesis of peptides 3
The corresponding amine (5 mmol) and COMPOUND LINKS
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Explore further on Open PHACTSacetone (10 mmol) were dissolved in 5 mL of COMPOUND LINKS
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Explore further on Open PHACTSisocyanide (5 mmol) were added at room temperature. The mixture was stirred for 24 h. The solvent was removed in vacuo and the residue was purified by column chromatography (COMPOUND LINKS
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Explore further on Open PHACTShexane–ethyl acetate 2 : 1). N-(tert-Butoxycarbonyl)-L-histidyl-N1-(2-azidoethyl)-N2-(2,4-dimethoxybenzyl)-2-methylalaninamide (3a). According to the general procedure for the synthesis of peptides, 3a was obtained from COMPOUND LINKS
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Explore further on Open PHACTS2,4-dimethoxybenzylamine, CH2O, N-Boc-L-histidine and 1-azido-2-isocyanoethane. White solid, yield 0.58 g (49%), mp 59–62 °C. IR (neat, ν/cm−1) 1707, 1760 (CO); 2110 (N3), 3350 (NH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 400 MHz) δ 1.22 (s, 3H), 1.39 (s, 3H), 1.44 (s, 9H), 2.80–2.86 (m, 1H), 3.03–3.07 (m, 1H), 3.33–3.50 (m, 4H), 3.70 (s, 3H), 3.77 (s, 3H), 3.99–4.18 (m, 2H), 4.86–4.90 (m, 1H), 5.84 (d, 1H, J = 4 Hz), 6.38 (s, 2H), 6.95–6.97 (d, 1H, J = 4 Hz), 7.30 (s, 1H), 8.12 (s, 1H), 8.86 (br. s, 1H). 13C NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 100 MHz) δ 21.8, 25.1, 27.9, 32.5, 38.8, 43.4, 50.3, 52.3, 54.7, 54.9, 62.9, 79.1, 98.2, 103.5, 115.3, 116.8, 129.0, 136.1, 138.4, 154.4, 157.9, 160.2, 171.8, 175.3. HRMS (ESI) calcd for C26H38O6N8 558.2909, found [M]+ 558.2906. Synthesis of betulinic acid derivatives 2a,b
Major isomer 2a. IR (neat, ν/cm−1) 1695 (CO); 2660 (OH); 3300 (CH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 600.30 MHz) δ 0.83 (H25), 0.85 (H23), 0.93 (H26), 1.00 (H27), 1.05 (H24), 1.07 (H12e), 1.15 (H5),1.19 (H15e), 1.26 (H11a), 1.30 (H1a), 1.35 and 1.49 (H6), 1.36 and 1.42 (H7), 1.36 (H9), 1.40 (H21β),1.41 (H16a), 1.44 (H11e), 1.49 (H22β), 1.54 (H15a), 1.63 (H1e), 1.63 (H18), 1.68 (H2e), 1.69 (CH3–C20), 1.69 (H12e), 1.93 (H2a), 1.96 (H22α), 2.00 (H21α), 2.18 (H13), 2.27 (H16e), 2.48 (H30), 3.00 (H19), 4.61 (CH2-trans), 4.74 (CH2-cis). 13C NMR δ 14.8 (C27), 15.9 (C26), 16.4 (C25), 17.3 (C23), 18.4 (C6), 19.3 (CH3–C20), 20.7 (C11), 25.36 (C12), 25.57 (C24), 29.66 (C15), 30.48 (C21), 32.07 (C16), 32.5 (C2), 34.1 (C7), 36.9 (C10), 37.1 (C22), 37.8 (C1), 38.4 (C13), 40.6 (C8), 41.2 (C4), 42.3 (C14), 46.8 (C19), 49.2 (C18), 50.4 (C9), 53.0 (C5), 56.3 (C17), 73.4 (C30), 75.6 (C3), 87.0 (C29), 109.6 (CH2C20), 150.3 (C20), 181.8 (COOH). HRMS (ESI) calcd for C32H48O3 + COMPOUND LINKS
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Explore further on Open PHACTSNa+ 503.3496, found [M + Na]+ 503.3496. Minor isomer 2b. IR (neat, ν/cm−1) 1687 (CO); 2108 (CC); 2869 (OH); 3306 (CH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 600.30 MHz) δ 0.85 (H25), 0.93 (H26), 0.97 (H23), 0.99 (H27), 1.03 (H12e), 1.04 (H24), 1.19 (H15e), 1.24 (H11a), 1.26 (H1a), 1.28 (H5), 1.35 and 1.42 (H7), 1.36 and 1.41 (H6), 1.39 (H21β), 1.39 (H9),1.41 (H16a), 1.42 (H11e), 1.42 (H1e), 1.48 (H22β), 1.53 (H15a), 1.62 (H18), 1.69 (CH3–C20), 1.69 (H12e), 1.75 (H2e), 1.96 (H22α), 1.98 (H21α), 2.10 (H2a), 2.17 (H13), 2.27 (H16e), 2.41 (H30), 3.00 (H19), 4.61 (CH2-trans), 4.74 (CH2-cis). 