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Novel Oleanolic Acid-Phtalimidines Tethered 1,2, 3 Triazole Hy-Brids As Promising Antibacterial Agents: Design, Synthesis, In Vitro Experiments and In Silico Docking Studies

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20 May 2023

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23 May 2023

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Abstract
As part of valorisation of agricultural waste into bioactive compounds, a series of structurally novel oleanolic acid ((3β-hydroxyolean-12-en-28-oic acid, OA-1)-phtalimidines (isoindolinones) conjugates 18a-v bearing 1,2,3-triazole moieties were designed and synthesized by treating a previously azide 4 prepared from OA-1 isolated from olive pomace (Olea europaea L.) with a wide range of propargylated phtalimidines using the Cu(I)-catalyzed click chemistry approach. OA-1 and its newly prepared analogues 18a-v were screened in vitro for their antibacterial activity against two Gram-positive bacteria, Staphylococcus aureus and Listeria monocytogenes; and two Gram-negative bacteria, Salmonella thyphimurium and Pseudomonas aeruginosa. Attractive results were obtained notably against L. monocytogenes. Compounds 18d, 18g and 18h exhibited the highest antibacterial activity when compared with OA-1 and other compounds in the series against tested pathogenic bacterial strains. Molecular docking study was performed to explore the binding mode of the most active derivatives against the target protein from L. monocytogenes. Results showed the importance of both hydrogen bonding and hydrophobic interactions with the target protein and are in favor to the experimental data.
Keywords: 
Subject: Chemistry and Materials Science  -   Organic Chemistry

1. Introduction

Agricultural waste has emerged as a huge pool of fine chemicals that can be turned into high-value compounds with many pharmaceutical applications [1]. Triterpenic acids such as Oleanolic Acid (OA-1, Figure 1), extracted from olive pomace [2], is a typical example of such high-value compounds. A number of studies demonstrated that OA-1 has a wide range of biological activities including anti- inflammatory [3], hepatoprotective [4], antioxidant [5], antitumor [6,7], anti-HIV [8], antidiabetic [9], and antiparasitic [10] effects. It has also found that OA-1 and its derivatives exhibit appreciable antibacterial activities with a broad spectrum of MIC values [11,12,13]. The antibacterial activity of OA-1 is thought to be due to its ability to influence peptidoglycan structure and composition, gene expression and biofilm formation, thereby preventing bacterial growth [14]. On the other hand, Nitrogen-containing heterocyclic compounds are omnipresent in bioactive molecules and are invaluable sources of therapeutic agents. Among them, Phtalimidine (1-isoindolinone and C3 1-isoindolinone-derived) derivatives [15] are playing an important role in drug discovery due to their broad and abundant biological activities [16,17,18].Many of them have been prepared and examined as antihypertensive [19], antipsychotic [20,21,22], anti-inflammatory [23], anesthetic [24], and vasodilatory [25] agents. Antiviral [26,27,28,29], anticancer [30,31,32,33], antimicrobial [34,35], and anxiolytic [36] activities have also been observed in this class of structures (Figure 1). Thus, enormous research has been taking place in the past few decades to synthesize and to explore various therapeutic prospective of this moiety [15,37,38].
Despite the many potential uses of OA-1, it has not been developed into pharmaceuticals due to its instability and low water solubility. To overcome these drawbacks, several studies have been designed to modify the structure of OA-1 with the hope of improving its physical properties for better bioavailability to enhance its bioactivity. Among the plethora of strategies used, molecular hybridization, which is a new concept in drug design and development based on the combination of two different bioactive compounds to produce a new hybrid substance, has emerged as an essential tool for design and construction of novel hybrid molecules with improved biological activities [39,40,41,42,43,44,45]. Considering the increasing incidence of multidrug resistant pathogens generated by extensive use of antibiotics [46], the interesting biological activities of isoindolinones and OA-1, our current research interest in the valorization of agricultural waste into eco-efficient, bioactive products, we are reporting herein the synthesis of novel triazole-tethered isoindolinones-oleanolic acid hybrids by means of click chemistry-mediated fusion between isoindolinones and oleanolic acid derivatives. To that end a Cu(I)-catalyzed azide alkyne Huisgen 1,3-cycloaddition was used [47,48,49,50]. Moreover, we hope that the introduction of triazole linkers, known by their broad and abundant biological properties (antiviral [51], antioxidant [52,53], antimicrobial [54], anticancer [55,56], antimalarial [57,58]...) could contribute to the improvement of the overall biological activity of the hybrid molecules. The antibacterial activity of OA-1 and the target com-pounds was screened followed by in silico molecular docking studies for the most potent derivatives to get a distinct insight about the interactions and binding mode in the active sites of the target protein.

2. Results and Discussion

2.1. Chemistry

2.1.1. Isolation of oleanolic acid OA-1

OA-1 (Figure 1) was isolated from olive pomace (Olea. europaea L.) cultivar: Chemlali, by using a solid-liquid and ultrasound-assisted extraction strategy as previously described by us [59]. This method is low-cost, selective and providing a large amount of OA-1 6.8 g (3.4 mg/g DW)

2.1.2. Synthesis

The new hybrid molecules 18 were designed to include OA-1 on one hand and isoindolinones 6-8 and (±)-12-17 on the other, by connecting them via linker chains of different length. The Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) was chosen as the linking methodology. Starting from the naturally occurring triterpene OA-1, azidoacetyl 4 as first precursor was synthesized in three steps according a previously reported procedure (Scheme 1) [60,61].
The second precursors, namely N/O-propargylated isoindolinones 6-8 were also synthesized using previously published protocols [62,63,64,65,66,67,68]. Substrates 6a-g were prepared through two synthetic routes involving base-assisted N-alkylation of phtalimide 5a, hydantoin 5b, succinimide 5c and saccharin 5d rings with propargyl bromide resulting in the isolation of the precursors 6a-d in good isolated yields (87 to 97%). Under the same conditions, deprotonation then alkylation of N-hydroxyphthalimide 5g afforded compound 6g in 67% isolated yield. Applied to isoindolinone 5a and successivelly but-3-yn-1-ol or but-3-yn-2-ol, mitsunobu reaction [69] (Ph3P/DEAD/THF) provided isoindolinones 6e and 6f in 97% and 57% isolated yields, respectively (Scheme 2). Wishing to study the effect of the presence of a hydroxy and or acetoxy group on the antibacterial activity of our molecules, we subsequently prepared the hydroxylactams 7a and 7e as well as the acetoxylactam 8g. In this sens, selective reduction of propargylated isoindolinones 6a and 6e was performed by using excess of NaBH4 (4 equiv) in a dry MeOH at 0 °C [70] leading to the hydroxylactams 7a and 7e in 95% and 50% isolated yields, respectively (Scheme 2). Using our recently reported one-pot reduction–acetylation protocol, imide 6g afforded the α-acetoxy-lactam 8g in a 98% overall yield (Scheme 2) [71].
Subsequently, phtalimidines (±)-12-17, the other precursors for the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction, were obtained straightforwardly starting from homophthalic acid (HPA-9) as previously described by our group [72,73,74]. Phtalimidines (±)-12a-f, as starting materials, were accomplished by using our well-known 3-step sequence including: (i) esterification of diacid (HPA-9) under reflux in the presence of gaseous HCl, (ii) radical bromination of the obtained diester 10 (e.g., NBS, AIBNcat, CCl4 at reflux), and (iii) condensation of excess of primary amine (α-bromophthalate 11, R-NH2, CH3CN, rt). Next, the C3-alkylated derivatives (±)-13 and (±)-14, were prepared by the deprotonation of the α-position of the nitrogen of phtalimidines (±)-12a-f with potassium carbonate, followed by reaction of various halogenated electrophiles. Under these conditions, substrates (±)-13a-d and (±)-14b-f, were obtained in good to excellent isolated yields 87% to 90% for (±)-13a-d and 81% to 93% for (±)-14b-f (Scheme 3).
In order to evaluate the binding mode, particulary the effect of an hydroxy, amide or carboxy group at C-3, on the antibacterial activity, acid (±)-15f, amide (±)-16b and alcohol (±)-17e were then prepared. Reduction of the ester group at C-3 of (±)-14e with LiBH4 in dichloromethane at room temperature gave phtalimidine alcohol (±)-17e in 80% isolated yield. Saponification of ester functions of esters (±)-14b and (±)-14f under standars conditions (excess of aqueous NaOH, EtOH, rt then diluted HCl at 0 °C) gave the corresponding carboxylic acids (±)-15b and (±)-15f in respectively 93% and 72% isolated yields. In order to obtain amide (±)-16b, acid (±)-15b was treated, in a next step, with methyl glycinate in the presence of EDCI/DMF at room tempreture leading to compound (±)-16b in 75% isolated yield (Scheme 3) [75]. With azide 4 and propargylated phthalimidines 6-8 and (±)-12-17 in hand, we next directed our attention to explore the 1,3-dipolar cycloaddition to form the new hybrid molecules 18. Thus, as shown in Scheme 4, treatement of 4 and 6a used as a model for our study under the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (e.g., CuSO4.5H2O, sodium ascorbate (NaC6H7O6), DCM/H2O, rt 24h) [76] resulted in the formation of the 1,2,3-triazole conjugate 18a in 84% isolated yield.
The above selected conditions were used with the rest of the substrates 6-8 and (±)-12-17, which cyclized to afford the hybrids molecules 18b-t with yields of 70 to 98% after silica gel column chromatography, and the results are summarized in Scheme 5.
It is worth noting that in the presence of the C,N-bispropargylated phtalimidine (±)-13d under standard conditions, the 1,3-dipolar cycloaddition became non selective and lead to a mixture of non separable products, including the mono-cycloaddition product on the C-propargylated alkyne, the mono-cycloaddition on the N-propargylated alkyne and the bis cycloadduct 18v. After rigorous investigations, it was observed that the double cycloaddition occurred seamlessly to give 18v in 80% yield when CuSO4.5H2O (0.2 equiv), sodium ascorbate (0.4 equiv) and 2 equivalents of 4 were used (Scheme 6).
The structures of all the synthesized compounds 18 were confirmed by spectroscopic analysis. For example, the IR spectra of 18e, shows a sharp intense band at 1714 cm−1 attributed to the four C=O moities; the 1H NMR shows a singlet signal at δH 7.56 corresponding to the triazole proton and two triplets at δH 4.02 and 3.17 with coupling constant of 7.3 Hz corresponding to the two methylene groups at the junction of the phthalimide and the triazole moities. The 13C NMR spectra, shows two characteristic signals at δC 177.5 and 168.2 corresponding to the C=O of the ester and the acetoxy groups, respectively, strong signal at δC = 166.1 ppm corresponding to the two C=O moieties of the Pht group was also showed.

2.2. Antibacterial avtivity

All the newly synthesized compounds were evaluated in vitro for their antibacterial activity against the following human pathogenstrains: two Gram-positive bacteria, Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 19115; two Gram-negative bacteria, Salmonella thyphimurium ATCC 14080 and Pseudomonas aeruginosa ATCC 27853. The activity of all the tested derivatives was compared with that of Tetracycline used as standard reference antibiotic and the determination of the inhibition zone “IZ” (in mm), MIC and MBC (in µM) was carried out in this study. Overall, attractive results were obtained against certain strains. Indeed, the values of the IZ diameters, as a preliminary test, given in Table 1 for certain compounds were found to be in agreement with those of the MIC and MBC described in Table 2. These results showed that the starting product OA-1 was active against S. aureus, S. thyphimurium and P. aeruginosa but inactive towards L. monocytogenes. The activity of this compound in several cases exceeds that of certain derivatives and of the reference antibiotic. It has been found to be more active than all its dervivatives 18a-v against S. aureus and P. aeruginosa, but only more active than 18f, 18k and 18q against S. typhimurium.The results showed, on the other hand, that most of the compounds tested proved to be quasi-selective towards L. monocytogenes. For more details, the most interesting results in terms of MIC were noted with the derivatives 18a-h in addition to 18k, 18m and 18q which showed good inhibitory effects on the growth of L. monocytogenes, compared to that of the reference antibiotic (MIC = 576.01 µM). Thus, from a structural point of view, the compound 18g exhibits the highest activity (9.48 µM) towards this Gram-positive starin compared to the rest of the active compounds followed by derivatives 18d (9.56 µM) and 18h (9.89 µM). It appears that the 3-hydroxyisoindolin-1-one fragment in compound 18h contributes to this activity compared to its analog 18a with an isoindoline-1,3-dione moiety (MIC = 12.4 μM). This finding shows that the additional hydroxyl group in 18h instead of the carbonyl function in 18a may account for the noted difference in activity. On the other hand, the higher activity of compound 18a against L. monocytogenes (Table 2), compared to that of its analog 18e allows to conclude that a single methylene binding the phthalimide to the trizole was better than two methylenes giving, perhaps, to this system more free rotation and therefore less possibility of interaction with the target proteins of the bacterium. Also, we noticed that when the methylene linker directly bonded to the phthalimide nitrogen atom in 18g (MIC = 39.01 µM and MBC = 2496.67 µM) is replaced by an oxygen atom in 18e (MIC = 9.48 µM and MBC = 155.65 µM), the antibacterial potential towards L. monocytogenes was much improved, hence the importance of this new linker (-O-CH2-) between phatlimide and triazole to better inhibit this strain. In addition, branching of the ethyl linker in 18e was found to significantly improve the MBC of its analog 18f (MBC = 624.16 µM). The additional methyl group in 18f compared to 18a, more than doubled the activity. Moreover, we noticed that compound 18a with an isoindoline-1,3-dione moiety (MIC = 12.4 µM) remains more active against L. monocytogenes than its analogue 18c with pyrrolidine-2,5-dione moiety (MIC = 12.4 µM). This finding serves as evidence that the antibacterial potential is affected by the additional aromatic ring.Interestingly, against P. aeruginosa, compound 18m exhibited the highest antibacterial activity (MIC = 42.79 µM and MBC = 171.18 µM) compared to the other active compound 18h (MIC = 158.41 µM and MBC = 633.66 µM) and also to the reference antibiotic. The results discussed above demonstrated the assumptions about the structural activity relationship (SAR) and also can be supported with some in silico studies.

2.3. Molecular docking study

The molecular docking simulations applied to antibacterial agents tested in vitro gives a descriptive explanation of the ligand’s binding mode for the inhibition of the target bacterium [77,78]. In this context, to pick up the mode of action of the tested derivatives for their antibacterial potentials, the molecular docking study has been used to determine the binding modes against ABC substrate-binding protein Lmo0181 from L. monocytogenes (PDB ID: 5F7V). As depicted in Table 3, the listed binding affinities of the formed complex were found to be in the range of -12.3 to -10.6 kcal/mol. Thus, from these results, it can be suggested that all tested compounds interact favorably with the target protein and especially for derivatives 18c, 18d, 18h and 18k which showed the best binding scores (-12.3 to -11.6 kcal/mol) even better than the docked antibiotic “Tetracycline” (-11.1 kcal/mol) used as a reference in the in vitro test. Interestingly, as Figure 2 and Figure 3 (A, B, C and D) show, the binding modes for these compounds demonstrate that each ligand was located inside the binding cavity, similar to the co-crystalized inhibitor. Therefore, the best docking score of compound 18h could be attributed to its correct orientation in the receptor cavity (Figure 3: A) and it depends on its structure containing the 3-hydroxyisoindolin-1-one moiety, which could contribute to the stability of the receptor-ligand complex by the formation of three intermolecular conventional hydrogen bonds: two by its hydroxyl group with the residues Asn380 (2.55 Å) and Asp384 (2.12 Å), and another one by its carbonyl function with Thr75 (3.13 Å) amino acid (Figure 4: C’). In addition, the stability of the complex is also perceptible through other hydrophobic interactions formed via the hydrocarbon skeleton of the molecule: Alkyl, Pi-Alkyl and Pi-Pi as depicted in Table 4. The second-best score was designated to compound 18d with -12.0 kcal/mol which is also involved in three conventional H-bonds formed through its benzo[d]isothiazol-3(2H)-one 1,1-dioxide fragment with Glu391 (3.27 Å) (3.38 Å) and its ester function with Trp271 (3.15 Å). As docking results of compound 18h, the derivative 18d displayed also some hydrophobic interactions (Figure 4: B’). On another hand, besides to other types of interactions, the ligand 18c (-11.7 kcal/mol) showed a single hydrogen bond through the carbonyl function of its pyrrolidine-2,5-dione pharmacophore with Trp271 (3.13 Å) (Figure 4: A’), while the derivative 18k (-11.6 kcal/mol) besides forming an hydrogen bond with Trp271 (3.25 Å), it displayed diverse interactions which additionally explain the stability of the docking complex as well as its inhibitory effect towards L. monocytogenes (Figure 4: D’). Attempt to validate QSAR model by using docking results demonstrated a high degree of correlation in terms of the ability to form hydrogen bonds, to display good binding scores and the antibacterial potentials of our synthesized compounds.

3. Materials and Methods

3.1. General experimental procedure

Commercially available compounds were used without further purification (suppliers: Thermo Fisher Scientific Inc., and Sigma-Aldrich Co.). All glass apparatus was oven dried and cooled under vacuum before use. Before their usage, precautions were taken to eliminate moisture by refluxing over CaH2 while distilling the solvents (CH3CN, CH2Cl2). Column chromatographies were carried out with a BUCHI Pure Flash/Prep C-850 chromatography system using puriFlash® packed columns. Distilled solvents were employed as eluents for column chromatography in all cases. Thin layer chromatographies (TLC) was realized on sheets of silica gel 60 precoated with fluorescent indicator UV254 (Merck). Detection was performed by irradiation with a UV lamp and by using an ethanolic solution of p-anisaldehyde. Melting points were measured using a Stuart Scientific SMP10 apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer FT-IR Paragon 1000 spectrometer. 1H NMR and 13C NMR spectra were recorded on a Bruker AvanceIIITM 300 MHz spectrometer at room temperature (rt) with tetramethylsilane (TMS) serving as internal standard. Chemical shifts are expressed in parts per million (δ). Splitting patterns are designed: s, singlet; d, doublet; dd, doublet of doublet; t, triplet; m, multiplet; br s, broaden singlet. Coupling constants (J) are reported in Hertz (Hz). Mass spectra (GC-MS) were obtained on a ThermoFinnigan Automass III spectrometer coupled with a gas chromatograph Trace GC 2000. An agilent 6530 Q-Tof MS system was used to conduct the measurment of high resolution mass spectra (HRMS).

3.2. Chemistry

3.2.1. General procedure for the preparation of compounds 18

To a mixture of equimolar amounts of azide 4 and propargylated phtalimidines 6-8 and (±)-12-17 in CH2Cl2 (1 mL) and H2O (1 mL) were added CuSO4.5H2O (0.2 equiv.) and sodium ascorbate (0.4 equiv). After stirring at room temperature for 24h, the resulting residue was concentrated under vacuum then extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were washed with brine, dried using MgSO4, filtered, and concentrated. The residue was then purified by flash column chromatography using a mixture of cyclohexane/EtOAc as eluent.

3.2.1.1. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-((1,3-dioxoisoindolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18a)

This derivative was isolated as a white solid in 84% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 84-86 °C; [α]D 20 +57 (c 0.95 mg/mL, CH2Cl2); IR (νmax/cm−1) 2922.91 (CH str.), 1716.51 (4 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.90–7.83 (m, 2Haro), 7.79–7.71 (m, 3Haro), 7.41–7.30 (m, 5Haro), 5.29 (t, J = 3.8 Hz, 1H), 5.09 (m, 6H), 4.54 (dd, J = 10.8, 5.2 Hz, 1H), 2.91 (dd, J = 14.2, 4.4 Hz, 1H) 1.12 (s, 3H), 0.92 (m, 6H), 0.85 (s, 3H), 0.79 (s, 3H), 0.64 (s, 3H), 0.60 (s, 3H); 13C NMR (75 MHz, CDCl3) δC 177.57 (C=O), 167.71 (C=O), 165.88 (2 x C=O), 143.88 (Cq), 136.57 (2 x Cq), 134.23 (2 x CHaro), 132.19 (2 x Cq), 128.55 (2 x CHaro), 128.10 (2 x CHaro), 128.04 (CHaro), 124.55 (CHaro/trz), 123.59 (2 x CHaro), 122.41 (CH), 83.97 (CH), 66.05 (CH2), 55.27 (CH), 51.31 (CH2), 47.61 (CH), 46.87 (Cq), 45.98 (CH2), 41.80 (Cq), 41.50 (CH), 39.39 (Cq), 38.07 (CH2), 37.79 (Cq), 36.95 (Cq), 33.98 (CH2), 33.23 (CH3), 33.09 (CH2), 32.69 (CH2), 32.49 (CH2), 30.83 (Cq), 28.15 (CH3), 27.73 (CH2), 25.97 (CH3), 23.78 (CH3), 23.49 (2 x CH2), 23.16 (CH2), 18.22 (CH2), 16.98 (CH3), 16.54 (CH3), 15.41 (CH3); HRMS (+ESI) calculated for C50H63N4O6 [M+H+]: 815.4703, found: 815.4775.

3.2.1.2. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 2,2,6a,6b,9,9,12a-heptamethyl-10-(2-(4-((3,4,4-trimethyl-2,5-dioxoimidazolidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18b)

This derivative was isolated as a white solid in 92% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.4; mp = 92-94 °C; [α]D20 +51 (c 1 mg/mL, CH2Cl2); IR (νmax/cm−1) 2923.14 (CH str.), 1770.86 (2 x C=O), 1711.07 (2 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.72 (s, 1H, CHaro/trz), 7.38–7.28 (m, 5Haro), 5.27 (t, J = 3.5 Hz, 1H), 5.17–5.00 (m, 4H), 4.81 (s, 2H), 4.55 (dd, J = 9.9, 6.0 Hz, 1H), 2.94–2.83 (m, 4H), 1.36 (s, 6H), 1.10 (s, 3H), 0.93–0.78 (m, 12H) 0.71 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.45 (C=O), 176.03 (C=O), 165.81 (C=O), 154.68 (C=O), 143.75 (Cq), 143.01 (Cq), 136.43 (Cq), 128.42 (2 x CHaro), 127.98 (2 x CHaro), 127.92 (CHaro), 124.38 (CHaro/trz), 122.29 (CH), 83.79 (CH), 65.93 (CH2), 61.39 (Cq), 55.17 (CH), 51.12 (CH2), 47.49 (CH), 46.73 (Cq), 45.84 (CH2), 41.68 (Cq), 41.36 (CH), 39.27 (Cq), 37.97 (CH2), 37.73 (Cq), 36.85 (Cq), 33.80 (CH2), 33.11 (CH3), 32.57 (CH2), 32.36 (CH2), 30.71 (Cq), 29.71 (CH2), 28.09 (CH3), 27.59 (CH2), 25.85 (CH3), 24.41 (CH3), 23.65 (CH3), 23.38 (2 x CH2), 23.03 (CH2), 22.00 (2 x CH3), 18.14 (CH2), 16.85 (CH3), 16.56 (CH3), 15.31 (CH3); HRMS (+ESI) calculated for C48H68N5O6 [M+H+]: 810.5125, found: 810.5169.