13C NMR δ 14.7 (C27), 15.8 (C25), 15.9 (C26), 18.7 (C6), 19.3 (CH3–C20), 20.6 (C11), 21.3 (C23), 25.2 (C24), 25.4 (C24), 29.6 (C15), 30.5 (C21), 31.9 (C2), 32.1 (C16), 31.9 (C2), 32.1 (C16), 34.0 (C1), 34.1 (C7), 36.9 (C10), 37.0 (C22), 38.3 (C13), 40.4 (C4), 40.6 (C8), 42.4 (C14), 46.9 (C19), 49.0 (C5), 49.2 (C18), 50.1 (C9), 56.3 (C17), 71.9 (C3), 74.1 (C3), 87.5 (C29), 109.6 (CH2C20), 150.3 (C20), 181.5 (COOH). HRMS (ESI) calcd for C32H48O3 480.3598, found [M]+ 480.3605. N-[(1,1-Dimethylethoxy)carbonyl]-L-histidyl-N1-[2-[4-[3,28-dihydroxy-28-oxolup-20(29)-en-3-yl]-1H-1,2,3-triazol-1-yl]ethyl]-N2-[(2,4-dimethoxyphenyl)methyl]-2-methyl-alaninamide (4a). A mixture of 103 mg (0.21 mmol) of alkyne 2a, 120 mg (0.21 mmol) of azide 3a and 3 mg of COMPOUND LINKS
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Explore further on Open PHACTStoluene was kept for 11 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel and washed with aqueous ammonia to remove the copper salts. The organic layer was separated, dried over COMPOUND LINKS
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Explore further on Open PHACTSNa2SO4 and filtered. The solvent was removed in vacuo. COMPOUND LINKS
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Explore further on Open PHACTSHexane was added and the resulting precipitate was filtered off to afford 170 mg (77%) of the triazole 4a as a white solid, mp 150–153 °C. IR (neat, ν/cm−1) 1710, 1758 (CO); 3423 (NH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 400 MHz) δ 0.58 (s, 3H), 0.93–0.95 (m, 6H), 1.00–1.02 (m, 6H), 1.44 (s, 9H), 1.68 (s, 3H), 2.94–3.05 (m, 2H), 3.69 (s, 3H), 3.76 (s, 3H), 3.91–3.95 (m, 1H), 4.10–4.14 (m, 1H), 4.54–4.58 (m, 3H), 4.73 (s, 1H), 4.87–4.90 (m, 1H), 5.97 (d, 1H, J = 8.0 Hz), 6.36–6.38 (m, 2H), 6.94 (d, 1H, J = 8.0 Hz), 7.25 (s, 1H), 7.67 (s, 1H), 8.02 (s, 1H), 9.02 (br. s, 1H). 13C NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 100 MHz) δ 14.8, 15.5, 16.1, 18.2, 18.9, 18.9, 20.4, 21.6, 25.1, 25.3, 28.0, 29.4, 30.1, 31.8, 32.4, 33.8, 36.1, 36.6, 36.7, 38.0, 39.5, 40.2, 41.0, 42.0, 43.4, 46.5, 48.5, 48.8, 50.1, 51.0, 52.2, 54.9, 54.7, 55.9, 63.0, 75.6, 79.2, 98.2, 103.5, 109.2, 115.3, 116.6, 122.5, 129.0, 136.3, 138.0, 146.3, 150.0, 152.5, 154.4, 157.9, 160.2, 171.8, 175.9, 180.6. HRMS (ESI) calcd for C58H86N8O9 + COMPOUND LINKS
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Explore further on Open PHACTSH+ 1039.6596, found [M + H]+ 1039.6568. N-[(1,1-Dimethylethoxy)carbonyl]-β-alanyl-N1-[(1S)-1-[[4-[3,28-dihydroxy-28-oxolup-20(29)-en-3-yl]-1H-1,2,3-triazol-1-yl]methyl]-2-methylpropyl]-N2-[(2,4-dimethoxyphenyl) methyl]-2-methyl-alaninamide (4b). A mixture of 100 mg (0.21 mmol) of alkyne 2a, 111 mg (0.21 mmol) of azide 3b, 5 mg of CuSO4·5H2O and 16 mg of sodium ascorbate in 10 mL of a mixture of COMPOUND LINKS
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Explore further on Open PHACTSCH2Cl2–COMPOUND LINKS
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Explore further on Open PHACTSH2O (10 : 1 ratio) was stirred at 40 °C for 7 h. Then the mixture of COMPOUND LINKS
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Explore further on Open PHACTSCH2Cl2–COMPOUND LINKS
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Explore further on Open PHACTSH2O was replaced by aqueous COMPOUND LINKS