3.2.1.3. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-((2,5-dioxopyrrolidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18c)

This derivative was isolated as a white solid in 83% yield; Rf (cyclohexane/EtOAc: 50/50) = 0.3; mp = 145-147 °C; [α]D20 +108 (c 0.5 mg/mL, CH2Cl2); IR (νmax/cm−1) 2926.82 (CH str.), 1741.39 (C=O), 1721.47 (C=O), 1701.29 (2 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.71 (s, 1H, CHaro/trz), 7.38–7.28 (m, 5Haro), 5.27 (t, J = 3.6 Hz, 1H), 5.17–4.99 (m, 4H), 4.80 (s, 2H), 4.54 (dd, J = 9.8, 6.1 Hz, 1H), 2.88 (dd, J = 13.6, 4.5 Hz, 1H), 2.71 (s, 4H), 1.10 (s, 3H), 0.93–0.85 (m, 9H), 0.81 (s, 3H), 0.71 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.51 (C=O), 176.54 (2 x C=O), 165.90 (C=O), 143.82 (Cq), 142.37 (Cq), 136.49 (Cq), 128.49 (2 x CHaro), 128.06 (2 x CHaro), 127.99 (CHaro), 124.72 (CHaro/trz), 122.35 (CH), 83.91 (CH), 66.00 (CH2), 55.24 (CH), 51.16 (CH2), 47.56 (CH), 46.79 (Cq), 45.91 (CH2), 41.75 (Cq), 41.43 (CH), 39.33 (Cq), 38.03 (CH2), 37.80 (Cq), 36.92 (Cq), 33.92 (CH2), 33.68 (CH2), 33.18 (CH3), 32.63 (CH2), 32.43 (CH2), 30.78 (Cq), 28.30 (2 x CH2), 28.16 (CH3), 27.66 (CH2), 25.93 (CH3), 23.73 (CH3), 23.46 (2 x CH2), 23.10 (CH2), 18.22 (CH2), 16.92 (CH3), 16.60 (CH3), 15.40 (CH3); HRMS (+ESI) calculated for C46H63N4O6 [M+H+]: 767.4703, found: 767.4751.

3.2.1.4. (4aS,6aS,6bR,10S,12aR)-benzyl 10-(2-(4-((1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18d)

This derivative was isolated as a white solid in 89% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 113-116 °C; [α]D20 +80 (c 0.7 mg/mL, CH2Cl2); IR (νmax/cm−1) 2921.46 (CH str.),1725.07 (3 x C=O), 1181.72 (2 x S=O); 1H NMR (300 MHz, CDCl3) δH 8.12–7.78 (m, 5Haro), 7.39–7.28 (m, 5Haro),5.27 (t, J = 3.69 Hz, 1H),5.21–4.97 (m, 6H), 4.53 (dd, J = 10.2, 5.6 Hz, 1H), 2.89 (dd, J = 13.7, 4.4 Hz, 1H), 1.10 (s, 3H), 0.93–0.87 (m, 6H), 0.83 (s, 3H), 0.78 (s, 3H), 0.65 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.56 (C=O), 165.77 (C=O), 158.59 (C=O),143.87 (Cq), 137.87 (Cq), 136.56 (2 x Cq), 135.09 (CHaro), 134.56 (CHaro), 128.54 (2 x CHaro), 128.09 (2 x CHaro), 128.03 (CHaro), 127.31 (Cq), 125.48 (2 x CHaro), 122.40 (CHaro/trz), 121.24 (CH), 84.00 (CH), 66.04 (CH2), 55.28 (CH), 47.59 (CH), 46.85 (Cq), 45.97 (CH2), 41.79 (Cq), 41.49 (CH), 39.38 (Cq), 38.07 (CH2), 37.80 (Cq), 36.94 (Cq), 33.98 (2 x CH2), 33.21 (CH3), 32.67 (CH2), 32.47 (CH2), 30.82 (Cq), 28.18 (CH3), 27.72 (2 x CH2), 25.96 (CH3), 23.76 (CH3), 23.48 (2 x CH2), 23.15 (CH2), 18.22 (CH2), 16.97 (CH3), 16.59 (CH3), 15.38 (CH3); HRMS (+ESI) calculated for C49H63N4O7S [M+H+]: 851.4373, found: 851.4464.

3.2.1.5. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18e)

This derivative was isolated as a white solid in 92% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.4; mp = 94-96 °C; [α]D20 +65 (c 0.85 mg/mL, CH2Cl2); IR (νmax/cm−1) 2929.05 (CH str.), 1714.31 (4 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.86–7.79 (m, 2Haro), 7.74–7.67 (m, 2Haro), 7.56 (s, 1H, Haro/trz), 7.39–7.28 (m, 5Haro), 5.27 (t, J = 3.6 Hz, 1H), 5.14–4.99 (m, 4H), 4.54 (dd, J = 10.0, 5.9 Hz, 1H), 4.02 (t, J = 7.3 Hz, 2H), 3.17 (t, J = 7.3 Hz, 2H), 2.89 (dd, J = 14.0, 4.5 Hz, 1H), 1.11 (s, 3H), 0.92–0.86 (m, 9H), 0.81 (s, 3H), 0.71 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.55 (C=O), 168.28 (C=O), 166.13 (2 x C=O), 144.80 (Cq), 143.86 (Cq), 136.54 (Cq), 134.08 (2 x CHaro), 132.17 (2 x Cq), 128.53 (2 x CHaro), 128.09 (2 x CHaro), 128.03 (CHaro), 123.43 (2 x CHaro), 122.88 (CHaro/trz), 122.40 (CH), 83.84 (CH), 66.04 (CH2), 55.30 (CH), 51.20 (CH2), 47.60 (CH), 46.84 (Cq), 45.96 (CH2), 41.79 (Cq), 41.48 (CH), 39.38 (Cq), 38.08 (CH2), 37.84 (Cq), 37.45 (CH2), 36.96 (Cq), 33.96 (CH2), 33.21 (CH3), 32.68 (CH2), 32.47 (CH2), 30.81 (Cq), 28.19 (CH3), 27.71 (CH2), 25.96 (CH3), 24.96 (CH2), 23.76 (CH3), 23.49 (2 x CH2), 23.14 (CH2), 18.25 (CH2), 16.96 (CH3), 16.64 (CH3), 15.43 (CH3); HRMS (+ESI) calculated for C51H65N4O6 [M+H+]: 829.4859, found: 829.4959.

3.2.1.6. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-(1-(1,3-dioxoisoindolin-2-yl)ethyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18f)

This derivative was isolated as a white solid in 80% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 82-84 °C; [α]D20 +40 (c 1 mg/mL, CH2Cl2); IR (νmax/cm−1) 2942.80 (CH str.), 1776.94 (C=O), 17113.95 (3 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.84–7.77 (m, 3Haro), 7.73–7.66 (m, 2Haro), 7.38–7.28 (m, 5Haro), 5.81 (q, J = 7.3 Hz, 1H), 5.27 (t, J = 3.6 Hz, 1H), 5.21–5.00 (m, 4H), 4.53 (dd, J = 10.5, 5.4 Hz, 1H), 2.89 (dd, J = 13.6, 4.4 Hz, 1H), 1.86 (d, J = 7.3 Hz, 3H), 1.10 (s, 3H), 0.93–0.87 (m, 6H), 0.87–0.82 (s, 3H), 0.81–0.75 (s, 3H), 0.70–0.61 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.54 (C=O), 167.76 (C=O), 165.67 (2 x C=O), 147.82 (Cq), 143.85 (Cq), 136.53 (2 x Cq), 134.13 (2 x CHaro), 132.05 (Cq), 128.53 (2 x CHaro), 128.08 (2 x CHaro), 128.02 (CHaro), 123.84 (CHaro/trz), 123.40 (2 x CHaro), 122.39 (CH), 83.94 (CH), 66.03 (CH2), 55.26 (CH), 51.32 (CH2), 47.58 (CH), 46.83 (Cq), 45.94 (CH2), 42.62 (CH), 41.77 (Cq), 41.46 (CH), 39.35 (Cq), 38.05 (CH2), 37.78 (Cq), 36.93 (Cq), 33.95 (CH2), 33.21 (CH3), 32.65 (CH2), 32.45 (CH2), 30.81 (Cq), 28.15 (CH3), 27.70 (CH2), 25.95 (CH3), 23.75 (CH3), 23.46 (2 x CH2), 23.13 (CH2), 18.42 (CH3), 18.21 (CH2), 16.95 (CH3), 16.58 (CH3), 15.40 (CH3); HRMS (+ESI) calculated for C51H65N4O6 [M+H+]: 829.4859, found: 829.4942.

3.2.1.7. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-(((1,3-dioxoisoindolin-2-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18g)

This derivative was isolated as a white solid in 89% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.4; mp = 107-109 °C; [α]D20 +77 (c 0.7 mg/mL, CH2Cl2); IR (νmax/cm−1) 2945.81 (CH str.), 1791.82 (C=O), 1729.79 (3 x C=O); 1H NMR (300 MHz, CDCl3) δH 8.00 (s, 1Haro/trz), 7.82–7.69 (m, 4Haro), 7.38–7.28 (m, 5Haro), 5.39 (s, 2H), 5.28 (t, J = 3.5 Hz, 1H), 5.17 (s, 2H), 5.12–4.99 (m, 2H), 4.58 (t, J = 7.9 Hz, 1H), 2.89 (dd, J = 13.9, 4.3 Hz, 1H), 1.11 (s, 3H), 0.94–0.82 (m, 12H), 0.75 (s, 3H), 0.59 (s, 3H); 13C NMR (75 MHz, CDCl3) δC 177.56 (C=O), 165.87 (C=O), 163.56 (2 x C=O), 143.86 (Cq), 142.13 (Cq), 136.54 (Cq), 134.61 (2 x CHaro), 128.95 (2 x Cq), 128.53 (2 x CHaro), 128.09 (2 x CHaro), 128.03 (CHaro), 126.09 (CHaro/trz), 123.73 (2 x CHaro), 122.39 (CH), 84.05 (CH), 70.52 (CH2), 66.04 (CH2), 55.30 (CH), 51.30 (CH2), 47.60 (CH), 46.84 (Cq), 45.95 (CH2), 41.79 (Cq), 41.47 (CH), 39.37 (Cq), 38.09 (CH2), 37.87 (Cq), 36.96 (Cq), 33.96 (CH2), 33.21 (CH3), 32.67 (CH2), 32.46 (CH2), 30.82 (Cq), 28.22 (CH3), 27.70 (CH2), 25.96 (CH3), 23.76 (CH3), 23.51 (2 x CH2), 23.14 (CH2), 18.25 (CH2), 16.96 (CH3), 16.68 (CH3), 15.44 (CH3); HRMS (+ESI) calculated for C50H63N4O7 [M+H+]: 831.4652, found: 831.4703.

3.2.1.8. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-((1-hydroxy-3-oxoisoindolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18h)

This derivative was isolated as a white solid in 96% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.4; mp = 88-90 °C; [α]D20 +24 (c 1 mg/mL, CH2Cl2); IR (νmax/cm−1) 3350 (OH), 2928.90 (CH str.), 1699.10 (3 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.77 (s, 1Haro/trz), 7.70 (d, J = 7.4 Hz, 1Haro), 7.61–7.40 (m, 3Haro), 7.39–7.27 (m, 5Haro), 5.92 (s, 1H), 5.25 (t, J = 3.8 Hz, 1H), 5.17–4.98 (m, 4H), 4.90 (d, J = 13.4 Hz, 2H), 4.70 (d, J = 15.4 Hz, 1H), 4.49 (dd, J = 10.5, 5.5 Hz, 1H), 2.89 (dd, J = 14.0, 4.4 Hz, 1H), 1.09 (s, 3H), 0.92–0.86 (m, 6H), 0.84–0.78 (s, 3H), 0.77–0.72 (s, 3H), 0.64–0.59 (s, 3H), 0.57 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.53 (C=O), 167.40 (C=O), 165.89 (C=O), 144.25 (Cq), 143.80 (Cq), 136.49 (Cq), 132.42 (Cq), 131.46 (Cq), 129.65 (2 x CHaro), 128.50 (2 x CHaro), 128.04 (2 x CHaro), 127.99 (CHaro), 123.60 (2 x CHaro), 123.26 (CHaro/trz), 122.35 (CH), 83.93 (CH), 81.59 (CH), 66.00 (CH2), 55.19 (CH), 51.25 (CH2), 47.53 (CH), 46.79 (Cq), 45.91 (CH2), 41.73 (Cq), 41.42 (CH), 39.32 (Cq), 37.99 (CH2), 37.73 (Cq), 36.87 (Cq), 34.49 (CH2), 33.92 (CH2), 33.18 (CH3), 32.61 (CH2), 32.42 (CH2), 30.77 (Cq), 28.09 (CH3), 27.66 (CH2), 25.93 (CH3), 23.73 (CH3), 23.42 (2 x CH2), 23.10 (CH2), 18.15 (CH2), 16.91 (CH3), 16.51 (CH3), 15.35 (CH3); HRMS (+ESI) calculated for C50H64N4O6Na [M+Na+]: 839.4718, found: 839.4752.

3.2.1.9. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-(2-(1-hydroxy-3-oxoisoindolin-2-yl)ethyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18i)

This derivative was isolated as a white solid in 98% yield; Rf (cyclohexane/EtOAc: 40/60) = 0.5; mp = 111-113 °C; [α]D20 +40 (c 1 mg/mL, CH2Cl2); IR (νmax/cm−1) 3350 (OH), 2931.46 (CH str.), 1729.50 (3 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.68 (d, J = 7.4 Hz, 1Haro/trz), 7.59–7.40 (m, 4Haro), 7.37–7.28 (m, 5Haro), 5.83 (d, J = 6.6 Hz, 1H), 5.39 (br.s, 1H), 5.27 (t, J = 3.7 Hz), 5.14–4.98 (m, 4H), 4.52 (t, J = 8.0 Hz, 1H), 3.92 (t, J = 6.7 Hz, 2H), 3.17 (t, J = 6.8 Hz, 2H), 2.89 (dd, J = 13.8, 4.5 Hz, 1H), 1.11 (s, 3H), 0.92–0.85 (m, 9H), 0.81–0.77 (s, 3H), 0.73–0.69 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.57 (C=O), 167.70 (C=O), 166.06 (C=O), 144.23 (Cq), 143.87 (Cq), 136.52 (Cq), 132.26 (CHaro), 131.67 (2 x Cq), 129.63 (CHaro), 128.53 (2 x CHaro), 128.09 (2 x CHaro), 128.03 (CHaro), 123.48 (2 x CHaro), 123.20 (CHaro/trz), 122.38 (CH), 84.10 (CH), 82.51 (CH), 66.04 (CH2), 55.27 (CH), 51.39 (CH2), 47.59 (CH), 46.83 (Cq), 45.95 (CH2), 41.78 (Cq), 41.46 (CH), 39.36 (Cq), 38.93 (CH2), 38.06 (CH2), 37.84 (Cq), 36.94 (Cq), 33.95 (CH2), 33.22 (CH3), 32.65 (CH2), 32.46 (CH2), 30.82 (Cq), 28.20 (CH3), 27.70 (CH2), 25.97 (CH3), 24.87 (CH2), 23.76 (CH3), 23.49 (2 x CH2), 23.13 (CH2), 18.24 (CH2), 16.95 (CH3), 16.65 (CH3), 15.44 (CH3); HRMS (+ESI) calculated for C51H67N4O6 [M+H+]: 831.5016, found: 831.5037.

3.2.1.10. methyl2-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-oxoisoindoline-1-carboxylate (18j)

This derivative was isolated as a white solid in 95% yield; Rf (cyclohexane/EtOAc: 50/50) = 0.5; mp = 86-88 °C; [α]D20 +52 (c 0.9 mg/mL, CH2Cl2); IR (νmax/cm−1) 2926.94 (CH str.), 1732.76 (4 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.96 (s, 1Haro/trz), 7.79 (d, J = 7.8 Hz, 1Haro), 7.64–7.43 (m, 3Haro), 7.38–7.30 (m, 5Haro), 7.03 (s, 1H), 5.33 (d, J = 1.7 Hz, 2H), 5.28 (t, J = 4.0 Hz, 1H), 5.17 (s, 2H), 5.13–4.99 (m, 2H), 4.57 (dd, J = 9.4, 6.6 Hz, 1H), 2.90 (dd, J = 14.0, 4.4 Hz, 1H), 2.17 (s, 3H), 1.11 (s, 3H), 0.93–0.85 (m, 9H), 0.83 (s, 3H), 0.73 (s, 3H), 0.59 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.56 (C=O), 170.81 (C=O), 165.92 (C=O), 165.66 (C=O), 143.88 (Cq), 142.91 (Cq), 138.67 (Cq), 136.56 (Cq), 133.51 (CHaro), 130.71 (CHaro), 128.54 (2 x CHaro), 128.10 (2 x CHaro), 128.04 (CHaro), 125.85 (CHaro), 124.22 (CHaro), 124.06 (CHaro/trz), 122.41 (CH), 84.01 (CH), 80.82 (CH), 69.99 (Cq), 66.05 (CH2), 55.31 (CH), 51.27 (CH2), 47.61 (CH), 46.85 (Cq), 45.96 (CH2), 41.80 (Cq), 41.49 (CH), 39.39 (Cq), 38.09 (CH2), 37.87 (Cq), 36.97 (Cq), 33.98 (CH2), 33.22 (CH3), 32.69 (CH2), 32.48 (CH2), 30.83 (Cq), 29.83 (CH2), 28.22 (CH3), 27.72 (CH2), 25.97 (CH3), 23.77 (CH3), 23.52 (2 x CH2), 23.15 (CH2), 21.18 (CH3), 18.26 (CH2), 16.97 (CH3), 16.66 (CH3), 15.45 (CH3); HRMS (+ESI) calculated for C52H67N4O8 [M+H+]: 875.4914, found: 875.4983.

3.2.1.11. ethyl2-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxoisoindoline-1-carboxylate (18k)

This derivative was isolated as a white solid in 71% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 91-93 °C; [α]D20 +47 (c 0.95 mg/mL, CH2Cl2); IR (νmax/cm−1) 2943.22 (CH str.), 1702.18 (4 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.82 (d, J = 7.3 Hz, 1Haro), 7.74 (s, 1Haro/trz), 7.65–7.45 (m, 3Haro),7.39–7.29 (m, 5Haro), 5.43–5.34 (m, 1H), 5.31–5.23 (m, 2H), 5.14–5.00 (m, 4H),4.62–4.46 (m, 2H),4.40–4.20 (m, 2H), 2.91 (dd, J = 13.2, 4,1 Hz, 1H), 1.33 (t, J = 7.1 Hz, 3H), 1.09 (s, 3H), 0.93–0.86 (m, 6H), 0.84–0.77 (s, 3H), 0.77–0.70 (s, 3H), 0.61–0.52 (m, 6H); 13C NMR (75 MHz,CDCl3) δC 177.58 (C=O), 168.50 (C=O), 168.08 (C=O), 165.81 (C=O), 143.78 (2 x Cq), 139.72 (Cq), 135.56 (2 x Cq), 132.20 (CHaro), 129.32 (CHaro), 128.55 (2 x CHaro), 128.09 (2 x CHaro), 128.04 (CHaro), 124.52 (CHaro), 124.05 (CHaro), 123.16 (CHaro/trz), 122.40 (CH), 83.96 (CH), 66.05 (CH2), 62.45 (CH2), 62.01 (CH), 55.23 (CH), 51.29 (CH2), 47.58 (CH), 46.85 (Cq), 45.95 (CH2), 41.78 (Cq), 41.48 (CH), 39.37 (Cq), 38.03 (CH2), 37.76 (Cq), 36.92 (Cq), 36.60 (CH2), 33.98 (CH2), 33.22 (CH3), 32.66 (CH2), 32.48 (CH2), 30.83 (Cq), 28.13 (CH3), 27.71 (CH2), 25.96 (CH3), 23.77 (CH3), 23.47 (2 x CH2), 23.14 (CH2), 18.20 (CH2), 16.97 (CH3), 16.47 (CH3), 15.40 (CH3), 14.33 (CH3); HRMS (+ESI) calculated for C53H69N4O7 [M+H+]: 873.5122, found: 873.5161.

3.2.1.12. ethyl2-benzyl-1-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxoisoindoline-1-carboxylate (18l)

This derivative was isolated as a white solid in 83% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 105-106 °C; [α]D20 +13 (c 0.7 mg/mL, CH2Cl2); IR (νmax/cm−1) 2927.79 (CH str.), 1710 (2 x C=O), 1700.90 (2 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.78 (d, J = 7.5 Hz, 1Haro), 7.60–7.39 (m, 5Haro), 7.36–7.27 (m, 6Haro), 7.25–7.20 (m, 1Haro), 6.18 (d, J = 9.4 Hz, 1Haro), 5.27 (t, J = 3.7 Hz, 1H), 4.94–4.62 (m, 4H), 4.53–4.43 (m, 1H), 3.90–3.74 (m, 3H), 2.89 (dd, J = 13.6, 4.4 Hz, 1H), 1.25 (t, J = 7.5 Hz, 3H), 1.10 (s, 3H), 0.93–0.87 (m, 9H), 0.80–0.72 (s, 3H), 0.71–0.64 (s, 3H), 0.59 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.56 (C=O), 169.99 (C=O), 169.38 (C=O), 165.84 (C=O), 143.87 (Cq), 143.76 (Cq), 140.77 (Cq), 137.17 (Cq), 136.54 (Cq), 132.35 (CHaro), 129.37 (3 x CHaro) , 128.53 (4 x CHaro), 128.11 (2 x CHaro), 128.04 (CHaro), 127.69 (CHaro), 124.06 (CHaro), 123.08 (CHaro/trz), 122.41 (CH), 122.00 (CHaro), 83.72 (CH), 70.76 (Cq), 66.05 (CH2), 62.33 (CH2), 55.28 (CH), 50.94 (CH2), 47.61 (CH), 46.84 (Cq), 45.96 (CH2), 44.87 (CH2), 41.79 (Cq), 41.47 (CH), 39.38 (Cq), 38.08 (CH2), 37.86 (Cq), 37.82 (Cq), 36.95 (Cq), 33.97 (CH2), 33.22 (CH3), 32.68 (CH2), 32.47 (CH2), 30.82 (Cq), 29.85 (CH2), 28.18 (CH3), 27.71 (CH2), 25.96 (CH3), 23.76 (CH3), 23.48 (2 x CH2), 23.15 (CH2), 18.26 (CH2), 16.96 (CH3), 16.65 (CH3), 15.43 (CH3), 13.61 (CH3); HRMS (+ESI) calculated for C60H75N4O7 [M+H+]: 963.5591, found: 963.5641.