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Explore further on Open PHACTSethanol (4 : 1). The reaction mixture was kept for additional 7 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel and washed with aqueous ammonia to remove the copper salts. The organic layer was separated, dried over COMPOUND LINKS
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Explore further on Open PHACTSNa2SO4 and filtered. The solvent was removed in vacuo. COMPOUND LINKS
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Explore further on Open PHACTSHexane was added and the resulting precipitate was filtered off to afford 140 mg (65%) of the triazole 4b as a white solid, mp 135–137 °C. IR (neat, ν/cm−1) 1647, 1708 (CO), 3390 (NH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 400 MHz) δ 0.62 (s, 3H), 0.90–1.07 (m, 18H), 1.41 (s, 9H), 1.68 (s, 3H), 2.98–3.05 (m, 1H), 3.29 (br. s, 2H), 3.79, 3.80 (s, 6H), 4.48–4.54 (m, 4H), 4.60 (s, 1H), 4.73 (s, 1H), 5.55 (m, 1H), 6.00 (d, 1H, J = 8.0 Hz), 6.44 (d, 1H, J = 4.0 Hz), 6.49 (d, 1H, J = 8.0 Hz), 7.42 (d, 1H, J = 8.0 Hz), 7.85 (s, 1H). 13C NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 100 MHz) δ 13.8, 14.7, 15.5, 16.1, 18.1, 18.4, 19.0, 19.2, 20.4, 23.5, 25.1, 28.0, 29.1, 29.4, 30.2, 31.8, 32.7, 33.8, 34.2, 36.0, 36.6, 36.7, 38.0, 40.2, 40.9, 42.0, 46.5, 48.8, 49.9, 50.2, 50.7, 54.2, 54.8, 54.9, 55.9, 60.0, 62.2, 75.6, 78.5, 98.1, 103.8, 109.2, 117.7, 123.3, 127.5, 150.1, 153.6, 155.62, 156.7, 159.8, 173.0, 174.6, 180.8. HRMS (ESI) calcd for C58H90N6O9 + COMPOUND LINKS
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Explore further on Open PHACTSH+ 1015.6848, found [M + H]+ 1015.6842. N-[(1,1-Dimethylethoxy)carbonyl]-L-seryl-N1-[(1S)-2-[4-[3,28-dihydroxy-28-oxolup-20(29)-en-3-yl]-1H-1,2,3-triazol-1-yl]-1-(phenylmethyl)ethyl]-N2-[(2,4-dimethoxyphenyl)methyl]-2-methyl-alaninamide (4c). 120 mg (0.25 mmol) of compound 2a, 149 mg (0.25 mmol) of azide 3c, 3 mg COMPOUND LINKS
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Explore further on Open PHACTSCuCl were added to 5 mL of COMPOUND LINKS
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Explore further on Open PHACTSbutan-1-ol. The reaction mixture was kept at 115 °C for 7 h, diluted with 10 mL of COMPOUND LINKS
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Explore further on Open PHACTStoluene and then washed with aqueous ammonia. The organic layer was separated, dried over COMPOUND LINKS
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Explore further on Open PHACTSNa2SO4 and filtered. The solvent was removed in vacuo. COMPOUND LINKS
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Explore further on Open PHACTSHexane was added and the resulting precipitate was filtered off to afford 193 mg (71%) of the triazole 4c as a white solid, mp 130–132 °C. IR (neat, ν/cm−1) 1654, 1710 (CO); 3360 (NH, OH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 400 MHz) δ 0.64 (s, 3H), 0.93–0.94 (m, 6H), 1.00–1.01 (m, 6H), 1.43 (s, 9H), 1.70 (s, 3H), 2.99–3.05 (m, 1H), 3.61–3.65 (m, 1H), 3.74, 3.78 (s, 6H), 3.88–3.91 (m, 1H), 4.42–4.48 (m, 3H), 4.61–4.77 (m, 4H), 4.93 (br. s, 1H), 6.00 (d, 1H, J = 8.0 Hz), 6.41–6.43 (m, 2H), 7.11 (d, 1H, J = 8.0 Hz), 7.18–7.31 (m, 5H), 7.71 (s, 1H). 13C NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 100 MHz) δ 13.8, 14.6, 15.5, 16.2, 18.1, 18.9, 19.0, 21.5, 24.8, 25.2, 27.9, 29.4, 30.2, 31.1, 31.8, 32.6, 33.7, 36.1, 36.6, 38.0, 40.2, 41.0, 42.0, 43.7, 46.5, 48.8, 49.9, 50.8, 50.9, 51.2, 53.1, 54.7, 54.9, 55.9, 60.0, 63.2, 76.0, 79.4, 98.2, 103.6, 109.2, 117.0, 123.7, 126.4, 128.2, 128.8, 136.8, 150.0, 152.8, 154.8, 157.7, 160.2, 172.3, 175.1, 180.9. HRMS (ESI) calcd for C62H90N6O10 + H+ 1079.6797, found [M + H]+ 1079.6791. N-[(1,1-Dimethylethoxy)carbonyl]-3-(1H-indol-2-yl)alanyl-N1-[(1S)-2-[4-[3,28-dihydroxy-28-oxolup-20(29)-en-3-yl]-1H-1,2,3-triazol-1-yl]-1-(phenylmethyl)ethyl]-N2-[(2,4-dimethoxyphenyl)methyl]-2-methyl-alaninamide (4d). A mixture of 140 mg (0.29 mmol) of alkyne 2a, 202 mg (0.29 mmol) of azide 3d and 5 mg COMPOUND LINKS
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Explore further on Open PHACTSCuCl in 7 mL of COMPOUND LINKS
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Explore further on Open PHACTStoluene was kept for 9 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel and washed with aqueous ammonia to remove the copper salts. The organic layer was separated, dried over COMPOUND LINKS
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Explore further on Open PHACTSNa2SO4 and filtered. The solvent was removed in vacuo. COMPOUND LINKS
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Explore further on Open PHACTSHexane was added and the resulting precipitate was filtered off to afford 260 mg (76%) of the triazole 4d as a white solid, mp 142–144 °C. IR (neat, ν/cm−1) 1697, 1702 (CO), 3423 (NH). 1H NMR ((CD3)2SO, 400.13 MHz) δ 0.48 (s, 3H), 0.84–0.87 (m, 12H), 1.24–1.28 (m, 15H), 1.64 (s, 3H), 2.64–2.82 (m, 2H), 2.93–3.02 (m, 3H), 3.73 (s, 3H), 3.77 (s, 3H), 4.21–4.42 (m, 5H), 4.55 (s, 1H), 4.65–4.68 (m, 2H), 4.83 (d, 1H, J = 20.0 Hz), 6.38–6.44 (m, 1H), 6.59 (s, 1H), 6.74–6.83 (m, 2H), 6.94–7.09 (m, 2H), 7.23–7.26 (m, 3H), 7.32–7.34 (m, 4H), 7.60–7.62 (m, 1H), 7.76–7.80 (m, 1H), 10.73 (s, 1H), 12.06 (s, 1H). 13C NMR ((CD3)2SO, 100 MHz) δ 14.0, 14.8, 15.7, 16.4, 18.1, 18.9, 19.7, 20.5, 22.1, 24.2, 25.4, 27.2, 28.1, 29.4, 30.2, 31.0, 31.8, 32.6, 33.9, 35.5, 36.7, 37.7, 38.2, 40.9, 40.9, 41.9, 42.0, 46.7, 48.6, 49.7, 49.8, 51.0, 53.3, 55.2, 55.4, 55.4, 61.8, 74.5, 78.0, 98.1, 104.3, 109.6, 109.9, 111.1, 118.0, 118.1, 119.0, 120.7, 123.9, 124.5, 126.3, 127.1, 128.4, 129.3, 136.0, 138.2, 150.4, 154.8, 155.5, 156.8, 159.7, 173.3, 174.0, 177.3. HRMS (ESI) calcd for C70H95N7O9 + COMPOUND LINKS
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Explore further on Open PHACTSH+ 1178.7270, found [M + H]+ 1178.7264. N-[(1,1-Dimethylethoxy)carbonyl]-L-isoleucyl-N1-[(1S)-2-[4-[3,28-dihydroxy-28-oxolup-20(29)-en-3-yl]-1H-1,2,3-triazol-1-yl]-1-methylethyl]-N2-[(2,4-dimethoxyphenyl) methyl]-2-methyl-alaninamide (4e). 140 mg (0.29 mmol) of compound 2a, 160 mg (0.29 mmol) of azide 3e, 5 mg of COMPOUND LINKS
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Explore further on Open PHACTSCuCl were added to 7 mL of COMPOUND LINKS
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Explore further on Open PHACTStoluene. The reaction mixture was kept at 110 °C for 24 h, diluted with 10 mL of COMPOUND LINKS
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Explore further on Open PHACTStoluene and then washed with aqueous ammonia. The organic layer was separated, dried over COMPOUND LINKS
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Explore further on Open PHACTSNa2SO4 and filtered. The solvent was removed in vacuo. COMPOUND LINKS