3.2.1.13. ethyl1-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxo-2-phenethylisoindoline-1-carboxylate (18m)

This derivative was isolated as a white solid in 90% yield; Rf (cyclohexane/EtOAc: 70/30) = 0.5; mp = 93-95 °C; [α]D20 +73 (c 0.75 mg/mL, CH2Cl2); IR (νmax/cm−1) 2936.05 (CH str.), 1708.51 (4 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.77 (d, J = 7.4 Hz, 1Haro), 7.62–7.54 (m, 2Haro), 7.53–7.45 (m, 1Haro),7.40–7.27 (m, 9Haro), 7.25–7.18 (m, 1Haro), 6.53 (d, J = 18.8 Hz, 1Haro), 5.27 (t, J = 3.7 Hz, 1H), 5.12–5.01 (m, 2H), 4.88 (qd, J = 17.43, 17.43, 17.42, 2.38 Hz, 2H), 4.52–4.43 (m, 1H), 4.28–4.10 (m, 2H), 4.03–3.93 (m, 1H), 3.87–3.77 (m, 1H), 3.67 (t, J = 7.9 Hz, 2H), 3.18–2.98 (m, 2H), 2.90 (dd, J = 13.7, 4.5 Hz, 1H), 1.20 (t, J = 7.1 Hz, 3H), 1.10 (s, 3H), 0.93–0.84 (m, 9H), 0.76 (s, 3H), 0.68 (s, 3H), 0.59 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.55 (C=O), 170.31 (C=O), 169.22 (C=O), 165.83 (C=O), 143.86 (Cq), 143.28 (Cq), 140.92 (Cq), 139.24 (Cq), 136.54 (Cq), 132.29 (CHaro), 129.61 (CHaro), 129.05 (2 x CHaro), 128.68 (2 x CHaro), 128.54 (2 x CHaro), 128.11 (2 x CHaro), 128.04 (CHaro), 126.54 (CHaro), 123.81 (CHaro), 122.84 (CHaro/trz), 122.41 (CH), 122.24 (CHaro), 83.79 (CH), 71.45 (Cq), 66.05 (CH2), 62.77 (CH2), 55.27 (CH), 51.06 (CH2), 47.60 (CH), 46.85 (Cq), 45.96 (CH2), 44.25 (CH2), 41.79 (Cq), 41.48 (CH), 39.37 (Cq), 38.06 (CH2), 37.83 (Cq), 37.81 (Cq), 36.94 (Cq), 34.36 (CH2), 33.97 (CH2), 33.22 (CH3), 32.68 (CH2), 32.47 (CH2), 30.82 (Cq), 30.69 (CH2), 28.17 (CH3), 27.71 (CH2), 25.96 (CH3), 23.77 (CH3), 23.48 (2 x CH2), 23.15 (CH2), 18.25 (CH2), 16.96 (CH3), 16.65 (CH3), 15.39 (CH3), 14.09 (CH3); HRMS (+ESI) calculated for C61H77N4O7 [M+H+]: 977.5748, found: 977.5807.

3.2.1.14. methyl2-allyl-1-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxoisoindoline-1-carboxylate (18n)

This derivative was isolated as a white solid in 70% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.4; mp = 98-100 °C; [α]D20 +80 (c 0.7 mg/mL, CH2Cl2); IR (νmax/cm−1) 2935.43 (CH str.), 1710.09 (4 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.75 (d, J = 7.4, 1.3 Hz, 1Haro), 7.61–7.43 (m, 3Haro), 7.37–7.29 (m, 5Haro), 6.55 (d, J = 21.3 Hz, 1Haro), 5.98–5.81 (m, 1H), 5.36–5.16 (m, 3H), 5.14–4.99 (m, 2H), 4.98–4.77 (m, 2H), 4.53–4.35 (m, 2H), 4.11–4.00 (m, 1H),3.88 (qd, J = 15.72, 15.71, 15.71, 4.49 Hz, 2H), 3.65 (s, 3H), 2.89 (dd, J = 13.5, 4.3 Hz, 1H), 1.10 (s, 3H), 0.94–0.82 (m, 9H), 0.77 (s, 3H), 0.73–0.65 (m, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.56 (C=O), 170.80 (C=O), 168.70 (C=O), 165.87 (C=O), 143.86 (2 x Cq), 140.86 (Cq), 136.53 (Cq), 133.11 (CHaro), 132.35 (CHaro), 129.61 (CHaro), 128.53 (2 x CHaro), 128.10 (2 x CHaro), 128.04 (CHaro), 124.03 (CHaro), 122.91 (CHaro/trz), 122.40 (CH), 121.93 (CH) 118.54 (CH2), 83.80 (CH), 70.35 (Cq), 66.04 (CH2), 55.28 (CH), 53.10 (CH3), 51.04 (CH2), 47.60 (CH), 46.84 (Cq), 45.95 (CH2), 44.15 (CH2), 41.78 (Cq), 41.47 (CH), 39.37 (Cq), 38.07 (CH2), 37.86 (Cq), 37.82 (Cq), 36.94 (Cq), 33.96 (CH2), 33.22 (CH3), 32.68 (CH2), 32.46 (CH2), 30.82 (Cq), 30.02 (CH2), 28.16 (CH3), 27.70 (CH2), 25.95 (CH3), 23.76 (CH3), 23.47 (2 x CH2), 23.14 (CH2), 18.25 (CH2), 16.95 (CH3), 16.64 (CH3), 15.42 (CH3); HRMS (+ESI) calculated for C55H71N4O7 [M+H+]: 899.5278, found: 899.5327.

3.2.1.15. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-((2-benzyl-1-((2-methoxy-2-oxoethyl)carbamoyl)-3-oxoisoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18o)

This derivative was isolated as a white solid in 94% yield; Rf (cyclohexane/EtOAc: 40/60) = 0.5; mp = 120-122 °C; [α]D20 +41 (c 1 mg/mL, CH2Cl2); IR (νmax/cm−1) 3300 (NH), 2931.02 (CH str.), 1726.01 (3 x C=O), 1677.77 (C=O); 1H NMR (300 MHz, CDCl3) δH 7.72 (d, J = 7.4 Hz, 1Haro), 7.63–7.39 (m, 6Haro), 7.36–7.27 (m, 6Haro), 7.25–7.19 (m, 1Haro), 6.32 (d, J = 11.7 Hz, 1Haro), 5.86 (br.s, 1H), 5.27 (t, J = 3.8 Hz, 1H), 5.12–4.76 (m, 5H), 4.65 (dd, J = 15.2, 6.7 Hz, 1H), 4.01–3.91 (m, 1H), 3.96 (d, J = 15.6 Hz, 1H), 3.85–3.56 (m, 5H),3.28–3.14 (m, 1H), 2.89 (dd, J = 13.8, 4.4 Hz, 1H), 1.10 (s, 3H), 0.92–0.87 (m, 9H), 0.78 (s, 3H), 0.72–0.67 (s, 3H), 0.59 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.55 (C=O), 169.83 (C=O), 169.18 (2 x C=O), 165.83 (C=O), 144.31 (Cq), 143.85 (Cq), 141.05 (Cq), 137.54 (Cq), 136.52 (Cq), 132.81 (CHaro), 130.95 (Cq), 129.66 (CHaro), 129.42 (2 x CHaro), 128.83 (2 x CHaro), 128.52 (2 x CHaro), 128.09 (2 x CHaro), 128.02 (CHaro), 127.83 (CHaro), 124.19 (CHaro), 123.54 (CHaro), 122.63 (CHaro/trz), 122.39 (CH), 83.73 (CH), 71.66 (Cq), 66.04 (CH2), 55.27 (CH), 52.34 (CH3), 50.98 (CH2), 47.60 (CH), 46.83 (Cq), 45.94 (CH2), 44.84 (CH2), 41.78 (Cq), 41.46 (CH), 41.29 (CH2), 39.36 (Cq), 38.07 (CH2), 37.86 (Cq), 36.94 (Cq), 33.95 (CH2), 33.20 (CH3), 32.67 (CH2), 32.46 (CH2), 30.81 (Cq), 29.81 (CH2), 28.16 (CH3), 27.69 (CH2), 25.95 (CH3), 23.75 (CH3), 23.47 (2 x CH2), 23.13 (CH2), 18.24 (CH2), 16.95 (CH3), 16.64 (CH3), 15.41 (CH3); HRMS (+ESI) calculated for C61H75N5O8Na [M+Na+]: 1028.5508, found: 1028.5584.

3.2.1.16. (4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)-benzyl 10-(2-(4-((2-((1,5-dimethyl-1H-pyrrol-2-yl)methyl)-1-(hydroxymethyl)-3-oxoisoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)acetoxy)-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (18p)

This derivative was isolated as a yellow solid in 70% yield; Rf (cyclohexane/EtOAc: 40/60) = 0.5; mp = 85-87 °C; [α]D20 +64 (c 0.85 mg/mL, CH2Cl2); IR (νmax/cm−1) 3400 (OH), 2923.75 (CH str.), 1726.83 (C=O), 1678.10 (2 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.78 (d, J = 7.5 Hz, 1Haro), 7.63–7.40 (m, 3Haro), 7.39–7.28 (m, 6Haro), 6.44 (d, J = 16.9 Hz, 1Haro), 6.16 (t, J = 2.6 Hz, 1H), 5.84 (d, J = 3.3 Hz, 1Haro), 5.27 (t, J = 3.7 Hz, 1H), 5.19–4.79 (m, 5H), 4.64–4.45 (m, 2H), 3.82–3.70 (m, 2H), 3.53–3.36 (m, 5H), 2.89 (dd, J = 14.0, 4.6 Hz, 1H), 2.18 (s, 3H), 1.11 (s, 3H), 0.93–0.86 (m, 9H), 0.79 (s, 3H), 0.74–0.68 (s,3H), 0.59 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.55 (C=O), 168.51 (C=O), 165.93 (C=O), 146.14 (Cq), 143.86 (Cq), 141.70 (Cq), 136.53 (Cq), 132.29 (Cq), 132.10 (CHaro), 130.78 (Cq), 128.90 (CHaro), 128.52 (2 x CHaro), 128.09 (2 x CHaro), 128.03 (CHaro), 127.19 (Cq), 123.92 (CHaro), 123.09 (CHaro/trz), 122.39 (CH), 121.92 (CHaro), 108.56 (CHaro), 105.93 (CHaro), 83.83 (CH), 70.12 (Cq), 66.04 (CH2), 55.29 (CH), 51.05 (CH2), 47.60 (CH), 46.84 (Cq), 45.96 (CH2), 41.79 (Cq), 41.47 (CH), 39.38 (Cq), 38.08 (CH2), 37.87 (Cq), 36.95 (Cq), 35.98 (CH2), 33.96 (CH2), 33.21 (CH3), 32.68 (CH2), 32.46 (CH2), 30.88 (CH3), 30.81 (Cq), 29.82 (CH2), 29.48 (CH2), 28.19 (CH3), 27.71 (CH2), 25.96 (CH3), 23.75 (CH3), 23.49 (2 x CH2), 23.14 (CH2), 18.26 (CH2), 16.96 (CH3), 16.67 (CH3), 15.44 (CH3), 12.66 (CH3); HRMS (+ESI) calculated for C58H75N5O6Na [M+Na+]: 960.5610, found: 960.5708.