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Explore further on Open PHACTSHexane was added and the resulting precipitate was filtered off to afford 264 mg (88%) of the triazole 4e as a white solid, mp 148–150 °C. IR (neat, ν/cm−1) 1708, 1714 (CO), 3434 (NH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 400 MHz) δ 0.58 (s, 3H), 0.80–0.87 (m, 6H), 0.93–1.02 (m, 12H), 1.41 (m, 15H), 1.69 (s, 3H), 2.98–3.05 (m, 1H), 3.79 (s, 6H), 4.30–4.68 (m, 7H), 4.73 (s, 1H), 5.36 (d, 1H, J = 8.0 Hz), 5.81–5.83 (m, 1H), 6.44–6.49 (m, 2H), 7.37 (d, 1H, J = 8.0 Hz), 7.62 (m, 1H). 13C NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 100 MHz) δ 11.0, 14.7, 15.2, 15.5, 16.1, 17.2, 18.1, 18.9, 20.4, 23.1, 23.4, 25.1, 27.9, 29.4, 30.2, 31.8, 32.6, 33.8, 36.0, 36.6, 36.7, 38.0, 38.2, 40.2, 40.9, 42.0, 42.2, 45.5, 46.5, 48.9, 50.0, 50.9, 53.3, 54.9, 55.4, 55.9, 62.6, 75.6, 78.9, 98.3, 103.8, 109.2, 117.8, 122.4, 128.4, 150.1, 153.4, 155.0, 156.9, 159.9, 173.5, 174.4, 180.7. HRMS (ESI) calcd for C59H92N6O9 + COMPOUND LINKS
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Explore further on Open PHACTSH+ 1029.7004, found [M + H]+ 1029.6999. N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl-N1-[(1S)-2-[4-[3,28-dihydroxy-28-oxolup-20(29)-en-3-yl]-1H-1,2,3-triazol-1-yl]-1-methylethyl]-N2-[(2,4-dimethoxyphenyl) methyl]-2-methyl-alaninamide (4f). A mixture of 120 mg (0.25 mmol) of alkyne 2a, 145 mg (0.25 mmol) of azide 3f, 5 mg of CuSO4·5H2O and 7 mg of sodium ascorbate in 10 mL of a mixture of COMPOUND LINKS
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Explore further on Open PHACTSTHF–COMPOUND LINKS
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Explore further on Open PHACTSH2O (1 : 1 ratio) was kept for 7 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel and washed with aqueous ammonia to remove the copper salts. The organic layer was separated, dried over COMPOUND LINKS
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Explore further on Open PHACTSNa2SO4 and filtered. The solvent was removed in vacuo. COMPOUND LINKS
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Explore further on Open PHACTSHexane was added and the resulting precipitate was filtered off to afford 160 mg (60%) of the triazole 4f as a white solid, mp 157–159 °C. IR (neat, ν/cm−1) 1654, 1708 (CO), 3429 (NH, OH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 400 MHz) δ 0.61 (s, 3H), 0.93–1.03 (m, 12H), 1.35 (s, 9H), 1.69 (s, 3H), 2.78–2.83 (m, 1H), 2.95–3.03 (m, 2H), 3.78 (s, 6H), 4.24–4.65 (m, 7H), 4.74 (s, 1H), 5.39 (d, 1H, J = 8.0 Hz), 5.87 (d, 1H, J = 8.0 Hz), 6.43 (s, 2H), 7.06 (s, 2H), 7.21 (m, 4H), 7.69 (s, 1H). 13C NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 100 MHz) δ 13.7, 14.7, 15.5, 16.1, 17.3, 18.1, 18.9, 19.0, 23.2, 23.4, 25.1, 27.9, 29.4, 30.2, 31.2, 32.7, 33.8, 36.0, 36.7, 38.0, 39.3, 40.2, 40.9, 42.0, 45.5, 46.5, 48.9, 49.9, 50.8, 52.7, 53.2, 54.8, 54.9, 55.9, 60.0, 62.4, 75.7, 79.1, 98.2, 103.9, 109.2, 117.9, 122.7, 126.3, 128.0, 129.2, 131.7, 136.2, 150.1, 153.5, 154.6, 156.8, 159.8, 173.1, 174.3, 180.8. HRMS (ESI) calcd for C62H90N6O9 + H+ 1063.6848, found [M + H]+ 1063.6842. N-[(1,1-Dimethylethoxy)carbonyl]-L-isoleucyl-N1-[(1S)-2-[4-[3,28-dihydroxy-28-oxolup-20(29)-en-3-yl]-1H-1,2,3-triazol-1-yl]-1-methylethyl]-N2-[(2,4-dimethoxyphenyl) methyl]-2-methyl- alaninamide (5). A mixture of 70 mg (0.146 mmol) of alkyne 2b, 80 mg (0.146 mmol) of azide 3e and 3 mg of COMPOUND LINKS
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Explore further on Open PHACTSCuCl in 5 mL of COMPOUND LINKS