3.2.1.17. 1-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(furan-2-ylmethyl)-3-oxoisoindoline-1-carboxylic acid (18q)

This derivative was isolated as a yellow solid in 60% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.4; mp = 81-83 °C; [α]D20 +76 (c 0.5 mg/mL, CH2Cl2); IR (νmax/cm−1) 2924.69 (CH str.), 1724.90 (3 x C=O), 1711 (C=O); 1H NMR (300 MHz, CDCl3) δH 7.79 (d, J = 7.4 Hz, 1Haro), 7.55–7.27 (m, 10Haro), 6.34 (dd, J = 6.9, 2.8 Hz, 2Haro), 5.27 (t, J = 4.2 Hz, 1H), 5.23–4.78 (m, 6H), 4.58–4.35 (m, 2H), 3.51 (s, 1H), 2.89 (dd, J = 14.1, 4.5 Hz, 1H), 1.10 (s, 3H), 0.93–0.86 (m, 9H), 0.80 (s, 3H), 0.72 (s, 3H), 0.59 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.53 (C=O), 171.42 (C=O), 168.26 (C=O), 165.83 (C=O), 150.45 (Cq), 144.47 (Cq), 143.85 (2 x Cq), 142.66 (CHaro), 136.51 (Cq), 132.25 (Cq), 131.88 (CHaro), 128.56 (2 x CHaro), 128.51 (2 x CHaro), 128.08 (2 x CHaro), 128.02 (CHaro), 123.91 (CHaro), 122.68 (CHaro/trz), 122.37 (CH), 110.65 (CHaro), 108.94 (CHaro), 83.87 (CH), 66.02 (CH2), 55.28 (CH), 47.59 (CH), 46.82 (Cq), 45.94 (CH2), 41.77 (Cq), 41.46 (CH), 39.36 (Cq), 38.07 (CH2), 37.86 (Cq), 37.83 (Cq), 37.06 (2 x CH2), 36.94 (Cq), 33.94 (CH2), 33.20 (CH3), 32.66 (CH2), 32.44 (CH2), 30.80 (Cq), 29.80 (CH2), 28.18 (CH3), 27.69 (CH2), 25.94 (CH3), 23.74 (CH3), 23.48 (2 x CH2), 23.12 (CH2), 18.24 (CH2), 16.94 (CH3), 16.65 (CH3), 15.42 (CH3).

3.2.1.18. ethyl2-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-1-(2-ethoxy-2-oxoethyl)-3-oxoisoindoline-1-carboxylate (18r)

This derivative was isolated as a white solid in 98% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 97-99 °C; [α]D20 +55 (c 1 mg/mL, CH2Cl2); IR (νmax/cm−1) 2936.99 (CH str.), 1710.10 (5 x C=O); 1H NMR (300 MHz, CDCl3)δH 7.88–7.77 (m, 2Haro), 7.61–7.45 (m, 3Haro), 7.39–7.28 (m, 5Haro), 5.27 (t, J = 3.7 Hz, 1H), 5.15–4.85 (m, 6H), 4.53 (dd, J = 9.5, 6.5 Hz, 1H), 4.26–4.05 (m, 2H), 4.03–3.92 (m, 2H), 3.52 (d, J = 17.1 Hz, 1H), 3.13 (dd, J = 17.0, 2.6 Hz, 1H), 2.89 (dd, J = 14.2, 4.4 Hz, 1H), 1.19 (td, J = 7.1, 2.5 Hz, 3H), 1.11–1.03 (m, 6H), 0.92–0.83 (m, 9H),0.80 (s, 3H), 0.69 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.55 (C=O), 169.08 (C=O), 169.40 (C=O), 168.92 (C=O), 165.91 (C=O), 143.85 (Cq), 143.57 (Cq), 136.54 (2 x Cq), 132.40 (2 x CHaro), 131.20 (Cq), 129.52 (CHaro), 128.53 (2 x CHaro), 128.09 (2 x CHaro), 128.03 (CHaro), 123.74 (CHaro), 122.40 (CHaro/trz + CH), 83.83 (CH), 69.23 (Cq), 66.03 (CH2), 62.77 (CH2), 61.12 (CH2), 55.28 (CH), 51.17 (CH2), 47.59 (CH), 46.84 (Cq), 45.95 (CH2), 41.78 (Cq), 41.47 (CH), 40.38 (CH2), 39.37 (Cq), 38.07 (CH2), 37.83 (Cq), 36.94 (CH2 + Cq), 33.96 (CH2), 33.21 (CH3), 32.67 (CH2), 32.46 (CH2), 30.81 (Cq), 28.18 (CH3), 27.70 (CH2), 25.95 (CH3), 23.76 (CH3), 23.48 (2 x CH2), 23.14 (CH2), 18.23 (CH2), 16.96 (CH3), 16.63 (CH3), 15.40 (CH3), 14.00 (2 x CH3); HRMS (+ESI) calculated for C57H75N4O9 [M+H+]: 959.5489, found: 959.5543.

3.2.1.19. ethyl 1-allyl-2-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxoisoindoline-1-carboxylate (18s)

This derivative was isolated as a white solid in 90% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 100-102 °C; [α]D20 +53 (c 1.5 mg/mL, CH2Cl2); IR (νmax/cm−1) 2943.31 (CH str.), 1731.34 (2 x C=O), 1701.17 (2 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.93 (s, 1Haro/trz), 7.80 (d, J = 7.4 Hz, 1Haro), 7.62–7.44 (m, 3Haro), 7.39–7.28 (m, 5Haro), 5.26 (t, J = 3.7 Hz, 1H), 5.17–4.67 (m, 9H), 4.55 (t, J = 8.0 Hz, 1H), 4.18–4.01 (m, 2H),3.26–3.11 (m, 2H), 2.89 (dd, J = 13.6, 4.4 Hz, 1H), 1.14 (dt, J = 7.1, 3.5 Hz, 3H), 1.10 (s, 3H), 0.93–0.85 (m, 9H),0.82 (s, 3H), 0.73 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.58 (C=O), 169.97 (C=O), 169.28 (C=O), 165.87 (C=O), 144.46 (Cq), 143.85 (Cq), 143.60 (Cq), 136.55 (Cq), 132.35 (CH=), 131.49 (Cq), 129.82 (CHaro), 129.27 (CHaro), 128.54 (2 x CHaro), 128.10 (2 x CHaro), 128.04 (CHaro), 125.88 (CHaro), 123.69 (CHaro), 122.42 (CHaro/trz + CH), 120.29 (CH2=), 83.89 (CH), 72.31 (Cq), 66.05 (CH2), 62.57 (CH2), 55.30 (CH), 51.23 (CH2), 47.60 (CH), 46.85 (Cq), 45.95 (CH2), 41.79 (Cq), 41.48 (CH), 39.38 (Cq), 38.09 (CH2), 37.86 (Cq), 37.77 (CH2), 37.02 (CH2), 36.96 (Cq), 33.97 (CH2), 33.22 (CH3), 32.69 (CH2), 32.48 (CH2), 30.82 (Cq), 28.22 (CH3), 27.71 (CH2), 25.96 (CH3), 23.77 (CH3), 23.51 (2 x CH2), 23.15 (CH2), 18.25 (CH2), 16.97 (CH3), 16.69 (CH3), 15.43 (CH3), 14.03 (CH3); HRMS (+ESI) calculated for C56H73N4O7 [M+H+]: 913.5435, found: 913.5466.

3.2.1.20. ethyl2-((1-(2-(((3S,4aR,6aR,6bS,8aS,12aS,14aR,14bR)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-1-(3-methoxybenzyl)-3-oxoisoindoline-1-carboxylate (18t)

This derivative was isolated as a white solid in 79% yield; Rf (cyclohexane/EtOAc: 60/40) = 0.5; mp = 100-102 °C; [α]D20 +70 (c 1.5 mg/mL, CH2Cl2); IR (νmax/cm−1) 2936.46 (CH str.), 1702.26 (4 x C=O); 1H NMR (300 MHz, CDCl3) δH 7.75, (s, 1Haro/trz), 7.69 (d, J = 7.5 Hz, 1Haro),7.63–7.53 (m, 2Haro), 7.50–7.42 (m, 1Haro), 7.40–7.28 (m, 5Haro), 6.86 (td, J = 7.9, 2.5 Hz, 1Haro), 6.56 (d, J = 7.8 Hz, 1Haro), 6.16 (t, J = 6.3 Hz, 1Haro), 5.99 (br. s, 1Haro), 5.27 (t, J = 3.9 Hz, 1H), 5.10–4.85 (m, 4H), 4.86 (s, 2H), 4.54 (t, J = 7.9 Hz, 1H), 4.12–4.01 (m, 2H), 3.83 (d, J = 14.7 Hz), 3.57 (d, J = 14.7 Hz, 1H), 3.49 (s, 3H), 2.89 (dd, J = 13.8, 4.4 Hz, 1H), 1.10 (s, 3H), 1.08–1.02 (m, 3H), 0.93–0.79 (m, 12H), 0.72 (s, 3H), 0.58 (s, 3H); 13C NMR (75 MHz,CDCl3) δC 177.52 (C=O), 169.96 (C=O), 169.28 (C=O), 165.83 (C=O), 158.96 (Cq), 143.95 (Cq), 143.81 (Cq), 136.50 (Cq), 135.19 (Cq), 132.01 (CHaro), 131.55 (2 x Cq), 129.28 (CHaro), 128.86 (CHaro), 128.50 (2 x CHaro), 128.06 (2 x CHaro), 127.99 (CHaro), 125.74 (CHaro), 123.71 (CHaro), 122.89 (CHaro), 122.38 (CH), 122.17 (CHaro/trz), 114.87 (CHaro), 113.00 (CHaro), 83.81 (CH), 72.80 (Cq), 66.00 (CH2), 62.64 (CH2), 55.25 (CH), 54.97 (CH3), 51.14 (CH2), 47.55 (CH), 46.80 (Cq), 45.91 (CH2), 41.74 (Cq), 41.43 (CH), 39.54 (CH2), 39.33 (Cq), 38.04 (CH2), 37.81 (Cq), 37.65 (CH2), 36.90 (Cq), 33.92 (CH2), 33.18 (CH3), 32.64 (CH2), 32.43 (CH2), 30.78 (Cq), 28.17 (CH3), 27.66 (CH2), 25.92 (CH3), 23.73 (CH3), 23.45 (2 x CH2), 23.10 (CH2), 18.20 (CH2), 16.92 (CH3), 16.63 (CH3), 15.38 (CH3), 13.84 (CH3); HRMS (+ESI) calculated for C61H77N4O8 [M+H+]: 993.5697, found: 993.5744.