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Explore further on Open PHACTStoluene was kept for 13 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel and washed with aqueous ammonia to remove the copper salts. The organic layer was separated, dried over COMPOUND LINKS
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Explore further on Open PHACTSNa2SO4 and filtered. The solvent was removed in vacuo. COMPOUND LINKS
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Explore further on Open PHACTSHexane was added and the resulting precipitate was filtered off to afford 110 mg (73%) of the triazole 5 as a white solid, mp 151–153 °C. IR (neat, ν/cm−1) 1643, 1714 (CO), 3435 (NH). 1H NMR (COMPOUND LINKS
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Explore further on Open PHACTSCDCl3, 400 MHz) δ 0.61 (s, 3H), 0.79–0.85 (m, 12H), 0.92 (s, 3H), 0.99 (s, 3H), 1.37 (s, 12H), 1.67 (s, 3H), 2.96–3.03 (m, 1H), 3.76–3.77 (m, 6H), 4.24–4.58 (m, 7H), 4.72 (s, 1H), 5.19 (d, 1H, J = 8.0 Hz), 5.66 (d, 1H, J = 8.0 Hz), 6.41–6.46 (m, 2H), 7.41 (d, 1H, J = 8.0 Hz), 7.76 (s, 1H). 13C NMR δ 11.3, 14.7, 15.5, 15.8, 15.9, 17.4, 18.7, 19.2, 20.6, 21.2, 23.0, 23.7, 23.9, 25.3, 28.2, 29.6, 30.4, 31.0, 32.0, 34.1, 34.4, 37.0, 37.1, 38.2, 38.8, 40.6, 42.3, 42.4, 45.6, 46.8, 49.1, 50.1, 50.2, 53.4, 55.2, 55.6, 56.2, 60.3, 62.7, 74.9, 79.2, 98.5, 104.1, 109.5, 118.1, 122.6, 128.5, 150.5, 152.6, 155.2, 157.0, 160.2, 173.6, 174.4, 180.9. Anal. calcd for C59H92N6O9:C, 68.84; H, 9.01; N, 8.16. Found: C, 68.54; H, 8.99; N, 7.92. Pharmacological experiments
The experiments were carried out using outbred male mice housed in standard environmental conditions. The animals were given standard granulated food and COMPOUND LINKS
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Explore further on Open PHACTSwater ad libitum. All experimental procedures were approved by the Bio-Ethical Committee of Medicine Chemistry Department of the Novosibirsk Institute of Organic Chemistry SB RAS in accordance with European Communities Council Directive 86/609/EEC. Inflammatory edema was induced by subplanar injection of 0.05 mL of 0.1% histamine in COMPOUND LINKS
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Explore further on Open PHACTSwater solution into the hind paw of male mice. The test compounds were administered intraperitoneally at a dose of 50 mg kg−1 b.w. as aqueous-Tween-80 suspension (COMPOUND LINKS
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Explore further on Open PHACTSwater : Tween-80 100 : 1, v/v) 1 h before the histamine injection. This route of administration was selected to exclude possible metabolic transformation of conjugates in the gastrointestinal tract. The reference agent COMPOUND LINKS
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Explore further on Open PHACTSindomethacin (Fluka BioChemica) was administered at two doses and regimens, one of which corresponded to the dose and regimen of agents administration (50 mg kg−1 i. p.), and the second – to a routine administration and dosage of COMPOUND LINKS
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Explore further on Open PHACTSindomethacin in mice – 20 mg kg−1 orally. Docking. Hardware and software