3.2.1.21. ethyl1,2-bis((1-(2-(((3S,4aR,6aR,6bS,8aS,12aR,14aS,14bS)-8a-((benzyloxy)carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-yl)oxy)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)-3-oxo-2,3-dihydro-1H-isoindole-1-carboxylate (18v)

This derivative was isolated as a white solid in 80% yield; Rf (cyclohexane/EtOAc: 50/50) = 0.4; mp = 143-145 °C; [α]D20 +63 (c 0.9 mg/mL, CH2Cl2); IR (νmax/cm−1) 2938.94 (CH str.), 1728.35 (6 x C=O);1H NMR (300 MHz, CDCl3) δH 7.92 (d, J = 6.6 Hz, 1Haro), 7.73–7.61 (m, 2Haro), 7.46–7.28 (m, 11Haro), 6.31 (d, J = 16.1 Hz, 1Haro), 5.26 (t, J = 3.6 Hz, 2H), 5.24–4.99 (m, 7H), 4.85 (t, J = 3.8 Hz, 2H), 4.67 (dd, J = 15.8, 4.5 Hz, 1H), 4.60–4.33 (m, 2H), 4.30–4.05 (m, 3H), 3.89 (dd, J = 15.3, 4.3 Hz, 1H), 2.92 (dd, J = 13.6, 4.4 Hz, 2H), 1.11 (s, 6H), 0.94–0.80 (m, 24H), 0.77–0.67 (m, 6H), 0.61 (s, 6H); 13C NMR (75 MHz,CDCl3) δC 177.55 (2 x C=O), 169.68 (C=O), 169.61 (C=O), 166.61 (C=O), 166.04 (C=O), 144.52 (Cq), 143.85 (2 x Cq), 142.98 (Cq), 140.25 (Cq), 136.53 (2 x Cq), 132.45 (CHaro), 130.98 (Cq), 129.39 (CHaro), 128.53 (4 x CHaro), 128.09 (4 x CHaro), 128.03 (2 x CHaro), 126.01 (CHaro), 123.51 (2 x CHaro/trz), 123.26 (CHaro), 122.38 (2 x CH), 84.31 (CH), 83.33 (CH), 72.40 (Cq), 66.04 (2 x CH2), 62.92 (CH2), 55.24 (2 x CH), 51.03 (2 x CH2), 47.59 (2 x CH), 46.84 (2 x Cq), 45.97 (2 x CH2), 41.80 (2 x Cq), 41.48 (2 x CH), 39.37 (2 x Cq), 38.08 (2 x CH2), 37.87 (Cq), 37.74 (Cq), 37.23 (CH2), 36.95 (2 x Cq), 33.96 (2 x CH2), 33.23 (2 x CH3), 32.68 (2 x CH2), 32.46 (2 x CH2), 30.81 (2 x Cq), 30.11 (CH2), 28.20 (2 x CH3), 27.72 (2 x CH2), 25.96 (2 x CH3), 23.76 (2 x CH3), 23.50 (4 x CH2), 23.14 (2 x CH2), 18.27 (2 x CH2), 16.98 (2 x CH3), 16.63 (2 x CH3), 15.46 (2 x CH3),14.10 (CH3); HRMS (+ESI) calculated for C95H125N7O11Na [M+Na+]: 1562.9329, found: 1562.9335.

3.3. Antibacterial activity

In the present study, the antimicrobial activity of the starting product OA-1 and its derivatives 18a-v was screened by agar disc diffusion method according to the protocol described by Dbeibia et al. (2022) [79] against four bacteria, namely Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19115, Salmonella thyphimurium ATCC 14080 and Pseudomonas aeruginosa ATCC 27853. The inoculums of the microorganisms were adjusted to 0.1 at OD600 and then streaked onto Muller Hinton (MH) agar plates using a sterile cotton mop. Sterile filter discs (diameter 6 mm, Biolife Italy) were placed at the surface of the appropriate agar mediums and 20 µL of the product was dropped onto each disc. Tetracycline (10 mg/mL; 10 μL/disc) was used as reference antibiotics. After incubation at 37 °C for 24h, the antibacterial activities were evaluated by measuring an inhibition zone formed around the disc. Each assay was performed in triplicate. The minimum inhibitory concentration (MIC) was evaluated as recommended by Dbeibia et al. (2022) [79]. Briefly, serial dilutions of the synthesized compounds (3.9–2000 µg/mL) were filled in 96 U bottomed-wells plates (Nunc, Roskilde, Denmark) with MH broth and the target bacteria. The treated plates were left to incubateovernight at 37 °C. The MIC was reported as the lowest concentration of the sample that did not allow the growth of microorganisms and do not show visible turbidity of the broth medium. The minimum bactericidal concentration (MBC) was evaluated by transferring 10 µL from the well showing no bacteria growth after MIC assay, on MH agar. After 24h period of incubation at 37 °C, the bacterial growth was examined and the MBC was determined as the lowest concentration of the sample having bactericidal activity.

3.4. Molecular docking procedure

Molecular docking studies were performed by using Auto Dock 4.2 program package [80]. The optimization of all the geometries of ligands was carried out by ACD (3D viewer) software (http://www.filefacts.com/acd3d-viewer-freeware-info) and the three dimensional structure of PDB (PDB: 5F7V) [81] was obtained from the RSCB protein data bank (https://www.rcsb.org/). Before docking, the water molecules have been erased and the missing hydrogens besides to Gasteiger charges were then added to the system during the receptor preparation input file. Then, the AutoDock Tools were used for the preparation of all ligands and protein files (PDBQT). Pre-calculation of grid maps was performed by Auto Grid for saving a lot of time during docking procedure and the docking calculation was carried out by a grid per map with 40 × 40 × 40 A˚ points of all PDB used besides to the grid-point spacing of 0.375 A ˚, that was centered on the receptor structure in order to determine the active site and the visualization and analysis of interactions were performed using Discovery Studio 2017R2 (https://www.3dsbiovia.om/products/collaborative-science/biovia-discovery-studio/). Further, all molecular docked models for the cavities 3D were prepared via PyMOL viewer v. 0.99 [82].

3.5. Statistical analysis

Statistical analysis was carried out using Graph Pad Prism 7.0 (Graph Pad Software Inc., CA, USA). The experimental data of the antibacterial activity expressed in inhibition zone are presented as mean ± standard error of the mean (SEM). Student’s t-test was used to assess the difference between two groups. For significant differences among three or more groups, one-way ANOVA with post hoc analysis was performed.

4. Conclusions

In summary, oleanolic acid (OA-1) isolated from olive pomace (Olea europaea L.) was used as a starting material to prepare a series of new (OA-1)-phtalimidines coupled 1,2,3-triazole derivatives by application of the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. All the synthesized compounds were obtained in good yields (70–98%). The designed click products were assessed for their antibacterial activity toward four bacterial strains (gram-positive: S. aureus and L. monocytogenes; gram-negative: S. thyphimurium and P. Aeruginosa). Significant antibacterial activities were observed notably against L. Monocytogenes. Interestingly, compound 18g exhibited the highest activity toward this bacterium (MIC = 9.48 µmol/L) followed by derivatives 18d (MIC = 9.56 µmol/L) and 18h (MIC = 9.89 µmol/L), compared to the reference antibiotic Tetracycline. In silico molecular docking study revealed that the chosen lead compounds 18c, 18d, 18h and 18k can fit in well with the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes. This study lays a strong starting pointfor future efforts of optimization and expansion of the antibacterial spectrum of the (OA-1)-Phtalimidines derivatives against gram-positive bacteria, as well as gaining deeper insights into the mechanism of action and resistance potential of thesenewly developed semi-synthetic compounds.

Supplementary Materials

The following supporting information can be downloaded at: Preprints.org.

Author Contributions

Conceptualization, Methodology and Data curation, G.L.; Software, Visualization, Validation, M.H.; Writing—Original draft preparation, G.L. and M.H.; Conducting the testing of antibacterial activity, A.D. and A.M.; Validation, A.M. and A.H.H.; Supervision, A.R., A.M.L. and A.Da.; Writing- Reviewing and Editing, H.B.J. and M.O. The manuscript was a collaborative effort by all authors. The final version of the manuscript has received approval from all the authors.

Funding

We acknowledge the financial support within Hubert Curien-Utique program (PHC-Utique) by Campus France, the Ministry of Higher Education and Scientific Research of Tunisia and Le Havre Normandie University, France. We are also grateful to King Saud University, Riyadh, Saudi Arabia for the Researchers Supporting Project number (RSP202317).

Institutional Review Board Statement

Not applicable

Informed Consent Statement

Not applicable

Data Availability Statement

The source data underlying tables and figures are available from the authors upon request.

Acknowledgments

We thank Campus France; Ministry of Higher Education and Scientific Research of Tunisia and Le Havre Normandie University, France for financial support from Hubert Curien-Utique program (PHC-Utique). Also our thanks go to King Saud University, Riyadh, Saudi Arabia for Researchers Supporting Project number (RSP2023R17).

Conflicts of Interest

The authors declare no financial competing interest.

Sample Availability

Samples of the compounds are available from the authors upon request.