Quad-Core AMD Phenom II X4 925 based configuration running on Linux 3.0.0-14 OS was used for calculations. All structures were visualized using UCSF Chimera 1.8 program.20 Docking was performed using AutoDock Vina 1.1.2 algorithms.21 PyRx 0.9 and MGL Tools 1.5.6 GUIs were utilized for the convenient docking preparation. Visualization of ligand–receptor complexes was carried out by using the PoseView web interface.22 The ProFit web interface [Martin, A.C.R. and Porter, C.T., http://www.bioinf.org.uk/software/profit] was applied to calculate the root mean square deviation (RMSD) of the ligands' coordinates. To minimize the energy of structures of novel derivatives and to record them in different chemical formats the OpenBabel 2.3.2 program was used.23 Models
4IQK is the X-ray crystallographic model of Keap1 Kelch-domain.24 In the binding site of 4IQK the Cpd16 molecule is coordinated, the structure of which was obtained by the authors of the model as a result of high-throughput screening of the MLPCN chemical database. The models of new betulinic acid derivatives were obtained by using the mmff94 energy minimization algorithm. Receptor and ligands preparation
A model of Keap1 Kelch-domain is available in the Protein Data Bank (http://www.pdb.com), PDB ID 4IQK. The model represents a symmetric subunit of a cylindrical shape with a distinct binding site, in which the Cpd16 molecule is coordinated. For the realization of docking it was necessary to adapt the 4IQK model for UCSF Chimera 1.8 program. The Cpd16 molecule was removed from binding sites, and was stored separately without changing the coordinates and conformation for docking validation and accurate mapping of the binding site by re-docking into the adapted model 4IQK. The adapted model 4IQK and all ligands were prepared for docking in AutoDock Vina by conversion to the pdbqt format, which contains the coordinates of the atoms, charges, solubility data and descriptions of rigid and flexible parts of the molecules. Algorithms validation, search space optimization and docking
The docking procedure was carried out for the unchanged conformation of the receptor and flexible ligand molecules. The standard parameters of the AutoDock Vina program were used. Re-docking of Cpd16 into the adapted model of 4IQK was done to validate the docking algorithms of AutoDock Vina. The docking algorithm is adequate upon the condition that the standard deviation (RMSD) between the coordinates of the native ligand and the best configuration obtained by re-docking satisfies RMSD <2 Å. The AutoDock Vina search area was configured exactly to the coordinates of the native ligand and it presents a required minimum that significantly increases the accuracy of the result. The docking of all studied ligands at the binding site of the adapted model of 4IQK was performed after the validation of docking algorithms was carried out. The results of ligands docking were compared with the minimum energy of binding of Cpd16 obtained as a result of re-docking. Acknowledgements
This work was supported by Interdisciplinary Grant no. 41 of SB of the Russian Academy of Sciences (2012–2014), the Chemical Service Centre of SB RAS, the Russian Foundation for Basic Research (Grants no. 12-03-31582, 12-03-00292a, and 13-03-00129a) and a grant of the Ministry of Education and Science of the Russian Federation (2014-2016). Notes and references
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(a) S. F. Vasilevsky, A. I. Govdi, I. V. Sorokina, T. G. Tolstikova, D. S. Baev, G. A. Tolstikov, V. I. Mamatuyk and I. V. Alabugin, Bioorg. Med. Chem. Lett., 2011, 21, 62 CrossRef CAS PubMed;
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