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Figure 1. Oleanolic acid (OA-1) and representative biologically active molecules including Isoindolinone and 1,2,3-Triazole.
Figure 1. Oleanolic acid (OA-1) and representative biologically active molecules including Isoindolinone and 1,2,3-Triazole.
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Scheme 1. Synthesis of the azidoacetyl 4. Reagents and Conditions: (i) K2CO3, BnBr, DMF, rt; (ii) ClCH2COCl, DMAPcat., DCM, 0 °C; (iii) NaN3, DMF, 75 °C.
Scheme 1. Synthesis of the azidoacetyl 4. Reagents and Conditions: (i) K2CO3, BnBr, DMF, rt; (ii) ClCH2COCl, DMAPcat., DCM, 0 °C; (iii) NaN3, DMF, 75 °C.
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Scheme 2. Straightforward synthesis of substrates 6-8. Reagents and Conditions for 6a-d and 6g: (i) imides (5a-d, R = H and 5g, R = OH) (1 equiv), NaH or K2CO3 (1.1 equiv), propargyl bromide (1.2 equiv), DMF, reflux; Reagents and Conditions for 6e and 6f: (ii) imide (5a, R = H) (1 equiv), but-3-yn-1-ol or but-3-yn-2-ol (1.2 equiv), Ph3P (1 equiv), DEADcat, THF, rt; Reagents and Conditions for 7a and 7e: iii) imides 6a and 6e (1 equiv), NaBH4 (4 equiv), MeOH, 0–20 °C. Reagents and Conditions for 8g: iv) a) 6g (1 equiv), NaBH4 (4 equiv), MeOH, 0–20 °C; b) Ac2O (2 equiv), Et3N (2 equiv), DMAPcat, DCM, rt.
Scheme 2. Straightforward synthesis of substrates 6-8. Reagents and Conditions for 6a-d and 6g: (i) imides (5a-d, R = H and 5g, R = OH) (1 equiv), NaH or K2CO3 (1.1 equiv), propargyl bromide (1.2 equiv), DMF, reflux; Reagents and Conditions for 6e and 6f: (ii) imide (5a, R = H) (1 equiv), but-3-yn-1-ol or but-3-yn-2-ol (1.2 equiv), Ph3P (1 equiv), DEADcat, THF, rt; Reagents and Conditions for 7a and 7e: iii) imides 6a and 6e (1 equiv), NaBH4 (4 equiv), MeOH, 0–20 °C. Reagents and Conditions for 8g: iv) a) 6g (1 equiv), NaBH4 (4 equiv), MeOH, 0–20 °C; b) Ac2O (2 equiv), Et3N (2 equiv), DMAPcat, DCM, rt.
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Scheme 3. Straightforward synthesis of substrates (±)-12-17. Reagents and Conditions for synthesis of phtalimidines (±)-12: (i) homophtalic acid 9, gaseous HCl, reflux; (ii) ethyl homophtalate 10 (1 equiv), NBS (1.5 equiv), AIBNcat, CCl4, 60 °C. (iii) diethyl α-bromophthalate 11 (1 equiv), amine (2 equiv), CH3CN, rt; Reagents and Conditions for synthesis of phtalimidines (±)-13 and (±)-14:(iv) phtalimidines (±)-12a-f (1 equiv), K2CO3, alkylyl bromide (1.2 equiv), CH3CN, reflux; Reagents and Conditions for (±)-15f: (v) phtalimidine (±)-14f (1 equiv), NaOH (2 equiv), EtOH/H2O, rt then aqueous 1M HCl, 0 °C; Reagents and Conditions for (±)-16b: (vi) acid (±)-15b (1 equiv), EDCI (1 equiv), DMF, rt; Reagents and Conditions for (±)-17e: (vii) phtalimidine (±)-14e (1 equiv), LiBH4 (2 equiv), DCM, rt.
Scheme 3. Straightforward synthesis of substrates (±)-12-17. Reagents and Conditions for synthesis of phtalimidines (±)-12: (i) homophtalic acid 9, gaseous HCl, reflux; (ii) ethyl homophtalate 10 (1 equiv), NBS (1.5 equiv), AIBNcat, CCl4, 60 °C. (iii) diethyl α-bromophthalate 11 (1 equiv), amine (2 equiv), CH3CN, rt; Reagents and Conditions for synthesis of phtalimidines (±)-13 and (±)-14:(iv) phtalimidines (±)-12a-f (1 equiv), K2CO3, alkylyl bromide (1.2 equiv), CH3CN, reflux; Reagents and Conditions for (±)-15f: (v) phtalimidine (±)-14f (1 equiv), NaOH (2 equiv), EtOH/H2O, rt then aqueous 1M HCl, 0 °C; Reagents and Conditions for (±)-16b: (vi) acid (±)-15b (1 equiv), EDCI (1 equiv), DMF, rt; Reagents and Conditions for (±)-17e: (vii) phtalimidine (±)-14e (1 equiv), LiBH4 (2 equiv), DCM, rt.
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Scheme 4. Click synthesis of 1,2,3-triazole conjugate 18a. Reagents and Conditions: (i) Phtalimidine 6a (1 equiv), azide 4 (1 equiv), CuSO4.5H2O (0.2 equiv), sodium ascorbate (0.4 equiv), DCM/H2O, rt, 24 h.
Scheme 4. Click synthesis of 1,2,3-triazole conjugate 18a. Reagents and Conditions: (i) Phtalimidine 6a (1 equiv), azide 4 (1 equiv), CuSO4.5H2O (0.2 equiv), sodium ascorbate (0.4 equiv), DCM/H2O, rt, 24 h.
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Scheme 5. Click synthesis of 1,2,3-triazole hybrids 18a-t. Reagents and Conditions: (i) substrates 6-8, (±)-12-17 (1 equiv), azide 4 (1 equiv), CuSO4.5H2O (0.2 equiv), sodium ascorbate (0.4 equiv), DCM/H2O, rt, 24 h.
Scheme 5. Click synthesis of 1,2,3-triazole hybrids 18a-t. Reagents and Conditions: (i) substrates 6-8, (±)-12-17 (1 equiv), azide 4 (1 equiv), CuSO4.5H2O (0.2 equiv), sodium ascorbate (0.4 equiv), DCM/H2O, rt, 24 h.
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Scheme 6. Click synthesis of 18v. Reagents and Conditions: (i) diproporgylated substrate (±)-13d, azide 4 (2 equiv), CuSO4.5H2O (0.2 equiv), sodium ascorbate (0.4 equiv), DCM/H2O, rt, 24.
Scheme 6. Click synthesis of 18v. Reagents and Conditions: (i) diproporgylated substrate (±)-13d, azide 4 (2 equiv), CuSO4.5H2O (0.2 equiv), sodium ascorbate (0.4 equiv), DCM/H2O, rt, 24.
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Figure 2. 3D binding modes of “Tetracycline” (green color) superimposed with the co-crystalized ligand “Cycloalternan” (yellow color) in the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
Figure 2. 3D binding modes of “Tetracycline” (green color) superimposed with the co-crystalized ligand “Cycloalternan” (yellow color) in the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
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Figure 3. 3D binding modes of (A) co-crystalized ligand; (B) derivative 18c; (C) derivative 18d; (D) derivative18h and (E) derivative 18k within the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
Figure 3. 3D binding modes of (A) co-crystalized ligand; (B) derivative 18c; (C) derivative 18d; (D) derivative18h and (E) derivative 18k within the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
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Figure 4. 2D binding modes of “Tetracycline”, (A’) derivative 18c; (B’) derivative 18d; (C’) derivative 18h and (D’) derivative 18k within the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
Figure 4. 2D binding modes of “Tetracycline”, (A’) derivative 18c; (B’) derivative 18d; (C’) derivative 18h and (D’) derivative 18k within the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
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Table 1. Antibacterial activity of 18a-v expressed in Zone of Inhibition (mm).
Table 1. Antibacterial activity of 18a-v expressed in Zone of Inhibition (mm).
Compound S. aureus
ATCC 25923		 L. monocytogenes
ATCC19115		 S. thyphimurium
ATCC14080		 P. aeruginosa
ATCC27853
OA-1 12.2 ± 1.7a na 12.8 ± 1.1c,d 14.0 ± 1.0c
18a na 12.0 ± 1.4b na na
18b na 15.0 ± 0.0e na na
18c na 9.5 ± 2.1a na na
18d na 12.0 ± 0.0b na na
18e na 14.0 ± 0.0d na na
18f na 9.0 ± 0.0a 12.0 ± 0.7c na
18g na 15.0 ± 0.0e na na
18h na 12.0 ± 0.0b na 16.0 ± 0.0d
18i na na na na
18j na na na na
18k na 14.0 ± 0.7d 10.0 ± 1.4a na
18l na na na na
18m na 13.0 ± 0.0c na 13.0 ± 0.0b
18n na na na na
18o na na na na
18p na na na na
18q na 12.0 ± 0.7b 11.0 ± 0.0b na
18r na na na na
18s na na na na
18t na na na na
18v na na na na
TET 18.0 ± 0.0b 12.5 ± 0.0b,c 12.0 ± 0.1c 11.0 ± 1.4a
TET: tetracycline; na: not actif.
Table 2. Antibacterial activity of compounds 18a-v expressed in MIC and MBC (µM).
Table 2. Antibacterial activity of compounds 18a-v expressed in MIC and MBC (µM).
Compound S. aureus L. monocytogenes S. thyphimurium P. aeruginosa
ATCC 25923 ATCC19115 ATCC14080 ATCC 27853
MIC MBC MIC MBC MIC MBC MIC MBC
OA-1 78.02 171.18 147.9 1353.18 59.97 633.66 21.39 85.59
18a na na 12.4 1588.22 na na na na
18b na na 59.97 1917.99 na na na na
18c na na 21.13 1353.18 na na na na
18d na na 9.56 151.86 na na na na
18e na na 39.01 2496.67 na na na na
18f na na 39.01 624.16 78.02 312.08 na na
18g na na 9.48 155.65 na na na na
18h na na 9.89 633.66 na na 158.41 633.66
18i na na na na na na na na
18j na na na na na na na na
18k na na 18.48 591.63 147.9 591.63 na na
18l na na na na na na na na
18m na na 21.39 85.59 na na 42.79 171.18
18n na na na na na na na na
18o na na na na na na na na
18p na na na na na na na na
18q na na 16.88 540.44 135.11 540.44 na na
18r na na na na na na na na
18s na na na na na na na na
18t na na na na na na na na
18v na na na na na na na na
TET 4.5 288 576.01 1152.02 9 576.01 576.01 1152.02
TET: tetracycline; na: not actif.
Table 3. Binding energy of the docked compounds in the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
Table 3. Binding energy of the docked compounds in the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
Compound Binding Energy (kcal/mol)
18a -10.7
18b -10.8
18c -11.7
18d -12.0
18e -10.7
18f -10.9
18g -10.9
18h -12.3
18k -11.6
18m -10.6
18q -11.0
Tetracycline -11.1
Table 4. Docking results of derivatives with lowest binding energy score and interacting residues the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
Table 4. Docking results of derivatives with lowest binding energy score and interacting residues the binding cavity of ABC substrate-binding protein Lmo0181 from L. monocytogenes.
Docked Compounds Interacting Residues Binding Energy (kcal/mol)
18c van der Waals: Gln193, Val195, Thr196, Asn198, Leu249, Gln252, Leu272, Phe299, Glu391; H bond: Trp271*(3.13); Alkyl/Pi–Alkyl:Pro194(3.92)(4.90)(5.06), Pro246(5.14), Leu253(4.11)(5.26), Pro254(4.85)(5.21), Leu394(5.37) ; Pi-Pi: Phe70(4.10), Trp271(4.33). -11.7
18d van der Waals: Phe38, Asn71, Val148, Val195, Thr196, Asn198, Glu199, Pro246, Leu253, Thr268, Gly269, Asn301, Asn387, Leu394; H bond: Trp271*(3.15), Glu391**(3.27)(3.38); Alkyl/Pi–Alkyl: Phe70(5.14), Pro194(4.86), Pro254(4.77), Trp271(4.89), His297(4.07), Phe299 (4.43) (4.76); Pi-Pi: Phe70(3.87), Trp271(4.66) ; Pi-Anion: Glu391(3.81). -12.0
18h van der Waals: Phe38, Asn71, Asp72, Phe74, Thr196, Asn198, Glu199, Thr268, Gly269, Asn301, Glu338, Ser383, Asn387 ; H-bond: Thr75*(3.13), Asn*380(2.55), Asp384*(2.12); C-H bond: Thr75(3.28), Asp384(3.71)(3.76); Alkyl/Pi–Alkyl: Phe70(5.05)(5.26), Pro194(4.27)(5.19); Pi-Pi: Phe70(5.06), Trp271(4.47), Phe299(5.16). -12.3
18k van der Waals: Phe38, Asn71, Val148, Val195, Thr196, Asn198, Glu199, Pro246, Leu249, Gln252, Leu253, Thr268, Gly269, Asn301, Asn387 ; H-bond: Trp271*(3.25); Alkyl/Pi–Alkyl: Phe70(5.28), Trp271(4.89), Pro194(4.69)(4.89)(5.34) (5.36), Pro254(4.58), His297(3.99), Phe299(4.40)(4.72); Pi-Pi: Phe70(3.77), Trp271(4.45) ; Pi-Anion: Glu391(3.68). -11.6
Tetracycline van der Waals: Phe38, Ser39, Pro194, Asn198, Glu199, Leu253, Leu272, Asn301, Asn387; H-bond: Thr196*(2.12), Glu338*(2.82); Alkyl/Pi-Alkyl: Phe70(4.42), Trp271(5.47);Pi-Pi: Trp271(4.09) -11.1
*: One H-bond; **: Two H-bonds.
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